13th European ISSX Meeting Abstracts

ABSTRACTS FROM THE 13 EUROPEAN ISSX MEETING TH

June 22 – 25, 2015 Glasgow, Scotland

13th European ISSX Meeting June 22 – 25, 2015 Glasgow, Scotland Contents Monday, June 22, 2015 Short Course 1: Mass Spectrometry in Drug Metabolism Studies Abstracts: SC1.1 – SC1.4

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Short Course 2: Stem Cell Derived Tissues in Toxicology Abstracts: SC2.1 – SC2.4

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Short Course 3: Physiologically-based Pharmacokinetic Modelling Abstracts: SC3.1 – SC3.4

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Short Course 4: New Horizons in Mass Spectrometry Abstracts: SC4.1 – SC4.4

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Opening Keynote Lecture: Protein Kinase Inhibitors: Major Drugs of the 21 Century Abstract: S1

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Tuesday, June 23, 2015 Parallel Symposium 1: Recent Developments in Stem Cell Technology and its Application to Drug Metabolism and Drug Safety Testing 8 Abstracts: S2 – S5; P84; P18 Parallel Symposium 2: Expanding the Boundaries of PKPD Modelling Abstracts: S6 – S9; P164; P2

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Parallel Symposium 3: Systems Toxicology Abstracts: S10 – S13

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Parallel Symposium 4: Clinical Implementation of Pharmacogenomics Abstracts: S14 – S17

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Parallel Symposium 5: Role of Drug Transporters: Current and Future Perspectives (an ITC Sponsored Symposium) 14 Abstracts: S18 – S21 Parallel Symposium 6: Advances in Pathways of Chemically-induced Oxidative Stress and DNA Damage Abstracts: S22 – S25

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Wednesday, June 24, 2015 Parallel Symposium 7: Humanised Animal Models in Drug Development Abstracts: S26 – S29; P131; P136 Parallel Symposium 8: The Contribution of the Gut Microbiome to Drug Metabolism, Drug Interactions and Drug Toxicity Abstracts: S30 – S33; P40; P69 Parallel Symposium 9: Epigenomics in Drug Metabolism and Disease Abstracts: S34 – S37 Parallel Symposium 10: Proteins as Targets for Covalent Modification by Xenobiotics: Therapeutic Opportunities versus Toxicological Risk Abstracts: S38 – S41

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Thursday, June 25, 2015 Parallel Symposium 11: Predicting Human Responses During Drug Discovery and Development Abstracts: S42 – S45; P29; P82

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Parallel Symposium 12: Conjugating Enzymes and Hydrolases in Endogenous and Foreign Compound Metabolism 23 Abstracts: S46 – S49; P77; P17 Plenary Session: Inflammation: Effects on Drug Metabolism and Disease Aetiology Abstracts: S50 – S53

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Poster Details

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Poster Information

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Finalists for the Graduate / Predoctoral Poster Awards Competition (Tuesday, June 23 – Thursday, June 25, 2015)

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Finalists for the Postdoctoral Poster Awards Competition (Tuesday, June 23 – Thursday, June 25, 2015)

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Poster Presentations (Tuesday, June 23 – Thursday, June 25, 2015)

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Abstracts: A1 – A6

Abstracts: A7 – A12

Abstracts: P1 – P166

Author Index

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Notes Pages

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13th European ISSX Meeting

Speaker Abstracts

SC1.1 - LC-MS IN METABOLITE IDENTIFICATION IN HIGH-THROUGHPUT DRUG DISCOVERY Anthony Dickie Evotec (UK) Ltd., Abingdon, United Kingdom The structural identification of metabolites (Met ID) is employed throughout the discovery and development of New Chemical Entities (NCEs). In the early phases of optimising the DMPK properties of a small molecule chemical series, following a high-throughput screening (HTS) approach, Met ID can be used efficiently to direct the chemical synthesis effort, by identifying and redesigning areas of weakness where metabolic clearance is too rapid, or alternatively (where metabolic clearance is too slow), by confirming that a newly introduced functional group has conferred some metabolism that is both predictable and safe. Met ID can be used to complement scaling of clearance to in vivo preclinical species where clearance is primarily hepatic, thus increasing confidence that clearance mechanisms are understood. In terms of safety it is important to reveal pharmacologically active or potentially damaging reactive metabolites, metabolic routes that may be implicated in clinical drug-drug interactions (DDI), as well as metabolites that are likely to be uniquely or disproportionately present in humans compared with pre-clinical species. There are benefits in terms of cost, time and effort if some of these studies can be performed faster and earlier in the discovery cascade. Since metabolism generally involves a change in both mass and hydrophobicity, the hybrid analytical technique of Liquid Chromatography coupled with Mass Spectrometry (LC-MS) is ideally placed to rapidly separate and characterise metabolites. The two functions used to provide information from such a system can be broadly described as 1) LC-MS, by which one may separate the NCE and its metabolites in the time dimension, semiquantitatively estimate the relative abundance of the drug-related material, and qualitatively describe the biotransformations according to changes in the mass dimension from the NCE; and 2) LC-MSn, which in addition fragments the metabolite into its constituent parts to provide an insight into where the biotransformation has taken place on the molecule. Time-of-flight (TOF) and ion trap mass spectrometers are still the instruments of choice in the field, due to their excellent mass resolution and accuracy. Coupled with ultra-high performance liquid chromatography (UHPLC) analytical run-times are much reduced. Various high-throughput approaches have been evaluated in order to reduce the onerous timelines traditionally associated with information-rich Met ID studies, and hence aid the acceleration of a route to molecules with optimised DMPK properties. This short course topic will focus primarily on the use, advantages and limitations of LC-MS in a high-throughput drug discovery setting, to glean structural information on small molecule metabolites.

SC1.2 - DETECTING AND IDENTIFYING REACTIVE METABOLITES Sara Amberntsson AstraZeneca, Innovative Medicines and Early Development, Drug Safety and Metabolism, DMPK, Mรถlndal, Sweden This lecture will describe methods for detection and characterization of reactive metabolites (RM) in drug discovery.

RM formation is an indicator of adverse drug reactions, which with other components provides a basis for a risk assessment of hepatic safety of drug candidates. A number of in vitro tools are available to predict RM of drug candidates during drug optimization phase. Trapping experiments with glutathione, cyanide and methoxylamine are commonly used for identification of soft and hard electrophiles. Experiments are performed in human lever microsomes incubated with drug compound and trapping agent. The choice of analytical method is critical for the selective and specific detection of relevant RM conjugates. Mass spectrometry (MS) methods based on neutral loss or precursor ion scanning with triple quadrupole or ion-trap MS are useful techniques. However, the advantages of high resolution MS are particularly applicable when analyzing drug compounds and their metabolites in biological samples.

The rich and high resolution full-scan data provided requires powerful data processing tools. Carboxylic acids containing drugs have also been associated with adverse reactions linked to activation via metabolism to acyl glucuronide- or acylCoA conjugates. Prediction of acyl glucuronide stability is performed by determining the rate of isomerization during a degradation incubation. Lately it has been shown that the acylCoA conjugate, can be more reactive than the corresponding acyl glucuronide and further investigation in this area is therefore required. In an attempt to understand bioactivation pathways, trapping agents can be incubatated with radio-labelled compound.

Similarly determination of covalent binding to proteins may give an indication of the reactive intermediaries.

Development of human based in vitro methods with improved correlation/predictive power to toxicity risk assessment are continuously ongoing. In this are MS, particularly Time of Flight- MS (TOF-MS), an analytical technique that in many cases can give the most comprehensive information. Continuous improvement of MS analysis and data processing are essential.

SC1.3 - THE USE OF LC-MS FOR COMPREHENSIVE METABOLITE CHARACTERISATION AND IDENTIFICATION Russell Mortishire-Smith Waters Corporation, Wilmslow, United Kingdom This presentation will cover the basic rationale for undertaking drug metabolism studies, and review a variety of approaches for acquiring and interpreting the kinds of datasets which are commonly used in contemporary qualitative analysis of DMPK samples. Included in this will be search strategies, accurate mass approaches, chemical intelligence, mass defect filtration, site of metabolism localization, ion mobility experiments, and molecular modelling.

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SC1.4 - TIPS, TRICKS AND LC-ICPMS FOR METABOLITE DETECTION AND IDENTIFICATION Ian Wilson Imperial College, London, United Kingdom The detection and identification of xenobiotic metabolites can be undertaken using a variety of strategies. One of the most common methodologies employed for metabolite hunting is LC-MS and this has been shown to provide an efficient method for the detection, characterization and identification of metabolites present in cell cultures, biological fluids and tissue extracts etc. However, this is a highly skilled and uncertain process (especially in the absence of a radiolabel) and arguably anything that gives an experimental “edge” to the metabolism scientist is to be welcomed.

One such technique that has been proposed for metabolite detection is the use of multivariate statistical analysis of the data to find signals resulting from the administration of the xenobiotic and it metabolites and this approach can, in addition to detecting drug metabolites, also potentially reveal changes in endogenous metabolites that are the result of both pharmacological and toxicological effects. It is also well known that the presence of a halogen, such as bromine or chlorine, in a molecule can (providing that the halogen is in a metabolically stable site) be especially useful as it provides a characteristic isotopic signature that can be used to identify drug-derived metabolites. Further the specific mass difference and ratio of the isotopes can be used to generate an “isotopogram” that highlights the metabolites in the chromatogram. It is also possible to make artificial isotope signatures by the incorporation of stable isotopes into the compound to be studied and mixing this with the unlabeled parent compound at a fixed ratio. All of this can be performed using a standard (molecular) mass spectrometer. However, if an inductively coupled plasma mass spectrometer (ICPMS) is used specific and quantitative detection of halogen, sulphur, phosphorous and metalcontaining drugs and metabolites can be performed (allowing both excretion balance and metabolite profiling studies to be performed). In essence the ICPMS can be used to detect drug-related material in an atom specific way, analogous to e.g., using a radioactivity detector. By combining the sensitivity and specificity of the ICPMS for detection and a conventional “molecular” mass spectrometer in a single multiply-hyphenated system, metabolite profiling, quantification and identification can be performed. Examples of the use of all of the above techniques will be provided, together with a discussion of their limitations.

SC2.1 - APPLICATION OF STEM CELLS IN SAFETY ASSESSMENT Chris Goldring MRC Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom Adverse drug reactions (ADRs) are a burden to the public, the pharmaceutical industry and regulators. Not only do we typically lack clear mechanisms that can explain the injury, but the manifestations of ADRs are extremely diverse, they are difficult to diagnose, and also they can take weeks or even months to develop. Whilst there are few good animal models of ADRs, there are similarly few good in vitro models at present. Efforts at improving predictability of druginduced tissue injury in man have included using stem cell technology to generate human cells for screening for ADRs in man. This general introduction will explain the background to this area, why stem cells may be useful tools in the assessment tool-box to predict basic cellular perturbation, what we need from the cells, how to compare them with their terminally-differentiated human counterparts and what techniques are used to carry out this phenotyping.

Examples of what we understand about human prediction, mechanisms, and mechanism-based biomarkers will be used to illustrate what is needed from stem cell-derived models.

SC2.2 - AUTOMATED STEM CELL PLATFORMS TO MODEL HEART DISEASE Chris Denning University of Nottingham, Nottingham, United Kingdom Over the last 15 years, human pluripotent stem cells (hPSCs) have progressed from an academic curiosity into a technology that underpins commerce, understanding of disease, drug development and clinical translation. With an emphasis on the heart, this presentation will consider development and in vitro phenotyping of patient-specific hiPSC models of genetic conditions that affect structure, function and survival of cardiomyocytes, including long QT syndrome, Duchenne muscular dystrophy and CPVT. It will discuss how we are using Cas9/CRISPR gene editing to expand the repertoire of genotypes available for understand heart disease and development, including in relation to drug development and safety assessment. We will show how high efficiency cardiomyocyte differentiation has been integrated with robotic platforms, and how this is evolving to overcome phenotyping bottlenecks by incorporating high content platforms that allow assessment of structure (e.g. confocal plate reader imaging) and function (mitochondrial activity, contractility and electrophysiology). Despite these advances, numerous challenges remain, including improved maturation of hPSC-cardiomyocytes, and our current approaches will be described.

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SC2.3 STEM CELL-DERIVED RENAL CELLS AND SAFETY ASSESSMENT 1 1 1 2 1 1 Yao Li , Jacqueline Kai Chin Chuah , Karthikeyan Kandasamy , Ran Su , Karthikeyan Narayanan , Kim Guan Eng , 1 1 2 1 1 Peng Huang , Sijing Xiong , Lit-Hsin Loo , Jackie Y. Ying , and Daniele Zink 1 2 Institute of Bioengineering and Nanotechnology, Singapore, Singapore, Bioinformatics Institute, Singapore, Singapore Methods for the differentiation of pluripotent stem cells into various organ- and tissue-specific cell types have been developed during the past years. However, the approaches were limited success with respect to the kidney. This situation has recently changed and we have published the first protocol for the differentiation of human embryonic stem cells (hESCs) into human renal proximal tubular (HPTC)-like cells (Narayanan et al., Kidney Int, 2013). Recently, various other protocols for the differentiation of human pluripotent stem cells into renal precursor structures were developed, which will be discussed. One of the main areas of interest with respect to stem cell-derived renal cells is predictive safety screening. The kidney is a major target organ for drug-induced toxicity and most frequently affected are the cells of the renal proximal tubule. Prediction of nephrotoxicity during preclinical drug development is challenging, and the development of in vitro models for the prediction of nephrotoxicity in humans has been difficult (reviewed in Tiong et al., Mol. Pharm., 2014). We developed recently the first in vitro model that predicts nephrotoxicity in humans with high accuracy (Li et al., Toxicol. Res., 2013; Su et al., BMC Bioinformatics, 2014). By combining this model with hESC-derived HPTC-like cells we demonstrated the first successful application of stem cell derived-renal cells (Li et al., Mol. Pharm. 2014). More recently, we have developed the most rapid and efficient protocol for the differentiation of human induced pluripotent stem cells (hiPSC) into HPTC-like cells. Such cells display typical marker gene expression patterns, as well morphological and functional characteristics of HPTC. By combining the hiPSC-based in vitro model with machine learning methods, proximal tubular toxicity in humans could be predicted with a training accuracy of 99% and a test accuracy of 87%. Currently we are developing fully automated approaches based on high content screening.

SC2.4 - HEPATOCYTE STEM CELLS IN TOXICITY TESTING Matt Wright Newcastle University, Newcastle Upon Tyne, United Kingdom Hepatocytes are the major functional cells in the liver and play a significant role in the metabolism and toxicity and drugs and chemicals. Hepatocytes in vitro, are often used to study drug and chemical toxicity and their use is likely to increase in the EU, since the REACH regulations require toxicity testing for chemicals used above certain levels.

These tests should ideally be performed in human hepatocytes, but supply is severely limited. Stem cell-derived or other cell sources hepatocytes are therefore being developed as an alternative. A brief overview of hepatocyte generation from stem cells will be given, followed by the state of the art with respect to their application in metabolism and toxicity studies. Given the high costs of stem cell-derived hepatocytes and the barriers often encountered to differentiation beyond a foetal-like hepatocyte, some alternative approaches will be presented.

SC3.1 - USE OF PBPK MODELLING FOR FIRST IN HUMAN DOSE PREDICTION – INCORPORATION OF UNCERTAINTY IN EXPERIMENTAL DATA AND IN-VITRO IN-VIVO EXTRAPOLATION STRATEGIES Michael Gertz F. Hoffmann-La Roche, Basel, Switzerland The application of physiologically based pharmacokinetic (PBPK) modelling has increased over the recent years in pharmaceutical companies. In vitro to in vivo extrapolations (IVIVE) of drug clearance, distribution and absorption are based on first principles integrating physiological and biochemical system data together with drug specific parameters.

Combining IVIVE within the PBPK modelling framework has made this modelling approach the gold standard for extrapolation of human pharmacokinetics from in vitro data. Furthermore, the mechanistic and extrapolative nature of PBPK models allows its application to various other challenges (predictions of drug-drug interactions, formulations/ food effects, PK in special populations and others). In this session, the basic concepts of PBPK modelling as well as the principles of IVIVE will be discussed.

SC3.2 - PBPK CASE STUDIES Karen Rowland Yeo Simcyp Limited, Certara, Waterford, Massachusetts, United States Modelling and simulation of the processes (absorption, distribution, metabolism and excretion; ADME) that define the concentration-time course of a drug can help to predict the potential exposure of a drug at a given dose in individual patients. Integral to this mechanistic approach is in vitro-in vivo extrapolation (IVIVE) in which in vitro systems act as surrogates for delineating various elements contributing to ADME processes in vivo. The development of IVIVE, a ‘bottom-up’ approach, to predict pharmacokinetic parameters and drug–drug interactions (DDIs) has accelerated mainly due to an increase in the understanding of the complex mechanisms involved in these interactions. In addition, recent advances in the knowledge and quantification of population variables required for IVIVE (demographic, anatomical, genetic and physiological parameters) indicating the potential sources of variability have led to wider application of this approach for different scenarios within the pharmaceutical industry. IVIVE used in conjunction with

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Speaker Abstracts

physiologically based pharmacokinetic (PBPK) modelling can help inform the exposure and potential interactions in individual subjects, including those who for ethical reasons cannot be investigated in formal clinical trials. Thus, development teams and regulators are relying on this approach to fill knowledge gaps and support decision making during discovery, development and regulatory review. Indeed, the numbers of submissions to regulatory bodies including PBPK modelling has increased significantly over the past few years. PBPK modelling has been used to determine dose selection in populations of interest including those with organ impairment, to assist with the design of clinical DDI studies as well as inform decisions related to product labelling. An overview of IVIVE and PBPK modelling will be covered in the presentation. This will be followed by a description of the approach generally used and the in vitro and clinical data required to derive a robust PBPK model for a drug in development. Several case studies involving the application of PBPK modelling in drug development will be presented.

