Essp v5e2

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EPSA Newsletter

Bringing Pharmacy Knowledge and Students together.

Volume 25 | Edition 2 | March 2018 www.epsa-online.org | @EPSA_Online

Liaison Secretaries Day First touch of LS love!

Advocacy How are our member associations working on local level?

Do not miss the new issue of EPSA Newsletter Find out more:

EU Health Award How did it happen?


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Dear reader, It is unbelievable how time flies! This is already the second edition of the EPSA Students’ Science Publication (ESSP) for the mandate 2017/2018. My name is Anja Šribar, Science Coordinator 2017/2018 and even though I stand behind this project, it would not exist without pharmacy students, who submitted their abstracts. Five of many, who have performed research during their studies, decided to take this chance, write an abstract and learn through the whole process until it is ready to be published.

Table of contents 4 Interdisciplinary seminar: Learning to work together 6 Iodine levels in Portuguese cow’s milk 8 Effects of erythropoietin therapy in the treatment of anemia in patients with chronic renal faliure 10 Drug-related problems in elderly nursing home patients 12 Evaluation of the influence of the storage conditions on the viability of different groups of microorganisms in homogenized kefir 14 The involvment of adrenergic receptors in the antiociceptive effect of eslicarbazepine acetate 16 The effect of over the counter drugs on urine amphetamine screening

This is what ESSP is all about – an opportunity to get the experience in writing abstracts, to get professional feedback from the European Federation of Pharmaceutical Sciences (EUFEPS) and to gain valuable skills which help students in their future research projects and professional development. Please, take your time and read all about the research projects that are being performed around Europe. Through the ‘’Question & Answer’’ section you will get to know better who stands behind these abstracts and have a better insight into their work. At the end I would like to thank EUFEPS for this collaboration, for the work they always put into ESSP and for helping us create it on a higher professional level; to students, who submitted their abstracts and to my dear EPSA Team, especially the Educational and Public Relations departments. EPSA Students’ Science Publication, V5E2 would not exist without your hard work and support. Luckily, this was not the last opportunity, so if you are thinking about publishing your abstract, I am looking forward to see your submissions for ESSP V5E3! Best wishes, Anja Šribar, EPSA Science Coordinator 2017/2018


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European Pharmaceutical Students’ Association

Total synthesis of human 6-hydroxysphingosine Majcher A.1, Kovácik A.1, Opálka L.1,Vávrová K.1

