DDL2018 Digital Proceedings by info-ddl-conference

The Aerosol Society 2018 DRUG DELIVERY TO THE LUNGS 2018

DDL - Shaping the future of nasal and pulmonary Drug Delivery

12th, 13th & 14th December 2018

Proceedings

www.ddl-conference.com

Thank you to our Platinum Sponsors

Drug Delivery to the Lungs Volume 29 2018 DRUG DELIVERY TO THE LUNGS, VOLUME 29, 2018 - CONFERENCE PAPERS Summaries: Journal of Aerosol Medicine and Pulmonary Drug Delivery, VOL. 32, NO.2

Page Numbers

Mechanistic Understanding of Microparticle Formation in Respiratory Applications Reinhard Vehring1 1University

1-3

of Alberta, Department of Mechanical Engineering, Edmonton, Alberta, T6G 1H9, Canada

Re-defining Asthma – What’s all the fuss about? Kian Fan Chung

National Heart & Lung Institute, Imperial College London, & Royal Brompton Hospital, London, UK Three drugs in one inhaler for COPD: where are we going with triple therapy Dr Brian Lipworth Scottish Centre for Respiratory Research University of Dundee Ninewells Hospital and Medical School University of Dundee, DD1 9SY

The Cystic Fibrosis ‘explosion’ - New medicines and unmet needs Bruce K. Rubin

6-8

Children's Hospital of Richmond at Virginia Commonwealth University, 1000 E Broad St, Richmond, VA 23298 USA Inspired or Expired Jane Scullion

University Hospitals of Leicester and Education Lead UKIG. Member ADMIT (Inhlaers4U) The problem with critical and non-critical inhaler errors Federico Lavorini Department of Experimental and Clinical Medicine University of Florence, Largo Brambilla 3, 50134 Florence, Italy

10-11

Advocacy in Respiratory Medicine – getting our voice heard for change Bronwen Thompson

UK Inhaler Group Quantification of beclomethasone dipropionate in living respiratory epithelial cells using Infrared Spectroscopy Wachirun Terakosolphan, Ali Altharawi, K. L. Andrew Chan & Ben Forbes Institute of Pharmaceutical Science, King’s College London, 150 Stamford St, London SE1 9NH, UK

13-16

Respiratory Development: Thinking Outside the Box - Big Data for Respiratory Medicines? David Mannino1,2 1GlaxoSmithKline,

5 Crescent Drive, Philadelphia, PA, 19112, USA 2University of Kentucky, 111 Washington Avenue, Lexington, KY, USA

17-19

Robust Method to Predict Inspiratory Flowrate from the Acoustic Signature of Swirl-Based DPIs using Deep Learning Lim K M1, Harris D S1, Sail P1 & Parry M2

20-23

1PA

Consulting, Global Innovation and Technology Centre, Back Lane, Melbourn, Herts, SG8 6DP, UK 2 Intertek Melbourn, Saxon Way, Melbourn, Herts, SG8 6DN, UK Bioengineering Strategies for Targeted Therapeutic Delivery to the Lungs Dr JosuĂŠ Sznitman

24-26

Sc. Department of Biomedical Engineering, Technion, Israel-Institute of Technology Cannabis, Cigarette Smoking and Lung Function â&#x20AC;&#x201C;not all downhill? Philip W. Ind

27-29

Respiratory Medicine Imperial College London Gene Delivery to Lung Epithelial Cells Using a Cell Penetrating Peptide Larissa Gomes dos Reis1, Maree Svolos1, Lyn M Moir1, Rima Jaber2, David Fecher2, Norbert indhab2 Paul M Young1 & Daniela Traini1 1Respiratory

Technology, Woolcock Institute of Medical Research and Discipline of Pharmacology, Faculty of Medicine and Health, The University of Sydney, NSW, 2037, Australia 2 Evonik Nutrition and Care GmbH, Kirschenallee, 64293, Darmstadt, Germany

30-33

Assessing Central and Peripheral Pulmonary Deposition of Three Fluticasone Propionate (FP) Dry Powder Inhaler (DPI) formulations with Different Aerodynamic Particle Size Distributions (APSD) in Healthy Subjects via Population Pharmacokinetics Modeling Stefanie K Drescher1, Brandon Seay1, Mong-Jen Chen1, Abhinav Kurumaddali1, Uta Schilling1, Yuanyuan Jiao1, Jie Shao1, Lawrence Winner2, Sandra M Baumstein3, Mutasim N. Abu-Hasan2, Renishkumar Delvadia4, Christine Tabulov1, Murewa Oguntimein1, Bavna Saluja4, Jag Shur6, Robert Price6, Michael Hindle7, Xiangyin Wei7, Denise S. Conti4, Juergen Bulitta1 & Guenther Hochhaus1 1Department

of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, Orlando, FL, USA Division of Pediatric Pulmonary and Sleep Medicine, Department of Paediatrics, College of Medicine, University of Florida, Gainesville, FL, USA 3Department of Pharmacotherapy and Translational Research, College of Pharmacy, University of Florida, Gainesville, FL, USA 4 Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA 5 Department of Statistics, College of Liberal Arts & Sciences, University of Florida, Gainesville, FL, USA 6 University of Bath, Bath, Great Britain 7Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, USA 2

34-38

In Vitro Activity of Inhalable Microparticles Containing Anti-Tb Drugs and an Efflux Pump Inhibitor against Mycobacteria Infections Irene Rossi1, Francesca Buttini, Elliott KMiller1, Filippo Affaticati1, Fabio Sonvico1, Marco Pieroni1,2, Nitesh K Kunda2, Pavan Muttil2 & Ruggero Bettini University of Parma, Food and Drug Department, Parco Area delle Scienze 27/A, Parma, PR, Italy of New Mexico, College of Pharmacy, Department of Pharmaceutical Sciences, 2705 Frontier Avenue NE, Albuquerque, NM, USA

2University

39-43

Dry Powder Inhalation of Glucagon-like Peptide-1 for Management of Type-2 Diabetes Mellitus Sanketkumar Pandya1,2, Durgesh Kumar1,2, Swati Gupta1, Kalyan Mitra1, Anil N Gaikwad1 & Amit Misra1 1CSIR-Central

Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, 226031, India 2Academy of Scientific and Innovative Research, New Delhi - 110025, India

44-47

Understanding the Inspiratory Manoeuvre and Why it’s Important to DPI Design David Harris

48-51

Cambridge Healthcare Innovations Controlling the size of nebulised droplets by pinning surface waves for precise delivery of aerosolised medicine Elijah Nazarzadeh1, Nikita Lomis2, Xi King1, Rab Wilson, Satya Prakash1, Manlio Tassieri1, Julien Reboud1, Jon Cooper1,2

52-55

1Division

of Biomedical Engineering, University of Glasgow, Rankine Building, Glasgow G12 8LT, UK Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, 3775 University Street, Montreal H3A 2B4, Canada

Efficient Engineering Simulation to Inform and Optimise Capsule Inhaler Design Stuart Abercrombie1 1Team

56-59

Consulting, Abbey Barns, Duxford Road, Ickleton, Cambridge, CB10 1SX, UK

The future beyond inhalers – endobronchial intervention in COPD Pallav L Shah Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK Chelsea & Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK 3National Heart & Lung Institute, Imperial College, DoveHouse Street, London SW3 6LY, UK 1

60-61

From modelling to synthesis to formulation to microbe: A multi-disciplinary approach to developing treatment for multi-drug resistant respiratory infection Arcadia Woods1, Mark Laws2, Kazi Nahar2, Shirin Jamshidi & Khondaker, Miraz Rahman2, Charlotte Hind3, Mark Sutton3,2 Development Group, School of Cancer and Pharmaceutical Sciences, King’s College London, 150 Stamford Street, London SE1 9NH, UK 2 Drug Discovery Group, School of Cancer and Pharmaceutical Sciences, King’s College London, 150 Stamford Street, London SE1 9NH, UK 3Research and Development Institute, National Infections Service, Public Health England, Salisbury SP4 0JG, Wiltshire, United Kingdom 1Medicines

62-65

Fluidised Powder Blending to Control Particle-Particle Interaction – The Future of DPI Formulation Afzal Mohammed1, Jasdip Koner2, Olaitan Abiona1 & David Wyatt2 1Aston

University, Aston Triangle, Birmingham, B4 7ET, United Kingdom, 2 Aston Particle Technologies Ltd, Aston University, Aston Triangle, Birmingham, B4 7ET, United Kingdom

66-69

Probing the Aerodynamic Particle Size Distribution of Dry Powder Inhaler Combination Products Foster® NEXThaler® and Seretide® Diskus® using Single Particle Aerosol Mass Spectrometry (SPAMS) Bradley Morrical, Martin Jetzer & Stephen Edge Novartis Pharma AG, Global Development, Novartis Campus, Lichtstrasse 35, Basel, 4056, Switzerland

70-74

Antisense Technologies – An Emerging Field for the Development of New Therapeutic Molecules: Anti-GATA3 DNAzyme as a Prototypic Example Harald Renz

Institute of Laboratory Medicine, Philipps University Marburg, Germany

Development of a theoretical model to predict pMDI spray force, using alternative propellant systems Barzin Gavtash1, Andy Cooper2, Sarah Dexter2, Chris Blatchford2 & Henk Versteeg1 Loughborough University, Epinal Way, Loughborough, LE11 2TL, United Kingdom 2 3M United Kingdom PLC, Charnwood Campus, 10 Bakewell Road, Loughborough, LE11 5RB, United Kingdom 1

76-79

In vitro testing of the new Space Chamber Slim with salbutamol sulfate, fluticasone propionate, and ipratropium bromide pressurized metered dose inhalers Michael Nairn1, Andrew Lorbeer1, Kurt Nikander2, Scott Courtney1 1Medical

Developments International Limited, 4 Caribbean Drive, Scoresby, 3179, Australia 2 InDevCo AB, Handbollsvagen 1B, Nykoping, 61164, Sweden

80-84

Evaluation of Pressurised Metered Dose Inhaler (pMDI) Plume Spray Force When a Valved Holding Chamber (VHC) is Present: A Proof of Concept Investigation to Identify Propensity for Premature Inhalation Valve Opening Mark W Nagel 1, Jolyon P. Mitchell2 & Jason A Suggett 1 1 2

85-88

Trudell Medical International, 725 Third Street, London, Ontario, NV5 5G4, Canada Jolyon Mitchell Inhaler Consulting Services Inc., 1154 St. Anthony Road, London, N6H 2R1, Canada

Hpmc Capsules In Dpis. Evaluation Of The Puncturing Force Using Capsules Made With Different Manufacturing Methods And Compositions Deepak Patil1, Jnanadeva Bhat1, Sanjay Powale1, Ajay Giripunje1, Justin Kalafat2, Fernando Diez3 ACG SciTech Center, 7 Prabhat Nagar Patel Estate, Jogeshwari (West), Mumbai, 400102, India 2 ACG North America, LLC, 262 Old New Brunswick Rd, Piscataway, NJ, USA 3 ACG Europe, 159 Princes Gardens, W3 0LS, London, United Kingdom 1

89-92

Comparison of Aerosol Characteristics and Nicotine Delivery by Conventional Pharmaceutical Inhalation Devices and Electronic Nicotine Delivery Systems (ENDS) Weiling Li, Qiang Wang, and Raymond W Lau

93-95

Altria Client Services LLC, 601 E Jackson Street, Richmond, VA, 23219, U.S.A

Number size distribution of particles dosed by MDI and DPI inhalers Lucie Ondráčková1, Jana Kozáková1, Jakub Ondráček1, Vladimír Ždímal1, Ludmila Mašková1 & Stavros Kassinos2 Department of Aerosol Chemistry and Physics, Institute of Chemical Process Fundamentals of the CAS, Rozvojová 135, Prague, 165 02, Czech Republic 2 Department of Mechanical & Manufacturing Engineering, University of Cyprus, 1 Panepistimiou Avenue, 2109 Aglantzia, Nicosia, Cyprus 1

96-99

Biopharmaceutics of (R)-roscovitine by Inhalation Magda Swedrowska1, Zachary Enlo-Scott1, Laurent Meijer2 & Ben Forbes1 Institute of Pharmaceutical Science, King’s College London,150 Stamford Street, London SE1 9NH, UK 2 ManRos Therapeutics, Centre de Perharidy, Roscoff, 29680, France 1

100-103

Interpretation of manual actuation profiles from nasal unit-dose spray devices Benedicte Grosjean1, Gerallt Williams1, Fabien Adam1, David Chopard1, Pierre Schwartz1, Janick Cabiddu1 1

104-107

Aptar Pharma, Route des Falaises, Le Vaudreuil, 27100, France

Albumin as an alternative dispersion enhancer for inhalable siRNA spray dried powders Michael Y.T. Chow,1 Philip C.L. Kwok2, Hak-Kim Chan2 & Jenny K.W. Lam1 Department of Pharmacology & Pharmacy, LKS Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong 2Sydney Pharmacy School, Faculty of Medicine and Health, Pharmacy and Bank Building A15, The University of Sydney, Camperdown, NSW 2006, Australia 1

108-111

Understanding the motion of rotating hard shell capsules in dry powder inhalers Benedict Benque1 & Johannes Khinast1 1

112-116

Institute of Process and Particle Engineering, Graz University of Technology, Inffeldgasse 13, Graz 8010, Austria

Impact of Budesonide Particle Shape on Uptake by Respiratory Cells and Macrophages Sarah Zellnitz1, Marie-Theres Müller1,2 & Eleonore Fröhlich1,2 Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, Graz, 8010, Austria 2 Center for Medical Research, Medical University of Graz, Stiftingtalstraße 24, Graz, 8010, Austria 1

117-121

In silico Prediction of Pharmacokinetic Parameters after Cisplatin Intravenous and Endotracheal Administration Using GastroPlusTM Software Selma Chraibi1, Jessica Spires2, Karim Amighi1 & Nathalie Wauthoz1 Laboratory of Pharmaceutics and Biopharmaceutics, Faculty of Pharmacy, Université libre de Bruxelles (ULB), Boulevard du Triomphe, B-1050 Brussels, Belgium, schraibi@ulb.ac.be 2 Simulation Plus, Inc., 42505 10th Street West, Lancaster 93534, United States of America (USA) 1

122-125

Dissolution Of Crystalline And Amorphous Particles In The Aerosol Phase Natalie Armstrong Green1, Allen Haddrell1, Jonathan Reid1, David Lewis2 & Tanya Church2 School of Chemistry, University of Bristol, Bristol, BS81TS, United Kingdom 2Chiesi Limited, Bath Road Industrial Estate, Chippenham, Wilts, United Kingdom 1

126-129

Characterisation of Nanomaterial in Nebulised Formulations for Clinical Products: Impact of Particle Size on Dissolution and Predicted Deposition Pattern. Carolyn Stevenson, 1Henrik Kristensson1, Karin Carlsson1, Pia Mattsson1, John Salomonsson1, Jan Olof Svensson1 and Ulrika Tehler1 1

Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca Gothenburg, Sweden

130-133

Directly Probing the Dynamic Behaviour of Particles Originating from DPI and MDI Starting Formulations Allen Haddrell1, David Lewis2, Tanya Church2, Jonathan Reid1 University of Bristol, Bristol, UK 2 Chiesi Farmaceutici S.p.A., Chippenham, UK 1

134-137

Relationship Between The Secondary Structure Of The Peptide Base siRNA Carrier And Effective Gene Silencing Effect On Lung Epithelial Cells Yingshan Qiu ,1 Bui Tam2, Winne Y.W. Chung1, James Mason2 & Jenny K.W. Lam1 1 Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong 2 Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London, United Kingdom

138-142

Ranking In Vitro Dissolution Of Orally Inhaled Drug Substance Powders In A Time-Efficient Manner Frans Franek,1 Linn Nilsson1, Helena Thörn1, Rebecca Fransson2, Ulrika Tehler2 AstraZeneca R&D, Pepparedsleden 1, Mölndal, 43183, Sweden 1 Pharmaceutical Technology & Development Inhalation, Operations 2Pharmaceutical Sciences, IMED

143-146

Targeted PEG-Poly(glutamic acid) Polymers For The Delivery of Proteins Into The Lung Epithelium Huitong Lucy Li,1 Alejandro Nieto-Orellana1, Franco H. Falcone1, Cynthia Bosquillon1, Gemma Keegan2, Nick Childerhouse2, Giuseppe Mantovani*1 and Snow Stolnik*1 1School 2

of Pharmacy, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.

147-150

Vectura Group plc, One Prospect West, Chippenham, SN14 6FH, UK

Development of an In Vitro Solubility Test as a Tool for Predicting Lung Retention of Poorly Water Soluble Compounds Stefani Mariarita1, Cesari Nicola 1, Corsaletti Roberto1, Fioni Alessandro1, Saccani Francesca1, Volta Roberta1, Brogin Giandomenico1 & Puccini Paola1 1

151-154

Chiesi Farmaceutici S.p.A., Largo Belloli 11/A, Parma, 43122, Italy

Systematic Development of an Inhaler Device for the Use in Horses: The EquiHaler ® Herbert Wachtel1, Marcus Rahmel1 Guido Endert2 & Benjamin Franzmann3 Boehringer Ingelheim Pharma GmbH & Co KG, Binger Strasse 173, 55216 Ingelheim, Germany DESIGNquadrat GbR, Schmiedhofsweg 1, 50769 Köln, Germany 3 Boehringer Ingelheim Vetmedica GmbH, Binger Strasse 173, 55216 Ingelheim, Germany 1 2

155-158

Safety and effectiveness of sodium-colistimethate-loaded nanostructured lipid carriers (SCM-NLC) against P. aeruginosa: in vitro and in vivo studies following pulmonary and intramuscular administration C. Vairo1,2, J. Basas3, M. Pastor1, G. Gainza1, M. Moreno-Sastre2,4, X. Gomis3, A. Fleischer5,6, E. Palomino5,6, D. Bachiller5,6, F.B. Gutiérrez7, J.J. Aguirre1,7, A. Esquisabel2,4, M. Igartua2,4, E. Gainza1, R.M. Hernandez2,4, J. Gavaldà3 & J.L. Pedraz2,4 1

BioPraxis Research AIE, R&D Department, Hermanos Lumière, 5, 01510 Miñano (Araba), Spain

NanoBioCel Group, Laboratory of Pharmaceutics, University of the Basque Country, School of Pharmacy, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain

3Antimicrobial Resistance Laboratory, Vall d’Hebron Research Institute (VHIR). Infectious Diseases Department, Hospital Universitari Vall d’Hebron, Passeig de la Vall d'Hebron, 119-129, 08035 Barcelona

159-163

Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). VitoriaGasteiz, Spain

Fundación Investigaciones Sanitarias Islas Baleares (FISIB), Development and Regeneration Program, Ctra. Sóller km 12, 07110 Bunyola (Balearic Islands), Spain

6Consejo

Superior de Investigaciones Científicas (CSIC), Ctra. Sóller km 12, 07110 Bunyola (Balearic Islands), Spain

Department of Pathological Anatomy, Hospital Universitario de Álava (HUA). José Atxotegi, 01009 Vitoria-Gasteiz, Spain

Telehealth Ready: Performance of the Amiko Respiro Sense connected technology with Merxin DPIs Philippe Rogueda1, Martijn Grinovero2, Luca Ponti2, Graham Purkins1 & Oliver Croad1 Ltd, King's Lynn Innovation Centre, Innovation Way, King’s Lynn, PE30 5BY, UK 2 Amiko Digital Health Limited, Salisbury House 31 Finsbury Circus, London, EC2M 5QQ, UK 1Merxin

164-167

Characterisation of Zinc Oxide as an Alternative to Aluminium Hydroxide in Nasal Vaccination Marie Hellfritzsch & Regina Scherließ

168-172

Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany

Cannabidiol in a DPI – maximising the spray drying yield of HPMC matrix particles Tobias Gutowski & Regina Scherließ

173-176

Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118, Kiel, Germany

Spray dried powders for nasal application - Influence of particle morphology and filling process on aerosol generation Angelika Jüptner1, Ségolène Sarrailh2 & Regina Scherließ1

177-180

Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany 2 Aptar Pharma, Route des Falaises, 27100 Le Vaudreuil, France 1

Assessment of Aerosol Delivery during Simulated Invasive Ventilation, Non-invasive Ventilation and High Flow Nasal Therapy Gavin Bennett1, Mary Joyce1, Louise Sweeney1 & Ronan MacLoughlin1 1

Aerogen, IDA Business Park, Dangan, Galway, Ireland

181-184

Softpellets for high dose pulmonary delivery Christian Etschmann, Regina Scherließ

185-188

Department of Pharmaceutics and Biopharmaceutics, Kiel University Grasweg 9a, 24118 Kiel, Germany

The Technical Challenges of the Developing Existing Inhalation Drug Products into New Metered Dose Inhaler Designs Neha Patel, 1 Alex Slowey1 & Lester Harrison2 3M Drug Delivery Systems Division (DDSD), Charnwood Campus, 10 Bakewell Road, Loughborough, Leicestershire, LE11 5RB, UK. 2 3M Drug Delivery Systems Division (DDSD), 3M Center, St. Paul, MN 55144-1000, USA 1

189-192

Assessment of the effect of cannula choice and gas flow rate on aerosol delivery during high flow nasal therapy Louise Sweeney1, Mary Joyce1, Gavin Bennett1 & Ronan MacLoughlin1 1

193-196

Aerogen, IDA Business Park, Dangan, Galway, Ireland

High dose spray-dried powders for treating tuberculosis Mohammad Abdul Motalib Momin, Shubhra Sinha, Ian G Tucker & Shyamal C Das

197-200

School of Pharmacy, University of Otago, 18 Frederick Street, P.O. Box 56, Dunedin 9054, New Zealand

Impact of real time feedback from inhalation devices on patient satisfaction and adherence Bernhard Müllinger1, Tobias Kolb1 & Tobias Gessler2 1Vectura

GmbH, Robert-Koch-Allee 29, Gauting, 82131, Germany 2 University of Giessen and Marburg Lung Center, Klinikstraße 33, Gießen, 35392, Germany

201-204

Performance of a New Polymer-Based Vibrating Mesh Nebulizer: A Comparison to Metal-Based Mesh Nebulizer Chen-Hsiang Sang, Shih-Cherng Lin, Huang-Fei Chen, Hsin-Hua Tseng & Hsiao-Hui Lo

205-208

Department of R&D, Medical Division, MicroBase Technology Corp., No.756, Jiadong Rd., Bade Dist., Taoyuan City, 33464, Taiwan (R.O.C.)

Developing clinically relevant methodology for existing pMDI aerosol products when coupled to the Intelligent Control Inhaler Russell Down1, Hayden Beresford1, Chris Blatchford1, Andy Cooper1 & Stewart Griffiths1

209-212

13M

Drug Delivery Systems Ltd, Charnwood Campus, 10 Bakewell Road, Loughborough, Leicestershire LE11 5RB

Experimental and Theoretical Investigation of a New Approach to In-Use Impactor Quality Specifications Daryl L. Roberts1, Mårten Svensson2, Karolina Sandell2, Dennis Sandell3 1Applied

Particle Principles LLC, 17347 Westham Estates Court, Hamilton, VA 20158, USA Emmace Consulting AB, Medicon Village, S-223 81, Lund, Sweden 3S5 Consulting, Ekvägen 8, S-275 62, Blentarp, Sweden 2

213-216

Analytical Technology to Improve the Efficiency of a Through-Life Analysis on DPI Products Stefano Campo1, Lorena Gasparini1, Federica Polimeni1, Tatiana Salvo1 1

217--220

CMC, Drug Product Development Department, Chiesi Farmaceutici, Largo Belloli 11/A - 43122 - Parma - Italy

A closer look on the dispersion behaviour of Parteck® M DPI – based interactive mixtures: improving the fine particle fraction by the addition of fines Nancy Rhein1, Gudrun Birk2 & Regina Scherließ1

221-224

1Department 2Merck

of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany KGaA, Frankfurter Straße 250, 64293 Darmstadt, Germany

Impact of Relative Humidity and Powder Filling Level on the Electrostatic Charging Behaviour of Different Capsule Types Thomas Wutscher1,2, Sarah Zellnitz1, Mirjam Kobler3, Francesca Buttini1,4, Laura Andrade5, Veronica Daza5, Alberto Mercandelli6, Stefano Biserni6, Susana Ecenarro Probst7, Johannes Khinast1,2 & Amrit Paudel1,2 Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, 8010 Graz, Austria ² Institute of Process and Particle Engineering, Graz University of Technology, Inffeldgasse 13, 8010 Graz, Austria ³ MEGGLE Excipients and Technology, Megglestraße 6-12, 83512 Wasserburg, Germany 4 Food and Drug Department, University of Parma, Parco delle Scienze 27, 43121 Parma, Italy 5 Laboratorios Liconsa, S.A. C/ El tejido 2, 19200 Guadalajara, Spain 6 MG2, Via del Savena 18, 40065 Pianoro, Bologna, Italy 7 Qualicaps Avda. Monte Valdelatas 4, 28108 Alcobendas, Madrid, Spain 1

225-228

Establishing the Vitrocell® Powder Chamber as a particle size-selective platform for in vitro dry powder testing S. Steiner1, M. Hittinger2, K. Knoth2, H. Gross2, S. Frentzel1, A. Kuczaj1, J. Hoeng1, M. Peitsch1, T. Krebs3 PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchâtel, Switzerland 2 PharmBioTec GmbH, Science Park 1, 66123 Saarbrücken, Germany 3 Vitrocell® Systems GmbH, Fabrik Sonntag 3, 79183 Waldkirch, Germany 1

229-232

Physicochemical characterisation of inhalation grade lactose after the removal of intrinsic fines. Ioanna Danai Styliari1, Arian Mobli1 & Darragh Murnane1 1

233-236

School of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield, AL10 9AB, UK

Acoustic Spectral Analysis on Lactose for inhaled formulation using UA-AFM Cedric Thomas1, N. Pocholle1, E. Bourillot1, L. Kerriou², V. Gamerre², E. Lesniewska1 ICB UMR CNRS 6303, Univ. Bourgogne Franche-Comté, Dijon, France 2Armor Pharma, Armor Proteines SAS, Saint Brice-en-Cogles, France 1

237-240

Probing Subtle Variabilities in Seretide/Advair Batches by Evaluation of Drug-Lactose Aerosol Interaction Larissa Gomes dos Reis 1, Michele Pozzoli1 Paul M Young1,2, Robert Johnson2 1Woolcock

Institute of Medical Research, University of Sydney, 431 Glebe Point Road, Sydney, NSW 2037, Australia 2 Oz-UK Ltd, Unit 15, Lansdowne Court, Bumpers Way Chippenham Wiltshire, SN14 6RZ, UK

241-244

Application of Void Forming Index (VFI): Detecting agglomeration of mannitol with different physical properties Sunao Maruyama1, Shuichi Ando1 and Etsuo Yonemochi2 Formulation Technology Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan 2Department of Physical Chemistry, School of Pharmacy and Pharmaceutical Sciences, Hoshi University, 2-4-41, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan 1

245-248

Safety cabinet for droplet size measurement by laser diffraction Aurélie Doméné1, Maria Cabrera1, Michael Pasteur2, Marguerite Tulli3 & Laurent Vecellio 1 CEPR, INSERM U1100, University of Tours, 10 boulevard Tonnellé, Tours, 37032, France Pharmacie Centrale des Armées, TSA 30004, Fleury-les-Aubrais cedex, 45404, France 3Stratégie Santé, 19 rue Georges Clémenceau, Versailles, 78000, France 1

249-252

Insights into DPI sensitivity to humidity and its correlation to formulation physicochemical characteristics: a temporal study using two commercial budesonide products J T Pinto1, S Radivojev1,2, E Fröhlich1,2 & A Paudel1,3 Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, Graz, 8010, Austria 2 Center for Medical Research, Medical University of Graz, Stiftingtalstraße 24, Graz, 8010, Austria 3 Institute of Process and Particle Engineering, Graz University of Technology, Inffeldgasse 13, Graz, 8010, Austria 1

253-256

Insights into humidity-induced changes on the in-vitro pulmonary deposition and the predicted plasma levels of budesonide from commercial DPIs: a pharmacokinetic model assisted risk assessment approach S Radivojev1,2, J T Pinto1, E Fröhlich1,2 & A Paudel1,3 Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, Graz, 8010, Austria 2Center for Medical Research, Medical University of Graz, Stiftingtalstraße 24, Graz, 8010, Austria 3Institute of Process and Particle Engineering, Graz University of Technology, Inffeldgasse 13, Graz, 8010, Austria 1

257-260 257-230

A European Pharmaceutical Aerosol Group (EPAG)-Led Cross-Industry Assessment of Inlet Flow Rate Profiles of Compendial DPI Test Systems: Part 1 – Experimental Data Jolyon P Mitchell1, Chris Blatchford2, Roland Greguletz3, Daryl L. Roberts4, & Henk Versteeg5 1 Jolyon

Mitchell Inhaler Consulting Services Inc., 1154 St. Anthony Road, London, N6H 2R1, Canada 3M United Kingdom plc, Loughborough, LE11 5RB, UK 3 Sofotec GmbH, Benzstraße 1-3, Bad Homburg, D-61352, Germany 4 Applied Particle Principles LLC, 17347 Westham Estates Court, Hamilton, VA 20158, USA 5 Mechanical and Manufacturing Engineering, Loughborough University, Loughborough, LE11 3TU, UK 2

261-264 231-234

A European Pharmaceutical Aerosol Group (EPAG)-Led Cross-Industry Assessment of Inlet Flow Rate Profiles of Compendial DPI Test Systems: Part 2 – First-Order Impactor Model Daryl L. Roberts1, Henk Versteeg2, Chris Blatchford3, Roland Greguletz4, Jolyon P. Mitchell5 Applied Particle Principles LLC, 17347 Westham Estates Court, Hamilton, VA 20158, USA and Manufacturing Engineering, Loughborough University, Loughborough, LE11 3TU, UK 3 3M United Kingdom plc, Loughborough, LE11 5RB, UK 4 Sofotec GmbH, Benzstraße 1-3, Bad Homburg, D-61352, Germany 5 Jolyon Mitchell Inhaler Consulting Services Inc., 1154 St. Anthony Road, London, N6H 2R1, Canada 1

2 Mechanical

265-268 235-238

Formulating amikacin for dry powder inhalation I. Sibum, F. Grasmeijer, P. Hagedoorn & H.W. Frijlink Department of Pharmaceutical Technology and Biopharmacy, Ant. Deusinglaan 1, Groningen, 9713 AV, the Netherlands

269-272 239-242

Effect of spray dried formulation on the aerosol performance of a novel dry powder inhaler Serena Bonasera1, Dale R. Farkas2, P. Worth Longest1,2 Bryce Beverlin II3 & Michael Hindle1 1

Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, USA

Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University, Richmond, VA, USA

3Quench

273-276 243-246

Medical Inc., St Paul, MN, USA

Spray dried composite powders: capsule filling process optimization and aerodynamic performance characterization Maria Braga1, Raquel Barros1, Bruno Ladeira1, Mariana F. Silva1,2, Joana Tavares1 & Eunice Costa1 Hovione FarmaCiencia SA, Sete Casas, 2674 – 506 Loures, Portugal 2Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Portugal

277-280 247-250

Innovation in manufacturing as applied to dry powder inhaler formulation. David A. Wyatt,1 Jasdip S. Koner1, Eman Z. Dahmash1 & Afzal R. Mohammed1,2 1 Aston

Particle Technologies Ltd., Aston University, Birmingham, B4 7ET, United Kingdom 2 Aston Pharmacy School, Aston University, Birmingham, B4 7ET, United Kingdom

281-284 251-254

Formulation of Inhalable Voriconazole Dry Powders Using Spray Freeze-Drying Technique Qiuying Liao1, Long YIP1,2 & Jenny K.W. LAM1 Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong 2 Department of Pharmacy and Forensic Science, King’s College London, 150 Stamford Street, London, SE1 9NH, United Kingdom 1

285-288 255-258

An In Vitro Study to Rationalise Combination Therapies for Cystic Fibrosis Treatment Zara Sheikh1, Hui Xin Ong1, Michele Pozzoli1, Paul Young1, Daniela Traini1 1Woolcock

Institute of Medical Research and Faculty of Medicine and Health, University of Sydney, 431 Glebe Point Road, Glebe NSW 2037, Australia

289-292 259-262

The Effect of Using a Turbulence Grid on Fluidization of Pharmaceutical Lactose Powder K. Elserfy1, S. Cheng1, H-K. Chan3, G. Hebbink2, M. Mehta2 & A. Kourmatzis4 School of Engineering, Macquarie University, NSW 2109 DFE Pharma, Klever Strasse 187, 47568 Goch, Germany 3 Advanced Drug Delivery Group, School of Pharmacy, The University of Sydney, NSW 2006 4 School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, NSW 2006 1 2

293-296 263-266

Treating Lymphangioleiomyomatosis with Inhaled Rapamycin Solid Lipid Nanoparticles Emelie Land1, Lyn M. Moir1, Daniela Traini, Paul M. Young1 & Hui Xin Ong1 Respiratory Technology, Woolcock Institute of Medical Research and Faculty of Medicine and Health The University of Sydney NSW, 2037, Australia 1

267-270 297-300

The Impact of Device Handling Errors upon Inhaled Medication Delivery from Pressurized Metered Dose Inhalers (pMDIs) Used with and without a Valved Holding Chamber (VHC) Jason A Suggett1, Mark W. Nagel1 & Jolyon Mitchell2 1

301-304 271-274

Trudell Medical International, 725 Third Street, London, Ontario, NV5 5G4, Canada Mitchell Inhaler Consulting Services Inc., 1154 St. Anthony Road, London, N6H 2R1, Canada

2Jolyon

Glycemic Profile in Rats After Pulmonary Administration of a Pure Insulin Powder Francesca Buttini,1 Veronica Chierici1, Eride Quarta1, Lisa Flammini1, Adryana Rocha Clementino1, Susana Ecenarro2, Massimiliano Tognolini1, Elisabetta Barocelli1, Paolo Colombo1 & Ruggero Bettini1 1

Food and Drug Department, University of Parma, Parco Area delle Scienze 27a, 43124, Parma, Italy

Qualicaps Europe, S.A.U., Alcobendas, Madrid, Spain

305-308 275-278

Inhaler Resistance, Flowrate and Duration of Inhalation: The Effort to Use an Inhaler Adequately Mark Sanders & Ashley Green

309-312 279-282

Clement Clarke International Limited, Edinburgh Way, Harlow, CM20 2TT, UK

Triple Therapy Aerosol Delivery from an Integrated Inhaler Technique Training Device Mark Sanders1 & Cuong Tran2 1Clement

Clarke International Limited, Edinburgh Way, Harlow, CM20 2TT, UK 2i2c Pharma Services, Cardiff Medicentre, Heath Park, Cardiff, CF14 4UJ, UK

313-316 283-286

Manufacturing DPIs: an engineering perspective Pietro Piera & Mathieu Pfeiffer IMA S.p.A., Via Emilia 428-442, 40064 Ozzano dell’Emilia (BO), Italy

317-320 287-290

Comparison of Aerosol Delivery Methods for Spontaneously Breathing Tracheostomy Patients Mary Joyce, Sorcha Murphy, Gavin Bennett, Louise Sweeney & Ronan MacLoughlin

321-323 291-293

Aerogen, Galway Business Park, Dangan, Galway, H91 HE94, Ireland

A Metal-Organic Framework (MOF) dry powder technology for antibiotic deep lung delivery and imaging Gabriela Wyszogrodzka1, Przemysław Dorożyński2, Piotr Kulinowski3 & Stefano Giovagnoli4 Department of Pharmacobiology Jagiellonian University Medical College, Medyczna 9, 30-068 Kraków, Poland Research Institute, Rydygiera 8, 01-793 Warszawa, Poland 3 Department of Magnetic Resonance Imaging, Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Kraków, Poland 4 Department of Pharmaceutical Sciences, via del Liceo 1, University of Perugia, Perugia, 06123, Italy 1

2Pharmaceutical

324-327 294-297

Comparison of human cell lines for risk assessment of aerosolised pesticides Zachary Enlo-Scott1, Magda Swedrowska1, Alex Charlton3, Leona Merolla3, Ian Mudway2 & Ben Forbes1 Drug Delivery Research Group, Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London, SE1 9NH, United Kingdom 2 MRC-PHE Centre for Environment and Health, Analytical, Environmental and Forensic Sciences, King’s College London,150 Stamford Street, London, SE1 9NH, United Kingdom 1

328-331 298-301

Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire, RG42 6EY, United Kingdom

Cyclodextrin-stabilised ion-pair complexes enhance drug lung uptake via the polyamine transporter Zarif M. Sofian1, Julie T. W. Wang1, Yuan L1, Paul Royall1, Ben Forbes1, David J. Barlow1, Clive Page 1,2, Khuloud T. Al-Jamal1, Stuart Jones1 & Faiza Benaouda1 1

332-335 302-305

School of Cancer and Pharmaceutical Science, King’s College London, 150 Stamford Street, London, SE1 9NH Institute of Pulmonary Pharmacology, King’s College London, 150 Stamford Street, London, SE 1 9NH

2Sackler

Developing alternative models for in vitro investigation of excipient influence on drug transport Precious Akhuemokhan1, Magda Swedrowska1, Josie Williams1, Richard Harvey2, Ben Forbes1 1

Institute of Pharmaceutical Science, School of Cancer and Pharmaceutical Sciences, King’s College London

2Institut

336-339 306-309

für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany

Nasal Dry Powder Delivery: Implementing a formulation independent Spray Drying Process Mariana F. Silva1,2, Diana A. Fernandes1,2, Maria Braga1, António Eloy1, João Marques1, M. Luísa Corvo2 & Eunice Costa1 1Hovione

FarmaCiência SA, Lumiar, 1649-038, Portugal 2 Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003, Portugal

340-343 310-313

Therapeutic Intranasal Drug Delivery: needleless treatment for epistaxis Larissa Gomes1, Daniela Traini1, Paul Young1, & Maliheh Ghadiri1, 2 Respiratory Technology, Woolcock Institute of Medical Research and Discipline of Pharmacology, Faculties of Medicine and Health, Sydney, Australia 2 Faculty of Engineering and Information Technology, University of Technology Sydney (UTS) 1

344-347 314-317

Deposition in three nasal cast models with a new concept of nasal administration (Retronose) vs nasal spray Laurent Vecellio1,2, Deborah Le Pennec1, Guillaume Grevin2 & Alain Regard2 CEPR, INSERM U1100, University of Tours, Tours, France 2Nemera, La Verpilliere, France 1

348-351 318-321

Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; Reinhard Vehring. Mechanistic Understanding of Microparticle Formation in Respiratory Applications Reinhard Vehring1 1University

of Alberta, Department of Mechanical Engineering, Edmonton, Alberta, T6G 1H9, Canada

Summary This contribution summarizes recent advances in the mechanistic understanding of particle formation processes that provides the basis for microparticle based products with respiratory applications. Key mechanisms are heat and mass transport on evaporating or condensing droplets, internal redistribution of components in droplets by diffusion, phase separation processes like nucleation and crystal growth, and particle plasticity, such as shell buckling or collapse of porosity. Since the parameter space governing these processes is much too large for empirical studies, systematic experimental and modelling studies need to be undertaken. Because of the complexity of actual manufacturing processes, experimental studies of particle formation are best conducted on idealised model systems. Progress on a variety of such experimental models is presented, ranging from highly idealised systems, like single particle levitation and droplet chains, to more representative ones like monodisperse spray drying and well-instrumented process equipment in combination with process models. Analytical and numerical models for particle formation in single solvent and co-solvent systems provide predictive parameters that can be used in the engineering of microparticles. Application examples are shown for glass stabilization in amorphous systems, surface modifications in partially crystalline systems, and encapsulation of nano-emulsions. Key Message Fundamental understanding of the physico-chemical processes underlying microparticle formation allows development of predictive tools for particle design, thereby greatly accelerating product development, reducing risk and enabling sophisticated products based on nano-structured microparticles. Introduction In recent years, inhalation products have been commercialized that rely on structured microparticles for their functionality; several more are in development. Such products cannot be developed with simple micronized material, but rather require a more sophisticated particle morphology that cannot be designed by trial and error. Consequently, the discipline of particle engineering has seen a marked expansion, both in industry and in academia. Particle design for pulmonary applications uses predictive process models to find and control the critical parameters of the manufacturing process, adds solid phase modelling to formulation design to achieve the desired storage stability and environmental robustness, and is based in a mechanistic understanding of the physical processes that govern particle formation.1 Many of these processes are still poorly understood, and modelling attempts are hampered by the lack of material properties, especially in non-equilibrium and supersaturation regimes. Hence, much systematic experimental work needs to be undertaken to advance the field. This contribution will briefly describe several important sub-processes that occur in particle formation during spray drying and explain why they are important to master. Next, experimental techniques will be described that can be used to learn more about these processes. Finally, selected applications of a predictive particle design framework will be presented. Particle Formation Processes in Spray Drying One of the first tasks that one encounters when trying to describe a droplet drying process is to model the size change of the droplet as a function of time. For a single component, this is a simple task, requiring only the application of established textbook theory. More complex is the situation for co-solvent systems and in propellant droplet evaporation. In these cases the composition of the droplets can change over time, evaporation rates are no longer constant, and condensation of water onto the droplet can occur through evaporative cooling.2 Knowledge of evaporation rates is essential for residence time calculations in spray dryers, since these rates determine the size of the equipment for a given production scale. The droplet size evolution of aerosols from pressurized metered dose inhalers, which can be treated as a special case of spray drying, is needed for lung deposition calculations, which can support dose finding. Furthermore, it is necessary to know the solvent composition in the droplets as a function of time, because otherwise it cannot be determined when individual actives or excipients begin to precipitate due to supersaturation. Adequate models need to take heat and mass transfer between droplet and gas phase into account and can only be solved numerically, except in the most simple cases.

Drug Delivery to the Lungs, Volume 29, 2018 - Mechanistic Understanding of Microparticle Formation in Respiratory Applications The next step is to describe what happens inside the droplets during evaporation. In general, spray drying can be used for solutions, suspensions or even more complex feed stock like nanoemulsions. This fact means that the internal distribution of dissolved or suspended components with hugely varying mobility needs to be described. A semi-analytical model can be developed for single solvent systems, if only the diffusion of components and the surface recession rate of the droplet are considered, e.g. in the form of a dimensionless number.3 The results from such a model inform the particle designer about the sequence of precipitation events in the shrinking droplets and indicate which components tend to accumulate on the surface. They also provide information about the time available for precipitation events. Diffusion models are very helpful in designing the radial distribution of the dried particles, e.g. for encapsulation or dispersibility enhancement. In the future, more comprehensive models will be required that also incorporate the effects of surface activity of excipients like leucine or trileucine.4 Once dissolved components reach critical supersaturation, nucleation of a solid phase commences and the formation of the solid phase begins. This step is poorly understood because most of the theory developed for heterogeneous nucleation is applicable only for much slower processes, in part because material properties for highly supersaturated phases are unavailable. In many cases, the drying of microparticles produces particles with a non-equilibrium solid phase, which is undesirable for long term stability. Currently, this important step of the drying process is primarily studied experimentally5 and by careful solid phase analysis of the final particles.6 In the case of suspension or colloidal systems, an additional complication arises because the packing and assembly of nanoscale sub-structures need to be considered as well. The last group of processes occurring during particle formation is related to changes in the mobility of the solid phase as the solvent content decreases. Initially the precipitated phases may be strongly plasticized, or ordered sub-structures may have enough mobility to re-arrange. Both of these effects can lead to plastic deformation of particles, manifesting itself in shell buckling or collapse of internal porous structures. Some attempts at describing these processes theoretically have been published 7, but in most cases experimental work is unavoidable, in particular careful ultramicroscopic analysis of whole or sectioned particles.8 Hybrid processes such as atmospheric spray freeze drying 9 have no evaporation phase, because droplets are frozen right after atomization and the water is removed mainly by sublimation. Here, new challenges are found in modelling the super-cooling and ice crystallization steps and in describing the desiccation of the freeze concentrate under near-collapse conditions. This technique is less developed but provides opportunities for particle design that are unavailable with standard spray drying. Experimental Model Systems Particle formation studies require a more controlled experimental environment than what is presented by a commercial spry dryer. The most idealised and arguably best-controlled model system is single particle levitation. Single droplets and dry particles in the respirable size range can be suspended acoustically, by light pressure, or via electrodynamic forces. It is advantageous to study individual particles in a gas phase that is well controlled and not influenced by the presence of the particle. Studies on single particles have contributed greatly to the understanding of aerosol microphysics10, but they have two important limitations. First, it is difficult to study very fast processes in single particle traps because of the time it takes to trap a particle and to change the gas phase conditions. Second, only a single particle can be analysed at a time, precluding techniques that need a larger sample mass. Droplet chain techniques partially address these limitations. Microdispensers can inject monodisperse microdroplets into a gas stream with extremely repeatable trajectories, in effect forming a chain of droplets whose temporal evolution can be measured along the length of the chain, making faster precipitation kinetics analytically available.11 The dried particles can be collected and analysed by ultramicroscopy, but since the production rate of particles in this technique is limited to a few hundred Hertz, the amount of material produced is still insufficient for many analytical techniques. Larger sample masses can be provided by a newly developed monodisperse spray dryer. 12 This instrument uses a vibrating orifice generator in place of the customary twin fluid nozzle of a standard size spray dryer. Some control over particle size and trajectory is lost, because the droplets from the vibrating orifice generator are closely spaced and need to be dispersed in a secondary gas nozzle. This dispersion leads to partial agglomeration, which is monitored with an in-process aerodynamic particle sizer. However, the resulting particles are still monodisperse and because the feed flow is much smaller than in a regular spray dryer, the drying gas conditions are largely unaffected by the presence of the droplets. This technique enables batch sizes of up to a gram of monodisperse test particles created in well described, homogeneous conditions. Further scale-up of monodisperse spray drying will require multiplexing of nozzles. If larger batch sizes are necessary, e.g. to set up long term stability studies, moving to a bench top or pilot scale spray dryer will be at the expense of much of the control that the more idealised experimental test beds provide. However, it is still possible to improve on typical commercial systems by adding sensors to the dryer. This approach allows the development of process models,13 which yield outlet conditions as a function of process parameters and describe the particlesâ&#x20AC;&#x2122; exposure to temperature and humidity during the process. This rather minor effort is highly recommended, because it makes it feasible to correlate pilot scale process conditions with the controlled drying conditions of the model systems described above.

Drug Delivery to the Lungs, Volume 29, 2018 – Reinhard Vehring. Application Examples Some advanced particle designs have been successfully commercialized, for example the COPD medication Bevespi Aerosphere®, which is based on the co-suspension technology.14 Others are in various stages of development. An area of particular interest is combination products, which after addition of suitable excipients may require the development of particles with many components. It has been shown that multi-component particle design benefits greatly from a systematic approach based on mechanistic understanding. 15 Another very active area is that of inhalable biologics. Particles that are suitable for this application provide glass stabilization for the biological and typically some dispersibility enhancement via a non-cohesive shell.16 Such shells can be achieved by early crystallization of leucine, a process that can be described quite accurately by existing particle formation models.17 Even sensitive biologicals, such as bacteriophages, can be processed with acceptable losses, stabilized for storage, and made dispersible for inhalation purposes.18 In summary, the mechanistic understanding of particle formation that has been gained over the last two decades is now put to use in the design of highly functional dosage forms. The advantages these particles hold in terms of storage stability and robustness will hopefully be of benefit to patients globally. References 1

Vehring, R.: Understanding Particle Formation Kinetics: Linking Device and Process with Particle Morphology and Product Performance. Respiratory Drug Delivery Europe 2017, Vol. 1, pp 167-174, 2017.

Ordoubadi, M., Gregson, F., Finlay, W. H., Vehring, R., Reid, J. P.: Interaction of Evaporating Multicomponent Microdroplets with Humid Environments. Respiratory Drug Delivery 2018, Vol 2, pp 569–572, 2018.

Boraey, M. A., Vehring, R.: Diffusion Controlled Formation of Microparticles. Journal of Aerosol Science, 67, 131-143, 2014.

Lechuga-Ballesteros, D., Charan, C., Stults, C. M., Stevenson, C. L., Miller, D. P., Vehring, R., Tep, V., Kuo, M.-C.: Trileucine Improves Dispersibility, Aerosol Performance and Stability of Spray-Dried Powders for Inhalation. Journal of Pharmaceutical Sciences, 97, 287-302, 2008.

Baldelli, A., Boraey, M. A., Nobes, D., Vehring, R.: Analysis of the Particle Formation Process of Structured Microparticles. Molecular Pharmaceutics, 12 (8), 2562-2573, 2015

Wang, H., Boraey, M. A., Williams, L., Lechuga-Ballesteros, D., Vehring, R.: Low-frequency Shift Dispersive Raman Spectroscopy for the Analysis of Respirable Dosage Forms. International Journal of Pharmaceutics, 469 (1), 197-205, 2014.

Tsapis, N., Dufresne, E. R., Sinha, S. S., Riera, C. S., Hutchinson, J. W., Mahadevan, L., Weitz, D. A.: Onset of Buckling in Drying Droplets of Colloidal Suspensions. Physical Review Letters, 94, 018302, 2005.

Baldelli, A. Vehring, R.: Control of the Radial Distribution of Chemical Components in Spray Dried Crystalline Microparticles. Aerosol Science and Technology, 50 (10), 1130-1142, 2016.

Wang, Z. L., Finlay, W. H., Peppler, M. S., Sweeney, L. G.: Powder Formation by Atmospheric Spray-Freeze Drying. Powder Technology, 170, 45-52, 2006.

Reid, J. P., Haddrell, A. E., Gregson, F., Lewis, D. A., Church, T., Ordoubadi, M., Vehring, R.: Hygroscopic Growth of Drugs and Excipients: An In Vitro Assessment of the Importance of Aerosol Growth in Deposition Calculations. Respiratory Drug Delivery 2018. Vol. 1, pp 113-122, 2018.

Baldelli, A., Power, R., Miles, R., Reid, J. P., Vehring, R.: Effect of Crystallization Kinetics on the Properties of Spray Dried Microparticles. Aerosol Science and Technology, 50 (7), 693-704, 2016.

Ivey, J.W., Bhambri, P., Church, T.K., Lewis, D.A., Vehring, R.: Experimental Investigations of Particle Formation from Propellant and Solvent Droplets Using a Monodisperse Spray Dryer. Aerosol Science and Technology, 52 (6), 702-716, 2018.

Ivey, J., Vehring, R.: The Use of Modeling in Spray Drying of Emulsions and Suspensions Accelerates Formulation and Process Development. Computers & Chemical Engineering, 34 (7), 1036-1040, 2010.

Vehring, R., Lechuga-Ballesteros, D., Joshi, V., Noga, B., Dwivedi, S. K.: Cosuspensions of Microcrystals and Engineered Microparticles for Uniform and Efficient Delivery of Respiratory Therapeutics from Pressurized Metered Dose Inhalers. Langmuir, 28 (42), 15015 - 15023, 2012.

Hoe, S., Ivey, J. W., Boraey, M. A., Shamsaddini-Shahrbabak, A., Javaheri, E., Matinkhoo, S., Finlay, W. H., Vehring, R.: Use of a Fundamental Approach to Spray-Drying Formulation Design to Facilitate the Development of Multi-Component Dry Powder Aerosols for Respiratory Drug Delivery. Pharmaceutical Research, 31, 449-465, 2014.

Hoe, S., Boraey, M. A., Ivey, J. W., Finlay, W. H., Vehring, R.: Manufacturing and Device Options for the Delivery of Biotherapeutics. Journal of Aerosol Medicine and Pulmonary Drug Delivery, 27 (5), 315-328, 2014.

Feng, A. L., Boraey, M. A., Gwin, M. A., Finlay, P. R., Kuehl, P. J., Vehring, R.: Mechanistic Models Facilitate Efficient Development of Leucine Containing Microparticles for Pulmonary Drug Delivery. International Journal of Pharmaceutics, 409 (1-2), 156-163, 2011.

Matinkhoo, S., Lynch, K. H., Dennis, J. J., Finlay, W. H., Vehring, R.: Spray Dried Respirable Powders Containing Bacteriophages for the Treatment of Pulmonary Infections. Journal of Pharmaceutical Sciences, 100 (12), 5197-5205, 2011.

Drug Delivery to the Lungs, Volume 29, 2018 - Kian Fan Chung et al. Re-defining Asthma – What’s all the fuss about? Kian Fan Chung National Heart & Lung Institute, Imperial College London, & Royal Brompton Hospital, London, UK

Asthma is a descriptive term that has been used over the centuries to denote a condition that is defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over time and in intensity, without any assumption of pathophysiology. Under this definition many other conditions apart from asthma may be diagnosed but with the later ability to measure lung function, the addition of variable expiratory airflow obstruction brought more precision to its diagnosis. More recently, recognising the importance of airway inflammation as associated with causing the symptoms of asthma, its presence was another useful differentiating property. The recognition that eosinophilic inflammation was commonly associated with symptomatic asthma led to the use of inhaled steroids as being the most important anti-inflammatory management of asthma, and together with inhaled long-acting beta-agonist bronchodilators formed the basis of treatment for all asthmatic patients with the dose of these anti-asthma drugs dosed according to the degree of severity of the asthma (https://ginasthma.org/2018-gina-report-global-strategy-for-asthma-management-and-prevention/). This led to the recognition that there was a group of asthma patients whose disease remained uncontrolled despite taking high doses of these inhaler therapies plus other asthma medications including oral corticosteroids who were therefore resistant to these existing therapies, the severe refractory asthma (1). In the search for new therapies, the development of antibody therapies targeted at specific components of the Type 2 inflammation led to the definition of specific groups of patients within this T2, such as anti-IgE antibody directed at severe allergic asthma and of antiIL5 antibody directed at severe eosinophilic asthma associated with recurrent exacerbations (2). This has led to the recognition of these phenotypes that would respond specifically to these targets, bringing precision medicine to asthma (3). The severe eosinophilic asthma phenotype is now well recognised phenotype of high T2 with IL-4, IL13 and IL5 being important drivers in this phenotype. Clinically it is recognised as being associated with severe asthma (ERS/ATS definition), an exacerbation frequency ≥ 2/year, a dependence on oral corticosteroids for asthma control and high circulating eosinophils. However, those phenotypes with a low T2 have remained poorly studied and these phenotypes do not have any specific targeted treatments. Omics data analysis has now allowed us to define T2 and non-T2 phenotypes. Using an analysis of sputum transcriptomics has allowed us to define a T2-high and 2 non-T2 high with airway neutrophilia with activated inflammasome and IL-1β signalling, and another with little inflammation but associated with elements of oxidative stress(4). What does this mean regarding the taxonomy for asthma? The idea that the disease is heterogeneous has been recognised over the centuries and recently used in the GINA definition. While the definition from the clinician’s point of view with a collection of symptoms will remain (physician-diagnosed asthma), it needs to be more granular with the definition of molecular phenotypes underlined by the driving mechanisms (leading to the endotype), since these will attach specific therapies to which these phenotyped patients will respond to. The definition of asthma according to treatable traits is not a useful concept as these classifications are not based on mechanisms and pathophysiology and therefore do not allow one to judge whether the trait is treatable or not . This new definition will not only apply to those with severe refractory asthma but to the whole spectrum of the disease from mild to severe disease. A redefinition of asthma is imperative: it needs to contain the concept of precision medicine, and this will represent the long journey from a cluster of symptoms to efficacious targeted therapies in specific well-defined endotypes.

References: 1. Chung KF, Wenzel SE, Brozek JL, Bush A, Castro M, Sterk PJ, et al. International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma. Eur Respir J. 2014;43(2):343-73. 2. Chung KF. New treatments for severe treatment-resistant asthma: targeting the right patient. The lancet Respiratory medicine. 2013;1(8):639-52. 3. Chung KF. Asthma phenotyping: a necessity for improved therapeutic precision and new targeted therapies. J Intern Med. 2016;279(2):192-204. 4. Kuo CS, Pavlidis S, Loza M, Baribaud F, Rowe A, Pandis I, et al. T-helper cell type 2 (Th2) and non-Th2 molecular phenotypes of asthma using sputum transcriptomics in U-BIOPRED. Eur Respir J. 2017;49(2):443-55.

Drug Delivery to the Lungs, Volume 29, 2018 - Dr Brian Lipworth et al. Three drugs in one inhaler for COPD :where are we going with triple therapy Dr Brian Lipworth Scottish Centre for Respiratory Research University of Dundee Ninewells Hospital and Medical School University of Dundee, DD1 9SY

Summary Single inhaler triple therapy comprising ICS/LABA/LAMA for COPD have been developed to improve patient adherence. The ICS moiety reduces eosinophilic inflammation while the LABA/LAMA improves airway calibre. Current guidelines advocate triple therapy for frequently exacerbating patients in GOLD group D.Evidence suggests that single inhaler triple is superior to either ICS/LABA or LABA/LAMA in reducing exacerbations while having a much smaller effect on lung function and quality of life . Patients with an eosinopihilic COPD phenotype fare better in terms of exacerbation reduction with triple therapy. The ICS moiety is also related to an increased pneumonia risk especially for fluticasone furoate compared to either budesonide or beclometasone dipropionate, which in turn is related to their relative lipophilicity and associated lung retention in the presence of impaired mucociliary clearance and altered lung microbiome. This is in turn may determine which triple inhaler is optimal in terms of the overall benefit-risk equation for treating COPD. Other factors such as device preference (DPI vs pDMI), particle size and dosing regimen (od vs bid) will also influence individual prescribing . Triples currently available or in include fluticasone furoate /vilanaterol/umeclidinium (od DPI), becometasone development dipripionate/formoterol/glycopyrronium (bid extra fine solution pMDI) and budesonide/formoterol/glycopyrronium (bid co-suspension pMDI) .

Key Message Single inhaler triple therapy for COPD should be considered in frequently exacerbating patients with the eosinophilic phenotype.

Drug Delivery to the Lungs, Volume 29, 2018 - Bruce K. Rubin The Cystic Fibrosis â&#x20AC;&#x2DC;explosionâ&#x20AC;&#x2122; - New medicines and unmet needs Bruce K. Rubin Children's Hospital of Richmond at Virginia Commonwealth University, 1000 E Broad St, Richmond, VA 23298 USA

Summary Cystic fibrosis (CF) is a multi-system illness caused by abnormalities in the CF transmembrane conductance regulator (CFTR) gene and protein. CFTR is an ion channel regulating transport of chloride, bicarbonate, and water, and influencing sodium resorption. Advances in CF care have led to dramatic increases in life expectancy and quality of life. We have entered an era of precision medicine, with therapy targeted to modify the CFTR protein, increasing production (premature termination codon read through), preventing degradation in the endoplasmic reticulum, improving folding, and chaperoning to the cell surface (correction), and increasing the open channel probability (potentiation). There are many new entrants into this therapeutic area, including combination therapy to enhance the recovery and effectiveness of ion transport through the most common CF gene defect â&#x20AC;&#x201C; phe508del. Challenges for modulator therapies include targeting uncommon CFTR defects, assessing the long term effects of these therapies, and identifying clinical trial outcomes to evaluate the potential for very early therapy of infants with CF. An additional need is effective immunomodulator therapy that can safely decrease the overwhelming neutrophil dominated inflammation that damages the CF airway. While glucocorticosteroids are effective in suppressing the eosinophil dominant inflammation characteristic of asthma, these appear to have little or no value in treating CF airway inflammation and their use comes at a significant cost in adverse events and current therapies for neutrophil dominant inflammation have limited effectiveness. Introduction Cystic fibrosis (CF) is a multi-system illness caused by abnormalities in the CF transmembrane conductance regulator (CFTR) gene and protein. CFTR is an ion channel regulating transport of chloride, bicarbonate, and water, and influencing sodium resorption. CF is inherited as an autosomal recessive disorder, and with about 70,000 CF patients worldwide, it is the most common life shortening disease among persons of European descent. Although there are more than 2,000 CFTR abnormalities reported - not all known to be disease causing - approximately 83% of patients with CF carry at least one phe508del allele. CFTR disease causing mutations are commonly classified into six classes depending on the mechanism of dysregulation (Table 1). These abnormalities can lead to complete failure to produce CFTR protein due to the presence of a premature termination codon, production of a misfolded and rapidly degraded CFTR (eg p.phe508del), and failure of the CFTR protein to effectively transport ions at the cell surface. In recent years, small molecule targeted therapy for specific classes of CFTR abnormalities have led to the development of CFTR potentiator medications that increase channel open probability enhancing chloride transport at the cell surface for Class III and Class IV defects, and CFTR correctors that decrease protein degradation for Class II defects like phe508del.

Therapy of cystic fibrosis The major goal in treating CF is to clear the abnormal and excess secretions that lead to persistent airway infection and inflammation. For patients with advanced stages of disease, a lung transplant may be necessary. Treatment of gastrointestinal disease is also important. A diet rich in fat and protein, supplemented with digestive pancreatic enzymes for the 85% of patients who have pancreatic malabsorption, will lead to weight gain and better health outcomes. Because of fat malabsorption, the fat-soluble vitamins A, D, E, and K are supplemented using water soluble forms of these vitamins. Patients with significant hepatic dysfunction will benefit from therapy with medications such as ursodiol and those with CF related diabetes mellitus may require insulin supplementation to maintain health. Together, treatment of GI and pulmonary disease is the mainstay of the medical management of CF.

For pulmonary disease, therapy consists of early identification of chronic bacterial infection with attempts at eradication using both intravenous and inhaled antibiotics (1). There is an ongoing need to develop new antibiotics for the treatment of bacteria that eventually become resistant under pressure of chronic infection and intermittent antibiotic therapy. Chronic Pseudomonas aeruginosa infection is characteristic of CF, with over 90% of adult patients being chronically infected (not colonized) with this organism. However, data suggests that infection by multiply resistant Staphylococcus aureus may lead to a more rapid decline in lung function (2). Patients with CF are also prone to infection with resistant strains of Burkholderia cepacia, Stenotrophomonas maltophilia and Achromobacter xylosoxidans. By adulthood, many patients with CF are on chronic rotating cycles of inhaled antibiotics with intermittent therapy using high-dose oral or intravenous antibiotics to treat acute declines in pulmonary function and increase in cough and sputum reduction referred to as a pulmonary exacerbation.

Drug Delivery to the Lungs, Volume 29, 2018 - The Cystic Fibrosis ‘explosion’ - New medicines and unmet needs

Accompanying chronic infection is retention of infected sputum. The use of peptide mucolytic medications, such as Pulmozyme (dornase alfa) and expectorants (sometime referred to as hydrators) such as 7% inhaled hypertonic saline have become a mainstay of therapy. These therapies are usually administered in conjunction with airway clearance, maneuvers or devices that can include chest physical therapy, huff coughing, breathing maneuvers such as autogenic drainage, high frequency chest wall compression devices such as therapy Vests and airway positive expiratory pressure (PEP) or oscillating positive airway pressure (OPAP) devices (3). Hybrid devices that can produce a positive expiratory and allow inhalation of nebulized medications at the same time have more recently been introduced to the market.

Another important and unmet need is therapy to treat the chronic airway inflammation that accompanies this infection. There is evidence that inflammatory response in the CF airway is dysregulated with failure to resolve a predominantly neutrophilic inflammation leading to an extensive neutrophil necrosis and NETosis. Although medications such as corticosteroids are often used, these are ineffective in treating neutrophil dominated inflammation. Ibuprofen therapy for neutrophilic inflammation was introduced some two decades ago but is used in few centers because of the need for careful monitoring of serum levels. Chronic low dose 14- and 15-member macrolide antibiotics are immunomodulatory medications primarily acting through the extracellular signaling regulated kinase to decrease neutrophil dominated inflammation (4). Most commonly used in CF is azithromycin, given three times a week as a chronic therapy. This inevitably leads to macrolide resistant organisms including atypical Mycobacteria. Thus, there is an unmet need for effective anti-inflammatory medications that can be given chronically, safely, and will not stimulate antimicrobial resistance.

CFTR modulator therapy CFTR modulator medications include potentiators, such as ivacaftor that increase channel opening probability when even a defective CFTR is chaperoned to the airway surface (5), premature termination codon, read through medications, and corrector medications such as lumacaftor that will prevent endoplasmic reticulum degradation of abnormally folded CFTR protein permitting more of this protein to come to the airway surface where a concomitantly administered potentiator can increase the channel open probability (6).

The first of these medications was ivacaftor, sold as Kalydeco (Vertex Pharmaceuticals). It is a small molecule orally administered potentiator of CFTR channel function active against Class III and Class IV CFTR mutations. In patients with these mutations, ivacaftor is as close as we have seen to a cure for C, producing significant improvement in pulmonary function, normalization of sweat chloride, improved weight gain, decreased airway inflammation, and decreased airway infection. However, low grade infection with bacteria such as Pseudomonas persists meaning that the need for antimicrobial therapy is not obviated even in patients who have a dramatic clinical response to ivacaftor. The combination of ivacaftor and lumacaftor (Orkambi – Vertex Pharmaceuticals) in patients who are homozygous for the phe508del CFTR mutation, has been shown to improve nutritional status, decrease the rate of pulmonary exacerbation, and decrease the rate of lung function decline by over 40% over a period of 2 years of therapy (7).

The second combination CFTR modulator of Tezacaftor (corrector) and /ivacaftor was approved in the United States in February 2018 and is marketed as Symdeko. Unlike Orkambi, Symdeko is approved for both patients over the age of 12 years who are homozygous and heterozygous for phe508del. Symdeko is reported to improve pulmonary function as least as well as Orkambi and appears to have fewer side effects – in particular the flu-like illness with chest tightness that has been reported in up to 20% of patients when starting Orkambi. This chest discomfort and dyspnoea is more common in patients with more severe lung disease.

Triple therapy is also in late stage clinical trials combining and early corrector (designated C1) and a late or C2 corrector with a potentiator. These include VX-445 + tezacaftor + ivacaftor, VX-659 + tezacaftor + ivacaftor, as well as early phase clinical trials by Vertex, Galapagos/AbbiVie. Recently guidelines for the use of CFTR modular therapy have been published (8) however this is so rapidly changing that updates may be needed as often as annually. There are also trials of inhibitors of the epithelial sodium channel (ENaC) that is overactive when CFTR is dysfunctional, CFTR mRNA delivery, and oligonucleotide therapy to repair CFTR mRNA. Most of these are orally administered therapies. Beyond these innovations are trials of new inhaled antibiotics, bacterial biofilm disrupters, and anti-inflammatory medications. Fifteen years ago, an editorial was published in the journal CHEST entitled “So many drugs, so little time. The future challenge of cystic fibrosis care” (9). Fifteen years on this is truer than ever (Figure 1). Unfortunately, the therapeutic burden for patients with CF is already quite complex, taking up a great deal of time. It has been shown that adherence to prescribe therapy is inversely correlated with therapeutic burden. Thus, studies are urgently needed to see if patients can safely discontinue some of their existing therapies as these new medications are added. The ultimate goal is to find a cure for CF, perhaps by adopting gene editing techniques to correct this autosomal recessive, monogenetic defect.

Drug Delivery to the Lungs, Volume 29, 2018 - Bruce K. Rubin

Figure 1: CF Treatment Landscape U.S. References 1. Blanchard AC, Horton E, Stanojevic S, Taylor L, Waters V, Ratjen F. Effectiveness of a stepwise Pseudomonas aeruginosa eradication protocol in children with cystic fibrosis. J Cyst Fibros. 2017;16(3):395-400 2. Dasenbrook EC, Merlo CA, Diener-West M, Lechtzin N, Boyle MP. Persistent methicillin-resistant Staphylococcus aureus and rate of FEV1 decline in cystic fibrosis. Am J Respir Crit Care Med. 2008;178(8):81421. 3. Rubin BK Mucus, phlegm, and sputum in cystic fibrosis. Respir Care. 2009;54(6):726-32 4. Shinkai M, Foster GH, Rubin BK. Macrolide antibiotics modulate ERK phosphorylation and IL-8 and GM-CSF production by human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2006;290(1):L75-85 5. Ramsey BW, Davies J, McElvaney NG, Tullis E, Bell SC, DĹ&#x2122;evĂnek P, Griese M, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med. 2011;365(18):1663-72. 6. Boyle MP, Bell SC, Konstan MW, McColley SA, Rowe SM, Rietschel E, Huang X, et al. A CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor) for treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial. Lancet Respir Med. 2014;2(7):527-38. 7. Konstan MW, McKone EF, Moss RB, Marigowda G, Tian S, Waltz D, Huang X, et al. Assessment of safety and efficacy of long-term treatment with combination lumacaftor and ivacaftor therapy in patients with cystic fibrosis homozygous for the F508del-CFTR mutation (PROGRESS): a phase 3, extension study. Lancet Respir Med. 2017;5(2):107-118 8. Ren CL, Morgan RL, Oermann C, Resnick HE, Brady C, Campbell A, DeNagel R, Guill M, Hoag J, Lipton A, Newton T, Peters S, Willey-Courand DB, Naureckas ET. Cystic Fibrosis Foundation Pulmonary Guidelines. Use of Cystic Fibrosis Transmembrane Conductance Regulator Modulator Therapy in Patients with Cystic Fibrosis. Ann Am Thorac Soc. 2018;15(3):271-280. 9. Boyle MP. So many drugs, so little time: the future challenge of cystic fibrosis care. Chest. 2003;123(1):3-5.

Drug Delivery to the Lungs, Volume 29, 2018 - Jane Scullion Inspired or Expired Jane Scullion University Hospitals of Leicester and Education Lead UKIG. Member ADMIT (Inhlaers4U)

Before the invention of the MDI, asthma medication was delivered using a squeeze bulb nebulizer which was fragile, unwieldy and for the main part unreliable.1 The relatively crude nature of the bulb nebulizer meant that the particles generated were comparatively large, and could be considered too large for effective drug delivery to the lungs. 2 Nonetheless these nebulizers paved the way for inhalation drug delivery and provided the inspiration for the development of the MDI. MDIs were first developed in 1955 by Riker Laboratories, later a subsidiary of 3M Healthcare.1 At that time MDIs represented a convergence of two relatively new technologies, the CFC propellant and the Meshburg metering valve which was originally designed for dispensing perfume. 3 The initial design by Riker used a glass canister coated with a vinyl plastic to improve its resilience.1 By 1956 Riker had developed two MDI based products, the Medihaler-Ept containing epinephrine and the Medihaler-Iso containing isoprenaline.2 Both products are agonists which provide short term relief from asthma symptoms and have now largely been replaced in asthma treatment by salbutamol which is more selective. Inhaler device technique is fundamental to reducing morbidity and more importantly mortality in Asthma and reducing symptoms and possibly mortality in COPD. The current plethora of devices available on the market today should improve our choices and options Unfortunately, a systematic review of 144 studies over 40 years showed that we hadn’t made any strides forward so it has to be about more than the inhaler device. 5 A recent Cochrnae review looked at 39 studies including more than 16,000 adults and children with asthma who were taking a steroid inhaler looked at strategies to improve adherence, and although there were some positive interventions unfortunately nothing appeared to improve outcomes in terms of control and attacks.6 All inhalers regardless, require inspiration but patients’ ideas concerns and expectations and lifestyles are not always compatible with good inhaler use. UKIG supports the concept that inhalers are medication and the medication essential in asthma and COPD symptom control and for preventing asthma deaths. If we consider that a third of asthma deaths in the UK can be directly linked to non-adherence we have to do something differently. If we do what we have always done we will get what we have always got we have to rethink our consultations with patients and make them effective and efficient. We have to change the perception of inhalers as ‘just an inhaler’. We have a lot of inhaler devices but essentially no change in adherence or outcomes Perhaps it’s time to consider whether devices are inspired or expired.

References 1. 2. 3. 4. 5. 6.

Purewal, Tol S.; D. Grant (1997). Metered Dose Inhaler Technology (Illustrated ed.). Informa Health Care. ISBN 1-57491-065-5. Swarbrick, James (2007). Encyclopedia of Pharmaceutical Technology (3rd Illustrated ed.). Informa Health Care. p. 1170. ISBN 0-8493-9394-9. Clark, A. R (1995). "Medical Aerosol Inhalers: Past, Present, and Future". Aerosol Science and Technology. 22 (4): 374–391. doi:10.1080/02786829408959755. Finlay, W. H., The Mechanics of Inhaled Pharmaceutical Aerosols: An Introduction, Academic Press, 2001. Sanchis J et al. Chest 2016; 150(2): 394–406 Normansell R, Kew KM, Stovold E Strategies to help people with asthma take their steroid inhaler as prescribed 18th April 2017 Cochrane Library

Drug Delivery to the Lungs, Volume 29, 2018 - Federico Lavorini The problem with critical and non-critical inhaler errors Federico Lavorini Department of Experimental and Clinical Medicine University of Florence, Largo Brambilla 3, 50134 Florence, Italy Summary Incorrect use of inhalers is common in real life despite advances in inhaler technology. Inhaler misuse, a consequence of device-related and/or patient-related factors, is associated with worsened health outcomes poorer disease control and increased use of health-care resources. The errors in the use of inhalers can be identified as ‘critical’ (sometimes defined as ‘essential’ or ‘crucial’) errors, which are likely to significantly impair the delivery of adequate medication to the lungs, and ‘non-critical’ errors, which are likely to result in a reduced amount of drug reaching the lungs compared with that attained using the correct technique. However, there is a wide variation in how critical errors are defined. Given the negative impact diminished disease outcomes impose on resource use, correct inhaler technique (mastery) is fundamental for effective therapy, and that inhaler device type and mastery play important roles in improving adherence, clinical outcomes, quality of life, and use of healthcare resources. There is a need for a consensus on defining critical and non-critical errors. Key Message The multitude of definitions of inhaler “errors” indicates that there is an urgent need for a consensus in the way in which critical (and non-critical) inhaler errors are defined. Inhaled drug delivery is the cornerstone treatment for asthma and chronic obstructive pulmonary disease (COPD). In the majority of patients, inhaled therapy can be given via hand-held inhaler devices such as pressurised metered dose inhalers (pMDIs) or dry powder inhalers (DPIs). However, the use of inhaler devices can be challenging, potentially leading to errors in handling that can reduce drug delivery to the lungs and effectiveness of treatment. Indeed, mastering an inhaler device involves correct preparation and handling of the device before inhalation, and an optimal inhalation technique; an error in any step of this process may lead to inadequate drug delivery to the lungs. There is no one ‘perfect device’ and several studies have shown that inhaler technique errors made by patients with asthma and COPD are common in real life with both pMDIs and DPIs despite advances in inhaler device technology. In a cross-sectional study of more than 3,500 asthma patients, inhaler errors were found to be common and not exclusive to a specific type of inhaler (Figure 1) 1. Specifically, insufficient inspiratory effort with both the Turbuhaler and Diskus inhalers was associated with an increased likelihood of uncontrolled asthma and exacerbation. By contrast, a lack of knowledge, incorrect preparation, timing or inhalation, incorrect head position and hand-breath dis-coordination with pMDIs was associated with an increased likelihood of uncontrolled asthma, but not exacerbation.1 Data from a real-life study of almost 3,000 COPD patients have indicated that inhaler handling errors are also frequent in patients with COPD (only 25% of whom did not make any error) and are associated with an increased rate of severe COPD exacerbations2. The societal and health-economic burden of poor inhaler technique is increasingly being recognised3. Worryingly, in three countries (the UK, Spain and Sweden) poor inhaler technique accounted for over €750 million in direct and indirect costs in 2015, for the two most commonly used DPIs3. These cost data, together with the increasing prevalence of obstructive lung diseases and restriction in healthcare spending is propagating the imperative need for correct and effective inhaler use. Recently, a systematic review was conducted to define ‘critical’ errors and their impact on health outcomes and resource in asthma and COPD4. Astonishingly, about 300 different descriptions of critical inhaler errors were observed. Even for the same inhaler device type, different terminology was used between different study authors to describe the same inhaler error, thus increasing the confusion observed in clinical practice concerning best inhaler practice and the limitations in determining associations with inhaler errors4. Furthermore, with the variety of definitions identified, difficulties arise in determining whether a particular inhaler type is inherently more vulnerable to critical inhaler errors. Taken in the context of a systematic review which found that inhaler use has not improved among patients over the past 40 years (1975–2014)5, these results should collectively serve as an urgent call to action for clinicians to ensure that they include patient education as an essential component of disease management. Overcoming problems with the use of inhalers starts with the prescriber choosing the most appropriate device for the individual patient 26,27. This must be followed by educating and training the patient in the use of the device (alongside more general asthma and COPD education and training). A system of monitoring should be in place, along with ongoing training in order to maintain an appropriate level of patient skill. The multitude of definitions cited within the literature indicates that there is an urgent need for a consensus in the way in which critical (and non-critical) inhaler errors are defined. There is a real need for an independent international panel of inhalation experts to collectively determine, through evidence and consensus, the definitions of critical and non-critical inhaler errors. If done for each device type, this would demystify the current confusion within the respiratory community. Hopefully, future studies will classify individual errors into categories such as inhalation manoeuvre, dose preparation, inhaler handling, device-specific or generic, in order to make comparison and analysis simpler in order to ultimately help healthcare professionals help their patients.

Drug Delivery to the Lungs, Volume 29, 2018 - The problem with critical and non-critical inhaler errors

Figure 1 - Percentage of patients making errors by device type in the CRITIKAL study

References 1.

Price DB, Roman-Rodriguez M, McQueen RB, Bosnic-Anticevich S, Carter V, Gruffydd-Jones K, et al. Inhaler Errors in the CRITIKAL Study: Type, Frequency, and Association with Asthma Outcomes. J Allergy Clin Immunol Pract. 2017;5(4):1071-81 e9. 2. Molimard M, Raherison C, Lignot S, Balestra A, Lamarque S, Chartier A, et al. Chronic obstructive pulmonary disease exacerbation and inhaler device handling: real-life assessment of 2935 patients. Eur Respir J. 2017;49(2). 3. Lewis A, Torvinen S, Dekhuijzen PN, Chrystyn H, Watson AT, Blackney M, Plich A. The economic burden of asthma and chronic obstructive pulmonary disease and the impact of poor inhalation technique with commonly prescribed dry powder inhalers in three European countries. BMC Health Serv Res. 2016;16:251 4. Usmani OS, Lavorini F, Marshall J, Dunlop WCN, Heron L, Farrington E, Dekhuijzen R. Critical inhaler errors in asthma and COPD: a systematic review of impact on health outcomes. Respir Res. 2018 Jan 16;19(1):10. 5. Sanchis J, Gich I, Pedersen S, Aerosol Drug Management Improvement T. Systematic Review of Errors in Inhaler Use: Has Patient Technique Improved Over Time? Chest. 2016;150(2):394-406.

Drug Delivery to the Lungs, Volume 29, 2018 - Bronwen Thompson. Advocacy in Respiratory Medicine – getting our voice heard for change Bronwen Thompson UK Inhaler Group It is important that patients optimise the benefit they receive from their inhalers for two reasons; to ensure that their respiratory condition is optimally controlled and to ensure that the healthcare system derives maximum benefit from the budget it commits to inhaled treatments. Ultimately, we want to see respiratory patients having the best quality of life that they can. Traditionally, efforts to maximise the benefit once an inhaler is on the market, have focused on educating patients to use their inhaler correctly, and ensuring prescribers and dispensers are familiar enough with the inhalers they are giving patients, to coach the patients in correct inhaler technique. Since the molecule and the device are integral components of the inhaled treatment, optimal usage of the device is required to deliver the molecule. The UK inhaler group (UKIG) has been exploring other ways to drive optimal use of inhalers, and one of these is to look at how policy makers can influence professionals and patients to take the use of inhalers more seriously and derive maximum benefit from them. We have identified organisations that shape relevant policy, and the mechanisms through those organisations which could be used as a channel to drive improved use of inhaler devices. These include the Department of Health and Chief Pharmaceutical Officer, Medicines and Healthcare products Regulatory Agency (MHRA) and National Institute for Health and Care Excellence (NICE). The mechanisms we seek to influence include clinical guidelines, best practice guidance, incentive schemes for hospitals and GPs, and regulation over prescribing and dispensing practices. Our work is organised around several themes: -

Colour coding of inhalers – how to maximise the benefit of colour coding so that patients understand the role of different inhalers and use them appropriately Prescribing by brand name, and dispensing the product intended by the prescriber – if the generic name is on a prescription, the patient may not always receive the same product each time Inhalers are medicines too – sometimes inhalers are treated less seriously than other medicines as they are not taken orally and swallowed Green/ environmental issues – selecting inhalers to minimise environmental harm by considering e.g. global warming potential of propellants, recycling of inhaler devices, refillable devices. The UK has set a target of reducing use of inhalers which have high global warming potential by 50% by 2022 Use of inhalers in emergencies – advice given to patients when they dial 999 for emergency services, guidance for paramedics and ambulance staff, the role of spacers with MDIs.

In these themes – which have been prioritised after consultation with our members – we may undertake research to explore and understand an issue; we may educate healthcare professionals and/or patients; we may seek to raise awareness amongst the general public, and we may influence policy makers and seek to create levers at policy level to drive optimal use of inhalers. The UK inhaler group is a coalition of not-for-profit organisations and professional societies with a common interest in promoting the correct use of inhaled therapies. There are currently 11-member organisations.

Drug Delivery to the Lungs, Volume 29, 2018 – Wachirun Terakosolphan et al. Quantification of beclomethasone dipropionate in living respiratory epithelial cells using Infrared Spectroscopy Wachirun Terakosolphan, Ali Altharawi, K. L. Andrew Chan & Ben Forbes Institute of Pharmaceutical Science, King’s College London, 150 Stamford St, London SE1 9NH, UK Summary The in situ quantification of drug in living cells is a burgeoning interest in pharmaceutical research because this information can be informative regarding drug efficacy and toxicity in patients. Infrared (IR) spectroscopy was selected as method to quantify the drug concentration entering living cells in this study. The IR technique is a nondestructive tool, widely available, but requires an additional sample compartment to conventional configurations for the investigation of living cells. The purpose of this study was to assess the suitability of IR spectroscopy for the quantification of the inhaled corticosteroid, beclomethasone dipropionate (BDP), in cultured respiratory epithelial cells (Calu-3). First, the detection limit of the instrument was evaluated by varying concentration of BDP in cell culture medium from 10 to 100 μM containing 1% v/v DMSO. A calibration curve was constructed by plotting the peak area against drug concentration from triplicate measurement. Second, the cells cultivated on the sample compartment of IR instrument were assessed for their viability. Thereafter, the cells were treated with drug solution, then monitored the change of spectrum over 24 h. We determined that it was possible to detect ~10 μM of BDP in cell culture medium using the IR instrumentation and quantify BDP over tested concentration range with a linear correlation coefficient greater than 0.99. Application of the method to cell uptake measured the uptake of BDP in living Calu-3 exposed to 80 μM drug. Key Message Ab initio studies into the application of infrared spectroscopy have demonstrated the feasibility of the technique to measure the uptake of drug into living cells and quantify intracellular concentrations. This provides a valuable analytical capability that can be utilised to study mechanisms that may influence lung retention and drug engagement with intracellular targets. Introduction In drug development, an in vitro drug concentration is informative for determination of drug efficacy and toxicity because the intracellular concentration is difficult to measure in vivo[1]. Accordingly, a number of analytical methods have been developed to estimate intracellular drug concentrations [2]. Regarding inhaled pharmaceuticals, Grainger et al. and Haghi et al. employed HPLC-MS technique to quantify the amount of inhaled beclomethasone dipropionate (BDP) that permeated across the cultured epithelial cell layers after mucosal application, and the intracellular drug concentration was quantified at the end of the experiment[3,4]. However, the real-time rate and extent of intracellular uptake of inhaled drug by living respiratory epithelial cells require the development of a sensitive, non-destructive analytical technique. Various analytical techniques have been developed to investigate the cellular dynamics in pharmaceutical research; for example, imaging microscopic platforms [5,6], Raman spectroscopy[7], and Fourier transform infrared (FTIR) spectroscopy[8,9]. Many of these methods are complicated and costly, whereas FTIR spectroscopy offers a labelfree and non-destructive chemical analysis and widely available in many laboratories. To use FTIR with living cells requires a multibounce attenuated total reflection (ATR) accessory (Figure 1) that is a minimal cost when compared to additional requirements for other techniques [10]. The ATR measurement has been demonstrated to be a useful technique to study drug accumulation in living cells previously by quantifying 20 μM of fluorouracil in cell culture medium and measuring the uptake by living cells (insulin-secreting beta cells) treated with 80 μM of fluorouracil[10]. Moreover, another study has also reported that the spectra exhibited only signal from the attached live cell on the plate without a significant contribution from the medium above the cell due to the relatively small depth of IR beam penetration (2 – 3 μm) through the ATR trough plate[11], while the thickness of cultured respiratory epithelial cell layer was 15 – 20 μm[12]. Accordingly, ATR-FTIR spectroscopy was selected as an analytical tool to measure temporal intracellular uptake of BDP by living cells in this study. BDP is a widely used inhaled corticosteroids for several respiratory diseases in recent guidelines [13,14]. As a suitable model for the respiratory epithelium, Calu-3 cells which are an adherent cell line derived from a bronchial adenocarcinoma were selected[15]. Therefore, this study aimed to evaluate the suitability of the FTIR spectroscopy for use to measure in vitro intracellular uptake of BDP by Calu-3 cell monolayers by determining the cumulative amount of BDP inside the cells as a function of time after drug exposure.

Drug Delivery to the Lungs, Volume 29, 2018 - Quantification of beclomethasone dipropionate in living respiratory epithelial cells using Infrared Spectroscopy Materials The Calu-3 human bronchial epithelial cells were from the European Culture Collection. L15 medium (Leibovitz) supplemented with 10% v/v fetal bovine serum, 1% v/v Penicillin-Streptomycin, 1% v/v non-essential amino acids (100x) and 1% v/v L-glutamine (all from Sigma-Aldrich, UK). Dimethyl sulfoxide (DMSO) was also purchased from Sigma-Aldrich, UK. 9-Chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl dipropanoate (beclomethasone dipropionate; BDP) and 9-Chloro-11β,21-dihydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17-yl dipropanoate (beclomethasone 17-propionate; 17-BMP) were supplied from Medchem Express (US). Experimental methods Drug solutions preparation: BDP and 17-BMP were separately dissolved in DMSO to make a 20 mM stock solution. The standard solutions of each component were produced by diluting the 20 mM stock solution in L15 media to obtain 10, 25, 50, 75, and 100 μM; the final solution of all concentrations contained 1% v/v DMSO. Calibration curve: The standard solutions of BDP were added onto the multibounce, temperature-controlled horizontal ATR trough (PIKE Technologies, USA) with a ZnS ATR element (Crystan Ltd., U.K.) which was then wellsealed and maintained at 37°C and covered with a warm blanket to prevent evaporation. All the samples were measured using the continuous scan FTIR spectrometers (Frontier, PerkinElmer) with a scanning time of 10 min for each sample. All measurements were acquired with a spectral resolution of 8 cm-1 and a spectral range of 3000 – 900 cm-1. Spectrum 10 (PerkinElmer) was used for all data processing. Three calibration curves were plotted between drug concentration (μM) and the peak area at the specific absorption band (~1190 cm-1). The linear equations were determined and used for calculating several parameters; a limit of detection (LOD) and linearity. Live cell preparation: The Calu-3 cells were cultivated in cell culture flasks with 75 cm2 (Greiner bio-one, Frickenhausen, Germany) using a supplemented L15 medium, in 5% CO2 environment at 37°C. For subcultivation, they were trypsinised and harvested at a confluence of 80%, and then centrifuged into a pellet. Thereafter, the cell pellet was resuspended in a supplemented L15 medium and diluted in 10 mL of medium to make a cell density of 1.5 x 106 cell per mL. 2.0 mL of the diluted cell dispersion were seeded onto the ATR trough with the warm blanket as described above (Figure 1). The IR spectrum was monitored at a 10 min interval until a steady but small increase in the absorbance of the cells was established, indicating that the cells attached and formed a monolayer on the trough plate.

Figure 1. A multibounce ATR FTIR measurement setup for live cells study.

Drug uptake measurement: The cell monolayer on the ATR element was exposed to 80 μM of the drug by adding 8 μL of the 20 mM BDP stock solution into 2.0 mL of the remaining medium on the trough plate. FTIR spectra were monitored using the same condition and setting as described above, using the function of multiple data collection with a scanning time of 10 min for 24 h per measurement. The drug concentrations at each time point were calculated from the integrated absorbance of 1190 cm-1 using the linear equation from the calibration curve. Results and Discussion IR Spectrum of BDP: The FTIR spectrum of BDP exhibited a number of major spectral bands with major identifying bands at 1730 cm-1 and 1664 cm-1, corresponding to C=O stretch for ester group and conjugated C=O stretch[16], but both bands could not be used due to an overlap with the strong adsorbed water signal (1640 cm-1)[17] and water vapour. Nevertheless, the BDP spectrum also contained a sharp band at 1190 cm-1 with relatively low absorbance of water (Figure 2A), which was a suitable marker to build the calibration curve in this study. Calibration curve of the integrated area of the peak at 1190 cm-1 plotted against BDP concentration was linear over the concentration range examined (10 – 100 μM) with a correlation coefficient greater than 0.99 (Figure 2B). The standard deviation of y-intercept for three curves was 5.8620, and the slope was 2.1975, resulting in the calculated LOD of 8.8 µM. Cell culture: Calu-3 cells developed a monolayer on the surface of ATR element and exhibited steady absorbance after 48 h incubation. The spectra of Calu-3 cells are shown in Figure 2C, with principal bandwidth of amide I and amide II at 1645 – 1545 cm-1 which are the typical vibrational bands of the protein backbone, corresponding to living cells[18]. The morphology of the cell layer on the ATR element was normal (Figure 2D).

Drug Delivery to the Lungs, Volume 29, 2018 – Wachirun Terakosolphan et al.

Figure 2. (A) FTIR spectra of BDP in the medium at various concentrations levels, which were indicated in figure. (B) Calibration curves for BDP generated from three different measurements of a set of standard solutions of known drug concentration, demonstrating linearity and repeatability of the test (mean ± SD; n = 3). The integrated absorbance is calculated based on the band at 1190 cm-1. (C) Overlay of FTIR spectra for the living cell over 48 h. (D) Typical microscopic image of the cellular layer of Calu-3 cells on the ATR element at 48 h.

Figure 3. (A) FTIR spectra of living cells before and after the exposure of 80 μM BDP in the medium. (B) The intracellular concentration of BDP measured in the cells layer as a function of time after the drug exposure.

Quantification and uptake of BDP: The first measurement of BDP 80 μM in cell culture medium was acquired immediately after adding the drug and was used as the reference spectrum to subtract from all the subsequent measurements, which were acquired at 10 min intervals. The FTIR spectra of the cells, before and after the addition of BDP, are shown in Figure 3A. A number of responses of the living cells such as the amide I – III bands (1640 – 1240 cm-1) and the DNA band (1080 cm-1)[10] were still detected in the spectra, implying that the cells were alive over the period of drug exposure. Early on, the BDP band at 1190 cm-1 in the cells can be seen that it was gradually increasing as a function of time, implying that the drug was entering the living cells (illustrated in the inset in Figure 3A). Figure 3B exhibited that the intracellular concentration of BDP gradually increased to the highest concentration of 14 μM at 6 h then started decreasing, indicating that BDP might be intracellularly metabolised to other forms of beclomethasone such as 17-monopropionate, 21-monopropionate, and inactive beclomethasone (BOH)[19] or excreted out of the cells. Regarding the metabolisation, the standard solutions of 17-BMP were measured using the same condition as the BDP standard solutions. The spectrum of 17-BMP which did not show the same major peak at 1190 cm-1 as that of the BDP confirmed that the parent drug and its metabolite in the cells could be distinguished using the FTIR spectra. Nonetheless, other spectral bands in the 17-BMP spectrum were also found in the BDP spectrum, so that the intracellular amount of 17-BMP could not be quantified explicitly in the data at this phase of the study.

Drug Delivery to the Lungs, Volume 29, 2018 - Quantification of beclomethasone dipropionate in living respiratory epithelial cells using Infrared Spectroscopy Conclusion Our preliminary study demonstrated that the FTIR technique with the multibounce ATR accessory was suitable for an in situ quantification of BDP in living respiratory epithelial cells. The lowest concentration of BDP in medium which was detected by such modified FTIR instrument was 8.8 μM. In the presence of living cells, FTIR spectral data showed chemical changes inside the cells indicative of drug uptake after the addition of 80 μM BDP solution. Moreover, the beneficial output of this technique is that uptake was measured continuously in living cells providing the capability to study temporal cellular phenomena which are hard to study from other techniques. In addition to Calu-3 cell and BDP, the modified FTIR is suitable for other cell lines and active pharmaceutical ingredients [10,20]. Thus, the FTIR method has potential to be a powerful tool in medicine development process by elucidating the rate and extent of drug uptake in cells as well as other drug-cell interactions. Interestingly, method validation, intracellular drug metabolism, and anti-inflammatory effect would be the promising focuses for further investigation. References 1.

Chien H-C, Zur AA, Maurer TS, Yee S-W, Tolsma J, Jasper P, Scott DO, Giacomini KM. Rapid Method to Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in OCT1-transfected HEK Cells. Drug Metab Dispos. 2016;44:356–64.

Chu X, Korzekwa K, Elsby R, Fenner K, Galetin A, Lai Y, Matsson P, Moss A, Nagar S, Rosania GR, Bai JPF, Polli JW, Sugiyama Y, Brouwer KLR. Intracellular drug concentrations and transporters: Measurement, modeling, and implications for the liver. Clin Pharmacol Ther. 2013;94(1):126–41.

Grainger CI, Saunders M, Buttini F, Telford R, Merolla LL, Martin GP, Jones SA, Forbes B. Critical characteristics for corticosteroid solution metered dose inhaler bioequivalence. Mol Pharm. 2012;9(3):563–9.

Haghi M, Bebawy M, Colombo P, Forbes B, Lewis DA, Salama R, Traini D, Young PM. Towards the bioequivalence of pressurised metered dose inhalers 2 . Aerodynamically equivalent particles (with and without glycerol) exhibit different biopharmaceutical profiles in vitro. Eur J Pharm Biopharm. 2014;86:38–45.

Frigault MM, Lacoste J, Swift JL, Brown CM. Live-cell microscopy - tips and tools. J Cell Sci. 2009;122(6):753–67.

Isherwood B, Timpson P, Mcghee EJ, Anderson KI, Canel M, Serrels A, Brunton VG, Carragher NO. Live cell in vitro and in vivo imaging applications: Accelerating drug discovery. Pharmaceutics. 2011;3(2):141–70.

Smith R, Wright KL, Ashton L. Raman spectroscopy: an evolving technique for live cell studies. Analyst. 2016;141(12):3590–600.

Schmidt M, Wolfram T, Rumpler M, Tripp CP, Grunze M. Live cell adhesion assay with attenuated total reflection infrared spectroscopy. Biointerphases. 2007;2(1):1–5.

Minnes R, Nissinmann M, Maizels Y, Gerlitz G, Katzir A, Raichlin Y. Using Attenuated Total Reflection-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy to distinguish between melanoma cells with a different metastatic potential. Sci Rep. 2017;7(1):1–7.

10.

Chan KLA, Fale PL V. Label-Free in Situ Quantification of Drug in Living Cells at Micromolar Levels Using Infrared Spectroscopy. Anal Chem. 2014;86:11673–9.

11.

Kazarian SG, Chan KLA. ATR-FTIR spectroscopic imaging: recent advances and applications to biological systems. Analyst. 2013;138(7):1940–51.

12.

Grainger CI, Greenwell LL, Lockley DJ, Martin GP, Forbes B. Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier. Pharm Res. 2006;23(7):1482–90.

13.

Adams NP, Lasserson TJ, Cates CJ, Jones P. Fluticasone versus beclomethasone or budesonide for chronic asthma in adults and children. In: Group. CA, editor. Cochrane Database of Systematic Reviews 2007. John Wiley & Sons, Ltd.; 2007.

14.

Asthma GI for. Global Strategy for Asthma Management and Prevention. 2016.

15.

Steimer A, Haltner E, Lehr C-M. Cell Culture Models of the Respiratory Tract Relevant to Pulmonary Drug Delivery. J Aerosol Med. 2005;18(2):137–82.

16.

Sahib MN, Abdalwahed S, Abdulameer, Darwis Y, Peh KK, Tan YTF. Solubilization of beclomethasone dipropionate in sterically stabilized phospholipid nanomicelles (SSMs): Physicochemical and in vitro evaluations. Drug Des Devel Ther. 2012;6:29–42.

17.

Liu Y, Gamble G, Thibodeaux D. Two-dimensional attenuated total reflection infrared correlation spectroscopy study of the desorption process of water-soaked cotton fibers. Appl Spectrosc. 2010;64(12):1355–63.

18.

Kong J, Yu S. Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures Protein FTIR Data Analysis and Band Assignment. Acta Biochim Biophys Sin (Shanghai). 2007;39(8):549–59.

19.

Nave R, Fisher R, McCracken N. In vitro metabolism of beclomethasone dipropionate, budesonide, ciclesonide, and fluticasone propionate in human lung precision-cut tissue slices. Respir Res. 2007;8:1–9.

20.

Fale PL, Altharawi A, Chan KLA. In situ Fourier transform infrared analysis of live cells’ response to doxorubicin. Biochim Biophys Acta - Mol Cell Res. 2015;1853(10):2640–8.

Drug Delivery to the Lungs, Volume 29, 2018 –David Mannino Respiratory Development: Thinking Outside the Box - Big Data for Respiratory Medicines ? David Mannino1,2 GlaxoSmithKline, 5 Crescent Drive, Philadelphia, PA, 19112, USA University of Kentucky, 111 Washington Avenue, Lexington, KY, USA 1

Summary The concept of big data, which is typically characterized by volume, variety, veracity, and velocity has the capacity to transform the scientific approach to respiratory disease. This transformation ranges from a better understanding of the public health or population health approach to disease, to a drug safety and drug development, to alternative approaches to monitoring outbreaks and epidemics. Medical big data can generally be thought of as being in one of three classes: large numbers (often millions) of people with small numbers of parameters (such as mortality data or other administrative data); smaller numbers of people with large amounts of data ( such as micro array or genetic data); and, more recently large numbers of people with large amounts of data. Each of these classes present their own challenges and opportunities. For example, administrative data is often complicated by issues such as missing or incorrect data and potential biases, such as residual confounding or reverse causality. In addition, big data may be useful for hypothesis generation but is not rally able to test causality. Big data approaches have been used in respiratory in several applications: identification of asthma mortality patterns across different countries over many years (which pointed to certain classes of medications as being responsible); determining the relation between area of residence and respiratory mortality (and those changes over time); and identification of the relation between air pollution exposure and mortality. Future applications may help to define better targets for respiratory therapy development. Key Message

Big data provides both promise and challenges in better understanding trends and patterns of respiratory disease, along with drug development and safety. The promise of big data includes identifying patterns of disease, finding potential drug targets, and improving the efficiency of new discoveries. The challenges of big data include missing or incorrect data and the inability to test causality with it. Introduction This paper reviews the role of big data in the understanding of respiratory disease and its potential role in the drug development and drug safety process. The components of this paper include: 1. Defining medical big data 2.

The promise of big data a.

Understanding asthma mortality i.

International comparisons over time

ii.

Small area changes in asthma deaths over time

Treatment of COPD and Asthma in a real world setting i.

Identifying potential drug targets or genetic variants i.

Salford lung study Asthma example

The challenges of big data a.

Missing and dirty data

Bigger is not always better i.

Cautionary tale of influenza tracking

Defining Medical Big Data The concept of “medical big data” has emerged in recent years as means of transforming medicine.[1] This data, by itself, is not transformative, but its subsequent analysis, interpretation, and the actions that may follow. The factors that make data “big” include the 4 V’s - volume, variety, velocity, and veracity [2] is important to consider in the application to healthcare. This provides the potential to represent what happens, in a nearly unbiased way, what happens in the real world. Medical big data can arise from a number of different sources, including public health databases ( birth and death certificates), administrative claims data, clinical registries, biometric data, electronic medical records, patient reported data, imaging data, clinical trials, and prospective cohorts, to name several sources.[3]

Drug Delivery to the Lungs, Volume 29, 2018 - Respiratory Development: Thinking Outside the Box - Big Data for Respiratory Medicines ? Medical big data can be broadly classified in to one of three forms, based on sample numbers and parameter numbers: those with very large samples and a small number of parameters, those with smaller samples and a very larger number of parameters, and, more recently, those with large samples and larger parameters. The first example has been used in public health applications for many years to analyze mortality and administrative data, and classical statistical techniques can typically be applied in the analyses. The second classification is newer and can be seen in applications where hundreds or thousands of parameters are evaluated in individuals ( such as genetic data from micro-arrays). Classical statistical testing is typically not useful in the interpretation of this type of data. The final type of big data, comprising large amounts of data on large numbers of people, present some of the issues seen in both the first and second types as described above. The Promise of Big Data Some of the potential value of medical big data has been demonstrated in the delivery of personalized medicine, the use of clinical decision support systems, tailoring diagnostic and treatment decisions, data driven population health analyses using large databases, and fraud detection and prevention. [4] Rumsfeld summarized eight areas of applicationn of big data analytics in the improvement of healthcare as predictive modelling for risk and resource use, population management, drug and medical device safety surveillance, disease and treatment heterogeneity, precision medicine and clinical decision support, quality of care and performance measurement, public health, and research applications.[5] Some of these applications have proven useful in the study of respiratory disease.

Asthma-related mortality provides two examples. A recent study looked at over 50 years of asthma deaths from 46 countries. [6] While there has been a downward trend over the this time period, there is also considerable differences between countries. Of particular interest is the historical peaks in asthma mortality observed in New Zealand in the 1960s and again in the 1970s and 1980s. This was thought to be due to the overuse of the beta agonists isoprenaline in the former period and fenoterol in the later period. A separate study examined asthma mortality over a 35 year period in the US counties.[7] This study found an overall decrease in asthma deaths over this period of time. The exception to this decrease occurred in a swath of counties in the southern part of the US that represents a population where poor blacks are overrepresented. Thus, this study points toward social and economic factors being important in asthma mortality.[8] A different kind of example of the use of big data in respiratory medicine is the Salford Lung Study, which was designed as a pragmatic, randomized real-world effectiveness study. [9] This unique study recruited patients from clinical practices, but followed them using electronic medical records and administrative data ( rather than the classical follow-up using study monitors). The COPD trial found a reduced rate of exacerbations in the intervention group ( by 8.4%).[10] The asthma study found a higher rate of response in the intervention group ( 71% vs. 56% ).[11] Both of these trials used big data to examine effectiveness of therapy as opposed to efficacy, which is what standard randomized controlled trials can test.[12] Another example of how big data can be used to inform response to therapeutic agents is the application of genetic analysis to a cohort of children with asthma to determine what factors might explain bronchodilator response. [13] In this study, five different loci associated with bronchodilator responsiveness were identified, but these showed substantial differences when looking at ethnic subgroups ( Puerto Ricans, African Americans, and Mexicans). The Challenges of Big Data Medical big data also can present significant challenges. [3] In the real world, data may be missing or incorrect. In classical statistical analyses subjects with missing data are often excluded from the analysis- although this assumes subjects with missing data do not differ from those without missing data. In the real world of big data, this is assumption is probably not true ( i.e. those with complete data and follow-up may not be similar to those with missing data and incomplete follow-up). Thus, different analytic techniques need to be used to account for this difference. Another challenge in big data analysis is the “curse of dimensionality”, which a problem seen when there are too many attributes relative to the number of observational units. This can manifest as multicolinearity, where two or more variables in a model are not independent. Although there are techniques designed to deal with this problem, they can result in the loss of important information. [3] Another challenge in the world of big data is avoiding the trap that “ bigger is better” and that these newer approaches provide more valuable information compared to traditional approaches. An example of this phenomenon can be seen in the story of Google Flu Trends, aninfluenza tracking system that was thought to be a better way for tracking flu outbreaks compared to the traditional surveillance reports that the US Centers for Disease Control and Prevention ( CDC) has been using for many years. In late 2012 and early 2013, the Google Flu Trends estimates of flu prevalence ended up being more than double what CDC had estimated.[14] While this was thought to be related to issues in the algorithm used to determine flu, an important factor was thought to be a function of the “media-stoked panic” related to influenza during the 2012-2013 flu season.[14] In this example, the traditional approach that CDC has used for years proved more accurate, although taking advantage of other approaches, including Google Flu Trends, may provide some additional unique and helpful information. Finally, in most application, medical big data is useful in hypothesis generation but is not able to demonstrate causality

Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C;David Mannino Summary

Big data provides both promise and challenges in better understanding trends and patterns of respiratory disease, along with drug development and safety. The promise of big data includes identifying patterns of disease, finding potential drug targets, and improving the efficiency of new discoveries. The challenges of big data include missing or incorrect data and the inability to test causality with it. References 1. Obermeyer Z, Emanuel EJ. Predicting the Future - Big Data, Machine Learning, and Clinical Medicine. N Engl J Med. 2016;375:pp 1216-9. 2.

Bellazzi R. Big data and biomedical informatics: a challenging opportunity. Yearb Med Inform. 2014;9:pp

8-13. 3.

Lee CH, Yoon HJ. Medical big data: promise and challenges. Kidney Res Clin Pract. 2017;36:pp 3-11.

Roski J, Bo-Linn GW, Andrews TA. Creating value in health care through big data: opportunities and policy

implications. Health Aff (Millwood). 2014;33:pp 1115-22. 5.

Rumsfeld JS, Joynt KE, Maddox TM. Big data analytics to improve cardiovascular care: promise and

challenges. Nat Rev Cardiol. 2016;13:pp 350-9. 6.

Ebmeier S, Thayabaran D, Braithwaite I, Benamara C, Weatherall M, Beasley R. Trends in international

asthma mortality: analysis of data from the WHO Mortality Database from 46 countries (1993-2012). Lancet. 2017;390:pp 935-45. 7.

Dwyer-Lindgren L, Bertozzi-Villa A, Stubbs RW, Morozoff C, Shirude S, Naghavi M, Mokdad AH, Murray

CJL. Trends and Patterns of Differences in Chronic Respiratory Disease Mortality Among US Counties, 1980-2014. JAMA. 2017;318:pp 1136-49. 8.

Mannino DM, Sanderson WT. Using Big Data to Reveal Chronic Respiratory Disease Mortality Patterns

and Identify Potential Public Health Interventions. JAMA. 2017;318:pp 1112-4. 9.

Bakerly ND, Woodcock A, New JP, Gibson JM, Wu W, Leather D, Vestbo J. The Salford Lung Study

protocol: a pragmatic, randomised phase III real-world effectiveness trial in chronic obstructive pulmonary disease. Respir Res. 2015;16:pp 101. 10.

Vestbo J, Leather D, Diar Bakerly N, New J, Gibson JM, McCorkindale S, Collier S, Crawford J, Frith L,

Harvey C, Svedsater H, Woodcock A, Salford Lung Study I. Effectiveness of Fluticasone Furoate-Vilanterol for COPD in Clinical Practice. N Engl J Med. 2016;375:pp 1253-60. 11.

Woodcock A, Vestbo J, Bakerly ND, New J, Gibson JM, McCorkindale S, Jones R, Collier S, Lay-Flurrie

J, Frith L, Jacques L, Fletcher JL, Harvey C, Svedsater H, Leather D, Salford Lung Study I. Effectiveness of fluticasone furoate plus vilanterol on asthma control in clinical practice: an open-label, parallel group, randomised controlled trial. Lancet. 2017;390:pp 2247-55. 12.

Ryan D, Blakey J, Chisholm A, Price D, Thomas M, Stallberg B, Lisspers K, Kocks JWH, Respiratory

Effectiveness G. Use of electronic medical records and biomarkers to manage risk and resource efficiencies. Eur Clin Respir J. 2017;4:pp 1293386. 13.

Mak ACY, White MJ, Eckalbar WL, Szpiech ZA, Oh SS, Pino-Yanes M, Hu D, Goddard P, Huntsman S,

Galanter J, Wu AC, Himes BE, Germer S, Vogel JM, Bunting KL, Eng C, Salazar S, Keys KL, Liberto J, Nuckton TJ, Nguyen TA, Torgerson DG, Kwok PY, Levin AM, Celedon JC, Forno E, Hakonarson H, Sleiman PM, Dahlin A, Tantisira KG, Weiss ST, Serebrisky D, Brigino-Buenaventura E, Farber HJ, Meade K, Lenoir MA, Avila PC, Sen S, Thyne SM, Rodriguez-Cintron W, Winkler CA, Moreno-Estrada A, Sandoval K, Rodriguez-Santana JR, Kumar R, Williams LK, Ahituv N, Ziv E, Seibold MA, Darnell RB, Zaitlen N, Hernandez RD, Burchard EG, Consortium NTOfPM. Whole-Genome Sequencing of Pharmacogenetic Drug Response in Racially Diverse Children with Asthma. Am J Respir Crit Care Med. 2018;197:pp 1552-64. 14.

Lazer D, Kennedy R, King G, Vespignani A. Big data. The parable of Google Flu: traps in big data analysis.

Science. 2014;343:pp 1203-5.

Drug Delivery to the Lungs, Volume 29, 2018 – Lim K M et al. Robust Method to Predict Inspiratory Flowrate from the Acoustic Signature of Swirl-Based DPIs using Deep Learning Lim K M1, Harris D S1, Sail P1 & Parry M2 1PA

Consulting, Global Innovation and Technology Centre, Back Lane, Melbourn, Herts, SG8 6DP, UK 2 Intertek Melbourn, Saxon Way, Melbourn, Herts, SG8 6DN, UK

Summary We present a novel method to predict the inspiratory flowrate through swirl-based dry powder inhalers (DPIs), using deep learning techniques to analyse acoustic data. We collected extensive acoustic data from several DPIs using a Copley Scientific BRS3000 breath simulator producing three distinct flowrate profiles to represent a range of typical inspiratory manoeuvres: weak (~2 kPa), moderate (~4 kPa), and strong (~8 kPa). The data was then processed and fed into a bidirectional Recurrent Neural Network (RNN), with the output metrics being the predicted flowrate profile measured by the mean square error (MSE), a binary classification of whether the inhaler was used or not, and the time of detection deviation in milliseconds. A MSE of 5.34 LPM was achieved with 97.5% detection accuracy, and 3% deviation in duration. The preliminary trained RNN model shows that it is possible to predict the inspiratory flowrate solely from the sound emitted by the inhaler in representative use environments, even with high levels of background noise. It is also able to robustly detect usage and the total inspiratory duration. The use of deep learning techniques for the analysis of acoustic signals from inspiratory manoeuvres has great potential value, including: Characterisation of inhaler usage to provide valuable feedback to the patient to improve their inhalation technique, understanding the efficiency of drug delivery and usage patterns over time. Key Message Accurate and robust measurement of inspiratory flowrate is currently achieved using expensive electronics that need to be retrofitted to existing dry powder inhalers (DPIs). We propose an alternative method that requires no modification to the inhaler device and no electronics. This simple, non-invasive method uses the power of a standard smartphone to listen to, and interpret, sound data emitted from an inhaler during normal use. The application of a tailored deep learning algorithm can then predict inspiratory flowrate accurately and robustly. We have effectively connected an inhaler to a smartphone for zero cost. Introduction Poor adherence amongst asthmatic and COPD (chronic obstructive pulmonary disease) patients is commonly accepted yet still inadequately addressed. The net cost of poor adherence is huge [1] and consequently numerous companies are developing - and in some cases already produce - electronically enabled add-ons, or alternative inhaler devices to address this issue. These technologies are often referred to as “smart” or “connected”, as they use electronics and wireless communication means (e.g. Bluetooth) to transfer data to and from smartphones or tablets. It is understood that monitoring inhaler usage and providing valuable feedback to the patient, payer and / or healthcare provider, is likely to improve adherence and reduce the overall cost burden. Whilst the benefits of smart inhalers are widely accepted, there is still significant challenge with the additional upfront cost of electronically enabling inexpensive DPIs. Most DPIs are comprised of simple moulded components, a spring or two, and foil / cold-form drug storage - and even the most expensive cost little over a dollar to produce. Conversely, the cost of the electronics alone to connect an existing DPI is of the order $3-4, and it is unclear how this additional expense is covered. In this study we aim to demonstrate the feasibility of a novel and alternative method to connect existing DPIs to a smartphone [2] which requires no modification to the inhaler device whatsoever, and requires no additional electronics. In collaboration with Intertek Melbourn we have used a Copley Scientific BRS3000 breath simulator to create thousands of inspiratory manoeuvres through Sun Pharma’s Starhaler DPI. Our foundation inspiratory manoeuvres are weak (~2 kPa), moderate (~4 kPa), and strong (~8 kPa). We have written code to subtly vary (or perturb) every inhalation event around the nominal, creating a total of 900 different (but known) inspiratory manoeuvres that all fall into a Weak, Moderate or Strong category. We have recorded the sound of these manoeuvres, and have used the audio data to train a deep learning algorithm to predict the inspiratory flowrate from the emitted sound alone. This is a significant advancement over previous work presented at RDD2018 in Tucson, Arizona focused on using deep learning to simply detect whether a DPI had been used.

[3],

which was

Drug Delivery to the Lungs, Volume 29, 2018 - Robust Method to Predict Inspiratory Flowrate from the Acoustic Signature of Swirl Based DPIs using Deep Learning Method The bidirectional RNN requires many data points for training. To acquire these data an experimental setup was configured at the Intertek laboratory in Melbourn. This setup consisted of connecting a Sun Pharma Starhaler DPI to the flow pipe of the Copley Scientific BRS3000 breath simulator, and placing a Zoom H4n Pro audio recorder (recording at 96 kHz, 24 bits) at a distance of 2 cm directly in front of the inhaler. The control software used was the proprietary breath simulator software and the GUI automation software, AutoIT. This automation software was chosen for its ability to run on the 32 bit windows XP OS, installed on the BRS3000, and allows the automation of highly manual processes. Weak, Moderate and Strong profiles (Figure 1) were generated according to the following model:

Where t is time (s), T is profile duration (s), I1 and I2 are inflection points (s), and P0 is the pressure (kPa), dependent on the profile used. From these nominal profiles, 300 additional profiles were generated for each nominal, using variations of Âą5% on the model parameters. These parameter changes incorporate expected variation into the generated data.

Figure 1 â&#x20AC;&#x201C; Idealised inspiratory flowrate profiles used in the BRS3000: Weak, moderate & strong

In addition to signal data, background audio was also recorded for the purpose of synthesising realistic data. A total of approximately 30 minutes of background audio was recorded and broken down into 10 second blocks. Training examples for the RNN were generated by superimposing signal audio randomly in time, on a randomly selected 10 second background clip. A total of 5000 audio clips were synthesised covering three flowrate models, weak, moderate, and strong. The ground truth information was generated concurrently with the synthesised files, with the dimensions chosen to be 2996, or 0.003 s. The flowrate values presented as the ground truth were interpolated from the original profile data. To study the collected data a Bidirectional Recurrent Neural Network (RNN) was constructed using the Keras frontend API, with the Tensorflow platform as the backend. This type of network was employed due to its ability to analyse time sequenced data, and to provide highly accurate classification and predictions. [4] The synthesised audio and ground truth data was provided to the RNN and trained, Figures 2 and 3.

Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; Lim K M et al.

Figure 2 - Architecture of the Recurrent Neural Network used to generate the classification and prediction model

Figure 3 - Depiction of how current deep learning model produce an inference for the flow rate (The flow rate is for illustration only)

Results and Discussions

Figure 4 - Deep learning prediction performance on a weak flow profile

Excellent agreement between predicted (inferred) flowrate and actual flowrate was achieved for the weak flow rate profile, Figure 4. Measurement on the flow rate accuracy was performed in terms of MSE across 3 sections of a profile: first third, middle and last third, as this corresponds to the ramp-up, sustain and decay of an inspiratory manoeuvre, Table 1.

Drug Delivery to the Lungs, Volume 29, 2018 - Robust Method to Predict Inspiratory Flowrate from the Acoustic Signature of Swirl Based DPIs using Deep Learning Table 1 - Flow rate inference accuracy

MSE (LPM)

First third

Middle

Last third

Average

8.25

0.93

6.83

5.34

The first and last third of the profile contributed the largest error components towards the MSE. This agrees with the data shown in Figure 4, where the deep learning algorithm accurately captures the peak inspiratory flowrate. However, it over predicts the beginning and the end of the inspiratory manoeuvre. The performance of the algorithm to detect if the inhaler usage is presented in the confusion matrix below: Table 2 - Confusion matrix for detecting inhaler use Actual positive Prediction

negative

positive

100

negative

The detection performance metrics were generated using 200 randomly selected unbiased data points, and the summary performance for the detection is provided below: Table 3 - Performance for detecting inhaler use Accuracy

Recall

Precision

F1-score

97.50%

100.00%

95.24%

97.56%

The current algorithm can accurately predict if the inhaler has been used to 97.5% accuracy, with a total recall and 95.24% precision, giving an F1 score of 97.6%. This algorithm has a similar detection accuracy compared to that reported previously.[3] Table 4 - Time detection accuracy Deviations Start time (s) 0.40

Stop time (s) 0.30

Duration (s) -0.11

Duration (%) 3.07%

The time deviation between predicted and the actual start and end time is less than 0.5s. The algorithm systematically under predicts the duration by 0.11s, translating to ~3% error from total deviation. Conclusion The use of supervised deep learning methods to predict, with high accuracy, inhaler flowrate from a naturally generated acoustic signature has been demonstrated. The current model uses a bidirectional recurrent neural network sequence model, in which the immediate past results are used to enhance current estimates. The model generated in these studies was found to be robust against time varying backgrounds. Moreover, the RNN model provides a similar detection rate to a previous implementation [3] which used a Convolutional Neural Network (CNN ~98%) to predict inhaler usage. The use of deep learning techniques for the analysis of acoustic signals from inspiratory manoeuvres has great potential value, including: Characterisation of inhaler usage to provide valuable feedback to the patient to improve their inhalation technique, and understanding the efficiency of drug delivery and usage patterns over time. References [1]

B G Bender, C Rand, “Medication non-adherence and asthma treatment cost”, Current Opinion in Allergy and Clinical Immunology: June 2004 - Volume 4 - Issue 3 - p 191-195

[2]

S M Lee, K M Lim, D S Harris and P Seeney, “A Novel Method Using a Smartphone to Capture and Analyse Patient Inspiratory Profiles”, Edinburgh, 2017.

[3]

K M Lim, S M Lee, D S Harris and P Seeney, “Robust Characterization of Inhalation Information Using a Deep Neural Network and Smartphone”, Tucson, Arizona, 2018.

[4]

M Schuster and P Kuldip K, “Bidirectional recurrent neural networks”, IEEE Transactions on Signal Processing, vol. 45, no. 11, pp. 2673-2681, 1997.

[5]

S Hochreiter and J Schmidhuber, “Long short-term memory”, Neural computation, vol. 9, no. 8, pp. 1735-1780, 1997.

Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; JosuĂŠ Sznitman Bioengineering Strategies for Targeted Therapeutic Delivery to the Lungs Dr JosuĂŠ Sznitman Sc. Department of Biomedical Engineering Technion, Israel-Institute of Technology

Our research lies at the crossroads of biomedical engineering and respiratory medicine. Driven by a desire to uncover the scientific underpinnings of respiratory phenomena, we leverage such knowledge towards novel strategies for delivering pulmonary therapies, including foremost inhalation aerosols. Our interdisciplinary team bridges expertise spanning bioengineering, nanotechnology and cell physiology. Our methodologies integrate cellular in vitro experiments and in silico simulations to devise biologically relevant lung models, with broad applications to drug delivery as well as cytotoxicity. Given the complexity of the lungs, our strength revolves around a multiscale approach, from the alveolar microscales to macroscopic whole-organ models. We exemplify below some of our leading contributions and ongoing efforts in the field. Biomimetic in vitro lung-on-chip platforms: With formidable progress undergone in microfabrication, lung-on-chips provide profound new opportunities to probe at true scale the pulmonary environment and deliver biomimetic platforms of the in vivo milieu1. Microfluidic airway platforms have transformed the landscape to explore in vitro respiratory physiology and advance both basic research and translational medicine. Since inhalation drug screening methods are still overwhelmingly conducted in animal models, organ-on-chips offer the prospect of tangible alternatives. Lined with human cells, within a physiologically-faithful architecture, these can help reduce the need for animal studies and importantly offer more relevant human models. We are leading major developments of the most advanced lung-on-chips to date. We have designed the first artificially-breathing acinar networks (trees) that capture physiologicallyrealistic respiratory flows2. Our acinuson-chips represent the first in vitro diagnostic tool enabling quantitative monitoring of the dynamics of inhaled aerosols directly at the acinar scales.

Figure 1: (a) Example of microfluidic alveolated airway model. (b) Closeup of airways with alveolar epithelial cells grown on a porous membrane. Inset: Staining of cell nuclei (blue) and tight junctions (red) of monolayer.

. The life-size model lung allows direct and time-resolved observations of airborne particle trajectories and their patterns of deposition in alveoli. Supported by the European Commission (ERC, 2016-2021) and via European collaboration (GermanIsrael Foundation, 20172020), we are expanding our platforms to advance precision medicine applications (Fig. 1). Patient cells can be collected from a biopsy, expanded and grown inside our devices allowing advanced diagnostic capabilities in addition to monitoring patient cell response to inhaled therapeutics for optimizing match. Ultimately, our in vitro models may provide off-the-shelf in vitro kits geared for end-users in biology, pharmacology and the pharmaceutical industry, interested in a wide range of cytotoxicity and drug screening assays.

Drug Delivery to the Lungs, Volume 29, 2018 - Bioengineering Strategies for Targeted Therapeutic Delivery to the Lungs Optimizing inhalation therapy in children: Inhalation therapy is a hallmark in treating pediatric respiratory disorders. Yet, drug deposition in the lungs of young populations remains adversely low (~5-40%). Inhalation devices and guidelines are typically derived from adult studies with dosages adapted for children according to body weight. Motivated by such shortcomings, we leverage in silico computational fluid dynamics (CFD) to explore opportunities for augmenting aerosol deposition, driven by physiological determinants (anatomy, breathing patterns) and aerosol transport dynamics3,4. Following multiscale approaches we have explored the fate of inhalation aerosols in models of the developing acinar airways and the evolution of aerosol deposition with growth5. Most recently, we have identified how deposition efficiency is captured by the existence of a narrow window of aerosol sizes optimizing deposition in upper airways. In the context of pediatric asthma therapy, our results translate to the existence of an optimal aerosol size range that evolves with age and inhalation device6 (Fig. 2). Supported by the European Commission (ERC Starting Grant), our ongoing efforts are suggesting a fresh outlook for pediatric inhalation therapies and support the prospect of child-specific inhalation therapies.

Figure 2: Aerosol deposition patterns in upper airways of a 5 yr. old and adult, following a dry powder inhalation (DPI) maneuver. Lungs shown at same scale for ease of comparison. Optimal Inhalation therapy in children advocates smaller particle sizes (relative to adults).

Point-targeted delivery of magnetic aerosols: We are developing a therapeutic platform for targeted aerosol delivery to selected points in the lungs. Our first embodiment is motivated by lung cancer (e.g. carcinoma), with ~2M patients yearly and ~90% mortality. With the prospect of point- targeted therapeutic delivery to airway tumors we aim to increase effective doses and dramatically reduce chemotherapeutic side effects. Our technology7 combines a smart inhaler delivering short pulsed aerosol boluses of magnetically-loaded therapeutic aerosols using FDA- approved SPIONs (superparamagnetic iron oxide nanoparticles), where aerosols are deposited using a designed external magnetic field. Our early in silico efforts have supported the prospect of such inhalation technique8,9. Following endorsement by the Israel Innovation Authority, we have developed a first prototype where a short aerosol bolus was successfully delivered to a desired point in 3D printed bronchial models using an external magnet, while all other aerosols were exhaled. Our longer term goals are to expand to ex and in vivo experiments. As a versatile inhalation platform, our technology may be further envisioned for early-stage treatment of severe infections, e.g. bacterial (pneumonia) and fungal (e.g. aspergilloma).

Liquid-foam therapy (LIFT) for Acute Respiratory Distress Syndrome (ARDS): ARDS is an inflammatory lung condition caused for example by sepsis, pneumonia and head/chest injury, affecting annually 255,000 in the US alone. ARDS is characterized by depletion of the lungsâ&#x20AC;&#x2122; inner liquid coating (pulmonary surfactant), which reduces surface tension forces and allows the lungs to expand. Patients lay in the intensive care unit with mortality rates ~40%. Although surfactant replacement therapy (SRT) is life-saving in neonates, administration methods for adults and even children remain inadequate. Traditional endotracheal surfactant liquid instillations used in newborns fail in larger lungs: liquid drains into pools, drowning these regions while leaving others untreated.

Drug Delivery to the Lungs, Volume 29, 2018 – Josué Sznitman To overcome these hurdles, our novel method uses liquid foam as a carrier for surfactant delivery10. Such an approach represents a paradigm shift in SRT, and more broadly, in the field of therapeutic pulmonary delivery. Unlike liquid installations, foam is “gravity defiant”: LIFT distributes homogeneously throughout the lungs, capable of delivering sufficient doses. In contrast to aerosols and liquid instillations, LIFT can flow through narrowed airways and penetrate beyond airway obstructions. In a proof-of-concept, we have demonstrated the feasibility of our technology, both in vitro in 3D printed and microfabricated airway models, and ex vivo in excised pig lungs using fluoroscopy (Fig. 3). Our promising technology has drawn considerable attention supported by the European Commission (Proof-of-Concept, 20182020), and will fund our first pre-clinical in vivo experiments. LIFT holds tremendous potential beyond ARDS treatment and may be leveraged for other lung therapies, including stem cell delivery to treat Idiopathic Pulmonary Fibrosis (IPF) and Chronic Obstructive Pulmonary Disease (COPD).

(a)

(b)

Figure 3: Experiments ex vivo in porcine lungs. (a) Right lung after liquid installation with pooling (red circle). (b) Left lung following LIFT with homogenous delivery. Inset: LIFT reaches all small airways in an in vitro microfluidic model.

1mm

Bibliography 1. Tenenbaum-Katan J, Artzy-Schnirman A, Fishler R, Korin N, and Sznitman J (2018). “Biomimetics of the pulmonary environment in vitro: A microfluidics perspective”. Biomicrofluidics 12: 042208. 2. Fishler R, Hofemeier P, Etzion Y, Dubowski, and Sznitman J (2015). “Particle dynamics and deposition in true-scale pulmonary acinar models”. Scientific Reports 12071: 1-13. 3. Hofemeier P and Sznitman J (2015). “Revisiting pulmonary acinar particle transport: convection, sedimentation, diffusion and their interplay”. Journal of Applied Physiology 118: 1375-1385. 4. Hofemeier P, Koshiyama K, W ada S and Sznitman J (2018). “One (sub-)acinus for all: Fate of inhaled aerosols in heterogeneous pulmonary acinar structures”. European Journal of Pharmaceutical Sciences 113: 53-63. 5. Tenenbaum-Katan J, Hofemeier P and Sznitman J (2016). “Computational models of inhalation therapy in early childhood: therapeutic aerosols in the developing acinus”. Journal of Aerosol Medicine and Pulmonary Drug Delivery 29: 288-298. 6. Das P, Nof E, Amirav I, Kassinos SC and Sznitman J (2018). “Optimizing inhaled aerosol delivery to upper airways in children: insight from Computational Fluid Dynamics (CFD)”. Under review. 7. Ostrovski Y and Sznitman J (2017). “Targeted delivery of aerosols of magnetized active agents”. PCT/IL2017/050472. 25 April 2017. 8. Ostrovski Y, Hofemeier P, and Sznitman J (2016). “Augmenting regional and targeted delivery in the pulmonary acinus using magnetic particles”. International Journal of Nanomedicine 11: 1-11. 9. Ostrovski Y, Dorfman S, Mezhericher M, Kassinos SC, and Sznitman J (2018). “Targeted drug delivery to upper airways using a pulsed aerosol bolus and inhaled volume tracking method”. Flow, Turbulence and Combustion, https://doi.org/10.1007/s10494-018-9927-1 (in press). 10. Ostrovski Y and Sznitman J (2017). “Foam for pulmonary drug delivery”, PCT/IL2017/051208. 6 November 2017.

Drug Delivery to the Lungs, Volume 29, 2018 - Philip W. Ind et al. Cannabis, Cigarette Smoking and Lung Function –not all downhill? Philip W. Ind Respiratory Medicine Imperial College London Summary The adverse effects of cigarette (tobacco) smoking are well established and well known. Cannabis (also called marijuana) is the most widely used illicit drug and the second most smoked substance. In its various forms it has been increasingly licensed for recreational as well as medical use. Cannabis contains many pharmacologically active substances and cannabinoid pharmacology is very complex. Much is known about its psychoactive properties but rather less about its pulmonary effects. Cannabinoids have well documented anti-inflammatory and immunomodulatory effects. This review explores the information available regarding its effects on the lungs. In normal subjects acute exposure to inhaled and oral marijuana produces substantial bronchodilatation. This also occurs in asthmatic subjects and patients recovering from an asthma exacerbation. Less information is available in COPD. Epidemiological information about chronic exposure to marijuana is compounded by the difficulties of studying an illicit substance, the problems of variability in composition and quantification of consumption and the fact that it is usually smoked in combination with tobacco. Available data suggest that cannabis smoking commonly leads to chronic bronchitis (cough and phlegm). However, surprisingly it does not generally cause progressive airflow obstruction and COPD. It produces an increase in vital capacity rather than a fall in FEV 1. It has been associated with the development of bullous lung disease but not definite emphysema in the limited studies. It does not seem to be associated with lung cancer nor with frequent respiratory infections. Key Message Cannabis smoking frequently causes bronchitis with symptoms of productive cough but unlike cigarette smoking does not generally lead to COPD. Long term exposure increases vital capacity rather than reducing FEV 1. Acutely, marijuana produces bronchodilatation in normal subjects and in asthmatics. The pharmacology of cannabinoids is complex but merits further investigation. Introduction Cannabis is the most widely used illicit drug in the world. Cannabis, commonly called marijuana, is the second most smoked substance, after tobacco. Cannabis comes from a flowering plant, native to central Asia and the Indian sub-continent. The genus includes 3 different species, Cannabis sativa, Cannabis indica and Cannabis ruderalis. They contain 2 major active compounds, delta-9-tetrahydrocannabinol (d-9-THC) and cannabidiol (CBD). Altogether about 90 cannabinoids and over 400 compounds are produced. The psychoactive compound is THC but this is modulated by CBD. Higher THC content occurs in sativa-dominant strains while the indica-dominant strains have more CBD. Recently, skunk-like cannabis containing very high THC concentrations has become prevalent.

The flower tops of the plant, are called ‘bud’, the resin ‘hash’ and other common names include ‘weed’, ‘dope’, ‘grass’, ‘hemp’, ‘ganga’, ‘reefer’, ‘spliff’, ‘toke’ and ‘blunt’. Cannabis can be smoked in a variety of ways, usually without a filter and burned at a higher temperature, and with users generally holding their breath for longer periods of time, compared to tobacco smokers. Joints can be made using just cannabis leaves or can be mixed with tobacco in ‘spliffs’. Many cannabis users also concurrently smoke tobacco cigarettes. Routes of administration vary by geographical region as well, with European countries mostly smoking spliffs while Americans largely smoke cannabis-only joints. Aside from joint smoking, users may also use water bongs, pipes and, more recently, vaporisers [1]. Recreational cannabis use, estimated at 240 million world-wide, has been legalized in Uruguay since 2013, in 5 US states and, very recently, in Canada. Medicinal use is legal in 31 US states, at least 30 countries and is imminent in the UK. However, we still know very little about the long term effects of smoking cannabis on the respiratory system and on health in general. This review focuses on effects on the lungs.

Tobacco smoking is well known to increase the risk of chronic bronchitis, emphysema, and small airways disease (all components of chronic obstructive pulmonary disease -COPD) as well as the development of various forms of lung cancer and other neoplasia. Chronic cannabis smoking might be expected to have similar effects, considering that the contents and properties of tobacco and cannabis smoke are similar [2].

Drug Delivery to the Lungs, Volume 29, 2018 - Cannabis, Cigarette Smoking and Lung Function â&#x20AC;&#x201C;not all downhill? Symptoms Respiratory symptoms such as cough, sputum production, and wheeze are increased in current cannabis use [3,4,5,6]. However, surprisingly, associations with shortness of breath were not found in the larger studies [4,5,6]. This suggests that cannabis smoke causes chronic bronchitis in current smokers but not shortness of breath or irreversible airway damage. Studies examining the effect of quitting marijuana smoking support this showing a significant reduction in morning cough, sputum production, and wheeze compared to continuing smokers [4]. No increased risk of developing chronic bronchitis was found in quitters compared to non-smokers at 10 years follow up [4]. In young adults vaping cannabis is increasingly popular but long-term respiratory health effects are not known. Acute airway effects of cannabis Experimentally, the acute bronchodilator effect of inhaled cannabis, seen in normal and asthmatic subjects and asthmatics recovering from exacerbations is well described as an effect of THC [7,8]. However, since cannabinoids can have partial agonist, or even antagonist, effects little is known about differences in airway effects from different strains of cannabis containing varying concentrations of cannabinols. Chronic effects on Lung function COPD is conventionally diagnosed when a patient has an irreversible reduced forced expiratory volume in 1 sec (FEV1) compared with forced vital capacity (FVC) on spirometry. Several large, recent, observational studies have reported an increase in FVC with little or no change in FEV 1 in long-term marijuana-only users, even after 20 joint years of smoking (1 joint-year is equivalent to 365 joints per year) [3,4,6,9]. A reduced FEV 1/FVC ratio due to the increased FVC, clearly differs from the classical spirometric changes seen in tobacco smoking. The mechanism(s) of this increase in FVC are not clear. Respiratory muscle training by the breath-holding techniques used during marijuana smoking has been proposed. However, there is little evidence that training can increase FVC. Additional lung function measurements have only been examined in smaller studies. Several studies have reported very small, not always consistent, changes in total lung capacity, functional reserve capacity, residual volume and specific airways conductance and resistance. Small reductions in carbon monoxide transfer factor have been reported only in smokers of cannabis and tobacco. Interestingly, a 13% reduction in exhaled nitric oxide, was recorded after marijuana use within 0 to 4 days [10]. Rather conflicting results on airway responsiveness have been reported in 3 studies with significant methodological differences. We do not know why cannabis smoking does not produce COPD. Possible explanations include a persistent bronchodilator effect (offsetting airway narrowing) or anti-inflammatory or immunomodulatory effects of THC [11]. Bullous Lung Disease Bullous lung disease, predominantly upper lobe involvement with added peripheral emphysema usually presenting with pneumothorax, is widely recognised in heavy marijuana smokers. However, there are actually few relevant data with only 57 cases in 7 case series and 11 case reports up to 2018 [1]. They are not representative of the general marijuana smoking population. A single cross-sectional study examined radiological changes among New Zealand marijuana smokers reporting an increase in macroscopic emphysema in tobacco smokers compared with non-smokers but not in cannabis-only smokers. Low density lung regions on HRCT in cannabis smokers were interpreted as hyperinflation rather than microscopic emphysema [6]. A study in spontaneous pneumothorax recording smoking status and emphysema on CT found no difference in emphysema prevalence among tobacco smokers and tobacco + cannabis smokers (there were no cannabis only smokers) [12]. No definite association of cannabis smoking and early emphysema has been established. Cannabis and Lung Cancer Although some studies have suggested precancerous histological changes in the bronchi of marijuana smokers no definite association of cannabis smoking and lung cancer has been established. Pooled analysis of 6 case-control studies including 2,159 lung cancer cases and 2,958 controls found little or no association between cannabis smoking and lung cancer. A large retrospective cohort study of 64,855 subjects found no increased risk of cancer after 8.6 years, although mean age was relatively young [13]. One 40-year longitudinal cohort study of 49,321 Swedish conscripts reported a doubling of lung cancers in those who had smoked cannabis more than 50 times [14].

Drug Delivery to the Lungs, Volume 29, 2018 - Philip W. Ind et al. Conclusions Acutely marijuana is a bronchodilator. Chronic cannabis smoking is associated with chronic bronchitis but not progressive airflow obstruction and COPD. A small unexplained increase in VC occurs. Heavy marijuana smoking is associated with bullous disease but there is no definite association with emphysema, lung cancer or recurrent infections. The general paucity of data, the evolving nature of available marijuana (newer, stronger forms, different modes of inhalation etc) and the important confounding factor of tobacco use have led to difficulties interpreting the health impact of marijuana on the lungs. More research, particularly prospective studies disentangling the effects of concurrent tobacco smoking are required.

References 1. Russell C, Rueda S, Room R, Tyndall M, Fischer B. Routes of administration for cannabis use; basic prevalence and related health outcomes: A scoping review and synthesis. Int J Drug Policy [Internet]. Elsevier; 2018 Feb 1;52:87–96. Available from: http://dx.doi.org/10.1016/j.drugpo.2017.11.008 2. Ribeiro LIG, Ind PW. Effect of cannabis smoking on lung function and respiratory symptoms: a structured literature review. Npj Prim Care Respir Med [Internet]. The Author(s); 2016 Oct 20;26:16071. Available from: http://dx.doi.org/10.1038/npjpcrm.2016.71 3. Moore BA, Augustson EM, Moser RP, Budney AJ. Respiratory effects of marijuana and tobacco use in a U.S. sample. J Gen Intern Med. United States; 2005 Jan;20(1):33–7. 4. Hancox RJ, Shin HH, Gray AR, Poulton R, Sears MR. Effects of quitting cannabis on respiratory symptoms. Eur Respir J. England; 2015 Jul;46(1):80–7. 5. Kempker JA, Honig EG, Martin GS. The effects of marijuana exposure on expiratory airflow. A study of adults who participated in the U.S. National Health and Nutrition Examination Study. Ann Am Thorac Soc. United States; 2015 Feb;12(2):135–41. 6. Aldington S, Williams M, Nowitz M, Weatherall M, Pritchard A, McNaughton A, et al. Effects of cannabis on pulmonary structure, function and symptoms. Thorax. England; 2007 Dec;62(12):1058–63. 7. Tashkin DP, Shapiro BJ, Frank IM. Acute pulmonary physiologic effects of smoked marijuana and oral 9 tetrahydrocannabinol in healthy young men. The New England journal of medicine. 1973;289(7):336-41. 8. Williams SJ, Hartley PR, Graham JP. Bronchodilator effect of tetrahydrocannabinol administered by aerosol to asthmatic patients. Thorax 1976;31`:720-23. 9. Tetrault JM, Crothers K, Moore BA, Mehra R, Concato J, Fiellin DA. Effects of Marijuana Smoking on Pulmonary Function and Respiratory Complications: A Systematic Review. Archives of internal medicine. 2007. p. 221–8. 10. Papatheodorou SI, Buettner H, Rice MB, Mittleman MA. Recent Marijuana Use and Associations With Exhaled Nitric Oxide and Pulmonary Function in Adults in the United States. Chest. 2016. p. 1428–35. 11. Atakan Z. Cannabis, a complex plant: different compounds and different effects on individuals. Therapeutic Advances in Psychopharmacology. 2012;2(6):241-54. 12. Ruppert AM, Perrin J, Khalil A, Vieira T, Abou-Chedid D, Masmoudi H, et al. Effect of cannabis and tobacco on emphysema in patients with spontaneous pneumothorax. Diagn Interv Imaging [Internet]. 2018; Available from: http://www.sciencedirect.com/science/article/pii/S2211568418300391 13. Callaghan RC, Allebeck P, Sidorchuk A. Marijuana use and risk of lung cancer: a 40-year cohort study. Cancer Causes Control. Netherlands; 2013 Oct;24(10):1811–20. 14. Zhang LR, Morgenstern H, Greenland S, Chang S-C, Lazarus P, Teare MD, et al. Cannabis smoking and lung cancer risk: Pooled analysis in the International Lung Cancer Consortium. Int J cancer. United States; 2015 Feb;136(4):894–903.

Drug Delivery to the Lungs, Volume 29, 2018 – Larissa Gomes dos Reis et al. Gene Delivery to Lung Epithelial Cells Using a Cell Penetrating Peptide Larissa Gomes dos Reis1, Maree Svolos1, Lyn M Moir 1, Rima Jaber2, David Fecher2, Norbert Windhab2, Paul M Young1 & Daniela Traini1 1

Respiratory Technology, Woolcock Institute of Medical Research and Discipline of Pharmacology, Faculty of Medicine and Health, The University of Sydney, NSW, 2037, Australia 2 Evonik Nutrition and Care GmbH, Kirschenallee, 64293, Darmstadt, Germany

Summary In this study, plasmid DNA (pDNA)-encapsulated nanoparticles (NPs) were manufactured (<200 nm). Addition of a novel proprietary cell penetrating peptide (CPP) to the formulation was essential for efficient internalisation. Cellular uptake in Beas-2B occurred rapidly (less than 3h) and efficiently (83.9% of the cell), with NP-DNA-CPP localised in the nucleus. Low level of protein expression was observed after 96 h of incubation. The NP-DNA-CPP did not affect mitochondrial respiration or membrane integrity. Reactive Oxygen Species (ROS) and Interleukin (IL-8) concentrations were reduced for formulations containing CPP. The increase in necrosis observed for NPDNA-CPP is likely to be related to the endosomal-escape mechanism that occurs when exposed to these particles. Although further investigation with other lung cells is needed, this system appears to be a safe delivery method for pDNA to the lungs. Key Message This study provides valuable information regarding the toxicology of polymeric systems in pDNA delivery to lung epithelial cells. Addition of CPP to pDNA-encapsulated NPs, resulted in protein expression, reduced ROS and IL8 production, with minimal effects on apoptosis and necrosis. NP-DNA-CPP seems to be safe for pDNA delivery to the lung. Introduction Intracellular delivery of pDNA is an essential requirement for gene delivery. Efficiency and transport of nanoparticle based-pDNA systems is directly correlated to the physico-chemical characteristics of the particles, due to their adhesion and interaction with the cells. In addition, following internalisation, particles need to escape the lysosomal-endocytic pathway to be released into the cytoplasm and enter the nucleus to induce protein expression. Although many studies have been carried out to characterise and optimise intracellular delivery [1], very little is known in regards to the effects of these formulations in lung epithelial cells. This study assessed the effects of pDNA-encapsulated NPs formulated with a CPP to enhance intracellular delivery. These particles were characterised and toxicological effects investigated in a healthy-derived lung epithelial cell line. Method Nanoparticles were prepared using a double emulsion technique. RESOMER® 502H (PLGA, Evonik, Germany) was used for NPs production. Bovine serum albumin 2% (w/v), and calcium salts were included in the first emulsion, while polyvinyl alcohol 1% (w/v) was used in the second aqueous solution [2]. A 5446 bp plasmid pcDNA3-EGFP (Addgene plasmid # 13031 - a gift from Doug Golenbock), encoding for enhanced green fluorescent protein (eGFP) was included during particle manufacturing for encapsulation. For flow cytometric and microscopy studies, Rhodamine B (Sigma Aldrich, Australia) at concentration 0.1% of polymer weight was added to the first aqueous phase. To remove any dye excess, after NP manufacturing, samples were centrifuged at 13,200 rpm for 20 min at 4°C. For the treatment with CPP (Supplied and Proprietary of Evonik Nutrition and Care GmbH, Germany [3, 4]), NPs were re-suspended in CPP solution to get a final 1:1.5 ratio of peptide: PLGA (mg:mg). Studies were performed at 0.4 mg/mL of PLGA. Particle size distribution and surface charge was assessed by dynamic light scattering (DLS) using the Malvern Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). Encapsulation efficiency (%) was determined using Pico Green Kit (Thermo Fisher), via an indirect method. Particles were visualised by transmission electron microscope (TEM) to confirm size and assess particle agglomeration. To assess cellular uptake, Beas-2B cells (ATCC, USA) were incubated with NPs formulated with (NP-DNA-CPP) or without CPP (NP-DNA) for 3 h and analysed by flow cytometer (BD Accuri C6, BD, USA) and confocal microscopy (Nikon A1R Confocal Fluorescence Microscope with Apo LWD 40x, Nikon, USA). For microscopic studies, to track the NPs-DNA inside of the cell, the cellular membrane (Cell Mask Deep Red Membrane Stain, 5 min at 37˚C, Thermo Fisher, Australia) and nucleus (DAPI, 1 µg/mL for 10 min, ThermoFischer, Australia) were also stained. Images were analysed for colocalisation using Huygens Software. Protein expression was assessed using an eGFP SingleStep ELISA® assay (eGFP Elisa, Abcam, USA). Beas-2B cells were incubated for 3 h with the NPs-DNA. Protein expression was assessed following 48, 72 and 96 h of the treatment being added. The expression of eGFP protein was normalised for the concentration of total protein, which was quantified using a Bicinchoninic Acid Protein Assay Kit (BCA1, Sigma Aldrich, Australia).

Drug Delivery to the Lungs, Volume 29, 2018 - Gene Delivery to Lung Epithelial Cells Using a Cell Penetrating Peptide To further investigate the potential use of this delivery system, the nanotoxicological effects of the NPs was assessed by metabolic assay (MTS Kit, Promega) and membrane integrity (LDH assay, Raybiotech, USA) after 24 h incubation. Apoptosis and necrosis (Annexin-V and Propidium Iodide, ThermoFischer, Australia) was assessed by flow cytometry (BD Accuri C6, BD, USA). Oxidative stress (DCFDA 2 µM for 15 min, Sigma, Australia) was investigated in a spectrophotometer using hydrogen peroxide (0.03% v/v; Sigma, Australia) as a positive control, while inflammatory markers (IL6, IL8 and TNF-α, BD OptEIATM, USA) were assessed using ELISA, both after 24 h of incubation. Results were analysed using two-way ANOVA. Means were compared using Tukey’s test, unless otherwise stated, at 95%CI. Differences were considered significant when * P<0.05; ** P<0.01; ***P<0.001; ****P<0.0001. Results The NPs manufactured were of 157.3 ± 1.86 nm (n=3). Both pDNA encapsulation and addition of CPP significantly increased particle size (p<0.0001) to 175.3 ± 0.36 nm for NP-DNA and 167.8 ± 0.49 nm for NP-DNACPP and were considered suitable for intracellular delivery [3]. The addition of CPP significantly increased the surface charge from -12.57 ± 0.21 mV for NP-DNA, to ~ -0.13 ± 0.05 mV in NP-DNA-CPP (P<0.0001), respectively. Particle size was confirmed by TEM. A high encapsulation efficiency was obtained, 96.72 ± 0.36 %. The CPP ability to enhance intracellular delivery was assessed by flow cytometry after 3h incubation with Beas2B cells. When the NPs were formulated with CPP and DNA, 83.85 ± 1.2% of the cells had NPs internalised, in comparison to 2.53 ± 0.5% internalisation when only NP-DNA was used. Internalization was confirmed by visualisation under confocal microscope (Figure 1) using a z-stack, in which the NP-DNA-CPP (yellow) can be clearly visualised within the cellular membrane limits (brown), and colocalising with the cell’s nucleus (blue).

Figure 1: Internalisation of NP-DNA and NP-DNA-CPP in Beas-2B assessed after 3h incubation by confocal microscopy (yellow, NP-DNA-CPP; blue, nucleus; brown, cellular membrane). Due to the high internalisation efficiency and the colocalisation with the nucleus observed in the confocal studies, protein expression in the Beas-2B cell line was assessed after 24, 48 and 96 hours of incubation with NP-DNACPP (Figure 2). Results expressed as a fold-increase response in relation to placebo particles (no pDNA included) showed no significant increase (p>0.05) after 48 and 72 h of incubation. However, a 7.4-fold increase to a very low level of eGFP expression (p<0.05) was observed after 96 h of incubation with NP-DNA-CPP in the ELISA, but could not be detected in the confocal microscope.

Figure 2: pDNA expression at 48, 72 and 96h in Beas-2B cells after incubation with NP-DNA-CPP

The ability of CPP to translocate a cargo across the cellular membrane can be associated to the effect of these molecules on the cellular membrane integrity. The relationship between the effect of CPP on the cellular integrity and the increased in internalisation promoted by these peptides was assessed using Lactate Dehydrogenase (LDH) assay. Results showed that at all concentrations tested (from 0.1 mg/mL to 1.2 mg/mL PLGA), no LDH leakage was observed for both NP-DNA and NP-DNA-CPP. The effects of both formulations on mitochondrial Image of NP-DNA-CPP activity was also investigated and showed no toxic effects (cell viability higher than 80%).

Drug Delivery to the Lungs, Volume 29, 2018 – Larissa Gomes dos Reis et al. Inducing the cell into an apoptotic state has been associated with NPs exposure to different cell systems [5, 6]. Results for the percentage of cells in early or late apoptosis are shown in Table 1. No effects were observed when investigating early apoptosis, however a significant increase of cells in late apoptosis occurred for Beas-2B exposed to NP-DNA-CPP (p<0.0001). An increase in necrosis was also observed after 24 h from exposure to NPDNA-CPP (P<0.0001), this effect was, however, reversed after 48h with a significant decreased in necrosis in comparison to the media control (P<0.0001) Table 1: Apoptosis and Necrosis levels of Beas-2B cells exposed to both NP-DNA and NP-DNA-CPP Live (PI-/Annexin -)

Early Apoptosis (PI-/Annexin V+)

Late Apoptosis (PI+/Annexin V-)

Necrosis (PI+/Annexin V-)

24h

48h

24h

48h

24h

48h

24h

48h

Cell

97.3±0.3

83.3±0.1

0.35±0.1

0.28±0.1

1.38±0.1

7.29±0.08

1.03±0.01

9.15± 0.01

NP-DNA

97.7±0.3

97.0±0.3A

0.29 ±0.1

0.06±0.1

1.12±0.1

1.89±0.24A

0.80±0.12

1.05± 0.06A

5.62±0.48A

14.05±1.0A

2.97± 0.40A

NP-DNA81.1±0.4A 91.3±0.7A 0.41±0.2 0.16±0.1 4.48±0.7A CPP Means were compared using two-way ANOVA and Tukey’s Test (A, p<0001), n=3

The increase in surface area that occurs with smaller particle size often affects the reactivity of these particles in biological systems, impacting on elevated ROS production [5-7]. The effect of the NPs on ROS production was assessed over 90 min and showed significant changes between particles formulated with or without CPP (Figure 3). For NP-DNA, oxidative stress was similar to those observed in Serum Free Media (SF):NaCl control (vehicle), however, when exposed to NPDNA-CPP the oxidative stress was significantly reduced (P<0.05) at all time points. The effect on eliciting cytokine release was also investigated and showed no changes in IL-6 and TNF-α (P>0.05). However, when IL-8 levels were investigated, a fold decrease to 0.73 and 0.54 compared to media control was observed for NP-DNA-CPP after 24 and 48h incubation, respectively.

Discussion

Figure 3: ROS Production in Beas-2B cell line exposed to NP-DNA and NP-DNA-CPP

The physicochemical properties of the NPs have direct impact on its toxic effect due to higher surface-to-volume ratio, increasing the reactivity between particles and cells [7]. The particle size and morphology of the manufactured NPs were characterised and showed suitable size (less than 200 nm) and shape (spherical) for intracellular delivery. A high encapsulation efficiency was observed using Pico Green assay, indicated that pDNA was still in a double stranded form, despite its exposure to high shear forces during particle manufacture. This effect is likely to be related to the calcium salts used in the first emulsion that, by complexing with the pDNA, protect the nucleic acid structure during homogenisation [2]. Increase in surface charge is a tool often used to enhance particle internalisation, with undesirable increase in toxic effects [8]. In this study, the addition of CPP to the NPs-DNA, increased the surface charge of the NP-DNA-CPP to neutrality, however no effect on cell viability was observed for either formulations (with or without CPP), corroborating the results from Platel et al [8] that showed no effects on cell viability for negatively charged and neutral particles. Particles with surface charge close to neutrality are generally associated with high particle aggregation and low stability, however, TEM images showed minimal agglomeration, which could be correlated to steric hindrance provided by the peptide on the outer surface of the NP-DNA-CPP [9]. Surface modification was shown to directly impact NPs internalisation [7] as it changes both specific (via receptors) and non-specific (i.e. Ionic strength, electrostatic forces, pH) interactions between NPs and cellular membrane. In this study, the addition of CPP to the formulation was essential for internalisation in Beas-2B, with rapid (less than 3 hours) and efficient (83.85% of the cells) internalisation for NP-DNA-CPP. Adding the CPP increased the surface charge, and may have influenced its interaction with the heparin sulphate molecules present in the membrane, as previously described in the literature [10]. As a result of high NPs internalisation, as well as the colocalisation of NP-DNA-CPP in the nucleus, expression of eGFP protein at low level occurred after 96 h of incubation.

Drug Delivery to the Lungs, Volume 29, 2018 - Gene Delivery to Lung Epithelial Cells Using a Cell Penetrating Peptide The cytotoxicity was first investigated via a metabolic and cellular membrane integrity assay, which showed that internalisation did not affect mitochondrial respiration or damage the cellular membrane, at the concentrations tested. A key mechanism for nanotoxicity is ROS generation, which occurs mainly due to the increased reactivity between cells and NPs [5]. In Beas-2B cells, the increase in oxidative stress observed for NP-DNA was reversed when CPP was added to the formulation (Figure 3), agreeing with other studies showing that surface modifications can impact on NPs properties [8]. This effect may be related to the nature of the CPP, which decreased ROS production in the same cell line. ROS generation is also an important mediator of inflammatory responses (IL-8, IL-1Î˛) [11], however in this study, although NP-DNA increased ROS production, IL-8 was reduced in these cells compared to media control. The same reduction was observed for NP-DNA-CPP at both 24 and 48h, indicating that the IL-8 pathway was not associated to ROS production when exposed to these NP-DNACPPs. Lysosomal escape is a common aspect of gene delivery, as most of the NPs enter the cells via an endocytic pathway. In this process, NPs induce the endocytic vesicles to burst whilst in the process of maturation into the lysosomal compartment. This bursting mechanism not only released the NPs into the cytosol but also lysosomal hydrolases, which have a direct effect on inflammation, necrosis and apoptosis [5,6]. The significant increase in cells in late apoptosis and necrosis compared to media control observed at 24 h was reversed at 48 h, indicating the cells quickly recovered from this stressor. Conclusion pDNA-encapsulated NPs were manufactured and showed efficient internalisation when CPP was added to the formulation. Low protein expression was observed after 96 h of incubation, indicating a first hint of effective gene delivery that should be further investigated. No effects on mitochondrial respiration or membrane integrity were observed. ROS and IL-8 concentrations were reduced for formulations containing CPP. Although an increase in necrosis and late apoptosis occurred after 24h, Beas-2B cells quickly recover from this stress with further incubation. Although further investigation is needed with other lung cells, this system appears to be a safe delivery method for pDNA to the lungs.