SC3.3 - PAEDIATRIC PBPK MODELLING Kay Ogungbero The University of Manchester, Manchester, United Kingdom The last two decades have witnessed an increased use of physiologically based pharmacokinetic (PBPK) models in drug development in academia and industry. PBPK models describe a quantitative mechanistic framework behind the absorption, distribution, metabolism and excretion of drugs, and consist of compartments that represent different tissues/organs/spaces in the body which are connected by circulating blood flow. A PBPK model is specified by two sets of parameters: system parameters and drug specific parameters. The system parameters define anatomical and physiological components of the model such as organ/tissue volume and blood flows that are specific to the organ/tissue they represent. Drug specific parameters define drug properties such as tissue affinity, plasma protein binding affinity, membrane permeability and enzyme and transporter activities, which again may be specific to organs/tissues represented in the model. One of the main advantages of PBPK models is that due to their mechanistic rationale they can be used to extrapolate outside the studied population and experimental conditions used to develop them. Due to the ethical and logistical constraints associated with conducting clinical trials in children, paediatric clinical pharmacology therefore represents an area of opportunity for applying PBPK models, especially with the use of traditional methods such as allometry in scaling preclinical and adult data to children having achieved limited success in drug development. Children have been shown to be different from adults anatomically and physiologically, and vary widely physiologically from birth to adulthood. The mechanistic nature of PBPK models, based on their structure and parameterisation allows different components of the model to be modified taking into account known anatomical and physiological changes, allowing extrapolation between and within species with greater rationale. Specifically, PBPK models developed in adults can be extrapolated to children by modifying age specific changes in organ/tissue volumes and blood flows, and also by incorporating enzyme ontogeny and maturation functions to account for differences in drug elimination. The focus of this presentation will be on development of a PBPK model for prediction of plasma concentrations in children. Methodologies that have been proposed for this purpose will be reviewed and workflows that have been proposed for paediatric PBPK model development will also be presented. Examples will be given to demonstrate the use of these workflows.

SC3.4 - ABSTRACT UNAVAILABLE SC4.1 - NEW APPLICATIONS OF PROTEOMICS IN BIOMEDICAL RESEARCH Andy Pitt Aston University, Birmingham, United Kingdom In many cases the clinical effects of drugs and the toxic effects of xenobiotics results from their interactions with proteins. Identifying these interactions is crucial to many areas such as streamlining the drug discovery pipeline and studying environmental toxins, and will provide a better overall understanding of the positive and negative effects of xenobiotics. However, doing this against the complexity of biological samples is challenging. Mass spectrometry has now become the method of choice for the analysis of proteins. In recent years the speed and sensitivity of instrumentation has increased, and new methods for sample preparation, separation and analysis have been developed. MS is now provides a platform for the rapid and sensitive detection of modifications to proteins, including adducts formed with xenobiotics and their metabolites, and other changes caused during disease. It can even be used to study the dynamics of small molecule interactions with proteins and the structural changes these induce. This wealth of information has revolutionized the ways in which we can use mass spectrometry in biomedical research.

This session will describe the recent advances in using mass spectrometry for the analysis of protein modifications in biological samples. It will look at sampling and sample handling and preparation to maximise successful analysis, and both peptide and protein level measurement and identification of modifications. It will discuss specific methods that can be used to detect small changes in complex mixtures, and to monitor these rapidly from complex biological samples.

SC4.2 - ABSTRACT UNAVAILABLE

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SC4.3 - DATA INDEPENDENT ACQUISITION: A NEW ERA FOR QUANTITATIVE PROTEOMICS? Rosalind Jenkins University of Liverpool, Liverpool, United Kingdom Conventional proteomic studies employ liquid chromatography coupled with mass spectrometry to identify and quantify proteins in complex mixtures. Despite advances in instrumentation, capturing quantitative proteomes from large numbers of samples in a rapid and consistent manner remains extremely challenging. Differential labelling of proteins or peptides with stable isotopes has proven to be a very effective tool for quantitative protein profiling, but it suffers from numerous drawbacks, not least the cost and the limitations on sample selection. Recent developments in data acquisition and processing software have lead to new approaches for quantitative proteomics which start to overcome some of these issues. SWATH is a data-independent MS method for label-free quantification which enables thousands of proteins to be quantified retrospectively. It relies on the rapid acquisition of composite MS/MS spectra which may be deconvoluted subsequently by alignment with an exhaustive protein database. This presentation will describe the technology and illustrate the utility of the approach using samples from mice exposed to model inducers of different cytochrome P450 isoforms. The P450s share extensive sequence homology, so that antibodies are incapable of discriminating every isoform, plus mRNA levels do not correlate well with protein levels.

The ability of SWATH to provide highly parallel and exquisitely discriminatory quantification of protein expression is thus exemplified by analysis of P450 induction.

SC4.4 - LARGE SCALE DATA ANALYSIS IN METABOLOMICS Yoann Gloaguen, Jeni Haggarty, Stefan Weidt, Simon Rogers, Ronan Daly, Mike Barrett, and Karl Burgess Glasgow Polyomics, University of Glasgow, Glasgow, United Kingdom Metabolomics is a new discipline that aims to comprehensively analyse the small molecule complement of biological samples. The most promising technology for the analysis of the metabolome is mass spectrometry coupled to chromatography, either gas or liquid. While the capabilities of mass spectrometry for quantitation and detection of complex compound mixtures are extremely powerful, the technology is limited in a number of ways. Reproducibility is a key issue, especially for large datasets, as the instruments drift or lose sensitivity over time; dynamic range constrains the upper and lower limits of detection for each compound; individual ions have complex responses to ionization, whether fragmentation or the formation of adducts that complicate detection tremendously; the plethora of structures that belong to each mass make identification a significant challenge in any metabolomics experiment; and most importantly the biological interpretation of the results is extremely challenging and requires expert biochemical knowledge. All of these challenges conspire to limit the impact and confidence ascribed to a metabolomics study. In this presentation, we describe the issues and present methodologies and algorithm development that compensate for or solve these challenges.

S1 - PROTEIN KINASE INHIBITORS: MAJOR DRUGS OF THE 21ST CENTURY Professor Sir Philip Cohen FRS, FRSE, University of Dundee, Dundee, United Kingdom Most cellular processes are regulated by the attachment and removal of phosphate from proteins, a process known as "reversible phosphorylation". Although this biological control mechanism was discovered over 50 years ago, the idea that drugs could be developed to treat diseases by targeting protein kinases, the enzymes that attach phosphate to proteins, was considered to be an impossible dream until 15 years ago. The situation changed in 1998 when the first such drug entered clinical trials and was shown to transform a fatal leukaemia into a manageable condition. This caused the field to explode so that today 30 new drugs that inhibit protein kinases have been approved for the treatment of cancers with hundreds more undergoing clinical trials. The first part of the lecture will chart the development of this field into what has become the pharmaceutical industry's most important area of cancer drug discovery. However following the approval of the first kinase inhibitor for the treatment of rheumatoid arthritis two years ago, there is now huge interest in developing many more kinase inhibitors to improve the treatment of chronic inflammatory and autoimmune diseases. My current research is therefore now focused on the dissection of innate immune signaling networks with the aim of identifying the most appropriate kinases to target for therapeutic intervention. Finally, I will discuss the Division of Signal Transduction Therapy that I set up in 1998 to help the pharmaceutical industry launch and accelerate kinase drug discovery programmes . Now in its 17th year, it has become Europe’s largest research collaboration between academia and the pharmaceutical industry and a model for effective interaction between academia and industry.

S2 - PLURIPOTENT STEM CELL HEPATOCYTES AND TISSUE ENGINEERING David Hay University of Edinburgh, Edinburgh, United Kingdom The development of renewable human liver models, from genetically defined origins, is likely to be a game-changing addition to the field. However, for these models to have significant impact they must be stable in character and capable of manufacture at scale. For this reason, we have chosen to use pluripotent stem cells (PSCs). We have developed highly efficient, scalable, and reliable hepatocyte differentiation procedures for use with PSCs, which demonstrate promising qualities. In collaboration with industry and academic laboratories, we provide proof of concept

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that stem cell derived hepatocytes metabolise drugs, possess an intact innate immune system and are capable of predicting human drug-induced liver injury, in line with current gold standard human models. While these are promising advances, there are key deficiencies in the stem cell derived hepatocyte. Our approach to resolve this and our most recent advances will be discussed at the meeting.

S3 - PLURIPOTENT STEM CELL HEPATOCYTES AND CHOLANGIOCYTE MODELS Ludovic Vallier Wellcome Trust - MRC Stem Cell Institute, Cambridge, United Kingdom Human pluripotent stem cells can be generated from embryos at the blastocyst stage (human Embryonic stem Cells or hESCs) or from reprogrammed somatic cells (human Induced Pluripotent Stem Cells or hIPSCs). These cells combine the property to grow indefinitely in vitro and the capacity to differentiate into a broad number of cell types.

Thus, human pluripotent cells represent a unique opportunity for regenerative medicine since they could enable to production of infinite quantity of cell types with a clinical interest such as liver and pancreatic cells. Importantly, hIPSCs could allow the production of patient specific cell types which are fully immuno-compatible with the original donor thereby avoiding the use of immune suppressive treatment during cell based therapy. In addition, hIPSCs can be generated from somatic cells isolated from patients with diverse diseases. Then, the resulting “diseased” hIPSCs can be differentiated into the cell type targeted by the disease and thus provide an in vitro model useful for basic studies and drug screening. By deriving more than 400 lines from 60 patients, my group has acquired a solid expertise in hIPSC production. For that, we have developed a robust method of reprogramming allowing the derivation of hIPSCs from 8 of 10 patients independently of their age or sex. In addition, we have developed fully defined culture systems to differentiate human pluripotent stem cells into broad number of cells type including hepatocytes and cholangiocytes. The resulting cells display characteristics specific of their in vivo counterparts and can function once transplanted in animal model. More importantly, this approach can be used with hIPSCs derived from patients with inherited metabolic disorders to develop in vitro models allowing large scale experiments impossible to perform with primary culture or biopsy material. Here, I will give a summary of recent progress concerning the use of hIPSCs derived liver cells for basic studies and drug development.

S4 - RECENT ADVANCES IN DERIVING HEPATOCYTE LIKE CELLS FROM INDUCED PLURIPOTENT STEM CELLS IN DRUG METABOLISM AND TOXICITY STUDIES Tommy Andersson AstraZeneca R&D, Cardiovascular and Metabolic Diseases DMPK, Mölndal, Sweden Human induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep) hold great promise as an unlimited cell source for metabolism and toxicity testing as well as models for drug targets in drug discovery. However the results from hiPSC-Heps published so far have shown limited drug metabolism functionality thus restricting their use. We recently reported on critical differences in toxicity pathways in hiPSC-hep demonstrating low metabolic capacity as compared with primary human hepatocytes and cell lines limiting the predictive and translational potential of the cells (Sjogren et al., 2014). In this study we compare hiPSC-hep from two sources, Cellular Dynamics International and Cellartis by Takara Bio. The metabolic capacity in cells from novel differentiation protocols showed considerably improved metabolic functions. The drug metabolizing activities for the major Cytochrome P450 isoenzymes (CYP1A2, 2B6, 2C9, 2C19, and 3A4) were close to the activities in primary human hepatocytes. We also investigated the capacity of cells to metabolize a cocktail of drugs, metabolized by several CYP and conjugating enzymes. The results indicated an intrinsic clearance capacity close to primary human hepatocytes. Induction of CYP enzymes by certain drugs is another important characteristic of the hepatocyte. However, the results indicated a limited response to model inducers of the hiPSC-heps from both sources thus limiting the usefulness of the cells in drug metabolism studies. A detailed analysis of gene expression revealed important differences of metabolic pathways and expression of individual gens in hiPSC-heps, primary human hepatocytes and hepatoma cell lines. These differences will likely have an important impact when considering the application of this technology in drug discovery research.

Reference: Sjögren et al. (2014) Critical differences in toxicity mechanisms in induced pluripotent stem cell-derived hepatocytes, cell lines and primary hepatocytes. Arch Toxicol. 88:1427-1437 S5 - ABSTRACT UNAVAILABLE S6 - SYSTEMS PHARMACOLOGY 1&2 Meindert Danhof 1 2 Leiden University, Leiden Academic Centre for Drug Research, Leiden, The Netherlands, President, European Federation of Pharmaceutical Sciences Systems pharmacology is an emerging discipline, which connects systems biology to quantitative pharmacology. A specific feature of systems pharmacology is the focus on biological networks as the basis for drug action. This is important as the network concept explains the well-known plasticity of biological systems with regard to i) drug action (i.e. the often observed lack of efficacy) and ii) disease (i.e. the resilience of disease progression to degeneration). As

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such systems pharmacology constitutes a novel scientific basis for 1) the identification of novel pathways of disease, 2) the discovery of novel drug targets and 3) the design of novel therapeutic interventions. Application of systems pharmacology concepts leads to novel therapeutic interventions which are often referred to as “systems therapeutics”.

Systems therapeutics interventions are: a) personalized, both with regard to the selection of the drug(s) and the dosing regimen, b) disease modifying rather than symptomatic, with emphasis on pre-emptive and preventive treatments, and c) complex, including the use of multi-target drugs or rational drug combinations, to overcome the plasticity of biological systems. Systems therapeutics interventions are “precision treatments”, which due to their inherent complexity, cannot be designed, nor be applied in clinical practice, by trial and error. A model-based approach is crucial to the successful implementation of systems therapeutic interventions. In recent years important progress has been made in the field of quantitative pharmacology. Novel mechanism-based pharmacokineticpharmacodynamics (PKPD) modeling concepts have been proposed for the prediction of in vivo drug effects, including the effects on disease progression. Sofar, mechanism-based PKPD has focused mainly on the modeling of drug effects through single transduction pathways. However, in theory, these concepts can be extended to the modeling of drug effects through biological networks, by considering the interactions between pathways. They constitute a unique scientific basis for the development and the clinical implementation of “systems therapeutic” interventions.

S7 - ABSTRACT UNAVAILABLE S8 - INCORPORATING TRANSPORTER KINETICS INTO PBPK MODELS Aleksandra Galetin Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom Physiologically-based pharmacokinetic (PBPK) modelling is a useful predictive tool for transporter-mediated pharmacokinetics and complex drug-drug interactions (DDIs) involving interplay between multiple transporters and enzymes in different tissues. However, implementation of in vitro uptake transporter kinetic data into PBPK models often results in under-prediction of hepatic clearance regardless of the cellular source of in vitro transporter data.

Factors contributing to this trend are discussed in the current presentation, with a particular focus on the impact of emerging proteomic data and use of clinical data to refine the developed PBPK models. The application of reduced PBPK models for optimization of transporter kinetic parameters is illustrated, together with limitations of this process associated with the use of plasma rather than tissue concentration-time data.

S9 - PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELLING OF MONOCLONAL ANTIBODIES Linzhong Li Simcyp Limited, Certara, Sheffield, United Kingdom The concept of predicting human pharmacokinetics from in vitro and in silico data using mechanistic PBPK models has been implemented widely in drug discovery and development with respect to small molecules. A challenge is to repeat this success for large molecules, including monoclonal antibodies (mAbs).

The PBPK modelling of mAbs requires attention to processes additional to those that operate on small molecules.

Thus, their movement from plasma/blood into tissue depends on convection and diffusion through pores in the endothelial vessels rather than transcellular diffusion, and the lymphatic system plays an important role in their return from the interstitial space of tissues to plasma. The neonatal Fc receptor (FcRn) contributes to the transport of mAbs across endothelial layers while limiting their lysosomal degradation. In addition, clearance mechanisms for mAbs are distinct from those that operate on small molecules, and binding to their targets can lead to dose-dependent pharmacokinetics. A major advantage of PBPK modelling with respect to understanding the kinetics and dynamics of mAbs is that binding to tissue targets can be related directly to the concentration of the mAb in relevant interstitial fluids rather than to that in the plasma. The presentation will consider: 1. Data requirements for the construction of minimal and full PBPK models for mAbs. 2. The impact of FcRn binding on the kinetic behaviour of mAbs. 3. Extension of the concept of target-mediated drug disposition to include target shedding and the use of bispecific antibodies.

S10 - UNRAVELLING MECHANISMS CONTRIBUTING TO DRUG-INDUCED LIVER INJURY BY DYNAMIC PATHWAY MODELLING Bob van de Water Leiden University, Leiden, The Netherlands Drug-Induced Liver Injury (DILI) remains an important problem in drug development and clinical practice. The very heterogenic group of chemicals inducing idiosyncratic DILI cannot be predicted in vitro, in vivo or even in clinical trials.