Skin Barrier Research Group, Department of Organic synthesis of H involved an alkynylation of (S)and Bioorganic Chemistry; Faculty of Pharmacy in Hradec Garner’s aldehyde (a protected L-serinal) (1) Králové, Charles University, Czech Republic with protected (R)-pentadec-1-yn-3-ol (2) followed by selective two-step trans-reduction Ceramides (Cer), as members of sphingolipid of triple bond. In this step, a mild and selective family, play an important role in cell signalling. [Cp*Ru(CH3CN)3]PF6-catalyzed Trost’s Furthermore, Cer also occur in the epidermis. hydrosilylation followed by protodesilylation In the uppermost skin layer (stratum corneum) was used.3 Cer, along with free fatty acids and cholesterol In conclusion, physiological 6-hydroxylated (in equimolar ratio), form an intercellular multi- sphingoid base has been prepared in lamellar lipid matrix. The key function of seven reaction steps. Moreover, Cer NH stratum corneum is to act as a permeability (N-lignoceroyl 6-hydroxysphingosine) and barrier, thus, to provide water and electrolyte Cer EOH (with ester-linked linolenic acid) homeostasis, and to prevent entry of harmful have been prepared as well. In the future, free substances into the organism1. 6-hydroxylated sphingoid base will serve as Cer are composed of a sphingoid base a precursor for the synthesis of the all known (e.g., sphingosine; C18) and an acyl part physiological H-Cer subclasses, which have derived from long-chain fatty acid (e.g., not been prepared (Cer AH and Cer OH). lignoceric acid; C24). Cer, based on Additionally, to elucidate another function 6-hydroxysphingosine (signed by Motta´s of (H) in healthy human skin, antimicrobial nomenclature as H)2, are the most unusual activity of this sphingoid base will be studied. sphingolipids. In contrast to sphingosine- This work was supported by the Czech based Cer, 6-hydroxysphingosine-based Cer Science Foundation (16-25687J) and Charles (H-Cer) are unique only for the epidermis. University (SVV 260 401). Moreover, the function and biosynthesis of H-Cer in the skin are still enigmatic. Several 1. Kovácik A., Roh J., Vávrová K. ChemBioChem. 2014, dermatological studies showed that lower 15, 1555-1562. concentrations of H-Cer in skin correlate with 2. Kovácik A., Opálka L., Šilarová M., Roh J., Vávrová K. skin diseases, such as atopic dermatitis1. RSC Adv. 2016, 32, 12894–12904. Therefore, the aim of this work was to explore 3. Motta S., Monti M., Sesana S., Caputo R., Carelli S., synthetic route towards H as a precursor of all Ghidoni R. Biochim. Biophys. Acta. Mol. Basis Dis. 1993, known H-Cer subclasses. 1182-1147. The total synthesis of (H) was based on the 4. Breiden B, Sandhoff K. Biochimica et Biophysica Acta reaction of commercially available tridecanal 1841 (2014) 441–452 with trimethylsilyl acetylene. The strategy for Scheme 1. Structure and retrosynthesis of physiological 6-hydroxysphingosine, i.e., (2S,3R,4E,6R)-2aminooctadec-4-ene-1,3,6-triol. Scheme 2. (Pie Chart) Relative proportion of Ceramides in healthy human skin4 1


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of my work was to explore a synthetic route towards 6-hydroxysphingosine as a precursor of all known H-Cer subclasses.

Questions & answers Please tell us a little bit more about yourself. My name is Adam Majcher and I am a third year student of Master’s degree Pharmacy programme at the Faculty of Pharmacy in Hradec Kralove, Charles University, Czech Republic.I am also a member of the Skin Barrier Research Group under supervision of prof. PharmDr. Katerina Vavrova Ph.D. I have started conducting my research project in 2016 as an undergraduate student. In EPSA, I am currently actively engaged as an EPSA Trainer (graduated from Reims, France 2017). Tell us a bit more about your research and its significance. As mentioned above, I am a member of the Skin Barrier Research Group, Department of Bioorganic and Organic chemistry, Faculty of Pharmacy in Hradec Kralove. Our group focuses on studying biophysics, composition and synthesis of molecules naturally occurring in the stratum corneum (uppermost layer of human skin). Therefore, we are studying functions of physiological as well as pathological human skin. My work is focused on the synthesis and biophysical evaluation of one very specific family of ceramides called H-ceramides (H-Cer, ceramides derived from 6-hydroxysphingosine). H-Cer are not typically present in all mammals. Moreover, their function and biosynthesis are unclear. However, various studies showed a relationship between lower concentrations of H-Cer (relative to the healthy skin) and pathophysiologic conditions, such as atopic dermatitis. The major limitation in understanding the nature of these very specific molecules is that they are not commercially available. Therefore, the aim

In your opinion, what is the benefit of joining ESSP and what advice do you have for students undertaking research in the future? I would definitely recommend conducting a research project to anybody. It is a great opportunity to show the world the importance of your research, and science in general. Additionally, it is an opportunity for young scientists/students to practice their scientific writing. Finally, you can never evaluate and think about the purpose and importance of your work enough. Let’s finish this with a quote. As Oscar Wilde once said, “I am so clever that sometimes I don’t understand a single word of what I am saying.” Cheers.