References [1] Azevedo C, Macedo M H, Sarmento B: Strategies for the enhanced intracellular delivery of nanomaterials, Drug discovery today 2018;23:944-59. [2] DĂśrdelmann G, Kozlova D, Karczewski S, Lizio R, Knauer S, Epple M: Calcium phosphate increases the encapsulation efficiency of hydrophilic drugs (proteins, nucleic acids) into poly(d,l-lactide-co-glycolide acid) nanoparticles for intracellular delivery, Journal of Materials Chemistry B 2014;2:7250-9. [3] Brock R F R, Fotin-Mleczec M, Hufnagel H, Windhab N: Lactoferrin peptides useful as cell-penetrating peptides, In: Patent I, editor.2011. [4] Duchardt F, Ruttekolk I R, Verdurmen W P, Lortat-Jacob H, Burck J, Hufnagel H, et al: A cell-penetrating peptide derived from human lactoferrin with conformation-dependent uptake efficiency, Journal Biological Chemistry 2009;284:36099-108. [5] Park M V, Neigh A M, Vermeulen J P, de la Fonteyne L J, Verharen H W, Briede J J, et al: The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles, Biomaterials 2011;32:9810-7. [6] Wang Y, Santos A, Evdokiou A, Losic D: An overview of nanotoxicity and nanomedicine research: principles, progress and implications for cancer therapy, Journal of Materials Chemistry B 2015;3:7153-72. [7] Zhang B, Sai Lung P, Zhao S, Chu Z, Chrzanowski W, Li Q. Shape dependent cytotoxicity of PLGA-PEG nanoparticles on human cells. Scientific Reports. 2017;7:7315. [8] Platel A, Carpentier R, Becart E, Mordacq G, Betbeder D, Nesslany F: Influence of the surface charge of PLGA nanoparticles on their in vitro genotoxicity, cytotoxicity, ROS production and endocytosis. Journal of Applied Toxicology 2016;36:434-44. [9] Fang C, Bhattarai N, Sun C, Zhang M: Functionalized nanoparticles with long-term stability in biological media, Small 2009;5:1637-41. [10] Wallbrecher R, Verdurmen W P, Schmidt S, Bovee-Geurts P H, Broecker F, Reinhardt A, et al: The stoichiometry of peptide-heparan sulfate binding as a determinant of uptake efficiency of cell-penetrating peptides, Cellular and Molecular Life Science 2014;71:2717-29. [11] Yang D, Elner S G, Bian Z M, Till G O, Petty H R, Elner V M: Pro-inflammatory cytokines increase reactive oxygen species through mitochondria and NADPH oxidase in cultured RPE cells, Experimental eye research 2007;85:462-72.

Drug Delivery to the Lungs, Volume 29, 2018 - Stefanie K Drescher et al. Assessing Central and Peripheral Pulmonary Deposition of Three Fluticasone Propionate (FP) Dry Powder Inhaler (DPI) Formulations with Different Aerodynamic Particle Size Distributions (APSD) in Healthy Subjects via Population Pharmacokinetics Modeling Stefanie K Drescher1, Mong-Jen Chen1, Abhinav Kurumaddali1, Uta Schilling1, Yuanyuan Jiao1, Jie Shao1, Brandon Seay2, Sandra M Baumstein3, Mutasim N. Abu-Hasan2, Renishkumar Delvadia4, Lawrence Winner5, Christine Tabulov1, Bavna Saluja4, Jag Shur6, Robert Price6, Michael Hindle7, Xiangyin Wei7, Murewa Oguntimein4, Denise S. Conti4, Juergen Bulitta1 & Guenther Hochhaus1 Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, Orlando, FL, USA Division of Pediatric Pulmonary and Sleep Medicine, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA 3 Department of Pharmacotherapy and Translational Research, College of Pharmacy, University of Florida, Gainesville, FL, USA 4 Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA 5 Department of Statistics, College of Liberal Arts & Sciences, University of Florida, Gainesville, FL, USA 6 University of Bath, Bath, Great Britain 7 Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, USA 1

Summary This study aimed to evaluate whether pharmacokinetic (PK) data in healthy subjects can quantitatively compare the regional deposition of different fluticasone propionate (FP) dry powder inhaler (DPI) formulations. Three experimental FP formulations which differed in lactose fines, mass median aerodynamic diameter (MMAD) and fine particle dose (FPD), were assessed in a randomized, double-blind, four-period, four-way crossover study in 24 healthy subjects. Each of the three formulations was dosed as 5 capsules each containing a nominal dose of 100 Âľg of FP, with one formulation being replicated. Inhalation profiles during drug administration and FP plasma concentrations over 24 hours were quantified. The developed population PK model consisted of three body compartments. Absorption was described by two parallel first-order processes, i.e., a slow absorption presumably from central lung and fast absorption from peripheral lung. High peak inspiratory flow rate (PIFR) values significantly increased pulmonary absorbed doses. The three formulations were estimated to have a similar extent of bioavailability from central lung. However, formulations differed significantly in the extent of bioavailability from peripheral lung. This agreed well with in vitro Next Generation Cascade Impactor (NGI) data of these three DPI formulations. Therefore, human PK data in healthy subjects assessed via population PK modeling provided meaningful information on the rate and extent of drug deposition and absorption in the lungs for these three DPI formulations. The insights from this study may be leveraged to help the development of orally inhaled drug products. Key Message Population pharmacokinetic modeling of PK data in healthy subjects is suitable to differentiate regional lung absorption for different FP dry powder inhaler formulations. This approach improves our understanding of the performance of inhaled medications in-vivo and may be useful to determine the similarity of the performance of various DPI formulations. Introduction A quantitative assessment of the pulmonary fate of inhalation drugs is challenging, especially if information on the regional drug deposition is of interest. Pharmacokinetic (PK) studies are often performed to evaluate the pulmonary available dose and pulmonary residence time during the development of orally inhaled drug products. Here, we evaluated whether PK studies can differentiate between formulations that are expected to differ in regional deposition and absorption. Three FP DPI formulations which differed in MMAD and FPD<5Âľm were prepared and evaluated in a clinical PK study. This study aimed to investigate whether a detailed population pharmacokinetic (PopPK) analysis can quantitatively assess pulmonary absorption processes from different regions of the lungs.

Drug Delivery to the Lungs, Volume 29, 2018 - Assessing Central and Peripheral Pulmonary Deposition of Three Fluticasone Propionate (FP) Dry Powder Inhaler (DPI) Formulations with Different Aerodynamic Particle Size Distributions (APSD) Modeling Experimental methods (including materials) Data: The data used to establish this population pharmacokinetic (PopPK) model were collected from a singlecenter, single-dose, double-blinded, four-way, four-period crossover clinical study in 24 healthy subjects. Prior to dosing, the healthy subjects were trained to correctly inhale through the DPI device by using Vitalograph AIMTM (Aerosol Inhalation Monitor). For each DPI formulation, each subject inhaled five 100 µg of FP capsules via a capsule-based DPI device (RS01 Monodose, Plastiape). Three FP DPI formulations (15-MM-015, 15-MM-016, 15-MM-017) which differed in MMAD and FPD<5µm were manufactured and characterized through in vitro NGI testing with a flow rate of 60 L/min, by Catalent pharma solutions (Morrisville, NC, USA). To prepare these DPI formulations, the first step was to combine a coarse lactose carrier (sieve grade) with fine lactose (milled or microfine grades, which each differed in their particle size distribution corresponding to different formulation attributes). The second step involved mixing the lactose blend with the FP. Three different DPI formulations were manufactured by controlling relative amount of coarse lactose and lactose fines in each formulation. The PK study was performed at the University of Florida Clinical Research Center (Gainesville, FL, USA). Formulation 15-MM15 was given twice (i.e., repeated at two study periods) to assess intra-subject variability. At each study visit, complete inhalation profiles (IP), were captured while administering the formulation to the subjects by attaching a calibrated pressure traducer (Phidgets, Calgary, Canada) to the mouth-piece of the DPI device. Inhalation volume (IT), inhalation time (IV), peak inspiratory flow rate (PIFR), and time to peak inspiratory flow rate (TPIFR), were recorded during drug administration; blood samples were obtained at pre-dose, 5, 10, 15, 20, 30, 45 and 60 minutes, as well as 1.5, 2, 3, 4, 6, 8, 10, 12, 14, and 24 hours after dosing. Drug concentrations were analyzed via LC-MS/MS at Worldwide Clinical Trials (Austin, Taxes, USA).

Modeling: The developed population PK model consisting of three body compartments. Pulmonary absorption was described by two parallel first-order processes (i.e., one slow, presumably central, and one fast, presumably peripheral absorption process). Modeling was performed in the S-ADAPT software (version 1.57) using the importance sampling algorithm. Total body clearance was fixed based on the mean of literature values after intravenous (iv) administration.1–4 Age, sex, body weight, and inhalation specific measurements such as IT, IV, PIFR, TPIFR, were explored as potential covariates for each PK parameters. The models were evaluated by goodness-of-fit plots, visual predictive checks (VPCs) and normalized prediction distribution error plots. Parameters reporting: The model-predicted absorbed dose was calculated as: Absorbed dose (µg) = model predicted absorbed dose fraction (%) x nominal dose (µg)/100. The nominal dose was 100 µg of FP per capsule. The associated absorption half-life was estimated and reported. Results

Results of the in vitro assessments of the three formulations (Table 1 and Figure 1) indicated that two formulations showed similar cascade impactor profiles while formulation 15-MM-017 had the largest MMAD and smallest FPD. The developed PopPK model (Figure 2) provided excellent curve fits and had good predictive performance for the three DPI formulations (Figure 3).

Table 1: The summary of in vitro characterization of three tested formulations. 15-MM-015

15-MM-016

15-MM-017

MMAD (µm) GSD FPD < 5µm (µg)

3.72 2.05 15.82

3.78 2.01 18.71

4.51 1.89 12.23

FPD < 3µm (µg)

8.57

9.98

5.27

Stage 2 to 3 (µg)

11.54

14.40

12.48

Stage 4 to 7 (µg)

8.14

9.37

4.83

Note: GSD: Geometric standard deviation.

Drug Delivery to the Lungs, Volume 29, 2018 - Stefanie K Drescher et al.

Figure 1: APSD profile of three formulations actuated into the NGI with a flow rate of 60 L/min (Impactor Size Mass deposited sites) In brief, the model was parameterized in terms of volume of distribution in the central (V c), shallow peripheral (Vp1) and deep peripheral (Vp2) compartment, as well as clearance (CL) and inter-compartmental clearance into the shallow (CLd) and deep (CLd2) peripheral compartment. The following population mean PK parameters were estimated: Vc = 119 L, Vp1 = 110 L, Vp2 = 280 L, CL = 71 L/h (fixed), CLd = 263 L/h, CLd2 = 24 L/h. Pulmonary absorbed doses and absorption half-lives were estimated for each DPI formulation. The estimates of absorbed doses from the central regions of the lungs (FC) were 4.8 µg, 4.4 µg and 6.4 µg for formulation 15-MM-015, -016, 017, respectively; while estimates of absorbed doses from the peripheral region of the lungs (FP) were 9.9 µg, 9.9 µg, and 5.1 µg, respectively. Further, the estimates of the pulmonary absorption half-life of the central lung (T1/2_C) were 9.1 h, 7.9 h and 6.2 h, respectively. The estimated pulmonary absorption half-lives from the peripheral lungs (T1/2_P) were 0.10 h, 0.11 h, and 0.2 h (Table 2). Table 2: The summary of estimated pulmonary absorbed doses and absorption half-lives for three tested FP DPI formulations. Parameters

15-MM015

15-MM016

15-MM017

Absorbed Dose in Central Lungs (µg)

4.8

4.4

6.4

Absorbed Dose in Peripheral Lungs (µg)

9.9

5.1

T1/2_C (h)

9.1

7.9

6.2

T1/2_P (h)

0.096

0.114

0.241

Figure 2: The model structure of the developed PopPK model with central as well as deep and shallow peripheral compartment (CMT): Fc and Fp are the fraction absorbed and kabs_C and kabs_P are the absorption rates from the central and peripheral lung, respectively. CLd and CLd2 are the distribution clearance into the shallow and deep peripheral compartment, whereas CL is the elimination clearance from the central compartment. Parameter labelled with a star (*) were estimated for each formulation separately.

Figure 3: The visual predictive check (VPC) for the performance of the proposed popPK model for three DPI formulations.

Figure 4: Individual model prediction of the regional absorbed doses in the central and peripheral regions of the lungs for formulations 15-MM-015, 15-MM-016, and 15-MM-017. Peak inspiratory flow rate (PIFR) was found to be a significant linear covariate on F C and FP, suggesting absorbed fraction increases as PIFR increases. The slopes of the covariate implemented as a linear function of PIFR on FC and FP were estimated as 0.72 and 1.06, respectively. Body weight (BW) was included via an allometrically scaled model to affect clearances and volumes of distribution. The absorption half-life from the central regions of the lungs ranged from approximately 9-6 hours among all formulations (T1/2_C in Table 2), while the absorption half-life of the peripheral regions was approximately two-fold longer for formulation 15-MM-017 compared to the other formulations (T1/2_P in Table 2); 15-MM-017 was the formulation with the largest MMAD and slowest in vitro dissolution rate (data not shown). Similarly, the absorbed doses of all formulations were similar in the central region of the lung; however, in the peripheral region the absorbed dose was substantially smaller for formulation 15-MM-017 compared to the other formulations (Figure 4). By comparison of deposited drug mass in grouped NGI stages and model-predicted pulmonary absorption doses, a consistent trend was observed between formulations. By grouping stage 2 to 3 and stage 4 to 7, the mass deposition in stages 2-3 were very similar between all formulations (11.54 to 14.40 µg). However, mass deposition in stage 4-7 was substantially smaller for formulation 15-MM-017 (4.83 µg) compared to formulations 15-MM-015 or 15-MM-016 (8.14 and 9.37 µg, respectively). Formulation 15-MM-15 was repeated at two study periods to assess intra-subject variability, which was found to be approximately 15% for area under the plasma concentration-time curve, and 29% for peak plasma concentration. These results can generally be considered as low variabilities.

Drug Delivery to the Lungs, Volume 29, 2018 - Stefanie K Drescher et al.

Conclusion Human PK data in healthy subjects assessed via population PK modeling provided meaningful information on the rate and extent of drug deposition and absorption in the lungs for three FP DPI formulations with different MMAD. These insights can be leveraged to help the development of orally inhaled drug products. The developed PopPK model could quantitatively estimate differences in regional lung deposition (i.e., the fraction deposited and absorption, and the absorption half-lives) for different FP DPI formulations. This model estimates correlated well with in vitro characteristics. It is proposed to apply this novel approach for comparing the regional deposition of inhaled corticosteroid formulations. Acknowledgments Funding for this work was made possible, in part, by the Food and Drug Administration through contract HHSF223201610099C. Views expressed in this abstract do not necessarily reflect the official policies of the U.S. Food and Drug Administration, nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government.

References 1. Mackie, A. E., Ventresca, G. P., Fuller, R. W. & Bye, A. Pharmacokinetics of intravenous fluticasone propionate in healthy subjects. Br. J. Clin. Pharmacol. 41, 539–542 (1996). 2. Brutsche, M. H. et al. Comparison of pharmacokinetics and systemic effects of inhaled fluticasone propionate in patients with asthma and healthy volunteers: a randomised crossover study. Lancet Lond. Engl. 356, 556–561 (2000). 3. Allen, A., Bareille, P. J. & Rousell, V. M. Fluticasone furoate, a novel inhaled corticosteroid, demonstrates prolonged lung absorption kinetics in man compared with inhaled fluticasone propionate. Clin. Pharmacokinet. 52, 37–42 (2013). 4. Thorsson, L., Edsbäcker, S., Källén, A. & Löfdahl, C. G. Pharmacokinetics and systemic activity of fluticasone via Diskus and pMDI, and of budesonide via Turbuhaler. Br. J. Clin. Pharmacol. 52, 529–538 (2001).

Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; Irene Rossi et al.

In Vitro Activity of Inhalable Microparticles Containing Anti-Tb Drugs and an Efflux Pump Inhibitor against Mycobacteria Infections Irene Rossi1, Francesca Buttini1, Filippo Affaticati1, Fabio Sonvico1, Marco Pieroni1, Elliott K Miller2, Nitesh K Kunda2, Pavan Muttil2 & Ruggero Bettini1 1University 2

of Parma, Food and Drug Department, Parco Area delle Scienze 27/A, Parma, PR, Italy University of New Mexico, College of Pharmacy, Department of Pharmaceutical Sciences, 2705 Frontier Avenue NE, Albuquerque, NM, USA

Summary Mycobacterial efflux pumps inhibitors (EPIs), along with first-line antibiotics, are widely studied as a new strategy to increase intracellular drug concentration, enhance the efficacy of the treatment and reduce the generation of drug-resistant strains. More than 80% of Mycobacterium tuberculosis (Mtb) reside in the lungs after infection, especially inside macrophages [1]. The aim of this work is to demonstrate the in vitro bactericidal activity of an innovative spray-dried formulation composed of low molecular weight hyaluronic acid nanoparticles encapsulating two first-line antibiotics and an efflux pump inhibitor, verapamil HCl. These formulations were evaluated for their efficacy against an intracellular nontuberculous mycobacterium (NTM; Mycobacterium smegmatis), used as a surrogate to Mtb. Powder 1 (P1), that contained the EPI verapamil HCl, was compared with the same formulation without the EPI (Powder 2, P2). The aerodynamic performance of P1 powder was better compared to P2 powder: (Fine Particle Fraction 78% (P1) vs 19% (P2)), although both powders presented a similar particle size distribution. Moreover, P1 powder showed increased bactericidal activity against Mycobacterium smegmatis after 48h of exposure, compared to P2. However, P1 powder was more toxic to macrophages than P2 powder at the 0.5 mg/mL concentration. In addition, P1 powder showed higher killing of the intracellular mycobacteria, even at concentrations below the minimum inhibitory concentrations of the encapsulated antibiotics. Key Message Considering the clinical importance of tuberculosis (TB) and non-tuberculous mycobacterial infections, and the increase in drug-resistant TB cases; this study showed the superior in vitro activity of a highly respirable controlled release formulation consisting of first-line antibiotics along with an efflux pump inhibitor to inhibit Mycobacterium smegmatis growth. Introduction Mycobacterium tuberculosis (Mtb) are intracellular pathogens that reside inside alveolar macrophages (AMs) [1]. The microorganisms enter the human lung through inhalation of sufficiently small (1-5 Âľm) bacilli-carrying droplets that usually deposit in the alveolar space [2]. AMs are known to provide the firstline of defence against pathogens with their intracellular bacterial killing activity and antigen presentation capability to lymphocytes; however, they also represent the habitat where Mtb can survive, reside, and subsequently proliferate to other organs. Conventional therapy consists of administering antimicrobial agents by the oral (first-line drugs) or parenteral route (second-line drugs) in very large doses. Treatment for both Mtb and nontuberculous mycobacteria (NTM) is not only expensive and prolonged, but also leads to the generation of drug-resistant strains. Moreover, long treatment time with antibiotics is known to cause patient non-compliance that further leads to the generation of multi-drug resistant (MDR) and extensively drug resistant (XDR) mycobacterial strains. A global increase in MDR and XDR cases emphasizes the need for novel treatment approaches to extend the longevity of existing first-line and second-line drugs [3]. Many studies, with limited success, reported different antibiotics delivery strategies targeting AMs [4] to increase the efficacy of antibiotics, reduce resistance and, at the same time, lower the dose of drugs in blood, and, consequently the systemic side-effects. The lungs, as a primary and main site of infection, are a logical target for therapy [1]. In order to develop an ideal pulmonary formulation, two main issues need to be addressed: achieving high drug payload in the powder to be inhaled [5], and the need to improve AMs uptake. It has been reported that low molecular weight hyaluronic acid sodium salt (HYA), an endogenous compound, is capable of efficient internalization by the macrophages via binding the CD44 receptor present on the AMs surface. The other point that needs to be evaluated is the role of efflux pumps inhibitors, such as the calcium channel blockers verapamil and thioridazine, on enhancing the activity of first-line anti-TB drugs. This combination therapy was reported to increase the intracellular drug concentration, and thus decrease the duration of treatment [6]. However, the side effects of verapamil and thioridazine are severe for systemic administration. The aim of this work was to demonstrate the superior in vitro activity of a highly respirable HYA-based dry powder, containing an efflux pump inhibitor (verapamil HCl) and two first line anti-TB drugs (isoniazid and rifampicin). These powder formulations showed efficient delivery and uptake by the AMs that were infected with a model NTM, Mycobacterium smegmatis (M. smeg). This powder was compared to the same type of formulation without verapamil.

Drug Delivery to the Lungs, Volume 29, 2018 - In Vitro Activity of Inhalable Microparticles Containing Anti-Tb Drugs and an Efflux Pump Inhibitor against Mycobacteria Infections Materials and Methods Formulation and characterization of microparticles: Two dry powders were formulated by spray drying (B-290, BÜCHI, Switzerland) using a low molecular weight (average MW= 29.5 kDa) sodium hyaluronate (HYA) hydroalcoholic (ethanol:water = 70:30 v/v) nanosuspension (0.59 w/v) containing two first-line antibiotics, rifampicin (RIF) and isoniazid (INH), with (P1) or without (P2) the efflux pump inhibitor (EPI) verapamil HCl (VER) (Table 1). Actual composition of the microparticles obtained was analysed by HPLC. The two powders were characterised in terms of particle size distribution (Spraytec, Malvern, UK) and aerodynamic behaviour. Briefly, 5 mg of each powder were loaded inside a hypromellose capsule size 3 (Quali-V-I, Qualicaps, Spain) and aerosolized by Turbospin (PH&T, Italy) device inside a Fast Screening Impactor (FSI, Copley scientific, UK) at 65 L/min for 3.7 s, so that a volume of 4 L of air was drawn through the inhaler during the experiment. In vitro cytotoxicity: In vitro cytotoxicity of the powders, after resuspension and sonication for 10 min in RPMI 1640 medium (high glucose with L-Glutamine with HEPES, ATCC, USA) at different concentrations (1.0-0.0125 mg/mL) were studied in monocyte-like THP-1 cells. The THP-1 cells were differentiated into macrophages by phorbol 12-myristate 13-acetate (PMA) 20 nM for 24h. Loss in cell viability was measured by a reduction in metabolic activity using sodium 2,3,-bis(2-methoxy-4-nitro-5sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium) (XTT) assay. Absorbance was read at 450 nm at two-time points, 24 and 48h, after exposing THP-1 cells to the formulations. Bactericidal activity: The efficacy of the dried powders against M. smeg was tested in vitro after 24 and 48h of incubation at 37°C. The two time points were chosen based on the in vitro release data [7]; approximately 40h were necessary to achieve complete release of each drug. Different concentrations of the powders were prepared in Middlebrook 7H9 broth medium (Sigma Aldrich, USA) and added to 4x105 CFU/mL of M. smeg. The M. smeg CFU was chosen based on our preliminary studies of in vitro infection of macrophages: we showed that the best multiplicity of infection (MOI) was one (4x105 THP-1 cells/mL cells confluency in a 12 well plate). Intracellular killing of M. smeg. in macrophages: An in vitro infection and treatment was performed on THP-1 cells differentiated into macrophages. Macrophages were infected for 3h (MOI 1), and wells were washed three-times with PBS to remove extracellular M. smeg. The formulations resuspended in RPMI medium (for the 48h test, powders were sonicated for 10 min to achieve a homogenous suspension) were added to the infected macrophages. Two powders concentrations were tested: a higher (0.125 mg/mL) and a lower (0.05 mg/mL) RIF concentration with respect to the minimum inhibitory concentration (MIC) of the antibiotic reported for M. smeg. (31.8 µg/mL) [8].

Results and Discussion Spray-dried microparticles (yield around 58-59%) were constructed by agglomeration of HYA nanoparticles (mean diameter: 480  150 nm) [7]. The spray-dried powders presented a drug content that reflected the theoretical value (Table 1).

Table 1. Powders theoretical and actual composition (mean value ± sd, n=3; * not quantified). Powder 1 (P1) Theoretical composition (%) Sodium Hyaluronate LMW Rifampicin Isoniazid Verapamil HCl

Powder 2 (P2)

Content assay (%)

Theoretical composition (%)

Content assay (%)

38*

35.00 ± 0.03

37.99 ± 0.03

17.71 ± 0.03

22.36 ± 0.02

12.00 ± 0.02

35*

Drug Delivery to the Lungs, Volume 29, 2018 – Irene Rossi et al.

The aerodynamic performance indicated a substantial difference in the respirability of the two formulations: Fine Particle Fraction (FPF, the percentage of particles with a diameter < 5 µm of the total amount of powder emitted) was very high for P1 (78.37%  5.79) compared to P2 (18.83%  7.77). This difference was due to a greater macroscopic state of agglomeration of microparticles in Powder 2, likely caused by the different components and, in particular, by the polymer content in the starting nanosuspension (slightly higher for P2). Figure 1 illustrates that the Powder 2 deposition was maximum in the Coarse Fraction Collector (CFC), which has a cut-off diameter > 5 µm. This was despite the laser diffraction data highlighting that the particle size distribution for both powders, resuspended in isopropyl alcohol, was < 5 µm (Dv,10-Dv,90= 0.38-1.96 µm and 0.71-3.11 µm, for P1 and P2, respectively). This could possibly be due to the device employed for the impaction studies that was not able to generate sufficient airflow to efficiently deagglomerate P2. The Emitted Fraction (the percentage of loaded powder that leaves the device) was > 68% in both cases.

Figure 1. Drugs distribution (% of the loaded amount) upon aerosolization in the Fast Screening Impactor with Turbospin (Device and Capsule, Dev + Caps; Induction Port and Rubber Adaptor, IP + RA; Coarse Fraction Collector, CFC; Fine Fraction Collector, FFC). Mean value obtained for each drug in each analysis, n=3  sd.

The THP-1 cells (macrophage) viability (Figure 2) for P 1 remained at 100% after 24 and 48h exposure for powder concentrations equal or lower than 0.125 mg/mL (RIF=43.75 µg/mL; INH=22.5 µg/mL; VER=15 µg/mL; HYA=43.75 µg/mL). No significant difference in cell viability was observed at 24 and 48h exposure indicating that each component at the above concentrations was not toxic to the cells. For P2, unlike P1, significant cell toxicity was observed only at 1 mg/mL concentration after exposure for 24h. However, cell toxicity was similar for P1 and P2 at 0.5 and 1 mg/mL concentration after exposure for 48 h. Pow der 1

Pow der 2 150

24h

V ia b ilit y (% o f c o n tr o l)

125

24h

125

48h

100 75 50 25

75 50 25

C o n c e n t r a t io n m g / m L

0 .0

0 1

0 .5 0

0 .1 0

0 0

5 0

0 0

0 .0 1

0 .5 0

0 0 0

5 0

2 .0 0

48h

100

V ia b ilit y (% o f c o n tr o l)

150

C o n c e n t r a t io n m g / m L

Figure 2. Biocompatibility of powders with THP-1 cells (macrophages) using the XTT assay for cells viability. Six different concentrations were tested at two time points, 24 and 48h (mean value ± sd, n=3).

The in vitro efficacy of the powders against M. smeg., a model mycobacteria, showed superior activity for P1 in inhibiting M. smeg. growth, where a concentration of 0.05 mg/mL was able to suppress bacterial growth after 48h of incubation in culture medium. It is worth mentioning that pure VER toxic concentration on M. smeg was measured in vitro and corresponded to 0.12 mg/mL, which was the concentration of VER in 1 mg/mL of P1. Therefore, any toxicity observed for P1 and P2 at the high concentration (1.0 mg/ml) could potentially be due to VER alone. In addition, our investigation was important to evaluate the ability of the antibiotics to kill M. smeg., since previous studies have reported resistance for this NTM against the antibiotics employed here [9].

Drug Delivery to the Lungs, Volume 29, 2018 - In Vitro Activity of Inhalable Microparticles Containing Anti-Tb Drugs and an Efflux Pump Inhibitor against Mycobacteria Infections

Pow der 1

Pow der 2

1 .0 0

2 0

5 2

2 .1 0

0 .0 0

0 .2 5

0 .0 0

0 .5 0

0 .2 5

0 .7 5

0 .5 0

0 .7 5

1 .2 5

1 .0 0

48h

1 .5 0

R a tio

1 .2 5

24h

1 .7 5

48h

1 .5 0

24h

1 .7 5

2 .0 0

C o n c e n t r a t io n m g / m L

Figure 3. In vitro efficacy of spray-dried powders to inhibit M. smeg. growth after 24 or 48h of incubation (n=1). Data are expressed as ratio between the mean value (CFU/mL) found for each powder concentration studied and the amount of bacteria (CFU/mL) growing in the positive control without any formulation added (0 mg/mL).

Finally, P 1 showed bactericidal activity in the treatment of macrophages infected with M. smeg. (Figure 4), after 48h of incubation (ratio of bacterial growth/control < 0.01 for concentration 0.05 mg/mL); this could be due to the slow release of drugs from the spray-dried powder. Pow der 1

Pow der 2

2 .0 0

1 .7 5

24h

48h

1 .7 5

1 .5 0

1 .0 0 0 .7 5

.0 0

2 .1

0 .0 0

0 .2 5

0 .0 0

0 .5 0

0 .2 5

0 .5 0

0 .7 5

R a tio

1 .2 5

1 .0 0

R a tio

1 .2 5

C o n c e n t r a t io n m g / m L

Figure 4. In vitro infection of macrophages with M. smeg. and consecutive treatment with the two powders for 24 or 48h. Data are expressed as ratio between the mean value (CFU/mL) for each powder concentration studied (n=3 ď&#x201A;ą sd) and the amount of bacteria (CFU/mL) growing in the positive control without any formulation added (0 mg/mL). Kanamycin at MIC was used as a negative control.

Conclusion This work showed the in vitro activity of our formulation developed against M. smeg. The presence of VER as an EPI in the spray-dried powder afforded a different behavior. P1, containing the EPI, not only reported higher respirable particles (> 78% of the emitted dose), but also showed a greater ability to inhibit M. smeg. growth at lower concentrations (0.05 mg/mL) compared to P2. Although P1 was more toxic to macrophages at higher concentrations (0.5 mg/mL), it showed, a better bactericidal activity against intracellular bacteria, even at sub-MIC concentration of the antibiotics encapsulated in the microparticles (0.05 mg/mL) after 48h exposure. Future studies will focus on increasing the time of macrophage exposure to M. smeg. and treatment duration of formulations with the infected cells. Moreover, we will conduct preclinical studies to demonstrate the efficacy of these formulations when delivered by the pulmonary route.

Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; Irene Rossi et al.

References Muttil P, Wang C, Hickey AJ: Inhaled drug delivery for tuberculosis therapy, Pharm. Res. 2009; 26(11): pp24012416. 1

Griffith DE, Aksamit TR: Understanding nontuberculous mycobacterial lung disease: itâ&#x20AC;&#x2122;s been a long time coming, F1000Research. 2016; 5: pp2797-2802.

Global Tuberculosis Report 2017.

Zhou H, Zhang Y, Biggs DL, Manning MC, Randolph TW, Christians U, Hybertson BM, Ng KY: Microparticlebased lung delivery of INH decreases INH metabolism and targets alveolar macrophages, Journal of Controlled Release 2005; 107: pp288-299.

Mortensen NP, Durham P, Hickey AJ: The role of particle physico-chemical properties in pulmonary drug delivery for tuberculosis therapy, J Microencapsul. 2014; 31: pp785-795.

Adams KN, Szumowski JD, Ramakrishnan L: Verapamil, and Its Metabolite Norverapamil, Inhibit Macrophageinduced, Bacterial Efflux Pump-mediated Tolerance to Multiple Anti-tubercular Drugs, J. infect. Dis. 2014; 210(3): pp456-466.

Rossi I, Buttini F, Affaticati F, Sonvico F, Pieroni M, Bettini R: Sodium Hyaluronate microparticles for Pulmonary Delivery of Anti-mycobacterial Drugs. (Abstract). Presented at: Respiratory Drug Delivery Congress, Tucson, Arizona, US, April 22-26 2018.

Gupta K, Singh S, van Hoek ML: Short, Synthetic Cationic Peptides Have Antibacterial Activity against Mycobacterium smegmatis by Forming Pores in Membrane and Synergizing with Antibiotics, Antibiotics 2015; 4(3): pp358-378.

Teng R, Dick T: Isoniazid resistance of exponentially growing Mycobacterium smegmatis biofilm culture, FEMS Microbiol Lett. 2003; 227(2): pp171-174.

Drug Delivery to the Lungs, Volume 29, 2018 - Sanketkumar Pandya et al. Dry Powder Inhalation of Glucagon-like Peptide-1 for Management of Type-2 Diabetes Mellitus Sanketkumar Pandya1,2, Durgesh Kumar1,2, Swati Gupta1, Kalyan Mitra1, Anil N Gaikwad1 & Amit Misra1 1

CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, 226031, India 2Academy of Scientific and Innovative Research, New Delhi - 110025, India

Summary Background: Glucagon-like peptide 1 (7-36) amide (GLP-1) is a bioactive peptide that regulates glucose homeostasis through multiple actions. The present study aims to develop a particulate delivery system for administration of the prototype incretin GLP-1 fragment for the management of Type 2 diabetes mellitus. Methods: Particles incorporating 5% and 10% GLP-1 were engineered using spray freeze drying (SFD) employing L-leucine and trehalose as cryoprotectants. The particles were extensively characterized for physicochemical properties such as mean particle size, surface morphology, mass median aerodynamic diameter (MMAD), specific surface area (SSBET) and porosity. Circular Dichroism (CD) spectroscopy was done to assess the structure integrity of peptide. Pharmacokinetic and pharmacodynamic studies with GLP-1 particles were performed on C57/BL6 mice. Results: Particles prepared by SFD in high yield (>90 %) exhibited good aerodynamic performance (MMAD =1.1 ± 0.35 µm, Fine Particle Fraction (FPF) =60.5±0.5%). Particles were porous and possessed high SSBET of 195 m2/g and low density (0.03 g/ml). In-vitro MTT assay on A549 cell-line showed that the particles did not exert any significant lung toxicity. CD analysis indicated that the secondary structure of GLP-1 fragment in particulate formulation was intact. Maximum circulating GLP-1 levels in mice were achieved in 20-30 min. Intraperitoneal glucose tolerance test revealed that a dose dependent response in terms of plasma glucose concentration was achieved following pulmonary administration of GLP-1 particles. Particles with 10% GLP-1 provided good control over glucose homeostasis. Conclusion: A suitable inhalable formulation of GLP-1 was developed for its feasible use in management of type 2 diabetes mellitus. Key Message GLP-1 particles administered via pulmonary route are efficacious in a mouse model of type-2 diabetes mellitus. Spray freeze drying is a suitable technique to prepare inhalable particles containing native GLP-1 for diabetes therapy. Introduction Diabetes Mellitus is a chronic metabolic disorder which is characterized by hyperglycaemia, hyperlipemia, excessive weight loss, polyurea, polydipsia, polyphagia and sometimes ketonemia. The current chemotherapy includes insulin injections as well as oral hypoglycemic agents. Afrezza® (inhaled insulin) has recently been approved by the USFDA after the withdrawal of Exubera® from the market. But, these agents are not so effective in preventing the progression of disease. Glucagon-like peptide 1 (7-36) amide (GLP-1), a 30-amino acid incretin hormone GLP-1 is an incretin hormone secreted by intestinal L-cells [1]. It inhibits glucagon release from the liver, interfering in the feedback-inhibition of insulin release. The effects of GLP-1 are glucose-dependent. It promotes not only insulin release, but also its biosynthesis. GLP-1 induces β-cell growth and differentiation and inhibits β-cell apoptosis; leading to increased βcell mass. Delivering proteins/peptides by inhalation route can eliminate first pass metabolism and thus can increase its systemic bioavailability. However, pulmonary delivery of peptides is challenging due to its labile nature. To minimize risk of denaturing GLP-1 while formulation processing, cryoprotectants/stabilizers such as L-leucine and trehalose are used that can also enhance dispersibility of dry powder inhalation (DPI). The peptide is extremely short lived (t1/2=1-2 min) as it gets rapidly metabolized by the enzyme dipeptidyl-petidase-IV (DPP-IV). SFD is a novel formulation strategy that produces particles having high porosity, large geometric size (>5 µm) and remarkably low density (<0.1 g/cc), thus displaying aerodynamic diameters (1-5 µm) eminently appropriate for deep lung delivery. Due to their relatively large size, they evade macrophage uptake [2] which ultimately increases overall bioavailability of native peptide. AIM: (1) Development of formulations containing glucagon-like peptide-1 (GLP-1) for rapid release (RR) on pulmonary delivery, using GRAS (Generally regarded as safe) excipients. (2) Establishment of the suitability of the prepared particles for deep lung delivery using characterization techniques which includes size distribution, morphology & surface characteristics, drug content, cascade impaction analysis, flow property measurement, dose emission studies, storage stability etc. (3) Determination of efficacy in terms of effects on blood glucose levels after oral glucose in diabetic C57/BL6 mice and comparison with daily injections of unformulated GLP-1.

Drug Delivery to the Lungs, Volume 29, 2018 - Dry Powder Inhalation of Glucagon-like Peptide-1 for Management of Type-2 Diabetes Mellitus Materials and Methods GLP-1 (7-36) amide fragment was purchased from ChemPep Inc. (FL, USA). L-Leucine was obtained from Merck KGaA (Germany) and Trehalose dihydrate from Spectrochem Pvt. Ltd. (India). NIR-797 isothiocyanate, Insulin (bovine pancreas), protease inhibitor cocktail and Sigmacote® (siliconizing reagent for glass) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Streptozotocin was purchased from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). Valine Pyrolidide a DPP IV inhibitor, was synthesized in our laboratory. Formulation: The optimized aqueous feed had 2 %w/v solids, which consisted of 5 %w/w and 10 % w/w GLP-1 excipients (Leucine:Trehalose=75:25), were directly sprayed into liquid nitrogen contained in a 5 L three-necked round bottom flask using a 0.5 mm diameter two fluid nozzle at a solution feed rate of 5 ml/min and nozzle pressure of 25 psi. The resulting suspension of frozen droplets was lyophilized for 48 h under 0.01 mBar at -80°C (Labconco, USA) to obtain dried powder. In the next step, 0.5 %w/w magnesium stearate was added to the dried powder, mixed well and finally stored at -20°C. All glassware was siliconized (3 coatings) using Sigmacote® prior to use. Particle size distribution: The median particle size (volume-mean diameter) and particle size distribution of the powder was determined by laser diffraction using a wet sample dispersion unit (Malvern Mastersizer 2000, Hydro 2000MU unit, Malvern Instruments, UK). Tapped density measurement: The density of the powder was measured by tapped density measurements. The ratio of powder (g) weight to tapped volume (ml) gives the tapped density (ρ t). Particle aerodynamic behaviour analysis: The in-vitro aerosol performance of the porous particles was experimentally determined in terms of MMAD using an eight-stage Andersen Cascade Impactor (ACI) and a Rotahaler® (Cipla Ltd., Mumbai, India) powder inhalation device. Geometric size distribution (GSD) represents the ratio between 84% undersize and 50% undersize (MMAD). The total emitted dose (TED) is the total mass of drug emitted from the inhaler. The fine particle dose (FPD) corresponds to the dose of aerosolized particles having size less than 5 µm. FPF is the percentage ratio of FPD to the total dose. Particle morphology: Particle shape, surface characteristics, porosity and self-aggregation of particles were observed using scanning electron microscopy (SEM). Samples were deposited on pin mounts having conductive aluminum tape with adhesive and coated with Au−Pd (80:20) using a Polaron E5000 sputter coater. Samples were examined at 1-5K magnification and imaged in a FEI (Hillsboro, OR, USA) Quanta 250 SEM at an accelerating voltage of 5 kV using a secondary electron (SE) detector. Surface area analysis: SSBET and total pore volume of inhalable particles was estimated from nitrogen adsorptiondesorption isotherm using linearized multi-point Brunauer, Emmet and Teller (BET) method. The measurement was carried out at cryogenic temperature (-196°C) on Autosorb® 1C instrument (Quantachrome instruments, FL, USA).). The mesopore size distribution was also calculated using Barrett Joyner Halenda (BJH) desorption method while the density functional theory (DFT) method was used to determine the micro-porosity of the powder samples. Circular Dichroism (CD): The CD analysis was done using Jasco J-1500 CD spectrometer (Jasco, USA) and the secondary structure of the peptide was analyzed using the spectral manager software (version 2.15.02, Build 1). All CD measurements were taken at a temperature of 20 °C at a wavelength ranging from 190 to 260 nm at a scan rate of 100 nm/min (1nm data interval, 71 data points) using a high transparency rectangular stoppered quartz cuvette (Spectrosil, Jasco type J/21/Q/2/CD) with path length of 2 mm. In-vitro cell viability assay: The in-vitro cytotoxicity of inhalable formulations was measured by the methyl tetrazolium (MTT) assay using air-liquid interface cell culture method. A549 cells (human lung adenocarcinoma) were cultured at the air-liquid interface of a Transwell® 24 well plate permeable supports (polystyrene, 6.5 m insert, 0.4µm pore size polyester membrane, Costar) at a density of 1.5 x 105 cells/well with 400 µl medium in apical chamber and 1.5 ml in the basolateral chamber. After appropriate culturing procedure, powder with different peptide concentration was sprayed over the insert using the modified syringe dispenser [3] followed by incubation for 12 h. The apical chamber containing drug particles was washed three times with incomplete RPMI medium. Then, 1 mg/ml MTT solution was added to both apical (200µl) and basolateral chambers (1.2 ml) followed by incubation at 37 °C for 4 h. Dimethylsulfoxide (150 µl) was added to each Transwell insert to solubilize formazan crystals. The optical density was read using a Biotek Microplate Reader at 550 nmm.

Drug Delivery to the Lungs, Volume 29, 2018 - Sanketkumar Pandya et al. Pharmacokinetics of GLP-1 in mice: The experimental protocols were approved by Institutional Animal Ethics Committee (IAEC) of CSIR-Central Drug Research Institute, Lucknow. Male C57/BL6 mice (four per group) with an average weight of 26 g were used in this study and randomized into three groups of 24 mice each. Mice were administered GLP-1 LD (2.3 µg) and GLP-1 HD (4.55 µg) powders for inhalation using the in-house inhalation apparatus. Another group was administered 50 µl GLP-1 standard solution (2.3 µg) via intra-tracheal (IT) route. Plasma was collected in eppendorf tubes containing DPP-IV inhibitor cocktail and the samples were analyzed for GLP-1 concentrations (pM) using total GLP-1 Elisa kit (Merck Millipore). In vivo studies: STZ induced and HFD diabetic mice (male C57BL/6 mice 10-12 week old) were used in the study. Intraperitoneal glucose tolerance test (IPGTT) and Insulin tolerance test (ITT) was done before and after drug dosing; blood glucose was monitored using Accu-check glucometer (Roche Diagnostics). Immunofluorescence (IF) technique was used to detect insulin positive β-cells and β-cell proliferative activity (BrdU staining). TUNEL assay was done to determine apoptosis. The lung deposition pattern after pulmonary administration of particles (incorporating NIR-797 isothiocyanate) was ascertained using an in vivo imaging system (IVIS; IVIS® spectrum, PerkinElmer, USA). Results and Discussion Table 1 shows the physical parameters of particles prepared by SFD. SFD particles with 25% trehalose had a MMAD of 3.7±0.1 µm (GSD=1.96±0.1) with fine particle fraction (FPF<4.6 µm) of 60.5±0.5 %. SFD particles showed good dispersibility and aerosol performance (Table 1). The yield of SFD particles was above 90%. Table 1: Physicochemical characteristics of inhalable GLP-1 particles prepared by SFD. Values represent mean±SD (n=3)

Median particle size (µm) 10.37±0. 4

Densit y (g/cc) 0.013

% yield

MMAD (µm)

GSD

Total emitted dose (µg)

93.4±2.3

3.7±0.1

1.9±0.1

490.2±5.7

*data represents only one determination

Fine Particle Dose (µg) 302.4±2. 3

FPF (%)

SSBET (m2/g)

Pore volum e (cc/g)

60.5±0.5

192.52*

0.71*

SEM (Figure 1A) shows that SFD particles were porous (specific surface area SS BET195.52 m2/g) and displayed spheroid morphology (‘micro-chrysanthemums’) with infinite micropore. In-vitro cytotoxicity studies on human epithelial A549 cells using Transwell permeable inserts (modified air-liquid interface culture method) indicated that the particles did not exert significant cytotoxicity (Figure 1B). CD analysis show that the 2° structure of peptide was intact during processing of particles. Plasma GLP-1 concentration (pM)

60000

GLP-1 LD GLP-1 HD GLP-1 IT

40000

20000

20 30 40 50 60 70 80 90

Time (min)

Placebo Placebo DPI DPI GLP-1 DPI (S-CM) GLP-1 DPI (S-CM) GLP-1 FD (S-CM) GLP-1 FD (S-CM) GLP-1 DPI (ALI-CM) GLP-1 DPI (ALI-CM)

10 % w/w 5 % w/w10 % w/w 5 % w/w 2.5% w/w 2.5% w/w

GLP-1 formulations

B 10 µm 10 µm 1 µm 1 µm 0.1 µm 0.1 µm 10 µm 10 µm 1 µm 1 µm 0.1 µm 0.1 µm 10 µm

10 µm

100

40 60 80 100 Cell viability (% of control)

120

Cell viability (% of control)

Figure 1: (A) SEM image of SFD particle. (B) Effect of GLP-1 and its DPI formulations (Placebo DPI=Blank particles, GLP1 DPI (S-CM)=cytotoxicity by submerged culture method, GLP-1 FD (S-CM)=cytotoxicity of GLP-1 standard solution by submerged culture method, GLP-1 DPI (ALI-CM)= cytotoxicity of GLP-1 particles by air-liquid interface culture method) on the viability of A549 cells. All data are shown as mean±SD (n=3). (C) Mean plasma concentrations of GLP-1 (pM) in male C57/BL6 mice receiving inhalations of low dose GLP-1 particles (GLP-1 LD; 2.3 µg), high dose particles (GLP1 HD; 4.55 µg) and intra-tracheal instillation of GLP-1 standard solutions (GLP-1 IT; 2.3 µg in 50 µl). Data represents group mean ± Standard error mean (n=3).

Drug Delivery to the Lungs, Volume 29, 2018 - Dry Powder Inhalation of Glucagon-like Peptide-1 for Management of Type-2 Diabetes Mellitus Pharmacokinetics of GLP-1 fragment following inhalation of particles versus intra-tracheal instillation in mice suggested that a Cmax of ~20-50 nMoles/L was achieved in 20-30 min following inhalation; while comparable levels were achieved upon intra-tracheal instillation of the peptide (Figure 1C). Values of AUC0-t and AUC(0-inf_obs) were in good agreement and showed clear dose dependence. A

IPGTT, GLP-1 (7-Day)

B AUC (Blood Glucose)

80000 ***

60000

***

40000 20000

C on C on tr ol tr +G ol L D Pia 1 be SS tic D ia C be on tic tro +G l LP -1 SS G LP -1 LD G LP -1 H D

ITT, GLP-1 (7-Day)

C AUC (Blood Glucose)

40000 30000

***

20000 10000

C on tr ol +G

LP D -1 ia SS be tic C on tr ol D +G LP -1 SS G LP -1 LD G LP -1 H D

Figure 2: (A) Fluoroscence dual staining of pancreatic insulin (red) and BrdU (green) positive cells, nuclei stained with DAPI (blue). Scale bar represents 50µm and 100µm. Mice were randomly assigned to six groups with five mice in each group; namely: (1) control (7 days), (2) control+GLP-1 standard (Control+GLP-1 SS), (3) diabetic, (4) diabetic+GLP-1 standard (D+GLP-1 SS), (5) diabetic+low dose DPI (GLP-1 LD), (6) diabetic+high dose DPI (GLP-1 HD). Mean glucose concentrations (AUC) following (B) intraperitoneal glucose tolerance test (IPGTT) and (C) insulin tolerance test (ITT). Data are represented in terms of mean ± Standard error mean (n=5).

In vivo efficacy studies showed that a dose dependent response in terms of plasma glucose reduction (until euglycemic levels) can be achieved by delivering GLP-1 by inhalation route. IPGTT (Figure 2B) and ITT (Figure 2C) indicates that high dose (4.5 µg) dry powder formulation of GLP-1 provided a better control over glucose homeostasis as compared to low dose formulation (2.3 µg). GLP-1 inhalation formulation promotes β-cell proliferation, RNA transcription in the β-cells and downregulates their apoptosis, as apparent in fluorescence staining for DNA (DAPI) RNA (BrDU) and protein (fluorescence immunohistochemistry) (Figure 2A). In vivo imaging (IVIS spectrum) provided useful insights on the lung deposition pattern of GLP-1 particles. Uniform fluorescence was observed throughout all the lung lobes suggesting that uniform deep lung delivery of particles was achieved. Conclusion SFD engineered particles possessed physicochemical and aerodynamic characteristics suitable for deep lung delivery. GLP-1 can be effectively delivered via pulmonary route for type 2 diabetes mellitus. Acknowledgements Funded by CSIR grant BSC0101. SP is grateful to CSIR for Senior Research Fellowship, Dr. AN Misra (MSU, Vadodara) for access to ACI and IIT-K for BET analysis. References 1. 2. 3.

Ahren B. GLP-1 for type 2 diabetes. Exp Cell Res. 2011;317:1239-1245. Healy AM, Amaro MI, Paluch KJ, and Tajber L. Dry powders for oral inhalation free of lactose carrier particles. Adv Drug Deliv Rev. 2014;75:32-52. Asai A, Okuda T, Yamauchi T, Sugiura Y, and Okamoto H. Safety Evaluation of Dry Powder Formulations by Direct Dispersion onto Air-Liquid Interface Cultured Cell Layer. Biological & pharmaceutical bulletin. 2016;39:368-377.

Drug Delivery to the Lungs, Volume 29, 2018 - David Harris Understanding the Inspiratory Manoeuvre and Why itâ&#x20AC;&#x2122;s Important to DPI Design David Harris Cambridge Healthcare Innovations Introduction The field of respiratory drug delivery involves complex science for which the fundamental principles are often not well understood. Current inhalers generally have poor performance, typically delivering more drug to the mouth and throat of the patient rather than their lungs, the target site. During this presentation we will cover the basics of fluid dynamics, which is key to successful inhaler design; how patientsâ&#x20AC;&#x2122; lungs function and provide the necessary energy for the aerosolisation of powdered drug formulation, and how this impacts device design and performance. The aim is to cover basic fluid dynamics and how lungs function, as the understanding of both is absolutely fundamental to good inhaler design. Every single Dry Powder Inhaler (DPI) in the world is powered by the energy available from the userâ&#x20AC;&#x2122;s inspiratory manoeuvre. Why does air flow? Differential pressure causes air to flow â&#x20AC;&#x201C; to achieve equilibrium, air (generally) moves from areas of high pressure to areas of low pressure. Think of an inflated balloon â&#x20AC;&#x201C; the air inside it is at slightly higher pressure than the air outside it due to the tension in the stretched rubber. If you burst it, the air rushes out â&#x20AC;&#x201C; usually quite quickly. Why do we have air pressure? Air has massâ&#x20AC;Ś The mass of the air in the atmosphere experiences gravity, like any other mass, so creates a force on the Earthâ&#x20AC;&#x2122;s surface according to: đ?&#x2018;?đ?&#x2018;? = đ?&#x153;&#x152;đ?&#x153;&#x152;đ?&#x153;&#x152;đ?&#x153;&#x152;â&#x201E;&#x17D;

Where Ď is density (kg m-3), g is acceleration due to gravity (m s-2) and h is the height above sea level (m) What is the air density? đ?&#x153;&#x152;đ?&#x153;&#x152;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D; =

đ?&#x2018;?đ?&#x2018;? đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;

Where p is pressure (Pa); R (Specific gas constant for dry air) is 287.05 J / kg K and T is temperature in K. For example, at 20Â°C and standard atmospheric pressure: đ?&#x153;&#x152;đ?&#x153;&#x152;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D; =

101,325 = 1.204 đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC; đ?&#x2018;&#x161;đ?&#x2018;&#x161;3 287.05 Ă&#x2014; (273.15 + 20)

So a cubic metre of air has a mass of about 1.2 kg at STP.

What is the weight of air that rests on a typical personâ&#x20AC;&#x2122;s shoulders? ď&#x192;&#x160; About a tonne â&#x20AC;&#x201C; 10.33 tonnes per m2 or 14.7 lbs / square inch. Think how much harder it is to cycle over 20 mph than 15 mph â&#x20AC;&#x201C; you need almost twice the power simply to push the air out of the way, as aerodynamic drag is proportional to the velocity squared. What is aerodynamic diameter? Take six balloons, inflated to the size of a golf ball, tennis ball â&#x20AC;Ś up to fully inflated. Which balloon is the largest? Ask for a volunteer. Ask them to drop the balloon from as high as they can hold it, and time the fall of each. Ask which balloon is aerodynamically larger! Ask if anyone knows why the larger balloon, with even more mass, falls more slowlyâ&#x20AC;Ś Explain that as the balloon is inflated to become geometrically larger and more massive, aerodynamically its size is reducing â&#x20AC;&#x201C; the largest balloon behaves as though it were the smallest particle, aerodynamically.

Drug Delivery to the Lungs, Volume 29, 2018 - Understanding the Inspiratory Manoeuvre and Why itâ&#x20AC;&#x2122;s Important to DPI Design

Aerodynamic diameter of irregular particle = diameter of unit density sphere with the same settling velocity.

Settling velocity? Does anyone know what this is? What would be the settling velocity of a person who jumps out of an airplane? Not sure â&#x20AC;&#x153;settlingâ&#x20AC;? is a good description â&#x20AC;&#x201C; more like â&#x20AC;&#x153;unsettlingâ&#x20AC;?! For living objects in freefall, this is perhaps more aptly known as â&#x20AC;&#x153;terminalâ&#x20AC;? velocity. A butterfly does not hit a car windscreen, even though it is geometrically larger than a fly, which usually does hit a car windscreen, especially when youâ&#x20AC;&#x2122;re out of screenwash.

Characterising the inspiratory manoeuvre Orifice plates restrict the airflow, so measuring only the pressure drop across an orifice plate the flowrate can be calculated reasonable accurately (assuming Cd is known) according to the following equation: 2Î&#x201D;đ?&#x2018;&#x192;đ?&#x2018;&#x192; đ?&#x2018;&#x201E;đ?&#x2018;&#x201E; â&#x2030;&#x2026; đ??śđ??śđ?&#x2018;&#x2018;đ?&#x2018;&#x2018; đ??´đ??´â&#x2C6;&#x161; đ?&#x153;&#x152;đ?&#x153;&#x152;

Where, Q is the volumetric flowrate in m 3/s Cd is the discharge coefficient and is dimensionless - ~0.6 for a sharp-edged inlet orifice plate and ~0.95 for a radiused inlet orifice plate. A is the open cross-sectional area of the orifice in m2 Î&#x201D;P is the differential pressure in Pa Ď is the upstream air density in kg/m3

Orifice Diameter

Pressure Drop

Flowrate

(mm)

(kPa)

(LPM)

ÎŚ2.50 mm

10.4

24.0

ÎŚ3.54 mm

9.9

46.8

ÎŚ4.33 mm

9.2

67.8

ÎŚ5.00 mm

8.1

84.9

ÎŚ5.95 mm

6.6

108.3

ÎŚ7.07 mm

5.0

133.1

If we now plot pressure drop against flowrate, fit a trendline, we can extrapolate data to find P.MIP and PIFR.

Drug Delivery to the Lungs, Volume 29, 2018 - David Harris

Considering the differences between a child’s and an adult’s lungs, what do you think a typical child’s Pressure-Flow plot would look like?

Drug Delivery to the Lungs, Volume 29, 2018 - Understanding the Inspiratory Manoeuvre and Why it’s Important to DPI Design Why is this so important for dry powder inhalers? Plotting typical DPI pressure-maps on the lung characteristic plot, we can work out the operating points by the intersections with the inspiratory pressure – flow curves.

Anyone’s’ lungs can be characterised and any inhaler with a known resistance can be plotted on interpolated lung data. Remember people’s lung characteristics are variable within themselves – e.g. may be more powerful early in the morning, or after exercise. Notice that there is much more consistency in Peak Maximal Inspiratory Pressure than there is in Peak Inspiratory Flowrate. The muscles that power lungs are generally quite similar (typically), whereas the internal resistance and capacity of each individual’s lungs is more variable. This indicates that designing high resistance DPIs will improve the consistency between users of varying lung capability – as there is much higher variability in inspiratory flowrate than there is in P⋅MIP. It is worth noting that increasing the resistance also promoted longer (and slower) inhalation – though increasing the resistance too far (e.g. less than 30 LPM @4 kPa) can cause discomfort with some users, particularly as they are instructed to inhale strongly and deeply. As all passive DPIs rely solely upon the user’s lungs to power them and ultimately deagglomerate the formulation to provide a fine, respirable aerosol, the first step to optimising their performance should be to build a good understanding of lung function in the target patient group. Once this data has been acquired, finding the optimal operating point (i.e. device airflow resistance) to minimise variability across the target patient group will be straightforward.

Drug Delivery to the Lungs, Volume 29, 2018 - Elijah Nazarzadeh et al. Controlling the size of nebulised droplets by pinning surface waves for precise delivery of aerosolised medicine Elijah Nazarzadeh1, Nikita Lomis2, Xi King1, Rab Wilson1, Manlio Tassieri1, Julien Reboud1, Jon Cooper1, Satya Prakash2 1

Division of Biomedical Engineering, University of Glasgow, Rankine Building, Glasgow G12 8LT, UK Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, 3775 University Street, Montreal H3A 2B4, Canada

2Biomedical

Summary The effective delivery of medication to the lungs via the inhalation of aerosols is strongly dependent upon the droplet size distribution; i.e., optimum pulmonary drug delivery occurs when the size falls between 1 and 5 Îźm in diameter. In this study, we investigated the formation of aerosols by means of surface acoustic waves (SAWs) and developed a new solution to control the droplet size distribution within the required range. The acoustic waves were coupled into a microfabricated array of microconfinements of the liquid sample to be nebulised. The therapeutic suspension also advantageously act as the acoustic coupling agent. SAWs were generated on the surface of a piezoelectric substrate and the nebulisation process was monitored by a high-speed camera (100,000 fps) and the particles size distribution measured by a Spraytec (Malvern Panalytical). We show that the physical confinement within the microstructures controls the wavelength of the deformation of the liquid, which allows us to precisely tailor the droplet size distribution of the aerosols to the optimum range for drug delivery (1 to 5Îźm). We also validated the applicability of the technique, by nebulising drug-loaded nanoparticles. Importantly, the control of the acoustic energy into the microstructures enables the direct formulation of encapsulated compounds from base materials (i.e. the compound and the shell). This new capability offers the possibility of nebulising formulations that have a short shelf-life when encapsulated. As a proof-of-concept, we demonstrate this by forming and nebulising the cancer drug Paclitaxel, encapsulated in ca. 250 nm human serum albumin particles. Key Message We demonstrate that surface acoustic waves can efficiently control of the size distribution of a nebulised aerosol, when coupled with microstructured surfaces. This capability allows to increase the proportion of drug nebulised that is delivered at the right location in the lungs to potentially increase efficacy. Additionally, we have shown the potential of the technique to form drug loaded of nanoparticle directly from the microstructured surface. Introduction Surface acoustic waves can be generated on the surface of a piezoelectric material to create a Rayleigh wave of nanometers in amplitude[1], which couple into the materials placed in their propagation path. In case of a fluid, the ultrasonic wave refracts into it and generates fluid streaming flows (Fig. 1a). The latter process could led to many phenomena including the dispersion of the liquid in form of a mist[2] (i.e., nebulisation) which has a wide range of applications including printing of micro-protein arrays[3], spray drying, mass spectrometry[4], nanoparticles synthesis and pulmonary drug delivery. In the latter case, It is well established that a narrow distribution of aerosol droplet diameters, between 1 to 5 Îźm, is required for an efficient and effective drug delivery to the lungs. Early studies on droplet formation showed that the propagation of waves on a liquid jet generates instabilities[5] that break-up the liquid jet into many droplets when the instabilities wavelength exceeds a critical value (ÎťCr ). These studies underpin the current understanding of drop formation due to instabilities at liquid-air interface[6]. The instability waves are categorised based on the dominant force between the gravitational force or the surface tension force. The latter is shown to be the dominant one when the length scale of liquid is very small (< 2cm) and these waves are known as capillary waves.

Earlier studies, using low frequency bulk acoustic waves (<1MHz), showed a correlation between the median diameter of the aerosol droplet size (đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x161;đ?&#x2018;&#x161; ) and the frequency of the capillary waves (đ?&#x2018;&#x201C;đ?&#x2018;&#x201C;đ?&#x2018;?đ?&#x2018;? ) formed on the liquid surface[7]. Using the assumption made by Kelvin[8] that đ?&#x2018;&#x201C;đ?&#x2018;&#x201C;đ?&#x2018;?đ?&#x2018;? = đ?&#x2018;&#x201C;đ?&#x2018;&#x201C;/2, Lang et al.[7] corroborated the existence of a correlation between the mean droplet size, capillary wavelength (đ?&#x153;&#x2020;đ?&#x153;&#x2020;đ?&#x2018;?đ?&#x2018;? ) and the acoustic excitation frequency (đ?&#x2018;&#x201C;đ?&#x2018;&#x201C;): 1â &#x201E; 3

8đ?&#x153;&#x2039;đ?&#x153;&#x2039;đ?&#x153;&#x2039;đ?&#x153;&#x2039; đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x161;đ?&#x2018;&#x161; â&#x2C6;? đ?&#x153;&#x2020;đ?&#x153;&#x2020;đ?&#x2018;?đ?&#x2018;? = ( 2 ) đ?&#x153;&#x152;đ?&#x153;&#x152;đ?&#x2018;&#x201C;đ?&#x2018;&#x201C;

(1)

Drug Delivery to the Lungs, Volume 29, 2018 - Controlling the size of nebulised droplets by pinning surface waves for precise delivery of aerosolised medicine This expression was also derived later by other authors[9]. However, recent studies suggest that Kelvinâ&#x20AC;&#x2122;s assumption on the capillary frequency (i.e., đ?&#x2018;&#x201C;đ?&#x2018;&#x201C;đ?&#x2018;?đ?&#x2018;? = đ?&#x2018;&#x201C;đ?&#x2018;&#x201C;/2) is not valid for high frequency excitations.

In general, it has been shown that depending on the geometry of the parent drop (e.g. diameter of a spherical cap droplet), nebulisation SAWs generates aerosols with a multimodal size distribution [10]. This has been explained by the acoustic deformation of the liquid volume, creating a thin layer of liquid adjacent to the bulk of the fluid, which results in the generation of small droplets (<10Âľm), while larger droplets (>10 Âľm) detach from the bulk of the liquid. These studies conclude that the final droplet size distribution of the nebulised liquid depends on a characteristic length scale of the liquid sitting within the SAW propagation path, which could be either its width or its height[11]. In this communication, we present a method to control the final aerosol droplet size distribution and its dispersity by filtering low frequency capillary waves within micro-fabricated cavities, which act as a low-pass filter and impose a well-defined characteristic length scale to the nebulisation system. Moreover, we demonstrate that these microfabricated cavities can be used to produce nano-particle formulations directly from the platform. Methods and materials SAWs device fabrication and nebulisation: SAWs were generated on the surface of the piezoelectric material, lithium niobate (LiNbO3), using an interdigitated transducer (IDT), as schematically shown in Figure 1a. The IDT was prepared by standard photolithography and microfabrication process. Cylindrical cavities were etched in Si, with diameters of 100, 200, 400, 600, 800 and 1500 Îźm. The silicon wafer was patterned using a standard photolithography and dry etched to a depth of 250 Îźm. The resulting silicon chip (22 x 22 mm) was then coupled to the surface of lithium niobate, as a thin plate (Figure 1a), using a small volume (ca. 10Âľl) of ultrasound transmission gel (Aquasonic 100, Parker Laboratories INC., USA). The cavities were fully filled with water before conducting each experiment. The SAW actuated nebulisation was performed using 2.0 đ?&#x153;&#x2021;đ?&#x153;&#x2021;đ?&#x153;&#x2021;đ?&#x153;&#x2021; volume of deionised water. A high-speed camera (Phantom V2511, Vision Research, USA) captured images in order to monitor formation of capillary waves, measure the capillary wavelength and monitor the nebulisation process. Several hundred measurements were carried out for the capillary waves on the surface of both free and confined liquids. Images were acquired at a frame rate of 100,000s-1 with a resolution of 384 x 288 pixels, unless otherwise stated. A laser diffraction instrument (Spraytec, Malvern, UK) was used to measure the aerosol droplet size distribution. Laser diffraction method is capable of measuring droplet sizes of 0.1 to 2000 Îźm in diameter. Mean value of 3 replicates is reported. The Bovine Serum Albumin (BSA) nanoparticles were prepared by emulsion solvent evaporation process [12]. Control emulsions were prepared though the conventional method by dispersion of 100 Îźl of chloroform in 1mg/ml solution of BSA in water (containing 12 đ?&#x153;&#x2021;đ?&#x153;&#x2021;g/ml of the anti-cancer drug Paclitaxel). The emulsion was then subjected to 5 minutes of tip sonication and subjected to rotary evaporation for 30 minutes at 90 rpm. For BSA nanoparticle formation, the same initial mixture was prepared and nebulised after a rigorous hand shake. A Malvern Nano-sizer was used to characterise nano-particles. The Paclitaxel concentration in nano-particles was measure using UVspectrophotometric method as reported previously[13]. Results In order to investigate the relationship between the characteristic length scale of the liquid and the SAW induced capillary waves at the water-air interface, two sets of experiments were performed: (I) a sessile (free) drop was placed directly on the lithium niobate wafer or (II) the liquid was constrained by a micro-structured filter. The capillary wavelengths were measured by means of image analysis. Results show a direct proportionality with the characteristic length scale of fluid (i.e., either with the diameter of sessile drop or with the characteristic length of the micro-structured filter). These results provide a new strategy for controlling the formation of capillary waves by means of engineered micro-structured filters. The aerosol droplet size distributions of nebulised liquids by using various confinements of different size were measured by using a laser diffraction technique and the results are reported in Figure 1b. These are consistent with earlier observation of the formation of capillary waves with short wavelength within small confinements. The droplet size distributions of aerosols yielded from confined liquids show a large decrease in distribution width and formation of aerosols with relatively sharper distributions compared to those performed with a free water droplet (Figure 1b). A decrease in the formation of large droplets is visible for cavities with a size of 800 Îźm in diameter. Further decrease of cavity size to 400 Îźm and 200 Îźm produce an aerosol with a mono-modal distribution and peaks in the range of 15 Îźm and 2 Îźm, respectively. The latter value lies within the desirable range of aerosol droplet size for pulmonary drug delivery.

Drug Delivery to the Lungs, Volume 29, 2018 - Elijah Nazarzadeh et al.

Figure 1. (a) A schematic showing propagation of surface acoustic waves on the surface of a material and generates fluid streaming. (b) Droplet size distribution with cavity size for droplets formed from 2 Îźl droplet (i.e., 1.6 mm which is diameter of sessile droplet) and liquid confined within cavities of 800 Îźm, 400 Îźm and 200 Îźm.

Finally, the formation of nano-particle formulation via these cavities was investigated. Conventionally, this process is done by means of emulsion solvent evaporation[12]. A volatile oil (e.g., chloroform) in water emulsion is formed in presence of a protein that acts as stabiliser (such as BSA). Usually, the production process starts with a tip sonication, followed by multiple passages of the emulsion through a high-pressure homogeniser to reduce the droplet size. Consequently, the volatile oil is evaporated using rotary evaporator to form BSA nanoparticles. In this work, the initial emulsion was prepared by adding 100Îźl chloroform into a 1ml of 1mg/ml BSA water solution (containing 12 đ?&#x153;&#x2021;đ?&#x153;&#x2021;g/ml of Paclitaxel), which was consequently shaken vigorously. The mixture was then fed into the platform and dispersed as an aerosol. Upon dispersion of mixture, the aerosol was collected in a water reservoir and measured by using dynamic light scattering (Malvern Zetasizer). The comparison between droplet size and zeta potential of particles produced by means of both the conventional method and the SAW based nebulisation shows that both methods produce particles in the range of 230nm and have comparable zeta-potential values (Figure 2a). The nanoparticle drug load efficiency was measured by measuring the drug concentration using a UVspectrophotometric method. Nanoparticles were concentrated using centrifugal filtration and the drug load concentration in nanoparticles produced through the conventional method and nebulisation method were measured. Comparison of drug loaded concentration in these nanoparticles and initial solution are shown in Figure 2b. It reveals that nano-particles produced by the means of SAW nebulisation contains a comparable amount of drug and low residue of the drug on platform was measured.

Figure 2. (a) Particle size and zeta potential comparison of BSA nanoparticles formed by a conventional method and by means of a SAW based nebuliser. (b) Measured concentration of Paclitaxel in nano-particles produced via the conventional method and SAW nebulisation. (c) SEM image of prepared nebulised BSA nanoparticles. Conclusion We have corroborated that the wavelength of capillary waves on the surface of a liquid is proportional to the characteristic length scale of the liquid. This is achieved by nebulising different volumes of water on the surface of an interdigitated transducer and by controlling the liquid characteristic length scale by means of a series of microfabricated disposable filters having different cavity size. Moreover, we employ the same technology to produce and nebulise BSA nano-particles; enabling a single step formulation of nano-particles for potential applications in pulmonary drug delivery. These experiments were carried out using a lab-prototype and we are currently seeking to commercialise the technology.

Drug Delivery to the Lungs, Volume 29, 2018 - Controlling the size of nebulised droplets by pinning surface waves for precise delivery of aerosolised medicine References 1. J. Reboud, Y. Bourquin, R. Wilson, G. S. Pall, M. Jiwaji, A. R. Pitt, A. Graham, A. P. Waters, andJ. M. Cooper, "Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies," Proceedings of the National Academy of Sciences of the United States of America 109, 15162 (2012). 2. Y. Bourquin, R. Wilson, Y. Zhang, J. Reboud, andJ. M. Cooper, "Phononic Crystals for Shaping Fluids," Advanced Materials 23, 1458 (2011). 3. J.-W. Kim, Y. Yamagata, M. Takasaki, B.-H. Lee, H. Ohmori, andT. Higuchi, "A device for fabricating protein chips by using a surface acoustic wave atomizer and electrostatic deposition," Sensors and Actuators B: Chemical 107, 535 (2005). 4. K. Tveen-Jensen, F. Gesellchen, R. Wilson, C. M. Spickett, J. M. Cooper, andA. R. Pitt, "Interfacing low-energy SAW nebulization with Liquid Chromatography-Mass Spectrometry for the analysis of biological samples," Scientific Reports 5, 9736 (2015). 5. L. Rayleigh, "On The Instability Of Jets," Proceedings of the London Mathematical Society s1-10, 4 (1878). 6. L. Duchemin, S. Popinet, C. Josserand, andS. Zaleski, "Jet formation in bubbles bursting at a free surface," Physics of Fluids 14, 3000 (2002). 7. R. J. Lang, "Ultrasonic Atomization of Liquids," The Journal of the Acoustical Society of America 34, 6 (1962). 8. J. W. S. Rayleigh, The Theory of Sound (Dover Publications, New York, 1954). 9. J. Ju, Y. Yamagata, H. Ohmori, andT. Higuchi, "High-frequency surface acoustic wave atomizer," Sensors and Actuators A: Physical 145â&#x20AC;&#x201C;146, 437 (2008). 10. A. Qi, L. Y. Yeo, andJ. R. Friend, "Interfacial destabilization and atomization driven by surface acoustic waves," Phys Fluids 20, (2008). 11. J. Blamey, J. R. Friend, andL. Y. Yeo, Acoustically induced micro-scale capillary wave turbulence (Sydney, Australia, 2010). 12. N. Lomis, S. Westfall, L. Farahdel, M. Malhotra, D. Shum-Tim, andS. Prakash, "Human Serum Albumin Nanoparticles for Use in Cancer Drug Delivery: Process Optimization and In Vitro Characterization," Nanomaterials 6, 116 (2016). 13. S. Sebak, M. Mirzaei, M. Malhotra, A. Kulamarva, andS. Prakash, "Human serum albumin nanoparticles as an efficient noscapine drug delivery system for potential use in breast cancer: Preparation and in vitro analysis," Int. J. Nanomed. 5, 525 (2010).

Drug Delivery to the Lungs, Volume 29, 2018 - Stuart Abercrombie Efficient Engineering Simulation to Inform and Optimise Capsule Inhaler Design Stuart Abercrombie1 1Team

Consulting, Abbey Barns, Duxford Road, Ickleton, Cambridge, CB10 1SX, UK

Summary Capsule dry powder inhalers (cDPIs) available on the market today face a number of performance challenges in delivering the best possible treatment to patients. This study demonstrates the benefits of using efficient engineering simulation tools including computational fluid dynamics (CFD) and Lagrangian particle tracking to optimise the design of a novel prototype cDPI for improved aerosol performance. Simulations were used to inform iterative design updates by increasing understanding of the airflow and particle dynamics within the device and quantifying key performance metrics such as the cumulative impulse acting on carrier particles during dose delivery. In vitro aerosol performance was evaluated using Next Generation Impactor (NGI) apparatus and commercially available Asthalin Rotacaps capsules containing a carrier-based formulation. Aerodynamic particle size distribution (APSD) results were significantly improved for the updated prototype design and demonstrated correlation with CFD quantified metrics. The results of this study support the use of efficient engineering simulations that maintain a balance between complexity and overall accuracy as valuable tools in the development of inhaler devices. Key Message The use of efficient engineering simulation tools during inhaler device development can significantly increase the effectiveness of design iterations. This study demonstrates that the in vitro aerosol performance of a novel prototype capsule dry powder inhaler can be improved using simulations to inform the design. Introduction Several cDPIs exist on the market today for the delivery of carrier-based dry powder formulations. These devices benefit from pharmaceutical capsules and the associated filling technologies being well-established in the industry for the packaging and stability of dry powders. A cDPI can often represent a good option as a platform device for the delivery of novel drug formulations during early stage clinical trials. However, cDPIs face several challenges as effective drug delivery devices such as a potential for poor or variable aerosol performance, the inclusion of relatively expensive metal capsule piercing components and usability issues caused by the quantity and complexity of use steps. Team Consulting has developed a novel prototype cDPI, known as the ‘TriAxEase’ (TAE) prototype, intended to address the challenge of aerosol performance and improve fine particle delivery. A key principle of the TAE prototype is to utilise a greater proportion of the energy available from a patient’s inhalation towards powder deagglomeration compared to currently available cDPIs. Most current cDPIs use the majority of inhalation energy for spinning or vibrating the capsule to release the powder through small pierced holes or slits. This is known to provide a level of powder deagglomeration due to inertial effects in the capsule and shear through the pierced holes [1]. However, it is proposed that this can be further improved with a downstream deagglomeration engine acting on the aerosolised powder. The TAE prototype includes a capsule splitting mechanism as an alternative to piercing, a 1st and 2nd swirl chamber to spin the separated capsule parts to release the powder and a 3rd swirl chamber to deagglomerate the powder (see Figure 1). A flow straightener is also included at the downstream end of the mouthpiece to reduce the swirl velocity of particles exiting the device, which can otherwise contribute to fine particle deposition in the mouth and throat [2]. This study demonstrates the use of engineering simulations to inform iteration of the TAE prototype from the original Gen1 design to Gen2 with the aim of improving fine particle delivery. Design updates for Gen2 were guided by learnings from Gen1 simulations and were carried out to improve CFD quantified performance metrics. Increasing the device efficiency to deliver drug particles in the size range 1 to 5 µm is intended to increase overall lung deposition for the patient and help maintain good performance across variable inhalation efforts. The engineering simulation approach presented here is intended to be efficient and flexible so that it can accommodate substantial design changes during early-stage development and remain a practical and informative tool. Materials and Methods Materials Capsules used were size 3 gelatine Asthalin Rotacaps (batch number SA74856, Cipla Ltd, India), each containing 15 mg carrier-based formulation with a labelled dose of 200 µg salbutamol sulphate. All high-performance liquid chromatography (HPLC) solvents and materials for cascade impactor testing were provided by Intertek Melbourn (Melbourn, UK). The Gen1 TAE prototype was injection moulded in Makrolon 2858 polycarbonate material (Covestro, Germany) using prototype tooling. The Gen2 TAE prototype was manufactured in Ultra 10122 material (DSM, Netherlands) using stereolithography apparatus (SLA). Metrology of the prototypes was carried out using computerised tomography (CT) scans provided by Carl Zeiss Ltd (Rugby, UK). A commercial sample HandiHaler device was used as a benchmark comparator.

Drug Delivery to the Lungs, Volume 29, 2018 - Efficient Engineering Simulation to Inform and Optimise Capsule Inhaler Design Computational Fluid Dynamics The commercial CFD solver Fluent 18.2 (ANSYS Inc, USA) was used to simulate the flow of air and particles through the cDPI. A mesh dependence study was conducted to ensure that the computational mesh resolution was sufficiently high to not affect results. Due to the highly swirling nature of the flow, a PRESTO scheme was used for pressure discretisation and a Reynolds Stress Model (RSM) was required for turbulence modelling [3]. The flow was found to include a significant unsteady component due to precession of the swirling flow, and so initial steady state solutions were extended to transient solutions to achieve sufficient convergence of numerical residuals. Lagrangian particle tracking was carried out using a Discrete Phase Model (DPM) [3] to compute samples of 6,000 particle trajectories based on individual spherical particles of diameter 3 µm representing drug fines and 50 µm representing carrier particles. A stochastic model was included to account for turbulence effects on trajectories. Wall collisions were represented using a Newtonian formulation with normal and tangential restitution coefficients of 0.8 and 0.9 respectively. A custom user defined function (UDF) was written to compute metrics that have previously been shown to inform the fine particle performance of DPIs, including the cumulative impulse acting on each carrier particle along its trajectory (J, Ns) [4] and the peak normal wall impact velocity experienced by each carrier particle (Vn, m/s) [5]. Cascade Impactor Measurements The airflow resistance of each device was measured using a Copley flow controller Model TPK (Copley Scientific, UK) and associated apparatus. Resistance results were used to determine the required flowrates for NGI testing at 2, 4 and 6 kPa pressure drop. A flow volume of 4 L was used for each device actuation. The analytical method for NGI testing was provided and carried out by Intertek Melbourn using HPLC. Five Asthalin Rotacaps capsules were delivered for each NGI test point and three repetitions of each test point were carried out. Analysis of the data to provide APSD results was carried out using Copley CITDAS software. Fine particle dose (FPD) was defined as the mass of drug in the size range <5 µm as a percentage of the labelled dose and fine particle fraction (FPF) as the mass of drug in the size range <5 µm as a percentage of the emitted mass. Mass median aerodynamic diameter (MMAD) was calculated as a measure of the average drug particle size deposited in Stages 1 to 8 of the NGI. Results The Gen1 prototype airflow resistance was measured as 1.67 √Pa.min.L-1 and the Gen2 resistance was measured as 1.96 √Pa.min.L-1. Predictions of device resistance from CFD simulations showed good agreement with measurements. The predicted flowrate at 4 kPa was within 5% of that measured for both the Gen1 and Gen2 designs, which contributed to validation of the CFD approach and provided confidence in the simulation results. Airflow pathlines for the Gen1 and Gen2 designs from CFD simulations are shown in Figure 1. A significant portion of the pressure drop through the Gen1 design occurred through the inlets to the 3rd swirl chamber. In response to this learning from Gen1 simulations the cross-sectional areas of the inlets were increased for Gen2, allowing for an increased pressure drop across the main body of the 3rd swirl chamber using a steeper cone angle. This generated a more highly swirling flow for Gen2 that increased the potential for powder deagglomeration based on improved CFD performance metrics. The Gen2 design increased the value of cumulative impulse, J, acting on carrier particles whilst maintaining a similar value of peak normal impact velocity, Vn, for carrier particles compared to Gen1. The pathlines through the 1st and 2nd swirl chambers for spinning the separated capsule halves and aerosolising the dose were similar for both the Gen1 and Gen2 designs. APSD results for the prototypes and a benchmark Handihaler are given in Figure 2. The Gen1 prototype produced a lower FPD at 4 kPa (29.4%) compared to HandiHaler (39.6%) but also achieved a finer MMAD (2.15 µm) compared to HandiHaler (2.80 µm). The Gen2 prototype maintained a low MMAD at 4 kPa (2.17 µm) and significantly increased the FPD (39.7%) and FPF (56.4%) compared to Gen1, exceeding the HandiHaler FPF (48.9%). The Gen2 prototype exhibited a good level of flowrate independence across the range 2 to 6 kPa with FPD varying between 38.7 – 39.7% and FPF varying between 52.3 – 57.1%. Fine particle results from experimental testing are plotted against CFD performance metrics in Figure 3. Increasing the mean value of cumulative impulse, J, acting on 50 µm carrier particles from CFD correlated with an increase in FPF. Increasing the mean value of peak normal impact velocity, Vn, for 50 µm carrier particles from CFD correlated with a decrease in MMAD. Discussion Iteration of the TAE prototype cDPI design from Gen1 to Gen2 was informed by engineering simulations to generate a more highly swirling flow and improve CFD performance metrics. This was shown to successfully improve in vitro aerosol performance.

Drug Delivery to the Lungs, Volume 29, 2018 - Stuart Abercrombie

1st Swirl Chamber: Spins 1st separated half of capsule and aerosolises powder

2nd Swirl Chamber: Spins 2nd separated half of capsule and aerosolises powder

3rd Swirl Chamber: Deagglomerates powder

Outflow Flow straightener Figure 1 – CFD pathlines plotted for the TAE prototype capsule dry powder inhaler Gen1 (left) and Gen2 (right) at 4 kPa coloured by static pressure (top, Pa) and velocity magnitude (bottom, m/s).

Figure 2 – Aerodynamic particle size distributions (left) and fine particle results (right). Error bars show ±1 standard deviation for N=3 runs. FPD fine particle dose, FPF fine particle fraction, MMAD mass median aerodynamic diameter.

Figure 3 – Comparison of fine particle results with CFD quantified performance metrics. Error bars show ±1 standard deviation for N=3 runs. FPD fine particle dose, FPF fine particle fraction, MMAD mass median aerodynamic diameter.

Drug Delivery to the Lungs, Volume 29, 2018 - Efficient Engineering Simulation to Inform and Optimise Capsule Inhaler Design Fine particle delivery as FPD and FPF at 4 kPa were both significantly increased for Gen2 (39.7%, 56.4%) compared to Gen1 (29.4%, 46.4%). This is expected to be beneficial for increasing the proportion of the dose reaching a patient’s lungs and reducing any potential side effects associated with oropharyngeal deposition. The improved fine particle efficiency of Gen2 also contributed to a good level of flowrate independence across the range of 2 to 6 kPa pressure drop, especially in terms of FPD which varied between 38.7 – 39.7% across this range. This is expected to be beneficial for improving the consistency of the dose reaching a patient’s lungs across variable inhalation efforts. The Gen2 prototype compared favourably with the benchmark commercial HandiHaler device. FPD at 4 kPa was similar for both devices but FPF was greater for Gen2 (56.4%) compared to HandiHaler (48.9%) and MMAD at 4 kPa was lower for Gen2 (2.17 µm) compared to HandiHaler (2.80 µm). The finer drug aerosol from the Gen2 prototype may be beneficial for certain therapies by enabling a shift in drug deposition towards deeper regions of the lung. A reduction in the delivered drug aerodynamic particle size in the range 1 to 6 µm has previously been shown to reduce oropharyngeal deposition, increase overall lung deposition and increase the penetration of deposition into deeper regions of the lung [6]. Increasing the CFD metric of cumulative impulse, J, for carrier particles correlated with an increase in FPF. This suggests that exposing carrier particles to significant forces over an increased residence time within the cDPI acts to increase the proportion of drug particles that are separated from carriers. Increasing to the CFD metric of peak normal impact velocity, Vn, for carrier particles correlated with a decrease in MMAD. This suggests that exposing carrier particles to greater magnitude impact forces acts to decrease the size of drug agglomerates released during impacts. In reality, the aerosol performance will depend on a combination of many different factors related to particle dynamics and formulation properties. For the case of the TAE prototype, the quantification of J and Vn from CFD has provided a valuable indication of performance differences to inform design iterations. The CFD approach presented here is limited in its complexity and its ability to accurately capture all the physical processes and interactions that occur during deagglomeration and aerosolisation of powder within a cDPI. A more complex approach such as that involving a discrete element method (DEM) could improve the resolution of impact forces and deagglomeration mechanisms [7]. However, this would also incur a cost due to greater complexity and computational time and would likely be less efficient for informing fast-paced design changes during early-stage device development. This study provides evidence that a CFD approach that increases complexity in a stepwise fashion whilst maintaining a balance between simulation efficiency and accuracy can contribute to significant in vitro performance increases during cDPI development. Conclusions This study has demonstrated the use of efficient engineering simulations that maintain a balance between complexity and overall accuracy as effective tools to inform the design of a prototype cDPI for improved in vitro aerosol performance. The CFD quantified metrics of cumulative impulse and peak normal impact velocity for carrier particles were shown to correlate with improved in vitro fine particle delivery. The updated Gen2 design achieved significantly improved FPD and FPF compared to Gen1 and achieved a greater FPF and finer MMAD compared to a benchmark HandiHaler. Gen2 also provided a good level of flowrate independence for fine particle delivery across the range of 2 - 6 kPa pressure drop. Acknowledgements The author would like to thank the inventors of the TAE prototype and contributors to its continued development: David Harris, Jamie Greenwood, Oliver Harvey and Philip Canner. The author would also like to thank Intertek Melbourn for carrying out NGI testing and Carl Zeiss Ltd for providing CT scan metrology of prototypes. 1

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Drug Delivery to the Lungs, Volume 29, 2018 - Pallav L Shah The future beyond inhalers â&#x20AC;&#x201C; endobronchial intervention in COPD Pallav L Shah 1 2

Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK

Chelsea & Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK

3National

Heart & Lung Institute, Imperial College, DoveHouse Street, London SW3 6LY, UK

Summary Lung volume reduction surgery (LVRS) was first reported by Otto Brantigan in 1957, but was not widely adopted until Joel Cooper perfected the technique of stapled resection in the 1990s. Several bronchoscopic therapies using differing strategies for reducing hyperinflation in emphysema have been developed. The greatest experience has been with endobronchial valves which have now been in use for almost 15 years. A wealth of clinical trial data has been produced in recent years with some key randomised clinical trials. The clinical trials with randomised endobronchial valves have demonstrated significant improvements in pulmonary function, quality of life and exercise capacity providing patients with heterogenous disease with absence of collateral ventilation are selected. 1-4 The responder rates are improved by valve adjustment or replacement where necessary. The results for endobronchial coils have been mixed with clinically meaningful results for pulmonary function and quality of life but at one year the benefits in walk tests have been marginal. 5-6 Vapor therapy appears to promise and has the capacity for more targeted and staged therapy. 7,8 There has traditionally been a rather nihilistic attitude toward emphysema and COPD, but recent technology developments mean this approach is no longer appropriate. The safety concerns over mortality and morbidity from LVRS has driven the development of BLVR, and whilst LVRS remains a very important, evidence-based treatment, BLVR has the potential to increase the availability of treatment to those with severe emphysema. The emergence of pivotal trial and longer-term follow-up data is likely to lead to more widespread and routine use of BLVR technologies, with the range of technologies allowing a suite of interventions that can be tailored to each individual patient. However, long-term benefit and cost-effectiveness needs to be demonstrated, and more work is needed to determine patient characteristics that best predict response to each individual technique. Patients with severe heterogenous emphysema, evidence of hyperinflation and intact lobar fissures. Patients without collateral ventilation may be considered for endobronchial valves. Those with collateral ventilation or homogenous distribution of emphysema may be considered for endobronchial coils. However, exciting advances have been made in interventions for non-emphysematous COPD, although technology is still at an early stage. Targeted lung denervation (TLD) aims to disrupt the parasympathetic nerve supply to the lung, which controls the release of acetylcholine and hence smooth muscle activity, by thermal ablation of the vagus nerve complexes around the main bronchi. Cryotherapy works by inducing cell death through repeated rapid freezethaw cycles with the subsequent regeneration of normal mucosa, and probe-based cryotherapy is an established bronchoscopic intervention. There are now a number of reports of the successful use of a catheter-based liquid nitrogen cryospray in both malignant and benign airway.

Drug Delivery to the Lungs, Volume 29, 2018 - The future beyond inhalers â&#x20AC;&#x201C; endobronchial intervention in COPD References 1: Criner GJ, Sue R, Wright S, Dransfield M, Rivas-Perez H, Wiese T, et al. A Multicenter RCT of ZephyrÂŽ Endobronchial Valve Treatment in Heterogeneous Emphysema (LIBERATE). Am J Respir Crit Care Med. 2018 May 22. doi: 10.1164/rccm.201803-0590OC. [Epub ahead of print] 2: Kemp SV, Slebos DJ, Kirk A, et al. A Multicenter Randomized Controlled Trial of Zephyr Endobronchial Valve Treatment in Heterogeneous Emphysema (TRANSFORM). Am J Respir Crit Care Med. 2017 Dec 15;196(12):15351543. 3: Klooster K, ten Hacken NH, Hartman JE, Kerstjens HA, van Rikxoort EM, Slebos DJ. Endobronchial Valves for Emphysema without Interlobar Collateral Ventilation. N Engl J Med. 2015:10;373(24):2325-35. 4: Shah PL, Herth FJ, van Geffen WH, Deslee G, Slebos DJ. Lung volume reduction for emphysema. Lancet Respir Med. 2017;5(2):147-156. 5. Shah PL, Zoumot Z, Singh S, Bicknell SR, Ross ET, Quiring J, Hopkinson NS, Kemp SV; RESET trial Study Group. Endobronchial coils for the treatment of severe emphysema with hyperinflation (RESET): a randomised controlled trial. Lancet Respir Med. 2013;1(3):233-40. 6. Sciurba FC, Criner GJ, Strange C, Shah PL, Michaud G, Connolly TA, et al. Effect of Endobronchial Coils vs Usual Care on Exercise Tolerance in Patients With Severe Emphysema: The RENEW Randomized Clinical Trial. JAMA. 2016: 24-31;315(20):2178-89. 7: Shah PL, Gompelmann D, Valipour A, McNulty WH, Eberhardt R, Grah C, et al. Thermal vapour ablation to reduce segmental volume in patients with severe emphysema: STEP-UP 12 month results. Lancet Respir Med. 2016 Sep;4(9):e44-e45. 8: Herth FJ, Valipour A, Shah PL, Eberhardt R, Grah C, Egan Jet al. Segmental volume reduction using thermal vapour ablation in patients with severe emphysema: 6-month results of the multicentre, parallel-group, open-label, randomised controlled STEP-UP trial. Lancet Respir Med. 2016;4(3):185-93

Drug Delivery to the Lungs, Volume 29, 2018 - Arcadia Woods et al. From modelling to synthesis to formulation to microbe: A multi-disciplinary approach to developing treatment for multi-drug resistant respiratory infection Arcadia Woods1, Mark Laws2, Kazi Nahar2, Shirin Jamshidi2, Charlotte Hind3, Mark Sutton3 & Khondaker Miraz Rahman2 Medicines Development Group, School of Cancer and Pharmaceutical Sciences, Kingâ&#x20AC;&#x2122;s College London, 150 Stamford Street, London SE1 9NH, UK 2 Drug Discovery Group, School of Cancer and Pharmaceutical Sciences, Kingâ&#x20AC;&#x2122;s College London, 150 Stamford Street, London SE1 9NH, UK 3 Research and Development Institute, National Infections Service, Public Health England, Salisbury SP4 0JG, Wiltshire, United Kingdom 1

Summary Multi-drug resistant (MDR) respiratory infections represent a significant challenge for healthcare. In the race to develop new effective treatments, a multi-disciplinary approach can enable rapid development of new medicines and accelerate translation from concept to pre-clinical testing. Liposomal formulations have been widely reported to improve the efficacy of inhaled antimicrobials and may enhance the already promising activity of novel antimicrobials against challenging respiratory infections. The aim of this project was to prepare a liposomal formulation of a lead candidate novel antimicrobial molecule (AB1) and to test its antimicrobial activity against clinically relevant pathogenic species for respiratory infection. A new antimicrobial scaffold (AB1) was synthesised following molecular modelling-led design and optimisation of a pharmacophore. Liposomes were prepared by lipid-film hydration, followed by extrusion. AB1 was loaded using the ammonium sulphate gradient method. Drug loading was quantified using high-performance liquid chromatography (HPLC) and liposome size and stability monitored using dynamic light scattering (DLS). Antimicrobial efficacy against a panel of Gram-positive strains was determined by minimum inhibitory concentration (MIC) determination. Liposomes had a size of 140 nm and with a narrow polydispersity (P.d.I.<0.1). Liposomal-AB1 had a drug loading capacity (DLC) of 21.8%, representing ~22 mg AB1 per 100 mg formulation. AB1 retained antimicrobial activity following liposomal encapsulation against two known respiratory pathogens, Staphylococcus aureus and Enterococcus faecalis. These promising early results indicate that liposomal-AB1 is worthy of further investigation for use as an inhaled formulation, which will include in vivo efficacy studies and dosage form design. Key Message A novel formulation comprising of liposome-encapsulated AB1, a new lead candidate antimicrobial, has been successfully developed and shows improved activity compared to liposomal levofloxacin against drug-resistant strains of respiratory pathogens, including Staphylococcus aureus, which in future may provide a valuable new tool to in the treatment of multi-drug resistant respiratory infection. Introduction Respiratory infection represents a major burden to global health, in particular to the health of children and infants 1. The challenge of treating these infections is made greater by the rapid increase in antimicrobial resistance (AMR) and emergence of multi-drug resistant (MDR) respiratory pathogens 2, 3. The inhaled route has demonstrated much success for the treatment of respiratory infection, particularly for cysticfibrosis-related infections 4 and for ventilator-acquired pneumonia 5. This is due to the ability to deliver higher concentrations of drug directly to the site of infection, reducing systemic exposure and plasma levels 6. In recent years, formulation advances, including the use of liposomal encapsulation, have been employed to improve the efficacy of inhaled treatments as they enable the retention of higher concentrations of drug in the lung for longer 7. This is vital to ensure that the local concentration within the lung exceeds both the Minimum Inhibitory Concentration (MIC) and Mutant Prevention Concentration (MPC) to ensure bacterial killing and reduced opportunity for AMRemergence due to sub-optimal therapy 8. The aim of this project was to prepare a liposomal formulation of a novel antimicrobial molecule, AB1, which would be suitable for development for inhaled use in the treatment of MDRrespiratory infection. AB1 was developed by molecular-modelling informed drug design. Using available crystal structures or homology models built with related antibiotic target enzymes, both wild type and mutant, the binding of the antibiotic core scaffold within the target identifies key residues that can be targeted to develop the novel modified antibiotics. This is followed by in silico screening of small-molecule fragment libraries to assess available scaffolds that are compatible with developing covalent modifiers for that antibiotic class. Molecular dynamics simulations are carried out on fragment-linked lead molecules to confirm their interaction with the mutant enzymes and ability to maintain interactions with the target topoisomerase. Top-ranked fragment-linked antibiotics are synthesized and tested for their ability to reverse target mediated resistance.

Drug Delivery to the Lungs, Volume 29, 2018 - From modelling to synthesis to formulation to microbe: A multidisciplinary approach to developing treatment for multi-drug resistant respiratory infection AB1 demonstrated promising activity against a range of respiratory pathogens (including MDR strains) in its free form. However, the molecular properties of this drug (molecular weight, logP) indicate liability for rapid absorption following administration to the lung 9. In this project, a collaborative, multi-disciplinary approach between medicinal chemists, formulation scientists and microbiologists was employed in the design, development and testing of the antimicrobial efficacy of a new liposomal formulation of AB1. It was hypothesised that this approach could overcome the challenges of its molecular properties without changing its activity. This project was a vital first stage in the development of this molecule, and its liposomal formulation, for use in inhaled drug delivery to the infected lung. Experimental Methods Synthesis of AB1 AB1 is a levofloxacin core modified fluroquinolone containing a 4-(piperazin-1-yl)pyrimidine covalent modifier. It was synthesized from commercially available (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4ij]quinoline-6-carboxylic acid in two steps using solution phase chemistry. The compound was purified using liquid column chromatography and fully characterised using NMR and Mass spectrometric techniques before formulation and biological studies. Preparation and characterisation of liposomal formulation Liposomes were prepared by thin-film hydration method 10 from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol in a 65:35 mass ratio. The lipid film was hydrated with 0.3M ammonium sulphate solution and heated with stirring for 2h at 65°C to form large multilamellar vesicles (LMV). Liposomes were passed 11 times through a high-pressure extruder (Avanti Mini Extruder with 0.1 μm filters, Avanti Polar Lipids, USA) at 65°C to form large unilamellar vesicles (LUV). Liposomes were purified by dialysis (Float-a-Lyzer®, 3-5 kDa molecular weight cut-off) against 150 mM NaCl overnight to remove excess ammonium sulphate. Liposome size and polydispersity index (P.d.I.) were analysed using dynamic light scattering (DLS) with the Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK) at a dilution of 1:100 in 150 mM NaCl (N=3 preparations). Drug loading of liposome formulation Liposomes were loaded with AB1 using an ammonium sulphate gradient in a modified method from that previously reported 10. The drug loading conditions were optimised using a factorial design with two variables (temperature and time) to achieve maximum drug loading and liposome stability. Liposomes were incubated with AB1 at a 2:1 lipid: drug ratio at 55°C for 70 minutes. Unencapsulated drug was removed by dialysis overnight against 150 mM NaCl. Liposomes were loaded with levofloxacin, a fluoroquinolone frequently used for treating respiratory infection in CF, as a control. Encapsulated drug was removed from liposomes by extraction in acetonitrile, followed by centrifugation (10,000 rpm, 10 min) to precipitate lipids. The resulting supernatant was analysed using HPLC. Drug loaded liposomes were analysed using DLS and compared to drug-free liposomes (N=3 preparations). Biological testing of the AB1-liposome formulation Biological activity of free and liposomal AB1 was assessed by determination of the minimum inhibitory concentration (MIC) using the broth microdilution method and compared to activity of free and liposomal levofloxacin. Bacterial growth in tryptic soy broth (TSB) was determined by measurement of optical density (Abs 600 nm). The MIC was determined as the lowest concentration of drug at which bacterial density (Abs 600 nm) was below 0.1 following 20h exposure. Biological activity was tested (N=3 replicates) against a Gram-positive panel containing strains of S. aureus and Enterococcus spp., both of which are known respiratory pathogens associated with cystic fibrosis 11 and ventilator-associated pneumonia12, respectively. Results Physicochemical characterisation of liposome formulation for drug loading The use of the high-pressure extruder resulted in a significant (P<0.001, unpaired t-test) reduction in liposome size from 3694 ±1804 nm to 142.8 ±5.1 nm (Figure 1a). The polydispersity index (P.d.I.) was also reduced significantly (P=0.005) following extrusion from 0.446 ±0.364 to 0.055 ±0.013, indicating an increase in the overall quality of the suspension when prepared using this technique. The liposomes demonstrated stability of size and polydispersity upon incubation in 150 mM NaCl at 4°C from 0-28 days and at 37°C for 0-7 days (Figure 1b), confirming that the formulation was stable during storage and during biological assay timeframes e.g. MIC testing.

Drug Delivery to the Lungs, Volume 29, 2018 - Arcadia Woods et al. Figure 1 - Liposome characterisation during and post-production a) the effect of extrusion on particle size and polydispersity as shown using particle size distribution measured using DLS (N=3) and b) stability of liposome formulation following incubation in 150 mM NaCl at 4°C and 37°C as measured using DLS (N=3).

Liposome drug loading efficiency Purified liposomes were successfully loaded with AB1 with a drug loading capacity (DLC) of 21.8 ±3.3%, which was comparable to the drug loading achieved with levofloxacin, 22.0 ±1.6% (Figure 2a). Dynamic light scattering confirmed that the drug loading procedure did not result in any significant change in particle size or polydispersity (Figure 2b). Figure 2 - Drug loading properties of the liposomal formulation a) drug loading capacity of the liposomal formulation to encapsulation AB1 and control fluoroquinolone, levofloxacin. b) Comparison of the particle size distributions of liposome formulation pre- and postloading with AB1 (N=3).

Biological testing of antimicrobial-loaded liposomes Biological testing indicated that antimicrobial activity of AB1 was retained following encapsulation in the liposome formulation against a range of Gram-positive pathogenic strains including two MRSA-S.aureus strains, NCTC 13616 and NCTC 13277 (Table 1). The formulation also demonstrated modest improvement in antimicrobial activity (>2fold decrease in MIC) compared to levofloxacin controls (liposomal and free drug). Table 1 - Antimicrobial activity comparison of liposomal AB1 vs free AB1, liposomal levofloxacin and free levofloxacin as illustrated by MIC values (μg/mL) for against a panel of Gram-positive strains. Strains NCTC 13616 and NCTC 13277 are methicillin-resistant; strains NCTC 12201 and NCTC 12204 are vancomycin-resistant.

Drug Delivery to the Lungs, Volume 29, 2018 - From modelling to synthesis to formulation to microbe: A multidisciplinary approach to developing treatment for multi-drug resistant respiratory infection Discussion A liposomal formulation of AB1 was prepared, demonstrating reproducible size (~140 nm) and narrow polydispersity. The formulation was loaded with AB1 with a high drug loading capacity (~21%), comparable to that previously reported for fluoroquinolone-type molecules 13. This high DLC can enable the delivery of high drug concentrations of drug without the need for high lipid payloads. Liposomal-AB1 retained antimicrobial efficacy compared to free AB1 and demonstrated improved activity compared to free levofloxacin and liposomal levofloxacin controls against several respiratory pathogenic strains, including those demonstrating known antimicrobial resistance. This confirmed that the formulation did not affect the activity of the molecule in vitro. Preliminary studies (not reported) have demonstrated retention of AB1 in the liposomal formulation over 24h, which has been previously demonstrated to correlate with enhanced retention in the lung following inhalation 14. We hypothesise that the enhanced retention time afforded by the liposomal formulation coupled with the low MIC values can contribute to enhanced efficacy in in vivo experiments. Therefore, the next stage in the development of this formulation will be to test the effect of encapsulation on treatment efficacy in vivo, and to investigate the aerosol properties of the formulation following nebulisation as the preferred route of delivery to the infected lung. Conclusion MDR-respiratory infection is a growing concern for clinicians worldwide, and new treatments are urgently needed. A multi-disciplinary approach between medicinal chemists, formulation scientists and microbiologists has enabled the development and testing of a new drug and formulation innovation, designed to improve its efficacy in the infected lung and improve treatment outcomes. Initial results indicate that liposomal-AB1 is a promising new formulation for inhaled treatment of respiratory infection and is worthy of further testing to establish its efficacy in treating infection in the lung in vivo. References 1

Wang HD, Naghavi M, Allen C, Barber RM, Bhutta ZA et al: Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016;388: pp1459-1544. 2

Enne VI, Personne Y, Grgic L, Gant V, and Zumla A: Aetiology of hospital-acquired pneumonia and trends in antimicrobial resistance. Curr Opin Pulm Med. 2014;20: pp252-258. 3

Jansen G, Mahrt N, Tueffers L, Barbosa C, Harjes M, Adolph G, Friedrichs A, Krenz-Weinreich A, Rosenstiel P, and Schulenburg H: Association between clinical antibiotic resistance and susceptibility of Pseudomonas in the cystic fibrosis lung. Evol Med Public Health. 2016;2016: pp182-194. 4

Ramsey BW, Pepe MS, Quan JM, Otto KL, Montgomery AB, Williams-Warren J, Vasiljev KM, Borowitz D, Bowman CM, Marshall BC, Marshall S, and Smith AL: Intermittent administration of inhaled tobramycin in patients with cystic fibrosis. Cystic Fibrosis Inhaled Tobramycin Study Group. N Engl J Med. 1999;340: pp23-30. 5

Kang CH, Tsai CM, Wu TH, Wu HY, Chung MY, Chen CC, Huang YC, Liu SF, Liao DL, Niu CK, Lee CH, and Yu HR: Colistin inhalation monotherapy for ventilator-associated pneumonia of Acinetobacter baumannii in prematurity. Pediatr Pulmonol. 2014;49: pp381-388. 6

Goldstein I, Wallet F, Nicolas-Robin A, Ferrari F, Marquette CH, and Rouby JJ: Lung deposition and efficiency of nebulized amikacin during Escherichia coli pneumonia in ventilated piglets. Am J Respir Crit Care Med. 2002;166: pp1375-1381. 7

Meers P, Neville M, Malinin V, Scotto AW, Sardaryan G, Kurumunda R, Mackinson C, James G, Fisher S, and Perkins WR: Biofilm penetration, triggered release and in vivo activity of inhaled liposomal amikacin in chronic Pseudomonas aeruginosa lung infections. J Antimicrob Chemother. 2008;61: pp859-868. 8

Blondeau JM, Hansen G, Metzler K, and Hedlin P: The role of PK/PD parameters to avoid selection and increase of resistance: mutant prevention concentration. J Chemother. 2004;16 Suppl 3: pp1-19. 9

Patton JS, Fishburn CS, and Weers JG: The lungs as a portal of entry for systemic drug delivery. Proc Am Thorac Soc. 2004;1: pp338-344. 10

Zhang X, Sun P, Bi R, Wang J, Zhang N, and Huang G: Targeted delivery of levofloxacin-liposomes for the treatment of pulmonary inflammation. J Drug Target. 2009;17: pp399-407. 11

Besier S, Smaczny C, von Mallinckrodt C, Krahl A, Ackermann H, Brade V, and Wichelhaus TA: Prevalence and clinical significance of Staphylococcus aureus small-colony variants in cystic fibrosis lung disease. J Clin Microbiol. 2007;45: pp168-172. 12

Bonten MJ, van Tiel FH, van der Geest S, Stobberingh EE, and Gaillard CA: Enterococcus faecalis pneumonia complicating topical antimicrobial prophylaxis. N Engl J Med. 1993;328: pp209-210. 13

Furneri PM, Fresta M, Puglisi G, and Tempera G: Ofloxacin-loaded liposomes: in vitro activity and drug accumulation in bacteria. Antimicrob Agents Chemother. 2000;44: pp2458-2464. 14

Ong HX, Benaouda F, Traini D, Cipolla D, Gonda I, Bebawy M, Forbes B, and Young PM: In vitro and ex vivo methods predict the enhanced lung residence time of liposomal ciprofloxacin formulations for nebulisation. Eur J Pharm Biopharm. 2014;86: pp8389.

Drug Delivery to the Lungs, Volume 29, 2018 - Afzal Mohammed et al. Fluidised Powder Blending to Control Particle-Particle Interaction â&#x20AC;&#x201C; The Future of DPI Formulation Afzal Mohammed1, Jasdip Koner2, Olaitan Abiona1 & David Wyatt1,2 1Aston

University, Aston Triangle, Birmingham, B4 7ET, United Kingdom Aston Particle Technologies Ltd, Aston University, Aston Triangle, Birmingham, B4 7ET, United Kingdom

Summary Traditional blending processes for dry powder inhaler (DPI) manufacture involve use of equipment that relies upon the solid-solid mixing of powders to deagglomerate and disperse cohesive micronised active pharmaceutical ingredients (APIs) onto the surface of coarser carrier particles, which often generate mechanical shear and are generally inefficient. Researchers at Aston University have developed a novel dry coating process which avoids the limitations of solid-solid blending by micro-fluidising powders held at the surface of a rotating chamber by a strong centrifugal field (high G) through the application of a nitrogen air-blade. Cohesive API is dispersed into primary particles in the fluidised state and coated onto the surface of much larger co-fluidised lactose particles. In a model study, cohesive micronised Rhodamine B was coated onto an inhalation grade lactose to mimic a typical DPI formulation. Blends were manufactured under a range of different processing conditions and the time course of the coating process was studied using confocal microscopy and scanning electron microscopy. This work exposed the nature of the coating process and the transitions through which the fine particles pass to achieve efficient coating. In a further study, a genuine DPI formulation, micronised fluticasone propionate was coated onto lactose (at 0.71%w/w) following a design of experiment scheme. The results demonstrate how the technology can control the formulation of DPI blends through manipulation the three process parameters; process speed, air-flow rate and process time. Two-dimensional response maps at set processing times were constructed highlighting how the critical process parameters of the novel methodology control two blend responses, content uniformity and fine particle fraction performance, and illuminate a standard to which DPI performance of the future might be designed and controlled.

Key Message Particle micro-fluidisation through the novel dry coating process produces consistent dispersion of the API/carrier system to primary particles and delivers concise deposition of API onto the lactose carrier. This results in a controlled DPI formulation process with exquisite control of the critical quality attributes of the resultant blend.

Introduction DPI formulations are dispersions of fine API particles in carefully engineered coarser inhalation grade lactoses (IGLs) originally added to the formulation to bulk up the dose to facilitate the dispensing of highly potent APIs. It was soon discovered that coating fine API onto carefully selected lactose carriers could enhance aerosolisation [1]. IGLs are typically chemically pure grades of alpha-lactose monohydrate with highly specialised particle size distributions but, despite more than 40 years of research and development, the performance of passive DPI formulations remains poor. Much research has been executed to understand the coating of fine particles on carriers. When fine (guest) particles are coated onto the surface of coarser (host) particles, composite particles result with enhanced properties different from those of the individual particles [2,3]. Current state of the art DPI blending mechanisms, such as high shear blending and turbula mixing, utilise solid-solid interaction between the constituent particles and the physical components of the mixing equipment to disperse API over the carrier to deliver an acceptable aerosol performance [2]. The link between aerosol performance of individual API/lactose systems and the manufacturing process is often poorly understood, which drives a trial and error approach to batch manufacture, with poorly predictable outcomes. Current practice in the formulation of DPIs is to seek to maximise the aerosol performance, by enhancing fine particle fraction (FPF). This is often achieved by the incorporation of fine material into the formulation (termed ternary formulations) either as a fine percentage of the lactose carrier or of other compounds such as magnesium stearate or leucine, known as force control agents. The latter dampen high energy sites on the lactose carrier prior to blending with the API, which results in higher percentages of the API being removable from the carrier during aerosolisation [3]. Additional components in the formulation add further unpredictability to nature of the blending process. A theoretical picture of the dry particle coating process is relatively simple to deliver schematically. It has been described as follows [4]: (1) deagglomeration of fine guest particles into individual particles; (2) attachment of the individual guest particles onto the host particle surface in close proximity; and (3) - redistribution/rearrangement of the fines between already coated and non-coated host particle surfaces to attain an even distribution of guest on host particles. This is presented in Figure 1.

Drug Delivery to the Lungs, Volume 29, 2018 - Fluidised Powder Blending to Control Particle-Particle Interaction – The Future of DPI Formulation

Figure 1: A schematic dry particle coating process

In DPI formulation the critical step in this process is the complete separation of any agglomerated API particles into primary particles. The API primary particles will have been engineered to be in the respirable size range (0.5 – 5 µm) so delivery of a respirable dose requires that the API is formulated and maintained as individual particles. If this level of dispersion is achieved the efficiency of the other two steps in the coating process will also be enhanced. The purpose of this study is to demonstrate that the unique dry particle coating process developed at Aston [5], achieves all 3 steps in the coating process. The unique dry coating process utilises rotation to generate a centrifugal force at the surface of a chamber which is continuously swept by a dry nitrogen gas air-blade. Powders inside the chamber are forced to this surface during processing. The centrifugal force and the sweeping air-blade cause disruption of any agglomerates in the powder into primary particles. Pockets of micro-fluidisation are formed at the surface in which the fine particles come into contact with the larger particles and are adsorbed. In effect the fine particles are mopped up by the larger particles. Since there is very low shear in the system and little energy is generated by collisions of particles near the surface of the chamber, the coating process occurs at ambient temperature. No increase in temperature is observed during processing due to the significantly reduced solid-solid interactions when compared with a typical blender and the absence of physical attrition. This is mediated through the relatively small set of critical process parameters (CPP) utilised by the process, and it is the intimate control of these CPP’s which provides controlled fluidisation and blending of the DPI formulations. The key objective of this work was to investigate the fluidisation principle using a model system and then to show how this can be applied to the controlled manufacture of a real DPI formulation. Experimental Methods Materials Rhodamine B Micronised (VMD – 6.27 ± 0.45 μm) (Sigma-Aldrich, Dorset, UK) used as a model guest cohesive material (utilised for fluorescent nature and resolution under confocal microscopy (CLSM)). Alpha-Lactose Monohydrate (Inhalac 251™ (Meggle, Wasserburg Am Inn, Germany) IGL (non-fluorescent). Fluticasone Propionate (FP) Micronised (VMD 3.22± 0.02 μm) (Discovery Fine Chemicals, Wimborne, UK) Methods Model Study: An APT benchtop dry particle coater was used to manufacture 10 g batches of 0.5%w/w rhodamine /IGL powder blend. The coater was operated at 201 G, at 3 different air-flows (0 l/min, 25 l/min and 50 l/min). Blend processing times were varied from 4 to 28 minutes (at 4-minute intervals). CLSM (Leica Microsystems Confocal Microscope TCS SP5 II, 10X dry objective lens - images were obtained between 528-560nm) was used to determine the state of dispersion of rhodamine particles. The blends were also analysed by scanning electron microscopy to visualise the coating of the rhodamine particles attached to the lactose surface. FP study: A D-optimal design of experiments (DoE) was conducted using FP (0.71%w/w) in the IGL and MODDE 10 software was used to design and assess the model. Manufacture of the formulations was conducted using an APT pilot scale dry particle coater, with 500 g batch sizes. Each of the FP blends was tested for content uniformity of 10 micro-samples and the aerosol particle size distribution from a capsule based delivery system (Aerolizer™) incorporating a notional dose of the blended material was measured. The doses were manually filled into size 3 gelatin capsules and tested using a Next Generation Impactor (NGI - Copley Instruments, UK) at 60 l/min.

Drug Delivery to the Lungs, Volume 29, 2018 - Afzal Mohammed et al. Results and Discussion Samples from the model blends containing Rhodamine B were analysed by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM). CLSM has a major advantage in that the position of the adsorbed rhodamine particles can clearly be identified in the images, and fluorescent image intensity of these particles is a measure of their state of dispersion whether present as agglomerates or primary particles. Figures 2A, B and C present images of powders blended for 4 mins at 0 l/min, 25 l/min and 50 l/min fluidising air-flow respectively. At this short processing time, the adsorbed rhodamine still contains agglomerates at all airflow rates even though at the highest flow rate, the lactose particles have already adsorbed a significant coating of fine particles. At the longer blending time of 20 minutes the agglomerates have completely disappeared and an excellent degree of coating has occurred (the images presented in 2D, E and F). This is true even when no fluidising air-flow was applied. A

Figure 2: CLSM images of Rhodamine B dry coated at 201 G onto IGL

Figure 3: Fluorescence Intensity as a function of processing time

This demonstrates that if the centrifugal force is applied for sufficient time even without the air-blade, the cohesive rhodamine will be reduced to primary particles. With increasing airflow rate, the coating builds up faster due to more rapid and efficient deagglomeration of the fine material through the micro-fluidisation mechanism. A fuller analysis of the intensity of the CLSM images is shown in Figure 3. The variation in fluorescence intensity clearly shows three phases of coating, independent of the fluidising air-flow rates investigated. A first phase, at short processing duration up to 8 minutes, shows a fluorescence intensity maximum most pronounced at low airflow rates. This suggests that although the coating process has commenced many of the rhodamine particles are present as agglomerates. This effect is less pronounced at the highest air-flow perhaps an indication that the presence of agglomerates is less pronounced under these conditions. At intermediate processing times, the fluorescence intensity decreases to a plateau value which suggests that the bulk of the particles have been reduced to primary particle size and are adsorbed onto the carrier particles. At the longest process times investigated, a 2nd peak in fluorescent intensity is seen which suggests that the adsorbed particles are once again in close proximity to each other on the surface. This phase could reflect a re-ordering of the particles on the surface and also that the coating is now becoming more coherent. At the longest process times investigated the intensity has again diminished which suggests that the adsorbed layer has obtained a new equilibrium. A further image analysis of the rhodamine coating at this intermediate processing time is presented in the scanning electron micrographs of Figure 4.

Figure 4: SEM images rhodamine coated lactose (16 minutes processing time) at fluidising airflows (A 0 l/min, B 25 l/min and C 50 l/min airflow at 1000x magnification and D, E, F at the same respective air-flows at 5000x magnification

Figure 5: Design space generation using the pilot scale dry particle coating equipment with FP (0.71%w/w)

Drug Delivery to the Lungs, Volume 29, 2018 - Fluidised Powder Blending to Control Particle-Particle Interaction – The Future of DPI Formulation By contrast to the CLSM images, the state of the adsorbed particles shown in the SEM images can only be discerned by changes in monochrome topography. Nevertheless, agglomerates of rhodamine particles are clearly visible on the surface of the IGL in the blend prepared without fluidising air whereas the other samples show the adsorbed rhodamine as discrete particles. Another important observation in all the images is that all the fine particles are attached to the larger carrier particles, which is evidence that the process causes the carrier particles to ‘mopup’ both fine rhodamine particles and the fines from the IGL. Each of the above observations is qualitative in nature and was made with a model cohesive material that had a particle size distribution only barely reflective of a typical respiratory API, so in a second suite of experiments, a commercially sourced API was used to generate quantitative data representative of a true DPI. The formulation of a commercially available API, fluticasone propionate, at 0.71%w/w was chosen to mimic a product designed to deliver 100 µg/dose with a commercially available grade of IGL, the same grade used in the rhodamine study. The study was deliberately carried out at larger scale (500 g batch size) to be more representative of the scale of batches that might be routinely expected/used in formulation development and product scale up. The seventeen randomized manufacturing runs were performed to investigate whether the new process could be modelled against two chosen responses, critical quality attributes, that would demonstrate how well the new process could control particle-particle interactions in a DPI formulation. With his in mind, content uniformity of the blends and fine particle fraction (FPF) performance of samples from the blends when dispensed via a unit dose capsulebased inhaler were the chosen response factors. These factors were measured with representative samples of each blend and a working performance model was created from these data and validated with statistical significance. Results from the DoE demonstrate the ability of the technology to deliver dry powder coating of FP on IGL such that the FPF of all the blends was between 21 32% of the nominal dose, whilst also maintaining an excellent homogeneity (<3%RSD). A primary finding of the study was that univariately increasing processing speed resulted in increased FPF performance, indicative of a potential to dial up respirable dose through manipulation of the critical process parameters; process speed, air-flow rate and process time. In addition, it was possible to identify a wide and flexible design space for manufacture the FP formulation at various process times as shown in the two-dimensional response maps (Figure 5). Optimal process control settings can be read from these control maps to ensure that the blend attributes fall within the target product profile. The green area at the centre of the model for 30 minutes processing time, delineated by the probability contours, demonstrates a wide degree of flexibility in the optimum processing parameters. It is likely that routine manufacture of batches of the FP formulation will meet the defined critical quality attributes with very small chance of failure. This the level of control is undoubtedly required for DPI formulations of the future. Conclusion Researchers at Aston University have developed a novel dry coating process which avoids the limitations of solidsolid blending by micro-fluidising powders held at the surface of a rotating chamber by a strong centrifugal field (high G) through the application of a nitrogen air-blade. Cohesive API is dispersed into primary particles in the fluidised state and coated onto the surface of much larger co-fluidised lactose particles. In a model study, cohesive micronised Rhodamine B was coated onto an inhalation grade lactose to mimic a typical DPI formulation. Blends were manufactured under a range of different processing conditions and the time course of the coating process was studied using confocal microscopy and scanning electron microscopy. This work exposed the nature of the coating process and the transitions through which the fine particles pass to achieve efficient coating. In a further study, a genuine DPI formulation, micronised fluticasone propionate was coated onto lactose (at 0.71% w/w) following a design of experiment scheme. The results demonstrate how the technology can control the formulation of DPI blends through manipulation the three process parameters; process speed, air-flow rate and process time. Twodimensional response maps at set processing times were constructed highlighting how the critical process parameters of the novel methodology control two blend responses, content uniformity and fine particle fraction performance, and illuminate a standard to which DPI performance of the future might be designed and controlled. References 1

Jones M, Santo J, Yakub B, Dennison N, Master H, Buckton G: The relationship between drug concentration, mixing time, blending order and ternary dry powder inhalation performance, Int, Journal Pharmaceutics 2010; 39: pp137 -147

Begat P, Morton D A V, Shur J, Kippax P, Staniforth J N, Price R: The role of force control agents in high‐dose dry powder inhaler formulations, J Pharm Sci 2008; 98(8): pp2770-2783

Gera M, Sahran V, Kataria M, Kukkar V: Mechanical methods for dry particle coating processes and their applications in drug delivery and development, Recent Pat Drug Deliv Formul 2010; 4(1): pp58-81

Bannister P, Harnby N: Colorimetric Technique for Assessing the Mixture Quality of Fine Particle Mixtures, Powder Technol 1983; 36: pp275-270

Mohammed A R, Dahmash E, Ahmed J, Drew T: Patent WO2016066462A1 Coating apparatus and method. 2016

Drug Delivery to the Lungs, Volume 29, 2018 - Bradley Morrical et al. Probing the Aerodynamic Particle Size Distribution of Dry Powder Inhaler Combination Products Foster® NEXThaler® and Seretide® Diskus® using Single Particle Aerosol Mass Spectrometry (SPAMS) Bradley Morrical, Martin Jetzer & Stephen Edge Novartis Pharma AG, Global Development, Novartis Campus, Lichtstrasse 35, Basel, 4056, Switzerland Summary The in-vitro aerosol performance of two combination dry powder inhaler (DPI) products, Foster® NEXThaler® and Seretide® Diskus® were investigated with single particle aerosol mass spectrometry (SPAMS). The in-vitro pharmaceutical performance was markedly different for both inhalers. Foster® NEXThaler® generated a higher fine particle fraction (FPF <5 μm) and a much higher relative extra-fine particle fraction (eFPF <2 μm). In terms of the composition of the aerodynamic particle size distribution (APSD), it could be verified with SPAMS that overall Foster® NEXThaler® emitted a significantly higher number of fine and extra fine particles. Additionally, the interactions between the two active pharmaceutical ingredients (APIs) in both products were different. While Seretide® Diskus® emitted a significant (37%) number of co-associated API particles, only a negligible number of co-associated API particles were found in Foster® NEXThaler® (<1%). A major difference with Foster® NEXThaler® is that it contains magnesium stearate (MgSt) as a second excipient besides lactose in a so-called ‘dual excipient’ platform. The data generated using SPAMS suggested that nearly all of the beclomethasone (FDA) (de Boer, Gjaltema, Hagedoom, & Frijlink, 2015)dipropionate particles in Foster® NEXThaler® also contain MgSt and must therefore be co-associated with this additional excipient. This finding may help explain why beclomethasone dipropionate in Foster® NEXThaler® forms less particle co-associations with the second API, formoterol fumarate, shows a lower cohesive strength in respect to beclomethasone itself and why both APIs exhibit superior detachment from the carrier as evidenced by the increased eFPF and smaller count median aerodynamic diameter (MAD). Key Message The degree of co-association in combination therapies can be manipulated with force control agents. For the first time this process could be directly observed with SPAMS due to its ability to directly characterize single aerosol particles. Introduction Dry powder inhalers (DPIs) are used to deliver an efficacious dose of active pharmaceutical ingredients (API) to a target region of the lung. The delivery of such a dose to the target region of the lung is dependent on the fine particle dose (FPD), or more precisely, the aerodynamic particle size distribution (APSD) of the API emitted from the DPI during its use by the patient. Aerodynamic particle sizes of <5 μm are usually considered optimal for drug delivery to the lungs [1]. However, there is an increasing interest in the development and use of the so-called ‘extra-fine’ aerosols to target the small airways in the treatment of peripheral airways in asthma and chronic obstructive pulmonary disease (COPD) [2-5]. It has been reported that the inhalation of very small drug particles (<2μm) leads to an increased total lung deposition, improved distal airway penetration and a higher peripheral lung deposition [6]. The chemical characterization of API particulates in a DPI formulation is achievable using mass spectrometry [7]. More recently, the technique was further developed and improved for the specific characterization of single aerosol particles, namely single particle aerosol mass spectrometry (SPAMS). SPAMS allows the determination of the qualitative chemical composition for the particulates. Importantly for DPIs, the technique is also capable of studying APIs and excipients. The APSD results generated using SPAMS can be transformed and compared to APSD data generated using cascade impactors. But, importantly, SPAMS allows the possibility to evaluate the chemical nature of the particulates in the APSD without the need for offline chemical analysis, such as HPLC [7]. In order to investigate product performance and interactions in DPIs and to test the applicability of SPAMS for the study of different DPIs, this study reports an investigation into the particulate and chemical composition of the emitted particles from two DPI combination products: one containing a single excipient (lactose monohydrate), Seretide® Diskus®, and one containing two excipients (lactose and MgSt), Foster® NEXThaler®, with particular focus on the interactions of the two APIs. Particle interactions may have a significant influence on the performance of inhaled formulations as, for example, fine particles exhibit a much faster dissolution in-vitro [8]. Another aim of this work was to determine the fate of the aerosolized MgSt in Foster® NEXThaler® and whether it also interacts with the APIs in the formulation. The effect of the force control agent MgSt on the aerosol performance in commercial combination DPI formulations was further investigated. Materials and Methods Seretide® Diskus® (250/50 mcg) and Foster® NEXThaler® (100/6 mcg) DPIs were used for the study. Seretide® Diskus® contains the APIs fluticasone propionate (FP) and salmeterol xinafoate (SX) in a blister and Foster® NEXThaler® contains the APIs beclomethasone dipropionate (BDP) and formoterol fumarate (FF) in a reservoir.

Drug Delivery to the Lungs, Volume 29, 2018 - Probing the Aerodynamic Particle Size Distribution of Dry Powder Inhaler Combination Products Foster® NEXThaler® and Seretide® Diskus® using Single Particle Aerosol Mass Spectrometry (SPAMS) A SPAMS 3.0 (Livermore Instruments Inc., USA) was used for this study. The experimental setup of the SPAMS instrument and sample testing has been described earlier by Morrical et al [7] and Jetzer et al [9]. Polystyrene (PLS) microspheres (Thermo-Fisher Scientific, USA) were used to calibrate the SPAMS instrument in the region from 0.110 μm, The DPI-derived powder aerosol samples were acquired by actuating the inhaler device via an induction port through a pre-separator (MSP corp.) into the relaxation chamber. This assembly of pre-separator and relaxation chamber was then fitted to the SPAMS inlet. The sampling chamber allows for dilution of the particles and flow rate matching. A test flow rate of 60 L/min was used for both devices [10], since it had been reported that Seretide® and NEXThaler® are relatively unaffected by flow rate. The same number of particles was analysed for each sample run (ca. 10,000 individual particles each). SPAMS experiments were alternated with background blank runs for one minute each. The desorption/ionization laser was set to operate at a wavelength at 248 nm. The SPAMS was able to analyse up to 64 particles per second in the configuration being operated, The laser energy was maintained at approximately 12 mJ per pulse to obtain a high and consistent acquisition rate of particles with mass spectral data. Results and Discussion Figures 1-3 show mass spectra of particles acquired from the tested combination DPI formulations Seretide® Diskus® and Foster® NEXThaler® acquired with SPAMS. Lactose and MgSt are not readily ionized at a wavelength of 248 nm using the SPAMS technique, therefore will not give a mass spectrum. However, it was found that if MgSt is co-associated with certain APIs, a large peak at M/Z=+24 (for Mg+) will be detected in the mass spectral data (Figure 2 and Figure 3).

Figure 1: Mass spectrum of co-associated particles of FP and SX from Seretide® Diskus®.

Figure 2: Mass spectrum of a single BDP particle from Foster® NEXThaler®.

Figure 3: Mass spectrum of co-associated particles of BDP and FF from Foster® NEXThaler®.

Drug Delivery to the Lungs, Volume 29, 2018 - Bradley Morrical et al. Additionally, it can be seen in Figures 1-3 that the APIs in both formulations were readily identified, which allowed for correct classification of particles as mono API particles, co-associated particles or mono or combination particles that contained MgSt (for the Foster®NEXThaler®). Interestingly, the presence of MgSt was identified clearly in the mass spectrum of single API particles of BDP (Figure 2) but not with the single API particles of FF (not shown) where the large Mg+-ion signal was not present. This finding suggests that an API treatment step may have been performed for BDP, perhaps to enhance the ability of these particles to detach from the lactose carrier and be less cohesive in combination with the FF particles. Even though both products are established commercial products, the pharmaceutical performance was markedly different with Foster® NEXThaler®, which exhibited a higher fraction of particles < 2µm as shown in Figure 4. Such a relatively high number of ultra-fine particles have been assigned to the presence of the breath-actuated mechanism (BAM) in Foster® NEXThaler® [11]. Farkas et al. recently reported that the BAM associated with this DPI leads to a reduction of emission of larger particles (lower upper airway deposited doses) and an increase in the available therapeutic fine particle fraction, resulting in a significantly higher lung dose [11]. The BAM appears to delay the emission of the drug until the inhalation flow rate of the patient is sufficiently high to detach the drug particles from their carriers. However, the generation of such a high FPF would also indicate that the inter-particulate interactions are such that they allow a very high level of detachment. It has also been reported that the BAM is capable of providing a reproducible and similar emitted dose and FPF of FF and BDP when activated using inhalation profiles of asthmatic patients[11]. The SPAMS APSD and chemical composition of particles in the aerosolized DPI doses results for Seretide® Diskus® and Foster® NEXThaler® are also presented in Figure 4. It is clearly evident that there were significant differences between the two tested products. In contrast to Seretide® Diskus® where there was a high number of co-associated particles (37%) emitted (Table 1) there appeared to be a negligible number of co-associated API particles (<1%) in the aerosol from the Foster® NEXThaler® DPI, with the vast majority of the particles present either as single API particles (FF) or in co-association with MgSt in case of BDP (Table 1 and Figure 4). FP

Co-associated particles (FP+SX)

BDP

800

400

600

300

Number of particles

400

200

Co-associated particles (BDP+FF)

200

100

0.3 0.7 1.1 1.5 1.9 2.3 2.7 3.1 3.5 3.9 4.3 4.7 5.1 5.5 5.9 6.3 6.7 Aerodynamic diameter [µm]

0.3 0.7 1.1 1.5 1.9 2.3 2.7 3.1 3.5 3.9 4.3 4.7 5.1 5.5 5.9 6.3 6.7 Aerodynamic diameter [μm]

Figure 4: A) APSD Seretide® Diskus® DPI (250/50 mcg) obtained by SPAMS (n=3 DPI actuations; error bars represent one standard deviation). The plot shows the distribution of number of particle co-associations (FP+SX), pure FP and pure SX. B) APSD of Foster® NEXThaler® (100/6 mcg) obtained by SPAMS (n=3 actuations; error bars represent one standard deviation). The plot shows the distribution of number of particle co-associations (BDP+FF), pure BDP and pure FF. Table 1: Percentage of individual particles measured in the DPI products (10’240 particles per run). [%] FP SX FP-SX co-associated

Seretide® Diskus®

[%] BDP FF BDP-FF co-associated

51.3 11.8 36.9

Foster ® NEXThaler® 92.6 7.0 0.4

It has been reported that the formulation in Foster® NEXThaler® comprises a coarse lactose (212–355 μm) blended with a micronized lactose/MgSt mixture [10, 12]. Even though quantification of the relative Mg-content is not possible, it was clear from the present study that MgSt is in some way ‘associated’ with BDP, but apparently not with FF in the aerosol emitted from the Foster® NEXThaler® DPI. However, it could not be ascertained whether the MgSt had simply ‘transferred’ to the BDP particles during actuation, storage or processing. It has also been reported that if any additional MgSt is added in any other part of manufacturing to either the carrier or API itself, such as in co-milling or mechanofusion of API and MgSt, such processing also leads to improved fine particle performance [13, 14]. Finally yet importantly, it is well known that the device itself can play an important role in aerosolization and de-agglomeration mechanisms of the product through differences in resistance, geometry, turbulence and collisions within the device [16].

Drug Delivery to the Lungs, Volume 29, 2018 - Probing the Aerodynamic Particle Size Distribution of Dry Powder Inhaler Combination Products Foster® NEXThaler® and Seretide® Diskus® using Single Particle Aerosol Mass Spectrometry (SPAMS) Conclusions These studies suggest that the SPAMS technique may be used to investigate the nature of API particulates and provide feedback control for the level of particle co-association (API-API or API-excipient). SPAMS was able to identify the presence of different API assemblies in Seretide® Diskus® and Foster® NEXThaler®, perhaps reflecting the use of different excipient platforms. Such a tool may not only be useful in early-phase development of inhaled products but also for the reverse engineering of generic/orphan products. A SPAMS apparatus with separate desorption and ionization lasers firing at a different wavelengths could potentially detect co-associations not only between APIs, but also between API and excipients (lactose or force control agents) [15]. This makes the SPAMS technique an attractive additional approach in the field of pharmaceutical aerosol characterization and is complementary to the current standard of cascade impactor/HPLC-analysis for the assessment of DPI aerosol aerodynamic particle size distribution. Acknowledgements This work was funded by Novartis Pharma AG’s internal research funds. 1. Draft Guidance for metered dose inhaler (MDI) and dry powder inhaler (DPI) drug products. Chemistry, manufacturing, and controls documentation. [http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm070573.pdf] 2. de Boer AH, Gjaltema D, Hagedoorn P, Frijlink HW: Can ‘extrafine’ dry powder aerosols improve lung deposition? European Journal of Pharmaceutics and Biopharmaceutics 2015, 96:143-151. 3. Usmani OS, Biddiscombe MF, Barnes PJ: Regional lung deposition and bronchodilator response as a function of beta2-agonist particle size. Am J Respir Crit Care Med 2005, 172:1497-1504. 4. Hamid Q, Song Y, Kotsimbos TC, Minshall E, Bai TR, Hegele RG, Hogg JC: Inflammation of small airways in asthma. J Allergy Clin Immunol 1997, 100:44-51. 5. Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, Cherniack RM, Rogers RM, Sciurba FC, Coxson HO, Pare PD: The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 2004, 350:2645-2653. 6. De Backer W, Devolder A, Poli G, Acerbi D, Monno R, Herpich C, Sommerer K, Meyer T, Mariotti F: Lung deposition of BDP/formoterol HFA pMDI in healthy volunteers, asthmatic, and COPD patients. J Aerosol Med Pulm Drug Deliv 2010, 23:137-148. 7. Morrical BD, Balaxi M, Fergenson D: The on-line analysis of aerosol-delivered pharmaceuticals via single particle aerosol mass spectrometry. International Journal of Pharmaceutics 2015, 489:11-17. 8. Rohrschneider M, Bhagwat S, Krampe R, Michler V, Breitkreutz J, Hochhaus G: Evaluation of the Transwell System for Characterization of Dissolution Behavior of Inhalation Drugs: Effects of Membrane and Surfactant. Molecular Pharmaceutics 2015, 12:2618-2624. 9. Jetzer MW, Morrical BD, Fergenson DP, Imanidis G: Particle interactions of fluticasone propionate and salmeterol xinafoate detected with single particle aerosol mass spectrometry (SPAMS). Int J Pharm 2017, 532:218-228. 10. Buttini F, Brambilla G, Copelli D, Sisti V, Balducci AG, Bettini R, Pasquali I: Effect of Flow Rate on In Vitro Aerodynamic Performance of NEXThaler((R)) in Comparison with Diskus((R)) and Turbohaler((R)) Dry Powder Inhalers. J Aerosol Med Pulm Drug Deliv 2016, 29:167-178. 11. Farkas Á, Lewis D, Church T, Tweedie A, Mason F, Haddrell AE, Reid JP, Horváth A, Balásházy I: Experimental and computational study of the effect of breath-actuated mechanism built in the NEXThaler® dry powder inhaler. International Journal of Pharmaceutics. 12. JN. S, DA. VM, R. G, G. B, R. M, L. F: Pharmaceutical formulations for dry powder inhalers in the form of hard-pellets. EP1274406 B1 2006. 13. Begat P, Price R, Harris H, Morton DA, Staniforth JN: The influence of force control agents on the cohesive-adhesive balance in dry powder inhaler formulations. KONA Powder and Particle Journal 2005, 23:109121. 14. Lau M, Young PM, Traini D: Co-milled API-lactose systems for inhalation therapy: impact of magnesium stearate on physico-chemical stability and aerosolization performance. Drug Development and Industrial Pharmacy 2017, 43:980-988. 15. Morrical BD, Fergenson DP, Prather KA: Coupling two-step laser desorption/ ionization with aerosol time-of-flight mass spectrometry for the analysis of individual organic particles. Journal of the American Society for Mass Spectrometry 1998, 9:1068-1073. 16. Mendes, PJ, Pinto JF, Sousa JMM: Non-dimensional functional relationship for the fine particle fraction produced by dry powder inhalers. Journal of Aerosol Science 2007, 38: References Begat, P., Price, R., Harris, H., Morton, D., & Staniforth, J. (2005). The influence of force control agents on the cohesive-adhesive balance in dry powder inhlater formulations. KONA Powder and Particle Journal, 23, 109-121.

Drug Delivery to the Lungs, Volume 29, 2018 - Bradley Morrical et al. Buttini , F., Brambilla, G., Copelli, D., Sisti, V., Balducci, A., Bettini, R., & Pasquali, I. (2016). Effect of Flow Rate on in-vitro Aerodynamic Performance of NEXThaler((R)) in Comparison with Diskus ((R)) and Turbohaler((R)) Dry Powder Inhalers. Journl of Aerosol Medicine and Pulmonary Drug Delivery, 29, 167-178. De Backer, W., Devolder, A., Poli, G., Acerbi, D., Monno, R., Herpich, C., . . . Mariotti, F. (2010). Lung deposition of BDP/fomoterol HFA pMDI in healthy volunteers, asthmatic and COPD patients. Journal of Aerosol Medicine adn Pulmonary Drug Delivery, 23, 137-148. de Boer, A., Gjaltema, D., Hagedoom, P., & Frijlink, H. (2015). Can "extrafine" dry powder aersols improve lung deposition? European Journal of Pharmaceutics and Biopharmaceutics, 96, 143-151. Farkas, A., Lewis, D., Church, T., Tweedie, A., Mason, F., Haddrell, A., . . . Balashazy, I. (n.d.). Experimental and computational study of the effect of breath-actuated mechanism built in the NEXThaler(R) dry powder inhaler. International Journal of Pharmaceutics. FDA.

(n.d.). FDA. Retrieved from FDA: www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm07573.pdf

Hamid , Q., Song, Y., Kotsimbos, T., Minshal, E., Bai, T., Hegele, R., & Hogg, J. (1997). Inflammation of small airways in asthma. Journal of Allergy Clinical Immunology, 100, 44-51. Hogg, J., Chu, F., Utokaparch, S., Woods, R., Elliott, W., Buzatu, L., . . . Pare, P. (2004). The nature of small-airway obstruction in chronic obstructive pulmonary disease. New England Journal of Medicine, 350, 2645-2653. Jetzer, M., Morrical, B., Fergenson, D., & Imanidis, G. (2017). Particle interactions of fluticasone propionate and salmeterol xinafoate detected with single particle aerosol mass spectrometry (SPAMS). International Journal of Pharmaceutics, 532, 218-228. JN, S., DA, VM, R, G., G, B., R, M., & L, F. (2006). Pharmaceutical formulatoins for dry powder inhalers in the form of hard-pellets. EP 1274406, B1. Lau, M., Young, P., & Traini, D. (2017). Co-milled API-lactose systems for inhalation therapy: impact of magnesium stearate on physico-chemical stability and aerosolization performance. Drug Development and Industrial Pharmacy, 43, 980-988. Mendes, P., Pinto, J., & Sousa, J. (2007). Non-dimensional functional relationship for the fine particle fraction produced by dry powder inhalers. Journal of Aerosol Science, 38, 612-624. Morrical, B., Balaxi, M., & Fergenson, D. (2015). The on-line analysis of aerosol-delivered pharmaceuticals via single particle aerosol mass spectrometry. International Journal of Pharmaceutics, 489, 11-17. Morrical, B., Fergenson, D., & Prather, K. (1998). Coupling two-step laser desorption/ionizatoin with aerosol timeof-flight mass spectrometry for the analysis of individual organic particles. Journal of the American Society for Mass Spectrometry, 9, 1068-1073. Rohrschneider, M., Bhagwat, S., Krampe, R., Michler, V., Breitkreutz, J., & Hochhaus, G. (2015). Evaluation of the Transwell System for Characterization of Dissolution Behavior of Inhalation Drugs: Effects of Membrane and Surfactant. Molecular Pharmaceutics, 12, 2618-2624. Usmani, O., Biddiscombe, M., & Barnes, P. (2005). Regional lung deposition and bronchodilator response as a function of beta-2-agonist particle size. American Journaol of Respiratory Critical Care Medicine, 172, 1497-1504.

Drug Delivery to the Lungs, Volume 29, 2018 - Harald Renz et al. Antisense Technologies â&#x20AC;&#x201C; An Emerging Field for the Development of New Therapeutic Molecules: AntiGATA3 DNAzyme as a Prototypic Example Harald Renz Institute of Laboratory Medicine, Philipps University Marburg, Germany

Transcription factors play a central role in the regulation of cellular function and activities. In this context GATA3 has been recognized as the master-transcription factor in the regulation of Th2 development and Th2 functions. GATA3 is expressed not only in CD4-Th2 cells, but also in ILC2 cells, eosinophils mast cells, and basophils, among others. We have developed an antisense molecule belonging to the DNAzyme family which specifically and selectively targets GATA3 mRNA. DNAzymes are characterized by target-specific binding domains and an intrinsic catalytic nucleic acid sequence. Following extensive preclinical analysis, toxicology studies and three clinical safety studies in healthy and asthmatic individuals, a phase IIa proof-of-concept clinical trial was performed in mild asthmatic patients. Treatment was carried out for 28 days by daily inhalation of a single dose of 10 mg SB010. Allergen provocations were performed before and after the treatment cause. The primary endpoint of this study, significant reduction of the late phase asthmatic response, was reached, and highly surprisingly, also significant effects on the early phase allergic response was measured. The degree of improvement could be furthermore related to the level of blood eosinophils before treatment. Based on these encouraging results, SB010 treatment has been recently clinically tested in patients with atopic dermatitis (NCT02079688), eosinophilic COPD (DRKS00006087), and ulcerative colitis (NCT02129439), all of these conditions with predominant type 2 inflammation. Topical administration of SB010 on lesional skin for 14 days significantly improved skin barrier function. 28 days of inhalation of SB010 in eCOPD patients significantly reduced sputum eosinophils and shifted systemic IL-5 and IFN- production; following encouraging results in animal models we have recently completed a clinical trial in UC patients with highly significant effects on the total Mayo score as the primary endpoint. In addition, target regulation was observed in local tissues. These data indicate for the first time that the transcription factor GATA3 represents potential targets for topical pharmatherapy in asthma, eosinophilic COPD, atopic dermatitis, and ulcerative colitis. However, additional clinical trials are warranted to firmly establish this new treatment modality.

Drug Delivery to the Lungs, Volume 29, 2018 - Barzin Gavtash et al. Development of a theoretical model to predict pMDI spray force, using alternative propellant systems Barzin Gavtash1, Andy Cooper2, Sarah Dexter2, Chris Blatchford2 & Henk Versteeg1 1Loughborough

University, Epinal Way, Loughborough, LE11 2TL, United Kingdom 3M United Kingdom PLC, Charnwood Campus, 10 Bakewell Road, Loughborough, LE11 5RB, United Kingdom

Summary Continued success in the treatment of asthma and COPD requires new pMDI propellants for delivering aerosols with good patient comfort and acceptable levels of oral cavity deposition. The purpose of this work is to develop a theoretical model capable of predicting pMDI spray force as a function of metering valve geometric parameters and different propellant systems: HFA134a, HFA227ea and HFA152a. Such theoretical tool can be used in combination with lab-based measurements for device characterisation and potentially to reduce the number of experimental trials. The outcome of the model is compared against measurements of plume force with Copley Scientific Spray Force Tester SFT 1000. Results suggest that the size of the spray orifice has a significant direct effect on the spray force. We have also observed HFA134a and HFA152a generates similar magnitude of spray force and velocity where HFA227ea generates the lowest velocity and force values. These findings could potentially mean HFA152a sprays are expected to show similar levels of mouth-throat deposition to HFA134a sprays rather than HFA227ea sprays. Key Message Predicted and measured values suggest that spray orifice size had a significant effect on metered dose inhaler spray force. It was also observed that HFA152a and HFA134a show similar spray force values where HFA227ea generates the lowest force values. 1.

Introduction

Kigali 2016 amendment of Montreal Protocol has delivered international agreement to phase out hydrofluorocarbons on the grounds of their high global warming potential. Replacements for pressurised metered dose inhaler (pMDI) propellants HFA134a and HFA227ea will need to be developed for inhalation therapies. Recently HFA152a has received attention as an alternative propellant to be used in pMDI [1, 3, 4] due to its much lower global warming potential (around 124) compared with HFA134a and HFA227ea (around 1400 and 3200 respectively). However, the feasibility of formulating pMDIs using HFA152a is still under rigorous investigation mainly from a safety and patient acceptability standpoint [1]. One important plume characteristic which influences patient acceptability is maximum spray force which also directly correlates with oropharyngeal deposition [1, 5, 6]. Therefore, the aim of this paper is to develop a theoretical model capable of predicting pMDI spray force as a function of metering valve geometric parameters and different propellant systems: HFA134a, HFA227ea and HFA152a. In past work [2, 9], results with two existing propellants have been validated. The present work includes mathematical predictions and experimental validation of spray force for proposed alternative propellant HFA152a with low global warming potential. Such theoretical tool can be also useful to minimise number of trials when it comes to pMDI device design/characterisation. We also compare model outcomes for different spray orifice sizes and propellants against measured values to increase confidence of the modelling activity. 2.

Mathematical model

Prediction of pMDI spray force requires calculation of two-phase propellant mass flow rate and velocity inside twinorifice system of pMDI [2]. Such model was originally developed by Fletcher [7] and Clark [8] and has been recently improved, validated and comprehensively discussed by Gavtash et al. [2, 9, 10]. Here we briefly discuss the key components of the model and underlying equations. Therefore, the reader is advised to refer to the references for further details. Our internal flow model is quasi-steady and assumes the propellant expansion is adiabatic and involves thermodynamic and thermal equilibrium between the propellant liquid and vapour phase, inside the metering and expansion chambers. The two-phase propellant flow through valve and spray orifices in a pMDI is evaluated with the homogeneous frozen model (HFM), which assumes that no evaporation takes place along the flow path through the orifices and the flow is choked for almost 90% of the actuation time. The spray force, đ??šđ??š, can be calculated using one-dimension steady momentum equation: đ??šđ??š = đ??şđ??şđ??şđ??şđ?&#x2018;&#x2030;đ?&#x2018;&#x2030;đ?&#x2018;&#x203A;đ?&#x2018;&#x203A;đ?&#x2018;&#x203A;đ?&#x2018;&#x203A;

2 /4 (đ?&#x2018;&#x161;đ?&#x2018;&#x161;2 ) is the spray orifice cross section area and đ??şđ??ş = đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;/đ??´đ??´ (đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC;đ?&#x2018;&#x161;đ?&#x2018;&#x161;2 /đ?&#x2018; đ?&#x2018; ) is the two-phase propellant Where đ??´đ??´ = đ?&#x153;&#x2039;đ?&#x153;&#x2039;đ??ˇđ??ˇđ?&#x2018; đ?&#x2018; đ?&#x2018; đ?&#x2018; mass flux through spray orifice which can be calculated using the following expression [2]:

Drug Delivery to the Lungs, Volume 29, 2018 - Development of a theoretical model to predict pMDI spray force, using alternative propellant systems

đ??şđ??ş = đ??śđ??śđ?&#x2018;&#x2018;đ?&#x2018;&#x2018;

(

đ?&#x2018;?đ?&#x2018;?0 đ?&#x2018;Ľđ?&#x2018;Ľ0 đ?&#x2018;Łđ?&#x2018;Łđ?&#x2018;&#x201D;đ?&#x2018;&#x201D;,0

2 2đ?&#x153;&#x201A;đ?&#x153;&#x201A;đ?&#x203A;žđ?&#x203A;ž

1 â&#x2C6;&#x2019; đ?&#x2018;Ľđ?&#x2018;Ľ0 đ?&#x2018;Łđ?&#x2018;Łđ?&#x2018;&#x2122;đ?&#x2018;&#x2122;,0 đ?&#x203A;žđ?&#x203A;ž1 [( )( ) đ?&#x153;&#x201A;đ?&#x153;&#x201A; + 1] đ?&#x2018;Ľđ?&#x2018;Ľ0 đ?&#x2018;Łđ?&#x2018;Łđ?&#x2018;&#x201D;đ?&#x2018;&#x201D;,0

đ?&#x203A;žđ?&#x203A;žâ&#x2C6;&#x2019;1 1 â&#x2C6;&#x2019; đ?&#x2018;Ľđ?&#x2018;Ľ0 đ?&#x2018;Łđ?&#x2018;Łđ?&#x2018;&#x2122;đ?&#x2018;&#x2122;,0 đ?&#x203A;žđ?&#x203A;ž [( )( ) (1 â&#x2C6;&#x2019; đ?&#x153;&#x201A;đ?&#x153;&#x201A;) + (1 â&#x2C6;&#x2019; đ?&#x153;&#x201A;đ?&#x153;&#x201A; đ?&#x203A;žđ?&#x203A;ž )] đ?&#x2018;Ľđ?&#x2018;Ľ0 đ?&#x2018;Łđ?&#x2018;Łđ?&#x2018;&#x201D;đ?&#x2018;&#x201D;,0 đ?&#x203A;žđ?&#x203A;ž â&#x2C6;&#x2019; 1

1 2

)

In equation 2, subscript 0 denotes the flow condition in the upstream reservoir (expansion chamber) and subscripts đ?&#x2018;&#x201D;đ?&#x2018;&#x201D; and đ?&#x2018;&#x2122;đ?&#x2018;&#x2122; denote the gas phase and liquid phase thermodynamic properties, respectively. đ?&#x203A;žđ?&#x203A;ž (â&#x2C6;&#x2019;) is the propellant heat capacity ratio, đ?&#x2018;Łđ?&#x2018;Ł (đ?&#x2018;&#x161;đ?&#x2018;&#x161;3 /đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC;) is the specific volume and đ?&#x2018;?đ?&#x2018;? (đ?&#x2018;&#x192;đ?&#x2018;&#x192;đ?&#x2018;&#x192;đ?&#x2018;&#x192;) is the saturated vapour pressure. đ?&#x2018;Ľđ?&#x2018;Ľ (â&#x2C6;&#x2019;) denotes the twophase flow quality and đ??śđ??śđ?&#x2018;&#x2018;đ?&#x2018;&#x2018; (â&#x2C6;&#x2019;) is the discharge coefficient. Finally, đ?&#x153;&#x201A;đ?&#x153;&#x201A; = đ?&#x2018;?đ?&#x2018;?đ?&#x2018; đ?&#x2018; đ?&#x2018; đ?&#x2018; /đ?&#x2018;?đ?&#x2018;?0 (â&#x2C6;&#x2019;) , denoting the ratio of spray orifice pressure to upstream reservoir pressure. Due to the choked nature of the emitted two-phase flow from spray orifice, đ?&#x2018;?đ?&#x2018;?đ?&#x2018; đ?&#x2018; đ?&#x2018; đ?&#x2018; is generally higher than the ambient downstream pressure. This pressure further accelerates the flow in nearorifice region. Corresponding velocity in near-orifice region can be calculated using equation 3, which is onedimensional axial momentum balance in a diverging control volume close to the spray [2]: đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;đ?&#x2018;&#x203A;đ?&#x2018;&#x203A;đ?&#x2018;&#x203A;đ?&#x2018;&#x203A; = đ??şđ??şđ?&#x2018;Łđ?&#x2018;ŁĚ&#x2026; +

đ?&#x2018;?đ?&#x2018;?đ?&#x2018; đ?&#x2018; đ?&#x2018; đ?&#x2018; â&#x2C6;&#x2019; đ?&#x2018;?đ?&#x2018;?đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D; đ??şđ??ş

Where đ?&#x2018;?đ?&#x2018;?đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x17D; is the ambient pressure and đ?&#x2018;Łđ?&#x2018;ŁĚ&#x2026; is the average two-phase flow specific volume at the spray orifice exit. Assuming isentropic expansion of the propellant vapour phase inside the spray orifice, đ?&#x2018;Łđ?&#x2018;ŁĚ&#x2026; reads as follows [2]: đ?&#x2018;Łđ?&#x2018;ŁĚ&#x2026; = đ?&#x153;&#x201A;đ?&#x153;&#x201A;

â&#x2C6;&#x2019;

1 đ?&#x203A;žđ?&#x203A;ž đ?&#x2018;Ľđ?&#x2018;Ľ đ?&#x2018;Łđ?&#x2018;Ł 0 đ?&#x2018;&#x201D;đ?&#x2018;&#x201D;,0

1 1 â&#x2C6;&#x2019; đ?&#x2018;Ľđ?&#x2018;Ľ0 đ?&#x2018;Łđ?&#x2018;Łđ?&#x2018;&#x2122;đ?&#x2018;&#x2122;,0 [( )( ) đ?&#x153;&#x201A;đ?&#x153;&#x201A; đ?&#x203A;žđ?&#x203A;ž + 1] đ?&#x2018;Ľđ?&#x2018;Ľ0 đ?&#x2018;Łđ?&#x2018;Łđ?&#x2018;&#x201D;đ?&#x2018;&#x201D;,0

Experimental set-up

Measurements are performed with a commercial spray force tester instrument (SFT 1000, Copley Scientific), as shown in Figure 1. The sledge which holds the pMDI is positioned such that the mouthpiece edge is 50mm from the load cell. Nine configurations of placebo inhalers, configured as per Table 1, are assessed in triplicate. The load cell records the maximum force observed during the spray event. Parameter

Value

Parameter

Value

Metering valve volume (Âľl)

Discharge coefficient (-)

0.6[2]

Expansion chamber volume (Âľl)

Ambient temperature (K)

295

Valve orifice diameter (mm) Spray orifice diameter (mm)

0.6[2] 0.3

0.4

Ambient pressure (Pa) 0.5

Propellant (HFA)

Table 1 â&#x20AC;&#x201C; Experimental and modelling parameters

Figure 1 â&#x20AC;&#x201C; Experimental setup (SFT 1000)

101300 Pa 134a

227ea

152a

Drug Delivery to the Lungs, Volume 29, 2018 - Barzin Gavtash et al. 4. Result and discussion 4.1 Spray velocity and mass flowrate Figure 2 shows the mass flow rate through the 0.3 mm spray orifice, predicted for three propellants as a function of actuation time. Initially, propellant starts to flow from the metering chamber to the expansion chamber of the pMDI, causing a rapid rise of the pressure in the expansion chamber and the spray mass flow rate. During this phase, the mass flow rate entering the expansion chamber is greater than the mass flow rate exiting this space. Hence, propellant evaporation in the expansion chamber becomes inhibited, which leads to a greater fraction of liquid in the discharged mixture. As a consequence, the average density of the emitted mixture increases and the mass flow rate through spray orifice rises up to a maximum value. After the maximum, the mass flow rate leaving the expansion chamber through the spray orifice becomes larger than the mass flow rate entering from the metering chamber. Evaporation increases, and the mass flow rate decreases continuously, until the discharge concludes. It can be observed that the peak mass flow rates are approximately similar for all three propellants considered in this study. It should be noted that similar temporal trends are observed for 0.4 mm and 0.5 mm spray orifice diameter cases with different mass flow rate magnitude and spray duration. Figure 3 shows the predicted near-orifice spray velocity of the 0.3 mm spray orifice. Results suggest that the velocity almost instantaneously rises which corresponds to the discharge of high quality vapour/liquid propellant mixture through the spray orifice. After this point a subsequent fall in the velocity is observed at around 10 ms after the actuation time. This time corresponds to when the peak mass flow rate occurs. From this minimum point, velocity almost linearly increases as a result of expansion chamber emptying, until it reaches a second maximum. Finally, the mass in the chambers depletes completely and the velocity sharply decays until it reaches zero.