This illustrates the necessity of a better understanding of underlying mechanisms. Drug exposure combined with proinflammatory TNF cytokine stimulation causes synergistical cell death, supporting a pivotal role for cytokine signalling in idiosyncratic DILI progression. TNF activates NF-ĸB supporting survival signaling, but TNF can also induce proapoptotic signaling depending on the cellular set point. To gather more insight in the dynamic activation of NF-κB and its importance in DILI, we have generated HepG2 Bacterial Artificial Chromosome (BAC) GFP-based reporter cell

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lines of the most important players in the NF-κB signalling cascade. We established and carefully characterized GFPBAC reporter cell lines of IκBα, RelA and ICAM1 and determined the effect of DILI-inducing compounds on proinflammatory cytokine signalling by live confocal microscopy. The time-resolved dynamics of the expression and/or localization of these genes was determined at a single cell level at different cytokine and DILI compound concentrations. A large panel of DILI compounds affected the kinetics of IκBα degradation, RelA nuclear translocation and ICAM1 expression induced by TNF. For various compounds this was directly associated with enhanced cytotoxicity of combined DILI/cytokine treatment. Moreover, we applied RNA interference sceening to determine signaling components that determine the dynamic TNF signaling response and modulaten DILI compound/TNFmediated cell killing. We propose that our novel dynamic high content imaging approach would be an effective means to pin-point DILI liability for novel drug candidates and unravel underlying molecular mechanism.

S11 - A QUANTITATIVE SYSTEMS BIOLOGY ANALYSIS OF SIGNAL TRANSDUCTION NETWORKS UNDERLYING DILI Ursula Klingmueller German Cancer Research Center, Heidelberg, Germany Deciphering drug induced liver injury (DILI) not only needs to consider direct toxic effects of the parent compound or its metabolites on hepatocytes, but also their impact on intracellular signalling networks enabling hepatocytes to respond to inflammatory responses occurring for example during liver regeneration. Accordingly, in studies with the human hepatocellular carcinoma cell line HepG2 it was shown that the combined treatment of diclofenac with TNFα caused an increased caspase-‐3 activity, whereas treatment with either diclofenac or TNFα alone did not. However, the mechanism how TNFα enhances diclofenac-‐induced toxic effects remains elusive. Mathematical modelling approaches offer powerful tools to study these complex and highly dynamic interrelations and to assess mechanisms underlying the synergistic drug-‐cytokine effects. We developed and validated an integrated dynamic pathway model based on time-‐ resolved, quantitative data of TNFα–induced NFκB signalling combined with diclofenac treatment.

Studies were performed with HepG2 cells and primary human hepatocytes and showed immediate, early effects on the NFκB signalling pathway by diclofenac. Supported by the dynamic pathway model we are currently testing different hypotheses to elucidate processes leading to enhanced hepatotoxicity and in particular to the early effects on intracellular responses.

S12 - SYSTEMS MODELING APPROACHES TO PREDICT IDIOSYNCRATIC DILI Paul B. Watkins The Hamner-UNC Institute for Drug Safety Sciences, Research Triangle Park, North Carolina, United States Drugs can cause liver injury (DILI) through a variety of mechanisms. Understanding and predicting the effects of multiple toxicity pathways as a function of time and exposure is difficult without systematic organization. Quantitative systems modeling can combine multiple drug effects to address this challenge. The DILIsim Initiative is a publicprivate partnership involving scientists from 14 major pharmaceutical companies and the FDA; it is now entering its fourth year. In addition to financial support, companies often provide unpublished data and perform in kind research to fill gaps in knowledge. The software produced by the initiative, DILIsym®, is a highly specified, mechanistic, hepatic model that utilizes extensive kinetic information among interrelated biological processes to explore the hepatotoxic underpinnings via simulations. Mechanisms currently included in the model are oxidative stress, mitochondrial dysfunction, bile acid transporter inhibition, and lipotoxicity. The Dilisym® software was originally developed to explain and predict interspecies differences in dose dependent hepatotoxicity to help inform first in man dosing. However, the modeling effort has expanded to improve interpretation of traditional and mechanistic serum biomarkers including miR122, CK18 and its caspase-cleaved fragment, and HMGB1. It is now possible to utilize DILIsym® to predict the range of percent hepatocyte loss through necrosis or apoptosis from measurements of these biomarkers in serial serum samples archived from clinical trials. By varying parameters within Dilisym®, it is possible to create simulated patient populations that mimic selected clinic populations in terms of susceptibility to DILI. This approach successfully recently predicted the latency and incidence of serum ALT elevations that were observed in the clinical trials of troglitazone (1). Systems modeling tools such as DILIsym® will increasingly be used to support decision making throughout the life cycle of new drug candidates.

1) Yang, K, Woodhead, JL, Watkins, PB, Howell, BA, Brouwer, KL. Systems Pharmacology modeling predicts delayed presentation and species differences in bile acid-mediated Troglitazone hepatotoxicity. Clin Pharmacol Ther 96(5):589-98, 2014.

S13 - INTEGRATION OF QSAR AND PK-PD MODELLING IN CARDIAC DRUG SAFETY ASSESSMENT Sebastian Polak Simcyp Limited, Certara, Sheffield, United Kingdom and Jagiellonian University Medical College, Krakow, Poland Proarrhythmic risk remains a major concern during drug development. The current approach based on the ICH S7B non-clinical and E14 based clinical methodologies is conservative and can result in false positives. Thus, while effective, the current paradigm may be inappropriately assigning TdP liability to some drugs, especially in the discovery realm. Proposed shift moves focus from QT interval prolongation towards proarrhythmic risk assessment

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with the use of nonclinical in vitro human models based and solid mechanistic considerations of TdP proarrhythmia.

The latter namely relatively good understanding of the complex electrophysiological phenomena allows for the mechanistic in silico models development and utilization as in inevitable component of this new paradigm. These include various approaches, starting from screening methods (i.e. QSAR based models), up to the utilization of the biophysically detailed cardiac myocyte models. The latter techniques vary with regard of the level of complexity of the mathematical description of the cardiac physiology at the ion channel (Hodgkin-Huxley or Markovian notation) and cell level (single cell up to the 3-dimensional heart structure). Such methods offer the possibility to incorporate variability of either stochastic or deterministic nature. This can further allow for the drug cardiac safety analysis at the population level and quantitative assessment of the combination of drug and non-drug related parameters.

Cardiac Safety Simulator (CSS) is a systems biology driven modelling and simulation platform for the assessment of the pro-arrhythmic potency of drugs and drug candidates. The platform facilitates detailed evaluation of factors influencing potential cardiac risk by utilizing drug-triggered cardiac ion-current disruption data (a) in combination with either in vivo or PBPK simulated drug exposure data (b) within the targeted clinical population (c). By default the inhibition data come from the in vitro electrophysiological studies (manual or automated Patch Clamp technology) but QSAR models including those build-in to the CSS platform can be also utilized as a source of input data. Drug exposure can be of various origin depending on the level of drug development advancement – clinical studies, scaled from the animal data or IVIVE platforms. Cardiac Safety Simulator is connected with Simcyp platform and its predictions of the parent compound, its’ metabolites and interacting drugs for the virtual individuals can be directly transferred to the CSS simulator. CSS operates at the population level incorporating and mimicking various sources of inter- and intra-individual variability (anatomical, genetic, circadian). Simulated and initially analyzed endpoints include action potential, pseudoECG and contractility and their time derivatives including APD50/90, QRS/QT/Tpeak-Tend/JTpeak, electro-mechanical window respectively as well as their drug triggered modifications (i.e. ΔQT). They can be further and more thoroughly analyzed for other pro-arrhythmia surrogates like early after depolarizations (EADs).

Lecture includes presentation and discussion of the case studies and simulation results comparison against the clinical trial results.

S14 - PHARMACOGENOMIC APPROACHES TO TREATMENT OF CYSTIC FIBROSIS Margareta Amaral Faculty of Sciences of the University of Lisbon, Lisbon, Portugal Cystic fibrosis (CF) is a major life-shortening genetic disease leading to severe respiratory symptoms caused by mutations in CF transmembrane conductance regulator (CFTR), a chloride/bicarbonate channel expressed at the apical membrane of epithelial cells. Absence of functional CFTR from the surface of respiratory cells reduces mucociliary clearance, promoting airways obstruction, chronic infection and ultimately lung failure [1]. For the establishment a definite CF diagnosis proof of CFTR dysfunction is also required, more commonly through the socalled "sweat chloride test" [2]. However, more recently other CFTR functional tests, like measurements of CFTRmediated chloride secretion in rectal biopsies, have been validated as robust biomarkers to prove CFTR dysfunction [3]. Newer assays of CFTR function like swelling assay in intestinal organoids [4] or measurement of CFTR chloride currents in polarized primary cultures of nasal cells [5] have been developed being now under validation for CF diagnosis, prognosis and also as good functional predictors of individual patient’s response to drugs [1]. Major clinical advances in treating CF symptoms (with mucolytics, antibiotics, etc) have significantly increased survival beyond the second decade (~25 years in Europe). However, to further increase CF patients life expectancy, CF needs to be treated beyond its symptoms i.e., through treatments addressing the basic defect associated with each CFTR gene mutation [1]. One new drug, potentiator VX-770 (ivacaftor/Kalydeco) has hit the clinical setting but only for patients bearing G551D and 8 other mutations causing a similar defect in the channel, i.e., applicable to only ~5% of all CF patients [6]. However, to date ~2,000 CFTR mutations were reported [7] but one single mutation, F508del occurs in ~85% of CF patients worldwide in at least one allele [8] and is associated with intracellular CFTR protein retention and a severe clinical phenotype. For the F508del mutation a new drug (Orkambi), combining corrector VX-809 (lumacaftor) that rescues F508del-CFTR to the cell surface with potentiatior ivacaftor is expected to be in the clinic still this year, following proven efficacy in a recent phase III clinical trial for F508del/ F508del patients [9]. As these therapies correcting defective CFTR become available, we should quickly pre-assess how other CFTR mutations respond to such new drugs. This is the way forward to extend them more CF patients, namely to those with very rare mutations in an effective and expedite way. Indeed, for such very rare mutations, "classical" clinical trials are not possible due to low numbers of patients and their geographic dispersion. It is thus crucial to use the above novel methods to preassess directly on patient’s cells/tissues how each individual responds to these novel drugs as a basis for their clinical use, i.e., in a precision medicine approach.

Work in the author’s lab is supported by strategic grant PEst-OE/BIA/UI4046/2011 centre grant (to BioISI, Centre Reference: 4046) from FCT/MCTES, Portugal; and by research grants PTDC/SAU-GMG/122299/2010 from FCT/MCTES/PIDDAC, Portugal, "INOVCF" from CF Trust, UK (Strategic Research Centre Award No. SRC 003) and Ref: AMARAL15XX0 from CFF-Cystic Fibrosis Foundation, USA. [1] [2] [3]

Amaral & Kunzelmann (2007) Trends Pharmacol Sci 28: 334-41,; Amaral (2015) J Intern Med 277:155-66 De Boeck et al (2011) J Cyst Fibros 10 Suppl 2: S53-S66.

Hirtz et al (2004) Gastroenterology 127:1085-95; Sousa et al (2012) PLoS One 7: e47708.

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Dekkers et al (2013) Nat Med 19: 939-45.

Beekman et al (2014) J Cyst Fibros 13: 363-72.

Ramsey et al (2011) N Engl J Med 365:1663-72 Cystic Fibrosis Centre HfSC. The CFTR Mutation Database. http://www.sickkids.on.ca/cftr. 2015.

Bobadilla et al (2002) Hum Mutat 19: 575-606.

Wainwright et al (2015) N Engl J Med. Epub May 17. [PMID: 25981758].

S15 - IMPLEMENTATION OF PHARMACOGENOMICS IN CLINICAL PRACTICE-FOCUS ON EVIDENCE-BASED MEDICINE AND COST-EFFECTIVENESS Munir Pirmohamed Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, United Kingdom Pharmacogenetics/genomics has been around for a long time: although this has led to many discoveries, i.e. associations between phenotypes (drug efficacy and safety) and genotypes, translation of these discoveries into clinical practice has been poor. Lack of robust evidence is cited as the main reason. In other clinical areas, the randomised controlled trial is often regarded as the top of the evidence hierarchy, but very few trials have been undertaken in pharmacogenomics. Undertaking a RCT for a pharmacogenomics phenotype is more complicated than undertaking a conventional RCT – apart from the usual factors such as design, sample size, clinical outcome measures, and follow-up, additional factors such as how patients will be genotyped, how genotype will affect drug dose/choice, whether it will be cost-effective, and how many patients will need to be screened to identify those that fit with the inclusion criteria, all need to be considered. These additional factors inevitably also make it much more expensive to undertake pharmacogenomics-based RCTs. An additional factor that needs to be considered is the frequency of the phenotype, especially in the case of rare adverse events, where it may be impossible to undertake a RCT or even a prospective cohort study. In order to improve the translation of laboratory findings into the clinic, we need to consider different forms of evidence in a more intelligent, rather than purely relying on a hierarchy of evidence.

S16 - IMPORTANCE OF DRUG TRANSPORTER POLYMORPHISMS AND SCOPE FOR CLINICAL IMPLEMENTATION Matthias Schwab Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany and Department of Clinical Pharmacology, University Hospital, Tuebingen, Germany Variation in drug disposition and response is a major concern associated with many drugs used in all medical disciplines. Variants in genes that are relevant for ADME processes such as drug transporters have been identified as important confounders affecting patient outcome. In addition non-genetic, gene regulatory as well as epigenetic factors contribute to the expression and function of human membrane transporters. Hepatic drug metabolism and elimination requires drug uptake that is determined by transporters in the sinusoidal membrane of hepatocytes.

Organic Anion Transporting Polypeptides (OATP) and Organic Cation Transporters (OCTs) are expressed in human liver (OATP1B1, OATP1B3, OATP2B1, OCT1, OCT3), but also in kidney (OCT2) and other tissues, mediating the uptake of endogenous compounds (e.g. bile acid by OATPs) and of several drugs (e.g. statins, anti-diabetic and anticancer drugs). Numerous clinical studies support the relevance of common/rare variants in the respective genes (SLCO1B1, SLC22A1, SLC22A2) altering either pharmacokinetics and/or drug response of substrates. SLC22A1 variants have been considered to contribute to the anti-hyperglycemic effect of metformin. Human organic anion transporter 7 (OAT7, SLC22A9), a hepatic transport protein, is poorly characterized so far. We recently identified variants in SLC22A9 including missense variants. These variants did not significantly affect hepatic SLC22A9/OAT7 expression. The missense variants, however, showed functional consequences when expressed in vitro. Although polymorphisms and haplotypes of transporter genes have been associated with alterations in drug disposition and drug response, including adverse events, the results have been conflicting, with limited clinical relevance. Thus in addition to genetic variation of transporter genes a more comprehensive approach including several -omics approaches (e.g. genomics, epigenomics, transcriptomics) are required to identify further putative targets for better prediction of drug response. The system’s pharmacology approach will support the integration process of the systemslevel understanding of drug response and therefore promotes also the drug discovery process for personalized medicine.

S17 - APPLICATION OF PHARMACOGENOMICS IN DRUG DEVELOPMENT Martin Armstrong UCB, Brussels, Belgium With increasing pressure on the pharmaceutical industry from patients, payers, regulators as well as from financial perspectives, the paradigm of how drug development is performed is changing. This talk will present how UCB is using pharmacogenomics to address some of these challenges. The primary focus of the presentation will be on the patient focus required early in the drug development pipelines. The talk will also cover examples of how pharmacogenomics is used in the later phases of drug development.

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S18 - REGULATORY VIEW Eva Gil Berglund Medical Products Agency, Uppsala, Sweden In June 2012, a revised Guideline on Drug Interactions was adopted by the CHMP and published at the EMA webpage for external consultation. The guideline has gone through major revisions to reflect the current regulatory and scientific knowledge. The document included new requirements in many areas, including identification of transporters involved in drug disposition and transporters to screen for inhibition and thereby predict interactions with drugs significantly affected by these transporters. The guideline gives detailed advice on study methodology when possible but in areas where there is much scientific development, advices are general. The guideline has now been into force for 2.5 years. The talk intends to give highlights of present requirements and lessons learned during this initial phase.

S19 - IN VITRO ASSESSMENT OF TRANSPORTER-MEDIATED DRUG INTERACTIONS AND DISTRIBUTION PROCESSES Christoph Funk Roche Innovation Center Basel, F. Hoffmann-La Roche, Basel, Switzerland Drug absorption, distribution, metabolism and elimination of new chemical entities is typically governed by physicochemical properties and multiple, interrelated drug metabolism and transport steps. The involved processes are nowadays studied and optimized as integral part of drug research and development. This results often in compounds with low metabolic clearance, complemented by elimination of unchanged drug via multiple drug transporters. Therefore, drug transporters gain increasing importance for the reduction or prediction of adverse events and variable efficacy due to transporter-mediated drug-drug interactions or polymorphism.