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European Pharmaceutical Students’ Association

Development and characterization of an atropine potentiometric sensor Ana Catarina Ferreira(up201305083@ff.up.pt), Célia Amorim, Conceição Branco da Silva Faculdade de Farmácia da Universidade do Porto, Rua de Jorge Viterbo Ferreira nº. 228 4050-313 Porto, PORTUGAL

Potentiometry with Ion-Selective Electrodes (ISEs) is an analytical technique used for direct determination of ions activities with application in multiple areas, namely life sciences and pharmaceutical control. When compared to other techniques, ISEs show advantages, mainly because, besides of being selective to a certain chemical species, they are economic, with small dimensions and consume low energy. Therefore, in this work, considering the request from partnership with Brazil, the construction and evaluation of an atropine selective electrode is proposed for application in the atropine content control of a pharmaceutical product, injectable, frequently used in surgeries. The electrodes were built with a membrane using cucurbituril (CB[6]) as a chemical recognition species, 2-nitrophenyl octyl ether (2-NPOE) as mediator solvent and plasticizer, and potassium tetrakis(4-chlorophenyl)borate (KTpCIPB) as additive, all immobilized in carboxylated polyvinyl chloride (PVC-COOH) of high molecular weight. After membrane optimization, the electrodes were evaluated in solutions with pH set to 6, using the buffer MES (2-(N-Morpholino) ethane sulfonic acid)NaOH, showing a lower limit of linear response of, a limit of detection of and a calibration curve slope of 58.5 mV. This sensitivity fits the analytical task because the atropine content of the pharmaceutical products is of the order of magnitude 10−3 mol/L. Interference degree determination of many ions, namely, sodium chloride, calcium chloride, magnesium chloride, potassium chloride, ammonium chloride and lithium chloride, and excipients, like sodium citrate dihydrate, polysorbate 80, disodium EDTA, boric acid, dibasic sodium phosphate, sodium phosphate and benzyl alcohol, show that electrodes were quite selective for the analyte, due to the low values

of (<1) found for each ion species. Key-words: potentiometry; ISE; plasticized membrane electrodes; cucurbituril; atropine; injectables.


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be a persistent and patient person to achieve the results of this work. Some examples are: faulty equipment, mistakes made by myself...

Questions & answers Please tell us a little bit more about yourself. I have always been interested in the fields of science and health. Then, I chose Pharmaceutical Sciences as my foundation in order to gain knowledge in both areas. Currently, I’m in my last year of studies and, at this point, I can say that I really liked all the competencies that this master’s degree gives. Besides the experience in research (from where this work resulted) and all the laboratory work developed, I involved myself in activities related with people care, basic support of life and multiple formations from various areas, like clinical trials and I participate even in formations for schools. Tell us a bit more about your research and its significance. The importance of my research was related to the current necessity of saving. Nowadays, a huge waste of atropine injectables occurs. These pharmaceutical formulations are used in emergency situations, namely in children. However, the current regulatory standards are: open the injectable before a surgery onset and, if not necessary, it will be rejected, which represented a great cost for the economy, mainly in Brazil, where this idea emerged. Then, in this work, the construction and evaluation of an atropine selective electrode for determination of this molecule is proposed. What was the biggest challenge whilst carrying out the research and how did you overcome that? The biggest challenge was, absolutely, to not give up when something does not occur as expected. It happens all the time and I had to

In your opinion, what is the benefit of joining ESSP and what advice do you have for students undertaking research in the future? It is a honour to see my work published in the ESSP. It is a great opportunity for all the pharmaceutical students of Europe to exhibit their research. For that, I appreciate this EPSA initiative. What I can say to the younger students who are interested in the research field, is that they can never give up and never forget that our schools provide all the knowledge that we need to go on to a successful career in research and all the conditions to initiate it while we attend the course. Besides, we have to work hard and commit as effort and organisation are crucial for the path of success.