Figure 2 â&#x20AC;&#x201C; Time dependent average mass flow rate of two-phase propellant flow issued from the spray orifice (Dso=0.3 mm)

Figure 3 â&#x20AC;&#x201C; Time dependent spray velocity at the exit plane of spray orifice (Dso=0.3 mm)

Comparison of the traces suggests that HFA152a shows the highest spray velocity whereas HFA227ea has the lowest velocity amongst the three propellants. Such trend can be best described by investigating the propellants vapour pressure as the main driving force of the flow, and density as the main opposing parameter to acceleration. Over a typical range of pMDI operating temperature of around -20 to 20 â °C in this study, saturated vapour pressure of HFA152a and HFA134a are very similar in terms of magnitude (less than 8% difference), with HFA152a being slightly lower. However, the liquid density of HFA152a is around 25% less than the one of HFA134a. Therefore, propellant pressure is used to accelerate a lighter mass which overall results in higher velocity of HFA152a compared with HFA134a. 4.2 Spray force Figure 4 shows the temporal behaviour of spray force for 0.3 mm orifice diameter. It can be observed that the force initially rises to a maximum and then more gradually decreases until the spray event finishes. This trend in time is very similar to the previous temporal force measurements [5, 8]. The results also suggest that the maximum force is experienced during the filling stage of the expansion chamber where liquid-rich two-phase propellant flow exits the spray orifice. Figure 5 shows comparison of predicted and measured spray force for different propellants and different spray orifice diameters. The predicted force values are averaged over the duration which 95% of pMDI propellant mass is emitted. It can be observed that the trend in variation of spray force with respect to propellant and orifice diameter are very well captured by our model prediction. Data shows that for any propellant, increase in spray orifice diameter results in a significant increase in spray force. This is mainly due to release of more mass per unit time as orifice diameter increases. Amongst the three propellants considered in this study, for any orifice diameter, HFA227ea spray produces the smallest force. The generated force by HFA134a and HFA152a propellants appears to be very similar in magnitude with HFA134a being slightly higher. When compared with measured values, the model outcomes are predicted within the correct order of magnitude.

Drug Delivery to the Lungs, Volume 29, 2018 - Development of a theoretical model to predict pMDI spray force, using alternative propellant systems

Figure 4 – Time dependent spray force at the exit plane of spray orifice (Dso = 0.3 mm)

Figure 5 – Comparison of predicted and measured plume force for different propellant systems and orifice sizes

Conclusion In this paper we have reported the findings of a theoretical model, capable of predicting spray force as function of metering valve geometric parameters and various propellant systems. Such theoretical tool can be used in combination with lab-based measurements for device characterisation and potentially to reduce the number of experimental trials. Amongst the studied propellants, our theoretical predictions suggest that the spray velocity is lowest for HFA227ea whereas it is relatively similar for HFA134a and HFA152a, being slightly higher for HFA152a. Our theoretical and measured force values demonstrate that orifice size is the most influential factor on, and directly correlates with spray force. Data also suggest that HFA227ea has the lowest force where HFA134a and HFA152a exhibit similar force values for any particular orifice size. Assuming there is a direct correlation between spray force and oropharyngeal deposition [1, 5, 6], these findings could potentially mean HFA152a sprays are expected to show similar levels of mouth-throat deposition to HFA134a sprays. However further investigations are required to confirm such hypothesis. Acknowledgement The authors would like to acknowledge Copley Scientific for use of their photograph of their SFT 1000 instrument. References [1] [2]

[3] [4] [5] [6]

[7] [8] [9]

[10]

Myrdal, P.B., Sheth, P. and Stein, S.W., 2014. Advances in metered dose inhaler technology: formulation development. AAPS PharmSciTech, 15(2), pp.434-455. Gavtash, B., Versteeg, H.K., Hargrave, G., Myatt, B., Lewis, D., Church, T. and Brambilla, G., 2017. Transient flashing propellant flow models to predict internal flow characteristics, spray velocity, and aerosol droplet size of a pMDI. Aerosol Science and Technology, 51(5), pp.564-575. Corr S, Noakes TJ, inventors; Mexichem Amanco Holding S.A DE C.V, assignee. Compositions comprising salbutamol sulfate. World Intellectual Property Organization patent WO 2013/054135. 2013 Apr 18. Corr S, Noakes TJ, inventors; Mexichem Amanco Holding S.A DE C.V, assignee. Pharmaceutical compositions. World Intellectual Property Organization patentWO 2012/156711. 2012 Nov 22. Gabrio, B.J., Stein, S.W. and Velasquez, D.J., 1999. A new method to evaluate plume characteristics of hydrofluoroalkane and chlorofluorocarbon metered dose inhalers. International journal of pharmaceutics, 186(1), pp.3-12. McCabe, J.C., Koppenhagen, F., Blair, J. and Zeng, X.M., 2012. ProAir® HFA delivers warmer, lower-impact, longer-duration plumes containing higher fine particle dose than Ventolin® HFA. Journal of aerosol medicine and pulmonary drug delivery, 25(2), pp.104-109. Fletcher, G.E. PhD thesis, Loughborough University of Technology, Loughborough, UK, 1975. Clark, A.R. PhD thesis, Loughborough University of Technology, Loughborough, UK, 1991. Gavtash, B., Versteeg, H.K., Hargrave, G., Myatt, B., Lewis, D., Church, T. and Brambilla, G., 2017. Transient aerodynamic atomization model to predict aerosol droplet size of pressurized metered dose inhalers (pMDI). Aerosol Science and Technology, 51(8), pp.998-1008. Gavtash, B., Versteeg, H.K., Hargrave, G., Myatt, B., Lewis, D., Church, T. and Brambilla, G., 2018. A model of transient internal flow and atomization of propellant/ethanol mixtures in pressurized metered dose inhalers (pMDI). Aerosol Science and Technology, 52(5), pp.494-504.

Drug Delivery to the Lungs, Volume 29, 2018 - Michael Nairn et al. In vitro testing of the new Space Chamber Slim with salbutamol sulfate, fluticasone propionate, and ipratropium bromide pressurized metered dose inhalers Michael Nairn1, Andrew Lorbeer1, Kurt Nikander2, Scott Courtney1 1

Medical Developments International Limited, 4 Caribbean Drive, Scoresby, 3179, Australia 2InDevCo AB, Handbollsvagen 1B, Nykoping, 61164, Sweden

Summary In vitro aerodynamic particle size distribution testing was performed on a new disposable cardboard spacer – the Space Chamber Slim (SCS; Medical Developments International Limited, Australia) – along with a similar marketed valved holding chamber – the LiteAire (LA; Thayer Medical Corporation, USA). The devices have similar holding chamber volumes (SCS ~200 mL; LA 184 mL), but their designs differ in terms of internal geometry and vent/valve organisation. Therefore, it is important to quantify how these variations affected the drug delivery characteristics of the devices. Pressurized metered dose inhalers (pMDIs) were used to deliver three common aerosolised medications through the devices into a cascade impactor at constant paediatric (for salbutamol sulfate) and adult flow rates (for salbutamol sulfate, fluticasone propionate, and ipratropium bromide). Both devices substantially reduced the total mass of drug delivered to the impactor, while increasing the proportion of fine (≤4.7 µm) particles within the delivered aerosols by at least two-fold (e.g. 37 % with pMDI alone versus 78 % with the SCS for salbutamol at 30 L/min). The SCS and LA devices performed similarly (within 15 % of one another at the 90 % confidence interval) with regard to their fine particle fractions. However, the fine particle dose was slightly (but significantly; P<0.05) higher with the Space Chamber Slim in all instances. The results indicate that, at the conditions tested, the Space Chamber Slim is highly effective in reducing the delivery of undesirable coarse particles compared with pMDIs alone, while either not affecting or increasing the delivery of fine particles. Key Message There is an unmet need for high quality, compact, and disposable spacers for use in ambulances, hospitals, and first aid kits. Here we assess the in vitro delivery characteristics of a new spacer across three common inhalation drugs, and draw comparisons against the pMDIs alone and a comparable marketed product. Introduction Spacers and valved holding chambers (VHCs) have been developed to assist patients in using pressurized metered dose inhalers (pMDIs). Their benefits generally include the improved targeting of aerosol to the lower respiratory tract, and a reduction in the amount of aerosol deposited in the oropharyngeal region, thus mitigating the potential for undesirable side-effects [1,2]. There is a need for disposable cardboard devices as an alternative to conventional plastic spacers/VHCs and nebulisers typically used in the hospital (emergency room, pulmonary function reversibility tests) and by ambulance services. Cardboard spacers can be compact, economical, and eco-friendly, and are non-electrostatic, so they do not need ‘priming’ and may have less variability in the amount of aerosol delivered (unlike conventional electrostatic plastic spacers) [2,3]. A new disposable cardboard spacer – the Space Chamber Slim (SCS; Medical Developments International Limited, Australia) – in the form of a flat-stored pop-up container, has been developed for short-term use by adults and children who do not require a facemask. Due to its size and design, it would also fit into first aid/emergency kits. The SCS features two internal compartments, including a holding chamber (~200 mL) with an internal baffle and inhalation vent, and a delivery chamber (~60 mL) with a one-way exhalation valve and a mouthpiece (Figure 1). The purpose of this study was to compare the in vitro aerosol delivery characteristics of the SCS with the pMDIs when used alone, and with a similar marketed device: the LiteAire VHC (LA; Thayer Medical Corporation, USA).

Figure 1. The design of the Space Chamber Slim is shown, with (1) the actuation of aerosol into the spacer and its subsequent distribution within the holding chamber, and (2) the delivery of aerosol to the patient during inhalation.

Drug Delivery to the Lungs, Volume 29, 2018 - In vitro testing of the new Space Chamber Slim with salbutamol sulfate, fluticasone propionate, and ipratropium bromide pressurized metered dose inhalers Experimental Methods A Next Generation Pharmaceutical Impactor (NGI; Copley Scientific, UK) and high-performance liquid chromatography system (HPLC; Agilent, USA) were used to size aerosol particles as they were delivered from pMDIs through the test devices, as per the guidelines outlined within the CSA Standards [4]. The study included salbutamol sulfate (salbutamol; Ventolin, GlaxoSmithKline, Australia; 108 µg/actuation ex-actuator), fluticasone propionate (fluticasone; Flixotide, GlaxoSmithKline, Australia; 125 µg/actuation) and ipratropium bromide (ipratropium; Atrovent, Boehringer Ingelheim, Australia; 17 µg/actuation ex-actuator) pMDIs. Each test consisted of a set of three pMDIs from the same manufacturing lot being actuated a specified number of times (twice for salbutamol and fluticasone, and six times for ipratropium) into the NGI through a test device taken from its original packaging and mounted to the induction port (or ‘throat’) of the NGI via a custom-made siliconecoated adaptor (Figure 2). The NGI was operated under a constant inhalation air flow rate (with no delay between pMDI actuation and induction of the aerosol into the NGI) of 15 L/min to simulate paediatric use (for salbutamol only) and 30 L/min to simulate adult use (for all three drugs), as recommended within the draft USP Chapter <1602> [5]. Three samples of each device were tested three times (n=9) for each drug/flow rate. Each set of pMDIs utilized during spacer/VHC testing was also tested three times in the absence of an accessory device, by actuating into the induction port of the NGI via a silicone adaptor tailored to fit the pMDI housing. Drug delivery data for the pMDIs alone was averaged over the total number of pMDI sets utilized for each drug and flow rate, which differed as a result of their lifespan and prior use. One set (n=3) was used for ipratropium at 30 L/min; two sets (n=6) for fluticasone at 30 L/min and salbutamol at 15 L/min; and three sets (n=9) for salbutamol at 30 L/min.

Figure 2. The induction port and adaptor of the NGI alone (left) and with the Space Chamber Slim in place (right).

After delivery from a pMDI/test device, the aerosol was drawn through the induction port, followed by the seven impaction stages and micro-orifice collector (MOC) of the NGI (with increasing velocity at each stage), and terminally through a glass fiber filter. Aerosol particles deposited within the various stages of the system (including within the spacer/VHC itself), depending largely on their aerodynamic particle size.

Inhalation airflow was ceased 20 s after the final actuation (as dictated by the CSA Standards [4]), after which drug residues were quantitatively desorbed from the impaction surfaces of the NGI and the glass fiber filter using 25 mL of HPLC-grade solvent (50 % aqueous methanol for salbutamol and ipratropium, and 100% methanol for fluticasone; Chem Supply, Australia). The mass of drug within each sample was then quantified by HPLC, following calibration of the system using reference standards (salbutamol hemisulfate and ipratropium bromide monohydrate from Sigma Aldrich, Australia; and fluticasone propionate from Chem Supply, Australia) of known concentrations. The drug depositing within the devices was not quantified, as the paperboard material was not suitable for desorption with solvents. As such, it was not possible to ascertain whether the overall drug recovery fell within the limits deemed acceptable by the CSA Standards [4] during device testing, though they did for the pMDIs alone.

Certified validated software (CITDAS, Copley Scientific) was used to generate aerosol delivery metrics, including the total dose delivered to the NGI (TDD), the fine particle dose delivered to the NGI (FPD) and the fine particle fraction (FPF; expressed as % of TDD) from the stage-wise drug mass data, with particles having an aerodynamic diameter of ≤4.7 µm being considered ‘fine’ [4]. Drug depositing within the glass fiber filter (which cannot be sized) was taken into account when calculating the TDD, but not the FPD or FPF. These metrics are presented as mean values ± 95 % confidence intervals (calculated according to the t-distribution, which takes sample size into account). An ANOVA with post-hoc Tukey HSD test was used to identify statistically significant differences between the mean values. The 90 % confidence intervals of ratios ([pMDI or LA]/SCS %) were used to assess the relative differences between the SCS and the other devices, and were calculated according to the method of Fieller [6]. The mass median aerodynamic diameters (MMAD) of the particles depositing within the impaction stages and MOC of the NGI were also generated by CITDAS, along with their geometric standard deviations (GSD) as a measure of spread.

Drug Delivery to the Lungs, Volume 29, 2018 - Michael Nairn et al. Results The in vitro test results presented in Figure 3 show that the TDD was substantially higher with the pMDI alone for all drugs and flow rates in comparison with the test devices. Meanwhile, it was significantly higher for the SCS compared with the LA for all drugs apart from ipratropium (in which case they were not significantly different). The FPF, on the other hand, was slightly higher for the LA compared with the SCS, but the difference only reached statistical significance (P<0.05) in the case of salbutamol at 30 L/min. At the 90% confidence interval, the FPF of the two devices fell within 15 % of one another in all cases. The FPD was significantly higher with the SCS compared with the pMDI alone and the LA in every instance apart from salbutamol at 30 L/min, in which case there was not a significant difference between the SCS and the pMDI alone. Meanwhile, as shown in Table 1, the MMAD of the particles depositing on the impaction stages and MOC of the NGI was only slightly reduced when using the test devices. Relative to the pMDIs alone, the MMAD was on average 11 % lower with the SCS (6 % lower with salbutamol at 30 L/min to 17 % with ipratropium) and 17 % lower with the LA (13 % with salbutamol at 15 L/min to 24 % with ipratropium). The GSD was also reduced in most cases by a similar factor.

Figure 3. The drug delivery characteristics (per actuation) of the pMDIs alone, and when paired with the test devices. Error bars denote 95 % confidence intervals (t-distribution) of the means. Bars within the same cluster that have different adjacent letters (a, b, or c) are significantly different (P<0.05). Bracketed values to the right of the letters indicate the size of the mean relative to that of the SCS, at the 90% confidence interval. Table 1. The mass median aerodynamic diameters (MMAD; Âľm) of the particles depositing within the impactor (after the induction port), and their geometric standard deviations (GSD).

pMDI alone Space Chamber Slim LiteAire

Salbutamol; 15 L/min MMAD GSD 2.65 2.06 2.40 1.60 2.31 1.56

Salbutamol; 30 L/min MMAD GSD 2.30 2.22 2.17 2.23 1.98 1.73

Fluticasone; 30 L/min MMAD GSD 2.65 1.85 2.35 1.62 2.24 1.61

Ipratropium; 30 L/min MMAD GSD 1.12 1.89 0.93 1.68 0.85 1.63

Drug Delivery to the Lungs, Volume 29, 2018 - In vitro testing of the new Space Chamber Slim with salbutamol sulfate, fluticasone propionate, and ipratropium bromide pressurized metered dose inhalers Discussion The devices tested in this study are relatively similar in terms of body shape and holding chamber volumes, although the SCS is slightly larger (~200 mL compared with ~184 mL [7]) and wider. The internal format of the two holding chambers differ somewhat, as the inhalation vent in the SCS is located close to the bottom of the holding chamber behind a fold in the wall between the holding chamber and the delivery chamber (Figure 1). In contrast, the wall between the holding chamber and delivery chamber of the LA is relatively flat and the inhalation vent, which contains a one-way valve, is located closer to its upper side. Given these subtle design differences, it was of interest to assess for differences in the amount and characteristics of aerosol delivered through the two devices. The CSA Standards suggest that spacers and VHCs should limit the delivery of course particles, while maintaining the delivery of fine particles (thus increasing the FPF) [4]. Both the SCS and the LA substantially reduced the TDD of the pMDIs, whilst increasing the proportion of fine particles within the aerosols delivered to the NGI by at least two-fold, in all cases. This was most likely due to a large proportion of coarse and high-velocity particles depositing instead within the devices themselves, along with some shrinking (by evaporation of propellant) and deceleration of the remaining particles [1], and is consistent with the observed effects of using anti-static plastic spacers [8]. In a clinical scenario, the coarse, high-velocity particles are likely to impact in the oral cavity and the oropharyngeal region of the upper airways, with potential for causing undesirable side-effects such as hoarseness, cold freon effect, metallic taste, and local side-effects associated with some inhaled medications like corticosteroids [1,2]. Despite the lower TDD, the mainly-respirable FPD delivered from the cardboard devices was either higher than, or not significantly different to, that of the pMDI alone in all cases aside from salbutamol at 30 L/min, where the pMDI had a slightly higher FPD than the LA (34.8 compared to 28.5 µg). Thayer Medical’s white papers report an FPD that ranged from substantially higher than [9], to not significantly different to [10], the pMDI alone for salbutamol at 28.3 L/min. Meanwhile, another cardboard device – the DispozABLE Spacer (Clement Clarke International Ltd, UK) – has also been shown to deliver an equivalent FPD compared with a pMDI alone [3]. A previous study showed that four anti-static plastic VHCs (including the Compact Space Chamber Plus, AeroChamber Plus Flow-Vu, OptiChamber Diamond, and VORTEX) also either slightly increased or did not change the FPD delivered by a range of pMDIs alone [8]. The slightly higher FPD observed with the SCS compared to the LA might be explained, to some degree, by the differences in design and volume of the holding chambers within the devices, as larger and wider spacers/VHCs have been shown to increase the FPD in some cases [2]. It should be noted that there was no delay between the actuation of pMDIs and induction of the aerosol into the NGI in this study, thus simulating fully-coordinated use. It may therefore be valuable to pursue future studies to assess the drug delivery characteristics of the SCS under circumstances of uncoordinated use, by introducing a delay between actuation and air flow induction, and/or assessing the TDD with simulated uncoordinated tidal breathing. When the DispozABLE Spacer was tested with suboptimal use (a one-second delay between actuation and induction), its mean FPD was shown to be 3.7 times greater than that of the pMDI alone [3]. Conclusions The relatively high FPF (2.0 to 4.9-fold higher than the pMDI alone) and FPD (1.0 to 2.1-fold higher than the pMDI alone) observed for the SCS indicate that, at the conditions tested, it substantially reduces the delivery of undesirable coarse particles from pMDIs, while maintaining or increasing that of the mainly-respirable fine particles. References 1

Vinken W, Levy M L, Scullion J, Usmani O S, Dekhuijzen P N R, Corrigan C J on behalf of the ADMIT group: Spacer devices for inhaled therapy: why use them, and how? ERJ Open Res 2018; 4: 00065-2018.

Nikander K, Nicholls C, Denyer J, Pritchard J: The evolution of spacers and valved holding chambers, J Aerosol Med Pulm Drug Deliv 2014; 27 (Suppl 1): ppS4-S23.

Sanders M, Bruin R: A rationale for going back to the future: use of disposable spacers for pressurized metered dose inhalers, Pulm Med 2015; Article ID 176194, 6 pages.

Canadian Standards Association: CAN/CSA-Z264.1-02 Spacers and Holding Chambers for Use with Metered-Dose Inhalers (reaffirmed 2011).

US Pharmacopeial Convention: <1602> Spacers and valved holding chambers used with inhalation aerosols: In Process Revision.

Fieller E C: The biological standardization of Insulin, Suppl to J R Statist Soc. 1940; 7: pp1–64.

Thayer Medical: LiteAire Additional Product Information, Thayer Medical Website 2017: Accessed 30Jul2018 at http://thayermedical.com/products/liteaire/liteaire-additional-product-information/

Courtney S, Pratt B: Investigation into the in vitro performance of anti-static VHCs by particle size distribution using the next generation pharmaceutical impactor (NGI). (Abstract). Presented at Drug Delivery to the Lungs (DDL26), Edinburgh, Scotland, Dec 9-11, 2015.

Drug Delivery to the Lungs, Volume 29, 2018 - Michael Nairn et al. 9

Johnson J L H: Performance of Thayer Valved Holding Chambers compared to Make-shift Spacers with ProventilÂŽ HFA pMDI. Document Number: PUB0511, Revision B.

Johnson J L H: Performance of Thayer Valved Holding Chambers with an albuterol sulfate MDI Product Marketed in the USA. Document Number: PUB0510, Revision A.

Drug Delivery to the Lungs, Volume 29, 2018 - Mark W Nagel et al. Evaluation of Pressurised Metered Dose Inhaler (pMDI) Plume Spray Force When a Valved Holding Chamber (VHC) is Present: A Proof of Concept Investigation to Identify Propensity for Premature Inhalation Valve Opening Mark W Nagel 1, Jolyon P. Mitchell2 & Jason A Suggett

Trudell Medical International, 725 Third Street, London, Ontario, NV5 5G4, Canada Jolyon Mitchell Inhaler Consulting Services Inc., 1154 St. Anthony Road, London, N6H 2R1, Canada 1

Summary A proof-of-concept experimental study has been undertaken to quantify the force of the aerosol plume emitted from the same pMDI product (Flovent-125 HFA-Evohaler), in combination with VHCs having different inhalation valve types. A sensitive force transducer (0 to 2000 mN) attached to a circular disc-shaped target was mounted perpendicular and on-axis with the horizontally-supported VHC, with its mouthpiece exit plane located 1.0  0.1 mm from the target. The force transducer was initially calibrated by actuating three pMDIs without a VHC, with the mouthpiece exit set 25.0  0.1 mm from the target, confirming that a force of 81.3  6.8 mN was delivered, comparable with previously reported measurements. The following VHCs (n=5 devices/group; 3-replicates/VHC) were subsequently evaluated: 1. 2. 3. 4.

antistatic SpaceChamber (Medical Developments International, Scoresby, Australia) Vortex (PARI Respiratory Equipment, Midlothian, VA, USA) OptiChamber Diamond (Philips-Respironics Inc., Parsippany, NJ, USA) antistatic AeroChamber Plus Flow-Vu VHC (Trudell Medical International, London, Canada)

Although the VHCs attenuated the plume pulse by almost an order of magnitude, all add-ons transmitted a detectable force from the inhaler-generated plume pulse via their inhalation valves to reach the target. The peak force measurements (mean  S.D.) for the antistatic AeroChamber Plus VHC group (2.0  0.5 mN) were lower than values for the other VHC groups (ranging from 3.7  1.7 to 8.3  2.7 mN), possibly because the toroidal valve deflected much of the incoming energy radially to the chamber walls. This new test method may be useful in VHC development as well as quality control. Key Message This laboratory-based investigation utilized a sensitive force transducer to assess premature VHC inhalation valve opening in response to plume expansion upon inhaler actuation. Ballistic component ejection through this valve could result in increased oropharyngeal deposition, especially with inhaled corticosteroids . Introduction pMDIs deliver a substantial portion of the emitted dose to patients as ballistic particles upon actuation 1, as a result of the propellant flash-evaporating upon exposure to ambient pressure in the vicinity of the actuator orifice of the inhaler 2. Particles contained in the ballistic fraction therefore have high initial forward velocities as they exit the mouthpiece 1 that result in most depositing in the oropharyngeal region of the patient 3. Tuohy et al. have observed that a less forceful plume may decrease drug impaction at the back of the throat and improve lung delivery 4. VHCs modify the emitted aerosol by largely capturing the ballistic fraction that typically comprises a large portion of the coarse particle mass fraction of the dose, that would otherwise deposit in the oropharynx 5. Oropharyngeal deposition may have both topical (i.e., oral candidiasis in the case of inhaled corticosteroids 6) and systemic effects, which are dependent on the metabolic pathway of the API(s) associated with the formulation, through swallowing and subsequent absorption via the gastrointestinal tract 7. It is tacitly assumed that the inhalation valve of a VHC remains closed to the incoming expanding plume, however data in support of this assumption are scant. Quantification of momentary inhalation valve opening in association with the passage of the ballistic component is difficult because the process is very rapid and any attempt to capture the aerosol burst is likely to interfere with the valve opening process. Several years ago, Gabbrio et al. published a method involving the impaction of the expanding plume containing the aerosol from pMDI products onto a metal plate coupled mechanically to a load cell force transducer, with the target plate arranged orthogonally to the axis of plume generation from the inhaler mouthpiece 8. This technique was more recently developed further by Blatchford et al., who used a metal stage of an Andersen cascade impactor as the target 9. The present proof-of-concept study was an adaptation of the approach of Blatchford et al. 9 to determine the instantaneous force exerted by the emergent aerosol pulse from VHCs having a variety of different inhalation valve designs. It was assumed likely that the volume of air within the VHC would dampen the pulse, but we hypothesized that sufficient force would be present at the distal end of the chamber to penetrate past the valve. The peak force was used as a surrogate for comparing relative amounts of particulate capable of potentially penetrating the oropharynx as the result of valve opening in association with the passage of the ballistic component from the inhaler.

Figure 1. Ballistic Component Force Measurement Apparatus Arranged to Evaluate Momentary Valve Opening in Association with Passage of the Ballistic Component of the Aerosol Emitted from the Inhaler Upon Actuation via the Inhalation Valve of the VHC diameter circular stainless-steel collection plate (solid surface) of an Andersen cascade impactor could be set to 1.0 mm (Figure 2). Measurements of the instantaneous force detected by the transducer were made at 50ms intervals beginning a few seconds before pMDI actuation. These measurements were automatically downloaded to an Excel spreadsheet (Microsoft Corp, Redmond, WA, USA), and the peak force detected was used as the indicator of the magnitude of the pulse of aerosol that had escaped from the inhaler mouthpiece.

Figure 2: Close-up Showing VHC Mouthpiece Exit and Target Disc Locations Before making measurements with the VHCs, the arrangement was calibrated by evaluating the plume force emitted from a Flovent-125 HFA Evohaler (125 g/actuation fluticasone propionate GSK Canada plc) pMDI alone, with the gap between inhaler mouthpiece exit plane and the target set to 25 mm, as described by Blatchford et al. 9. After verifying that similar measures of maximum spray force were achievable with the present apparatus configuration, the following VHCs (n = 5 devices/group, 3 replicates/device) were evaluated with the distance between VHC mouthpiece exit and target reduced to the nominal value of 1.0 mm. The measurement procedure was as described for the system calibration measurements, and the following VHCs were evaluated, each device being tested immediately after removal from its packaging: 1. antistatic SpaceChamber (Medical Developments International, Scoresby, Australia) - cross-cut valve 2. Vortex (PARI Respiratory Equipment, Midlothian, VA, USA) - duck-bill valve 3. OptiChamber Diamond (Philips-Respironics Inc., Parsippany, NJ, USA) - duck-bill valve

Drug Delivery to the Lungs, Volume 29, 2018 - Mark W Nagel et al. 4.

antistatic AeroChamber Plus Flow-Vu VHC (Trudell Medical International, London, Canada - toroidal valve.

The closer distance was required because of the greatly reduced force of the plume after transiting the length of the VHC-on-test. All the VHC-based measurements were undertaken with Flovent-125 (125 g/actuation fluticasone propionate, GSK Canada plc), as a representative inhaled corticosteroid, as this class of medication, is more likely to be associated with side effects associated with oropharyngeal deposition. Results Figure 3a illustrates a typical force-time profile obtained with one of three different canisters of Flovent  pMDIs. The initial peak force (mean  S.D.) of 81.3  6.8 mN was comparable with the Maximum Spray Force of about 90 mN reported by Blatchford et al. 8 for Flixotide pMDI, containing the same active pharmaceutical ingredient (API) and HFA 134a propellant.

Figure 3: Plume Force-Time Profiles (A) for a Measurement with Flovent-125 HFA Evohaler pMDI Alone to Calibrate the Measurement System; (B) for a Measurement with one of the pMDI-VHC Combinations The Magnitude of the Peak Force was Typically in the Range 2 to 10% of That From the pMDI Alone A corresponding profile for one of the pMDI-VHC combinations chosen to represent the group of measurements reported in Table 1 is shown in Figure 3b. Table 1: Plume Expansion-Related Peak Force Following Actuation of Flovent-125 Evohaler at the Mouthpiece of Various pMDI-VHC Combinations Representing Potential Emission of Particles in Ballistic Fraction to a Patient (n = 5 VHCs/group; 3 replicates/device) Peak Force (mN) Mean S.D. 1 2.9 1.0 2 4.7 0.9 3 7.6 1.4 antistatic SpaceChamber/ 4 3.7 2.0 cross-valve 5 4.3 3.3 Group 4.7 2.3 1 2.7 0.8 2 2.5 2.6 3 3.0 0.4 Vortex/duck-bill valve 4 3.6 0.3 5 4.3 3.0 Group 3.7 1.7 1 7.8 0.5 2 7.9 3.0 3 4.3 0.5 OptiChamber Diamond/ duck-bill valve 4 10.6 2.3 5 9.2 0.6 Group 8.3 2.7 1 2.2 0.3 2 2.2 0.5  antistatic AeroChamber Plus 3 2.6 0.5  Flow-Vu / 4 1.8 0.5 fixed torus baffle valve 5 1.9 0.4 Group 2.0 0.5 The attenuation of the plume pulse by passage through the VHC is evident in the reduction in peak force to < 10 mN, as in the case illustrated. The peak pulse with either the pMDI alone (less evident due to the coarser scale setting) or with a VHC present was followed by a series of oscillations whose magnitude steadily dampened towards zero. This behaviour is associated with the mechanical response of the target and is therefore not pertinent to understanding plume-inhalation valve interactions. VHC/inhalation valve type

Device Number

Drug Delivery to the Lungs, Volume 29, 2018 - Evaluation of Pressurised Metered Dose Inhaler (pMDI) Plume Spray Force When a Valved Holding Chamber (VHC) is Present: A Proof of Concept Investigation to Identify Propensity for Premature Inhalation Valve Opening Discussion In the present study, all of the VHCs were found to transmit some of the plume pulse emitted from the inhaler via their inhalation valves and mouthpiece to reach the target to which the force transducer was attached. In general, the replicate measurements of peak force for different devices of the same type were reproducible, with a few exceptions, such as the high device mean value of 7.6 mN for the third antistatic SpaceChamber  VHC and the low and repeatable value of 4.3  0.5 mN for the third OptiChamber Diamond VHC compared with the mean peak force of 8.3  2.7 mN for that group. In more general terms, the peak force measurements for the antistatic AeroChamber Plus VHC as a group were lower than almost all values for the other VHC types, having mean peak force of 2.0  0.5 mN (1-way ANOVA on ranks followed by multiple comparison with AeroChamber Plus  VHC group as control; p < 0.05). In explanation, it is conjectured that the leading edge of the fixed toroidal baffle comprising the inhalation valve for this VHC type may deflect the velocity of the incoming aerosol plume pulse from the pMDI, entering on-axis with the interior of the chamber, radially towards the chamber walls. Such a wall interaction could then result in a dampening of the amount of energy available to project the flow further into and beyond the mouthpiece. However, computational fluid dynamic analysis would be needed to confirm this suggestion and was beyond the scope of the present proof-of-concept approach. Gabbrio et al. have demonstrated by means of cascade impactor-based measurements of APSD for a range of different pMDIs that the magnitude of the spray force and inlet (throat) deposition are correlated, with the lowest spray force associated with the smallest inlet deposition 8. However, the present force-based measurements cannot be used quantitively to determine the relative mass of API capable of reaching the oropharynx of a patient because other factors, in particular whether the user is breath-holding, inhaling or exhaling at the time of pMDI actuation, will influence how much plume penetration actually takes place. The merit of this non-invasive approach is therefore likely to be as a further simple-to-use diagnostic aid to support VHC device development as well as possibly in product quality control. Conclusions A proof-of-concept experimental study has been undertaken to quantify the magnitude of the force of the expanding aerosol plume emitted from a pMDI in combination with VHCs having a range of different inhalation valve types. All of the VHCs were found to transmit some of the plume pulse emitted from the inhaler via their inhalation valves and mouthpiece to reach the target to which a highly sensitive force transducer was attached, but to differing extents. There were detectable and generally reproducible differences in the magnitude of the peak force observed from one device to another of the same type and between VHCs of different construction. This approach has the potential to be used as a method to evaluate inhalation valve performance both in VHC development and in product quality control, but further work will be needed to establish the clinical relevance of such measurements. References Hickey AJ, Evans RE: Aerosol generation from propellant-driven inhalers. In: A J. Hickey (ed): Inhalation Aerosols - Physical and Biological Basis for Therapy. Marcel Dekker , N.Y., USA; pp417-439 1

Hickey AJ. Controlled delivery of inhaled therapeutic agents. J Control Release 2014; 190: pp182-188.

Rubin BK, Fink JB. Optimizing aerosol delivery by pressurized metered-dose inhalers. Respir Care 2005; 50(9): pp1191–1200.

Tuohy J, Marshall J, Danagher H. Plume spray force of three HFA-propelled ICS/LABA combination inhalers. ERJ 2017;50:524.

Dolovich MB, MacIntyre NR, Andersen PJ. Consensus statement: aerosols and delivery devices. Respir Care 2000; 45(6): pp589–596.

Rachelefsky GS, Liao Y, Faruqi R: Impact of inhaled corticosteroid-induced oropharyngeal adverse events: results from a metaanalysis. Ann Allergy Asthma Immunol. 2007; 98(3): pp225–238.

Barnes NC: The properties of inhaled corticosteroids: similarities and differences. Prim Care Resp J 2007;16: pp149–154.

Gabbrio BJ, Stein SW, Velasquez D.J. A new method to evaluate plume characteristics of hydrofluoroalkane and chlorofluorocarbon metered dose inhalers. Int J Pharm 1999; 186: pp.3-12. 8

Blatchford C, Nixon G, Hargrave G, Justham T, Long E. An Evaluation of Analytical Techniques for Characterising the Plume Velocity and the Plume Force of Pulmonary and Nasal Products. Drug Delivery to the Lungs-18, The Aerosol Society, Edinburgh, UK. 2007: pp. 243-246. 9

Drug Delivery to the Lungs, Volume 29, 2018 - Deepak Patil et al. HPMC Capsules In Dpis. Evaluation Of The Puncturing Force Using Capsules Made With Different Manufacturing Methods And Compositions Deepak Patil1, Jnanadeva Bhat1, Sanjay Powale1, Ajay Giripunje1, Justin Kalafat2, Fernando Diez3 1

ACG SciTech Center, 7 Prabhat Nagar Patel Estate, Jogeshwari (West), Mumbai, 400102, India 2ACG 3

North America, LLC, 262 Old New Brunswick Rd, Piscataway, NJ, USA

ACG Europe, 159 Princes Gardens, W3 0LS, London, United Kingdom

Summary Capsules based inhalers belong to the category of dry powder inhalers. Their importance arises as a drug delivery component when high drug doses must be delivered directly to the lungs. The process consists of two steps; capsule puncturing and aersolization. The use of this platform is not only restricted to pulmonary diseases, as new products for Parkinson disease and acute migraines are being developed using these types of inhalers. Most of these new products use HPMC capsules with a gelling agent in the formulation. These capsules perform well in the puncturing process, and most of the studies reviewed in the reference section refer to these capsules. [1] In this study we focus on another type of HPMC capsules, with two noticeable differences from the legacy product. First is the composition, as the standard capsules use a gelling agent (normally carrageenan) and a gelling promoter. The second generation of HPMC capsules only consist of the polymer, hypromellose, and water. The other difference refers to the manufacturing process (hot versus cold) in which the temperature of the pin bars are drastically changed. The standard capsules are made with pin bars at a temperature of 20-25 ยบC, and the absence of the gelling promoter makes it necessary to elevate the temperature to 65-70 ยบC for the capsules to form on the pins. Puncturing forces are measured for both types of HPMC capsules, detailing the correlation between capsule thickness and puncturing force . There are not significative differences between both capsules. This capsule is an additional tool for the formulator to design innovative systems in product development of generic and new products. Key Message Capsule based inhalers are becoming more popular in the pharmaceutical industry [3,4], as the capsule plays a key role in the device efficiency [2,3]. In this work, two different types of HPMC capsules (gelling agent contained and gelling agent absent) are compared in terms of puncturing. There are not any significant differences in terms of puncturing forces between the capsule types. Introduction Introduction One attractive platform for inhalation drug delivery is capsule-based inhalers. Apart from cost considerations and the possibility of delivering high doses in new product development research, they are extremely simple. The formulation only consists of the API alone, or as a mixture of the API with a carrier particle such as lactose or mannitol. Formulators are considering utilizing larger capsules than the traditional Size 3, and research is expanding to many new therapeutic areas beyond COPD and asthma treatment. For hard shell capsules to function effectively as drug reservoirs in the inhaler, the capsule must be capable of a clean puncture. Sharpened pins or blades cut the capsule to release the powdered medication upon inspiration out of the inhaler. Shedding capsule fragments or flaps at the puncturing site can create hindrances in the outflow of the powder to the patient. The capsule must be cut or punctured in a reproducible and easy manner. This enables the powder to be emptied as completely as possible. The challenge in this process is to ensure a minimum amount of shells broken off during cutting or puncturing. Although unlikely due to the filter on the inhalation device, these capsule shreds could be inhaled even they are too large to be deposited in the lungs [2,3]. Standard HPMC capsules used in inhalation are based on a hypromellose polymer, a gelling agent (normally carrageenan) and a gelling promoter. The capsules are manufactured at room temperature by dipping two sets of stainless steel mold pins into the raw material mixture. The goal of this work is to compare the values of the forces to penetrate a hard-shell capsule for standard HPMC capsules and for another type of HPMC capsule. The second HPMC capsule type is only composed by hypromellose and water with an increased pin bar temperature [3,4].

Drug Delivery to the Lungs, Volume 29, 2018 - HPMC Capsules In Dpis. Evaluation Of The Puncturing Force Using Capsules Made With Different Manufacturing Methods And Compositions Experimental methods Size 0 hypromellose empty hard-shell capsules, with and without gelling agent, for inhalation manufactured by ACG were utilized in this study. An Autograph TM testile testing machine in compression force measurement mode was used. The capsules were positioned into the recess of a stainless-steel bushing, which was orientated appropriately and subsequently secured in a fixed position. The metallic pins used in this study were attached to the chuck and the software( Trapazium-2 TM) was programmed to conduct a compression test at a speed of 25 mm/min. The displacement of the pin and the resulting force were recorded and registered on a load-displacement curve. The parameters chosen for analysis were the max force (N) and the displacement at the max force (mm). (Figure 1) . Forces were measured for each range of dome thickness ( 110-190 μ) for cap and body shell.

Force/ Deformation for Hard Capsules 3.5 3

Force(N)

2.5 2 1.5 1 0.5 0

0.5

1.5

2.5

3.5

Deformation (mm) Figure 1: Load-displacement curve. Arrows show max force and displacement at max force. Redrawn from [2]. Rupture corresponds to the maximum

Results The results of the maximal force (puncturing force) vs capsules thickness (µ) corresponding to the two types of capsules are collected in Tables 1 and 2.

Size

Capsules

Moisture (%)

130

140

150

160

170

180

190

HPMC S1 (Body)

5.5 - 7.0

2.31

2.5

2.85

2.75

3.23

3.74

3.81

HPMC S2 (Body)

5.5 - 7.0

2.17

2.19

2.47

2.72

3.27

3.30

HPMC S3(Body)

5.5 - 7.0

2.61

2.70

3.58

3.94

3.98

3.91

HPMC S1 (Cap)

5.5 - 7.0

3.51

3.72

3.68

3.72

3.91

4.48

4.55

HPMC S2 (Cap)

5.5 - 7.0

2.87

2.17

2.54

2.99

3.17

3.14

3.53

HPMC S3 (Cap)

5.5 - 7.0

2.87

3.00

3.49

4.01

4.32

4.29

4.49

Table 1: Values of maximal force for capsules without gelling agent (N) vs thickness (µ). S refers to one sample

Drug Delivery to the Lungs, Volume 29, 2018 - Deepak Patil et al. Size

Capsules

Moisture (%)

120

130

140

150

160

170

HPMC S4 (Body)

5.5 - 7.0

2.29

2.300

2.74

2.84

3.34

HPMC S5 (Body)

5.5 - 7.0

2.82

3.02

3.15

3.23

3.32

HPMC S6 (Body)

5.5 - 7.0

2.22

2.30

2.41

2.73

3.04

HPMC S4 (Cap)

5.5 - 7.0

3.06

3.16

3.26

3.49

3.37

HPMC S5 (Cap)

5.5 - 7.0

2.64

2.75

2.84

2.96

3.16

HPMC S6 (Cap)

5.5 - 7.0

2.26

2.35

2.41

2.71

3.21

Table 2: Values of maximal force for capsules with gelling agent (N) vs thickness(µ). S refers to one sample

Non gum HPMC capsules 5

Force(N)

4 3 2

y = 0.0263x - 1.1786 R² = 0.938

1 0

100

120

140

160

180

200

Capsules thickness (µ) Figure 2: Correlation puncturing force vs capsule thickness for HPMC capsules without gelling agent

HPMC capsules with gelling agent Force (N)

4 3 2

y = 0.0264x - 0.994 R² = 0.9184

1 0

100

110

120

130

140

150

160

170

Capsules thickness (µ) Figure 3: Correlation puncturing force vs capsule thickness for HPMC capsules with gelling agent

Discussion Considering the standard capsules thickness (140-160µ), the puncturing force values for HPMC without gelling agent are in the range 2.96 – 3.57 N for the cap and 2.43 – 2.93 N for the body. For HPMC capsules with gelling agent present the values are in the range 2.84 – 3.24 N for the cap and 2.76 – 3.23 N for the body. Capsules did not show splintering, as the punctures were clean. Because of the low number of measurements, it is not possible to make a strict statistic analysis of the data. The values are aligned with the data found in bibliography that shows maximal forces of 3 - 4 N for HPMC capsules with gelling agent [2].

Drug Delivery to the Lungs, Volume 29, 2018 - HPMC Capsules In Dpis. Evaluation Of The Puncturing Force Using Capsules Made With Different Manufacturing Methods And Compositions As expected, there is a correlation between thickness and puncturing force, where the higher thickness yields a higher puncturing force. This correlation is linear, in that every 10 Âľ thickness increase, the puncturing force is increased around 0.25 N. Conclusion Capsules based inhalers represent the most rapidly expanding field in pulmonary drug delivery. Largely as a result of the perceived limitations in pMDIs and nebulizers, capsule based inhalation has formulators intrigued globally on many new products and disease treatments. Although the fact of loading the capsules may not be easily accomplished by a patient who requires an immediate delivery of the drug, capsule based inhalers avoid problems inherent in the use of propellant gases and the need for coordination between inhalation and actuation. Capsule based devices are also very portable, patient friendly, easy to use and adaptable to different capsule sizes. The standard capsule used for these devices is HPMC containing a gelling agent. In this study this capsule is compared in terms of puncturing with an HPMC capsule without the gelling agent, a simpler composition containing only solely hypromellose and water. The results show that there are no significant differences between the puncturing forces necessary for both capsules. Moreover, the influence of the capsule thickness on puncturing forces is small. This enables manufacturing capsules with reproductible values for puncturing feasible and adds to patient familiarity in the feel of puncturing the capsules with their device.

1234-

References N. Islam, E. Gladky. DPIs review of device reliability and innovation. International Journal of Pharmaceutics. 360 (2008). 1-11 B. Torrisi, J. Birchall, B. Jones, F. Diez. The development of a sensitive methodology to characterize hard shell capsules puncture by dry powder inhaler pins. International Journal of Pharmaceutics. 456 (2013). 545-552 F. Diez, J. Kalafat, J. Bhat. The science behind capsules-based dry powder inhalation technology. On Drug Delivery. 2017. November issue D. Patil, J. Kalafat. Bhat, S. Powale, A. Giripunje, F. Diez. Mapping performance of inhalation-based capsules with appropriate puncturing for optimum dose delivery. Poster submitted in RDD 2018 Arizona

Drug Delivery to the Lungs, Volume 29, 2018 - Weiling Li et al. Comparison of Aerosol Characteristics and Nicotine Delivery by Conventional Pharmaceutical Inhalation Devices and Electronic Nicotine Delivery Systems (ENDS) Weiling Li, Qiang Wang, and Raymond W Lau Altria Client Services LLC, 601 E Jackson Street, Richmond, VA, 23219, U.S.A Summary An understanding of the aerosol characteristics and delivery of nicotine is important in the continuous development of a class of products called electronic nicotine delivery systems (ENDS) that has the potential to reduce the harm associated with tobacco use, particularly cigarette smoking. We compared the physical characteristics (aerosol mass, particle size distribution) and nicotine delivery of aerosols from three nebulizers, a multi-dose propellant-free inhaler device (no nicotine) and two ENDS products – one generating aerosols thermally and one non-thermally. Aerosols from inhalation devices, which are also generated by non-thermal means, had lower mass deliveries compared to aerosols from ENDS products that generate aerosols thermally. Inhalation devices aerosols also have larger particles which usually deposit in the upper airways mostly, and deliver a lower level of nicotine compared to aerosols from ENDS. Key Message Conventional pharmaceutical inhalation devices deliver aerosols with supermicron particles (˃ 1µm) compared to the submicron particles from electronic nicotine delivery systems (ENDS). From formulations with the same concentration of nicotine, inhalation devices deliver less nicotine in the aerosols compared to ENDS. Introduction Whilst evidence suggests that a class of tobacco products called electronic nicotine delivery systems (ENDS) are likely to be far less harmful than conventional cigarettes [1], effort is on-going to continue to develop devices that would offer nicotine and sensory satisfaction comparable to that of cigarette smoking, but with much reduced harm. Such devices may facilitate transition from cigarette smoking and contribute to tobacco harm reduction. Pharmaceutical inhalation devices have been traditionally used with success in the delivery of drugs via the inhalation pathway. A comparison of aerosol characteristics (aerosol mass, particle size distribution) and nicotine delivery from these two classes of inhalation products may yield insights that inform the development of next generation ENDS. Experimental Methods Four pharmaceutical inhalation devices were compared to two ENDS products in terms of aerosol mass, nicotine delivery, and particle size. Detailed descriptions of the devices and formulations (aerosol precursors) being studied are shown in Table 1. Also described in Table 1 are the aerosol collection conditions. Given the devices had very different aerosol generation mechanisms, a best attempt was made in adjusting the formulations to enable each device to deliver similar amount of aerosol so they could be meaningfully compared. For example, Inhalation Devices 2 and 3 would only work with aqueous nicotine solutions, Inhalation Device 1 would not work with formulations with high propylene glycol and glycerine content. Inhalation Device 4 was a closed system prefilled with a drug formulation not containing nicotine. The formulation in ENDS 2 product was close to that in ENDS 1 product, except that ENDS 2 would not work properly with any water in the formulation. The air flow rate and aerosol collection time for ENDS 1 followed a standard recommended method [2]. An initial trial of aerosol collection indicated that the air flow used to entrain the aerosol had to be modified for ENDS 2 and Inhalation Device 4 compared to that for the other devices, again, to achieve comparable aerosol and also to reduce aerosol loss during transit from the device to measurement instrument. Aerosol from five puffs was collected from all devices. To minimize variability in aerosol mass measurements the duration of each puff of some of the devices was extended to 10s (Table 1). Five puffs were collected using filter pad[2] for aerosol mass measurement per replicate. The Cambridge filter pad was then extracted with isopropanol using Carvol as internal standard to determine nicotine delivery. For particle size, Spraytec (Malvern Panalytical) was used to measure aerosols generated by the four inhalation devices and Fumex[3] (Fraunhofer Institute, Germany), which is based on light scattering and designed for characterizing ENDS, was used to measure ENDS 1 and ENDS 2. Results Table 2 shows a summary of data collected. Due to the varying aerosol collection regimes used, the mass delivery and nicotine delivery of each of the devices were normalized to unit volume and time to facilitate comparison. ENDS 1 and Inhalation Device 3 generated the highest normalized aerosol mass. The two ENDS products delivered higher normalized level of nicotine. The particle size, characterized by mass mediandiameter (MMD), is submicron for both ENDS products and supermicron (˃ 1µm) for all the pharmaceutical inhalation devices. The

Drug Delivery to the Lungs, Volume 29, 2018 - Comparison of Aerosol Characteristics and Nicotine Delivery by Conventional Pharmaceutical Inhalation Devices and Electronic Nicotine Delivery Systems (ENDS) difference in particle size between these two classes of inhalation products exceeds what would be expected because of the use of the two optical devices. Formulation (% total weight)

Air Flow Rate lpm (litres per minute)

Aerosol Collection time, s/puff

2.5% nicotine, 15% water, 49.5% glycerin, 33% propylene glycol

1.10

2.5% nicotine, 58.5% glycerin, 39% propylene glycol

0.66

2.5% nicotine in 48.75% glycerin, 48.75% water

1.10

Inhalation Device 2

• Vibrating mesh nebulizer • 180 kHz • Button activated delivery • Battery operated

2.5% nicotine in water

1.10

Inhalation Device 3

• Ultrasonic nebulizer • 2.4 MHz • Button activated delivery • Battery operated

2.5% nicotine in water

1.10

Non-nicotine containing drug formulation

2.60

Device

ENDS 1

ENDS 2

Inhalation Device 1

Inhalation Device 4

Description • Ciga-like E-vapor product • Non-refillable liquid cartridge • Thermal aerosol generator • Puff activated delivery • Battery operated • Refillable tank E-vapor product • Ultrasonic aerosol generator (2.4 MHz) • Button activated delivery • Battery operated • Jet nebulizer • Compressed air operated

• Multi-dose propellant-free inhaler • Aerosol generated by colliding liquid jets • Non-refillable cartridge • Mechanical activation • No battery

Table 1 - Devices, Formulations Studied and Aerosol Collection Conditions Aerosol mass, mg/puff

Normalized Mass delivery, mg/cc/s

Nicotine delivery, g/puff

Normalized nicotine delivery, g/cc/s

Particle size (MMD), m

ENDS 1

3.0±0.1

0.0167

100±6

0.606

0.60±0.03

ENDS 2

3.6±0.9

0.0033

590±220

0.536

0.87±0.03

Inhalation Device 1

22.0±2.1

0.0014

627±48

0.041

2.40±0.05

Inhalation Device 2

13.4±1.5

0.0073

389±60

0.212

6.20±0.52

Inhalation Device 3

27.0±4.1

0.0147

749±84

0.408

4.95±0.14

Inhalation Device 4

10.0±1.7

0.0090

N/A

4.80±0.04

Table 2 – Measured Aerosol Properties from ENDS and Inhalation Devices (3 replicates , mean  SD values shown)

Drug Delivery to the Lungs, Volume 29, 2018 - Weiling Li et al. Discussion and Conclusion Data from this study clearly illustrate some fundamental differences between aerosols from conventional pharmaceutical inhalation devices and from ENDS products. Aerosols from inhalation devices, which are generated by non-thermal means, had lower normalized mass deliveries compared to aerosols from ENDS products that generate aerosols thermally, i.e. ENDS 1. Inhalation devices aerosols generated larger particles which usually deposit in the upper airways mostly and deliver a lower level of nicotine compared to aerosols from ENDS. Reference 1

National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. https://doi.org/10.17226/24952.

No. 81 - Routine Analytical Machine for E-Cigarette Aerosol Generation and Collection - Definitions and Standard Conditions, 2015. CORESTA (Cooperation Centre for Scientific Research Relative to Tobacco)

Dunkhorst W, Lipowicz P, Li W, Hux C, Wang Q, Koch W: In-situ characterization of e-cigarette aerosols by 90Â°-light scattering of polarized light, Aerosol Science and Technology, 2018; 52: pp 717-724.

Drug Delivery to the Lungs, Volume 29, 2018 – Lucie Ondráčková et al. Number size distribution of particles dosed by MDI and DPI inhalers Lucie Ondráčková1, Jana Kozáková1, Jakub Ondráček1, Vladimír Ždímal1, Ludmila Mašková1 & Stavros Kassinos2 Department of Aerosol Chemistry and Physics, Institute of Chemical Process Fundamentals of the CAS, Rozvojová 135, Prague, 165 02, Czech Republic Department of Mechanical & Manufacturing Engineering, University of Cyprus, 1 Panepistimiou Avenue, 2109 Aglantzia, Nicosia, Cyprus 1

Summary The purpose of this research was to determine the influence of inspiratory flow rate and relative humidity on particle size distributions (PSDs) generated by three MDI inhalers (Flutiform, Fullhale and Ventolin) and one DPI inhaler (Spiriva). Particle size distribution was measured by APS 3321 (TSI, USA) for three different inspiratory flow rates – 30, 60 and 90 l/min. Aerosol from individual inhalers was led through a stainless steel tubing simulating the geometry of the human respiratory tract. The hygroscopicity of the particles was determined by comparing data obtained at laboratory conditions and at relative humidity of 90%. During the measurements of number PSDs by using of APS spectrometer, we measured bimodal distributions for Flutiform and Fullhale and monomodal distributions for Ventolin and DPI Spiriva. The increasing inspiratory flow rate had a minimal effect on the position of the modes of the individual distributions. The differences in PSD, measured under ambient conditions and at RH of 90%, were minimal and did not significantly affect the assumed probability of drug deposition. The results showed that determination of the PSDs by online spectrometer offers an alternative to NGI impaction testing to develop mechanistic understanding of factors affecting the particle size distributions of inhalation aerosol. Key Message The increasing inspiratory flow rate had a minimal effect on the position of the modes of the individual distributions. The differences in PSD, measured under ambient conditions and at RH of 90%, were minimal and did not affect the assumed probability of inhalant deposition. Introduction Pressurized metered dose inhalers (MDI) and dry powder inhalers (DPI) are an integral part of the treatment of lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) [1, 2]. An important characteristic of particles dosed by MDI and DPI is particle size distribution (PSD) [3]. This may vary depending on the chemical composition of the inhalation drug (the active substance and the excipient), but also on the temperature and humidity changes during the transport from the inhaler to the human respiratory tract. Measurement of the aerodynamic size of aerosol allow us to predict its deposition efficiency and behaviour in the lungs. The aerosol generated by inhalers is most often characterized by the particle mass size distribution measured by the cascade impactors [4, 5]. The disadvantage of using impactors is a highly time-consuming analysis and a low resolution of the size spectrum. In addition, the dynamics of volatile or pressurized aerosols (MDI) include problems that do not occur when characterizing solid or liquid particles alone. Another alternative is the measurement of particle size distribution using online spectrometers based on time-of-flight principle [6, 7]. The data obtained in realtime sampling compared to measurement of an aged aerosol better describes the circumstances that occur during clinical use of the inhalers. However, there are also many limitations on the use of TOF spectrometers, e.g. only a fraction of the particles is measurable, variable density with particle size required to calculate mass distribution, coincidence error when the concentration is high etc. [8] Mathematical models of particle deposition are useful for interpreting experimental data as well as making predictions for the conditions not covered by experiments. All developed models make simplifying assumptions on lung morphometry and use theoretical or empirical equations for deposition efficiency of airway segments. Among any others, two advanced and widely used models are those of the International Commission on Radiological Protection (ICRP) [9] and the National Council on Radiation protection and Measurement (NCRP) [10]. The aim of this study was to characterise in detail the PSD of three MDIs and one DPI described in Tab.1 during different RHs (laboratory conditions and 90% RH) and inspiratory flow rates 30, 60, and 90 lpm simulating conditions in the human respiratory tract. In addition, the main size modes were used to estimate the region of the particle deposition in the human respiratory tract using the deposition model of the International Commission on Radiological Protection. The respirability of the formulation allows drug to deposit in the target region (the tracheobronchial region) of the respiratory system (RS). Particle deposition in the upper airways or in the alveolar region may cause unwanted systemic effects.

Drug Delivery to the Lungs, Volume 29, 2018 - Number size distribution of particles dosed by MDI and DPI inhalers Experimental methods Four commercially available inhalers used to treat asthma and COPD were tested, three MDIs and one DPI. The list of inhalers together with manufacturer information is given in Table 1. To measure particle number size distribution an on-line spectrometer APS 3321 (TSI, USA) was used. Inhalation aerosol samples from individual inhalers were led through a stainless steel tubing simulating the geometry of the human respiratory tract - the first bend behind the oral cavity (Fig. 1). The total air flow rate through the inhaler was 30, 60 and 90 lpm, with 5 lpm sampled with the APS spectrometer.Size distributions were measured in 52 size channels in the range of 0.5 - 20 μm. All the above measurements were performed under laboratory conditions t = 25 ± 0.5 ° C and RH = 35 ± 5%, that were measured by using of Tinytag logger. The influence of increased relative humidity was measured under conditions t = 25 ± 0.5 ° C, RH = 90 ± 1% and flow rate 30 lpm. For these measurements, a humidifier connected to the cryostat and T/RH sensor were installed between the elbow and the isokinetic sampling for the APS spectrometer.

Table 1 - List of inhalers with manufacturer information Inhaler No.

Type

Active substance (µg/dose)

Excipient

Flutiform

MDI

Fluticasone propionate (50) Formoterol Fumarate Dihydrate (5)

Ethanol, Apaflurane

Fullhale

MDI

Salmeterol xinafoate (25) Fluticasone propionate (125)

Norflurane

Ventolin

MDI

Sulbutamol sulfate (120,5)

Norflurane

Spiriva

DPI

Tiotropium bromide (18)

Lactose monohydrate

The individual particle sizes from the measured distributions were used to estimate the proportion of the particle number deposited in three main regions of RS (UA, TB, AL) using the ICRP deposition model [10].

Inhaler

Flow meter

APS 3321

Vacuum pump

Fig. 1 – Experimental setup for PSD measurement at three different inspiratory flow rates (30, 60 and 90 l/min)

Results Number PSDs measured by APS spectrometer at different inspiratory flow rates are shown in Fig. 2 in the left column. Comparison of PSDs obtained under ambient condition and at higher relative humidity is also in Fig 2 in the right column. It is obvious, that we got bimodal distribution for Flutiform and Fullhale and monomodal distribution for one Ventolin and DPI Spiriva. The increasing inspiratory flow rate had a minimal effect on the position of the modes of the individual distributions. The differences in PSD, measured under laboratory conditions and at higher relative humidity, were minimal and did not affect the assumed probability of inhalant deposition. Number median aerodynamic diameters (NMAD) and geometric standard deviation (GSD) for PSDs under different experimental conditions are listed in Table 2. The number of replicate measurements was 3. In terms of deposition fraction, 3-5% of the number concentrations were deposited in the target tracheobronchial (TB) region. Compared to the literature, deposition fraction of aerosol mass in TB region was 8.1% for pMDI and 4.2% for DPI measured by in vitro experiments [11] and in the central airways about 10% for MDI predicted by the model [12].

Drug Delivery to the Lungs, Volume 29, 2018 – Lucie Ondráčková et al.

Laboratory condition

Comparison of laboratory condition and condition with 90% RH (30 lpm)

dN/dLogDp (%)

12 10

60 lpm

90 lpm

6 4 2 0

Fullhale

30 lpm lab. condition

30 lpm

dN/dLogDp (%)

Flutiform

30 lpm 90% RH

10 8 6 4 2

0.1

dp (µm)

100

0.1

dp (µm)

0.1

1 dp (µm)

dN/dLogDp (%)

4 2

0.1

30 lpm 60 lpm 90 lpm

100

dp (µm)

100

30 lpm lab. condition 30 lpm 90% RH

dN/dLogDp (%)

90 lpm

Ventolin

60 lpm

100

30 lpm lab. condition 30 lpm 90% RH

30 lpm 6

5 4 3 2 1

dp (µm)

dN/dLogDp (%)

4 2 0

0.1

dp (µm)

0.1

dp (µm)

30 lpm 60 lpm 90 lpm

100

dN/dLogDp (%)

Spiriva

0.1

100

30 lpm lab. condition

30 lpm 90% RH

4 2 0

0.1

dp (µm)

Figure 2 - Number particle size distributions of the individual inhalers measured by APS 3321

100

Drug Delivery to the Lungs, Volume 29, 2018 - Number size distribution of particles dosed by MDI and DPI inhalers Table 2 – Size distribution parameters obtained from APS 3321 under different measurement conditions

Flutiform Fullhale Ventolin Spiriva

30 lpm NMAD (µm) GSD 1,06 ± 0,13 1,99 ± 0,04 1,88 ± 0,07 1,76 ± 0,09 1,61 ± 0,02 1,64 ± 0,01 2,81 ± 0,21 1,69 ± 0,01

60 lpm NMAD (µm) GSD 1,03 ± 0,06 1,99 ± 0,02 1,76 ± 0,22 1,71 ± 0,06 1,56 ± 0,08 1,62 ± 0,02 2,03 ± 0,18 1,71 ± 0,03

90 lpm NMAD (µm) GSD 0,78 ± 0,06 1,90 ± 0,05 1,50 ± 0,35 1,74 ± 0,08 1,44 ± 0,05 1,60 ± 0,01 2,39 ± 0,11 1,67 ± 0,03

30 lpm, RH 90 % NMAD (µm) GSD 1,12 ± 0,11 1,85 ± 0,02 1,48 ± 0,42 1,64 ± 0,07 1,54 ± 0,04 1,53 ± 0,01 2,43 ± 0,21 1,74 ± 0,19

Discussion and conclusion The differences in PSD, measured under ambient conditions and at higher humidity, were minimal and did not affect the assumed probability of inhalant deposition. Slight movement of the mode to the left at higher humidity can be explained by adsorption of a thin film of water at the rough surface, particles became more spherical and it results into lower APS aerodynamic diameter. However, the measurements showed that the inhalation particles generated by the selected inhalers are not hygroscopic at RH of 90%. DF in the TB seems to be low, however according to the model, the respirable formulation has an optimal size distribution which allows the maximal possible deposition in the target region. When measuring inhalation aerosols using the APS spectrometer, it is important to bear in mind some limitations affecting the resulting shape of the measured distributions. The number of large particles (in our case around 2-3 µm) can be underestimated due to coincidence with small particles at high total concentrations. It is also important to mention that the experimental setup we used allows us to measure only the non-ballistic particle distribution, as we assume that particles of about 20 µm are evaporated. We are well aware of the deficiencies and imperfections of the APS 3321 spectrometer. Therefore we intend to compare it using parallel measurements by a Berner low pressure impactor that has been well characterized in our laboratory, concerning both it’s collection efficiency on individual stages, and particle bounces and inter-stage losses. Acknowledgement This work was supported by the MEYS project LTC17010 and COST-European Cooperation in Science and Technology, to the COST Action MP1404 (SimInhale). References 1

Virchow J C., Crompton G K, Dal Negro R, Pedersen S, Magnan A, Seidenberg J, Barnes P J: Importance of inhaler devices in the management of airway disease, Respir Med 2008; 102 (1): pp10-19.

Choräo P, Pereira A M, Fonseca J A: Inhaler devices in asthma and COPD--an assessment of inhaler technique and patient preferences, Respir Med 2014; 108(7): pp968 - 975.

Byron P R: Respiratory drug delivery, CRC Press, Boca-Raton; 1990.

Weda M, Zanen P, De Boer A H, Gjaltema D, Ajaoud A, Barends D M, Frijlink H W: Equivalence testing of salbutamol dry powder inhalers: in vitro impaction results versus in vivo efficacy, Int J Pharm 2002; 249: pp247-255.

Taki M, Marriott C, Zeng X-M, Martin G P: Aerodynamic deposition of combination dry powder inhaler formulations in vitro: a comparison of three impactors, Int J Pharm 2010; 388: pp40-51.

Mitchell J P, Nagel M W: Time-of-flight aerodynamic particle size analyzers: their use and limitations for the evaluation of medical aerosols, J Aerosol Med 1999; 12(4): pp217-240.

TSI Incorporated: Model 3321 Aerodynamic Particle Sizer® Spectrometer Rapid Tool for Accelerated Inhaler Development. TSI, USA 2008.

Dolovich M,: Measurement of particle size characteristics of meterd dose inhaler (MDI) aerosol , J Aerosol Med 1991; 4 (3): pp251-263.

ICRP, 1994: Human Respiratory Tract Model for Radiological Protection, ICRP Publication 66. Ann. ICRP 24 (1-3).

NCRP, 1997: National Council on Radiation Protection and Measurement, Deposition Retention and Dosimetry of inhaled radioactive substances, Report S.C. 57-2, NCRP, Bethesda.

Longest P W, Tian G, Walenga R L, Hindle M: Comparing MDI and DPI aerosol deposition using in vitro experiments and a new stochastic individual path (SIP) model of the conducting airways, Pharm Res 2012; 29, pp1670–1688.

Smyth H D C, Martonen T B, Isaacs K K, Hickey A J:. Estimation of particle deposition in the airways from different inhaler formulations using an In Silico model, KONA Powder Part J 2011; 29, pp107–117.

Drug Delivery to the Lungs, Volume 29, 2018 – Magda Swedrowska et al. Biopharmaceutics of (R)-roscovitine by Inhalation Magda Swedrowska1, Zachary Enlo-Scott1, Laurent Meijer2 & Ben Forbes1 1Institute

of Pharmaceutical Science, King’s College London,150 Stamford Street, London SE1 9NH, UK 2 ManRos Therapeutics, Centre de Perharidy, Roscoff, 29680, France

Summary A novel therapeutic agent (R)-roscovitine is under investigation for delivery by inhalation for the treatment of cystic fibrosis. Inhaled therapy has the potential to reduce systemic side effects, enhance drug availability (lower doses may be required for optimal effect) by delivering a drug directly to the target organ. However, the pharmacokinetics and safety of (R)-roscovitine after delivery to the lungs by inhalation have not yet been evaluated. This study aims to assess the biocompatibility and permeability of (R)-roscovitine in a human airway epithelial cell line to provide a preliminary non-clinical evaluation of the drugs inhalation biopharmaceutics before further testing using an isolated perfused lung (IPL) model. The bronchial epithelial cell line, Calu-3 was used as an absorption model. Different concentrations of (R)-roscovitine (up to 250 µM, exposure for 24 h) were evaluated for biocompatibility using the MTT assay. The absorptive transport of (R)-roscovitine (100 and 280 µM) was evaluated across polarised epithelial cell layers with the transepithelial electrical resistance (TEER) was measured to ensure the integrity of the cell monolayer. (R)-Roscovitine showed a moderate dose-dependent reduction of the metabolic activity of Calu-3 cells, but the half maximal inhibitory concentration (IC50) was not reached at the concentrations tested. There was no significant difference in the absorptive transport between 100 and 280 µM (R)-roscovitine (Papp = 5.6 ± 0.56 and 5.9 ± 0.72 × 10-5 cm/s, respectively) and TEER was not affected by the 2 h transport experiment. Based on physicochemical properties and the absorptive permeability of (R)-roscovitine in the Calu-3 respiratory epithelial cell drug transport model, the molecule is expected to be rapidly absorbed from the lungs. Further investigations will be performed using the IPL model. Key Message A human bronchial epithelial cell line was used to evaluate the biocompatibility and permeability of (R)-roscovitine, a drug candidate under investigation for delivery by inhalation as a novel treatment for cystic fibrosis patients. On the basis of the absorptive permeability, Papp = 5.6 ± 0.56 × 10-5 cm/s, (R)-roscovitine can be predicted to be rapidly absorbed from the lungs, similarly to propranolol in previous studies [1], [2]. IPL studies are being performed to test this hypothesis. Introduction (R)-Roscovitine acts as an inhibitor by competing with adenosine triphosphate (ATP) for binding to the active site of target enzymes and has been in phase II clinical trials as a drug candidate for the treatment of Cushing disease (Cedars-Sinai Medical Centre, Los Angeles, USA), various cancers (Cyclacel Pharmaceutical, Inc., UK) and rheumatoid arthritis (University of Edinburgh, UK). (R)-roscovitine is also considered as potential novel therapeutic agent for the treatment of cystic fibrosis (CF) [3], [4]. CF is a rare genetic disease caused by different mutations of the CF transmembrane conductance regulator (CFTR) gene. Patients suffering from CF develop life-threatening or debilitating lung complications including in many cases, chronic bacterial pulmonary infections and associated pulmonary inflammation. Due to complexity of the disease there is still no curative treatment available for CF patients, despite extensive research efforts [5], [6]. Delivery as an inhaled aerosol medicine has the potential to enhance drug availability for target engagement in the lungs. (R)-Roscovitine is a low molecular weight compound (Figure 1) with a low aqueous solubility, high tissue binding and rapid systemic metabolism making it promising candidate for achieving lung selectivity by obtaining prolonged and elevated lung concentrations compared to those in plasma and other organs. (R)-roscovitine has a pKa of 4.4 making the drug a weak mono-base. (R)-roscovitine is a lipophilic compound with partition coefficient (logP) of 3.2 and it complies with the ‘rule of five’ by Lipinski [7], [8]. Therapeutically, (R)-roscovitine offers novel treatment to CF patients through mechanism of action and targets which are not covered by existing drugs [4].

Figure 1 - Structure of the isomer of (R)-roscovitine.

100

Drug Delivery to the Lungs, Volume 29, 2018 - Biopharmaceutics of (R)-roscovitine by Inhalation Although (R)-roscovitine has been evaluated extensively pre-clinically and in human volunteers, this will be the first evaluation of biocompatibility and permeability of the compound in human airway epithelial cell culture system. Prediction of the inhalation biopharmaceutics will reduce the risk of failure due to poor pharmacokinetics or unexpected interactions during drug development. The main focus of the research thus far has been the prediction of the drug transport across the intestinal- and blood-brain barriers in-vitro and in-vivo models in animals and humans. The absorption kinetics from the lungs in relation have not been subjected to research. In the present study, the Calu-3 cell line was utilised to model the airway epithelial permeability barrier in vitro. This absorption model was established in the late 1990’s and has become a standard as human respiratory absorption model, as the cell line displays relevant morphology, forms tight junctions, provides a suitable polarised barrier and produces a mucociliary barrier when grown in air-liquid interface conditions. Additionally, the drug permeability in Calu-3 has been correlated with absorption from the lung in-vivo and ex-vivo [1], [9], [10]. The aim of this study was to evaluate the biocompatibility and permeability of (R)-roscovitine across human bronchial epithelial cell line.

Experimental Methods Cell culture The cells used for this work were Calu-3 cells, which are human lung epithelial cells derived from lung adenocarcinomas. Cells were cultured in 75 cm 2 tissue culture-treated flasks (Greiner bio-one) and grown in Dulbecco’s modified Eagle’s medium F-12 Ham (Sigma-Aldrich) supplemented with 10% (v/v) foetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Gibco), 1% (v/v) Non-essential amino acids (Sigma-Aldrich), and 5% (v/v) Penicillin-Streptomycin (Sigma-Aldrich). MTT Cell Viability Assay The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) viability assay is a colorimetric assay that uses the conversion of a yellow tetrazolium salt (MTT) into purple formazan crystals to measure cellular metabolic activity, which serves as a marker of cell viability. For this assay, Calu-3 cells were seeded at a density of 40,000 cells/well in 96-well plates (Costar) and cultured for 2 days in a humid cell culture incubator at 37˚C, 5% CO 2. The effect of different concentration of roscovitine (250 - 1 µM) was measured as a reduction in metabolic activity. Cells were were exposed to 100 µL of the compound at varying concentrations suspended in 2% FBS in DMEM solution incubated for 24 hours. After the exposure period, the medium was aspirated and replaced by 200 µl fresh 2% DMEM. MTT (50 µl; 5 mg/mL) was added to the wells and the plate was incubated for further 4 h. The medium was removed and the resulting intracellular formazon crystals were dissolved over 24 h in 100 µl of 10% SDS prepared in 1:1 water:DMF, after which the absorbance from the solubilized formazan was measured spectrophotometrically at an absorbance wavelength of 560 nm. The relative cell viability (% viability) was calculated as follows: Viability (%) = (A-S)/(CM-S) × 100

Equation 1.

Where A is the absorbance obtained for each concentration of the test drug, S is the absorbance obtained for positive control (1% v/v Triton-X) and CM is the absorbance obtained for untreated cells (negative control, DMEM alone). The latter reading was defined as 100 % cell viability. Permeability assay For permeability studies, the Calu-3 cells were seeded at a density of 2 × 10 5 cells/well on the porous polyester membrane inserts of the Transwell system with a 0.4 μm pore size and a surface area of 0.33 cm2, placed in 24well cell culture plates (Corning). Air-liquid interface (ALI) culture was established by removing the medium from the apical compartment 48 h after seeding. The medium in the basal compartment was changed every 2-3 days throughout the 14 days culture period to allow cells to form a confluent and functional mucociliary barrier. On the day of the experiment, the cell culture medium was removed and warm transport medium (Hanks’ Balanced Salt Solution (HBSS) supplemented with 10 mM HEPES, pH 7.4) at 37˚C was added: 0.2 mL to the apical chamber and 0.5 mL to the basolateral chamber, then the plate was incubated for 30 min to equilibrate. After equilibration, the pre-experimental Trans-Epithelial Electrical Resistance (TEER) reading was taken from all wells prior to treatment using an EVOM voltohmeter and STX2 electrode (World Precision Instruments). The HBSS was removed from all wells and replaced with 550 µl of HBSS in the basal compartment and 250 µl of drug solution in the apical compartment. Within, 1 min, 50 µl from both compartments were removed to establish the donor concentration (C 0). Samples (250 µL) were withdrawn from the receiver chamber at 30, 60 and 90 min. The volume withdrawn was replaced with 250 µL preheated fresh transport medium, which was corrected for further calculation. After each sample, the cell layer was returned to 37˚C incubator under stirring conditions (50 rpm). After 120 min, 100 µL and 250 µL samples were removed from both the receiver and donor chamber, respectively. The integrity of the monolayer was confirmed by measuring TEER. Collected samples were analysed using HPLC system.

101

Drug Delivery to the Lungs, Volume 29, 2018 – Magda Swedrowska et al. Permeability data analysis The apparent permeability coefficient (Papp. cm/s) for the roscovitine was calculated according to equation 2, derived from Fick’s first law: Papp=dQ/dt × (1/C0.A)

Equation 2.

Where dQ/dt is the gradient of the slop of flux versus time (µmoles/s), C 0 is the initial drug concentration applied in the donor chamber (µmoles/cm3) and A is the surface area of the Transwell® filter (cm2). HPLC method A Waters 2796 Separations Module HPLC was used, equipped with Waters 2489 Photodiode Array Detector. The column was Zorbax SB-CN (3.5 µm x 4.6 mm x 150 mm). Data were digitalized by Empower program (Waters, Elstree, UK) for chromatograms and integration. The UV detection wavelength was set at 305 nm. An isocratic mobile phase consisting of tetrahydrofuran and 25 mM phosphate buffer pH 2.6 (20:80 v/v) degassed and filtered was used in HPLC analysis. The flow rate was 0.9 mL/min [11] and injection volume was set to 50 µL. Samples were analysed in duplicate with run time of 12 min. The calibration curve was constructed by the standard (R)-roscovitine solution and it was used to quantify each sample peak obtained. Stock solution of (R)-roscovitine (5 mg/mL) was prepared and serial dilutions (100 – 1 µg/mL) were performed for construction of calibration curve. Results and Discussion The biocompatibility of varying concentrations of (R)- roscovitine with Calu-3 cells was tested by MTT assay after 24 h exposure to compound. (R)-Roscovitine showed a dose-dependent toxicity profile (Figure 2). The highest concentration (250 µM) decreased cell viability by 48.5% compared to control. (R)-Roscovitine concentration in the range of 0 to 5 µM showed a reduction of 5% in cell viability which was not statistically different compared to control. The reduction of 30% in cell viability was observed only above 30 µM, however an IC50 was not reached at the concentration range tested. The absorptive permeability of tested compound was measured in Calu-3 airway epithelial cell drug transport model. Beforehand, to measure any significant effect of (R)-roscovitine on Calu-3 cell monolayer integrity, TEER measurements were taken at the beginning (t=0 min) and at the end (t=120 min) of transport experiment conducted in absorptive direction. The integrity of monolayer was unaffected by the presence of varying concentrations of (R)roscovitine, as the TEER values did not decrease significantly even at the highest tested concentration compared to control (untreated cells). TEER values derived from subsequent transport experiments were expressed as an average (98.6 ± 5.1% compared to control) as no significant difference was observed for (R)-roscovitine. The recovery (mass balance) of (R)-roscovitine was > 93.2 ± 5.1% and cumulative drug transport vs time profiles were linear in all tested concentrations (R2> 0.99) (Figure 3a). The absorptive permeability of (R)-roscovitine was concentration independent (Figure 3b). The absorptive permeability of (R)-roscovitine was 5.6 ± 0.56 and 5.9 ± 0.72 × 10-5 cm/s at 100 µM and 280 µM, respectively.

Figure 2 - The effect of different concentrations of (R)-roscovitine on Calu-3 cells after 24 h of exposure using the MTT assay. Cell viability was calculated on the basis of the cells metabolic activity as percentage of the control (cells with medium only). The data represent the mean ± SD n=3; each experiment was performed in sextuplicate.

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Figure 3 - Absorptive transport of (R)-roscovitine. A, left graph) The cumulative drug transported in the absorptive direction vs time profiles of (R)-roscovitine 100 µM (red) and 280 µM (blue) across Calu-3 cell monolayers, B, right graph) Absorptive permeability of varying concentrations of (R)-roscovitine (100 and 280 µM) over 2h experiment across Calu-3 cell monolayers. Data represents mean ± SD from three independent experiments.

Conclusion (R)-Roscovitine reduced the metabolic activity of the Calu-3 cells, however an IC50 was not reached at the concentration range tested (up to 250 µM). The absorptive permeability was independent of concentrations used, 5.6 ± 0.56 and 5.9 ± 0.72 × 10-5 cm/s at 100 µM and 280 µM, respectively. The absorptive permeability of (R)roscovitine (Log P 3.24) was consistent with that expected from the physicochemical properties of the molecule and on the basis of similar investigations may be predicted to show lung absorption to that of propranolol (Log P 3.56). IPL studies are being performed to test this hypothesis. References [1] [2] [3] [4]

[5] [6] [7] [8] [9] [10] [11]

N. R. Mathias, J. Timoszyk, P. I. Stetsko, J. R. Megill, R. L. Smith, and D. A. Wall, “Permeability Characteristics of Calu-3 Human Bronchial Epithelial Cells: In Vitro - In Vivo Correlation to Predict Lung Absorption in Rats,” J. Drug Target., vol. 10, no. 1, pp. 31–40, Jan. 2002. F. Manford, A. Tronde, A.-B. Jeppsson, N. Patel, F. Johansson, and B. Forbes, “Drug permeability in 16HBE14o- airway cell layers correlates with absorption from the isolated perfused rat lung,” Eur. J. Pharm. Sci., vol. 26, no. 5, pp. 414–420, Dec. 2005. L. Meijer and E. Raymond, “Roscovitine and Other Purines as Kinase Inhibitors. From Starfish Oocytes to Clinical Trials,” Acc. Chem. Res., vol. 36, no. 6, pp. 417–425, Jun. 2003. L. Meijer, D. Nelson, V. Riazanski, A. G. Gabdoulkhakova, G. Hery-Arnaud, R. Le Berre, N. Loaëc, N. Oumata, H. Galons, E. Nowak, L. Gueganton, G. Dorothee, G. Rault, and L. Meijer, “Modulating innate and adaptative immunity by (R)-roscovitine: potential therapeutic opportunity in cystic fibrosis,” J Innate Immun, vol. 8, no. 4, pp. 330–349, 2016. L. J. Caverly, J. Zhao, and J. J. LiPuma, “Cystic fibrosis lung microbiome: Opportunities to reconsider management of airway infection,” Pediatr. Pulmonol., vol. 50, no. S40, pp. S31–S38, Oct. 2015. N. Rieber, A. Hector, M. Carevic, and D. Hartl, “Current concepts of immune dysregulation in cystic fibrosis,” Int. J. Biochem. Cell Biol., vol. 52, pp. 108–112, Jul. 2014. M. Vita, M. Abdel-Rehim, C. Nilsson, Z. Hassan, P. Skansen, H. Wan, L. Meurling, and M. Hassan, “Stability, pKa and plasma protein binding of roscovitine,” J. Chromatogr. B, vol. 821, no. 1, pp. 75–80, Jul. 2005. S. Wang, S. J. McClue, J. R. Ferguson, J. D. Hull, S. Stokes, S. Parsons, R. Westwood, and P. M. Fischer, “Synthesis and configuration of the cyclin-dependent kinase inhibitor roscovitine and its enantiomer,” Tetrahedron: Asymmetry, vol. 12, no. 20, pp. 2891–2894, Nov. 2001. B. Forbes, “Human airway epithelial cell lines for in vitro drug transport and metabolism studies,” Pharm. Sci. Technolo. Today, vol. 3, no. 1, pp. 18–27, Jan. 2000. C. Bosquillon, M. Madlova, N. Patel, N. Clear, and B. Forbes, “A Comparison of Drug Transport in Pulmonary Absorption Models: Isolated Perfused rat Lungs, Respiratory Epithelial Cell Lines and Primary Cell Culture,” Pharm. Res., vol. 34, no. 12, pp. 2532–2540, Dec. 2017. H. Sallam, A. T. El-Serafi, E. Filipski, Y. Terelius, F. Lévi, and M. Hassan, “The effect of circadian rhythm on pharmacokinetics and metabolism of the Cdk inhibitor, roscovitine, in tumor mice model,” Chronobiol. Int., vol. 32, no. 5, pp. 608–614, May 2015.

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Drug Delivery to the Lungs, Volume 29, 2018 - Benedicte Grosjean et al. Interpretation of manual actuation profiles from nasal unit-dose spray devices Benedicte Grosjean1, Gerallt Williams1, Fabien Adam1, David Chopard1, Pierre Schwartz1, Janick Cabiddu1 1Aptar

Pharma, Route des Falaises, Le Vaudreuil, 27100, France

Summary There has been increasing interest lately in Human Factors studies and these are becoming a critical part of aerosol product development. In this case study, a panel of ten volunteers each actuated six unit-dose devices filled with water. The displacement and force over time during the actuation were recorded. The data were analysed to understand qualitatively the curves with regard to the mechanical events that occur to deliver a spray. Two phases could be observed. A first one was very fast, a second one was much slower, during which the liquid enters narrow sections of the flow path within the device and the spray is emitted. The curves did not show neither constant force nor constant velocity as is obtained with an automated machine. However, the second phase would be the one to consider in order to define parameters that would impact the spray performance. Several critical output parameters, like the force to trigger the device, the delivery force, the velocities and the stroke length, were qualitatively analysed. The force to trigger the device and the stroke length were found to be very reproducible (less than 5%) while the delivery force and velocities were quite variable (around 20%). The former are governed by mechanical features in the device, while the latter depend strongly on the specific energy the user applies during actuation. The range of parameters can be used to define suitable levels for automated in vitro tests or for quality control tests. Key Message Verification of user ability to actuate an OINDP (Orally Inhaled and Nasal Drug Product) is a typical element of Human Factors studies. Hand actuation profiles are a valuable tool in understanding user interaction with the device. Here is an example showing how these profiles can be interpreted and how key parameters can be analyzed to justify automated actuation parameters in in vitro Quality Control tests. Introduction There has been increasing interest lately in Human Factors studies (1-4) and these are becoming a critical part of aerosol product development. Nasal unit-dose devices are currently used for the delivery of many aerosolized therapies for treatment such as migraine and opioid overdosing (5). Investigating human factor issues and understanding how patients manually actuate such device involves clarifying aspects such as user device interfaces, key parameters and assuring safe & effective device use by the users. There is also a need to justify and correctly apply quality control (QC) actuation parameters for assuring relevant specifications and QC control methods for drug delivery devices. This work is a case study looking into detailed actuation parameters for nasal unit-dose delivery devices and how these can help satisfy regulatory expectations with regard to human factor studies. The actuation profiles, i.e. the force and displacement over time, of a panel of persons were recorded during actual use of unit-dose devices in order to better understand the events that occur during an actual use of the device and which parameter may then be critical in the delivery of a spray with this type of device. Materials, method and results In order to gain as much insight as possible into the manual actuation profiles of the nasal devices by human volunteers, specific prototype R&D equipment was developed and employed comprising of force sensor (Flintec, range 0-200 N, resolution 0.01 N) and displacement sensor (TE Connectivity, range 0-50 mm, resolution 0.01 mm), see Figure 1, coupled with data capturing and display software. Calibrated parameters such as force and displacement over time could be measured and translated into representative actuation profile curves for each manual actuation.

Mini force sensor

Displacement sensor

Figure 1 - the component parts put together to capture and represent the output of the manual actuation measurements, mini force sensor, displacement sensor, R&D manual actuation apparatus and data interpretation software.

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Drug Delivery to the Lungs, Volume 29, 2018 - Interpretation of manual actuation profiles from nasal unit-dose spray devices The volunteers (n=10) used for the manual actuation studies were comprised of 5 males and 5 females in age groups 21-30 years old (n=5), 31-40 years old (n=3) and 51-60 years old (n=2) and they were suitably trained in the use of the manual data-capturing R&D apparatus. The unit-dose nasal devices employed for the study were Aptar Pharma standard items, which are currently used in marketed therapies such as Imitrex™, Imigran™, and Narcan™ etc. The test liquid used for this study was distilled water and a 100 µl dose was delivered from each nasal spray device.

Figure 2: Unit-dose liquid nasal spray device

The unit-dose spray devices do not need priming before use. The dose is delivered when the patient applies force to the button component. Once enough force is applied, the small plastic safety clips between the actuator and the body of the device (used for protecting the dose during transport & storage) are broken and the actuator and cannula moves towards the dose chamber. The cannula pierces the rubber stopper and accesses the liquid dose in the dose chamber. Further movement of the button upwards by the patient causes the liquid dose to be expelled via the nozzle generating a spray on exiting the device. The resulting manual actuation data captured and interpreted from the volunteers allowed for the deconstruction of the actuation profile to its various constituent parts related to device actuation phases as well as spray generation and delivery, see Figures 3 and 4.

Figure 3 – Manual actuation profile from a nasal spray unit-dose device: typical representation of the force versus the displacement

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D A B E

Figure 4 â&#x20AC;&#x201C; Manual actuation profile from a nasal spray unit-dose device: typical representation of the force and displacement versus time, with a zoom in the region where the peaks of force occur. The total duration from the mechanical bridge breaking point A to the end of the spray is typically 50 - 100 ms.

Phase A represents the first force applied by the patient and the start of the actuation. Phase B represents the breaking of the safety retention clip in the device to begin the actuation movement of the device, with an increase of force corresponding to the plunger breakloose. Phase C represents the piercing of the dose chamber to give access to the drug dose within. Phase D represents the expulsion of the small air bubble present within the dose chamber and the filling of the cannula with the liquid dose. Phase E represents the expulsion of the dose and the generation of the spray via the device nozzle exit and is the most critical part of assuring the delivery of a suitable and consistent spray for dosing. Some of the output parameters, which may be considered key to the understanding, and quantification of manual actuation parameters are detailed in Table 1.

Parameter (units)

Mean results

Std dev

Bridge breaking force (N)

26.5

1.1

Delivery force (N)

39.5

7.6

Actuation velocity before spray delivery (mm/s)

429

Spray phase velocity (mm/s)

12.9

0.3

Stroke (mm)

Table 1 â&#x20AC;&#x201C; Key output parameters from manual actuation profiles, n=60 (10 volunteers, n=6 per volunteer)

The force necessary to break the safety retention clips (bridge breaking force) is very reproducible between users and is due to the fact it is a controllable mechanical point. Not surprisingly, delivery force is quite variable from one person to another. This force depends on the energy each person applies the device, and how quickly they actuate the device. The velocity changes over the course of the manual actuation. One can observe that there are mainly two stages: before and during delivery out of the device. A first stage has a relatively high velocity, while the second stage has somewhat lower with a velocity in the order of 70 mm/s, during which the spray is generated and emitted. Both velocities are quite variable from one person to another.

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Drug Delivery to the Lungs, Volume 29, 2018 - Interpretation of manual actuation profiles from nasal unit-dose spray devices Discussion Qualitatively, the manual actuation profiles of the unit-dose device displayed two distinct phases with radically different velocities. A first mechanical phase showed a high velocity, and a second phase showed a much slower movement when the spray is emitted, due the passage of the fluid through a restricted section of the device. The profile is thus not linear, with neither a constant force nor a constant velocity, as would behave many automated machines used for QC testing of devices. The definition of parameters to be used in these types of machines will need to be established with a good understanding of what is critical to characterize device function and quality attributes. In this example, the velocity of interest will be the one obtained while the spray is emitted as it will be the phase where product performance characteristics such as spray droplet size may depend on this parameter. Quantitatively speaking, some parameters like the bridge breaking force and the stroke length are very reproducible. They are parameters controlled mechanically, thus they will not depend on the patient. However, this type of study is useful to check that a target population of patients can actually achieve the level of force required by the device. The other parameters, such as velocities before and during spray delivery and the delivery force, are found to be quite variable and depend on the user. This is explained by the fact that each person will apply a specific energy to the device actuation, which will lead to specific velocities and thus forces. The variability of these parameters is not necessarily critical. Their impact on the performance such as delivered dose or droplet size will need to be further assessed. In any case, the range obtained will support justification of the selection of suitable automated parameters even if the actuation mode cannot be exactly the same. Conclusion Measuring the detailed phases of manual actuation of drug delivery devices is essential in order to interpret and understand key parameters related to real-world use of the device. This in turn will assist in satisfying regulatory expectations for inclusion of comprehensive and complete human factor assessments in drug submission dossiers. This case study with nasal unit-dose devices describes how to gather and interpret the different phases of manual actuation which can then allow one to develop quality control methods coupled with suitable control equipment and with appropriate specifications and controls for realistic in vitro performance testing.

References 1.

US FDA, Human Factors Studies and Related Clinical Study Considerations in Combination Product Design and Development, Draft Guidance for Industry and FDA Staff, Feb 2016.

US FDA, Final Rule-Current Good Manufacturing Practice Requirements for Combination Products (ยง21CFR4).

US FDA, Final Rule-Quality System Regulation (ยง21CFR820).

US FDA, Final Rule-Current Good Manufacturing Practice Requirements for Combination Products (ยง21CFR4).

Williams G, Suman J: Repurposing CNS Drugs: Opportunities and Challenges via the Nose, RDD Asia, Goa, India, 2016, p 37-44.

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Drug Delivery to the Lungs, Volume 29, 2018 – Michael Y.T. Chow et al. Albumin as an alternative dispersion enhancer for inhalable siRNA spray dried powders Michael Y.T. Chow,1 Philip C.L. Kwok2, Hak-Kim Chan2 & Jenny K.W. Lam1 Department of Pharmacology & Pharmacy, LKS Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong 2 Sydney Pharmacy School, Faculty of Medicine and Health, Pharmacy and Bank Building A15, The University of Sydney, Camperdown, NSW 2006, Australia 1

Summary Respiratory diseases such as asthma or infections are often attributed to the (over)expression of disease-causing genes. Exploiting RNA interference (RNAi), local administration of RNAi molecules has become an attractive treatment strategy. Our previous study has shown that leucine could promote the aerosol performance of otherwise poorly dispersed siRNA powders to achieve a fine particle fraction (FPF) of 44.4%. However, the need of a large amount of leucine (50% w/w) and its hydrophobic nature posed limitations such as restricting siRNA load and solubility issues. In this study, we investigated human serum albumin (HSA) as an alternative dispersion enhancer to leucine in improving the aerosol performance of spray dried siRNA powders. At 2% w/w siRNA, the highest siRNA concentration in an inhalable solid formulation ever reported, we prepared and characterised siRNA powders cospray dried with HSA (5% or 35% w/w) and mannitol as the bulking agent. The solutions were prepared at 1% or 2% w/v solute concentrations. The result of cascade impactor assay showed that at 35% HSA and at 1% solute concentration, the resultant powder exhibited a FPF of 68.9%. Scanning electron microscopy images revealed that particles with higher HSA composition exhibited less regular shape with wrinkled surfaces. The median physical size of the particles was between 1.5 to 2.2 µm as measured by laser diffraction. The structure of siRNA was also preserved as shown in gel retardation assay. This study demonstrated that HSA could serve as an effective dispersion enhancer of spray dried siRNA powders. Key Message Human serum albumin was shown to serve as an effective dispersion enhancer to improve spray dried powders of siRNA for inhalation. The spray dried formulation containing 1% w/w siRNA and 35% w/w human serum albumin exhibited the highest FPF of 68.9%. Introduction Many respiratory diseases are associated with the expression of disease-causing genes, and/or the expression of foreign genes from pathogens, with examples including the overexpression of inflammatory regulators in chronic asthma and chronic obstructive pulmonary disease (COPD), as well as the expression of viral proteins during influenza infection. RNA interference (RNAi) represents an effective mechanism to transiently silence the expression of genes as targeted by RNAi mediators such as small interfering RNA (siRNA) and micro RNA (miRNA). Local administration of the RNAi molecules has become an attractive therapeutic strategy to combat these diseases [1-3] . Our group has previously reported the spray dried powder formulation of siRNA for inhalation using leucine as dispersion enhancer [4]. While the formulation exhibited satisfactory aerosol performance with FPF of 44.4%, the need of large amount (50% w/w) of hydrophobic leucine poses solubility issue and limits the siRNA load in the formulation. The current study explored the use of human serum albumin (HSA) as an alternative dispersion enhancer to prepare inhalable spray dried powder formulations of siRNA. It is hypothesized that during the drying process, the macromolecular albumin competed with siRNA to stay on the particle surface, modify the surface and reduce cohesiveness, leading to improved dispersion and delivery efficiency. Although HSA is yet to be approved as excipient for inhalation product, it has been frequently used in different studies as an excipient for inhalable formulations [5, 6]. It has also been inhaled in human subjects as a radiolabeled aerosol for the study of aerosol distribution in lungs [7, 8]. Experimental Methods Materials Human serum albumin (HSA) (Sigma-Aldrich; Poole, UK), mannitol (Pearlitol 160C; Lestrem, France) and model siRNA targeting monocyte chemoattractant protein 1 (MCP-1; Integrated DNA Technologies; IL, US) (Antisense sequence: 5′- CCG UAA UCU GAA GCU AAU TT-3′) was dissolved in ultrapure water in the compositions as shown in Table 1. Spray drying A Büchi B-290 Mini spray dryer in tandem with a B-296 dehumidifier (Büchi Labortechnik AG; Postfach, Switzerland) was used to spray dry the solution using the conditions as stated in Table 2. The powder formulations were stored in glass vials inside a desiccator at ambient temperature. The mass of powder recovered from the collecting vial relative to the initial mass input constituted the production yield.

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Table 1 – Compositions of siRNA/HSA/mannitol formulations used for spray drying.

Composition (% w/w)

Formulations

siRNA

2S5H-1 2S5H-2

HSA

Mannitol

Solute Concentration (% w/v) 1 2

2S35H-1 2S35H-2

1 2

Table 2 – Spray drying conditions used for the preparation of siRNA/HSA/mannitol dry powder formulations.

Parameters

Inlet temperature

Feed rate

Atomization

Aspiration

Configuration

80°C

1.4 ml/min

742 L/h; Air

35 m /h

Closed loop; Suction mode

Aerosol performance study and particle size measurement To study the in vitro aerosol performances of the formulations, the Next Generation Impactor (NGI; Copley Scientific, UK) was used with a low resistance capsule-based inhaler as the dispersing device (Breezhaler; Novartis Pharmaceuticals, Hong Kong). Approximately 6 mg of powder was used for each dispersion. The dispersion flow rate was 90 L/min and duration was 2.7 seconds. Since mannitol was the most abundant component in all the formulations, the amount of powder deposited on each compartment of the NGI was quantified with mannitol using high performance liquid chromatography upon recovering the powders with ultrapure water. An ion-exchange ligand-exchange column (Agilent Hi-Plex Ca, 7.7 × 50 mm, 8µl; Agilent Technologies, CA, USA) was used to resolve mannitol which was detected with a refractive index detector (G1362A; Agilent Technologies). The emitted particle (powders that exited the inhalers) and the fine particle (particles with aerodynamic diameter under 5 µm) fraction, with respect to the total recovered powder from the assay, was calculated. The primary particle size of the powder formulations was determined using laser diffractometry (Mastersizer 2000; Malvern Instruments Ltd., Worcestershire, UK) and the dispersion pressure was 4 bar. Gel retardation assay and morphology study Gel retardation assay was performed to study the integrity of siRNA in spray dried powders. Particle morphology was visualized using scanning electron microscopy (Hitachi S-4800 FEG Scanning Electron Microscope; Hitachi; Tokyo, Japan). Results and Discussion From our previous studies, it was demonstrated that siRNA could remain intact at the present spray drying condition [4] . The present study aimed to investigate HSA as an alternative dispersion enhancer to leucine in spray dried powder, with siRNA load up to 2% by mass. This is the highest siRNA concentration in a solid formulation that has been reported to date [9]. It is anticipated that there is an enrichment of HSA on the particle surface during spray drying due to its large molecular size and low mobility, and hence modifying the particle surface properties. Two levels of HSA and two levels of solute concentration were selected. Under the current spray drying condition, the outlet temperature reached was 50°C. The highest production yield achieved was 69% (2S35H-2) and the lowest was 46.5% (2S35H-1) (Table 3). The EF and the FPF results are shown in Figure 1. All formulations achieved an EF of above 85%, suggesting the formulations could be readily dispersed. The use of a high level of HSA resulted in a higher FPF, despite only the difference between 2S5H-1 and 2S35H-1 was significant (one-way ANOVA, Tukey’s post-hoc test, p < 0.05). The latter also gave the highest FPF value of 68.9%. The effect of solute concentration on aerosol performance was not conclusive. As a comparison, the FPF of siRNA dry powder using 50% w/w leucine as dispersion enhancer has been reported to be 44.4%. Here, a better aerosol performance could be achieved with a lower amount of dispersion enhancer, potentially allowing a higher siRNA load in the formulation.

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Figure 1 – Emitted fraction (EF) and fine particle fraction (FPF) expressed as percentage relative to recovered dose. EF was similar among all formulations. A higher FPF was achieved for formulations with higher HSA composition. Data were presented as mean ± standard deviation (n = 3). The results were analysed by one-way ANOVA followed by Tukey’s post-hoc test (* represents p < 0.05).

The image of gel retardation assay was shown in Figure 2. The integrity of siRNA in spray dried powders was preserved. Our preliminary data of in vitro transfection study also showed that the bioactivity of the siRNA was successfully maintained (data now shown). Furthermore, it was confirmed that siRNA remains in its free form without complexing with HSA. Although the integrity of HSA in the spray dried powder was not evaluated here, it has been reported that proteins could be spray dried at high temperature (100-130 ºC) without compromising their structures and bioactivity10, 11. The particle surface morphology of the formulations was visualised by SEM and was shown in Figure 3. At low HSA level, the particles were spherical in shape with minor ridges on the surface. As HSA level increased to 35% w/w, the particles transformed from spherical to irregular shape with wrinkled surface. It is hypothesised that the wrinkles reduced the effective surface area between particles for interparticle interactions, thus reducing particle aggregation and promoting powder dispersibility.

Figure 2 – Gel retardation assay (non-denaturing 15% PAGE) of reconstituted samples after spray drying. DS and SS represent unformulated siRNA and the antisense RNA (a single strand RNA) from siRNA as control, respectively. The gel image was sliced and reordered from the same retardation assay.

Figure 3 – Scanning electron microscope images of the formulations at 5,000× magnification. Scale bar equals 10 µm.

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The 10th, 50th (median) and 90th percentile of the particle size distribution is shown in Table 3. Overall, all the powder formulations prepared had a median diameter between 1.5 to 2.2 µm, and all their 90th percentile were under 4 µm. It was noticed that particles prepared from feed solution with higher feed solute concentration were larger in size, because a higher solute concentration implied a higher solid content and thus a larger particle for droplets with the same volume. The span measured the dispersity in size distribution and is defined as (D 90 – D10) / D50. All four formulations exhibited a narrow distribution as indicated by the small span value. Table 3 – Production yield and particle size distribution of the powder formulations (unit: µm). Values in brackets refer to standard deviation. n = 3

Sample

Yield (%)

D10

D50

D90

Span

2S5H-1

65.0

0.93 (0.008)

1.77 (0.023)

3.20 (0.041)

1.29 (0.003)

2S35H-1

46.5

0.77 (0.012)

1.52 (0.002)

2.80 (0.024)

1.34 (0.022)

2S5H-2

68.2

0.96 (0.002)

2.00 (0.017)

3.78 (0.030)

1.41 (0.005)

2S35H-2

69.0

1.05 (0.003)

2.17 (0.011)

3.99 (0.018)

1.35 (0.004)

Conclusions The present study investigated the use of HSA as alternative dispersion enhancer to improve the aerosol performance of (naked) siRNA spray dried powders for inhalation. Compared to previous reports, where a substantial amount of leucine was used, it was shown here that powders with higher FPF could be achieved with HSA despite a lower amount was employed. The best performing formulation was 2S35H-1, with FPF value of 68.9%. Future study direction includes further increasing siRNA load in the formulations to clinical relevant levels, demonstrating bioactivity of siRNA using in vitro or in vivo transfection experiment, and long term stability of the formulations. References 1

Qiu Y, Lam JK, Leung SW, and Liang W. Delivery of RNAi Therapeutics to the Airways-From Bench to Bedside. Molecules. 2016;21.

Liao W, Dong J, Peh HY, Tan LH, Lim KS, Li L, and Wong WF. Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases. Molecules. 2017;22.

Moschos SA, Usher L, and Lindsay MA. Clinical potential of oligonucleotide-based therapeutics in the respiratory system. Pharmacol Ther. 2017;169:83-103.

Chow MYT, Qiu Y, Lo FFK, Lin HHS, Chan HK, Kwok PCL, and Lam JKW. Inhaled powder formulation of naked siRNA using spray drying technology with l-leucine as dispersion enhancer. Int J Pharm. 2017;530:40-52.

Woods A, Patel A, Spina D, Riffo-Vasquez Y, Babin-Morgan A, de Rosales RT, Sunassee K, Clark S, Collins H, Bruce K, Dailey LA, and Forbes B. In vivo biocompatibility, clearance, and biodistribution of albumin vehicles for pulmonary drug delivery. J Control Release. 2015;210:1-9.

Choi SH, Byeon HJ, Choi JS, Thao L, Kim I, Lee ES, Shin BS, Lee KC, and Youn YS. Inhalable self-assembled albumin nanoparticles for treating drug-resistant lung cancer. J Control Release. 2015;197:199-207.

Fleming J, Conway J, Majoral C, Tossici-Bolt L, Katz I, Caillibotte G, Perchet D, Pichelin M, Muellinger B, Martonen T, Kroneberg P, and Apiou-Sbirlea G. The Use of Combined Single Photon Emission Computed Tomography and X-ray Computed Tomography to Assess the Fate of Inhaled Aerosol. J Aerosol Med Pulm Drug Deliv. 2011;24:49-60.

Elsadek B and Kratz F. Impact of albumin on drug delivery - New applications on the horizon. J Control Release. 2012;157:428.

Chow MY and Lam JK. Dry Powder Formulation of Plasmid DNA and siRNA for Inhalation. Curr Pharm Des. 2015;21:38543866.

Rohani SSR, Abnous K, and Tafaghodi M. Preparation and characterization of spray-dried powders intended for pulmonary delivery of Insulin with regard to the selection of excipients. Int J Pharm. 2014;465:464-478.

Varshosaz J, Hassanzadeh F, Mardani A, and Rostami M. Feasibility of haloperidol-anchored albumin nanoparticles loaded with doxorubicin as dry powder inhaler for pulmonary delivery. Pharm Dev Technol. 2015;20:183-196.

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Drug Delivery to the Lungs, Volume 29, 2018 – Benedict Benque et al. Understanding the motion of rotating hard shell capsules in dry powder inhalers Benedict Benque1 & Johannes Khinast1 1

Institute of Process and Particle Engineering, Graz University of Technology, Inffeldgasse 13, Graz 8010, Austria

Summary In certain dry powder inhalers (DPI), the discharge and dispersion of carrier-based or carrier-free formulations from a pierced and rotating capsule is crucial for the delivery of small drug particles to the patient’s peripheral airways. The dispersion of drug particles depends on turbulence and on particle collisions in the inhaler. The present study examined the three-dimensional motion and the resulting wall collisions of a size three capsule in an Aerolizer® inhaler using high-speed photography. A correlation between the capsule-wall collision rate and the capsule’s rotation was observed. The capsule collided predominantly with the mouthpiece grid, frequently in a bouncing motion. Computational Fluid Dynamics (CFD) and Discrete Element Method (DEM) simulations were used to investigate how this motion affects the powder behavior and powder release from the capsule. The CFD simulations were conducted using ANSYS Fluent and neglected the lateral and vertical displacement of the capsule. The transient air flow through the pierced capsule was shown to be the dominant force on small drug particles, while the motion of the larger carrier particles was governed by inertial forces. DEM simulations were conducted using XPS® (eXtended Particle Simulation) to study the influence of capsule-wall collisions on the discharge of cohesive and non-cohesive powder from the rotating capsule. The collisions were found to increase the powder release of non-cohesive and slightly cohesive material, while decreasing it in the case of highly cohesive materials. Key Message Collisions between the rotating capsule and the inhaler wall were found to decrease the powder discharge of highly cohesive powders, suggesting that this inhaler design is most suited for non-cohesive and slightly cohesive powders. Introduction To be able to reach narrow lung regions, the drug particles for dry powder inhalation have to be very small (aerodynamic diameters of 1-5 µm) 1,2. Particles in that size range have a high surface free energy and tend to stick to each other or to other surfaces. This results in poor flowability and a tendency to be retained in the inhaler device 3–5. These small particles are often blended with carrier particles in the range of 50-500 µm to improve flowability and dispersion 3,5. Another advantage of carrier-based systems is that the handling of the API during manufacturing becomes easier, which is especially relevant for low dose formulations 6. The attractive force between the active pharmaceutical ingredient (API) and the carrier particles has to be large enough to ensure that the powder can be easily handled, but low enough so the API detaches from the carrier during inhalation 4. The powder blend is aerosolized in the DPI device, where the API particles are dispersed from the carrier particles so that the small API particles may enter the lungs, while the larger carrier particles impact in the mouth and upper airways 6. This process is highly dependent on particle size and flow properties, formulation, drug-carrier interaction, respiratory flow rate and design of the inhaler 7. The two primary dispersion mechanisms are the air flow and the particle-particle and particle-wall collisions 8. The detachment due to the airflow around the carrier particle can be further characterized according to the underlying detachment mechanism, which can be rolling, sliding or lift-off. The forces acting on an attached API particle at different positions on a carrier in an air flow have been determined by Cui and Sommerfeld using Lattice Boltzmann Method simulations 9. Comparing them with the measured Van der Waals forces showed that in a typical inhaler flow, the probability of lift-off is negligible. The probabilities of rolling and sliding were described in dependence of the carrier particle Reynolds number 1,9. The rotation around the primary axis has been experimentally studied before for size three capsules in an Aerolizer® geometry and was found to be proportional to the flow rate. The rotation axis as well as the center point of the rotation were found to show large variations, and the collisions of the capsule with the inhaler walls were described to reduce the angular velocity 10. So far, the collisions of the capsule with the inhaler have not been studied extensively in computational or experimental studies. In this study, the rate of collisions and the collision velocity as well as the collision locations were determined from high-speed photography data, and the information obtained from the experiments was used for computational studies regarding the impact on the powder discharge from the capsule.

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Drug Delivery to the Lungs, Volume 29, 2018 - Understanding the motion of rotating hard shell capsules in dry powder inhalers Experimental methods A high-speed camera (Integrated Design Tools Os8) was used to analyze the motion of the filled capsule in an AerolizerÂŽ inhaler. The capsule was a CyclocapsÂŽ Salbutamol 200 Âľg, which is a size three gelatine capsule. All analyses were conducted using the open source video analysis software Tracker by Douglas Brown. The AerolizerÂŽ inhaler geometry only allowed for two types of rotary motion of the capsule due to the geometric restraints of the swirl chamber. The primary rotation axis is roughly parallel to the axis of the inhaler tube, the secondary axis is the capsuleâ&#x20AC;&#x2122;s symmetry axis (see figure 1). A transparent inhaler was employed to be able to capture the capsuleâ&#x20AC;&#x2122;s motion in the swirl chamber. A pump (Copley Scientific SCP5) attached to the inhaler outlet provided a constant ďŹ&#x201A;ow rate. Flow rates of 50-100 L/min were studied. The camera took 5000 frames per second, with LED lighting providing sufficient light for the short exposure times. To obtain the angular velocity around the primary rotation axis, full rotations of the capsule were counted. The rotation of the capsule around the secondary axis was studied by tracking the imprint on the capsule. Figure 1: inhaler

AerolizerÂŽ

The rate of capsule-wall collisions was determined by tracking sudden changes in the capsuleâ&#x20AC;&#x2122;s motion, and the change of direction revealed the estimated location of 11 the l collision. This methodology is similar to the work of Behara et al. on other inhaler devices . The collision velocity was obtained by tracking the capsule movement in the image series. The collision velocity was defined to be the velocity normal to the wall prior to the collision. Discrete Element Method The particle motion was simulated using the discrete element method (DEM) from the commercial software eXtended Particle System (XPS), which uses the parallelization technology of CUDA provided by Nvidia graphics cards 12. The discrete element method (DEM) solves the equations of Newtonâ&#x20AC;&#x2122;s second law of motion, i.e. the total force and the torque acting on a particle are calculated from the contact forces and the gravitational force 13. The software uses the soft-particle approach where the force between particles is calculated from the overlap between the particles. Two colliding particles are assumed to deform, with the spring stiffness correlating the contact force and the deformation 13â&#x20AC;&#x201C;15. For simplicity, a model carrier powder of monodisperse 200 Âľm particles and a Gauss-distributed carrier powder (Âľ=200 Âľm, Ď&#x192;=80 Âľm) were simulated. A linear macroscopic elasto-plastic adhesive (MEPA) contact model was employed to simulate the cohesion between carrier particles. The loading parameter k2 was chosen to be 2.5 times higher than the virgin loading parameter k1, and the adhesive parameter đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC;đ??´đ??´đ??´đ??´â&#x201E;&#x17D; was three times higher for the particleparticle interaction than for the particle-wall interaction. These values yielded a powder behavior similar to the one observed in experiments. Only the large carrier particles were simulated, since the small drug particles are mostly attached to the carriers while in the capsule. To evaluate the relevance of collisions on the emptying of powders from a rotating capsule, a capsule geometry was introduced that was rotated with a constant rotational speed of 4500 rpm (equivalent to a flow rate of 100 L/min) 10. The collisions between the capsule and the inhaler grid were approximated by a repeated movement pattern. Computational fluid dynamics The computational fluid dynamics software ANSYS Fluent was used to simulate the transient fluid flow. It solves the spatially and temporally discretized momentum and continuity equations. The turbulence was modeled using a Large Eddy Simulation (LES) approach. The capsule motion was approximated by implementing a stator-rotor model as described by Coates et al. 10. This method limited the capsule motion to a rotation around a fixed axis with a fixed center point is allowed. The angular velocity was prescribed as a constant value. The particles were ignored for these calculations, as their volume fraction in the capsule amounted to only 3.5%.

Figure 2: Capsule-wall collision rate

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Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; Benedict Benque et al. Results The high-speed photography analysis confirmed previous findings by Coates et al. by showing a linear relation between the rotation rate around the primary axis and the flow rate 10. The axis of rotation was not constant over time. Instead, its angle and center position changed. The rotation rate around the secondary axis was highly irregular and did not exhibit any clear dependence on the flow rate. Most capsule collisions took place with the part of the inhaler that leads to the outlet, sometimes in a rapid succession of collisions. The air flow carried the capsule until it collided with the walls, bounced off, and was carried back to collide again. Only few of the observed collisions took place with the sides of the chamber, and even fewer with the bottom. The collision rate varied significantly but correlated with the inhaler flow rate (see figure 2), with collision speeds mostly around 2 m/s and up to 3 m/s. In CFD simulations of dry powder inhalers with rotating capsules, the fluid flow in the capsule is usually neglected. The location and size of the pierced holes for the simulation geometry were established from experiments. Simulating the flow in the rotating capsule showed air velocities of up to about 10 m/s. Figure 3 shows the major forces acting on spherical lactose particles in the capsule. The relative velocity between the particle and the fluid was assumed to be the maximum observed air velocity in the capsule, 10 m/s. The drag force was calculated as đ??šđ??šđ??ˇđ??ˇ = đ?&#x2018;?đ?&#x2018;?đ??ˇđ??ˇ đ??´đ??´đ?&#x2018;&#x192;đ?&#x2018;&#x192; đ?&#x2018;Łđ?&#x2018;Ł2 đ?&#x153;&#x152;đ?&#x153;&#x152;đ?&#x2018;&#x201C;đ?&#x2018;&#x201C;

Figure 3: Dominant forces on particles in the rotating capsule

spherical

with the drag coefficient calculated according to Brown and Lawler 16, which is valid at đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026; < 2 â&#x2C6;&#x2014; 105 : đ?&#x2018;?đ?&#x2018;?đ??ˇđ??ˇ =

24 0.407 (1 + 0.15 đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019; 0.681 ) + đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026; 1 + 8710 đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019; â&#x2C6;&#x2019;1

The characteristic inertial force was set to

đ??šđ??šđ?&#x2018;&#x2013;đ?&#x2018;&#x2013; = đ?&#x2018;&#x161;đ?&#x2018;&#x161;đ?&#x2018;&#x192;đ?&#x2018;&#x192; đ?&#x2018;&#x17D;đ?&#x2018;&#x17D;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;&#x; = đ?&#x2018;&#x161;đ?&#x2018;&#x161;đ?&#x2018;&#x192;đ?&#x2018;&#x192; đ?&#x153;&#x201D;đ?&#x153;&#x201D;2 đ?&#x2018;&#x;đ?&#x2018;&#x;đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?

Figure 3 shows that the drag force is not negligible except for very large particles. All drug particles and small carrier particles can be assumed to experience significant drag. The gravity force is two orders of magnitude below the inertial force due to the capsule rotation and can be neglected. DEM simulations of powders of varying cohesiveness showed the importance of the capsule-wall collisions for the powder release from the capsule. The effects were studied for monodisperse 200 Âľm particles and Gaussdistributed particles (Âľ=200 Âľm, Ď&#x192;=80 Âľm), with very similar results. The collision rate was set to 176.4 1/s, which was the average of the observed collision rates at a flow rate of 100 L/min in the experiments. Since the number of collisions in any but the main direction were negligible in the experiments, they were also disregarded in the simulations. The collision speed (the maximum translational velocity of the capsule just prior to the collision) was set to 2 m/s. Without cohesion, the collisions decreased the capsule retention by about 30%. At low cohesions, the collisions still decreased the final capsule retention noticeably. At moderate and high cohesions, the capsule retention was higher with collisions (see figure 4).

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Drug Delivery to the Lungs, Volume 29, 2018 - Understanding the motion of rotating hard shell capsules in dry powder inhalers Discussion and Conclusion

Figure 4: Capsule retention at different cohesions for monodisperse powder (dP=200 µm)

For small drug particles in the capsule, the drag from the air flow was found to be the dominating force, while inertial forces dominate the behavior of carrier particles. At a flow rate of 100 L/min, the Reynolds number of a 100 µm carrier particle in the capsule reached about 30. According to the detachment probabilities suggested by Cui, 5 µm-sized drug particles are not expected to detach from the carrier through lift-off or sliding in the capsule, although a low probability of detachment through rolling is given 4. According to Cui, no sliding occurred for 5 µm drug particles on a 100 µm carrier with untreated surfaces at Re<140, and for treated surfaces sliding began at a Reynolds number of roughly 70. Rolling detachment was found to occur at Reynolds numbers as low as 30 for surface treated material and around 100 for untreated carriers 4.

It was found that the effect of the capsule-wall collisions on the powder discharge depends on the cohesiveness of the system. The capsule retention was up to almost 50% lower with collisions as compared to simulations without collisions for moderately cohesive material. For more cohesive material the collisions increased the retention by up to 45%. This suggests that for very cohesive blends, the powder discharge could be inhibited by the capsule translation and collision along the rotation axis. References 1.

Cui Y, Schmalfuß S, Zellnitz S, Sommerfeld M, Urbanetz N. Towards the optimisation and adaptation of dry powder inhalers. Int J Pharm [Internet]. 2014;470(1–2):120–32.

Tong ZB, Yang RY, Yu AB. CFD-DEM study of the aerosolisation mechanism of carrier-based formulations with high drug loadings. Powder Technol. 2016;

Tong Z, Kamiya H, Yu A, Chan HK, Yang R. Multi-scale modelling of powder dispersion in a carrier-based inhalation system. Pharm Res. 2015;32(6):2086–96.

Cui Y. Application of the Lattice Boltzmann Method for Analysing the Detachment of Micro-Sized Drug Particles from a Carrier Particle. Martin-Luther-Universität Halle-Wittenberg; 2016.

Healy AM, Amaro MI, Paluch KJ, Tajber L. Dry powders for oral inhalation free of lactose carrier particles. Adv Drug Deliv Rev [Internet]. 2014 Aug;75:32–52.

Pilcer G, Wauthoz N, Amighi K. Lactose characteristics and the generation of the aerosol. Adv Drug Deliv Rev [Internet]. 2012;64(3):233–56.

Islam N, Gladki E. Dry powder inhalers (DPIs)-A review of device reliability and innovation. Int J Pharm. 2008;360(1–2):1–11.

Yang J, Wu C-Y, Adams M. Three-dimensional DEM-CFD analysis of air-flow-induced detachment of API particles from carrier particles in dry powder inhalers. Acta Pharm Sin B [Internet]. 2014;4(1):52–9.

Cui Y, Sommerfeld M. Forces on micron-sized particles randomly distributed on the surface of larger particles and possibility of detachment. Int J Multiph Flow [Internet]. 2015;72:39–52.

10.

Coates MS, Fletcher DF, Chan H-K, Raper JA. The role of capsule on the performance of a dry powder inhaler using computational and experimental analyses. Pharm Res. 2005;22(6):923–32.

11.

Behara SRB, Farkas DR, Hindle M, Longest PW. Development of a High Efficiency Dry Powder Inhaler: Effects of Capsule Chamber Design and Inhaler Surface Modifications. Pharm Res [Internet]. 2014 Feb 16;31(2):360–72.

12.

Boehling P, Toschkoff G, Dreu R, Just S, Kleinebudde P, Funke A, et al. Comparison of video analysis and simulations of a drum coating process. Eur J Pharm Sci. 2017;104(September 2016):72–81.

13.

Radeke CA, Glasser BJ, Khinast JG. Large-scale powder mixer simulations using massively parallel GPUarchitectures. Chem Eng Sci [Internet]. 2010;65(24):6435–42.

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Böhling P. Modeling of non-spherical particles in the Discrete Element Method (DEM) simulations. Graz University of Technology; 2014.

15.

Siegmann E, Jajcevic D, Radeke C, Strube D, Friedrich K, Khinast JG. Efficient Discrete Element Method Simulation Strategy for Analyzing Large-Scale Agitated Powder Mixers. Chemie-Ingenieur-Technik. 2017;89(8):995–1005.

16.

Brown PP, Lawler DF. Sphere Drag and Settling Velocity Revisited. J Environ Eng. 2003 Mar;129(3):222– 31.

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Drug Delivery to the Lungs, Volume 29, 2018 –Sarah Zellnitz et al. Impact of Budesonide Particle Shape on Uptake by Respiratory Cells and Macrophages Sarah Zellnitz1, Marie-Theres Müller1,2 & Eleonore Fröhlich1,2 Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, Graz, 8010, Austria Center for Medical Research, Medical University of Graz, Stiftingtalstraße 24, Graz, 8010, Austria 1

Summary For administering active pharmaceutical ingredients (APIs) to the lung, a particle size of 1-5 µm is desirable. Consequently, during dry powder inhaler (DPI) formulation development usually a processing/size reduction step of the API is needed. The most common processing technique is milling, however, spray drying has shown to be a suitable alternative. Both techniques lead to inhalable sized particles but other properties like shape and/or solidstate can vary. Previous work on salbutamol sulphate has shown that for highly water soluble APIs the distinct particle properties generated during processing do not affect the dissolution behaviour. However, cellular uptake and permeability as well as in vitro aerosolization performance could be influenced [1]. In order to get more insight on the impact of particle properties (especially the shape, on the biological action of the inhaled particles) budesonide, an API with lower solubility, was chosen as model API for the present work. Jet milled and spray dried budesonide formulations were evaluated for dissolution, permeation, and preferential uptake by epithelial cells compared to macrophages. Spray dried spherical particles showed lower respirable fractions and dissolved slower compared to jet milled needle/rod shaped particles. However, permeability and cellular uptake were higher for the spray dried API. This could possibly be due to the action of one or more uptake mechanisms; i.e. passive diffusion and/or pinocytosis.

Key Message API particles with intermediate solubility may exploit two routes for uptake and permeation across the respiratory epithelium, diffusion and non-specific adsorptive pinocytosis. As shape of the API particles has an influence on particle uptake, the technique selected for particle processing appears important.

Introduction In order to reach the lung, the raw active pharmaceutical ingredient (API) of an inhalable formulation ideally needs to be processed to a size of 1 µm to 5 µm, as only particles of such small size are able to reach the deep lung. This can be done by techniques such as milling or spray drying. Both these techniques are suitable to generate inhalable sized particles; however, particles with disinct properties regarding their shape, size, density and/or solid-state are produced. For example, spray drying of salbutamol sulphate leads to X-ray amorphous spherical particles whereas jet milling to needle shaped predominantly crystalline particles [2]. This study presents a follow up study to work that was shown at DDL 2017, where the influence of API shape on the aerosolization performance and biological effects in the lung were analysed specifically related to salbutamol sulphate [1]. The previous results indicated that differences in salbutamol sulphate properties lead to different results concerning aerosolization performance. Spray dried spherical API particles overall showed lower fine particle fractions (FPFs) compared to the jet milled counterpart, when mixed with a lactose model carrier [1]. However, no difference regarding dissolution profiles was obtained, as after 2 minutes full dissolution was observed. Cellular uptake studies suggested that the needle-shaped particles were ingested by macrophages to a lower extent. However, the extremely fast dissolution contradicted against a prominent role of particle shape and size because it was reported that only 20-40% of 2 µm particles had been ingested by macrophages after 2 minutes of incubation [3]. Consequently, in the present study an API with lower solubility was tested in order to evaluate the influence of shape on cellular uptake. The model API used in this study was budesonide, a synthetic glucocorticoid, frequently used in the long-term treatment of asthma and COPD (chronic obstructive pulmonary disease). In order to investigate the effect of particle shape on biological effects in the lung, permeability studies and cellular uptake studies were performed with distinct cell types: alveolar epithelial cells and macrophages. Additionally, adhesive mixtures were prepared with jet milled and spray dried budesonide particles and lactose as model carrier in order to evaluate the aerodynamic performance and dissolution behaviour of fractions collected from impaction inserts of a next generation impactor (NGI). This study complements the data presented last year by investigating a different model API with lower solubility, and the impact of API shape on biological effects in the lung is considered.

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Drug Delivery to the Lungs, Volume 29, 2018 - Impact of Budesonide Particle Shape on Uptake by Respiratory Cells and Macrophages Materials and Methods Particle engineering Budesonide, used as model API was purchased from Sigma Aldrich GmbH (Munich, Germany) and engineered to the inhalable size using jet milling (Spiral Jet Mill 50 AS, Hosokawa Alpine AG, Augsburg, Germany) and spray drying (Nano Spray Dryer B-90, Buchi Labortechnik AG, Flawil, Switzerland). Budesonide was fed manually into the spiral jet mill; the injection pressure was set to 6 bar and the milling pressure to 3 bar. Spray drying conditions were adapted from Boraey et al. [4]. A 1.5% (w/w) ethanolic solution (EtOH/Aqua pur., 75/25, w/w) of budesonide was sprayed through a 7 µm nozzle at an inlet temperature of 120 °C with an intensity of 40% and a drying gas flow rate of 110 L/min. Particle characterization Particles were characterized with respect to particle size (laser diffraction, HELOS/KR system, Sympatec, Clausthal-Zellerfeld, Germany) and surface morphology/shape (Scanning electron microscopy (SEM), Zeiss Ultra 55, Zeiss, Oberkochen, Germany). In vitro aerodynamic performance coupled with dissolution Alpha-lactose monohydrate (Lactohale 100, DFE Pharma, Goch, Germany) was selected as model carrier for the present study and adhesive mixtures with 2% (w/w) API content of either spray dried or jet milled budesonide particles were produced in a tumble blender TC2 (Willy A. Bachofen Maschinenfabrik, Muttenz, Switzerland). Mixing time was 60 minutes at 60 rpm, followed by a sieving step through a 400 µm sieve and subsequent blending for 30 minutes at 60 rpm. After blending, 10 samples were taken and the budesonide content in samples was determined with a validated HPLC method. The homogeneity of the mixtures was expressed by the relative standard deviation (RSD) of the mean sample API content. Deposition in the lung and dissolution in lung lining fluid are important parameters for the effectiveness of an inhalable mixture. The in vitro studies obtaining predictors for these values were performed via Next Generation Impactor (NGI, Copley Scientific, Nottingham, United Kingdom), according to the European Pharmacopoeia. For each NGI experiment four capsules were filled manually with 25 mg of each formulation and discharged via the Cyclohaler® (PB Pharma GmbH, Meerbusch, Germany). The budesonide content in each part of the impactor was quantified using a validated HPLC method. For the Dissolution experiments, the stage where most budesonide was deposited in standard NGI experiments was selected (Stage 3 for jet milled budesonide and Stage 2 for spray dried budesonide) and adjusted with an impaction insert. NGI dissolution tests were performed (n=3) based on a method previously described1, displaying more physiologically relevant dissolution conditions. The dissolution was started in 55 mL simulated lung fluid ((SLF) composed of PBS (Phosphate Buffered Saline, Lonza, Verviers, Belgium) + 0.02% (w/v) DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, TCI Deutschland GmbH, Eschborn, Germany) and 500 µl samples were taken immediately (0 minutes), and at 2, 5, 10, 20, 40, 60, 120 and 180 minutes. The total dissolution volume was kept constant, by replenishing with 500 µL SLF at each sampling time. The API content in all samples was analyzed using a validated HPLC method and the emitted dose (ED), the fine particle fraction (FPF), the mass median aerodynamic diameter (MMAD), and the amount of budesonide dissolved over time were calculated. Permeability and cellular uptake studies Biological cell studies were performed according to procedures described by Zellnitz et al [1]. For permeability studies Calu-3 bronchial epithelial cells, common models for the determination of pulmonary apparent permeability coefficient (Papp) values [5] were used. Cell uptake studies were performed using human A549 alveolar epithelial cells, murine DMBM-2 and J774 macrophages and primary human macrophages isolated from buffy coat residues obtained from the Department of Blood Group Serology and Transfusion Medicine of Medical University of Graz (Auenbruggerplatz 3, Graz, Austria). Results and Discussion SEM images show that spherical particles were generated with spray drying (Figure 1A) and more cubic particles were produced with jet milling (Figure 1B). Particle size analysis demonstrated that both engineering techniques resulted in inhalable sized particles, x50 = 1.75 µm for jet milled particles and 1.63 µm for spray dried budesonide. Moreover, the sauter mean diameter (SMD) of the differently engineered particles is in a comparable range (1.07 µm for jet milled, and 1.28 for spray dried budesonide). As generally needle shaped particles have a higher surface to volume ratio, the comparable SMD allows to attribute results mostly to particle shape.

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Figure 1 - SEM images of spray dried (A) and jet milled (B) budesonide particles (image width 57.16 µm)

After blending for both mixtures, the deviation from the mean API content was below 5%, indicating a homogenous distribution of API over the carrier surface.

Table 1 summarizes the parameters obtained with the NGI; the results show that the FPF for jet milled budesonide is higher compared to the FPF for spray dried budesonide, 14.45% and 10.36% respectively (significant different, p<0.05, 2-tail t-test for equal variances). The same trend was observed in the previous study for salbutamol sulphate (FPF for jet milled API and spray dried API, 27.7% and 12.49%, respectively) [1]. However, the difference in FPF for jet milled and spray dried particles was less pronounced compared to salbutamol sulphate, where the jet milled API resulted in double the FPF of spray dried material. This could possibly be explained by the cohesive nature of budesonide where entrainment of higher forces, or ternary agents are often necessary to properly disperse the API [6,7] . Table 1 –

Assessment of aerodynamic performance (FPF, ED, MMAD) for formulation containing spray dried or jetmilled budesonide and Lactohale 100 (n=6, mean ± SD)

Sample

LH100 + Budesonide jet milled LH100 + Budesonide spray dried

FPF / % 14.45 ± 1.72 10.36 ± 3.47

ED / % 84.15 ± 2.95 85.69 ± 5.75

MMAD / µm 3.15 ± 0.24 4.61 ± 0.51

As in the previous salbutamol sulphate investigation [1], the ED was in the same range for blends containing jet milled or spray dried API, approximately 85% regardless of the API used. This again indicated that API detachment from the carrier is more inefficient for spray dried API particles compared to the jet milled API, as the same quantity leaves the inhaler, but the FPF is lower for spray dried material. Again, this could possibly be explained by the MMAD of the differently engineered API particles. The MMAD for jet milled budesonide is smaller than the MMAD of spray dried budesonide. The larger MMAD of the latter could indicate the formation of particle agglomerates that are not dispersed or not entirely dispersed during inhalation. Further, the MMADs for jet milled as well as spray dried budesonide are larger compared to the MMADs of salbutamol sulphate, although the initial particle size is slightly lower for engineered budesonide particles [1]. This could further be attributed to the more cohesive nature of budesonide compared to salbutamol sulphate [8]. B

Figure 2 - Dissolution curve (A, amount of budesonide dissolved over time) and permeation across Calu-3 monolayers (B) of jet milled and spray dried budesonide from stage 2 and stage 3, respectively, of the NGI (n=3, mean ± SD)

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Drug Delivery to the Lungs, Volume 29, 2018 - Impact of Budesonide Particle Shape on Uptake by Respiratory Cells and Macrophages Results for the dissolution (Figure 2) showed a slightly different dissolution behaviour between the two formulations. Spray dried budesonide dissolved slower compared to jet milled budesonide. However, the final API content dissolved after 180 minutes is comparable. The larger MMAD could play a role in the different behaviour and explain the slower dissolution for spray dried material. Moreover, compared to salbutamol sulphate, were jet milled and spray dried API rapidly dissolve in SLF (almost after 2 minutes nearly everything is dissolved [1]), budesonide dissolves more slowly over time. Despite the slower dissolution in the time frame used for permeation studies (120 minutes), permeability values were higher for spray dried than for jet milled budesonide (Figure 2B). A possible explanation may be that in addition to passive diffusion of partially dissolved budesonide particles, permeation by transcytosis can increase the amount of budesonide in the acceptor compartment, resulting in slightly higher P app values. Additionally, the cellular content of budesonide is expected to be higher for spray dried budesonide; as was seen for all epithelial cells (A549 and Calu-3) and macrophages (J774, DMBM-2, PBMC) (Figure 3). The fact that both Papp values and cellular content are similar when the particles were dissolved in DMSO (Dimethyl sulfoxide) supports the hypothesis that differences are caused by particle parameters.

Figure 3 – Cellular uptake for jet milled and spray dried budesonide by different cell types (n=3, mean ± SD)

Transcytosis is a well-known process for macromolecules and occurs via receptor-binding. Unspecific, adsorptive endocytosis of particles has been studied in detail for nanoparticles as a strategy for targeted drug delivery. The extent of cellular uptake is linked to various particle properties, including shape [9]. Spherical particles performed better for most cells, because uptake was independent of the orientation of the particles, while orientation was important for irregularly formed particles and particles with different overall shape characteristics (e.g. rods, disks, squares, etc.). As seen in Figure 1, jet milled budesonide has a non-spherical, more irregular shape, and even for a partially dissolved particle, orientation may play a role in cellular uptake. Conclusion and Outlook It may be suggested that APIs with intermediate solubility are most efficiently transported across the respiratory barrier because they may easily undergo passive diffusion after dissolution and non-specific adsorptive pinocytosis of partially dissolved API for transport. For APIs with minimal solubility dissolution may be too slow to generate particles that can be ingested by non-specific adsorptive pinocytosis. To determine whether the findings for budesonide are API-specific or can be generalized other APIs with similar and lower solubility should be investigated. References 1

Zellnitz S, Zellnitz L, Roblegg E, Fröhlich E: Impact of Salbutamol Sulphate Particle Properties on Biological Effects in the Lung (Abstract). Presented at: Drug Delivery to the Lung 2017 Conference, Edinburgh, United Kingdom, December 6-8 2017, pp. 274–7.

Pinto JT, Radivojev S, Zellnitz S, Roblegg E, Paudel A: How does secondary processing affect the physicochemical properties of inhalable salbutamol sulphate particles ? A temporal investigation, Int J Pharm 2017; 528 (1–2): pp416–28.

Paul D, Achouri S, Yoon Y, Herre J, Bryant CE, Cicuta P: Phagocytosis Dynamics Depends on Target Shape. Biophys J 2013;105(5): pp1143–50.

Boraey MA, Hoe S, Sharif H, Miller DP, Lechuga-Ballesteros D, Vehring R: Improvement of the dispersibility of spray-dried budesonide powders using leucine in an ethanol-water cosolvent system. Powder Technol 2013; 236: pp171–178.

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Shur J, Harris H, Jones MD, Kaerger JS, Price R: The role of fines in the modification of the fluidization and dispersion mechanism within dry powder inhaler formulations. Pharm Res 2008; 25(7): pp1631–40.

Kinnunen H, Hebbink G, Peters H, Huck D, Makein L, Price R: Extrinsic lactose fines improve dry powder inhaler formulation performance of a cohesive batch of budesonide via agglomerate formation and consequential co-deposition. Int J Pharm 2015; 478(1): pp53–9.

Kaialy W, Nokhodchi A: The use of freeze-dried mannitol to enhance the in vitro aerosolization behaviour of budesonide from the Aerolizer®. Powder Technol 2016; 288: pp291–302.

Liu Y, Tan J, Thomas A, Ou-Yang D, Muzykantov VR: The shape of things to come: importance of design in nanotechnology for drug delivery. Ther Deliv 2012;3(2): pp181-94.

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Drug Delivery to the Lungs, Volume 29, 2018 – Selma Chraibi et al. In silico Prediction of Pharmacokinetic Parameters after Cisplatin Intravenous and Endotracheal Administration Using GastroPlusTM Software Selma Chraibi1, Jessica Spires2, Karim Amighi1 & Nathalie Wauthoz1 1

Laboratory of Pharmaceutics and Biopharmaceutics, Faculty of Pharmacy, Université libre de Bruxelles (ULB), Boulevard du Triomphe, B-1050 Brussels, Belgium, schraibi@ulb.ac.be 2Simulation Plus, Inc., 42505 10 th Street West, Lancaster 93534, United States of America (USA)

Summary Introduction. Governmental health agencies encourage the development of in silico models that will bring in vitro data closer to in vivo data in a shorter time. GastroPlus TM (Simulation Plus, Lancaster, USA) is software that can simulate lung physiology to predict in vivo behaviour relying on in vitro data. The aim of this work was to use this tool to develop a cisplatin (CIS) model that can predict pharmacokinetic (PK) parameters after intravenous (IV) or endotracheal (EN) administration of a CIS solution or a CIS-based dry powder for inhalation (CIS-DPI). These simulations will help to develop new inhalable treatments against lung cancer. Methods. In vivo data came from the study made by Levet et al where a CIS-DPI and a CIS solution were administered to mice at 1.25 mg/kg using the EN or IV routes[1]. PK parameters were generated from the in vivo data using the PKPlusTM module (Simulation Plus, Lancaster, USA) and were compared to the GastroPlusTM simulated results generated using only in vitro data. The pulmonary permeability (PP) and the systemic absorption rate (SAR) were optimized to develop a pulmonary model that best fits the in vivo data. Results. Default parameters provided by GastroPlusTM were suitable in obtaining IV and PK parameters that were close to the in vivo data. After adjusting PP and SAR, both the EN administration of CIS solution and CIS-DPI agreed with the pulmonary in vivo data. Conclusion. GastroPlusTM can predict CIS in vivo behaviour after IV or EN administration of CIS solution or CIS-DPI. Key Message We succeeded in developing a pulmonary model that allows GastroPlus TM to predict CIS PK parameters after intravenous and endotracheal administrations of a CIS solution and a CIS-based dry powder for inhalation. This finding will help to develop optimized CIS-DPIs against lung cancer more quickly. Introduction Nowadays, pharmaceutical development and manufacturing aims to develop high quality products as quickly as possible using the latest techniques. Developing an in vitro-in vivo correlation using an in silico tool is one of the most promising strategies for achieving this goal[2]. GastroPlusTM software (Simulation Plus, Lancaster, USA) helps to adapt the drug formulation by predicting its likely in vivo behaviour in the human body using in vitro data only. GastroPlusTM has an Additional Dosage Routes Module (ADRMTM), which has a nasal-pulmonary module that can simulate solution and powder distribution after pulmonary administration. This module considers four pulmonary compartments, namely the extra-thoracic, thoracic, bronchiolar and alveolar-interstitial compartments. These are connected to the stomach, to the systemic compartment and/or to the lymph in the model. This module takes into account specifications for the compound (i.e. pulmonary permeability, PP and systemic/lymph absorption rate, SAR) and the specific physiology of each compartment, along with its enzymes and transporters. The compound that was selected is cisplatin (CIS). This is one of the most widely used drugs in chemotherapy against lung cancer[3]. However, CIS is also well-known for its nephrotoxicity, requiring massive hydration of patients before, during and after intravenous administration[3]. Considering both the benefit and the toxicity of CIS, the use of the pulmonary route to deliver the drug directly into the lung has become one of the most promising therapeutic strategies[1]. This administration route concentrates the drug in the lungs and reduces the amount of CIS that goes to the bloodstream and causes nephrotoxicity. Consequently, the efficacy of CIS can be higher and systemic side effects limited. The aim of our work was to develop a pulmonary model that predicts CIS pharmacokinetic (PK) parameters considering only in vitro data. This simulation could help to develop more quickly a more advanced CIS formulation presenting a suitable PK profile to treat lung cancer more efficiently. Materials and methods In vitro and in vivo data The in vivo data came from the study made by Levet et al where a CIS-DPI and a CIS solution were administered to mice at 1.25 mg/kg using the endotracheal (EN) or intravenous (IV) routes. CIS-DPI was prepared by suspending CIS in isopropanol at 5% (w/v) and then the particle size was reduced using an Emulsiflex C5 high-pressure homogenizer (Avestin Inc., Ottawa, Canada). Then, D-α-Tocopherol poly(ethylene glycol) 1000 succinate (TPGS) was added to the CIS suspension at 2% (w/v) before being spray dried by a B-290 Mini Spray Dryer (Buchi Labortechnik AG, Flawil, Switzerland). The composition, drug content and dissolution profiled are reported in Table 1. Platinum quantification and in vitro dissolution profiling were performed using a validated electrothermal atomic absorption spectrometry (ETAAS) method and an in vitro dissolution test adapted for inhalation products [5].

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Drug Delivery to the Lungs, Volume 29, 2018 - In silico Prediction of Pharmacokinetic Parameters after Cisplatin Intravenous and Endotracheal Administration Using GastroPlusTM Software Table 1. Theoretical composition, measured platinum content determined by ETAAS and the in vitro released percentages from the dry powder for inhalation (DPI)1.

DPI

Percentage release vs time (% w/w)

Theoretical composition (%w/w)

Platinum content (%w/w ± SD, n=9)

5 min

CIS/TPGS 95/5

95.6 ± 2.6 %

93.6 ± 8.6%

97.1 ± 0.6%

100.0 ± 0.0%

CIS administration was performed via the IV or EN route using a CIS solution (EN-SOL) or a CIS-DPI (EN-DPI) to CD-1 female mice]. Briefly, CIS solution was prepared in saline and was then administrated at 1.25 mg/kg via IV (200 µl by tail vein) or EN (50 µl using a MicroSprayer© Aerolizer - model 1A-1C for mouse (Penn-Century Inc., USA)) route. Secondly, to deliver a 1.25 mg/kg dose of CIS-DPI, the CIS-DPI was first mixed with a dry diluent (spray-dried mannitol-Leucine (90:10 w/w)) and then administered using a DP-4M Dry Powder InsufflatorTM for a mouse (Penn-Century Inc., USA). Converting in vivo data into PK parameters using PKPlus TM The in vivo data that illustrate the evolution of the plasma concentration vs time (Figure 1) after IV, EN-SOL and EN-DPI administrations of 1.25 mg/kg of CIS was loaded into the PKPlusTM module. The PKPlusTM was able to convert the in vivo data into PK parameters using non-compartmental but also one-, two- or threecompartment models. Then, it selected the compartment that best fitted the in vivo data. These parameters were then considered as the observed values derived from the experimental in vivo data and were compared to the values simulated by GastroPlusTM. Furthermore, the selected IV PK parameters were used for the EN administration of SOL and DPI to describe the CIS distribution and clearance when it arrives in the bloodstream from the lung.

Figure 1. Plasma concentration vs time profiles of CIS after an IV, an EN administrations of a CIS solution and CIS-DPI of 1.25 mg/kg of CIS in mice[3].

In silico prediction using GastroPlusTM We used the GastroPlusTM v.9.5 (SimulationPlus Inc., Lancaster, USA) with ADRMTM to perform the in silico simulations. Compound characteristics The molecular formula (Cl2H6N2Pt), the molecular weight (300.05 g/mol) and the logD (-2.19 at pH=7) of the drug were entered into the selected compound tab. The dosage form and the initial dose were selected depending on the ongoing simulation (IV or pulmonary (PL); PL: Solution, PL: Powder). The initial dose was adapted to the mean weight of the mice. The solubility value (2.53 mg/mL) was found in the Merck Index[6]. We used the default values provided by GastroplusTM for the mean precipitation time (900 sec), and the drug particle density values (1.2 g/mL). The diffusion coefficient (0.768 x 105 cm2/s) was estimated by GastroPlusTM, relying on CIS molecular formula. The default effective permeability was kept. Loaded files To compare the calculated results to the observed ones, the experimental in vivo plasma-concentration-time profiles were loaded for the IV, EN-CIS and EN-DPI formulations. These data were used only for comparison. The software did not take them into account to run the simulation. The experimental values were then automatically plotted graphically results. Furthermore, we loaded the in vitro dissolution profile vs time for the DPI simulation. The GastroPlusTM used this profile to perform the in vivo DPI-simulation. ADRM: nasal-pulmonary module The nasal-pulmonary module developed by SimulationPlus TM was used for the EN-SOL and EN-DPI. The animal model, called “physiology”, was switched to mice when the default lymph volume (1.985×10-3mL) and the total lung volume (0.49987 mL) values were kept. Regarding to the parameters related to the drug, “the compound” parameters, the lymph transit was set to 0 as the default value given by GastroPlus TM. The “pulmonary deposition model” describes the percentage deposited in each compartment . It was defined for each formulation based on the drug recovery in the lung published by Levet et al.[1].The actual Pt content was quantified on the lung lobes 2 minutes after administration and compared to the theoretical emitted dose from the

The DPI was fractionated on a fast screening impactor filter (fraction below 5 m) and packaged on a watch glass covered with a PTFE and polycarbonate membrane and a PTFE mesh screen in simulated lung fluid (400 ml, 37.0 0.2°C, pH= 7.35  0.05) under 50 rpm of stirring (mean  SD, n=3)[4].

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Drug Delivery to the Lungs, Volume 29, 2018 – Selma Chraibi et al. device (EN-SOL: 21  9%, EN-DPI: 35  22%)[1]. The drug recovery values were entered directly into the bronchiolar and alveolar-interstitial compartments (50% each). Values for all the characteristics related to the pulmonary module were set up by default. These characteristics included the PP, metabolic clearance, SAR, bounding to mucus or cells, and physiologic characteristics of each compartment. To develop a model that best fits the CIS formulations, two pulmonary parameters – the PP and the SAR – were optimized for the EN-SOL administration before being transposed to the EN-DPI form. Each of these two pulmonary parameters was decreased or increased by a factor of 10 separately (PP: from 10 -6 to 106, SAR: from 4.82.10-5 to 4.82.105). Then, the PP and the SAR were both changed using the same factor. As soon as the PK parameters were reached, the pulmonary parameters were considered as optimized and the simulation results were named “optimized values”. Results and discussion

Figure 2. Plasma concentration vs time profile simulated by GastroPlusTM using default (blue) and optimized (red) parameters after IV (A) and EN administrations of a CIS solution (B) and CIS-DPI (C) compared to the in vivo data (grey dots).

The aim of our work was to develop an in silico model that best fits the CIS-DPI. The first step was to assess an IV model to check whether the simulation results matched the experimental data. As shown in Figure 2.A, the default values corresponded to all the in vivo experimental plotted values. Furthermore, the Area Under the Curve (AUC) were similar for both default and observed values (5.42 g.h/mL vs 6.31 g.h/mL), meaning that the curves are alike. However, the Tmax is slightly different (0.08 h vs 0.00 h) for the calculated values (Figure 2.A). This 5-minute shift is explained by the fact that this timing corresponds to the time required to take the samples from the mice during the experiment. This can also explain why the C max is slightly higher for the default values than for the observed ones (1.20 g/mL vs 0.87 g/mL). Considering the standard deviation (SD) described by Levet et al [3], the simulated Cmax is in the observed range (0.87  0.042 g/mL). The similarity between the curves is illustrated in similar PK parameters, showing that our model is supported by the software and that the pulmonary simulation can be conducted. To simplify the process, the pulmonary simulation was first assessed with a CIS-solution and only after with the CIS-DPI, which was administered using the EN route, as explained previously. In this case, the software did not start by taking into account the drug dissolution from the powder, but started with its systemic distribution. The default values illustrated in Figure 2.B and Figure 2.C show that the observed Cmax is not reached by the default simulation for EN administration of a CIS solution and a CIS-DPI (EN-SOL: 0.03 g/mL vs 0.17 g/mL and EN-DPI: 0.05 g/mL vs 0.13 g/mL). Besides, a longer Tmax is observed (EN-SOL: 0.50 h vs 1.70 h and EN-DPI: 0.50 h vs 1.60 h). Nevertheless, it seems clear that from 8h, the curves meet the 24h and 48h experimental data for both pulmonary formulations. This showed that GastroPlusTM was able to manage the clearance from the blood compartment, but the permeability from the lungs to plasma is underestimated when the default pulmonary parameters are used. This underestimation leads to a longer time to reach a lower blood plasma concentration, which explains why the Cmax and the Tmax are both different from the default values. The assessment of the pulmonary parameters was previously reported by Salar-Behzadi et al with their study on inhaled budesonide[7]. They reported that better results were obtained when the pulmonary parameters were adapted to the specific drug. During our optimization, we noticed that if only one parameter was changed, the result remained the same. This means that the Cmax, Tmax, and AUC were not affected (data not shown). This result can be explained by the fact that even if a drug can easily cross the pulmonary membrane (characterized by a high PP), it can reach the bloodstream only if the SAR is high enough. Consequently, both pulmonary parameters were changed. The PP is 10-fold higher (10-6 vs 10-5) and the SAR 100-fold higher for this case (10-3 vs 10-5) (Table 2). This result is not surprising because CIS is a small drug (300.05 g/mol) compared to others. CIS permeability is high when the chloride concentration is high. For this drug, 85% is present in its dichloride form when the chloride concentration is equal to 110 mM (extracellular physiological compartment). This neutral charge form makes the diffusion across the lipid membranes easier [8]. The drug molecule is reported to use a passive diffusion pathway as well as active transport mediated by numbers of transporters, mainly copper transporter 1, ATP7A and ATP7B, organic cation transporters and multidrug and toxin extrusion proteins (MATEs)[9]. As a complement, the PP could be verified using in vitro tests on Calu-3 cells. As soon as the PP and SAR were higher, the Cmax was higher and the Tmax was lower. These results mean that a larger amount of CIS reached the bloodstream from the lungs faster. The optimized curves illustrated in Figure 2.B and Figure 2.C after EN administration of a SOL and DPI show a much better fit to the experimental data than the default curves. This is also illustrated with the PK parameters: the optimized C max is more than 3-fold higher with the optimized pulmonary parameters than with the default ones (EN-SOL: 0.11g/mL vs 0.03 g/mL and EN-DPI:

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Drug Delivery to the Lungs, Volume 29, 2018 - In silico Prediction of Pharmacokinetic Parameters after Cisplatin Intravenous and Endotracheal Administration Using GastroPlusTM Software 0.17g/mL vs 0.05 g/mL). This leads to a better evaluation of the Tmax, which is lower than in the default simulation (EN-SOL: 0.10 vs 1.70 h and EN-DPI: 0.16 vs 1.60 h). Consequently, the optimized PK parameters after EN-SOL administration were closer to the observed values than the default ones. Nevertheless, the Cmax is slightly lower than the observed value (0.11 g/mL vs 0.17g/mL), but is still in the observed range (0.17  0.06 g/mL)[3]. Also, the Tmax is lower than observed, but is still closer to the observed values than the default values. On the other hand, the C max was slightly higher for the EN-DPI administration but remains in the SD range (0.13  0.05 g/mL). The Tmax was also slightly higher during this optimized simulation, but was in the same range as the T max after the EN-SOL administration. Nevertheless, it is important to note that the initial lung half-life is low (EN-SOL: 5.0 min and EN-DPI: 2.6 min). This result means that both CIS-DPI and CIS solution are absorbed very quickly, as reported by Levet et al [1], and that the simulated values are consistent with the in vivo data. GastroPlusTM showed that the Cmax after EN administration of CIS solution and CIS-DPI are close, as reported by Levet et al[1]. The software also showed that the powder takes a little more time to be dissolved before being recovered in the bloodstream. This may be something that the GastroPlusTM indicates and that can barely be observed in vivo because of the experimental difficulties at the first dosing times, as explained before. It also shows that the AUC is higher with the EN-DPI than with the EN-SOL (1.71 g.h/mL vs 1.05 g.h/mL), which is also similar to the experimental data. Moreover, comparing the optimized pulmonary results (EN-SOL and EN-DPI) to the IV results, there is no doubt that the plasma Cmax is much higher after an IV administration than after EN administration of CIS solution or CIS-DPI (1.20 g/mL vs 0.11 and 0.17 g/mL). This result is because all the dose goes directly into the bloodstream. It also means that the Tmax is much lower (0.00 h vs 0.10 or 0.16h) and that the AUC is 5- to 3-fold higher (5.42 g.h/mL vs 1.05 or 1.71 g.h/mL), as reported by the experimental data. Table 2. Pulmonary permeability (PP) and systemic absorption rate (SAR) values used for the default and optimized simulation. Pulmonary compartment

Default values

Optimized values

SAR

Extra-thoracic

1.38.10-6

4.82.10-5

1.38.10-5

4.82.10-3

Thoracic

7.38.10-6

4.82.10-5

7.38.10-5

4.82.10-3

Bronchiolar

5.57.10-6

4.82.10-5

5.57.10-5

4.82.10-3

Alveolar-interstitial

3.05.10-4

4.82.10-5

3.05.10-2

4.82.10-3

Conclusion The cisplatin pharmacokinetic was successfully predicted by the GastroPlusTM after an intravenous administration. The pulmonary route required additional optimization with the adjustment of the pulmonary permeability and the systemic absorption rate considering the specific characteristics of the active compound. Now that the solution and the powder with immediate release administered via the endotracheal route are established, we aim to develop a model that can simulate results from a controlled-release form of the powder. The actual results are promising for the use of GastroPlusTM as software that can predict pharmacokinetic parameters by establishing an in vitro-in vivo correlation after pulmonary administration. References Levet V, Merlos R, Rosière R, Amighi K, Wauthoz N: Platinum pharmacokinetics in mice following inhalation of cisplatin dry powders with different release and lung retention properties, Int J Pharm 2017; 517 : pp359-372. 2 Fisher AC, Lee SL, Harris DP, Buhse L, Kozlowski S, Yu L, Kopcha M, Woodcock J: Advancing pharmaceutical quality: An overview of science and research in the U.S. FDA’s Office of Pharmaceutical Quality, Int J Pharm. 2016; 515 :pp390-402. 3 Sakaida E, Iwasawa S, Kurimoto R, Ebata T, Imai C, Oku T, Sekine I, Tada Y, Tatsumi K, Takiguchi Y: Safety of a short hydration method for cisplatin administration in comparison with a conventional method-a retrospective study, Jpn J Clin Oncol 2016; 46 : pp370-377. 4 Levet V, Rosière R, Merlos R, Fusaro L, Berger G, Amighi K, Wauthoz N: Development of controlled-release cisplatin dry powders for inhalation against lung cancers. Int J Pharm 2016; 515 : pp209-220. 5 Son YJ, McConville JT: Development of a standardized dissolution test method for inhaled pharmaceutical formulations, Int J Pharm 2009; 382 : pp15-22. 6 Budavari S, O'Neil MJ, Smith A, HeckelmanPE, Kinneary JF: The Merck Index : An Encyclopedia of Chemicals, Drugs and Biologicals (12th Edition). Merck & Co; pp390-391,1996. 7 Salar-Behzadi S, WS, Mercuri A, Meindl C, Stranzinger S, Fröhlich E: Effect of the pulmonary deposition and in vitro permeability on the prediction of plasma levels of inhaled budesonide formulation. Int J Pharm 2017; 532 : pp337-344. 8 D.Eljack N, Ho-Yu M, Drucker J, Shen C, Hamb ley TW, New EJ, Friedrich T, Clarke RJ: Mechanisms of cell uptake and toxicity of the anticancer drug cisplatin, Metallomics 2014; 6: pp2126-33. 9 Spreckelmeyer S, Orvig C, Casini A : Cellular transport mechanisms of cytotoxic metallodrugs: An overview beyond cisplatin. Molecules 2014; 19: pp15584-15610. 1

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Drug Delivery to the Lungs, Volume 29, 2018 - Natalie Armstrong Green et al. Dissolution Of Crystalline And Amorphous Particles In The Aerosol Phase Natalie Armstrong Green1, Allen Haddrell1, Jonathan Reid1, David Lewis2 & Tanya Church2 School of Chemistry, University of Bristol, Bristol, BS81TS, United Kingdom Chiesi Limited, Bath Road Industrial Estate, Chippenham, Wilts, United Kingdom 1

Summary Dry powder inhalers have become a popular inhalation delivery system due to good drug stability and a minimal need for patient coordination.[1] The powdered mixture consists of large, course carrier particles, e.g. lactose, and an active pharmaceutical ingredient (API), e.g. salbutamol sulphate. Given that the efficacy of the drug is dependent on where the dose is delivered, it is important to understand the physicochemical properties of the aerosol to predict dose. Once inhaled, the dry particles experience a warm humid environment where, depending on their solubility properties, they can take up water. A particle that fully dissolves in the aerosol phase will grow during inhalation, the magnitude of growth is controlled by its hygroscopicity. This dynamic size change influences the deposition mechanism, thus the deposited fraction. A particle that partially dissolves or remains solid, will experience a different deposition pattern, and deposit on the lung surface in a different physical state. Here, we report for the first time, dissolution measurements in the aerosol phase of a pharmaceutical aerosol comprising: sodium chloride and salbutamol sulphate. An adapted comparative kinetics electrodynamic balance (CK-EDB) has been used to replicate the dissolution process of dry powder particles in the aerosol phase experienced in the lung. The CK-EDB traps individual particles within its core and monitors size/phase change as a function of time (0.01 second resolution) and environmental conditions (relative humidity and temperature which can be changed <0.1 seconds). Key message An adapted electrodynamic balance was used to measure dissolution dynamics of crystalline and amorphous particles in the aerosol phase. It accurately replicates the dynamic size change of aerosolised drug mixtures during generation and inhalation. Primary results of model systems: salbutamol sulphate and sodium chloride, have been explored. Introduction Aerosolised drugs are most commonly used to treat respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). New medicines, aerosol formulations, and devices have enhanced our ability to start treating diseases such as diabetes and cancer, by aerosol delivery.[1] The large surface area of the lung offers full cardiovascular output, and paired with targeted site delivery, reduces the dose needed for a pharmacological effect.[2] However, the physicochemical processes from aerosol generation to inhalation and finally deposition, are poorly understood.[3] Understanding the time-dependent dynamic processes of aerosol administration would enhance our ability to control their deposition pattern in the lung and optimise drug delivery. [3] The dynamic size change of a hygroscopic particle influences the aerosol deposition pattern in the lung.[3] The mechanism of aerosol deposition onto the lung surface occurs via impaction (particles radius>2.5 µm), sedimentation (particles >0.5µm) or diffusion (particles <0.1µm). [4] Large, fast moving particles (>2.5 µm) are likely to deposit via impaction in the upper respiratory tract, the desired target site for treating respiratory diseases. [4] Smaller particles (<2.5 µm), deposit in the lower regions of the lung, administering drugs that may be available for systemic absorption. To optimise drug delivery, the rate and magnitude of size change during inhalation can be manipulated to control the deposition pattern. There are three main delivery systems used for administering an aerosolised drug; metered-dose inhaler (MDI), dry powder inhaler (DPI) and nebuliser. In an MDI, the drug is suspended or dissolved in a liquid propellent and mixed with other excipients, such as surfactants or aerosol enhancers. Immediately following aerosolization, the propellant evaporates off the aerosol cloud, forming the particles ready for inhalation. In a DPI however, the drug is inhaled as a cloud of fine dry particles. A DPI mixture often consists of the drug and large carrier particles, often lactose mono-hydrate. They are breath operated, making it easier for patients to administer the drug, their main disadvantage over MDIs is that they are exposed to ambient conditions, most significantly temperature and relative humidity (RH). These are both dependent on numerous variables: time of day, season, geographical location, and the immediate environment (in and outside). Currently, DPIs are less efficient at drug delivery than MDIs. [5] To improve their efficiency, it is vital to understand the dynamic size change of the powdered mixture, and therefore the deposition pattern. The dissolution kinetics and hygroscopic properties of drug particles in the aerosol phase control size change during inhalation, influencing the deposition pattern. Once deposited, the dissolution kinetics in the bulk phase on the cell surface governs pharmacokinetic rates. If the deposited aerosol is homogenous (i.e. solute is fully dissolved) this process will be much faster than if the aerosol still has solute dissolving within it. Therefore, it is essential to know the state of the aerosol before and during inhalation, up to the point of deposition.