A number of challenges limit the assessment of transporter functions and their relevance to clinics. Many of the key drug transporters exhibit multiple binding sites and have overlapping substrate specificities. Selective substrates and inhibitors are therefore rarely available, impeding straightforward mechanistic in vitro and in vivo studies. One of the approaches for the extrapolation of transporter inhibition is the “calibration” of in vitro assays with multiple model compounds with known clinical inhibition potentials. This approach also helps to reduce variability related to the individual assay set-up. This lack of standardization across labs is currently one of the major limitations for the broader application of mechanistic tools to assess the relevance of in vitro results to in vivo. In addition to reliable transporter kinetic in vitro data, such PBPK-based models further need the appropriate scaling of transport rates to in vivo. Those scaling factors can be established either by calibrating in vitro studies (relative transport factors) or by applying proteomics technologies (absolute transporter expression levels). Finally, thorough validation of the approach using selective transporter substrates is essential. In conclusion, the impact of major transporters on the pharmacokinetics and safety profile of potential clinical development candidates is nowadays studied early-on in drug research and development. By this, unfavorable compound properties, which later-on might lead to significant adverse events, can be recognized and eliminated during lead optimization, additionally the improved mechanistic understanding helps to guide clinical compound development.

S20 - PREDICTIONS OF CELLULAR DRUG BINDING AND IMPLICATIONS FOR INTRACELLULAR DRUG CONCENTRATIONS AND EFFECTS Pär Matsson Department of Pharmacy, Uppsala University, Uppsala, Sweden Many pharmacological targets, off-targets, and drug-metabolizing enzymes are located in the cell interior. Therefore, accurate determination of the intracellular exposure to a drug molecule is paramount for the successful prediction of its pharmacokinetic, pharmacological, and toxicological profile. The unbound intracellular drug concentration is particularly important, because it is generally considered to be the pharmacologically relevant concentration. In this presentation, I will discuss our work to develop a simple and straightforward experimental technique for rapidly determining unbound intracellular drug concentrations in cultured cell lines and primary cells, how cellular drug binding compares across multiple cell types and tissues, and how computational models can be used to predict and explain cellular drug binding and intracellular drug accumulation.

S21 - N1-METHYLNICOTINAMIDE: AN ENDOGENOUS PROBE FOR DRUG INTERACTIONS BY RENAL CATION TRANSPORTERS? Martin Fromm Friedrich-Alexander-University Erlangen-Nuremberg, Institute of Experimental and Clinical Pharmacology and Toxicology, Erlangen, Germany Multiple (mostly cationic) drugs are taken up via the basolateral membrane of renal proximal tubular cells by organic cation transporter 2 (OCT2). The second step of renal tubular secretion of (cationic) drugs can be mediated by multidrug and toxin extrusion proteins 1 (MATE1) and 2-K (MATE2-K), which are expressed in the apical membrane.

Inhibition of these transporters by concomitantly administered drugs reduces renal clearance of the victim drug and might lead to increased plasma concentrations and an increased risk of adverse drug reactions.

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It would be desirable to have endogenous markers during clinical drug development in order to predict the likelihood of transporter-mediated drug-drug interactions. The endogenous metabolite N1-methylnicotinamide (1-methylpyridinium3-carboxamide, NMN), which is formed within the nicotinate and nicotinamide metabolism complex from nicotinamide by the nicotinamide N-methyltransferase (NNMT), is such a candidate. Ito et al. (Clin Pharm Ther 2012) showed that NMN is a substrate of OCT2, MATE1, and MATE2-K with Km values around 350 μM. The antiprotozoal drug pyrimethamine preferentially inhibited MATE1- and MATE2-K-mediated NMN transport. Based on these in vitro data and the results of a clinical study in humans showing a pronounced reduction in renal clearance of NMN from 403 to 119 ml/min by pyrimethamine, Ito et al. concluded that NMN could be a suitable in vivo probe for drug–drug interactions with involvement of MATEs [and OCT2; Ito et al. (Clin Pharm Ther 2012)]. Further evidence for a potential role of NMN was recently generated by Müller et al. (Eur J Clin Pharmacol 2015) using in vitro experiments and a clinical study on the metformin-trimethoprim interaction. The potential limitations for using NMN as an endogenous probe for drug interactions by renal cation transporters will also be discussed.

S22 - CONTROL OF REDOX HOMEOSTASIS BY THE NRF2 REGULATORY NETWORK John D. Hayes Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom The transcription factor NF-E2-related factor 2 (Nrf2) plays a major role in mediating cellular adaptation to oxidative stress. It controls the basal and inducible expression of genes encoding glutathione-based and thioredoxin-based antioxidant proteins, as well as aldo-keto reductases, glutathione S-transferases and quinone reductase, along with multi-drug resistance-associated efflux pumps, each of which contain an antioxidant response element (ARE) sequence in its promoter region. Under non-stressed conditions, Nrf2 is a short-lived protein that is principally controlled at the level of protein stability by Kelch-like ECH-associated protein-1 (Keap1) and beta-transducin repeatcontaining protein (beta-TrCP), which serve as substrate adaptors for Cul3 and Cul1 based E3 ubiquitin ligases, respectively. However, upon exposure to oxidative stress or metabolic cues, Nrf2 protein is stabilized, and ARE-driven genes induced. It is upregulated in 34% of lung tumours, where it augments antioxidant capacity, thereby conferring drug resistance and increased cell proliferation. The upregulation of Nrf2 in tumours often results from somatic mutations that prevent its repression by Keap1. Knockdown of Nrf2 prevents drug resistance and diminishes cell proliferation in tumours that harbour Keap1 mutations. The discovery that Nrf2 is targeted for proteasomal degradation independently of Keap1 by beta-TrCP provides a pharmacological means of overcoming Nrf2-mediated resistance to oxidative stress and drugs, and diminishing cell proliferation. One of the Nrf2 destruction motifs (DSGIS-338) recognised by beta-TrCP is part of a phosphodegron formed by glycogen synthase kinase-3 (GSK-3), a kinase that is active in resting cells but inhibited by induction of various signalling pathways. Typically, GSK-3 requires its substrates to be ‘primed’ by another kinase before it can phosphorylate them. We will present emerging evidence from in vitro kinase assays that Nrf2 is primed before it is phosphorylated by GSK-3.

S23 - REGULATION OF PROTEIN FUNCTION FOLLOWING S-GLUTATHIONYLATION Kenneth Tew Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina, United States Cysteine is a malleable amino acid susceptible to many types of post-translational modifications. One of these, Sglutathionylation, occurs through the reversible addition of a proximal donor of glutathione (GSH) to thiolate anions of cysteines in target proteins - where the modification alters molecular mass and charge, structure/function and/or prevents degradation from sulfhydryl over-oxidation or proteolysis. Catalysis of both the forward (glutathione Stransferase P; GSTP) and reverse (glutaredoxin; sulfiredoxin; GST omega) reactions creates a practical cycle that can regulate certain functional protein clusters including those involved in redox-dependent cell signaling events and protein folding. Pertinent to the latter, GSTP can exist as an endoplasmic reticulum (ER)-resident protein where it demonstrates both chaperone and catalytic functions. Redox based proteomic analyses identified a number of proteins cooperatively involved in regulation of ER stress (immunoglobulin heavy chain-binding protein [Bip], protein disulfide isomerase [PDI], calnexin, calreticulin, endoplasmin, sarco/endoplasmic reticulum Ca2+-ATPase [SERCA]) that both co-immunoprecipitate with GSTP (implying protein complex formation) and are subject to S-glutathionylation following treatment with drugs such as disulfiram. S-glutathionylation of each of these six proteins was found to be attenuated in cells (liver, embryo fibroblasts or bone marrow dendritic) from mice lacking GSTP compared to wild type.

Knockout cells were also significantly more sensitive to the cytotoxic effects of the ER-stress inducing drugs, thapsigargin (7-fold) and tunicamycin (3-fold). Contextually, these results provide mechanistic evidence that GSTP can exert redox regulation in the highly oxidative ER environment and indicate that, within the ER, GSTP influences the lethality of the unfolded protein response (UPR) through S-glutathionylation of a series of key interrelated proteins that control UPR pathways. In clinical applications, quantitative and qualitative levels of S-glutathionylated serum proteins (e.g. serine proteinase inhibitors [serpins] A1 and A3) have been shown to be useful biomarkers in predicting exposure of individuals (who may also have polymorphic expression of GSTP) exposed to agents that cause oxidative or nitrosative stress (ROS/RNS). In individuals exposed to hydrogen peroxide mouthwash, human buccal cells are found to have increased levels of S-glutathionylated proteins suggesting that these cells are useful surrogates for predicting exposure to ROS in the oral cavity. In future, these biomarkers may be adaptable as extrapolated pharmacodynamic indicators for assessing the impact of other systemic drugs that cause ROS and/or impact redox

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homeostasis in humans. The cyclical nature of the S-glutathionylation process allows rapid regulation of signaling pathways to occur in response to changes in cellular ROS homeostasis. Compared to the total proteome, the number of proteins susceptible to S-glutathionylation is not large (hundreds), but improved detection will likely identify further candidates. There is a nascent opportunity to study the importance of polymorphisms (e.g. GSTP; sulfiredoxin; glutaredoxin; GSTÎ&#x;) and how these might influence an individual’s capacity to regulate the S-glutathionylation cycle.

In turn, this could help to codify differences in population response to environmental stress (drug treatments) and susceptibility to disease.

S24 - THE REDOX CHEMISTRY OF ANTIOXIDANTS Claus Jacob University of Saarland, Saarland, Germany The field of antioxidants in general, and their various potential roles in the prevention and treatment of diseases in particular, have recently witnessed a certain renaissance, in part fueled by the demographic changes characteristic of many Western Societies. Natural products with a suspected "antioxidant" activity, in particular, have gained considerable prominence in the field of cancer prevention, in the stimulation of the immune system, in the fight against free radicals and other suspects, and even in the practical area of cosmetics. Still, the underlying chemistry and biochemistry of such compounds is often ignored, a matter that frequently results in misconceptions and misunderstandings, especially when crossing the boundaries of disciplines. Whilst it is certainly correct to assign a particular reducing power to electron donors such as vitamins C or E, other antioxidants act via more subtle avenues.

Many suspected antioxidants, such as the Organic Sulfur Compounds (OSC) and Reactive Sulfur Species (RSS) found in garlic, are de facto not reducing but cause oxidative posttranslational (cysteine) modifications in proteins of the cellular thiolstat, which in turn trigger an antioxidant cellular response - a response which amazingly often runs in parallel with more sinister signalling cascades heading towards cell death. On the other hand, we also find double agents, i.e. classic chemical reducing agents with pro-oxidant potential, such as hydroquinones (which may well donate electrons but at the same time form highly reactive and toxic quinones) or oxygen- or sulfur-centered radicals (which donate electrons to dioxygen, hence generating a pro-oxidant, ROS chemistry detrimental to the cell affected).

Indeed, the chemistry of substances considered as antioxidants can be rather complicated. Oligomeric polyphenols, such as the proanthocyanidins and the tannins, possess a considerable redox activity in vitro, yet seem to act preferably via extensive "non-redox" hydrogen bonding in vivo. Here, redox activity is not essential for biological activity, but may in part control it via a switch mechanism. In sharp contrast, antioxidants such as zinc ions are not even redox active, but can still act as antioxidants, for instance via specific cellular antioxidant response elements and mechanisms. Ultimately, the dichotomy which exists between chemical redox activity and biological antioxidant action needs to be considered with great care, and methods such as redox-specific fluorescent staining are now available to study the relevant cellular redox responses in form of a comprehensive "intracellular diagnostics". Eventually, such studies will enable us to decide if, when, where and especially how the redox chemistry of antioxidants and related sensor/effector agents also translates into a cellular response, which may be protective and antioxidant, but possibly also detrimental and pro-oxidant.

Literature: C. Jacob, Special Issue: Redox Active Natural Products and Their Interaction with Cellular Signalling Pathways, Molecules, 19, 19588-19593 (2014).

C. Jacob, G. Kirsch, A. Slusarenko, P.G. Winyard, T. Burkholz (eds.), Recent Advances in Redox Active Plant and Microbial Products: From basic chemistry to widespread applications in Medicine and Agriculture, Springer Science, ISBN: 978-9401789523 (2015).

S25 - CONTROL OF H2O2 TRANSPORT IN AND BETWEEN CELLS Roberto Sitia Division of Genetics and Cell Biology, Milan, Italy The hormetic properties of H2O2 - essential for cell survival, but toxic at high concentrations - require a tight cellular control on its production, transport and removal. In physiologic conditions, most H2O2 is generated in the outer face of the plasma membrane, in the endoplasmic reticulum and in mitochondria. In all cases, H2O2 has to cross a membrane to reach its cytosolic targets. In HeLa and murine B lymphoma (I.29Âľ+) cells, aquaporin 8 (AQP8) is necessary to negotiate H2O2 transport across the plasma membrane. H2O2 inhibits tyrosine phosphatases, thus regulating tyrosine kinase signalling pathways. Evidence will be presented that diverse physical or chemical stresses inhibit entry of H2O2 into cells. Transport is rescued by DTT, implying the existence of reversible redox modifications of AQP8-dependent H2O2 fluxes. Therefore, the control of H2O2 transport emerges as a novel mechanism to regulate tyrosine kinase signalling during cell stress.

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S26 - HUMANISED MOUSE MODELS OF DRUG METABOLISM 1 1 2 1 Colin Henderson , Yury Kapelyukh , Nico Scheer , and Roland Wolf 1 2 University of Dundee, Dundee, United Kingdom, Taconic Biosciences, Cologne, Germany Transgenic models have contributed significantly to our appreciation of the role of drug metabolising enzymes (DME) and transporters in the disposition of xenobiotics. Mouse – and now also rat – models, where genes have been deleted or modified, have allowed us to ascribe functions to individual DMEs or transporters and to better understand substrate specificities. The application of increasingly sophisticated reporter systems also allows us to determine where DME expression occurs, and in response to which type of chemicals, in vivo and in real time, for example with bioluminescent imaging. However, one issue that has become increasingly apparent with such rodent models is that of species specificity. It is well recognised not only that xenobiotic metabolism is around 10-fold faster in mice and rats than in humans, but also that the DMEs (and transporters) found in rodents are significantly different from those in man in terms of both expression and functionality. These factors seriously handicap the use of transgenic systems in their ability to model human drug metabolism and disposition, and in recent years there have been a number of efforts to overcome such shortcomings by generating humanised models where murine genes have been deleted and replaced by their appropriate human counterparts. In our laboratory, this has culminated in the generation of a mouse model where 33 functional mouse genes (the murine Cyp2c, Cyp2d, and Cyp3a gene clusters and the nuclear receptors Car and Pxr) have been replaced by six human genes CYP2C9, CYP2D6, CYP3A4/3A7, CAR, PXR), representing one of the most complex genetically humanised mouse models to date and involving the human cytochrome P450s that contribute ~80% to the Phase I metabolism of marketed drugs. Initial characterisation of this model will be presented, with evidence for its utility in assessing complex drug-drug interactions. Alternative approaches to the generation of humanised mouse models will also be discussed; the pros and cons of the different models will be considered.

S27 - MOUSE MODELS FOR ELUCIDATION OF CYP2D6 AND CYP2C19 FUNCTION IN THE DEVELOPING AND ADULT BRAIN Magnus Ingelman-Sundberg Karolinska Institutet, Stockholm, Sweden CYP2D6 and CYP2C19 metabolize many endogenous and synthetic psychoactive compounds. In addition to the liver, they are also expressed in the brain, suggesting their role in brain biochemical homeostasis. Genetic polymorphisms in CYP2D6 and CYP2C19 are determining the level of enzymatic capacity and they are also associated with various brain phenotypes. We have previously identified an ultrarapid phenotype of CYP2C19, an enzyme responsible for the metabolism of several psychoactive drugs including antidepressants. We have found that humans lacking CYP2C19 exhibit a less depressive symptoms and that in a transgenic mouse model, mice overexpressing CYP2C19 have smaller hippocampi and phenotypically less doublecortin positive and parvalbumin positive neurons in the hippocampus (HC). Whereas the homozygous transgenic CYP2C19 (2C19Tg-Hom) mice did not survive, we found that adult hemizygous CYP2C19 transgenic (2C19Tg) mice demonstrate behavior indicative of increased stress and anxiety based on four different tests (Persson et al., Mol Psychiatry 2014;19:733-41). The CYP2C19 brain expression in both human and 2C19Tg mice was restricted to fetal life and we hypothesized that expression of the CYP2C19 enzyme prenatally affects brain development by metabolism of endogenous compounds and that CYP2C19 polymorphism may have a role in inter-individual susceptibility for psychiatric disorders. Recently we found a significant increase of Beck’s SIS objective circumstances score in suicide victims with increased CYP2C19 capacity and that the frequency of suicide victims with Beck’s SIS objective circumstances higher than 75th percentile is significantly higher in a group with increased CYP2C19 enzymatic capacity. Also the polymorphism of CYP2D6 has been linked to important alterations in brain perfusion and behaviour including increased risk for suicide among ultrarapid metabolisers. Also transgenic mice expressing human CYP2D6 show pronounced behavioral phenotypes and biochemical alterations in the brain. The lecture will summarize the field of the roles of CYP2C19 and CYP2D6 for CNS function.