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European Pharmaceutical Students’ Association

Determination of oleuropein content and antioxidant capacity of olive leaves extracts obtained by supercritical CO2 extraction Student: Kaker Rok (rok.kaker@gmail.com), Supervisor: Pajk Stane, Ph. D, Assist. Prof. The Chair of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Slovenia

Introduction: Olive tree (Olea europaea L.) is a traditional mediterranean plant. Its products have been widely used both in diet and in traditional medicine in Mediterranean countries. Olive leaves represent a by-product obtained from pruning of olive trees and harvesting olives. They are rich in bioactive polyphenols, with secoiridoid oleuropein being the main active constituent. Aim/Objective: The aim was to determine the yields of oleuropein in extracts from olive leaves obtained by supercritical CO2 extraction and to assess the suitability of this process in comparison to classical extraction methods. Firstly, we developed HPLC methods for quantitation of oleuropein and for identification of other co-extracted phenolic compounds. Secondly, we quantified oleuropein, identified any major co-extracted phenols and determined antioxidant capacity of the extracts. Thirdly, we assessed the results and determined the suitability of supercritical CO2 extraction for oleuropein from olive leaves. Materials and methods: In order to identify phenolic constituents and determine oleuropein content, RP-HPLC analysis was performed (Thermo Scientific). We used C18 columns (150 x 4.6 and 250 x 4.6) with 5 µm and 4 µm particles, respectively. Moreover we measured antioxidant capacity of the extracts by performing DPPH· (2,2-diphenyl1-picrylhydrazyl) reduction assay and by using Folin-Ciocalteau’s phenol reagent with Synergy™ H4 Hybrid Multi-Mode Reader (BioTek instruments). Results: After comparing retention times of peaks of standard compounds to those on the chromatogram of our samples we were able

to identify several compounds in the extracts from SFE. However, two of the extracts were almost without any phenolic compounds. Furthermore, the highest content of oleuropein was found in the extracts obtained from maceration with ethanol (16.9 and 15.2 % w/w), while the oleuropein content in SFE extracts did not exceed 4.4 % w/w. 4.9 % w/w of oleuropein was also found in the plant residue after SFE had been completed, which suggests a poor extraction. We acquired similar results from testing extract’s antioxidant capacity. Both assays showed that the extracts obtained with classic procedures had a considerably higher antioxidant capacity. Conclusion: Our results show that classical extraction method is superior for the extraction of oleuropein in comparison to SFE, due to a much lower oleuropein yield, significantly poorer phenolic profile and overall much lower antioxidant capacity of SFE extracts.


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with the quantity of the available standard phenolic compounds which left little room for error and required the work to be done in a very precise manner.

Questions & answers Please tell us a little bit more about yourself. My name is Rok Kaker. I am currently in my final semester as a student at the Faculty of Pharmacy, University of Ljubljana. Science is my passion and so is discovering and learning new things. I enjoy working both in research and in community pharmacy. That way I can explore new ideas, develop new skills in the laboratory and still stay in contact with patients in everyday life. Apart from my scientific work, I enjoy traveling, learning new languages and playing guitar. Tell us a bit more about your research and its significance. The work represented in the abstract is a part of my research for my master thesis. Although superfluid CO2 extraction (SFE) has been around for many years and is proved to be a good method for the extraction of several phytochemicals, there is not a lot of information about the suitability of the method for the extraction of olive leaf polyphenols. It was therefore necessary to evaluate the composition and properties of such extracts and to determine any possible benefits of such extraction techniques. What was the biggest challenge whilst carrying out the research and how did you overcome that? The biggest obstacle that I came across during my work was to establish suitable HPLC methods for the identification of phenolic compounds. We overcame that by reviewing several scientific articles, through which we were able to develop methods that produced good results. We were also limited

In your opinion, what is the benefit of joining ESSP and what advice do you have for students undertaking research in the future? It is a wonderful way for students to learn how to write scientific articles, gain important feedback and above all to share our research and knowledge with others. My advice to students is to work on a matter you are passionate and deeply interested about. Something you enjoy exploring and something that makes you work harder and research deeper.