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Drug Delivery to the Lungs, Volume 29, 2018 - Dissolution Of Crystalline And Amorphous Particles In The Aerosol Phase This work highlights two parts of the dissolution process. Firstly, the time taken for a particle to start taking up water after an increase of RH in the gas phase and secondly, the time taken for a particle to reach an equilibrated size with the surrounding gas phase RH. If the substance takes a significant amount of time to reach size equilibrium, then its size at deposition will be smaller than its potential final size, thus it is likely to experience different deposition mechanisms depending on how long it takes to size equilibrate. However, if the equilibrated size is reached rapidly the deposition mechanism can be predicted and controlled. These two variables are vital for lung models and significantly influence the deposition fraction. To be reported for the first time are the dissolution dynamics for two model systems comprising: sodium chloride and salbutamol sulphate. Materials and methods A CK-EDB was used to look at the dissolution dynamics of different substances (Fig. 1).This system has been used to explore physical properties of individual particles in the aerosol phase for a range of applications: atmospheric aerosol, spray drying, bioaerosol, and aerosol inhalation.[6] Through probing the drug while in the aerosol phase, it is possible to explore states not readily reached in the bulk phase (e.g. the supersaturated state), that are commonly experienced by an inhaled aerosol in the environment/lung. A detailed description of the operation of the CK-EDB has been published previously and will be briefly described here.[6] Firstly, two droplet on demand dispensers are filled with solutions of known composition. They are set up to sequentially dispense probe and sample droplets (Fig. 1). Droplets from the probe dispenser are used to accurately measure the RH of the trap (Âą0.1%), and the droplets are either water, for RH >80%, or sodium chloride solution, for RH<80%. The evaporation kinetics of the probe droplets and the equilibrated size are used to accurately infer the RH within the CK-EDB chamber. The sample dispenser is filled with the solution that is being analysed. Single droplets, with a radius of ~20-30Âľm, are generated on demand by applying a pulse voltage to the filled micro dispenser. Upon generation, a net charge is imparted on each droplet by an induction electrode. Within 100 milliseconds the droplet is tightly confined in the centre of the electrodynamic field inside the chamber. The alternating current (AC) and direct current (DC) voltages applied to the cylindrical electrodes in the chamber, create an electric potential in which the droplet with net charge is held at the centre.[6] Laser illumination of the droplets results in an elastic scattering pattern, collected in the form of a phase function over a specific angular range. The average fringe separation in the phase function is used to determine the radius of the droplet using the simplified geometrics optics approximation.[6] The time-dependent size change of an aerosol, as it reaches equilibrium with the gas phase RH, is used to determine the mass flux of water, giving information on the vapor pressure of water above the droplet surface. The hygroscopic properties are estimated by comparing the evaporation kinetics of the probe and sample droplets using the mass and heat transport equations from Kulmala and co-workers.[6]

Figure 1. Comparative Kinetics Electrodynamic Balance. A single charged droplet is confined within the electrodynamic field in the chamber. The particle is illuminated with a laser and the resulting elastic scattering light pattern is used to determine the radius as a function of time.

Results and discussion The dissolution dynamics were explored for two model systems. The first was salbutamol sulphate (SS), commonly used in MDIs and DPIs to treat respiratory diseases. Therefore, understanding its dissolution properties in a spray dried and solution state is necessary for optimising its deposition pattern. The second system studied was sodium chloride, a well understood system in the CK-EDB. Our aim is to provide a clear comparison of the timescales for dissolution of crystalline aerosol particles when exposed to high humidity to the equilibration for solution droplets and amorphous dry particles. This is a first step towards understanding the complex phase behaviour of true drug formulations used in treating respiratory diseases.

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Drug Delivery to the Lungs, Volume 29, 2018 - Natalie Armstrong Green et al. Figure 2A illustrates the efflorescence and deliquescence cycle of a solution sodium chloride droplet. Once the has phase RH is reduced below 45% RH, a sodium chloride droplet forms a crystalline particle. Then, the RH must exceed75% for the sodium chloride to re-dissolve and form a homogenous, liquid droplet, otherwise it will remain a solid crystal particle. For comparison, the equilibrium hygroscopic curve for SS is also shown. At any particular RH, the hygroscopic growth of SS is less than that of NaCl, confirming that it is less hygroscopic. The time it takes for a substance to dissolve is essential to drug delivery efficacy. This work investigates the kinetics that govern the varying dissolution times for these two systems. For homogenous solution droplets for both systems, the data in Figure 2B show a quick response time to a change in gas phase RH, i.e. water uptake is rapid and is limited by gas diffusional transport. The time taken to reach an equilibrated size, however, differs for each system (SS ~8 seconds and sodium chloride ~12 seconds), determined by the hygroscopicity of the particle and the amount of water that must condense.

Figure 2. Dissolution kinetics of salbutamol sulphate and sodium chloride. A) Hygroscopic growth curves for salbutamol sulphate (orange) and sodium chloride (green) B), C) and D) show the dissolution dynamics of salbutamol sulphate and sodium chloride for solution droplets (61%-72/83%RH) and crystalline (NaCl) or amorphous (salbutamol sulphate) particles (0%-72/83%RH) respectively. The solid black line at T=0 denotes the time that the RH in the chamber is switched from one gas flow to the other. I.e. from 61% - 83%RH and 0%RH(Dry) - 83%RH.

In Figure 2C, we compare the timescales for the combined dissolution and water condensation of a crystalline sodium chloride particle and for a SS droplet dried into <5 %RH. Under dry conditions, the SS particle no longer retains water but light scattering indicates that it has not crystallised, suggesting an amorphous particle is formed just from the SS. Crystalline sodium chloride reaches its equilibrated solution droplet size considerably slower (~25 seconds) than SS (~8 seconds). Indeed, the response time for the amorphous SS is similar to the timescale for condensation and equilibration on a solution droplet, suggesting that the kinetics are limited again by gas diffusion. The dissolution of a crystalline particle is an extremely complicated process. Once the RH is increased past the solubility limit/deliquescence RH, water must begin to condense to form a saturated solution layer and this must be followed by continuing dissolution of the remaining crystalline solid and water condensation. Further work is needed to better understand the dissolution kinetics from the time of switching the RH to when the droplet is homogenous and reaches equilibrium with the gas phase RH. In Figure 2D, at an upper RH of 72%, the crystalline sodium chloride particle does not dissolve, which is as expected from its deliquescence point of ~75%RH, i.e. there is insufficient water in the gas phase to permit the formation of a saturated solution droplet. By contrast, SS does grow, again consistent with the starting particle being amorphous rather than a crystal. It should be noted in all of these measurements that when the particle is inhomogeneous or crystalline, the elastic light scattering phase function is irregular and the particle cannot be sized with high accuracy. This leads to considerable noise in the reported particle size. [6]

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Drug Delivery to the Lungs, Volume 29, 2018 - Dissolution Of Crystalline And Amorphous Particles In The Aerosol Phase

Figure 3. Fitting the Kulmala model to the dissolution kinetics of salbutamol sulphate and sodium chloride. A) The model fits well for the dissolution of a solution droplet, however the model greatly over estimates the dissolution rate for a crystalline particle. B) The Kulmala model fits the dissolution of both particle states similarly. Symbols, solid blue lines; dissolution of solution droplet from 61% to 83%RH, solid red lines; dissolution of dry particle from 0% to 83%RH, light blue and orange lines; the Kulmala model using the heat and mass flux properties.

Experimentally, the dissolution process of a crystalline sodium chloride particle is much slower than model predictions of gas diffusional condensation kinetics (as predicted from the model of Kulmala et al. [9]), indicating that there are kinetic limitations preventing instantaneous water uptake onto the particle and induction period is observed (Fig. 3A). By comparison, the models for condensation of water on a dried particle and solution droplet of salbutamol sulphate show similar differences when compared with the measurements (Fig. 3B). Conclusion Studying dissolution dynamics with an electrodynamic balance allows us to simulate the size changes of particles that may occur during inhalation in the aerosol phase. Rapid change in the gas phase RH within the EDB chamber (<0.1 seconds) means a single particle may experience similar environmental conditions to that of the lung. Understanding particle size dynamics gives more control of the deposition mechanism, leading to a more efficient deposition pattern of inhaled drug mixtures. Dissolution results of sodium chloride clearly show a time lag between the switch in RH and when the particle begins taking up water from the gas phase, implying that the water condensational kinetics are not purely limited by gas phase diffusional transport. References 1

Rubin, B. K. & Williams, R. W. Emerging aerosol drug delivery strategies: From bench to clinic. Adv. Drug Deliv. pp141–148, 2014.

Dolovich, M. B. & Dhand, R. Aerosol drug delivery: Developments in device design and clinical use. Lancet. pp1032–1045, 2011.

Haddrell, A. E., Davies, J. F., Miles, R. E. H., Reid, J. P., Dailey, L. & Murnane, D. Dynamics of aerosol size during inhalation: Hygroscopic growth of commercial nebulizer formulations. Int. J. Pharm. pp50–61, 2014.

Ariyananda, P. L., Agnew, J. E. & Clarke, S. W. Aerosol delivery systems for bronchial asthma. Postgraduate medical journal. pp151–156, 1996.

Kolanjiyil, A. V., Kleinstreuer, C. & Sadikot, R. T. Computationally efficient analysis of particle transport and deposition in a human whole-lung-airway model. Part II: Dry powder inhaler application. Comput. Biol. Med. pp247–253, 2017.

Rovelli, G., Miles, R. E. H., Reid, J. P. & Clegg, S. L. Accurate Measurements of Aerosol Hygroscopic Growth over a Wide Range in Relative Humidity. J. Phys. Chem. A. pp4376-4388, 2016.

Davies, J. F., Haddrell, A. E., Miles, R. E. H., Bull, C. R. & Reid, J. P. Bulk. Surface, and gas-phase limited water transport in aerosol. J. Phys. Chem. pp10987–10998, 2012.

Ali, H. S., York, P., Blagden, N., Khoubnasabjafari, M., Acree Jr, W. E., & Jouyban, A. Solubility of salbutamol and salbutamol sulphate in ethanol + water mixtures at 25 °c. J. Mol. Liq. pp62–65, 2012.

Kulmala, M., Vesala, T., Wagner, P. E. An analytical expression for the rate of binary condensational particle growth. Proc. R. Soc. Lond. A. pp589-605, 1993.

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Drug Delivery to the Lungs, Volume 29, 2018 Carolyn Stevenson, et al. Characterisation of Nanomaterial in Nebulised Formulations for Clinical Products: Impact of Particle Size on Dissolution and Predicted Deposition Pattern. Carolyn Stevenson, 1Henrik Kristensson1, Karin Carlsson1, Pia Mattsson1, John Salomonsson1, Jan Olof Svensson1 and Ulrika Tehler1 1

Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca Gothenburg, Sweden

Summary Selection of a poorly soluble active pharmaceutical ingredient (API) for clinical development can be a strategic decision to achieve lung retention. To attain desirable exposure for these types of APIs, it is possible to optimise the particle size to modify the retention in the lung. We have investigated three different suspension formulations with different API particle size (d50 of 0.13, 0.23 and 2.2 µm) to be delivered by nebulisation. For the different nanosized products, no significant difference was observed in output rate or mass median aerodynamic diameter, (MMAD). However, for microsized particles a lower output rate and higher MMAD was observed in comparison to the nanoproducts using an experimental inhalation flow rate of 15 L/min. It was found that dissolution rate was altered by modification of the particle size. Prediction of the lung deposition however, suggested no difference between the particle sizes investigated. From a drug product development perspective this means that the particle size of the API can be optimised to achieve the desired exposure of a dissolved API. Additionally, knowledge of the manufacturing window, i.e. the limit of particle size that will achieve desirable exposure, can be gained by evaluating differences in particle sizes. Key Message Predicted lung deposition is not changed when altering particle size of a poorly soluble API in nebulised suspensions but the dissolution profile can be manipulated. From a development perspective, this means that the API particle size could be optimised to achieve the desired exposure of a dissolved API. Introduction For an active pharmaceutical ingredient (API) that is poorly soluble, lung absorption may be limited by slow dissolution. Altering the particle size of the API in a product can lead to a change in dissolution rate and the exposure of the drug product.[1] Processing of API particles to the nanosize range has been widely reported with a variety of techniques.[2] By altering the particle size of a poorly soluble API in a nebulised product different lung retention could be expected if the deposition pattern in the lung remains unchanged. The size characteristics of the nebulised aerosol droplets from a suspension is not expected to significantly change if the size of the API particles is varied, and are significantly smaller than the aerosol droplets.[3] Hence, the lung deposition is not expected to change when the particle size of the API is altered since the aerosol droplet size drives the lung deposition. Further, by understanding the impact of particle size of the API a view of the “manufacturing window”, i.e. the limit of particle size that will achieve desirable exposure, for the drug product can be established for development. Use of nebuliser products during the early clinical development phase is an efficient approach to reduce development time and to provide greater flexibility with respect to achieving a wide range of delivered dose. Once the particle size parameters of the API have been established, the commercially desirable drug product formulation could be developed that delivers the API in the same way. Other principles could potentially be applied to increase the solubility of the API, such as development of co-crystals or salts. However, even if such options would be possible it could be challenging to reach enough solubility improvement and achieve a stable product, i.e. control over the precipitation of the free base for example during storage.[4] We have investigated three different particle sizes of one poorly soluble API. 1) Nebuliser suspension with particle size of d50 = 0.13 µm, “nano1”. 2) Nebuliser suspension with particle size of d50= 0.23 µm, “nano2”. 3) Nebuliser suspension with particle size d50 = 2.2 µm, “micro”. Characterisation of the nebuliser aerosol has been conducted and the output rate and aerodynamic particle size distribution (APSD) determined. Assessment of the in vitro dissolution profile has been performed. In-silico prediction of the lung deposition using in-house software, Lung-Sim has been made. This prediction is done using clinically relevant ventilation parameters in combination with the experimentally determined aerosol characteristics. The aim of this work is to establish the feasibility of altering particle size in nebulised inhalation formulation development for clinical delivery.

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Drug Delivery to the Lungs, Volume 29, 2018 - Characterisation of Nanomaterial in Nebulised Formulations for Clinical Products: Impact of Particle Size on Dissolution and Predicted Deposition Pattern. Experimental Methods The nebuliser suspensions were manufactured by suspending the active substance in a vehicle containing a stabiliser and water. The suspension is further processed to achieve the different particle sizes. The nano suspension “nano1”, d50 = 0.13 µm, is manufactured by processing in a bead mill. The nano suspension “nano2”, d50= 0.23 µm, is manufactured by high pressure homogenisation in a microfluidiser. The micro suspension was prepared using an ultrasonic probe, “micro”, d50 = 2.2 µm. After processing to different sizes, glucose was added to the suspension as a tonicity adjuster and the suspension is diluted to 5 mg/mL concentration. Particle sizes of the suspension products have been determined by laser diffraction principle using Malvern Mastersizer 2000 instrumentation. Measurement was made on the API in the suspension diluted in deionised water. The d50 values of the products have been reported. Viscosity measurements of the nanosuspension formulations have been conducted using Anton Paar Modular Compact Rheometer MCR 302, equipped with a Viscotherm VT2 temperature regulating accessory. Isothermal measurement was conducted at 25 °C. A shear rate ramp of 1-100 1/S was used. A commercial jet nebuliser was used to generate the aerosol with an inhalation flow of 15 l/min. To determine the output rate and APSD for each suspension, internal standard methodology (to compensate for potential evaporation loss) with Liquid Chromatography (LC) was used. Repsigard filters were used for output determination. To determine APSD, a cooled next generation impactor (NGI) was used.[5] The dissolution of the crystalline particles was followed using fluorescence analogous to a method previously described in the literature.[6] In brief, a small volume of a diluted crystalline suspension (e.g. 4 µL of 15µM) was transferred to a cuvette containing 3 mL of solvent (PBS) under continuous stirring to achieve a final concentration of 20 nM. The time resolved fluorescence was measured using a Cary-Eclipse fluorescence spectroscopy, (Agilent, Santa Clara, CA, USA). The dissolution kinetics was recorded over an approximate 5 hour period and reported as fraction of crystalline material versus time. Dissolution data was evaluated using a Weibull fit and expressed as T63, the time to dissolution of 63% of the initial dose.[7, 8] T63 was selected as the timepoint for statistical comparison between the different products. Lung-SIM has been previously described in detail and is a physiologically based biopharmaceutical prediction model based on the Weibel lung model of a healthy lung.[9] Lung-SIM uses aerosol properties such as bolus volume, MMAD and geometric standard deviation (GSD) (from the NGI investigations) and assumed inhalation manoeuvre appropriate for each intended clinical device. Results Suspension formulations with different geometric API particle sizes have been produced, refer to Figure 1 and characterisation of the formulations has been conducted. The viscosity of the nanosuspensions and the zeta potential data are presented, refer to Table 1. Further, XRPD data and chemical stability have been monitored over the study period and no significant change was observed, (data not shown). Output rate and aerosol characterisation data of the suspension formulations was collected and is detailed in Table 2. Dissolution data for the suspensions show T63 (time when 63% of API is dissolved) of Nano 1- 2 min < Nano 2- 12 min< Micro- 107 min respectively, refer to Figure 2 below. Lung deposition using Lung-SIM model predicts a deposition of approximately 80% for the three suspensions, refer to Figure 4 below.

Figure 1 - Particle Size Distribution of API in Nano Suspensions, (nano 1 green and nano 2 blue) and Micro Suspension Product (red). Table 1: Mean Particle Size Distribution, Viscosity and Zeta Potential Data of Suspension Formulations PSD (d 50/µm)

Viscosity (cP)

Zeta potential (mV)

Nano 1 (0.13 µm)

0.13

1.2

-54.6

Nano 2 (0.23 µm)

0.23

1.3

-55.4

Micro (2.2 µm)

2.2

-55.6

NT. -Not Tested PSD – Particle Size Distribution

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Drug Delivery to the Lungs, Volume 29, 2018 Carolyn Stevenson, et al. Table 2: Mean Output Rate, MMAD and GSD Data of Suspension Formulations, (n= ≥ 3) Output Rate, µL/s (%RSD)

MMAD (%RSD)

GSD

Nano 1 (0.13 µm)*

3.9 (6.4)

4.4 (3)

1.8

Nano 2 (0.23 µm)*

4.4 (9.4)

3.8 (9)

1.9

Micro (2.2 µm)

3.0 (5.0)

5.3 (3)

1.7

* Data is cumulative mean of 2 samples MMAD – Mass Median Aerodynamic Diameter GSD – Geometric Standard Deviation

Fraction of Crystalline Material

1.0 0.9

Micro (2.1 µm)

0.8

Nano 2 (234 nm)

0.7

Nano 1 (131 nm)

0.6 0.5 0.4 0.3 0.2 0.1 0.0

100

150

200

250

300

350

Time (mins)

Predicted Lung Deposition (% total delivered dose)

Figure 2 - Variation of the Fraction of Crystalline Drug verses Time in Dissolution Experiments (Plotted average of n=3).

100 90 80 70 60 50 40 30 20 10 0

Nano1

Nano2

Micro

Figure 3 - Predicted Lung Deposition by Lung-SIM for the Three Formulations Investigated.

Discussion Suspensions with different particle sizes of API have been formulated and characterised. The viscosity was not significantly different between the suspensions and the zeta potential data suggested good stability of all the suspensions during the course of this study. The average aerosol output rate between the nanoproducts was slightly different but with the variation observed this is not considered significant. Further, review of both the MMAD and GSD data suggested that the aerosol distribution was similar. It was observed that the MMAD was smaller with the larger nanoproduct (nano 2), however it was assumed that this is due to experimental variability during APSD measurements as the formulation composition and characteristics are similar and this size of the API should not influence the nebuliser droplet. It was therefore considered that there is no significant difference in the two nanoparticle sizes with respect to aerosol characterisation. A decrease in the output rate and increase in the MMAD was observed with the microsized product in comparison to the nanoproducts. It is known that the nebuliser droplets have an approximate gaussian distribution [10] thus, the difference in the output rate and MMAD for the microsized product was assumed to be due to the reduction in the API amount within the smaller droplets. In practical terms,

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Drug Delivery to the Lungs, Volume 29, 2018 - Characterisation of Nanomaterial in Nebulised Formulations for Clinical Products: Impact of Particle Size on Dissolution and Predicted Deposition Pattern. for nebuliser clinical studies, where there is a decrease in output rate, this would mean that a larger number of inhalations is needed to achieve the same dose. Differences in the predicted dissolution profiles were observed between all the products. The predicted lung deposition for the different suspensions are similar regardless of mean particle size (0.1 µm to 2 µm). This was considered primarily due to the control of the low flow rate used for the nebulisation (15 L/min) and therefore the differences observed in the MMAD when related back to the predicted lung deposition, were not significant in this experimental set-up. The assumption therefore is that it is possible to alter the in-vitro dissolution rate and thereby the exposure profile of the API without impacting the lung deposition. For higher flow rates the differences in MMAD would be expected to result in larger difference in the lung distribution due to inertial impaction. In a clinical setting, it may therefore be important to control a low flow rate when assessing different particle size products. Conclusions It has been demonstrated for suspensions with particle sizes in the nano range (0.13 to 0.23 µm) that there is no significant influence on the aerosol characteristics that impacts the predicted lung deposition. Comparison between the suspension with microsized API (2.2 µm) and the nanosuspensions (0.13 and 0.23 µm) shows a decrease in output rate and MMAD and it is hypothesised that this is due to reduction in API within the smaller droplets. The invitro dissolution rate can be manipulated by altering particle size (0.13 to 2.2 µm) of the API in the suspensions meaning that we anticipate a difference in exposure profile. No significant difference is observed in the predicted total lung deposition for the nebulised suspensions when altering the size of the API from 0.13 to 2.2 µm. Acknowledgements The author wishes to acknowledge the following people who have assisted with the generating the data and for helping with technical discussion and advice. Mikael Brülls, Tulasi Kirla, and Rebecca Fransson. References 1.

Allen A, Bareille P, Rousell V. Fluticasone Furate, A Novel Inhaled Corticosteroid, Demonstrates Prolonged Lung Absorption Kinetics in Man Compared with Inhaled Fluticasone Propionate. Clinical Pharmacokinetics, 2013, 52: pp 37-42

Zhang J, Wu L, Chan K-H, Watanbe W. Formation, Characterisation and Fate of Inhaled Drug Nanoparticles, Advanced Drug Delivery Reviews, 2011, 63, pp 441-455.

Wiedmann T, DeCastra L, Wood W. Nebulisation of Nanocrystals. Production of a Respirable Solid-in-Liquid-in-Air Colloidal Dispersion. Pharmaceutical Research, 1997, 14:1, pp 112-116

Karashima M, Sano N, Yamamoto S, Arai Y, Yamamoto K, Amano N, Ikeda Y. Enhanced Pulmonary Delivery of Poorly Soluble Itraconazole by Micronized Cocrystal Dry Powder Formulations. European Journal of pharmaceutics and biopharmaceutics, 2017, 115, pp 65-72.

Berg E, Svensson J, Asking, L. Determination of Nebulizer droplet size: A method based on Impactor Refrigeration, Journal of Aerosol Medicine, 2007, 2: pp 97-104

Lindfors L, Skantze P, Skantze U, Westergren J, Olsson U. Amorphous Drug Nanosuspensions. 3. Particle Dissolution and Crystal Growth, Langmuir, 2007, 23: pp 9866-9874

Langenbucher, F. Letters to the Editor: Linearization of Dissolution Rate Curves by the Weibull Distribution. Journal of Pharmacy and Pharmacology 1972, 24:12, pp 979–981

Dokoumetzidis A, Papadopoulou V, Macheras, P. Analysis of Dissolution Data Using Modified Versions of Noyes–Whitney Equation and the Weibull Function. Pharmaceutical Research, 2006, 23:2, pp 256–261.

Tehler U, Fransson R, Thörn H, Franek F, Westergren J. Lung-Sim: A Physiologically Based Biopharmaceutical Prediction Tool. Proceedings of Drug Delivery to the Lungs [ISBN: 978-0-9957688-0-2], pp90-93, Edinburgh 6-8th December 2017.

10. Ryan G, Dolovich M, Obminski B, Cockcroft D, Juniper E, Hargreave F, Newhouse M: Standardization of Inhalation Provocation Tests: Influence of Nebulizer Output, Particle Size and Method of Inhalation. Journal of Allergy and Clinical Immunology, 1981, 67:2, pp 156-161

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Drug Delivery to the Lungs, Volume 29, 2018 Allen Haddrell et al. Directly Probing the Dynamic Behaviour of Particles Originating from DPI and MDI Starting Formulations Allen Haddrell,1 David Lewis2, Tanya Church2, Jonathan Reid1 1 2Chiesi

University of Bristol, Bristol, UK Farmaceutici S.p.A., Chippenham, UK

Summary How deep in the respiratory tract a pharmaceutical aerosol penetrates is dependent on the size of the particle at the point of inhalation, its composition, and the microphysical processes that occur during inhalation. The aerosol composition influences the degree to which particles grow when inhaled directly, affecting the deposited dose and pattern. An understanding of the dynamic behaviour of inhalable aerosol is expected to be critical to predicting regional and total dose, which in turn will affect overall efficacy. Meaning, there is the potential to tailor the dynamics of pharmaceutical aerosol, through including trace quantities of additives to the starting formulation, to deliver the desired dose to a specific region in the lung. Previously, precise measurements of pharmaceutical aerosolâ&#x20AC;&#x2122;s thermodynamics (e.g. hygroscopic growth from dry to >99% relative humidity (RH)) and dynamic (rapid size change resulting from changes in RH) has been demonstrated for nebulizer formulations. To be presented here is the expansion of this technology to directly measure the dynamic behaviour of aerosol originating from metered dose inhaler (MDI), and dry powder inhaler (DPI) starting formulations. These measurements include: (1) the dissolution, in the aerosol phase, of DPI particles and, (2) the rapid evaporation of volatile species from droplets from MDI starting formulation, to saturated water droplet, to final particle. The time scale for these processes was observed to be on the same order as that of a single breath, meaning they will directly impact their deposition pattern in the lung. Key Message We have developed the instrumentation to directly probe the dynamic behaviour (e.g. rapid evaporation and dissolution) of pharmaceutical aerosol produced from DPI and MDI starting formulations in relevant environmental conditions (e.g. ambient pressure, temperature and RH). Introduction The deposition pattern within the respiratory tract is dependent on the time-dependence of the aerosol size distribution as the aerosol penetrates deeper into the lung. The size of a droplet at the point of generation, the reduction in droplet size resulting from the net loss of propellant/solvent during transfer from the device to the mouth, and any hygroscopic growth on inhalation all could have an effect on total and regional dose. To design active pharmaceutical ingredient (API) formulations for targeted dose, one must have a detailed understanding of these three phases, and the interplay between them. As an example, for metered-dose inhalers (MDI), the rapid evaporation of HFA and ethanol will drastically decrease the temperature of an aerosol droplet. The reduction in temperature will cause it simultaneously to take up water while still losing solvent. Such a complex process in an MDI plume is not well understood, yet vitally important to understanding particle size distributions and the deposited dose. Following the change in MDI propellants from chlorofluorocarbons (CFC) to hydrofluoroalkanes (HFA) dictated by the Montreal Protocol, the average starting droplet diameter was reduced from 2.5-3.8 microns to 1-1.2 microns, improving dose delivery from 10-20% to 52-68%[1]. A consensus forum of industrial, academic and regulatory experts identified a poor understanding of the relationships between physicochemical characteristics of drug formulations and performance in the humid environment of the respiratory tract as one key barrier to progress in inhalation therapeutics[2]. Thus, quantifying the properties of pharmaceutical aerosol that govern dynamic behaviour prior to and during inhalation will yield the potential for the rational design of new formulations for drug delivery to the lung. The delivery of an API from a starting formulation to the deep lung involves four phases: aerosol generation, transport from the aerosolization device to the mouth, inhalation, and concludes with deposition. The deposition rate in the lung is controlled by the aerodynamic diameter of the droplet at the time of deposition. Thus, the size of the droplet at the point of generation, the reduction in droplet size resulting from the net loss of volatile mass (if present) during transfer from the device to the mouth, and any hygroscopic growth on inhalation will have an effect in total and regional dose. The development of an instrument capable of directly measuring the interplay between these complex processes will be reported here.

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Drug Delivery to the Lungs, Volume 29, 2018 - Directly Probing the Dynamic Behaviour of Particles Originating from DPI and MDI Starting Formulations Experimental The comparative kinetic electrodynamic balance (CK-EDB, figure 1A) was used in this study to measure the hygroscopic properties/dissolution dynamics of surrogates of ambient aerosol. Our aim in this paper is to present and benchmark the most recently developed capabilities of this instrument. As such, we provide a brief summary of the instrument and its origins, summarise the new capabilities, and then provide benchmarking data of these new capabilities in subsequent sections. The ability of a direct current (DC) field to suspend droplets has been used since the days of the Millikan oil droplet experiment, with marked improvements in the method being continually made over the century since. Notable developments have included the coupling of an alternating current (AC) field to the DC field to add a restoring force that focuses the droplet to a confined space. Once confined, the droplet size, relative mass and composition can be readily probed. In this work, we have shaped the electrodes into two pairs of concentric cylinders. As a consequence, the strength of the restoring force in the trap that holds the droplet increases. This device, the CKEDB, can trap and confine a droplet dispensed only <100 milliseconds before. The trapped droplet is illuminated with a laser beam and the angularly scattered light collected by a camera. The angular spacing of elastic light scattering fringes is used to estimate the droplet size. Once trapped the absolute radius of the droplet (when the droplet is spherical) can be measured at a frequency of 100 Hz (Figure 1B). The absolute radius of a rapidly drying droplet can be accurately measured (size accuracy under +/-50 nm).

Figure 1. (A) Key components of the CK-EDB. (B) The measured radius of the droplet as a function of time. This data is then used to calculate (C), the hygroscopic properties.

A droplet dispenser is used to generate the droplet and, thus, absolute chemical composition of the droplet is known throughout the entire process, allowing for the direct comparison between the experimental data with a mass flux model[3]. The airflows in the CK-EDB enable the RH and temperature that the droplet experiences to be rapidly changed, making direct observation of aerosol growth kinetics possible, analogous to that experienced in the lung. The droplet dispenser is functional at temperatures under -100 oC. This makes the analysis of the rapid mass flux of droplets originating from metered dosed inhalers (MDI) formulations possible. The ability to directly monitor the mass flux from a single droplet made up of the starting MDI formulation is unique to this technology. In these experiments, the focus will be on the kinetics of water/co-solvent loss and gain. Dry particles are directly injected and trapped in the CK-EDB. A dry needle is first coated with the DPI powder, then positioned near the centre of the CK-EDB. 1 kV pulse is then applied to the needle causing the particles on the needle immediately be ejected from the needle and into the trap. The capabilities that are unique/advantageous to the CK-EDB include: 1) Use of a water droplet to probe the RH of the airflow that the sample droplet is subsequently studied in with accuracy of Âą0.1%, an order of magnitude better than conventional RH probes[4]. 2) Direct measurement of the dynamic behaviour of a droplet of known composition in a static and/or variable environment (<100 milliseconds, figure 1B)[4]. 3) Accurate determinations of the radial growth factor, a measure of the equilibrium hygroscopic growth of the aerosol as a function of gas phase RH across a range of RH from dry to >100%, and with temperature ranging from <50oC to <-30oC (figure 1C). Reaching supersaturation has yet to be published. 4) The conditions, both temperature and/orRH, that the droplet experiences in the trap can be rapidly changed (<0.1 seconds). These features are unique to this technology and are critical in being able to directly mimic the conditions that a pharmaceutical aerosol experiences prior to and during inhalation. Rapid changes in temperature has yet to be published. 5) The dynamics of a single pharmaceutical aerosol droplet/particle originating from DPI and MDI solutions can be measured. This is a newly reported feature of this technology and enables the detailed parameterization of the physicochemical properties that govern the dynamic properties of these inhalable species, and that key to be able to predict total and regional dose. This has yet to be published.

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Drug Delivery to the Lungs, Volume 29, 2018 Allen Haddrell et al. Results

80 60

5 Time / Seconds

2 4 Time / seconds

Temperature / C

100

Radius / Microns

Relative Humidity / %

Radius / Microns

We now provide new benchmarking data of the performance of the instrument. The ability to simultaneously control and rapidly change both the relative humidity and temperature that the droplet experiences is a unique feature of the newly designed CK-EDB. The impact of isolated changes in RH and temperature on the evaporation rates of pure water droplets are shown in Figure 2. The change in environmental condition is evident from the change in the evaporation rate of a single droplet as the conditions are change.

Figure 2. The rapid change of relative humidity (left) and temperature (right) that a single levitated water droplet in a CKEDB experiences. The orange line on the right indicates a model prediction for an evaporating water droplet into air set at a temperature of 0oC and a relative humidity of 0%.

Such measurements also allow for detailed measurements of particle dissolution as there is a clear, and measurable, point in time that the particle experiences a change in relative humidity. Preliminary data demonstrating the timescale for the dissolution of a single crystalline sodium chloride particle is shown in Figure 3, the very first dynamic measurement of the dissolution kinetics of a crystalline aerosol particle.

Radius / Microns

30 25 20 15 10 5 0

5 10 Time / Seconds

Figure 3. The dissolution of a sodium chloride particle. The relative humidity is increased from dry to >80% at 0 seconds (<0.1 seconds for the RH change to occur). When the particle is non-spherical, it is not accurately sized. It takes 7 seconds for the dry sodium chloride particle to completely dissolve into a spherical droplet.

The ability to accurately determine the size the droplet in the CK-EDB is dependant on the droplet being spherical. As a result, when the particle is dry, much more noise in the data is observed (all data under ~5 seconds). This does, however, give a clear indication of the point where the solid particle becomes a spherical droplet (in this case, ~7 seconds), after which, itâ&#x20AC;&#x2122;s physicochemical properties are much more well understood. The rapid evaporation of the volatile components within an MDI starting formulation will result in the droplet rapidly cooling to temperatures below 0oC. The resulting change in vapor pressure causes water vapour to rapidly deposit onto the particle. Direct measurements of this complex process are readily made with the CK-EDB (figure 4). Here we present measurements of the evaporation kinetics of a volatile ethanol droplet containing an API in a humid atmosphere.

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Droplet Radius / Microns

Drug Delivery to the Lungs, Volume 29, 2018 - Directly Probing the Dynamic Behaviour of Particles Originating from DPI and MDI Starting Formulations

Water Experimental

15 10 5

10 Time / Seconds

Figure 4. Size of a droplet originally made of Formoterol Fumarate Dihydrate dissolved in ethanol (orange Xs) injected into an airflow with a relative humidity or 81%. The grey line is a model of the size change of a water droplet injected into an airflow with a relative humidity of 81%.

The ethanol present in the starting droplet rapidly evaporates, as indicated by the curve in the first half second (figure 4). This rapid evaporation directly leads to the uptake of water onto the droplet. Note that no water was present in the starting formulation despite the majority of the dynamic behaviour of the aerosol being best described as that of a water droplet. This data suggests that inhaled aerosol from an MDI starting formulation will contain a significant amount of water throughout its lifetime and will likely never be a dry particle in the respiratory system. Discussion and Summary The ability to probe the particles and droplets originating from MDI and DPI starting formulations has been developed. Early notable observations found using this novel technology include: -

Dissolution of sodium chloride particles is a slow process (relative to the time scale of inhalation); this may dramatically affect the overall deposition pattern of DPI particles in the lung.

Droplets containing highly volatile solvents (including ethanol) such as originate from an MDI are likely to never be dry and will rapidly transition into water droplets during their evaporation into a humid environment (such as the respiratory system). This will have a consequential effect on where the dose is delivered in the lung.

References 1. Leach, C.L.: The CFC to HFA Transition and Its Impact on Pulmonary Drug Development. Respiratory Care, 2005: pp1201-1208. 2. Forbes, B. et al.: Challenges in inhaled product development and opportunities for open innovation. Adv. Drug Del. Rev. 2010; 63: pp69-87. 3. G. Rovelli, R.E.H. Miles, J.P. Reid and S.L. Clegg: Accurate Measurements of Aerosol Hygroscopic Growth Over a Wide Range in Relative Humidity. J. Phys. Chem. A 2016; 120: pp4376â&#x2C6;&#x2019;4388. 4. J.F. Davies, A.E. Haddrell, A.M.J. Rickards and J.P. Reid: Simultaneous Analysis of the Equilibrium Hygroscopicity and Water Transport Kinetics of Liquid Aerosol. Anal. Chem.; 2013; 85 (12): pp5819-5826.

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Drug Delivery to the Lungs, Volume 29, 2018 – Yingshan Qiu et al. Relationship Between The Secondary Structure Of The Peptide Base siRNA Carrier And Effective Gene Silencing Effect On Lung Epithelial Cells Yingshan Qiu ,1 Bui Tam2, Winne Y.W. Chung1, James Mason2 & Jenny K.W. Lam1 1

Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong 2 Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London, United Kingdom

Summary Pulmonary delivery of small interfering RNA (siRNA) has potential in treating many lung diseases. KL4 is a synthetic surfactant peptide that was initially designed to imitate the function of surfactant protein B (SP-B). The safety and the cationic nature of the KL4 make it an attractive candidate for siRNA delivery. Previous data showed that KL4 peptide could mediate efficient gene silencing effect of siRNA in human lung epithelial cells without signs of cytotoxicity at concentrations used for transfection. However, one of the problems associated with KL4 is poor aqueous solubility. To overcome this problem, five KL4-modified peptides were introduced by replacing leucine with other less hydrophobic amino acid residues such as valine and alanine. The siRNA binding affinity of these modified peptides was examined using a gel retardation assay, and the transfection efficiency was evaluated on A549 cells using siRNA targeting Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The secondary structure of the modified peptides was investigated by circular dichroism (CD). The results showed that only the KL4 peptide which adopted an alpha helix structure could transfect the siRNA into the cells and mediate an efficient gene silencing effect. Key Message The relationship between peptide secondary structure and siRNA transfection efficiency was investigated in this study. Only KL4 with an alpha helix conformation could transfect the siRNA into cells among all the studied peptides. Thus, the alpha helix structure of the peptide was critical for good transfection efficiency. Introduction Pulmonary delivery of the small interfering RNA (siRNA) has great potential for the treatment of many lung diseases, such as lung cancer, asthma, chronic obstructive pulmonary disease (COPD) and influenza [1]. One of the major barriers of siRNA therapeutics development is the lack of a safe and effective delivery system that is suitable for clinical applications, because siRNA is incapable of crossing a biological membrane unassisted[2]. KL4 is a 21residue peptide containing hydrophobic leucine interspersed with cationic lysine. This synthetic peptide is an active component in the pulmonary surfactant product Surfaxin® (Windtree Therapeutics, Inc. USA), which is a FDA approved intratracheal suspension to prevent respiratory distress syndrome in premature infants [3]. KL4 is structurally similar to pulmonary surfactant protein B (SP-B) and designed to imitate its function. The safety and the cationic nature of the KL4 makes it a potential candidate for siRNA delivery. A previous study showed that the KL4 peptide could mediate an efficient gene silencing effect of siRNA in both cancer and normal lung epithelial cells without signs of cytotoxicity at concentrations used for transfection[4]. Furthermore, the transfection efficiency of KL4 was not affected by the presence of pulmonary surfactant, which hinders the transfection of many lipid-based delivery vectors. However, one of the problems associated with KL4 is poor aqueous solubility, hence a co-solvent such as dimethyl sulfoxide (DMSO) is often needed to help dissolve the peptide. Here, in order to improve the efficacy of the system, the KL4 peptide was modified by replacing leucine with other less hydrophobic amino acid residues such as valine and alanine. The sequence and physicochemical properties of the peptides used in this study are shown in Table 1. The secondary structures of the KL4 and modified peptides were investigated by circular dichroism (CD). CD is a form of light absorption spectroscopy that measures the difference in the absorption of left‐handed circularly polarised light (L‐CPL) and right‐handed circularly polarised light (R‐CPL) and occurs when a molecule contains one or more chiral chromophores (light‐absorbing groups)[5]. CD spectroscopy is widely used to study the secondary structure of macromolecules; mostly proteins and peptides. The aim of this study was to compare the siRNA binding affinity, transfection efficiency and secondary structure of the KL4 peptide and the modified analogues, in order to understand how secondary structure affect the transfection efficiency of the peptide base siRNA delivery vector.

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Drug Delivery to the Lungs, Volume 29, 2018 - Relationship Between The Secondary Structure Of The Peptide Base siRNA Carrier And Effective Gene Silencing Effect On Lung Epithelial Cells Experimental methods Materials The peptides (Table 1) were purchased from ChinaPeptides (Shanghai, China) with over 80% purity. Peptides were dissolved in 5mM Tris-HCl buffer at a final concentration of 0.1 mg/mL in 0.1% (v/v) DMSO. siRNAs (SilencerSelect GAPDH positive control siRNA and SilencerSelect negative control siRNA) were purchased from Ambion (Austin, TX, USA). GelRed™ nucleic acid stain was purchased from Biotium (Hayward, CA, USA). Dulbecco’s modified eagle medium (DMEM), Trypsin-EDTA (0.25%), Fetal Bovine Serum (FBS), Antibiotic-Antimycotic (100X) and Lipofectamine 2000 were purchased from ThermoFisher Scientific (Waltham, Massachusetts, USA). All other reagents were obtained from Sigma Aldrich (Saint Louis, MO, USA) and of analytical grade or better. Table 1 Sequence, average hydrophobicity, molecular weight of peptides used in this study Peptides

Sequence

Average Hydrophobicity

Molecular weight (Da)

KA1

KALLLKALLLKALLLKALLLK-NH2

0.37

2300

KA2

KAALLKAALLKAALLKAALLK-NH2

0.28

2132

KA3

KAAALKAAALKAAALKAAALK-NH2

0.20

1964

KA4

KAAAAKAAAAKAAAAKAAAAK-NH2

0.12

1795

KV4

KVVVVKVVVVKVVVVKVVVVK-NH2

0.47

2244

KL4

KLLLLKLLLLKLLLLKLLLLK-NH2

0.45

2468

* K = Lysine; V = Valine, A = Alanine; L = Leucine siRNA binding affinity Peptide/siRNA complexes were prepared at various peptide to siRNA weight ratios from 5:1 to 30:1 (w/w) with 0.4 μg siRNA in 10 μl TAE buffer. The samples containing gel loading buffer were loaded into a 2% w/v agarose gel stained with GelRed™. Gel electrophoresis was run in TAE buffer at 125 V for 25 min and the gel was visualised under the UV illumination. Particle size analysis The hydrodynamic size of the KL4/siRNA (20:1 w/w), KA1/siRNA (15:1, w/w), KA2/siRNA (10:1, w/w) and KV4/siRNA (15:1, w/w) complexes were measured by dynamic light scattering (DLS) (Delsa Nano C, Beckman Coulter, CA, USA). The complexes were prepared with 4 μg siRNA in 100 μl of ultra pure water and incubated in room temperature for 30 min before particle size measurement. siRNA Transfection A549 cells were seeded in 6-well plates at a density of 1.5 x 105 cells per well one day before transfection. The complexes at various peptide to siRNA ratios were prepared in OptiMEM I reduced serum medium with 50 pmol GAPDH or scramble siRNA per well. Lipofectamine 2000 was used as positive control. After 4 hours of incubation at 37 °C, the cells were washed with PBS and fresh DMEM supplemented with 10% FBS were added to the cells. After 72 hours, the cells were washed and lysed. The GAPDH expression was detected by Western blotting assay. Secondary structure analysis Peptides were dissolved in 5mM Tris-HCl buffer at a final concentration of 0.1 mg/mL in 0.1% (v/v) DMSO. CD spectra were acquired on a Chirascan™ Spectrometer, Applied Photophysics (Leatherhead, UK). Far-UV CD spectra were obtained with the peptides solution incubated at room temperature. Spectra were recorded from 190 to 260 nm using a 0.5 mm path length and were processed using Chirascan software where a spectrum of the peptide free solution was subtracted and Savitzky-Gorlay smoothing applied.

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Drug Delivery to the Lungs, Volume 29, 2018 – Yingshan Qiu et al. Results The siRNA binding affinity of the peptides was examined by gel retardation assay (Figure 1). There was a gradual decrease in the intensity of siRNA band as the KA1, KA2, KV4 and KL4 to siRNA ratio (w/w) increased. The disappearance of the siRNA band at 10:1 ratio for KA1; 15:1 ratio for KA2 and KL4; and 20:1 for KV4 indicated that complete binding of siRNA occurred around these ratios. However, the siRNA failed to bind to the KA3 and KA4 peptides as the siRNA bands were still present at high ratios. The hydrodynamic diameter of different peptide/siRNA complexes was shown in Table 2. The size of the KA1/siRNA complexes was much larger thant the others and the polydispersity index (PDI) was also high. The KA2, KV4 and KL4 peptide could form much smaller size particles while the KL4/siRNA was the smallest in size.

The transfection efficiency of peptides was performed on A549 cells using siRNA targeting GAPDH (Figure 2). The complexes were prepared at the ratios that the peptides could completely bind to siRNA or above based on the gel retardation assay. At 72 hours post-transfection, the GAPDH protein expression was not affected by all the modifiedpeptide/siRNA complexes at all ratios, while the KL4 peptide could show significant gene silencing effect at all tested ratios from 10:1 to 30:1 (w/w).

The secondary structures of different peptides were studied at room temperature by CD (Figure 3). The KL4 peptide adopted an -helix conformation as a positive band at 195 nm and two negative bands at 208 nm and 222 nm were observed, which is the typical spectra of alpha helix structure. The positive band at 195 nm and the negative band at 215 nm of the KV4 peptide indicated that the peptide adopted a -sheet conformation. The other peptides had lower intensity of the positive band and the negative bands, which indicated disordered average structures. KA1 siRNA

5:1 10:1

15:1

KA2

20:1

25:1

30:1

5:1

KL4 siRNA

15:1

10:1

15:1

KA3

20:1

25:1

KA4

20:1

25:1

5:1

10:1

30:1

5:1

10:1

20:1

30:1

15:1

20:1

25:1

30:1

KV4

20:1

30:1

5:1

10:1

Figure 1 - siRNA binding study of KA1, KA2, KA3, KA4, KV4 and KL4 by gel retardation assay. Peptide/siRNA complexes were prepared at 5:1, 10:1, 15:1, 20:1, 25:1, and 30:1 ratio (w/w). Naked siRNA was used as control. Electrophoresis was carried out at 125 V for 25 min, and the gel was visualized under UV illumination.

b-actin

10:1 -

15:1 +

10:1 +

KV4:siRNA

KA4:siRNA

KA2:siRNA

KA1:siRNA Lipo 2k +

15:1 +

Lipo 2k +

b-actin

30:1

20:1 -

30:1

20:1 +

GAPDH

KL4:siRNA

b-actin

Lipo 2k +

10:1 +

15:1 +

20:1 +

25:1 +

30:1 +

GAPDH

Figure 2 - In vitro transfection efficiency of the modified peptide on A549 cells. Peptide/siRNA complexes were prepared at various ratios (w/w) with 50 pmol of GAPDH siRNA (+) or negative control siRNA (−) per well in a six-well plate (50 nM of siRNA). Lipofectamine 2000 (Lipo 2k)/ siRNA at 2:1 ratio (v/w) was used as positive control. Western blot analysis of GAPDH protein was performed at 72 h post-transfection, with β-actin used as internal control.

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Drug Delivery to the Lungs, Volume 29, 2018 - Relationship Between The Secondary Structure Of The Peptide Base siRNA Carrier And Effective Gene Silencing Effect On Lung Epithelial Cells

KA1

KA2

KV4

KL4

Hydrodynamic diameter ± standard deviation (nm)

7404.9 ± 508.2

564.0 ± 21.1

492.6 ± 56.0

283.3 ± 7.9

Polydispersity index (PDI) ± standard deviation

0.50 ± 0.03

0.06 ± 0.03

0.28 ± 0.03

0.28 ± 0.02

KA1 KA2 KA3 KA4 KL4 KV4

-1

Mean Residue Molar Ellipticity (deg cm dmol )

5x10

-2

4x10

3x10

2x10

1x10

0 4

-1x10

-2x10

-3x10

190

200

210

220

230

240

250

260

Wavelength (nm)

Figure 3 - Far-UV CD spectra of different peptides in 5mM Tris-HCl buffer at room temperature. Spectra were recorded from 190 to 260 nm using a 0.5 mm path length and were processed using Chirascan software where a spectrum of the peptide free solution was subtracted and Savitzky-Gorlay smoothing applied.

Table 2. Particle size of peptide/siRNA complexes measured by Dynamic Light Scttering Discussion The KL4 peptide could efficiently deliver the siRNA into lung epithelium after forming nano complexes with siRNA [4]. However, the modified peptides behaved differently in terms of siRNA binding affinity and transfection efficiency. The KA1, KA2 and KV4 had similar binding affinity as the KL4 peptide, while the KA3 and KA4 peptide did not show any binding with the siRNA at the tested ratios. None of the modified peptides could transfect the siRNA into the cells. The structure of the peptides affects their stability, siRNA binding affinity and the cellular uptake efficiency of the complexes. Therefore, the transfection efficiency of the delivery system would also be affected. CD is an important tool for studying the secondary structure of the proteins or peptides. Alpha helix and beta sheets are two types of typical structures of peptides. In the alpha helix structure, every backbone N−H group donates a hydrogen bond to the backbone C=O group of the amino acid located three or four residues earlier along the protein sequence[6]. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. A β-strand is a stretch of polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation[7]. The KL4 and KV4 peptides adopt alpha helix and beta sheet structure, respectively. As the number of alanine residues increased, the peptides became more disordered. The binding affinity of the siRNA and the peptides may be affected by the level of disorder of the peptides. Although all the three peptides could bind to the siRNA with similar binding affinity, KL4 peptide was the only peptide that could mediate gene silencing effect. The size of the peptide/siRNA complexes was also measured and it was found that the KA1/siRNA, KA2/siRNA and KV4/siRNA complexes were significantly larger than the KL4/siRNA complexes. Thus, the alpha helix structure of the peptide was critical for good transfection efficiency by affecting the siRNA binding, size of the complexes and possibly cellular uptake.

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Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; Yingshan Qiu et al. Conclusions This study investigated the relationship between binding affinity, transfection efficiency and secondary structure of the peptide vectors for siRNA delivery. The peptide KL4, KV4, and KA2 could bind to the siRNA. However, only KL4 could successfully transfect siRNA into the cells. The KL4 peptide has alpha helical structure, and the KV4 peptide has beta sheet structure. Other peptides adopt a disordered structure. The KL4 peptide with alpha helical conformation could mediate effective siRNA transfection, and the replacement of 25% of leucine with alanine in the sequence could dramatically change the structures, resulting in poor transfection efficiency. Therefore, cationic peptides with an alpha helical structure have the potential to be developed as siRNA carriers.

References 1. 2. 3. 4. 5. 6. 7.

Qiu Y, Lam JK, Leung SW, and Liang W: Delivery of RNAi Therapeutics to the Airways-From Bench to Bedside. Molecules 2016; 21(9). Ruigrok MJR, Frijlink HW, and Hinrichs WLJ: Pulmonary administration of small interfering RNA: The route to go? J Control Release 2016; 235: pp14-23. Cochrane CG, Revak SD, Merritt TA, Heldt GP, Hallman M, Cunningham MD, Easa D, Pramanik A, Edwards DK, and Alberts MS: The efficacy and safety of KL4-surfactant in preterm infants with respiratory distress syndrome. Am J Respir Crit Care Med 1996; 153(1): pp404-10. Qiu Y, Chow MYT, Liang W, Chung WWY, Mak JCW, and Lam JKW: From Pulmonary Surfactant, Synthetic KL4 Peptide as Effective siRNA Delivery Vector for Pulmonary Delivery. Mol Pharm 2017; 14(12): pp4606-4617. Yao H, Wynendaele E, Xu X, Kosgei A, and De Spiegeleer B: Circular dichroism in functional quality evaluation of medicines. J Pharm Biomed Anal 2018; 147: pp50-64. Scholtz JM and Baldwin RL: The mechanism of alpha-helix formation by peptides. Annu Rev Biophys Biomol Struct 1992; 21: pp95-118. Dubey RC: Advanced Biotechnology. 2008, Ram Nagar, New Delhi: S. CHAND & COMPANY PVT. LTD.

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Franek et al.

Ranking In Vitro Dissolution Of Orally Inhaled Drug Substance Powders In A Time-Efficient Manner Frans Franek,1 Linn Nilsson1, Helena Thörn1, Rebecca Fransson2, Ulrika Tehler2 AstraZeneca R&D, Pepparedsleden 1, Mölndal, 43183, Sweden Technology & Development Inhalation, Operations 2 Pharmaceutical Sciences, IMED

1Pharmaceutical

Summary For poorly soluble drug substances, dissolution is likely to be the rate-limiting step for absorption and an important parameter to consider when evaluating drug product performance. Currently, in vitro characterization of orally inhaled powder dissolution is time-consuming, often requiring separate (“off-line”) quantification of concentration. In this study, a time-efficient method to rank in vitro dissolution of poorly soluble orally inhaled drug substance powders was developed. Micronized budesonide, fluticasone propionate (FP), fluticasone furoate (FF) and a candidate drug (CD) were deposited onto filters, either by direct weighing onto filters or by being dispersed onto filters using a modified Andersen cascade impactor. Filter holders were designed and 3D-printed, which allow for drug powder placed on filters to be added into the µDiss, a dissolution apparatus with built-in UV-probes for “online” quantification of concentration. Dissolution started with addition of dissolution media (Phosphate buffer pH 6.8 with 0.5% SDS). Concentrations were quantified using the µDiss probe and via separate analysis using UPLC-UV. The dissolution profiles were fitted to a Weibull equation which allowed for statistical comparison of time when 63% of drug substance dose is dissolved (t63). All non-dispersed powders dissolved slower than dispersed powders and only the budesonide dissolution rate differed from the other substances. For dispersed powders a ranking was successfully established: budesonide (t63 (SD) = 11 (1) min) < FP (45 (27) min) = CD (55 (8) min) < FF (96 (12) min).The rank-order of dissolution for budesonide, FP and FF corresponded to the rank order of lung absorption expressed as mean absorption time (MAT) in man for the same substances formulated as drug products. Key Message In this study, a method for on-line in vitro dissolution characterization and ranking of poorly soluble orally inhaled drug substance powders was developed. The method can rapidly rank in vitro dissolution of poorly soluble drug substance powders, which may be important for understanding drug product performance. Introduction For poorly soluble inhaled drug substances, dissolution is likely to be the rate-limiting step for lung absorption. Dissolution can thus be considered an important quality attribute of drug product performance of poorly soluble drug substances[1]. Although dissolution testing is considered a valuable technique in the development of inhaled dosage forms, there is currently no recommended method from regulatory agencies for measuring dissolution rates for inhaled drug products[2]. Dispersion of the powder prior to dissolution deaggregates the powder and is likely important for differentiation of dissolution profiles[3]. Powders may be dispersed on filters prior to dissolution using e.g. impactors[2], modified impactors[4, 5] or dedicated systems. We have previously presented a method inspired by May et al.[5], which utilizes a modified Andersen cascade impactor to disperse a powder of lung-relevant particle size onto filters prior to dissolution[4]. Surfactants can also be used to aid deaggregation and differentiation of dissolution profiles [2, 3]. Furthermore, micelles may facilitate sink conditions during in vitro dissolution[2, 3]. Rohrschneider et al., proposed a method that uses sodium dodecyl sulphate (SDS) which shows promise in differentiating between the poorly soluble drug substances while providing sink conditions[2]. Although more in vitro dissolution methods are becoming available that are successful in discriminating between dissolution profiles, many require manual sampling and subsequent (off-line) quantification of drug substance using e.g. UPLC-UV[2–4]. To the author’s knowledge, there are no methods that manage to satisfactorily differentiate between poorly soluble inhaled drug substance (DS) powders while using on-line analysis. Consequently, in this study we have sought to develop a method that can (1) differentiate between drug substance with respect to dissolution and (2) perform concentration quantification during dissolution (i.e. “on-line”). To achieve this, we have adapted an on-line apparatus (µDiss)[6] to be able to analyse dispersed drug powder (which is important for differentiation) by designing and 3D-printing filter holders that allow for dispersed drug powder on filters to be added into the apparatus.

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Drug Delivery to the Lungs, Volume 29, 2018 - Ranking In Vitro Dissolution Of Orally Inhaled Drug Substance Powders In A Time-Efficient Manner Method Prior to dissolution, budesonide, fluticasone furoate (FF), fluticasone propionate (FP) and a candidate drug (CD) powder were deposited onto filters (Figure 1). The powders had similar median particle size (MMD ~2-3µg). The powders were either weighed in onto filters directly (approximately 100 µg) or dispersed onto filters (15-75 µg) using a previously described modified Andersen Cascade Impactor method (mACI)[4]. Furthermore, to investigate optimal stirring rate, 100 µl of 150 µg/mL budesonide solution were pipetted onto filters and µDiss stirring rate was varied from 200-600 rpm. After dose deposition, two filters were placed together with the deposited drug substance side facing each other, enclosing deposited DS within the filters. The filters were then assembled into the 3D printed filter holders which were placed into the µDiss. Dissolution started with addition of dissolution media (phosphate buffer pH 6.8 with 0.5% SDS). Dissolution occurred under sink conditions (Dose/Volume < 1/5 x Solubility). Concentrations were quantified using the µDiss probe and AuPro™ Software version 5.1.6.0 (pION INC., Woburn, MA) and via separate analysis using UPLC-UV (Figure 1). Fraction dissolved was determined by setting the highest cumulative amount to 1 at the end of experiment (18 hours for powders, 2 hours for solution). To control for complete dissolution, filter holders (with filters) were removed and extracted with methanol. The methanol extraction was analysed using UPLC-UV. The dissolution profiles were fitted to a Weibull equation [7] and compared based on time at which 63% of dose is dissolved (t63).

Drug substance between filters as · Solution · Weighed in powder · Dispersed powder

Manual samples for UPLC-UV control UV Probe 3D printed filter holder Magnetic Stirrer

Figure 1. Study overview – 3D printed filter holders were used to introduce drug substance solution or dispersed and non-dispersed (“weighed in”) powder into the µDiss. Concentrations were quantified using the µDiss probe (online) and via UPLC-UV of withdrawn samples (off-line). Results UPLC-UV and µDiss standard concentrations between 0.08-5 µg/mL were linear with respect to absorbance at drug substance wavelength maximum absorbance (R2>0.999). Consequently, most of the dissolution profile concentration quantification occurred within the linear range (Figure 2). Diffusion into bulk solution reached a maximum at 400 rpm; indicated by a more rapid diffusion than at 200 rpm and by profile similarity between 400 and 600 rpm stirring rates (Figure 3). Furthermore, the profile with stirring rate at 400 rpm contained less “noise” than at 600 rpm. Dispersed powder dissolved more rapidly than non-dispersed powder (Figure 4). Furthermore, there was better differentiation between dissolution profiles with dispersed drug substance powder than with non-dispersed powder. This was also indicated by statistical comparisons between t63 values, where non-dispersed powder only differentiated between budesonide and the other drug substances. For the dispersed powders however, a rank order for t63 (p<0.05) was established where budesonide (t63 (SD) = 11 (1) min) < FP (45 (27) min) = CD (55 (8) min) < FF (96 (12) min), indicating that budesonide dissolved most rapidly and FF least rapidly of the investigated powders. There was no significant difference between FP and CD (p>0.05).

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Drug Delivery to the Lungs, Volume 29, 2018 - Frans Figure 2. Average concentrations determined by UPLC-UV or µDiss 15mm probe during dissolution of dispersed Budesonide (red), Fluticasone propionate (orange), Fluticasone Furoate (blue) and Candidate Drug (green) micronized powder.

Franek et al. Figure 3. Average fraction of budesonide solution diffused into bulk at increasing stirring rates.

Figure 4. Budesonide (red), Fluticasone propionate (orange), Fluticasone furoate (blue) and candidate drug (green) micronized powder dissolution profiles. Full lines denote dissolution profiles of dispersed (using the modified Andersen Cascade impactor) powder and dashed lines denote non-dispersed (directly weighed) powder. The black profile denotes budesonide diffusion of solution into the bulk. Mean values (n=3) are shown with standard-deviation denoted by error-bars.

Discussion The aim of the study was to develop a time-efficient method to rank dissolution rates of poorly soluble DS powders using the mACI, µDiss and 3D-printed filter holders. The method was successful in ranking dispersed poorly soluble drug substance powders (Figure 4). The µDiss UV-probes are sufficient for concentration quantification and, consequently, for ranking drug substance dissolution (Figure 2). Although nominal concentrations between UPLC-UV and µDiss UV-probes differed for some substances, concentration-absorbency linearity within detection methods was observed for the majority of the dissolution profile concentration ranges. Consequently, once concentration profiles are normalized to fraction dissolved, the differences between detection methods decreased; t63 ranking remained the same, the average difference in t63 was 13% and was not statistically significant between the two detection methods. The quantification limit could, at least theoretically, be improved by increasing the path length of the probe [6]. The probes used had a 15 mm path length, and it is known that probes with at least a 20 mm path length are available[6]. Diffusion layer thickness may influence and rate-limit dissolution, this is inversely proportional to stirring rate. To minimize the risk for diffusion layer thickness limiting dissolution rates of poorly soluble DS powder dissolution, an appropriate stirring speed was determined by varying stirring rates (Figure 3). A rate of 400 rpm was considered appropriate, because diffusion layer thickness and UV-probe disturbances reached a minimum. Consequently, powder dissolution experiments were all performed at 400 rpm.

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Drug Delivery to the Lungs, Volume 29, 2018 - Ranking In Vitro Dissolution Of Orally Inhaled Drug Substance Powders In A Time-Efficient Manner The dispersion of the drug substance has been shown to be important prior to dissolution in order to differentiate between dissolution profiles (Figure 4)[3]. Here, all non-dispersed powders were found to dissolved slower than dispersed powders and most failed to be differentiated, despite sink conditions and presence of the surfactant SDS. Consequently, SDS is not enough for sufficient primary particle wetting and the non-dispersed powders were likely dissolving as aggregates. However, SDS does facilitate sink conditions because of micellar formation. Rohrschneider et al. suggest that the use of the synthetic surfactant SDS may be considered more biorelevant than using no surfactant[2]. Lung lining fluids also contain surfactants that increase particle wettability[1, 3]. The addition of surfactants for in vitro dissolution testing of poorly soluble substances may therefore be reasonable. Interestingly, the rank-order of dissolution for budesonide, fluticasone propionate and fluticasone furoate corresponds to the rank order of human lung absorption for the same substances (but formulated as drug products); expressed as mean absorption time (MAT) in man[8, 9]. The rank-order agreement between dissolution and absorption is logical because the investigated drug substances are poorly soluble and dissolution should thus be the rate limiting step for absorption[1]. Conclusions In this study, a method for on-line in vitro dissolution characterization and ranking of poorly soluble orally inhaled drug substance powders was developed. The method can rapidly rank in vitro dissolution of poorly soluble drug substance powders, which may be important for understanding drug product performance. References 1

Hastedt JE, Bäckman P, Clark AR, Doub W, Hickey A, Hochhaus G, Kuehl PJ, Lehr CM, Mauser P, McConville J, Niven R: Scope and relevance of a pulmonary biopharmaceutical classification system AAPS/FDA/USP Workshop March 16-17th, 2015 in Baltimore, MD, AAPS Open, 2016; 2.

Rohrschneider M, Bhagwat S, Krampe R, Michler V, Breitkreutz J, Hochhaus G: Evaluation of the Transwell System for Characterization of Dissolution Behavior of Inhalation Drugs: Effects of Membrane and Surfactant, Mol Pharm, 2015; 12: pp2618–2624.

Velaga SP, Djuris J, Cvijic S, Rozou S, Russo P, Colombo G, Rossi A: Dry powder inhalers: An overview of the in vitro dissolution methodologies and their correlation with the biopharmaceutical aspects of the drug products, Eur J Pharm Sci, 2017;113: pp18-28.

Franek F, Yousef G, Kinga BS, Thörn H, Fransson R, Tehler U: Biorelevant and flexible dose deposition of drug substance powders and nebulised liquids onto filters and cell-cultures, Proc Drug Deliv Lungs 2017, [ISBN: 978-0-9957688-0-2]: pp182– 185.

May S, Jensen B, Weiler C, Wolkenhauer M, Schneider M, Lehr CM: Dissolution testing of powders for inhalation: influence of particle deposition and modeling of dissolution profiles, Pharm Res, 2014; 31: pp3211–3224.

Schatz C, Ulmschneider M, Altermatt R, Marrer S: Evaluation of the Rainbow Dynamic Dissolution MonitorTM semi-automatic fiber optic dissolution tester, Dissolution Technol, 2000; 7: pp8–19.

Dokoumetzidis A, Papadopoulou V, Macheras P: Analysis of dissolution data using modified versions of Noyes–Whitney equation and the Weibull function, Pharm Res, 2006; 23: pp256–261.

Thorsson L, Edsbäcker S, Källén A, Löfdahl CG: Pharmacokinetics and systemic activity of fluticasone via Diskus® and pMDI, and of budesonide via Turbuhaler®, Br J Clin Pharmacol, 2001; 52: pp529–538.

Allen A, Bareille PJ, Rousell VM: Fluticasone Furoate, a Novel Inhaled Corticosteroid, Demonstrates Prolonged Lung Absorption Kinetics in Man Compared with Inhaled Fluticasone Propionate, Clin Pharmacokinet, 2012; 52: pp37–42.

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Drug Delivery to the Lungs, Volume 29, 2018 – Huitong Lucy Li, et al. Targeted PEG-Poly(glutamic acid) Polymers For The Delivery of Proteins Into The Lung Epithelium Huitong Lucy Li,1 Alejandro Nieto-Orellana1, Franco H. Falcone1, Cynthia Bosquillon1, Gemma Keegan2, Nick Childerhouse2, Giuseppe Mantovani*1 and Snow Stolnik*1 1

School of Pharmacy, University of Nottingham, University Park, Nottingham, NG7 2RD, UK. 2

Vectura Group plc, One Prospect West, Chippenham, SN14 6FH, UK

Summary Pulmonary delivery via inhalation offers a potential route for non-invasive delivery of proteins and peptides. However, protein degradation, pulmonary clearance and immunogenicity issues are challenges that need to be addressed. Non-covalent polymer-protein conjugation has emerged as a potential strategy to overcome these challenges. In this work, we propose B12 targeted non-covalent polymer-protein nanocomplexes (consisting of linear PEG-polyglutamic acid polymers (PEG-poly(GA) ionically interacting with positively charged amino acid groups in proteins) in an attempt to increase the cellular internalisation. The expression of TCN2 B12 receptor gene by lung epithelial Calu-3 cells and the receptor’s involvement in the B12 cellular internalisation was confirmed by a series of experiments. Initial studies strongly indicated that polymerprotein nanocomplexes can be formed between the polymer and lysozyme protein through electrostatic interaction. Comparisons of cellular uptake of polymer–protein nanocomplexes were assessed in vitro between B12 targeted and non-targeted nanocomplexes. The study showed B12-targeted polymer-protein nanocomplexes had enhanced cell uptake in vitro by lung Calu-3 cells, making this PEG-poly(GA) polymer a promising alternative for targeted delivery of biologicals in the lung.

Key Message Successfully synthesised B12 polymers and complexed with lysozyme. Investigations are ongoing to determine their ability to transport the protein into lung epithelial cells using an in vitro model. Initial studies confirm the presence of TCN2 receptors at gene level by the cells and the receptor’s role in the B12 uptake.

Introduction Pulmonary delivery is increasingly seen as an attractive, non-invasive route for the delivery of protein therapeutics [1]. The presence of mucus acts as an almost impassable barrier for macromolecular therapeutics such as proteins. Poor protein bioavailability and immunogenicity are also challenges that still need to be addressed [2, 3]. However, there are a number of biological transport pathways that are present at mucosal surfaces and lung epithelium. Nanocarriers conjugated with specific ligands can increase the interaction with epithelial cells, provided that suitable receptors can be identified [4, 5]. In fact, they have a crucial role on the uptake of molecules such as vitamin B 12, albumin and IgG [4, 6]. This knowledge has been utilised in designing specific ligands to decorate the surface of drug delivery systems and to increase the cellular uptake in the airway mucosa epithelium [7]. Although the B12 pathway has been shown to transport nanoparticles across intestinal Caco-2 cells information can be found regarding its presence and functions in the pulmonary system [9].

[8]

, limited

In this study we utilised vitamin B12 as targeting ligand for our polymers, and confirmed the presence of specific vitamin B12 receptors in Calu-3 lung epithelial cells by checking the gene specific mRNA expression and competition studies in the presence of free vitamin B12. After the initial confirmation of the receptor expression, we further showed cellular internalisation of B12-targeted protein-polymer nanocomplexes.

Experimental methods Materials Unless otherwise stated, all chemicals and materials were supplied by Sigma-Aldrich® (UK) or Fisher Scientific® (UK) and used without further purification. Fluorescent Vitamin B12 probe Pyranine and Vitamin B12 were first modified to possess either a carboxyl or amino group respectively, then they were conjugated together in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM).

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Drug Delivery to the Lungs, Volume 29, 2018 - Targeted PEG-Poly(glutamic acid) Polymers For The Delivery of Proteins Into The Lung Epithelium Preparation of polymer-protein nanocomplexes The non-targeted copolymers used in this study possessed linear A-B structures where the hydrophilic A block consists of polyethylene glycol (PEG) and that of protein-binding B arms (Poly-glutamic acid) have about 30 repeating units [10]. The targeted polymer possesses the same A-B structure with a slightly larger A block and a B12 molecule synthetically attached to the end of A block. (mPEG2k-poly(GA))-protein nanocomplexes were prepared at relative molar charge ratios r = 1.25 where r is the charge ratio between the number of glutamic acid residues present in the copolymer (negatively charged at physiological pH) and the positively charged residues in the protein at physiological pH (surface charges: lysozyme +7.5). Polymers and protein were separately dissolved in phosphate buffer (PB, 10 mM, pH 7.4) and appropriate aliquots were mixed to achieve r = 1.25. To prepare targeted polymer-protein nanocomplexes, mPEG2k-poly(GA) and vitB12-PEG3K-poly(GA) copolymers were mixed at relative molar ratios of 85/15 (% mol/mol), respectively. Polymer mixture and lysozyme were then mixed at relative molar charge ratio of 1.25 as previously described. Cell culture Human airway epithelial cells (Calu-3) were obtained from the ATCC (Manassas, Virginia, USA) and used between passages 30-40. Cells were grown to confluence in 75 cm2 flasks (canted neck and vented caps, Corning, New York, USA) in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 supplemented with Foetal Bovine Serum (FBS, 10% v/v), L-glutamine (1% v/v), penicillin (100 units mL -1) and streptomycin (100 µg mL-1). Upon confluence, cells were detached from the flasks and 100,000 cells were seeded on permeable supports (Transwell ®, Corning, 12 mm diameter, 1.1 cm2, 0.4 µm pore size). Calu-3 cells were grown in air-liquid interface (media in the bottom of Transwells® only) culture condition with media replaced every other day. Transepithelial Electrical Resistance (TEER, Ωcm2) was measured using a portable epithelial voltohmmeter (EVOM, World Precision Instruments) and cell monolayers with TEER exceeding 500 Ωcm2 on day 14 of culture were deemed suitable for experiments. mRNA expression of cubilin and TCN2 receptor in Calu-3 cells The RNeasy Plus Mini Kit (Qiagen, Hilden, German) was used to extract mRNA from Calu-3 cells following manufacturer’s instructions. cDNA were prepared using QuantiTect Reverse Transcription Kit (Qiagen). Real-time polymerase chain reaction (qPCR) was performed using a standard quantitation-comparative Ct procedure as set by the manufacturer. Triplicate reactions (25 μL) of each experimental sample were prepared using CYBR master mix. Reactions were subjected to an initial 10-minutes denaturation step at 95 °C, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 60 seconds. Reference gene GAPDH was used as internal control. Cellular uptake of fluorescent B12 to check receptor presence Calu-3 cells were cultured on Transwell® inserts over 14 days in air-liquid interface condition. Culture medium was removed from filter-cultured cells and replaced with Hank’s Balanced Salt Solution (HBSS). Following a routine measurement of TEER, fluorescent B12 probe were suspended in media at concentration of 200 µg/ml; 500 µL were added to the apical chamber of the Transwell® insert. Cells were then incubated at 37 °C for 3h and analysed by flow cytometry in at least triplicate experiments, with more than 10,000 cells measured in each sample. The effect of a “competitor” on receptor-mediated uptake of fluorescent B12 probe was studied in the presence of excess free vitamin B12. 0.3 mL of 1 mg/mL vitamin B12 dissolved in DMEM/F12 media solution were added to the apical chamber of Transwell® inserts. Cells were then incubated at 37 °C for 30 minutes followed by addition of the fluorescent B12 probe suspension in the presence of excess free B12 to reach a final volume of 0.5 mL and final probe concentration of 200 µg/mL. Cells were incubated again at 37 °C for 3h and analysed by flow cytometry. Experiments were made in at least triplicates, with more than 10,000 cells measured in each sample. Cellular uptake of lysozyme by forming polymer-protein nanocomplexes Calu-3 were cultured on Transwell® inserts over 14 days as previously described. Polymer-protein nanocomplexes prepared as previously described were suspended in media at protein concentration of 50 µg/ml; the nanocomplex suspension (0.5 mL) was added to the apical chamber of the Transwell ® insert. Cells were then incubated at 37 °C for 3h and analysed by flow cytometry in at least triplicate experiments, with more than 10,000 cells measured in each sample.

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Drug Delivery to the Lungs, Volume 29, 2018 – Huitong Lucy Li, et al. Results and Discussion There are two main types of receptors mediating B12 uptake: B12-intrinsic factor (IF) (a glycoprotein produced by the stomach) complexes via the cubilin receptor and B 12 uptake via the transcobalamin II receptor [11]. qPCR analysis of Calu-3 cells was performed as an initial screen to determine the mRNA expression of cubilin (human CUBN gene) and transcobalamin II (human TCN2) (Figure 1). Breast cancer cell line MCF-7 was used as a negative control for cubilin expression and positive control for TCN2 expression [12, 13]. The data confirmed the expression of both cubilin and TCN2 receptors in Calu-3 cells, but the cubilin expression was very low and almost negligible. The mechanism of B12 uptake via cubilin receptor requires the complexation between B 12 and gastric intrinsic factor (IF), as IF is not present in the lungs, this receptor could have been down regulated due to its lack of purpose. For this reason, IF was not used in subsequent uptake studies.

0 .0 0 2 0 .0 0 1

0 .0 0 0 3 0 .0 0 0 2 0 .0 0 0 1

il b

0 .0 0 0 0

u C

a C

00030 00025 00020 0 .0 0 0 4

-3

-7

0 .0 0 0

-3

0 .0 0 0 0 0

0 .0 0 3

00040 00035

0 .0 0 0 0 2

R e la tiv e m R N A

0 .0 0 0 0 4

0 .0 0 4

n o r m a lis e d to G A P D H

0 .0 0 0 0 6

-7

0 .0 0 0 0 8

r e la tiv e to G A P D H

T C N 2 m R N A e x p r e s s io n

b) 0 .0 0 0 1 0

r e la tiv e to G A P D H

C u b ilin m R N A e x p r e s s io n

Figure 1. Cubilin and TCN2 receptor mRNA expression in Calu-3 and MCF-7 (used as negative control for cubilin and positive control for TCN2) cells. a) mRNA expression of cubilin relative to GAPDH and b) mRNA expression of TCN2 relative to GAPDH. c) mRNA expression of TCN2 compared to cubilin in Calu-3 cells relative to GAPDH.

Cells were incubated with 200 µg/mL of fluorescent B12-probe suspension for 3h with and without free B12 competitive ligand. Following 3h incubation, uptake of the fluorescent B12 probe was found to be suppressed by up to about 25% in the presence of free B12 as quantified by flow cytometry (Figure 2a). This ‘competition’ indicates that the fluorescently labelled B12 is entering cells through the receptor mechanism and possibly some other less well-characterised mechanisms as well, which require further exploration. To investigate the ability of the targeted nanocomplexes to engage with specific endocytic receptors on the mucosal surfaces of the airway epithelium, protein uptake by Calu-3 cells was evaluated, in analogy with what was already tested with the fluorescent B12 probe. Bronchial Calu-3 cells were chosen here as an airway epithelium cell model able to promote mucus secretion when they are grown in air-interface culture (AIC) conditions. After the success formation of polymer-protein nanocomplexes, Figure 2b shows the mean fluorescence intensity (MFI) as an indication of cellular protein uptake at 50 μg/mL protein concentration. Targeted vitB12-PEG3k-poly(GA) nanocomplexes were able to significantly increase the lysozyme uptake (up to 50% more) by Calu-3 cells when compared to free lysozyme and non-targeted mPEG2k-poly(GA) nanocomplexes. In addition, the formation of the complexes did not appear to hinder the uptake of lysozyme, which might indicate that the use of polymer does not adversely affects protein bioavailability while providing the benefit of preventing protein degradation extracellularly.

ge p te l d 12 ex es C t om ar ge p te le d xe s

b ro

12 B

e b ro

eB re F

-t

* 15

( 5 0  g /m l) b y C a lu - 3 c e lls 20

M e a n F lu o r e s c e n c e I n t e n s ity

C e ll u l a r u p t a k e o f l a b e ll e d ly s o z y m e

F lu o r e s c e n t B 1 2 u p t a k e b y C a l u - 3 c e l ls M e a n F lu o r e s c e n c e I n t e n s ity

Figure 2. a) Competition study: Calu-3 cellular uptake of 200µg/ml fluorescent B12 probe in the presence of free B12. Control: untreated cells. b) Cellular uptake in Calu-3 layers of free lysozyme compared to lysozyme complexed with targeted polymer-protein nanocomplexes (targeted / non-targeted polymer ratio: 15/85%). Protein concentration at 50µg/ml. “Lysozyme” corresponds to samples without polymers. All samples were added in solution on top of the cells. One-way ANOVA was used for statistical analysis. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001.

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Conclusions In this study, we confirmed the presence of TCN2 receptor on lung epithelial Calu-3 cells. Competition studies with the uptake of fluorescent B12 probe suggest that B12 were taken up through receptor mechanism. In addition, the absence of cubilin receptors implicates that the uptake of B12 was through the TCN2 pathway. Nanocomplexes were successfully formed between protein lysozyme and the linear PEG-poly(GA) polymers; cells were internalising up to 1.5 times more of the nanocomplexes with targeted B 12 polymers. Furthermore, the nanocomplexes may be formulated into dry powder formulations for the targeted inhalation therapy to the lungs.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Patil, J.S. and S. Sarasija, Pulmonary drug delivery strategies: A concise, systematic review. Lung India : Official Organ of Indian Chest Society, 2012. 29(1): p. 44-49. Baker, M.P., H.M. Reynolds, B. Lumicisi, and C.J. Bryson, Immunogenicity of protein therapeutics: The key causes, consequences and challenges. Self Nonself, 2010. 1(4): p. 314-322. Hussain, A., J.J. Arnold, M.A. Khan, and F. Ahsan, Absorption enhancers in pulmonary protein delivery. Journal of Controlled Release, 2004. 94(1): p. 15-24. Tuma, P. and A.L. Hubbard, Transcytosis: crossing cellular barriers. Physiol Rev, 2003. 83(3): p. 871-932. Russell-Jones, G.J., L. Arthur, and H. Walker, Vitamin B12-mediated transport of nanoparticles across Caco-2 cells. Int J Pharm, 1999. 179(2): p. 247-55. Russell-Jones, G.J., S.W. Westwood, and A.D. Habberfield, Vitamin B12 mediated oral delivery systems for granulocyte-colony stimulating factor and erythropoietin. Bioconjug Chem, 1995. 6(4): p. 459-65. Fowler, R., D. Vllasaliu, F.H. Falcone, M. Garnett, B. Smith, H. Horsley, C. Alexander, and S. Stolnik, Uptake and transport of B(12)-conjugated nanoparticles in airway epithelium(). Journal of Controlled Release, 2013. 172(1): p. 374-381. Chalasani, K.B., G.J. Russell-Jones, S.K. Yandrapu, P.V. Diwan, and S.K. Jain, A novel vitamin B12nanosphere conjugate carrier system for peroral delivery of insulin. J Control Release, 2007. 117(3): p. 421-9. Vortherms, A.R., A.R. Kahkoska, A.E. Rabideau, J. Zubieta, L.L. Andersen, M. Madsen, and R.P. Doyle, A water soluble vitamin B12-ReI fluorescent conjugate for cell uptake screens: use in the confirmation of cubilin in the lung cancer line A549. Chem Commun (Camb), 2011. 47(35): p. 9792-4. Nieto-Orellana, A., M. Di Antonio, C. Conte, F.H. Falcone, C. Bosquillon, N. Childerhouse, G. Mantovani, and S. Stolnik, Effect of polymer topology on non-covalent polymer-protein complexation: miktoarm versus linear mPEG-poly(glutamic acid) copolymers. Polymer Chemistry, 2017. 8(14): p. 2210-2220. Kozyraki, R. and O. Cases, Vitamin B12 absorption: Mammalian physiology and acquired and inherited disorders. Biochimie, 2013. 95(5): p. 1002-1007. Rowling, M.J., C.M. Kemmis, D.A. Taffany, and J. Welsh, Megalin-Mediated Endocytosis of Vitamin D Binding Protein Correlates with 25-Hydroxycholecalciferol Actions in Human Mammary Cells. The Journal of Nutrition, 2006. 136(11): p. 2754-2759. Viola-Villegas, N., A.E. Rabideau, M. BartholomĂ¤, J. Zubieta, and R.P. Doyle, Targeting the Cubilin Receptor through the Vitamin B12 Uptake Pathway: Cytotoxicity and Mechanistic Insight through Fluorescent Re(I) Delivery. Journal of Medicinal Chemistry, 2009. 52(16): p. 5253-5261.

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Drug Delivery to the Lungs, Volume 29,2018 - Stefani Mariarita et al. Development of an In Vitro Solubility Test as a Tool for Predicting Lung Retention of Poorly Water Soluble Compounds Stefani Mariarita1, Cesari Nicola 1, Corsaletti Roberto1, Fioni Alessandro1, Saccani Francesca1, Volta Roberta1, Brogin Giandomenico1 & Puccini Paola1 1Chiesi

Farmaceutici S.p.A., Largo Belloli 11/A, Parma, 43122, Italy

Summary Background: The prediction of pulmonary exposure after inhalation of a drug is one of the key objectives for the success of a novel inhalable drug candidate. This study focuses on the determination of an apparent solubility (Sapp) value that may be used to study the impact of solubility on the drug candidate lung mean retention time (MRT) in order to support lead candidate selection. Methods: An in vitro solubility test in simulated lung fluid (SLF) was developed to measure Sapp. The Sapp value was determined by the diffusion of the dissolved amount from a drugsaturated solution through a membrane, which provides the separation between the donor and the receiver (sampling) compartments. In order to evaluate the validity of the test, different compounds with poor water solubility and different solid state were tested. Results: The test can provide different values of Sapp for the same compound, based on the different solid states of the powder. The values correlated well with the in vivo lung mean residence time (MRT) after intra-tracheal administration in rats. Conclusion: The Sapp value seems promising in predicting the impact of solubility on drug candidate lung MRT in the drug discovery phase. Key Message We developed an in vitro solubility test that provides a value of apparent solubility. This value correlated well with the lung mean residence time and, therefore, may be useful in predicting lung retention of inhaled compounds. Introduction Interest in the inhalation of drugs is increasing, but the knowledge about the involved processes and all the variables to be considered for the prediction of drug pharmacokinetics is still incomplete.The prediction of pulmonary exposure after inhalation of a drug is one of the key objectives for the success of a novel inhalable drug. One of the most difficult parameters to set up consists of the determination of the solubility of drug particles in the lung, especially for poorly soluble compounds. The aim of this study was to find a Sapp value that may be used to study the impact of solubility on drug candidate lung retention. This could play an important role in the prediction of pulmonary exposure of drugs. Materials and methods Materials Slide-A-Lyzer™ G2 Dialysis Cassettes, 10K MWCO, 15 mL (Fisher Scientific Italia, Milan, Italy) was used as cassette membrane. The natural porcine surfactant Curosurf® (Chiesi Farmaceutici S.p.A., Parma, Italy) was used as surfactant in simulated lung fluid (SLF). Saline buffer was purchased from Eurospital (Trieste, Italy). The reagents for the samples preparation, the mannitol and the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Sigma-Aldrich (Milan, Italy). The filter vials 0.2µm PTFE were purchased from Thomson (Restek Italia, Milan, Italy). All the solvents used for the LC-MS/MS bioanalysis, suitable for UHPLC instrument, were HiPerSolv CHROMANORM® from VWR International Srl (Milan, Italy). Preparation of simulated lung fluid (SLF) The PBS tablets were dissolved in Milli-Q water to obtain a final concentration of 10mM and the pH was adjusted to 6.9 by adding few drops of 1M HCl. Curosurf® was added to obtain final concentration of 0.02% w/v (namely 2.5 mL of Curosurf® 80 mg/mL suspension to 1L PBS). Experimental setting The experimental setting consists of a glass container filled with 300 mL of SLF at 37 °C under slow stirring. The cassette with the membrane was put into the container for about 5 min to hydrate the membrane; then 10mL of test compound SLF solution at 5 μg/mL (diffusion with solution) or 5 mg of test compound powder (diffusion with powder) were added to 10 mL of SLF into the inner compartment. The cassette was closed and put again into the glass container, as shown in Figure 1.

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Drug Delivery to the Lungs, Volume 29, 2018 - Development of an In Vitro Solubility Test as a Tool for Predicting Lung Retention of Poorly Water Soluble Compounds

Figure 1 â&#x20AC;&#x201C; Experimental setting: the cassette was put into a glass container filled with SLF for 5 min to hydrate the membrane (a), the cassette was removed from buffer (b), the test compound solution was added into the inner compartment (c), the cassette was closed and immersed again into the glass container (d).

Aliquots for analysis were withdrawn from the outside compartment before the immersion of the cassette and a series of time points up to 2h after the immersion. Sample preparation and analysis The sample preparation was performed by precipitation with acetonitrile using filter vials and analysed by LCMS/MS system (Agilent 1290 UHPLC system, Triple Quad 6500, AB Sciex S.r.l., Milan, Italy). Analyte quantitation was performed by applying the internal standard method. The area ratios between the analyte peak area and the internal standard peak area were used for the calculations. A calibration curve of at least 6 standard and 2 quality control samples for each 3 different concentration levels were used in each analytical session. Calculations The test consisted on the measurement of the diffusion of the dissolved drug through a membrane, which provided the separation between the donor and receiver (sampling) compartment, avoiding any issues due to separation of the solution from the undissolved powder and the biological matrix of the test, which was SLF at pH 6.9[1]. The rate of diffusion depends on the permeability across the membrane and the concentration gradient. The relationship that guides the diffusion rate under sink conditions is: đ?&#x2018;&#x192;đ?&#x2018;&#x192; â&#x2C6;&#x2014; đ??śđ??ś = đ??žđ??žđ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018; .

where P is permeability and C the concentration in the donor compartment. The diffusion rate (Kdiff) is represented by the slope of the linear portion of the curve, resulting from the plot of the concentration found in the receiver compartment versus time, after the diffusion with solution. The same process is involved when the drug powder, instead of a solution, is tested in the same condition. The dissolved compound generates a concentration in the donor compartment, that is, S app, which drives the diffusion in the receiving compartment. The occurrence of a linear increase of the drug amount in the receiver compartment supports the hypothesis that a constant Sapp is obtained in the donor compartment. The relationship that described this process is: đ?&#x2018;&#x192;đ?&#x2018;&#x192; =

đ??žđ??žđ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;_đ?&#x2018; đ?&#x2018; đ?&#x2018; đ?&#x2018; đ?&#x2018; đ?&#x2018;

đ??śđ??ś đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;

đ??śđ??ś đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘ (Sapp)

Where kdiff_sol is the rate of diffusion measured when a solution of the drug is placed in the donor compartment, Cdiffusion is the concentration of the solution and kdiff_powder is the rate of diffusion measured when the drug powder is placed in the donor compartment.

Therefore: đ??śđ??ś đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘đ?&#x2018;˘ (Sapp) =

đ?&#x2018;&#x2DC;đ?&#x2018;&#x2DC; đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x201C;đ?&#x2018;&#x201C;_đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;?đ?&#x2018;? â&#x2C6;&#x2014; đ??śđ??ś đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;

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Drug Delivery to the Lungs, Volume 29,2018 - Stefani Mariarita et al. Results In order to support the validity of the test, the following aspects were investigated: 1.

The permeability is constant in the concentrations that include the Sapp for each powder tested. Three diffusion experiments were performed with Compound 1, using solutions at different concentrations in the donor compartment. The rate of diffusion was proportional with the concentration, which indicates the constancy of the permeability, as shown in Figure 2.

Figure 2 - Three diffusion experiments with three different concentrations of the solution in the donor compartment were performed; the rate of diffusion resulted proportional with the concentration.

The system was at sink conditions. The concentration in the receiving compartment was always <10% of the donor one for the whole experiment.

The Sapp is the same when different amounts of powder are put in the donor compartment. The same value of Sapp was obtained for Compound 2 when two different experiments were performed using 1 mg and 5 mg of powder, as described in Table 1.

Table 1 - The Sapp of Compound 2 was obtained using two different amounts of powder, resulting the same value of rate of diffusion and Sapp.

Compound 2

Kdiff_powder (ng/min)

Sapp (ug/mL)

1 mg

33.2 (n = 2)

1.86

5 mg

34.6 ± 5.63 (n = 3)

1.94 ± 0.32

The test provides reproducible values of Sapp when determinations are made in different days: The Sapp of the anhydrous form of Compound 3 was calculated on three different days: the values obtained, the mean value and the SD are shown in Table 2.

Table 2 -

The Sapp of Compound 3 resulted reproducible on experiments conducted on different days.

Compound 3

Day

Sapp (µg/mL)

Anhydrous form Anhydrous form Anhydrous form

Day 1 Day 2 Day 3

48.1 47.4 49.2 48.2 0.907 1.88

Mean SD %CV

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Drug Delivery to the Lungs, Volume 29, 2018 - Development of an In Vitro Solubility Test as a Tool for Predicting Lung Retention of Poorly Water Soluble Compounds

The test provides different values of Sapp for the same compound, based on the different solid states or formulation of the powder considered. The Sapp of Compound 4 was calculated for its crystalline form, its amorphous form and for a spray-dried formulation with mannitol and DPPC (Compound 4:mannitol:DPPC 35:62:3 w/w)[2, 3] The resulted values of Sapp were 2.56, 14.3 and 54.1 ug/mL for the crystalline form, the amorphous form and the formulation, respectively.

Discussion The test was performed in a concentrations range assuring constant permeability through the membrane. The system was in sink conditions and the Sapp value did not change when different amounts of powder were used. Moreover, the test provided different values of Sapp for the same compound, based on the different solid states or formulation of the powder considered. A preliminary evaluation of the utility of the Sapp values obtained from this test was conducted by comparing the values with the lung MRT obtained after intra-tracheal administration in rats[3]. The different characteristics of the three different formulations of Compound 4 affected the lung retention after inhalation and the Figure 3 strongly suggest a trend of correlation between the values of Sapp and the value of lung MRT. Further investigations will be performed to confirm this correlation.

Figure 3 â&#x20AC;&#x201C; Correlation between the values of Sapp in SLF and the lung MRT found after intra-tracheal administration of Compound 4.

Conclusion The test described in this study can provide different values of Sapp for the same compound, based on different solid states/formulations. The good correlation obtained with the lung MRT determined in vivo in rats suggests that the Sapp could be used in a rat lung PBPK model to predict the lung disposition of low soluble inhaled drugs. 1

Ng AW, Bidani A, Heming T A: Innate host defense of the lung: effects of lung-lining fluid pH, Lung 2004; 182(5):297-317

Duret C, Merlos R, Wauthoz N, Sebti T, Vanderbist F, Amighi K: Pharmacokinetic evaluation in mice of amorphous itraconazole-based dry powder formulations for inhalation with high bioavailability and extended lung retention, Eur. J. Pharm. Biopharm. 2014; 86(1):46-54

Fioni A, Bagnacani V, Miozzi M, Balducci A, Saccani F, Benetti C, Stefani M, Brogin G, Pappani A, Lipreri M, Amadei F, Catinella S, Puccini P: Evaluation of Dry Powder Formulation for the Improvement of Lung Pharmacokinetic of NCEs during Preclinical In vivo Experiments (Poster). Presented at: World Preclinical Congress 2018, Boston, Massachusetts, 18-21 June, 2018.

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Drug Delivery to the Lungs, Volume 29, 2018 – Herbert Wachtel et al. Systematic Development of an Inhaler Device for the Use in Horses: The EquiHaler® Herbert Wachtel1, Marcus Rahmel1 Guido Endert2 & Benjamin Franzmann3 1

Boehringer Ingelheim Pharma GmbH & Co KG, Binger Strasse 173, 55216 Ingelheim, Germany 2 DESIGNquadrat GbR, Schmiedhofsweg 1, 50769 Köln, Germany 3 Boehringer Ingelheim Vetmedica GmbH, Binger Strasse 173, 55216 Ingelheim, Germany

Summary Soft Mist Inhalers™, like the Respimat® SMI™, are officially called non-pressurized metered dose inhalers and provide unique medicinal aerosol generated features such as: a slowly moving aerosol cloud, a small droplet particle size, high fine particle fractions, as well as an intuitive user interface. A high medical need for an advanced aerosol treatment has been identified in horses. Based on the idea that Respimat ® technology should be available across species, equine feasibility studies were conducted by Boehringer Ingelheim Vetmedica GmbH. Considering the upper airway geometry of horses and their typical breathing patterns as well as the user habits, a novel and innovative device has been built around the functional core units of the Respimat ®. A set of relevant interfaces and characteristics has been defined, these include: the nostril adapter (connecting the nostril of the horse with the inhaler containing a breath indicator), and the handle with a dual-functional lever enabling single hand operation by the user. Here we describe the interdisciplinary cooperation of the design team, the resolution for the equine anatomical puzzle, aerosol science, human operational factors, and last but not least the coordination challenge of treating an animal. As a result of these studies, the EquiHaler® was designed to give relief to many suffering horses. In this inhaler an ethanolic solution, containing the 3rd generation corticosteroid ciclesonide, is used to treat clinical symptoms associated with equine asthma, formerly known as RAO (recurrent airway obstruction) and SPAOPD (summer pasture associated pulmonary disease). The combination of EquiHaler® and the ethanolic formulation generates an extra fine aerosol characterized by an adaptable therapeutic dose of 2.7 mg (8 actuations) and a mass median aerodynamic diameter of 1.5 µm, depending on the operating conditions of the impactor. Key Message The EquiHaler® is the first soft mist device developed for inhalation treatment of respiratory diseases in horses. The Equihaler device is based on the Respimat® technology, and demonstrates features that consider the specific anatomical considerations of horses as well as the ease of use for the operator. Introduction Many horses suffer from equine asthma, formerly also known as RAO (Recurrent Airway Obstruction) and SPAOPD (Summer Pasture Associated Pulmonary Disease). The inhaler design is based on the well-established Respimat® soft mist inhaler, and here the first tests are described. A multi-disciplinary team was formed and challenged with developing the prototype. Several design variants were created, using rapid prototyping technology (Fig. 1). In parallel, with the development of the handling principle, fully functional technical building blocks were assembled; paving the way for analytical method development.

Figure 1 - Development history of the EquiHaler®: A first trial in horses was supported by the prototype at left. A conventional Respimat® device is attached to a transparent tube which is connected to the nostril adapter, to direct dose aerosol emission. The clear tube contains a one-way valve and a breath indicator in order to ensure coordination of inhalation and dose release. The same features were built into the analytical test device (center) which was used during development of the analytical tests. A circular valve plate is located in the circumference of the nozzle system. The horse's breathing is indicated by a diaphragm at the nostril adapter (breath indicator).

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Drug Delivery to the Lungs, Volume 29, 2018 - Systematic Development of an Inhaler Device for the Use in Horses: The EquiHalerÂŽ The endpoint of the development (right) considers usability engineering as single hand operation is possible. In addition to the breath indicator, a dose indicator is incorporated into the design. Experimental methods Usability engineering was applied in order to define the requirements, and the final design of the inhaler. It was known that horsemen guide their horses by the right hand and stay at the horse's left side. Thus, the inhaler must be activated using the left hand, even if the majority of horsemen are right handed (see Fig. 2).

Figure 2 - Horsemen stand on the horse's left side and therefore the horseman's left hand should be used to prepare and to release the inhaler (double action feature). The necessary force has been tuned that also a smaller hand can tension the spring and then actuate the device by a second small movement of the lever. As shown by the Figures 1 and 2, the design of the EquiHaler ÂŽ relies on a single nostril adapter. The aerosol is inhaled through the left nostril while the other nostril remains open. There is sufficient air flow during inhalation and furthermore, the one-way valve in the device is not stressed because the horse can exhale through the open nostril at any time. The analysis of the breathing pattern is important, as it is intended to treat horses at rest, therefore typical tidal breathing profiles have been recorded. The Pneumotachograph used was a MasterScope (Jaeger, Hoechberg, Germany). Also the inspiratory and expiratory pressure was measured at the blocked nostril (keeping the right nostril open all the time). Figure 3 gives an example of a typical pattern.

Figure 3 - Inhalation air flow through one nostril (the other nostril stays open). Typical pattern measured with an adult horse (~500 kg). High flow rate and fast breathing are observed if the inhalation is new to the horse. Airflow and deposition simulations were carried out using the computational fluid dynamics program Fluent (Ansys, Canonsburg, Pennsylvania, USA). The internal cross section area of the inhaler was enlarged during development, thus avoiding internal losses of aerosol. The spray direction was aligned with the airflow and the valve design was modified to generate a circular sheath flow which further increased drug delivery efficiency.

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Drug Delivery to the Lungs, Volume 29, 2018 – Herbert Wachtel et al. In order to speed up analytical method development, functional sub-units were manufactured and measurements of delivered dose and particle size distribution were optimized at an early development state even without the complete inhaler being available. The laser diffraction apparatus consisted of a commercial analyser (Sympatec GmbH, Clausthal-Zellerfeld, Germany) with a measurement cell designed by Boehringer Ingelheim (BI)[1]. The air flow rate was set to 15 L/min in order to minimize dilution of the aerosol and to optionally link the analysis to an aerodynamic measurement method using the next generation cascade impactor which has a calibration at that flow rate available. Materials were selected according to experiences made with the Respimat ® platform (Boehringer Ingelheim, Ingelheim, Germany). The nozzle assembly consisting of modified uniblock, nozzle holder, gaskets, and the central tube was copied from the Respimat® design, additionally the capillary with cambushing was re-used. Thus the metered volume was approximately the same as that of Respimat®. The formulation was switched from aqueous to ethanolic (90 vol.-% ethanol).

Results The aerodynamic layout was optimized and combines nostril adapter, breath indicator, and valve holding chamber. This facilitates coordination of dose release with the horse's inhalation cycle. The cross section area inside the inhaler was large by design, as inhalation through the device and a single nostril competes with the open second nostril.

Figure 4 – Computational Fluid Dynamic (CFD) simulations indicates low flow velocity of the air entering through the valve (right) and surrounding the slightly faster spray cloud, which is also slowed down through the device (graph shows outlet flow rate 40 L/min). Handling results indicate that the device can be well used in daily practice. This was checked during three workshops (one in Germany, two in the USA) with a total of 25 horsemen (BI employees). Coordination based on the breath indicator is simplified because the user can observe the breathing action by the movement of the diaphragm located at the nostril adapter (see also Figure 1). After user training, the dual-functional lever was used without difficulty. Analytical results indicate the reliability of the delivery system: The delivered dose consists of 8 or 12 actuations administered at a time. This results in an error-tolerant administration scheme, as the individual single inhalation has low statistical weight. The particle size distribution reflects an extra-fine soft mist (mass median diameter < 2 µm, see Figure. 5) which should enable the drug-laden particles to follow the inhaled air through all regions of the horse's respiratory tract [2]. Tests with different spring forces characterize the robustness of the aerosol generation principle (Figure 5).

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mass median diameter (µm)

2.5

y = 0.0002x - 0.0292x + 2.6351 1.5 without nostril adapter with nostril adapter 1 20

spring force (N)

Figure 5 - Mass median diameter of the soft mist measured by laser diffraction (Sympatec Helos BF, R2 lens) with and without nostril adapter. Conditions: Air flow rate 15 L/min, T=22°C, RH=38.5%. Error bars indicate standard deviation. Discussion This development work was additionally characterized by a short feasibility phase which intentionally de-risked the project by first checking the clinical effects using a very simple prototype (data not shown). During the development phase, the device design was modified considerably, mainly based on CFD and usability considerations. Functional inhaler elements already known from the human inhaler platform were tested separately, as soon as functional samples were available. This integrated approach shortened the time required for development. Usability engineering was guided by international test groups of horse handlers, who confirmed the position of the horseman, the selected dexterity, and the overall applicability of the concept. The final design was also tested for mechanical robustness and ease of application. Other device configurations were also considered [3]. Studies of the horses’ inhalation pattern (see Figure 3 for an example) guided the selection of the spray duration and the detection of flow as well as the handling of the release action. On purpose, the breathing patterns of different horses with different conditions were investigated. Investigations were carried out to establish the robustness of the pharmaceutical parameters, e.g. the mass median diameter (MMD) of the aerosol leaving the inhaler. It was shown that the MMD only weakly depends on the mechanical spring force (Figure 5). As the device actively generates the aerosol after release, the generation of the aerosol is decoupled from the physical ability and performance of the user. Next Generation Impactor data were in reasonable agreement with the laser diffraction data, e.g. a mass median aerodynamic diameter (MMAD) of 1.5 µm has been determined. The small difference may be explained by shrinking of the particles due to evaporation inside the impactor. The extra-fine soft mist is indicated for the treatment of horses because the airway from the nostril to the lungs is long and premature deposition can be avoided by the small particle size. Controlled particle growth by hygroscopic properties can result in the desired deposition inside the lungs [4]. Conclusion We report on the development of a soft mist inhaler designed to treat horses. In our view, data driven design decisions are very important and here they were taken in a timely manner. The modular understanding of building blocks and functional sub-units allowed to speed-up development timelines and to adapt the Respimat® platform device to a new application in animal health. In a concurrent approach, the analytical method development was supported to keep pace with the state of development of the device.

Ziegler J and Wachtel H: Comparison of Cascade Impaction and Laser Diffraction for Particle Size Distribution Measurements, J Aerosol Med. 2005; Vol. 18(3): pp 311-324,

Funch-Nielsen H, Roberts C A, Weekes J S, Deaton C M and Marlin D J: Evaluation of a new spacer device for delivery of drugs into the equine respiratory tract, Poster and web-link: https://www.jorvet.com/wp-content/uploads/2012/01/Equinehaler.pdf (last visited May 28, 2018).

Niedermaier G and Gehlen H: Benefit of inhalative therapy in horses with RAO, Pferdeheilkunde 2009; 25: pp1-6.

Longest P, Tian G, Son Y, Li X, Hindle M: Engineered Drug Targeting to the Lungs: Ensuring Growth and Deposition of Submicrometer Aerosols Through Device Design and Formulation, Respiratory Drug Delivery 2012; Vol 1: pp 61-72.

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Drug Delivery to the Lungs, Volume 29, 2018 – C. Vairo et al. Safety and effectiveness of sodium-colistimethate-loaded nanostructured lipid carriers (SCM-NLC) against P. aeruginosa: in vitro and in vivo studies following pulmonary and intramuscular administration C. Vairo1,2, J. Basas3, M. Pastor1, G. Gainza1, M. Moreno-Sastre2,4, X. Gomis3, A. Fleischer5,6, E. Palomino5,6, D. Bachiller5,6, F.B. Gutiérrez7, J.J. Aguirre1,7, A. Esquisabel2,4, M. Igartua2,4, E. Gainza1, R.M. Hernandez2,4, J. Gavaldà3 & J.L. Pedraz2,4 1BioPraxis 2NanoBioCel

Research AIE, R&D Department, Hermanos Lumière, 5, 01510 Miñano (Araba), Spain

Group, Laboratory of Pharmaceutics, University of the Basque Country, School of Pharmacy, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain

Antimicrobial Resistance Laboratory, Vall d’Hebron Research Institute (VHIR). Infectious Diseases Department, Hospital Universitari Vall d’Hebron, Passeig de la Vall d'Hebron, 119-129, 08035 Barcelona 4Biomedical

Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). Vitoria-Gasteiz, Spain

Fundación Investigaciones Sanitarias Islas Baleares (FISIB), Development and Regeneration Program, Ctra. Sóller km 12, 07110 Bunyola (Balearic Islands), Spain

Consejo Superior de Investigaciones Científicas (CSIC), Ctra. Sóller km 12, 07110 Bunyola (Balearic Islands), Spain 7Department

of Pathological Anatomy, Hospital Universitario de Álava (HUA). José Atxotegi, 01009 VitoriaGasteiz, Spain

Summary In an effort to make a step forward optimising the administration of antibiotics mainly improving their effectiveness and safety, in the present study we have developed a novel formulation against different Pseudomans aeruginosa (Pa) multiresistant clinical isolates based on sodium colistimethate-loaded nanostructured lipid carriers (SCMNLC), administered by the pulmonary and intramuscular routes. In the present study the effectiveness, safety and biodistribution of SCM-NLC have been tested against non-encapsulated SCM, using both in vitro and in vivo models. The aims of this study were the follows; first, to prepare SCM-NLC and prove their in vitro antimicrobial activity against different multiresistant P. aeruginosa (MR-Pa) clinical isolates; second, to evaluate the toxicity, the biodistribution and the effectiveness of the pulmonary administration of nebulised SCM-NLC and intramuscular (IM) administration of SCM-NLC against an extensively drug-resistant (XDR)-Pa strain in an acute pneumonia model in mice. With this interdisciplinary research, we found that (i) encapsulation process enhances the pharmacological efficacy compared to free SCM in vivo; (ii) nanoencapsulation resulted in reduced toxicity, especially nephrotoxicity; (iii) the pulmonary administration resulted in low systemic absorption while the intramuscular administration showed greater systemic absorption. Thus, the pulmonary route might be selected in case of maintenance therapy for its effect in lungs, whereas, intramuscular route may be more useful in an emergency as it showed a faster absorption. In conclusion, the results showed herein may represent an important demonstration of the therapeutic potential of SCM-NLC against P. aeruginosa in vivo. Key Message Sodium colistimethate-loaded nanostructured lipid carriers (SCM-NLC) are effective against P. aeruginosa in vivo, non-toxic and distribute efficiently to the lung and liver after pulmonary or intramuscular administrations. Overall, the results showed herein may represent an important advance in treatment of resistant P. aeruginosa infection.

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Drug Delivery to the Lungs, Volume 29, 2018 - Safety and effectiveness of sodium-colistimethate-loaded nanostructured lipid carriers (SCM-NLC) against P. aeruginosa: in vitro and in vivo studies following pulmonary and intramuscular administration Introduction Antimicrobial resistance has emerged as a result of the use and misuse of antibiotics, leading to the beginning of a post-antibiotic era where minor injuries and previously treatable illnesses will be fatal again. Indeed, antibiotic resistance is a serious threat for global public health, jeopardizing the latest improvements in control of diseases, such as organ transplants, cancer chemotherapy or major surgery (1). It is estimated that 51,000 health careassociated P. aeruginosa infections occur in the United States every year. More than 6,000 (or 13%) of these are multidrug-resistant, with roughly 400 deaths per year attributed to these infections. Alarmingly, some of these strains were not susceptible to any antipseudomonal compound (2). In this framework, colistin, and its pro-drug, sodium colistimethate, were proposed as rescue therapy in late 1990s and 2000s but cases of colistin-resistance are becoming widely reported and there are even cases of resistance to colistin in patients that have never previously used this antibiotic (3). Moreover, the difficulty of using colistin, mainly in severely ill patients, and its high nephroand neuro-toxicity should be highlighted (4). In this regard, the use of nanotechnology has arisen as a strategy against multidrug-resistant bacteria, making it possible to reduce drug dose as well as to prevent resistance and toxic effects. Among different drug delivery systems, nanostructured lipid carriers (NLC) have gained the attention of many research groups as promising second-generation lipid nanoparticles. These nanoparticles present a lipid core made of liquid and solid lipids leading to disordered matrix and providing more space for drug loading. NLC offers high drug loading and stability as well as good tolerability as the lipids selected for the preparation are under GRAS (Generally Regarded As Safe) FDA-denomination. Overall, the aims of this study were the follows; first, to prepare sodium colistimethate-loaded nanostructured lipid carriers (SCM-NLC) and prove their in vitro antimicrobial activity against different multiresistant P. aeruginosa (MRPa) clinical isolates; second, to evaluate the toxicity, the biodistribution and the effectiveness of the pulmonary administration of nebulised SCM-NLC and intramuscular (IM) administration of SCM-NLC against an extensively drug-resistant (XDR)-Pa strain in an acute pneumonia model in mice. Experimental methods SCM-NLC preparation SCM-NLC were prepared using hot melt homogenisation process. Briefly, Precirol® ATO 5 and Miglyol 182 N/F were melted with the drug to achieve the oily phase (10:1:1). Simultaneously, the aqueous phase was prepared dissolving Tween® 80 at 1.3% (w/v) and Poloxamer 188 at 0.6% (w/v) and then the solution was tempered. Both phases were mixed and emulsified under sonication. Afterwards, the resulting emulsion was gradually cooled down and kept under 5 ± 2ºC overnight. 15% (w/w) of trehalose was added to the formulation as cryo-preserving agent prior to the freeze-drying step. SCM-NLC characterization First, size, polydispersity index (PDI) and zeta potential were analysed by means of Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Then, encapsulation efficiency (EE) was calculated indirectly by HPLC, quantifying the amount of the drug in the supernatant. Briefly, the chromatographic conditions were set as follows, a Novapak C18 x 150 mm column with a 4 μm pore size, the mobile phase consisted of 77 % of an aqueous solution and 23 % of acetonitrile, and detection wavelength of 206 nm. Once quantified, the EE was estimated according to this equation: EE (%) = ((initial amount of drug - non-encapsulated drug)/ initial amount of drug) x 100 Antimicrobial test Antimicrobial activity of SCM-NLC was tested against eight clinical isolates of Pseudomonas aeruginosa, more precisely against three (Pa1016, Pa46, Pa54) extensively drug-resistant strains (XDR), four (Pa116, Pa166, Pa167, Pa179) multidrug-resistant strains (MDR) and one (Pa17) moderate drug-resistant (modR). The minimum inhibitory concentration (MIC) values of SCM-NLC and free SCM were determined by broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines and breakpoints. In vivo efficacy study in acute pneumonia model Briefly, Balb/c female mice (n=40) weighing 20 to 22 g were infected with 1·108 CFU/mL of XDR P. aeruginosa 1016R strain by nasal instillation of 30 μ in order to create the acute pneumonia model. Therapy was initiated 1.5 h after infection. The mice were treated for 3 days with: nebulised saline q12 h (every 12 hours); nebulised free SCM 15 mg/kg q12 h; nebulised SCM-NLC 2.8 mg/kg q12 h or q24 h (every 24 hours); IM free SCM 80 mg/kg q12 h; IM SCM-NLC 2.8 mg/kg q12h or q24h. The groups receiving the free drug were administered 324 µg (15 mg/kg) and 1,700 µg (80 mg/kg) of SCM q12h, whereas the SCM-NLC groups received 70 µg (2.8 mg/kg) of SCM per dose.

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Drug Delivery to the Lungs, Volume 29, 2018 – C. Vairo et al. In vivo toxicity studies in healthy mice To evaluate the potential toxicity of the SCM-NLC, three different studies were carried out in healthy mice. On the one hand, three groups (T1.1. to T1.3.) of mice were maintained under hyperoxia conditions and received SCMNLC 2.8 mg/kg either q12h or q24h pulmonary administered (n=10). At the end of the study at the third day, the lungs were dissected and fixed in paraformaldehyde 4 %. On the other hand, six groups (T2.1. to T2.6.) received SCM-NLC 2.8 mg/kg, free SCM 2.8 mg/kg or the equivalent amount of blank-NLC by the IM route (n=40). Finally, six additional experimental groups (T3.1. to T3.6.) were tested to evaluate whether higher doses could exert toxic effects (n=30). In this regard, the toxicity of SCM-NLC 5.6 mg/kg and 11.2 mg/kg, blank-NLC, free SCM 8 mg/kg and saline solution were assessed by administering them daily by the IM route. At the end of the study, after four days of treatment, lungs, spleen, liver and kidneys were dissected and fixed, dehydrated and paraffin embedded to obtain 4 µm slide cuts. Then, inflammation was graded using a scale of 0 to 4; the score was set as, 0 no leukocytes, 1 a few leukocytes, 2 a ring of single-cell layer leukocytes, 3 a ring of 2-4 leukocytes layer deep and 4 a ring of leukocytes deeper than four cell layers. In vivo biodistribution studies in healthy mice Biodistribution of NLC was studied in healthy mice, after pulmonary and IM administration (n=28). Mice were administered infrared-labelled NLC (IR-NLC) 2.8 mg/kg by the pulmonary or IM route using a Microsprayer® aerosolizer. At pre-established time points (5 min, 30 min, 24h and 48h), the mice were sacrificed and observed under LI-COR Pearl® impulse small animal imaging system (LI-COR Corporate). In Groups 6-7, additional doses of IR-NLC (at 24 and 48 h) were administered to study the cumulative effect that NLC might present; mice were sacrificed 2 hours after the repeated dose administration. Results SCM-NLC characterization Nanoparticles displayed a particle size of 354.10 ± 2.85 nm with 0.29 ± 0.01 polydispersity index (PDI) and negative zeta potential of -20.35 ± 0.05 mV. Moreover, a high encapsulation efficiency of 94.94 ± 0.04 % was obtained, giving 70 μg of SCM per formulation mg. Antimicrobial test Using the CLSI interpretative standards, microbiological tests revealed that MIC values of SCM-NLC and free SCM were between 0.125 and 2 mg/L for all P. aeruginosa MDR/XDR/modR strains. Antimicrobial test Using the CLSI interpretative standards, microbiological tests revealed that MIC values of SCM-NLC and free SCM were between 0.125 and 2 mg/L for all P. aeruginosa MDR/XDR/modR strains. In vivo efficacy study in acute pneumonia model The groups nebulised with free SCM 15 mg/kg and SCM-NLC 2.8 mg/kg, either q12h or q24h, presented significant (p<0.05) lower bacterial density in the lungs than the NaCl control group. SCM-NLC 2.8 mg/kg q24h achieved the highest reduction of more than 1 log10 CFU/ g lung (p<0.05). Regarding the IM route, free SCM 80 mg/kg and SCM-NLC 2.8 mg/kg q24h showed a significant (p<0.05) lower bacterial count in the lungs than the control group. In other words, by the pulmonary route, 70 or 140 µg of SCM encapsulated in the nanoparticles achieved the same results as 648 µg of free SCM. Moreover, 140 µg of SCM encapsulated in NLC by the IM route presented a similar bacterial count to 3,400 µg of free SCM.

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Figure 1 - Left, lung inflammatory scores for SCM-NLC given q12h or q24h when maintained under hyperoxia. Right, histological slides of two samples scored as 1 and 4

When toxicity was studied via the IM route, in this case under environmental oxygen concentration (T2.1. to T2.6.), the animals grew healthily without significant differences (p > 0.05) between animal weights prior to the treatment and at the end of the study. Also, free SCM 2.8 mg/kg (T2.4. and T2.5.) was administered by the IM route in order to ensure that low free SCM doses were innocuous. The histological analysis revealed no toxicity in tissues. Finally, no damage was found in the kidneys and brain in any case, indicating the lack of toxicity of SCM-NLC in these organs. When the dose was increased in the IM route (groups T3.1. to T3.6.), no significant toxic (p > 0.05) effect was detected. Weights increased slightly as the study progressed. In vivo biodistribution studies in healthy mice After nebulisation, IR-NLC were mainly biodistributed in the lungs and to a lesser extent in liver. After 48 h, the signal of the liver decreased sharply, whereas the signal in the lung was still high. In addition, lungs displayed broad, homogeneous signal, meaning that IR-NLC distributed throughout the lungs. IR-NLC were also detected in the kidneys and spleen, although at significantly lower concentrations. When extra doses were administered at 24 and 48 h, it could be observed that the signal in the lung had increased. Likewise, the signal from the liver, kidneys and gallbladder was also raised but to a lesser extent. When IR-NLC were administered by the IM route, firstly the main signal was located at the injection site. However, within the 0.5-2 h period, the signal was transferred to other organs, such as the liver and gallbladder. After 24 h, the signal spread to other organs such as the lungs, kidneys and heart. When no additional doses were administered, this signal was detectable for 48 h, while when repeated doses were administered the signal stayed the same and increased over the study. Spleen displayed very low intensity. Discussion Size, zeta potential and encapsulation efficiency of SCM-NLC were appropriate to dose SCM efficiently and administer nanoparticles using both administration routes; pulmonary and intramuscular. MIC values of SCM-NLC were similar to the free SCM value used as reference data, which demonstrate that nanoencapsulation did not affect the antimicrobial activity of SCM. The effect of SCM-NLC during the efficacy study was similar to that of the free SCM group via pulmonary or IM route. This highlighted that the SCM-NLC in vivo were more active than the free SCM because the same effect was observed using at least five times lower dose. SCM-NLC administered by pulmonary route and under hyperoxia condition resulted to be non-toxic (T1.1. to T1.3.). In addition, SCM-NLC, blank-NLC or low doses of free SCM, administered by IM route (T2.1. to T2.6.), resulted in an absence of histological damage. Therefore, the non-toxic effect of SCM-NLC might be related to the low dosage of the drug. When the higher doses were assessed (T3.1. to T3.6), almost no toxicologically important events that could be attributed to the treatments were detected. In the biodistribution study, nebulisation gave rise to a predominant lung disposition of NLC with a small IR signal detected in other organs, suggesting low systemic absorption. In particular, the signal detected in the kidneys was low which suggests minimising exposure may mitigate against nephrotoxicity. In contrast, after IM administration NLC were detected predominately at the injection site, but also distributed to other organs including liver, gallbladder, lungs, kidneys and heart, suggesting a high systemic absorption of the NLC after administration by the IM route.

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Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; C. Vairo et al. Conclusion In conclusion, we found that (i) encapsulation enhanced the pharmacological efficacy of SCM in vivo; (ii) nanoencapsulation reduced toxicity, especially nephrotoxicity; (iii) the pulmonary administration resulted in low systemic absorption while intramuscular administration resulted in a high systemic absorption. Thus, the pulmonary route might be selected in case of maintenance therapy for its main effect in lungs, whereas, intramuscular route may be more useful in an emergency as it showed a faster absorption. References 1

World Health Organization. Antimicrobial resistance. 2018; Available at: http://www.who.int/en/news-room/factsheets/detail/antimicrobial-resistance.

Centres for Disease Control and Prevention. Antibiotic Resistance Threats in the United States, 2013. 2018; Available at: https://www.cdc.gov/drugresistance/threat-report-2013/.

Olaitan AO, Morand S, Rolain J: Emergence of colistin-resistant bacteria in humans without colistin usage: a new worry and cause for vigilance, International Journal of Antimicrobial Agents 2016; pp1-3.

Wolinsky E, Hines JD: Neurotoxic and nephrotoxic effects of colistin in patients with renal disease, The New England journal of medicine JID â&#x20AC;&#x201C; 0255562.

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Drug Delivery to the Lungs, Volume 29, 2018 – Philippe Rogueda et al. Telehealth Ready: Performance of the Amiko Respiro Sense connected technology with Merxin DPIs Philippe Rogueda1, Martijn Grinovero2, Luca Ponti2, Graham Purkins1 & Oliver Croad1 Ltd, King's Lynn Innovation Centre, Innovation Way, King’s Lynn, PE30 5BY, UK Amiko Digital Health Limited, Salisbury House 31 Finsbury Circus, London, EC2M 5QQ, UK 1Merxin

Summary Data presented in this abstract demonstrate the use of Amiko Respiro Sense technology in the form of an add-on attachment to the blister and capsule based Merxin DPIs, MRX001 and MRX003. The technology can capture clinical and real-world adherence data used to steer treatment regimens and improve patient inhaler technique. Key device usage metrics such as peak inspiratory flow, inhalatory volume, inhalation time and the whole flow profile were collected and assessed using breath simulation apparatus and compared to estimations of these parameters provided by the Respiro Sense technology. Accuracy values were calculated to be around 90% or above for all these respiratory parameters, indicating that the combination of Respiro Sense with the Merxin devices can be used to accurately estimate key measures of inhaler usage and lung health. Key Message The Amiko Respiro Sense technology was combined with the Merxin blister and capsule based dry powder inhalers. It can be used to monitor true adherence, helping to improve inhaler technique and provide valuable lung health information to the patient and physician. Introduction Asthma and COPD are severe chronic conditions that often require life-long therapies from onset creating huge health and economic burdens.[1] Many clinical trials have demonstrated that the currently available therapies are effective at reducing exacerbations and improving outcomes in controlled clinical environments, however a disconnect between these studies and real-world results has been observed. [2] Sub-optimal patient adherence and inhaler technique are thought to be key drivers for this disconnect and can easily be improved with the intervention of a medical professional.[2,3] Successful interventions start with the physician having detailed, reliable information about the patient’s real-world usage of their inhaled therapy and an understanding of the best methods to improve inhaler technique and adherence.[4] State of the art electronic monitoring devices or “connected devices” have been regarded as one of the most exciting leaps forward for inhaler development in recent years. Their ability to monitor adherence, track patient usage and provide detailed objective information to the physician make them ideal tools for aiding, informing and assessing the success of interventions. Most currently available devices simply track basic usage and adherence metrics, however successful actuation and reliable delivery of the required dose for respiratory medications often requires multiple, non-trivial steps. Many patients make technique errors when using their inhaler, resulting in compromised dose delivery leading to poor overall effectiveness of the treatment. A solution is required that provides a combined measure of correct dosing and accurate inhaler technique and has often been referred to as ‘true adherence”. [5] A thorough understanding of true adherence in both clinical and realworld settings is crucial for our understanding of effective inhaler usage and will inform personalised interventions promoting adherence and accurate inhaler technique. Amiko have developed the proprietary Respiro Sense technology that collects advanced data on respiratory medication use and adherence. The Respiro Sense portfolio covers a whole range of integrated smart inhalers and add-ons to marketed and developmental inhalers focussing on the key drivers of functionality, low power consumption and cost-efficiency. Each integrated or add-on sensor uses the same off-the-shelf MEMS sensors and real-time machine learning to monitor digital signals, making the sensor readily transferable to a whole range of respiratory devices from pMDIs and DPIs to nasal devices, soft mist inhalers and nebulisers. The unique digital fingerprint of each event of the inhalation manoeuvre for each device can be used to monitor when and how accurately a patient follows the user instructions, and the digital signal during each inhalation can be used to determine key measures of device performance and to determine indicators of lung health [6,7,8,9]. Critically, the add-on sensors allow the patient to continue using their accustomed therapy and physicians can use the data to optimise the patient’s technique. The same handling steps are used to administer each therapeutic dose, the air flow path is not interrupted, and delivery performance is unaffected when using a Respiro sensor. The Merxin DPI devices are designed to provide 505j substitutable device platforms for the delivery of APIs to the lungs and in particular fluticasone/salmeterol and tiotropium. The devices, MRX001 a blister based dry powder inhaler and MRX003 a capsule based inhaler, have the same user steps, performance characteristics to the respective originator reference devices. MRX001 and MRX003 are currently under clinical evaluation in different settings, from pilot PK to pivotal PD bio-equivalence studies, as well as in vitro setting for new molecules. The purpose of integrating the Merxin devices with the Respiro Sense technology was to test the compatibility of the Respiro Sense technology and its effectiveness in providing the Merxin clients and users with a telehealth option that would increase their chances of success in bio-equivalence studies by spotting outlier performers that could skew the results, and offer new commercial perspectives for new APIs formulated in the Merxin devices, including nicotine and cannabinoid applications.

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Drug Delivery to the Lungs, Volume 29, 2018 - Telehealth Ready: Performance of the Amiko Respiro Sense connected technology with Merxin DPIs Experimental methods and materials Bespoke Respiro Sense attachments were developed for each device, MRX001 and MRX003, as shown in Figure 1. Each attachment contains the same MEMS (micro electro mechanical systems) sensors that provide consistent digital feedback for each critical step in the operation of the device. The requirements of the Respiro Sense attachments were: i) not to interfere with the normal patient handling of the device, ii) not to alter the airflow pathway, and iii) not to affect the effectiveness of dose delivery. For example, with MRX001, the Respiro Sense attachment was engineered not to hinder the operation of the cap and the lever and not to obstruct the air flow pathway. Likewise, use and air flow for the MRX003 device, shown in Figure 1d, should not be hindered by the addition of the Respiro Sense attachment.

Figure 1 â&#x20AC;&#x201C; A graphical representation of the MRX001 device (a) and the MRX003 device (c) with the Respiro Sense device attachments (b & d).

In order to test the sensitivity, compatibility and performance of the Respiro Sense attachments with the Merxin devices, digital fingerprint responses were acquired for each step of the inhalation manoeuvres. The following four key steps were recorded with MRX001 (inclusive of pre and post actuation steps): (i) open the device by rotating the cap to reveal the mouthpiece; (ii) push the lever away from the mouthpiece to move the dose from the blister into the mouthpiece; (iii) performing one inhalation via the mouthpiece; (iv) close the device by sliding the cap back to the original position until you hear a click. For MRX003, the inhalation manoeuvre was defined as the following (i) opening the lid and mouth-piece; (ii) inserting the capsule; (iii) closing the mouth-piece; (iv) piercing the capsule; (v) performing one inhalation; (vi) disposing of the capsule. An example of the digital signal recorded for the inhalation step of MRX001 is shown in Figure 2 along with the flow rate estimation.

Figure 2 â&#x20AC;&#x201C; A digital signal recorded by the Respiro Sense device (top left) translated into a flow rate estimation (bottom left, dotted red line) compared to the flow rate measured by an ASL 5000 instrument. A schematic of the experimental set-up for the Respiro sensor attached to the MRX001 or the MRX003 device connected to a breathing simulator via a vibration compensation tube with a pressure sensor. All data fed data into the acquisition laptop.

In order to assess the performance of the Respiro Sense device a set of test apparatus was designed to simulate human inhalation as depicted in Figure . The Merxin devices with the Respiro Sense attachments were connected to a lung/breath simulator (ASL 5000, Active Servo Lung, IngMar Medical, Pittsburgh, USA) via a vibration compensation tube with a pressure sensor attached. Both the breath simulator and the pressure sensor fed data into the acquisition laptop, which was then compared to data recorded by the Respiro Sense detector attached to the device. Respiro Sense uses real-time machine learning to identify the events in the inhalation manoeuvre and to translate the digital signal into an estimation of flow rate and inhalation volume.

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Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; Philippe Rogueda et al. Inhalers such as MRX001 and MRX003 require a forceful deep inhalation to ensure maximum dose delivery.[10] It has previously been reported that many patients with asthma and COPD cannot produce a sufficiently forceful inhalation for successful dose delivery. We investigated whether the Respiro Sense technology could successfully estimate key parameters that could be indicators of accurate/true delivery of the dose and be indicators of lung health, such as peak inspiratory flow (PIF) and inhalation volume (IV) (depicted in Error! Reference source not found.). This was achieved by first creating large datasets of inhalation manoeuvres for each device over a clinically relevant range of peak inspiratory flow rates (28 L/min to 102 L/min for MRX001 and 21 L/min to 61 L/min for MRX003). During these inhalation manoeuvres, peak inspiratory flow and inhalation volume were monitored. The Respiro Sense attachment was then trained by correlating the true values of peak inspiratory flow and inhalation volume to adjust the complex algorithms to accurately estimate the parameters. Each parameter measured by the Respiro sense technology (Pest) was compared to the experimentally determined value (Ptrue) using the following error formula, where N is the total number of inhalation manoeuvres and i is the manoeuvre index. The percentage accuracy of the Respiro Sense measurement over all manoeuvres was then used as an indicator of the performance of the Respiro Sense technology with MRX001 and MRX003. đ?&#x2018; đ?&#x2018;

1 đ?&#x2018;&#x192;đ?&#x2018;&#x192;đ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ą (đ?&#x2018;&#x2013;đ?&#x2018;&#x2013;) â&#x2C6;&#x2019; đ?&#x2018;&#x192;đ?&#x2018;&#x192;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019; (đ?&#x2018;&#x2013;đ?&#x2018;&#x2013;) Error(đ?&#x2018;&#x192;đ?&#x2018;&#x192;đ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ą , đ?&#x2018;&#x192;đ?&#x2018;&#x192;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019; ) đ?&#x2018;&#x2013;đ?&#x2018;&#x2013;đ?&#x2018;&#x2013;đ?&#x2018;&#x2013; % = â&#x2C6;&#x2018; | | Ă&#x2014; 100 % đ?&#x2018; đ?&#x2018; đ?&#x2018;&#x192;đ?&#x2018;&#x192;đ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ąđ?&#x2018;Ą (đ?&#x2018;&#x2013;đ?&#x2018;&#x2013;) Results and Discussion

đ?&#x2018;&#x2013;đ?&#x2018;&#x2013;=1

Inhalation manoeuvre recognition was tested for both the MRX001 and MRX003 devices. 200 out of 200 manoeuvres were recorded accurately and therefore it was determined that the Respiro Sense technology is capable of identifying 100% of inhalation manoeuvres for the operation of both MRX001 and MRX003. This result shows that the Respiro Sense technology can now be used to monitor basic patient adherence when using the MRX001 and MRX003 devices and the combined connected device monitors inhaler actuations, equally or better than other inhaler electronic monitoring, such as the SmartTurbo[11] and the SmartInhaler[12]. The measured and estimated values for peak inspiratory flow (PIF), flow, inspiratory volume (IV) and inhalation time (IT) for the MRX001 device are plotted in Error! Reference source not found.. Each graph shows results of the full 200-point dataset except for the flow plot, which shows all flow estimates for each temporally partitioned window of 0.064 s. All graphs show a strong correlation of the measured parameter with the parameter estimated by the Respiro Sense device. The accuracy of flow reconstruction (89.40%), inspiratory volume (91.20%), peak inspiratory flow (88.03%) and inhalation time (94.84%) were all measured to be around 90% or above. This data is comparable to accuracy data acquired for MRX003 device for flow reconstruction (89.41%), inspiratory volume (90.88%), peak inspiratory flow (90.11%) and inhalation time (97.33%). The accuracy of the combined connected device is again better than or equal to other sensors based on auditory response determination of PIF and IC (inspiratory capacity).[13].

Figure 3 â&#x20AC;&#x201C; Values for peak inspiratory flow (PIF), flow, inspiratory capacity (IC) and inhalation time for MRX001 measured using the experimental apparatus and plotted against equivalent values estimated by the MRX001 Respiro Sense detector. Red lines indicate where the measured value is equal to the estimated value.

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Drug Delivery to the Lungs, Volume 29, 2018 - Telehealth Ready: Performance of the Amiko Respiro Sense connected technology with Merxin DPIs The accuracy for peak inspiratory flow, inhalation volume and flow reconstruction in both the MRX001 and MRX003 are equal to or better than the of detection for MDIs[13] or the Ellipta[6] or Diskus[14] DPIs when using similar auditory methods. Conclusion The Respiro Sense technology has been successfully integrated with the MRX001 and MRX003 devices showing that the devices can be connected with Respiro Sense technology and can provide a range of measures of inhaler usage and true adherence. The Respiro Sense technology and the Merxin blister and capsule devices are fully compatible. Respiro Sense not only allows basic adherence monitoring but can also provide detailed information about key metrics for inhaler technique and lung health such as peak inspiratory flow and inhalation volume, more accurately than other devices on the market. This detailed information can be used in both clinical research and clinical practice to monitor the effectiveness of a therapy. Clinical researchers can obtain detailed information about the use of the therapy across the whole study facilitating thorough investigation of clinical data sets and monitoring of key indicators of lung health. For physicians, the Respiro Sense device represents a fantastic tool that can monitor ongoing patient use and indicators of lung health that can be used to effectively tailor improved interventions and treatment regimes. Combined with well-established device platforms like the Merxin DPI devices and the Respiro Sense technology provides product developer and clinician with a ready solution for patient monitoring and adherence improvement. References “Cost of Asthma on Society”. Asthma and Allergy Foundation of America. (www.aafa.org/page/cost-of-asthma-onsociety.aspx, Accessed Aug 2018) 2 Mukherjee M, Stoddart A, Ramyani G P, Nwaru B I, Farr A, Heaven M, Fitzsimmons D, Bandyopadhyay A, Aftab C, Simpson C R, Lyons R A, Fischbacher C, Dibben C, Shields M D, Phillips C J, Strachan D P, Davies G A, McKinstry B, Sheikh A: The epidemiology, healthcare and societal burden and costs of asthma in the UK and its member nations analyses of standalone and linked national databases. BMC Med. 2016, Vol 14(1), p 113. 3 Craven V E, Morton R W, Spencer S, Devadason S G, Everard M L: Electronic monitoring and reminding devices for improving adherence to inhaled therapy in patients with asthma (Protocol). Cochrane Database of Systematic Reviews 2015, 3: 1-11. 4 Morton R W, Elphick H E, Rigby A S, Dew W J, King D A, Smith L J, Everard M L: STAAR: a randomised controlled trial of electronic adherence monitoring with reminder alarms and feedback to improve clinical outcomes for children with asthma, Thorax 2016; 0: 1-8. 5 Nikander K, Turpeinen M, Pelkonen A S, Bengtsson T, Selroos O, Haahtela T: True adherence with the Turbuhaler in young children with asthma; Arch Dis Child. 2011 Feb;96(2):168-73 6 Taylor T E, Lacalle Muls H, Costello R W, Reilly R B: Estimation of inhalation flow profile using audio-based methods to assess inhaler medication adherence. PLoS ONE 2018; 13(1): 1-14. 7 Janson C, Lööf T, Telg G, Stratelis G: Impact of Inhalation Flow, Inhalation Volume and Critical Handling Errors on Delivered Budesonide/Formoterol Dose in Different Inhalers: An In Vitro Study. Pulmonary Therapy, 2017; 3(1): 243–253. 8 Mahler D A: Peak Inspiratory Flow Rate as a Criterion for Dry Powder Inhaler Use in Chronic Obstructive Pulmonary Disease: Ann Am Thorac Soc. 2017, 14(7): 1103-1107 9 Mahler D A, Waterman L A, Gifford A H: Prevalence and COPD phenotype for a suboptimal peak inspiratory flow rate against the simulated resistance of the Diskus® dry powder inhaler. J Aerosol Med Pulm Drug Deliv. 2013;26(3):174-179 10 Laube B L, Janssens H M, de Jongh F H C, Devadason S G, Dhand R, Diot P, Everard M L, Horvarth I, Navalesi P, Voshaar T, Chrystyn H: What the pulmonary specialist should know about the new inhalation therapies, Eur. Resp J. 2011; 37 (6):13081417. 11 Pilcher J, Shiftcliffe P, Patel M, McKinstry S, Cripps T, Weatherall M and Beasley R: Three-month validation of a turbuhaler electronic monitoring device: implications for asthma clinical trial use. BMJ Open Respir Res. 2015; 2(1): e000097. 12 Patel M, Pilcher J, Chan A, Perrin K, Black P, Beasley R: Six-month in vitro validation of a metered-dose inhaler electronic monitoring device: implications for asthma clinical trial use. J Allergy Clin Immunol 2013;130:1420–2. 13 Taylor T E, Zigel Y, Egan C, Hughes F, Costello R W, Reilly R B: Objective Assessment of Patient Inhaler User Technique Using an Audio-Based Classification Approach. Sci Rep. 2018; 8(1):2164 1

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Drug Delivery to the Lungs, Volume 29, 2018 - Marie Hellfritzsch Characterisation of Zinc Oxide as an Alternative to Aluminium Hydroxide in Nasal Vaccination Marie Hellfritzsch & Regina ScherlieĂ&#x; Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany Summary To evoke a mucosal immune response via nasal administration a safe antigen needs an adjuvant. Because aluminium hydroxide, a commonly used adjuvant for intramuscular administration, is not effective on mucosa and has other disadvantages, Zinc Oxide (ZnO) is evaluated as an alternative in this study. The aim of the present work is to characterise ZnO, especially with regard to adsorption capacity of Ovalbumin (OVA) and aerodynamic characterisation for nasal administration. That is why adsorption capacity, desorption profile and particle size distribution of ZnO are measured. With lower adsorption capacity but similar desorption profile ZnO shows comparable properties to the reference material aluminium hydroxide. Due to its particle size in the nanoscale and its resulting high surface area, ZnO has a high tendency to build agglomerates. In this study, a nasal dry powder device (UDS powder device) is used to disperse and apply ZnO. Particle sizes that are more suitable for nasal administration than for pulmonary administration are the result of the low dispersion forces in the device. Aerodynamic characterisation confirms these results with almost 90 % ZnO in the nasal expansion chamber of the NGI by using the UDS powder device. Part of the characterisation are also saturation solubility and pH studies at different pH levels. ZnO is slightly soluble in acidic solutions, but solubility is decreasing with increasing pH. At neutral pH, ZnO is practical insoluble. Compared to solubility the effect of increasing pH to an almost neutral pH after 24 h is independent of starting pH. The study gives a first idea of ZnO as a compound for adsorbed vaccines. Key Message ZnO is a multifunctional material and a potential alternative to nowadays commonly used aluminium hydroxide as adjuvant in vaccine formulations, especially for nasal administration. Adsorbing a lower amount of protein in comparison to aluminium hydroxide while showing a comparable desorption profile seems promising and will be followed up by further characterisation. Introduction Active immunisation, which can achieve a lifelong immunity, is one chance to prevent infectious diseases. Because of a systemic immune response and exact dosing possibilities, intramuscular administration is the mostly used one. Due to the fact, that it is an invasive procedure a usage of a needle is always necessary. Hence, a sterile dosage form is indispensable and undesired effects can occur as a result, e.g. accidental needle sticks. Dermal and mucosal immunisation are possibilities of non-invasive administration, especially mucosal immunisation in the nose, because antigens have their first contact with the nasal mucosa when entering the human body. Besides a systemic immune response, a local immune response is achieved. The nose-associated lymphoid tissue (NALT) is a part of the mucosal immune system in the upper airways and of particular importance for nasal vaccination [1]. To achieve immunity a safe antigen needs an adjuvant. Aluminium salts are the most commonly used adjuvants both in human and veterinary vaccines due to their low cost and good adjuvanticity if combined with various types of antigens. Despite that aluminium salts have been used for a long time, since 1926, the mechanism of immune-potentiation is not yet fully understood and negative effects of aluminium salts such as accumulation in the body and neurotoxic effects, while not being effective as mucosal adjuvant, are also frequently discussed [2]. For this reason, alternatives should be sought for. One possible alternative may be ZnO, which is a useful multifunctional material and well characterised in a number of clinical studies. ZnO is believed to be non-toxic, biodegradable, biocompatible, available at low cost and very simply processable. That is why ZnO is already used in various applications in daily life, e.g. in cosmetics and in medical materials [3, 4]. Besides, ZnO seems to have immune adjuvant and antiviral properties [5]. A few years ago, another working group from Kiel, in cooperation with a group from Chicago, investigated specially designed ZnO for the prevention and treatment of genital herpes. Herpes virus bound to ZnO was taken up by antigen presenting cells available in mucosal tissues and presented to lymphocytes evoking an immune response. It is therefore of great interest to investigate whether ZnO can exhibit adjuvant activity in mucosal vaccine formulations, intended for nasal administration. In this study, ZnO is characterised to get an idea about the raw material, its protein adsorption capacity and desorption profile. Moreover, first aerodynamic tests with regard to nasal application of ZnO are conducted. Materials and Experimental Methods Imaging Visualisation of ZnO (CAESAR & LORETZ GmbH, Germany) was performed via Scanning Electron Microscopy (SEM) with a Phenom World XL (Phenom-World B. V., The Netherlands). Samples were fixed on aluminium stubs with double-sided carbon tapes and then covered with a thin conductive gold layer.

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Drug Delivery to the Lungs, Volume 29, 2018 - Characterisation of Zinc Oxide as an Alternative to Aluminium Hydroxide in Nasal Vaccination Size measurements Particle size analysis was performed by laser diffraction using the HELOS (Sympatec, Germany) in combination with the Windox 5 software (Sympatec, Germany). Two different dispersion methods were applied: the RODOS module dispersing particles with pressurised air (3 bar) and the SPRAYER module in combination with a Unit Dose System (UDS; Aptar, France) powder device. For those measurements, 90 mg ZnO powder were filled in a device. The automated actuator with an actuation force of 60 N was placed between the laser and the detector at 60° to imitate the usual angle of nasal applicators in vivo. Measurements were performed in triplicate. Solubility tests in combination with pH studies A 24-h-solubility test was performed to investigate saturation solubility of ZnO. For this 150 mg ZnO were suspended in different solutions (25 mL each) comprising 2.922 g sodium chloride (AppliChem GmbH, Germany) and being adjusted to pH 1.2, 2.2, 4.5, 5.6, 6.6 and respectively 7.4 with hydrochloric acid (HCl), dilute R (73 g/L HCl) [6] or sodium hydroxide solution (NaOH), dilute R (85 g/L NaOH) [6]. The samples were placed in a shaker at room temperature for 24 h. Before adding ZnO and after the experiment pH was monitored. After separating undissolved ZnO, zinc concentration in the supernatant was measured via spectrophotometric determination of a zinc-zincon complex at 620 nm. Zincon was acquired from Carl Roth GmbH & Co KG, Germany. Measurements were performed in duplicate. Adsorption capacity and desorption profile Adsorption of OVA (Sigma-Aldrich Chemie GmbH, Germany) as a model protein was performed to determine adsorption capacity of ZnO. 2 mg OVA were dissolved in 1 mL cell buffer (PBS Dulbeco without calcium and without magnesium; Merck KGaA, Germany). After adding the solution to 1 mg ZnO, the sample underwent a turnincubation, a constant 360° rotation, with 20 rpm at room temperature for 1 h. Dissolved OVA in the supernatant was determined after centrifugation using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc., USA). Afterwards, the corresponding desorption profile was examined. For this purpose, 1 mL of the sample with 1 mg ZnO was placed in a prepared dialysis tube (Float-A-Lyzer G2 100 kDa; Spectrum Laboratories Inc., USA). The dialysis bag was transferred in 20 mL cell buffer. The dialysis volume was stirred with a magnetic stirrer for a period of 6 h at room temperature and samples were taken at predefined time points. The concentration of dialysed OVA was determined with a BCA protein assay kit as well. In both experiments aluminium hydroxide (VAC 20; SPI Pharma SAS, France) was used as reference. Measurements were performed in duplicate. Aerodynamic assessment In vitro aerodynamic performance of ZnO was evaluated based on impaction using the Next Generation Pharmaceutical Impactor (NGI; Copley Scientific, United Kingdom) in combination with a 2 L nasal expansion chamber and the UDS powder device as nasal applicator at a fixed flow rate of 15 L/min. 90 mg ZnO were manually filled in the device. All stages and nasal expansion chamber were coated with a mixture of 2-propanol (AppliChem GmbH, Germany) and 1,2-propanediol (Sigma-Aldrich Chemie GmbH, Germany). Samples on all stages, in the nasal expansion chamber as well as in the device were dissolved in 1 N HCl and analysed with spectrophotometric determination of a zinc-zincon complex at 620 nm. Fine particle fraction was evaluated according to Ph.Eur. 9.1 [7]. Measurements were performed in duplicate. Results and Discussion Characterisation and aerodynamic performance of ZnO as an alternative to aluminium hydroxide in mucosal vaccines are shown and discussed below. Imaging and size measurements As the SEM picture shows (Figure 1), ZnO has no spherical morphology. On closer inspection, it is more reminiscent of a needle-shaped or tetrapodial structure. In addition to some larger agglomerates, single particles in the nanoscale are visible. Particle size measurements after dispersion with pressurised air give a mean particle size of 0.92 µm ± 0.06 µm (Figure 2), confirming particle sizes in the nanoscale as observed in the SEM picture. It should be noted that there is a second smaller peak in a higher particle size range. This indicates that not all agglomerates of ZnO can be dispersed at a pressure of 3 bar. This results in an x90 value of 3.71 µm ± 0.59 µm and a broad span of 3.62. As the main aim is to create a formulation for nasal application it is interesting to analyse the particle size of ZnO when dispersed from a nasal device (ZnO UDS). With a single almost symmetrical peak using a logarithmic scale, ZnO has an x50 of 51.10 µm ± 7.19 µm and a span of 1.32 by using the SPRAYER module. That shows the low dispersion capacity of the UDS powder device, but its relatively high standard deviation also indicates uncontrolled agglomerats of ZnO, which should be avoided.

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density distribution q3*

Drug Delivery to the Lungs, Volume 29, 2018 - Marie Hellfritzsch

Figure 1

SEM picture of ZnO

2.5

ZnO ZnO UDS

2.0 1.5 1.0 0.5 0.0

0.1

10 particle size, µm

100

1000

Figure 2 Particle size distribution of ZnO measured with the RODOS module (ZnO) and with the SPRAYER module (ZnO UDS) in combination with the UDS powder device (n = 3; error bars = standard deviation)

Solubility tests in combination with pH studies To investigate saturation solubility of ZnO a 24-h-solubility test was performed. Results of the test are shown in the diagram as yellow bars (Figure 3). In a solution with pH 1.2 the highest solubility with 399.1 mg/100 mL ± 16.4 mg/100 mL is found. With 50.0 mg/100 mL ± 0.3 mg/100 mL the saturation solubility at pH 2.2 is lower. That means ZnO is slightly soluble in a solution with a pH of 1.2, very slightly soluble with a pH of 2.2 and practically insoluble in less acidic solutions according to the classification of Ph. Eur. 9.1 [8]. The pH at the beginning (pH 0 h, black line) and at the end of the experiment (pH 24 h, light yellow line) are depicted in the diagram (Figure 3). Noticeable pH changes by addition of ZnO to a pH of approximately 7 independently of the starting pH. 7 6

300

5 4

200 ZnO pH 0 h pH 24 h ZnO

100 0

pH 1.2

pH 2.2

pH 4.5

pH 5.6

pH 6.6

pH 7.4

concentration in mg/100 mL

8 400

3 2 1 0

Figure 3 Saturation solubility (ZnO) – yellow bar – and pH studies of ZnO as a function of different pH. At the beginning (pH 0 h) – black line – and at the end of the experiment (pH 24 h ZnO) – light yellow line – pH values are shown (n = 2; error bars = min/max)

Solubility and pH value are of interest at various steps from application to immune reaction. Nasal formulations are applied and deposited on the nasal mucosa. The solubility in combination with the pH has among other things an influence on the subsequent uptake of the formulation. Dissolved substances can be absorbed directly. Insoluble particles are deposited in the nose and transported towards the nasopharynx by mucociliary clearance. The pH of the nasal mucosa is approximately 5.5 to 6.5 [9]. As ZnO is practically insoluble in less acidic solutions (Figure 3), it should keep its solid form and serve as a depot for adsorbed antigen. However, if the formulation has been taken up, the pH is of greater interest for the activation of the adaptive immune system. If the innate immune system, which in most cases detects and removes a pathogen or antigen, does not react or does not react sufficiently, an inflammatory reaction occurs. The secretion of different cytokines upon uptake causes an inflammation reaction. In addition to the four typical symptoms: robur (redness), calor (heat), tumor (swelling) and dolor (pain), there is also a decrease of the pH in the tissue. This results in accumulation of cells of the immune system and at the same time induction of the adaptive immune system. Consequently, antigenspecific antibodies are produced via a chain reaction [10]. As ZnO increases the pH in the experiments independently of the environmental pH, there are theoretically two possibilities with regard to the pH. On the one hand, ZnO could also increase the pH value in vivo and thus prevent inflammatory and immune reactions. On the other hand, the body could regulate against increasing pH and possibly intensify the immune reaction. Nonetheless, with all the theoretical consideration, those experiments must be interpreted carefully. Values of the pH are not measured in physiologically relevant buffered media but merely in simple sodium chloride solutions. To gain a deeper understanding of this pH change further experiments including buffered media and in vitro cell experiments will be performed.

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Drug Delivery to the Lungs, Volume 29, 2018 - Characterisation of Zinc Oxide as an Alternative to Aluminium Hydroxide in Nasal Vaccination Adsorption capacity and desorption profile

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

1.5

desorbed OVA, mg

mg OVA/mg adjuvant

To achieve a sufficient immune response after vaccination with a subunit vaccine not only a sufficient amount of antigen, but an adjuvant is necessary. Adsorption capacity and desorption profiles of ZnO in comparison to aluminium hydroxide are shown in Figure 4.