S28 - KNOCKOUT AND HUMANISED MOUSE MODELS FOR DETOXIFYING PROTEINS Alfred Schinkel The Netherlands Cancer Institute, Amsterdam, The Netherlands Multidrug transporters of the ATP binding cassette (ABC) protein family and multidrug-metabolizing enzymes such as cytochrome P450 3A (CYP3A) can limit the oral bioavailability of many drugs. ABC transporters can further drastically limit the accumulation of substrate drugs into tissues such as brain and testis, and fetal penetration across the placental fetal-maternal barrier. Studies in knockout and humanised transgenic mouse strains for these detoxifying systems have greatly contributed to insights into their mechanism of action in reducing oral drug bioavailability and tissue accumulation, but also how they interact with each other, and how they can be modulated by co-administered inhibitor drugs. Also for the organic anion-transporting polypeptide (OATP)1A/1B family of drug uptake transporters many insights have been obtained through analysis of knockout and humanised mouse strains. Functions of Oatp1a/1b proteins in hepatic uptake and clearance of many drugs and other compounds from plasma have been well documented. However, postulated roles of Oatp1a/1b proteins in the intestinal uptake of drugs have turned out to be enigmatic so far, at least as tested in well-defined mouse models. More work will be necessary to establish whether

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Oatp1a/1b proteins can play a significant role in this process. Several of our recent studies revealed that pharmacokinetic analyses in knockout and humanised mouse strains may sometimes be complicated by the upregulation or occasionally downregulation of alternative detoxifying proteins, such as cytochrome P450 2C (CYP2C) proteins in Cyp3a knockout mice. In a couple of recent studies, we found that upregulation of a number of carboxylesterases (CES) of the CES1 or CES2 protein families in some of the knockout mouse strains that we use can dramatically alter the behavior of some drugs. This may either occur through metabolism of the analysed (pro)drug by these CES enzymes or, more unexpectedly, by tight binding of the drug to one or more upregulated CES enzymes that circulate in the plasma of knockout mice. As transgenic expression of the equivalent human proteins also often reverses the CES upregulation seen in the knockout strains, great caution is required in proper interpretation of pharmacokinetic experiments done in these mouse models. However, so far it seems that only a minority of drugs is substantially affected by these CES changes, and critical analysis of data, bearing in mind this potential complication, usually quickly reveals whether this plays a role for a certain drug or not. Therefore, even considering this potential CES confounder, knockout and humanised mouse strains remain extremely useful tools to understand mechanisms underlying drug pharmacokinetics and metabolism.

S29 - ABSTRACT UNAVAILABLE S30 - ABSTRACT UNAVAILABLE S31 - IMPACT OF HOST-MICROBIAL INTERACTIONS ON TOXICOKINETICS OF TACRINE Eric Chan National University of Singapore, Singapore The ability to elucidate the mechanisms underlying the heterogeneous responses to drug therapy are essential for personalized medicine. Considering the superorganismic nature of the human host, the influence of the gut microbiota and its associated metabolic activities on inter-individual responses to pharmacotherapy has only been explored in a limited way. As proof-of-principle, we investigated the complex influence of host-gut microbial interactions on the toxicokinetics of tacrine, a probe eliciting idiosyncratic hepatotoxicity. Pharmacokinetic studies in rats demonstrated 3.3-fold higher systemic exposure to tacrine and a double maximum plasma concentration-time profile in strong responders that experienced transaminitis, revealing an enhanced enterohepatic recycling pattern of deglucuronidated tacrine which is not attributable to host variation in gene expression. Metabolic phenotyping studies implicated variation in gut microbial activities that mapped onto tacrine-induced transaminitis. Metagenomics delineated greater deglucuronidation capabilities in strong responders, based on differential gut microbial composition (e.g. Lactobacillus, Bacteroides and Enterobacteriaceae) and higher β-glucuronidase gene abundance compared to non-responders. Coadministration with oral β-glucuronidase significantly augmented the susceptibility of rats to tacrine-induced transaminitis, confirming the role of liver-gut microbiota axis in defining inter-individual variability in tacrine toxicokinetics. Our results raise the possibility that in vivo modulation of β-glucuronidation may be useful in regulating toxicology of drugs that exhibit narrow therapeutic index and large inter-individual variation in pharmacokinetics.

S32 - THE GUT MICROBIOME AND ITS CROSSTALK WITH THE XENOBIOTIC RECEPTOR, THE ARYL-HYDROCARBON RECEPTOR (AHR) Sven Pettersson Karolinska Institutet, Stockholm, Sweden and LKC School of Medicine NTU, Singapore, Singapore Current understanding holds that our indigenous gut microbiome (all its genes and metabolites) has co-evolved with its host to satisfy, in part, mutually biochemical and biological needs. This symbiosis has established that gut microbes are important components in the harvesting and generation of energy for its host. This can be direct by digestion of food derivatives or indirect by influencing nutrient absorption and/or liver metabolism. The liver also express the Arylhydrocarbon-Receptor (AhR), a well known ligand induced transcription factor that regulate xenobiotic metabolism of dioxin. In the first part of my presentation, the existence of a microbiome-AhR signalling pathway that impacts on liver metabolism will be presented. Host metabolism is also regulated by skeletal muscles. In the second part of my talk, I will present the existence a gut microbiome-muscle axis that regulate muscle growth and function. These two sets of data will be discussed in the context of the Holobiont (coined to illustrate the coexistence of microbes inside our body and its capacity to support host metabolism) and microbiome mediated mechanisms influencing host physiology.

S33 - THE IMPACT OF THE METAEXOMETABOLOME ON GUT FUNCTION AND HEALTH – A CASE STUDY OF SHORT CHAIN FATTY ACIDS Bernard Corfe Molecular Gastroenterology Research Group, Department of Oncology, University of Sheffield, Sheffield, United Kingdom The gut microbiome ferments dietary residues entering via the ileo-caecal vcalve to produce a range of exometabolites. If particular interest are the short-chain fatty acids (SCFAs), predominantly acetate, propionate and butyrate, which are produced in millimolar quantities and are therefore available to the host within their pharmacologically active window. The SCFAs, most notably butyrate, have been implicated in a number of biological

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processes, and this will form the focus of the majority of the seminar. Butyrate is a primary energy source for the colon mucosa; butyrate is also a potent regulator of gene expression and drives modified expression of detoxifying enzymes; butyrate has furthermore been implicated as a regulator of both cell death and cell cycle pathways. We have shown that both of these cell fate determinations are also achieved through modification in gene expression profiles. Our work has further demonstrated that this is effected through the actions of butyrate as an inhibitor of deacetylating enzymes which thereby modify the activity of transcription factors: specifically butyrate drives acetylation of Sp1 transcription factor which is associated with a range of promoters of core effectors of cell cycle and apoptosis.

We will present a unified model of activity of butyrate demonstrating the interaction and continuum between metabolic and regulatory functions, integrating the actions of this xenobiotic as a driver of health outcomes. The role of acetate and propionate in competition with butyrate is relatively unexplored and we will review ongoing work in this area, including through metabolic modelling. Finally we will explore the relationship between SCFA and host function at the tissue level, using multicellular compartmental models.

S34 - EPIGENOMICS – IMPACT FOR TRANSLATIONAL SCIENCES Jonathan Moggs Novartis Institutes for Biomedical Research, Basel, Switzerland Recent advances in the mapping and functional characterisation of mammalian epigenomes provide a unique opportunity to gain novel insight into the molecular basis of long-lasting xenobiotic-induced cellular perturbations.

Predicting non-genotoxic carcinogenesis (NGC) during preclinical development of novel therapeutics intended for chronic administration in humans is a major challenge for drug safety scientists. In particular, species-specific molecular, biochemical, cellular and pathophysiologic differences between human and preclinical species confound the extrapolation of rodent carcinogenicity findings to humans. The identification of early NGC mechanisms and biomarkers would provide industry and regulatory scientists with improved tools for earlier decision-making, mitigation of positive rodent carcinogenicity findings and human cancer risk assessment. Integrated epigenomic and transcriptomic profiling of a well-characterized phenobarbital rodent model for drug-induced nuclear-receptor dependent liver tumour promotion reveals novel early biomarkers including dynamic changes in 5-hydroxymethylation and increased expression of Dlk1-Dio3 imprinted gene cluster noncoding RNAs. Mice expressing human CAR and PXR support liver proliferative responses to phenobarbital indicating that the cancer phenotypic outcome of CAR activation may be dependent on species differences in the chromatin landscape of target genes. DNase-seq mapping of liver transcription factor-chromatin interactions is providing novel insights into the molecular basis for speciesspecific differences in NGC mechanisms.

Antonio Vitobello1, Karen Kapur2, Arne Mueller3, Johanna Beil1, Sarah Brasa1, Katharina Hoeland1, Florian Hahne3, Philippe Marc3, Edward Oakeley4, Marc Altorfer4, Nicholas Kelley4, Mark Borowsky2, Jiang Zhu2, Brant Peterson2, Sebastian Hoersch2, Salah-Dine Chibout1, Alberto Del Rio Espinola1, Philippe Couttet1, Olivier Grenet1, Rémi Terranova1, Jonathan Moggs1 and the IMI MARCAR consortium5, 1 Discovery & Investigative Safety, Preclinical Safety, Novartis Institutes for Biomedical Research (NIBR), 2 NIBR Informatics - Scientific Data Analytics, 3 Preclinical Safety Informatics, 4 NIBR Analytical Sciences and Imaging, 5 Innovative Medicines Initiative MARCAR consortium (www.imi-marcar.eu).

S35 - EPIGENETIC DYSREGULATION IN THE LIVER DURING THE PROGRESSION OF NON-GENOTOXIC CARCINOGENESIS 1 1 2 3 3 3 John Thomson , Raffaele Ottaviano , Harri Lempiäinen , Arne Muller , Remi Terranova , Jonathan Moggs , Roland 4 5 Wolf , and Michael Schwarz 1 2 3 University of Edinburgh, Edinburgh, United Kingdom, Genedata, Basel, Switzerland, Novartis Institutes for 4 5 BioMedical Research, Basel, Switzerland, University of Dundee, Dundee, United Kingdom, University of Tübingen, Tübingen, Germany The molecular events which arise during toxicological insult - particularly in cases whereby exposure result in the progression of a disease and or cancerous state - are still poorly understood and present a massive hurdle to the pharmaceutical and research community. As part of an IMI funded European wide initiative (entitled the “MARCAR project”) we are investigating molecular and pathological perturbations which arise during the progression of at early stages of non-genotoxic carcinogenesis through phenobarbital (PB) mediated liver tumor promotion in vivo. We have previously shown that epigenetic DNA modification (both 5-methly-cytosine and the recently characterized 5hydroxymethyl-cytosine marks) and histone modification (H3K4me2, H3K27me3 and H3K36me3) are reproducibly perturbed in a temporal manner following PB exposure which allows us to define specific “barcodes” of drug exposure.

Subsets of genes were found to exhibit an altered promoter epigenetic state following PB exposure such as those associated with the metabolism of xenobiotics in the liver (i.e. the cytochrome P450 genes). Through our recent work we have found that genome wide promoter epigenetic changes appear at predefined loci prior to tumour formation.

We find that in the healthy livers a cohort of promoters are transcriptionally silent and marked by both 5hmC and H3K27me3. Upon chronic PB exposure (91 days) the DNA modification status of these promoters as dramatically perturbed, largely through loss of 5hmC. Interestingly these same promoters are found to become hyper-methylated in the resulting PB driven liver tumours indicating that disruption of active demethylation pathways are likely involved in the process of drug driven hepatocarcinogenesis.

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S36 - ABSTRACT UNAVAILABLE S37 - SPATIAL ORGANIZATION OF THE NUCLEUS AND INFLUENCE ON HUMAN DISEASE Wendy Bickmore MRC Human Genetics Unit, University of Edinburgh, Edinburgh, United Kingdom The human genome is not randomly organised in the cell nucleus. Some sequences are preferentially found at the nuclear periphery, others in the nuclear interior. Some genome regions are packaged more tightly than others and chromosome folding may juxtapose, in the nucleus, sequences that are far apart on the linear DNA. Therefore beyond the human DNA sequence and epigenetic maps of chromatin modifications, there needs to be an understanding of 3D genome organisation in order to understand how the genome is regulated in development and disease. This lecture will also discuss the various ways in which 3D chromosome folding can be analysed, and how this is contributing to an understanding of how genes are repressed during development and how cis-acting regulatory sequences can act on their target genes located hundreds or thousands of megabases away in the genome.

S38 - PHARMACOLOGICAL, PHYSIOLOGICAL AND PATHOLOGICAL CONSEQUENCES OF DRUG BIOACTIVATION AND COVALENT BINDING Kevin Park Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom The generation of reactive intermediates is of concern in drug development because of the perceived risk of a variety of drug toxicities including hepatotoxicity, hypersensitivity and blood dyscrasias (Park et al, 2011). A wide range of therapeutic agents that are able to bind covalently to proteins and other macromolecules either through bioactivation or by direct chemical reactivity in vitro are associated with such toxicities in man. Acetaminophen is the best paradigm for understanding the role of drug bioactivation in liver toxicity. Acetaminophen undergoes 5-10% bioactivation at therapeutic doses in man, to a quinone imine which undergoes spontaneous bioinactivation by direct conjugation with glutathione and also triggers cell defence through the Keap1-Nrf2 system. Integrated in vitro, in vivo and clinical studies have revealed that supratherapeutic concentrations can induce apoptosis, necrosis and an inflammatory response leading to drug-induced liver injury (Antoine et al, 2014). The chemical mechanism underlying acetaminophen-induced hepatotoxicity in man and mouse involves mitochondrial damage and nuclear DNA fragmentation (McGill et al, 2012). The liver has the capacity to respond to such chemically induced injury through tissue regeneration. Studies with penicillins have defined the role of T cells, which recognise covalently bound drug, in immune mediated skin (piperacillin; Whitaker et al, 2011) and liver injury (flucloxacillin; Monshi et al, 2013). The role of chemically reactive metabolites in drug hypersensitivity remains incompletely understood. A large number of drugs associated with idiosyncratic drug toxicity in man undergo bioactivation in vitro and have been shown to form covalent adducts in vitro and more recently in vivo in patients (Meng et al, 2015). However, covalent binding per se does not lead to an immune response and subsequent hypersensitivity, which can show pronounced genetic restriction.

Moreover certain drugs, such as abacavir, can stimulate T cells through non-covalent interactions. Thus the induction of idiosyncratic immunological reactions is presently thought to be a complex function of various chemical and patient factors which precludes prediction of toxicity on chemical grounds alone.

S39 - ABSTRACT UNAVAILABLE S40 - (DMPK) CHALLENGES IN DEVELOPING AZD9291: AN IRREVERSIBLE INHIBITOR OF EGFR SELECTIVE FOR SENSITISING AND T790M RESISTANCE MUTATIONS Peter Ballard AstraZeneca Oncology iMed, Alderley Park, Chesire, United Kingdom Small molecule epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), for example gefitinib and erlotinib, have demonstrated clinical benefit in patients with advanced non-small cell lung cancer (aNSCLC) with EGFR sensitizing mutations (EGFRm). However, most aNSCLC patients treated with a currently approved EGFR-TKI develop resistance resulting in disease progression. The most common resistance mechanism (in up to 60% of cases1,2,3) is the T790M mutation and there are no currently approved treatments specifically for patients with T790M tumours. AZD9291 (N-[2-[2-(dimethylamino)ethyl-methyl-amino]-4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2yl]amino]phenyl]prop-2-enamide) is an oral, potent, selective, irreversible inhibitor of both EGFRm and resistance mutation (T790M) in aNSCLC that has been shown in a Phase 1 study (AURA; NCT01802632) to be clinically active and well tolerated as monotherapy at all dose levels tested. AZD9291 has been designed to achieve nanomolar potency through a direct covalent binding mechanism to a cysteine residue in the EGFR kinase ATP binding site.

While AZD9291 binds covalently to target, it can also bind to other proteins containing cysteine residues (plasma albumin for example) and other tissues including hepatocytes, which may influence drug distribution and excretion characteristics. Notably, AZD9291 demonstrates differential cross-species stability in plasma resulting in the absence of protein binding data and the impact of this on assessing DDI potential will be discussed. Although AZD9291 is primarily metabolised in vitro by cytochrome P450 3A4 to two de-methylated metabolites which retain the covalent binding functional group in all species, other metabolic processes did demonstrate cross species differences. The demethylated metabolites are active and present in the plasma of pre-clinical species and patients. The influence that

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these metabolites had on describing the pre-clinical pharmacokinetic/pharmacodynamic/efficacy relationship in mouse xenograft models and the subsequent simulation of potential efficacious clinical doses for candidate drug selection will be presented along with the impact of the actual clinical pharmacokinetics on these simulations.

1. Arcila ME et al. Rebiopsy of Lung Cancer Patients with Acquired Resistance to EGFR Inhibitors and Enhanced Detection of the T790M Mutation Using a Locked Nucleic Acid-Based Assay. Clinical Cancer Research 2011;17:1169–1180 2. Oxnard GR et al. Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in EGFR-Mutant Lung Cancer: Distinct Natural History of Patients with Tumors Harboring the T790M Mutation. Clinical Cancer Research 2011;17:1616–1622 3. Yu HA et al. Analysis of Tumor Specimens at the Time of Acquired Resistance to EGFR-TKI Therapy in 155 Patients with EGFR-Mutant Lung Cancers. Clinical Cancer Research 2013;19:2240–2247 S41 - INHIBITORS OF CATHEPSIN C – NOT YOUR TYPICAL ORAL SMALL MOLECULES Catherine Booth-Genthe, Michael Palovich, Brian Bolognese, Alison Churchill, Joseph Foley, Philip Landis, Gregory Logan, and Edward Long III GlaxoSmithKline, Collegeville, Pennsylvania, United States Cathepsin C is a cysteine protease localized in immune cells and has been identified as the required activator of numerous serine proteases, particularly neutrophil serine proteases, whose biological functions include degradation of extracellular matrix, inflammatory cell recruitment and chemokine/cytokine activation.1,2 As such, inhibitors of Cathepsin C may serve as a therapy for a variety of diseases know to be impacted by protease activity (such as nonCF bronchiectasis).3 Efforts to date to discover oral small molecule inhibitors of Cathepsin C have been unsuccessful due to an inability to mirror good in vivo activity with good oral PK properties.4 We have been able to succeed by adopting a covalent irreversible inhibition approach. This strategy has resulted in the identification of compounds that have good oral efficacy and good physical properties. We will describe the events that led the team to choose this inhibitory profile as well as the activities the team undertook to elucidate the risk in the development of compounds with this mode of inhibition.