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European Pharmaceutical Students’ Association

A novel cyclic peptide discovered in the plant Leonurus cardiaca S. Pakzad, A. Backlund and U. GĂśransson Division of Pharmacognosy, Dept. of Medicinal Chemistry, Uppsala University, BMC - Biomedical Center, Box 574, 75123 Uppsala, Sweden

Introduction: During the course of evolution, plants and other organisms have diversified and developed their chemical defence lines. This has resulted in plants creating advanced metabolites, among them biologically active polypeptides, such as cyclotides. The typical cyclotide consists of 28 to 37 amino acids and has a head-to-tail cyclic backbone. Leonurus cardiaca is a perennial plant from the family Lamiaceae. The aerial parts and its fruits have been used in traditional medicine in Sweden, with the main application to treat heart conditions. Previous studies about Leonurus have resulted in the isolation of the cyclic peptides, cycloleonuripeptides A-F. Based on the hypothesis that evolutionary close neighbours might display similar metabolites and hence bioactivities, L. cardiaca could be assumed to possess the ability to produce peptides. Aim/Objective: The aim of this project is to isolate novel peptides from L.cardiaca. This will also test the hypothesis that closely related plants contain similar constituents and possibly the same biological activity. CONCLUSION The project resulted in the isolation of the first peptide retrieved from L.cardiaca. The hypothesis that close neighbours in the phylogenetic tree possess similar metabolites is in this case true. By surviving in different high and low temperatures it is clear that the retrieved peptide is a stable cyclic peptide.

higher lipophilicity. All fractions were analysed with MS in positive mode by direct infusion. 3 of the fractions contained cycloleocardi A and were pooled together and subjected to analytical HPLC and also TOF-MS, to confirm that it was an peptide.

Results: Due to the short time-schedule, the whole structure could not be determined. But it could be confirmed that it was an peptide with the aminoacids proline, aspargine and aspartic acid. The purified sample of cycloleocardi A was subjected to analytical HPLC and also TOF-MS, this to confirm that Materials/Methods: The aerial parts of it was a peptide with the molecular weight of the plant material were collected in August 933. 2000 in Uppsala, Sweden and later air-dried. A large scale extraction was done on the material. RESULT The material was extracted with 60% methanol by maceration.The extract was defatted with dichloromethane 2:1 in a separatory funnel. The aqueous part was saved. The methanol was removed from the extract with a rotary evaporator. Preparative RP-HPLC was used to fractionate the large scale extraction. 72 fractions were obtained out of the crude extract, the higher the fraction-number, the


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In your opinion, what is the benefit of joining ESSP and what advice do you have for students undertaking research in the future. ESSP is an excellent platform to find new connections and friends, publish your research and get feedback on it, and also get inspired to do more. My advice to students that want to get into research is; if you don’t Please tell us a little more about love it with all your heart, don’t do it. yourself. Hello! My name is Shaden and I did this research during my final year of studying pharmacy in Sweden. My dream is to continue doing research in this field and hopefully discover something that can make the world a better place.

Questions & answers

Tell us a bit more about your research. My research is in the field of pharmacognosy. Pharmacognosy is the study of the physical, chemical, biochemical and biological properties of drugs, drug substances or potential drugs or drug substances of natural origin, such as plants or other natural sources. It also encompasses the search for new drugs from natural sources. I wanted to ‘discover’ a new drug substance, and what better way is there to do that than to analyse a plant that has been used in traditional medicine centuries ago? I actually found four new drug substances, but due to my very limited time schedule I could not investigate them further. What was the biggest challenge whilst carrying out the research and how did you overcome it? A plant consists of a tremendous amount of constituents. My biggest challenge was to ´’know’’ where to look. Even if I knew where to look, there was a very big risk that the plant I chose did not contain anything out of the ordinary. In situations like this, you need to be patient.