(A)

ZnO

Al(OH)3

(B) 1.0 0.5 0.0

ZnO Al(OH)3 0

100

200 time, min

300

400

Figure 4 Adsorption capacity (A) and desorption profile of ZnO (B) – yellow – in comparison to aluminium hydroxide – grey (n = 2; error bars = min/max)

As diagram (A) shows, ZnO (yellow bar) with an adsorption of 0.099 mg ± 0.009 mg OVA/mg adjuvant has a much lower adsorption capacity for OVA than aluminium hydroxide (grey bar) with an adsorption of 0.736 ± 0.012 mg OVA/mg adjuvant. In diagram (B) the corresponding desorption profiles are shown. At the beginning ZnO (yellow curve) shows a slightly faster desorption of OVA, but even though the adsorption capacity is very different, the desorption profiles of ZnO and aluminium hydroxide (grey curve) are quite similar. Small differences can be explained by the lower adsorption capacity of ZnO. Aerodynamic assessment The in vitro assessment of the aerodynamic characterisation using a UDS powder device in combination with the NGI shows that 83.5 % ± 8.6 % of ZnO is deposited in the nasal expansion chamber. On stages 1 through 5 27.3 % ± 11.9 % of the emitted dose is found (stage 1: 15.6 % ± 6.3 %, stage 2: 5.9 % ± 3.0 %, stage 3: 2.8 % ± 1.6 %, stage 4: 1.7 % ± 1.0 %, stage 5: 1.3 % ± 0.0 %). With a flow rate of 15 L/min the fine particle fraction (FPF) is evaluated according to Ph. Eur. 9.1 [7]. That results in a FPF of 2.1 % ± 1.3 %. All stages equal and below stage 6, including the micro-orifice collector (MOC), show no detectable amount of ZnO. With a zinc-zincon complex it is possible to measure dissolved zinc concentrations greater than 0.02 mg/100 mL (LOD). 4.8 % ± 3.2 % ZnO is left behind in the UDS powder device. Previous measurements of the particle size distribution with the SPRAYER module are confirmed by predominant nasal deposition in the used setup.

deposited zinc, %

100 80 60 40 20 0

Figure 5

In vitro aerodynamic characterisation of ZnO using an NGI and the UDS powder device (n = 2, error bars = min/max)

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Drug Delivery to the Lungs, Volume 29, 2018 - Marie Hellfritzsch Conclusion and Outlook In the present work, first experiments to characterise ZnO as adjuvant in vaccine formulations are performed to get an idea of the material, investigate adsorption capacity, desorption profile and do first aerodynamic tests of the raw material. Besides the high tendency of ZnO in the nanoscale to build agglomerates, the results of imaging and size measurement with high span values show a necessity to create defined particles, e.g. spray dried agglomerates with uniform particle size and morphology. The effect of ZnO on the pH independently of the starting pH is an interesting aspect in terms of cell toxicity, immunological effect and biodegradability of ZnO in in vitro and in vivo experiments. Not only pH changes are important aspects, also the possibility of ZnO having a similar desorption profile beside of showing big differences in adsorption capacity in comparison to aluminium hydroxide should be investigated further. Therefore, live imaging could be one way to visualise and explain the mechanism and degradation of ZnO. Furthermore, experiments relating to immune effects on mucosa should be performed. References 1 2 3 4 5 6 7 8 9 10

Murphy, K. M.; Travers, P.; Walport, M.: Janeway Immunologie, 7. Auflage, Springer Berlin (2014) Huang, M.; Wang, W.: Factors affecting alum-protein interactions, International Journal of Pharmaceutics 466, pp139-146 (2014) Papavlassopoulos, H.; Mishra, Y. K.; Kaps, S.; Paulowicz, I.; Abdelaziz, M. E.; Maser, E.; Adlung, R. and Röhl, C.: Toxicity of Functional Nano-Micro Zinc Oxide Tetrapods: Impact of Cell Culture Conditions, Cellular Age and Material Propertis, PLoS One 9(1): e84983 (2014) Huang, Z.; Zheng, X.; Yan, D.; Yin, G.; Liao, X.; Kang, Y.; Yao, Y.; Hao, B.: Toxicological Effect of ZnO Nanoparticles Based on Bacteria, Langmuir 24, pp4140-4144 (2008) Antoine, T. E.; Hadigal, S. R., Yakoub, A. M.; Mishra, Y. K.; Bhattacharya, P.; Haddad. C.; Valyi-Nagy, T.; Adelung, R.; Prabhakar, B. S.; Shukla, D.: Intravaginal Zinc Oxide Tatrapod Nanoparticles as Novel Immunoprotective Agents against Genital Herpes, The Journal of Immunology (2016) European Pharmacopoeia, 9.1: 4.11 Reagenzien, Deutscher Apotheker Verlag (2018) European Pharmacopoeia, 9.1: 2.9.18 Zubereitung zur Inhalation: Aerodynamische Beurteilung feiner Teilchen, Deutscher Apotheker Verlag (2018) European Pharmacopoeia, 9.1: 5.11 Zum Abschnitt „Eigenschaften“ in Monographien, Deutscher Apotheker Verlag (2018) England, R. J.; Homer, J. J.; Knight, L. C. and Ell, S. R.: Nasal pH measurement: a reliable and repeatable parameter, Clinical Otolaryngology, Volume 24, Issue 1, pp67-68 (1999) Schütt, C.; Bröker, B.: Grundwissen Immunologie, 2. Auflage, Springer Berlin (2009)

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Drug Delivery to the Lungs, Volume 29, 2018 – Tobias Gutowski & Regina Scherließ Cannabidiol in a DPI – maximising the spray drying yield of HPMC matrix particles Tobias Gutowski & Regina Scherließ Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118, Kiel, Germany Summary The purpose of this study was the evaluation of the ability of hydroxypropyl methylcellulose (HPMC) to form solid cannabidiol dispersions at 25 % drug load with high loading efficiency and maximised yield in the inhalable range (< 5 µm). Cannabidiol (94 % purity) was chosen as model drug for highly lipophilic, resinous materials. A preliminary experiment showed that a loading efficiency of > 85 % is feasible. To maximise the yield, a software supported experimental design (3³) was carried out for pure HPMC. The results of the Design of Experiments (DoE) show that the best yields are achieved with a high solid content and aspirator setting and a low inlet temperature. Since the aerodynamic behaviour could not be evaluated without the active pharmaceutical ingredient (API), two parameter settings, which initially showed promising results, were selected in addition to the theoretical optimum to evaluate the loading efficiency of the optimised spray drying process. All three parameter settings were suitable to achieve the targeted loading dose (25 %) with an efficiency of > 95 %. Powders were dispersible from a DPI device; aerodynamic assessment is still ongoing. Key Message It is possible to formulate resinous cannabidiol into a dry powder with a relatively high drug load of 25 % using spray drying and hydroxypropyl methylcellulose as a matrix. This could be of interest for the formulation of lipophilic plant extracts and other resinous substances. Introduction Cannabinoids are very intriguing substances for a multitude of diseases and symptoms due to their wide variety of effects. The physico-chemical properties of cannabis plant extracts, however, are problematic, as cannabinoids show a distinct lipophilicity (Δ9-Tetrahydrocannabinol (THC) logP 6.97 & Cannabidiol (CBD) logP 5.79 [1]) and, as long as they are not of exceptional purity (i.e. CBD ≥ 98 %), a sticky and resinous form. Due to the advantage of not being psychoactive CBD was used as a model substance in this study. CBD has been reported to show antinausea, anti-inflammatory, anti-psychotic, anticonvulsive, sedative, hypnotic and anxiolytic effects [2]. It is already used in the therapy of the Lennox-Gaustaut- and Dravet-Syndromes, two rare forms of epilepsy, where it has orphan drug status. A combination of CBD and THC (Nabiximols) is used in the therapy of multiple sclerosis. As CBD shows a higher bioavailability when administered pulmonary (mean value 31 %) than orally (13 % - 19 %) [3] inhalation appears to be the more promising administration route. The aim of this study is the formulation of resinous CBD into a dry powder, suitable for inhalation. Materials Cannabidiol (94 % purity) was supplied by Ai Fame GmbH (Wald-Schönengrund, Switzerland). Methocel (Hydroxypropyl methylcellulose; HPMC) E5 was obtained from Colorcon GmbH (Idstein, Germany). Ethanol (J.T. Baker; HPLC Grade) was purchased from Walter CMP (Kiel, Germany). Methods Experimental design was realised utilising the Modde 10.1 Software (Umetrics, Malmö, Sweden). Spray drying was carried out with a Büchi B-290 Mini Spray Dryer equipped with a high performance cyclone and a B-295 Inert Loop (Büchi Labortechnik AG, Flawil, Switzerland). HPMC E5 and later on CBD were dissolved in 80 % Ethanol (wt/wt) and processed according to the DoE. During spray drying it was ensured that the outlet temperature never exceeded 60 °C (melting point of CBD: 66 °C) by adjusting the spray rate. Table 1: Variable parameters and levels of DoE

Parameter

Level 1

Level 2

Level 3

Inlet temperature

100 °C

110 °C

120 °C

Aspirator rate

50 %

75 %

100 %

Solid Content (HPMC)

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Drug Delivery to the Lungs, Volume 29, 2018 - Cannabidiol in a DPI – maximising the spray drying yield of HPMC matrix particles Particle size distributions (PSD) of the spray drying batches were analysed in triplicate with a Sympatec HELOS combined with a RODOS dispersing module (Sympatec GmbH, Clausthal-Zellerfeld, Germany) at 3 bar dispersing pressure. Scanning electron microscopy: Particles were visualised with a Phenom-World PhenomXL (Phenom-World BV, Eindhoven, Netherlands) with a backscatter detector and at 10 kV acceleration voltage. To prevent charging effects the samples were gold coated with a SCD 005 Sputter Coater (Bal-Tec AG, Balzers, Liechtenstein). API quantification was performed with a HP Agilent series 1100 HPLC. Acetonitrile / Aqua bidest. (85:15) was used as the mobile phase. The oven temperature was set to 55 °C and an RP-18 column with a precolumn was utilised. Peak evaluation was done with an external 8-point calibration in the range from 0.1 µg/mL – 100 µg/mL. Emitted dose was investigated with the dose uniformity sampling apparatus (DUSA). 10 ± 0.5 mg powder were weighed into size 3 HPMC hard capsules. Each capsule was measured individually with a Cyclohaler. For the measurement a flowrate of 100 L/min was chosen as it corresponds to 4 kPa pressure drop. To achieve an airflow of 4 L measurement duration was set to 2.4 seconds. Sample analysis was done with aforementioned HPLC method and an external 9-point calibration in the range from 0.1 µg/mL – 250 µg/mL. For each batch 10 capsules were measured. Methanol / Aqua bidest. (75:25) was used as the solvent. One successful “inhalation” was performed per capsule. A successful inhalation was signalled by the sound of the capsule rotating in the inhaler. Results & Discussion There are three different types of inhalative formulations: Dry powders for inhalation (DPIs), liquid formulations in pressurised containers (pMDIs) and liquid formulations for nebulisers. The first provides some benefits over the latter two, the key advantage being an increased storage stability, which is vital when working with cannabinoids as it was shown that cannabinoids undergo a rapid degradation process in standardised preparations [4]. Additionally DPIs offer simpler handling than MDIs [5] as no additional coordination between inhalation and actuation is required. Inhalation with a nebuliser is often far less convenient than inhalation with a DPI (or pMDI) reducing the attractiveness of these inhalation devices. The used CBD is a resinous substance which requires additional processing to be formulated as a dry powder. A possible approach to formulate sticky/resinous substances as a dry powder is the preparation of solid dispersions. Solid dispersions can be formulated by several different methods, one of which is spray drying. The advantages of spray drying over other methods (e.g. freeze drying) are short process durations and relatively low costs. Due to the distinct lipophilicity of CBD spray drying needs to take place from an aqueous suspension, an emulsion or from an organic solvent-based system. To minimise the use of additional excipients like emulsifiers or (suspension) stabilisers a solvent based system was chosen. HPMC E5 was selected as the polymer for the matrix, as it has a common solvent with CBD (namely ethanol 80 % wt/wt). A 3³ full factorial experimental design was performed for pure Methocel E5 to evaluate its optimal spray drying parameters from ethanol 80 % (wt/wt). Three additional center points were added, to verify the reproducibility of the process, resulting in a total of 30 runs. A number (N1, N2 etc) identifies each run.

Figure 1: DoE contour plots depicting the correlation between solid content and inlet temperature at 100 % aspirator setting; A – Response: x50; B – Response: yield

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Drug Delivery to the Lungs, Volume 29, 2018 – Tobias Gutowski & Regina Scherließ Figure 1 shows that yield and mean size mainly depended on the solid content. Two runs were excluded for Figure 1B as the yield was distorted by a clogged cyclone in two runs. Data also revealed that an aspirator setting of 100 % is beneficial to achieve a maximum yield (not shown). SEM images (Figure 2) showed wrinkled particles both with and without CBD. This appearance was expected, as ethanol evaporates much faster than water. Faster solvent evaporation reduces the time that is available for diffusion in the droplet, resulting in a shell formation and therefore hollow spheres. These particles collapse due to the vacuum that occurs when the solvent vapour escapes the particle, if the substance is flexible. Polymeric substances often form more flexible particles than small molecules, explaining the observed appearance. Images also revealed that exchanging 25 % of the matrix substance with API resulted in smaller particles (Fig. 2 D, E, F).

Figure 2: SEM images 5000x magnification; pure HPMC-particles from DoE (A - C) & HPMC + CBD particles (D - E): A & D - parameters N13; B & E - parameters N20; C & F – parameters N25 (optimal)

The “Summary of Fit” for this DoE showed that particle size and yield are very reproducible (reproducibility > 0.85). The resulting model, however, only has a moderate validity for the particle size (~ 0.65). The validity for the yield is very low (~ 0.4). However, after evaluation, following settings for input parameters were considered optimal: an inlet temperature of 100 °C, an aspirator setting of 100 % and a solid content of 5 % (equal to run N25). Two additional, promising parameter settings were selected (Table 2) for API incorporation, as the influence of the API on the process and the aerodynamic behaviour of the particles was unknown. Table 2: Chosen parameter combinations for API incorporation

N13

N20

N25 (Optimal)

Inlet temperature

100 °C

110 °C

100 °C

Aspirator rate

75 %

100 %

Solid Content

For API incorporation 25 % of the matrix was replaced with CBD, maintaining the total solid content. Both x50 and yield are below the values that were observed with pure HPMC (Table 3) suggesting that CBD has a major influence on the spray drying of HPMC.

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Drug Delivery to the Lungs, Volume 29, 2018 - Cannabidiol in a DPI – maximising the spray drying yield of HPMC matrix particles Table 3: x50 and yield comparison of selected runs ± CBD

Parameter

N13

N13 + CBD

N20

N20 + CBD

N25

N25 + CBD

x50 (µm) (n=3)

2.99 ± 0.14

1.15 ± 0.03

2.38 ± 0.04

1.67 ± 0.05

4.49 ± 0.10

2.76 ± 0.06

Yield (%) (n=1)

66.78

21.63

64.98

15.72

75.15

39.01

Analysis of the API content of the resulting powder showed that all three batches had a very high loading efficiency between 95.69 % and 102.45 % signalling that HPMC E5 is a viable matrix substance for CBD.

Figure 3: Emitted dose assessment of run N25 (A) & N13 (B)

Delivered dose evaluation revealed that 84.31 ± 3.59 % of the recovered API are emitted from batch N25 while batch N13 emits 58.92 ± 6.34 %.This shows that the powder in general is dispersible from a DPI device, but efficiency should be increased. All ten measurements from both batches were within a 25 % range around the corresponding mean emitted dose. The yield of batch N20 was too low for measurement after PSD analysis. As these aerodynamic experiments exhibited uneven recoveries, the homogeneity of batch N25 was checked. Data shows that the powder is homogeneous. However, it could be seen, that the incorporation of CBD in a HPMC matrix does not protect the API from degradation, as the CBD content decreased from 25 % to 20 % over the course of four months. Investigation of the uneven recoveries and further aerodynamic assessment are ongoing. Conclusion Experiments showed that HPMC E5 is very suitable to form solid dispersions of the used CBD quality. Spray drying Methocel E5 from ethanol, however, resulted in some difficulties, namely a very unpredictable yield. Another problem is that the spray dried API is not protected from degradation. Further aerodynamic characterisation will be done in ongoing experiments. Delivered dose data is promising as a large percentage is delivered without additional excipients to enhance flowability. Additionally, other polymers such as HPMC-acetate succinate, polyvinylpyrollidone vinyl acetate or modified starches will be investigated for their potential to form solid dispersions of CBD, as CBD might exhibit less influence on their spray drying behaviour, enabling higher yields and thereby a more economic process. Due to CBD’s lipophilicity solid lipid particles might be another possible way to formulate the API as a dry powder.

Thomas B F, Compton D R, Martin B R: Characterization of the lipophilicity of natural and synthetic analogs of delta 9tetrahydrocannabinol and its relationship to pharmacological potency, J Pharmacol Exp Ther, 1990; 2: pp151-156

Mechoulam R, Parker L A, Gallily R: Cannabidiol: An overview of some pharmacological aspects, J Clin Pharmacol, 2002; 11 Suppl: pp11-19

Scuderi C, Filippis D D, Iuvone T, Blasio A, Steardo A, Esposito G: Cannabidiol in medicine: A review of its therapeutic potential in CNS disorders, Phytoter Res, 2009; 5: pp597-602

Pacifici R, Marchei E, Salvatore F, Guandalini L, Busardo F P, Pichini S: Evaluation of cannabinoids concentration and stability in standardized preparations of cannabis tea and cannabis oil by ultra-high performance liquid chromatography tandem mass spectrometry, Clin Chem Lab Med, 2017; 10: pp1555-1563

Crompton G K: Dry powder inhalers: Advantages and limitations, J Aerosol Med, 1991; 3: pp151-156

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Drug Delivery to the Lungs, Volume 29, 2018 - Angelika Jüptner et al. Spray dried powders for nasal application - Influence of particle morphology and filling process on aerosol generation Angelika Jüptner1, Ségolène Sarrailh2 & Regina Scherließ1 1

Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany 2Aptar Pharma, Route des Falaises, 27100 Le Vaudreuil, France

Summary Spray drying is an interesting formulation alternative to interactive blends, which are commonly used in oral inhalation. Particles of spherical morphology can be obtained by this technology. By changing process parameters or adding excipients morphology, density and size can be tailored to modify dispersion characteristics and location of deposition. Being already used in pulmonary delivery they are also an opportunity in nasal application. In this study it was shown that spray dried formulations can be filled with equipment like a vacuum drum system. The nasal deposition of spherical particles is affected by the filling process, if the assessment of deposition in a nasal cast is conducted without airflow. During the filling process powder plugs are formed which are less dispersible than the manually filled powder. A similar deposition profile to manually filled powders is seen with an airflow of 15 L/min because the dispersion of the built plugs is improved. Besides the filling process the morphology of particles also has an impact on the deposition profile. Particles with a wrinkled morphology tend to have a lower deposition in the middle and upper turbinates at an airflow of 15 L/min than at 0 L/min compared to the spherical particles. Also the postnasal fraction is increased markedly compared to spherical particles. Wrinkled particles with HPMC show the same trend as particles with leucine but they have a lower postnasal fraction. For targeting the upper turbinates (e.g. for nose-to-brain delivery via the olfactory region) a wrinkled morphology can be disadvantageous, if administered to an airflow of 15 L/min. Key Message Spray dried powders can be tailored for nasal delivery. Depending on particle morphology and post-processing steps, such as filling, aerodynamic characteristics are altered. The influence on the aerodynamic characteristics depends on the administration procedure and the particle morphology. Introduction Dry powder formulations are typical in pulmonary delivery but can also be used for nasal application. A commonly known strategy to formulate dry powders is an interactive mixture but engineered powders are gaining more attention as an alternative. They provide the possibility of high dose application and the particles can be designed as required. Spray dried powders exhibit mostly a large specific surface area due to their small size and have low bulk density as particles are often hollow and highly porous [1]. The cohesiveness of these powders can be challenging during the filling process, as oftentimes they are not flowable and difficult to process. The formed plug during filling process may further affect the dispersion during application. The objective of this study is to look at the filling process of spray dried formulations, the impact on the aerosol generation and to point out the influence of particle morphology on nasal delivery. Materials and Methods Preparation of model formulations A batch of spherical particles for nasal delivery (N/S/0.5%) was obtained by spray drying an aqueous 10 % solution of mannitol (Roquette, Lestrem, France) and erioglaucine disodium (Sigma-Aldrich, Saint Louis, USA), also known as brilliant blue (BB), in the composition 99.5 % and 0.5 %. A Mini Spray Dryer B-290 with an ultrasonic nozzle (60 kHz) set to 1.6 Watt and a high performance cyclone (Büchi Labortechnik AG, Flawil, Switzerland) were used. The inlet temperature (IT) was set to 130 °C, the aspirator rate to 20 m3/h and the outlet temperature (OT) was adjusted with the spray flow to 61 °C. BB is a watersoluble dye that was used in this study as a model drug. Another batch of nasal particles with a partly indented morphology (N/S/Leu10%) was developed by adding leucine (Leu, Sigma-Aldrich, Saint Louis, USA) to the solution. The final amount of leucine in the solid mass accounted to 10 %. The leucine concentration was increased to 18 % in the solid mass to obtain a batch with a wrinkled morphology (N/W/Leu18%). The same setup as already mentioned was used. For a second batch with wrinkled morphology (N/W/HPMC1%) hydroxypropylmethylcellulose (HPMC; Shin-Etsu, Wiesbaden, Germany) was added to the solution of mannitol and BB, so the concentration in the solid mass was 1 %. The batch was spray dried with an IT of 150 °C and an OT of 72 °C with the same settings as described previously.

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Drug Delivery to the Lungs, Volume 29, 2018 - Spray dried powders for nasal application - Influence of particle morphology and filling process on aerosol generation Characterisation methods The particle size distribution was measured by using a HELOS laser diffractometer (Sympatec GmbH, ClausthalZellerfeld, Germany) connected to the RODOS dispenser system. Particles were dispersed at 0.2 bar and data evaluation was performed with the Windox 5.8.0.0 software (Sympatec GmbH, Clausthal-Zellerfeld, Germany) utilising the Fraunhofer theory. The average of 3 measurements is given. The span was calculated by subtracting the x10 from x90 and divided by the x50 to evaluate the distribution. Particle morphology was assessed by scanning electron microscopy (SEM, Smart SEM Supra55VP, Zeiss, Germany; Phenom XL, Phenom-world, Netherlands). Particles were fixed on carbon stickers and sputter-coated with a thin gold layer prior to SEM evaluation. A BS detector and working voltage of 10 kV were used. The bulk density was calculated from the volume, measured with a 10 mL measuring cylinder, and the weight (Sartorius analytic balance A200S, Goettingen, Germany) of 3 g formulation. The flow function coefficient as a dynamic measure of powder flowability was determined using a FT4 Powder Rheometer (FT4, Freeman Technology, Worcestershire, UK) with the 1 ml shear cell. The shear head inserts the blades into the powder and induces normal stress upon contact with the powder until the required normal stress is established. Due to the slow rotation, shear stress is induced and increases until the powder bed fails. The maximum shear stress is measured as the yield point. The yield loci are constructed by plotting the failure points against the normal stress. The major principal stress and the unconfined yield strength are calculated by fitting Mohr circle to the yield locus. The ratio of the major principal stress to the unconfined yield strength is the flow function coefficient (ffc). Below ffc = 4 a powder has a poor flowability, above 10 it is free flowing and between 4 and 10 good flowing. The nasal deposition was assessed with a nasal cast (Boehringer Ingelheim, Ingelheim, Germany) modeled after an adult male. The nasal cast consists of five different parts, the nostrils, nasal vestibule, the lower turbinates, middle and upper turbinates and the nasopharynx. The parts were coated with a mixture of isopropanol and propylene glycol (1+1). The tests were carried out with the UDS Powder (Unit Dose System Powder) device (Aptar Pharma) and 40 mg in one shot per nostril per run was administered. The UDS Powder is an active device, which is able to create an aerosol without airflow. The measurement was performed without air flow and at 15 L/min. A delivery angle of 45 ° to horizontal plane and an insertion depth of 10 mm were used. The airflow of 15 L/min was held constant with a vacuum pump connected to the nasopharynx of the nasal cast. All parts of the nasal cast were rinsed with water and the powder content was assessed spectroscopically at 630 nm. The possible fine particle fraction (FPF) was assessed with the next generation impactor and a 2 liter expansion chamber (Copley Scientific, United Kingdom), coated with a mixture of propyleneglycol and Brij dissolved in ethanol. The measurement was performed at a flow rate of 15 L/min and two devices filled with 40 mg per run of spray dried formulation. The quantification was performed as described previously. The FPF was calculated of the mean of 3 runs. The UDS Powder devices were either filled manually or semi-automatically. The semi-automatic filling was performed with an Omnidose machine (Harro Höfliger, Germany) utilising the drum system. The powder is given into a powder tank above the drum system. The dosing bore fills in assistance of vacuum. Remaining powder is scraped off, the drum turns and ejects the powder plug into the powder chamber of the UDS powder. A target of 40 mg was set and the relative standard deviation should be below 10 % for the filling session to be successful. Results and Discussion Bulk properties of model formulations Spray drying resulted in particles with different morphologies (Figure 1). Mannitol and BB formed uniform spherical particles with a mean geometric diameter of 22.23 ± 0.08 µm, which meets the requirements of > 10 µm for powders for nasal deposition[2]. An addition of 10 % leucine altered the morphology slightly but did not affect the particle size. Leucine is known to precipitate at the droplet surface during spray drying and forms a hydrophobic shell around the particle [3]. With the hydrophobic shell the interparticle interactions and cohesiveness are reduced and therefore the flowability and the bulk density are increased (Table 1). The addition of 18 % leucine or HPMC leads to an indented morphology. These particles have a much larger surface than the spherical ones and a lower bulk density due to their shape. Flowability measurements indicate a good flowing powder for N/S/0.5%. A comparable formulation for pulmonary application with an x50 ~ 2.6 µm showed poor flowing behaviour[4]. Because of the larger particle size for nasal delivery the flowability increased markedly. The moderate addition of leucine increased the flowability further to a free flowing powder. With the change in morphology the ffc dropped because the particles interlink and interfere with each other, even though the powder is still free flowing. This could also be observed in pulmonary particles5 but not as prominent as shown here. The ffc of particles with HPMC did not drop but increased slightly. Particles containing HPMC do not get a hydrophobic shell thus this effect can only be attributed to morphology.

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Drug Delivery to the Lungs, Volume 29, 2018 - Angelika Jüptner et al. Table 1 – Characteristics of the model formulations (n=3 ± standard deviation)

N/S/0.5%

N/S/Leu10%

N/W/Leu18%

N/W/HPMC1%

x50 [µm]

22.23 ± 0.08

21.21 ± 0.09

24.70 ± 0.02

25.99 ± 0.07

Span

1.01

1.05

1.89

1.67

Bulk density [g/cm3]

0.49 ± 0.00

0.51 ± 0.00

0.31 ± 0.01

0.43 ± 0.01

Specific surface area [m2/g]

0.381 ± 0.032

0.963 ± 0.075

0.953 ± 0.042

0.861 ± 0.050

ffc

7.36 ± 0.12 → good flowing

22.5 ± 3.4 → free flowing

14.63 ± 0.25 → free flowing

8.82 ± 1.36 → good flowing

FPF [%]

< LOD

0.29 ± 0.04

Figure 1- scanning electron microscopy images of the prepared formulations. N/S/0.5% on the top left, N/S/Leu10% top middle, N/W/Leu18% top right, N/W/HPMC1% bottom left

Filling trials and influence of filling procedure on aerosol generation

deposition in %

The influence of filling was evaluated exemplarily for N/S/0.5%. Filling was performed with the Omnidose and the drum system with the N/S/0.5%. A mean filling weight of 43.02 mg and a relative standard deviation below 2 % was achieved. This was evaluated as a successful filling session. For comparison the same formulation was filled manually as done with all other formulations. 60 50 40 30 20 10 0

nostrils

nasal vestibule lower turbinates

N/S/0.5 % manually 0 L/min

middle and upper turbinates

N/S/0.5 % drum 0 L/min

nasopharynx

N/S/0.5% manually 15 L/min

postnasal fraction

device

N/S/0.5% drum 15 L/min

Figure 2 – deposition in the nasal cast; comparison of manually and semi-automatically filled devices with N/S/0.5% at airflow of 0 L/min and 15 L/min (n=3; error bars are standard deviation)

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Drug Delivery to the Lungs, Volume 29, 2018 - Spray dried powders for nasal application - Influence of particle morphology and filling process on aerosol generation In the assessment with the nasal cast an overall high deposition in the middle and upper turbinates (MUT) could be found (Figure 2). This is an interesting region to be targeted, because of its high inner surface and the localisation of the olfactory region. This can be of interest for systemic drug delivery or nose to brain transport. A high deposition is also found in the frontal regions of the nasal cast which is not preferable. If the device was filled with a semiautomatic filling system, the deposition profile without airflow indicated that the built plugs could not be dispersed well and the deposition in the nasal vestibule increased. If the measurement was conducted with airflow of 15 L/min the trend to a higher deposition in the MUT and less in the nasal vestibule was observed. So the plugs from filling procedures were better dispersed with airflow. The airflow also increased the postnasal fraction and reduced the deposition in the lower turbinates. Influence of particle shape on nasal delivery

deposition in %

At breath hold the particles with HPMC have a higher deposition in the MUT (Figure 3) than hydrophilic spherical particles (N/S/0.5%). The cohesion went down with addition of HPMC as seen in the ffc, so the powder is better dispersed. Thus the deposition in the anterior nasal parts is reduced which is preferable. A moderate addition of leucine reduces the cohesion markedly with its hydrophobic shell. The wrinkle of N/W/Leu18% can interlink with each other resulting in a higher deposition in frontal nasal parts and a higher variability. The deposition in the MUT decreases with these constrictions. At an airflow of 15 L/min the better flowability of N/S/Leu10% and N/W/Leu18% increased the postnasal fraction to ~ 20 % and decreased the deposition in the MUT. The formulation with HPMC showed the same tendency as the N/W/Leu18% with less deposition in the MUT and a higher postnasal fraction, but the postnasal fraction was lower than for the formulations with leucine and the deposition in the MUT tended to be higher. This correlates with the very low cohesion of the leucine particles, while the cohesion for HPMC particles is higher. A fine particle fraction, however, was not determinable for all formulations but the N/W/HPMC1%. All of this showed that the wrinkled morphology had a remarkable influence on the aerodynamic properties and could be favourable for a high deposition in targeted zones with no air flow but can also induce a possible lung fraction. 60 50 40 30 20 10 0

nostrils

nasal vestibule lower turbinates

middle and upper turbinates

nasopharynx

postnasal fraction

N/S/0.5 % 0 L/min

N/S/Leu10% 0 L/min

N/W/Leu18% 0 L/min

N/W/HPMC1% 0 L/min

N/S/0.5% 15 L/min

N/S/Leu10% 15 L/min

N/W/Leu18% 15 L/min

N/W/HPMC1% 15 L/min

device

Figure 3 - deposition of the different formulations in the nasal cast at 0 L/min and 15 L/min, filling weight of 40 mg, manual filling (n=3; error bars are standard deviation)

Conclusion Spray dried formulations are fillable with standard equipment. The impact of this procedure is more pronounced for measurements with no airflow. The addition of leucine increases the flowability to free flowable and with it the postnasal fraction at 15 L/min. The deposition in targeted regions decreases. At breath hold free flowing powders with low cohesion can have a favourable influence on the deposition profile. A wrinkled morphology without a hydrophobic shell increases the flowability a bit but the powder is still good flowing. The reduced cohesion compared to spherical particles leads to a higher deposition in the MUT at breath hold. The postnasal fraction at 15 L/min is not as high as for free flowing formulations and the deposition in the MUT not as decreased. To further differentiate the influence of hydrophobicity and morphology a more hydrophilic formulation without additives and a wrinkled morphology will be looked at. References 1

Vehring R, Foss W R, Lechuga-Ballesteros D, Particle formation in spray drying. In: Journal of Aerosol Science 38, pp. 728746, 2007. 2

Ozsoy Y, Cevher E: Nasal delivery of high molecular weight drugs. In: Molecules 14 (9), pp. 3754-3779, 2009.

Vehring R: Pharmaceutical Particle Engineering via Spray Drying. In:Pharmaceutical Research 25 (5), pp. 999-1022, 2008.

Scherließ R, Jüptner A, Hellfritzsch M, Kaj C, Vimont A, Sarrailh S, Williams G: Spray dried formulations for pulmonary delivery – challenges in filling, aerosol generation and delivery, poster at DDL 2017 4

Jüptner A, Scherließ R: Spray dried formulations for inhalation – influence of particle shape, poster at 11th PBP world meeting 2017 5

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Drug Delivery to the Lungs, Volume 29, 2018 – Gavin Bennett et al. Assessment of Aerosol Delivery during Simulated Invasive Ventilation, Non-invasive Ventilation and High Flow Nasal Therapy Gavin Bennett1, Mary Joyce1, Louise Sweeney1 & Ronan MacLoughlin1 1Aerogen,

IDA Business Park, Dangan, Galway, Ireland

Summary Patients receiving mechanical ventilation are often prescribed nebulised therapy. Non-invasive ventilation and high flow nasal therapy are increasingly used for ventilatory support in patients with acute respiratory failure. Some patients receiving these therapies may also benefit from inhaled drug delivery. The objective of this study was to evaluate aerosol delivery during simulated adult mechanical ventilation, non-invasive ventilation and high flow nasal therapy using a vibrating mesh nebuliser. Simulated adult mechanical ventilation assessed the filter dose beyond the endotracheal tube across four potential nebuliser placement positions within the circuit. Simulated non-invasive ventilation assessed the filter dose when the nebuliser (Aerogen Solo, Aerogen, Ireland) was positioned between the facemask (Pneumocare Health, India) and the patient circuit with continuous positive airway pressure (10cmH 20 and 15cmH20). Simulated high flow nasal therapy assessed the filter dose downstream of the model oropharyngeal region at two clinically relevant gas flow rates, in line with the Fisher & Paykel Airvo 2. For each ventilation type, a 2 mL dose of albuterol sulphate (2 mg/mL) was nebulised using a vibrating mesh nebuliser (Aerogen Solo, Aerogen, Ireland) of similar droplet size (4.5µm). The drug captured on a filter was eluted using a 10 mL buffer solution of 0.1M HCI. The mass of drug eluted was determined using UV spectroscopy at 276nm. The greatest aerosol delivery was observed during simulated non-invasive ventilation (26.79%), in comparison with invasive ventilation (22.81%) and high flow nasal therapy (21.00%). A one way analysis of variance resulted in a pvalue of 0.0012, indicating that there was a statistically significance difference in aerosol delivery between each type of ventilatory support. This study suggests that continuity of high efficiency aerosol delivery is possible across both invasive and noninvasive patient interventions using a vibrating mesh nebuliser. Key Message To the authors’ knowledge, this is the first study to evaluate and directly compare aerosol delivery across invasive ventilation, non-invasive ventilation and high flow nasal therapy in vitro. This study illustrates the potential for efficient aerosol delivery with each ventilation type, with a significantly greater dose inhaled during simulated noninvasive ventilation. Introduction Patients receiving mechanical ventilation are often prescribed nebulised therapy [1]. However, non-invasive ventilation (NIV) and high flow nasal therapy (HFNT) are increasingly used for ventilatory support in patients with acute respiratory failure. Some patients receiving these therapies might also benefit from inhaled drug delivery [2]. HFNT provides sufficient flow rates to equal or exceed inspiratory flow and reduces the inspiratory resistance associated with the nasopharynx, thus reducing the work of breathing [3]. In addition, HFNT is utilized to improve weaning in ventilator dependent patients [4]. To our knowledge, no study has compared aerosol efficiency across simulated invasive and non-invasive ventilation in vitro. The objective of this study was to evaluate aerosol delivery during simulated adult mechanical ventilation, NIV and HFNT using a vibrating mesh nebuliser. Materials and Methods Invasive (ETT) Mechanical ventilation Simulated adult mechanical ventilation assessed the filter dose beyond the endotracheal tube across four potential nebuliser placement positions within the circuit; at the ventilator, at the wye, at the dry side of the humidifier and at the wet side of the humidifier (Figure 1). The F&P MR850 humidifier (Fisher & Paykel, New Zealand) was used. A 2 mL dose of 2 mg/mL albuterol sulphate was nebulised (n=3) using a vibrating mesh nebuliser (Aerogen Solo, Aerogen, Ireland). The dose was aerosolised and the deposited fraction was captured on an absolute filter (RespirGard II 303, Baxter), which was positioned between the endotracheal tube and lung. Drug was eluted using a 10 mL buffer solution of 0.1M HCI. The mass of drug eluted was determined using UV spectroscopy @ 276nm. Results were expressed as the percentage of the nominal dose placed in the nebuliser’s medication cup. The Maquet Servo-i breathing parameters employed for testing are outlined in Table 1.

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NIV Simulated non-invasive ventilation assessed the filter dose when the vibrating mesh nebuliser (Aerogen Solo, Aerogen, Ireland) was positioned between the facemask (Pneumocare Health, India) and the patient circuit. Continuous positive airway pressure (CPAP) at 10cmH 20 and 15cmH2O was supplied through a single limb circuit using a ventilator (Bellavista 1000, IMT Medical, Switzerland). A 2 mL dose of 2 mg/mL albuterol sulphate was nebulised (n=3) using a vibrating mesh nebuliser (Aerogen Solo, Aerogen, Ireland). The dose was aerosolised and the deposited fraction was captured on an absolute filter (RespirGard II 303, Baxter), which was positioned between the facemask and the breathing simulator. A standard adult breathing pattern was used (15BPM, Vt 500 mL, I:E 1:1). Drug was eluted using a 10 mL buffer solution of 0.1M HCI. The mass of drug eluted was determined using UV spectroscopy @ 276nm. HFNT Tracheal deposition at each gas flow rate under test (10 and 60LPM) was recorded using the Fisher & Paykel Airvo 2. The Airvo 2 system features a humidifier with an integrated flow source and was used in conjunction with the bespoke nebuliser adapter with an adult nasal cannula (Optiflow™ OPT544, Fisher & Paykel). Tracheal deposition was characterised by quantifying the mass of drug captured on a filter positioned downstream of the model oropharyngeal region, as an indication of aerosol that could potentially reach the lung. Adult high flow nasal cannula were positioned on a previously described adult head model (LUCY) in accordance with manufacturers’ instructions. The head model was connected to a breathing simulator (Ingmar ASL 5000) via an absolute filter (RespirGard II 303, Baxter), using a standard adult breathing pattern; (15 BPM, Vt 500 mL, I:E 1:1). A 2 mL dose of albuterol sulphate (2 mg/mL) was nebulised (n=3) using a vibrating mesh nebuliser (Aerogen Solo, Aerogen, Ireland). The drug captured on the filter was eluted using a 10 mL buffer solution of 0.1M HCI. The mass of drug eluted was determined using UV spectroscopy @ 276nm.

Regimen

Adult

Ventilator Mode

Volume-Controlled Ventilation

Flow Pattern

Ramp

Tidal Volume (mL)

500

Respiratory Rate (BPM)

Inspiratory:Expiratory Ratio

1:1

Ventilator circuit (mm ID)

Endotracheal tube (mm ID)

8.0

Table 1 – Maquet Servo-i Ventilator Breathing Parameters

A Figure 1 – Illustration of invasive ventilation test set-up. The Aerogen Solo nebuliser and standard t-Piece were positioned at the ventilator (A), dry side of the humidifier (B), wet side of the humidifier (C) and wye (D).

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Figure 2 – NIV test set-up. The Aerogen Solo nebuliser and standard t-piece was placed between the facemask and the patient circuit.

Filter

Figure 3 – Illustration of HFNT test set-up. The Aerogen Solo nebuliser was placed at the humidification chamber, using a nebuliser adapter.

Results The results of aerosol delivery are outlined in Tables 2-4 across invasive ventilation, NIV and HFNT.

Nebuliser position

Filter dose (%)

Ventilator

22.81 ± 1.63

Wye

17.71 ± 1.08

Dry side

17.08 ± 0.27

Wet side

19.42 ± 0.72

P-value

0.0007

Table 2 – Average ± Standard Deviation values of the filter dose (%) beyond the endotracheal tube, during simulated mechanical ventilation. The Aerogen Solo was positioned at the ventilator, wye, dry side and wet side of the humidifier under normal adult breathing settings.

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Drug Delivery to the Lungs, Volume 29, 2018 – Assessment of Aerosol Delivery during Simulated Invasive Ventilation, Non-invasive Ventilation and High Flow Nasal Therapy Filter dose (%) CPAP 10cmH20

26.00 ± 0.80

CPAP 15cmH20

26.79 ± 0.39

P-value

0.1990

Table 3– Average ± Standard Deviation values of the filter dose (%) between the facemask and the breathing simulator, during simulated NIV (CPAP of 10cmH2O and 15cmH2O) when using the Aerogen Solo in combination with the Pneumocare facemask. Litres Per Minute

Filter dose (%)

10 60

21.00 ± 0.56 1.00 ± 0.20

P-value

<0.0001

Table 4– Average ± Standard Deviation values of the filter dose (%) downstream of the model oropharyngeal region, during simulated HFNT at 10LPM and 60LPM.

For simulated NIV, results were substantially equivalent for a CPAP level of 10cmH20 and 15cmH20 (p = 0.1990). During simulated invasive ventilation, the mean filter dose (%) beyond the endotracheal tube was compared between four potential nebuliser placement positions within the humidification system, with the highest aerosol efficiency seen when the nebuliser was placed at the ventilator. A one way analysis of variance (ANOVA) resulted in a p-value of 0.0007, indicating that there was a statistically significance difference in mean filter dose between positions. A decreasing gas flow rate was associated with a significantly greater tracheal deposition during simulated HFNT (p = <0.0001). Overall, the greatest aerosol delivery was observed with simulated NIV (26.79%), in comparison to invasive ventilation (22.81%) and HFNT (21.00%). A one way analysis of variance (ANOVA) resulted in a p-value of 0.0012, indicating that there was a statistically significance difference in aerosol delivery between ventilation types. Discussion Optimal aerosol delivery was achieved when the nebuliser was positioned at the ventilator during simulated invasive ventilation, with a CPAP of 10cmH20 during NIV and at a gas flow rate of 10LPM during HFNT. Our findings are consistent with those in the literature. Ari et al. reported that optimal drug delivery depends on the aerosol generator position during adult mechanical ventilation [5]. Similarly, MacLoughlin et al. previously reported that higher flow rates are associated with reduced efficiency of drug delivery through an adult HFNT system [6]. Bennett et al. showed that to optimize the amount of aerosol exiting the nasal cannula interface during HFNT, it is necessary for gas flow rate to be low [7]. The greatest aerosol delivery was observed with simulated NIV, in comparison to invasive ventilation and HFNT. This may be explained by the position of the nebuliser between the facemask and patient circuit. White et al. previously showed that greater drug delivery was observed with a vibrating mesh nebuliser placed proximally to the mask during simulated NIV [8]. During simulated HFNT, nasal aerosol inhalation may promote deposition due to the turbulent flow in the nose, thus reducing delivery [9]. In invasive ventilation, aerosol losses may be caused by bias flow within the patient circuit [5]. This study demonstrates efficient aerosol delivery during various types of simulated ventilation whilst using a vibrating mesh nebuliser. References 1

Berlinski A, Willis JR. Albuterol Delivery by 4 Different Nebulizers Placed in 4 Different Positions in a Pediatric Ventilator In Vitro Model. Respiratory care. 2013;58(7):1124-33

Hess DR. Aerosol Therapy During Noninvasive Ventilation or High-Flow Nasal Cannula. Respiratory care. 2015;60(6):880-93.

Dysart K, Miller TL, Wolfson MR, Shaffer TH. Research in high flow therapy; mechanisms of action. Respiratory medicine. 2009;103(10): 1400-5

Hernandez G, Vaquero C, Colinas L, Cuena R. Effect of Post Extubation High Flow Nasal Cannula vs Noninvasive on Reintubation and POstextubation Respiratory Failure in High-Risk Patients: A Randomized Clinical Trial. JAMA. 2016; 16(15): 1565-1574

Ari A, Areabi H, Fink JB. Evaluation of Aerosol Generator Devices at 3 Locations in Humidified and Nonhumidified Circuits During Adult Mechanical Ventilation. Respiratory care. 2010;55(7):837-44.

Bennett G, Joyce M, Sweeney L, MacLoughlin R. In Vitro Determination of the Main Effects in the Design of High-Flow Nasal Therapy Systems with Respect to Aerosol Performance. Pulmonary Therapy. 2018. White CC, Crotwell DN, Shen S, Salyer J, Yung D. Bronchodilator delivery during simulated pedaitric noninvasive ventilation. Respiratory care. 2013: 58(9): 1459-66

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Reminiac F, Vecellio L, Heuze- Vourc’h N, Petitcollin A, Respaud R. Aerosol Therapy in Adults Receiving High Flow Nasal Cannula Oxygen Therapy. Journal of Aerosol Medicine. 2016; 29(2): 134-41

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Drug Delivery to the Lungs, Volume 29, 2018 – Christian Etschmann Softpellets for high dose pulmonary delivery Christian Etschmann, Regina Scherließ Department of Pharmaceutics and Biopharmaceutics, Kiel University Grasweg 9a, 24118 Kiel, Germany

Summary High dose formulations are increasingly gaining attention in the field of inhalation. Soft agglomerates seem to be a suitable technique to produce good flowing powders that are stable enough to allow powder handling and dosing, but are soft enough to be deagglomerated and get dispersed during inhalation utilising a suitable inhaler. Typically softpellets are produced by preagglomeration of micronised powders using a screw feeder. Thereafter the preagglomerates are rounded in a spheroniser. In this study a new technique utilising vibration is applied to produce placebo and active pharmaceutical ingredient (API) softpellets. In the first step of this method pre-agglomerates are formed by forcing the powder through a sieve resulting in oblong powder agglomerates. The particle size of the resulting product can be controlled by the selection of the sieve for pre agglomeration. Afterwards the preagglomerates were rounded on a vibrating slide. In this study three different sizes of placebo softpellets were compared with respect to particle size and sphericity. In a last step the fines and coarse particles were separated with sieves. It has been shown that smaller particles show a higher sphericity and higher stability than larger particles. Furthermore the bulk density of larger particles was higher compared to the smaller ones. First results of aerodynamic characterisation of an API batch consisting of rifampicin are very promising. They seem to get well deagglomerated with the Turbohaler and produce a high fine particle fraction of 52.90 %. Key Message In this study placebo and API soft powder agglomerates were produced utilising a new method based on vibration. The particle sizes of the batches were easily controlled and show narrow distributions between 200 µm and 400 µm. Furthermore the API soft pellets were investigated due to their aerodynamic behaviour showing their potential for high dose delivery. Introduction High dose dry powder inhalation increasingly gets in the focus of scientific efforts. There are several reasons for that. The driving force to deliver active pharmaceutical ingredients (APIs) to the lungs was motivated by the treatment of asthma and COPD. However there is still a large area to expand the field of application on other diseases too. Patients suffering from tuberculosis or cystic fibrosis (often being associated with bacterial infections of the lung), for instance, are treated with high doses of antibiotics over a long time interval. This long treatment period and the high doses administered are often associated with side effects affecting patient adherence. To reduce the required dose of API local administration seems to be a possible method that can at the same time increase the drug concentration in the infected tissue compared to oral administration. For example Santoshi et. al. demonstrated that a single dose of 30 mg rifampicin reaches higher API concentrations in the alveolar macrophages (the place where mycobacterium tuberculosis reside and multiply) than 500 mg dose orally [1]. As consequence of the decreased dose the frequency of side effects can be reduced making the therapy more comfortable for patients. However the dose of 30 mg API is still very high for pulmonal delivery constituting a challenging task for formulation technology. Softpellets are micronised powder agglomerates held together by Lifshitz-van der Waals attraction, capillary forces, electrical forces and electrostatic forces [2] and they seem to be a very promising strategy to deliver high doses of API to the lungs. These pellets have a much higher API to excipient ratio than an interactive blend and can even be prepared from pure API. The biggest problem in softpellet production is the manufacturing method itself. In the method established in the industry the powder is pre agglomerated with a screw feeder. Thereafter a rounding procedure in a spheroniser creates round softpellets that can be classified with sieves. The disadvantage of this discontinuous method is the limitation caused by the capacity of the spheroniser and the high losses in the classification step. In this study a new continuous production method exploiting the auto adhesion mechanism of micronised powders based on the method described by Hartmann [3,4] utilising vibration was adopted to form soft agglomerates of lactose and rifampicin. Materials and Methods Materials: Inhalac 500 was a kind gift of Meggle (Molkerei MEGGLE GmbH & Co. KG, Wasserburg Germany). Rifampicin was purchased from Caesar & Loretz (Caeser & Loretz GmbH, Hilden, Germany). Particle size distribution / sphericity / convexity was measured by dynamic image analysis (QicPic, Sympatec GmbH, Clausthal Zellerfeld, Germany, lens M5). Moreover the Windox 5.8.0.0 software (Sympatec GmbH, Clausthal-Zellerfeld, Germany) calculated the sphericity and convexity of the sample. The softpellets were fed to the device with the GRADIS freefall shaft with deflector plate cascade to enable gentle dispersion. In every measurement a number of about 5000 particles were analysed.

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Drug Delivery to the Lungs, Volume 29, 2018 - Softpellets for high dose pulmonary delivery Softpellet production: At first powders were micronised with an air jet mill (Fluid Energy Aljet, Plumsteadville, USA). Hereafter the powder was pre agglomerated by forcing it through a sieve of defined mesh size. In this study the mesh sizes 180 µm, 355 µm and 500 µm where used resulting in the batches Lac-180, Lac-355, Lac-500 (consisting of Inhalac 500) and Rif-355 (consisting of the API rifampicin). The surfaces of these agglomerates were smoothened and the softpellets were rounded by sliding down a vibrating slide (Fig. 1) connected to a stereo system (RX V 365 5.1 AV-Receiver, Yamaha, Japan) controlled by a computer with the Test-Ton-Generator software (Test-Tone-Generator, Timo Essers Audio Software, Germany). Vibration was generated by an audio speaker (E-Series ML-1715E Power 8, Mac Audio, Pulheim, Germany) under the slide. The frequency of the vibration was set to 100 Hz and a sine wave function was chosen. The volume of the windows software was set to 100 % and the amplification of the stereo system was set to – 5 dB. This smoothening step was repeated six times with every batch. The whole construction was mounted on a plate with a slope of about 7°. After the rounding procedure the fines were separated with 90 µm, 180 µm and 250 µm sieves and the coarse fraction was separated with 300 µm, 500 µm and 1000 µm sieves respectively.

Figure 1: Device for softpellet preparation.

The bulk density was calculated from the volume of 1 g formulation as measured by weighing (Sartorius analytic balance A200S, Goettingen, Germany). The volume was measured with a 10 mL measuring cylinder. Particle morphology was assessed by scanning electron microscopy (SEM, Phenom XL, Phenom-World BV, Netherlands). Particles were fixed on carbon stickers and sputter-coated with a thin gold layer prior to SEM evaluation. The following magnifications were used: 500x, 750x, 1000x, 2500x and 4000x. All pictures were taken at 5 kV. Laser light diffraction: To evaluate the stability of the softpellets the method of Thorsten Hartmann was adopted [3]. In brief to determine the dispersion behaviour of the softpellets as an indirect measure for particle stability the particle fraction < 20 µm was measured by using a HELOS laser diffractometer (Sympatec GmbH, ClausthalZellerfeld, Germany) connected to the RODOS dispenser system with a VIBRI set to 50% vibration. Particles were dispersed at 1 bar and data evaluation was performed with the Windox 5.8.0.0 software (Sympatec GmbH, Clausthal-Zellerfeld, Germany) utilizing the Fraunhofer theory. The average of 3 measurements is given. Dose uniformity was investigated by the dose sampling apparatus (DUSA) utilising a reservoir inhaler (Turbohaler, Astra Zeneca, Wedel, Germany) and a flow rate corresponding to 4 kPa pressure drop over the device according to Ph. Eur. (60 l/min). The tube and the filter were washed thoroughly with an acetonitrile/buffer mixture. Rifampicin content was determined by RP-HPLC. Impaction analysis was performed with the NGI (apparatus E, European Pharmacopoeia 9.0) utilising the Turbohaler. To avoid re-entrainment of particles the cups were coated with a stage coating consisting of Brij 35, Ethanol and Glycerol. Flow rate was adjusted to 60 l/min. 5 doses per run were delivered. Rifampicin content was determined by RP-HPLC. Data were evaluated with the Colpley Inhaler Testing Data Analysis Software (Copley Scientific, Nottingham, United Kingdom). Fine particle fraction below 5 µm (of emitted) is calculated from the resulting aerodynamic particle size distribution. Reported data is one run and was measured at constant conditions (21 °C and 45 % relative humidity). Results and discussion The x50 values of the primary particles were 2.90 µm (Span: 2.45) for InhaLac 500 and 2.09 µm (Span: 2.11) for rifampicin respectively. Three different batches of placebo softpellets were produced to establish the production method. For the batch Lac-180 starter pellets were produced with the 180 µm sieve, for Lac-355 µm with the 355 µm sieve and for Lac-500 with the 500 µm sieve respectively. Furthermore one batch of API softpellets was manufactured. The batch Rif-355 was pre agglomerated with the 355 µm sieve. After the production the fines and coarse particles were separated in a classifying step. The x50 values of the three batches is shown in Table 1. This data demonstrates that the particle size of the product can be controlled by the selection of the sieve for starter pellet production. The span values below 1 indicate a narrow particle size distribution. A reduction in sphericity can be observed for larger particles. This is also observed in the SEM images (Fig. 2). The softpellets of the Lac-180 batch form round spheres whereas Lac-500 looks more like irregularly formed stones. In the SEM batch Lac-355 had the most homogenous appearance whereas in batch Lac-180 and Lac-500 small fragments of particles could be observed. Therefore the API batch Rif-355 was also produced in this size.

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Drug Delivery to the Lungs, Volume 29, 2018 – Christian Etschmann Table 1: X50-values of three different placebo batches and the API softpellets, sphericity and span (span = (x90-x10)/x50), ± standard deviation, Lac-180, Lac-355, Lac-500: n = 6; Rif-355: n=1.

Batch

Lac-180

Lac-355

Lac-500

Rif-355

X50 [µm]

240.23 ± 4.54

324.64 ± 1.88

382.78 ± 3.06

290.15

Span

0.48 ± 0.029

0.52 ± 0.014

0.73 ± 0.082

0.72

Sphericity

0.880 ± 0.000

0.878 ± 0.002

0.869 ± 0.001

0.877

Figure 2: SEM images of placebo softpellets Lac-180 (A,B), Lac-355 (C,D), Lac-500 (E,F) (A: 1000x magnification, B: 4000x, C: 750x, D: 2500x, E: 500x, F: 2500x).

140

99.50

120

99.00

API Content [µg]

Particle fraction <20 µm [%]

The particle fraction < 20 µm was determined utilising the HELOS at a dispersion pressure of 1 bar. It was observed that particle fraction < 20 µm increases for larger particles (Fig. 3). This observation was seen as indirect measure for stability and therefore larger particles are assumed to be less stable than smaller ones. Dose uniformity of batch Rif-355 is shown in figure 4. The mean dose had a content of only 91.07 µg due to the small size of the Turbohaler dosing cavity only dispersing few softpellets per shot. There was no dose outside the allowed thresholds. The recovery in aerodynamic characterisation was 91.61 %. Fine particle dose was 52.90 % and mean aerodynamic diameter 2.41 µm. Drug deposition profile is illustrated in figure 5.

98.50 98.00 97.50

100 80 60 40 20

Lac-180

Lac-355

Lac-500

Figure 3: Particle fraction <20 µm [%] of placebo softpellets, n=3

Shot 1 Shot 2 Shot 3 Shot 4 Shot 5

Figure 4: Dose uniformity of batch Rif-355.

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Concentration API [µg] 65 % of average 75 % of average 125 % of average 135 % of average

Drug deposition, µg

Drug Delivery to the Lungs, Volume 29, 2018 - Softpellets for high dose pulmonary delivery

160 140 120 100 80 60 40 20 0

Figure 5: Deposition profile of batch Rif-355, n=1.

Conclusion Formation of softpellets is a versatile alternative to a classical binary mixture in the area of dry powder inhalation especially if high dose administration is desired. In this study it has been demonstrated that a new manufacturing method based on vibration provides a product with narrow particle size distribution. Initial characterisation of aerodynamic properties looks promising and the concept of a carrier free formulation seems to work with rifampicin. The Turbohaler used in this study is the only device on the market containing softpellets and was therefore used for initial experiments with dispersing an agglomerated rifampicin formulation. Unfortunately, the Turbohaler is designed to deliver doses up to 1 mg and the target dose of this project is 20 mg. Thus, another device has to be utilized to increase the delivered dose. On the one hand there are already capsule based devices on the market able for high dose delivery, for instance the Tobi Podhaler (Novartis) and the Turbospin for use with Colobreathe (PH&T S.p.A.). On the other hand there are also some promising new developments, for example the Orbital with a dose range between 50 mg to 400 mg or the Cyclops being able to disperse powder formulations up to 50 mg pure API (tobramycin)5. Further experiments have to find out which device is the most suitable for this project. Moreover it has to be investigated if excipients could improve powder deagglomeration and with this increase the fine particle fraction. Possible excipient candidates are magnesium stearate and lactose with very high surface area. References 1 2 3 4 5

Santoshi JA, Pallepati SCR, Thomas BP. Low-dose inhaled versus standard dose oral form of anti-tubercular drugs: Concentrations in bronchial epithelial lining fluid, alveolar macrophage and serum. J Postgrad Med. 2008; 54: pp245–246. Podczeck F, Newton JM, James MB. Influence of Relative Humidity of Storage Air on the Adhesion and Autoadhesion of Micronized Particles to Particulate and Compacted Powder Surfaces. J Colloid Interface Sci. 1997; 187: pp484–491. Hartmann T. Agglomeration feiner Pulver: Ein neues Verfahren zur Softpellet-Produktion. Dissertation, Kiel University 2008. Hartmann T, Müller, Bernd,W., Steckel H. Europäisches Patent. Apparatus and process for continously producing spherical powder agglomerates. Harro Höfliger European Patent Office - EP 2217360 B1. 2008. Scherließ R, Etschmann C. DPI formulations for high dose applications - Challenges and opportunities. International Journal of Pharmaceutics. 2018; 548: pp49–53.

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Drug Delivery to the Lungs, Volume 29, 2018 - Neha Patel, et al. The Technical Challenges of the Developing Existing Inhalation Drug Products into New Metered Dose Inhaler Designs Neha Patel,1 Alex Slowey1 & Lester Harrison2 1

3M Drug Delivery Systems Division (DDSD), Charnwood Campus, 10 Bakewell Road, Loughborough, Leicestershire, LE11 5RB, UK. 2 3M Drug Delivery Systems Division (DDSD), 3M Center, St. Paul, MN 55144-1000, USA.

Summary Many of the best-selling inhaler products currently on the market deliver drugs that have been around for decades, yet the complex regulatory pathway required to develop generic inhalers has resulted in few generic inhalers being available on the market. In general, companies have chosen to develop ‘branded generic’ products using the same drug substances and inhaler design as the reference product on the market using the 505(b)(2) regulatory pathway in the US or Article 10.3 submission in the EU. An alternative approach is to develop existing drug substances into new inhaler designs that contain the same drug substances currently in the reference inhaler product, but within an entirely new delivery system (e.g. developing a new metered dose inhaler (MDI) that delivers drugs which are currently delivered via a dry powder or soft mist inhaler (SMI)). This approach may be chosen due to intellectual property considerations, as a product lifecycle management strategy or to provide market differentiation (e.g. using improved inhaler technologies). This abstract will illustrate some of the technical challenges associated with developing existing drug substances into new inhaler designs, with the consideration of more complex in-vitro and in-vivo relationships. An approach for establishing in-vitro-in-vivo relationships for different delivery formats, through the careful design of an early proof of concept (POC) pharmacokinetic (PK) study, is demonstrated in the form of a case study. POC PK data are presented alongside in-vitro data sets, thus emphasizing the importance of clinically relevant in-vitro testing and the role that this has in determining the viability of developing existing drug substances in the MDI format.

Key Message Careful design of an early proof of concept PK study, with the consideration of clinically relevant in-vitro testing to enhance the prediction of a successful clinical outcome, can be utilised to provide confidence in the ability to define a target dose for new MDI test products developed with existing drug substances which are not currently available in the MDI format.

Introduction Many of the best-selling inhaler products currently on the market deliver drugs that have been around for decades, yet the complex regulatory pathway required to develop generic inhalers has resulted in few generic inhalers being available on the market. In general, companies have chosen to develop ‘branded generic’ products using the same drug substances and inhaler design as the reference product on the market using the 505(b)(2) regulatory pathway in the US or Article 10.3 submission in the EU. An alternative approach is to develop existing drug substances into new inhaler designs that contain the same drug substances currently in the reference inhaler product, but within an entirely new delivery system (e.g. developing a new metered dose inhaler (MDI) that delivers drugs which are currently delivered via a dry powder or soft mist inhaler (SMI)). This approach may be chosen due to intellectual property considerations, as a product lifecycle management strategy or to provide market differentiation (e.g. using improved inhaler technologies). The MDI has a prominent place in the market, however there are many inhaled drugs which are not available in this format. There are several benefits from patient use of an MDI including MDIs are essentially functionally uniform, independent of the manufacturer, they are familiar, and the majority of patients will have experience of using an MDI as a rescue inhaler. In addition to this, MDIs provide multiple doses in each device and have a low resistance, which is particularly beneficial for those patients with limited airway capacity. Ultimately the availability of an MDI provides patients and providers with a choice in the case where this may be their preferred format. This abstract will illustrate some of the technical challenges associated with developing existing drug substances into new inhaler designs, with the consideration of more complex in-vitro and in-vivo relationships, in the form of a case study: Can an MDI be developed to be comparable to Combivent® Respimat®?

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Drug Delivery to the Lungs, Volume 29, 2018 - The Technical Challenges of the Developing Existing Inhalation Drug Products into New Metered Dose Inhaler Designs The need to employ clinically relevant testing – for example, anatomically relevant throat models (ATM), has previously been reported as being critical when in-vitro data are being used to predict in-vivo behaviour of an MDI product, particularly when comparing different inhalation dosage forms1. The Combivent® Respimat® product is an aqueous SMI, which delivers 20mcg of ipratropium bromide and 100 mcg of salbutamol per dose. It is significantly different compared to an MDI in terms of both the delivery system and the formulation. Historically Combivent was available on the market in a CFC-containing MDI format. The CFC MDI required essentially double the dose compared to the Respimat, yet these products were shown to be clinically comparable. In assessing the viability of an HFA MDI version of this product, the strategy was to target a product that would be comparable to the CFC MDI in-vitro, specifically focused on FPM. Additionally, clinically relevant testing with an ATM was included to aid understanding of the in-vitro-in-vivo relationship. Due to the significant differences between these dosage forms and the expected lack of in-vitro-in-vivo correlation, a proof of concept (POC) PK study was carefully designed to establish an in-vitro-in-vivo relationship and to allow interpolation of the data for future product optimisation if required.

In-Vitro Investigations Experimental Methods 3M HFA MDI product variants were formulated to be comparable to Combivent® CFC MDI in terms of aerodynamic particle size distribution (APSD). The inhalers were tested using a Next Generation Impactor (NGI) (Copley, UK) with both a standard induction port (SIP) and an anatomical throat model (ATM, Emmace Consulting AB) at 3M Drug Delivery Systems Division (DDSD, Loughborough, UK). The NGI test methodology was adapted to ensure its suitability for testing the aqueous SMI, and all testing was performed at a constant flow rate of 30Lmin -1.

Results and Discussion During initial feasibility investigations, in-vitro investigations compared the aerodynamic particle size distribution (APSD) using a Standard Induction Port (SIP) and using an anatomical throat model (ATM) for both the 3M MDI and the existing Respimat product. These data are presented in Figure 1. Throat and FPM data are presented for the HFA MDI and the SMI, for both drug substances. Albuterol

Ipratropium Bromide FPM

SIP ATM

100 80 60 40 20 0

HFA-MDI

SMI

HFA-MDI

Throat

Drug Deposition (mcg/dose)

Throat

SIP ATM

20 15 10 5 0

SMI

FPM

HFA-MDI

SMI

HFA-MDI

SMI

Figure 1 - Aerodynamic Particle Size Distribution (APSD)

The data presented in Figure 1 clearly demonstrate that clinically relevant in-vitro testing is key if these data are to be helpful in predicting expected PK or clinical outcomes. Throat deposition is higher with the anatomical throat compared to the standard induction port for the MDI with a corresponding decrease in the FPM. Use of an ATM for APSD testing has a major impact in terms of clinical relevance for the MDI in particular. It was also demonstrated that the FPM of the MDI and the SMI are comparable but not the same when tested with the ATM. The FPM of the Respimat SMI is slightly higher than the MDI, which agrees with data recently presented by Wei et al.2, where it was demonstrated that this ATM provides a reasonable prediction of lung dose for MDIs, but that it overestimates the lung dose for the Respimat® SMI.

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Drug Delivery to the Lungs, Volume 29, 2018 - Neha Patel, et al. These data were considered to provide an acceptable in-vitro baseline to progress to a POC PK study to determine the potential for PK comparability between the HFA MDI and the SMI products.

In-Vivo Investigations: Proof of Concept (POC) Pharmacokinetic (PK) Study Strategy Experimental Methods The POC PK study was designed to compare the HFA MDI to the Reference (Combivent® Respimat®) to provide confidence in the ability to define the target dose for HFA MDI. The study was conducted open label in healthy adult male and female subjects (Novum PRS, Houston TX, USA). All subjects received Test Treatments A, B and C and one reference product (Reference Treatment), according to a balanced four-period randomized cross-over design. Subjects were fasted overnight (no food or beverages except water) for at least 10 hours before and until 3 hours following each dose. Subjects were required to demonstrate proper inhalation technique with a placebo pMDI inhaler. For each study treatment and at each specified time, two blood samples were collected for the pharmacokinetic analyses of ipratropium bromide and albuterol in the plasma. Pharmacokinetic parameters were calculated for each drug and compared. Subsequent doses were given following 7-14 days wash out. An FPM range of approx. 80% – 160% compared to CFC MDI target were selected in order to establish the in-vitroin-vivo relationship. In summary, the POC PK study was designed as follows: •

4-arm, 4-period crossover with 47 subjects

•

Reference: Combivent® Respimat®

•

Test: 3 x test products HFA MDIs (A, B & C) were included to allow evaluation of 3 different doses

•

Test A FPM below-target (approx. 80% compared to the CFC MDI)

•

Test B: FPM on-target compared to the CFC MDI and

•

Test C FPM above-target (approx. 160% compared to the CFC MDI)

•

Acceptance Criteria: The mean Cmax and AUC0-t for a test product was considered bioequivalent to the respective mean values for the reference product if the 90% confidence interval for the ratio of geometric means is completely contained within the interval 80% to 125%

Results and Discussion A POC PK study was performed as detailed above. The data are presented in Figure 2

Figure 2 – PK Study data

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Drug Delivery to the Lungs, Volume 29, 2018 - The Technical Challenges of the Developing Existing Inhalation Drug Products into New Metered Dose Inhaler Designs The data presented in Figure 2 demonstrate that for albuterol, Test product B, with FPM comparable to previous CFC MDI product, demonstrated bioequivalence for both Cmax and for AUC0-t. The corresponding data for ipratropium bromide, demonstrated that Test Product B was slightly lower than the reference product for both Cmax and AUC0-t.

Conclusions In-vitro testing of the test products using an ATM in combination with an NGI allowed the FPM and throat deposition to be assessed alongside the marketed reference product. This in turn allowed a meaningful prediction to made of the potential outcome of a POC PK study. The subsequent POC PK study demonstrated that bioequivalence was achieved for both Cmax and for AUC0-t for the albuterol component of the 3M MDI. Whilst the ipratropium bromide component was determined to be slightly lower than the reference product in this instance, the study design allows interpolation of data, based on the established in-vitro-in-vivo relationship, to closely predict a product configuration that can achieve bioequivalence for both drug substances if required.

Acknowledgements The authors would like to thank Principle Investigator, Robert A. Weaver (Novum Pharmaceutical Research Services, 11300 Richmond Ave, Houston TX, 77082, USA).

References 1

Holmes S, Slowey A: A Comparison of Different Anatomical Throats vs The USP Throat. Drug Delivery to the Lungs (DDL2017), 2017 Wei X, Hindle M, Kaviratna A, Huynh B, Delvadia R, Sandell D, Byron P: In Vitro Tests for Aerosol Deposition. VI: Realistic Testing with Different Mouth-Throat Models and In Vitro-In Vivo Correlations for a Dry Powder Inhaler, Metered Dose Inhaler, and Soft Mist Inhaler. J Aerosol Med Pulm Drug Deliv. 2018; 31: pp1-14 2

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Drug Delivery to the Lungs, Volume 29, 2018 –Louise Sweeney et al. Assessment of the effect of cannula choice and gas flow rate on aerosol delivery during high flow nasal therapy Louise Sweeney1, Mary Joyce1, Gavin Bennett1 & Ronan MacLoughlin1 1

Aerogen, IDA Business Park, Dangan, Galway, Ireland

Summary Non-invasive Nasal High Flow therapy is increasingly used across a variety of patient populations. This study evaluated the effects of gas flow rates across a range of nasal cannulas (adult and junior) and head models using the Airvo™ 2 high flow system in conjunction with the Aerogen solo ® vibrating mesh nebuliser. A 2 mL dose of albuterol sulphate (1 mg/mL) was nebulised to determine % dose distal to the trachea for both an adult and paediatric (5 year old) head model (available online: www.rddonline.com). A total of three adult cannulas and two paediatric cannulas were used in this study.The Aerogen Solo nebuliser was positioned in the Airvo™ 2 nebuliser adapter at the humidification chamber. Tracheal dose (dose delivered to a filter beyond the trachea) was characterised by quantifying the mass of drug captured on a filter placed distal to the head models during simulated High Flow Nasal Therapy. Results for tracheal dose were expressed as the percentage of the nominal dose placed in the nebuliser’s medication cup. Statistically significant differences in tracheal dose were observed across the range of gas flows under test for each individual cannulas. There was also a significant difference between all three adult cannulas for each individual gas flow rate, with the exception of 30 & 60LPM. There was no significant difference between the junior cannulas for the individual gas flow rates. In addition, increasing gas flow rates were associated with a reduced tracheal dose. Key Message This study demonstrates that the choice of cannulas size and prescribed gas flow rate has a significant effect on the amount of aerosol delivered to the patient. The dose delivered distal to the trachea decreases as gas flow increases and this observation holds true regardless of cannula size. These findings provide insight to both the potential of, but also the possible limitations of concurrent aerosol therapy during high flow therapy. Introduction In most circumstances, a nasal route for the delivery of pulmonary aerosol medications is rarely considered; however, in specific instances, this route may be quite useful [1]. HFNT is easy to apply and easy to use (with Airvo™ 2 not requiring an additional oxygen or gas supply). It is generally comfortable for patients and it is not usually interrupted or discontinued because of intolerance [2]. Several factors influence the amount of aerosolised medication a patient receives during HFNT. The objective of this study was to determine the effects of cannulas size and gas flow rates for both adult and paediatric patient types. Materials and Methods The Aerogen Solo was connected to Airvo™ 2 humidification chamber (see Figure 1). Appropriate model and nasal cannulas under investigation were attached to Airvo™ 2 (Figure 2 & 3). Airvo™ 2 was powered on and appropriate mode was selected (Adult or Junior). Gas flow rate was selected; Adult 10-60L/Min, Junior 2-25L/Min. The ASL 5000 breathing simulator was used to generate the infant (BPM 25, Vt 155 mL, I:E 1:2) and Adult (BPM 15, Vt 500 mL, I:E 1:1) breath. A 2.0 mL dose of albuterol sulphate (1 mg/mL) was nebulised (n=3 for each gas flow rate). At the end of each dose the drug was extracted from the filter and quantified using UV spectrophotometry (at 276 nm). The mass of drug eluted from the filters was determined using spectrophotometry and interpolation on a standard curve of albuterol sulphate concentrations (100 µg/mL to 3.125 µg/mL). Results were expressed as the percentage of the nominal dose placed in the nebuliser’s medication cup that was delivered beyond the trachea. Paired t-test and one-way anova was carried out to determine significance. P-values <0.05 were considered significant.

Testing was carried out for the adult and paediatric patient types, using the head models and patient breathing parameters provided in Table 1. NOTE: these breathing patterns are defined in the US pharmacopeia <1601>. Patient type Adult Paediatric

Head Model LUCY 5 year old

Tidal Volume (mL) 500 155

Table 1: Head models and associated patient breathing parameters.

193

Breaths per minute 15 25

I:E ratio 1:1 1:2

Drug Delivery to the Lungs, Volume 29, 2018 – Assessment of the effect of cannula choice and gas flow rate on aerosol delivery during high flow nasal therapy

Figure 1: Assembly of Aerogen Solo nebuliser into Airvo nebuliser adapter. Note: This Airvo adapter is designed

specifically for inclusion of vibrating mesh nebulisers.

Figure 2: Tracheal dose setup. NOTE: Adult head ‘LUCY’ model shown here. Nasal cannula placed onto face and

model attached to a breathing simulator. The capture filter is placed at the distal end of the models’ airway.

Figure 3: Tracheal Dose set-up; Airvo™ 2 with 5 Year old head model) and Optiflow™ Junior cannula.

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Drug Delivery to the Lungs, Volume 29, 2018 –Louise Sweeney et al. Results The results of testing for ‘’LUCY’’ Adult head model are presented in Table 2.

Gas Flow

%Tracheal Dose

OPT 942

OPT 944

OPT 946

(Small adult cannula)

(Medium adult cannula)

(Large adult cannula)

18.26 ± 1.61

19.81 ± 0.93

21.67 ± 1.03

<0.05

11.14 ± 0.50

11.29 ± 1.22

15.71 ± 2.30

<0.05

12.35 ± 2.51

14.61 ± 0.91

14.05 ± 1.36

0.001

5.07 ± 0.25

3.67 ± 0.44

4.14 ± 0.14

<0.05

5.12 ± 0.33

N/A

1.86 ± 0.29

1.86 ± 0.38

0.001

P-value

<0.05

N/A

LPM

P-value

Table 2: Average ± Standard Deviation values of tracheal dose (%) for the Aerogen Solo nebuliser across a range

of gas flow rates for ‘’LUCY’’ Adult head model.

Statistically significant differences in tracheal dose were observed between all three adult cannulas for each individual gas flow rate, with the exception of 30 & 60LPM where there is no statistical difference (p= 0.32 and p=0.74, respectively). Increasing gas flow rates were associated with a reduced tracheal dose ranging from 18.26 ± 1.61 at 10LPM to 5.12 ± 0.33 at 50LPM for OPT 942, 19.81 ± 0.93 at 10 LPM to 1.86 ± 0.29 at 60LPM for OPT 944 and 21.67 ± 1.03 at 10 LPM to 1.86 ± 0.38 at 60 LPM for OPT 946. There is also a statistical difference between the range of gas flows under test for each individual cannula (p= <0.05).

The results of testing for 5 year old nose/throat head model are presented in Table 3. Gas flow (LPM)

% Tracheal Dose

P-value

OPT 316

OPT 318

(Large Junior cannula)

(X-Large Junior cannula)

7.97 ± 1.25

9.68 ± 0.42

0.001

5.26 ± 0.56

6.21 ± 0.98

0.001

4.20 ± 0.58

N/A

3.97 ± 0.58

N/A

P-value

<0.05

N/A

Table 3: Average ± Standard Deviation values of tracheal dose (%) for the Aerogen Solo nebuliser across a range of gas flow rates for 5 year old nose / throat head model.

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Drug Delivery to the Lungs, Volume 29, 2018 – Assessment of the effect of cannula choice and gas flow rate on aerosol delivery during high flow nasal therapy There was no significant difference between the OPT 316 and OPT 318 for the individual gas flow rate (p= 0.20, p=0.49). There is a statistical difference (p=<0.05) between the range of gas flows under test for each individual cannula, with efficiencies of 7.97 ± 1.25 at 5 LPM to 4.20 ± 0.58 at 20 LPM for the OPT 316 and 9.68 ± 0.42 at 5LPM to 3.97 ± 0.58 at 25 LPM for the OPT 318 cannula.

Discussion Our findings show that we have successfully demonstrated efficient aerosol drug delivery using the Aerogen Solo nebuliser in conjunction with the Airvo ™2 and Optiflow™ nasal cannulas across both infant and adult simulated spontaneously breathing patients. As expected higher gas flow rates are in general associated with reduced efficiencies, due to particle collisions in the circuit and also within the head model in the case of dose delivered distal to the trachea. MacLoughlin et. al (2013) previously reported that higher flow rates are associated with reduced efficiency of drug delivery through an adult NHF system [3]. Paired t-test and one-way anova results for the adult model indicate statistically significant differences in tracheal dose between all three adult cannulas for each individual gas flow rate, with the exception of 30 & 60LPM. There is also a statistical difference between the range of gas flows under test for each individual cannula (p= <0.05). For, the paediatric model, there was no significant difference between the OPT 316 and OPT 318 for the individual gas flow rate’s, and again there is a statistical difference (p=<0.05) between the range of gas flows under test for each individual cannula. In addition to this, increasing gas flow rate is associated with decreased efficiency. Although, the Airvo ™2 nebuliser adapter is bespoke for this system, the findings in this study are in line with a previous study on High-Flow Nasal Therapy Systems [4]. In conclusion, this study demonstrates that aerosol drug delivery during NHF is dependent on cannula size, with a larger cannula yielding a greater tracheal dose.

References Bhashyam AR, Wolf MT, Marcinkowski AL, Saville A, Thomas K, Carcillo JA, Corcoran TE. Aerosol delivery through nasal cannulas: an in vitro study. J Aerosol Med Pulm Drug Deliv - June 1, 2008; 21 (2); 181-8. 1

Sztrymf B, Messika J, Bertrand F, Hurel D, Leon R, Dreyfuss D, Ricard JD. Beneficial effects of humidified high flow nasal oxygen in critical care patients: a prospective pilot study. Intensive Care Med 2011; 37(11): 1780-1786. 2

MacLoughlin R, Power P, Wolny M, Duffy C. Evaluation of vibrating mesh nebulizer perfromance during Nasal High Flow therapy. International Society for Aerosols in Medicine International Congress 2013. :https://www.researchgate.net/publication/275967693_Evaluation_of_vibrating_mesh_nebulizer_perform ance_during_Nasal_High_Flow_therapy. 3

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Drug Delivery to the Lungs, Volume 29, 2018 - Mohammad Abdul Motalib Momin et al. High dose spray-dried powders for treating tuberculosis Mohammad Abdul Motalib Momin, Shubhra Sinha, Ian G Tucker & Shyamal C Das School of Pharmacy, University of Otago, 18 Frederick Street, P.O. Box 56, Dunedin 9054, New Zealand Summary High dose delivery of drugs to the lung using a dry powder inhaler (DPI) is an emerging approach to treat tuberculosis and other respiratory infections. To achieve a high dose in the lung, a highly aerosolizable powder is required which is often difficult for a hygroscopic drug. This study was designed to develop powders of a hygroscopic drug with surfaces enriched in the hydrophobic material by manipulating the spray-drying conditions and to investigate the effect of hydrophobic surface enrichment on aerosolization and stability of the hygroscopic drug. Using a 23 full factorial design, inhalable size (3.1 to 3.9 µm) composite powders of kanamycin (hygroscopic drug) and rifampicin (hydrophobic drug) were produced by varying three spray drying conditions: drug ratio, co-solvent composition and inlet temperature. The powders were wrinkled, flake-shaped and amorphous. Hydrophobic surface enrichment was significantly affected by co-solvent composition as confirmed by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToFSIMS). Increase in hydrophobic surface enrichment (from 80.8 to 100%) increased aerosolization (from 48.0 to 77.2% fine particle fraction- FPF) determined using a Next Generation Impactor (NGI) and decreased density The composite powders were stable at 15% and 43% RH and 25 ± 2°C during one-month storage in an open Petri dish, and non-toxic (up to 50 µg/mL) to human alveolar and bronchial cell-lines. This systematic study has reported the manipulation of spray-drying conditions for hydrophobic surface enrichment in composite powder particles. Surface enrichment of kanamycin by hydrophobic rifampicin improved aerosolization and stability. The surface modification approach by spray drying can be used for developing high dose powders for treating tuberculosis and other lung infections. Key Message By manipulating spray drying conditions, hydrophobic surface enrichment of a hygroscopic drug can be achieved. Hydrophobic surface enrichment can improve the aerosolizability and stability. This approach has potential to develop high dose DPI formulations for treating tuberculosis and other lung infections. Introduction Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a leading cause of death worldwide despite the availability of antibiotic treatment. Since 70-80% of Mtb is localized in the lung, pulmonary delivery of anti-TB drugs could offer advantages over oral and parenteral delivery via high drug concentrations in low doses reducing systemic toxicity, drug resistance and treatment duration while increasing efficacy. Despite these potential benefits, there is still no inhaled, dry powder anti-TB drug on the market. The hope for anti-TB DPIs has been rising since the introduction of Tobi Podhaler by Novartis and Colobreathe by Teva for treating lung infections. The doses of drugs (e.g., 4x28 mg, i.e. 112 mg tobramycin or 125 mg colistimethate sodium) in such DPIs are higher compared with the doses of drugs (e.g., 5-500 µg) used for the treatment of asthma and chronic obstructive pulmonary disease [1, 2]. The delivery of these high doses of drugs requires the development and innovation in both devices and formulations. The production of highly aerosolizable powders with high drug loading is required for high dose DPI. The development of highly aerosolizable powder with a hygroscopic drug is extremely difficult due to their cohesiveness and agglomeration tendency [3, 4]. There are several particle engineering techniques such as mechanical dry coating, co-milling, crystallization, spray freeze drying and spray drying are used to develop particles for high dose delivery [5]. Among all these techniques, spray-drying is most widely used since particle properties can be optimized through manipulation of spray drying conditions. However, spray-dried powder particles are often amorphous leading to physical instability [6]. There is also a concern whether delivery of such high doses will be non-toxic to the lung. It is hypothesized that coating of a hygroscopic drug with a hydrophobic material may improve aerosolization and stability of the spray-dried powders. Spray drying conditions such as the solubility and ratio of formulation components, solvent volatility and composition, initial droplet size, drying temperature and rate significantly influence the surface composition of the spray-dried composite particles. Although reports on surface composition and the effect of a hydrophobic surface on powder properties have been published, a systematic study on manipulation of spray-drying conditions to achieve hydrophobic surface enrichment in dry powder particles is lacking. The objectives of this study were, therefore, to engineer inhalable powders of a hygroscopic drug with surfaces enriched in the hydrophobic material by manipulating spray-drying conditions using a factorial design; and to investigate the effect of hydrophobic surface enrichment on aerosolization and stability of the hygroscopic drug. In this study, kanamycin sulfate and rifampicin were used to represent a hygroscopic drug and a hydrophobic compound. The influence of drug ratio, feed solvent composition (water and ethanol), and drying temperature on the hydrophobic surface enrichment of composite particles was investigated. Kanamycin is currently administered intravenously (1g once daily) for treating drug-resistant tuberculosis [7]. This high dose of kanamycin can cause serious side effects. The kanamycin-rifampicin combination is synergistic against Mycobacterium aviumintracellular complex [8]. Since respiratory delivery of kanamycin-rifampicin will require lower doses than their current recommended doses, this combination DPI could potentially reduce side effects and improve the therapeutic activity [9].

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Drug Delivery to the Lungs, Volume 29, 2018 – High dose spray-dried powders for treating tuberculosis

Experimental methods including materials Materials Kanamycin sulphate and rifampicin were purchased from Hangzhou Dayangchem Co., Ltd., Zhejiang, China. Acetonitrile, ethanol and methanol (High performance liquid chromatography, HPLC, grade) were purchased from Merck, Germany. Sodium dihydrogen phosphate, phenylisocyanate, triethylamine, orthophosphoric acid (analytical reagent grade) and silicone oil (viscosity 10 cSt) were purchased from Sigma–Aldrich, St. Louis, USA. Size 3 hard gelatin capsules were kindly donated by Capsugel Co., Ltd., Tokyo, Japan. Fresh Milli-Q water was collected and filtered through 0.45 μm membrane filter before use. Table 1 The 23 full factorial design for powder preparation Formulation Level of factor A B C KR1 -1 -1 -1 KR2 1 -1 -1 KR3 -1 1 -1 KR4 1 1 -1 KR5 -1 -1 1 KR6 1 -1 1 KR7 -1 1 1 KR8 1 1 1 Note: A: Rifampicin: kanamycin (mass ratio), -1 (40:60) and 1 (60:40) B: Ethanol: water (volume ratio), -1 (30:70) and 1 (70:30) C: Inlet temperature (°C ), -1 (70) and 1 (170)

Preparation of powders A 23 full factorial design (Table 1) was used to prepare the composite powders using a Buchi B-290 Mini Spray-Dryer (Buchi Labortechnik AG, Flawil, Switzerland) with a high performance cyclone in a closed-mode. Feed solutions of kanamycin sulphate and rifampicin combinations (at different ratios) were prepared in a co-solvent system of ethanol and water (at different ratios). The following factors were constant during spray drying: solid contents (0.67% w/v), feed rate (2 mL/min), aspiration (100%), spray-gas flow rate (670 L/h) and nozzle diameter (0.7 mm). Duplicate batches of the optimized powder formulations based on the surface composition studies were prepared to evaluate the inter-batch variability in aerosolization and related properties. The spray-dried powders were collected in a glass jar connected to a cyclone at the outlet and transferred into a screw-capped glass scintillation vials and stored in a desiccator over silica gel at room conditions until used.

Surface composition Surface compositions (to a depth of 5-10 nm) of the single-drug and combination powders were evaluated using XPS (AXIS Ultra DLD Spectrometer, Kratos Analytical Ltd., Manchester, UK). The elemental distribution on the surface (top 1-2 nm) was determined using ToFSIMS (PHI TRIFT V nanoTOF instrument, Physical Electronics Inc., Chanhassen, MN, USA). Since surface analysis techniques are expensive, a single batch (Batch 1) of each powder was prepared for surface composition analysis, and the main effects of the spray-drying and formulation factors on surface enrichment were analysed by balanced ANOVA using the statistical software mentioned in the statistical analysis section below. Physicochemical properties The powders were characterized for particle size, morphology, water content, and crystallinity by laser diffraction, scanning electron microscopy, Karl Fischer titration, and X-ray diffractometry, respectively. Both bulk and tapped density of the powders were also measured. In vitro aerosolization The in vitro aerosolization performance of the spray-dried powders was determined using an NGI (Copley Scientific Ltd., Nottingham, UK). The samples were collected from different stages (1 to 7 and micro-orifice collector, MOC) and analyzed using a validated HPLC method. The aerosolization efficiency was represented as FPF, defined as a quotient of drug deposited on stages 2 to MOC and drug emitted from the inhaler device. Storage stability The influence of humidity on the aerosolization of the spray-dried powders was investigated after storing the samples in an open Petri dish at 15%, 43% and 75% RH and 25±2 °C for one-month. After storage, the aerosolization, morphology, crystallinity and moisture content were also investigated. Cytotoxicity studies Powders were also evaluated for cytotoxicity on human respiratory cell-lines (Calu-3 and A549 cells) by MTT assay. Statistical analysis Statistical analyses were conducted using Minitab (Minitab Inc., Version 16, State College, PA, USA) and Instat GraphPad Prism (GraphPad Software, Version 4.00, San Diego CA) softwares at a significance level of P < 0.05. Data were presented as mean ± standard deviations of triplicate measurements. Results and Discussion All co-spray-dried powder particles were in the inhalable size range (3.1 to 3.9 µm), wrinkled and flake shaped and X-ray amorphous. Hydrophobic surface enrichment was achieved in kanamycin-rifampicin composite (KR) powders as revealed by XPS (Table 2). Complete hydrophobic surface enrichment was achieved in one formulation (KR7) (Table 2 and Fig 1). Hydrophobic surface enrichment was significantly affected by co-solvent composition used during spray-drying. Complete hydrophobic surface enrichment was achieved at the high level of co-solvent composition when high inlet temperature was used.

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Table 2 Molar fractions of kanamycin (k) and rifampicin (R) on the surface of cospray dried particles (KR1 to KR8) calculated based on the presence of distinguishing element S determined by XPS. Powder Molar fraction (%) K

100.0 − 15.4

− 100.0 84.6

15.4

84.6

7.7

92.3

3.8

96.2

KR5

19.2

80.8

KR6 KR7 KR8

7.7

92.3

− 7.7

100.0 92.3

SD-K SD-R KR1 KR2 KR3 KR4

Fig. 1. Elemental distribution of kanamycin (red) and rifampicin (green) on the surface of composite particles (KR7) determined by ToFSIMS (arrows indicate red dots)

Three composite powders based on low (KR5), medium (KR3) and high (KR7) levels of hydrophobic surface enrichment were selected for aerosolization and stability studies. Aerosolization efficiency of kanamycin from freshly prepared kanamycin-rifampicin composite powder (FPF: ~48.0%) was higher than that of kanamycin-only powder (FPF: ~27.0%) (Table 3). No difference was observed between the aerosolization of rifampicin-only and completely rifampicin-enriched (KR7) powder (P > 0.05). Increase in hydrophobic surface enrichment (from 80.8 to 100%) increased FPF (from 48.0 to 77.2%) and decreased bulk density (Fig. 2).

Fig. 2. Effects of surface enrichment on bulk density of powders (error bars represent intra-batch standard deviations, n = 3) During storage at different RH (15, 43 and 75%) for one month, the FPF of hydrophobic rifampicin-only powder was unaffected but the FPF of hygroscopic kanamycin-only powder significantly decreased even at 43% RH. The kanamycin-only particles fused together, crystallized and formed hard cakes at 75% RH. The aerosolization of kanamycin and rifampicin in the composite powders remained unchanged at 15% and 43% RH, but it significantly decreased at 75% RH. Hydrophobic surface enrichment of the particles prevented particle agglomeration up to 43% RH. At 75% RH, the moisture uptake led agglomeration of the particles resulting in an increase in aerodynamic diameter. The formulations were non-toxic to both of the cell-lines (calu-3 and A549) up to 50 µg/mL.

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Drug Delivery to the Lungs, Volume 29, 2018 – High dose spray-dried powders for treating tuberculosis Table 3. Fine particle fraction (FPF) of spray-dried powders kanamycin-only, rifampicin-only and kanamycinrifampicin composite powders, both fresh and after one-month storage at 15%, 43% and 75% relative humidity (RH) and 25°C (data are means ± intra-batch standard deviations, n = 3). Powder Fine particle fraction (%) Batch 1 Batch 2 SD-K

Fresh 27.6 ± 2.8

15% RH 27.0 ± 2.1

43% RH 22.2 ± 0.5

75% RH −

Fresh 28.0 ± 2.6

15% RH 27.0 ± 1.9

43% RH 21.9 ± 0.4

75% RH −

SD-R K-KR7

82.1 ± 1.2 77.4 ± 1.9

82.3 ± 1.4 76.9 ± 1.7

81.2 ± 4.9 75.3 ± 1.0

81.1 ± 1.0 56.7 ± 1.8

81.5 ± 0.8 77.2 ± 1.8

82.6 ± 0.8 77.0 ± 2.6

82.4 ± 2.8 75.4 ± 1.4

80.9 ± 0.8 56.4 ± 1.8

R-KR7 84.9 ± 5.5 81.9 ± 0.4 81.4 ± 1.0 67.4 ± 1.6 84.8 ± 5.6 81.6 ± 0.7 81.2 ± 1.0 66.8 ± 1.5 K-KR3 60.6 ± 2.3 61.7 ± 1.6 61.0 ± 0.7 52.1 ± 1.3 59.2 ± 2.2 61.2 ± 1.7 60.9 ± 0.2 51.0 ± 1.3 R-KR3 60.1 ± 1.1 59.9 ± 4.8 58.9 ± 0.6 50.8 ± 0.9 59.4 ± 0.7 60.7 ± 5.7 59.0 ± 0.4 50.3 ± 0.9 K-KR5 51.3 ± 1.7 52.0 ± 1. 50.6 ± 1.8 43.7 ± 0.7 48.0 ± 1.8 51.0 ± 1.2 51.1 ± 1.9 43.6 ± 0.3 R-KR5 50.4 ± 0.7 51.9 ± 0.8 52.7 ± 2.5 44.0 ± 0.3 49.8 ± 0.3 51.8 ± 1.1 52.3 ± 3.1 42.1 ± 0.4 Note: SD means spray-dried; K and R mean kanamycin and rifampicin, respectively; KR5, KR3 and KR7 represent kanamycin-rifampicin composite powders; K-KR5, K-KR3 and K-KR7 represent kanamycin from KR5, KR3 and KR7 powders; R-KR5, R-KR3 and R-KR7 represent rifampicin from KR5, KR3 and KR7 powders. Conclusion Hydrophobic surface enrichment in composite dry powder particles containing kanamycin and rifampicin was successfully achieved by manipulation of formulation and spray-drying conditions (drug ratio, co-solvent composition, inlet air temperature). The hydrophobic surface enrichment was significantly affected by co-solvent composition. Complete hydrophobic surface enrichment (100% rifampicin on the surface) was achieved in formulation KR7. The hydrophobic surface enrichment improved aerosolization (FPF) of the hygroscopic drug, kanamycin by decreasing density. Hydrophobic surface enrichment could protect aerosolization and agglomeration at RH up to 43% for composite powders, but it significantly decreased at high RH (75%). The formulation was non-toxic to pulmonary cell lines up to 50 µg/mL. This is the first systematic study that focused on the manipulation of spray-drying conditions to achieve hydrophobic surface enrichment in composite dry powder particles and the effect of hydrophobic enrichment on aerosolization. Improved aerosolization may help to deliver high doses of these drugs to the deep lung to treat tuberculosis. Further studies are required to explore particle interactions of the composite powders to understand the mechanisms for improved aerosolization.

References Claus S, Weiler C, Schiewe J, Friess W: How can we bring high drug doses to the lung? Eur J Pharm Biopharm 2014; 86: pp16. 1

Yeung S, Traini D, Lewis D, Young P M: Dosing challenges in respiratory therapies. Int J Pharm 2018; 548: pp659-671.

Aquino R P, Prota L, Auriemma G, Santoro A, Mencherini T, Colombo G, Russo P: Dry powder inhalers of gentamicin and leucine: formulation parameters, aerosol performance and in vitro toxicity on CuFi1 cells. Int J Pharm 2012; 426: pp100-107. 3

Hoppentocht M, Akkerman O W, Hagedoorn P, Frijlink H W, de Boer A H: The Cyclops for pulmonary delivery of aminoglycosides; a new member of the Twincer family. Eur J Pharm Biopharm 2015; 90: pp8-15. 4

Brunaugh A D, Smyth H D: Formulation techniques for high dose dry powders. Int J Pharm 2018; 547: pp489-498.

Vehring R: Pharmaceutical particle engineering via spray drying. Pharm Res 2008; 25: pp999-1022.

WHO: Treatment of Tuberculosis: Guidelines. World Health Organization 2010.

Zimmer B L, DeYoung D R, Roberts G D: In vitro synergistic activity of ethambutol, isoniazid, kanamycin, rifampin, and streptomycin against Mycobacterium avium-intracellulare complex. Antimicrob Agents Chemother 1982; 22: pp148-150. 8

Pham D D, Fattal E, Tsapis N: Pulmonary drug delivery systems for tuberculosis treatment. Int J Pharm 2015; 478: pp517–529.

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Drug Delivery to the Lungs, Volume 29, 2018 - Bernhard Müllinger et al. Impact of real time feedback from inhalation devices on patient satisfaction and adherence Bernhard Müllinger1, Tobias Kolb1 & Tobias Gessler2 Vectura GmbH, Robert-Koch-Allee 29, Gauting, 82131, Germany University of Giessen and Marburg Lung Center, Klinikstraße 33, Gießen, 35392, Germany 1

Summary Inhaler use errors and poor compliance are the most common reasons for failures in inhalation treatments. Correct inhaler technique is key for effective treatment with inhalation devices, but many patients fail to use inhalation devices correctly even after training and education. Inhalation devices, which guide patients on correct inhalation technique by design may reduce inhalation errors resulting in a higher patient satisfaction, better adherence and improved treatment outcomes. Smart nebulisers such as Breelib™ and AKITA® JET improve inhalation technique by providing real-time feedback to patients on correct inhalation steps. Both inhalers guide patients to an optimum inhalation inspiratory flow rate and volume and help them to avoid errors to achieve an effective delivery of aerosols to periphery of the lungs. This analysis assesses patient satisfaction and treatment compliance from two different studies. Patient satisfaction and ease of use of the device was assessed in a study in PAH patients inhaling Iloprost via Breelib™, a novel smart nebuliser. The study showed that patients were highly satisfied with the use of Breelib™. Patients found that the device was easy to use and the treatment time per treatment session was reduced by 8.3 min when using Breelib compared to a standard device. In a second study treatment compliance was assessed in patients with severe asthma being treated with budesonide inhalation suspension via AKITA JET. Total dose compliance over all patients was 82%. Together, these findings suggest that smart nebulisers that guide patients on correct inhalation technique and monitor treatment adherence, can improve patient satisfaction, which may result in better adherence and improved clinical outcomes in patients. Key Message Inhalation devices such as AKITA® JET and Breelib™ guide patients on correct inhalation technique, improve drug delivery to peripheral airways [1-6], and monitor treatment compliance. Observed ratings in relation to the inhalation feedback from the device indicate that patients positively perceived guidance on flow rate and inhalation volume. Particularly in indications such as severe asthma and PAH, the smart nebulisers studied herein provide meaningful advantages for patients in their daily therapy, which might result in higher patient satisfaction, better adherence, and more favourable treatment outcomes. Introduction Incorrect inhaler use and poor compliance are the most common reasons for failure to achieve good asthma control.[7] Recent studies show that critical use errors are very common with inhalation devices and have negative effects on outcomes and economic burden. Achieving correct inhalation technique is key for effective asthma therapy.[8] Inhalation devices which guide the patients on inhalation technique during the dosing, may play a significant role in improving inhalation technique and patient satisfaction. Such new sensing inhalation devices can also help patients and healthcare professionals monitor compliance and manage the disease.[9] This analysis assesses patient satisfaction and treatment compliance collected in two studies. Patient satisfaction including ease of use was assessed in a study[6] with PAH patients inhaling Iloprost with the Breelib® device and compliance data was assessed in a study that evaluated the sparing of oral steroids by inhalation of budesonide suspension administered using the AKITA® JET system in adult patients with severe asthma.[10] Experimental Methods Patient satisfaction was analysed through patient questionnaires from a crossover design study including 27 patients with PAH receiving 2 weeks of iloprost 5g via Breelib™(Vectura plc, Chippenham, UK), (Figure 1), and INeb® (Philips-Respironics Ltd.), two breath-actuated, vibrating mesh smart nebulisers.[6]. BreelibTM is breathactuated and guides the patient’s inhalation flow rate and volume using coloured lights to illuminate the mouthpiece of the device to provide direct feedback to the patient on their inhalation. The device incorporates a air control valves to further guide patient inhalation flow rate and volume. This allows the patient to take a slow deep inhalation manoeuvre during which the aerosol is delivered. After assessing patient satisfaction questionnaires after initial treatment patients were offered to enter into a 30 month long-term extension study, in which they assessed patients satisfaction after two weeks of treatment.

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Drug Delivery to the Lungs, Volume 29, 2018 -Impact of real time feedback from inhalation devices on patient satisfaction and adherence Compliance data was analysed from a randomised, double-blind, placebo-controlled, parallel-group study[10] conducted in 200 patients aged 18–65 years with severe asthma. The AKITA® JET (Vectura plc, Chippenham, UK), (Figure 1) guides the patient to inhale with a slow inspiratory flow rate and a deep inhalation. Aerosol delivery is controlled via a programmable Smart Card that allows the therapy to be tailored to specific drugs and indications, by managing drug dose, breathing pattern, and the point at which the aerosol bolus is delivered during inhalation. For targeting the aerosol to the small airways of the lungs, an air bolus is inhaled after the aerosol. The AKITA ® JET provides feedback to the patient via an LCD display that helps guide the patient through the inhalation. During the study period, patients were allowed to adjust inhalation volume after consulting the healthcare professional at the study site. Inhalation volume selection was recorded on the Smart Card. Daily use of the inhalation device was recorded, and the steroid-sparing effect of budesonide (1 and 0.5 mg) administered twice daily for 18 weeks was compared with that of placebo. A Smart Card attached to the device recorded each treatment, its duration, and the breathing patterns during inhalation. These data were collected on the Smart Card for review by the treating physician to assess the compliance values Daily Dose Compliance (number of successful treatment days/total number of treatment days) and Total Dose Compliance (number of successful treatments/total number of expected treatments).

Figure 1. Pictures of the smart nebuliser Breelib™ (left) and AKITA® JET (right)

Results In the study in PAH patients, rating of patient satisfaction was assessed after the first inhalation with the Breelib™. 85% of patients rated the operation of the device as easy to use. (Figure 2). The LED-light feedback guiding the inhalation flow rate and providing error feedback was rated as helpful by 88% of patients. The limitation of the inspiration flow was well perceived by 85% of the patients.(Figure 3)

Figure 2. Results from patient satisfaction questionnaire after initial inhalation with Breelib

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Figure 3. Patient satisfaction in relation to inhalation flow LED-feedback and the interactive inhalation flow limitation of Breelib, which guide the patient on the correct flow rate and inhalation volume Breelib™ resulted in a reduction of treatment time (Figure 4) vs I-Neb. The treatment time for an inhalation of 5µg Iloprost with Breelib™ was 2.6 min compared with 10.9 min with I-Neb® (26 patients). Daily treatment times calculated for 9 Iloprost treatments per day were 23.4 min and 98.1 min for Breelib™ and I-Neb, respectively. Out of 26 patients 25 decided to enter into the 30 month long-term extension study with the Breelib device. 89% of patients (23 out of 26) in long term use reported that Breelib was easy or adequate to use.

Figure 4. Treatment time from start to end of inhalation

In the study with asthmatic patients, Daily dose compliance (number of successful treatment days/total number of treatment days) was 81% for all patients on AKITA® Jet treatment and 87% for those who completed treatment (Figure 5). Total dose compliance (number of successful treatments/total number of expected treatments) was 82% and 89%, respectively. A treatment was deemed successful when the patient inhaled at least 80% of the prescribed dose.

Figure 5. Treatment time from start to end of inhalation

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Figure 4. Treatment time from start to end of inhalation

Discussion and Conclusion Smart nebulisers such as AKITA® JET and Breelib™, which guide patients on correct inhalation technique, can improve patient satisfaction and treatment outcomes [6, 10, 12] and can significantly reduce treatment time for an equivalent delivered dose. Observed ratings in relation to the inhalation feedback from the device indicate that patients positively perceived guidance on flow rate and inhalation volume. Particularly in indications such as severe asthma and PAH, the smart nebulisers studied herein provide meaningful advantages for patients in their daily therapy, which might result in higher patient satisfaction, better adherence, and more favourable treatment outcomes. References 1

2 3

5 6

8 9

11 12

Brand P, Schulte M, Wencker M, Herpich C H, Klein G, Hanna K, Meyer T: Lung deposition of inhaled alpha1-proteinase inhibitor in cystic fibrosis and alpha1-antitrypsin deficiency, Eur Respir J 2009; 34: pp354-360. Brand P, Friemel I, Meyer T, Schulz H, Heyder J, Haubetainger K: Total deposition of therapeutic particles during spontaneous and controlled inhalations, J Pharm Sci 2000; 89: pp724-731. Brand P, Beckmann H, Maas Enriquez M, Meyer T, Mullinger B, Sommerer K, Weber N, Weuthen T, Scheuch G: Peripheral deposition of alpha1-protease inhibitor using commercial inhalation devices, Eur Respir J 2003; 22: pp263-267. Fischer A, Stegemann J, Scheuch G, Siekmeier R: Novel devices for individualized controlled inhalation can optimize aerosol therapy in efficacy, patient care and power of clinical trials, Eur J Med Res 2009; 14 Suppl 4: pp71-77. Scheuch G, Brand P, Meyer T, Müllinger B, Sommerer K: Regional drug targeting within the lungs by controlled inhalation with the AKITA-inhalation system., Respiratory Drug Delivery 2002; 8: pp471-474. Gessler T, Ghofrani H-A, Held M, Klose H, Leuchte H, Olschewski H, Rosenkranz S, Fels L, Li N, Ren D, Kaiser A, Schultze-Mosgau M-H, Müllinger B, Rohde B, Seeger W: The safety and pharmacokinetics of rapid iloprost aerosol delivery via the BREELIB nebulizer in pulmonary arterial hypertension, Pulmonary Circulation 2017; 7: pp505-513. Global Initiative for Asthma. Global strategy for asthma management and prevention. Available at: https://ginasthma.org/wpcontent/uploads/2016/01/wms-GINA-2017-main-report-tracked-changes-forarchive.pdf. Accessed July 6, 2018. Usmani OS, Lavorini F, Marshall J, Dunlop WCN, Heron L, Farrington E, Dekhuijzen R: Critical inhaler errors in asthma and COPD: a systemic review of impact on health outcomes, Respir Res. 2018;19(1):10. Sulaiman I, Greene G, MacHale E, Seheult J, Mokoka M, D'Arcy S, Taylor T, Murphy DM, Hunt E, Lane S, Diette GB, FitzGerald JM, Boland F, Bhreathnach AS, Cushen B, Reilly RB, Doyle F, Costello RW: A randomised clinical trial of feedback on inhaler adherence and technique in patients with sever unctrolled asthma, Eur Respir J. 2018;51(1):[Epub ahead of print]. Vogelmeier C, Kardos P, Hofmann T, Canisius S, Scheuch G, Muellinger B, Nocker K, Menz G, Rabe K F: Nebulised budesonide using a novel device in patients with oral steroid-dependent asthma, Eur Respir J 2015; 45: pp1273-1282 Bennett W D: Controlled inhalation of aerosolised therapeutics, Expert Opin Drug Deliv 2005; 2: pp763767. Richter M, Wan J, Ghofrani HA, Rieth A, Seeger W, Tello K, Gall H: Acute response of rapid Ilopprost inhalation using the Breelib nebulizer in pulmonary aterial hypertension: the Breelib acute study, ERS Congress Paris 2018, Thematic Poster Presentation:TP-39 Pulmonary hypertension therapy

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Drug Delivery to the Lungs, Volume 29, 2018 - Chen-Hsiang Sang et al. Performance of a New Polymer-Based Vibrating Mesh Nebulizer: A Comparison to Metal-Based Mesh Nebulizer Chen-Hsiang Sang, Shih-Cherng Lin, Huang-Fei Chen, Hsin-Hua Tseng & Hsiao-Hui Lo Department of R&D, Medical Division, MicroBase Technology Corp., No.756, Jiadong Rd., Bade Dist., Taoyuan City, 33464, Taiwan (R.O.C.)

Summary It is important to understand the performance of vibrating mesh nebulizers, since it is related to the efficacy of inhalative drug delivery. The present study compared the distribution of droplet size, nebulization output rate, and vibration modal pattern during nebulization among the polymer-based mesh nebulizer from MicroBase Technology (MBTC), and metal-based mesh nebulizer from Aerogen® Solo, and PARI VELOX®. The results revealed that these nebulizers generated particle diameter distribution similarly. The volumetric-median-diameter of MBTC nebulizer, Aerogen® Solo, and PARI VELOX® were 5.11±0.44 μm, 5.02±0.67 μm, and 4.11±0.30 μm, respectively. The averaged output rate of the nebulizers from MBTC was 0.73±0.15 ml/min for normal saline (0.9%), which was at around 1.4 times greater than Aerogen® Solo (0.53±0.12 ml/min), and was similar to that of PARI VELOX® (0.76±0.18 ml/min). Moreover, these three nebulizers showed similar modal pattern during operations, and the resonant frequency was similar between MBTC and, Aerogen® Solo (125.9 kHz vs. 128.2 kHz, respectively) yet lower than the PARI VELOX® (160.0 kHz). Accordingly, further development and application of polymer-based mesh nebulizer could be implemented in different designs. Further studies will also be carried out to assess the delivery of other liquid medications from the MBTC nebulizer.

Key Message In this study, a performance comparison was conducted on the new polymer-based vibrating mesh nebulizer from MBTC, and commercialized nebulizers from Aerogen (Aerogen® Solo) and PARI (PARI VELOX®). Through the measurements of droplet size distribution, output rate, vibrational modal pattern, it is demonstrated that the new polymer-based vibrating mesh nebulizer could present a comparable performance to current metal-based mesh nebulizer. A low-cost mesh nebulizer could be expected and achieved in the future.

Introduction Inhaled medication plays a crucial role in treating patients with pulmonary and some systemic diseases. In order to improve the delivery efficacy, the liquid medication must be transformed into medical aerosol with specific droplet size distribution. Some researchers revealed that the droplet size of medical aerosol should be controlled within 1 to 6 μm because the droplets could deposit in the mouth and throat when the size is larger than 6 μm, and may be exhaled when the size is smaller than 2 μm [1-3]. For this reason, the nebulization device must generate aerosol that meet these demanding properties. In addition to droplet size, nebulization output rate is another factor which could affect the treating efficacy. The low output rate for aerosol delivery can extend therapeutic duration and reduce the treatment compliance for patients. Polymeric material has the advantage in the cost of manufacturing than metal material, such as Pd-Ni, and Ni-Ti alloy. In addition, the polymeric material can be drilled easily through precisely laser processing. Yet, the performance of a polymer-based mesh nebulizer should be comparable to the metal-based mesh nebulizer on the current market, the cost to the users/patients could be reduced. In this study, a new polymer-based vibrating mesh nebulizer was fabricated through a specific process technique. Vibration modal pattern during nebulization, droplet size, and nebulization output rate of the nebulizer were determined and used to compare with those of commercially available vibrating mesh nebulizers, Aerogen® Solo and PARI VELOX®. Experimental Materials & Methods A 248 nm UV range excimer laser source (with output power 300 mJ and firing frequency of 200 Hz), optical path system, and laser dynamic control module were integrated to explore the relationship between different laser shots and the geometry of apertures. After that, the polymer-based meshes with specific aperture design were fabricated. The new polymer-based vibrating mesh module consists of a stainless steel plate in ring shape, vibrating mesh, and piezoelectric actuator (Figure 1).

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Figure 1 – Illustration of the new polymer-based vibrating mesh module In order to compare the performances of nebulizers, the ability to aerosolize normal saline (0.9 %) of 4 new polymerbased vibrating mesh nebulizers from MBTC, 4 Aerogen® Solo and 4 PARI VELOX® were tested in this study. In addition to the output rate, the distribution of droplet size was assessed by Spraytec Analyzer (Malvern Panalytical Ltd., Malvern, UK) with 300 mm lens system using analysis ranging from 0.1 to 900 μm. The vibration modal pattern and resonance frequencies of each nebulizer during nebulization were determined by the Laser Doppler Scanning Vibrometer, Polytech PSV-500 (Polytec GmbH, Waldbronn, Germany). Results and Discussion The volume droplet size distribution of the new polymer-based vibrating mesh nebulizers from MBTC, and commercialized nebulizers from Aerogen and PARI are shown in Figure 2. Three nebulizers present a similar distribution of particle diameter pattern that approximately 75% of droplets were smaller than 6 μm. The output rate and volumetric-median-diameter of each nebulizer are shown in Figure 3. The output rate from MBTC ranges from 0.53 to 0.92 ml/min, while that of Aerogen ® Solo and PARI VELOX® ranges from 0.33 to 0.66 ml/min and 0.60 to 1.07 ml/min, respectively. The volumetric-median-diameter of the nebulizers from MBTC ranges from 4.62 to 5.81 μm, while that of Aerogen® Solo ranges from 3.99 to 5.85 μm and PARI VELOX® ranges from 3.60 to 4.39 μm. The volumetric-median-diameter of the droplet, output rate, and resonant frequency of the nebulizer from MBTC, Aerogen® Solo and PARI VELOX® are summarized in Table 1. In addition to output rate, all the nebulizers present a similar result in distribution of particle size. The output rate 0.73±0.15 ml/min of MBTC’s nebulizer is similar to PARI VELOX® at 0.76±0.18 ml/min , in which both are greater than the output rate 0.53 ml/min of Aerogen® Solo. It is believed that a nebulizer having a high output rate as well as fine particle size of aerosol can shorten the treatment time for treatment and increase the treatment compliance of patients. The Figure 4 shows the vibration modal pattern among three nebulizers during operations. The vibration modal pattern of the MBTC nebulizer features with a concentric circle pattern, same as the Aerogen® Solo and PARI VELOX®. That might be a possible reason to explain the similarity in droplet size distribution between these nebulizes.

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Figure 2 – The droplet size distribution of the new polymer-based vibrating mesh nebulizers from MBTC (a), Aerogen® Solo (b) and PARI VELOX® (c)

Figure 3 – The experimental results of volumetric-median-diameter and output rate among different Nebulizers

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Drug Delivery to the Lungs, Volume 29, 2018 - Performance of a New Polymer-Based Vibrating Mesh Nebulizer: A Comparison to Metal-Based Mesh Nebulizer Table 1 – Comparison in volumetric-median-diameter of the droplet, output rate and resonant frequency of each nebulizer Dv(10) (μm)

Dv(50) (μm)

Dv(90) (μm)

output rate (ml/min)

Resonant frequency (kHz)

MBTC nebulizer

2.24±0.12

5.11±0.44

11±1.08

0.73±0.15

125.9

Aerogen® Solo

2.08±0.19

5.02±0.67

10.84±1.6

0.53±0.12

128.2

PARI VELOX

1.97±0.12

4.11±0.30

8.53±1.15

0.76±0.18

160.0

Figure 4 – Vibration modal pattern of the new polymer-based vibrating mesh module from MBTC (a), Aerogen® Solo (b) and PARI VELOX® (c)

Conclusion In this study, nebulizer performance comparisons were conducted on the new polymer-based vibrating mesh nebulizer from MBTC, and commercialized nebulizers of Aerogen (Aerogen® Solo) and PARI (PARI VELOX®). The MBTC nebulizer presents a similar aerosol distribution to Aerogen® Solo and PARI VELOX®. The output rate of MBTC nebulizer was comparable to that of PARI VELOX®, but 40% greater than that of Aerogen® Solo. In addition, these three nebulizers showed similar modal pattern (concentric circle pattern) during operations. As a result, the new polymer-based vibrating mesh nebulizer illustrated a comparable performance to current metal-based mesh nebulizers. A low-cost mesh nebulizer could be achieved in the future. Microbase will further study in assessing the delivery of other liquid medications from the MBTC nebulizer. Reference 1. 2. 3.

Olszewski OZ, MacLoughlin R, Blake A, O’Neill M, Mathewson A, Jackson N. A Silicon-based MEMS Vibrating Mesh Nebulizer for Inhaled Drug Delivery. Procedia Engineering. 2016;168:1521-1524. Barnes P, Godfrey S, Godfrey S. Asthma Therapy. Informa Healthcare. 1998. Nikander K, von Hollen D, Larhrib H. The size and behavior of the human upper airway during inhalation of aerosols. Expert Opin Drug Deliv. 2017 May;14(5):621-630.

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Drug Delivery to the Lungs, Volume 29, 2018 - Russell Down, et al. Developing clinically relevant methodology for existing pMDI aerosol products when coupled to the Intelligent Control Inhaler Russell Down1, Hayden Beresford1, Chris Blatchford1, Andy Cooper1 & Stewart Griffiths1 1

3M Drug Delivery Systems Ltd, Charnwood Campus, 10 Bakewell Road, Loughborough, Leicestershire LE11 5RB

Summary The Intelligent Control Inhaler (ICI) is a next generation breath actuated pressurised metered dose inhaler (pMDI) device. A rapid screening method has been developed to compare the ICI to the commercial press and breathe (P&B) actuator devices for Dulera® and QVAR® aerosols. The methodology is designed to be clinically relevant, including accelerated impactor measurements (AIM), an anatomical throat and breath inhalation profile, in order to gain understanding of the in vitro-in vivo relationship (IVIVR). For Dulera®, no statistical differences were determined for FPM and MMAD between the ICI and P&B devices. For QVAR®, no statistical differences were determined for MMAD; however, there was a statistically significant difference in FPM. There was also a statistically significant lower throat deposition using the ICI, in comparison to the P&B device, for both Dulera® and QVAR®. It is not clear how these differences would impact clinical equivalence. The analysis shows that further method development is required to potentially allow a prediction of clinical equivalence between ICI and commercial P&B actuator devices for Dulera® and QVAR®. Key Message A rapid screening method has been developed to compare the Intelligent Control Inhaler device to the commercial P&B actuator devices for Dulera® and QVAR® aerosols. Although some initial results appear promising, further method development is required to potentially allow a prediction of clinical equivalence. Introduction The Intelligent Control Inhaler (ICI) is a next generation breath actuated pressurised metered dose inhaler (pMDI) device (Fig. 1).[1]

Figure 1 – The Intelligent Control Inhaler, a next generation breath actuated pressurised metered dose inhaler device. The ICI could be used with new pMDI aerosol products to improve adherence/compliance, which is a problem for all inhalation products,[2,3] and could also be used with existing press and breath (P&B) pMDI aerosol products as part of a lifecycle product management strategy. To transfer existing P&B products to the ICI, it is necessary to demonstrate equivalence in terms of in vitro performance, via clinically relevant testing,[4-6] to develop understanding of the in vitro in vivo relationship (IVIVR). Work was performed here to develop rapid screening methodology to assess in vitro equivalence to complement standard in vitro testing. To introduce more clinically relevant testing parameters than pharmacopoeial methods, accelerated impactor measurements (AIM) were performed in conjunction with an anatomical throat model (ATM) and breath inhalation profile simulation, using a reduced next generation impactor (rNGI) setup. As this methodology will be more complex than traditional pharmacopoeial methods, increased variability may be expected. Testing was performed for both Dulera® and QVAR® aerosols, chosen as model suspension and solution pMDI products, respectively.

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Drug Delivery to the Lungs, Volume 29, 2018 - Developing clinically relevant methodology for existing pMDI aerosol products when coupled to the Intelligent Control Inhaler Methodology A reduced next generation impactor (rNGI) was set up with an internal filter placed into stage 4 (to test Dulera®) or stage 7 (to test QVAR®) of the NGI stack (Copley Scientific). A mixing inlet (Copley Scientific) was connected to the inlet of the NGI stack, with an anatomical throat model (Emmace consulting AB, medium size) placed on top, coupled to the inhaler device. The air inlet of the mixing inlet was connected to a BRS 3000 breath simulator (Copley Scientific) and supplied by a compressed air cylinder (BOC). The outlet of the NGI stack was connected to a high capacity pump (Copley Scientific). The interior surfaces of the Emmace throat and the NGI cups were coated with a solution of ethanolic Brij 35 and glycerol and left to dry before assembling the equipment. The equipment setup is summarised in Figure 2. The flow through the stack was kept constant at 60 L/min, and the flow through the inhaler device controlled by the BRS 3000 to give the breath profile shown in Figure 2. This single profile is an artificial simplification of a median breath profile reported in the literature and is a slight variant on a steady-state flow system used in pharmacopoeial testing.[5]

Anatomical throat Flow controlled by breath simulator (F2)

Breath profile (F1 – F2)

Mixing inlet

rNGI stack

60 L/min constant flow (F1)

Figure 2 – Schematic diagram of the IVIVR equipment setup. Commercial pMDIs were obtained and either used with the P&B actuators provided, or the aerosols were harvested and inserted into the ICI. The ICI actuator mouthpiece shape was different in comparison to the corresponding P&B actuator mouthpiece (ICI – oval, Dulera® – rounded rectangular, QVAR® – circular). Samples using the ICI were generated through breath actuation; P&B samples were actuated at an equivalent point in the breath profile as the ICI actuated. All inhalers underwent 4 priming shots prior to analysis before 5 shots were actuated into the rNGI. Inhalers were shaken appropriately prior to firing. Samples were recovered using an appropriate diluent and analysed by UHPLC methods that were validated for specificity and linearity.[7] Mass balances were all within USP <601> limits[8] of 85-115%, demonstrating the accuracy of this methodology. Quantification was performed using Empower 3 (Waters). Mass median aerodynamic diameter (MMAD) and fine particle mass (FPM) were estimated via interpolation/extrapolation, in accordance with the calculations detailed in the CITDAS 3.10 instruction manual (Copley Scientific). A cut-off of 5 µm was used for FPM. Statistical analysis was performed using a two-sample t test of the means at the 95% confidence interval. Predicted therapeutic equivalence analysis was performed for QVAR® by expressing the impactor sized mass (ISM) data for the individual ICI analyses as a percentage of the mean ISM data for the P&B device. A 95% confidence interval is then calculated for these data, and compared to limits of 85-115%.[9] For this QVAR® rNGI setup, ISM is defined as the mass deposited on cups 5 and 6 and the filter (<1.7µm). Results For Dulera® aerosols, no statistical differences were determined for fine particle mass (FPM) and mass median aerodynamic diameter (MMAD) between the ICI and P&B devices (p = 0.2 and p = 0.3, respectively; Fig. 3), suggesting the devices are comparable. Dulera® aerosols actuated through the ICI also had a statistically significant lower throat deposition than those actuated through the P&B devices (p = 0.001). With %RSD > 10 for both the ICI and P&B (Table 1), the FPM is more variable than standard in vitro testing conducted as per USP <601> due to the added complexity of the method; it should be noted that variability can also be high in vivo.[10] The data shown is for mometasone furoate only, but the findings also apply to formoterol fumarate.

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160 140 120 100 80 60 40 20 0

MMAD (µm)

Amount (µg/act)

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Throat Deposition

2 1 0

FPM ICI

MMAD

P&B

Figure 3 – Comparison of throat deposition, FPM and MMAD for mometasone furoate in Dulera®, fired through ICI (blue) and the P&B actuator (orange). Error bars are standard deviation, n = 3. As with Dulera®, QVAR® aerosols actuated through the ICI demonstrated equivalent MMAD (p = 0.9) versus aerosols actuated through the standard P&B devices (Fig. 4). 1

MMAD (µm)

Amount (µg/act)

15 10 5 0

Throat Deposition

FPM ICI

0.5

MMAD

P&B

Figure 4 – Comparison of throat deposition, FPM and MMAD for beclomethasone dipropionate in QVAR®, fired through the ICI (blue) and the P&B device (orange). Error bars are standard deviation, n = 3. QVAR® aerosols actuated through the ICI had a statistically significant lower throat deposition than those actuated through the P&B devices (p = 0.002) as well as a statistically significant lower FPM (p = 0.004). These results are likely due to the statisically lower delivered dose (p = 0.001), which may be linked to the differences in the actuator mouthpiece. Despite these results, the impactor sized mass (ISM) of QVAR® fired through the ICI is within 85115% of the mean ISM via P&B at the 95% confidence interval (Fig. 5), which is an indication of therapeutic equivalence in accordance with European guidance,[9] again suggesting the devices are comparable.

Figure 5 – Predicted therapeutic equivalence analysis of QVAR®. Error bars are the 95% confidence interval, n = 3.

211

Drug Delivery to the Lungs, Volume 29, 2018 - Developing clinically relevant methodology for existing pMDI aerosol products when coupled to the Intelligent Control Inhaler It is also noted that, using this clinically relevant methodology, the FPM appears less variable for QVAR® than with Dulera® (Table 1). This may be due to the increase in relative dose that penetrates the anatomical throat for the QVAR® aerosols (53 %), in comparison to Dulera® (33 %). Product Dulera (MF) QVAR

Device

Throat (µg/act)

FPM (µg/act)

MMAD (µm)

Mean

%RSD

Mean

%RSD

Mean

%RSD

ICI

133.2

1.7

59.3

10.1

2.9

0.5

P&B

145.2

0.3

50.3

16.4

3.1

7.4

ICI

17.9

1.6

20.1

0.7

0.54

1.1

P&B

20.1

2.2

22.5

3.1

0.54

0.3

Table 1 – Summary table of throat deposition, FPM and MMAD for each product/device combination. Conclusion The ICI is a next generation breath actuated pMDI device. A rapid screening method has been developed to compare the ICI to the commercial P&B actuator devices for Dulera® and QVAR® aerosols. The APSD methodology is designed to be clinically relevant, including AIM, an anatomical throat and breath inhalation profile, in order to gain understanding of the IVIVR. For Dulera®, no statistical differences were determined for FPM and MMAD between the ICI and P&B devices. For QVAR®, no statistical differences were determined for MMAD; however, there was a statistically significant difference in FPM. There was also a statistically significant lower throat deposition using the ICI, in comparison to the P&B device, for both Dulera® and QVAR®. It is not clear how these differences would impact clinical equivalence. Further work is proposed to further refine the IVIVR by introducing more clinically relevant testing parameters, including reducing the number of shots to match patient dosage, using different ATMs and introducing different, more realistic breath profiles. In silico modelling may also further develop understanding of the IVIVR. Such methodological refinements could potentially allow a prediction of clinical equivalence. References 1. https://www.3m.com/3M/en_US/drug-delivery-systems-us/technologies/inhalation/intelligentcontrol/ 2. Federico Lavorini, Antoine Magnan, Jean Christophe Dubus, Thomas Voshaar, Lorenzo Corbetta, Marielle Broeders, Richard Dekhuijzen, Joaquin Sanchis, Jose L. Viejo, Peter Barnes, Chris Corrigan, Mark Levy, Graham K. Crompton, Effect of incorrect use of dry powder inhalers on management of patients with asthma and COPD, Respir. Med. 2008; 102; pp593-604 3. Henry Chrystyn, Inhaler device and errors, in Drug Delivery to the Lungs 2016 4. Jolyon Mitchell and Jason Suggett, Developing Ways to Evaluate in the Laboratory How Inhalation Devices Will Be Used by Patients and Care-Givers: The Need for Clinically Appropriate Testing, AAPS PharmSciTech 2014; 15; pp1275–1291 5. Bo Olsson, Lars Borgström, Hans Lundbäck, Mårten Svensson, Validation of a General In Vitro Approach for Prediction of Total Lung Deposition in Healthy Adults for Pharmaceutical Inhalation Products, J. Aerosol Med. Pulm. Drug Deliv. 2013; 26; pp355-369

6. Samantha Holmes and Alex Slowey, A Comparison of Different Anatomical Throats vs the USP Throat, in Drug Delivery to the Lungs 2017

7. ICH Harmonised Tripartite Guideline Q2(R1). (1994): Validation of Analytical Procedures: Text and Methodology 8. USP chapter (601): Aerosols, Nasal Sprays, Metered-Dose Inhalers, and Dry Powder Inhalers 9. CPMP/EWP/4151/00 Rev. 1 10. Peter Hirst, Gary Pitcairn, Jeff Weers, Thomas Tarara, Andrew Clark, Luis Dellamary, Gail Hall, Jolene Shorr, Stephen Newman, In Vivo Lung Deposition of Hollow Porous Particles from a Pressurized Metered Dose Inhaler, Pharm. Res. 2002; 19; pp258-264

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Drug Delivery to the Lungs, Volume 29, 2018 – Daryl L. Roberts et al. Experimental and Theoretical Investigation of a New Approach to In-Use Impactor Quality Specifications Daryl L. Roberts1, Mårten Svensson2, Karolina Sandell2, Dennis Sandell3 1

Applied Particle Principles LLC, 17347 Westham Estates Court, Hamilton, VA 20158, USA 2Emmace Consulting AB, Medicon Village, S-223 81, Lund, Sweden 3 S5 Consulting, Ekvägen 8, S-275 62, Blentarp, Sweden

Summary The behavior of cascade impactors has been studied under non-ideal nozzle conditions, including those that can arise in practical laboratory use. This study is a first step towards establishing a new method of setting specifications on used impactor nozzle dimensions, one that is derived from the drug product’s mass-per-stage specifications established, for example, at the time of regulatory approval, rather than on the original as-manufactured nozzle dimensions. Next Generation Impactors (NGIs) were tested with purposely occluded nozzles with a commercial beclomethasone dipropionate (BDP) metered-dose inhaler (MDI), measuring the collected drug mass and the pressure drop across stages with ordinary (unblocked) and blocked nozzles and noting changes in these quantities. The pressure drop across unblocked stages varied with flow rate in accord with theory, as did the pressure drop across any given stage when nozzles were increasingly blocked at a fixed flow rate. To quantify the change in collected particle mass when nozzles were blocked, this study focused on NGI stage 5 at an inlet flow rate of 30 L/min. The collected drug mass on stage 5 increased with increasing proportion of blocked nozzles, but generally less than expected from a first-order model, possibly because of shifts in the individual nozzle behavior, especially when > 10% of the nozzles were blocked. Nevertheless, this model enables an approach to impactor quality that is independent of as-manufactured nozzle dimensions and that can rely upon stage pressure drop values. The overall approach rests firmly on known impaction principles and is worth further development. Key Message Mass-per-stage specifications established at the time of regulatory approval or by other used-defined means, can assist with setting specifications for in-use cascade impactors. These in-use specifications are independent of asmanufactured nozzle dimensions and can be monitored by stage pressure drop measurement. Introduction Quality control testing of inhalable drug products includes measuring the size distribution of the aerosol generated by representative samples of the commercial batches of these devices. Cascade impactors are key to this testing, one of several instruments necessary for measurement of the aerodynamic particle size distribution (APSD; other equipment like flow meters and means of drug assay are also necessary). All analytical equipment needs to be calibrated. For this reason, the NGI was calibrated with particles, at its inception, and with quantitative specifications on the dimensions of the nozzles, so that users know the D50 cut-points of each stage.1 The common question asked by the user community is whether the same quantitative constraints on the NGI nozzle dimensions must be maintained with in-use impactors, and if not, then what specifications may be acceptable. The task of the user is to define the quality specifications necessary for an impactor to meet that user’s stated purpose. In contrast, the task of the impactor manufacturer is to make equipment that is quantitatively controlled and generally meets the needs of the global user community. The question arises whether the user community can establish inuse quality specifications less strict than those of the manufacturer. This article explains, along with supporting experimental data for a specific solution-MDI product, an approach to quantitative constraints on the NGI stage D50 cut-points based on user-established mass-per-stage (or grouped stage) specifications, such as those of a registered drug product. Materials and Methods The study included NGIs (serial numbers 323, 446, 465, 501) used under controlled laboratory conditions for approximately a decade and with nozzles within specifications of used impactors, determined by periodic optical inspection. The flow resistance was measured of each stage of these impactors, before the nozzles were purposely occluded, at inlet flow rates from 15 to 80 L/min. Also with up to 25% of the nozzles on stages 4, 5, or 6 blocked, the stage pressure drops were measured at inlet flow rates of 30 and 60 L/min (±2%). Developmental equipment from FIA AB (Södra Sandby, Sweden), with two differential pressure transducers with full-scale ranges of 600 Pa and 6 kPa, allowed measurement of the pressure drop of stages 2 to 7 to an accuracy of less than 1% of typical values, with methods similar to those described by previous investigators. 2,3 In the particle sizing portion of the study, a BDP MDI was tested (solution in ethanol and HFA-134a propellant; 100 µg per dose; Teva GmbH, Ulm, Germany) at an inlet flow rate of 30 L/min and with the impactors “as is” and then with purposely blocked nozzles on stage 5 (nominally 2%, 4%, 6%, 10% and 25% blockage). For blocking nozzles, transparent tape (3M Scotch Crystal tape) was used at the entrance to the nozzles, for example, as shown in Figure 1 for stage 5 with eight nozzles blocked (blue marks added for clarity).

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Drug Delivery to the Lungs, Volume 29, 2018 – Experimental and Theoretical Investigation of a New Approach to In-Use Impactor Quality Specifications

Figure 1 – Blocking of Nominally 5% of the Nozzles on Stage 5 (8 blocked out of 152 nozzles) Results – Pressure Drop Tests The pressure drop across each stage, with no blocked nozzles, increased with the square of the inlet flow rate, in accord with Bernoulli theory (reference 4, Appendix A4). This theory predicts that the pressure drop depends on the square of the air velocity in the nozzles and in its simplest form can be written ΔP = K*Q2

(1)

Equation 1 applies to each individual stage, with Q equal to the inlet impactor flow rate. Table 1 gives the average value of K for stages 2 to 7 in the flow range 15 to 80 L/min (least-square fit of each impactor’s data to equation 1). Table 1 – Pressure Drop Flow Coefficients K† for NGI Stages Two to Seven Stage Number NGI Serial 2 3 4 5 Number 0323 0.0207 0.0329 0.0724 0.1459 0465 0.0211 0.0323 0.0714 0.1427 0501 0.0202 0.0322 0.0716 0.1387 Average 0.0207 0.0325 0.0718 0.1424 †R2 values greater than 0.99 in all cases; pascal per (L/min)2

6 0.2955 0.2920 0.2973 0.2949

7 0.6722 0.7061 0.7821 0.7201

Bernoulli theory also predicts that the open nozzle area, squared, times the pressure drop ∆P across any individual stage should be a constant at a given inlet air flow rate. Regarding the open area of each nozzle on a stage to be the same leads to equation 2, where N is the number of open nozzles: ΔP*N2 = Constant

(2)

Equation 2 was confirmed for all stages with purposely blocked nozzles (stages 4, 5, and 6, and at inlet flow rates of 30 L/min and 60 L/min; up to 22% of the nozzles blocked; R2 values ≥ 0.99 for all cases). Figure 2 shows that data collected for three NGIs follow equation 2 for both 30 L/min and 60 L/min. Twenty-two percent blocked nozzles is an extreme condition compared to the mildly partially occluded nozzles more representative of in-use impactors. The confirmation of the Bernoulli relationship (equation 2), even in such an extreme case, verifies that the same relationship will apply to normal in-use impactors.

Figure 2 – Pressure Drop at Fixed Inlet Flow Rate Follows Theory (Stage 5; 30 L/min and 60 L/min)

214

Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; Daryl L. Roberts et al. Theoretical Development of Impactor Specifications: Because the Bernoulli-style relationships hold both for changing flow rates and for blocked nozzles at a fixed flow rate, conventional impaction theory should apply when nozzles become partially or completely occluded, So, the D50 cut-point of the stage with blocked nozzles should still follow Stokesian theory5, which indicates that D50 divided by the square root of the number of open nozzles will remain constant, as in equation 3: đ??ˇđ??ˇ50 = Constant â &#x201E; â&#x2C6;&#x161;đ?&#x2018; đ?&#x2018;

(3)

Equation 3 indicates that the mass on a stage will increase when nozzles get blocked. To derive a first-order model of the effect of occluded nozzles, we treat the particle capture efficiency curves of each impactor stage as step-functions. If the aerosol is log-normally distributed with a mass-median aerodynamic Ě&#x2026; and a geometric standard deviation (GSD) of đ?&#x153;&#x17D;đ?&#x153;&#x17D;đ?&#x2018;&#x201D;đ?&#x2018;&#x201D; , the fraction of the impactor-sized mass diameter (MMAD) of đ??ˇđ??ˇ collected on stage â&#x20AC;&#x153;nâ&#x20AC;? is given by 1

đ?&#x2018;&#x201C;đ?&#x2018;&#x201C;đ?&#x2018;&#x203A;đ?&#x2018;&#x203A; = {đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019; [ 2

ln(

đ??ˇđ??ˇ50,đ?&#x2018;&#x203A;đ?&#x2018;&#x203A;â&#x2C6;&#x2019;1 ) Ě&#x2026; đ??ˇđ??ˇ

â&#x2C6;&#x161;2 ln đ?&#x153;&#x17D;đ?&#x153;&#x17D;đ?&#x2018;&#x201D;đ?&#x2018;&#x201D;

] â&#x2C6;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019; [

ln(

đ??ˇđ??ˇ50,đ?&#x2018;&#x203A;đ?&#x2018;&#x203A; Ě&#x2026; ) đ??ˇđ??ˇ

â&#x2C6;&#x161;2 ln đ?&#x153;&#x17D;đ?&#x153;&#x17D;đ?&#x2018;&#x201D;đ?&#x2018;&#x201D;

(4)

]}

Here, D50,k (k=n or n-1) denotes the cut-point for stage k, and the error function (erf(x)) is a known tabulated function6 and is available for example in Microsoft EXCELâ&#x201E;˘. Since the D50 value of stage n changes with blocked nozzles in accord with equation 3, the expected change in the mass fraction on the stage with blocked nozzles, â&#x2C6;&#x2020;f, is given by equation 5: 1

â&#x2C6;&#x2020;đ?&#x2018;&#x201C;đ?&#x2018;&#x201C; = {đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019; [ 2

ln(

đ??ˇđ??ˇ50,0 Ě&#x2026; ) đ??ˇđ??ˇ

â&#x2C6;&#x161;2 ln đ?&#x153;&#x17D;đ?&#x153;&#x17D;đ?&#x2018;&#x201D;đ?&#x2018;&#x201D;

] â&#x2C6;&#x2019; đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019; [

ln(

đ??ˇđ??ˇ50,0 đ?&#x2018; đ?&#x2018; Ě&#x2026; â&#x2C6;&#x2014;â&#x2C6;&#x161;đ?&#x2018; đ?&#x2018; 0) đ??ˇđ??ˇ

â&#x2C6;&#x161;2 ln đ?&#x153;&#x17D;đ?&#x153;&#x17D;đ?&#x2018;&#x201D;đ?&#x2018;&#x201D;

(5)

]}

Here, we have dropped the stage subscript â&#x20AC;&#x153;nâ&#x20AC;? to eliminate confusion with the number of open nozzles, N, and we use the subscript 0 to denote the initial, clean stage condition with zero blocked nozzles. [We have also taken D 50 for stage n-1 to be a constant, which was the case in our experiments; simultaneous change in D 50,n-1 and D50,n can be accommodated in the theory for real in-use impactors but lengthens the algebra]. In practice, â&#x2C6;&#x2020;f can be monitored by measuring the stage pressure drop â&#x20AC;&#x201C; which we can see by inserting equation 2 (which we verified experimentally) into equation 4: 1

â&#x2C6;&#x2020;đ?&#x2018;&#x201C;đ?&#x2018;&#x201C; = {đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019; [ 2

ln(

đ??ˇđ??ˇ50,0 Ě&#x2026; ) đ??ˇđ??ˇ

â&#x2C6;&#x161;2 ln đ?&#x153;&#x17D;đ?&#x153;&#x17D;đ?&#x2018;&#x201D;đ?&#x2018;&#x201D;

] â&#x2C6;&#x2019; đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019;đ?&#x2018;&#x2019; [

ln(

1 đ??ˇđ??ˇ50,0 â&#x2C6;&#x2020;đ?&#x2018;&#x192;đ?&#x2018;&#x192;0 â &#x201E;4 ) Ě&#x2026; â&#x2C6;&#x2014;( â&#x2C6;&#x2020;đ?&#x2018;&#x192;đ?&#x2018;&#x192; ) đ??ˇđ??ˇ

â&#x2C6;&#x161;2 ln đ?&#x153;&#x17D;đ?&#x153;&#x17D;đ?&#x2018;&#x201D;đ?&#x2018;&#x201D;

]}

(6)

In this way, monitoring changes in â&#x2C6;&#x2020;P can indicate if the impactor is suitably within specifications. Results â&#x20AC;&#x201C; API Mass per Stage Five series of measurements were made of the mass of active pharmaceutical ingredient (API) on stage 5 with 30 L/min inlet flow rate. With no blocked nozzles, we found that the BDP inhaler emitted an aerosol particle sizes typical of those reported by others7 (MMAD of 1.0 micron and a GSD of 1.6). The series of blocked-nozzle testing consisted of blocking nominally 2%, 4%, 6%, 10%, and 25% of the nozzles on stage 5 (3, 6, 9, 15, and 38 blocked nozzles; total of 152 nozzles on stage 5). Figure 3, comparing the measured and theoretical increase in mass on stage 5 when blocking of nozzles is increasing, shows that equation 5 reasonably represents the measured results for nozzle blockages of 5% or less, a shift of about 2.5% in the D50 value of stage 5 (per equation 3). However, the observed change in collected drug mass remains significantly smaller than predicted when the blocked area reaches double-digit percentages, surprisingly only a 4% increase for 10% blockage and an 8% increase for 25% blockage. A previous report of approximately uniform blocking of nozzles, but with the Andersen impactor, showed reasonable adherence to impactor theory (nozzles blocked fully or partially, up to 25%8). The drift away from the theory observed in the present study may result from the blocking pattern in combination with physical differences between the NGI and the Andersen. For example, the deposits under NGI nozzles are largely not circular, a result of the non-axisymmetric design of the NGI cup. In the current study it was observed that more blocked nozzles enables more circular deposits (Figure 4a,b), indicating possibly a tightening and shifting of the stage efficiency curve. Also, the first-order model assumes that the MMAD and GSD of the aerosol has not changed, an approximation that will need to be examined more fully for a generalized theory. Nevertheless, in a practicing laboratory, nozzle blockage is likely to be in the single-digit percentages where the first-order model is adequate.

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Drug Delivery to the Lungs, Volume 29, 2018 – Experimental and Theoretical Investigation of a New Approach to In-Use Impactor Quality Specifications

Figure 3 – Change in Collected Mass with Nozzle Blockage, Stage 5, 30 L/min Flow Rate

Figure 4a, b – Stage 5 deposits with 2% blockage (3 nozzles; L) and 25% blockage (38 nozzles; R) Conclusions – Toward a New Method of Impactor Qualification for Registered Products A satisfactory impactor is one that does not, by itself, change the mass per stage by a significant portion of the product quality specifications. Allowed shifts in D50 can be expressed in terms of effective diameter derived from traditional stage mensuration data3 or from more readily measured stage pressure drop data2 (equation 6) and notably with no reference to as-manufactured quantitative nozzle specifications. The initial experimental results shown here support a first-order model that reasonably predicts the mass-per-stage changes that would occur if nozzle area were occluded in the single-digit percentages typical of well-maintained impactors in routine use, such as those used in quality control for registered drug products. Further development of this model could lead to quality specifications for in-use cascade impactors that are rooted in the product quality specifications and less strict. References: Marple, V. A., B. A. Olson, K. Santhanakrishnan, J. P. Mitchell, S. C. Murray, B. L. Hudson-Curtis, “Next Generation Pharmaceutical Impactor, Part II: Archival Calibration,” J. Aerosol Med., 16(3), 301-24 (2003). 2 Milhomme, K., C. Dunbar, C. Lavarreda, D. L. Roberts, F. J. Romay, “Measuring Effective Diameter with the Flow Resistance Monitor,” Respiratory Drug Delivery 2006, Boca Raton, FL, April 23-27, 2006, pages 405-7. 3 Roberts, D. L., N. Maidment, M. A. Copley, “Improved Protocol for Relating Impactor Stage Pressure Drop to the Suitability for Routine Use,” Drug Delivery to the Lung 2017, Edinburgh, UK, The Aerosol Society, December 6 to 8, 2017, pg. 94-7. 4 Roberts, D. L., F. J. Romay, “Relationship of Stage Mensuration Data to the Performance of New and Used Cascade Impactors, J. Aerosol Med., 18(4), 396-413 (2005). 5 Rader, D. J., V. A. Marple, “Effect of Ultra-Stokesian Drag and Particle Interception on Impaction Characteristics,” Aerosol Sci. Tech., 4, 141-56 (1985). 6 Matthews, J. R., L. Walker, “Mathematical Methods of Physics,” 2 nd Edition, Benjamin Cummings Press, 1970. 7 Hampel, F., E. Lisberg, J. C. Guérin, “Effectiveness of Low Doses of Beclomethasone Dipropionate Delivered as a CFC-free Extrafine Aerosol in Adults with Mild to Moderate Asthma,” J. Asthma, 37(5), 389-98 (2000). 8 Kadrichu, N., N. Rao, G Sluggett, B. Fong, G. Jones, T. Perrone, S. Seshadri, P. Shao, “Sensitivity of Andersen Cascade Impactor Response to Stage Nozzle Dimensions,” Respiratory Drug Delivery IX, pg. 561-4 (2004). 1

216

Drug Delivery to the Lungs, Volume 29, 2018 – Stefano Campo et al Analytical Technology to Improve the Efficiency of a Through-Life Analysis on DPI Products Stefano Campo1, Lorena Gasparini1, Federica Polimeni1, Tatiana Salvo1 1

CMC, Drug Product Development Department, Chiesi Farmaceutici, Largo Belloli 11/A - 43122 - Parma - Italy

Summary A through-life (TL) analysis consists of the determination of Fine Particle Mass (FPM) by Next Generation Impactor (NGI) and Mean Delivered Dose (MDD) by Dosage Unit Sampling Apparatus (DUSA) during the entire life of a device. In order to minimize the electrostatic charges due to device handling during the standard regimen, the waste shots are fired manually with the completion of a TL requiring more than one day, where a specific number of doses are collected per day. The aim of the study was to improve the efficiency of a TL analysis in terms of timing and analytical resources by automating the analytical procedure, but assuring good data quality. For this aim, the automatic workstation Xelair® 1 Series was implemented in Chiesi laboratories. It is an automatic equipment manufactured by Astech Projects and specifically customized in order to perform the waste shots of Dry Powder Inhaler (DPI) products filled in NEXThaler® devices. For this study a one day regimen was investigated analysing different DPI products and performing the waste shots with Xelair® 1 Series, equipped with a probe to reduce the electrostatic charges. The data quality was guaranteed by comparing the FPM, MDD and mean shot weights obtained with both the regimens which resulted comparable. By applying the one-day regimen using Xelair® 1 Series, improvement of efficiency in terms of analytical time was demonstrated to be ~40% per TL, while in terms of involved analytical resources, the time saving of a single analyst per TL was estimated to be ~25%. Key Message Improvement of the efficiency of a through-life analysis of DPI products filled in NEXThaler® device, both in terms of timing and analytical resources by introducing the workstation Xelair® 1 Series in order to automate the analytical procedure to perform the required waste shots. Introduction The aim of the study was to improve the efficiency of a through-life analysis in terms of timing and analytical resources, assuring good data quality. A through-life analysis consists in the determination of Fine Particle Mass and Mean Delivered Dose obtained by NGI and DUSA respectively, during the entire life of the device. The predefined through-life scheme follows the Eur.Ph. requirements that foresee the collection of the NGI samples at the beginning and end of the device life[1]. In some cases, the NGI-based measurements are collected also at the middle of the device life, while the DUSA samples are collected at the beginning, middle and end of the device life[2]. For a standard regimen the remaining doses of the device are fired manually, on different days (from 3 up to 5 days) according to the analytical procedure in place, in order to minimize the influence of electrostatic charge[3,4]. The study was carried out by analysing placebo and different DPI-delivered drug products having different strengths, all filled in NEXThaler® devices. Each through-life analysis has been performed on a one-day regimen, discharging the waste shots with the workstation Xelair® 1 Series, an automatic equipment manufactured by Astech Projects and specifically customized for Chiesi. Xelair® 1 Series can perform the waste shots of DPI products filled in NEXThaler® devices, and it is equipped with a specific antistatic system. In order to consider appropriate the oneday regimen using the Xelair® 1 Series, the FPM, MDD and mean shot weights data obtained from the analysis of the products across days and in one day regimens have been compared. The experimental plan is reported below. Experimental Plan Table 1– Experiments performed to compare the standard and the one day regimens of a through-life analysis

PRODUCT

Nº OF ANALYSED DEVICE / REGIMEN Manual SW

Automatic SW

Placebo DPI

2 devices in more than 1 day

2 devices in one day

Compound A low strength

6 devices in more than 1 day

2 devices in one day

Compound A high strength Compound B low strength Compound B high strength

3 devices in more than 1 day 4 devices in more than 1 day 2 devices in more than 1 day

2 devices in one day 4 devices in one day 2 devices in one day

217

PERFORMED ANALYSES (FOR EACH DEVICE) Shot weight 10 DUSA 3 NGI 10 DUSA 3 NGI 2 NGI 10 DUSA 2 NGI

ACCEPTANCE CRITERIA Mean SW 10mg ±1.5mg RSD ≤ 5.0% The FPM results should not differ more than 15.0% and the MDD should not differ more than 10.0% by comparing the devices analyzed with manual and automatic shot waste

Drug Delivery to the Lungs, Volume 29, 2018 - Analytical Technology to Improve the Efficiency of a Through-Life Analysis on DPI Products Experimental Methods The chromatographic method employed a RP-HPLC/UV method (HPLC/UV 2690/2695 Alliance and chromatographic column Atlantis dC18; 150 x 3.9 mm; 3µm; Waters, Milford, MA, USA). The NGI (Figure 1) equipped with an additional pre-separator (Copley Scientific, Nottingham, UK) was used for the aerodynamic particle size distribution (APSD) determination and the DUSA (Figure 2) for the delivered dose determination.

Figure 1 – Next Generation Impactor

Figure 2 – DUSA

The waste shots were performed using, the workstation Xelair® 1 Series (Figure 3) for the one day regimen and a dedicated DUSA for the “more than one day” regimen. Workstation Xelair® 1 Series analysed up to ten devices using a different analytical procedure for each device. It was equipped with an analytical balance and a specific system for electrostatic charge reduction which consists of a probe provided with a direct static elimination structure that locates the ion generation point at the tip of the head. It enables high-speed and high-precision static elimination, where it is needed most. The number of actuations, the flow rate and the time between the actuations of each device were set for each of the devices. The response of the workstation was the shot weight of each actuation.

Figure 3 – Automatic workstation Xelair® 1 Series

The HPLC data elaboration was carried out using Waters® EmpowerTM 2. It was a quantitative determination which used an external standard calibration to calculate the amount of active ingredient in each sample. The aerodynamic particle size calculations were performed using C.I.T.D.A.S. software (Copley Scientific Ltd) and for the qualitative data comparison a Principal Component Analysis (PCA)[5] was used, which converts a set of responses of possibly correlated variables into a set of values of linearly uncorrelated variables called Principal Components. PCA defines a new orthogonal coordinate system that optimally describes the experimental variance into a single dataset. Results In all the graphs reported below the error bars have been calculated using the confidence interval of 95%. As shown in Table 1, 2 Placebo DPI devices were analysed manually by applying the “more than one day” regimen and 2 devices using workstation Xelair® 1 Series in “one day” regimen. Figure 4 shows the obtained mean shot weights and RSD for each device. Figure 5 shows all actuation shot weights. The results clearly show that the two regimens are comparable.

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Drug Delivery to the Lungs, Volume 29, 2018 – Stefano Campo et al

Figure 4 - Placebo

Figure 5 – Placebo

In the next figures are reported the results for compound A and B. Since the considered dosage strengths for both compounds showed the same behaviour, only the highest strengths of each of them (of those stated in Table 1) are reported in the graphs below. In terms of Inhalation Performance, Figures 6 and 7 show the similarity between the APSD curves obtained with both regimens for both compounds. The particle mass stage grouping (Figures 8 and 9) show the same behaviour for both compounds. The Mean Delivered Dose results and its relative RSD are comparable as shown in Figures 10 and 11. All the Inhalation Performances, obtained with both regimens for each compound, can be considered comparable also in terms of variability. In addition, a qualitative analysis of the two regimens was evaluated. The PCAs (Figures 12 and 13) do not show significant difference between the experiments performed with the two different regimens. Instead, all the experimental results were well randomized, indicative that the two regimens were comparable.

Figure 6 – Compound A High Strength

Figure 7 – Compound B High Strength

Figure 8 – Compound A High Strength

Figure 9 – Compound B High Strength

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Drug Delivery to the Lungs, Volume 29, 2018 - Analytical Technology to Improve the Efficiency of a Through-Life Analysis on DPI Products

Figure 10 – Compound A High Strength

Figure 11 – Compound B High Strength

Figure 12 – Compound A

Figure 13 – Compound B

Conclusion All the acceptance criteria stated in Table 1 were satisfied. The results reported above demonstrated that the two investigated regimens are comparable and the one-day regimen can be adopted for the through-life DPI routine analyses. The workstation Xelair® 1 Series is very useful during a through-life analysis since it replaces the analyst during the waste shots collection and, at the same time, reduces the electrostatic charges due to the device handling. By applying the one day regimen using Xelair® 1 Series, the improvement of efficiency in terms of analytical time was demonstrated to be ~40% per through-life, while in terms of involved analytical resources, the time saving of a single analyst per through-life was estimated to be ~25%. In conclusion the use of workstation Xelair® 1 Series is a valid help to improve the efficiency of a through-life analysis in terms of time saving, analytical resources, equipment and materials necessary for the analysis, assuring a good data quality. References 1

European Pharmacopoeia 9.4 (2.9.18): Preparations for inhalation: aerodynamic assessment of fine particles

European Pharmacopoeia 9.4: Preparations for inhalation - Inhalanda

Stefan Karner, Nora Anne Urbanetz: The impact of electrostatic charge in pharmaceutical powders with specific focus on inhalation-powders, Journal of Aerosol Science 42 (2011) 428-445

Watanabe H., Ghadiri M., Matsuyama T., Ding Y., Pitt K.G., Maruyama H., Matsusaka S., and Masuda H. (2007): Triboelectrification of Pharmaceutical Powders by Particle Impact, Int. J. Pharm., 334, 149-155

A.M.C. Davies T. Fearn: “Back to basics: the principles of principal component analysis”, Spectroscopy Europe, 16, 20-23 (2004)

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Drug Delivery to the Lungs, Volume 29, 2018 – Nancy Rhein et al A closer look on the dispersion behaviour of Parteck ® M DPI – based interactive mixtures: improving the fine particle fraction by the addition of fines Nancy Rhein1, Gudrun Birk2 & Regina Scherließ1 1

Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany 2Merck KGaA, Frankfurter Straße 250, 64293 Darmstadt, Germany

Summary Different methods are known to improve the fine particle fractions of dry powder inhalation formulations. One example is the addition of fines. It is a simple approach to saturate active sites, reduce press-on forces or form easy-to-disperse API (active pharmaceutical ingredients)-fines-agglomerates. But this process is often a disadvantage for the consistency in dosing delivery, because an increase of fines leads to poorer powder flow behaviour. In this study the addition of up to 70 % fines to the Parteck® M DPI carrier was investigated with the aim to maximise fine particle fraction (FPF). Rheological measurements were performed to get a closer into the process of surface coverage with fines. The addition of fines maximised FPF for budesonide as well as for salbutamol sulphate blends. These increases of the FPF component were independent of the used inhaler devices. In this context the saturation of the indentations (active sites) was the most important effect. Also, an increase of the fluidisation energy was observed, indicating a rise in cohesion forces for the blends with a higher amount of fines. The small standard deviation associated with cascade impactor-based measurements of powder aerodynamic particle size distribution as well as with the measurements of the total metered dose are indicative that the Parteck® M DPI has a constant dosing behaviour even with the addition of high amounts of fines. Key Message The Parteck® M DPI preserved consistent dosing behaviour associated with increased FPF for salbutamol sulphate blends up to 67 % by adding up to 70 % of fines. The optimal content of fines was between 10 and 40 %, depending on the efficiency of the inhaler dispersion process. Introduction Dry powder inhalation utilising carrier-based blends is a well-known formulation strategy in local treatment of asthma and chronic obstructive pulmonary disease [1]. Currently, lactose is the most established carrier in such carrier-based powder blends [2]. Mannitol as an alternative to lactose has been discussed for some time [3]. In previous studies it was shown that the commercial mannitol quality Parteck® M DPI is a suitable mannitol carrier and exhibits good storage stability with constant fine particle fractions (FPFs) [4]. An average FPF of 30-40 % (depending on the inhaler used) [5] was achieved. To improve these FPFs different methods like adding fines, making the surface of the carrier particles hydrophobic with magnesium stearate [6] or by modifying the active pharmaceutical ingredient (API) as well as the carrier material can be used. A simple approach is the addition of fines to saturate active sites, reduce press-on forces or form API-fines-agglomerates to maximise the FPF [7]. However, this process is often a disadvantageous for consistency in dosing delivery, because an increase in fines results in higher cohesion forces and associated poor powder flow behaviour. The addition of InhaLac® 500 as model fines to the Parteck® M DPI was investigated in the present study, with the aim to maximise FPF content. Rheological measurements were also performed to get a closer look into the process of surface coverage with fines. The impact of the used dispersion principle was examined by using three different inhaler devices (one capsule-based inhaler and two reservoir-based inhalers). Materials and Methods Materials: Parteck® M DPI (Merck KGaA, Darmstadt, Germany; mean particle size  S.D. of 114 ± 9 µm, span: 2.3) served as carrier material in all blends. Micronised budesonide (BUD, mean particle size  S.D. of 1.45 ± 0.03 µm; Farmabios,S.p.A., Cropello Cairoli, Italia) and micronised salbutamol sulphate (SBS, mean particle size  S.D. of 1.61 ± 0.03 µm; Lusochimica S.p.A, Peschiera Borromoo, Italy) were used as model APIs in blend preparation, aerodynamic performance experiments and shot weight testing. InhaLac® 500 was selected as fines (mean particle size  S.D. of 3.16 ± 0.09 µm; Meggle Excipients and Technology, Wasserburg, Germany). Scanning electron microscopy (SEM): Particle morphology and surface were examined by SEM. Samples were investigated with a PhenomXL (Phenom-World BV, Eindhoven, the Netherlands) using the BS detector and a working voltage of 10 kV.