References 1. Pham, Christine T. N.; Ivanovich, Jennifer L.; Raptis, Sofia Z.; Zehnbauer, Barbara; Ley, Timothy J; PapillonLefevre Syndrome: Correlating the Molecular, Cellular, and Clinical Consequences of Cathepsin C/Dipeptidyl Peptidase I Deficiency in Humans. Journal of Immunology 2004, 173 (12), 7277-7281.

2. Kirstin M. Heutinck; Ineke J.M. ten Berge, C; Erik Hack, Jörg Hamann; Ajda T. Rowshani, Serine proteases of the human immune system in health and disease. Molecular Immunology 2010, 47, 1943-1955.

3. Daniel Guay; Christian Beaulieu; M. David Percival; Therapeutic Utility and Medicinal Chemistry of Cathepsin C Inhibitors. Current Topics in Medicinal Chemistry 2010, 10, 708-716.

4. Daniel Guay; Christian Beaulieu; T. Jagadeeswar Reddy; Robert Zamboni; Nathalie Methot; Joel Rubin; Diane Ethier; M. David Percival; Design and synthesis of dipeptidyl nitriles as potent, selective, and reversible inhibitors of cathepsin C. Bioorganic & Medicinal Chemistry Letters 2009, 19, 5392-5396 S42 - APPLICATION OF IN VIVO REPORTERS FOR STRESS RESPONSES IN DRUG DEVELOPMENT Roland Wolf, C.J. Henderson, T. Frangova, L.A. Jimenez, and M.J.M. McMahon University of Dundee, Dundee, United Kingdom A wide range of adaptive response systems have evolved to protect cells in multi-cellular organisms from toxic environmental exposures. These systems increase the rate of chemical detoxication, antioxidant response and the repair of damage to DNA. By definition the activation of these pathways precede the onset of cell death and therefore provides biomarkers of cell stress before the advent of overt toxicity. In our current research programme, funded by the European Research Council, we have been developing a range of transgenic animal models where specific genes activated by these stress response pathways have been linked to invasive (Lac Z) and non-invasive (luciferase) reporters. These systems allow both the in vivo real-time assessment of the activation of stress responses in different tissues as well as more detailed histopathological analysis. A range of stress response models have been created through the insertion of the reporter into various endogenous gene loci of interest. These models allow the pharmacological activities as well as potential toxic liabilities of drugs to be assessed, in addition to the potential deleterious/beneficial effects of environmental chemicals. The models allow the measurement of DNA damage through the transcriptional regulation of p21, oxidative stress using heme oxygenase-1 and superoxide dismutase 2, and an IL6 reporter to measure inflammatory response. As a part of a European Union collaborative project to identify biomarkers for non-genotoxic carcinogens we have used the in vivo heme oxygenase-1 reporter to evaluate the capacity of a range of non-genotoxic carcinogens to induce oxidative stress in the liver and have demonstrated that the capacity of these agents to activate these pathways varies very significantly between compounds (Toxicol. Sci. (2015) 145, 138-48). We have also used the p21 reporter system to study the effects of ionising radiation and the DNA repair enzyme PARP inhibitor, olaparib, on the activation of the DNA damage response pathway in normal mouse tissues. We find that both the agents can activate the p21 reporter in multiple tissues and that the coadministration of ionising radiation with PARP inhibitors exacerbates toxicity. These finding are important in view of

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the proposed use of these agents in combination in the clinic. These and other applications of stress reporter systems for drug development and toxicology studies will be described in this presentation.

Acknowledgement: Financial Support from: Cancer Research UK, Grant no: C4639/A10822, The European Research Council, Grant no: REDOX 294533, The EU Innovative Medicine Initiative Joint Undertaking MARCAR programme, Grant no: 115001.

S43 - PREDICTING HUMAN RESPONSES TO DRUGS: CHALLENGES AND OPPORTUNITIES OF IN VITRO APPROACHES 1 1 2 3 Jen-Yin Goh , Libby Dixon , Richard Weaver , and Ruth Roberts 1 2 The Association of the British Pharmaceutical Industry (ABPI), London, United Kingdom, Institut de Recherches 3 Servier, Croissy-sur-Seine, France, AstraZeneca, Wilmslow, United Kingdom Preclinical safety testing is a crucial step in pharmaceutical drug development and depends on a series of in silico, in vitro and in vivo tests before administration to humans. Currently, in vivo testing is a vital part of safety assessment, and is a regulatory requirement before a drug can progress into clinical trials and onwards to a marketing application.

However, many in vitro approaches have been developed and validated for early stage screening aimed at filtering out molecules with a higher potential for toxicity. We examined the use of in vitro approaches in preclinical safety testing between 1980 and 2013 to determine drivers, opportunities and challenges. Data were collected via a survey sent to the Association of the British Pharmaceutical Industry (ABPI) member companies requesting use and utility of in vitro screens at 5-year intervals between 1980 and 2005 then yearly from 2008 onwards. Four pharmaceutical companies and 3 contract research organisations (CROs) responded, providing >895000 data points across all years and all assays. Overall, there was a year-by year increase in use of in vitro approaches with >190 000 tests reported in 2012 alone. Trends and step changes in uptake were most notable in ADME, safety pharmacology and genotoxicity, probably explained by adoption of the relevant International Committee on Harmonisation (ICH) guidelines. Trends in uptake were also correlated with perceptions of utility where scores varied from poor (Eye Irritation) to excellent (Genotoxicity and Skin irritation). Examples of success included EpiSkin, validated and endorsed by organizations including ECVAM as a replacement for the rabbit skin irritation test and the Bovine Corneal Opacity and Permeability (BCOP) assay which offers an excellent opportunity to fully replace the rabbit Draize eye irritation. Despite progress, availability of reliable and relevant models remains a challenge: in vitro approaches in areas such as genetic toxicology and electrophysiology scored low on utility possibly due to a high level of false positive results making extrapolation to humans difficult. The reliability of in silico testing to predict safety signals remains in its infancy and has even been called into question in a recent paper from Cook et al (2014). Implementation can also be a challenge since even in vitro approaches with high scores on utility took many years to implement. Overall, the evidence demonstrates development and uptake of in vitro approaches with significant success in key areas such as genetic toxicology, skin absorption and reproductive toxicology but there is still much to be done. There is a pressing need to improve success rates in the pharmaceutical industry and also to make failure less costly perhaps via the development and validation of further in silico and in vitro laboratory tests that could address the main reasons for failure: unexpected toxicity and/or lack of efficacy.

Cook, D., Brown, D., Alexander, R, March, R., Morgan, P., Satherwaite, G and Panagalos, M. (2014) Lessons learned from the fate of AstraZeneca's drug pipeline: a five-dimensional framework. Nat Rev Drug Disc, 2014, 13, 419–431.

S44 - ESTABLISHING POTENTIAL SAFETY LIABILITIES IN EARLY DRUG DEVELOPMENT Bill Pennie Takeda, Cambridge, Massachusetts, United States The incorporation of predictive toxicology approaches in early preclinical drug discovery is a scientifically, technically and operationally challenging opportunity to reduce early the stage attrition of drug candidates. Multiple approaches utilizing in silico and in vitro modeling have been brought to bear in this area. The ability of a compound to cause toxicity can be considered a consequence of physico-chemical properties, specific structural alerts within the molecule or molecular interaction with specific primary or secondary pharmacology targets. The specific presentation of the toxicology in vivo is also likely to be driven by the absorption, distribution and metabolism of the molecule. Multiple laboratories have developed models to identify toxicological risks e.g. targeted in vitro assays have been integrated with structure activity-relationship rules and physicochemical properties to predict in vivo effects such as the probability of undesirable low exposure toxicity findings for a given molecule. The results have demonstrated that selecting appropriate cell systems that predict the cellular mechanisms of toxicity are key factors that determine the successful prediction of broad in vivo effects, although predicting organ-specific toxicity remains a challenge. In profiling drug-like molecules, the need to balance an understanding of transporter interactions, metabolism and intended dose of the compound are all shown to have an effect on predictive model performance. Multi-factorial computational models that incorporate dose prediction as well as chemical properties in an attempt to guide early medicinal chemistry to safer chemical space have been developed.

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S45 - THE APPLICATION OF EPIGENOME AND KINOME-BASED BIOASSAYS FOR RISK ASSESSMENT Ivonne Rietjens and Jochem Louisse Division of Toxicology, Wageningen University, Wageningen, The Netherlands One mechanism that has emerged as a major regulator of the organization and function of chromatin are histone posttranslational modifications. Several different covalent modifications exist on histones, including for example acetylation, methylation, ubiquitylation, phosphorylation and sumoylation. These modifications work together in the form of a ‘histone code’ to regulate chromatin-based activities. This histone code functions, in part, through the direct recruitment of protein coregulators to the sites of modification, which then alter the organizational state of chromatin and/or facilitate a biological process (e.g. transcription). A large number of chromatin modifying enzymes have been identified and these include histone acetyl transferases (HATs) that acetylate specific lysine residues in histones and are reversed by the action of histone deacetylases (HDACs), the histone kinases that phosphorylate specific serine or threonine residues and the phosphatases (PPTases) which can remove these phosphorylation marks. In addition, two classes of methylating enzymes are involved being protein arginine methyltransferases (PRMTs) modifying arginine residues and histone lysine methyltransferases (HKMTs), methylating lysine residues. Demethylation activity has been described for histone lysine demethylases (KDMs), which demethylate various histone lysine residues. To obtain insight in the recruitment of some of these histone modifying coregulators we have been pioneering the use of the socalled Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) assay. This Pamgene assay is able to detect the ligand-induced nuclear receptor-mediated recruitment or loss of coactivators and corepressors from protein complexes that regulate and modulate gene transcription. Coregulators that can be detected include for example histone acetyltransferase p300, methylated-DNA-protein-cysteine methyltransferase, histone-lysine N-methyltransferase, serine/threonine-protein kinase PAK 6, histone acetyltransferase PCAF, histone acetyltransferase KAT5, and 60 other coregulators involved in regulation of gene expression. To what extent modulation of these coregulators influences the histone code remains to be established. The array technology also allows for the detection of the activity of both tyrosine and serine/threonine kinases providing possibilities to define the so-called cellular kinome. Using this Pamgene array technology modifications in coregulator activation and in the cellular kinome upon exposure to toxic compounds of interest can be detected and were shown to be concentration dependent, providing the possibility to define concentration-response curves for the effects observed. Current work is focused on defining the links between the effects observed and the ultimate adverse effects of the compounds under study. It is expected that the Pamgene array-based assays provide a novel in vitro technique to support future hazard and risk assessment of toxic compounds, providing insight in their mode of action.

S46 - A HYPERACTIVE mEH VARIANT: SEVERE TRADE-OFF FOR FASTER DETOX Anne Marowsky University of Zurich, Zurich, Switzerland Microsomal epoxide hydrolase (mEH) plays a well-documented role in detoxification by metabolizing genotoxic compounds to less harmful metabolites. A more active mEH variant that leads to accelerated detoxification should therefore present a major benefit. A highly active mEH variant in which the glutamic acid of the catalytic triad is replaced by an aspartic acid (mEH E404D) exists, but exclusively in lower species such as Aspergillus niger. To answer the question why this variant has not persisted in mammals, we generated mice harboring the mEH E404D point mutation. In liver microsomes from these mice, turnover of the xenobiotic compound phenantrene-9,10-oxide was five times faster compared to liver microsomes from WT, confirming accelerated detoxification. In addition, mEH E404D animals also showed faster metabolization of endogenous substrates, i.e. of arachidonic acid-derived epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). Increased DHET/EET ratios were found in mEH E404D liver indicating increased turnover of EETs relative to WT liver. Moreover, DHET/EET ratios were enhanced in mEH E404D urine, plasma and brain compared to WT controls, which suggests a broad impact of the mEH mutant on EETs metabolism. As EETs are strong vasodilators, hemodynamics were assessed in cerebral cortex and hippocampus using cerebral blood volume (CBV)-based functional magnetic resonance imaging (fMRI). Basal CBV 0 levels were similar between mEH E404D and control mice in both brain areas. However, vascular reactivity and vasodilation in response to the vasodilatory drug acetazolamide was significantly diminished in mEH E404D cortex and hippocampus compared to controls. These results reveal a hitherto unexpected role for the detoxifying enzyme mEH in cerebral vasodynamics. The alterations in EETs metabolism brought about by the mEH E404D mutation offer a plausible explanation for the absence of this highly active variant in mammals.

S47 - DRUG-DRUG AND DRUG-ENDOBIOTIC INTERACTIONS ARISING FROM INHIBITION OF UDPGLUCURONOSYLTRANSFERASES John Miners Flinders University School of Medicine, Adelaide, Australia The UDP-glucuronosyltransferases (UGTs) comprise a superfamily of enzymes that metabolise structurally diverse compounds that include drugs and non-drug xenobiotics, as well as a variety of endogenous compounds such as bilirubin, fatty acids and hydroxy-steroids. Like the cytochromes P450, the individual human UGT enzymes differ in terms of substrate and inhibitor selectivities. Although it was originally believed that drug-drug interactions arising from inhibition of UGT enzymes were rare, there is increasing evidence that inhibition of drug and endobiotic

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glucuronidation may be of clinical significance. Selective substrates for most human UGT enzymes are available, and this permits the screening of new chemical entities for inhibition drug and endobiotic glucuronidation using recombinant UGT proteins, human liver microsomes, and hepatocytes as the enzyme sources [1]. The utility of this approach will be demonstrated using tyrosine kinase inhibitors (TKIs) as potential perpetrators of drug-drug and drugendobiotic interactions. When corrected for non-specific binding to the enzyme source, the TKIs lapatinib, pazopanib, regorafenib and sorafenib potently inhibit several UGT enzymes, especially UGT1A1 (which is responsible for bilirubin glucuronidation). Ki values for inhibition of recombinant and human liver microsomal UGT1A1 range from approximately 20 nM (regorafenib and sorafenib) to 1.7 µM (pazopanib). Ki values for the polymorphic UGT1A1*6 variant (G71R) are similar. In addition to UGT1A1, the four TKIs are potent inhibitors of UGT1A9 (Ki values 150 nM to 2 µM), and ‘moderate’ inhibitors of UGT1A6 (Ki values < 10 µM). TKIs are known to variably cause jaundice, and these data provide a mechanistic basis for the clinical observations. Moreover, the results indicate the possibility of drug-drug interactions arising from inhibition of UGT1A6 and UGT1A9.

[1] JO Miners, PI Mackenzie and KM Knights. Drug Metab Rev 42:196-208 (2010).

S48 - ROLE OF SULPHOTRANSFERASES IN METABOLIC ACTIVATION Hansruedi Glatt, Gitte Barknowitz, Carolin Bendadani, Gisela Dobbernack, Wolfram Engst, Simone Florian, Kristin Herrmann, and Walter Meinl German Institute of Human Nutrition, Berlin, Germany Sulphotransferase (SULT)-mediated activation is ignored in standard in vitro genotoxicity tests due to the lack of SULT expression in the target cells and the absence of the cofactor 3'-phosphoadenosine-5'-phosphosulphate (PAPS) in S9 preparations. Supplementation of PAPS is no remedy, as many sulpho conjugates do not penetrate cell membranes owing to their charge, in particular if their half-life time is short. SULT-mediated activation is also largely disregarded in tests conducted in bone marrow cells in vivo. During the last decades, we expressed numerous mammalian SULTs in target cells of in vitro mutagenicity assays (Salmonella typhimurium strains and Chinese hamster V79 cells). We detected > 100 compounds activated by this class of enzymes. Activation of a given promutagen often required the expression of a particular SULT form from a given species (out of ca. 13 forms), varying for different promutagens. Not rarely, the individual enantiomers of a compound were activated by different SULT forms. We also noticed substantial differences in the bioactivation among orthologous SULT forms from different species. Subsequently, we constructed knockout mouse lines for Sult1a1 and Sult1d1, transgenic lines for human SULT1A1/2 and SULT1B1, and combinations thereof. Using isotope-dilution LC-MS/MS techniques we studied the levels and the tissue distribution of DNA adducts formed by various compounds: Knockout of Sult1a1 drastically reduced the adduct formation by methyleugenol (a carcinogenic constituent of many herbs and spices) and its metabolite 1’-hydroxymethyleugenol, Nmethoxyindole-3-carbinol (a breakdown product of neoglucobrassicin, a glucosinolate contained in Brassica species), furfuryl alcohol (a carbohydrate pyrolysis product) and 1-hydroxymethylpyrene (major primary metabolite of a common carcinogenic polycyclic aromatic hydrocarbon). Replacement of the endogenous Sult1a1 by human SULT1A1/2 strongly enhanced the adduct formation by these compounds and expanded the target tissues compared to the wildtype. The latter effects were also seen with 1-methylpyrene (a carcinogenic polycyclic aromatic hydrocarbon) and 1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, a carcinogenic heterocyclic aromatic amine). Interestingly, knockout of Sult1d1, but not Sult1a1, reduced the adduct formation by PhIP. Adduct formation in the bone marrow by SULTdependent genotoxicants was weak in any mouse model, as far as studied. This work demonstrates that SULTs play pivotal roles in the bioactivation of many genotoxic carcinogens and that there are marked species-dependent differences in this bioactivation. Finally, we analysed human tissue specimens (liver, lung) and blood samples for the presence of DNA and protein adducts, respectively, of some SULT-dependent genotoxicants using LC/MS-MS techniques. DNA adducts of methyleugenol were detected in nearly all human 151 liver samples and 10 lung samples tested, whereas adducts of 4-aminobiphenyl were not found in any samples despite a similar detection sensitivity.