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European Pharmaceutical Students’ Association

Core-shell nanofibers: iinovative drug delivery system for controlled release Špela Vicc*, Špela Zupancc1, Julijana Kristl Department of Pharmaceutical technology, Faculty of Pharmacy, University of Ljubljana, Aškerceva cesta 7, SI-1000 Ljubljana, Slovenia ** spelca.vicic@gmail.com

Introduction: Periodontal disease is a chronic inflammation of gums and periodontal tissue. One of treatments for periodontal disease are antimicrobial drugs in form of tablets, which provide less effective concentration at the targeted site, more side effects and possibility for antimicrobial resistance. Core-shell nanofibers are an innovative material, used in different fields of industry. They can be used as a pharmaceutical drug delivery system with sustained release and present a solution, because they can be applied locally into a periodontal pocket and provide sustained release of the active ingredient.

Aim/Objective: Our goal was to develop core-shell nanofibers with metronidazole, which we could apply locally on the target tissue, where it would release the active ingredient through 7 to 14 days. Materials and Method: Core polymer solution was prepared with polycaprolactone or polyvinyl alcohol as a polymer, dissolved in different organic solvents and hydrophilic drug, metronidazole, was added to the mixture. Shell polymer solution consisted only of polymer and organic solvents. Core and shell polymer solutions were electrospun simultaneously with the use of coaxial needle on Bioinicia Fluidnatek LE100 machine. Scanning and transmission electron microscope were used to characterize the morphology of nanofibers. An in vitro test was used to predict the release of metronidazole. Nanofiber mats were dissolved in buffer solution with pH of 7.4 and absorbance of metronidazole was measured with UV-VIS. Results: Organic solvents from core and shell polymer solution were mixing during electrospinning process and therefore, we did not always obtain a core-shell structure

of nanofibers. Polyvinyl alcohol as a polymer in core solution released metronidazole in 8 hours, when exchanged for polycaprolactone, release was prolonged to 2 days. Higher concentration of polycaprolactone (10%) prolonged release of metronidazole up to 7 days. Core-shell nanofibers with 10% of polycaprolactone in dichloromethane and dimethylformamide in core and 15% of polycaprolactone in formic and acetic acid in shell polymer solution resulted in a sustained release of metronidazole for 14 days. Conclusion: Mixing of organic solvents influenced the morphology of nanofibers. Core-shell nanofibers were successfully electrospun and the release of metronidazole was sustained for up to 14 days. Important parameters affecting the release of metronidazole were the concentration of polymer in core and organic solvents used.


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Questions & answers Please tell us a little bit more about yourself. My name is Špela Vičič, I come from Faculty of Pharmacy, University of Ljubljana and I am a recently graduated Master of Pharmacy. I did research for my Master thesis at the Department for Pharmaceutical Technology and did mandatory traineeship as part of our studies at a community pharmacy. Recently I started working as a Regulatory Affairs Trainee at Lek Pharmaceuticals, which is my first real job. Tell us a bit more about your research and its significance. My research was focused on developing coreshell nanofibers for treatment of periodontal disease. Periodontal disease is a very severe disease, common in older population and one of the main reasons for loss of teeth. With nanofibers as a local drug delivery system, treatment could be less painful and more effective. What was the biggest challenge whilst carrying out the research and how did you overcome that? The biggest challenge was to identify the core-shell structure of nanofibers and point out the location of the active substance. We managed to overcome this problem by adding nanoparticles in the core polymer solution. With a transmission electron microscope, we could see the nanoparticle distribution in core and shell part of the nanofibers and distinguish the core from the shell part of the nanofibers.

In your opinion, what is the benefit of joining ESSP and what advice do you have for students undertaking research in the future? I think the most important thing when doing any kind of research is presenting your results and sharing them with others. This way, people can read about current ideas, approaches of research and have a quick insight into the so called »lab work«. Young students who are reading this, go ahead and try out research, take an opportunity or create one yourself. It is a great way to indulge in fulfilling learning experiences.


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P. S

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Topic “Pharmacoeconomics

& the affordability of healthcare”

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2nd Period

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the Netherlands

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2018

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