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Drug Delivery to the Lungs, Volume 29, 2018 - A closer look on the dispersion behaviour of Parteck ® M DPI – based interactive mixtures: improving the fine particle fraction by the addition of fines Preparation of powder blends: In this study the process of blend preparation consisted of two steps. In the first step the InhaLac® 500 was blended with Parteck® M DPI for 3 x 5 min in the Turbula® tumble blender (Type T2C, Willy A. Bachhofen AG Maschinenfabrik, Basel, Switzerland) at 42 rpm. After every 5 min a sieving step was introduced to destroy potential agglomerates. In the second step the API (in a concentration of 2 % by mass) was added by using the same blending conditions. Blends were produced with an increasing amount of fines up to 70 % by mass in 10 %-steps. Placebo blends only containing fines and a binary blend of BUD and carrier and of SBS and carrier, respectively, were prepared for the powder rheology measurements. A double-sandwichweighing method (weighing order: carrier-API/fines-carrier-API/fines-carrier) was used for all blending steps. Ten samples (8-12 mg) of each powder mixture were taken for homogeneity testing and API content was analysed by reversed phase high performance liquid chromatography (RP-HPLC). Blends were judged to be homogeneous at a relative standard deviation (RSD) of less than 5 % and a recovery above 95 %. Shot weight testing for the Novolizer®: The mass of one metered dose could directly be weighed on an analytical balance (AT106 Comparator, Mettler Toledo, Switzerland) by using a disassembled Novolizer® device (without mouthpiece and cyclone). For each blend 10 doses were evaluated and results are given together with their standard deviations. Shot weight testing was done with API blends. Cascade Impaction analysis: Investigation of the aerodynamic particle size distribution was performed with a Next Generation Impactor (NGI, apparatus E, European Pharmacopoeia 9.1) utilising three commercially available inhaler devices (two reservoir-based devices (Easyhaler® and Novolizer®) and one capsule-based device (Cyclohaler®)). Flow rates were adjusted to ensure 4 kPa pressure drop over the devices according to the Ph. Eur. procedure and cutoffs were calculated accordingly by the software. The stage collection cups were coated with a Brij 35, ethanol and glycerol (15:51:34 % by mass) mixture to avoid re-entrainment of particles. Samples were collected by a methanol-double distilled water mixture (75:25) for BUD and with pure double distilled water for SBS. BUD and SBS content was identified by RP-HPLC. The cascade impactor-measured data were evaluated with the CITDAS 3.0 software (Copley Scientific, Nottingham, UK). The fine particle fraction below 5 µm (of emitted dose) is calculated from the resulting aerodynamic particle size distribution. All impaction tests were done in triplicate and measured at constant conditions (21 °C and 45 % relative humidity). Aeration testing: Fluidisation energy measurements were conducted with a Freeman FT4 powder rheometer (Freeman Technology, Tewkesbury, UK). 10 g of the placebo blends were filled in a 25 mm x 35 mL bore borosilicate vessel and connected to an aeration control unit. Each analysis consisted of 8 test cycles. During these cycles the air velocity was increased stepwise in a scheme adapted to each powder, until the powder was completely fluidised and the fluidisation energy level was no longer affected by a change in air flow. At this point the required energy during the downward movement to move the 23.5 mm blade through the powder reached a plateau, which corresponded to the fluidisation energy. Reported data are the average of three replicate tests. Results and Discussion Morphology of the powder blends, content uniformity and shot weight testing The Parteck® M DPI is a spray granulated mannitol with a wide particle size distribution and a unique, large, rough surface area which exhibits many indentations. Figure 1 presents the pure Parteck® M DPI and all placebo blends up to 70 % of fines. InhaLac® 500 was used, because of its commercial availability, in further studies this product will be replaced with mannitol fines. It was visible from SEM images that a high amount of fines adhered to the carrier particles and only few fines were unbound. With higher concentration of fines, it was evident that the surface of the carrier became increasingly saturated with fines. This principle aligns with knowledge from ternary mixtures with lactose as carrier material. Furthermore, it was assumed that the dosing behaviour was not affected by the low amount of unbound fines. This hypothesis was verified via shot weight testing (Fig. 2 right).

Figure 1: SEM pictures of placebo blends with Parteck® M DPI and an increasing amount of InhaLac® 500 (indicated by percentage), magnification 500x

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Drug Delivery to the Lungs, Volume 29, 2018 – Nancy Rhein et al All API blends had an RSD of less than 5 % (Fig. 2, left). The mean value for BUD blends was 3.2 % and for SBS blends 2.2 % RSD. A tendency to higher RSD was seen in blends with an increasing amount of fines (≥ 30 %). Figure 2 right depicts the average metered dose including the standard deviation from the Novolizer ® as a function of fines content. The metered dose increased in comparison to the binary API carrier blends with the addition of 10-30 % InhaLac® 500. This outcome is believed to have happened because the indentations (active sites) became saturated and the density of the powder increased thereafter. Therefore the mass of one dose increased even though the same volume was metered from the Novolizer®. At higher concentrations, the metered dose declined, because of the formation of agglomerates as a possible explanation. The air contained in these agglomerates caused the density of the powder to drop again. Consequently, the metered dose decreased, this was also seen by Hertel et al [8]. Despite the amount of fines, the standard deviation remained low. Thus, over a very large concentration range of InhaLac® 500, a constant good metering capacity was reached.

BUD

Metered dose/mg

SBS

RSD/%

4 3 2 1 0

Amount of InhaLac 500/%

SBS

10 8 6 4 2 0

BUD

Amount of InhaLac 500/%

Figure 2 – left: Relative standard deviation (RSD) in % of all investigated blends based on ten randomly taken samples; right: metered dose in mg tested manually with the Novolizer® device ± standard deviation, n = 10

Aerodynamic performance in comparison to the powder rheology measurements The cascade impactor-based analysis performed with the three different inhalers showed that the addition of fines increased the FPF. The optimal concentration of InhaLac® 500 was between 10 and 40 %, depending on the inhaler type (Fig. 3). 30 % by mass fines content was the ideal amount to maximise the FPF for both drugs (from 28.0 to 53.9 % for BUD and from 35.0 to 61.6 % for SBS) for the Novolizer®. Higher fines contents caused the FPF value to drop. This decrease can be explained by the formation of agglomerates, which the Novolizer ® cannot disperse adequately at a concentration of about 40 % of fines content. When the indentations were saturated with fines, the fines were distributed on the surface of the carrier particles and could form API-finesagglomerates. Also based on the fluidisation energy, this formation of agglomerates was indicated by an increase in the cohesive forces at 40 % fines [9].

50 40

10 0

Novolizer - SBS Easyhaler - BUD

30 40 50 Amount of InhaLac 500/%

Easyhaler - SBS Cyclohaler - BUD

Cyclohaler - SBS Fluidisation energy

Fluidisation energy/mJ

Fine particle fraction/%

Novolizer - BUD

Figure 3: - Fine particle fraction in % (of emitted dose) of all powder blends depending on the amount of InhaLac 500 under use of three different devices (Novolizer®, Easyhaler® and Cyclohaler®) compared to the fluidisation energy in mJ of placebo blends; n = 3, error bars = standard deviation

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Drug Delivery to the Lungs, Volume 29, 2018 - A closer look on the dispersion behaviour of Parteck ® M DPI – based interactive mixtures: improving the fine particle fraction by the addition of fines In the case of the Cyclohaler®, the optimal concentration of fines was observed at 40 % increasing the FPF from 34.0 to 54.0 % for BUD and from 42.0 to 67.0 % for SBS blends. Afterwards, no significant changes were seen and no further increase in the FPF values. This stagnation of the FPF showed that the operation of the Cyclohaler® was more efficient than that of the Novolizer® in dispersing the agglomerates consisting of API and fines. The largest increase in FPF was observed with an addition of 10% InhaLac® 500 with these inhalers, both of which have an efficient dispersing mechanism. Saturation of the active sites, the buffer effect and the formation of agglomerates are considered important factors [7] in this concentration range. The ideal concentration of fines was 10 to 20 % for the Easyhaler®, which generates the powder aerosol without any dispersing baffles. A rise in FPF from 3.0 to 15.0 % for BUD and from 5.0 to 13.5 % for SBS blends could be seen. Subsequently, the FPF dropped again, because the Easyhaler® was unable to disperse the interactive mixture with higher fines contents. SBS achieved higher FPFs than BUD when comparing the performance of both drugs in these inhalers. This outcome can be explained by the different morphology of both micronised APIs. SBS had a needle-like shape so that particles possessed a smaller aerodynamic size compared to their physical size based on the longest dimension. In contrast, budesonide particles were almost spherical. Despite this difference in absolute FPF, SBS and BUD showed the same trends in the respective inhalers. Again, the low standard deviation reflected the good and constant dosing capability of Parteck® M DPI in reservoir-based inhalers. Conclusion With this study it has been shown that the addition of fines to tune aerodynamic performance, already established for lactose-based interactive blends, also works in the rough, spray-granulated mannitol when dispersed by the Parteck® M DPI. The addition of fines maximised the FPF for both APIs (SBS and BUD) studies. Saturation of the indentations (active sites) by the adhesion of fines appears to have been the most important effect observed. At a fines concentration of more than 40 % fine particles are distributed over the Parteck® M DPI carrier surface and agglomerates containing the API accumulate. These agglomerates are difficult to disperse from the inhalers that were studied. An increase of the fluidisation energy could be observed with the aid of powder rheological measurements and thereby a rise in cohesion forces for the blends with higher amounts of fines. The Parteck® M DPI has a good and constant dosing behaviour even with the addition of high amounts of fines, as demonstrated by relatively low standard deviations associated with the measures derived from cascade impaction analysis as well as replicate measurements of the metered dose show. Mannitol carrier particle-based technology could have potential for dispersing efficiently medium to high dose therapy utilising interactive mixtures. References 1

Timsina M P, Martin G P, Marriott C, Ganderton D, Yianneskis M: Drug Delivery to the respiratory tract using dry powder inhalers. In: International Journal of Pharmaceutics, Int J Pharm 1994; 101: pp1–13.

Rahimpour Y, Kouhsoltani M, Hamishehkar H: Alternative carriers in dry powder inhaler formulations. In: Drug Discovery Today 2014; 19 (5), pp. 618–626.

Steckel H, Bolzen N; Alternative sugars as potential carriers for dry powder inhalations. In: International Journal of Pharmaceutics 270 (1-2), pp. 297–306. DOI: 10.1016/j.ijpharm.2003.10.039.

Rhein N, Birk G, Scherließ R: Storage Stability of a Novel Mannitol Carrier in Dry Powder Inhalation Formulation. (Abstract). Presented at: RDD, Antibes, France, April 25-28, 2017

Rhein N, Birk G, Scherließ R: Influence of different inhalers on fine particle fraction of mannitol carriers in dry powder inhaler formulations. (Abstract). Presented at: World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology. Glasgow, UK; 2016.

Tay T, Das S, Stewart P; Magnesium Stearate increases salbutamol sulphate dispersion: What is the mechanism. In: Int J Pharm 383 2010, pp. 62–69.

Grasmeijer F, Lexmond A J, van den Noort M, Hagedoorn P, Hickey A J, Frijlink H W, de Boer A H; New Mechanisms to Explain the Effects of Added Lactose Fines on the Dispersion Performance of Adhesive Mixtures for Inhalation. In: PLoS ONE 9(1): e87825. doi:10.1371/journal.pone.0087825

Hertel M, Schwarz E, Kobler M, Hauostein S, steckel H, Scherließ R; Powder flow analysis: A simple method to indicate the ideal amount of lactose fines in dry powder inhalations. In: International Journal of Pharmaceutics 535 (2018), pp. 59–67.

Freeman Technology, 2006a. Instruction Documents: W7015 Aeration Method. Pp. 1-9.

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Drug Delivery to the Lungs, Volume 29, 2018 –Thomas Wutscher et al. Impact of Relative Humidity and Powder Filling Level on the Electrostatic Charging Behaviour of Different Capsule Types Thomas Wutscher1,2, Sarah Zellnitz1, Mirjam Kobler3, Francesca Buttini1,4, Laura Andrade5, Veronica Daza5, Alberto Mercandelli6, Stefano Biserni6, Susana Ecenarro Probst7, Johannes Khinast1,2 & Amrit Paudel1,2 Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, 8010 Graz, Austria ² Institute of Process and Particle Engineering, Graz University of Technology, Inffeldgasse 13, 8010 Graz, Austria ³ MEGGLE Excipients and Technology, Megglestraße 6-12, 83512 Wasserburg, Germany 4 Food and Drug Department, University of Parma, Parco delle Scienze 27, 43121 Parma, Italy 5 Laboratorios Liconsa, S.A. C/ El tejido 2, 19200 Guadalajara, Spain 6 MG2, Via del Savena 18, 40065 Pianoro, Bologna, Italy 7 Qualicaps Avda. Monte Valdelatas 4, 28108 Alcobendas, Madrid, Spain 1

Summary Electrostatic charging of powders and solid surfaces (e.g. capsule, inhalers) is a complex phenomenon that can negatively impact the performance of inhalation products by particle aggregation or segregation and adherence to surfaces. Tribo-charging plays a major role during cohesive/fine powder processing and might undesirably impact certain process steps during DPI manufacturing. The present study aimed to understand the charging tendencies of different types of HPMC (thermally and chemically gelled) and gelatin capsules when transported over stainless steel and polyvinylchloride (PVC) surfaces, after storage at relative humidity (RH) of 22% and 51%. Furthermore, the impact of powder fill level in the capsule on charging propensity was investigated. Results showed that all capsules charge positively on both steel and PVC surfaces. Charging tendency of capsules was in the range of 0.5 to 2.5 nC/g. Capsules appeared to get charged to the higher extent when passed over the PVC surface as compared to steel. Both HPMC capsules tend to attain the lower extent of charge at 22% RH as compared to that at 51% RH. The charge attained by gelatin capsules was independent to the storage RH. Filled capsules show a lower extent of charging compared with empty capsules. These varying charging tendencies for different capsules passing over different surfaces as a function of fill level and RH can provide valuable information for the capsule-based DPI product and process development. Key Message HPMC capsules charge less when stored dry, whereas charging of gelatin capsules is unaffected by the storage humidity. Filling of the capsules by DPI powder blends tends to reduce the charging tendency. Overall, capsules charge positively and to a higher extent when moving on PVC compared with steel surfaces. Introduction Tribo-charging is a complex phenomenon that can largely affect quality, safety and efficiency of pharmaceutical products1. For dry powder inhaler (DPI) products, tribo-charging can lead to particle agglomeration and particle adherence to surfaces and subsequently raise problems in terms of mixing homogeneity and dosing accuracy2. Further, tribo-charging can influence the release of the powder formulation from the inhaler resulting in a variable performance3. Moreover, besides its impact on formulation and product performance, tribo-charging plays a major role during processing and might negatively impact certain process steps during DPI manufacturing. So far, most studies have focused on the tribo-charging of powders and formulations used in DPIs. However, understanding of the process induced- tribo-charging of capsules is equally important. Tribo-charging occurs when two materials are brought into contact and subsequent frictional forces like rubbing, rolling, sliding and or impact act to create charge transfer4. During capsule filling, tribo-charging of the capsules induced by the contact with the capsule filler components could lead to rocking and jumping of the capsules further increasing the amount of rejected capsules during manufacturing. Likewise, charging of capsules when in contact with polymers e.g. PVC surface might impact the aerosolisation performance of the DPI in use. This phenomenon is still poorly understood due to the multitude of factors affecting tribo-charging, such as particle properties like size, shape, chemical structure and surface and solid-state, to environmental conditions and moisture content. These factors have been reviewed in detail elsewhere1,5. The present study focuses on the impact of different capsule types on the charging behaviour when transported over steel and PVC. A gelatin and two types of HPMC capsules (a thermally gelled and a chemically gelled) have been compared. Further, the impact of capsule storage conditioning on charging has been analysed by measuring the selected capsules after storage at 22% RH and 51% RH. Additionally, the charging tendencies of empty capsules and powder-filled capsules have been analysed in order to see if and how the capsule fill level affects the charging behaviour. This study was intended to get a better understanding of the charging behaviour of distinct capsule types depending on both the fill level and storage conditions.

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Drug Delivery to the Lungs, Volume 29, 2018 - Impact of Relative Humidity and Powder Filling Level on the Electrostatic Charging Behaviour of Different Capsule Types Materials and Methods Capsules Three different capsules of size 3 were used for the experiments in the present study (Quali-G® and Quali-V®-I obtained from Qualicaps Europe, S.A.U., Spain and VCaps® Plus, Capsugel, France). Quali-G® as a gelatin capsules were compared to the performance of the two hydroxypropylmethylcellulose (HPMC) capsules, chemically gelled Quali-V®-I and thermally gelled VCaps® Plus. Preparation of adhesive mixtures A model blend was prepared for evaluating the charging tendency of filled capsules for inhalation. This blend comprised of 1% (w/w) jet-milled budesonide and 99% (w/w) α-lactose monohydrate (IH230). Budesonide was obtained from Laboratorios Liconsa, S.A. (Chemo Iberica, S.A., Spain). The model carrier, InhaLac® 230 was contributed by Meggle (Molkerei MEGGLE Wasserburg GmbH & Co. KG, Germany). A total batch size of 200g was produced. Therefore, the carrier and the API were transferred to a 500ml steel container (30-35% fill level) and layered via the sandwich method. The container was then put into a low shear tumble blender (T2F, Willy A. Bachofen AG Maschinenfabrik, Switzerland) for 20 minutes at 20 revolutions per minute (rpm). This stage was followed by another sieving through a 400µm sieve and another blending step for 20 minutes at 20 rpm. The final blends were kept in the sealed metal containers until capsule filling was performed. Ten samples of the blend were taken (3 from the top, 4 from the middle and 3 at the bottom of the container), dissolved in 20ml of buffer (ACN:purified water, 60:40) and subsequently analysed for budesonide concentration in order to ensure mixture homogeneity. The budesonide content was quantified via a validated HPLC method. The blend homogeneity was expressed as the coefficient of variation of the mean drug content. Conditioning Capsules were placed in glass vials and stored in two separate desiccators at different humidity levels over saturated salt solutions of potassium acetate, for a RH of 22% at 20°C, and magnesium nitrate, for a RH of 51% at 20°C for at least seven days. Karl-Fischer-Titration The water content of the gelatin and HPMC capsules was determined with Karl-Fischer titration (Titroline 7500 KF, SI Analytics, Mainz, Germany) after one week of storage at 22% and 51% RH. The capsules were extracted for 300 seconds in a mixture of CombiMethanol and dry formamide (1:1) before being added to the titrator cell. As titrant Combititrant5 (Merck KGaA, Germany) was used. Capsule filling Capsule filling of the stored capsules, was done manually on an analytical scale (Denver SI- 234A, Denver Instrument GmbH, Germany) to get target capsule fill weights of 25mg and 75mg per capsule. Per fill weight, 15 capsules were filled within a range of 25 ± 5mg and 75 ± 5mg. Capsules were filled under ambient conditions of about 40% RH and 23°C. Afterwards, they were stored in desiccators at 22% and 51% RH over night before capsule measurements were done. Charge measurements A GranuCharge™ (Granutools, Belgium) was used in a conditioned room (RH of 50%± 3%) to measure the charging tendencies (n=5) of the capsules stored at different relative humidities. The influence of environmental conditions on the measurement was limited due to a very short exposure time of the capsules to the environment of about 5 minutes. The RH within the instrument chamber was monitored as well and remained constant within a range of 50%± 2% throughout the measurements. The initial charge to mass ratio (q0) of the capsules was evaluated by dispensing them into the inbuilt Faraday cup of the GranuCharge™. Then, 15 filled capsules with a total mass of 1.2g (25mg powder fill weight) and 1.9g (75mg powder fill weight), as well as a total mass of 1.2g of empty capsules were fed via rotational feeder into a steel or a PVC V-tube. After dropping the samples in the Faraday cup, the charge to mass ratio was calculated again (q1). The charging difference, namely the charging tendency was obtained by subtracting initial charge from total charge to mass ratio, represented as q1-q0. The limit of accuracy of the obtained charge values was ±0.5nC/g.

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Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C;Thomas Wutscher et al. Results and Discussion Figure 1 compares the charging tendency of the three capsule types over stainless steel and PVC after storage at two different RHs. Overall, all capsules have a negative initial charge (q0) (data not shown) and were charged positively when transported over steel as well as PVC. When transported over steel surface, chemically gelled HPMC capsules showed the lowest charging tendency, followed by gelatin and thermally gelled capsules. No effect of capsule storage conditions could be observed. However, all capsules showed higher positive charging when transported over PVC-tubes. The higher charging behaviour on the PVC surface can be explained by the fact that non-conductive materials, like PVC, can hold charge better than conductive materials like steel. This is also reflected by the work function values 4.4eV6 and 4.85-5.13eV7 for steel and PVC, respectively. For the latter, electrons of the capsules can be hold back better on PVC when transported over the tube wall (frictional forces), which leads to the higher charging tendency of all materials on PVC. When in contact with PVC, differences between the different capsule materials after storage at 51% RH can be observed. Here chemically gelled capsules tend to charge more compared to gelatin and thermally gelled capsules. No significant differences between the three capsule types were observed when stored at 22% RH.

Figure 1 - Charging tendencies (q1-q0) for empty capsules stored at different RH levels on stainless steel (A) and PVC (B), mean (n=5)

When HPMC capsules were transported over PVC, the capsules stored at 22% RH had a lower charge density than those stored at 51% RH. This trend is in contrary to commonly reported cases8,9 where materials with higher water content or generally a higher RH tend to decrease charge acquisition. However, most of these studies focused on charging behaviour of powder materials. Whereas others like Biegaj et al.10 worked on functionalized glass beads and state that with increasing RH a monolayer of water is built up depending on the functional groups on the surface. A reduction in charge by moisture was reported only after the complete formation of the monolayer. Before that point, the charge can rise with increasing humidity. For gelatin capsules, the charging propensity was independent of the storage RH. This outcome could possibly have been related to the higher moisture content of gelatin capsules. The gelatin capsules had a higher water content at both storage conditions (11.82% at 22% RH and 15.18% at 51% RH) compared to the HPMC capsules (Vcaps Plus 3.53% at 22% RH and 6.86% at 51% RH; Quali V I 3.59% at 22% RH and 7.74% at 51% RH). Even at 51% RH, the moisture content of HPMC capsules was lower compared with that observed for the gelatin capsules at 22% RH. Electrical conductivity as well as discharging rates are increased by a higher water content and therefore triboelectric effects are reduced and lead to a lower charge density11,12. The quality of the blend was assessed before capsule filling. A homogenous mixture having a relative standard deviation of 4.75% from the mean drug content was achieved. Figure 2 shows the comparison of the three different capsules after storage at 51% RH and filled with 25mg and 75mg of DPI blend, when transported over steel and PVC surface. Results over stainless steel showed a decreasing trend of charging tendency with increasing fill weight of the capsules, irrespective of the capsule material used (Figure 2A). Overall, the charging tendency of the three distinct capsule types over steel was in the range between 0.5 and 1nC/g. When transported over PVC, filled capsules charged to a higher extent compared to stainless steel (Figure 2B). For gelatin and thermally gelled capsules, no differences between 25mg and 75mg filled capsules were observed but compared to empty capsules, filled capsules showed lower charging tendencies. The net charge of the Inhalc230-budesonide blend was negative (â&#x20AC;&#x201C;1.03nC/g). The most likely explanation is that there was a transport of charge from the outer surface of the capsule to the inside and therefore charge is better distributed, which results in less net surface charge. So, the decrease of the charging tendency for the filled capsules could be possibly related to the negative charge (q0) of the DPI blend material. Another explanation for the decrease in the charge attained by the filled capsules could be that the filled capsules, being heavier than the empty ones are transported faster through the V-tube and therefore had less time for friction to occur against the wall material.

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Drug Delivery to the Lungs, Volume 29, 2018 - Impact of Relative Humidity and Powder Filling Level on the Electrostatic Charging Behaviour of Different Capsule Types

Figure 2 - Charging difference of capsules, conditioned at 51% RH with different fill levels (0mg, 25mg and 75mg) on stainless steel and PVC, mean (n=5)

Conclusion The overall capsule charge is positive and higher when transported over PVC. Further, it was shown that storage RH level had an impact on capsule tribo-charging, which could have an impact on the performance and quality of some DPI products. HPMC capsules charged lower when stored at 22% RH, contrary what is typically reported in literature. Further investigation is needed to understand the relatively lower charging of capsules stored at drier conditions compared to those stored at moderate humidity (i.e. 51% RH). Therefore, our future focus will be on the influence of varying RH (between 11 to 60% RH) systematically on capsule charging behaviour. Different materials for the V-tubes will also be investigated to better mimic capsule behaviour in either distinct inhaler designs, or during different powder processing steps. Experiments with different filling materials and fill weight levels are planned to provide a more detailed examination of the fill weight dependent charging effects. The goal is to understand and connect tribo-charging of capsules to powder release from the capsule and DPI performance as well as capsule performance during processing (e.g. ideal processing and capsule storage conditions). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Desai P, Jin Tan B, Liew C, Chan L, Sia Heng P. Impact of Electrostatics on Processing and Product Performance of Pharmaceutical Solids. Curr Pharm Des. 2015;21(40):5923–9. Karner S, Urbanetz NA. Arising of electrostatic charge in the mixing process and its influencing factors. Powder Technol [Internet]. 2012 Aug [cited 2015 Jan 9];226:261–8. Available from: http://linkinghub.elsevier.com/retrieve/pii/S003259101200304X Telko MJ, Kujanpää J, Hickey AJ. Investigation of triboelectric charging in dry powder inhalers using electrical low pressure impactor (ELPITM). Int J Pharm [Internet]. 2007 May 24 [cited 2015 Jan 9];336(2):352–60. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17218072 Wong J, Chan H-K, Kwok PCL. Electrostatics in pharmaceutical aerosols for inhalation. Ther Deliv. 2013;4(8):981–1002. Kaialy W. A review of factors affecting electrostatic charging of pharmaceuticals and adhesive mixtures for inhalation. Int J Pharm. 2016;503:262–76. Lee J-K, Shin J-H. Triboelectrostatic separation of pvc materials from mixed plastics for waste plastic recycling. Korean J Chem Eng. 2002;19(2):267–72. Common Work Functions. Pulsed Powder. 2018. Park CH, Park JK, Jeon HS, Chun BC. Triboelectric series and charging properties of plastics using the designed verticalreciprocation charger. J Electrostat [Internet]. 2008;66(11–12):578–83. Available from: http://dx.doi.org/10.1016/j.elstat.2008.07.001 Rowley G, Mackin LA. The effect of moisture sorption on electrostatic charging of selected pharmaceutical excipient powders. Powder Technol. 2003;135–136:50–8. Biegaj KW, Rowland MG, Lukas TM, Heng JYY. Surface Chemistry and Humidity in Powder Electrostatics: A Comparative Study between Tribocharging and Corona Discharge. ACS Omega. 2017;2(4):1576–82. Lumay G, Traina K, Boschini F, Delaval V, Rescaglio A, Cloots R. Effect of relative air humidity on the fl owability of lactose powders. J Drug Deliv Sci Technol. 2016;35:207–12. Galembeck F, Burgo TAL, Balestrin LBS, Gouveia RF, Silva CA, Galembeck A. Friction, tribochemistry and triboelectricity: Recent progress and perspectives. RSC Adv. 2014;4(109):64280–98.

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Drug Delivery to the Lungs, Volume 29, 2018 - S. Steiner Establishing the Vitrocell® Powder Chamber as a particle size-selective platform for in vitro dry powder testing S. Steiner1, M. Hittinger2, K. Knoth2, H. Gross2, S. Frentzel1, A. Kuczaj1, J. Hoeng1, M. Peitsch1, T. Krebs3 1

PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchâtel, Switzerland 2 PharmBioTec GmbH, Science Park 1, 66123 Saarbrücken, Germany 3 Vitrocell® Systems GmbH, Fabrik Sonntag 3, 79183 Waldkirch, Germany

Summary Inhaled aerosol particles deposit on the epithelia of the respiratory tract mainly due to sedimentation, impaction and diffusion that, in their kinetics, strongly depend on particle size and flow patterns. As a result, the deposition efficiency of different sized particles is variant in each regions of the respiratory tract 1. This has to be considered when testing dry powders for their biological activity in vitro, as the biological response to particle deposition on in vitro models is a function of the combined action of particle mass, surface and number 2. The Vitrocell® Powder Chamber was developed for the size-selective deposition of dry powders on in vitro test systems in order to simulate the regionally different deposition efficiency of different particle sizes. We tested the performance of the Vitrocell® Powder Chamber in cell-free exposure experiments using dry powders as test material. Our findings demonstrate that the system allows control of the size distribution of the deposited powder within the targeted ranges. The deposition was uniform across the area of the exposure chambers, but we observed relatively high variations in the powder mass deposition in individual exposure chambers. Taken together, our results demonstrate that the Vitrocell® Powder Chamber is a valuable tool for in vitro testing of dry powders that could be candidates for inhalable therapeutics or consumer products. Key Message We demonstrate that in the Vitrocell® Powder Chamber, dry powders can be tested in a particle size specific manner in vitro. The system therefore provides a valuable tool for screening inhalable dry powder formulations for their bioactivity or therapeutic potential in course of pre-clinical phases of product assessment. Introduction The pre-clinical product assessment typically includes in vitro studies in which a large number of candidate formulations are tested using high-throughput methods. If complex biological test systems, such as primary human cell cultures, are used, the relevance of the results obtained in in vitro studies is thereby strongly dependent on the way in which the tested formulations are applied. In fact, the applied physiological conditions need to be mimicked as closely as possible in order not to generate biological artefacts3. This is highly challenging if inhalable aerosolized therapeutics or consumer products are tested in vitro. Aerosol testing is preferably performed at the air-liquid interface where the biological test system is grown with their basal surfaces in contact with media, and the top of the cellular layer is exposed to the aerosol. This complex exposure mode requires the structural and functional complexity of the human respiratory tract to be accounted for. The main physical mechanisms governing aerosol deposition in the human respiratory tract are responses due to gravity, inertia and diffusion1. Whereas gravitation and inertia mainly act towards particles in the size range above 1 µm, particles in the size range below 0.1 µm are mainly affected by diffusion. Due to the regionally heterogeneous structure and function of the respiratory tract, the relative relevance of the influences of these three forces changes along the path from the nasal and oropharyngeal cavities through the airways of the lungs to the distal alveolar spaces. As a consequence, the size distribution of the particles depositing along this path varies as a function of the geometry and flow pattern in a given region of the respiratory tract and the particle size distribution of the aerosol fraction that reaches this region. If aerosols containing solid particles are tested, this has direct implications for aerosol dosimetry. Firstly, solid particles of different sizes may display different dissolution kinetics and the kinetics of the transfer of compounds from the epithelial surface into the bloodstream differ between the different regions of the respiratory tract. Hence, particle sizes have a profound impact on absorption kinetics and the overall pharmacokinetics of the active aerosol components. Secondly, it is known that cells of the airway or respiratory epithelia may respond to particle numbers, particle surface areas and particle masses and that neither of the three alone describes the deposited aerosol dose comprehensively 2. So far, in vitro aerosol exposure systems have therefore been unable to simulate the regiospecific aerosol deposition in the respiratory tract. The Vitrocell® Powder Chamber was recently developed by Vitrocell® GmbH (Waldkirch, Germany4) as an advanced version of the Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC5). It offers the possibility to sort particles in a dry powder aerosol by size prior to deposition on a cell culture. The system thereby takes advantage of the size specific difference in the settling velocity of aerosol particles. In the present work, we tested the performance of the system in absence of biological test systems. The objective was to determine the feasibility of the system for routine in vitro testing of aerosolized dry powders with a particular focus on the system’s ability to reliably select the particle size range to be used for exposures.

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Drug Delivery to the Lungs, Volume 29, 2018 - Establishing the Vitrocell® Powder Chamber as a particle sizeselective platform for in vitro dry powder testing Functional principle of the Vitrocell® Powder Chamber: The Vitrocell® Powder chamber and its working principle are described in Figure 1. The system consists of four main parts: the distribution head at which the inlet to the system is located, the sedimentation tubes, the movable exposure tray comprising four exposure chambers, and a control unit.

Figure 1: A) the Vitrocell® Powder Chamber comprising 1) the distribution head with powder inlet, 2) the sedimentation tubes, 3) the movable exposure tray and 4) the control unit. B) functional principle of the Vitrocell® Powder Chamber and the effect of the system on the particle size distribution: B1) the test powder is placed at the inlet and a vacuum is generated inside the system. B2) When the inlet is opened, the air entering the system aerosolizes the powder and transports it into the sedimentation tubes. Large particle agglomerates are removed by inertial impaction in the distribution head. B3) Large particles present in the sedimentation tubes display a higher settling velocity than small ones and are removed from the aerosol by sedimentation. The level to which the particle size distribution is shifted towards smaller sizes is thereby a function of the duration of the sedimentation time. When the targeted upper cut-off particle size is reached, the exposure tray is shifted, whereby the exposure chambers are moved below the sedimentation tubes and the exposure is started (B4). The lower particle size cutoff can be defined by the duration of the exposure time, for example by moving the exposure tray back again; the particles with the smallest settling velocity can be kept from reaching the exposure chambers.

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Drug Delivery to the Lungs, Volume 29, 2018 - S. Steiner The exposure tray provides four exposure chambers in which cell cultures (or cell-free test systems) present on transwell inserts can be placed. The basal side of the cell culture inserts can be kept in contact with cell culture medium, the apical side is in direct contact with the air in the exposure chambers. Alternatively, Quartz Crystal Microbalances (QCMs) can be placed in the exposure chambers for monitoring powder mass deposition during the exposure in real-time. For exposure experiments, a vacuum is generated inside the distribution head and the sedimentation tubes and the test powder is loaded to the inlet. When the inlet is opened, the air entering the system passes the test powder and thereby generates shear forces that result in its aerosolization. The formed dry powder aerosol is then ‘stored’ in the sedimentation tubes for defined periods of time, during which the particles sediment on the blind field of the exposure tray. The particles’ settling velocity is thereby a function of their size: with increasing sedimentation time, the particle size distribution in the aerosol is shifted towards smaller sizes. When the targeted upper cut-of particle size is reached, the exposure tray is shifted, whereby the exposure chambers are placed below the sedimentation tubes and exposure is started. The aerosol is allowed to deposit on the cell cultures for a defined period of time, after which the exposures can be terminated by shifting the exposure tray back again. The particles remaining in the aerosolized form in the sedimentation tubes at this time point will deposit on the blind fields of the exposure tray again and will not reach to the biological test system. Materials and Methods: The test substance used in the present study was a dry powder consisting of polysaccharides and 5% nicotine by mass. The mass median aerodynamic diameter of the primary particles in the powder was 2.6 µm and irregularly shaped agglomerates with sizes above 100 µm in their largest dimension and large aspect ratios were formed during storage of the powder. Three exposure modes were defined for testing the system’s ability to deliver dry powder aerosols in a particle size selective manner and for testing deposition uniformity across the four exposure chambers (Table 1). The system parameters resulting in the targeted, particle size selective powder deposition were determined empirically. Table 1: System parameters used during dry powder exposures. Exposure mode

Loaded powder mass (mg)

Sedimentation time (seconds)

Exposure time (minutes)

Sedimentation tube length (cm)

Mode A

Mode B

Mode C

Mode A: deposition of the whole range of particle sizes reaching the sedimentation tube Mode B: removal of particles larger than about 35 µm (in humans reaching the head airways with low efficiency 1) Mode C: removal of particles larger than about 15 µm (in humans exclusively deposited in the extrathoracic airways 1 ) Powder mass deposition in the exposure chambers was monitored in real-time during the exposures using Quartz Crystal Microbalances (QCMs), or determined off-line either by imaging the deposited particles by scanning electron microscopy (SEM) or by quantification of the deposited nicotine by high performance liquid chromatography. For SEM analyses, SEM discs were placed into cell culture inserts (12-well format, Greiner Bio-one, Frickenhausen, Germany) and exposed to the dry powder aerosol. Before SEM imaging, the powder loaded discs were sputtered with a gold layer of ̴ 15 nm thickness (Sputter coater: Quorum Q150R ES, Quorum Technologies Ltd, East Grinstead, UK). SEM imaging (EVO HD15 from Carl Zeiss Microscopy GmbH, Jena, Germany) was performed at 5 kV acceleration voltage. The secondary electron detector was used. Image analysis, such as the calculation of Feret diameters of the deposited particles, was performed using the image processing software ImageJ. For HPLC analyses, 100 µL Phosphate buffered saline (PBS, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland, Ref# D8537) was pipetted into cell culture inserts (12 well format, Greiner Bio-one) and exposed to dry powder aerosol under the settings described above. Samples were analysed on an Agilent 1290 Infinity LC System (Agilent Technologies, Basel, Switzerland) using a ACQUITY UPLC Peptide CSH C18 Column (Waters, Elstree, UK). Ammonium bicarbonate at 10 mM pH 10.0 and Acetonitrile (60:40, isocratic) was used as a mobile phase. Nicotine was detected fluorometrically at 254 nm using a 1290 Infinity Variable Wavelength Detector (Agilent).

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Drug Delivery to the Lungs, Volume 29, 2018 - Establishing the Vitrocell® Powder Chamber as a particle sizeselective platform for in vitro dry powder testing Results SEM analyses confirmed that the system allowed control of the size distribution of the deposited dry powder within the ranges targeted in this work (Figure 2). Visual inspection of the SEM micrographs furthermore confirmed uniform powder deposition across the surface of the inserts. The results obtained from HPLC analyses, as well as the QCM readout, revealed a high variation in the powder mass deposition between individual experimental repetitions (high inter-repetition variation), whereas the uniformity of powder mass deposition within individual experimental repetitions was considerably smaller (low intra-repetition variation).

Figure 2: Cumulative size distribution of dry powder particles in the exposure chambers of the Vitrocell® Powder Chamber, measured by SEM analyses. Exposure mode A aimed at maximal powder deposition (no removal of large particles), with a sedimentation time of 0 seconds. Exposure mode B aimed at intermediate removal of large particles, with a sedimentation time of 10 seconds. Exposure mode C aimed at depostion of the smallest particles only, with a sedimentation time of 30 seconds. Error bars indicate standard deviation across 8 measurements Discussion and Conclusion We demonstrated that using the Vitrocell® Powder Chamber, dry powders containing individual particles in the size range of 2 µm, as well as particle agglomerates of a broad size spectrum, could be deposited on cell culture inserts in a particle size-selective manner. The weakness of the system is the relatively large variation in powder deposition between individual repetitions of exposure. We consider this variation as the result of difficulties in loading accurate powder masses to the inlet of the system and suboptimal aerosolization of the loaded powder. Optimization of the system structure and function at the powder inlet may improve the performance of the system. This work is currently performed by the supplier. Despite the observed variation in powder mass deposition, the system is an interesting tool for in vitro aerosol testing as the differences between particle sizes that are expected to deposit in different regions of the human respiratory tract can actively be simulated. This may considerably increase the relevance of in vitro aerosol testing, as the kinetics of particle dissolution - and thereby the overall pharmacokinetics of the active aerosol constituents - as well as cellular responses to the deposition of the particles per se can be modelled more accurately. In addition, the system allows conducting test exposures with as little as 20 mg of the test substance. This is an advantage particularly in the early phases of product testing when usually only limited amounts of the test substances are available, usually at high costs. References 1 2 3

4 5

Carvalho TC, Peters JI, and Williams Iii RO: Influence of particle size on regional lung deposition – What evidence is there? International Journal of Pharmaceutics. 2011;406:1-10. Oberdorster G, Oberdorster E, and Oberdorster J. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environmental health perspectives. 2005;113:823-839. Paur HR, Cassee FR, Teeguarden J, Fissan H, Diabate S, Aufderheide M, Kreyling WG, Hanninen O, Kasper G, Riediker M, Rothen-Rutishauser B, and Schmid O. In-vitro cell exposure studies for the assessment of nanoparticle toxicity in the lung-A dialog between aerosol science and biology. J Aerosol Sci. 2011;42:668-692. www.vitrocell.com. Hein S, Bur M, Schaefer UF, and Lehr C-M. A new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to evaluate pulmonary drug absorption for metered dose dry powder formulations. European Journal of Pharmaceutics and Biopharmaceutics. 2011;77:132-138.

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Drug Delivery to the Lungs, Volume 29, 2018 – Ioanna Danai Styliari et al. Physicochemical characterisation of inhalation grade lactose after the removal of intrinsic fines. Ioanna Danai Styliari1, Arian Mobli1 & Darragh Murnane1 1School

of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield, AL10 9AB, UK.

Summary Lactose is a common excipient in Dry Powder Inhaler (DPI) formulations, used as a carrier for the micronized drug particles. The presence of intrinsic lactose fines in the formulation influences its performance and their role and interactions between the lactose carrier and the micronized drug is still not fully understood. As a first step towards this investigation, “clean” lactose, with removed fines, was produced via wet decantation. Ethanol and isopropyl alcohol have been used in wet decantation, successfully removing lactose fines from the surface of the coarse particles. Differential Scanning Calorimetry (DSC) was employed to show that the powders maintained their crystalline character. Scanning Electron Microscopy (SEM) showed tomahawk-shaped particles in all the powders and some surface alteration occurring after decantation. An airflow titration method using laser diffraction (LDA) allowed the estimation of the removal of fines as well as the particle size distributions, while the non-polar and the polar components of the surface energy of the powders were calculated via Inverse Gas Chromatography-Surface Energy Analysis (iGC-SEA). As both solvents successfully removed fines, we propose the addition of isopropyl alcohol in the list of organic solvents suitable for this purpose. Key Message Removal of intrinsic fines from inhalation grade lactose monohydrate can be achieved via wet decantation, using isopropyl alcohol instead of ethanol. Introduction Lactose is used as an excipient in a plethora of inhaled formulations; its role is that of a coarse carrier particle bulking agent where the micronized drug particles (usually in the range of 1-5 μm) are attached [1]. The physicochemical properties of lactose, as well as the addition, or absence, of lactose fines has been shown to affect the fine particle fraction of a formulation and as such, its success or failure.[2–4]. However, the underlying interactions that take place between the carrier particles and the drug fines are still not fully understood. In an attempt to shed light in these complex interactions, a step-by-step process has been proposed; clean coarse lactose particles can be prepared via wet decantation [4], in order to produce a “clean” platform where the addition of lactose fines can be achieved in a controlled manner, followed by the addition of the micronized drug. In this present work we focus on the solvent selection for wet decantation; although Ethanol has been used before for the surface modification of lactose, the use of other organic solvents have not been reported. We investigated the use of isopropyl alcohol, as well as the established ethanol, in wet-decantation and characterised the “clean” lactose powders produced via Laser Diffraction Analysis (LDA), Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC) and Inverse Gas Chromatography- Surface Energy Analysis (iGC-SEA). Materials and Methods Inhalation grade lactose monohydrate, namely Lactohale 100 (sieved) and Lactohale 200 (milled), was provided by DFE Pharma (Goch, Germany). Organic solvents (Isopropyl Alcohol and Absolute Ethanol) were purchased from Fischer Scientific (Loughborough, UK). Wet decantation was adjusted following the protocol proposed by Islam et al.[4]. Approximately 20 g of lactose was dispersed in 500 mL of solvent (ethanol, isopropyl alcohol) in a conical flask. During the first run, the flask was sonicated for 3 mins to make a homogeneous suspension. The suspension was then allowed to settle for 15 min. The supernatant liquid was decanted through grade 1 filter paper, with extra care to minimise the disturbance on the precipitated powder at the lower part of the suspension. The supernatant was filtered and recycled back into the flask as a saturated solvent. The flask was then vigorously mixed (manually) for 2 min prior to the next settling run. The process was repeated until the supernatant liquid was clear. The decantation process was done with extra care to have minimal disturbance on lower parts of the suspension and prevent the removal of larger particles. In the final step the powders were wet-sieved (63 μm) and allowed to air-dry for 24 h. Particle morphology was investigated via Scanning Electron Microscopy (SEM). The powders were deposited onto adhesive carbon tabs (Agar Scientific G3357N), which were pre-mounted onto aluminium stubs (Agar Scientific JEOL stubs G306). Samples were then sputter coated with gold for 1 minute to achieve a thickness of around 30 nm (Quorum SC7620). The images were acquired using a JEOL 5700 scanning electron microscope, operated at 20kV, and a working distance of 10 mm. Differential Scanning Calorimetry (DSC) experiments were conducted in a DSC 200 (TA Instruments), calibrated with Indium as a reference material, at a heating rate of 10 oC/min. In all measurements nitrogen was used as the purge gas, flowing at a rate of 50 ml/min. Tzero sample pans with a pinhole lid were used for all the samples.

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Drug Delivery to the Lungs, Volume 29, 2018 - Physicochemical characterisation of inhalation grade lactose after the removal of intrinsic fines. Particle size measurements were performed on a Sympatec HELOS/RODOS Laser Diffraction (LDA) unit, using the ASPIROS dispersing system (dispersing aperture diameter 4mm, feed velocity 25 mm/s) (Sympatec GmbH, Clausthal-Zellerfel, Germany). The R5 lens (measuring range 4.5–875 μm) was fitted for the measurements. Powder was filled into the ASPIROS glass vials and was dispersed via vacuum suction. Airflow titration measurements were performed following a previously established protocol [5]. The primary pressure (PP) was manually set using the adjustment valve in the range 0.2–5.0 Bar and three measurements were taken at each pressure setting using freshly loaded powder. Particle size distributions were calculated using the Fraunhofer theory and were analysed in WINDOX 5.3.1.0 software, while further analysis was performed in MATLAB. Particle size measurements for a complete airflow titration curve were conducted on a single day. Specific surface area (SSABET) and surface energy (SE) analysis were conducted using an Inverse Gas Chromatography Surface Energy Analyser (iGC-SEA, Surface Measurement Systems Ltd, UK). Approximately 1 g for the lactose was packed into silanised iGC glass columns (internal diameter 4mm). Prior to any measurements, the columns were conditioned using helium carrier gas at 10 scc/min for 2 h at 30 oC and 0% RH. Methane gas was injected at the start and the end of the experiments for the dead volume calculation. SSABET was calculated via the Brunauer-Emmett-Teller (BET) theory, based on the n-octane adsorption isotherm data [6]. For the surface energy, the columns were equilibrated as mentioned above. Non-polar probes (n-undecane, n-decane, n-nonane, n-octane and n-heptane) and polar probes (dichloromethane, ethyl acetate, ethanol, acetone and acetonitrile) were injected in the column at a range of surface coverages (n/n m). Following an analysis method previously reported[5], the dispersive (γd, non-polar) and acid-base (γab, polar) components of the surface energy were calculated using the Dorris-Gray method. All measurements were made in triplicate. Results Due to the different production method, milled Lactohale 200 has a higher percentage of fines, as can been seen in Figure 1. After wet decantation the surface fines have clearly been reduced, and a smoother surface can be seen in the larger particles that maintain their tomahawk shape. Solid bridging was observed in the initial wet decantation trials (data not shown), however it was significantly reduced thanks to the wet-sieving final step.

Figure 1 - SEM images of Lactohale 100 (top row left), and Lactohale 200 (bottom row left) decanted with isopropyl alcohol (IPA- middle) with ethanol (right) (x500 magnification). Some surface alteration can be seen using IPA and ethanol but they have both succeeded in the removal of the fines.

The solid-state of the powders was evaluated via DSC (Figure 2). The thermographs are in accordance with the expected α-lactose monohydrate behaviour, exhibiting an endothermic dehydration peak at 147 oC while the melting endotherm commenced at about 207oC [7].

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1 0 0 E tO H

1 0 0 IP A

200

2 0 0 E tO H

2 0 0 IP A

H e a t F lo w ( W /g )

100

-2

-4

-2

-4

-6 0

100

200

300

100

200

300

T e m p e r a tu r e ( C )

Figure 2 – DSC thermographs of the raw (blue line) Lactohale 100 (left) and Lactohale 200 (right), decanted with ethanol (light brown line) and isopropyl alcohol (black line). The powders exhibit the same thermal behaviour indicating that their crystalline state has not been altered.

LDA measurements were conducted to investigate the particle size distribution of the powders under flow (Figure 3, top row). D10 was found to be 55.1 ± 1 for Lactohale 100, 47.9 ± 1.1 for Lactohale 100 – Ethanol, and 66.2 ± 2.4 for Lactohale 100 – IPA. The same trend was observed for the Lactohale 200 powders as well with D10 = 13.2 ± 0.8 for Lactohale 200, 19.7 ± 0.5 for Lactohale 200 – Ethanol and 28.2 ± 0.7 for Lactohale 200 – IPA. In order to have a better understanding of the removal of fines, D2, reporting the particles in the lower 2% of the PSD was measured in the whole pressure range (Figure 3, bottom row). As the pressure increases, D2 decreases, as any smaller particles, loosely bound to the surface will de-agglomerate a result consistent with literature[5,8]. However, IPA has in both cases, the highest D2, suggesting a better removal of fines.

Figure 3 – Top row: Particle Size Distribution of the raw materials and the decanted powders when dispersed at 2.0 bar primary pressure. Bottom row: Airflow titration measurements in the range 0.2-5.0 bar, reporting the amount of particles smaller than the 2% of the distribution (D2), for Lactohale 100 (left) and Lactohale 200 (right). In both cases, the IPA treated lactose (light brown) has the highest D2, indicating the highest removal of fines. (70.3% for Lactohale 100 and 84.2% for Lactohale 200).

Surface Energy (SE) analysis was conducted for the Lactohale 200 powders using iGC (Table 1). An increase in the BETSSA of the decanted powders was observed; this could be attributed to surface modification as an effect of the solvents. The highest contribution in the total surface energy is through dispersive forces (45.2 ± 0.2 for Lactohale 200) as has been reported previously[9]. Wet decanted powders exhibit lower SE values; 39.2 ± 0.3 for Lactohale 200-Ethanol and 41.4 ± 0.1 for Lactohale 200 – IPA. The reduction in the SE indicates the successful removal of the high-energy fines from the surface.

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Drug Delivery to the Lungs, Volume 29, 2018 - Physicochemical characterisation of inhalation grade lactose after the removal of intrinsic fines. Table 1 – Surface Energy measurements via Inverse Gas Chromatograph (iGC-SEA) at 3% surface coverage (n=3, ± SD)

Lactohale 200

Lactohale 200 Ethanol

Lactohale 200 Isopropyl Alcohol

BETSSA (m2/g)

0.3071 ± 0.0008

0.391 ± 0.003

0.3703 ± 0.0008

γd (mJ/m2)

45.2 ± 0.2

39.2 ± 0.3

41.4 ± 0.1

γab (mJ/m2)

6.2 ± 0.1

2.59 ± 0.06

3.73 ± 0.05

Discussion It has been shown in the past that addition of fines in coarse lactose samples results in an overall increase in surface energy [10]. In a similar note, the removal of fines via wet decantation as presented in this work, has resulted in the decrease of the surface energy of the coarse lactose particles. The lower SE of the ethanol-treated lactose indicates that fewer high-energy fines are present in the powder i.e. a better removal of fines in comparison to IPA. This comes in contradiction with what was observed via Laser Diffraction, where the D2 values of IPA-treated lactose were consistently higher than the others. This could be explained by the remaining fines in the IPA-treated powders not being as readily dispersed under the airflow as the ethanol ones. An alternative explanation is the potential for surface-modification (e.g. localized surface etching and/or dissolution) due to dispersion in IPA solvent. The latter issue is currently being addressed through further surface characterization studies. The importance of iGC-SEA is highlighted, as it can facilitate the understanding of the absence/presence of fines in a formulation. Nevertheless, IPA has demonstrated a similar behaviour to ethanol, and is recommended from the authors as a second solvent of choice for wet-decantation. Conclusion This study investigated the use of isopropyl alcohol (IPA) instead of ethanol in wet decantation for the removal of intrinsic lactose fines. Both solvents have successfully removed lactose fines and in doing so a decrease in the Surface Energy of the lactose carriers was observed. Although LDA measurements suggested that IPA had removed more fines than ethanol, the significant difference in the Surface Energy measurements between the two implies that ethanol still removed more fines, while IPA-remaining fines were more strongly adhering to the particles’ surface, and were not as easily dispersed under the airflow. References 1

Pilcer G, Wauthoz N, Amighi K: Lactose characteristics and the generation of the aerosol. Adv. Drug Deliv. Rev. 2012;64(3):pp 233-256. doi:10.1016/j.addr.2011.05.003.

Jones MD, Price R: The influence of fine excipient particles on the performance of carrier-based dry powder inhalation formulations. Pharm. Res. 2006;23(8):pp 1665-1674.

Islam N, Stewart P, Larson I, Hartley P: Lactose surface modification by decantation: Are drug-fine lactose ratios the key to better dispersion of salmeterol xinafoate from lactose-interactive mixtures? Pharm. Res. 2004;21(3):pp 492-499.

Jaffari S, Forbes B, Collins E, Barlow DJ, Martin GP, Murnane D: Rapid characterisation of the inherent dispersibility of respirable powders using dry dispersion laser diffraction. Int. J. Pharm. 2013;447(1-2):pp 124-131.

Ramachandran V, Murnane D, Hammond RB, Pickering J, Roberts KJ, Soufian M, Forbes B, Jaffari S, Martin GP, Collins E, Pencheva K: Formulation pre-screening of inhalation powders using computational atom-atom systematic search method. Mol. Pharm. 2015;12(1):pp 18-33.

Larhrib H, Zeng XM, Martin GP, Marriott C, Pritchard J: The use of different grades of lactose as a carrier for aerosolised salbutamol sulphate. Int. J. Pharm. 1999;191(1):pp 1-14.

Calvert G, Hassanpour A, Ghadiri M: Analysis of aerodynamic dispersion of cohesive clusters. Chem. Eng. Sci. 2013;86:pp 146-150.

Thielmann F, Burnett DJ, Heng JYY: Determination of the surface energy distributions of different processed lactose. Drug Dev. Ind. Pharm. 2007;33(11):pp 1240-1253.

Ho R, Muresan AS, Hebbink GA, Heng JYY: Influence of fines on the surface energy heterogeneity of lactose for pulmonary drug delivery. Int. J. Pharm. 2010;388(1-2):pp 88-94.

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Drug Delivery to the Lungs, Volume 29, 2018 – Cedric Thomas et al. Acoustic Spectral Analysis on Lactose for inhaled formulation using UA-AFM Cedric Thomas1, N. Pocholle1, E. Bourillot1, L. Kerriou², V. Gamerre², E. Lesniewska1 1ICB 2

UMR CNRS 6303, Univ. Bourgogne Franche-Comté, Dijon, France Armor Pharma, Armor Proteines SAS, Saint Brice-en-Cogles, France

Summary The understanding of interactions in formulations is a major issue in the pharmaceutical industry. It is essential to understand the surfaces of excipients and active ingredients as well as their interactions in order to improve the stability, the reproducibility, the physico-chemical properties and the quality of lactose-based carrier particle-drug product mixtures. Identification of contaminants inside and on surface of excipients used in the pharmaceutical industry with non-destructive techniques is a challenging procedure. We propose coupling ultrasound acoustic investigation with atomic force microscopy for tomographic reconstruction. The new version of our prototype makes it possible to perform acoustic spectral analyses of the pharmaceutical lactose particles surface in order to identify non-detectable contaminants by conventional techniques (IR, XPS, EDX, UV...). The acoustic microscopy technique (1-20 MHz) operates in the attenuated total reflectance (ATR) configuration and is sensitive to the local density variation. A home-built platform was used to detect the variation of density properties of material in the bulk at specified depth of investigation by varying the ultrasound acoustic frequencies.

Key Message The UA-AFM (Ultrasound Acoustic Atomic Force Microscope) is probably the only non-destructive approach capable of acquiring both depth investigation, density and chemical information of pharmaceutical sample at the nanometer scale without specific labelling. This new technique offers the potential as a major technological breakthrough.

Introduction A growing interest in the improvement of inhalation devices has occurred due to the rise of chronic respiratory diseases (WHO). We investigated the interactions between the active pharmaceutical ingredient(s) and excipients. Since these interactions take place on the surface of the particles, it is relevant to characterise these surfaces. There is a demand for the characterisation of surfaces in a non-destructive manner and at high resolution. The Ultrasound Acoustic Atomic Force Microscope (UA-AFM), developed within our laboratory is an attempt to provide answers to this expectation (1).

Experimental methods The UA-AFM (Ultrasound Acoustic Atomic Force Microscope)(2), developed at the ICB laboratory, couples ultrasound acoustic analysis and AFM microscopy. The ultrasound acoustic microscopy component used an analogue Kretschmann-ATR configuration and allowed non-destructive in-depth investigation samples. Ultrasound that penetrate the sample are sensitive to local density of material. A change in density causes variations in phase and amplitude of the incident ultrasound wave. In our case, by coupling acoustic microscopy to the AFM microscope in order to circumvent the diffraction limit, we could probe the sample in depth while retaining the inherent resolution in the atomic force microscope (3). The principle of UA-AFM is based on the study of the phase and the amplitude of the modulated surface stationary beat wave generated by the composition of two ultrasonic waves. In the present study, the applied frequencies varied between 1 and 10MHz.

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Figure 1 : Illustration of Ultrasound Acoustic Atomic Force Microscope

UA-AFM was implemented on a Bioscope AFM platform (Bruker, Santa Barbara, CA). Two ultrasonic waves of respective frequencies f1 and f2 were generated by two wave function generators in a range between 1.0 MHz and 10.0 MHz. Through a lock-in amplifier (model SRS 850; Stanford Research Systems, Sunnyvale, CA), we were able to detect changes in the difference frequency |f1 - f2| fixed by the experimenter (heterodyne detection). The superposition of these two waves generated a beat wave with frequency Δf = |f 1-f2| which had an absorption minimum depending on the depth of investigation and the value of Δf (Figure 1). We performed acoustic spectral analyzes by spot measurements in order to identify all components on the surface and the subsurface of lactose particles. In the case of lactose as excipients for inhaled formulation, the known contaminants are lactulose (an isomer of lactose), glucose, galactose, riboflavin and small peptides. They are all present in milk or appear in the process of lactose extraction. Last year, we presented the impact of the presence of these impurities on interactions with different active ingredients(4). It was first necessary to determine the spectral signatures of each component on the surface of the inhalation-grade lactose particles, as to the best of our knowledge, the coefficients of acoustic absorption of our compounds do not exist in the literature. We use commercial riboflavin, lactulose, glucose and galactose on which we reproduced at least 10 times each spectrum under the same conditions of temperature and humidity (22°C and 30% RH). We also carried out 4 successive purifications (supersaturation at 80°C, filtration and return to ambient temperature) of the lactose. The acoustic amplitude detected was characteristic of our system and depended on how the sample was deposited on the prism. All impurities used (glucose, galactose, lactulose and riboflavin) were of European Pharmacopeia quality with purities higher than 99,8%. Results and discussion The spectral signature of pure lactose (Figure 2) showed 4 characteristic locations: 4.10MHz with Δf=80kHz and 500kHz, 4.25MHz with Δf=240kHz, and 4.50MHz with Δf =80kHz. We have used three frequencies to identify lactulose: 2.50MHz with Δf=100kHz, 2.90MHz with Δf=100kHz, and 3.70MHz with Δf=100kHz. For galactose, we have used three locations: 3.10MHz with Δf=240kHz, 2.10MHz with Δf=240kHz, and 2.30MHz with Δf=240kHz.

Glucose and riboflavin absorbed more acoustic wave energy than lactose. For glucose we have used four frequencies: 2.20MHz with Δf=500kHz, 2.50MHz with Δf=500kHz, 2.70MHz with Δf=120kHz, and 2.80MHz with Δf=80kHz. For riboflavin, we have used 3 frequencies for identification: 2.40MHz with Δf=800kH, 2.60MHz with Δf=520kHz and 720kHz.

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Figure 2 : Acoustic responses of different constituents of the surface of pharmaceutical lactose particles

All results are summarized in Error! Reference source not found.. The spots of maximum amplitude characteristic of pure lactose were at frequencies between 4 and 5MHz. All impurities had responses below 3MHz. Riboflavin was visible for high differential frequencies between 500 and 800kHz Table 1: Characteristic frequencies of each constituent of the inhalation-grade lactose particle surface

We show the acoustic response of commercial inhaled lactose in Figure 3 (ordered at Armor Pharma). The characteristic spots for pure lactose were found at: 4.10MHz with a Δf=500kHz, 4.25MHz with Δf=240kHz, and 4.50MHz with Δf=80kHz. The characteristic frequencies of lactulose were not visible on the spectral signature of commercial lactose. We can therefore affirm that the surface of these particles does not contain lactulose. We found the same situation for riboflavin. The three characteristic galactose frequencies are visible on this spectrum which makes it possible to detect the presence of this particular contaminant. Finally, for the glucose, which was also detectable, only two frequencies were visible.

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Figure 3: Acoustic response of commercial pharmaceutical lactose

Conclusion This technique, which has been in development for five years, shows results of great interest for the analysis of powders for inhalation. We have implemented a new mode for performing point-to-point acoustic spectroscopy. The objective of this work was to show that each compound has its own acoustic signature and that it is possible to identify their presence on a complex mixture. UA-AFM opens a new way in the characterisation of the surfaces of excipients for pharmaceutical use. It is the only non-destructive technique for such characterisation, which makes it possible to identify component on the surface of particle and to measure the surface accessible to active ingredients. Acknowldegments This work was funded by Armor Pharma, and the French National Research Agency: ANR-15-IDEX-03 PIA2/iSiteBFC, ANR-15-CE09-0002-02 grants, and FEFER â&#x20AC;&#x201C; Region of Bourgogne Franche-Comte grants.

References 1

Vitry P, Bourillot E, Tetard L, Plassard C, Lacroute Y, Lesniewska E: Modeâ&#x20AC;?synthesizing atomic force microscopy for volume characterization of mixed metal nanoparticles. J. Microscopy 2016; 263(3): pp307-311. 2

Vitry P, Rebois R, Bourillot E, Deniset-Besseau A, Virolle MJ, Lesniewska E, Dazzi A: Combining infrared and mode synthesizing atomic force microscopy: Application to the study of lipid vesicles inside Streptomyces bacteria. Nano Research 2016; 9(6), pp1674-1682. 3

Vitry P, Bourillot E, Plassard C, Lacroute Y, Calkins E, Tetard L, Lesniewska E: Mode-synthesizing atomic force microscopy for 3D reconstruction of embedded low-density dielectric nanostructures. Nano Research 2015; 8 (7), pp2199-2205. 4

Thomas C: Identification of contaminants on excipients using Ultrasound Acoustic-AFM. Impact on the interaction drug-carrier. Oral presentation, DDL 2017

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Drug Delivery to the Lungs, Volume 29, 2018 – Larissa Gomes dos Reis et al. Probing Subtle Variabilities in Seretide/Advair Batches by Evaluation of Drug-Lactose Aerosol Interaction Larissa Gomes dos Reis 1, Michele Pozzoli1 Paul M Young1,2, Robert Johnson2 1Woolcock

Institute of Medical Research, University of Sydney, 431 Glebe Point Road, Sydney, NSW 2037, Australia 2Oz-UK Ltd, Unit 15, Lansdowne Court, Bumpers Way Chippenham Wiltshire, SN14 6RZ, UK

Summary A sensitive high-pressure liquid chromatography (HPLC) and HPLC/mass spectrometry (MS) method was developed to determine the aerosol properties of fluticasone propionate (FP) and salmeterol xinafoate (SX) active ingredients and excipient (lactose (LAC)) from two batches (lots) of Advair/Seretide (500/50 g). The aerosol properties of all components were evaluated using cascade impaction by Next Generation Impactor (NGI). Both lots had similar deposition profiles and fine particle fractions. Statistical analysis of conventional aerosol parameters indicated no significant differences between lots when evaluating the aerosol performance of FP, SX or LAC. However, analysis of the LAC/FP ratio as a function of aerodynamic diameter (Dae) indicated differences in the LAC/FP ratio, as a function of both aerodynamic diameter and lot number. For example, on Stage 3 of the NGI (Dae 4.46 µm) the ratio of LAC/FP was approximately 1:1, indicating that there is approximately one LAC particle to every FP particle. Importantly, significant differences in this ratio were observed. Electron microscopy was conducted to evaluate morphology of the particles captured on NGI collection cups 2, 3 and 5. Particles appeared as loose agglomerates, presumable as LAC/FP mixtures. While conventional aerosol analysis showed no significant difference the LAC/FP ratio appears to be an important tool for batch-to-batch differentiation of multi-component DPIs, such as Seretide/Advair. This variation in LAC/FP ratio may drive variations in drug wettability, dissolution and ultimately pharmacokinetics (PK). Key Message Variability in FP and SX from different batches of Advair/Seretide were not observed using cascade impactor studies. However significant differences between batches were observed when analysing lactose/FP aerosolisation ratios using a sensitive analytical method. This may have implications on FP dissolution and PK of Advair/Seretide products. Introduction Batch-to-batch differences in formulation parameters of dry powder inhalers (DPIs) can influence aerosol properties resulting in variation in lung deposition1,2,3. FDA and EMEA are aware that batch‐to‐batch variability, among inhaled medicines4. Additionally, interaction between different formulation components of high and low solubility can influence drug dissolution3. Ultimately, the dissolution rate for a given drug will impact its pharmacokinetic (PK) profile and efficacy. Furthermore, for poorly and insoluble drugs used in many DPI-delivered formulations, the presence of more soluble compounds in the fine particle fraction may impact on dissolution at the site of delivery. The majority of DPIs are formulated as drug/carrier blends where the carrier is a soluble lactose excipient. While the majority of carrier is too large to be inhaled into the airways, a small fraction may still be inhaled and/or associated with the drug particles as micro-agglomerates. Seretide/Advair contains FP, SX as drugs, as well as lactose (LAC) as an excipient. FP is insoluble, with reported aqueous solubility values for FP ranging from 0.14 mg/L and 0.51 mg/L,5,6. This equates to 140 ng/ml to 510 ng/ml, making FP practically insoluble. Additionally, small variations in blending of LAC/FP and interactions between FP and LAC in an agglomerated state may have a large effect on wettability and dissolution parameters 1,3. We propose that subtle differences in agglomerate and FP/LAC structure exist in products, such as Seretide/Advair, which may impact batch-to-batch product variability in vivo. Subsequently, we developed a sensitive method for determining the aerosol characteristics of all formulation components in Seretide/Advair, using cascade impactor methodology, with a view to identify differences that are not routinely observed when analysing the active pharmaceutical ingredients alone. Materials and Methods Materials: Two batches (lots) of Advair/Seretide dry powder inhalers (Seretide 500/50 g FP/SX dose) were purchased from a local pharmacy. Batch/Lot numbers were TG5E (hereafter referred to as Lot 1) and TD3H (hereafter referred to as Lot 2) All solvents were analytical grade and water purified by reverse osmosis. SX, FP and lactose monohydrate pharmacopeia standards were used for method development. and purchased from Sigma Aldrich.

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Drug Delivery to the Lungs, Volume 29, 2018 - Probing Subtle Variabilities in Seretide/Advair Batches by Evaluation of Drug-Lactose Aerosol Interaction Analytical methodology: One of the challenges of assessing the aerosol deposition of lactose or other excipient components in cascade impactors is the ability to routinely quantify low levels of excipient that are not detectable by conventional analytical techniques such as UV. Additionally, excipients often have very different solubility parameters to that of active pharmaceutical ingredients used in inhaled products, such as steroids and β2-agonists. Subsequently, a sensitive method had to be developed that enabled quantification of lactose in the presence of SX and FP. Quantification of FP/SX and LAC was determined using in-house developed and validated HPLC and HPLC/MS methods using C18 reverse phase chromatography. A Shimadzu ULPC system equipped with a diode array SPD-M20A UV-VIS detector was used in isocratic mode with a methanol:water buffered mobile phase for the assay of SX and FP. A buffered acetonitrile:water gradient method was employed for lactose assay, and detection was undertaken using a quadrupole mass spectrophotometer equipped with an electrospray ionization source (LCMS 2020) operated in negative mode (lactose quantification occurring at m/z 377). The retention times for FP and SX were 5.5 and 3.5 minutes, respectively. The limit of detection (LOD) and limit of quantification (LOQ) for FP were 0.088 and 0.268 µg/ml, respectively. LOD and LOQ for SX were 0.058 and 0.177 µg/ml, respectively. The retention time for lactose was 4.5 minutes. The LOD and LOQ were determined as 0.151µg/mL and 0.456 µg/mL, respectively. Standards and samples were prepared in a mixture of water and ethanol (50-50 % v/v) for all components. Standard calibration curves for FP, SX and LAC are shown in Figure 1. In vitro aerosol evaluation of drug and excipient deposition from Seretide/Advair lots: The in vitro aerodynamic assessment SX/FP and LAC from two lots of Advair/Seretide DPI products was conducted using an Apparatus E impactor (Next Generation Impactor (NGI), Copley Scientific Ltd, Nottingham, UK) following protocols outlined in the British Pharmacopoeia7. The NGI was connected to a critical flow controller (TPK200, Copley Scientific Ltd) and high capacity rotary vein pump. For each experiment, three doses were discharged into the NGI with at a flowrate of 60 l.min-1, calibrated using a flow meter (Model 3063; TSI Inc. St Paul, MN, USA), for 4 seconds.

The collection cups of the NGI were coated with 50 µl of 3% w/w Brij35 glycerol solution, in order to prevent particle bouncing. After each deposition study, the powder deposited on each stage of the impactor was collected into suitable volume, using sample collection solvent for HPLC/HPLC/MS quantification. Each lot was tested in triplicate. Data were processed using Copley CITDAS™ software and exported as stage deposition, delivered dose, fine particle dose and fine particle fraction (FPF), based on the mass and percent with an aerodynamic diameter < 5 µm. Figure 1 – Calibration curve for FP/SX and LAC (n=3 ± S.D. for each calibration curve).

Electron microscopy: Scanning electron microscopy (SEM) was used to investigate agglomeration and morphology of the SX/FP/LAC system. A JCM-6000 bench-top scanning electron microscope (SEM; Jeol, Tokyo, Japan) operating at 15 kV. Samples with different aerodynamic diameter were collected by placing carbon sticky tape on each collection cup of the NGI. Aerosolization of the powder from each batch was performed as for the routine cascade impaction studies described above. After aerosolisation, prior to imaging, samples were goldcoated for 2 minutes using an automated sputter coater (Smart Coater, Jeol, Tokyo, Japan). Results and Discussion: Aerosol performance of FP/SX and LAC from two Lots of Advair/Seretide: Stage-by-stage deposition profiles of each formulation component (FP/SX and LAC) from lots 1 and 2 are shown in Figures 2A and 2B respectively. In general, the majority of the mass of LAC was deposited in the lower-numbered stages of the impactor, with relatively small amounts being deposited on the higher-numbered stages. This outcome was expected, since the majority of the lactose component in the formulation was associated with the larger carrier fraction. Analysis of FP and SX deposition across the NGI indicated that the FP component had an FPF (%) of 18.6% ± 1.7% and 18.1% ± 0.9%, for lots 1 and 2 respectively. The SX component had an FPF of 17.7% ± 1.3% and 17.3% ± 1.1% for lots 1 and 2 respectively. Statistical analysis (2-tailed t test p <0.05) indicated no significant difference in performance for either FP or SX between lots. The lactose-related FPF values were 0.73% ± 0.06% and 0.76% ± 0.06% for lots 1 and 2 respectively. As expected, the lactose FPF was low since the majority of the carrier particles was deposited in the pre-separator (ca. 10 mg). As with the FP and SX, no significant difference was observed in FPF for LAC between batches (Figure 2).

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Figure 2 - NGI stage deposition profiles of FP, SX and Lactose from two different Lots (A = Lot TG5E B = Lot TD3H) of Seretide/Advair at 60 l.min-1 (n=3 replicates ± S.D.) As previously discussed FP is practically insoluble and the adhesive and cohesive interactions between LAC and FP may drive variation in dissolution and distribution of the FP component at the lung interface. This process may affect the PK profiles. Figures 3A and 3B shows the stage-by stage distribution for both Advair/Seretide Lots with respect to FP and LAC, respectively. Importantly, while the FPF for LAC is low, closer examination of the mass deposition of fine lactose on the NGI impactor stages (Figure 3B) indicates that on the upper stages the mass of lactose was in the same order of magnitude to that of FP. For example, on stage 2 of the NGI there appears to be almost double the mass of LAC to that of FP. The relative mass of LAC to FP was therefore calculated for each individual run to determine the ratio LAC/FP ratio in order to further investigate this observation.

Figure 3 – Comparison of Lot 1 (TG5E) and Lot 2 (TD3H) NGI stage deposition profiles for (A) FP and (B) lactose (n=3 replicates ± S.D.) Stage-by-stage mass ratio analysis of LAC/FP from different Lots of Advair/Seretide: LAC/FP ratio was plotted as a function of aerodynamic diameter (Figure 4) for both lots.

Figure 4 – Comparison of the ratio of Lactose to FP as a function of effective cut-off diameter for Lots 1 and 2 with linear regression (n=3 replicates ± S.D.)

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Drug Delivery to the Lungs, Volume 29, 2018 - Probing Subtle Variabilities in Seretide/Advair Batches by Evaluation of Drug-Lactose Aerosol Interaction It can be seen from Figure 4 that the ratio of LAC to FP changed significantly depending on the stage evaluated. For example, for ‘particles’ collected on stage 2 (Dae 8 µm) there is a LAC/FP ratio of ≥ 2, indicating that there would be approximately 2 particles of lactose to every 1 particle of FP. For particles collected on Stage 3 (Dae 4.5 µm) the LAC/FP ratio drops to approximately to 1. Importantly, statistical analysis of the LAC/FP ratios indicated significant differences between the two studied lots of Advair/Seretide that were not observable by analysis of the cascade impactor deposition profiles of the FP and SX components alone. For example, on Stage 2 of the NGI a LAC/FP ratio was 2.17 ± 0.04 and 2.36 ± 0.06, with a statistical significance of p value <0.05. However, these upper stages represent coarse aerosol fractions that are unlikely to impact PK significantly. Analysis of the lower stages (stage 5 Dae 1.6 µm) showed lower LAC/FP ratios of around 0.3 to 0.4 lactose particles to FP particles. Statistical analysis of the two batches on these stages indicated significant differences where p<0.05 and ratios were 0.29 ± 0.02 and 0.39 ± 0.03 for batch 1 and 2 respectively. Such subtle variations in LAC/FP ratio may influence the wetting and dissolution of FP upon deposition at the lung interface, thus having potential implications for PK that should be further investigated. Electron microscopy: SEM images of aerosol particles captured on stage 2 of the NGI, for lot 1 and lot 2 Seretide/Advair are shown in Figure 5A and 5B, respectively. It is interesting to note that for each stage, particles collected were often in the form of loose agglomerates that approximated the cut-off diameter for any given stage. This means that the majority of particles captured on a given stage are not necessary representative of the true drug size, but more of LAC drug agglomerates. Again, for stage 2 particle deposition (Figure 5) over half of the particles observed by SEM were LAC. Such agglomeration and interaction is likely to have impact on wetting and dissolution of insoluble components such as FP in such systems.

Figure 5 – Scanning electron microscopy images of Advair/Seretide aerosol samples captured from stage 2 of an NGI from (A) Lot 1 and (B) Lot 2. LAC/FP mass ratio = (A) 2.17 and (B) 2.36 Conclusions It was possible to probe further the drug-drug and drug-excipient interactions that may contribute to variations in aerosol and in vivo performance utilizing a sensitive method developed for the determination of both excipient and drug components in Advair/Seretide. We have shown that two lots of Advair/Seretide 500/50 had very similar aerosol aerodynamic diameter profiles when measured using cascade impactor methodology to assess FP and SX components. Interestingly, however, by evaluating the aerosol deposition of LAC also, subtle variations in the ratio of LAC to FP were observed. These variations may have an impact on in vivo performance since complex agglomeration and wetting properties may drive different dissolution and drug absorption profiles. However, this would need to be studied in future research. Subsequently, understanding LAC/FP profiles may provide a robust screening tool for batch-to-batch comparison. Further investigations should be conducted to increase statistical power and confirm these preliminary findings. References 1 2 3 4 5 6 7

Young, P. M. (2016). Lactose Specifications: Supplier and Process Defined Properties vs. Surface Characteristics Controlled and Specified In-house. Proceedings of Respiratory Drug Delivery. R. N. Dalby, P. R. Byron, J. Peart et al., Davis Healthcare Int'l Publishing. Illinois, USA.1-933722-94-0 pp165-174 V.N.P. Le, E. Robins, M.P. Flament. Agglomerate behaviour of fluticasone propionate within dry powder inhaler formulations European Journal of Pharmaceutics and Biopharmaceutics. 2012, 80, 596-603 Kalea K, Hapgood, Stewart, P. Drug agglomeration and dissolution – What is the influence of powder mixing? European Journal of Pharmaceutics and Biopharmaceutics. 2009, 72, 156-164 Burmeister GetzE, Carroll KJ, Jones B, Benet BZ. Batch‐to‐batch pharmacokinetic variability confounds current bioequivalence regulations: A dry powder inhaler randomized clinical trial. Clinical Pharmacology and Therapeutics.2016, 100, 223-231. http://www.drugbank.ca/drugs/DB00588 Baumann D, Bachert C, Högger P. Dissolution in nasal fluid, retention and anti‐inflammatory activity of fluticasone furoate in human nasal tissue ex vivo Clinical & Experimental Allergy. 2009, 39, 1540–1550 British Pharmacopoeia. London: Stationery Office; Appendix XII C. Consistency of Formulated Preparations. 2018.

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Drug Delivery to the Lungs, Volume 29, 2018 - Sunao Maruyama et al. Application of Void Forming Index (VFI): Detecting agglomeration of mannitol with different physical properties Sunao Maruyama1, Shuichi Ando1 and Etsuo Yonemochi2 Formulation Technology Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan 2 Department of Physical Chemistry, School of Pharmacy and Pharmaceutical Sciences, Hoshi University, 2-4-41, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan 1

Summary Mannitol is a promising candidate carrier for dry powder inhalers (DPIs). However, mannitol tends to form agglomerates by crosslinking between particles at high humidity. In a DPI formulation, fine particles are necessary to deliver the drug substance to the lungs, or to improve aerodynamics of the DPI, though smaller particles tend to form agglomerates. Therefore, in the case of using mannitol as a DPI carrier, controlling the risk of powder agglomeration is important. Here, we have focused on the relationship between agglomeration and pressure drop of the sample. Agglomeration ratio was defined as sample retain on the mesh and Void Forming Index at 4 hours (VFI 4 h) was defined as ratio of pressure drop of 4 h purged samples to initial samples. It was apparent that the correlation between the agglomeration ratio and VFI 4 h were clearly confirmed rather than that between the agglomeration ratio and other indices such as particle size, Hausner ratio and dispersive surface energy. We therefore propose that the VFI detection is a convenient method for evaluation of powder agglomeration capability not only for mannitol but it is also potentially useful for general DPI formulation development. Key Message Void Forming Index is a useful index for detecting the starting point and degree of powder agglomeration in DPI formulation development. This method can help to clarify the risk of agglomeration by elucidating parameters which affect this undesirable process. Introduction For the development of dry powder inhalers (DPIs) it is important to understand powder agglomeration behaviour, as powder agglomeration during storage causes a decrease in the aerosolisation performance of the DPI. Lactose is the most common and frequently used carrier in DPI formulations. However, lactose shows some limitations in formulations with certain drugs and peptides that prohibit its usage as a carrier in DPI formulations [1]. Mannitol has been frequently reported as suitable component for DPI [2, 3]. Mannitol is currently marketed in some countries as a pulmonary diagnostic DPI aerosol and as a therapeutic dry powder inhalation aerosol for the treatment of cystic fibrosis and chronic bronchitis [2]. Although, mannitol is a promising candidate carrier for DPI formulation development, it tends to form agglomerates in high relative humidity conditions by crosslinking between particles [4] . Mixing mannitol with particles of a hydrophilic drug in DPI formulation development might risk powder agglomeration in humidity condition, especially with drugs consisting of protein or peptide molecules. It is necessary to understand the physicochemical properties of the powder including its agglomeration behaviour to ensure the aerosolisation performance of DPIs is satisfactory, given that particle sizes smaller than 10 Âľm [5] are required. Coarse and fine carrier particles are intimately mixed with the drug substance in order to improve the resulting aerodynamic performance of the inhaled particles [3]. Since particle size affects agglomeration behaviour, the presence of fine carrier particles might increase the risk of forming agglomeration. Powder agglomeration during storage affects the aerodynamic performance of DPIs by reducing fine particle delivery which may reduce efficacy of DPI, it is important to understand the properties of powder agglomeration for the development of DPIs. For these reasons, controlling the risk of powder agglomeration is important if mannitol is chosen as a DPI carrier. Measuring the aerosolisation performance by cascade impactor at each time points are the direct way to evaluate powder agglomeration of DPI formulation. However, a convenient method to estimate the risk of agglomeration is also required in DPI formulation screening on the early stage development. Although Mannitol is suitable for carrier, few studies have evaluated the powder agglomeration risk. Therefore, the development of a method which can easily detect agglomeration is useful for clarifying the risk of this undesirable process, as well as for understanding the parameters which affect powder agglomeration. We have proposed the novel index Void Forming Index (VFI) as a method to detect powder agglomeration. VFI is related to the pressure drop of inverse gas chromatography (iGC) [7]. We have focused on agglomeration-induced structural changes by measuring the pressure drop in samples. As the powder forms agglomerates, micro-voids develop between these agglomerates. When a void is generated in the powder bed, the pressure drop decreases substantially. Our goal was to be able to evaluate the agglomeration behaviour of samples by comparing the pressure drop ratio of the initial sample with that of the agglomerated sample. In a previous study, we confirmed the relationship between VFI and starting point of agglomeration by using inhalation grade lactose powder [7]. The experimental results implied that VFI can be a way for easily detecting the onset of structural changes induced by agglomeration. Agglomeration behaviour was clearly detected in lactose particles by measuring VFI, though we have not yet confirmed that VFI can be applied to other similar carrier materials. As VFI indicates microstructural changes within the powder, we anticipated that this method has the ability to indicate agglomeration tendency irrespective of the chemical nature of the powder.

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Drug Delivery to the Lungs, Volume 29, 2018 - Application of Void Forming Index (VFI): Detecting agglomeration of mannitol with different physical properties In the present study we focused on the agglomeration behaviour of five mannitol samples with different physicochemical properties to evaluate the capability of VFI to other carrier compounds, as well as to extend knowledge of agglomeration behaviour with mannitol itself. Material and Methods Five different mannitol grades were used in this study (Mannitol A, B, C, D and E). All grades were passed through a 42-mesh sieve (screen size of 355 µm) prior to be used for the experiments. All other chemicals and solvents were of analytical reagent grade, and deionized water was used throughout the study. 25 ml samples of unagglomerated particles were used in the determination of bulk density of samples. Following 240 mechanical taps in a measuring cylinder, the Hausner ratio was calculated from the ratio of the tapped and bulk densities by the following equation: Hausner ratio = (Tapped bulk density of powder) / (Bulk density of powder) The volume-weighted particle size distribution of the unagglomerated powder samples was measured by low angle laser light diffraction using HELOS & RODOS apparatuses (Sympatec GmbH, Germany). Each powder sample was dispersed with an applied pressure of 4.0 bar. The lens was selected from the samples distribution, R3 lens for sample A, R4 lens for sample B and C, R5 lens for D and E. Size statistics in terms of X10, X50, and X90 are measured, which are the values of the particle diameter at 10%, 50%, and 90%, respectively, in the cumulative volumetric particle size distribution. (n = 3). The dispersive surface energy of the unagglomerated powder samples were measured using iGC (Surface Measurement Systems Ltd., U.K.). Samples of 600 mg were packed into silanized glass columns (same manufacturer) of the size 6 mm o.d., 3 mm i.d., and 200 mm length by vertical tapping. Tapping was continued until there were no visible cracks, hollows, or channels present in the body of the powder. Both ends of the column were loosely stoppered with silanized glass wool. Conditioning of the column packed with the sample powder was carried out at 40°C/0%RH, and the measurement was performed under the same conditions in order to measure the dispersive surface energy of the sample. Methane was used for the inert reference; n-decane, n-nonane, n-octane and n-heptane were used to determine the alkane series; and chloroform, ethyl acetate, acetone and ethanol were employed as polar probes. Dispersive surface energy profiles were calculated using the iGC standard analysis software suite (Revision: 1.41 SMS, London) following Schultz methods [8]. VFI measurement was carried out after measuring the dispersive surface energy using the same powder sample. Helium gas at 40°C/75%RH supplied at a flow rate of 5.1 mL/min was used as the carrier gas in order to encourage formation of agglomerates inside the column. The pressure drop change of the iGC was monitored during measurement using a 6-min program in order to detect the flow resistance during the purging of the helium gas. VFI was calculated by the following equation: VFI= (Pressure drop of sample at the point of time (Torr)) / (Pressure drop of initial sample (Torr)) The agglomeration ratio was measured from the mass of powder from samples (3 g) stored at 40°C/75%RH for 3 h retained by the 42-mesh sieve. 42-mesh sieve was selected according to previous report [9]. Agglomeration ratio was calculated by the following equation: Agglomeration ratio= (Mass of powder retained on 42-mesh sieve) / (Mass of total powder) Results and Discussion Agglomeration behaviour and VFI Micro-voids developed between individual particles as the powder formed agglomerates. When a void was generated in the powder bed, the pressure drop substantially decreased. We therefore expected to be able to evaluate the agglomerative behaviour of samples by comparing the pressure drop ratio of the initial sample and the agglomerated sample. When sample A was observed at 40°C/75%RH, the pressure drop started to decrease after 1 h period and had reached its plateau after 4 h (Fig.1). Since all samples reached the plateau after the same time period, VFI at 4 h period (VFI 4 h) was used to compare mannitol samples. The agglomeration ratio of the sample stored at 40°C/75%RH for 3 h was determined in order to confirm the relationship between VFI and mannitol agglomeration, (Fig.2). In cases where a VFI change was not detected, such as with samples D and E, no agglomeration was assumed to have occurred. However, in samples A, B and C, changes in VFI were observed, indicating that agglomeration was present. The agglomeration ratio and VFI 4 h were found to have a strong correlation (r2 = 0.967). It is clear from these results that the VFI can indicate agglomerative behaviour of mannitol powder over time. Given the high degree of correlation with agglomeration activity, VFI 4 h was used in the following sections as an index of this behaviour.

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Drug Delivery to the Lungs, Volume 29, 2018 - Sunao Maruyama et al. Physicochemical properties and VFI The relationships between VFI 4 h and particle size (x50), Hausner ratio and dispersive surface energy are shown in Figures 3, 4 and 5 respectively. A reduction of VFI 4 h was observed with decreasing particle size (x50). Within the range of 17.3 to 78.0 µm, mannitol less or equal to 32.8 µm showed clear decrease of VFI 4 h. Similar relationships were observed in x10 and x90 (data not shown). In Hausner ratio, within the range of 1.3 to 1.8, samples more or equal to 1.7 tended to form agglomeration. Although, particle size and Hausner ratio were influenced by the degree of agglomeration, a clear correlation was not observed, as a threshold existed below which agglomeration was not observed. A reduction of VFI 4 h was observed as the value of the dispersive surface energy increased, although the relation was only weak. However, the agglomeration degree could easily be detected by measuring VFI. Moreover, the threshold of agglomeration could also be detected, which is difficult to predict only from initial physicochemical properties. Agglomeration of mannitol in high humidity condition is reported to be formed by solid crosslinking following liquid crosslinking across primary powder [4]. In narrow clearance between particles, as humidity increase from external condition, liquid crosslinking formed by condensation of water [10]. On the point of forming liquid crosslinking, smaller particle and higher Hausner ratio will induce high humidity area [10] and higher dispersive surface energy will increase adsorption of water. From the mechanism of agglomeration, it is reasonable that the particle size, Hausner ratio and surface energy affect agglomeration behaviour of the samples, Although these physicochemical properties suggest the progress of agglomeration, as multiple factors affect the process, it is difficult to predict the degree of agglomeration only from physicochemical properties. From these results, we believe that the advantage of VFI detection is its simplicity and versatility, which can compare the agglomeration behaviour without necessarily describing the underlying mechanism. Application of VFI to DPI formulation development In DPI formulation development, it is important to estimate agglomerate behaviour, as the formation of agglomerated particles affects aerosolisation performance. The experimental results suggest that VFI is a useful index for easily detecting the degree of agglomeration and could be used to elucidate that the particle size, Hausner ratio and surface energy of the sample affect agglomeration behaviour. As VFI directly detects the microstructural changes that are not chemistry-related, such as void formation that are associated with powder particle agglomeration , we infer that VFI can detect agglomeration behaviour of not only mannitol and lactose but also other carrier powders. Although, aerosolisation performance measurements are necessary in long-term stability testing to ensure the quality control of DPI formulations, predicting the risk of agglomeration in early stage development, may provide a more efficient formulation development process. Moreover, as agglomeration behaviour can be easily detected by VFI, it might be possible to estimate agglomeration risk in accelerated condition and thereby improve aerosolisation performance.

Figure 1. Change in pressure drop of mannitol sample A purged with 40°C/75%RH helium gas. The dotted line indicates VFI 4 h.

Figure 3. Relationship between VFI 4 h and particle size (x50) of mannitol. Data represent mean± SD (n=3).

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Figure 2. Correlation between VFI 4 h and agglomeration ratio of 40°C/75%RH 3 h storage mannitol.

Figure 4. Relationship between VFI 4 h and Hausner ratio of mannitol.

Drug Delivery to the Lungs, Volume 29, 2018 - Application of Void Forming Index (VFI): Detecting agglomeration of mannitol with different physical properties

Figure 5. Relationship between VFI 4 dispersive surface energy of mannitol.

and

Conclusion The experimental results suggest that VFI is a useful index for easily detecting the degree of structural changes induced by DPI carrier particle agglomeration. Mannitol A, B and C which shows less or equal to 32.8 µm in particle size (x50), more or equal to 1.7 in Hausner ratio tended to formed agglomerate at 40°C/75%RH condition. Same as previous study on Lactose VFI change was observed, as the agglomeration of mannitol proceed. As VFI 4 h and agglomeration ratio showed clear correlation, we conclude that VFI can detect the degree of agglomerate. From the relationship between physicochemical properties of mannitol and VFI 4 h, VFI clarify the threshold of agglomeration, which is difficult to detect only from physicochemical properties. Additionally, on the point of agglomeration forming mechanism, the relation between VFI and physicochemical properties are suitable. The outcome from this study is indicative that VFI detection may be applicable to a wide variety of powder samples as well as being a convenient method for detecting agglomeration behaviour both directly and quantitively. As powder agglomeration affects aerosolisation performance of the powder when dispersed from a DPI, it is important to be able to estimate the tendency to form agglomerates in early stage development. Especially in the case of DPI formulation in protein drugs, as mannitol is candidate for DPI carrier, mixing mannitol with hydrophilic drug might have a risk of powder agglomeration. We are planning to investigate the application of VFI to formulations containing mixed mannitol/protein or mannitol/peptide constituents. References 1.

F. Depreter, G.Pilcer, K. Amighi: Inhaled proteins: Challenges and perspectives Int J Pharm 2013; 447 pp251-280

Y Rahimpour, M Kouhsoltani, H Hamishehkar: Alternative carriers in dry powder inhaler formulations, Drug Discovery Today 2014; 19(5): pp618-626.

M Mönckedieck, J Kamplade, P Fakner, N A Urbanetz, P Walzel, H Steckel, R Scherließ: Dry powder inhaler performance of spray dried mannitol with tailored surface morphologies as carrier and salbutamol sulphate, Int J Pharm 2017; 524: pp351–363.

Leon F, Gabriel I T, James N M: Micro-mechanical properties of drying material bridges of pharmaceutical excipients, Int J Pharm 2005; 306: pp41–55.

G Scheuch, R Siekmeter: Novel approaches to enhance pulmonary delivery of proteins and peptides, Journal of physiology and pharmacology 2007; 58 suppl 5(Pt 2): pp615–625.

Wanling Liang, Philip C L Kwok, Michael Y T Chow, Patricia Tang, A James Mason, Hak-Kim Chan, Jenny K W Lam: Formulation of pH responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids, Eur J Pharm Biopharm 2014; 86: pp64-73.

S Maruyama, E Yonemochi, S Hasegawa, S Suzuki, H Minami: Void forming index: A new parameter for detecting microstructural transformation caused by powder agglomeration, Int J Pharm 2017; 532: pp118–123.

J. Schultz, L. Lavielle, C. Martin: The role of the Interface in Carbon Fibre-Epoxy Composites, J.Adhes. 1987; 23 pp45-60

H. Steckel, P. Markefka, H. teWierik, R. Kammelar: Functionality testing of inhalation grade lactose, Eur J Pharm Biopharm 2004; 57: pp495-505.

10.

Podczeck F, Newton J, James M: The influence of constant and changing relative humidity of the air on the autoadhesion force between pharmaceutical powder particles, Int J Pharm 1996; 145: pp221-229.

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Drug Delivery to the Lungs, Volume 29, 2018 - Aurélie Doméné et al. Safety cabinet for droplet size measurement by laser diffraction Aurélie Doméné1, Maria Cabrera1, Michael Pasteur2, Marguerite Tulli3 & Laurent Vecellio 1

CEPR, INSERM U1100, University of Tours, 10 boulevard Tonnellé, Tours, 37032, France 2 Pharmacie Centrale des Armées, TSA 30004, Fleury-les-Aubrais cedex, 45404, France 3Stratégie Santé, 19 rue Georges Clémenceau, Versailles, 78000, France

Summary Laser diffraction is the recommended method to measure the droplet size distribution from aqueous nasal spray products. There is a risk in terms of Spraytec instrument contamination (lens and body) and air contamination by the sprayed drug product with the standard experimental set up. The use of drug products may require protective equipment such as fume cupboards or chemical hoods. Unfortunately, the measure of the droplet size distribution using the Spraytec cannot be carried out in such devices. In addition, the Malvern inhalation cell is not suitable for aqueous nasal spray due to the risk of lens contamination and spray impaction in the inhalation cell. In this context, we developed a safety cabinet allowing the measurement of the droplet size distribution of a new drug (aqueous nasal spray) using the Spraytec. To validate the cabinet, the first step was the particle size measurement with and without the safety cabinet. The second step was the evaluation of ambient air contamination using a chemical tracer (fluorescein) in a form of spray and aerosol. Results have showed that droplet size distributions were similar in terms Dv10, Dv50, Dv90, Span and percentage of particle smaller than 10 µm with and without safety cabinet. No contamination was detected in the ambient air. The safety cabinet is an efficient protection system allowing droplet size measurement by laser diffraction for aqueous nasal spray products. Key Message Droplet size produced by nasal spray can be measured by laser diffraction without risk of air contamination when using a safety cabinet. Introduction Aqueous nasal sprays are widely used to administer different drugs in the nasal cavities. Particle size ranges from 1 µm to 100 µm and is a key parameter to predict the deposition in the human airways (nasal and lungs). The cascade impactor is designed to measure aerosol aerodynamic particle size distributions in the size range from 0,1 µm to 10 µm but is not relevant for characterization of the entire aqueous nasal spray that contains much larger droplets. FDA guideline [1] recommends laser diffraction method to measure the droplet size distribution with this large range of particle size. Most of aqueous nasal droplets are larger than 10 µm, with only a tiny fraction in smaller particles. But these particles have the potential to remain airborne for long enough to contaminate the local environment and penetrate the nasopharynx reaching the lungs of the worker. The risk has to be removed regarding safety regulatories. Previous studies have been carried out in the laboratory showing that there is a risk in terms of Malvern Spraytec instrument contamination (lens and body) and air contamination by the sprayed drug product with the standard experimental set up [2] using an aqueous nasal spray (data not shown). Moreover, the Malvern inhalation cell is a suitable accessory for the measurement of the particle size produced by devices such as Dry Powder Inhalers (DPIs) and nebulizers [3] but not suitable for nasal sprays because of the risk of lens contamination and droplet impaction in the inhalation cell due to large spray angle. The objective of the study was to develop an experimental and measurement method ensuring the safety of the user and allowing the droplet size measurement by laser diffraction method. Experimental methods We developed a safety cabinet using a standard filtration method in accordance with the ISO 10648-2 [4]. A HEPA filter (BAG 32040100, Camfil, France) was located in the upper part of the cabinet and was connected to a constant aspiration flow rate at 11 m3/h in order to produce a negative pressure of –200 Pa inside the cabinet. Additional openings connected with absolute filters (PALL BB50T, Pall Medical, France) were located around the lateral wall of the cabinet in order to ensure good ventilation and to limit the interaction between the air and the spray. The air change rate was 30 volumes/h. Cabinet includes airtight lens to ensure the laser measurement with a Spraytec laser diffractometer (Malvern, UK). An additional aspiration at 40 L/min and an absolute filter was placed in the cabinet at 2 cm behind the laser to collect the produced spray (Figure 1). We performed two types of experiments.

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Drug Delivery to the Lungs, Volume 29, 2018 - Safety cabinet for droplet size measurement by laser diffraction The first part of the study was to evaluate the influence of the safety cabinet on the particle size measurement. Three aqueous nasal sprays were used to measure the droplet size distribution by laser diffraction. The nasal pump was placed close to the lens taking into account the spray angle to avoid lens contamination by the spray. This distance was 6 cm. Furthermore, the distance from the tip of the nasal spray pump was located perpendicularly 3 cm from the laser beam. Three measurements have been performed on the developed flow phase and the average of particle size distribution has been calculated, resulting in one result of measurement. Measurement provides size distribution at Dv10, Dv50, Dv90 (corresponding to diameter of the particle such as 10%, 50% and 90% respectively of particle volume is under this diameter), Span (corresponding to the dispersion of particle size distribution and defined by [(D90-D10)/D50)]) and %< 10 µm (percentage of particle smaller than 10 µm) were recorded for the 3 aqueous nasal pumps during the stable phase. Measurements were done in triplicate for each aqueous nasal spray pump both with and without the safety cabinet present, resulting in a total of 18 measurements. Statistical analysis was performed on Dv10, Dv50, Dv90, Span with and without cabinet (Mann and Whitney, GraphPad Prism 5, GraphPad software, La Jolla, USA). The second part of the study was to evaluate the risk of air contamination using the safety cabinet. Three nasal pumps were filled with fluorescein sodium (50 g/L) and 30 sprays were delivered in the safety cabinet. Fluorescein tracer was used for its high sensitivity in terms of detection. An absolute filter (ambient filter) (PARI Gmbh, Starnberg, Germany) connected to an aspiration pump (25 L/min) was located outside of the safety cabinet to simulate the worker exposure (Figure 2). A second type of risk of exposure was simulated with droplets smaller than those produced by aqueous nasal sprays. A mesh nebulizer (AeroNeb Solo®, Aerogen, Galway, Ireland) (Dv50 = 5 m), was filled with 4 mL of fluorescein sodium (50 g/L). The aerosol was generated at a distance of 1 cm from the laser beam An absolute filter (PARI Gmbh, Starnberg, Germany) connected to an aspiration pump (25 L/min) was located outside of the safety cabinet to simulate the worker exposure. Three replicate measurements were performed (Figure 2). The mass of fluorescein deposited on the absolute ambient filters was assayed by a spectrophotometry method.

Figure 1: Safety cabinet for nasal spray measurement with the Spraytec

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Figure 2: Experimental set up for ambient air contamination measurement using the safety cabinet

Results Results obtained in terms droplet size distribution (Table 1) have shown statistical similar results in terms Dv10, Dv50, Dv90, Span between experimental set up with safety cabinet and without safety cabinet (p<0,05). Statistical test could not perform with %< 10 µm due to zero value. No fluorescein was detected on the ambient filter after fluorescein spray or aerosol. Mean ± sd (3 devices) Dv10 (µm) Dv50 (µm) Dv90 (µm) Span %<10µm

Without safety cabinet

With safety cabinet

34,3 ± 2,1

34,0 ± 2,2

56,8 ± 3,5

56,3 ± 2,2

98,5 ± 7,8

94,9 ± 9,4

1,1 ± 1,1

1,1 ± 0,2

0,0 ± 0,0

Table 1: Particle size results by Spraytec using nasal pump with and without safety cabinet

Discussion Results have shown that there is no influence of the safety cabinet on the droplet size measurement using a laser diffraction method (Spraytec). Air flow and circulation in the safety cabinet allow an efficient operation of the safety cabinet without spray modification and influence on the measurement of the droplet size distribution using the Spraytec. Moreover, this airtight safety cabinet equipped with efficiently extraction air ensures user safety and airborne environment protection. In this experimental set up, the Spraytec was not contaminated because the safety cabinet includes lenses. The safety cabinet can be considered like an inhalation cell for spray, ie “spray cell” without risk of ambient air contamination. After safety cabinet use, the Spraytec can be used for standard experimental setup without risk of cross contamination.

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Drug Delivery to the Lungs, Volume 29, 2018 - Safety cabinet for droplet size measurement by laser diffraction Conclusion The safety cabinet is an efficient protection method which can be used with a laser diffraction method for measuring droplet size produced by aqueous nasal spray pump products. The safety cabinet has been validated for particle size measurement with nasal spray when using hazardous or new drugs regarding the safety regulatory. Additional measurements will be conducted using smaller particles to evaluate the ability of the safety cabinet to be used with other inhaler devices such as nebulizers. References 1. Guidance for Industry, Bioavailability and bioequivalence studies for nasal aerosols and nasal sprays for local action, Draft guidance, FDA, Center for Drug Evaluation and Research (CDER), 2003. 2. Copley M, Kipppax P: From actuation to deposition: Particle sizing techniques for characterizing nasal drug delivery systems. Inhalation, 2012. http://www.copleyscientific.com/files/ww/news/COP%20JOB%20183_From%20actuation%20to%20deposition.pdf 3. Lelong N, Junqua-Moullet A, Diot P, Vecellio L: Comparison of laser diffraction measurements by Mastersizer X and Spraytec to characterize droplet size distribution of medical liquid aerosols. J Aerosol Med Pulm Drug Deliv; 27: pp94-102, 2014. 4. ISO 10648-2 : Enceintes de confinement -- Partie 2: Classification selon leur étanchéité et méthodes de contrôle associées,1994.

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Drug Delivery to the Lungs, Volume 29, 2018 - J T Pinto et al. Insights into DPI sensitivity to humidity and its correlation to formulation physicochemical characteristics: a temporal study using two commercial budesonide products J T Pinto1, S Radivojev1,2, E Fröhlich1,2 & A Paudel1,3 Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, Graz, 8010, Austria Center for Medical Research, Medical University of Graz, Stiftingtalstraße 24, Graz, 8010, Austria 3Institute of Process and Particle Engineering, Graz University of Technology, Inffeldgasse 13, Graz, 8010, Austria 1

Summary Dry powder inhalers (DPIs) can present different resistances to humidity. Considering that patients do not necessarily store their inhalers as recommended, the present study aims to evaluate the impact of different storage conditions (25ºC/ 65% RH and 22ºC/ >90% RH) on the physicochemical characteristics of two budesonide dry powder inhalers, namely Easyhaler® and Novolizer®. Both formulations are composed of a mixture of drug (ca. 4 wt %) and α-lactose monohydrate. Easyhaler® powders were found to contain larger coarse excipient particles as well as a higher fraction of fines when compared to Novolizer®. Moreover, it was possible to identify distinct trends with respect to the impact of storage on the two products. It was evident that at >90% RH, the kinetics of change in the physicochemical characteristics of the formulations were accelerated. Particularly, particle size distribution of Easyhaler® was notably impacted by storage. While, only subtle, temporary differences could be identified in the solid-state and particle size of Novolizer®. The encountered changes in physicochemical properties and consequent in-vitro aerodynamic performance correlated well with the already documented higher sensitivity of Easyhaler ® to humidity than Novolizer®. This demonstrated that different formulation strategies can have distinct impacts on dry powders sensitivity to humidity. Key Message Comparison of two commercial DPI products revealed that formulations with a smaller particle size and higher amount of fines are more susceptible to humidity, resulting in a poorer in-vitro aerodynamic performance after storage. This demonstrated that some formulation strategies might be more efficient protecting powders against humidity than others. Introduction It is well known that powder performance can be influenced by environmental conditions. This is particularly true for DPIs, where humidity has shown to be able to critically impact product performance. After storage comparison of the in-vitro aerodynamic performance of Novolizer® (NOV) and Easyhaler® (EH) showed that the latter product was more sensitive to humidity [1]. This being particularly important if one considers that devices can be improperly handled. For instance, a patient-reported survey found that 42% of the subjects kept their primary maintenance inhaler in the bathroom, where humidity can rise substantially [2]. This study aimed to understand how the physicochemical characteristics of EH and NOV dry powders influence their sensitivity to humidity. Materials Two commonly used carrier based DPIs containing 400 µg dose of Budesonide, Giona Easyhaler® (Orion Pharma) and Novolizer® (Meda Pharma GmbH) were purchased and used to carry out the present study. Storage conditions The inhalers were stored in a climate cabinet at 25ºC/60% RH (a typical ICH condition) and in a desiccator at 22 ± 2ºC containing a saturated salt solution of potassium nitrate (93-94% RH). The high humidity condition was selected in order to mimic the high relative humidity (RH) conditions found in in a household bathroom. The inhalers were analysed as received (time 0) after which they were stored at two aforementioned conditions, with their protective dust caps on, and tested after 14 and 28 days. Formulation physicochemical characterization Modulated scanning calorimetry (MDSC) For the MDSC measurements, 9-11 mg of powder were weighted into aluminium pans that were sealed with pierced lids. These were heated in a DSC 204 F1 Phönix (Netzsch) at a rate of 5ºC/min from -25ºC to 250ºC with an isothermal step of 10 min at 100ºC. A modulation amplitude of ± 0.53ºC every 40 s was selected as adequate. Karl Fischer titration The water content of the formulations was determined according to coulometric Karl Fischer titration method using a TitroLine® 7500 KF trace (SI Analytics). For this 20-30 mg of powder were dissolved in the titration solvent and the water of the resulting solution extracted (90 min). The water content was determined by subtracting the concentration of water within the solution containing the sample to the one of the blank solvent.

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Drug Delivery to the Lungs, Volume 29, 2018 - Insights into DPI sensitivity to humidity and its correlation to formulation physicochemical characteristics: a temporal study using two commercial budesonide products Laser diffraction Particle size distribution (PSD) was measured by laser diffraction (HELOS/KR, Sympatec GmbH) using wet dispersion. Isopropanol was chosen as the dispersant media. This was added into a stationary cuvette and the powder added, step-wise to the solvent. The system was magnetic stirred (1000 rpm) in order to disperse the samples. Measurements were carried during 120 s, once an optical concentration above 0.5% was reached. Small angle X-ray scattering (SAXS) For SAXS the powders were filled into 2 mm diameter glass capillaries and analysed using a point-focusing camera system S3-MICRO (Bruker AXS GmbH). Measurements were performed in the real space resolution range of 10– 1500 Å with a total measuring time of 600 s at 30 counts/s. The K/Q was derived from SAXS data using Porod law. This parameter can be directly correlated to sample surface area as follows: φp φm S K = 104 π m Q ρs where, S is the specific surface area of the porosities (1-100 nm), m the mass of the sample, K the porod factor, Q the scattering vector, φm and φp the respective fractions of matrix and pores, and ρ s the true density of the solid. In-vitro aerodynamic performance The aerodynamic performance was conducted using a Next Generation Impactor (NGI) (Copley Scientific). The flow rate of the NGI was adjusted to 60 l/min and 4.0 L of air were made pass through the apparatus to discharge the inhalers. For each experiment, ten shots were discharged and the budesonide content was quantified by HPLC. Results and discussion Solid-state For MDSC measurements, both EH and NOV samples revealed the characteristic thermogram of α-lactose monohydrate (α-LH) with a dehydration peak ca. 140ºC and the melting of the respective α form at about 207-210ºC (Figure 1). Interestingly enough, at time 0 NOV revealed a melting point for the α-form some degrees lower than that in the EH. Also, an exothermic peak at about 168.5ºC, typically attributable to the crystallization of the stable anhydrous α-form was found to be only present in NOV. In turn, analysis of EH clearly showed two additional endothermic events at about 222ºC and 233ºC. These could have originated from the melting point depression of budesonide that melts at 246ºC (results not shown) or due to the melting of a mixture of the α and β (mp at 230ºC) anomers of lactose. Additionally, for EH it was also possible to identify a glass transition event (Tg) at ca. 40ºC. After 14 and 28 days, for the EH, it was clearly seen the disappearance the Tg and appearance of an exothermic peak at 162.9 ± 0.4ºC. More concretely at 14 days, 60% RH the dehydration peak of α-LH revealed a trend of decrease in its enthalpy (ΔH) in combination with an increase on the onset of the event. With respect to the melting of α-lactose, an increase of the onset was also observed, however no clear change could be identified in its melting ΔH. After 28 days no further changes were observed on the onset of both events however a slight increase in the ΔH of dehydration as well as in the enthalpy of melting of the α-form were seen. With regard to NOV after 14 days a trend for the increase of the dehydration peak onset as well as a decrease in its respective ΔH were detected. For the melting event of the α-form, an increase on the onset of the event as well as its ΔH were identified. After 28 days, the dehydration peak onset showed a tendency to decrease and its ΔH to increase; for the melting of α-lactose the same trend in the endotherm onset was seen however, for a decrease in its ΔH was observed instead. At >90% RH EH and NOV showed overly evidently the same trends identified at 60% RH. Karl-Fischer results (Table 1) revealed that at time 0 both the formulations had similar water contents (about 5 wt %). A trend of decrease in water content was identified over time (more prominent for NOV). It has been shown that amorphous materials can lose weight during recrystallization due to the release of water molecules that will eventually evaporate from the surface of the crystal solid [3]. If the former is taken into consideration with MDSC and Karl Fischer it can be hypothesized that in a first step water adsorbs to the surface of lactose leading to its dissolution and changes in its solid-state [4]. However, once this thin layer of solvent is saturated lactose recrystallizes and a loss of water is observed. Table 1 Karl-Fischer results (n=2).

Conditioning EH 60% RH >90% RH NOV 60% RH >90% RH

Time 0 5.35 ± 0.28

5.29 ± 0.37

14 days Water content (w/w %) 5.45 ± 0.40 5.49 ± 0.28 Water content (w/w %) 2.98 ± 0.33 2.58 ± 0.36

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28 days 3.04 ± 0.99 3.03 ± 1.15 1.70 ± 1.47 2.26 ± 0.24

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Figure 1 - MDSC thermograms (n=2) of (a) EH 60% RH, (b) EH >90% RH, (c) NOV 60% RH and (d) NOV >90%RH (dots mark the appearance of an exothermic event).

Particle size distribution and inner surface area (K/Q) PSD data (Figure 2) revealed that EH formulation is composed of particles with a smaller mean particle size and a larger amount of fines than the NOV powder. A gradual increase in PSD of EH was observed over time, the trend particularly evident after storage at >90%RH, where the Dv0.1 increased from to 8.5 Âľm to 20.9 and 23.9 Âľm , after 14 and 28 days, respectively. For the NOV a curious trend for the PSD to decrease after 14 days and increase again after 28 days was observed at both 60% RH and >90% RH. In respect to the K/Q parameter it was observed that at time 0 this was higher for EH than for NOV (Figure 3). This could be explained by the former formulation being composed of particles with a smaller size and thus expected to have a larger surface area. Over time both the formulations showed a gradual decrease in the K/Q parameter. For the EH the decrease was more evident at >90%RH and after longer times, for the NOV no clear difference could be seen between the chosen storage conditions and between 14 and 28 days. This was not surprising, considering that more notable changes were seen over time in the PSD of EH than NOV, particularly the accentuated loss of fine particles in the former formulation. Consequently, it is purposed that water condensation in the EH was slower due to its smaller particle size and consequent smaller capillary radii and explaining the more rapid occurring changes in NOV solid-state (see previous section) [5]. Notwithstanding, once condensation occurs the smaller particles present in EH will be susceptible to liquid bridging and agglomeration and a consequent decrease in powder surface area. In turn, for the NOV water adsorbs to the surface of coarser particles dissolving them and leading to a transient decrease in particle size, once recrystallization occurs particle size increases once again. However, the former could have led to smoothing of the particles surface causing an irreversible decrease of their surface area [4].

Figure 2 - PSD distribution (n= 1) (a) EH 60% RH, (b) EH >90% RH, (c) NOV 60% RH and (d) NOV >90%RH.

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Figure 3 - SAXS results (n= 3) for (a) EH and (B) NOV.

In-vitro aerodynamic performance As observed in Figure 4 the changes seen over time in the physicochemical properties of EH and NOV formulations resulted in some differences being found in their in-vitro aerodynamic performance. This was particularly critical for the EH where a striking decrease of the delivered and fine particle dose was seen after storage, these were even more accentuated after 28 days and at higher humidity storage conditions (>90%). In turn, for NOV slight differences were seen over time, interestingly enough the emitted dose decreased at 14 days to increase again at 28 days, the fine particle dose was decreased at both time points, however more accentuated at the early time point. Thus, it was interesting to notice how the transient physicochemical changes observed at 14 days were able to subtly impact NOV performance. For the EH the observed changes had an acute impact in aerodynamic performance, confirming the documented susceptibility of this formulation to humidity.

Figure 4 - Overview of the differences found in (a) delivered drug dose and (b) fine particle mass after evaluation of the in-vitro aerodynamic performance (n= 2).

Conclusions Findings in this work uncovered how different formulation properties can be correlated to distinct susceptibilities to humidity, supporting other works in literature. EH smaller particle size and higher amount of fines showed to be critically impacted by humidity, explaining the poorer in-vitro aerodynamic performance found after storage. In turn, NOV powder revealed to be more resistant to environmental conditions, resulting that only a transient effect could be seen in powder physicochemical characteristics after 14 days; these only subtly affecting the in-vitro aerodynamic performance. It is worth noticing that most prominent changes were observed at the first time point and that subsequent changes were small. It cannot be excluded, however, that incorrect use by the patient (e.g. exhalation into the mouthpiece) can lead to further decrease in performance at later time points. Thus, it becomes evident that some formulation strategies might be more efficient protecting powders against humidity than others. Therefore, these must be carefully considered when developing new products for inhalation delivery. References 1

C. Janson, Thomas Lööf, G. Telg, G. Stratelis, and F. Nilsson: Difference in resistance to humidity between commonly used dry powder inhalers: an in vitro study, NPJ Prim Care Respir Med 2016, 26: pp 16053.

N. Lærum, G. Telg and G. Stratelis: Need of Education for dry powder inhaler storage and retention – a patient-reported survey, Multidiscip Respir Med 2016; 11: pp 21.

R. L. Hassel and H. Nathan: Dynamic vapor sorption characterization of pharmaceutical recrystallization (Ref# TA335), retrieved from TA instruments website: http://www.tainstruments.com/applications-library-search/ .

C.P. Watling, J.A. Elliott, C. Scruton and R.E. Cameron: Surface modification of lactose inhalation blends by moisture, Int.J. Pharm. 2010, 391: pp 29-37.

J. Bronlund and T. Paterson: Moisture sorption isotherms for crystalline, amorphous and predominantly crystalline lactose powders, Int. Dairy J. 2004, 14: pp 247-254.

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Drug Delivery to the Lungs, Volume 29, 2018 – S Radivojev et al. Insights into humidity-induced changes on the in-vitro pulmonary deposition and the predicted plasma levels of budesonide from commercial DPIs: a pharmacokinetic model assisted risk assessment approach S Radivojev1,2, J T Pinto1, E Fröhlich1,2 & A Paudel1,3 1Research

Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, Graz, 8010, Austria Center for Medical Research, Medical University of Graz, Stiftingtalstraße 24, Graz, 8010, Austria 3Institute of Process and Particle Engineering, Graz University of Technology, Inffeldgasse 13, Graz, 8010, Austria 2

Summary In-silico studies are recognized as a strategy to facilitate the development of new pharmaceutical products and/or assess post-approval changes to the manufacturing process or formulation. In-silico predictions can be utilized to understand the interplay between the physicochemical characteristics of the formulation and drug pharmacokinetics (PK). The aim of this study was to explore the impact of different storage conditions on the in-vitro deposition profiles of two commercially available dry powder inhaler (DPI) formulations of budesonide, namely Easyhaler® and Novolizer®, predicting the potential changes on in-vivo PK parameters. Sensitivity of DPI formulations to humidity is increasingly being reported in the literature. However, their potential relevance on in-vivo performance is not yet clear. Utilization of distinct in-vitro deposition profiles found over time after storage under 60% and >90% relative humidity (RH) in the in-silico PK model resulted into the predicted PK parameters demonstrating different extents of impact depending on formulation characteristics. In general, a slight decrease in the in-vitro deposited fraction of drug in the peripheral lung (over time) was observed for Novolizer®. This yielded a subtle change in the predicted PK parameters for the samples stored at different time points, yet, no notable difference could be found between the two storage conditions. For Easyhaler®, a time-dependent decrease in the trend in delivered dose of the drug was identified, especially when inhalers were stored at >90% RH. This directly impacted budesonide plasma concentration profiles predicted in-silico. These first set of data demonstrated that during product development, the risk of the change in the in-vivo fate of DPI induced mishandling, namely storage at extreme RH can be accessed by combining in-vitro and in-silico approaches. Key Message In-vitro deposition profiles together with in-silico modelling revealed that some formulation strategies are more efficient protecting DPI products from humidity than others. DPIs that were more sensitive to moisture showed reduced dose consistency over time posing a potential risk in terms of the drug therapeutic effect. This study highlights how a combined in-vitro-in-silico approach can be used as a risk assessment tool in DPI formulation development. Introduction In the last years, different in-silico methodologies have been utilized as an important branch of clinical drug development. In-vitro-in-vivo correlations (IVIVC) can be applied to correlate the in-vitro performance with the invivo behavior of therapeutic products. DPIs are commonly used orally inhaled products (OIPs). While the effect of different manufacturing processes on the physicochemical characteristics of the drug, as well as different formulation strategies are intensively investigated, emphasis on products resistance to relative humidity (RH) has been less. Differences between DPIs resistance to humidity have been reported and shown to significantly affect their aerodynamic performance. For instance, a comparison of two generic products of inhaled budesonide, i.e. Easyhaler® (EH) and Novolizer® (NOV) showed the former to be more sensitive to humidity [1]. Considering that approximately 40% of patients improperly store their inhalers in the bathroom [2], it is important to understand the impact that extreme humidity conditions have on DPI performance. By applying an in-silico PK modelling approach, this study aimed to explore how the moisture sensitivities of different DPI formulations can impact their predicted in-vivo performance. For this, changes in the aerodynamic particle size distribution (APSD) were determined across different time points using a Next Generation Impactor (NGI) and used as inputs to simulate the plasma levels of inhaled budesonide using a Multiple-Path Particle Dosimetry Model in combination with a compartmental PK model developed using GastroPlusTM (GP) software integrated Pulmonary Compartmental Absorption and TransitTM route model (PCAT). Materials and methods The following commercially available budesonide inhalers were tested: Easyhaler, 400 µg per dose, 100 doses (Giona Easyhaler®, Orion Pharma, Espoo, Finland) and Novolizer, 400 µg per dose, 100 doses (Novolizer®, Meda Pharma GmbH, Vienna, Austria). The inhalers were stored in a climate cabinet at 25ºC/ 60% RH (a typical ICH condition) and at high relative humidity conditions in a desiccator at 22 ± 2ºC containing a saturated salt solution of potassium nitrate (93-94% RH) to mimic a household bathroom. The inhalers were analysed as received (time 0) after which they were stored at two aforementioned conditions, with their protective dust caps on, and analysed after 14 and 28 days. All the products were within their shelf-life expectancy.

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Drug Delivery to the Lungs, Volume 29, 2018 - Insights into humidity-induced changes on the in-vitro pulmonary deposition and the predicted plasma levels of budesonide from commercial DPIs: a pharmacokinetic model assisted risk assessment approach PK model development and validation GP version 9.6 (SimulationsPlus, Inc., USA) with integrated PCAT was used for in-silico modelling and predictions. A three-compartmental model was firstly developed for budesonide based on the intravenous (i.v.) in-vivo data available in the literature [3]. The data was obtained after administration of a 500 µg dose of budesonide as a 10 minutes infusion in humans with a mean age of 42 years and weight of 68 kg. The physicochemical properties of budesonide were generated using ADMET Predictor TM version 9.0 (SimulationsPlus, Inc., USA) (Molecular Weight=430.54 g/mol, logP 2.52). Afterwards, predictability of the model was estimated by simulating available invivo data for the Giona® Easyhaler DPI [4]. The lung deposition was predicted using Multiple-Path Particle Dosimetry Model (MPPD; Applied Research Associates, Inc, USA), using Mass Median Aerodynamic Diameter (MMAD) obtained from the literature [5]. In-silico prediction of budesonide from commercially available DPIs The developed model was further used for in-silico predictions of budesonide from EH and NOV. In combination with the APSD obtained experimentally, MPPD was used for the estimation of deposition fractions after storage. The used data is listed in Table 1. Table 1 Parameters obtained via in-vitro deposition studies used in simulations (MPPD Lung Geometry: stochastic lung, 60th percentile of airways)

Parameter Airflow [L/min] EH n=2 Storage conditions Time 0 14 days 28 days

NOV n=2 Storage conditions

Time 0 14 days 28 days

MMAD [µm]

Value 60 GSD

60% >90% RH RH 3.55 3.55 5.09 7.34 4.77 9.69 MMAD [µm]

60% >90% RH RH 2.24 2.24 1.94 2.23 2.18 2.24 GSD

60% RH 1.98 2.15 2.35

60% RH 2.04 2.23 2.19

>90% RH 1.98 1.73 2.30

>90% RH 2.04 1.96 2.21

Delivered Dose [mg] 60% >90% RH RH 0.400 0.400 0.210 0.081 0.180 0.047 Delivered Dose [mg] 60% >90% RH RH 0.400 0.400 0.349 0.380 0.409 0.418

Results and discussion Construction of the PK model for budesonide Plasma concentration profile after i.v. administration of budesonide is shown in Figure 1A. The calculated budesonide PK parameters were used subsequently in the pulmonary model. The budesonide model was validated using literature data [4], [5] correspondent to an intake of 1000 µg of budesonide as an inhalation powder at 45 L/min and an MMAD of 3.91 µm (Figure 1B). The deposited fractions of the drug were simulated using MPPD and input into GP; these were 67.71% for the extra-thoracic compartment (ET), 8.10% for the central (C) and 22.72% for the peripheral (P) lung. These findings were in line with others described in literature, where healthy volunteers using EH in combination with salbutamol showed by gamma scintigraphy to have 63.9±11.9 % of the drug deposited in the upper airways and 28.9±11.6% in the lungs [6]; thus MPPD was found appropriate to simulate particle deposition. The predicted Cmax after inhalation of budesonide was approximately 9 times higher than in-vivo values, while the AUC0-∞ was in a good agreement with the latter (Figure 1B). There are several possible reasons for the observed trend, particle size being reported as one of them [6]. Additionally, considering that budesonide is a low soluble compound, dissolution might be a rate-limiting step for absorption, potentially influencing in-silico results. Dissolution of aerosol particles generated from DPIs is dependent on particle size, API pulmonary distribution as well as the limited volume of the lung lining fluid (10-30 mL). At present, lung dissolution cannot be properly accounted for in the GP software possibly explaining the observed results [8]. To further improve the model, the unknown mass fractions of swallowed and expectorated budesonide from the ET compartment in the GP model were optimized to 2% and 98%, respectively. Considering the low solubility and slow dissolution rate of budesonide particles, it is reasonable to assume that all the amount deposited in the ET compartment gets swallowed or expectorated, thus this values were found appropriate to be used.

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Figure 1– A) Plasma concentration profile after i.v. administration of budesonide (■) vs. predicted plasma profile ( ), R2=0.97, B) Plasma concentration profile after inhalation of 1000 µg of budesonide from Giona® Easyhaler (■), predicted plasma concentration profile using calculated deposited fractions by MPPD (---), R2=0.83

In-silico predictions of budesonide from commercially available DPIs The MPPD deposition model resulted in a mass fraction of approximately 36% in the whole lung (C and P fractions) which agrees with the reported in-vitro values for EH [5]. Naturally, a slight increase in the whole lung deposited fraction was observed with the flow increase from 45 L/min to 60 L/min. The resulted deposited fractions of NOV across different time points slightly differed for both storage conditions (Figure 2).

Figure 2 – The deposited fractions, across different time points A) EH stored at 25ºC/ 60% RH; B) EH stored at 22 ºC/ >90% RH; C) NOV stored at 25ºC/ 60%; D) NOV stored at 22 ºC/ >90% RH in extra-thoracic (ET), central (C) and peripheral (P) compartments

Given the low model predictability of the Cmax, evaluation was focused on AUC0-∞ values. Moreover, considering that inhaled corticosteroids (ICs) are used in the long-term maintenance therapy of asthma, the AUC of the concentration-time curve is a more important parameter to consider when evaluating their therapeutic effect. Hence, this was taken as the relevant parameter to compare and evaluate the over time performance of the investigated DPIs. The AUC0-∞ values were quite constant for the NOV implying that the performance of this formulation is rather consistent, even under extreme conditions such as >90% RH (Figure 3A). Overall, EH displayed a trend towards inconsistency in its performance, which was especially noted after storage under high humidity (>90% RH). In the latter, MMAD changed from 3.55 µm (Time 0), to 4.77 µm (28 days, 60% RH) and 9.69 µm (28 days, >90% RH, Figure 3B). Moreover, it was also interesting to note a tendentiously progressive reduction of delivered dose throughout the time points. Consequently, the predicted AUC0-∞ values were, over time, notably lower (Figure 3A).

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Figure 3 – Predicted AUC0-∞ values (A) and in-vitro MMAD (B) for inhalers stored under different storage conditions and at different time points

For control of asthma symptoms, a twice daily 400 µg dose of budesonide is recommended for one month, followed by maintenance therapy using a lower dose. Reports show that lowering the dose from 800 µg to 400 µg or 200 µg after one month of therapy will have the same effect in asthma symptoms relief, not leading to any exacerbations. However, considering that an initial dose of 800 µg per day is needed within the first month [9], it is possible that the over time inferior drug delivered due to moisture sensitivity of DPI formulations stored under high relative humidity might affect therapeutic outcomes. Conclusion and outlook This study showed that, depending on the formulation strategy, patients mishandling of the device might have a critical impact on the therapeutic effect of inhaled drugs, as here demonstrated for budesonide. Generally, the observed trends confirmed that EH is more prone towards high humidity instability, causing substantially lower doses as well as smaller FPFs to be delivered after 28 days. On the other hand, NOV showed a consistent trend in terms of in-vitro aerosolization performance and solely minor changes that only subtly affected the predicted plasma concentration profiles were detected. Thus, it becomes evident that certain formulation approaches can be more successful than others in conferring DPIs resistance to humidity and lowering potential therapeutic effect variances. Moreover, coupling of in-silico PK modelling with in-vitro characterization appeared to be a useful tool to support the risk evaluation that patient mishandling of DPIs (such as storage at higher humidity) might have on product performance. References [1]

C. Janson, T. Loöf, G. Telg, G. Stratelis, and F. Nilsson, “Difference in resistance to humidity between commonly used dry powder inhalers: An in vitro study,” npj Prim. Care Respir. Med., vol. 26, no. March, pp. 1–5, 2016.

[2]

B. Norderud Lærum, G. Telg, and G. Stratelis, “Need of education for dry powder inhaler storage and retention - A patient-reported survey,” Multidiscip. Respir. Med., vol. 11, no. 1, pp. 1–5, 2016.

[3]

L. Thorsson and S. Edsbäcker, “Lung deposition of budesonide from a pressurized metered-dose inhaler attached to a spacer,” Eur. Respir. J., vol. 12, no. 6, pp. 1340–1345, 1998.

[4]

S. Lähelmä et al., “Equivalent lung deposition of budesonide in vivo: A comparison of dry powder inhalers using a pharmacokinetic method,” Br. J. Clin. Pharmacol., vol. 59, no. 2, pp. 167–173, 2005.

[5]

I. Parisini, “Improved Aerosol Deposition Profiles from Dry Powder Inhalers,” no. November, pp. 1–238, 2014.

[6]

M. Vidgren, M. Silvasti, P. Vidgren, H. Sormunen, K. Laurikainen, and P. Korhonen, “Easyhaler® multiple dose powder inhaler—Practical and effective alternative to the pressurized MDI,” Aerosol Sci. Technol., vol. 22, no. 4, pp. 335–345, 1995.

[7]

M. Frost, “ORALLY INHALED & NASAL DRUG PRODUCTS : Innovations from Major Delivery SUBSCRIPTIONS : PRODUCTION / DESIGN :,” Drug Deliv.

[8]

D. Arora, K. A. Shah, M. S. Halquist, and M. Sakagami, “In Vitro aqueous fluid-capacity-limited dissolution testing of respirable aerosol drug particles generated from inhaler products,” Pharm. Res., vol. 27, no. 5, pp. 786–795, 2010.

[9]

A. Foresi, M. C. Morelli, and E. Catena, “Low-dose budesonide with the addition of an increased dose during exacerbations is effective in long-term asthma control,” Chest, vol. 117, no. 2, pp. 440–446, 2000.

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Drug Delivery to the Lungs, Volume 29, 2018 - Jolyon P Mitchell et al. A European Pharmaceutical Aerosol Group (EPAG)-Led Cross-Industry Assessment of Inlet Flow Rate Profiles of Compendial DPI Test Systems: Part 1 â&#x20AC;&#x201C; Experimental Data Jolyon P Mitchell1, Chris Blatchford2, Roland Greguletz3, Daryl L. Roberts4, & Henk Versteeg5 Jolyon Mitchell Inhaler Consulting Services Inc., 1154 St. Anthony Road, London, N6H 2R1, Canada 2 3M United Kingdom plc, Loughborough, LE11 5RB, UK 3 Sofotec GmbH, BenzstraĂ&#x;e 1-3, Bad Homburg, D-61352, Germany 4 Applied Particle Principles LLC, 17347 Westham Estates Court, Hamilton, VA 20158, USA 5 Mechanical and Manufacturing Engineering, Loughborough University, Loughborough, LE11 3TU, UK 1

Summary We report outcomes from an EPAG-led cross-industry study, characterizing flow rate/elapsed-time profiles of equipment used for testing dry powder inhalers (DPIs). A thermal mass flow sensor was used by nine organizations in a round-robin approach to record inlet flow rate-time profiles of individual participant compendial test systems (TS) including either sample collection tubes (SCT) or a cascade impactor (either the Andersen 8-stage non-viable impactor, ACI, or the Next Generation Impactor, NGI) equipped with USP/PhEur induction port and pre-separator. An inlet orifice generated a 4-kPa pressure drop at each of the target flow rates (30, 60 and 90 L/min), simulating a pressure drop typical for high-, medium- and low-resistance DPIs respectively. Rise times to 90% of these target flow rates (t90) were longest with largest internal dead volume and followed the order NGI>ACI >SCT>TS. When the surrogate DPI (4-kPa orifice) was absent, t90 values generally lengthened with increasing target flow rate. In contrast, the opposite behaviour was observed when the surrogate DPI was present. A flow acceleration parameter was also calculated, expressed as the slope between the 20% and 80% flow rates of each final steady flow value (slopet20/t80). Greater flow acceleration occurred at higher final flow rates, irrespective of apparatus, but the presence of the surrogate DPI was associated with slower flow acceleration. These flow rate-rise time profiles will be useful for those involved in evaluating equipment for characterizing both existing and new DPIs. Key Message Flow rate rise times associated with DPI testing are correlated with the magnitude of the internal dead-volume and intrinsic resistance of the measurement apparatus. The resistance associated with a surrogate DPI has a marked influence on profile shape. These data will prove useful for evaluating and using DPI testing equipment. Introduction Compendial methods for testing DPIs require the rapid opening of a solenoid valve to start drawing air into and through the inhaler at the start of the test1. The flow-rate/rise-time profile has the potential to affect the measured in vitro characteristics of the dose that comes from the DPI, as the aerosol formation and subsequent transport of the bolus to the measurement apparatus takes place from the inhaler during this period. Furthemore, the cut-point sizes of the impactor stages are flow rate dependent. The objectives of the present multi-laboratory investigation were therefore as follows: 1.

to measure the influence of DPI resistance on flow-elapsed time profiles for a wide range of equipment used in the industry for DPI testing either for content uniformity or emitted aerosol APSD, following compendial procedures: a. without any resistance at the entry to the measurement apparatus (baseline condition); b. with resistance imposed by placing an orifice at the inlet of the apparatus whose aperture was sized to mimic a high-, medium- or low-resistance DPI by generating a pressure drop of approximately 4kPa at the chosen target final flow rates of 30, 60 and 90 L/min respectively.

to define and report the following measures of the flow-time profile that are believed to be helpful to the user community: a. the area under the flow rate-time profile (AUC), equivalent to the sampled volume; b. the associated time for the flow rate to achieve 90% of the final steady-state flow rate value, t90; c. a flow acceleration parameter derived from the slope of the rising flow rate profile, calculated by linear interpolation between the times where the flow rate attained 20% and 80% of the final flow rate values (slopet20/t80); d. the presence of any peak in the flow profile (Qpeak) before reaching the steady-state flow rate;

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Drug Delivery to the Lungs, Volume 29, 2018 - A European Pharmaceutical Aerosol Group (EPAG)-Led CrossIndustry Assessment of Inlet Flow Rate Profiles of Compendial DPI Test Systems: Part 1 â&#x20AC;&#x201C; Experimental Data Preliminary outcomes from this investigation were reported previously2. Since then, further examination of the data has been undertaken, including the exclusion of measurements made by a participant not evaluating DPI products, and repeating tests that had produced anomalous results. The present evaluation therefore reflects a more accurate assessment of the flow rate-rise time profiles as an ensemble. The interpretation has been extended by comparing the outcomes with internal dead volume (Vint) of the apparatuses, which is the apparatus-specific parameter deemed most likely to be correlated with the above metrics. Materials and Methods The procedures associated with this investigation were previously described2, so only a brief outline is given here. A series of flow rate profiles (n = 3 replicates at each condition per apparatus configuration) was therefore determined in a round-robin approach by each participating laboratory, using the same calibrated, high-resolution (Âą2% of actual reading) thermal mass flow sensor (model 4040, TSI Corp, Shoreview, MN, USA) located at the inlet of each of the following sampling apparatuses, including induction port and pre-separator: a.

individual participant flow rate test systems (TS) - average Vint = 87 mL;

sample collection tubes (SCT) - average Vint = 115 mL;

an Andersen 8-stage non-viable impactor (ACI) - Vint = 1155 mL3;

a Next Generation Impactor (NGI) - Vint = 2025 mL3.

The target sample volumes corresponding to the three final flow rates of 30 L/min, 60 L/min and 90 L/min were 2.0 L, 4.0 L and 6.0 L respectively, assuming an ideal rectangular flow-time profile for the 4.0-s time period of each measurement sequence. Flow rate-elapsed time profiles (n=3 replicates) were initially determined at each target flow rate, with the inlet to each apparatus fully unrestricted (i.e. open). The measurement sequence was subsequently repeated with an orifice-based flow restriction (surrogate DPI). This restriction, when fitted to the inlet of each apparatus, applied a fixed 4-kPa pressure drop to simulate either a high-, medium- or low-resistance DPI at the final flow rates of 30, 60 and 90 L/min respectively, by adjusting the size of the orifice aperture. The signal from the flow sensor was processed for flow-time data recording by a purpose-developed proprietary recording software (FlowMonitor version 1.2, Sofotec GmbH, Germany), that is based on the LabVIEW TM platform (National Instruments Corp., Austin, TX, USA). Each participant recorded the instantaneous flow rate once every five milliseconds. The recording software stored the flow-time raw data in csv-text file format and performed the following calculations: a.

integration of the resulting flow rate-elapsed time profile to enable the total volume sampled to be calculated as the area-under-the-curve (AUC);

determination of the maximum peak flow rate (Qpeak) from the entire flow rate-time profile for that particular measurement sequence.

The following calculations were undertaken subsequently using the collected data for each measurement: a. determination of characteristic rise time indicators in milliseconds, t20, t80 and t90, corresponding to times to attain 20%, 80%, and 90% of the final steady-state reference flow level respectively. b. determination of the slope of the rising flow rate (L/min/ms) by linear interpolation between t20 and t80, to evaluate a flow acceleration metric in the middle of the flow rate-time profile (slopet20/t80). Results Sampled air volumes (AUC) with or without the surrogate DPI were all close to the nominal values of 2.0 L, 4.0 L and 6.0 L, with most data within the ď&#x201A;ą5% interval for all apparatus configurations. The overall associated measures of variability (RSD) were low at 1.7%, 1.3% and 2.0% for target flow rates of 30 L/min, 60 L/min, and 90 L/min, respectively, confirming that a high degree of measurement accuracy and precision existed overall. A few values of Qpeak greater than 110% of the targeted flow rate (overshoot) were observed at all target flow rates. However, these incidences were confined almost exclusively to the measurements with TS alone (with no SCT nor impactor). The phenomenon was most apparent at the highest flow rate (Figure 1). At each target rate, values of t90 lengthened as the internal volume of the test setup increased when the surrogate DPI was present (Table 1). The NGI, having the largest Vint, in combination with the surrogate DPI having the highest intrinsic resistance (lowest target flow rate), was associated with the longest t90 values at each target flow rate. This cascade impactor was also associated with the greatest change in t90 when the target flow rate was increased from either 30 to 60 L/min or from 60 to 90 L/min. Significantly, when the surrogate inhaler was present, the opposite behaviour was evident for the t90 values associated with all sampling configurations; that is, t90% decreased as the final flow rate was increased. The presence of the surrogate DPI at the inlet also significantly increased the t90 values associated with each apparatus compared with corresponding values when that flow restriction was absent.

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Figure 1 - Participant-by-Participant Measures of Qpeak for target flow rate of 90 L/min, Organized by Identity Letter Code A to J; TS= Organization-Specific Test System; SCT = Sample Collection Tube; ACI = Andersen 8-Stage Cascade Impactor; NGI = Next Generation Impactor Longer rise times, in combination with decreased values of the flow acceleration parameter (Table 2), were associated with apparatus configurations having larger internal volumes. This behaviour is best illustrated by the data for the NGI, whose internal volume with pre-separator is almost twice that of the ACI with pre-separator. Table 1 - Mean Values of t90 for Each Apparatus Surrogate DPI

Apparatus Configuration

Final Flow Rate = 30 L/min

Average Vint (mL)

12 (n=45) 33 SCT 115 (n=27) Absent ACI 60 L/min* + 32 1155 preseparator (n=9) NGI 49 2025 + preseparator (n=27) 31 TS 87 (n=45) 62 SCT 115 (n=27) Present ACI 60 L/min* + 281 1155 preseparator (n=9) NGI 431 2025 + preseparator (n=27) * ACI 60 L/min stage configuration: stages -1, -0, 1 to 6, and back-up filter TS

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Final Flow Rate = 60 L/min t90 (ms) 13 (n=45) 34 (n=27) 46 (n=12) 91 (n=27) 20 (n=45) 46 (n=27) 158 (n=12) 266 (n=27)

Final Flow Rate = 90 L/min 13 (n=44) 34 (n=27) 65 (n=9) 106 (n=27) 17 (n=45) 41 (n=27) 131 (n=9) 197 (n=27)

Apparatus Configuration

Final Flow Rate = 30 L/min

Average Vint (mL)

3.72 (n=45) 2.37 SCT 115 (n=27) Absent ACI 60 L/min*+ 1.83 1155 preseparator (n=9) NGI 1.74 2025 + preseparator (n=27) 1.67 TS 87 (n=45) 0.85 SCT 115 (n=27) Present ACI 60 L/min*+ 0.14 1155 preseparator (n=9) NGI 0.08 2025 + preseparator (n=27) * ACI 60 L/min stage configuration: stages -1, -0, 1 to 6, and back-up filter TS

Final Flow Final Flow Rate = 60 Rate = 90 L/min L/min Slopet20 / t80 (L/min/ms) 7.15 10.30 (n=45) (n=44) 4.59 6.84 (n=27) (n=27) 2.18 2.00 (n=12) (n=9) 0.98 1.14 (n=27) (n=27) 4.42 8.46 (n=45) (n=45) 2.96 4.87 (n=27) (n=27) 0.49 0.88 (n=12) (n=9) 0.27 0.54 (n=27) (n=27)

Discussion and Conclusion: Rise time performance, both with and without the surrogate DPI present, was relatively undamped for either the TS or SCT apparatuses (where both Vint and intrinsic apparatus resistance were small), reflected in short t90 and large slopet20/t80 values. This behaviour was largely independent of whether the surrogate DPI was present or absent. The increased resistance imposed by the surrogate DPI resulted in slower rise times and reduced slopet20/t80 values. This behaviour was evident with all apparatus configurations, but was most apparent with both the CIs, that had substantially larger values of Vint. The greatly dampened flow rate rise time behaviour with these apparatuses requires further explanation, as does the reversal in the behaviour of the relationships between both rise time and slope and target flow rate for a given apparatus configuration, comparing the situations when the surrogate DPI was absent or present. The first few stages of either CI comprise at least half, if not more, of the total internal dead volume, and contribute only a small flow resistance compared to that imposed by the presence of the 4-kPa pressure drop associated with the surrogate device. When the surrogate DPI was absent, the CI volume encompassing these low-resistance stages filled up relatively rapidly, so that the magnitude of t90 was dictated almost exclusively by the resistance of the last one or two stages having the highest intrinsic resistance. When the 4-kPa surrogate device was present, however, the air flowing into the CI took much longer to fill up the half or more of the impactor internal volume that in the previous case had been almost instantaneously filled. This delay decreased as the target flow rate was increased. Regardless of the presence or absence of the surrogate device, the smallest values of the acceleration parameter were associated with configurations having the largest internal dead volumes and highest intrinsic apparatus resistance, an intuitive outcome. A simple two-compartment first-order model aiming to qualitatively interpret these and other observed effects, has been presented at this conference 4. References: 1

European Directorate for Quality in Medicines and Healthcare (EDQM). European pharmacopeia 9.0, monograph 2.9.18. Preparations for inhalation: Aerodynamic assessment of fine particles. EDQM, Strasburg, France; 2017.

Greguletz, R, Andersson P, Arp J, Blatchford C, Daniels G, Glaab V, Hamilton H, Hammond M, Mitchell J, Roberts D, Shelton C, Watkins A. A collaborative study by the European Pharmaceutical Aerosol Group (EPAG) to assess the flow-time profile of test equipment typically used for pMDI/DPI testing – Part 2: Flow-time profile testing. Drug Delivery to the Lungs-25, The Aerosol Society, Edinburgh, UK, 2014: pp. 146-149.

Copley M, Smurthwaite M, Roberts DL, Mitchell JP, Revised Internal Volumes of Cascade Impactors for Those Provided by Mitchell and Nagel, J. Aerosol Med., 2005;18(3): pp. 364-366.

Roberts D, Versteeg H, Blatchford C, Greguletz R, & Mitchell JP, A European Pharmaceutical Aerosol Group (EPAG)-Led CrossIndustry Assessment of Inlet Flow Rate Profiles of Compendial DPI Test Systems: Part 2 – First-Order Model. Drug Delivery to the Lungs-2018, The Aerosol Society, Edinburgh, UK, 2018: pp. TBA.

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Drug Delivery to the Lungs, Volume 29, 2018 - Daryl L. Roberts et al.

A European Pharmaceutical Aerosol Group (EPAG)-Led Cross-Industry Assessment of Inlet Flow Rate Profiles of Compendial DPI Test Systems: Part 2 – First-Order Impactor Model Daryl L. Roberts1, Henk Versteeg2, Chris Blatchford3, Roland Greguletz4, Jolyon P. Mitchell5 Applied Particle Principles LLC, 17347 Westham Estates Court, Hamilton, VA 20158, USA Mechanical and Manufacturing Engineering, Loughborough University, Loughborough, LE11 3TU, UK 3 3M United Kingdom plc, Loughborough, LE11 5RB, UK 4 Sofotec GmbH, Benzstraße 1-3, Bad Homburg, D-61352, Germany 5 Jolyon Mitchell Inhaler Consulting Services Inc., 1154 St. Anthony Road, London, N6H 2R1, Canada 1

Summary A simple two-compartment, first-order flow resistance model of a cascade impactor reveals the reasons for the major trends observed in the companion, cross-industry study of the transient behaviour of the inlet flow rate in compendial DPI test systems. This model is physically reasonable because most of the internal volume of compendial impactors is comprised of stages with rather small resistance to flow, and when no DPI is attached to the induction port, the major flow resistance is contributed by the final one or two stages of the impactor. The typical DPI, then, with approximately 4-kPa pressure drop at the sampling flow rate, changes this situation by placing a significant flow resistance upstream of the otherwise insignificant resistance of the bulk of the impactor volume. Results with the two-compartment model reasonably agree with the experimental data in three important aspects: (a) the substantial increase in rise time when a surrogate DPI is present, (b) the decrease in rise time as the steadystate flow rate increases but only if the surrogate DPI is present (and opposite to the observed trend when the surrogate DPI is absent), and (c) the increase in rise time with larger total internal volume of the test equipment. Compared with three-dimensional, unsteady-state numerical solutions of flow rate behaviour at start-up, the simple model intuitively conveys important physics that will assist users in understanding compendial DPI quality control test results, which could be very helpful when a user experiences unexpected trends or outliers in a data set. Key Message The role of the DPI and of the impactor volume in compendial testing for particle size can be physically understood by considering the impactor to consist of a low-resistance major volume and a high-resistance minor volume. This two-compartment model agrees qualitatively with nearly all of the EPAG cross-industry experimental data. Introduction Compendial methods for testing dry-powder inhalers DRAW air through the inhaler device and the cascade impactor by quickly opening a solenoid valve placed downstream of the test apparatus. The air flow begins by passing through the solenoid valve, and the point of forward air flow propagates upstream to the inlet of the DPI. Consequently, the air flow drawn into and through the inhaler device itself is delayed relative to the flow drawn through the solenoid valve. This inlet air flow increases with time at the outset of the test and reaches steady state in tens or hundreds of milliseconds after the solenoid valve opens, depending on the details of the test system and of the DPI. A major experimental study of these flow start-up kinetics is described in a companion publication 1. We report here on a simple first-order computational model designed to explain the major trends seen in these experimental data, specifically those with the Andersen impactor or the NGI, with and without a surrogate DPI with 4 kPa of flow resistance attached to the impactor’s induction port. We summarize in Table 1 the key experimental observations to which this model applies; we strongly advise the reader to take the time to understand the experimental set ups described in reference 1 before proceeding further. Table 1 – Selected Experimental Values of t90 for Flow Start-Up in DPI Testing (milliseconds) Target Flow Rate (L/min) 30 60 90 Test system only 12 13 13 ACI 32 46 65 NGI 49 91 106 Test system + 4kPa orifice* 31 20 17 ACI + 4kPa orifice 281 158 131 NGI + 4kPa orifice 431 266 197 *one of three fixed orifices described in reference 1 imparting 4 kPa of pressure drop at the target flow rate and therefore acting as a surrogate DPI Configuration

265

Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; A European Pharmaceutical Aerosol Group (EPAG)-Led Cross-Industry Assessment of Inlet Flow Rate Profiles of Compendial DPI Test Systems: Part 2 â&#x20AC;&#x201C; First-Order Impactor Model Several issues are apparent from these data. First, the rise time increased by a multiple of two to eight when the surrogate DPI was present, seemingly disproportionately to its 4-kPa flow resistance. Second, with this 4-kPa pressure drop, the rise time decreased when the flow rate increased, a behavior opposite to that observed when the surrogate DPI was absent. Third, the system with the largest internal volume exhibited the largest rise time. This third observation is likely the only one of the three that would fit the intuition of most inhaler testers. For this reason, we believe that a model that outlines the fundamental physics of these trends would go a long way toward educating the user community about the factors that control rise time in compendial DPI test systems. Physical Model The internals of a cascade impactor, along with the typical ancilliary tubing, valves, and fittings of a compendial DPI test system contain substantial details that may or may not be significant to the observed behavior. Surprisingly, no complete three-dimensional model of a compendial system has appeared in the literature. However, 3-D and one-dimensional computational models of the impactor itself have been described 2,3. Even so, in these models, no attempt has been made to study the effect of the DPI itself. To remedy this situation at the same time as conveying the important physics, we consider flow start up in a cascade impactor to consist simply of two volumes separated from the ambient air and each other by two arbitrary nozzle plates that provide a â&#x20AC;&#x153;low resistanceâ&#x20AC;? to flow, denoted by R1, and a â&#x20AC;&#x153;high resistanceâ&#x20AC;? to flow, denoted by R2 (Figure 1).

Figure 1: Two-compartment conceptual model of present analytical study These flow resistances can be contributed physically by anything in the flow path. For the purposes of this model, resistance r1 can include an inhaler device or not. This resistance can also be the aggregate resistance of several nozzle plates, with or without an inhaler device. Resistance r2 can be the resistance of a particular nozzle plate or can be the aggregate resistance of several nozzle plates. For the ACI and the NGI, the first five or six stages have typically less than 10% of the overall flow resistance 4,5. We postulate therefore that these impactors can be represented by two regions, one of which constitutes the majority of the internal volume (V1) with little flow resistance and one with a small portion of the total volume (V2) but with the bulk of the flow resistance. Mathematical Model Using the ideal gas law, the time rate of change of pressure in volume V1 and in volume V2 can be expressed in terms of the mass flow rate of air into and out of each chamber as follows: đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x192;đ?&#x2018;&#x192;1 đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026; đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;1

) . (đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;1 â&#x2C6;&#x2019; đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;2 )

and

đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x192;đ?&#x2018;&#x192;2 đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026; đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;2

(1a,b)

) . (đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;2 â&#x2C6;&#x2019; đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020; )

Here, đ?&#x2019;&#x17D;đ?&#x2019;&#x17D;Ě&#x2021;đ?&#x;?đ?&#x;? and đ?&#x2019;&#x17D;đ?&#x2019;&#x17D;Ě&#x2021;đ?&#x;?đ?&#x;? are the mass flow rates of air entering V1 and into V2, T is the absolute temperature of the air (assumed to be isothermal throughout), and R is the universal gas constant. The term đ?&#x2019;&#x17D;đ?&#x2019;&#x17D;Ě&#x2021;đ?&#x2018;şđ?&#x2018;şđ?&#x2018;şđ?&#x2018;ş is the mass flow rate of air leaving the impactor at steady-state flow conditions. We assume that this mass flow rate of air begins to leave volume V2 immediately at time zero, an assumption that is reasonably accurate because the velocity of air leaving the control valve just downstream of the solenoid valve reaches sonic speed nearly instantaneously under the compendial protocol conditions. A more accurate boundary condition would include an expression for the mass flow rate through a sonic control valve 3. However, such an approach would thwart development of an analytical solution to the relevant equations. Equations 1a and 1b can be rearranged into two â&#x20AC;&#x153;pressure differenceâ&#x20AC;? equations â&#x20AC;&#x201C; that is, the pressure drop across each of the two resistances -- as follows: â&#x2C6;&#x2019;

đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;(đ?&#x2018;&#x192;đ?&#x2018;&#x192;â&#x2C6;&#x17E; â&#x2C6;&#x2019;đ?&#x2018;&#x192;đ?&#x2018;&#x192;1 ) đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2021;đ?&#x2018;&#x2021;â&#x2C6;&#x17E; đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;1

) . (đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;1 â&#x2C6;&#x2019; đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;2 )

and

đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;(đ?&#x2018;&#x192;đ?&#x2018;&#x192;1 â&#x2C6;&#x2019;đ?&#x2018;&#x192;đ?&#x2018;&#x192;2 ) đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2021;đ?&#x2018;&#x2021;â&#x2C6;&#x17E; đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;1

) . (đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;1 â&#x2C6;&#x2019; đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;2 ) â&#x2C6;&#x2019; (

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2021;đ?&#x2018;&#x2021;â&#x2C6;&#x17E; đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;2

) . (đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;2 â&#x2C6;&#x2019; đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020; )

(2a,b)

Here đ?&#x2018;&#x192;đ?&#x2018;&#x192;â&#x2C6;&#x17E; is the (constant) ambient pressure; its time derivative is equal to zero. In general, đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;2 exceeds đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;1 , accounting for the negative sign on the left-hand side of equation 2a.