DNA adducts of furfuryl alcohol were found in most lung samples (not studied in liver). Haemoglobin and serum albumin adducts of neoglucobrassicin were readily detected after a broccoli-rich diet. Lower levels of these adducts were also found in many probands without intervention. These findings demonstrate that SULT-mediated bioactivation is relevant in humans in vivo.

S49 - CARBOXYLESTERASE 1 – SUBSTRATES, REGULATION OF ENZYME ACTIVITY AND PHARMACOGENETICS Henrik Rasmussen and Laura Ferrero-Miliani Institute of Biological Psychiatry, Mental Health Centre Sct. Hans., Roskilde, Denmark Carboxylesterase 1 (CES1) is a broad-specificity serine esterase which catalyzes the hydrolysis of ester, thioester, and amide bonds. Xenobiotic substrates metabolized by CES1 include several commonly used therapeutics, illegitimate drugs, pesticides, and environmental toxicants. Additionally, this enzyme appears to be involved in physiological functions, including metabolism of lipids such as cholesteryl esters and triglycerides.

CES1 is predominantly expressed in the liver, adipose tissue, and monocytes/macrophages. Importantly, the level of CES1 in liver microsomes from different humans has been found to vary by almost 5-fold suggesting that this level is an important contributor to individual variation in the kinetics of drugs and other xenobiotics metabolized by the enzyme. There is a complex genomic architecture at the CES1 locus with a wild-type gene (CES1A1), an inverted

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gene duplication (CES1A2) and two CES1-like pseudogenes, called CES1P1 and CES1P2. The most common variant of the duplicated gene, CES1A2, is transcribed at a low level. The complex genomic structure of CES1 leads to challenges in its genotyping. Several single nucleotide variants (SNVs) with a potential impact on the pharmacokinetics and response of drugs have been reported in CES1A1 and CES1A2. However, these SNVs either appear to be relatively rare or are of modest effect size. CES1 exists in an oligomeric form consisting of three 60-kDa units and a hexameric form with the former of these two being the enzymatically active form. There are three ligand binding sites in monomeric CES1, namely, the active site, a “side door” possibly serving as an additional entrance for substrates and the Z-site. The latter is a superficial and low affinity ligand-binding site potentially involved in regulating the enzyme activity. It is likely, that binding of endogenous small molecules to the Z-site affects enzyme activity.

Metabolomics has the potential to identify and quantitate such molecules thus informing about the hepatic CES1 activity in an individual. Additionally, this approach may be capable of detecting endogenous substrates of CES1 and their products in the blood that correlate with the activity of the enzyme in the liver. Research should aim at refining the CES1 genotyping, identify new variants in the gene encoding CES1, and unravel metabolomic signatures in the blood that can serve as proxies for CES1 activity in the liver. A pharmacogenetic profile provides information about an individual’s inherited response to a drug whereas a metabolomic profile contains information about environmental influences. Accordingly, the combination of CES1 genotyping with metabolomic profiling of the enzyme may provide a more accurate prediction of an individual’s capacity for metabolism of CES1-dependent drugs than either of these two approaches alone. This may help personalize treatment with drugs metabolized by CES1 such as methylphenidate and angiotensin-converting enzyme inhibitors. In this presentation substrates of CES1, substructures in this protein determining its activity, genetic variants of CES1 will be reviewed followed by a discussion of the prospects of personalizing treatments with drugs metabolized by CES1.

S50 - INFLAMMATORY REGULATION OF HUMAN DRUG METABOLIZING ENZYMES Ulrich Zanger Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany Inflammatory processes of diverse origin are associated with circulating proinflammatory cytokines such as IL-6, IL-1β, and TNFα, which elicit in the liver the acute phase response (APR), primarily resulting in the mass production of APRproteins to combat the cause of inflammation. Simultanesously to this positive APR a similarly extensive negative APR takes place that leads to the downregulation of many drug metabolizing enzymes and transporters, resulting in a broad and clinically relevant impairment of hepatic detoxification. Although data suggest that this reflects a coordinated response, it remained unclear, whether a common mechanism is involved. We used a combination of models (human livers from donors with elevated versus normal levels of the inflammation marker C-reactive protein; primary human hepatocytes and HepaRG cells stimulated with cytokines, chemical inhibitors, and microRNAs) and methods (expression profiling, phosphoproteomics, enzyme activity measurements, mathematical modeling) to characterize in depth the hepatic APR. Extensive data sets were generated and used to calibrate a prior knowledgebased fuzzy-logic model comprising signal transduction and gene regulation. Our model suggests a major role of MAPK and PI3K signaling, with the orphan nuclear receptor RXRα playing a central role. Validation experiments revealed a striking similarity of RXRα gene silencing and IL-6 stimulation with respect to negative gene regulation.

These results concur with RXRα functioning as obligatory heterodimerization partner for several nuclear receptors that regulate drug and lipid metabolism. Additional data suggest that some microRNAs are strongly elevated in cholestatic liver and during inflammation. We found that miR-130b downregulates enzyme activities of various cytochromes P450 (e.g., CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP3A4) and nuclear receptors CAR and FXRalpha. At least for CYP2C9 a direct interaction with the 3’UTR could be shown. These data support miR-130b as a potential negative regulator of drug metabolism by directly and/or indirectly affecting the expression of several ADME genes. In conclusion, several mechanisms may simultaneously contribute to a very effective downregulation of ADME genes during inflammation.

S51 - REGULATION OF DRUG TRANSPORTERS IN INFLAMMATION: IMPACT ON DRUG DISPOSITION Micheline Piquette-Miller Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada Underlying disease states are often key sources of inter-subject variability of drug response in patient populations.

Inflammation, which is a component of many diseases such as infection, arthritis, atherosclerosis, diabetes and cancer, has been reported to impart changes in drug disposition. Transport proteins which are highly expressed in epithelial membrane barriers can profoundly impact the absorption, distribution and clearance of numerous drugs, toxins and metabolic products and thus may play an important role in these changes. Indeed, our studies in rodent models of protozoan, bacterial and viral infection as well as chronic conditions have demonstrated significant changes in the expression of many of the ABC drug efflux transporters, the SLC family of uptake transporters as well as CYP3A drug metabolizing enzymes in the liver, intestine, blood brain barrier, kidney and placenta. These changes are associated with altered distribution and clearance of their substrates. NF-kappa B activation and nuclear-receptor mediated pathways play a key role. Recent studies in human placenta also indicate disease or infection induced changes in transporter expression. As transporters are involved in the distribution and elimination of a large number of clinically important drugs and endogenous compounds, our studies suggest that disease-induced changes is an

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important source of inter-subject variability and could contribute to adverse outcomes or therapeutic failure. This information is important in the prediction of drug-disease interactions.

S52 - ROLE OF NO IN INFLAMMATORY REGULATION OF DRUG METABOLIZING ENZYMES Edward Morgan Emory University, Atlanta, Georgia, United States Inflammatory signaling in the liver results in the down-regulation of many cytochrome P450 (P450) and other drug metabolizing enzymes. A role for nitric oxide in this down-regulation was proposed by several groups in the early 1990s, based on the ability of inhibitors of NO synthases (NOS) to block or attenuate the down-regulation. Most studies of P450 down-regulation in inflammation have focused on transcriptional mechanisms, which appear to be predominant for many or most enzymes. Evidence for a role of NO in transcriptional regulation is limited, and studies in our laboratory using NOS inhibitors in vitro and in vivo, or in NOS2-deficient mice showed that down-regulation of several P450 mRNAs occurred independently of NO production. However, studies in primary rat hepatocyte cultures revealed that the phenobarbital-inducible enzyme CYP2B1 undergoes NO-dependent proteolysis that is more rapid than the decline in CYP2B1 mRNA. This degradation proceeds via the canonical ubiquitin (Ub)-dependent proteasomal degradation pathway. Systematic mutation of critical Cys and Tyr residues on CYP2B1 suggested that the triggering event may be an accumulation of amino acid modifications by NO, rather than modification of a single sensitive residue. A proteomic study in cultured rat hepatocytes detected two additional P450 proteins regulated by NO: CYP3A1 and the retinoic acid-metabolizing enzyme CYP2C22. The degradation of CYP2C22 is not blocked by inhibitors of the proteasome, indicating that other proteolytic pathways may participate in the degradation of this enzyme, at least when the proteasome is inhibited. To determine whether human P450s undergo NO-dependent degradation, we studied the effect of NOS inhibition in cultured human hepatocytes. Down-regulation of CYP2B6 by a cytokine mixture was attenuated by NO inhibitors, and mimicked by a chemical NO donor. We have further investigated the mechanism of CYP2B6 down-regulation using cell lines expressing CYP2B6 or CYP2B6 with a Cterminal V5 tag from a lentiviral vector with a CMV promoter. In this system, CYP2B6 protein is degraded within 3 h of treatment with the NO donors DETA NONOate or DPTA NONOate, in the absence of changes in its mRNA levels.

The concentration of DPTA NONOate required to down regulate CYP2B6 was significantly lower than that for rat CYP2B1. Proteasome inhibitors had little effect on CYP2B6 down-regulation. However, calpain and lysosomal protease inhibitors also failed to inhibit the NO dependent CYP2B6 down-regulation. Studies are ongoing to investigate the mechanism of CYP2B6 down-regulation by NO, including the possible role(s) of novel proteolytic pathways. This work was supported by grant R01GM069971 from the National Institutes of Health.

S53 - ABSTRACT UNAVAILABLE

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Poster Details A12 - CAR TRANSCRIPTIONAL ACTIVITY IS UNDER THE INFLUENCE OF EGF IN PRIMARY HUMAN HEPATOCYTES. IDENTIFICATION OF CAR SPECIFIC TARGET GENES Hugues de Boussac, INSERM U1183, IRMB, Hôpital Saint-Eloi, Montpellier, France

GRADUATE / PREDOCTORAL POSTER FINALISTS (A1 – A6) A1 - SIMULTANEOUS QUANTIFICATION OF ESTROGENIC TAMOXIFEN METABOLITES AND SEX STEROID HORMONES IN HUMAN PLASMA VIA UHPLC-ESI-MS/MS Janina Johänning, Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and Universitiy of Tuebingen, Stuttgart, Germany

ABSORPTION (P1 – P2) P1 - A NEW APPROACH TO ASSESS PROCESSES THAT DETERMINE HUMAN ORAL BIOAVAILABILITY USING EX VIVO PORCINE INTESTINAL TISSUE Evita van de Steeg, TNO, Zeist, The Netherlands

A2 - IS THE USE OF 1-AMINOBENZOTRIAZOLE AS CYTOCHROME P450 INACTIVATOR IN RAT STUDIES APPROPRIATE? Marc-Olivier Boily, Centre de Recherche, Hôpital MaisonneuveRosemont, Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada

P2 - SIMULATION STUDY ON CONTRIBUTIONS OF MEMBRANE PERMEABILITY, METABOLIC CLEARANCE, AND EFFLUX TRANSPORT BY P-GLYCOPROTEIN TO INTESTINAL AVAILABILITY USING TRANSLOCATION MODEL Hirotaka Ando, Kyorin Pharmaceutical Co., Ltd., Nogi-machi, Shimotsuga-gun, Japan

A3 - PRECISION-CUT INTESTINAL SLICES AS EX VIVO MODEL TO STUDY THE UPTAKE OF BILE ACIDS BY ASBT IN THE INTESTINE Ming Li, Department of Pharmacy, University of Groningen, Groningen, The Netherlands

ANALYTICAL (P3 – P6) P3 - A SENSITIVE AND RAPID ANALYSIS OF ESCITALOPRAM IN HUMAN PLASMA BY UPLC/MS/MS SK Wo, School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong

A4 - INVOLVEMENT OF DIFFERENT CYTOCHROME P450 ISOFORMS IN THE SEQUENTIAL 2-STEP BIOACTIVATION OF DICLOFENAC AND THE PHYSIOLOGICAL RELEVANCE OF SUBSEQUENT DETOXIFICATION BY GLUTATHIONE STRANSFERASES Michiel W. den Braver, VU University Amsterdam, Amsterdam, The Netherlands

P4 - CONTRIBUTION OF QUANTITATIVE MASS SPECTROMETRY IMAGING (MSI) IN PHARMACEUTICAL FIELD: APPLICATION TO DRUG AND PEPTIDE ANALYSIS IN TISSUE Gregory Hamm, ImaBiotech, Loos, France

A5 - THE POLYMORPHIC VARIANT P24T OF UGT1A4 AND ITS UNUSUAL CONSEQUENCES Johanna Troberg, University of Helsinki, Helsinki, Finland

P5 - EVALUATION OF RESPONSE FACTORS OF DRUG METABOLITES ON CAPILLARY HPLC/MS WITH ELECTROSPRAY IONIZATION Joachim Blanz, Novartis Institutes for Biomedical Research / Analytical Sciences and Imaging, Basel, Switzerland

A6 - ABCB1, ABCC1 AND ABCG2 MODULATE TRANSPORT AND PHARMACOKINETICS OF DINACICLIB IN VITRO Daniela Cihalova, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic

P6 - INNOVATIVE APPROACH TO BLOOD SAMPLING USING DRIED BLOOD SPOTS. APPLICATION TO PHARMACOKINETICS AND CYTOCHROME P450 PHENOTYPING Youssef Daali, Geneva University Hospitals, Geneva, Switzerland

POSTDOCTORAL POSTER FINALISTS (A7 – A12) A7 - DEORPHANIZATION OF HUMAN CYTOCHROME P450 4X1 Michal Šiller, Palacky University, Olomouc, Czech Republic

BIOAVAILABILITY (P7 – P9) P7 - KEEPING AN EYE ON MOLECULAR IMAGING: ASSESSMENT OF DRUG TOXICITY IN SMALL OCULAR STRUCTURE USING MASS SPECTROMETRY IMAGING Gregory Hamm, ImaBiotech, Loos, France

A8 - ACTIVITIES OF CYTOCHROMES P450 IN HUMAN SKIN EXPLANTS Nenad Manevski, DMPK, Novartis, Basel, Switzerland

P8 - PHARMACOKINETICS OF GANODERMA ACID A IN RAT PLASMA AND BRAIN DETERMINED BY A DEVELOPED UPLC-MS/MS METHOD Fangrui Cao, Institute of Medicinal Plant Development, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China

A9 - TAB-METHYL-SEQ PROTOCOL FOR TARGETED NEXT-GENERATION SEQUENCING OF DNA (HYDROXY)METHYLATION: IMPLICATIONS FOR ADME GENE REGULATION Maxim Ivanov, Karolinska Institutet, Stockholm, Sweden A10 - IN SILICO PREDICTION OF HEPATOCELLULAR DRUG BINDING Frauke Assmus, Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom

P9 - PROTEOMIC INVESTIGATION OF SALIVA AS A SOURCE OF BIOMARKERS OF ENERGY INTAKE Joanna Chowdry, Department of Oncology, University of Sheffield, Sheffield, United Kingdom

CLEARANCE PREDICTION (P10 – P16)

A11 - COMPARISON OF RESPIRATORY DRUG ACCUMULATION AND LYSOSOMAL SEQUESTRATION IN NR8383 AND PRIMARY HUMAN ALVEOLAR MACROPHAGES Ayse Ufuk, Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom

P10 - ASSESSING METABOLIC COMPETENCE IN ISOLATED HEPATOCYTES: EXPLORING THE RELATIONSHIP BETWEEN ENZYME FUNCTION AND PLASMA MEMBRANE INTEGRITY VIA SAPONIN TREATMENT Francesca Wood, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom

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Poster Details

P11 - CYNOMOLGUS MONKEY AS A PRECLINICAL MODEL FOR THE INVESTIGATION OF HEPATIC UPTAKE OF OATP SUBSTRATES Tom de Bruyn, Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom

P22 - DEFINING SPECIES DIFFERENCES IN THE METABOLISM OF THE EGFR INHIBITOR AZD9291 USING TRANSGENIC MODELS Lesley McLaughlin, University of Dundee, Dundee, United Kingdom P23 - DISCOVERY OF A HIGHLY SELECTIVE PROBE FOR HUMAN CYTOCHROME P450 3A5 Jingjing Wu, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

P12 - DETERMINATION OF A HORSE HEPATIC MICROSOMAL SCALING FACTOR FOR PREDICTING IN VIVO METABOLIC CLEARANCE Khaled Shibany, The University of Nottingham, Loughborough, United Kingdom