266

Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; Daryl L. Roberts et al. The functional relationship of the mass flow rate to the resistances r1 and r2 and the pressure drop across a DPI is typically regarded as a square root relationship 6: (đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021; = đ??žđ??ž â&#x2C6;&#x2014; â&#x2C6;&#x161;â&#x2C6;&#x2020;đ?&#x2018;&#x192;đ?&#x2018;&#x192;). Cascade impactor stages also follow this â&#x20AC;&#x153;Bernoulli-typeâ&#x20AC;? relationship 7. The ratio of the resistances in any portion of the flow path is therefore independent of the flow rate (true for any power-law relationship of đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021; to â&#x2C6;&#x2020;đ?&#x2018;&#x192;đ?&#x2018;&#x192;, provided that the relationship is the same in each component of the flow path). Therefore, assuming a linear relationship is very likely to exhibit the proper trends, and, IMPORTANTLY, the linear assumption affords us an analytical solution that reveals much of the relevant physics. [Proper parameter selection is described below]. With the linear relationship of the pressure drop to the mass flow rate, equations 2a and 2b can be written in a nondimensional format as follows: đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x153;&#x2021;đ?&#x153;&#x2021;1 đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;

=â&#x2C6;&#x2019;

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x;đ?&#x2018;&#x;

đ?&#x153;&#x2021;đ?&#x153;&#x2021;1 +

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x;đ?&#x2018;&#x;

đ?&#x153;&#x2021;đ?&#x153;&#x2021;2

đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x153;&#x2021;đ?&#x153;&#x2021;2

and

đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;đ?&#x2018;&#x2018;

â&#x2C6;&#x2019;

1 đ?&#x153;&#x2021;đ?&#x153;&#x2021; đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; (1â&#x2C6;&#x2019;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; )(1â&#x2C6;&#x2019;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x;đ?&#x2018;&#x; ) 2

+ (1â&#x2C6;&#x2019;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;

(3a,b)

đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; )(1â&#x2C6;&#x2019;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x;đ?&#x2018;&#x; )

Here, the dimensionless mass flow rates are a fraction of the steady-state mass flow rate (đ?&#x153;&#x2021;đ?&#x153;&#x2021;1 = đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;1 â &#x201E;đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020; and đ?&#x153;&#x2021;đ?&#x153;&#x2021;2 = (đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; +đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; )(đ?&#x2018;&#x;đ?&#x2018;&#x; +đ?&#x2018;&#x;đ?&#x2018;&#x; ) đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;2 â &#x201E;đ?&#x2018;&#x161;đ?&#x2018;&#x161;Ě&#x2021;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020; ). The dimensionless (characteristic) time is given by đ?&#x153;?đ?&#x153;? = đ?&#x2018;Ąđ?&#x2018;Ąâ &#x201E;đ?&#x2018;Ąđ?&#x2018;ĄĚ&#x192; with đ?&#x2018;Ąđ?&#x2018;ĄĚ&#x192; = 1 2 1 2 . Finally, the dimensionless parameters Rv: (đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; =

đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;1

đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;1 +đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;2

) and Rr: (đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x;đ?&#x2018;&#x; =

đ?&#x2018;&#x;đ?&#x2018;&#x;1

đ?&#x2018;&#x;đ?&#x2018;&#x;1 +đ?&#x2018;&#x;đ?&#x2018;&#x;2

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2021;đ?&#x2018;&#x2021;â&#x2C6;&#x17E;

) show that the qualitative behavior of the system will

be governed by the ratio of the two volumes and the ratio of the two resistances, not the individual values, an intuitively reasonable outcome. We have developed an explicit analytical solution to these two simultaneous first-order differential equations (equations 3a and 3b), via Laplace transformations, leading to the following expression for the inlet flow rate Q(t):

đ?&#x2018;&#x201E;đ?&#x2018;&#x201E; (đ?&#x2018;Ąđ?&#x2018;Ą ) = đ?&#x2018;&#x201E;đ?&#x2018;&#x201E;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020; . [1 +

đ?&#x2018;Ąđ?&#x2018;Ą đ?&#x2018;Ąđ?&#x2018;Ą đ?&#x2018; đ?&#x2018; 1

đ?&#x2018;Ąđ?&#x2018;Ą đ?&#x2018;Ąđ?&#x2018;Ą đ?&#x2018; đ?&#x2018; 2

đ??ľđ??ľ.exp(â&#x2C6;&#x2019; Ě&#x192;â &#x201E; )+đ??śđ??ś.exp(â&#x2C6;&#x2019; Ě&#x192;â &#x201E; ) đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x;đ?&#x2018;&#x; (1â&#x2C6;&#x2019;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; )(1â&#x2C6;&#x2019;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x;đ?&#x2018;&#x; )

]

đ?&#x2018;&#x201E;đ?&#x2018;&#x201E; (đ?&#x2018;Ąđ?&#x2018;Ą )/đ?&#x2018;&#x201E;đ?&#x2018;&#x201E;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020;đ?&#x2018;&#x2020; â&#x2C6;&#x2019; 1 =

đ?&#x2018;Ąđ?&#x2018;Ą đ?&#x2018;Ąđ?&#x2018;Ą đ?&#x2018; đ?&#x2018; 1

đ?&#x2018;Ąđ?&#x2018;Ą đ?&#x2018;Ąđ?&#x2018;Ą đ?&#x2018; đ?&#x2018; 2

đ??ľđ??ľ.exp(â&#x2C6;&#x2019; Ě&#x192;â &#x201E; )+đ??śđ??ś.exp(â&#x2C6;&#x2019; Ě&#x192;â &#x201E; )

(4a,b)

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x;đ?&#x2018;&#x; (1â&#x2C6;&#x2019;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; )(1â&#x2C6;&#x2019;đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x;đ?&#x2018;&#x; )

Here, Qss is the steady-state flow rate, and the coefficients B and C are non-linear expressions involving Rv, Rr and the time-scaling coefficients S1 and S2. The rearrangement, equation 4b, reveals that the deviation of the inlet flow rate from the steady-state value decays to zero exponentially at a rate that is a complex combination of the several relevant physical parameters (proper values of the coefficients B and C ensure that Q(t) is less than Qss). Parameters for the Mathematical Model Two aspects of the parameter estimation derive from available data and are independent of assumptions intrinsic to the approximate model. First, values of V1 and V2 must be consistent with the internal volumes of the induction port, pre-separator, and impactor 8. Second, the dimensionless ratio Rr must be consistent with the reported pressure drop data for the impactors 4,5,7. The question becomes â&#x20AC;&#x153;what reasonable fractionâ&#x20AC;? of the total volume should be considered in the â&#x20AC;&#x153;low resistanceâ&#x20AC;? compartment as opposed to the â&#x20AC;&#x153;high resistanceâ&#x20AC;? compartment. If we take stages -1 to 5 of the ACI (60-L/min configuration) and stages 1 to 6 of the NGI to constitute the lowresistance, larger volume (including the induction port and pre-separator), we find that the fraction of the total flow resistance found in volume V2 is indeed less than 10%. Additionally, V1 is more than 75% of the system volume. Both of these results are intuitively sensible for the two-compartment model. Now, we calculate the linear resistance coefficient đ?&#x2018;&#x;đ?&#x2018;&#x;1 by dividing the actual, known pressure drop in V1 at the steady-state flow rate by the steady-state mass flow rate, and we calculate the dimensionless ratio Rr from the actual, known values for V1 and for the total impactor (Table 2). For 30 L/min and 90 L/min steady-state flow rates, the value of đ?&#x2018;&#x;đ?&#x2018;&#x;1 is 0.5 and 1.5 times the value at 60 L/min, respectively, but the ratio Rr is the same regardless of the steady-state flow rate. Table 2 â&#x20AC;&#x201C; Volume and 60-L/min Resistance Parameters for the ACI and the NGI

Impactor

V1 (cm3)

V2 (cm3)

ACIa

885

270

NGI a

1540

485

đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;1 đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;1 + đ?&#x2018;&#x2030;đ?&#x2018;&#x2030;2

0.77

đ?&#x2018;&#x;đ?&#x2018;&#x;1 (Pa-s/kg) 2.251x105

đ?&#x2018;&#x;đ?&#x2018;&#x;2 (Pa-s/kg)

0.76

8.176x105

9.026x106

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x2030;đ?&#x2018;&#x2030; =

60-L/min configuration of the ACI

267

1.556x107

đ?&#x2018;&#x2026;đ?&#x2018;&#x2026;đ?&#x2018;&#x;đ?&#x2018;&#x; =

đ?&#x2018;&#x;đ?&#x2018;&#x;1 đ?&#x2018;&#x;đ?&#x2018;&#x;1 + đ?&#x2018;&#x;đ?&#x2018;&#x;2

0.014 0.083

Drug Delivery to the Lungs, Volume 29, 2018 – A European Pharmaceutical Aerosol Group (EPAG)-Led Cross-Industry Assessment of Inlet Flow Rate Profiles of Compendial DPI Test Systems: Part 2 – First-Order Impactor Model Results and Discussion The results in Table 3 reveal the combined effect of surrogate device resistance, impactor volume, and steadystate flow rate on the behavior of t90: ➢ ➢ ➢

addition of the surrogate device is responsible for a significant increase of the rise time. the rise time for cases without surrogate device increases with target flow rate; for cases with the 4 kPa surrogate device, the rise time decreases with flow rate (although not smoothly for the ACI system). the rise time increases with impactor system volume. Table 3 – Predicted Values of t90 for Flow Start-Up in DPI Testing Configuration ACI NGI ACI + 4kPa orifice NGI + 4kPa orifice

30 56 67 228 407

Target Flow Rate (L/min) 60 112 131 164 272

90 168 212 192 261

Because linearization of the flow resistance means that the calculated flow resistance always exceeds the actual, the model should and does predict larger values of t90 than those observed experimentally. Also, the trends are very much the same, and the effects are of a magnitude that is in the same range as the experimental data. Conclusions A simple two-compartment, first-order flow resistance model of a cascade impactor anticipates the major trends in the experimental data described in the EPAG Cross-Industry study 1 and in a manner that conveys an intuitive understanding of the physics controlling the kinetics of the inlet flow to the inhaler in compendial DPI testing. The model reasonably agrees with the experimental data in three important aspects: (1) the substantial increase in rise time when a surrogate DPI is present; (2) the decrease in rise time as the steady-state flow rate increases but only if the surrogate DPI is present (and opposite to the observed trend when the surrogate DPI is absent); (3) the increased rise time for impactors with larger total internal volume. Compared with three-dimensional, unsteady-state numerical solutions of cascade impactor behavior, the current model conveys important physics that will assist users in understanding compendial DPI quality control test results, which can be very helpful when a user experiences unexpected trends or outliers in a data set. References Mitchell JP, Blatchford C, Greguletz R, Roberts DL, Versteeg H. A European Pharmaceutical Aerosol Group (EPAG)-led crossindustry assessment of inlet flow rate profiles of compendial DPI test systems: Part 1 – Experimental data. The Aerosol Society, Edinburgh, UK, 2018. 2 Dechraksa J, Suwandecha T, Maliwan K., Srichana T. The comparison of fluid dynamics parameters in an Andersen cascade impactor equipped with and without a preseparator. AAPS PharmSciTech. 2014: 15(3): pp.792-801. 3 Roberts, D. L., M. Chiruta, “Transient Impactor Behavior during the Testing of Dry-Powder Inhalers via Compendial Methods,” Drug Delivery to the Lung 18, The Aerosol Society, Edinburgh, Scotland, December 13-14, 2007. 4 Roberts DL, Lavarreda C, Milhomme K, Shelton C, Managing impactor quality with measurements of flow resistance and effective diameter. Drug Delivery to the Lungs-17, The Aerosol Society, Edinburgh, Scotland, November 30 – December 1, 2006, pp.. 243-246. 5 Roberts DL, Maidment N, Copley MA, Improved protocol for relating impactor stage pressure drop to the suitability for routine use. Drug Delivery to the Lungs-2017, The Aerosol Society, Edinburgh, UK, 2017, pp. 94-97. 6 Clark AR, Hollingworth AM. The relationship between powder inhaler resistance and peak inspiratory conditions in healthy volunteers – Implications for in vitro testing. J. Aerosol Med. 1993: 6(2); pp. 99-110. 7 Roberts DL., Romay FJ. Relationship of stage mensuration data to the performance of new and used cascade impactors. J. Aerosol Med., 2005; 18, pp. 396-413. 8 Copley M, Smurthwaite M, Roberts DL, Mitchell JP. Revised internal volumes of cascade impactors for those provided by Mitchell and Nagel. J. Aerosol Med., 2005; 18(3), pp. 364-366. 1

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Drug Delivery to the Lungs, Volume 29, 2018 - I. Sibum et al. Formulating amikacin for dry powder inhalation I. Sibum, F. Grasmeijer, P. Hagedoorn & H.W. Frijlink Department of Pharmaceutical Technology and Biopharmacy, Ant. Deusinglaan 1, Groningen, 9713 AV, the Netherlands Summary The aim of this study was to formulate amikacin as a dry powder inhalation product by spray drying with the least amount of excipients possible. Pure spray dried amikacin results in a primary particle size distribution suitable for pulmonary administration. However, due to the co- and adhesiveness of the powder, dispersion from our Cyclops® inhaler was poor. By adding 1 or 2% L-leucine dispersion was improved significantly without affecting the primary particle size distribution. Inhaler retention dropped from 50% to around 8% while the fine particle fraction improved from approximately 11% to 50%. This is likely a result of the fact that L-leucine has the tendency to form a coating around powder particles during spray drying. L-leucine is less co- and adhesive than amikacin and as a result dispersion is improved. As there was no significant difference between the 1 and 2% L-leucine samples the stability study was performed on the 1% L-leucine formulation. This stability study showed that the fine particle fraction stayed stable during 6 months under long-term and accelerated storage conditions. Inhaler retention was between 10.92 ± 2.75% and 22.48 ± 8.27% but no trend in time could be discerned. A pharmacokinetic evaluation and local tolerability clinical study is currently in preparation to study the suitability of this formulation for the treatment of tuberculosis patients. Key Message Amikacin spray dried with 1% L-leucine results in a powder that can be delivered successfully and consistently from our inhaler the Cyclops®. This powder and inhaler product might be of benefit over the standard treatment by infusion for multidrug resistant tuberculosis. This we aim to assess in a clinical study. Introduction According to the WHO an estimated 10.4 million patients were diagnosed with active tuberculosis (TB) in 2016.[1] These patients included an estimated 490.000 cases of multidrug resistant tuberculosis (MDR-TB). For 250.000 patients MDR-TB proved fatal in this year alone. New anti-tuberculosis drugs for MDR-TB, as well as better methods for therapeutic drug monitoring of existing anti-TB drugs have been investigated successfully.[2], [3] Exploring other routes of administration for existing drugs is another option. Amikacin is one of these existing drugs. Antibiotics administered by pulmonary administration are delivered directly to the port of entry for TB. This means that a higher local concentration can be achieved with the same dose. This higher local concentration could potentially surpass the minimal inhibitory concentration of the resistant strain, which results in the eradication of the bacteria considered to be resistant.[4] Additionally, in some countries amikacin is administered intramuscularly. This gives rise to neuromuscular blockade, which, given at high doses, results in loss of motor function and pain. This may be avoided by administering amikacin via the pulmonary route. Inhalation of amikacin and other aminoglycosides by nebulisation has already been studied in TB, non-tuberculous mycobacterial infections and other non-TB patients.[5]–[8] However, most cases of TB are seen in developing countries, which have limited capabilities to support a cold-chain. As such, a stable and cheap formulation which does not need to be refrigerated is preferred.[1] A dry, and therefore stable powder combined with a cheap inhaler would satisfy these requirements.[4] Antibiotics have to be administered in high doses. For these reasons we aimed to develop an amikacin dry powder with no or a limited amount of excipients suitable for pulmonary delivery. Materials and Methods Amikacin used during spray drying was purchased from Ofipharma (Ter Apel, The Netherlands). The L-leucine used was purchased from Sigma Aldrich (St. Louis, United States). For spray drying a B-290 mini spray dryer, supplied by Büchi (Flawil, Switzerland), was used. All samples were spray dried with an inlet temperature of 130 °C, and a feed rate of 2.5 ml/min. The flow meter valve for the atomizing airflow was opened till a value of 50 mm from the bottom of the indicator was read. The aspirator was set to 100%. Total feed concentration was 50 mg/ml. For batches of 2.5 gram a syringe pump was used to control the feed rate. For the 15 gram batch the B-290’s peristaltic pump was used. Amikacin was spray dried in its pure form and with 1% or 2% L-leucine w/w. Demineralised water was used. Differential scanning calorimetry (DSC) was performed with the Q2000 supplied by TA instruments (New Castle, United States). A sample of 2-4 mg was placed in a Tzero pan. Pans were loaded into the DSC at 20°C and a ramp from 20°C until 210°C was made at a rate of 20°C /min. Samples were always analysed in duplicate the same day as they were spray dried.

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Drug Delivery to the Lungs, Volume 29, 2018 - Formulating amikacin for dry powder inhalation Particle size distributions were obtained by using a HELOS BF diffraction unit equipped with a RODOS dry disperser (Sympatec, Clausthal-Zellerfeld, Germany). An R3 lens was used with a measuring range of 0.5 μm-175 μm. The dispersion pressure used was 3 bars and each measurement was performed in triplicate. The average and standard deviations are reported. Cascade impaction analyses were performed using a Next Generation Impactor (NGI) provided by Copley Scientific (Nottingham, United Kingdom). A modified protocol with glass filters was used to account for the high doses. [9] Measurements were performed at a pressure drop of 4 kPa and with a dose of 50 mg with 20 mg of sweeper crystals. The function of these sweeper crystals is to scrape any drug deposition loose from the inhaler and they consist solely out of lactose in a size range of 45 to 63 µm.[10] The inhalation time was 4 seconds. A 2,4,6Trinitrobenzene Sulfonic Acid (TNBSA) assay was used to determine the final Amikacin concentrations. To measure the storage stability, 15 g of amikacin with 1% L-leucine w/w was prepared in a single batch. With this batch Cyclops single dose cartridges (SDCs) were filled with 50 mg + 20 mg sweeper and sealed under GMP conditions. These cartridges were sealed per three units in a secondary aluminium laminate pouch. These pouches were stored either under long-term conditions (30 °C and 66% RH) or accelerated conditions (40 °C and 75% RH), as defined by the FDA.[11] Samples were tested right after sealing, after a week, one month, three and six months. For each time point one pouch was opened and the three SDCs were placed in a Cyclops inhaler and their dispersion analysed. Scanning electron microscopy (SEM) was performed using a JEOL (Tokyo, Japan) JSM 6301-F microscope. Samples were fixed on double sided adhesive carbon tape and coated with 10 nm of gold with the JFC-1300 Auto Fine Coater, also provided by JEOL. Results Figure 1 shows representative DSC data of the spray dried pure amikacin, amikacin + 1% L-leucine and amikacin + 2% L-leucine samples. Crystalline amikacin has a melting peak at 203-204°C.[12] The fact that all these samples lack this peak indicate that spray dried amikacin, pure or with L-leucine, is amorphous. In Table 1 the spray dried samples together with the resulting X10, X50 and X90 values of the primary particle size distributions are shown. The addition of L-leucine marginally increases the particle size distribution. However, all X90 values found where below 5 m. Furthermore, the particle size distributions are quite narrow. Figure 1 - representative DSC data from the spray dried samples (n = 2) twice. The red line is the pure spray dried amikacin sample, the green line is amikacin spray dried with 1% L-leucine while the red line is amikacin spray dried with 2% Lleucine. diffraction data of the spray dried amikacin dispersed at 3 bars (average ± SD, n = 3).

Table RODOS

laser

NGI results are displayed in Table 2. Pure amikacin resulted in an inhaler retention of 50.90 ± 3.22%. This was decreased to 7.58 ± 3.22% with the Pure 0.82 ± 0.01 2.09 ± 0.01 4.21 ± 0.01 addition of 1% L-leucine. Adding 2% L-leucine did not lower the inhaler retention any further. Inlet 1% Leu 0.79 ± 0.01 2.12 ± 0.04 4.66 ± 0.11 deposition, which is an indication of throat deposition, seems to increase by the addition of 1% 2% Leu 0.82 ± 0.03 2.23 ± 0.06 4.99 ± 0.07 or 2% L-leucine. However, the high inhaler retention seen for the pure spray dried amikacin contributes to a great extend to this. The fine particle fraction is remarkably increased by the addition of leucine. The leucine-containing samples had a fine particle fraction of around 50%, while for the pure drug this was 11.10 ± 3.18%. X10 (µm)

X50 (µm)

X90 (µm)

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Drug Delivery to the Lungs, Volume 29, 2018 - I. Sibum et al.

Table 2 - NGI data of spray dried amikacin from the Cyclops inhaler at 4 kPa. Data are presented as a fraction of the nominal dose (n = 2, average ± Δ min/max).

Scanning electron microscopy images of spray dried amikacin with and without 1% Lleucine are depicted in Figure 2. The spray dried amikacin without L-leucine shows round particles with dimples in them. The addition of leucine results in severely wrinkled particles.

Pure (%)

1% Leu (%)

2% Leu (%)

Inhalator

50.90 ± 4.55

7.58 ± 0.49

7.86 ± 1.26

Inlet

20.36 ± 5.42

30.74 ± 2.68

28.72 ± 1.99

FPF

11.10 ± 4.50

51.78 ± 0.72

50.81 ± 3.09

The effect of storage on the fine particle fraction and inhaler retention is presented in Figure 3. The fine particle fraction does not notably change under normal and accelerated conditions during 6 months of storage. All values found for the normal condition fell between 84.40 ± 2.15% and 87.87 ± 1.74%. For the accelerated condition the values fell between 85.81 ± 7.24 and Figure 2 - Scanning electron microscopy images of the spray dried samples. Ami is 97.61 ± 0.95 %. The inhaler the pure spray dried amikacin sample, while Ami + 1% Leu is the amikacin sample retention fluctuates slightly, spray dried with 1% L-leucine. especially under accelerated conditions, but no clear trend is apparent. For the normal storage condition, the values found where between 10.92 ± 2.75% and 17.83 ± 3.92%. The accelerated storage condition resulted in only a slightly higher inhaler retention between 11.80 ± 1.54% and 22.48 ± 8.27%.

Figure 3 - the fine particle fractions (left) and inhaler retentions (right) measured during storage of the amikacin + 1% Lleucine formulation under normal (30°C, 66% RH) and accelerated (40°C, 75% RH) conditions. Error bars display the standard deviation (n = 3).

Discussion Pure spray dried amikacin results in a particle size distribution suitable for inhalation (Table 1), but its dispersion from the Cyclops® is rather poor (Table 2). Less than 50% of the dose leaves the inhaler. Furthermore, with a fine particle fraction of 11.10 ± 3.18% the lung deposition is too low to result in a suitable product. This poor result is likely a result of the co- and adhesiveness of the powder, which is a known problem for dry powder aminoglycoside inhalation products. Amikacin spray dried with 1% L-leucine is comparable to the pure drug formulation where the primary particle size distribution is concerned (Table 1). However, there is a remarkable difference between the two formulations in dose emission and aerosolisation. Inhaler retention is lowered significantly to only 7.33 and 7.82% of the nominal dose. Furthermore, the fine particle fraction increases to over 50% of the nominal dose.

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Drug Delivery to the Lungs, Volume 29, 2018 - Formulating amikacin for dry powder inhalation The DSC data show that L-leucine does not change the solid state of amikacin particles and that the samples stay amorphous. Therefore, it is not a change in solid state that explains the differences in dispersibility. The differences seen are likely a result of the fact that L-leucine forms a coating during the spray dry process[13]. L-leucine is surface active, which means that during spray drying the compound enriches at the surface of the droplet. This then forms a coating upon drying. L-leucine is a lot less adhesive then amikacin, and as the surface of the powder particle likely consists of L-leucine, the powder sticks less to itself or the inhaler surfaces. Furthermore, as can be seen in Figure 2, the surface morphology changes by the addition of L-leucine. The L-leucine sample has a more corrugated surface, which is known to improve dispersion.[14] The addition of 2% L-leucine vs 1% L-leucine did not have a noteworthy influence. As a result, the 1% L-leucine sample was picked as the best formulation and the stability study was performed on this powder. The stability study shows that the 1% L-leucine formulation is stable. With no differences being seen in fine particle fraction over time for the long-term and accelerated conditions. The retention does change substantially. However, no trend is distinguishable and it looks more like random variation. A likely explanation for this is that the SDCs where not always stored flat but sometimes were stored in a vertical position, which seems to result in the powder compacting in the back of the SDC, which reduces its entrainment during dispersion. However, if compacted powder is seen by a patient after inhalation it can be easily tapped loose and inhaled during a second inhalation manoeuvre. Conclusion Amikacin is formulated successfully for dry powder pulmonary administration with the Cyclops by the use of 1% Lleucine as excipient during the spray drying process. The addition of 1% L-leucine is required to improve dose emission and dispersion to acceptable levels. The formulation is stable for at least 6 months under normal and accelerated storage conditions. A pharmacokinetic evaluation and local tolerability clinical study is now in preparation to determine the suitability of this formulation for actual treatment. References 1 2 3

4 5

6 7 8 9 10 11 12 13 14

WHO, Global Tuberculosis Report 2017. 2017. C. a Peloquin, “Therapeutic drug monitoring in the treatment of tuberculosis.,” Drugs, vol. 62, no. 15, pp. 2169–2183, 2002. J. W. C. Alffenaar, R. Van Altena, I. M. Harmelink, P. Filguera, E. Molenaar, A. M. A. Wessels, D. Van Soolingen, J. G. W. Kosterink, D. R. A. Uges, and T. S. Van Der Werf, “Comparison of the pharmacokinetics of two dosage regimens of linezolid in multidrug-resistant and extensively drug-resistant tuberculosis patients,” Clin. Pharmacokinet., vol. 49, no. 8, pp. 559–565, 2010. M. Hoppentocht, P. Hagedoorn, H. W. Frijlink, and A. H. De Boer, “Developments and strategies for inhaled antibiotic drugs in tuberculosis therapy: A critical evaluation,” Eur. J. Pharm. Biopharm., vol. 86, no. 1, pp. 23–30, 2014. K. N. Olivier, P. A. Shaw, T. S. Glaser, D. Bhattacharyya, M. Fleshner, C. C. Brewer, C. K. Zalewski, L. R. Folio, J. R. Siegelman, S. Shallom, I. K. Park, E. P. Sampaio, A. M. Zelazny, S. M. Holland, and D. R. Prevots, “Inhaled amikacin for treatment of refractory pulmonary nontuberculous mycobacterial disease,” Ann. Am. Thorac. Soc., vol. 11, no. 1, pp. 30–35, 2014. L. V Sacks, S. Pendle, D. Orlovic, M. Andre, M. Popara, G. Moore, L. Thonell, and S. Hurwitz, “Adjunctive salvage therapy with inhaled aminoglycosides for patients with persistent smear-positive pulmonary tuberculosis.,” Clin. Infect. Dis., vol. 32, no. 1, pp. 44–9, 2001. K. K. Davis, P. N. Kao, S. S. Jacobs, and S. J. Ruoss, “Aerosolized amikacin for treatment of pulmonary Mycobacterium avium infections: an observational case series.,” BMC Pulm. Med., vol. 7, p. 2, 2007. C.-E. Luyt, M. Clavel, K. Guntupalli, J. Johannigman, J. I. Kennedy, C. Wood, K. Corkery, D. Gribben, and J. Chastre, “Pharmacokinetics and lung delivery of PDDS-aerosolized amikacin (NKTR-061) in intubated and mechanically ventilated patients with nosocomial pneumonia.,” Crit. Care, vol. 13, no. 6, p. R200, 2009. F. Grasmeijer, P. Hagedoorn, H. W. Frijlink, and A. H. De Boer, “Characterisation of high dose aerosols from dry powder inhalers,” Int. J. Pharm., vol. 437, no. 1–2, pp. 242–249, 2012. A. H. De Boer, P. P. H. Le Brun, H. G. Van Der Woude, and P. Hagedoorn, “Dry powder inhalation of antibiotics in cystic fibrosis therapy , part 1 : development of a powder formulation with colistin sulfate for a special test inhaler with an air classifier as de-agglomeration principle,” vol. 54, pp. 17–24, 2002. Food and Drug Administration, “Guidance for Industry Q1A(R2) Stability Testing of New Drug Substances and Products,” no. November, pp. 1–22, 2003. “Amikacin - Drugbank.” [Online]. Available: https://www.drugbank.ca/drugs/DB00479. [Accessed: 13-Jun2018]. L. Li, S. Sun, T. Parumasivam, J. A. Denman, T. Gengenbach, P. Tang, S. Mao, and H.-K. Chan, “l-Leucine as an excipient against moisture on in vitro aerosolization performances of highly hygroscopic spray-dried powders.,” Eur. J. Pharm. Biopharm., vol. 102, pp. 132–141, 2016. C. Weiler, M. Egen, M. Trunk, and P. Langguth, “Force Control and Powder Dispersibility of Spray Dried Particles for Inhalation,” J. Pharm. Sci., vol. 99, no. 1, 2009.

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Drug Delivery to the Lungs, Volume 29, 2018 - Serena Bonasera et al. Effect of spray dried formulation on the aerosol performance of a novel dry powder inhaler Serena Bonasera1, Dale R. Farkas2, P. Worth Longest1,2 Bryce Beverlin II3 & Michael Hindle1 1Department 2

of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, USA

Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University, Richmond, VA, USA 3

Quench Medical Inc., St Paul, MN, USA

Summary Dry powder inhalers (DPIs) remain a widely used option for the delivery of aerosols despite their relatively low drug delivery efficiency to the lungs with fine particle fractions ranging from 20-40%[1]. Laser diffraction particle sizing and realistic in vitro mouth-throat aerosol delivery experiments were employed to characterize the emitted dose of aerosols using two capsule air inlet aperture positions with a novel dry powder inhaler (CC90-3D) for two spray dried excipient enhanced growth (EEG) powder formulations. For budesonide-EEG aerosols, the mean volumetric diameter was not affected by the position of the air inlet aperture. For the albuterol sulfate-EEG aerosols, positioning the air inlet aperture in the side wall of the capsule head produced a beneficial lower mean volumetric diameter compared to positioning the air inlet aperture in the perpendicular axis of the capsule head. Irrespective of capsule air inlet positioning, the duration of aerosol emission was different for the two powder EEG formulations. The budesonide-EGG formulation was delivered from the inhaler over a duration of 1.5-1.9s compared to the dose emission for the albuterol sulfate-EEG formulation being delivered over 0.6-0.7s. Capsule drug retention was lower when the air inlet aperture was positioned in the perpendicular axis of the capsule head for both EEG formulations. Mouth-throat depositions for all the EEG formulations were low (<7%) except for the poorly dispersed albuterol sulfate-EEG formulation which had a mouth-throat deposition of 16.2%. Aerosolization conditions should be optimized for each individual spray dried EEG formulation in order to ensure high efficiency aerosol performance. Key Message A combination of spray dried EEG formulations and the CC90-3D DPI generated powder aerosols with low capsule and mouth-throat drug deposition. Both capsule emptying and formulation aerosolization were affected by capsule air inlet aperture position and evaluation of these effects should be made for each spray-dried EEG formulation. Introduction Dry powder inhalers (DPIs) are a widely used option for the delivery of inhalation aerosols despite their relatively low drug delivery efficiency to the lungs with fine particle fractions ranging from 20-40%[1]. However, there remains a need for high efficiency dry powder inhalers that minimize drug deposition losses in the mouth-throat region and improve delivery to the tracheobronchial and alveolar regions. Recent in vitro studies have shown that dry powder aerosols generated using a combination of a novel dry powder inhaler (CC90-3D) and spray dried powder formulations developed for the excipient enhanced growth (EEG) application were shown to have high lung delivery efficiencies (fine particle fractions >90% of the emitted dose)[2,3]. The CC90-3D DPI has a number of features that enable high efficiency dispersion of the spray dried formulation including the motion and vibration of the capsule for internal fluidization of the powder and the 3D rod array for deagglomeration of the aerosol powder in the airstream [3]. The spray dried EEG formulation was developed to produce micrometer-sized dispersible powders and the approach has been applied to a number of drugs including albuterol sulfate (AS), terbutaline sulfate and ciprofloxacin[3–5]. In this study, we have evaluated a novel budesonide-EEG spray dried formulation and compared the aerosol performance with an AS-EEG formulation. Previous studies have shown that the capsule air inlet aperture position did not significantly affect device emptying however did affect powder dispersion [3]. In this study, we have employed laser diffraction and realistic in vitro mouth-throat aerosol delivery experiments to characterize the emitted dose of aerosols generated using two capsule aperture positions for the study formulations. Methods and Materials Spray Dried EEG Formulations Albuterol sulfate (AS) USP and Budesonide (BUD) USP were used as the active ingredients in two spray dried EGG formulations, respectively. The AS-EEG powder was formulated with mannitol as the hygroscopic excipient, leucine as the dispersion enhancer and poloxamer 188. The BUD-EEG powder was formulated with sodium chloride as the hygroscopic excipient and leucine as the dispersion enhancer. The ratio of components in the final spray dried ASEEG powder was 30:48:20:2 %w/w of AS:mannitol:leucine:poloxamer 188. Similar values for the BUD-EEG powder were 8:52:40 %w/w of BUD:sodium chloride:leucine. The AS-EEG powder was spray dried using the Büchi Nano spray-dryer B-90 HP[4]. The BUD-EEG powder was spray dried at Lonza’s Bend facility with the BLD-35 spray dryer. The primary particle size distribution of each formulation was determined by laser diffraction using the Sympatec HELOS (submicron R1 lens with 20 mm focal length) with RODOS/M disperser and ASPIROS sample feeder (Sympatec GmbH, Clausthal-Zellerfeld, Germany). The RODOS/M used compressed air to disperse the powders at 4 bar. The mean (SD) Dv50 for the BUD-EEG and AS-EEG formulations were 1.51 (0.01) µm and 1.01 (0.02) µm, respectively.

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Drug Delivery to the Lungs, Volume 29, 2018 - Effect of spray dried formulation on the aerosol performance of a novel dry powder inhaler CC90-3D Dry Powder Inhaler The CC90-3D inhaler as shown in Figure 1 (a) was created using Autodesk Inventor and exported as STL files to be prototyped. To ensure smooth interior surfaces of the inhaler, the parts were built by 3D Systems On Demand Manufacturing (3D Systems Inc., Rock Hill, SC) using Accura ClearVue stereolithography resin (3D Systems Inc.). Once the parts were prototyped, they were assembled by inserting 0.5 mm stainless steel rods for the rod array, inserting a stainless-steel mesh at the outlet of the capsule chamber, to prevent the capsule from being pulled into the mouthpiece, and joining all connections using epoxy to ensure a sealed device. The inhale resistance was determined using the method described by Clark and Hollingworth[6] to be 0.14 cm H2O1/2/L/min. The EEG formulations (1mg) were filled into size 3 hydroxypropyl methylcellulose (HPMC) capsules (Qualicaps, Whitsett, NC). Capsules were pierced as shown in Figure 1 (b) and (c) to produce two capsule air inlet aperture positions (AIP) with 0.5 mm holes. For AIP 1, which was previously optimized for use with the CC90-3D inhaler, the inlet aperture was positioned in the side wall of the capsule head [3]. For AIP 2, which was previously optimized for use with the inline N2L inhaler, the inlet aperture was positioned in the perpendicular axis of the capsule head [5].

Figure 1 (a) The CC90-3D dry powder inhaler. Capsule air inlet aperture position (AIP) previously optimized for use with (b) the CC90-3D inhaler (AIP 1) and (c) the inline N2L inhaler (AIP 2). Adapted from Behara et al., 2014[3]. Emitted Aerosol Characterization – Laser Diffraction The Malvern Spraytec (Malvern Instruments, Ltd., Worcestershire, UK) was used to characterize the emitted aerosol from the CC90-3D inhaler. For these studies, the Spraytec was fitted with the inhalation flow cell to enable breath actuation of the inhaler using a breath simulator (ASL 5000-XL, IngMar Medical, Pittsburgh, PA). The CC90-3D inhaler loaded with 1 mg of EEG formulation was connected using an airtight mouthpiece connector to one side of the inhalation cell and a simulated breath profile was drawn through the system to actuate the inhaler. The ASL 5000-XL breath simulator was used to generate a realistic breathing profile using the method described by Delvadia et al[7]. The medium breath profile was characterized with an average flow of 45 L/min, with a peak inspiratory flow rate of 65 L/min inhaled over 4 s with an inhalation volume of 2.8 L. At the exit of the inhalation flow cell, a PulmoGuard IITM filter was connected to collect the emitted dose and prevent contamination of the breath simulator. Spraytec data collection was triggered when the % transmission was less than 99% and data was collected for 4 s with a sampling frequency of 4000 Hz equivalent to one record every 0.4 ms. Finally, the drug retained in the capsule and captured on the emitted dose filter was determined using validated HPLC methods, for AS and BUD, respectively and expressed as a percentage of the nominal dose. The time-history profiles of the emitted dose aerosol particle size distributions were compared with respect to the % transmission of the aerosol, reflecting laser obscuration and aerosol cloud density. Particle size was presented as time averaged volumetric diameters (Dv). Results were presented for the 10th (Dv10), 50th (Dv50) and 90th (Dv90) percentiles of the volumetric diameter frequency, respectively, together with calculation of the aerosol duration and the % of particles less than 1 µm and 5 µm. Emitted Aerosol Characterization – In Vitro Mouth-Throat Aerosol Delivery For in vitro aerosol performance testing, 1 mg of EEG formulation was loaded into the novel CC90-3D DPI and aerosolized into a medium-sized VCU realistic mouth-throat model using a medium inhalation profile generated by a breath simulator[8]. The mouth-throat was coated with Molykote® 316 Silicone Release Spray to prevent particle bounce and re-entrainment. Aerosol penetrating the mouth-throat was collected on a respiratory filter (PulmoGuard IITM Queset Medica, North Easton, MA) positioned at the trachea as the estimated total lung dose. Drug deposition in the capsule, mouth-throat and respiratory filter was collected for quantitative analysis using a validated HPLC method to determine the regional deposition of drug following simulated inhalation and expressed as a percentage of the nominal dose.

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Drug Delivery to the Lungs, Volume 29, 2018 - Serena Bonasera et al. Results and Discussion Emitted Aerosol Characterization – Laser Diffraction Table 1 shows the particle size characteristics of the emitted aerosols generated from the two spray dried EEG formulations that were aerosolized using capsules pierced using two methods. For the BUD-EEG aerosols, the mean volumetric diameter was not affected by the position of the air inlet aperture, with mean (SD) values of 1.4 (0.0) µm and 1.5 (0.0) µm, respectively. For the AS-EEG aerosols, positioning the air inlet aperture in the side wall of the capsule head (AIP 1) produced a lower mean volumetric diameter (1.2 (0.0) µm) compared to positioning the air inlet aperture in the perpendicular axis of the capsule head (1.6 (0.1) µm). This was indicative of effective aerosol dispersion of the AS-EEG formulation using AIP 1. In addition, it was observed that for the AS-EEG formulation aerosolized using AIP 2 has a Dv90 of 38.2 (5.1) µm compared to Dv90 values of <4 µm for the BUD-EEG formulations and the AS-EEG formulation with AIP 1. Table 1. Mean (SD) particle size characteristics of the emitted aerosol from the two spray dried EEG formulations measured using the laser diffraction method (n≥3). Aerosol Duration (s)

Dv10 (µm)

Dv50 (µm)

Dv90 (µm)

%<1µm

%<5µm

Filter Dose (% of nominal dose)

BUD AIP 1

1.9 (0.1)

0.4 (0.0)

1.4 (0.0)

3.6 (0.0)

34.1 (0.2)

96.0 (0.0)

61.4 (3.3)

BUD AIP 2

1.5 (0.3)

0.4 (0.0)

1.5 (0.0)

3.8 (0.2)

34.3 (1.0)

95.1 (1.1)

74.6 (3.2)

AS AIP 1

0.7 (0.1)

0.4 (0.0)

1.2 (0.0)

3.0 (0.1)

43.1 (0.4)

96.8 (0.4)

54.5 (1.2)

AS AIP 2

0.6 (0.0)

0.4 (0.0)

1.6 (0.1)

38.2 (5.1)

31.7 (1.0)

81.2 (1.8)

68.6. (2.4)

Figure 2 shows the time history profile for a typical aerosol emission for each of the test conditions plotted as the % transmission (top) and Dv50 (bottom) against time for the BUD-EEG (left) and AS-EEG (right) aerosols. The duration of aerosol emission was different for the two powder EEG formulations. The BUD-EGG formulation was delivered from the inhaler over a duration of 1.5-1.9 s compared to the dose emission for the AS-EEG formulation being delivered over 0.6-0.7 s. It is hypothesized that density differences between the powder formulations were responsible for the differences in the device emptying profiles. Irrespective of the duration of emptying, the % transmission profiles revealed that AIP 2 appeared produce greater laser obscuration (lower transmission) for both formulations which was also more variable over the time course of dose emission compared to AIP 1. The lower transmission appeared to correlate with the higher filter doses that were collected in the inhalation cell filter and lower capsule retention using AIP 2 for both EEG formulations. The aerosol dispersion as shown by the Dv50 for the BUD-EEG formulation (both capsule air inlet positions) and AS-EEG (AIP 1) appeared relatively constant over the duration of dose emission while more variability in the measured Dv50 was observed for the AS-EEG formulation aerosolized using AIP 2. Emitted Aerosol Characterization – In Vitro Mouth-Throat Aerosol Delivery Mouth-throat deposition for the BUD-EEG formulations were low (<7%), as expected given the observed particle size distributions. Using AIP 1, capsule retention was observed to be high (mean (SD): 23.3 (4.0) %) and this was associated with low mouth-throat deposition (2.6 (0.6) %). For the BUD-EEG formulation, AIP 2 appeared to perform well, with lower capsule retention (13.2 (1.7) %), while maintaining low mouth-throat deposition (6.3 (1.9) %). The measured in vitro lung filter deposition for the optimized air inlet position 2 with the BUD-EEG formulation was 73.5 (1.1) % of the nominal dose and was indicative of high efficiency dispersion of this formulation. For the AS-EEG formulation, capsule drug retention was higher for AIP 1 (27.0 (5.0) %) compared to only 6.1 (0.2) % of the nominal dose for AIP 2, which was similar to the trend observed for the BUD-EEG formulation. However, for the AS-EEG formulation, AIP 2 was not able to effectively disperse the aerosol formulation with mouth-throat deposition of 16.2 (2.5) % of the nominal dose. This relatively high mouth-throat deposition agreed well with the observed high Dv90 (38.2 µm) indicating incomplete dispersion of the emitted aerosol dose with this air inlet position despite good capsule empting for the AS-EEG formulation.

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Figure 2. Typical time history profile of the aerosol emission for each of the two capsule air inlet positions plotted as the % transmission (top) and Dv50 (bottom) against time for the BUD-EEG (left) and AS-EEG (right) aerosols. Conclusions A combination of spray dried EEG formulations and optimized aerosolization conditions using the CC 90-3D dry powder inhaler can be employed to generate small particle aerosols with low capsule retention and low mouththroat deposition. In contrast to previous studies[3], this work demonstrates that both capsule emptying and formulation aerosolization can be affected by the capsule air inlet aperture positioning and that evaluation of these effects should be made for each spray-dried EEG formulation. References 1.

Steckel H, Müller BW: In vitro evaluation of dry powder inhalers I: drug deposition of commonly used devices, Int J Pharm 1997; 154: pp19–29.

Behara SRB, Farkas DR, Hindle M, Longest PW: Development of a high efficiency dry powder inhaler: effects of capsule chamber design and inhaler surface modifications, Pharm Res 2014; 31: pp360–372.

Behara SRB, Longest PW, Farkas DR, Hindle M: Development and comparison of new high-efficiency dry powder inhalers for carrier-free formulations, J Pharm Sci 2014; 103: pp465–477.

Son Y-J, Longest PW, Hindle M: Aerosolization characteristics of dry powder inhaler formulations for the excipient enhanced growth (EEG) application: Effect of spray drying process conditions on aerosol performance, Int J Pharm 2013; 443: pp137–145.

Longest PW, Golshahi L, Behara SRB, Tian G, Farkas DR, Hindle M: Efficient nose-to-lung (N2L) aerosol delivery with a dry powder inhaler, J Aerosol Med Pulm Drug Deliv 2015; 28 (3): pp189-201.

Clark AR, Hollingworth AM: The relationship between powder inhaler resistance and peak inspiratory conditions in healthy volunteers--implications for in vitro testing, J Aerosol Med Off J Int Soc Aerosols Med 1993; 6: pp99–110.

Delvadia RR, Wei X, Longest PW, Venitz J, Byron PR: In Vitro Tests for Aerosol Deposition. IV: Simulating Variations in Human Breath Profiles for Realistic DPI Testing, J Aerosol Med Pulm Drug Deliv 2016; 29: pp196– 206.

Wei X, Hindle M, Kaviratna A, Huynh BK, Delvadia RR, Sandell D, Byron PR: In Vitro Tests for Aerosol Deposition. VI: Realistic Testing with Different Mouth–Throat Models and In Vitro—In Vivo Correlations for a Dry Powder Inhaler, Metered Dose Inhaler, and Soft Mist Inhaler, J Aerosol Med Pulm Drug Deliv 2018; DOI: 10.1089/jamp.2018.1454.

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Drug Delivery to the Lungs, Volume 29, 2018 – Maria Braga et al. Spray dried composite powders: capsule filling process optimization and aerodynamic performance characterization Maria Braga1, Raquel Barros1, Bruno Ladeira1, Mariana F. Silva1,2, Joana Tavares1 & Eunice Costa1 FarmaCiencia SA, Sete Casas, 2674 – 506 Loures, Portugal Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Portugal 1Hovione

2Research

Summary The main objectives of this work were to assess precision capsule filling of a model spray dried composite powder using a dosator-based unit from MG2, optimize the filling process and evaluate its impact on powder in-vitro aerodynamic performance (AP). Size 3 capsules were efficaciously filled with three different fill weights (5, 10 and 20 mg), using different dosator diameters (1.9, 2.8, 3.4 and 3.7 mm), powder bed heights (4 – 10 mm) and filling speeds (1000 - 2000 capsules/h). The powder characteristics were also visually assessed throughout filling. The successful integration of tools for homogenizing the powder layer and for removing the excess powder from the dosator were key for filling. For the different fill weights, compaction was varied by adjusting the dosing-chamber-height-to-powder-layer-depth compression ratio. For capsules filled with 10 and 5 mg the most promising trials of were characterized. A fine particle fraction (FPF) of 72.7 % and 80.1% and a fine particle dose (FPD) of 6.5 mg and 3.3 mg were obtained for the 10 and 5 mg fill weight respectively. Aerodynamic performance results of 20 mg fill weight showed a higher performance for capsules with lower compaction level. The estimated FPF and FPD varied between 69.3 % and 13.4 mg for the lower compaction trial and 57.0 % and 11.1 mg for the trial with higher compaction. This study showed that the MG2 Flexalab is suitable for the capsule filling of spray dried composite powders with low rejection rates and very good aerodynamic performance which are in accordance with similar studies performed previously. Key Message The dosator-based MG2 Flexalab capsule filler is a suitable tool for filling challenging spray dried composite powders. In the capsule filling process, it is essential to perform a preliminary stage of process optimization, since the filling parameters have an impact on powders aerodynamic performance. Introduction In dry powders for inhalation (DPIs), engineering of carrier-free composite particles in which the API is embedded within an excipient matrix by spray drying is a suitable alternative to circumvent drug delivery uniformity challenges and overcome poor delivery efficiency occasionally observed with carrier-based powders [1 – 3]. Capsule filling processes via a dosator are widely applied in the pharmaceutical industry. However, little research has been performed regarding dosator-based precision capsule filling processes for inhalation applications. Some studies concerning the capsule filling of carrier-based powders have showed that the desired target fill weight of powderfilled capsules strongly depends on both the material characteristics and the instrumental settings [4, 5]. Authors have reported that the ratio between the dosing chamber length and powder layer height play a major role in terms of achieving a performant process over time with high yields. Additionally, when capsules are filled with high dosator diameters (3.4 mm or above) having a homogeny powder layer without air holes became critical particularly for smaller filling masses. However, no studies have been found in literature qualifying scientifically a dosator capsule filler capacity for the filling of spray dried composite powders, neither the impact of process parameters on the final powder aerodynamic performance. Thus, the aim of this study was to assess the capability of a pilot-scale dosator capsule-filling machine to fill spray dried composite particles. A pilot-scale MG2 Flexalab was selected to perform this study. The MG2 Flexalab machine is integrated with a 100% weight control system, MultiNETT, controlling in process the net weight contained in each single capsule. The effect of the process parameters on the aerodynamic performance was also studied. To achieve the objective some set-up modifications were performed in order to enable the capsule filling process: a cleaning system for the removal of the excess powder from the dosator and a special mixing powder rod to homogenize the powder bed and decrease the powder adhesion to the walls of the rotary container.

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Drug Delivery to the Lungs, Volume 29, 2018 - Spray dried composite powders: capsule filling process optimization and aerodynamic performance characterization Materials and Methods Placebo composite powder of trehalose:L-leucine (80/20% w/w) at 2% (w/w) in a water:ethanol (50/50% w/w) solvent system were manufactured under the best process conditions identified previously [6]. The powder was spray-dried using a BUCHI model B-290 Advanced spray dryer equipped with a high efficiency cyclone and operating at a feed flow of 7 g/min, an atomization gas flow of 50 mm in the rotameter and a drying gas flow of 35 kg/h. The outlet temperature (T_out) was set at 70 ºC. The inlet temperature (T_in) was readjusted in order to maintain T_out at the target value. The morphology of the dry powders was examined by Scanning Electron Microscopy (SEM) for particle shape and morphology. The particle size was measured by dry Laser Diffraction. HPMC size #3 capsules were automatically filled at relative humidity controlled conditions of < 30% using a pilot scale MG2 FlexaLab capsule filler (Figure 1). Capsules were filled with three different fill weights: 5 mg, 10 mg and 20 mg per capsule, applying an acceptance limit of ± 1.0 mg for the 10 and 20 mg fill weights and ± 0.5 mg for the 5 mg fill weight. Filling speed varied between 1000 and 2000 capsules/hour. Number of filled capsules varied from 195 up to 331, from 315 up to 343 and from 244 up to 298 for 20 mg, 10 mg and 5 mg fill weights, respectively. Four different dosator diameters were used: 1.9 mm, 2.8 mm, 3.4 mm and 3.7 mm. Powder layer height in the rotary container ranged from 4 mm up to 10 mm. The initial powder bed high and dosator diameter/dosing chamber high were adjusted manually until the targeted fill weight were reached. The compaction level inside the dosing chamber was also evaluated by visual observation during the manual adjustment process.

Figure 1 – MG2 capsule filling set-up.

For the selected capsules, the aerodynamic performance of the dry powders was assessed in-vitro using a gravimetric 8-stage Andersen Cascade Impactor (ACI) using a Plastiape HR model 7 inhaler at 60 L/min (4 kPa pressure drop). Each ACI stage was covered with a glass fibber filter where the sample was collected. The amount of powder in filter was determined gravimetrically. Three replicates were carried out for each filling condition. Results and Discussion The SEM micrograph of trehalose:leucine spray-dried system (Figure 2 A) showed a powder presenting spherical and slightly shriveled particles. The PSD data showed a Dv50 of 1.2 µm. The results were in agreement to what was observed in previous studies [6]. Capsule filling optimization At all, 25 experimental runs were performed in order to optimize the capsule filling process of spray dried composite powders. Table 1 presents the experimental results for seven selected runs for capsule filling of 20, 10 and 5 mg with various combinations of process parameters (dosator diameter; dosing chamber height, layer depth and filling speed).

Figure 2 – Visual observation performed during capsule filling process: SEM images of the spray dried composite powder (A); set-up and powder appearance before optimization (B); capsules filled with 20 mg for run 1 (C) and 3 (D).

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Drug Delivery to the Lungs, Volume 29, 2018 – Maria Braga et al. During the capsule filling process, the challenging flow properties of the spray dried powder led to some operational difficulties (Figure 2 B and C), in some cases resulting in the impossibility of filling capsules. To overcome the situation, engineering solutions were applied to the set-up, reducing powder adhesion to the walls and improving layer homogenization. After adjusting all machine parameters and creating a smooth powder layer without densification and/or agglomerates, capsule filling experiments were carried out for the seven experimental runs as presented in Table 1. From Table 1 it can be seen that there is no significant change in capsules weight average over runs, with SD varying between 0.04 up to 0.82 mg for the different fill weights, which indicates that no powder aggregation in the rotary container nor accumulation in the dosator has occurred during the capsule filling process. This study showed very promising results: rejection rates varied between 0 % up to 3 % for 20 mg, 0 % up to 20 % for 10 mg and 0 % up to 18 % for 5 mg fill weights. As expected, the rejection rate was relatively higher for lower target fill weights (even when applying a more stringent weight tolerance of 10% for the 5 and 10 mg fill weights, over a 5% for the 20 mg). The 10% acceptance limit was based on content uniformity as per the European Pharmacopoeia for a capsule fill content below 40 mg (Version 6.0, Chapter 2.9.6.), where not more that 1 out of 30 samples may be outside 15% of the average and all the capsules must be within 25% [6]. For the 10 and 5 mg, higher rejection rates corresponded to the highest level of compaction of the powder as visually assessed inside the dosing chamber. For capsule filled with 20 mg, rejection rates were very low and no evident relation was founded between the rejection rate and the compaction degree. Table 1 – SD powders capsule filling optimization: process parameters and results for seven experimental runs. Fill Average Acceptance Rejection Dosator Number weight SD weight limit rate diameter Run of filled (target) (mg) (mg) (mg) (%) (mm) caps. (mg)

Dosing chamber height (mm)

Layer Chamber depth / Layer (mm) ratio

1 2 3 4 5 6 7

7.0 6.0 4.75 4.5 3.25 2.5 4.5

7.5 9.0 9.0 7.0 6.0 5.5 6.0

280 331 196 315 343 244 298

20 20 20 10 10 5 5

20.91 20.06 20.05 10.65 9.74 4.86 5.1

0.71 0.57 0.4 0.04 0.32 0.82 0.33

±1 ±1 ±1 ±1 ±1 ± 0.5 ± 0.5

0 0 3 20 0 18 0

2.8 3.4 3.7 2.8 2.8 2.2 1.9

1:1.1 1:1.5 1:1.9 1:1.6 1:1.8 1:2.2 1:1.3

Compaction level in the dosing Speed chamber (caps./h) (visual observation) 1500 High 1500 Medium 2000 Low 2000 High 2000 Low 2000 High 2000 Low

The compaction of the powder visually observed in the dosing chamber would not correlate to the dosing-chamberheight-to-powder-layer-depth (chamber/layer) compression ratio, being actually mostly impacted by the contribution of the dosator diameter: the larger the dosator, the lower the compaction. On Figure 2 (C and D) it is possible to observe the differences obtained in terms of powder compaction in capsules for run 1 and run 3. For the different fill weights, the optimal chamber/layer compression ratio values were 1:1.9 for 20 mg, 1:1.8 for 10 mg and 1:1.3 for 5 mg, being above a ratio of 1:1 that would theoretically equate to no pre-compression of powder during filling. Overall, it could be observed that the ratio decreased while decreasing the fill weight. The same behaviour was observed in literature for lactose powders capsule filling [7]. As expected, the ratio was higher for 20 mg since to achieve the desired fill weight and minimize the compaction level in the capsule the largest dosator diameter of 3.7 mm was found to be the best choice. Larger dosator sizes require a larger compression ratio for a successful transfer of powder into the capsules. On the contrary, a lower ratio was necessary for 5 mg fill weight since the smaller dosator diameter of 1.9 mm was found suitable. In general, capsule filling speed had no significant impact on capsule compaction level or rejection rate. An optimum value of 2000 capsules per hour was set for the capsule filling of the produced SD composite powder. Regardless, additional experiments would be strictly required for a better understanding of the impact and interaction of the several capsule filling parameters on the process performance and final capsules characteristics. Aerodynamic performance characterization As observed in Table 2, the aerodynamic performance results for experimental run 1, 2 and 3, corresponding to capsule fill weight of 20 mg, are in accordance with visual observation on the powder compaction level in the capsule: FPD increased approximately 20% and FPF increased approximately 12% while decreasing the compaction degree (run 1 to run 3). For capsules filled with 10 mg and 5 mg, the results are within the expected for composite SD powders, with FPD of 6.5 mg and 3.3 mg and FPF of 72.7 % and 80.1 %, respectively for each fill weight. Overall, the performance was found to be lower for higher fill weights and chamber/layer ratios. Finally, the ACI results were in accordance with those found in previous work regarding SD composite powders that had been hand-filled into capsules (without any compaction), indicating that similar aerodynamic performance can be achieved through a scalable and high-throughput automated dosator-based precision capsule filling process [6].

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Drug Delivery to the Lungs, Volume 29, 2018 - Spray dried composite powders: capsule filling process optimization and aerodynamic performance characterization Table 2 – Aerodynamic performance results measured by ACI for the different fill weights with three repetition experiments. Run

Fill weight (target) (mg)

1 2 3 5 7

20 20 20 10 5

Moura C. et al. [6]

Compaction level (visual observation) High Medium Low Low Low Hand-filled (no compaction)

ED (%)

RSD_ED (%)

FPD (mg)

FPF_ED (%)

RSD_FPF (%)

97.4 103.1 96.7 88.7 83.1

6.0 1.2 5.2 3.0 1.5

11.1 12.1 13.4 6.5 3.3

57.0 58.5 69.3 72.7 80.1

4.2 0.8 4.7 2.7 5.3

Conclusions A capsule filling process of spray dried composite particles using a MG2 Flexalab machine was successfully achieved. By optimizing the dosing-chamber-height-to-powder-layer-depth ratio and by implementing appropriate engineering solutions it was possible to obtain a lower compaction level of the powder inside the capsule and low rejection rates. The aerodynamic performance results obtained for the capsules filled with 20 mg were in accordance with visually observation of the powder compaction inside the capsules. The aerodynamic performance results obtained for the different fill weights (20 mg, 10 mg and 5 mg) were aligned with previous studies on hand-filled spray dried composite particles. Further experiments would be required to precisely correlate process parameters (dosator size, chamber height and powder bed height) with the visual compaction of powder in the capsules and final performance. Also, additional SD composite powder characterization will be performed in order to correlate powder attributes to the filling and aerodynamic performance. In conclusion, this study allowed filling capsules of spray dried composite powder with good aerodynamic performances using a reliable and robust technology in a manufacturing environment, which is easily scaled-up. Acknowledgments We acknowledge MG2 for their technical support. References 1

Yadav N, Lohani A: Dry powder inhalers: A review, Indo Global Journal of Pharmaceutical Sciences 2013; 3(2): pp142-53.

Healy AM, Amaro MI, Paluch KJ, Tajber L: Dry powder for oral inhalation free of lactose carrier particles, Advanced Drug Delivery Reviews 2014; 75: pp32-52.

Nandiyanto ABD, Okuyama K: Progress in developing spray-drying methods for the production of controlled morphology particles: From the nanometer to submicrometer size ranges, Advanced Powder Technology 2011; 22(1): pp1-19.

Eva Faulhammer, Marlies Fink, Marcos Llusa, Simon M. Lawrence, Stefano Biserni, Vittorio Calzolari, Johannes G. Khinast: Low-dose capsule filling of inhalation products: Critical material attributes and process parameters, International Journal of Pharmaceutics 2014; 473: pp617-626.

S. Stranzinger, E. Faulhammer, V. Calzolari, S. Biserni, R. Dreu, R. Šibanc, A. Paudel, J.G. Khinast: The effect of material attributes and process parameters on the powder bed uniformity during a low-dose dosator capsule filling process, International Journal of Pharmaceutics 2017; 516: pp9-20.

European Pharmacopoeia, Edition 6.0, Chapter 2.9.6 – Uniformity of content of single-dose preparations, pp278.

Moura C, Vicente J, Palha M, Neves F, Aguiar-Ricardo A, Costa E: Screening and optimization of formulation and process parameters for the manufacture of inhalable composite particles by spray-drying, Drug Delivery to the Lungs 2014: pp74-77. 7

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Drug Delivery to the Lungs, Volume 29, 2018 - David A. Wyatt et al. Innovation in manufacturing as applied to dry powder inhaler formulation. David A. Wyatt,1 Jasdip S. Koner1, Eman Z. Dahmash1 & Afzal R. Mohammed1,2 1 Aston

Particle Technologies Ltd., Aston University, Birmingham, B4 7ET, United Kingdom Pharmacy School, Aston University, Birmingham, B4 7ET, United Kingdom

2 Aston

Summary An innovative process for development and manufacture of dry powder inhaler formulations is presented. The technology utilises a single stage, low mechanical-shear, ambient temperature, fluidisation process which conserves the physical and chemical integrity of all formulation components through control of well characterised process parameters. The technique’s utility is demonstrated with commercially sourced micronised fluticasone propionate and inhalation grade lactoses (IGLs). A bench-scale manufacturing process produces formulations with excellent content uniformity from which controllable respirable dose (RD) / fine particle fraction (FPF) is delivered when aerosolised. A designed experiment utilising the principles of Quality by Design (QbD) shows that broad design spaces can be found with two different IGLs. Most surprisingly, a single product formula manufactured by this process can be tuned for RD/FPF through controlled variations in the critical process parameters. This holds out the tantalising prospect of tuneable dry powder formulation performance (dial-a-dose). Key Message An innovative single step manufacturing method has been developed that delivers dry coating of particle surfaces in complex formulations of sensitive ingredients at ambient temperature, which are tuneable for use in dry powder inhalers. Introduction The basic elements of the development [1] of any medicinal product have been stated as; • • • • •

defining the Quality Target Product Profile identifying critical quality attributes of the drug product determining (critical) quality attributes of the starting materials (drug substance, excipients) selecting an appropriate manufacturing process identifying a control strategy

If the first three requirements are appropriately specified, the final two demand manufacturing processes that conserve the critical quality attributes of the starting materials, and the drug product whilst being highly controllable. To this end, a number of innovative technologies (often termed advanced manufacturing technologies) have been developed suitable for the production of functionalised particles by dry powder coating but each is limited in pharmaceutical application [2-4]. Firstly, they are predominantly high mechanical shear methodologies, which means that particle size can be adversely affected, a critical attribute for respiratory medicines. Secondly, the mechanics of these processes often generate heat, which can result in chemical or physical modification to both the coating particles and coated particles themselves. A third constraint for introduction of novel techniques may be concern over the necessary regulatory oversight of modern manufacturing technology [5] and general conservatism. For whatever reason, there is a very small cadre of ‘tried and tested’ technologies that are routinely used in the pharmaceutical industry such as high shear blenders or turbula mixers with narrow operating windows. These technologies remain ‘fit-for purpose’ solely because process validation and control strategies can be achieved with effort. For over a decade, the regulatory authorities have encouraged the industry to seek processes that are much less stochastic and less sensitive to variations in input material. The FDA have stated ‘pharmaceutical quality is assured by understanding and controlling formulation and manufacturing variables’ and support implementation of Quality by Design (QbD) to ‘enable transformation of the chemistry, manufacturing, and controls (CMC) review…into a science-based pharmaceutical quality assessment’ [6].. The new technique, APT-Hale™ [7], presented in this paper is designed on these principles and does not have the physicochemical limitations of existing technologies. The technique has broad applicability, here exemplified in the development of formulations for DPIs, because such formulations typically require uniform dispersal of minute quantities of microfine cohesive active pharmaceutical ingredients within a matrix of coarser carrier particles. Manufacture of such formulations requires a ‘systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management’ [8]. This is QbD. Experimental methods Manufacturing apparatus: A schematic representation of the operating principles of the novel coating apparatus is shown in Figure 1 beside a picture of the Mark IV bench-top apparatus. In these studies, blends were prepared at 20g scale, run times varying between 10 - 30 minutes and centrifugal force between 36 -202 G.

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Drug Delivery to the Lungs, Volume 29, 2018 Innovation in manufacturing as applied to dry powder inhaler formulation. Air Inlet Air Outlet

Nitrogen Gas Supply Rotating Processing Vessel

Cross Sectional View

Motor

View of Air Blade

Figure 1: Schematic diagram and picture of Mk IV dry coating apparatus

[9,10]

Operation: Powders (coarse host particles and fine guest particles) to be formulated are co-dispensed into the processing vessel which is then sealed and placed in its cradle. The vessel is then accelerated to rotate on its axis at high angular velocity generating a high centrifugal force. Whilst in operation, the internal surface of the vessel is continuously swept by an air-blade (dry nitrogen) at a set flow rate, which fluidises the powder at the vessel surface. The precise acceleration and deceleration of the vessel is computer controlled. Materials Each of the materials treated by this process was sourced commercially and processed without further conditioning. The materials were thus ‘off the shelf’. The physical specifications of the materials are shown in Figure 2. Micronized Fluticasone Propionate (FP) (Discovery Fine Chemicals) Inhalation Grade Lactose (Respitose™ SV003 and SV010) (DFE Pharma)

Particle Size Data Particle Size Fraction

FP Particle Size (μm)

SV003 Particle Size (μm)

SV010 Particle Size (μm)

D10

0.91 ± 0.02

19-43

25-65

D50

2.67 ± 0.03

55-66

95-125

D90

6.29 ± 0.02

75-106

160-190

VMD*

3.22 ± 0.02

*measured by laser particle diffraction using Sympatec Helos equipped with Rodos powder disperser

Figure 2: Commercially sourced ‘off the shelf’ input materials

Results and Discussion Principle of operation: By means of the centrifugal force, particles are accelerated against the inner surface of the vessel and aggregates, in both the fine and coarse materials, are broken into individual particles. As the vessel spins, this surface passes under the air-blade which fluidises the particles. Due to mass differences, the net residence time of the individual particles on the inner surface of the vessel varies. The larger particles effectively become bathed in a cloud of finer particles which, because of the cohesive nature of the finest particles, are adsorbed at suitable sites on the surface of the larger particles. Theory: The dispersion process occurs in three consecutive stages. The first phase is the breakup of all agglomerates to individual particles. The second is the dispersion of the fine particle ‘cloud’ around the coarser particles and the third is the attachment/adhesion of the fine particles onto the surface of the host to reach a uniform spatial distribution of the fine particles. These steps are shown schematically in Figure 3.

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Figure 3: The theoretical stages in the dry coating process The adhesion of the fine particles onto the host is primarily due to electrostatic or van der Waals forces of attraction. The critical process parameters that control this are the centrifugal force, the gas pressure (flow rate) and the processing time coupled to the composition of the formulation and the properties of the input materials. Process benefits: There is a complete absence of mechanical shear and the process occurs at ambient temperature, with a slight cooling effect due to the expansion of the gas as it passes through the array of entry jets. This protects the critical quality attributes of the input materials. The manufacturing process has high capability in terms of repeatability and reproducibility due to computer control. Content uniformity: Formulations of FP in Respitose™ SV003 (Inhalation Grade Lactose (IGL)) manufactured by the process were investigated over the concentration range 0.5%w/w to 10%w/w (Figure 4). The relative standard deviation of content at all concentrations was found to be <2% with excellent recovery of FP.

Figure 5: SEM images of an FP formulation

Figure 4: Content uniformity of FP batches in IGL

Formulation structure: Representative scanning electron micrographs from samples taken during this study are shown in Figure 5. The images show a uniform microfine coating of particles on the surface of the coarser particles. These fine particles are made up of individual particles of micronised FP and also of lactose fines from the carrier itself (the lactose fines being identifiable by the characteristic axe-head shape). Of note is that no free FP particles or agglomerates are detected in any of the micrographs. FP particles are bound as singlets mainly to the surface of the coarse lactose particles or, as shown at the highest magnification, to the co-adsorbed fine lactose particles. There is no evidence of cohesive agglomeration of FP. This confirms that the process has been able to disperse the most cohesive material completely into individual particles either prior to adsorption onto the carrier or, postadsorption, during a re-ordering of fine particulate material on the surface of the lactose. The absence of FP agglomerates possibly explains the very tight content uniformity. It should be noted that there is no evidence of Respitose™ carrier attrition. This is because the process uses centrifugal force to hold the larger particles predominantly at the vessel surface and, as a result, direct particle-particle collisions are minimised. Dosing performance: In another series of experiments with a single formulation of FP and IGL, selected from the previous study at 0.5%w/w, the critical process parameters were modified in a planned design of eight consecutive batches, designated APT-5 to APT-12. Each of the batches was then sampled and manually filled into size 3 gelatine capsules to mimic 100µg inhalation capsules. Three capsules from each batch were then actuated into a Next Generation Impactor (Copley Scientific) using an Aerolizer™ inhaler at 60 l/min airflow for 4 seconds. After HPLC analysis, the amount of respirable material in each actuation was calculated, as were the efficiency of dose delivery and the theoretical fine particle fraction (FPF) for each batch. These results are plotted in Figure 6. Although the efficiency of dose delivery is variable, as expected for a capsule based inhaler, there is a clear and increasing trend in the respirable dose and the FPF which correlate with the controlled changes in the critical processing parameters. From the same formulation it has been possible to change the FPF from 10% to nearly 50%. This demonstrates the ability of the manufacturing process not only to produce tight content uniformity but also to have strong influence on the ability of the resultant formulations to release the therapeutic dose. In effect, the process has transformed a single formula into one that delivers a low lung dose, a medium lung dose or a high lung dose by controlled variation of processing.

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Figure 6: NGI data generated for consecutive batches 0.5%w/w FP batches in IGL

Figure 7: Targeted Critical Quality Attributes (FPF 25-50 % and Content Uniformity RSD <5%)

Quality by Design: In a follow up study, the FP formulation was processed using a ‘design of experiments’ developed using MODDE software version 8.2 (Quadratic Model, D-optimal design, 16 runs, 3 repeat) in which design spaces were generated for both IGL carriers. The results shown in Figure 7 demonstrate at least two significant findings. Firstly, a sweet spot for formulation critical quality attributes (content uniformity and FPF) has been found under different processing conditions for both IGL carriers. This demonstrates that the technology can optimise performance with different input excipients. Carrier selection can be made on the basis of which carrier yields the broadest control space and hence the most robust performance in manufacturing. Secondly, further evidence has been generated that control of a small number of critical process parameters can deliver tailored dosing performance. When these findings are coupled to the fact that the process required no pre-treatment of starting materials, whether excipient or the active pharmaceutical ingredient, the power of this new dispersion method can be appreciated. Conclusion The utility of a novel bench-scale manufacturing process has been demonstrated using commercially sourced micronised fluticasone propionate and carriers (inhalation grade lactose) The technique produced formulations with excellent content uniformity across a wide range of concentrations. Batches manufactured to a single product formula, containing 100 µg FP / 20 mg blend, when dispensed and tested by delivery from an Aeroliser™ Inhaler delivered increasing respirable dose (FPF) as a direct function of the selected critical process parameters. This holds out the tantalising prospect of being able to tune dry powder formulation performance (dial-a-dose) with this novel technique. In addition, a designed experiment with the same formulation, utilising the principles of QbD identified broad design spaces with two different carriers demonstrating that the technique will aid formulation selection References 1

Welin, M.: GMP, Quality by Design and Validation, http://www.ema.europa.eu/docs/en_GB/document _library/Presentation/2015/05/WC500187355.pdf .

Pfeffer, R, Dave, RN, Wei, DG, Ramlakhan, M. 2001 Jun 4Synthesis of engineered particulates with tailored properties using dry particle coating. Powder Technol;117(1-2):40-67. PubMed PMID: WOS:000168905500002.

Gera, M, Saharan, VA, Kataria, M, Kukkar, V. 2010 Mechanical methods for dry particle coating processes and their applications in drug delivery and development. Recent patents on drug delivery & formulation;4(1):58-81.

Mullarney, MP, Beach, LE, Langdon, BA, Polizzi, MA. 2011 Applying dry powder coatings. Pharmaceutical Technology;35(10):94-102.

Yu, L.X.: Evolving the FDA’s Approach to Pharmaceutical Quality, IPAC-RS/University of Florida Orlando Inhalation Conference Approaches in International Regulation, March 18-20, 2014

Yu, L.X.: Pharmaceutical quality by design: product and process development, understanding, and control. Pharm Res. 2008 Apr;25(4):781-91

Registered Trademark for Aston Particle Technologies Ltd, Respiratory Formulation Technology

Yu, L.X.: Evolving the FDA’s Approach to Pharmaceutical Quality, IPAC-RS/University of Florida Orlando Inhalation Conference Approaches in International Regulation, March 18-20, 2014

Dahmash, E.Z.: Development of Novel Dry Coating Device for Pharmaceutical Application. Ph.D. Thesis, Aston University, June 2016

Patent WO2016066462A1 Coating apparatus and method. 2016

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Drug Delivery to the Lungs, Volume 29, 2018 - Qiuying Liao et al. Formulation of Inhalable Voriconazole Dry Powders Using Spray Freeze-Drying Technique Qiuying Liao1, Long YIP1,2 & Jenny K.W. LAM1 Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong 2 Department of Pharmacy and Forensic Science, King’s College London, 150 Stamford Street, London, SE1 9NH, United Kingdom 1

Summary Systemic administration of antifungal agents intended for the treatment of pulmonary aspergillosis is limited by the poor lung distribution and severe adverse effects. Pulmonary delivery is desirable as it allows deposition of drug at high concentrations directly in the site of infection. Voriconazole is the primary treatment of pulmonary aspergillosis with potent and wide-spectrum activity. This study aimed to develop inhalable voriconazole dry powder formulation with excellent aerodynamic performance by constructing porous particles using spray freeze-drying technique. Mannitol was included in the formulation as a bulking agent. Since voriconazole has a poor aqueous solubility, tertbutyl alcohol (TBA) was used as a co-solvent. A two-level full factorial design was employed to systematically investigate the effect of three factors and their interactions on the aerosol performance of the formulations. These factors were (i) solute concentration of the feed solution; (ii) the voriconazole concentration; and (iii) the co-solvent composition. The cascade impactor study revealed that spray freeze-dried powder containing high level of voriconazole concentration could reach the highest fine particle fraction (FPF, <5 µm) of 47.4%. After analysing the factorial design using Minitab® 18 statistical software, the voriconazole concentration was found to be the most significant factor that can positively affect the fine particle fraction and negatively affect the emitted fraction. This result suggests that, with current production method, increasing the voriconazole concentration in the feed solution could not only improve the delivery efficiency as higher percentage of voriconazole was included in same amount of powder, but also enhance the aerosol performance of powder formulation as higher FPF was achieved. Key Message Porous inhalable voriconazole powder formulation that showed good aerosolization performance (fine particle fraction > 40%) was successfully produced by spray freeze-drying. The results of a full factorial design for the formulation optimization suggests that the voriconazole concentration in the feed solution could significantly enhance the aerosol performance. Introduction Pulmonary aspergillosis caused by Aspergillus species has been showing a significant rise on morbidity and mortality especially in immunocompromised patients during the last decade [1]. While a second-generation triazole – voriconazole is the primary treatment of pulmonary aspergillosis, the currently available intravenous and oral formulations are suffering from poor lung distribution and severe side-effects [2]. Pulmonary delivery is an attractive approach as it allows deposition of high drug concentration at the infected site with reduced risk of systemic exposure. This study aimed to develop inhaled voriconazole powder formulation with excellent aerosol performance by spray freeze-drying technique. Mannitol and TBA served as a bulking agent and a co-solvent for voriconazole, respectively. A full factorial design was adopted to examine how the operating conditions affect the aerosol performance and to optimize the formulation for further pharmaceutical and clinical development. Experimental Methods Materials Voriconazole was purchased from Tecoland Corporation (Irwin, CA, USA). Mannitol (Pearlitol 160C) was obtained from Roquette (Lestrem, France). T-butyl alcohol (TBA) and formic acid were obtained from Sigma (Poole, UK). Acetonitrile was obtained from Anaqua Chemicals Supply (Cleveland, OH, USA). All solvents and reagents were of analytical grade or better unless otherwise stated. Experimental design – factorial design A 23 full factorial design generated by Minitab ® 18 was used for designing the experiments. The studied factors were: solute concentration (A); voriconazole concentration (B) and TBA concentration (C). The response studied were: fine particle fraction (FPF) and emitted fraction (EF), both were obtained from aerosol performance. The levels of each variable were designated as -1, 0 and +1, respectively, and the corresponding actual values for each factor are shown in Table 1.

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Drug Delivery to the Lungs, Volume 29, 2018 - Formulation of Inhalable Voriconazole Dry Powders Using Spray Freeze-Drying Technique Table 1 – A 23 full factorial experimental design. The levels of each factor are designated as -1 (low level), 0 (middle level), and +1 (high level).

-1

Levels 0

A – Solute concentration (w/v%)

2.5

B – Voriconazole concentration (w/w%)

C – TBA concentration (v/v%)

Factors

Preparation of spray freeze dried powders Solutions with different levels of solute concentration, voriconazole concentration and TBA concentration were prepared (table 2) and fed to an ultrasonic nozzle (130K50ST, Sonaer ®, Farmingdale, NY, USA) operating at 130 kHz powered by digital ultrasonic generator for atomizers (Sonaer ®, NY, USA). The atomized droplets were collected in liquid nitrogen instantly and transferred into freeze dryer for lyophilization. The samples were kept under vacuum with a chamber pressure below 0.133mBar at -25 C for 20 h, followed by secondary drying at 20 C for 20 h, and kept at room temperature for 25 h. The dried powders were collected and stored in desiccator with silica gel at ambient temperature until further analysis. Table 2 – Compositions of spray freeze dried formulations. The formulation S2.5-V30-T55 was prepared in triplicate as the centre point of factorial design. Formulation

Solute Concentration (%, w/v)

VRC percentage (%, w/w)

TBA Concentration (%, v/v)

S1-V20-T40

S1-V20-T70 S1-V40-T40

S1-V40-T70

S4-V20-T40

S4-V20-T70

S4-V40-T40

S4-V40-T70

S2.5-V30-T55

2.5

Morphology study Spray freeze dried powder morphology was observed using scanning electron microscopy (SEM, S4800 FEG SEM, Hitachi, Tokyo, Japan) at 5 kV. Quantification of voriconazole Voriconazole was quantified chromatographically using high performance liquid chromatography (HPLC; Agilent 1260 Infinity; Santa Clara, USA) equipped with a C18 column (Agilent Prep – C18, 4.6 x 250 mm, 5 m). The mobile phase was acetonitrile and 0.5% formic acid (50:50, v/v) running at an isocratic flow rate of 1 ml/min. Voriconazole was detected at the wavelength of 254 nm and quantified against a standard curve in the range of 3.125 to 100 g/ml at retention time of 6.8 min. In vitro aerosol performance evaluation The aerosol performance of the spray freeze dried powders were evaluated using Next Generation Impactor (Copley Scientific, United Kingdom) coupled with a Breezhaler® (Novartis Pharmaceuticals, Hong Kong) operated at 100 L/min for 2.4 s. For each dispersion, approximately 3.0  0.5 mg of powder were loaded in a size 3 hydroxypropyl methylcellulose (HPMC) capsule (Capsugel, West Ryde, NSW, Australia). Voriconazole deposited on each stage was assayed using the HPLC as described above. Recovered dose was defined as the sum of powder mass assayed on inhaler and all NGI stages in a single run. The EF was the fraction of powder exited the inhaler with respect to the recovered dose. FPF (<5 µm) was defined as the fraction of powder with aerodynamic diameter less than 5 µm of the recovered dose.

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Drug Delivery to the Lungs, Volume 29, 2018 - Qiuying Liao et al. Dissolution study Fine particle doses (FPD, dae < 5µm) of S1-V40-T40, S1-V40-T70, S4-V40-T40, and S4-V40-T70 were captured by a glass fibre filter paper using Fast Screening Impactor (Copley Scientific, United Kingdom) operated at 100 L/min for 2.4s. The paddle over disk method was adopted to examine the dissolution profile of the FPD of voriconazole after transferred into a dissolution apparatus containing 400 ml of PBS buffer (pH 7.4) as dissolution medium. Same method was also applied to study the dissolution profile of an equivalent amount of raw voriconazole power placed on a glass fibre filter paper. Results and discussion Spherical particles with porous structure and minor agglomeration were observed from all the spray freeze dried powder formulation by SEM. Figure 1 showed representative SEM images of three formulations that contained different voriconazole concentration (two corner points and one centre point of the factorial design), where the powders contain higher voriconazole concentration exhibited more porous structure and finer network. The powder formulations containing high voriconazole concentration (40%) had highest FPF and lowest EF (figure 2). Among all the formulations, S1-V40-T70 and S4-V40-T70 showed best aerosol performance with FPF of 47.4% and 41.1%, respectively. The results of in vitro aerosol performance were analysed systematically using statistical software Minitab® 18. The pareto charts identified the significant factors and interaction among factors, ranked the factors and their interactions from largest effects to smallest effect (figure 3). Any factor or their interaction that cross the vertical reference line is considered as significant. Voriconazole concentration was found to be the most significant factor affecting the FPF. At the same time, the EF was only significantly affected by voriconazole concentration. Interestingly, according to the results of aerosol performance (figure 2), it can be observed that the formulations with higher voriconazole concentration resulted in higher FPF (>40%) and lower EF (approx. 80%). A high FPF is desirable for a good aerosol powder formulation as it indicates effective deposition in the deep lung. On the other hand, since EF represents the portion of powder that exposed to the patient regardless of site of deposition, EF and FPF should be closed with each other. An inhalable powder formulation that has high FPF but relatively low EF will cause minimal exposure of antifungal drug at unintended site, such as the throat and the upper airways, but effective drug deposition in the lower respiratory tract. Additionally, including higher voriconazole concentration in the powder formulation can increase the delivery efficiency as a higher dose of voriconazole can be delivered with a lower powder dose. Overall, increasing the voriconazole concentration in the formulation can not only enhance its aerosol performance but also allow more drug to be deposited at the target site. The dissolution profile of FPD of S1-V40-T40, S1-V40-T70, S4-V40-T40, and S4-V40-T70 formulation, as well as the raw voriconazole were evaluated (figure 4). The dissolution rate of raw voriconazole was slow, as the accumulative percentage of dissolved drug reached 95% after 90 min. On the contrary, the FPD of the formulated powders containing high voriconazole concentration showed fast dissolution behaviour as 95% accumulative percentage of drug was dissolved within 5 min. Given the low aqueous solubility of voriconazole, it is important for voriconazole aerosol powder formulation to have a fast dissolution behaviour. The results showed that the dissolution rate of voriconazole can be accelerated by formulating inhalable powder using spray freeze drying technique, as particles with porous structure and increased surface area were successfully produced. Conclusions Inhaled voriconazole powders with mannitol as a bulking agent were prepared by spray freeze drying in this study. Spherical and porous particles were constructed, and the more porous structure and finer network were observed in the powders containing higher voriconazole concentration. According to the factorial design analysis, voriconazole concentration was found to be the most significant factor that can positively affect FPF and negatively affect EF in the in vitro aerosol performance evaluation. The FPD of powders containing high voriconazole concentration showed improved dissolution behaviour compared to unformulated voriconazole powders. Further studies will be conducted to evaluate the pharmacokinetic profiles and biodistribution of spray freeze dried powders following pulmonary delivery in animal models.

Figure 1 – Representative SEM images of spray freeze dried powders at 2,500 magnification. Scale bar represents 20 µm. Nomenclature: S – solute concentration (%, w/v); V – voriconazole concentration (%, w/w); T – TBA concentration (%, v/v).

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Figure 3 – in vitro aerosol performance of spray freeze dried powders evaluated by NGI. (A) Fine particle fraction and (B) emitted fraction were expressed as percentage relative to the recovered dose. Nomenclature: S – solute concentration (%, w/v); V – voriconazole concentration (%, w/w); T – TBA concentration (%, v/v). Data was presented as mean ± standard deviation (n=3).

Figure 4 – pareto charts of the standardized effects where the responses were (A) fine particle fraction and (B) emitted fraction. The vertical line represents minimum statistically significant effect magnitude for a 95% confidence level, any terms that cross the vertical line are considered statistically significant.

Figure 2 – dissolution profiles of raw voriconazole powder and fine particle dose (FPD) of the formulated powders containing 40% voriconazole concentration. Nomenclature: S – solute concentration (%, w/v); V – voriconazole concentration (%, w/w); T – TBA concentration (%, v/v). Data was presented as mean ± standard deviation (n=3).

References 1

Kousha M, Tadi R, Soubani AO. Pulmonary aspergillosis: a clinical review. Eur Respir Rev. 2011;20(121):156-74.

Beinborn NA, Du J, Wiederhold NP, Smyth HDC, Williams RO. Dry powder insufflation of crystalline and amorphous voriconazole formulations produced by thin film freezing to mice. European Journal of Pharmaceutics and Biopharmaceutics. 2012;81(3):600-8.

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Drug Delivery to the Lungs, Volume 29, 2018 – Zara Sheikh et al. An In Vitro Study to Rationalise Combination Therapies for Cystic Fibrosis Treatment Zara Sheikh1, Hui Xin Ong1, Michele Pozzoli1, Paul Young1, Daniela Traini1 1Woolcock

Institute of Medical Research and Faculty of Medicine and Health, University of Sydney, 431 Glebe Point Road, Glebe NSW 2037, Australia

Summary Chronic inflammation associated with recurrent infections that leads to pulmonary failure is the principal cause of morbidity and mortality in cystic fibrosis (CF) patients. Therefore current CF therapy is focused on inhaled antibiotics (e.g. tobramycin) in combination with oral anti-inflammatory drugs (e.g. high dose ibuprofen) and other oral/intravenous antibiotics (e.g. azithromycin). However, there is a gap in the understanding of potential interactions between these drugs in terms of their efficacy and mechanism of action, resulting in variable treatment outcomes, as stated in the current CF consensus treatment guidelines. For instance, a recent clinical trial of 263 CF subjects has shown that oral azithromycin antagonises the antimicrobial efficacy of inhaled tobramycin. Based on this recent evidence, this study aims to investigate other potential interactions between tobramycin and other anti-inflammatory drugs used in CF treatment, specifically, ibuprofen and azithromycin. To achieve this aim, an in vitro inflammation study of two combinations of tobramycin with either ibuprofen or azithromycin on IL-8 expression was performed using CuFi-1 and BEAS-2B pulmonary epithelial cell lines. Both combinations demonstrated a significant increase in IL-8 concentration in the two cell lines compared to the control using lipopolysaccharide (LPS) in CuFi-1 cells, suggesting an enhanced inflammatory effect. This study was a preliminary step in elucidating these drug-drug interactions with the aim to rationalize combination therapies for CF treatment that could potentially change future current clinical practice and overall, reduce treatment burden of CF patients. Key Message In this study, tobramycin, the most widely prescribed antibiotic for CF treatment, has demonstrated a reduction in anti-inflammatory efficacy of ibuprofen and azithromycin, suggesting a possible drug-drug interaction. This study is relevant to current clinical practice, enabling health practitioners to make evidence-based informed decisions about prescribing combinations for CF treatment. Introduction Cystic fibrosis is a congenital, life-limiting, orphan disease that affects 1/2500 to 1/3000 of the global population. The disease is most prevalent in Europe (more than 30,000), North America (approximately 30,000), United Kingdom (around 10,000) and Australia (around 3,200).[1] Children with CF hardly lived beyond adolescence in the 1980s but advancement in treatment options and innovative management strategies has improved life expectancy of CF patients’ up to 40 years.[2] This autosomal recessive condition occurs as a consequence of mutations within the gene that codes for the CF transmembrane conductance regulator (CFTR) protein. The CFTR protein is present in the epithelial lining of the respiratory tract, biliary tree, intestines, vas deferens, sweat ducts and pancreatic ducts, making CF is a multi-organ disease. However, the primary reason for 90% of morbidity and mortality of CF patients is lung failure.[3] The lack of CFTR function leads to an impaired secretion of chloride ions and simultaneous sodium hyperabsorption, causing depletion of the airway surface liquid, which in turn disrupts the mucociliary action and reduces mucus clearance. As a result, the accumulation of viscid mucus secretions creates an ideal environment for bacterial colonisation, predisposing the lungs to repeated infections, chronic inflammation, bronchiectasis and pulmonary exacerbations.[4] Subsequently, CF is characterized by a relentless cycle of inflammation with recurrent infections. The mainstay of current CF therapy is focused on a combination of antibiotics delivered through the inhalation route coupled with oral or inhaled anti-inflammatory drugs and other antibiotics either given orally or intravenously, as well as mucolytic agents.[5] Cycled, chronic use of inhaled antibiotics targeted at P. aeruginosa airway infection (the dominant infecting microorganism) greatly improves lung function and diminishes the frequency of pulmonary exacerbations. [3] Specifically, tobramycin is the most prescribed antibiotic for CF patients accounting for its use in two-thirds of the CF population in the U.S. (Cystic Fibrosis Foundation Registry, 2011), widely used in UK (approximately 49.1%), as well as in Australia.[6] Chronic inflammation with a surge of neutrophils infiltrating the airways is the hallmark of CF. To date, the only antiinflammatory drug approved for chronic use in CF is ibuprofen.[7] Long term use of high doses of oral ibuprofen (2030 mg/kg/dose twice daily targeting a peak plasma drug concentration of 50-100 µg/ml) can substantially reduce the progression of lung disease in CF patients between 6-17 years of age, with FEV1 predicted to be greater than 60%.[1] Approximately 1000 CF patients are currently using Ibuprofen worldwide. [8] Besides ibuprofen, the macrolide antibiotic azithromycin is also recommended for chronic administration to CF patients aged 6 years and older, owing to its combined beneficial anti-inflammatory and antibacterial effects.[9] The

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Drug Delivery to the Lungs, Volume 29, 2018 - An In Vitro Study to Rationalise Combination Therapies for Cystic Fibrosis Treatment anti-inflammatory activity of oral azithromycin in CF subjects was proven in a double-blinded, randomised controlled trial in terms of decrease in neutrophil counts and serum inflammatory markers. [10] In the U.S., oral azithromycin is prescribed to 71% patients infected with P. aeruginosa and 75% of CF patients already on inhaled tobramycin.[11] In the UK, 48.5% CF patients with chronic P. aeruginosa and approximately 56.7% patients in Australia take azithromycin on a long-term basis (UK Cystic Fibrosis Registry 2016, Australian Cystic Fibrosis Data Registry 2015). Although these anti-inflammatory drugs have proven to be effective in these trials when administered alone, a recent clinical trial of 263 CF subjects has shown that oral azithromycin antagonized the antimicrobial efficacy of inhaled tobramycin [11], when administered concurrently. The potential interactions between the concomitant drugs used in CF therapy leading to variability in treatment outcomes is poorly understood; this has been stated in the current CF consensus treatment guidelines as one of the key issues. [7] Based on this evidence, the present study aims to elucidate the anti-inflammatory efficacy of the combination of tobramycin with either ibuprofen or azithromycin. Since IL-8 is the prime neutrophil-attracting chemokine present in abundance in the airways of CF patients [12], the anti-inflammatory effect of tobramycin, in combination with ibuprofen or azithromycin, on IL-8 expression using established immortalized in vitro CF cellular model (CuFi-1 cell line) compared with normal pulmonary epithelial cell line BEAS-2B was investigated. Experimental Methods Materials Tobramycin and azithromycin were purchased from Shanghai TECH (Chemical Industry Testing Co., Ltd, China). Ibuprofen was used as supplied from HuBei (Granules-Biocause Pharmaceutical Co., Ltd, China.) CuFi-1 (Immortalised Epithelial cells of cystic fibrosis origin) and BEAS-2B (Epithelial cells isolated from normal human bronchial epithelium obtained from autopsy of non-cancerous individuals) were purchased from the American Type Culture Collection (VA, USA). Lipopolysaccharide (LPS) from Escherichia coli was obtained from Sigma-Aldrich (Sydney, Australia). All cell culture reagents including Dulbecco’s modified eagle’s medium (DMEM), Minimum Essential Medium Eagle (MEM), phosphate buffered saline (PBS), foetal bovine serum (FBS), trypsin-EDTA solution (2.5g/l trypsin, 0.5g/l EDTA), L-glutamine solution, non-essential amino acids were obtained from Invitrogen (Sydney, Australia). Human IL-8 ELISA Kit II BD OptEIATM for determination of the inflammation marker IL-8 were purchased from BD Bioscience (Sydney, Australia). In vitro anti-inflammatory activity of combination of tobramycin with ibuprofen and azithromycin The inflammatory effect of tobramycin, ibuprofen and azithromycin as single agents and in combination (tobramycin with ibuprofen and tobramycin with azithromycin) were tested on LPS-induced CuFi-1 cell line (passage number 29) and BEAS-2B (passage number 19). Approximately, 500,000 cells / well were seeded into 24 well plates with 10 µg/ml of LPS before incubation for 24 hours at 37°C in a humidified atmosphere at 5% CO 2 to allow for the production of the inflammatory cytokine, IL-8. The cells were subsequently treated with drugs at a concentration of 6 mg/ml of tobramycin, 1mg/ml of ibuprofen and 200 µg/ml of azithromycin, respectively, alone and in combination. It should be noted that the LPS concentration used in this experiment was not toxic to the cell (cell viability above 90%, data not shown). After 24 hours, samples of the culture medium were collected and stored at -20°C. Samples were analysed for expression of IL-8 using Human IL-8 ELISA Kit II BD OptEIATM according to the manufacturer’s instructions. All the experiments were performed in triplicate. Statistical analysis One-way ANOVA or unpaired 2-tailed t-tests were performed to determine significance (which was quoted at the level of p<0.05) between treatment groups and control. Results Drug-drug interaction on IL-8 levels were measured at 24 hours after stimulation of the CuFi-1 and BEAS-2B cells with LPS to simulate inflammatory process during a bacterial infection. Statistically significant differences were found between the samples and the control (p<0.05) (Figures 1 and 2).

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Figure 1 -

Inflammatory activity of tobramycin with ibuprofen on IL-8. Ibuprofen alone (IBU) inhibited the expression of IL-8 in CuFi-1 cells compared to untreated cells exposed to LPS but showed increased levels of IL-8 in BEAS2B. Tobramycin as a single agent (TOBI) increased IL-8 levels in both the cell lines. The notable factor is the combination of ibuprofen and tobramycin (IBU+TOBI) which markedly increase the concentration of IL-8 in CuFi-1 cells and in BEAS-2B, indicating a pronounced inflammatory inducing effect. Data represents mean ± SD (n=3). * p<0.05, **** p<0.01

Figure 2 - Inflammatory activity of Tobramycin with Azithromycin on IL-8. Azithromycin alone (AZI), an anti-inflammatory agent at low dose, did not show a reduction in IL-8 expression in either cell lines. Tobramycin as a single agent (TOBI) increased IL-8 levels in both the cell lines. The combination of tobramycin with azithromycin (TOBI + AZI) resulted in an enhanced inflammatory effect, demonstrated by an increase in IL-8 expression, compared to that of untreated cells exposed to LPS. Data represents mean ± SD (n=3). * p<0.05, **** p<0.01, (ns means not significant)

Discussion High levels of the inflammatory IL-8 marker are expressed in CF airways when exposed to lipopolysaccharide, which is responsible for the recruitment of neutrophils during an infection that in turn produces excessive inflammatory response in CF patients. Therefore, down-regulation of IL-8 expression could ease the neutrophil burden and reduce inflammation.

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Drug Delivery to the Lungs, Volume 29, 2018 - An In Vitro Study to Rationalise Combination Therapies for Cystic Fibrosis Treatment In our study, ibuprofen alone showed a decrease in IL-8 levels in CuFi-1 cells but not in BEAS-2B. A number of mechanisms have been proposed for the anti-inflammatory action of ibuprofen such as suppression of the major transcription factor that activates IL-8 production, known as nuclear factor kappa (NF-ÎşB), decrease of prostaglandin E2 synthesis in CF respiratory epithelium that stimulates IL-8 production by activating C/EBP homologous protein, downregulation of the cyclic adenosine monophosphate activation and/or stimulation of the peroxisome proliferatoractivated receptors, which is a negative regulator of inflammation [1]. Previously, downregulation of mitogen activated protein kinase phosphatase (MKP-1) which is an important negative regulator of inflammatory gene expression in pulmonary epithelial cells has been associated with increased IL-8 expression in A549 cells.[13] Therefore, the difference in response of ibuprofen in BEAS-2B could possibly be explained in terms of inability of ibuprofen to enhance MKP-1 pathway,[14] but this line of enquiry requires further study. Although anti-inflammatory effects of tobramycin have recently been addressed, however, in our experiment, tobramycin did not decrease IL8 concentration suggesting that its anti-inflammatory action is still debatable. The enhanced inflammatory effect when ibuprofen and tobramycin were in combination could be attributed to the formation of an ion pair, as ibuprofen is a weakly acidic drug with a pKa of 4.9 and tobramycin is basic in nature with pKa ranging from 6.9 to 9.9. This will also need to be further investigated. As for azithromycin, its anti-inflammatory activity on IL-8 in CF airway epithelium is through the inhibition of transcription of pro-inflammatory genes.[15] In our study however, we did not observe a decrease in IL-8 expression. On the contrary, when combined with tobramycin, azithromycin showed an increase in inflammatory response. This is in agreement with findings by Nick et al. on the reduced anti-microbial efficacy of combination of inhaled tobramycin with oral azithromycin in a cohort of 263 CF patients, further confirming a drug-drug interaction. [11] Conclusion This study indicates that tobramycin has a negative effect on the anti-inflammatory properties of ibuprofen and azithromycin, when administered concurrently, suggesting a possible drug-drug interaction. In general, with the exception of ibuprofen on the CuFi cell line, all drugs studied increased IL-8 production, indicating a synergic proinflammatory response, resulting from drug-drug interactions. This preliminary study, is an indication that potential adverse drug interactions may exist when these particular combination therapies are prescribed in CF. Further investigations are needed to deeply understand these mechanisms in order to rationalize combination therapies used for CF treatment. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Sheikh, Z., et al., Is there a role for inhaled anti-inflammatory drugs in cystic fibrosis treatment? Expert Opinion on Orphan Drugs, 2018. 6(1): p. 69-84. Kumar, S., A. Tana, and A. Shankar, Cystic fibrosis â&#x20AC;&#x201D; What are the prospects for a cure? European Journal of Internal Medicine, 2014. 25(9): p. 803-807. Amin, R. and F. Ratjen, Emerging drugs for cystic fibrosis. Expert Opin Emerg Drugs, 2014. 19(1): p. 14355. Elborn, J.S., Cystic fibrosis. The Lancet, 2016. 388(10059): p. 2519-2531. Prayle, A.P. and A.R. Smyth, From pipeline to patient: new developments in cystic fibrosis therapeutics. Expert Opin Pharmacother, 2013. 14(3): p. 323-9. Hennig, S., et al., Safety of inhaled (Tobi(R)) and intravenous tobramycin in young children with cystic fibrosis. J Cyst Fibros, 2014. 13(4): p. 428-34. Peter J. Mogayzel, J., et al., Cystic Fibrosis Pulmonary Guidelines. American Journal of Respiratory and Critical Care Medicine, 2013. 187(7): p. 680-689. Dhooghe, B., et al., Lung inflammation in cystic fibrosis: pathogenesis and novel therapies. Clin Biochem, 2014. 47(7-8): p. 539-46. Cantin, A.M., et al., Inflammation in cystic fibrosis lung disease: Pathogenesis and therapy. J Cyst Fibros, 2015. 14(4): p. 419-30. Ratjen, F., et al., Effect of azithromycin on systemic markers of inflammation in patients with cystic fibrosis uninfected with Pseudomonas aeruginosa. Chest, 2012. 142(5): p. 1259-1266. Nick, J.A., et al., Azithromycin may antagonize inhaled tobramycin when targeting Pseudomonas aeruginosa in cystic fibrosis. Ann Am Thorac Soc, 2014. 11(3): p. 342-50. Sagel, S.D., et al., Airway inflammation in children with cystic fibrosis and healthy children assessed by sputum induction. Am J Respir Crit Care Med, 2001. 164(8 Pt 1): p. 1425-31. Turpeinen, T., et al., Mitogen-activated protein kinase phosphatase-1 negatively regulates the expression of interleukin-6, interleukin-8, and cyclooxygenase-2 in A549 human lung epithelial cells. J Pharmacol Exp Ther, 2010. 333(1): p. 310-8. Dauletbaev, N., et al., Down-regulation of Cytokine-induced Interleukin-8 Requires Inhibition of p38 Mitogen-activated Protein Kinase (MAPK) via MAPK Phosphatase 1-dependent and -independent Mechanisms. The Journal of Biological Chemistry, 2011. 286(18): p. 15998-16007. Cigana, C., et al., Anti-inflammatory effects of azithromycin in cystic fibrosis airway epithelial cells. Biochem Biophys Res Commun, 2006. 350(4): p. 977-82.

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Drug Delivery to the Lungs, Volume 29, 2018 - K. Elserfy et al. The Effect of Using a Turbulence Grid on Fluidization of Pharmaceutical Lactose Powder K. Elserfy1, S. Cheng1, H-K. Chan3, G. Hebbink2, M. Mehta2 & A. Kourmatzis4 School of Engineering, Macquarie University, NSW 2109 DFE Pharma, Klever Strasse 187, 47568 Goch, Germany 3 Advanced Drug Delivery Group, School of Pharmacy, The University of Sydney, NSW 2006 4 School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, NSW 2006 1

Summary An experimental platform is presented to investigate the effect of grid-generated turbulence and turbulence decay on the fluidization of pharmaceutical powder for oral inhalation drug delivery. The flow consists of a fully developed channel flow which delivers a well-defined velocity profile over a bed of powder. An in-house laser extinction method has been developed to extract quantitative information on the global powder fluidisation time. Four lactose powders are examined which have a range of mass mean diameters and cohesiveness. Reynolds numbers from approximately 3,383 to 9,000 are examined. The grid location position was changed from being at the powder pocket edge (x=0) to a distance equivalent to approximately 10 times the mesh size (M), representing one of a few quantitative studies to address the role of a regular turbulence grid on the fluidization of pharmaceutical powder in a simple powder pocket geometry. The use of turbulence grids enables evacuation of powder at lower Reynolds numbers and complete evacuation of the powder from the pocket with minimal residue. Key Message Using a turbulence generating grid upstream of the powder bed leads to a decrease in the evacuation time of the powder with an increase in fluidisation randomness. Placing the grid at the edge of the powder pocket generates a significant wake which minimizes evacuation time most effectively. Introduction The fluidization of pharmaceutical powder is affected by the physicochemical properties of the formulation (e.g. powder cohesiveness, density, and particle diameter), the design of the device’s dose metering systems, and physical mechanisms (e.g. air flow rate, flow turbulence, shear and/or impactions) [1]. A number of studies have focused on altering powder formulation in order to improve fluidization. Shur et al investigated the effect of different blending techniques and the specific role of fine excipient material on fluidization [2] and other researchers focused on the design of dry powder inhaler (DPI) devices to improve the fluidization. As an example, Voss et al [3] studied the effect of turbulence and mechanical impaction on particle deagglomeration using a novel rig with a key conclusion that turbulence is effective as a powder deagglomeration mechanism, but that mechanical impaction was not as effective. Tuley et al [4] examined evacuation in simple geometries and noted two evacuation mechanisms; erosion, and fracture. Behara et al [5] studied the kinetics of powder evacuation of micronized salbutamol sulphate (SS) and Lactohale® (LH300) from different commercial inhaler devices (Rotahaler®, Monodosehalers®, and Handihaler®) using an air flow rate of 30 – 180 Lmin-1. More rapid aerosolisation was observed using a Rotohaler® compared to the other devices and the emitted mass and rate of emitted mass increased with the increase of air flow rate using different devices. While a number of studies have been conducted and resulted in improvements to DPI design, the systematic analysis of what fundamental fluid mechanical factors affect particle deagglomeration using basic canonical models is surprisingly uncommon, given the importance and usefulness of these studies to help with inhaler optimization. In addition to experimental studies, computational fluid dynamics have also been employed in several studies to understand the influence of grids on particle fluidization. For example, Tong et al [6] used coupled computational fluid dynamics (CFD), and a discrete element method (DEM) technique to simulate particle laden flow and showed that a grid helps to increase the fine particle fraction. Coates et al [7] showed that grids when implemented in inhalers helped to decrease the amount of powder retained in the device and Wong et al [9] studied the effects of grid structure on particle breakup and showed that the agglomerate impaction against the grid appears to play an important role in breaking up the agglomerate. Studies that systematically demonstrate the effects of turbulent mixing on particle fluidisation and de-agglomeration are however less common. The key aim of this study is to examine how varying intensities of grid generated turbulence in a canonical flow influence the fluidisation process of lactose powders with different cohesiveness, and volume weighted particle sizes. Control of the distance between the powder pocket and turbulence grid allows for a direct investigation of the influence of turbulence decay on the fluidization of lactose powder. Practically, this also enables determination of the optimum location of placement of the turbulence grid so as to reduce evacuation time.

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Drug Delivery to the Lungs, Volume 29, 2018 - The Effect of Using a Turbulence Grid on Fluidization of Pharmaceutical Lactose Powder Experimental Methodology A schematic of the experiment is shown in Fig. 1. A controlled flow of air is delivered over a powder containing pocket located within a channel 250mm long (upstream of the pocket). A schematic of the optically accessible rig, with key dimensions, is also shown in Fig. 1. The flow rate is controlled using a valve, pressure regulator and rotameter, such that a constant flow-rate up to 100 SLPM is achieved after the solenoid valve actuates. Once the solenoid is fully open, it remains open until the powder evacuation process is complete, which is easily ascertained through observation of the raw data, as when the signal voltage remains constant. The solenoid valve was controlled using a DAQ NI9401 digital output counter with an update rate of 100ns coupled to a transistor (model No 67R1070).

View on Plane A

Figure 1 - Schematic of full Experimental Layout (top), powder channel assembly (bottom left), and planar view of optically accessible glass powder 10X10 channel showing schematic of pocket and laser location inside powder bed. D is the powder pocket depth (5mm), and W is the powder pocket width equal to the channel width (B) (W=B=10mm)

A four beam line of sight attenuation method is used to provide real-time measurements of light attenuation caused by the fluidisation of powders. Two continuous wave 633nm 5mW R-30900 Helium Neon lasers with a beam diameter of 0.8mm were used with each beam being split evenly using a 50-50 splitter. Each of the four beams were delivered to four grounded large area silicon photodiodes, with a bandwidth sensitivity from 350-1100nm. The photodiode arrangement enabled a frequency sensitivity of up to approximately 300 KHz. The four photodiodes were connected to a 4 Channel NI 9223 DAQ with a 1MHz maximum simultaneous sampling frequency. The method used here utilizes four beams through inclusion of two beam splitters enabling a real-time monitoring of light intensity of each beam prior to its incidence through the channel. This enables a continuous normalization of intensity fluctuations by accounting for changes in beam power with time. Raw data obtained using this method therefore consists of a voltage signal vs. time from four different photodiodes which is processed to obtain information on powder fluidisation behaviour and concentration.

-Powders and Powder Loading Four lactose powders were supplied by DFE Pharma including Lactohale® 200 and Lactohale® 206 (LH200 and LH206) and two from the Respitose® series (SV003 and SV010). Both LH200 and LH206 are milled lactose, however LH200 has a higher percentage of fines and higher cohesiveness as defined through its Carr’s index. SV003 is a fine sieved inhalation grade powder and SV010 is a coarse sieved inhalation grade powder. SV010 has a significantly higher mass mean diameter than SV003, however their cohesiveness is similar. The sieved grades also have a smaller particle size span (x90-x10)/(x50) compared to the milled grades. Powders were loaded into the pocket following a similar process as described elsewhere [4] which involved inserting a fixed amount of powder into the pocket size and swiping clear excess powder. At least three repetitions of each test was performed for all the cases, and the average was used. -Turbulence grid A regular turbulence grid with mesh size (M) of 1.92mm is used, the blockage ratio of the grid is approximately 32%. The grid is placed before the powder pocket at distance (X) as shown in the section view of the channel in Fig. 1 and is manufactured using 3d printing.

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Drug Delivery to the Lungs, Volume 29, 2018 - K. Elserfy et al. Results In this section, the effect of using turbulence grids on the fluidisation characteristics of powders is presented and analysed for flow rates (80, 60, 40, 30 Lmin-1), and compared to the case where no turbulence grid was present. The effect of moving the grid (changing X) from the powder pocket is also presented to investigate turbulence decay and hence enable a direct observation of the influence of turbulence intensity in the channel for fixed mean conditions. Such an approach has not been used before in pharmaceutical lactose fluidisation studies. The Grid is placed at a distance of (x=0) to (xâ&#x2030;&#x2026; 10M), where M is the mesh size. It is important to note that the mesh size M has been kept fixed in these studies. Future work will focus on variation in M to investigate for a fixed powder pocket size the influence of varying the integral scales in the flow. The evacuation time for one of the powders (LH206) used in the study is illustrated in figure (2A) for different flow rates and different grid positions. It is noticed that the evacuation time decreased by nearly 96% when using the grid at the powder pocket edge. At 80 Lmin-1, evacuation time is nearly equal for both cases where x=0, and 0.8 M, but increasing the grid position (X) further leads to an increasing trend of evacuation time with the grid position (X). A similar trend is noticed for LH206 at a flow rate of 60 Lmin -1, except for x=0, and 0.8M. Decreasing the flow rate leads to an increase in the evacuation time due the decrease in the mean velocity and shear force acting on the powder bed. From figure (2B) it can be seen that at 80 Lmin -1, the evacuation time for different powders increases with an increase in the grid position (X). Sv010 has the fastest evacuation time in most of the grid position cases where Sv010 also has the highest tapped density, and the largest particle size compared to the other powders. LH200 is a very cohesive powder (high Carrâ&#x20AC;&#x2122;s index) which at this mean velocity, did not demonstrate significant evacuation without the turbulence grid placed at x=0.

Figure 2- the effect of using turbulence grid on evacuation time (A) for LH206 powder with and without grid for different flow rate and effect of different grid positioning (B) for different powder and different grid position at 80Lmin-1, and for fixed M.

Figure 3- the effect of using turbulence grid on standard deviation of signal fluctuation (A) for LH206 powder with and without grid for different flow rate and different grid positioning, (B) for different powder and different grid position at 80Lmin-1, and for fixed M.

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Drug Delivery to the Lungs, Volume 29, 2018 - The Effect of Using a Turbulence Grid on Fluidization of Pharmaceutical Lactose Powder Analysis of fluctuation of the laser extinction signal provides a direct measurement of how variable with time the evacuation process is, which may have important implications on drug delivery. This fluctuation measure is calculated by taking the difference between the raw output voltage signal and its best polynomial fit, the latter of which is taken to represent the equivalent “mean” of the output signal. The standard deviation of this difference is then calculated and utilized as one measure of fluctuation exhibited during the powder fluidisation process. In figure (3A) it can be seen that using the turbulence grid for the LH206 powder leads to an increase in the standard deviation of signal fluctuation at 80L/min compared to the case without the grid when the grid is placed at grid edge (X=0) till (X=1.6M). Increasing the grid position further leads to a decrease in standard deviation of signal fluctuation due to the decay of turbulent eddies with increase grid position. Decreasing the flow rate also leads to a decrease in the standard deviation as shown in the figure however the results indicate that addition of a turbulence grid can be utilized to maximize dispersion behaviour at lower mean velocities. From figure (3B), it can be seen that all the tested powder flows show a decreasing trend of standard deviation of signal fluctuation with increasing the grid position (X) for most of the cases. Conclusion This experiment is one of the first studies to investigate the effect of using a variable degree of grid generated turbulence on the fluidisation characteristics of pharmaceutical lactose in a well-defined geometry. This constitutes an important study for understanding how turbulent mixing (for fixed mean flow-rates) can be incorporated in dry powder inhaler devices to alter powder evacuation characteristics and can also form a platform for DEM model validation. Use of turbulence grids leads to favourable effects like a decrease in the evacuation time and improved downstream mixing. Further work is merited in order to fully quantify the influence of these grids on downstream dispersion, particle velocities, trajectories and deposition behaviour. Reference [1] Islam, N. and Cleary, M. J. Developing an efficient and reliable dry powder inhaler for pulmonary drug delivery– a review for multidisciplinary researchers. Medical engineering and physics, 34, 4 (2012), 409-427. [2] Shur, J., Harris, H., Jones, M. D., Kaerger, J. S. and Price, R. The role of fines in the modification of the fluidization and dispersion mechanism within dry powder inhaler formulations. Pharmaceutical Research, 25, 7 (2008), 1631-1640. [3] Voss, A. and Finlay, W. H. Deagglomeration of dry powder pharmaceutical aerosols. International journal of pharmaceutics, 248, 1-2 (2002), 39-50. [4] Tuley, R., Shrimpton, J., Jones, M. D., Price, R., Palmer, M. and Prime, D. Experimental observations of dry powder inhaler dose fluidisation. International journal of pharmaceutics, 358, 1-2 (2008), 238-247. [5] Behara, S. R. B., Kippax, P., Larson, I., Morton, D. A. and Stewart, P. Kinetics of emitted mass—a study with three dry powder inhaler devices. Chemical engineering science, 66, 21 (2011), 5284-5292. [6] Tong, Z., Zheng, B., Yang, R., Yu, A. and Chan, H. CFD-DEM investigation of the dispersion mechanisms in commercial dry powder inhalers. Powder technology, 240 (2013), 19-24. [7] Coates, M. S., Fletcher, D. F., Chan, H. K. and Raper, J. A. Effect of design on the performance of a dry powder inhaler using computational fluid dynamics. Part 1: Grid structure and mouthpiece length. Journal of pharmaceutical sciences, 93, 11 (2004), 2863-2876. [8] Coates, M. S., Chan, H.-K., Fletcher, D. F. and Raper, J. A. Effect of design on the performance of a dry powder inhaler using computational fluid dynamics. Part 2: air inlet size. Journal of pharmaceutical sciences, 95, 6 (2006), 1382-1392. [9] Wong, W., Fletcher, D. F., Traini, D., Chan, H. K., Crapper, J. and Young, P. M. Particle aerosolisation and break‐up in dry powder inhalers: Evaluation and modelling of the influence of grid structures for agglomerated systems. Journal of pharmaceutical sciences, 100, 11 (2011), 4710-4721.

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Drug Delivery to the Lungs, Volume 29, 2018 –Emelie Land et al. Treating Lymphangioleiomyomatosis with Inhaled Rapamycin Solid Lipid Nanoparticles Emelie Land1, Lyn M. Moir1, Daniela Traini, Paul M. Young1 & Hui Xin Ong1 1Respiratory

Technology, Woolcock Institute of Medical Research and Faculty of Medicine and Health The University of Sydney NSW, 2037, Australia

Summary Lymphangioleiomyomatosis (LAM) is a rare lung disease characterised by uncontrolled growth of smooth muscle cells in the lungs that can spread via the lymphatic system to other parts of the body. The current treatment of LAM is oral Rapamycin that is limited by its low oral bioavailability and side effects. Therefore, high-doses of rapamycin are required to achieve therapeutic doses to treat LAM in the lung and prevent its spread through the lymphatic system. The aim of this study is to develop an inhaled formulation of rapamycin solid lipid nanoparticles (SLNs) that will help the drug to by pass first -pass metabolism, increase its in-vivo half-life and facilitates entry into the lymphatic system through the lungs, thereby overcoming the problems associated with current oral treatment. Rapamycin and lipids for SLN were manufactured using a hot evaporation technique, before being freeze-dried overnight with 5% (w/v) mannitol. Particle characteristics were assessed using a Zetasizer and transmission electron microscope. The formulation’s in vitro aerosol performance was also evaluated using the next generation impactor (NGI) and in vitro release profile analysed via a dialysis bag experiment. The SLNs had an average particle size of 237.5±1.8nm (PDI=0.341), negative charge (-11.16) and high encapsulation efficiency (97.32 ±1.28%). The Fine Particle Fraction (FPF) and Mass Median Aerodynamic diameter (MMAD) was 44.127±5.1% and 5.317±0.38μm, respectively. This study successfully produced an inhaled formulation of rapamycin SLNs with the appropriate size, charge, in-vitro release performance and sustained release profile, making it suitable for pulmonary delivery and entry into the lymphatic system. This formulation is therefore a promising alternative treatment for LAM patients, as it could potentially reduce problems associated with low bioavailability and unpleasant side effects of current oral treatment. Key Message The current study successfully produced an inhaled formulation of rapamycin SLNs that is suitable for pulmonary delivery and further entry into the lymphatic system. This formulation is therefore a promising alternative treatment to LAM patients, as it will potentially reduce the problems associated with low bioavailability and side effects of current oral treatment. Introduction Lymphangioleiomyomatosis (LAM) is a rare lung disease affecting mainly women during their reproductive age. It is caused by a mutational inactivation of the tumour suppressor genes, tuberous sclerosis complex 1 (TSC1) and 2, resulting in constitutively activation of the mTOR pathway causing increased cell proliferation, migration and autophagy as well as the formation of new lymphatic vessels [1]. Histologically, the disease is characterised by neoplastic cysts in the lungs, which are formed by uncontrolled growth of smooth muscle cells. These cysts can further spread via the lymphatic system to other parts of the body affecting other organs such as the kidneys, liver and lymph nodes [2]. Although LAM is considered to be a multi organ disease with the primary causes of mortality and morbidity are airway obstruction, decreased lung function and collapsed lungs. The current oral treatment of rapamycin available for LAM disease has a low bioavailability of 15%, thus patients need to take large doses to reach the therapeutic effect, which in turn results in many unpleasant side effects such as lymphedema, hypercholesterolemia and latent malignancy [3, 4]. Formulating drugs with lipids carriers delivered via the lungs have been shown to be a successful way of targeting drugs to the lymphatic system [5]. In order for lipids particles to enter the lymphatic system they need to have an average particle size of ~200 nm and a negative surface charge [6, 7]. Therefore, the aim of this study is to develop an inhaled formulation of rapamycin encapsulated by a carrier of solid lipid nanoparticles (SLNs) that will act to increases the drug’s solubility and in-vivo half-life,[7] thereby help to overcome the low bioavailability issues associated with oral treatments. An inhaled SLN formulation will have the advantage of allowing the delivery of large doses directly to the pulmonary site, increasing the drug’s residency time in the lungs and facilitating drug uptake into the lymphatics, that will ultimately target other organs affected by the disease.

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Drug Delivery to the Lungs, Volume 29, 2018 - Treating Lymphangioleiomyomatosis with Inhaled Rapamycin Solid Lipid Nanoparticless Experimental methods The Rapamycin SLNs were produced by dissolving rapamycin and gliceryl behenate (Compritol 888) in methanol and dichloromethane, respectively. Subsequently, the organic solvents were evaporated using a rotary evaporator (RV8, IKA Rotary Evaporator). The resulting dry lipid-drug film was added to a hot aqueous surfactant (Tween 80, 1.5% w/v) solution and homogenised (T25, Ultraturrax) at 70 ºC for 15 minutes. The cooled suspension was mixed with 5% (w/v) mannitol solution, centrifuged and finally snap freezed in liquid nitrogen before freeze-drying (B. Braun, Melsungen, Germany) overnight. The encapsulation efficiency of the formulation was determined by measuring the initial amount of unfiltered rapamycin solution and untrapped (free) drug in aqueous media following centrifugation with Amicon Ultra centrifugal filters (Millipore) at 4000 rpm for 15 min. The encapsulation efficacy of the formulation was then estimated using the formula: Encapsulation efficiency (%) = Unfiltered rapamycin- Filtered Solution (free drug) x100 Unfiltered rapamycin The particle size, polydispersity index (PDI) and surface charge (zeta potential) of the SLNs (25 mg/mL) were assessed using the zetasizer (Nano SZ, Malvern Instruments) and morphology evaluated (0.1 mg/mL) using Transmission Electron Microscopy (TEM). The in vitro aerosol performance of the nebulised formulation (10mg/mL) was evaluated using the next generation impactor (NGI, Copley Scientific, Nottingham UK). The Pari Jet nebuliser reservoir was filled with 2 mL (10 mg/mL) of formulation and the aerosol delivery was measured for 3 min at a flowrate of 15 L/min. The drug release profile of Rapamycin from the SLNs matrix was evaluated using the dialysis bag (MW cut-off 6000-8000, CelluSep, Texas, USA) membrane technique, whereby the rapamycin-SLNs suspension and free rapamycin solution was enclosed in the dialysis bag and immersed in 60 mL of phosphate buffer solution (PBS) to ensure sink conditions. Samples were withdrawn from the sink medium every 60 min and analysed using a validated High Performance Liquid Chromatography (HPLC). Results Rapamycin SLNs were found to have an average size of 237.5 ± 1.8 nm (PDI + 0.41) and a negative surface charge (potential) of -11.16 ± 0.59. The particle size and morphology were further confirmed via TEM (Figure 1), which showed uniform spherical, smooth particles of approximately 200 nm. Further, the encapsulation study showed that most of the rapamycin was contained within the carrier lipids resulting in an encapsulation efficacy of 97.32 ± 1.28%. Figure 1 - TEM image of Rapamycin SLNs at a concentration of 0.1 mg/mL.

The nebulised formulation showed good in vitro aerosol performance (Table 1) with a Fine Particle Fraction (FPF), defined as aerosols with a diameter smaller than 5 μm [8], of 44.13 ± 5.15% and the aerosols having an average Mass Median Aerodynamic Diameter (MMAD) of 5.32 ± 0.38 μm, making the formulation suitable for pulmonary delivery. Table 1 - Summary of Rapamycin SLNs characteristics and In vitro aerosol performance results, (n=3, mean ± StDev) Particle characterization

In vitro aerosol Performance

Size (nm)

Surface Charge (Potential)

Polydispersity Index

Encapsulation Efficacy (%)

MMAD (μm)

FPF (%)

237.5 ± 1.8

-11.16 ± 0.59

0.41 ±

97.32 ± 1.28

5.32 ± 0.38

44.13 ± 5.1

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Drug Delivery to the Lungs, Volume 29, 2018 –Emelie Land et al. Figure 2 shows the drug release profile of the rapamycin SLNs in comparison to free rapamycin. During the 4h experiment, the free rapamycin demonstrated rapid drug diffusion from the dialysis bag, with >65% of the drug released, while the rapamycin SLNs showed a more sustained release, with approximately 50% of the Rapamycin being released from the SLNs.

Figure 2. In vitro drug release profiles of Rapamycin SLNs and free Rapamycin solution across a dialysis membrane (n=3, mean ± SD). Discussion and Conclusions The nebulised rapamycin SLNs formulation has shown to be suitable for pulmonary delivery, as the aerosols produced had MMAD and FPF values [8] required for deposition in the lower lungs. This will allow the administration of rapamycin directly to the lungs where the majority of LAM patient’s cysts are located, and reducing problems associated with low bioavailability when administered orally. Consequently, allowing patients to take smaller doses to reach the same therapeutic effect, as with the current oral treatments, and reducing the side effects [3]. Furthermore, the rapamycin SLNs produced had the desired average size of ~200 nm and anionic surface charge, which have shown by previous work [5, 7] to be important characteristics necessary for particles to enter into the lymphatic system. This will then allow the rapamycin to target LAM cells that have grown in the lymphatic vessels, as well as those that have spread from the lungs via the lymphatic system to other parts of the body [2]. Moreover, an earlier study [7] showed that using a solid lipid matrix to deliver radiolabelled nanoparticles via a nebuliser to the lungs of rats, resulted in high lymphatic uptake and lymph node distribution up to four hours after delivery. They also found a high degree of particle retention in the lungs of animals sacrificed after four hours, suggesting that use of lipid carriers improves the delivery profile of nanoparticles to the lungs [9] as well as facilitates entry into the lymphatic system [7]. The current study showed similar results, with the in-vitro drug release of the rapamycin SLNs showing a more sustained release of rapamycin compared to free drug over the time course of the experiment, with a significant difference (p<0.05) in the release rate of rapamycin SLN compared to free rapamycin after four hours (Fig 2). Together with the evidence of the formulation’s high encapsulation efficacy, with 97.32 ± 1.28% of the drug being encapsulated within the lipids (table 1), this data demonstrates that delivering the hydrophophic drug rapamycin, via a controlled nanostructured matrix with the lipid (gliceryl behenate) allows sustained drug delivery of the rapamycin. This in turn increases its retention time in the lungs and thereby increases its in vivo half-life and consequently helps to reduce the systemic side effects of the drug. Moreover, previous studies have showed that gliceryl behenate is a suitable lipid to use as a carrier for pulmonary drug delivery.[10,11] Mezzena et al., (2009) showed that formulation of budesonide solid lipid microparticles using gliceryl behenate as the carrier resulted in a formulation with good in-vitro aerosol performance, particles of suitable size range and controlled release profiles similar to the conventional spray-dried Budesonide[10]. In another study, Sanna et al., (2003) showed that gliceryl behenate microparticles delivered to the lungs of rats did not cause any changes to their alveolar macrophage, lymphocyte and neutrophile counts and did not cause any significant inflammatory response[11], thus indicating that gliceryl behenate has no toxic effect on the pulmonary system. In summary, an inhaled formulation of rapamycin SLNs was manufactured with good in vitro aerosol performance suitable for pulmonary delivery, with a controlled release profile and particles of the correct average size and negative surface charge necessary for lymphatic uptake through the lung. With this promising preliminary data, it has been shown that SLNs of rapamycin could potentially be used as a promising alternative to the current oral treatment available for LAM disease and further studies are currently on-going to demonstrate efficacy.

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Drug Delivery to the Lungs, Volume 29, 2018 - Treating Lymphangioleiomyomatosis with Inhaled Rapamycin Solid Lipid Nanoparticless References 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11.

Atochina-Vasserman EN, Goncharov DA, Volgina AV, Milavec M, James ML, and Krymskaya VP. Statins in lymphangioleiomyomatosis. Simvastatin and atorvastatin induce differential effects on tuberous sclerosis complex 2-null cell growth and signaling. Am J Respir Cell Mol Biol. 2013;49:704-709. Kumasaka T, Seyama K, Mitani K, Souma S, Kashiwagi S, Hebisawa A, Sato T, Kubo H, Gomi K, Shibuya K, Fukuchi Y, and Suda K. Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis. Am J Surg Pathol. 2005;29:1356-1366. Johnson SR, Cordier JF, Lazor R, Cottin V, Costabel U, Harari S, Reynaud-Gaubert M, Boehler A, Brauner M, Popper H, Bonetti F, Kingswood C, and Review Panel of the ERSLAMTF. European Respiratory Society guidelines for the diagnosis and management of lymphangioleiomyomatosis. The European respiratory journal. 2010;35:14-26. Desai N, Heenan S, and Mortimer PS. Sirolimus-associated lymphoedema: eight new cases and a proposed mechanism. Br J Dermatol. 2009;160:1322-1326. Singh I, Swami R, Khan W, and Sistla R. Lymphatic system: a prospective area for advanced targeting of particulate drug carriers. Expert opinion on drug delivery. 2014;11:211-229. Jain RK. Delivery of molecular and cellular medicine to solid tumors. Advanced drug delivery reviews. 2012;64:353-365. Videira MA, Botelho MF, Santos AC, Gouveia LF, de Lima JJ, and Almeida AJ. Lymphatic uptake of pulmonary delivered radiolabelled solid lipid nanoparticles. J Drug Target. 2002;10:607-613. Dolovich MB, MacIntyre NR, Anderson PJ, Camargo CA, Chew N, Cole CH, Dhand R, Fink JB, Gross NJ, Hess DR, Hickey AJ, Kim CS, Martonen TB, Pierson DJ, Rubin BK, and Smaldone GC. Consensus statement: Aerosols and delivery devices. J Aerosol Med. 2000;13:291-300. Barker S.A. TKMGaS, M.D. The deposition and clearance of liposome entrapped 99mTc-DTPA in the human respiratory tract. International journal of pharmaceutics. 1994;102:159-165. Mezzena M, Scalia S, Young PM, and Traini D. Solid lipid budesonide microparticles for controlled release inhalation therapy. The AAPS journal. 2009;11:771-778. Sanna V, Kirschvink N, Gustin P, Gavini E, Roland I, Delattre L, and Evrard B. Preparation and in vivo toxicity study of solid lipid microparticles as carrier for pulmonary administration. AAPS PharmSciTech. 2004;5:e27.

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Drug Delivery to the Lungs, Volume 29, 2018 - Jason A Suggett et al. The Impact of Device Handling Errors upon Inhaled Medication Delivery from Pressurized Metered Dose Inhalers (pMDIs) Used with and without a Valved Holding Chamber (VHC) Jason A Suggett1, Mark W. Nagel1 & Jolyon Mitchell2 Trudell Medical International, 725 Third Street, London, Ontario, NV5 5G4, Canada Mitchell Inhaler Consulting Services Inc., 1154 St. Anthony Road, London, N6H 2R1, Canada 1

2Jolyon

Summary Evaluations of inhaler use have demonstrated that patient mishandling of pMDIs is commonplace. This study investigated the potential impact on medication delivery associated with three errors: (a) delayed inhalation, (b) over-forceful inhalation, and (c) exhalation instead of inhalation at the time of pMDI actuation. The large adult Aerosol Delivery to an Anatomic Model (ADAM) model oropharyngeal airway was used to determine the penetration of pMDI-delivered salbutamol (Ventolin-HFA, 100-g salbutamol base equivalent/actuation) to a distal filter representing potential lung delivery. AeroChamber Plus Flow-Vu antistatic devices with mouthpiece (n = 3 devices, 1 measurement per device) were used as the test VHC. Delaying inhalation by 1-s resulted in a decrease of 80% in the mass penetrating as far as the filter compared to optimal no delay for the pMDI alone. When the VHC was present, the mass of salbutamol reaching the filter after a 2-s delay was comparable with optimum delivery of medication from the pMDI alone and further delays up to even 15-s resulted in less than 40% reduction. When a forceful inhalation was simulated, filter-collection of salbutamol for the pMDI alone and pMDI with VHC were both reduced in the order of 20-30% compared to the slow inhalation condition with the same configuration. No medication was collected on the filter when exhalation took place upon pMDI actuation when used alone. Adding the VHC resulted in filter collection comparable to the mass delivered under normal inhalation with a 2-s delay.

Key Message This study provides insight into the change in inhaled medication delivery associated with (a) delayed inhalation, (b) over-forceful inhalation, and (c) exhalation instead of inhalation upon pMDI actuation. The data illustrate the relative impact of such device handling deviations and how a VHC might mitigate such changes in certain instances.

Introduction pMDIs are ideally prescribed by clinicians to be used with a VHC for patients with obstructive lung diseases, such as asthma and chronic obstructive pulmonary disease [1]. However, there is strong evidence from the evaluation of inhaler use, particularly in Europe by the Aerosol Drug Management Improvement Team (ADMIT) of specialist respiratory clinicians [2] and the International Primary Care Respiratory Group (IPCRG) [3], that mishandling of pMDIs by patients is commonplace, even when a VHC is prescribed. The present study was designed to investigate, using a clinically relevant laboratory test, the potential changes in medication delivery associated with three commonplace patient errors [4}: (a) delayed inhalation; (b) over-forceful inhalation; (c) exhalation instead of inhalation at the time of pMDI actuation. Each simulated type of device handling error was assessed with and without the presence of a VHC with the aim of understanding the relative impact of such errors generally and the comparison of effect with and without VHC.

Materials and Methods The large adult model oropharyngeal airway of the Aerosol Delivery to an Anatomic Model (ADAM) series [5] (Figure 1) was used in order to provide a more clinically relevant laboratory determination of the total mass per actuation of a representative pMDI short-acting bronchodilator (Ventolin-HFA, GSK (Canada) Inc., 100 g salbutamol base equivalent/actuation ex metering valve). AeroChamber Plus Flow-Vu antistatic devices with mouthpiece ((AC+ AS Flow Vu); n = 3 devices, 1 measurement per device; Trudell Medical International, London, Canada) were used as the test VHC group.

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Drug Delivery to the Lungs, Volume 29, 2018 - The Impact of Device Handling Errors upon Inhaled Medication Delivery from Pressurized Metered Dose Inhalers (pMDIs) Used with and without a Valved Holding Chamber (VHC) The following device handling scenarios were evaluated, each representing realistic ‘in-use’ situations: a)

Impact of delayed inhalation:

Normal inhalation at a constant flow rate of 30 L/min

ii.

Actuation of pMDI alone with no delay (perfect coordination), 1s and 2s delays

iii.

Actuation of pMDI with VHC with 2s (normal), 5s and 15s delays

Impact of forceful inhalation: i.

Actuation of pMDI with no delay and pMDI/VHC with 2s delay at 80 L/min inhalation flow rate

Impact of exhalation on actuation, prior to inhalation: i.

Adult tidal breathing (tidal volume Vt = 500 ml; inspiratory/expiratory (I:E) ratio = 1:2; 13 respiration cycles/min

ii.

Actuation of pMDI alone at onset of exhalation

iii.

Actuation of pMDI / VHC at onset of exhalation

Following each measurement, the deposited particulates were recovered separately from the mouthpiece of the pMDI, the interior surfaces of the VHC-on-test (when present), the interior surfaces of the model oropharyngeal airway and on the filter (representative of medication available to lungs). Assay for salbutamol was undertaken by a validated HPLC-UV spectrophotometric procedure.

Figure 1 -

Test Arrangement Showing ADAM Adult Oropharyngeal Inlet Between pMDI with VHC and Filter for Collection of Aerosol Particles Capable of Penetrating to the Carinal Region and Therefore Available for Lung Deposition: The VHC was Removed for the Tests with the pMDI Alone

Results Table 1 is a summary of the distribution of recovered medication from the various components of the aerosol sampling system for each of the scenarios described above. In general, the total mass recovered from each measurement was within 15% of the label claim value. The slightly lower values for the pMDI alone obtained in the tidal breathing simulation with exhalation instead of inhalation at inhaler actuation, are likely associated with escape of aerosol to the environment during the period when the small internal volume of the model and pMDI mouthpiece was pressurized slightly by the ingress of air from the breathing simulator for the exhalation manoeuvre. The effect was not evident when the VHC was present, likely due to the presence of a larger internal volume introduced by the chamber itself.

302

Drug Delivery to the Lungs, Volume 29, 2018 - Jason A Suggett et al. Impact of inhalation delay: As expected, less salbutamol deposited in the oropharyngeal airway when the VHC was present, as the presence of the chamber increased the distance for the ballistic fraction of the emitted mass to expand before entering the model oropharynx (Figure 2A). For the pMDI alone, it is evident that virtually no delay to inhalation from pMDI actuation is possible without appreciably decreasing delivery of salbutamol to the filter and therefore potentially available for lung deposition (Figure 2B). Delaying inhalation by as little as 1-s, resulted in a decrease of about 80% in the mass penetrating as far as the filter to 5.7 ± 0.8 g/actuation, and a further 1-s delay reduced the filter-deposited mass still further to 4.3 ± 1.2 g/actuation (Figure 2B). In contrast, when the VHC was present with a 2-s delay following inhaler actuation and the onset of inhalation, the mass of salbutamol reaching the filter (29.1 ± 2.5 g/actuation) was comparable with optimum delivery of medication from the pMDI alone. Importantly, increasing the delay to 5-s decreased the filter-collected mass of salbutamol by only 24% to 22.0 ± 1.4 g/actuation, and extending the delay to as much as 15-s, representing an extreme instance of imperfect coordination, still resulted in the collection of a significant fraction of the optimum delivered mass, at 17.9 ±1.2 g/actuation. Table 1:

Distribution of Salbutamol Sulphate in Model of Adult Patient Using a pMDI with or without a VHC, Simulating Normal Inhalation at 30 L/min with Different Inhalation Delays, Over-Forceful Inhalation at 80 L/min and Tidal Breathing but Exhaling Instead of Inhalation at pMDI Actuation

Handling Scenario

Test Conditions

Model Oropharyngeal Airway

Filter*

pMDI mouthpiece

VHC Interior

Total Mass

No delay

55.1 ± 3.7

29.0 ± 4.8

13.9 ± 2.8

N/A

98.0 ± 5.8

1 sec delay

64.3 ± 5.2

5.7 ± 0.8

27.7 ± 2.7

N/A

97.7 ± 3.3

2 sec delay

65.7 ± 6.4

4.3 ± 1.2

31.0 ± 4.2

N/A

101.0 ± 4.3

2 sec delay pMDI with AC+ AS Flow Vu 5 sec delay VHC MP

16.2 ± 2.5

29.1 ± 2.5

29.5 ± 4.0

38.6 ± 7.0

113.3 ± 9.0

13.2 ± 0.2

22.0 ± 1.4

28.4 ± 1.3

52.3 ± 1.1

115.9 ± 0.9

15 sec delay

11.0 ± 0.7

17.9 ±1.2

29.9 ± 2.3

41.2 ± 6.8

100.0 ± 6.9

No delay

60.2 ± 4.2

23.1 ± 0.4

14.7±1.9

N/A

97.9 ± 3.2

pMDI with AC+ AS Flow Vu 2 sec delay VHC MP

18.2 ± 2.0

20.7 ± 0.2

27.1 ± 2.6

33. 1± 3.6

99.1 ± 7.0

Actuate on Exhalation

54.7 ± 5.3

0.0 ± 0.0

30.3 ± 4.2

N/A

85.0 ± 6.7

pMDI with AC+ Actuate on AS Flow Vu Exhalation VHC MP

2.1 ± 0.6

33.5 ± 4.1

27.6 ± 2.2

38.6 ± 3.0

101.8 ± 7.4

pMDI alone Impact of delayed inhalation

Impact of forceful inhalation

Impact of exhalation upon actuation

Mass Salbutamol Sulphate Recovered (g; mean  SD)

pMDI alone

* filter deposition represents mass of medication penetrating as far as the carina and therefore potentially available for lung deposition

Figure 2: Effect of Inhalation Delay; (A) Mass per Actuation Retained in Model Oropharynx; (B) Mass per Actuation Recovered from the Filter Distal to the Model, Representing Carinal Deposition

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Drug Delivery to the Lungs, Volume 29, 2018 - The Impact of Device Handling Errors upon Inhaled Medication Delivery from Pressurized Metered Dose Inhalers (pMDIs) Used with and without a Valved Holding Chamber (VHC) Impact of forceful inhalation: With the pMDI alone and optimal coordination, the mass of salbutamol collected on the filter (23.1 ± 0.4 g/actuation) was reduced by approximately 20% compared with the more ideal slow inhalation condition with the same configuration. A similar decrease in filter-collected mass from 29.1 ± 2.5 g/actuation (slow inhalation) to 20.7 ± 0.2 g/actuation was observed when the VHC was present and a ‘normal’ 2-s delayed inhalation was simulated. Interestingly, the amount of drug deposited in the oropharyngeal airway was not increased greatly for either the pMDI alone or pMDI with VHC, albeit the absolute amount still being much higher for the pMDI alone. Although in general terms there was not a large reduction in the amount of salbutamol reaching the filter under these circumstances, the results still support the recommendation 6, 7 that patients should inhale slowly from their inhaler, rather than attempt a more forceful inhalation. Impact of exhalation on actuation: The role of the VHC at retaining the aerosolized medication until the patient inhales is amply illustrated by the outcomes from the third part of the study, involving the simulation of inhaler actuation upon exhalation by a tidal-breathing adult. No medication was collected on the filter when the pMDI was used alone, with the aerosol depositing in the model oropharynx and the mouthpiece of the pMDI. In contrast, following an initial exhalation when the VHC was present, the mass of salbutamol recovered from the filter was 33.5 ± 4.1 g/actuation, comparable to the mass delivered in the first part of the study under constant flow inhalation and a 2-s delay (29.0 ± 4.8 g/actuation). The incremental impact of all three errors is illustrated in Figures 3A and 3B for the pMDI alone and the pMDI with VHC present respectively, and the overall decreases in medication potentially available for lung deposition are indicated.

Figure 3: Incremental overview of all three errors upon filter delivery for (A) MDI alone, and (B) MDI with VHC Present Conclusions The reported laboratory study explored the likely consequences of three commonly encountered patient errors associated with the use of pMDI-delivered medications. The findings highlight two errors that will likely greatly reduce the amount of the drug available to the lungs when using the pMDI alone, namely a delay in inhalation and exhaling on actuation. The use of the test VHC appreciably reduces the impact of inhalation delay and removes completely the impact of exhaling on actuation. The third patient error investigated, higher (more forceful) inhalation flow rates, appears to have a lesser impact upon the amount of drug available to the lungs, although there was still some decrease (for both pMDI alone and pMDI with VHC), confirming the validity of the advice to inhale slowly rather than more forcefully in order to achieve optimal medication delivery to the lungs. Although it is possible to achieve effective medication delivery to the lungs with a pMDI alone, provided that the onset of inhalation coincides with inhaler actuation, the findings confirm that without a VHC, the impact of potential patient handling errors will likely be much more severe and clinically relevant. References Global Initiative for Asthma (GINA). Global strategy for asthma management and prevention. 2018. Available from: www.ginasthma.org. 1

Lavorini F, Levy ML, Corrigan C, Crompton G. on behalf of the ADMIT Working Group: The ADMIT series – issues in inhalation therapy: 6) Training tools for inhalation devices. Prim. Care Respir. J., 2010; 19(4): pp 335-341. 2

Papi A, Haughney J, Virchow JC, Roche N, Palkonen S, Price D. Inhaler devices for asthma: A call for action in a neglected field. Eur. Respir. J., 2011; 37(5): pp.982-985. 3

Melani AS, Bonavia M, Cilenti V, Cinti C, Lodi M, Martucci P, Serra P, Scichilone N, Sestini P, Aliani M, Neri M. Inhaler mishandling remains common in real life and is associated with reduced disease control. Respir Med. 2011; 105: pp. 930-938. 4

Nagel MW, Suggett JA, Coppolo DP, Mitchell JP. Development and evaluation of a family of human face and upper airway models for the laboratory testing of orally inhaled products. Aerosol Sci Technol. 2017: 18(8): pp. 3182-3197. 5

Haughney J, Price D, Barnes NC, Virchow JC, Roche N, Chrystyn H. Choosing inhaler devices for people with asthma: Current knowledge and outstanding research need. Respir Med. 2010; 104: pp. 1237-1245. 6

Hesselink AE, Penninx BWJH, Wijnhoven HAH, Kriegsman DMW, van Eijk JTM. Determinants of an incorrect inhalation technique in patients with asthma or COPD. Scand J Prim Health Care. 2001; 19(4): pp. 255-260. 7

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Drug Delivery to the Lungs, Volume 29, 2018 – Francesca Buttini et al. Glycemic Profile in Rats After Pulmonary Administration of a Pure Insulin Powder Francesca Buttini,1 Veronica Chierici1, Eride Quarta1, Lisa Flammini1, Adryana Rocha Clementino1, Susana Ecenarro2, Massimiliano Tognolini1, Elisabetta Barocelli1, Paolo Colombo1 & Ruggero Bettini1 1Food

and Drug Department, University of Parma, Parco Area delle Scienze 27a, 43124, Parma, Italy 2

Qualicaps Europe, S.A.U., Alcobendas, Madrid, Spain

Summary The purpose of this study was to investigate the chemical stability and respirability of a pure insulin spray-dried (Ins_SD) powder. Moreover, the glycemic plasma profile of rats was measured after pulmonary insufflation of Ins_SD and Afrezza® at a dose of 10 IU/Kg. The results of this study show that a pure insulin pulmonary powder (Ins_SD), produced by spray drying from an acid aqueous solution of the peptide, presented a high respirable fraction (FPF=91.5%) and favourable stability behaviour. The powder, filled into HPMC Quali-V®-I capsules together with the PVC-PVDC packaging material, showed long term stability at room temperature whilst maintaining good aerodynamic performance. The in vivo study was conducted in rats and the glycemic plasma profile was determined after pulmonary insufflation of Ins_SD and Afrezza® powder. These profiles resulted to be similar to each other when the powder was administered and both of them were similar to the glycemic plasma profile registered after the subcutaneous insulin solution administration. Key Message A highly respirable insulin spray dried powder was demonstrated to be stable at room temperature for 6 months, this outcome opens the possibility of a diabetic therapy that is less dependent on the cold storage of drug product. The glycemic plasma profile after pulmonary insufflation was equal to the one obtained by Afrezza ®. Introduction Approximately 6 million people in the U.S. require insulin therapy. Insulin therapy allows for better glycemic control, but patients are often hesitant to make the transition to insulin because of its adverse-event profile (e.g., hypoglycemia, weight gain) and because of fear of injections. Afrezza® (insulin human) inhalation powder (MannKind Corp./Sanofi-Aventis US) was approved by the FDA in July 2014 for patients with type-1 and type-2 diabetes mellitus. Afrezza® employs the Gen2 inhaler and it is available in different IU strengths that are modulated loading augmented amount of powder in the device. This product contains 18% of insulin with fumaryl diketopiperazine used to manufacture respirable microparticles according to proprietary Technosphere® technology. Afrezza® has to be stored at 2-8°C and when the blister foil package is opened it must be used within 10 days.[1] A pure insulin pulmonary powder produced by spray drying was developed and patented by the Department of Pharmacy of the University of Parma.[2] The aim of this study was to investigate the chemical stability and respirability of this insulin spray-dried powder loaded into capsules packed in blister. High molecular weight covalent degradation products were searched at 0, 30, 150 and 180 days after powder production. The in vitro respirability of Ins_SD was compared to the one of the commercial Afrezza® products. Finally, the glycemic plasma profile of rats was measured after pulmonary insufflation of Ins_SD and Afrezza® at a dose of 10 IU/Kg. Experimental methods A human recombinant insulin powder for inhalation (Ins_SD) was prepared by spray drying using a mini Spray Dryer Büchi B-290 (Büchi, CH), as previously described.[3] The powder was characterized by SEM analysis (SUPRA 40, Zeiss, DE) and its particle size distribution measured (Spraytec, Malvern Instruments Ltd, UK). HPMC capsules size 3 (Quali-V®-I, Qualicaps Europe, ES) were semi-automatically filled with 2 mg of INS_SD powder using a Omnidose TT vacuum drum filler system (Harro Höfliger Verpackungsmaschinen GmbH, DE). Capsules were packed in PVC/PVDC 260 mm x 250 µm, 60 g transparent blister (Research Pharmaceutical Co., Ltd., Colombia) sealed with a standard 20 µm aluminum foil (Amcor Flexibles Soliera, Srl, IT). The in vitro respirability of Ins_SD was assessed using the Next Generation Impactor (NGI) (Copley Scientific, UK) and RS01® high resistance inhaler (Plastiape, IT) at 65 L/min to aerosolize the formulation. In comparison, the aerodynamic performance of Afrezza® (8 IU), corresponding to 0.70 mg of insulin, was assessed. The content of three Ins_SD capsules or six Afrezza® cartridges was discharged for each NGI test. Insulin collected in the impactor was quantified by HPLC, using a previously validated method.[3] Emitted Dose (ED), Median Mass Aerodynamic Diameter (MMAD), Fine Particle Dose (FPD<5 µm) and Fine Particle Fraction (FPF<5 µm), were calculated according to USP 36. The stability study was conducted storing the capsules in blisters at room temperature (25°C-60% RH) and at refrigerate conditions (4°C) up to 6 months. Samples were analysed for higher molecular weight protein (HMWP) impurities, according to insulin and impurities HPLC quantification methods reported in Ph. Eur. 9th Edition.

305

Drug Delivery to the Lungs, Volume 29, 2018 – Glycemic Profile in Rats After Pulmonary Administration of a Pure Insulin Powder Male Wistar rats weighing 280-320 g (Charles River, LC, Italy) were maintained in the animal facility of Parma University with a 12 h day and night schedule with food and water available ad libitum for 7 days before the experiment. Rats (4 per group) were fasted for 3 h prior to basal glycemic determination then they received a 1 g/Kg glucose injection (time zero) and 5 min later 10 IU/Kg of insulin were administered subcutaneously (SC) or intratracheally using a dry powder inhaler device DP-4 insufflatorTM (Penn-Century, Inc, Philadelphia, US). Ins_SD and Afrezza® were blended in order to achieve a mass of powder sufficient to be loaded in the device. Blends were prepared in Turbula® mixer (25 rpm for 5 min) with mannitol spray-dried powder in order to have 80 µg of peptide in 2 mg of powder. A group of rats received the carrier microparticles (mannitol spray-dried), as control. For SC administration insulin was dissolved in 0.01 N HCl/saline 1:9 (solution pH= 4.7). Blood samples (250 µl) were sublingually collected at time 15, 30, 60, 120 and 180 min after administration of insulin or carrier formulation. Plasma was separated by centrifugation and glucose concentration was spectrophotometrically determined using Glucose Oxidase reagent (Werfen) The rats were sacrificed under anaesthesia after completion of the experimental procedure by withdrawing the entire blood volume through their abdominal aorta. Data obtained were analyzed using a Two-way ANOVA and Bonferroni post test. Results Ins_SD was successfully prepared by spray drying with a yield process > 60%. The powder particle size distribution is reported in Figure 1. The SEM analysis showed that Ins_SD was constituted by microparticles having a wrinkled shape attributable to the insulin substance. Shrivelled microparticles derived from the collapse of inflated protein solution droplets during drying. Some spherical particles were present in the population.

Ins_SD

Figure 1 - Describe the figure above

dv 0.1 (µm)

dv 0.5 (µm)

dv 0.9 (µm)

1.20 ± 0.03

2.72 ± 0.14

7.91 ± 1.67

Figure 1

Particle size distribution of Ins_SD by laser diffraction. The dv 0.1, dv 0.5, dv 0.9 represent the volume diameter of the particles at 10, 50 and 90% on the cumulative undersize curve.

Figure 2.

SEM images of insulin spray-dried Ins_SD.

Ins-SD powder showed a high respirability, considering that the delivered dose was >95% and the FPF lower than 5 µm was 91%. The MMAD of 0.85 µm (Table 1) was lower than the median volume diameter because particles had corrugated and aerodynamic-favourable shape. The uniformity of content after the semi-automatic filling of Ins_SD powder was performed and the capsules resulted contain slightly less (1.70 ± 0.06 mg) than the target value of 2 mg. The data collected before and after the filling process were similar in terms of fine particle fraction showing that the mechanical manipulation of the powder did not affect the aerosolization behaviour. Afrezza® powder, tested for comparison, was successfully emitted from the device (more than 95%). The powder mainly deposited on stages 3, 4 and 5 of the impactor, with an MMAD of 3.2 µm.

306

Drug Delivery to the Lungs, Volume 29, 2018 – Francesca Buttini et al. Table 1 -

Aerodynamic parameters of Ins_SD when the capsule was filled manually and post Harro filling process (n=3) and Afrezza® powder (8 IU cartridge). Metered Dose

Emitted Dose

MMAD

FPD

FPF

(mg)

(µm)

(mg)

(%)

Extra-FPD (mg)

Extra-FPF (%)

Ins_SD Post production

2.35 ± 0.06

2.26 ± 0.07

0.85 ± 0.09

2.07 ± 0.04

91.5 ± 1.2

1.92 ± 0.04

85.1 ± 0.8

Ins_SD Post filling

1.62 ± 0.08

1.50 ± 0.05

0.89 ± 0.09

1.37 ± 0.04

91.5 ± 5.9

1.28 ± 0.07

85.2 ± 7.7

Afrezza®

0.64 ± 0.00

0.60 ±0.00

3.19 ± 0.03

0.41 ± 0.01

68.3 ± 0.6

0.20 ± 0.00

40.5 ± 0.6

The association of insulin monomers to dimers and larger aggregates (hexamers) is a complex, dynamic equilibrium and is a function of pH, ionic strength and protein concentration. The insulin aggregates have to preserve ability to readily dissociate into a rapidly absorbed monomeric/dimeric compound when dissolved in a biological fluid (Figure 3). Covalently linked aggregates of insulin, as a consequence of chemical degradation, are referred to as High Molecular Weight Products (HMWP) and are not able anymore to dissociate and release the monomers. Diabetic patients exposure at low HMWP levels cannot be prevented as they inevitably form in the formulation during use and storage of insulin products. The percentage of all Ins_SD covalent products (dimers and hexamers) was found to be below the USP limits of 2% (dotted line, Figure 3) in both storage conditions for the 6 months of the investigation. Similarly, the level of other degradation products as A21 desamido insulin + other related proteins (ORP) was maintained in the USP limit for 6 months as previously reported.[4]

Figure 3 - high molecular weight protein (HMWP) degradation products of insulin spray-dried powders INS_SD stored at room temperature (25 °C, 60% RH) for six months: (mean ± standard deviation, n = 3), USP limits are represented by the dotted lines. The schematic process of powder dissolution, hexamers dissociation and monomers absorption across capillary membrane is reported.

From the plasma analysis it was demonstrated that Ins_SD provided a similar profile of glucose control as Afrezza ® (p>0.05). The AUC profiles were not statistically different from the ones obtained after SC insulin injection (p>0.05). However, both the pulmonary insulin afforded a more rapid glucose decrease compared to SC which provided a longer lasting hypoglycaemic effect. The control group that received the carrier (mannitol spray-dried powder) reported the glucose profile correspondent to the physiological decrease after its assumption.

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Drug Delivery to the Lungs, Volume 29, 2018 – Glycemic Profile in Rats After Pulmonary Administration of a Pure Insulin Powder

Glucose plasma level (mg/dl)

600

Ins_SD Ins_SC

500

Mannitol_SD Afrezza

400 300 200 100 0

Basale

15min

30min

60min

120min

180min

Time Figure 4 - Plasma glucose level time curve of rats that received Ins_SD, Afrezza®, mannitol by pulmonary insulation and insulin subcutaneously. The insulin dose was 10 IU/Kg per animal. Data are expressed as mean ± ES (n=4).

Conclusions The results from this study show that pure insulin pulmonary powder, produced by spray drying from an acid aqueous solution of the peptide, presented a higher respirability compared to Afrezza ®. Furthermore, insulin spraydried powder stability data have shown that Qualicaps® Quali-V®-I capsules, together with the PVC-PVDC packaging material, can provide long-term stability, maintaining good aerodynamic performance. This specific spray dried powder formulation pre-metered in HPMC capsules and packaged in PVC/PVDC 260 mm x 250 µm, sealed with standard aluminum foil, opens the possibility of an inhalation therapy less dependent on the cold storage of drug product.

References 1.

Goldberg, T., Wong, E., 2015. Afrezza (Insulin Human) Inhalation Powder: A New Inhaled Insulin for the Management Of Type-1 or Type-2 Diabetes Mellitus. P T 40, 735–741.

Cagnani, S., Colombo, P., Ventura, P., 2004. Insulin highly respirable microparticles. Assignee: University of Parma; US Patent Number: 7625865 B2.

Balducci, A.G., Cagnani, S., Sonvico, F., Rossi, A., Barata, P., Colombo, G., Colombo, P., Buttini, F., 2013. Pure Insulin Highly Respirable Powders for Inhalation. Eur J Pharm Sci 51, 110–117. doi:10.1016/j.ejps.2013.08.009

Buttini F, Rossi I, Anakayana J, Dujovny G, Seyfang K, Colombo P. Investigation of Physicochemical Stability of a Pure Insulin Spray-dried Powder for Inhalation Semi-automatically Filled in Quali-V-I Capsules. Respiratory Drug Delivery Europe 2017.

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Drug Delivery to the Lungs, Volume 29, 2018 - Mark Sanders & Ashley Green Inhaler Resistance, Flowrate and Duration of Inhalation: The Effort to Use an Inhaler Adequately. Mark Sanders & Ashley Green Clement Clarke International Limited, Edinburgh Way, Harlow, CM20 2TT, UK Summary As chronic obstructive pulmonary disease (COPD) becomes more severe, the ability of the patient to perform the correct inhalation manoeuvre becomes progressively limited and may fall below that required for correct use. The physiological effort associated with achieving this has seldom been expressed with the focus on peak inspiratory flow and resistance: duration and flow profile are also important. We have quantified the effort of untrained and trained moderate (n=12) and very severe (n=10) COPD volunteers to use seven commonly used pMDI, DPI and SMI devices in a minimally adequate way (n=6: Evohaler, Respimat, HandiHaler, Breezhaler, Turbohaler, Ellipta, and NEXThaler). Patient flow profiles, at three resistances (0.04, 0.16 and 1.00 kPa½/L.min-1) were accessed from a database, enabling interpolation and modelling to the devices. Device resistances were measured and enumerated at the device-effective flowrate. Duration of inhalation was determined from the literature, combined with an assessment of time for actuation/coordination, aerosol delivery, ‘chase’ air, and capsule emptying. Energy (airWatts, aW), was calculated as volume (m3.sec-1) x pressure (kPa). To provide a comparable value for each inhaler, Energy (aW) was multiplied by duration (sec) to express the Effort of inhalation. The data show that training helps subjects to exert a greater effort. Untrained subjects who produced insufficient Effort or who were close to failure were able to deliver adequate Effort following training. Devices that propel the medication towards the patient (pMDI, SMI) require less inspiratory Effort. Post-training, all of the devices were within the theoretical capability of the subjects for successful use. Key Message A new patient-inhaler parameter (inspiratory Effort) has shown that untrained subjects exert less effort than trained subjects, and that some inhalers require an inspiratory effort that is beyond the capability of the untrained subject: for those devices training must be regarded as a necessity. Introduction Patients taking inhaled medication need to be able to use their inhaler correctly to ensure effective treatment. Many patients believe that they are doing so when, in reality, they are making important errors [1]. Correct inhaler use entails not only preparing the device but also making an inspiratory manoeuvre appropriate to receive the medication: the slow gentle deep inhalation for pressurised metered dose inhalers (pMDIs) and the fast, powerful, deep inhalation for dry powder inhalers (DPIs) [1]. Much has been written about the difficulties patients have with technique and particularly with the appropriate inspiratory flowrate [2]. As chronic obstructive pulmonary disease (COPD) and asthma—the latter particularly during periods of worsening control—become more severe, the ability of the patient to perform the correct inhalation manoeuvre becomes progressively limited and may fall below the level required for the correct use of a particular inhaler [2-3]. The physiological effort associated with achieving this has seldom been expressed. Previously studies have focused largely on peak inspiratory flowrate and resistance, ignoring the impact of duration or the full importance of the flow profile [4-6]. We have sought to quantify the patient effort required to use particular devices in a minimally adequate way, using the three elements that contribute to that effort: (i) the resistance of the inhaler to air flow, (ii) the inhalation flowrate necessary for acceptable use, and (iii) the duration of inhalation. By considering duration as well as flowrate and resistance we can express inhalation as an effort. We investigated the inspiratory effort that subjects with COPD could apply relative to the effort necessary for minimally successful inhaler use. We have completed these effort determinations for seven commonly used inhaler devices. Experimental methods Firstly, we accessed a database of inspiratory profiles for COPD subjects classified by severity (moderate: FEV1 50%-80% predicted, severe: 30%-50% and very severe <30%) [7]. The subjects had been asked to inhale (without training) through three different resistances (0.04, 0.16 and 1.00 kPa½/L.min-1). They were then trained to inhale forcefully, and asked again to inhale against the three resistances. These flow profile data enable interpolation and modelling to a range of different device resistances that cover marketed inhaler products. Secondly, we obtained six replicates of seven commercial inhaler devices (Table 1) and measured device resistance for each device using the Clark and Hollingworth method [8]. All measurements were made on the same calibrated equipment, by the same operator (AG) and in the same laboratory. The devices were a pMDI (Evohaler, GSK), a Soft Mist inhaler (SMI, Respimat, Boehringer Ingelheim), high resistance and low resistance capsule DPIs (HandiHaler, Boehringer Ingelheim and Breezhaler, Novartis), high resistance reservoir and low resistance blister DPIs (Turbohaler, AstraZeneca and Ellipta, GSK) and a breath actuated reservoir DPI (NEXThaler, Chiesi). Active pharmaceutical ingredients are provided in the footnote. The mean resistance for each device was interpolated at the flowrate considered effective for that device as determined from the literature [9-12].

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Drug Delivery to the Lungs, Volume 29, 2018 - Inhaler Resistance, Flowrate and Duration of Inhalation: The Effort to Use an Inhaler Adequately.

Table 1 – Details and properties of the Inhaler devices (c = capsule, b = blister, r = reservoir)

We then attributed values to each device for a minimum effective inspiratory flowrate based on manufacturers’ information and supported by studies at a range of different flowrates [9-10]. Similarly, published references were used where available for the duration of inhalation to achieve minimally correct usage [13], combined with a pragmatic assessment of the total timing for actuation/coordination, delivery of aerosol and the ‘chase’ air that drives the drug into the lung [9]. In the case of the capsule devices (Breezhaler and HandiHaler), we also conducted confirmatory testing by connection to a precision vacuum pump (Copley LCP5, Copley Scientific Limited, Nottingham, UK) to ensure that capsule rattle—the signal of capsule movement and thereby powder displacement—and capsule emptying were within pre-determined times at a particular flowrate. In the case of devices that are manually actuated (pMDI, Respimat) a time factor for coordination was considered: based on earlier work with Respimat we adopted a 0.5s coordination time [13]. The components of an inspiratory effort determination per device were therefore available. Inspiratory Effort (airWatts per inhalation, aW) was calculated as volume (m3.sec-1) x pressure (kPa) x duration (seconds). The modelling for moderate (n=12) and very severe (n=10) COPD patients both untrained and trained are presented. Results Flowrate, resistance, and duration data are given in Table 1. Ideal inspiratory Effort values (airWatts per inhalation), and those for untrained and trained COPD subjects are given in Table 2. Within inhaler, the untrained/ trained ideal values differ slightly owing to the zero to peak acceleration portion of the ideal profiles (which are based on the subject profiles). The ratio of ideal to subject ability to generate a minimally adequate Effort is given as a percentage: failure being represented by >100%. An example of an inspiratory flow profile in untrained and trained subjects (mean values) is given in Figure 1.

Figure 1 – Turbohaler: modelled flow profiles in untrained (left) and trained (right) very severe COPD subjects (mean, n=10). subject Effort; ideal Effort.

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Drug Delivery to the Lungs, Volume 29, 2018 - Mark Sanders & Ashley Green Moderate COPD (FEV1 50-80% predicted) Device

UNTRAINED

Type Ideal

TRAINED

Subject (mean)

Effort %

Ideal

Subject (mean)

Effort% 0.2

Evohaler

pMDI

0.0006

0.15

0.4

0.0008

0.47

Respimat

SMI

0.03

0.76

3.9

0.05

2.20

2.3

HandiHaler

c-DPI

0.70

2.53

27.7

0.80

6.90

11.6

Breezhaler

c-DPI

0.87

1.58

55.1

1.00

4.10

24.4

Ellipta

b-DPI

0.40

1.68

23.8

0.40

4.10

9.8

Turbohaler

r-DPI

0.87

2.44

35.7

0.90

5.70

15.8

NEXThaler

r-DPI

1.80

3.15

57.1

1.50

7.50

20.0

Very Severe COPD (FEV1 <30% predicted) Device

UNTRAINED

Type Ideal

Subject (mean)

TRAINED Effort %

Ideal

Subject (mean)

Effort % 0.2

Evohaler

pMDI

0.0005

0.06

0.8

0.0003

0.15

Respimat

SMI

0.03

0.29

10.3

0.03

0.80

3.8

HandiHaler

c-DPI

0.67

1.62

41.4

0.80

3.60

22.2

Breezhaler

c-DPI

0.70

0.57

122.8

0.90

1.70

52.9

Ellipta

b-DPI

0.37

0.61

60.7

0.40

1.90

21.1

Turbohaler

r-DPI

0.89

1.02

87.3

0.90

2.90

31.0

NEXThaler

r-DPI

1.80

1.42

126.8

1.50

3.80

39.5

Table 2 - Inspiratory Effort values

Discussion It is well recognised that the effort of using inhalers varies with their specific resistance but few studies have considered the influence of duration on the ability of the subject to generate the effort necessary to use a particular device. Inspiratory power and energy research in healthy paediatric and adult volunteer users of DPIs found age- and height-related effects and provided recommendations linking device resistance to comfort of use [14] . Determination of inspiratory manoeuvre duration is important and with a move to incorporate objective measures of inhaler performance this parameter will need to be properly mapped for future developments. Our work has been based on a pragmatic rationalisation that some chase air is needed to carry the medication deep into the lung and we attributed an approximate volume of one litre. For inhalers that deliver the dose very rapidly, the chase air duration follows delivery but for those with slower delivery the chase air begins during the delivery and extends, to some extent, post-delivery. We have attributed durations that are justified by pragmatic reasoning, but arguably different values could be rationalised. Known ‘optimal’ flowrates and capsule emptying are not necessarily a marker of adequate delivery because higher flowrates may be necessary to drive powder deagglomeration [2]. For example we did note that the Breezhaler capsule emptied at 21 L.min-1 but a minimum flowrate of 50 L.min-1 is recommended from the literature [9]. The higher resistance of the HandiHaler brings more separation force to the powder at a lower flowrate and clinical studies have identified 20 L.min-1 as adequate [9]. The implication of this is that a patient could achieve successful delivery from the HandiHaler at a rate at which Breezhaler would not work, and that to achieve successful minimally adequate delivery from Breezhaler the patient would be required to exert greater effort. Unsurprisingly, the two devices that propel the medication towards the patient (pMDI, SMI) require markedly less inspiratory Effort than the DPIs studied. Not all medications are available via the pMDI and the SMI delivery systems. The necessity of multiple inhaler use for any chronic patient leads not to the conclusion that all patients should use low Effort requirement inhalers but that a clearer appreciation of the different inspiratory Effort requirements and appropriate training are necessary to ensure minimally adequate delivery. This is underpinned by the finding that—post-training—all of the devices were within the theoretical capability of the subjects for successful use. The other immediate observation from the current data is that training helps subjects to exert a greater effort. The modelling indicates that subjects who could not produce sufficient Effort or who were close to failure when using a device in an untrained, spontaneous manner were then able to deliver adequate Effort following training. This appears most relevant to devices with a higher Effort requirement and in the very severe subjects; for example, Breezhaler, Turbohaler and NEXThaler.

311

Drug Delivery to the Lungs, Volume 29, 2018 - Inhaler Resistance, Flowrate and Duration of Inhalation: The Effort to Use an Inhaler Adequately. In general, our results reveal that untrained subjects exert less effort than trained subjects and this supports the call for inhaler technique training that is made in most guidelines. Some inhalers require an inspiratory effort that is beyond the capability of the untrained subject, and for those devices training must be regarded as an absolute necessity. Interestingly, a recent study of the cost-effectiveness of improving suboptimal inhaler adherence (use and technique) in COPD patients calculated that intervention—including training—would result in worthwhile economic and clinical benefits [15]. Conclusion The data indicate that COPD severity has an important influence on ability to use inhalers. Some inhalers may be beyond the capability of some COPD subjects to use correctly. While DPI inhalers appear similar in a higher Effort requirement, there are powder-presentation effects, e.g. duration of capsule emptying that may be clinically relevant. Inspiratory Effort assessments may represent an important consideration for inhaler device selection and merit further investigation. Footnote Evohaler pMDI (100g salbutamol); Respimat SMI (2.5g tiotropium / 2.5g olodaterol); HandiHaler capsule DPI (18g tiotropium); Breezhaler capsule DPI (85g indacaterol / 43g glycopyrronium); Ellipta blister DPI (55g umeclidinium / 22g vilanterol); Turbohaler reservoir DPI (160g budesonide / 4.5g formoterol); NEXThaler reservoir DPI (100g beclomethasone / 6g formoterol).

References 1

Price D, Bosnic-Anticevich S, Briggs A, Chrystyn H, Rand C, Scheuch G, Bousquet J: Inhaler competence in asthma: Common errors, barriers to use and recommended solutions, Respir Med 2013; 107: pp37-46. DOI: 10.1016/j.rmed.2012.09.017

Rau JL: Practical problems with aerosol therapy in COPD, Respir Care 2006; 51: pp158-172.

Al-Showair RAM, Tarsin WY, Assi KH, Pearson SB, Chrystyn H: Can all patients with COPD use the correct inhalation flow with all inhalers and does training help?, Respir Med 2007; 101: pp2395-2401. DOI: 10.1016/j.rmed.2007.06.008

Dunbar CA, Morgan B, Van Oort M, Hickey AJ: A comparison of dry powder inhaler dose delivery characteristics using a power criterion, PDA J Pharm Sci Tech 2000; 54: pp478-484.

Pohlmann G, Hohlfeld JM, Haidl P, Pankalia J, Cloes RM: assessment of the power required for optimal use of current inhalation devices, J Aerosol Med Pulmon Drug Del 2018; 31: pp1-8. DOI: 10.1089/jamp.2017.1376

Bischofberger J, Rhima CE, Tibbatts J, Christie E, Ahern D, Chhabildas VS, Danagher H, Marshall J: A study on the inspiratory flowrate and power required to trigger Flutiform K-haler, Qvar Easi-Breathe, and Fostair NEXThaler breath actuated inhalers, Respir Drug Del 2018; 2: pp363-368.

Wachtel H, Flüge T, Gössl R: Flow-pressure-energy-power: which is the essential factor in breathing patterns of patients using inhalers?, Respir Drug Del 2006; 2: pp511-514.

Clark AR, Hollingworth AM: The relationship between powder inhaler resistance and peak inspiratory conditions in healthy volunteers – implications for in vitro testing, J Aerosol Med 1993; 6: pp99-110.

Haidl P, Heindl S, Siemon K, Bernacka M, Cloes R-M: Inhalation device requirements for patients’ inhalation maneuvers, Respiratory Medicine 2016; 118: pp65-75.

Chodosh S, Flanders JS, Kesten S, Serby CW, Hochrainer D, Witek TJ: Effective delivery of particles with the HandiHaler dry powder inhalation system over a range of chronic obstructive pulmonary disease severity, J Aerosol Med 2004; 14: pp309-315.

Ghosh S, Ohar JA, Bradley DM: Peak inspiratory flowrate in chronic obstructive pulmonary disease: implications for dry powder inhalers, J Aerosol Med Pulm Drug Del 2017; 30: pp381-387.

Medicines & Healthcare products Regulatory Agency (MHRA): Braltus/Gregal 10 microgram per delivered dose inhalation powder, hard capsule (tiotropium bromide), Public Assessment Report (PAR) Decentralised Procedure August 2016: 28pp. www.mhra.gov.uk/home/groups/par/documents/websiteresources/con723050.pdf

Hochrainer D, Kreher HH, Spallek LS, Wachtel H: Comparison of the aerosol velocity and spray duration of Respimat Soft Mist inhaler and pressurized metered dose inhalers, J Aerosol Med 2005; 18: pp273-282.

Harris DS, Scott N, Willoughby A: How does airflow resistance affect inspiratory characteristics as a child grows into an adult?. (Abstract). Presented at: The Aerosol Society Drug Delivery to the Lungs 21 Conference, Edinburgh, Scotland, December 9-11, 2010; J Aerosol Med Pulm Drug Del 2011; 24: pp303-332; DOI: 10.1089/jamp.2011.ab01

van Boven JFM, Cushen B, Sulaiman I, Greene G, MacHale E, Mokoka MC, Doyle F, Reilly RB, Bennett K, Costello RW: Personalising adherence-enhancing interventions using a smart inhaler in patients with COPD :an exploratory costeffectiveness analysis, Prim Care Respir Med 2018; 28: Art 24, 3pp DOI: 10.1038/s41533-018-0092-8.

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Drug Delivery to the Lungs, Volume 29, 2018 - Mark Sanders & Cuong Tran Triple Therapy Aerosol Delivery from an Integrated Inhaler Technique Training Device. Mark Sanders1 & Cuong Tran2 1

Clement Clarke International Limited, Edinburgh Way, Harlow, CM20 2TT, UK Pharma Services, Cardiff Medicentre, Heath Park, Cardiff, CF14 4UJ, UK

2i2c

Summary The main barrier to asthma control is correct inhaler use and treatment adherence. Training can be variable in quality and occasional in delivery, but can be augmented by whistle training tools which also provide the opportunity for detection by smartphone app. A new actuator-integrated whistle Clip-ToneM (M for multi-therapy pMDIs) is app-detectable. This device modifies the upright portion of the pMDI actuator body via either a comanufactured or retro-fit ridging element that whistles at the appropriate flowrate. Proof of concept studies have shown aerosol characteristics and particle size distributions equivalent to use of the pMDI alone. The present Next Generator Impactor research studied aerosol performance of the triple therapy pMDI, Trimbow (100 µg beclometasone dipropionate [BDP], 6µg formoterol fumarate, 10 µg glycopyrronium bromide, n=5). The dose, aerosol characteristics and particle size distribution of BDP, as representative of the combination solution, were consistent between control pMDI and pMDI plus Clip-Tone M: mean emitted dose 83.75 µg and 80.64 µg; fine particle fraction 41.87% and 41.27%, and fine particle dose 35.17 µg and 33.42 µg, respectively. These in vitro data support previous Clip-Tone research carried out with single and dual API pMDIs, demonstrating that a retrofit device that forms part of the actuator has little effect on aerosol performance. Fundamental to this new device approach is that guidance/self-training need not cease once the patient has learnt correct technique. If linked to app technology, this low-cost modification to existing pMDIs is suitable to be extended into adherence monitoring and reinforcement of technique. Key Message The Clip-Tone inhaler technique training tool applied to the top of the pMDI avoids any change in aerosol dose delivery. This feature, plus app detection, suggests that the device may have broader applicability than as a placebo-only or add-on trainer; remaining in place—during daily use—on an active inhaler. Introduction Currently only self-management apps are available for asthma, typically logging medication usage and pulmonary function [1]. Without doubt however, the main barrier to asthma control is correct inhaler use and treatment adherence. This is the desired deliverable from inhaler training but unfortunately, despite being a Guidelinenecessity [2], it is variable in quality and objectivity, and importantly is largely occasional in delivery [3]. Face-toface tuition is known to be augmented by whistle training tools [4], and these can provide the opportunity for selfguidance via sound-detection apps. The Flo-ToneCR (CR, controlled resistance) is a mouthpiece add-on, whistle trainer/mini spacer device for pressurised metered dose inhalers (pMDIs), and is a development improvement to the pre-existing Flo-Tone, enabling it to be used with a broader range of pMDI mouthpiece actuator shapes without compromising drug delivery [5]. Flo-Tone CR (Figure 1) is now the preferred device and has demonstrated aerosol performance and spacer functionality [5-7]. The mini-spacer function does introduce potential regulatory requirements and has led to the device being used mainly during training, enabling multiple use of valuable placebo pMDI stock via use of the Flo-Tone CR as a patient-specific mouthpiece. To extend the capabilities of this type of training tool, a new actuator-integrated, solid-state whistle has been developed which is smartphone app detectable. In practical terms this device—ClipTone—modifies the upright portion of the pMDI actuator body via either a comanufactured or retro-fit ridging element that whistles at the inspiratory flowrate appropriate to use [8]. Various proof of Figure 1 – clockwise, top left: Flo-Tone CR, Clip-Tone V concept studies using tailored devices (Ventolin, ± canister), Clip-Tone M (multi-therapy, ± canister) (Figure 1) with 100 µg salbutamol (Ventolin Evohaler, GSK), 20 µg ipratropium (Atrovent, Boehringer Ingelheim), and dual therapy 5 g formoterol/125 g fluticasone (Flutiform®, Napp) have shown key aerosol characteristics and particle size distributions equivalent to use of the pMDI alone [9-11] .

313

Drug Delivery to the Lungs. Volume 29, 2018 - Triple Therapy Aerosol Delivery from an Integrated Inhaler Technique Training Device. Comparing the performance of Ventolin Evohaler pMDI with Flo-Tone CR and Clip-Tone devices confirmed previous findings of unimpaired aerosol characteristics, and reduced in vitro throat deposition with Flo-Tone CR only [12]. Clip-Tone, with the actuator whistle-location physically ahead of the point of aerosol generation, cannot affect valve delivery and therefore offers a simpler regulatory pathway. This feature, plus smartphone app detection, suggests that the Clip-Tone device may be more suited to regular patient-controlled training. We have now extended the previous research, looking at the aerosol performance of the only currently available triple therapy pMDI. Experimental Methods The beclometasone dipropionate (BDP) aerosol characteristics of Trimbow pMDI (100µg BDP, 6µg formoterol fumarate, 10µg glycopyrronium bromide, Chiesi) were determined using a Next Generator Impactor (NGI, Copley Scientific Limited, UK) operated at the standard flowrate of 30L.min-1 [13]. Five pMDIs were used and were assigned numbers 1–5. All five were tested with Clip-Tone M (M, suitable for multi-therapy pMDIs) and numbers 1, 2 and 3 were used for control mass balance testing. pMDIs were primed according to the patient information leaflet, and canister weights were recorded before and after priming. Once primed, pMDIs were transferred to clean actuators and the initial weight of the devices was recorded. Devices were held in an upright position and shaken for 5 seconds. Immediately following shaking the pMDI was actuated into the NGI. This process was repeated until five actuations in total were delivered. Following NGI deposition, the devices were re-weighed to determine actuation weights. The NGI components were quantitatively washed with recovery solution, with BDP quantified using a validated HPLC methodology. Metered dose (dose delivered from the valve, µg), emitted dose (dose delivered from actuator, µg), fine particle fraction (FPF, %<5.0 µm), fine particle dose (FPD, µg<5.0 µm), mass median diameter (MMAD, µm), and geometric standard deviation (GSD, σg) were calculated. Results The mean BDP dose and aerosol characteristics from control pMDI and pMDI plus Clip-Tone M were consistent (Table 1), with metered dose approximately 96 and 93 µg, and emitted doses approximately 84 and 81 µg, respectively. Mass balance recovery of BDP was within acceptable limits (± 25% of targeted metered dose). Mean FPF and FPD were, respectively, approximately 42% and 35 µg (control), and 41% and 33 µg (plus ClipTone M). MMAD and GSD were comparable. Aerosol particle size distributions are shown in Figure 2: again recovery was consistent if very slightly less with use of Clip-Tone M. Trimbow Control

Trimbow plus Clip-Tone M

Metered Weight (mg)

76.13 ± 0.91

75.86 ± 0.59

Metered Dose (µg)

95.94 ± 3.37

93.09 ± 5.00

Emitted Dose (µg)

83.75 ± 3.20

80.64 ± 4.77

FPF (%<5µm)

41.87 ± 4.94

41.27 ± 4.37

FPD (µg<5µm)

35.17 ± 5.35

33.42 ± 5.31

MMAD (µm)

0.99 ± 0.06

0.96 ± 0.07

GSD (σg)

1.89 ± 0.07

1.90 ± 0.06

BDP on Actuator (µg)

12.71 ± 0.77

13.40 ± 0.98

Table 1 - NGI dose characteristics of BDP (mean ± SD) from Trimbow Control (n=3) and Trimbow plus Clip-Tone M (n=5)

Discussion Trimbow pMDI is formulated as a combination solution. The aerosol particle size distribution of the solution was evaluated using the deposition profile of BDP alone. Using a single active pharmaceutical ingredient (API) from a triple API system as a surrogate for the other components is acceptable since all three will be homogenously distributed within the formulation and in the emitted aerosol droplets. BDP-only NGI analyses have also been completed with dual API Fostair (100 µg BDP/6µg formoterol fumarate, Chiesi) and with Trimbow used in combination with Flo-ToneCR, and have shown that drug delivery is unaffected [7]. The current in vitro data support the previous Clip-Tone research carried out with single and dual API pMDIs [9-12], demonstrating that a retro-fit device that forms part of the actuator has little effect on aerosol performance. Fundamental to this new approach is that guidance/self-training need not cease once the patient has learnt correct technique. If linked to app technology, this low-cost modification to existing pMDIs is suitable to be extended into adherence monitoring and reinforcement of technique. This capability already exists via a prototype sensing app: assessing inhaler skills objectively (timing of the actuation: pre- or post-inspiratory manoeuvre, or the time from the start of the manoeuvre; and the duration of the inhalation), improving technique and providing date-and-time scrutiny of technique [11, 14]. It is possible to envisage that prescription repeats could be linked to patient competency and engagement via data gathered through this type of app.

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Drug Delivery to the Lungs, Volume 29, 2018 - Mark Sanders & Cuong Tran

Figure 2 - Trimbow

BDP NGI deposition profile (mean values Âą SD)

Conclusion The Clip-Tone training tool is applied to the top of the pMDI and avoids any change in aerosol dose delivery. This feature, plus smartphone app detection, suggests that the Clip-Tone device may have broader applicability than as a placebo-only or add-on training device, remaining in place on the active inhaler. This upgrades patient training from being clinic-based, and conducted possibly only once per year, to being available each time the inhaler is used.

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Drug Delivery to the Lungs. Volume 29, 2018 - Triple Therapy Aerosol Delivery from an Integrated Inhaler Technique Training Device. References 1

Bender BG: Technology interventions for nonadherence: new approaches to an old problem, J Allergy Clin Immunol 2018; 6: pp794-800. DOI: 10.1016/j.jaip.2017.10.029

Global Initiative for Asthma: 2017 GINA Report, Global strategy for asthma management and prevention, 2017 Global Initiative for Asthma. http://ginasthma.org/2017-gina-report-global-strategy-for-asthma-management-and-prevention/

Capstick TGD, Clifton I: Inhaler technique and training in people with chronic obstructive pulmonary disease and asthma, Exp Rev Respir Med 2012; 6: pp91-103.

Ammari WG, Al-Hyari N, Obeidat N, Khater M, Sabouba A, Sanders M: Mastery of pMDI technique, asthma control and quality-of-life of children with asthma: A randomized controlled study comparing two inhaler technique training approaches, Pulmon Pharmacol Ther 2017; 43: pp46-54.

Sanders MJ, Bruin R, Tran C: Overcoming differences in pMDI actuator resistance to create a standardised training tool. (Abstract). Presented at: The Aerosol Society Drug Delivery to the Lungs Conference, Edinburgh, Scotland, UK, December 9-11, 2015; J Aerosol Med Pulmon Drug Del. 29:3: A-1—A-27; DOI: 10.1089/jamp.2016.ab01.abstracts.

Sanders M, Tran C: Technique improvement for a combination aerosol inhaler. (Abstract). Presented at: The International Society for Aerosols in Medicine Congress, Santa Fe, USA, June 3-7, 2017; J Aerosol Med Pulmon Drug Del. 30:3 A1-A-38; DOI: 10.1089/jamp.2017.ab01.abstracts

Sanders M, Tran C: Impact of a pMDI-technique training device on combination asthma therapy pharyngeal steroid deposition. (Abstract). Presented at: The International Primary Care Respiratory Group World Conference and 1st IberoAmerican Primary Care Respiratory Meeting, Porto, Portugal, May 31 – June 2, 2018; Prim Care Respir J. In press.

Sanders M, Bruin R, Tran C: Development of a non-electronic technique-based ‘smart’ inhaler. (Abstract). Presented at: The International Society for Aerosols in Medicine Congress, Santa Fe, USA, June 3-7, 2017; J Aerosol Med Pulmon Drug Del. 30:3; A1-A-38; DOI: 10.1089/jamp.2017.ab01.abstracts

Sanders M, Tran C: In vitro evaluation of a small, retro-fit training device for salbutamol pMDI. (Abstract). Presented at: The International Primary Care Respiratory Group World Conference and 1 st Ibero-American Primary Care Respiratory Meeting, Porto, Portugal, May 31 – June 2, 2018; Prim Care Respir J. In press.

Sanders M, Bruin R, Tran C: Pressurised metered dose inhalers (pMDIs) with integrated flowrate guidance. (Abstract). Presented at: European Respiratory Society Congress, Milan, Italy, September 9-13, 2017; Eur Respir J. 50 (Suppl 61) DOI: 10.1183/1393003.congress-2017.PA529.

Sanders M, Bruin R, Tran C: How to make a standard inhaler device into a ‘smart’ inhaler that teaches technique. (Abstract). Presented at: The Aerosol Society Drug Delivery to the Lungs Conference, Edinburgh, Scotland, UK, December 6-8, 2017; J Aerosol Med Pulmon Drug Del. 31:2; A-1—A-32; DOI: 10.1089/jamp.2018.ab02.abstracts.

Sanders MJ, Bruin R, Tran CT: Comparison of aerosol delivery from two inhaler technique-guidance devices. (Abstract). Presented at: European Respiratory Society Congress, Paris, France, September 15-19, 2018; Eur Respir J. 52 (Suppl 62): In press.

British Pharmacopoeia (BP): General Chapter 2.9.18 – Preparations for inhalation: aerodynamic assessment of fine particles,.Publisher: The Stationery Office; 2017 edition, ISBN: 978-0113230204.

Sanders M, Crawford E: Comparative assessment of tone-guided inhaler training with and without real-time app feedback. (Abstract). Presented at: Inhaler Research Workgroup Conference, Groningen, The Netherlands, March 15-16, 2018.

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Drug Delivery to the Lungs, Volume 29, 2018 - Pietro Piera et al. Manufacturing DPIs: an engineering perspective Pietro Piera1 & Mathieu Pfeiffer2 IMA S.p.A., Via Emilia 428-442, 40064 Ozzano dellâ&#x20AC;&#x2122;Emilia (BO), Italy Summary When developing new pharmaceutical products in DPI form, industrial manufacturing aspects must be considered from the very beginning to shorten the scale-up and optimization of the final manufacturing process, as well as to achieve a more efficient and cost effective production. Precise micro-dosing, weight control, containment measures and ease of device assembly are all issues that must be faced at an early stage. IMA draws on its extensive expertise to provide the most advanced solutions for DPI processing and assembly. Direct weight control performed in line on each single capsule or device, both before and after filling. Absence of mechanical powder compression for improved airway intake. Accurate micro-dosing and automatic feedback and adjustment. Highly flexible and precise inhaler assembly in accordance with design for manufacturing (DFM) and proof of principle studies at an early stage. This presentation investigates optimal process parameters for low-dose DPIs achieved by the dosator technology. The study proves that a major advantage of using this technology for processing DPIs is that the dosators can be accurately adjusted without any need to compress or aspirate the powder. Maintaining the free-flowing properties of the dispended powder within the capsule better ensures the release of powder from the capsule into the inhaler when the capsule is pierced, thereby better controlling both the emitted dose and the fine particle fraction of the dose discharged from the DPI. Key message How to achieve optimal low-dose Dry Powder Inhalers by combining the dosator technology and the direct net weight control both in a table-top device and in an industrial production scale capsule filler. Introduction In 1948, the first commercial dry powder inhalation device was launched on the market. This first technology seems archaic by todayâ&#x20AC;&#x2122;s standards: a deep inward breath would cause a ball to strike a cartridge containing powder and shake the powder into the airstream. Since then, changes in the drug delivery market and regulatory pressures have driven innovation of DPIs forward. It is estimated by the WHO that, worldwide, some 300 million people suffer from asthma and 240 million people suffer from chronic obstructive disease (COPD). DPIs represent 50% of the total asthma/COPD market by value worldwide. The latest patientfocused studies using DPIs indicated that the expectations regarding this technology have evolved. Patients and pneumologists are now increasingly focusing on convenience and ease of use, favouring a compact design. Indeed, DPIs have shown great promise in their ability to deliver drugs reliably and effectively, and novel designs can ensure that future cost, compliance and safety challenges are overcome. Some of the performance characteristics essential to DPIs are related to dose delivery, fine particle fraction content and performance levels at varying airflows. These characteristics can differ from one powder formulation to another, and some fine tuning of either device or formulation or a combination of both may be necessary to achieve optimal performance. Micro-dosing DPIs takes this challenge to extremes. IMA draws on its extensive expertise to provide the most advanced solutions for DPI processing and assembly.

Case study: investigating optimal process parameters for low-dose Dry Powder Inhalers. Aim of the study The aim of the study was to explore the best process parameters to achieve the 5.5 mg dose of a powder mix including a first Lactose type as carrier and a second one (4% in concentration by weight) of micro-fine lactose as API simulator. The process was carried out as a first approach in a table-top capsule filling device (Minima, IMA) and then up scaled to an industrial production scale capsule filling machine (Adapta with 100% gravimetric net weight control, IMA). Two types of Lactose were compared from different suppliers.

317

Drug Delivery to the Lungs, Volume 29, 2018 - Manufacturing DPIs: an engineering perspective Materials Components of the tested formulations are described below: A) Blend of placebo powder composed of Inhalac 251 (Meggle, Germany) and 4% in concentration of Lactochem microfine lactose (DFE, Germany). B) Blend of placebo powder composed of Respitose (DFE, Germany) and 4% in concentration of Lactochem microfine lactose (DFE, Germany). For the execution of the tests, HPMC capsules size 3 produced by Lonza Capsugel (USA) were used. Table 1: technological characteristics of the two kinds of powder mixes: Inhalac 251 (Meggle, Germany) and Respitose (DFE, Germany) with 4% Lactochem microfine lactose each.

Powder mix Inhalac 251 + 4% Lactochem microfine lactose Respitose SV003 + 4% Lactochem microfine lactose

Bulk Density (g/ml)

Tapped density (g/ml)

Carr Index (%)

Loss on drying (%)

0.593

0.780

23.9 (poor flowability)

0.04

0.658

0.812

18.9 (fairly good flowability)

0.08

Methods The Minima and Adapta capsule fillers are designed to process low-dose Dry Powder Inhalers. Minima is a table-top capsule filler designed to dose solid products in hard capsules. Extremely precise and with a single dosator, Minima can be equipped with the same dosing devices applied on IMA production machines. Minima comes factory-preset for use with Dry Powder Inhalers, thus being an efficient system for inhalation product optimization. The Adapta capsule filling machine covers medium and very high-speed production requirements and features exceptional design flexibility. Fitted with the 100% gravimetric net weight control, Adapta ensures maximum dosing precision and reliability even at very high speed. On the Minima machine, the target dosages of 5.5 mg, 15 mg and 25 mg was achieved with both formulations. Each lot consists of 20 samples. Reliability and consistency were assessed by replicating the acquisitions three times for each lot. The second step of the study was to up scale the experience gained on the bench top machine to the production equipment. Since the target dose was 5.5 mg the main work was concentrated on this target with both preparations. 100,000 capsules were produced with Adapta with 100% gravimetric net weight control for each blend. To determine the net weight of the samples dosed with Minima, the macro-analytical electronic scale Sartorius QUINTIX224-1S was used. Weighing range: 220 g, accuracy: 0.1 mg. To check the net weight of the samples dosed with the capsule filling machine, the total filling control system of Adapta was used. Experimental part In tables 2 and 3 the experiments of the Minima first screening are reported including machine setting, real weight achieved, tolerances, range between minimum and maximum sample weight obtained and relative standard deviation. Table 2: Inhalac 251 + 4% Lactochem microfine lactose, Minima trials.

Average net weight (mg)

Doser internal diameter (mm)

Dosing chamber position (mm)

Powder layer height (mm)

25.9 14.6 5.4

3.0 2.5 2.0

4.1 3.7 2.4

15 15 10

318

Min-max weight sample deviation (mg) 1.48 0.74 0.70

Tolerance obtained (%)

Relative Standard deviation (%)

+3.3/-2.3 +1.6/-3.4 +8.0/-6.1

2.03 1.48 3.0

Drug Delivery to the Lungs, Volume 29, 2018 - Pietro Piera et al. Table 3: Respitose SV003 + 4% Lactochem microfine lactose, Minima trials. Average net weight (mg)

Doser internal diameter (mm)

Dosing chamber position (mm)

Powder layer height (mm)

25.5 14.6 5.5

3.0 2.5 2.0

4.2 3.8 2.2

15 15 10

min-max weight sample deviation (mg) 1.75 0.46 0.67

Tolerance obtained (%)

Relative standard deviation (%)

+3.4/-3.4 +1.5/-1.6 +4.8/-8.1

2.24 0.90 2.9

Table 4 summarize the final results of the Adapta with 100% gravimetric net weight control: the powder mixes machine setting are reported including real weight achieved and relative standard deviation for an easy evaluation. Table 4: Powder mixes Adapta with 100% gravimetric net weight control trials.

Lactose kind Inhalac 251 + 4% Inhalac 500 Respitose SV003 + 4% Inhalac 500

Average net weight (mg)

Doser internal diameter (mm)

Dosing chamber position (mm)

Powder layer height (mm)

Relative standard deviation (%)

Machine speed (caps/h)

5.5

2.0

2.13

2.52

85,000

5.5

2.0

2.18

2.52

85,000

The graphs 1 and 2 under show the net weights of all 24 dosators of the Adapta with 100% gravimetric net weight control for both powder mixes.

Graph 1: Inhalac 251 + 4% Lactochem microfine lactose, behavior of the 24 dosators on Adapta with 100% gravimetric net weight control.

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Graph 2: Respitose SV003 + 4% Lactochem microfine lactose, behavior of the 24 dosators on Adapta with 100% gravimetric net weight control. Results The tests on Minima demonstrated that both formulation gave good results in terms of workability and tolerance obtained. No significant differences were observed by the operator. Both formulations demonstrated good behavior even on the machine Adapta with 100% gravimetric net weight control: no seizing and no empty capsules produced. It was confirmed that for 5.5 mg dosing the range between the minimum and maximum weight value in the table-top capsule filler was always lower than 1 mg. The results obtained once formulations were tested in the production scale capsule filling machine were even better: for both formulation the relative standard deviation was confirmed below 3%. Discussion and conclusion As proven by this study, a major advantage of using the dosator technology for processing low-dose Dry Powder Inhalers is that the system can dose very small amounts of powders into capsule. This powder dosing technology does not require powder compaction to transfer the powder to the capsule. This ensures that the powder within the capsule is less likely to form aggregates and is maintained as a free-flowing powder. Maintaining the freeflowing properties of the dispended powder within the capsule better ensures the release of powder from the capsule into the inhaler when the capsule is pierced, thereby better controlling both the emitted dose and the fine particle fraction of the dose discharged from the DPI. References

Edwards D., Applications of capsule dosing techniques for use in dry powder inhalers, in “Therapeutic Delivery”, July 2010. Rogueda P., Take a deep breath. Inhalable drug delivery, in “World Pharmaceuticals”, April 2016. Williams G., The future of DPIs: Aligning Design with Market Demands, in “Drug Development & Delivery”, December 2012.

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Drug Delivery to the Lungs, Volume 29, 2018 â&#x20AC;&#x201C; Mary Joyce et al. Comparison of Aerosol Delivery Methods for Spontaneously Breathing Tracheostomy Patients Mary Joyce, Sorcha Murphy, Gavin Bennett, Louise Sweeney & Ronan MacLoughlin Aerogen, Galway Business Park, Dangan, Galway, H91 HE94, Ireland Summary Tracheostomy procedures are continually being utilised as a method for artificial airway management. This procedure is commonly used to promote weaning from mechanical ventilation. It is necessary to deliver therapeutic inhaled aerosols to spontaneously breathing tracheostomy patients for respiratory care. In clinical practice, the drug delivery device required for optimal aerosol drug delivery for tracheostomy patients varies across institutes. One of the challenges to overcome with tracheostomy is the risk of accidental decannulation from the additional weight on the tracheostomy tube from the drug delivery device. There is no standard protocol for delivering aerosol to a spontaneously breathing adult tracheostomy patient using the vibrating mesh technology. This study investigated the use of a vibrating mesh nebuliser for aerosol delivery to a spontaneously breathing adult tracheostomy patient by utilising different methods of attachment to the tracheostomy tube. The delivery of aerosol was assessed by determining the % inhaled dose in a simulated adult patient by nebulising a 2.0 mL dose of Albuterol sulphate (2 mg/mL) using a vibrating mesh nebuliser attached to a T-piece (34.93 %), T-piece/22M-15F connector (32.06 %), T-piece/catheter mount (33.91 %), aerosol chamber/catheter mount (36.07 %) and aerosol chamber/tracheostomy mask (32.81 %). The mass of drug eluted from the filters was determined using spectrophotometry at 276nm. A one-way analysis of variance (ANOVA) was performed and an overall significant difference between the attachment methods to the tracheostomy tube was determined with a p-value of 0.02. Therefore, the method selection has a significant effect on the efficiency of aerosol delivery. Key Message This study outlines the potential methods available for clinicians for inhalation therapy in spontaneously breathing adult tracheostomy patients using the vibrating mesh technology. Therefore, allowing the clinician to select a method that reduces the risk of accidental decannulation without compromising the quantity of aerosol delivered to the patient. Introduction Tracheostomy procedures are continually being utilised as a method for artificial airway management. This surgical practice of creating an opening in the trachea to facilitate the insertion of a tracheostomy tube in order to maintain an airway is often used to assist in the weaning of a patient from mechanical ventilator support. [1] Limited research has been conducted to assess aerosol delivery to spontaneously breathing tracheostomy patients using the vibrating mesh technology. One of the factors influencing aerosol drug therapy to tracheostomy patients is the drug delivery device used. [2] In this study, aerosol performance was assessed using a vibrating mesh nebuliser attached directly or indirectly to the tracheostomy tube by various methods. One of the associated risks with tracheostomy is accidental decannulation. Some of the factors which can lead to this issue are patient movement and/or the tracheostomy tube not being securely fastened. [3] These are important factors to consider when deciding on the method for drug delivery device attachment to the tracheostomy tube in order to minimise accidental decannulation whilst maintaining efficient aerosol delivery. The aim of this study was to assess the method of attachment of the vibrating mesh nebuliser to a tracheostomy tube whilst maintaining an efficient aerosol delivery without compromising patient safety. Materials & Methods Aerosol delivery was evaluated by characterising the % inhaled dose [drug delivered beyond the tracheotomy tube] in a spontaneously breathing adult tracheostomy patient. The % inhaled dose was assessed using different methods of attaching the vibrating mesh nebuliser (VMN) (Aerogen Solo, Aerogen, Ireland) to the tracheostomy tube (TT) (Shiley, Covidien-Medtronic, Ireland). A 2.0 mL dose of Albuterol sulphate (2 mg/mL) was nebulised using a VMN attached to the TT with a). 15mm Tpiece (Aerogen paediatric T-piece, Aerogen, Ireland), b). 15mm T-piece and a straight connector (22M-15F connector, Intersurgical, UK), c). 22mm T-piece (Aerogen adult T-piece, Aerogen, Ireland) and a catheter mount (straight catheter mount 22F 170mm, Intersurgical, UK), d). Aerosol chamber (Aerogen Ultra, Aerogen, Ireland) and a catheter mount (flexible fixed elbow catheter mount 22F 170mm, Intersurgical, UK), or e). Aerosol chamber with a tracheostomy mask (Adult tracheostomy mask, Hudson RCI, UK). A supplemental gas flow rate of 2LPM was used in combination with the aerosol chamber. The end of the TT was inserted into an absolute filter (Respirgard II 303, Baxter, Ireland) and attached to a breathing simulator (ASL 5000, IngMar Medical, Pittsburgh, USA) set to simulate an adult breathing pattern of BPM 15, Vt 500mL and I:E ratio 1:1. All testing was completed n=3. The nebuliser had an average droplet size (volumetric mean diameter) of 4.02 Âľm (measured using the Malvern Spraytec).

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Drug Delivery to the Lungs, Volume 29, 2018 - Comparison of Aerosol Delivery Methods for Spontaneously Breathing Tracheostomy Patients The mass of drug eluted from the filters was determined using spectrophotometry at 276nm and interpolation on a standard curve of Albuterol sulphate concentrations (200 µg/mL to 3.125 µg/mL). Results were expressed as the percentage of the nominal dose placed in the nebuliser’s medication cup that was delivered beyond the tracheostomy tube. A one-way ANOVA was performed to determine whether there was a significant difference for aerosol delivery between the methods tested. Significance was determined at < 0.05.

Figure 1 – Test set up for spontaneously breathing tracheostomy patient using different methods of VMN attachment to TT.

Results Results are outlined in Table 1 and illustrated in Figure 2 below.

% Inhaled Dose

T-piece (A)

T-piece/ 22M-15F connector (B)

T-piece/ catheter mount (C)

Aerosol chamber /catheter mount (D)

Aerosol chamber/ tracheostomy mask (E)

P-Value

34.93 ± 0.94

32.06 ± 0.49

33.91 ± 1.89

36.07 ± 0.43

32.81 ± 1.81

0.02

Table 1 - Mean ± Standard Deviation values of % inhaled dose for a spontaneously breathing tracheostomy patient using different methods of attachment with a VMN for aerosol delivery.

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A e r o s o l D e liv e r y f o r A d u lt T r a c h e o s t o m y P a t ie n t 40 A : T -P ie c e

% In h a le d D o s e

B : T -P ie c e /2 2 M -1 5 F C o n n e c to r

C : T -P ie c e /C a th e te r M ount

D : A e ro s o l C h a m b e r/C a th e te r M o u n t

E : A e ro s o l

C h a m b e r/ T ra c h e o s to m y M a sk

28 A

A tta c h m e n t M e th o d

Figure 2 – Aerosol delivery for a spontaneously breathing tracheostomy patient with a VMN attached to a TT by: (A). T-piece; (B) T-piece with 22M-15F connector; (C). T-piece with straight catheter mount; (D). Aerosol chamber with fixed elbow catheter mount and (E). Aerosol chamber with tracheostomy mask.

Discussion Overall for vibrating mesh technology, results indicate that the quantity of drug being delivered to a spontaneously breathing adult tracheostomy patient is dependent upon the method of attachment of the drug delivery device to the TT. A statistically significant difference was observed overall between the attachment methods. Our findings are similar to previous studies that show the % inhaled dose for other types of drug delivery devices to be higher with a T-piece in comparison to a tracheostomy mask. [4] It has also been demonstrated that the use of a VMN for aerosol delivery to a tracheostomy patient delivers significantly more aerosol than a jet nebuliser. [5] However, the positioning of the nebuliser directly onto the TT may not be clinically applicable. [5] This study further investigated the attachment of the VMN indirectly onto the TT by various different methods and determined that the highest % inhaled dose was observed when the VMN was tested in combination with the aerosol chamber and the fixed elbow catheter mount. Conclusion For clinical use, these results highlight the potential methods available for aerosols to be delivered to spontaneously breathing adult tracheostomy patients in combination with a VMN. A one-way ANOVA showed there was a statistically significant difference overall between the attachment methods of the VMN to the TT. Therefore, the method selection plays a key role in the quantity of drug being delivered. References 1

Bové MJ, Afifi MS: Tracheotomy procedure: Morris L, Afifi MS (eds): Tracheostomies: the complete guide. Springer Publishing; pp 17 – 41, 2010.

Ari A, Fink JB: Inhalation therapy in patients with tracheostomy: a guide to clinicians. Expert Rev Respir Med. 2017 Mar; 11(3):201 – 208.

O’Connor HH, White AC: Tracheostomy decannulation: Respir Care 2010; 55(8) 1076 – 1081.

Piccuito CM, Hess DR: Albuterol delivery via tracheostomy tube. Respir Care 2005; 50(8) 1071 – 1076.

Kelly PM, O’Sullivan A, McKenna C, Sweeney L, MacLoughlin R: Effect of nebuliser type and position on aerosol drug delivery during support mechanical ventilation and spontaneously breathing for tracheostomized adult patients (Abstract). Presented at: Drug Delivery To The Lungs, Edinburgh, UK, Dec 2016.

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Drug Delivery to the Lungs, Volume 29, 2018 – Gabriela Wyszogrodzka et al. A Metal-Organic Framework (MOF) dry powder technology for antibiotic deep lung delivery and imaging Gabriela Wyszogrodzka1, Przemysław Dorożyński2, Piotr Kulinowski3 & Stefano Giovagnoli4 1Department

of Pharmacobiology Jagiellonian University Medical College, Medyczna 9, 30-068 Kraków, Poland 2Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warszawa, Poland 3Department of Magnetic Resonance Imaging, Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Kraków, Poland 4Department of Pharmaceutical Sciences, via del Liceo 1, University of Perugia, Perugia, 06123, Italy

Summary The use of the theranostic approach could allow effective delivery and imaging of dry powders in the lungs for a better control and personalization of antibiotic therapy. In this work, we employed the Metal-Organic Frameworks (MOFs) technology to develop a dry powder blend with suitable aerodynamic properties and drug release behavior. The presence of Fe can allow tracking of particle deposition in the airways while granting adequate drug delivery. Isoniazid was employed as a model drug and loaded onto Fe-MIL-101-NH2 MOF. This isoniazid-MOF complex was embedded into a blend of poly(lactide-co-glycolide) and leucine microparticles. A high loading and content uniformity was pursued in both microparticles. A factorial experimental design study determined the predominant effect of blending ratio compared to MOF content and blending time on the measured fine particle fraction. Final blends showed good aerodynamic properties (fine particle fraction > 50%) and isoniazid-MOF content uniformity (%RSD < 7%), as also observed by energy dispersive X-rays spectroscopy, in spite of a rather irregular morphology, certain fragility and broad size distribution of the particles. The in vitro isoniazid release was slower than expected with only 12% of drug released within the first 24 h. This was linked to a slow liberation of isoniazid from the MOF. Overall, the obtained blends showed promising features compatible with lung deposition imaging purposes even in light of the good Magnetic Resonance Imaging contrast capacity of MOF. This formulation strategy could be useful for the development of an effective personalized pulmonary therapy. Key Message This work successfully employed the MOF technology to develop a theranostic inhaled blend composed of heterogeneous particles for antibiotic sustained release in the lungs. Coupling delivery and imaging features into one formulation is feasible and could be advantageous for an improved personalized therapy of lung infectious diseases. Introduction The efficacy of inhaled antibiotic treatments is strictly dependent on drug deposition patterns within the lungs, which in turn is determined by device/formulation performances and pathophysiological factors. Even when proper susceptibility screening and optimal formulations are being adopted, disease-related impairment of conductive airways can cause insufficient delivery to infected regions, resulting in sub-effective local antibiotic levels. Such an issue is detrimental to treatment efficacy, raising serious resistance concerns. Moreover, the often neglected large individual variability in lung capacity and physiology reduces further the chances of success of inhaled antibiotic therapies [1]. Therefore, to maximize therapeutic outcomes and to grant control over lung deposition and local drug levels, treatment personalization is recommended [1,2]. Although theoretically feasible, inhalation treatment personalization is not an easy task and requires the development of proper tools to record patient’s conditions and inhalation behavior. In this regard, the application of theranostics is considerably growing in personalized medicine [3,4,5]. The term ‘theranostic’ was coined combining the two words: therapy and diagnostics. A classical theranostic agent is defined as a system enabling simultaneous monitoring of delivery and treatment outcome so as to allow proper therapy-patient match [6,7]. Nowadays, nanotechnology has allowed combination of such features into a single particle system, coupling an effective delivery approach with imaging capabilities. In this regard, functional imaging has the potential to provide new insights into the behavior of inhaled particles by allowing monitoring of deposition patterns upon administration. This may grant patient-centered therapeutic regimen and administration strategies. Recently investigated theranostic agents are Metal-Organic Frameworks (MOFs). MOFs are porous materials whose structure is built by inorganic single ion or ion cluster nodes joined together by organic linkers [8]. Their characteristic feature is the presence of a metal center, i.e. paramagnetic cations (eg. Fe), that makes possible their use as magnetic resonance imaging (MRI) contrast agents. Moreover, selection of proper building components allows tuning of MOF pore size and biocompatibility, thus affording drug delivery applications [9]. Although still largely unaddressed, iron carboxylate MOFs safety has been proved in vitro [10] and in vivo, with complete elimination after 3 months from i.v. injection of 220 mg kg-1 and no side effects [11]. In this study, Fe-MIL-101-NH2 MOF, consisting of iron and aminoterephthalic acid as a linker, was used as a delivery system for isoniazid (INH), a first line antitubercular drug, chosen as a model drug. The distinctive features of tuberculosis (TB) infection support targeting of anti-TB drugs directly to alveolar macrophages as a logical strategy for maintaining local therapeutically effective concentrations while avoiding massive systemic exposure. The possibility of tracking particles after inhalation may allow to assess the actual drug targeting to the infected sites and personalized dose adjustments.

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Drug Delivery to the Lungs, Volume 29, 2018 - A Metal-Organic Framework (MOF) dry powder technology for antibiotic deep lung delivery and imaging The aim of the study was to develop spray-dried INH-MOF formulations. Due to poor MOF aerodynamic properties, hydrophobic and hydrophilic microparticles (MP) loaded with INH-MOF were prepared and blended. This approach was intended to avoid drug dilution effect upon microencapsulation and to provide a proper release behavior. The obtained blends were characterized for their aerodynamic behavior, release and morphology. MATERIALS AND METHODS Materials INH, D-Leucine (LC), poly(lactide-co-glycolide) (PLGA, Resomer® RG504H), tetrabutylammonium hydroxide solution (tBAH) and HPLC-grade acetonitrile (ACN) were from Sigma-Aldrich (Germany) and hydroxypropylmethylcellulose (HPMC; Metolose, 90SH,400 cP) was from Shin-Etsu (Japan). MOF synthesis and drug incorporation Fe-MIL-101-NH2 was synthesized according to the procedure reported by Bauer et al. [12]. After drying, MOF was activated under vacuum at 100°C until no droplets were observed. INH was incorporated into the MOF matrix by mixing 5ml of INH solution in DMF with 1000mg of Fe-MIL-101-NH2 for 12h. The product was separated by centrifugation and washed with ethanol and vacuum dried. Preparation and characterization of spray-dried particles LC and PLGA MP were prepared by spray-drying using the B290 Mini spray-dryer (Buchi, Switzerland). For PLGA MP, MOF (with and without drug) was suspended in an 12ml of ACN PLGA solution. Total amount of solids concerned 400mg and the process parameters were as follows: air flowrate = 357 L/h, inlet temperature = 75°C, aspirator rate = 20 m3/h, feed rate = 2.4 mL/min. For LC MP, LC and MOF were solubilized in 15ml of water (total amount of solids concerned 300mg), and spray-drying was performed as follows: air flowrate = 473 (L/h), inlet temperature = 140°C, aspirator rate = 20 m3/h, feed rate = 2.4 mL/min. Table 1. Design of Experiment: composition and preparation Design of Experiment (DOE) and particle parameters of blends based on 23 factorial design. characterization The effects of MOF loading, PLGA/LC blend ratio and blending time were investigated on emitted dose (ED) and fine particle fraction (FPF) employing a 23 factorial design (Table 1). Blends were prepared by placing appropriate amounts of each powder in a tube (total amount 200mg) and mixed for a specified time at a speed of 60 rpm in a coclea mixer kept at an angle of 45°. In vitro aerodynamic characterization Aerodynamic properties were assessed according to the European Pharmacopeia Ed. IX, using a TwinStage Impinger (TSI). A 50:50 ACN:0.1M NaOH solution was used as extraction medium. Twenty mg of powder were loaded into a HPMC capsule (Type 3, Quali-V, Qaulicaps® S.A.U, Spain). Air flowrate was set at 60 ± 5 L/min and emission was performed for 5 seconds through a RS01 model dry powder inhaler. Samples were assayed by UV-vis analysis in triplicate. MOF and drug quantification MOF was analyzed by UV-vis spectrophotometry using an Agilent 8453 spectophotometer. Samples were sonicated during 20 s and analyzed (λmax= 325 nm). Calibration was performed in the 25-200 µg/mL concentration range in 0.1M NaOH solution (r2=0.9987). INH was assayed as follows: INH was extracted using a 50:50 ACN:0.01M phosphate buffer, pH 7.4 solution and assayed by HPLC using a HP1050 chromatograph equipped with UV-vis detector set at 254 nm and a GROM-SIL 120 Diol 250x4.6mm (Grace Davison Discovery Sciences) column. Operating conditions were: column temperature room/25 °C, flow rate 1ml/min, the mobile phase was 70:30 ACN:0.4 mM tBAH, pH 6. Calibration was performed in the 0.5-16 µg/mL concentration range in water (r2=0.9997). Sampling for content uniformity test 200 mg of formulation powder was spread over the surface of a circle with 5.5cm diameter. The circle was divided into three slices and three samples (about 3mg) were gently taken from each slice and assayed as reported above. Particle size and morphology Particle size analysis of PLGA MP was carried out by an Accusizer C770 particle counter (PSS, Santa Barbara, California USA) equipped with an autodilution system. Due to LC solubility in water and even partial solubility in organic solvents, particle size of LC MP was determined by Scanning electron microscopic (SEM) images, using Image J software. An average of 200 particles from at least six different SEM images were counted and size distributions and mean diameters were calculated.

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Drug Delivery to the Lungs, Volume 29, 2018 – Gabriela Wyszogrodzka et al. SEM analyses were performed by using a FEG LEO 1525 microscope (LEO Electron Microscopy Inc., NY). The acceleration potential voltage was 1 keV. Samples were placed onto carbon tape coated aluminum stubs and sputter coated with chromium for 20 s at 20 mA (Quorum Technologies, East Essex, UK). Additional observations were conducted on dispersed particles collected from the impinger immediately after actuation. Energy dispersive X-rays spectroscopy (EDX) was performed detecting Fe and N signals were measured at 10 kV with acquisition times of 20 min. Coating was performed with graphite at 20 mA for 22 sec. An MRI test was carried out on pure 2% HPMC solution as well as for MOF suspensions in 2% HPMC solution, using 9.4 T MRI research system and TopSpin 2.0 software (Bruker Biospin, Germany) and Multi-Slice Multi-Echo (MSME) imaging sequence. In vitro drug release In vitro drug release experiments were performed by the dialysis bag method (12-14 kDa MWCO). A 0.1 M phosphate buffer pH 7.4 solution was employed as release medium. Fifty mL conical Falcon tube incubated at 37°C were employed. Drug release quantification was performed in triplicate by HPLC using the method described above. Statistical analysis One-way ANOVA was employed to determine statistically significant differences for content uniformity test in blends at 95% statistical significance level. RESULTS The factorial study pointed out a dominant effect on FPF of the percentage of LC MP in the blends that reached values > 50% when the PLGA/LC ratio was < 30% (Fig. 1 Panel a). Blending PLGA MP with LC MP improved considerably respirability of the powders compared with the poor behavior of MOF alone and the PLGA MP (FPF << 30%), confirming once more the fundamental glidant role of LC. An intermediate MOF content value was found to improve the aerodynamic properties of the blend. On the other hand, ED was little influenced with values always > 91% and a small effect of MOF content. Blending time was generally irrelevant. Overall, the obtained model suggested about 30% MOF content and a PLGA/LC ratio between 30-40% that provided a FPF close to 60% and an ED > 93 %. MOF loading in LC and PLGA MP produced rather irregular particles due to the presence of MOF crystals that, in turn, showed a bipyramidal geometry (Fig. 1 Panel b). The presence of the MOF crystals produced fragile MP that, in particular LC MP, were partially fragmented as shown even after blending. This resulted in broad particle size distributions with average volume-weighed diameters between 11 for MOF and 20 µm for PLGA MP, while numberweighed diameters were from 0.9 µm for MOF to 6.7 µm for LC MP and 4.2 µm for PLGA MP. As expected, the dispersed blend behavior was a measure of the presence of large clusters in the powder. In fact, particle clusters > 10 µm deposited in the stage 1 of the TSI, while the smaller aggregates generally < 10 µm were all found in the stage 2 (Fig. 1 Panel c). Therefore, this size cutoff was considered discriminant in determining respirability of the obtained blends.

Figure 1 - Panel a. FPF, ED and desirability surface plots indicating the target conditions to achieve best aerodynamic properties (maximum FPF and ED); Panel b. SEM images of 1. INH-MOF, 2. INH-MOF loaded LC MP, 3. INH-MOF loaded PLGA MP and 4. INH-MOF loaded PLGA/LC MP blend; Panel c. SEM images of the dispersed INH-MOF loaded PLGA/LC MP blend showing the powders deposited in TSI stage 1 and stage 2; Panel d. EDX analysis of the chosen INH-MOF loaded PLGA/LC MP blend reporting the spectrum with the Fe and N signals and relative map images; Panel e. in vitro INH release profile from PLGA/LC blend performed in 0.1 M phosphate buffer, pH 7.4, at 37°C; Panel f. MRI signal of INH-MOF. System 9.4 T (Bruker Biospin, Germany). Sequence Multi Slice Multi Echo (MSME), echo time, TE= 3.5 ms, repetition time, TR= 0.7 s. a. negative control 2% w/v HPMC solution; b. INH-MOF suspension (1.7 mg/mL); c. INH-MOF suspension (3.3 mg/mL).

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Drug Delivery to the Lungs, Volume 29, 2018 - A Metal-Organic Framework (MOF) dry powder technology for antibiotic deep lung delivery and imaging INH content in the blend was determined as 3.7% wt. INH-MOF was partially found on the surface of particles, as shown by EDX analysis of the blends (Fig. 1 Panel d). This observation is a consequence of the high MOF loading employed in this study. Moreover, a rather homogeneous distribution can be inferred from EDX signals, which is consistent with the good INH content uniformity measured either in the blends or the PLGA and LC MP (%RSD = 2-7%). A high content and an evenly distributed MOF are important in order to ensure tracking capacity of the inhaled blend. Contrarily to expectations, the presence of INH-MOF close to the surface of particles did not provoke a high burst release, as it can be seen from the INH release profile over 4 days (Fig. 1 Panel e). Within the first 24 h, only about 12% of release was measured. Complete release occurred over about 4 days. This behaviour may be explained considering that, although LC MP dissolve immediately, MOF requires much more time to dissolve at neutral pH, thus releasing INH at a much slower rate than expected. Naturally, the choice of PLGA as a long term

releasing polymer was instrumental to the achievement of a sustained release behaviour and local accumulation, and it was dictated by the rather poor arsenal of accepted materials for lung drug delivery. To date, this is perhaps the major limitation to commercialization of new and more effective inhaled formulations able to grant at the same time sufficient local and systemic antibiotic levels.

Overall, the obtained INH-MOF loaded PLGA/LC MP blend showed good aerodynamic properties and promising features compatible with lung deposition imaging purposes. In this regard, the good MRI contrast capacity of MOF is given in Fig. 1 Panel f, where MOF suspensions clearly light up upon the application of a mild magnetic field. Moreover, the good INH loading of the obtained formulation supports its in vivo applicability in consideration of the suggested inhaled INH dosages (0.1-0.73 µg) administrated as microparticles with 4% wt. INH content[13].Therefore, inclusion of iron-MOF in the formulation can help development of theranostic formulations with better drug release control and enhanced imaging properties. The effectiveness of inhaled treatments depends not only on formulation properties but also on factors related to lung ventilation, therefore functional lung imaging has the potential to provide new insight into the assessment and development of inhaled treatments and thus contribute to increasing therapy success. This work was supported by the National Science Center Poland [grant number 2016/21/N/NZ7/02663]. References 1 2 3 4 5 6 7 8 9 10

Dubsky S, Fouras A: Imaging regional lung function: A critical tool for developing inhaled antimicrobial therapies, ADV DRUG DELIVER REV. 2015; 85: pp100-109. Sethi T, Agrawal A: Structure and function of the tuberculous lung: Considerations for inhaled therapies, Tuberculosis 2011; 91(1): pp67-70. Ahmed N, Fessi H, Elaissari A: Theranostic applications of nanoparticles in cancer, Drug Discov Today. 2012;17(17–18): pp928–34. Terreno E, Uggeri F, Aime S: Image guided therapy: the advent of theranostic agents, J Control Release. 2012;161(2): pp328–37. Picard FJ, Bergeron MG: Rapid molecular theranostics in infectious diseases, Drug Discov Today. 2002;7(21): pp1092–101. Funkhouser J: Reinventing pharma: the theranostic revolution, Curr Drug Discov. 2002;2: pp17–9. Sumer B, Gao J: Theranostic nanomedicine for cancer, Nano. 2008;3(2): pp137–40. Furukawa S, Reboul J, Diring S, Sumida K, Kitagawa S: Structuring of metal-organic frameworks at the mesoscopic/ macroscopic scale, Chem Soc Rev. 2014;43(16): pp5700–34. Huxford RC, Della Rocca J, Lin W: Metal-organic frameworks as potential drug carriers, Curr Opin Chem Biol. 2010;14(2): pp262–8. Wyszogrodzka G, Dorożyński P, Gil B, Roth WJ, Strzempek M, Marszalek B, Węglarz WP, Menaszek E, Strzempek W, Kulinowski K: Iron-Based Metal-Organic Frameworks as a Theranostic Carrier for Local Tuberculosis Therapy, Pharm Res. 2018;35(7):pp144. Horcajada P, Chalati T, Serre C, Gillet B, Sebrie C, Baati T, Eubank JF, Heurtaux D, Clayette P, Kreuz C, Chang J, Hwang Y, Marsaud V, Bories P, Cynober L, Gil S, Férey G, Couvreur P, Gref R: Porous metal–organic-framework nanoscale carriers as a potential platform for drug delivery and imaging, Nat mater. 2010;9(2);pp172 Bauer S, Serre C, Devic T, Horcajada P, Marrot J, Ferey G, Stock N: High-throughput assisted rationalization of the formation of metal organic frameworks in the iron(III) aminoterephthalate solvothermal system, Inorg Chem. 2008;47(17): pp7568–76. Sharma R, Saxena D, Dwivedi AK, Misra A: Inhalable microparticles containing drug combinations to target alveolar macrophages for treatment of pulmonary tuberculosis, Pharm Res. 2001;18(10):pp1405-1410.

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Drug Delivery to the Lungs, Volume 29, 2018 – Zachary Enlo-Scott et al. Comparison of human cell lines for risk assessment of aerosolised pesticides Zachary Enlo-Scott1, Magda Swedrowska1, Alex Charlton3, Leona Merolla3, Ian Mudway2 & Ben Forbes1 Drug Delivery Research Group, Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London, SE1 9NH, United Kingdom 2MRC-PHE Centre for Environment and Health, Analytical, Environmental and Forensic Sciences, King’s College London,150 Stamford Street, London, SE1 9NH, United Kingdom 1

Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire, RG42 6EY, United Kingdom

Summary Pesticide aerosols are not delivered deliberately to the lung and yet are of concern to regulatory bodies such as the European Food Safety Authority (EFSA) due to the potential health risk of inhalation through bystander or occupational exposure. EFSA guidelines currently assume that 100% of an inhaled pesticide is systemically absorbed and presently there are a lack of well-accepted in vitro models for quantitative prediction of systemic bioavailability and quantification of respiratory toxicity. This study aims to compare human respiratory epithelial cell lines for their suitability as models of different anatomical regions of the respiratory tract with regards to pesticide toxicity and transepithelial permeability. A549, Calu-3 and RPMI-2650 were used to represent the alveolar, bronchiolar and nasal regions of the respiratory tract, respectively. Seven pesticides with varying physicochemical properties were evaluated for their acute cytotoxicity (2.5-300 μM for 24 h) using the MTT assay, whilst epithelial cell layer permeability was evaluated using Transwell® inserts to measure transepithelial electrical resistance (TEER), with tight junction integrity assessed using confocal microscopy to visualise Zonula Occludens-1. Overall no significant difference in pesticide cytotoxicity between the cell lines was observed. The permeability barrier presented by A549 and RPMI-2650 was significantly different to that of Calu-3 and TEER values were similar to those described previously. Whilst an expanded matrix of cytotoxicity markers is required, the initial results illustrate the impact of using a suite of cell lines for inhaled toxicological assessments and the need for model validation, particularly for modelling regional differences in mucosal permeability. Key Message Human epithelial cell lines derived from different regions of the lungs were evaluated as experimental models for predicting respiratory toxicology and systemic exposure after pesticide inhalation. This may inform European regulatory guidelines for occupational pesticide exposure and is consonant with contemporary efforts to use in vitro data to inform computational modelling of inhaled drug delivery. Introduction Inhalation represents a major route of exposure to xenobiotics including pesticides. Although pesticide aerosols are not inhaled deliberately, these products are utilised in ways that produce respirable particles, for example crop spraying. The European Food Safety Authority (EFSA) regulate the potential health risk of occupational exposure by inhalation and guidelines currently assume that 100% of inhaled pesticides are absorbed systemically[1]. This assumption is conservative and does not consider exhalation of inhaled pesticides or non-absorptive clearance mechanisms such as mucociliary clearance or metabolism in the lungs. Within an occupational exposure setting, most pesticide aerosols will be inhaled as liquid droplets likely ≥ 20 μm, with the majority of the ‘dose’ being deposited in the nasal cavity or upper respiratory tract, where respiratory bioavailability is likely to be lower than within the alveolar region of the lung. Although this estimate is intended to safeguard workers and bystanders, it is neither based on scientific evidence, nor constitutes a data driven approach as applied to exposure to pesticides via the oral and dermal routes. In vitro models are becoming increasingly important in inhalation toxicology to replace, reduce and refine in vivo studies, whilst also allowing for increased throughput [2]. The utility of in vitro models is well accepted within some realms pharmaceutical and environmental science, although much of the research for various air pollutants has focused on modelling the effects within the lower respiratory tract [3, 4]. There is significant overlap in the interests of regulatory bodies such as EFSA, Medicines and Healthcare products Regulatory Agency and DEFRA [Department for Environmental, Food & Rural Affairs] regarding respiratory absorption and toxicity following inhalation of xenobiotic aerosols. From an occupational exposure perspective, there is the need to develop reliable in vitro models that may aid the characterisation and prediction of bioavailability and toxicity of inhaled pesticides. Aim To investigate the suitability of cell lines derived from different regions of the respiratory tract for assessing cytotoxicity and permeability following exposure to pesticides. Seven commonly used pesticides were selected for the investigation.

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Figure 1. A549, Calu-3 and RPMI-2650, representing different regions of the respiratory tract.

Experimental Methods Pesticide cytotoxicity studies Calu-3 and RPMI-2650 cells were seeded in 96-well plates at a seeding density of 4 x 104 cells/well in 100μL of the Eagles minimum essential medium (supplemented with 10% v/v FBS, 1% v/v non-essential amino acids, 100 units/ml penicillin-streptomycin). A549 cells were seeded by the same method at a seeding density of 4 x 10 3 cells/well. Different cell seeding densities were used between cell lines, to account for differences in the rate and amount of formazan crystal formation. All cells were grown for 24 h before exposure to 2.5-300 μM of the pesticides in fresh medium (2% FBS). 0.75% DMSO and 1% Triton-X were used as negative and positive controls, respectively. Pesticide containing medium was then aspirated and replaced with 25 μL MTT solution (5 mg/ml) diluted 1:5 in fresh medium, incubating for 4 hours, before removing the solution and solubilising formazan crystals with 100Μl 10% SDS, 45% DMF, 45% H2O. To calculate cell viability, absorbance was measured at 570nm and 650nm, with the latter being subtracted to remove background absorbance. The assay was performed with six replicates for each pesticide and repeated to give n=3. Transepithelial electrical resistance (TEER) A549, Calu-3 and RPMI-2560 were seeded in sterile 6.5 mm transwell inserts with 0.4 μm pore polyester membranes, cells were cultured on membranes with and without a pre-coating of rat tail collagen. Prior to seeding the cells on the apical side of the membrane at a density of 100,000 cells/well, the membrane was pre-wetted for 30 min with fresh medium. Medium was removed from the apical membrane after two days, to take the cells to air liquid interface. A549 and Calu-3 cells were cultured for 11-14 days and RPMI-2650 for 20-22 days, with 500 μL fresh medium being added to the basolateral side every 2 days. TEER readings were taken every 2 days using an EVOM™ epithelial voltohmmeter with silver “chopstick” electrodes. Final TEER values were calculated by subtracting the resistance of blank/cell-free transwell inserts and correcting for the surface area of the insert. All experiments were repeated to give an n=3. Zonula Occludens-1 After the final TEER values for A549, Calu-3 and RPMI-2650 had been measured, the samples were stained to visualise expression of the tight junction protein zonula occludens-1 (ZO-1), and DAPI was used to stain the cell nuclei. The cell layers were incubated for 2 minutes on ice, with a solution containing; 0.2% triton-X 100 in 100 mM KCl, 3 mM MgCl2, 1 mM CaCl2, 200 mM sucrose and 100 mM HEPES. Cell layers were then rinsed twice with PBS, and permeabilised with 0.05% triton-X 100 in PBS for 5 minutes, rinsed twice with PBS and once with 5% powdered milk in PBS, then incubated with AlexaFluor 488 chicken anti-rabbit IgG 10μg/ml for 60 minutes at room temperature, before rinsing 4 times with PBS, staining with Prolog Gold antifade reagent with DAPI. Slides were mounted, and refrigerated until imaging within 1 week using an A1 inverted confocal microscope with spectral detector. Fluorescent emissions from DAPI (λex= 205 nm, λem = 430-480 nm) and AlexaFluor 488 (λex= 488 nm, λem = 510-570 nm) were collected using separate channels at a magnification of x40 and then the images obtained were overlapped to give a multicoloured composite image. Results shown depict a representative image from at least an n=2 of each sample.

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Figure 2. Cytotoxicity assay based on MTT absorbance, following 24-hour exposure to (A) Abamectin, (B) AcibenzolarS-Methyl, (C) Chlorothalonil, (D) Diquat, (E) Isopyrazam, (F) Pinoxaden and (G) Prosulfocarb. Data represented as mean ±SEM (n=3). Details of physicochemical properties of the selected pesticides are provided in the table.

Figure 3. Transepithelial electrical resistance over 14 and 22-days, for A549, Calu-3 and RPMI-2650, cultured on transwells pre-coated with rat tail collagen. Data represented as mean ±SEM (n=2) Representative confocal microscopy images of ZO-1 fluorescently stained with AlexaFluor 488 and cell nuclei stained with DAPI, using A549, Calu-3 and RPMI-2650 cells grown on blank or collagen pre-coated transwells at air-liquid interface. (n=2)

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A549 and RPMI-2650 cells gave similar dose-response curves when exposed to the pesticides. Crucially, there was evidence of differences in cytotoxicity between the different pesticides, with the broad spectrum fungicide chlorothalonil, which is a known respiratory irritant in vivo[5] being the most cytotoxic in the in vitro model. The observed cytotoxicity in vitro corresponds to that in vivo with the known mechanism for the respiratory irritancy of chlorothalonil being linked to localised necrosis following inhalation. In contrast, Acibenzolar-S-Methyl was not significantly cytotoxic at <150 μM, a threshold that would not realistically be expected to occur in an occupational exposure setting therefore suggests a low risk of toxicity. The lack of observed cytotoxicity for this fungicide is likely to do its mode of action, which works by activating the plants systemic activated resistance (SAR) response, to stimulate its natural defence mechanisms, in contrast chlorothalonil reduces glutathione and inhibits key metabolic processes within fungi. However the in vitro LC50 values, for all of the pesticides assessed in this study are many magnitudes higher than those previously described using rodent nasal inhalation models. This suggests that a more sensitive in vitro model of cytotoxicity is required, potentially with a focus on oxidative stress or cytokine release. Alternatively, the use of primary cells or human non-cancer derived cell lines, such as those that are “normal” immortalised cell lines, may also have improved sensitivity to pesticide toxicity. RPMI-2650 and Calu-3 may be particularly useful for the assessment of transepithelial permeability. As shown in Error! Reference source not found., both may be cultured to give physiologically relevant transepithelial electrical resistance values, which suggests that they may form similar barriers to inhaled xenobiotics in vivo. After being cultured for 22 days on transwell inserts RPMI-2650 had a TEER value of 45.4±1.5Ωcm2, with measurements of excised human/animal nasal mucosa being approximately 40-120Ωcm2[6]. Although higher TEER values for RPMI2650 can be achieved by altering the culturing conditions, for the purposes of occupational exposure risk assessment, a slightly weaker barrier that may overpredict pesticide permeability is favourable to a less permeable barrier than may underpredict the risk of systemic absorption. Calu-3 TEER values were similar to those previously suggested by other researchers and also to previous in vivo and ex vivo models[7]. Conversely, under these culturing conditions A549 did not form TEER values representative of what may be expected in vivo, with A549 generally being less suitable for permeability studies[8]. The TEER values measured in this study correspond relatively well to the confocal images taken. The images for Calu-3 show clear and well defined tight junctions, which relate to the good barrier function and high TEER values of this in vitro model. In contrast to this, whilst A549 and RPMI-2650 do express the ZO-1 protein, as has been previously described by other researchers[8, 9], the images taken in this study show that clear well defined tight junctions with ZO-1 are absent in these models. Although the presence/absence of organised ZO-1 for the formation of tight junctions is just one factor in the barrier function of the epithelial membrane, as shown from the TEER values for these cell lines it is likely to be an important one. Conclusion This study demonstrated that Calu-3 and RPMI-2650 possess characteristics that make them suitable for development as in vitro models for the toxicological assessment of exposure to aerosolised pesticides. An expanded suite of toxicity assays is required to develop a matrix of cell lines and endpoints for evaluation using the selected pesticides. These findings provide a promising starting point for the development of models based on human cell lines for the purpose of inhaled occupational exposure risk assessments. References 1.

EFSA, G.O., Guidance on the assessment of exposure of operators, workers, residents and bystanders in risk assessment for plant protection products. EFSA J, 2014. 12(10): p. 3874.

Backman, P., et al., Advances in experimental and mechanistic computational models to understand pulmonary exposure to inhaled drugs. Eur J Pharm Sci, 2018. 113: p. 41-52.

Jarvis, I.W., et al., Genotoxicity of fine and coarse fraction ambient particulate matter in immortalised normal (TT1) and cancer‐derived (A549) alveolar epithelial cells. Environmental and molecular mutagenesis, 2018. 59(4): p. 290-301.

Klein, S.G., et al., Endothelial responses of the alveolar barrier in vitro in a dose-controlled exposure to diesel exhaust particulate matter. Particle and fibre toxicology, 2017. 14(1): p. 7.

MESHRAM, N., A. SINGH, and R. SHRIVASTAVA, ACUTE HUMAN LETHAL TOXICITY EFFECTS OF SOME PESTICIDE FAMILIES. Journal of Industrial Pollution Control, 2014. 30(2).

Kreft, M.E., et al., The Characterization of the Human Nasal Epithelial Cell Line RPMI 2650 Under Different Culture Conditions and Their Optimization for an Appropriate in vitro Nasal Model. Pharmaceutical Research, 2015. 32(2): p. 665-679.

Sakagami, M., In vivo, in vitro and ex vivo models to assess pulmonary absorption and disposition of inhaled therapeutics for systemic delivery. Advanced Drug Delivery Reviews, 2006. 58(9): p. 1030-1060.

Togami, K., et al., Evaluation of permeability alteration and epithelial–mesenchymal transition induced by transforming growth factor-β1 in A549, NCI-H441, and Calu-3 cells: Development of an in vitro model of respiratory epithelial cells in idiopathic pulmonary fibrosis. Journal of Pharmacological and Toxicological Methods, 2017. 86: p. 19-27.

Mercier, C., N. Perek, and X. Delavenne, Is RPMI 2650 a Suitable In Vitro Nasal Model for Drug Transport Studies? European Journal of Drug Metabolism and Pharmacokinetics, 2018. 43(1): p. 13-24.

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Drug Delivery to the Lungs, Volume 29, 2018 - Zarif M. Sofian et al. Cyclodextrin-stabilised ion-pair complexes enhance drug lung uptake via the polyamine transporter Zarif M. Sofian1, Julie T. W. Wang1, Yuan L1, Paul Royall1, Ben Forbes1, David J. Barlow 1, Clive Page 1,2, Khuloud T. Al-Jamal1, Stuart Jones1 & Faiza Benaouda1 1

School of Cancer and Pharmaceutical Science, King’s College London, 150 Stamford Street, London, SE1 9NH Sackler Institute of Pulmonary Pharmacology, King’s College London, 150 Stamford Street, London, SE 1 9NH

Summary Ion-pairs comprised of a drug (theophylline) and an active transporter substrate (spermine) have been shown in previous work to modulate drug transport into pulmonary cells via the polyamine transport system (PTS). However, the rapid ion-pair breakdown after dose administration (~2 min post dose application) resulted in the transport effects being short-lived. The aim of this work was to investigate if the formation of an ion-pair cyclodextrin inclusion complex could physically stabilise the theophylline-spermine ion-pair and prolong its enhanced uptake in the lung. The theophylline-spermine ion-pair formation was confirmed using Fourier transform infrared spectroscopy and the 1:1 ion-pair/cyclodextrin complex formation was characterised using NMR and 3-D modelling. In vitro, using A549 cells, the uptake of theophylline was increased 1.8-fold when it was formulated as the ion-pair compared to theophylline alone, but this effect decreased with time due to ion-pair dissociation. Complexation of the ion-pairs with cyclodextrins increased the ion-pair’s lifetime and enabled the ~2-fold cell uptake enhancement to be maintained over 20 min. When the cyclodextrin-stabilised ion-pair complex was administered in vivo (as a bolus intravenous injection), the theophylline lung concentration was 2.5-fold higher compared to theophylline alone, but the distribution of the complex in other tissues remained unchanged. The in vitro and in vivo data together suggested that a cyclodextrin stabilised theophylline-spermine ion-pair complex could use the PTS to target delivery to the lung after intravenous infusion, an effect that could be beneficial in the treatment of acute respiratory crisis. Key Message Formation of an ion-pair/cyclodextrin inclusion complex containing a polyamine transporter targeting moiety was able to specifically enhance the amount of drug delivered into the lung after intravenous administration. Introduction The polyamine transporter system (PTS) has been identified in a number of mammalian cells, but is predominantly expressed in the pulmonary tissue [1]. It transports polyamines in a saturable, carrier-mediated, time, temperature, pH, energy and concentration-dependent manner [2]. In the lung, the PTS displays a high capacity to sequester not only naturally occurring polyamines but also certain xeniobiotics, such as paraquat, into the pulmonary tissue [1,3]. Therefore, the PTS offers a potential means by which to target drug delivery to the lungs. Structure-activity studies have indicated that for a xenobiotic to be a good PTS substrate it requires two or more N-containing moieties that are protonated at physiological pH 7.4. Using this information, a number of polyamine drug analogues and covalent polyamine drug conjugates have been developed that attempt to use the PTS to target delivery to the lungs [4-6]. However, these studies have shown limited in vivo success because retaining the molecular features required for effective PTS-mediated active transport whilst not disturbing the drug’s pharmacological action has thus far proven to be problematic [4-6]. An alternative approach to drug structural modification, which could be used to target drug delivery to the lung via the PTS is to form non-covalent polyamine complexes such as ion-pairs. Ion-pairing is the spontaneous association of two oppositely charged molecules ((A-) and (B+)), in the solution state, to form a temporarily bound complex (A-B+). The formation of ion-paired complexes has been shown to alter the uptake of ionised molecules into the skin [7], across the nasal mucosa [8] and across the intestinal mucosa [9] through passive diffusion, but there is very little work that investigates the use of ion-pairs in the lung. In our previous work it was shown that the formation of an ion-pair between a polyamine substrate for the PTS, spermine, and a model drug, theophylline, enhanced drug uptake into pulmonary cells. However, the enhancement was short-lived (~2 min) due to the physical instability of the ion-pair post-dose administration [10]. The aim of the present study was to investigate the effect of forming a theophylline-spermine ion-pair cyclodextrin inclusion complex on the ability of the ion-pair to be delivered to the lung. The interaction between the ion-pair and cyclodextrins was tested using NMR and the effect of complex formation on theophylline cell uptake was tested using human alveolar epithelial cells (A549). This immortalised cell line provided a robust in-vitro model in which polyamine transport has been well characterised [2]. The capacity of the polyamine ion-pair to sequester theophylline to the lung was then assessed in vivo in rats following the intravenous injection of the ion-pair complex.

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Drug Delivery to the Lungs, Volume 29, 2018 - Cyclodextrin-stabilised ion-pair complexes enhance drug lung uptake via the polyamine transporter Experimental methods Materials Theophylline (anhydrous, ≥ 99%), spermine (≥ 99%), hydrochloric acid (HCl), cyclodextrins (beta-cyclodextrin, ßCD; hydroxylpropyl-beta-cyclodextrin, HP-ß-CD and gamma-CD, γ-CD) and Krebs buffer salts (NaCl, KCl, CaCl2, MgSO4, NaHCO3, KH2PO4, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and D-glucose) were supplied by Sigma Aldrich, UK. Methanol (high performance liquid chromatography (HPLC) grade and Optiphase “Safe” scintillation cocktail were obtained from Fisher Scientific International, UK. Heparin was purchased from Movianto UK Ltd, saline (0.9 %w/v) from Baxter, UK and tissue solubilizer Soluene-350, was obtained from PerkinElmer, UK. 8-14C theophylline (60 mCi/mmol, > 98%) and 1,4-14C spermidine trihydrochloride (3.7 mCi/mmol, > 98 %) were obtained from American Radiochemicals, USA. A549 cells were obtained from ATCC, USA and all reagents for the cell culture (Minimum Essential Medium Eagle, fetal bovine serum (FBS), Lglutamine, non-essential amino acids, gentamicin, trypsin-EDTA (0.25% trypsin, 0.05% EDTA), Hank’s Balanced Salt Solution, trypan blue and Triton X), and deuterated water were all purchased from Sigma. Ion-pair Characterisation Theophylline-spermine ion-pair formations was confirmed using using solution-state FTIR and the apparent affinity constant was determined as was described previously [10]. A universal transmission cell system (OmniCell, Specac Ltd., UK) fitted with CaF2 windows and a 25 m Mylar spacer (Specac Ltd., UK) was used for transmission measurements of the theophylline-spermine (molar ratios ranging from 1:0 to 1:10) in water (at pH 9.6 ± 0.2). Deuterated water (D2O) was employed in the measurements as it allowed accurate detection of theophylline absorption bands in the 1700-1500 cm-1 range. All infrared spectra were recorded using a Spectrum One spectrometer (Perkin Elmer Ltd., UK) and spectral analysis was performed with Spectrum version 10 software (Perkin Elmer Ltd., UK). Theophylline-spermine/cyclodextrin inclusion complexes were formulated and a continuous-variation method (Job’s plot) was employed to establish the stoichiometry of these complexes. This was determined by assessing a series of solutions containing mixtures of β-CD, HPβCD and γ-CD with theophylline-spermine ion-pair at different CD-ion-pair molar ratios using 1H-NMR. The total concentration of the interacting species [CD and (theophyllinespermine)] in the solutions was kept constant at 5 mM. The mole fraction (r) of [CD]/[CD mixture] varied in the range of 0.1 to 0.9. The final pH of all mixtures was kept at 9.6 ± 0.1. All 1H-NMR spectra were acquired at 300 ± 0.1 K using a Bruker Advance 400 MHz spectrometer with a broad band inverse probe equipped with x, y and z gradients. Using the 3D molecular docking technique, the possible structure of the complex was determined. Modelling of the ternary inclusion complex was carried out using HyperchemTM and the structure was optimised using Polak-Ribiere conjugate gradient minimisation of the potential energy to an rms gradient of 0.001 kcal/(mol.Å). Images were generated using Discovery Studio Visualizer (Accelrys Inc, CA, USA). Cell uptake A549 human alveolar epithelial cell line was cultured as described previously in Benaouda 2018 [10]. The PTS has been established to be functional under the current study conditions by assessing spermidine uptake kinetics (Km at 0.5 ± 0.1 M and Vmax at 1.6 ± 0.2 pmol.min-1 per 105 cells) [10]. In the current work, uptake studies were initiated by the addition of either theophylline control, theophylline-spermine ion-pair or theophylline-spermine ionpair complexed with -cyclodextrin, 2-hydroxypropyl- -cyclodextrin and -cyclodextrin to the HBSS submerged cells. After an incubation period ranging from 2-20 min, the experiment was stopped and a full mass balance performed. Each condition was tested in triplicate using three different passages of cells. Cell uptake following the application of theophylline alone and theophylline-spermine was conducted at both 37°C and 4°C. In vivo theophylline lung uptake Theophylline control, theophylline-spermine ion-pair and theophylline-spermine ion-pair/b-cyclodextrin complex were injected intravenously into the tail of Wistar Rats. After 10 min, the animals were anesthetized by intraperitoneal injection of pentobarbital sodium and the major organs were collected for the measurement of theophylline concentration (all organs were washed out through perfusion via the heart). 14C-theophylline was extracted from the lungs, kidney, spleen, liver, brain, blood and muscle tissue and quantified by scintillation counter. The intravenous route was selected for dosing the theophylline ion-pairs as this mimics the use of theophylline for the treatment of acute bronchoconstriction in the clinic. Results and Discussion The FTIR data generated in this study showed that theophylline and spermine formed ion-pairs at pH 9.6 through N1 amine of spermine and the C-N7 of theophylline (data not shown). The ion-pair was possibly also stabilised through a second hydrogen bond between spermine N2 and the theophylline C6=O. The apparent affinity constant (pKapp) was calculated as 0.91 ± 0.1. The affinity constant of the theophylline spermine ion-pair provided support to the hypothesis that the ion-pair complex had two points of interaction because it was held together more strongly than electrolytes held by a single electrostatic interaction such as KCl (pKapp is -0.7)[11].

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Drug Delivery to the Lungs, Volume 29, 2018 - Zarif M. Sofian et al. The NMR data was used to construct a Job’s plot (data not shown), which suggested that the stoichiometry of the theophylline-spermine/cyclodextrin complexe was 1:1. The structural modelling data indicated that only ~10% of the volume of the spermine, and ~10% of the volume of the theophylline was inserted into the cyclodextrin cavity upon complexation (Figure 1a). Full insertion of the ion-pair into the cyclodextrin was not possible because the sum cross-sectional areas of theophylline and spermine (~28 Å2,theophylline) and 25 Å2,spermine)) was greater than that of the cyclodextrin cavity (42 Å2). Theophylline uptake into A549 cells equilibrated at 2 min. The presentation of theophylline to the cells as the theophylline:spermine ion-pair increased the drug’s uptake at 2 min by ~1.8-fold (~75% increase) compared to theophylline alone at 37 °C (Figure 1b). Reducing the temperature from 37°C to 4°C negated the ion-pair enhancement effect on theophylline uptake into the cells (data not shown), which indicated that the uptake could be energy-dependent. The lack of uptake enhancement beyond 2 min suggested that the ion-pair dissociated rapidly upon mixing with Krebs buffer. The ion-pair was formulated at pH 9.6 and when it was dispersed in the Krebs the pH drop reduced the theophylline ionisation and probably drove the ion-pair break down. However, the administration of the ion-pair as a cyclodextrin complex to the cells resulted in the theophylline cell uptake enhancement being maintained over the full 20 min experiment period (Figure 1b). The cylodextrin alone had no effect on the cells in pre-treatment studies (data not shown) and thus the cell uptake data suggested that the formation of the ion-pair/cyclodextrin inclusion complex increased the ion-pair lifetime to sustain the increase in the cell uptake of theophylline. The PTS is a poorly characterised transporter, but it is thought to be responsible for the sequestering of polyamines into cells via active transport. In this work, the mechanism of uptake was not investigated and thus it was not possible to determine if cyclodextrin played any role in the drug transport beyond stabilising the ion-pair complex. It was evident however from the enhancement of drug uptake that the ion-pair was moving into the cells as an intact complex, which presumably broke down once it moved away from the high concentration of ion-pair polyamine counterions placed on the cell surface in the experiment into the relatively polyamine deficient cell interior.

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Figure 1- (A) a representation of a 1:1 theohylline-spermine ion-pair/β-cyclodextrin complex in water at pH 9.6. Atoms coloured blue = nitrogen, red = oxygen, white = hydrogen and dark grey = carbon. B) percentage increase of the total accumulated theophylline in the cells following the application of (i) theophylline-spermine ion-pair (THE-SP) (2.78:55.6 µM; 1:20 molar ratio) and (ii) THE-SP complexed with -cyclodextrin (GCD:THE:SP, 2.78:2.78:55.6 µM; 1:1:20 molar ratio), 2-hydroxypropyl--cyclodextrin (HPBCD:THE:SP, 2.78:2.78:55.6 µM; 1:1:20 molar ratio) and -cyclodextrin (BCD:THE:SP, 2.78:2.78:55.6 µM; 1:1:20 molar ratio) onto HBSS-submerged cells. The increase percentage was calculated using the total accumulation of free theophylline. All solutions were prepared in water pH adjusted to 9.6 (n=3 + SD).

The intravenous administration of the theophylline-spermine ion-pair in vivo was not found to alter the level of theophylline deposited in the lung tissue (p > 0.05). This was presumably due to rapid ion-pair dissociation upon entering the systemic circulation. Unlike in previous work using an isolated perfused lung (IPL) system, where the ion-pair was continuously injected into the perfusate to elicit effect [10], in the in vivo studies the theophyllinespermine dose was injected intravenously as a bolus injection into the rat tail and this change in delivery kinetics could explain the inconsistent results across the ex-vivo and in-vivo models. However, in addition, the ion-pair probably experiences more ‘binding’ competition in vivo where both plasma protein binding and the active cellular process could have been actively trying to separate the ion-pair. The intravenous (bolus) administration of the theophylline-spermine/-cyclodextrin complex in vivo resulted into a 2.5-fold increase (p < 0.05) in theophylline concentration in the lung after a 10 min washout period (Figure 2). The data supported the in vitro cell work which suggested that the cyclodextrin complex formation maintained the integrity of the ion-pair. In vivo the consequences of the increased ion-pair physical stability was that the ion-pair survived the blood circulation and reached the PTS in the lung in-tact, wherein, the theophylline-spermine was actively transported into the lung.

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THE Figure 2- In-vivo biodistribution50profiles following a slow intravenous injection of theophylline control, theophyllineTHE-SPand theophylline-spermine ion-pair/-cyclodextrin complex (BCD-THEspermine ion-pair (THE-SP) (1:20 molar ratio) BCD-[THE-SP] SP): (1:20:1 molar ratio). *significantly different when compared to control group (p < 0.05) (n=3 ± SD). 40 % ID (14C-theophylline) /organ

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Conclusion 30 3 The findings from the study indicate that2 therapeutic ion-pairs formed with a PTS substrate can increase uptake specifically in lungs after intravenous administration. Enhancing ion-pair lifetime using cyclodextrin was able to 20 1 translate the in vitro ion-pair effects on lung* cell uptake into lung targeting in vivo. This positive data suggests that 0 the use of non-covalently bound PTS targeting moieties on therapeutic agents warrant further investigation. The 10 non-covalent targeting approach offers opportunities not afforded by covalent targeting because the release of active in its original form in the tissue is very efficient. This mitigates the toxicity problems faced when the covalent linkage is not broken, 0i.e., a new chemical entity is formed and the poor pharmacological activity of the complexes when the targeting moiety is cleaved from the drug slowly. As a result it is envisaged that therapeutic ion-pairs that target the lung could be particularly useful when the subsequent pharmacological action needs to be immediate, for example, in critical care. Hoet P H M, Nemery B: Polyamines in the lung: polyamine uptake and polyamine-linked pathological or toxicological conditions, Am. J. Physiol. Lung Cel. Mol Physiol 2000; 278: pp L417-L433. Cullis P M, Green R E, Merson-Davies L, Travis N: Probing the mechanism of transport and compartmentalisation of polyamines in mammalian cells, Chem Bio 1999; 6: pp717–729. Rose M S, Smith L L, Wyatt I: Evidence for energy dependent accumulation of paraquat into rat lung, Nature 1974; 252: pp 314-315. Marton L J, Pegg A E: Polyamines as targets for therapeutic intervention, Annu Rev Pharmacol Toxicol 1995; 35: pp55-91. Tomasi S, Renault J, Martin B, Duhieu S, Cerec V, Le Roch M, Uriac P, Delcros J G: Targeting the polyamine transport system with benzazepine- and azepine-polyamine conjugates, J Med Chem 2010; 53: pp7647-7663. Wang C J, Delcros J G, Cannon L, Konate F, Carias H, Biggerstaff J, Gardner R A, Phanstiel O: Defining the molecular requirements for the selective delivery of polyamine conjugates into cells containing active polyamine transporters, J Med Chem 2003; 24: pp5129-5138. Neubert R: Ion pair transport across membranes,Pharmaceutical Research 1989; 6: pp743-747. Hatanaka T, Kamon T, Morigaki S, Katayama K, Koizumi T J J: Ion pair skin transport of a zwitterionic drug, cephalexin, J Control Release 2000; 66: pp63-71. Ivaturi V D, Kim S K: Enhanced permeation of methotrexate in vitro by ion pair formation with L-arginine, J Pharm Sci 2009; 98: pp3633-3639. Benaouda F, Jones S A, Chana J, Dal Corno B M, Barlow D J, Hider R C, Page C P, Forbes B: Ion-pairing with spermine targets theophylline to the lungs via the polyamine transport system, Mol Pharm 2018; 15: pp861-870. Martell A E, Smith R M: Critical stability constants. Plenum Press, New York, 1989. Sofian Z M: Evaluating drug delivery to the lung using polyamine ion-pair, King’s College London, PhD Thesis, 2018.

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Drug Delivery to the Lungs, Volume 29, 2018 – Precious Akhuemokhan et al. Developing alternative models for in vitro investigation of excipient influence on drug transport Precious Akhuemokhan1, Magda Swedrowska1, Josie Williams1, Richard Harvey2, Ben Forbes1 1

Institute of Pharmaceutical Science, School of Cancer and Pharmaceutical Sciences, King’s College London 2Institut

für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany

Summary This study explored the use of the Parallel Artificial Membrane Permeability Assay (PAMPA) to study the effect of excipients on the disposition of inhaled drugs. The main aim was to compare a PAMPA with the Calu-3 cell layer model by assessing the effect of glycerol on the permeability of radioactive small molecules utilised as probes for different routes of transport. The probes were a hydrophilic compound 14C-Mannitol for paracellular transport and the hydrophobic probes 3H-propranolol for transcellular transport. Results showed that the transport of both probes in the cell model was affected even at glycerol concentrations of 1% v/v and 2% v/v. Altered barrier permeability was evidenced by an increasing flux of mannitol due to disruption of tight junctions at these concentrations. In contrast, there was a reduction in propranolol permeability suggesting an effect of glycerol to reduce transcellular transport. PAMPA proved to provide suitable barrier properties as there was significant difference between the apparent permeability (Papp) of probes in the empty and PAMPA wells of the experiments. Further work will be carried out with PAMPA using higher glycerol concentrations (>5% v/v) as minimal effect on permeability was observed at concentrations up to 2% v/v in the PAMPA. Key Message This study aimed to compare PAMPA (Parallel Artificial Membrane Permeability Assay) to an established Calu-3 technique for evaluating the effect of glycerol on drug permeability. Whilst it was shown that glycerol at concentrations > 2% were detrimental to Calu-3 layer tight junction integrity, the PAMPA provides an alternative model for studying transport across lipid membranes. Introduction Studies of aerosols emitted from beclomethasone diproprionate (BDP) pressurised metered dose inhaler formulations and glycerol-free formulations have reported differences in the absorptive profiles of drug after deposition on respiratory epithelial cell models (1). Even formulations which were subsequently formulated to be aerodynamically equivalent and differ only in glycerol content produced differences in drug transport across epithelial cell lines in vitro (2). Calu-3 cells provide an established system for investigating the disposition of drug compound and excipients across epithelial cell layers in vitro and provide permeability data that correlates well with in vivo lung absorption data (3,4). The efficacy of inhaled steroids is dependent on penetrating lung tissue membranes which is in turn partly governed by the permeability of the drug. PAMPA provides an alternative platform to cell models and ex vivo techniques to evaluate the passive transcellular transport of drug molecules across a membrane (5). PAMPA has already been utilized as transport membrane surrogates for gastrointestinal delivery (GI-PAMPA) and delivery to and through the blood brain barrier (BBBPAMPA) (6,7). In this assay, first described by Kansy et al (8), an artificial membrane separates two compartments; donor and receiver compartments and consists of phospholipids and or lipids that produce bilayer structures which closely mimic the cell membranes of the absorptive surfaces of the gastrointestinal tract and blood brain barrier respectively. The drug is applied to the donor compartment and the flux through the membrane to the receiver compartment is evaluated. In addition to the cellular model described above to investigate the effect of glycerol on BDP passive transcellular transport, the parallel artificial membrane permeability assay (PAMPA) is proposed as a complementary technique in which to test the hypothesis that glycerol modifies drug permeability through an effect on phospholipid membrane stiffness (9). Materials All materials, unless otherwise specified were purchased from Sigma Aldrich, UK. Glycerol was purchased from Severn Biotech Ltd. Liquid scintillation cocktail was obtained from Fisher Chemical. Transwell plates with 6.5 mm diameter and 0.4 µm pore size were purchased from Coring Costar Incorporated NY. PAMPA solution consisted of POPC (-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) from Avanti polar lipids and cholesterol in dodecane. The molar ratio of lipid to cholesterol was 2:1 Cell culture Calu-3 cells were seeded at a density of 2 × 10 5 cells/well onto 0.4 μm pore size, 0.33 cm2 surface area polyester Transwell cell culture inserts (Corning), placed in 24-well cell culture plates (Corning). Air-liquid interface (ALI) culture was established by removing the medium from the apical compartment 48 h after seeding and incubated under standard conditions of 37C, 95% natural atmosphere, 5% CO2, 100% humidity. The medium in the basal compartment was changed every 2-3 days throughout the 14-day culture period to allow cells to form a confluent and functional mucociliary barrier.

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Drug Delivery to the Lungs, Volume 29, 2018 - Developing alternative models for in vitro investigation of excipient influence on drug transport Transport experiment. On the day of the experiment, the cell inserts were incubated for 30 minutes with HBSS (Hank’s balanced Salt Solution) and the untreated TEER was measured. This was done by preparing different glycerol concentrations test solutions via dilutions with HBSS which were then ‘spiked’ with 3H-propranolol (1:1000) and 14C-Mannitol (1:1600) before being used as ‘donor’ solutions. Transport studies were carried out in the apical to basolateral direction. Immediately after the introduction of donor solution to the apical chamber of the well 50 µL of the test solution was removed to determine the initial starting concentration. At each time point (30, 60, 90 and 120 min), 200 µL of sample was removed from the basolateral chamber and transferred to a scintillation vial. These samples were immediately replaced with equal volumes of warmed HBSS to maintain a constant volume in the receiver compartment. Theeffect platesof where shaken between time-points using a Wesbart orbital shaker at 50 rpm. At the end 3.2 Studying the effect of glycerol on cell tight junctions 3.2 Studying the glycerol on cell tight junctions 3.2 Studying the effect of glycerol on cell tight junctions 3.2 Studying the effect of glycerol on cell tight junctions 3.2 Studying the effect of glycerol on cell tight junctions 3.2 the effect of glycerol on cell tight junctions of Studying the experiment, liquid scintillant was added to the samples which were immunostaining then quantified viawith scintillation counting ToTo further investigate the effect on the tight junctions, anti-ZO1, To investigate the effect ofof glycerol on the tight junctions, immunostaining with anti-ZO1, thethe further investigate the effect ofglycerol glycerol on the tight junctions, immunostaining with anti-ZO1, the Tofurther further investigate the effect of glycerol on the tight junctions, immunostaining with anti-ZO1, the using afurther LS6500 Beckman Coulter Multipurpose scintillation counter (Beckman, UK). To To further investigate investigate the the effect effect of of glycerol glycerol on on the the tight tight junctions, junctions, immunostaining immunostaining with with anti-ZO1, anti-ZO1, the the tight junction protein, was performed onon Calu-3 cell layers after incubation with increasing junction protein, was performed on Calu-3 cell layers after 2 2hours incubation with increasing tight junction protein, was performed Calu-3 cell layers after 2hours hours incubation with increasing Fortight the PAMPA, 10 L of the solution was applied to the membrane and allowed to equilibrate at room temperature tight junction protein, was performed on Calu-3 cell layers after 2 hours incubation with increasing tight tight junction junction protein, protein, was was performed performed on on Calu-3 Calu-3to cell cell layers layers after after 22effect hours hoursbetween incubation incubation with with increasing increasing concentrations ofglycerol glycerol HBSS. There appears be decreasing postof ininin HBSS. There appears be a correlative effect between thethe decreasing postconcentrations of glycerol in HBSS. There appears todescribed bea acorrelative correlative effect between the decreasing postforconcentrations 30 min. Permeability experiments were carried outto as for the cell layers above. concentrations of glycerol HBSS. There appears to be acorrelative correlative effect between the decreasing postconcentrations concentrations of of glycerol glycerol in in HBSS. HBSS. There There appears appears to to be be a a correlative effect effect between between the the decreasing decreasing postpost2A) and the disappearance of tight junctions between cells experimental TEER readings (figure experimental TEER readings (figure 2A) and the disappearance of tight junctions between cells with experimentalTEER TEERreadings readings (figure (figure 2A) 2A) and and the the disappearance disappearance of of tight tight junctions junctions between between cells cellswith with experimental with 2A) and the disappearance disappearance of tight tight junctions junctions between cells cells with with experimental experimental TEER TEER readings readings (figure (figure 2A) and the of between increased diffuse staining throughout the cytoplasm from the breakdown tight junctions (figure 2B). Immunostaining cell layers for imagingthe increased diffuse staining throughout cytoplasm from the breakdown ofof tight junctions (figure 2B). increased diffuse staining throughout the cytoplasm from the breakdown of tight junctions (figure 2B). increaseddiffuse diffusestaining stainingthroughout throughoutthe the cytoplasm from the breakdown tight junctions (figure 2B). increased diffuse staining throughout the cytoplasm from the breakdown ofof tight junctions (figure 2B). increased cytoplasm from the breakdown of tight junctions (figure 2B). Calu-3 cell layers were stained for ZO-1 (rabbit anti-zona occludens-1, Zymed, Cambridge Bioscience) on day 14 In figure 2C the cell layers also appear bebe less confluent with big gaps throughout the glycerol In figure 2C the cell layers also appear tototo be less confluent with big gaps throughout as as the glycerol In figure 2C the cell layers also appear less confluent with big gaps throughout as the glycerol figure 2Cthe thecell celllayers layersalso also appear beless lessconfluent confluent with big gaps throughout the glycerol InInfigure figure 2C the cell layers also appear toto be less confluent with big gaps throughout asas the glycerol In 2C appear to be with big gaps throughout as the glycerol in content culture toincreases, visualise expression of the tight junction protein. Pre-extraction step: firstly, a just pre-extraction buffer was content increases, however these images are from one single experiment and may from however these images are from one single experiment and may just bebe artefacts from content increases, however these images are from one single experiment and may just beartefacts artefacts from content increases, however these images are from one single experiment and may just be artefacts from applied to increases, the cell layers for 2 minutes then washed offsingle with PBS. The cell layers were then fixedfrom in 4 % content increases, however these images are from one single experiment and may just be artefacts from content however these images are from one experiment and may just be artefacts the fixing and staining process. the fixing and staining process. the fixing and staining process. the fixing and staining process. paraformaldehyde on ice process. for 20 minutes.  After several washes in PBS, the cells were prepared for staining by the fixing and staining the fixing and staining process. permeabilizing with 0.05 % Triton X-100 in PBS for 5 minutes then washed again in PBS and BLOTTO solution. A)The A) B)B)B) and applied to cells overnight at A) primary antibody anti-zona occludens-1 was prepared in BLOTTO solution A) A) B)B) antibody (Alexa Fluor 488  chicken A) B) 4 ̊C.  The following day cells were washed 3x with PBS then the secondary anti-rabbit, Invitrogen) was prepared in BLOTTO solution at 10 μg/mL and applied to cells  for 1 h at RT in the dark.  The cell layers were washed several times in PBS then the membrane was cut from the transwell  insert and 2%2% 5%5% 2% 5% placed apical side up on microscope slides.  One drop of Prolog Gold antifade reagent with DAPI 2% 5%(Invitrogen) was 2% 2% 5% 5% applied to each membrane  then glass coverslips were mounted onto the membranes and sealed with clear nail polish once dry. Slides were stored at RT, protected from light and imaged within a couple of weeks.   Results 7.5% 7.5% 7.5% probes10% 10% The effect of glycerol on membrane permeability was investigated using radioactive in10% cell layers and the 7.5% 10% 7.5% 10% 10% artificial system, PAMPA. Below are results from imaging and transport studies7.5% performed in the presence of varying amounts of glycerol.

C)C) C) C) C) C)

Figure 2. Effect of glycerol tight junctions. Graph Ashows shows the pre (0min) min) and post (120 min) experimental TEER Figure 2. of on tight junctions. Graph AA shows the pre (0(0 min) and post (120 min) experimental TEER Figure 2.Effect Effect ofglycerol glycerol onon tight junctions. Graph the pre and post (120 min) experimental TEER readings of Calu-3 cells treated with 0–10 –1010 %glycerol glycerol inHBSS. HBSS. Bars represent the mean of three independent readings of Calu-3 cells treated with 0 %Graph in Bars represent the mean of three independent Figure Figure 2.2. Effect Effect of of glycerol glycerol on on tight tight junctions. junctions. Graph AAshows the the pre pre (0 (0 min) min) and and post post (120 (120 min) min) experimental experimental TEER TEER Figure of glycerol on tight junctions. Graph Ashows shows the pre (0 min) and post (120 min) experimental TEER readings ofEffect Calu-3 cells treated with %glycerol inHBSS. Bars represent the mean of three independent Figure 1: 2. Confocal microscopy images of0–the cell layers captured at 40x magnification after treatment for 2 hours with experiments performed in duplicate error bars ±SEM. SEM. Image Bshows shows zoomed insection section from each the performed in duplicate with error bars ± ±SEM. Image B shows a zoomed in section from each of the readings readings of ofofCalu-3 Calu-3 cells cells treated treated with with 00with 10 %%imaged glycerol glycerol inin HBSS. HBSS. Bars Bars represent represent the the mean mean of ofof three three independent independent readings Calu-3 cells with 0––were –10 10 % glycerol in HBSS. Bars represent the mean three independent experiments performed intreated duplicate with error bars Image B a azoomed in from each ofofthe theexperiments various glycerol concentrations. These at 40x on the Nikon Eclipse Ti-E inverted confocal microscope. images of Calu-3 layers after immunostaining for the tight junction protein. highlight the green intact tight images of layers after immunostaining for the tight junction protein. Arrows highlight thethe green intact tight experiments experiments performed performed in inin duplicate duplicate with with error error bars bars ± ±tight SEM. SEM. Image Image BBprotein. shows aaArrows zoomed zoomed ininin section section from from each each of ofof the the experiments performed duplicate with error bars ± SEM. Image Bshows shows aArrows zoomed section from each the The tight junction protein, ZO-1 was fluorescently stained with AlexaFluor 488 (green) and cell nuclei was stained with images ofCalu-3 Calu-3 layers after immunostaining for the junction highlight green intact tight junctions whereas the stars (*) highlight areas of diffuse staining where the tight junctions are beginning totight break junctions whereas the stars (*) highlight areas of staining where the tight junctions arethe beginning to break DAPI (Blue). Scale bars = 25m images images of ofof Calu-3 Calu-3 layers layers after after immunostaining immunostaining for for the the tight tight junction junction protein. protein. Arrows Arrows highlight highlight the green green intact intact tight images Calu-3 layers after immunostaining for the tight junction protein. Arrows highlight the green intact tight junctions whereas the stars (*) highlight areas ofdiffuse diffuse staining where the tight junctions are beginning to break down. Image C shows representative images of the cell layers captured at40X 40X magnification after treatment down. Image CCshows representative images ofof the cell layers captured atthe 40X magnification after treatment for 2for22 junctions junctions whereas whereas the the stars stars (*) (*) highlight highlight areas areas of of diffuse diffuse staining staining where where the tight tight junctions junctions are are beginning beginning to toto break break junctions whereas the stars (*) highlight areas of diffuse staining where the tight junctions are beginning break down. Image shows representative images the cell layers captured at magnification after treatment for hours with relative glycerol concentration. hours with the glycerol concentration. down. down. Image Image Cthe Crelative shows shows representative representative images images of ofof the the cell cell layers layers captured captured at atat 40X 40X magnification magnification after after treatment treatment for for 22 2 down. Image C shows representative images the cell layers captured 40X magnification after treatment for hours with the relative glycerol concentration.

hours hours with with the the relative relative glycerol glycerol concentration. concentration. hours with the relative glycerol concentration.

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Drug Delivery to the Lungs, Volume 29, 2018 – Precious Akhuemokhan et al.

2.0

20 Papp x 10-6 (cm/s)

14C-Mannitol

3H-Propranolol

Papp x 10-6 (cm/s)

1.5

1.0 0.5

10 %

% 7. 5

10 %

5% 7.

0.0

Glycerol concentration % v/v

Figure 2: Effect of glycerol concentration on apparent permeability (Papp) of transport probes. Graphs A and B show the permeability of transport probes, mannitol (paracellular) and propranolol (transcellular), across Calu-3 cell monolayers after incubation glycerol solutions for 2 h. For both probes, each data point represents a single well from three or four independent experiments performed in at least duplicate. Bars represent the mean of the data with error bars ± SEM.

B: 3H-Propranolol

A: 14C-Mannitol Empty Transwell PAMPA Transwell

Empty Transwell

PAMPA Transwell

Papp x 10-6 (cm/s)

40 0

0 1%

Papp x 10-6 (cm/s)

Glycerol concentration % v/v

Figure 3: Graphs A and B show the permeability of mannitol and propranolol across PAMPA, the cell-free model with varying glycerol concentrations. Each well was incubated with glycerol in HBSS solution for two hours. Each data point represents a single well from three independent experiments performed in duplicate. Bars represent the mean of the data with error bars ± SEM.

Discussion Glycerol has been shown to exhibit effects on paracellular and transcellular routes of transport. The paracellular route appears to be more affected as there is disturbance of the tight junctions evidenced by the confocal images shown (Figure 1) with increasing glycerol concentration. There appears to be an increasing loss of tight junctions’ proteins (green lines) and defined boundaries with increasing glycerol concentration. This is also supported by progressive decline in TEER values from 0% v/v glycerol to 10% v/v glycerol (data not shown). Thus, whilst glycerol appeared to enhance transport of mannitol across cell layers, the change in paracellular permeability can be attributed to the effect on tight junctions. A reduced transport was evident for the transcellular probe propranolol (Figure 2B & 3B). This phenomenon requires further investigation but the observations are consistent with the hypothesis that glycerol has a stiffening effect on biological membranes which may retard drug transport(9). PAMPA (Figure 3) almost exclusively represents passive transcellular transport which makes it suitable for the study of transport of drugs like inhaled steroids that utilize transcellular route as their main mode of transport. Thus, for PAMPA to be considered an adequate model for transport, differential permeability between hydrophobic and hydrophilic probes must be established, much like the relative transport rates that would be obtained from an established cell model. In this study, it was shown that the PAMPA provided a barrier to transport as drug permeability for both mannitol and propranolol was significantly reduced (Figure 3 A&B) in comparison with the empty Transwell (p < 0.05). The effect of glycerol on propranolol transport, at concentrations up to 2% v/v, was less pronounced using PAMPA compared to Calu-3. These studies are on-going but they suggest the use of PAMPA as artificial membrane model complementary to cell culture studies.

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Drug Delivery to the Lungs, Volume 29, 2018 - Developing alternative models for in vitro investigation of excipient influence on drug transport Conclusions Glycerol concentrations > 5% v/v have been shown to compromise cell layer integrity to a point where data on transcellular transport obtained from such experiments would be unreliable as there is a possibility of drug leakage through the disrupted tight junctions. This study has shown some evidence for glycerol’s proposed stiffening effect on biological membranes with the Calu-3 model and artificial membranes with the PAMPA. Further work is required using higher glycerol concentrations on the PAMPA to confirm the findings described above. Nevertheless, these studies are a step towards establishing conditions and methods for the investigation of the effect of glycerol on the transport of BDP and, more generally, the effect of other excipients on lung permeability. Acknowledgements Special thanks to Zachary Enlo-Scott for the confocal imaging.

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Grainger CI, Saunders M, Buttini F, Telford R, Merolla LL, Martin GP, et al. Critical Characteristics for Corticosteroid Solution Metered Dose Inhaler Bioequivalence. Mol Pharm . 2012;9(3):563–9. Lewis DA, Young PM, Buttini F, Church T, Colombo P, Forbes B, et al. Towards the bioequivalence of pressurised metered dose inhalers 1: Design and characterisation of aerodynamically equivalent beclomethasone dipropionate inhalers with and without glycerol as a non-volatile excipient. Eur J Pharm Biopharm [Internet]. 2014;86(1):31–7. Florea BI, Cassara ML, Junginger HE, Borchard G. Drug transport and metabolism characteristics of the human airway epithelial cell line Calu-3. J Control Release [Internet]. 2003 [cited 2017 May 15];87(1):131-8. Grainger CI, Greenwell LL, Lockley DJ, Martin GP, Forbes B. Culture of Calu-3 Cells at the Air Interface Provides a Representative Model of the Airway Epithelial Barrier. Pharm Res [Internet]. 2006;23(7):1482–90. Available from: http://dx.doi.org/10.1007/s11095-006-0255-0 Avdeef A. The rise of PAMPA. Expert Opin Drug Metab Toxicol [Internet]. 2005;1(2):325–42. Mensch J, Melis A, Mackie C, Verreck G, Brewster ME, Augustijns P. Evaluation of various PAMPA models to identify the most discriminating method for the prediction of BBB permeability. Eur J Pharm Biopharm [Internet]. 2010;74(3):495– 502. Avdeef A, Artursson P, Neuhoff S, Lazorova L, Gråsjö J, Tavelin S. Caco-2 permeability of weakly basic drugs predicted with the Double-Sink PAMPA pKaflux method. Eur J Pharm Sci [Internet]. 2005 [cited 2017 May 16];24(4):333–49. Kansy M, Senner F, Gubernator K. Physicochemical high throughput screening: parallel artificial membrane permeation assay in the description of passive absorption processes. J Med Chem . 1998;41:1007–10. Terakosolphan W, Trick JL, Royall PG, Rogers SE, Lamberti O, Lorenz CD, et al. Glycerol Solvates DPPC Headgroups and Localizes in the Interfacial Regions of Model Pulmonary Interfaces Altering Bilayer Structure. Langmuir. 2018 Jun 12;34(23):6941–54.

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Drug Delivery to the Lungs, Volume 29, 2018 – Mariana F. Silva et al. Nasal Dry Powder Delivery: Implementing a formulation independent Spray Drying Process Mariana F. Silva1,2, Diana A. Fernandes1,2, Maria Braga1, António Eloy1, João Marques1, M. Luísa Corvo2 & Eunice Costa1 Hovione FarmaCiência SA, Lumiar, 1649-038, Portugal Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003, Portugal 1

Summary The aim of this study is to assess the interactions between formulation composition and spray drying (SD) process parameters in enabling nasal powder formulations for the delivery of a diverse array of biopharmaceuticals. A design of experiments was performed on the composition in trehalose and atomization flow rate in order to compare the properties of the generated powders by SD with a Two-fluid nozzle (2FN) atomizing system. Additionally, a SD with a 25 kHz Ultrasonic Nozzle (USN) to primarily assess the main differences in the properties of the powders obtained with two different atomizing systems was used. SD powders suitable for nasal delivery were successfully generated using the 2FN with particle size (Dv50) between 11 and 39 μm. Furthermore, there appears to be no impact of the formulation composition in the powders properties, providing the ability to adjust the ratio between trehalose and hydroxypropyl methylcellulose E5 (HPMC E5), depending exclusively on the active pharmaceutical ingredient (API) requirements. However, SD powder produced by USN was outside the nasal size range. Future work will be developed using the latter atomizing technology in order to assess the ability of this atomizing system to produce suitable nasal powders. Key Message Nasal powders were successfully produced via SD using an excipient matrix of Trehalose/HPMC E5 that can incorporate a vast array of biopharmaceuticals for local or systemic delivery. Surprisingly, the formulation composition did not have a significant impact in the powders properties, allowing the adjustment of the excipients ratio to exclusively meet the API requirements. Introduction The nasal delivery, which is commonly used in symptomatic relief, prevention and treatment of topical nasal conditions, is now currently being considered as an attractive route for the treatment of systemic diseases and for the administration of biopharmaceuticals. This route presents a large surface area available for drug absorption and a porous, thin and highly vascularized epithelium, which allows a rapid absorption and onset of therapeutic action while avoiding the hepatic first-pass metabolism and the degradation mechanisms of the gastro-intestinal tract. [1][2] Additionally, nasal route can allow direct delivery to the brain, while circumventing the obstacles of the bloodbrain barrier through the olfactory neuroepithelium and may involve transcellular, paracellular, and/or neuronal transport.[1] Although the majority of nasal products consist of sprays or drops and dry powder formulations are more frequently used in oral inhalation, the delivery of nasal powders presents a considerable set of advantages, the most evident one being the stability of solid dosage forms over liquids. The nose presents barriers to drug delivery such as mucociliary clearance, enzymes and the low volume of nasal cavity, with the latter having an impact on the amount of drug formulation that can be administered.[1][2] The human nose can hold 10 - 25 mg of powder per nostril per shot and the particle size of the powder must be in the range 10 - 45 μm, which can be narrowed to 20 -30 μm, according to Jüptner et al., for an adequate nasal deposition.[3][4] Formulation wise, nasal powders such as dry powder inhalers can include fillers, stabilizers, mucoadhesive agents and absorption enhancers depending on the API potency, solid state properties, site of action, amongst other requirements.[3] There is a considerable number of particle engineering technologies that can be employed to produce nasal powders. In that context, SD is a well-established, scalable and flexible technology, which allows a tight control over the key features that impact the performance of nasal powders through precise manipulation of the process parameters and the formulation itself.[5] Regarding the atomizing system, the 2FN produces a spray through the interaction between a liquid feed with a high velocity compressed gas stream. On the other hand, when using USN, a high frequency electrical input is converted into a vibratory mechanical motion that is amplified, producing standing waves along the length of the nozzle, and hence atomizing the feedstock into homogeneous droplets. The USN is known to generate a more homogeneous droplet size distribution than conventional external TFN.[6] The aim of this study is to assess the interactions between formulation composition and SD process parameters in enabling nasal powder formulations for the delivery of a diverse range of biopharmaceuticals and thus creating a platform. For that purpose, a small molecule was used as a model API, Naproxen, embedded in an excipient matrix suitable for nasal powders and later on relevant for delivering more complex biopharmaceuticals.[6] The formulation comprised trehalose as a water soluble filler and stabilizing glass former. Additionally, HPMC E5, a water-soluble mucoadhesive agent, was added to prolong the residence time in the nasal cavity of the formulation.

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Drug Delivery to the Lungs, Volume 29, 2018 - Nasal Dry Powder Delivery: Implementing a formulation independent Spray Drying Process Materials and Methods Experimental design: In order to assess the interactions between formulation composition and the main SD process parameter with influence on powders particle size, composition in trehalose and the atomization flow rate (Fatom) were the selected factors to study the properties of the powder obtained by SD using a 2FN atomizing system (Niro, GEA Group, Denmark). For the process variables, two levels (low and high) were set out. Within the formulation, the API concentration was kept constant at 0.01 %, trehalose ranged between 20.00 – 50.00 % and, therefore, HPMC E5 between 49.99 - 79.99 %. The lower level of trehalose was defined based on the minimum concentration required to stabilize a model biopharmaceutical used in previous work[6]. In addition, when the administration of macromolecules is required, the increase of the Figure 1 – Design of formulation's nasal residence time is particularly favourable; therefore, the Experiments for the 2FN lower concentration of HPMC E5 was considered based on this atomizing system assumption[3]. The Fatom, ranged between 0.54 and 0.86 kgh-1. Fatom and the 2 percentage of trehalose were adjusted in agreement with a 2 full factorial experiment (Figure 1), described in Table 1, and an additional center point, resulting in a total of five experiments. Drying gas flow rate (F drying), outlet temperature (Tout), feed flow rate (Ffeed) and solids concentration (Csolids) of the feed solutions were kept constant across the trials (Table 1). An extra trial was performed with a 25 kHz USN (Sono-Tek Corporation, USA) to primarily assess the main differences in the properties of the powders obtained with two different atomizing systems. The operating conditions and excipients concentration details are described in Table 1. All the experiments were executed using a customised lab-scale spray drier (Hovione, Portugal). Table 1 – Set of test conditions performed according to the defined design of experiments Atomizing system

2FN

USN

Test condition 1

Fatom (kg.h-1) 0.54

Trehalose (%)

HPMC E5 (%)*

20.00

79.99

50.00

49.99

0.86

0.54

0.86

0.68

35.00

64.99

20.00

79.99

Csolids (%)

Tout (ºC)

Ffeed (g.min-1)

Fdrying (kg.h-1)

16.7

8.4

*composition in HPMC E5 is a dependent factor of composition in trehalose (100% - % Trehalose).

SD Feed Solutions Composition: To obtain 50g of powder, the SD feed solutions were at 10% (w/w) and 5% (w/w) in a deionized water, for the 2FN and USN, respectively. SD Powder Characterization: The morphology of the dry powders was examined by Scanning Electron Microscopy (SEM) for particle shape and morphology using a JSM7001F FEG – SEM scanning electron microscope (JEOL, Japan). Particle size distribution (PSD) by laser diffraction was measured with a SYMPATEC (Sympatec Inc., USA). Bulk Density was assessed by weighing the powder volume of 12.5 mL in a measuring cylinder. The solid state properties were analysed by X-ray Powder Diffraction (XRPD) with an Empyrean Alpha 1 X-ray diffraction system (Malvern PANalytical, Netherlands) and Differential Scanning Calorimetry (DSC) in a DSC 250 (TA Instruments, USA). Powders water content was determined through Thermogravimetric Analysis (TGA) in a TGA 550 (TA Instruments, USA). In order to understand the interaction between the formulation composition and Fatom on the particle size (Dv50), bulk density and water content of the powders, a multivariate statistical analysis based on partial least squares (PLS) regression model was performed using SIMCA v13.0.3.0 software (Umetrics). Results and Discussion SD powders were successfully generated in all trials using the 2FN atomizing system. Regarding USN atomizing system, an inability to continuously sustain a homogeneous spray wetting the equipment walls led to some product accumulation leading to a low process yield. According to the SEM micrographs (Figure 2), all powders generated with the 2FN are mainly composed by shrivelled particles and a particle size within the nasal size range of 10 – 45 μm. In Test 1 and 3, where Fatom was lower, the generated particles present higher particle size. Conversely, Tests 2 and 4 display a lower particle size for a higher Fatom. In Test 4, where trehalose and HPMC E5 were at a 50:50 concentration, the generated particles presented the lowest particle size. The powder produced by USN (Test 6) consisting also in shrivelled particles, but with a particle size larger than 45 μm and, therefore, outside the nasal size range. However, the PSD span is significantly narrower for the USN, supporting the suggested main advantage of using this nozzle over the 2FN (Table 2).

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Drug Delivery to the Lungs, Volume 29, 2018 – Mariana F. Silva et al.

Figure 2 – SEM micrographs of different formulation compositions, according with the test conditions of Table 1 with different magnifications of 200 and 1000, where the scale bar correspond to 100 μm and 10 μm, respectively.

The apparent particle size observed in the SEM micrographs is in agreement with the PSD results determined by laser diffraction (Table 2) for both 2FN and USN powders. Regarding the 2FN atomizing system, particle size increases when lower values of Fatom are used, as previously observed in other studies.[6] The test with the highest PSD span ((Dv90 – Dv10)/Dv50) was Test 2, corresponding to the one with the highest concentration of HPMC E5. In addition and contrary to the above, the PSD span of the USN powder is the smallest when comparing all experiments. Concerning the bulk density values, there is no significant difference between the 2FN tests. The highest values were obtained when the HPMC E5 was at a higher concentration and the Fatom lower or when the trehalose was at a higher concentration and the Fatom higher. Furthermore, the highest bulk density value of 0.221 gcm-3 was obtained with USN powder (Table 2). This result is in agreement with a previous study by Jüptner et al., suggesting that particles with a more defined size distribution (lower PSD span) have higher bulk density. Additionally, and according with the same study, the powders flowability seems to be improved with the increase of particle size. Thus, regarding the 2FN, the Tests 1, 3 and 5 should be the ones that generate powders with higher flowability.[4] Regarding the powders solid state properties, the XRPD patterns (data not shown) reveal two amorphous halos, which are attributed to the amorphous trehalose and HPMC E5 in all tests performed. As presented in Table 2, the reversible DSC thermograms (open pan) show two glass transition temperatures (Tg) for all SD powders. The first Tg value seems to correspond to the amorphous trehalose as experimental values were closed to the one determined for this raw-material (Tg ~117ºC). The second Tg value could be attributed to the amorphous HPMC E5 (Tg ~150ºC). These results suggest that a possible phase separation occurred during the SD process. Additionally, DSC thermograms in closed pan of test conditions 1 and 4 (formulations with lower and highest amount of trehalose, respectively) were analysed to assess the impact of water in Tg. The water seems to have a considerable influence on the first Tg, although little impact in the second one. In Test 1, the first Tg decreased about 76 % when the second Tg decreased only 20 % when comparing with values in open pan. The same trend was observed in Test 4, where the first and second Tg decreased 60 % and 15%, respectively. Since the SEM micrographs show the same morphology in all tests performed, it is possible that during the SD, the HPMC E5 had formed a shell containing the API and trehalose. Informal accelerated stability tests (40 ºC and 75% relative humidity) will be performed to assess the stability level of the formulations. Regarding the water content, it ranged from 2.8 to 4.0 %, values below 5 %, which in general is a contributing factor for ensuring long term stability of the formulation (Table 2).

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Drug Delivery to the Lungs, Volume 29, 2018 - Nasal Dry Powder Delivery: Implementing a formulation independent Spray Drying Process To strengthen the previous qualitative observations concerning the 2FN atomizing system, a multivariate statistical analysis was carried out to understand the interaction between the formulation composition and Fatom on the outputs as particle size (Dv50), BD and water content. For particle size (Dv50), the PLS regression model that displayed the best fit (R2 = 0.971) and prediction (Q2 = 0.965) was described only as a function of Fatom, showing that when the Fatom is higher, the Dv50 is lower. This is not surprising considering the small particle size variations between the different trehalose compositions for the same F atom. Contrary, no suitable PLS model could be derived for BD and water content as R2 values < 0.7 and Q2 values < 0.5. Furthermore, the assumptions based on the results previously described, suggest an agreement with the above multivariate statistical analysis. Table 2 – Overview of the morphology (Dv50, Span), bulk density, solid state properties and yield for the tested conditions. 2FN

USN

Test condition

Test 1

Test 2

Test 3

Test 4

Test 5

Test 6

Dv50 (µm)

37.2 ± 0.6

11.3 ± 0.4

39.2 ± 0.7

10.7 ± 0.3

19.6 ± 0.2

46.6 ± 0.3

Span

2.2 ± 0.0

3.0 ± 0.2

1.8 ± 0.0

2.6 ± 0.1

2.4 ± 0.1

1.0 ± 0.0

BD (g.cm )

0.197

0.149

0.177

0.191

0.157

0.221

XRPD

Amorphous

112 158 27 126

113 154 -

117 152 -

118 158 47 134

116 155 -

117 150 -

Water content (%w/w)

3.9

2.8

3.7

4.0

3.6

3.3

Yield (%)

54.5

73.3

78.8

88.2

84.8

23.9

-3

Open pan

DSC Tg (ºC)

Closed pan

For the 2FN, the process ran smoothly with good yields between 73.3 and 88.2 % for Test 2 to 5. Test 1 was the exception with a yield of 54.5 %, where some material coated the equipment walls. Regarding USN, the low yield of 23.9 % obtained resulted from the large amount of accumulated material in the bottom of the drying chamber and, therefore, not collected in the vessel. Based on the results, the formulation composition seemed not to have a statistically significant impact in the properties of the powders generated. This implies that, regardless of the ratio between the excipients of the matrix chosen for this study, the powders properties would only be affected by the SD process parameters within the explored experimental ranges. This would result in the possibility of adjusting the ratio between trehalose and HPMC E5 in relation to API requirements only and not for the benefit of powder properties or performance. Ultimately, considering the narrow range of 20 – 30µm, spray dried powders generated under conditions of Test 5 seem the most promising for nasal delivery, although further aerodynamic test would be strictly required to corroborate this hypothesis. Conclusions The impact induced by the composition in trehalose and the Fatom was assessed to produce powders within the nasal size range. With the 2FN, all experiments performed generated amorphous powders with a particle size between 10 - 45 μm. The experiment with the USN had produced a powder with a Dv50 value (47μm) slightly above the recommended upper limit for nasal delivery (45 μm). The generated powder had, simultaneously, an expected lower PSD span. More experiments using USN are required to assess the ability of nasal powders production using this atomizing system. This study also indicates that there would not be an impact of the formulation composition in the powders properties, therefore, allowing the adjustment of the ratio between trehalose and HPMC E5, according to the API requirements. Future work is required in order to strengthen and validate the outcomes of the present study, including research on the actual flowability and aerodynamic performance of the produced powders. References [1]

A. Pieres, A. Fortuna, G. Alves, and A. Falcao, “Intranasal Drug Delivery: How, Why and What for?,” J Pharm Pharm. Sci, vol. 12, no. 3, pp. 288–311, 2009.

[2]

Y. Ozsoy, S. Gungor, and E. Cevher, “Nasal delivery of high molecular weight drugs,” Molecules, vol. 14, no. 9, pp. 3754–3779, 2009.

[3]

L. Tiozzo Fasiolo et al., “Opportunity and challenges of nasal powders: Drug formulation and delivery,” Eur. J. Pharm. Sci., vol. 113, no. May, pp. 2–17, 2017.

[4]

A. Juptner et al., “Spray Dried Formulations for Nasal Applications - Challenges and Opportunities in Filling and Drug Delivery,” Respir. Drug Deliv., pp. 2–5, 2018.

[5]

D. A. Fernandes, R. Barros, C. Moura, E. Costa, and M. L. Corvo, “Impact of Spray Drying on Superoxide Dismutase Activity in Composite Systems with Optimal Aerodynamic Performance for Dry Powder Inhalers,” Drug Deliv. to Lungs, pp. 5–8, 2016.

[6]

D. A. Fernandes, R. Barros, C. Moura, J. Pereira, M. L. Corvo, and E. Costa, “Ultrasonic versus Two-Fluid Nozzle in a Spray Drying Process: A Comparative Study for the Production of Dry Powder Inhaler Formulations for Biopharmaceutical Delivery,” Drug Deliv. to Lungs, pp. 5–8, 2017.

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Drug Delivery to the Lungs, Volume 29, 2018 - Larissa Gomes et al. Therapeutic Intranasal Drug Delivery: needleless treatment for epistaxis Larissa Gomes1, Daniela Traini1, Paul Young1, & Maliheh Ghadiri1, 2 1

Respiratory Technology, Woolcock Institute of Medical Research and Discipline of Pharmacology, Faculties of Medicine and Health, Sydney, Australia 2 Faculty of Engineering and Information Technology, University of Technology Sydney (UTS)

Summary Epistaxis or nose bleeding is a common problem in emergency departments (ED) and ear nose and throat (ENT) surgeries. Reducing the need to progress beyond the simple topical application of medication, would simplify care for nose bleeding, and allow many to self-treat at home during similar spells, shorten their emergency or clinic length of stay, and reduce healthcare costs. The use of alternative agents for the treatment of epistaxis before the use of nasal packing may be reasonable. One such agent is tranexamic acid. Tranexamic acid (TXA, antifibrinolytic drug) in combination with hyaluronic acid (HA) (mucoadhesive agent) as nasal formulation can fulfil this need. Therefore, HA was co-freeze dried with TXA and a freeze dried TXA was prepared as a control. Two freeze dried formulations (TXA and TXA/HA) were characterised according to particle size, particle morphology, hygroscopicity, and nasal deposition. In vitro biological tests were carried out on RPMI 2650 human nasal epithelial cells on both formulation in terms of cytotoxicity and trans-epithelial electrical resistance (TEER) and drug transport. Results indicated that co-freeze dried TXA/HA formulation has the necessary in vitro characteristics for delivery to the nose, potentially for the treatment of epistaxis. Aim: The aim of this study was to formulate a novel nasal tranexamic/hyaluronic acid (TXA/HA) dry powder combination for epistaxis. Hyaluronic acid was added to the formulation of tranexamic acid to enhance residency time of TXA and healing of the nasal epithelia. Key Message: Utilising a dry powder intranasal formulation TXA/HA treatment may provide a simple, inexpensive and safe solution to treat emergency epistaxis. Introduction Epistaxis is a common problem in the ED. Although usually relatively benign, it can escalate to serious, lifethreatening situations. Patients with acute epistaxis frequently visit ED when unable to control or stop the bleeding. If patients require treatment beyond simple topical application of medication, they consume significantly more emergency or clinic resources in terms of supplies, physician time, ENT consultations and even operative intervention [1]. Nasal Packing is a common procedure involved in the treatment of epistaxis, which is not only uncomfortable for the patient, but also involves increased risk of sinus infection, hypoxia, septic shock, rebleeding when removing the pack, and mucosal injury [1]. Current treatments for epistaxis are not efficient and often painful. Reducing the need to progress beyond the simple topical application of medication, would simplify care for these patients, and allow many to self-treat at home during similar episodes, shorten their emergency or clinic length of stay and reduce healthcare costs. Tranexamic acid is an anti-fibrinolytic drug, which reversibly binds to plasminogen and prevents its interaction with fibrin, thus inhibiting the dissolution of fibrin clots [2]. Tranexamic acid as injection solution is commonly used in ED for the treatment of traumatic haemorrhage, and it is also useful for the management of anterior epistaxis [1]. Topical nasal spray of TXA has been already studied [2,3] for various types of bleeding, including epistaxis, and shown to be a more efficient treatment when compared to the nasal packing in a randomized controlled trial, with bleeding stopping within 10 minutes of treatment [3]. Hyaluronic acid is a hygroscopic macromolecule and its solutions are highly osmotic and able to trap large quantities of water and ions, providing hydration and tissue regeneration. HA also possess unique viscoelasticity and mucoadhesive capability, which makes it more attractive for nasal formulations. Mucoadhesive property of HA in a powder formulation also results in the dehydration, and decreases clearance rate in the nasal cavity [4]. Increase in the residency of the drug in the nasal cavity is crucial to enhance absorption across the mucosa or increase its effect locally. Additionally, the topical formulation of HA has also shown wound healing properties following nasal surgery [5, 6]. Even though TXA and HA have long been clinically studied and used as anti-fibrinolytic and mucosal hydrating/ mucoadhesive in separate nasal formulations, no study has yet investigated the possibility of delivering these two active ingredients in synergy to stop nose bleeding and promote wound-healing as a dry powder formulation for nasal delivery.

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Drug Delivery to the Lungs, Volume 29, 2018 - Therapeutic Intranasal Drug Delivery: needleless treatment for epistaxis Experimental methods Dry Powder Formulation: A solution of TXA/HA was prepared with 10% w/v (based on 40 mg per dose) TXA and 0.30% (w/v) HA in deionized water and freeze dried using a mini freeze dryer (B. Braun, Melsungen, Germany). The freeze dried (FD) powder was passed through a 400 sieve before use. TXA alone powder was also prepared with the same technique to compare with TX/HA formulation. Freeze drying technique was selected based on the concept that particles generated with freeze drying are flaky[7]; therefore residence time in the nasal cavity might be higher. Characterisation: The formulations were characterised according to particle size using dynamic laser diffraction (Spraytec, Malvern) to investigate their potential use in nasal delivery. Morphology of FD particles was analysed using a bench top scanning electron microscope (JEOL Benchtop Scanning Electron Microscopes - USA). Hygroscopicity of FD particles was measured using Dynamic Vapour Sorption Instrument (DVS intrinsic- Malvern- UK). Viscosity of both formulations in PBS was measured with the contact angle measurement using a Nikon camera. Aerosol deposition in vitro was investigated using the next generation impactor (NGI, Copley) equipped with a 2.1L glass expansion chamber, according to FDA Guidelines [8]. In this assay, each formulation was loaded (15 mg) into a unit-dose powder system device (Aptar) and tested at 15 L/min for 4s. The device was weighted before and after each shot to determine the emitted dose. Each stage of the NGI was washed with Mili-Q water and TXA was quantified using a validated method in LC/MS (Nexera System, Shimadzu LCMS 2020). Results are expressed as a percentage of the dose loaded to each device. In vitro biological tests were carried out on RPMI 2650 human nasal epithelial cells. Cells were cultured in minimum essential medium with phenol Red (MEM) supplemented with 10% foetal bovine serum (FBS), 1% nonessential amino-acids, and 1% L-glutamine (Gibco, Invitrogen, Australia). The cytotoxicity of both freeze-dried TXA/HA and TXA formulations on RPMI2650 cells were investigated using a MTS assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay from Promega). Briefly, cells were seeded in 96 well-plate at the density of 5 × 104 cell per well and incubated overnight at 37 ºC. The formulations were added to the seeded cells and incubated for 72 hours, followed by the addition of 20 μl of MTS reagents and incubation for 4 hours. The optical absorbance was determined at 490 nm with Spectromax micro-plate reader. To determine the TXA transport rate in both formulations, RPMI 2650 nasal cells were grown in air-liquid configuration (ALI) using Snapwell inserts (Corning, ThermoFisher) for 14 days, as previously described [9]. Formulations were deposited on the cells using a modified nasal chamber attached to the NGI [9]. Transported rate of TXA across an ALI grown RPMI cells was measured over 4 hours using LC/MS. The effect of the formulations on the tight junctions was assessed by measuring the changes in resistance every 5 min over 4 hours using Electric Cell-substrate Impedance Sensing (ECIS, Applied biophysics, USA). Statistical analysis Data presented as the mean ± standard deviation of at least three independent experiments. A student t-test used to compare data, with differences considered statistically significant where P< 0.05. Results Tranexamic acid and TXA/HA formulations showed suitable size distributions for nasal drug delivery, as shown from the Mastersizer data in Table 1. Morphology of FD particles was shown in Figure 1, demonstrated that particles are flaky in shape. Hence adding HA to the formulation increased particle size as observed in the mastersizer data (Table 1). Hygroscopicity of both formulation was measured with DVS and results indicated that FD TXA sample lost 0.36% of total mass during drying cycle, whereas TXA/HA lost 1.5%. These results indicate increased hygroscopicity when HA was present in the formulation. Contact angle measurement demonstrated TXA/HA formulation is significantly (P<0.005) more viscous than TXA alone (Figure 2). Aerosol deposition study’s using the expansion chamber attached to the NGI, demonstrated that over 90% of the powder deposited in the expansion chamber, with less than 10% of the TXA deposited in the NGI stages with a cut-off diameter smaller than 10 µm. There was no significant difference between % deposition of TXA and TXA/HA in the expansion chamber (Figure 3). More TXA (6%) was deposited in stage 1 compared to TXA/HA (P<0.0001). This could be due to the particle size of TXA are smaller than TXA/HA formulation allowing for a larger amount to be deposited in S1, since the cut off diameter of this stage is >14.1 µm. No significant differences (P>0.05) were observed in the amount of TXA remaining in the device, although a larger variation was observed with the addition of HA, which may related to the hygroscopic properties of HA.

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Drug Delivery to the Lungs, Volume 29, 2018 - Larissa Gomes et al. Table 1- Particle size distribution of the freeze-dried formulation of TXA and TXA/HA DV10 (µm)

DV50 (µm)

DV90 (µm)

TXA

3.02 ± 0.03

16.83 ± 0.25

97.03 ± 4.51

TXA/HA

20.9 ± 0.30

102.33 ± 1.15

261.66 ± 5.51

Data presented as Mean ± StDev (n=3)

Figure 1-SEM images of FD particles of TXA and TXA/HA

Figure 2- Contact angle measurements of TX and TXA/HA formulations in PBS, TXA/HA has higher viscosity than TXA alone formulation in PBS (P<0.05)

Figure 3- In vitro aerosol deposition of TXA (Black line) and TXA/HA (Green column) formulations (n=3, Mean ± Stdev)

Biological studies on RPMI2650 cells demonstrated that the nasal formulations were not toxic at the experimental concentrations used. Also, the formulations did not alter the resistance (ohm) of the cell layer, assessed by impedance (data not shown), indicating that the formulation did not affect the tight junctions. Transport data demonstrated that TXA, with or without HA, was transported across RPMI 2650 cells over 4 hours (less than 15% in total), and no significant differences were observed, with more than 85% of TXA remaining on the nasal cells (Figure 4).

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Drug Delivery to the Lungs, Volume 29, 2018 - Therapeutic Intranasal Drug Delivery: needleless treatment for epistaxis

Figure 4- Total TXA transported in RPMI 2650 cells when incubated to TXA and TXA/HA formulations for 4 hours (n=3, Mean ± Stdev)

Discussion The deposition profile of these formulations, delivered using a unit-powder dose device, showed that more than 90% of the formulation was deposited in the nasal fraction (expansion chamber), agreeing with the particle size distribution and corroborating its application for nasal delivery. No TXA was deposited below stage 1 of the NGI, confirming that the aerodynamic diameter of the formulations is suitable for nasal application. The particle size distribution of both formulations was analysed by laser diffraction and SEM, and showed TXA/HA particles to be larger than the TXA alone. These results suggest that adding HA increased the particle size, mostly due to the highly hygroscopic nature of HA molecule, observed in the TXA/HA formulation. Following deposition using the nasal device, transport studies were conducted showing that more than 85% of TXA remained on the surface of the cells after aerosol deposition, indicating the potential of using this formulation for a local effect in the nasal cavity to stop the bleeding and enhance healing. Conclusions In conclusion, the co-freeze dried TXA/HA formulation showed to have the necessary in vitro characteristics for delivery to the nose, potentially for the treatment of epistaxis. Further studies are necessary to investigate its effect in vivo, including assessing the wound healing properties in the nasal cavity. References 1. Zahed R, Moharamzadeh P, AlizadehArasi S, Ghasemi A, Saeedi M. A new and rapid method for epistaxis treatment using injectable form of tranexamic acid topically: a randomized controlled trial. Am J Emerg Med. 2013;31(9):1389-92. doi: 10.1016/j.ajem.2013.06.043. PubMed PMID: WOS:000324332900018. 2. Hilton L, Reuben A. Topical Intranasal Tranexamic Acid for Spontaneous Epistaxis. Emerg Med J. 2014;31(5):436-7. doi: 10.1136/emermed-2014-203763.3. PubMed PMID: WOS:000334673800027. 3. Tibbelin A, Aust R, Bende M, Holgersson M, Petruson B, Rundcrantz H, Alander U. Effect of Local Tranexamic Acid Gel in the Treatment of Epistaxis. Orl J Oto-Rhino-Lary. 1995;57(4):207-9. doi: Doi 10.1159/000276741. PubMed PMID: WOS:A1995RQ91400008. 4. Fransen N, Bjork E, Edsman K. Changes in the mucoadhesion of powder formulations after drug application investigated with a simplified method. Journal of pharmaceutical sciences 2008 Sep;97(9):3855-64 5. Soldati D, Rahm F, Pasche P. Mucosal wound healing after nasal surgery. A controlled clinical trial on the efficacy of hyaluronic acid containing cream. Drug Exp Clin Res. 1999;25(6):253-61. PubMed PMID: WOS:000085471300002. 6. Mayo L, Quaglia F, Borzacchiello A, Ambrosio L, La Rotonda MI. A novel poloxamers/hyaluronic acid in situ forming hydrogel for drug delivery: Rheological, mucoadhesive and in vitro release properties. Eur J Pharm Biopharm. 2008;70(1):199-206. doi: 10.1016/j.ejpb.2008.04.025. PubMed PMID: WOS:000259742600022. 7. Ghirişan, Adina & Miclaus, Vasile. (2017). Comparative study of spray-drying and freeze drying on the soluble coffee properties. Studia Universitatis Babeș-Bolyai Chemia. 62. 309-316. 10.24193/subbchem.2017.4.26. 8. US department of health and human services, food and drug administration, centre for drug evaluation and research. Bioavailability and bioequivalence studies of nasal aerosols and nasal sprays for local action. 2003; 1-37. 9. Pozzoli M, Ong HX, Morgan L, Sukkar M, Traini D, Young PM, Sonvico F. Application of RPMI 2650 nasal cell model to a 3D printed apparatus for the testing of drug deposition and permeation of nasal products. Eur J Pharm Biopharm. 2016;107:223-33. doi: 10.1016/j.ejpb.2016.07.010. PubMed PMID: WOS:000384388300023.

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Drug Delivery to the Lungs, Volume 29, 2018 - Laurent Vecellio et al. Deposition in three nasal cast models with a new concept of nasal administration (Retronose) vs nasal spray Laurent Vecellio1,2, Deborah Le Pennec1, Guillaume Grevin2 & Alain Regard2 1

CEPR, INSERM U1100, University of Tours, Tours, France 2Nemera, La Verpilliere, France

Summary Nasal drug delivery is a non-invasive method that allows a rapid and high local therapeutic effect. Deposition in the different target region of the nasal cavity is an important key factor for improving the drug efficacy. A new concept of nasal administration (Retronose) has been compared to a nasal spray pump on three different nasal cast models. Nasal casts were heated and humidified during nasal drug administration. A nasal pump (Flixonase®, France, GSK) was used for nasal drug administration to the nose. The Retronose prototype uses a pMDI to administer the drug through the buccal cavity model during the nasal expiratory phase. Drug deposition in the different anatomical regions was measured by spectrophotometry and scintigraphy measurements. Results show a more homogenous deposition in terms radioactivity distribution in the cast when using Retronose than nasal spray. The variability of drug deposition in the three nasal cast models was lower when using Retronose than nasal spray pump.

Key Message The Retronose® device reduces the variability of nasal deposition and allows a more homogenous deposition distribution than a nasal spray in nasal cast models. Introduction Nasal drug delivery is a non-invasive method that allows a rapid and high local therapeutic effect. It also offers significant opportunities for new drug development in order to deliver systemic drugs, vaccines and treatments for the central nervous system. A recent study on chronic rhinosinusitis (CRS) patients has shown how corticosteroids deposition distribution in the nasal cavities can have an impact on clinical outcomes [1]. This study has demonstrated the importance of a homogenous deposition in the different target regions of the nasal cavity to treat CRS. A different aerosol concept has been developed for better drug deposition in the distal region of the nose [2-4] without lung deposition. This new device (Retronose) uses a pMDI to administer the drug through the buccal cavity during the nasal expiratory phase. The drug particles enter in the nasal cavities through the rhino pharynx, which has a significant impact on drug deposition profile. The main objective of our study was to compare the deposition obtained by nasal spray pump to Retronose prototype using three different nasal casts.

Experimental methods The Retronose® prototype device was a pMDI (Inhalia®, Nemera, France) filled with HFA 134a gas (no surfactant) and a 12µm active compound (API-1) particle size, resulting in a 14.8 ± 0.4 µm in term of Mass Median Aerodynamic Diameter measured by cascade impactor [3]. A standard nasal pump (Flixonase®, France, GSK) was filled with API-1 solution resulting 48 ± 2 µm in terms of volume mean diameter measured by laser diffraction (n=3). API-1 deposition in the nasal casts was studied using three different anatomical models: a model developed by the Virginia Commonwealth University (VCU, Richmond, VA, USA) [5], and two recent printed models obtained from women scanners (FANI 1 & FANI 2, INSERM U1100, Tours, France). The trachea model was connected to an absolute filter and a humidified air source (Figure 1). A sinusoidal pump (Tidal volume= 750mL, I/E=1, frequency=15 breaths/min) was connected to the humidifier and the filter to mimic patient breathe. A vacuum pump was located near the nose model for collecting the totality of the exhaled aerosol from the model. Retronose device was connected to the mouth model and was actuated during the beginning of expiratory phase. Five breathes were done after every actuation. Four doses were delivered. A Similar experimental set up was used for nasal spray administration but without sinusoidal pump (Figure 1). Two sprays per nostrils were manually administrated in the nose of humidified nasal cast models.

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Drug Delivery to the Lungs, Volume 29, 2018 - Deposition in three nasal cast models with a new concept of nasal administration (Retronose) vs nasal spray Eight regions of interest were defined in the upper airway models: The mouth (Mo), the trachea (T), the oropharynx (O), the rhinopharynx (R), the upper part of the nasal cavity (U), the middle part of the nasal cavity (M), the lower part of the nasal cavity (B), the nose (N). 3 additional regions of interests were added for FANI 1 & 2 nasal casts (maxillary sinuses, frontal sinuses, sphenoids). The different regions were washed with different volumes of sodium hydroxyde using syringes. API-1 was assayed by a spectrophotometric method.

Figure 1: Experimental set up for nasal spray (A) and Retronose® (B) using the VCU upper airway model.

An additional experiment was performed using a second active product (API-2). Flixotide® (125µg, GSK, France) pMDI was used in the Retronose® prototype and was radiolabelled with Tc99m as previously described by Chan et al [5]. Flixonase® (50µg, GSK, France) was radiolabelled with 0.1mL of Tc99m. Nasal administration was performed on the VCU model (Figure 1). Deposition was imaged using gamma camera and was fused with nasal cast scan. Results No active compound was detected in the filter (lung model) for nasal spray and Retronose. About 50% of the drug was deposited in the nose for nasal spray and about 50% of the drug was deposited into the mouth using the Retronose prototype. Less than 5% of the delivered dose was exhaled in ambient air using the Retronose device. Results showed a major deposition in the middle part of the nasal cavity using Retronose device by contrast to the nasal spray highlighted the deposition in the nose. Depositions in maxillary sinuses, sphenoides and frontal sinuses were detected when using Retronose device. A higher variability in terms of deposited drug mass in the region of interest (M + U + R + sinuses) was observed for the nasal spray in comparison with the Retronose device (21% vs 10%) when using the three models. The deposition distribution variability in the different regions of nasal cavities was higher for nasal spray than Retronose, except for the middle part (Figure 2). For nasal spray, the rhinopharynx region is not likely to be reach by particles, whereas for Retronose a significant deposition rate is consistently observed. Deposition images show a more distal and homogeneous deposition in VCU nasal model when using Retronose than nasal spray (Figure 3).

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Drug Delivery to the Lungs, Volume 29, 2018 - Laurent Vecellio et al.

Figure 2: Drug deposition in the different regions of the nasal cavities (upper airway model) using three different nasal cast models for the Spray (A) and Retronose® (B) (Results expressed in terms of deposited fraction in nasal cavity, n=3 for eah nasal cast).

Figure 3: Radioactive deposition of fluticasone in the VCU model with Flixonase® nasal spray (A), and Flixotide® Retronose® prototype device (B) Discussion The deposition differences between nasal spray and Retronose can be explained by the particle size and the route of administration. Particle size generated by the Retronose device is smaller (12µm) than the nasal spray (47 µm) and the kinetic of the particles in the nasal cavities should be different, resulting in difference in term of deposition. The administration through the mouth induces a difference in term of air humidity/temperature effect and particle kinetic in comparison with nasal administration. When the particle is administrated through the mouth, there is an addition of the humidified air which can increase the size of the particle. Particle intake from the oropharynx instead of the nose can also explain the difference in terms of penetration and deposition in the different regions of the nasal cavities. Depositions in the mouth and oropharynx using Retronose device can be explained by the particle velocity generating by the pMDI. Similar deposition has been reported when using commercialized pMDI for inhalation. The homogenous deposition (Figure 3) obtained with Retronose® prototype device demonstrates the ability to address the corticosteroid directly into the different anatomical regions of interest including sinuses (Figure 2). This deposition is very different in comparison with the nasal spray where the drug is located in the first centimetres of the nose. Similar homogenous depositions using nebulizer have been reported and have demonstrated the increase of drug retention in nasal cavities and higher clinical efficiency than nasal spray [3]. In this study, the Retronose device uses a pMDI aerosol generator. It can be defined like a multidose and portable aerosol device for nasal delivery route without lung deposition. A recent clinical study using a similar concept to Retronose has reported positive results on nasal function for asthmatic patients with rhinosinusitis [7].

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Drug Delivery to the Lungs, Volume 29, 2018 - Deposition in three nasal cast models with a new concept of nasal administration (Retronose) vs nasal spray

Conclusion The Retronose device reduces the variability of nasal deposition and allows a more homogenous deposition distribution than the nasal spray in different nasal casts models. The Retronose device should be of interest for local, vaccine and systemic drugs delivery.

References. 1. Reychler G, Colbrant C, Huart C, Le Guellec S, Vecellio L, Liistro G, Rombaux P: Effect of three-drug delivery modalities on olfactory function in chronic sinusitis. Laryngoscope. 2015, 125: pp549-555. 2. Regard A, Vecellio L: Navigating the Nose. The Medicine Makers. 2017, 34: pp9 3. Vecellio L, Le Pennec D, Regard A: Nasal drug delivery by oral route using a pressurized Metered Dose Inhaler (pMDI): Influence of particle size and expiratory flow rate. Respiratory Drug Delivery 2018: pp541-544 4. Vecellio L, Chantrel G, Massardier JM: Device for oral administration of an aerosol for the rhinopharynx, the nasal cavities or the paranasal sinuses. 2011. Patent WO2011080473 5. Longest PW, Tian G, Hindle M: Improving the Lung Delivery of Nasally Administered Aerosols During Noninvasive Ventilationâ&#x20AC;&#x201D;An Application of Enhanced Condensational Growth (ECG). Journal of Aerosol Medicine and Pulmonary Drug Delivery 2011, 24: pp103-118. 6. Chand R, Kuehl P.J. , Leach C, McDonald JD: Radiolabeling Validation of Fluticasone Propionate/Salmeterol Xinafoate - HFA Pressurized Metered Dose Inhaler, Respiratory Drug Delivery 2012: pp573-575 7. Kobayashi Y, Asako M, Kanda A, Tomoda K, Yasuba H: A novel therapeutic use of HFA-BDP metered dose inhaler for asthmatic patients with rhinosinusitis: Case series. Int J Clin Pharmacol Ther. 2014, 10: pp914-919.

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