P24 - KINETIC PARAMETERS (KM AND KCAT) OF CYTOCHROMES P450: A COMPREHENSIVE SURVEY REVEALS A ROLE FOR SPECIFIC INTERACTIONS AS WELL AS LIPOPHILICITY IN SUBSTRATE AND TRANSITION STATE RECOGNITION. Stephen J. Messham, Liverpool John Moores University, Liverpool, United Kingdom

P13 - IMPACT OF AGE ON HEPATIC EXTRACTION RATIO OF DRUGS IN PAEDIATRIC POPULATION Yoshiteru Kamiyama, Analysis & Pharmacokinetics Labs, Astellas Pharma Inc., Tsukuba-shi, Japan P14 - LOW INTRINSIC CLEARANCE DETERMINATION USING PRIMARY HUMAN HEPATOCYTES IN MONOCULTURE COMPARED TO CO-CULTURE WITH NONPARENCHYMAL STROMAL CELLS. Petter Svanberg, RIA iMed DMPK, AstraZeneca R&D, Mölndal, Sweden

P25 - LITTLE EFFECT OF SOLIDAGO VIRGAUREA L. EXTRACT ON PHASE I. BIOTRANSFORMATION ENZYMES IN HUMAN HEPATOCYTES AND HUMAN LIVER MICROSOMES Veronika Tománková, Faculty of Medicine and Dentistry, Palacky University, Department of Medical Chemistry and Biochemistry, Olomouc, Czech Republic

P15 - PREDICTION OF PASSIVE RENAL TUBULAR REABSORPTION USING A MINIMAL PHYSIOLOGICALLYBASED MODEL Daniel Scotcher, Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom

P26 - MEDICINAL PLANTS EPILOBIUM HIRSUTUM L . AND VISCUM ALBUM L. ALTER PROTEIN AND MRNA EXPRESSIONS OF RAT LIVER BILE ACID SYNTHESIZING CYP7A1 Tuba Culcu, Middle East Technical University, Department of Biological Sciences and Joint Graduate Program in Biochemistry, Cankaya-Ankara, Turkey

P16 - UTILIZATION OF LIVER MICROSOMES FOR PREDICTION OF HEPATIC INTRINSIC CLEARANCE OF MONOAMINE OXIDASE SUBSTRATE DRUGS IN HUMANS Yukio Kato, Faculty of Pharmacy, Kanazawa University, Kanazawa, Japan

P27 - POSSIBLE MECHANISM(S) CAUSING INVERSE ENANTIOSELECTIVITY IN BUNITROLOL 4HYDROXYLATION BY CYP2D ENZYMES Shizuo Narimatsu, Okayama University, Okayama, Japan

CONJUGATION REACTIONS AND ENZYMES (P17 – P19)

P28 - SUBSTANTIAL INHIBITORY EFFECT OF DMSO ON IN VITRO INTRINSIC CLEARANCE MEASUREMENT – ROLE OF CYP3A4 ?

Hugues Chanteux, UCB Biopharma, Braine L'Alleud, Belgium

P17 - A NOVEL APPROACH IN MEASURING PROTEIN SGLUTATHIONYLATION IN STRESS RESPONSE SIGNALLING David McGarry, University of Dundee, Dundee, United Kingdom

DIFFERENCES IN METABOLISM (SPECIES, GENDER, AGE, DISEASES) (P29 – P32)

P18 - EFFECT OF DONOR VARIABILITY AND CULTURE CONDITIONS ON PHASE II ACTIVITY IN HUMAN HEPATOCYTES Shalenie P. den Braver-Sewradj, VU University Amsterdam, Amsterdam, The Netherlands

P29 - APPLICATION OF A MOUSE LINE HUMANISED FOR HPXR/CAR/CYP3A4/CYP3A7/CYP2D6/CYP2C9 TO INVESTIGATE THE REVERSIBLE AND TIME-DEPENDENT INHIBITION OF MIDAZOLAM 1’-HYDROXYLATION Yury Kapelyukh, University of Dundee, Dundee, United Kingdom

P19 - METABOLISM OF C-1748, 1-NTROACRIDINE ANTITUMOR AGENT, VIA HUMAN UDPGLUCURONOSYLTRANSFERASES IN PANCREATIC CANCER CELL LINES AND ITS EFFECTS ON THE MODULATION OF UGT1A9 AND UGT2B7 ACTIVITY Zofia Mazerska, Department of Pharmaceutical Technology and Biochemistry, Chemical Faculty, Gdańsk University of Technology, Gdańsk, Poland

P30 - DIFFERENCES OF BETAHISTINE PHARMACOKINETICS AND METABOLISM IN DOG, CAT AND MAN Matthias Olbrich, Abbott Laboratories , Hannover, Germany P31 - FORMATION OF THE CARBOXYLIC ACID METABOLITE OF AZD5069 Anja Ekdahl, RIA iMed DMPK, AstraZeneca R&D, Mölndal, Sweden

CYTOCHROME P450 (P20 – P28) P20 - A MULTIPLEX APPROACH TO INVESTIGATE DRUG INDUCED CHANGES IN P450 ENZYME GENE EXPRESSION Jason DeLoach, HTG Molecular Diagnostics, Inc., Tucson, Arizona, United States

P32 - IN-SITU METABOLITE PROFILING OF REMODELED ARTERIES IN PULMONARY ARTERIAL HYPERTENSION USING INNOVATIVE MASS SPECTROMETRY IMAGING TOOLS Gregory Hamm, ImaBiotech, Loos, France

P21 - ANTIOXIDANT ACTIVITIES OF PROPOLIS AND ITS BIOACTIVE COMPONENTS, AND THEIR EFFECTS ON CYP1A1 GENE EXPRESSION IN HT-29 ADENOCARCINOMA CELL LINE Deniz Irtem Kartal, Middle East Technical University, Ankara, Turkey

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Poster Details P44 - DIRECT AND CYTOKINE-MEDIATED EFFECTS OF ALBUMIN-FUSED HUMAN GROWTH HORMONE, TV-1106, ON CYP ENZYME EXPRESSION IN HUMAN HEPATOCYTES IN VITRO David Buckley, XenoTech LLC, Lenexa, Kansas, United States

DISPOSITION (P33 – P38) P33 - APPLICATION OF A MULTI-COMPARTMENT PERMEABILITY LIMITED LUNG MODEL TO PREDICT LUNG CONCENTRATIONS OF ANTI-TUBERCULOSIS DRUGS IN VIRTUAL HUMAN SUBJECTS Iain Gardner, Simcyp, Ltd., Sheffield, United Kingdom P34 - DICLOFENAC: BIOCONCENTRATION, TISSUE DISTRIBUTION AND METABOLISM IN FISH Andrew McEwen, Quotient Bioresearch Limited, Rushden, United Kingdom

P45 - EFFECT OF PARACETAMOL ON THE PHARMACOKINETICS AND TISSUE DISTRIBUTION OF SUNITINIB IN FEMALE MICE Ignacio Segarra, Clinical Pharmacy and Pharmacotherapy Unit, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Barcelona, Spain

P35 - NOVEL MULTIPLE ASSAY SYSTEM FOR HEPATOCELLULAR DRUG DISPOSITION Ryosuke Takahashi, Central Research Laboratory, Hitachi, Ltd., Saitama, Japan

P46 - EFFECTS OF METRONIDAZOLE AND TINIDAZOLE ON THE HEPATIC CYP EXPRESSION Kiyomi Ito, Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan

P36 - PROTEIN BINDING IN COMBINATION WITH PH, METALS AND INTERACTING DRUGS CAN AUGMENT THE DISPOSITION OF RALTEGRAVIR Darren Moss, University of Liverpool, Liverpool, United Kingdom

P47 - EVALUATION OF THE DRUG-DRUG INTERACTION BETWEEN ZIDOVUDINE AND PROBENECID BY USING A MECHANISTIC KIDNEY MODEL (MECH KIM) NESTED WITHIN A FULL PHYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODEL Hilary Kim Crewe, Simcyp, Ltd., Sheffield, United Kingdom

P37 - RODENT PHARMACOKINETICS AND IN VITRO ADME PROPERTIES OF IV3086, AN ORALLY AVAILABLE AND BRAIN PENETRANT NURR1/RXR ACTIVATOR Bruno Bournique, INVENTIVA, Daix, France

P48 - EVALUATION OF THE IN VITRO BINDING AFFINITY OF PHOTOTOXIC CHEMICALS TO SYNTHETIC MELANIN Jelle Reinen, WIL Research Europe, 's Hertogenbosch, The Netherlands

P38 - WHAT DATA CAN A 14C CLINICAL STUDY DELIVER? A DISPOSITION DASHBOARD: INNOVATIVE AND INTEGRATED 14C STUDY DESIGNS TO UNDERSTAND DRUG ABSORPTION AND DISPOSITION IN HUMAN SUBJECTS Iain Shaw, Quotient Clinical Ltd., Nottingham, United Kingdom

P49 - HERB-DRUG INTERACTION MEDIATED HEPATOTOXICITY OF TRIPTOLIDE: ROLES OF CYP AND EFFLUX TRANSPORTER Hua Li, Beijing Institute of Pharmacology and Toxicology, Beijing, China

DRUG DISCOVERY AND DEVELOPMENT (P39 – P42)

P50 - IN VITRO ASSESSMENT OF THE EFFECT OF GLUCURONIDES ON METABOLIC ENZYMES AND OATP1B1 Aleksandra Galetin, Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom

P39 - NOVEL PHTHALOCYANINES AND TETRAPYRIDOPORPHYRAZINES IN PHOTODYNAMIC THERAPY AND VASCULAR-TARGETED PHOTODYNAMIC THERAPY OF CANCER – IN VITRO STUDY. Miloslav Macháček, Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic

P51 - IN VITRO ASSESSMENT OF THE PHARMACOKINETIC DRUG-DRUG INTERACTION POTENTIAL OF RASAGILINE AND ITS MAJOR METABOLITE AMINOINDAN Lydia Vermeer, XenoTech LLC, Lenexa, Kansas, United States

P40 - ON-LINE ELECTROCHEMISTRY/MASS SPECTROMETRY (EC/MS) – A POWERFUL TOOL FOR THE PREDICTION OF DRUG OXIDATIVE METABOLISM REACTIONS AND THEIR MOLECULAR MECHANISM. Agnieszka Potęga, Department of Pharmaceutical Technology and Biochemistry, Chemical Faculty, Gdańsk University of Technology, Gdańsk, Poland

P52 - IN VITRO STUDY OF HERBAL CONSTITUENT INHIBITION ON HUMAN LIVER MICROSOMAL MORPHINE GLUCURONIDATION: A PREDICTION OF METABOLIC DRUG-DRUG INTERACTION ARISING FROM ANDROGRAPHOLIDE INHIBITION Verawan Uchaipichat, Khon Kaen University, Khon Kaen, Thailand

P41 - SMALL MOLECULE TARGETING OF THE PYRUVATE DEHYDROGENASE COMPLEX (PDC)/PYRUVATE DEHYDROGENASE KINASE (PDK) AXIS Peter Stacpoole, University of Florida, Gainesville, Florida, United States

P53 - IN-VITRO EVALUATION OF DDI WITH COBICISTAT AND RITONAVIR USING HEPARG CELL LINE Flavia Storelli, Geneva University Hospitals, Geneva, Switzerland

P42 - THE EFFECT OF CHRYSIN ON LYMPHANGIOGENESIS IN VITRO Orawin Prangsaengtong, Department of Biopharmacy, Faculty of Pharmacy, Srinakharinwirot University, Nakornayok, Thailand

P54 - POTENTIAL INTERACTION OF ANXIOLYTIC DRUG, BUSPIRONE, WITH HUMAN ORGANIC CATION TRANSPORTER 1 Chutima Srimaroeng, Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand

DRUG INTERACTION (P43 – P58)

P55 - PREDICTION OF IN VIVO DRUG-DRUG INTERACTIONS BETWEEN DRONEDARONE AND RIVAROXABAN BASED ON IN VITRO INHIBITION STUDIES Eric Chan, National University of Singapore, Singapore, Singapore

P43 - ‘TIME-DEPENDENT’ INHIBITION OF HEPATIC UPTAKE TRANSPORTERS IN HEPATOCYTES AND IMPLICATIONS ON THE ASSESSMENT OF TRANSPORTER-MEDIATED DRUG-DRUG INTERACTIONS Michiharu Kageyama, Center for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom

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Poster Details

P56 - SILENSOMES™: A NEW IN VITRO MODEL FOR HUMAN CYTOCHROMES P450 PHENOTYPING ASSAYS Christophe Chesné, R&D, Biopredic International, Saint Gregoire, France

P67 - IN VITRO ASSESSMENT OF DIRECT AND TIMEDEPENDENT INHIBITORY EFFECTS ON MAJOR HUMAN CYTOCHROME P450 ENZYMES BY SPASMOLYTICS Uwe Fuhr, Department of Pharmacology, Hospital of the University of Cologne, Cologne, Germany

P57 - THE LACK OF UTILITY OF PHARMACOLOGICAL INTERFERENCE FOR THE STUDY OF PROTEIN DEGRADATION Christina Chan, University of Liverpool, Liverpool, United Kingdom

P68 - REVERSIBLE INHIBITION AND MECHANISM-BASED INACTIVATION OF HUMAN CYP2J2 BY DRONEDARONE AND AMIODARONE Aneesh Karkhanis, National University of Singapore, Singapore, Singapore

P58 - TOWARDS QUANTITATIVE PREDICTION OF DISEASE-DRUG INTERACTIONS USING A PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELLING APPROACH: SUPPRESSION OF CYP1A2 BY IL-6 Krishna Machavaram, Simcyp, Ltd., Sheffield, United Kingdom

EXTRAHEPATIC METABOLISM (P69) P69 - IN VITRO ASSESSMENT OF SKIN METABOLISM: COMPARISON OF METHODS AND IMPLICATIONS FOR WIDER APPLICATION Katie Plant, Cyprotex, Macclesfield, United Kingdom

ENZYME INDUCTION (P59 – P63)

GENE EXPRESSION AND REGULATION (P70 – P79)

P59 - EVALUATION OF CYTOCHROME P450 ENZYME INDUCTION IN VITRO: TREND ANALYSIS AND CORRELATION OF ENZYME ACTIVITY DATA WITH MRNA EXPRESSION David Wilkinson, Covance Laboratories Ltd., Harrogate, United Kingdom

P70 - AHR, PXR, NRF2 AND GR INDUCE THE EXPRESSION OF THE HUMAN GLUTATHIONE S-TRANSFERASE ALPHA 1 (HGSTA1) IN HEPG2 CELLS María Del Carmen Martínez-Guzmán, Department of Cell Biology, CINVESTAV-IPN, México City, México

P60 - EVALUATION OF DIFFERENT NORMALISATION METHODS TO PREDICT CYP3A4 INDUCTION IN SIX FULLY CHARACTERIZED CRYOPRESERVED HUMAN HEPATOCYTE PREPARATIONS AND HEPARG CELLS Helene Vermet, Sanofi, Montpellier, France

P71 - ARYL HYDROCARBON RECEPTOR (AHR) UPREGULATES UBCM4 EXPRESSION IN THE MOUSE BRAIN Emmanuel González-Barbosa, Department of Cell Biology, CINVESTAV-IPN, México City, México

P61 - NUCLEAR RECEPTOR-MEDIATED CHANGES IN GENE EXPRESSION IN A HUMAN HEPATOCYTE MICROPATTERNED CO-CULTURE SYSTEM FOLLOWING TREATMENT WITH HEPATOTOXIC COMPOUNDS Mann Shoffner, Hepregen Corporation, Medford, Massachusetts, United States

P72 - DEFINING THE REGULATORY NETWORKS THAT CONTROL THE IN VIVO EXPRESSION OF HUMAN CYTOCHROME P450 CYP2B6 USING A NOVEL LACZ REPORTER MOUSE MODEL Aileen McLaren, University of Dundee, Dundee, United Kingdom

P62 - THE EFFECT OF CYP1A2 INDUCTION ON MELATONIN PHARMACOKINETICS IN HEALTHY THAI SUBJECTS Sirimas Kanjanawart, Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

P73 - DETERMINATION OF CELLULAR SIGNALING PATHWAYS MODULATED BY EXPOSURE OF HUMAN HEPATIC CELLS TO CYLINDROSPERMOPSIN Antoine Huguet, Anses, Fougeres, France P74 - DIRECT TRANSCRIPTIONAL REGULATION OF CYTOCHROME P450, CYP2C8, BY PPARΑ IN HUMAN LIVER Maria Thomas, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany

P63 - THE EFFECT OF PROTOTYPICAL INDUCERS ON MRNA EXPRESSION OF CYP1A1, CYP1A2, CYP2B1, CYP3A1 AND UGT1A1 IN CRYOPRESERVED RAT HEPATOCYTES David Stresser, Corning Gentest Contract Research Services, Woburn, Massachusetts, United States

P75 - NEW METHODOLOGICAL APPROACHES FOR DISTINGUISHING DIRECT AND INDIRECT CONSTITUTIVE ANDROSTANE RECEPTOR (CAR) ACTIVATORS Alejandro Carazo, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic

ENZYME INHIBITION / INACTIVATION (P64 – P68) P64 - ACTIVITY OF ANTIOXIDANT ENZYMES IN HUMAN COLORECTAL ADENOCARCINOMA CELL LINES: CAN IT BE MODIFIED BY SELECTED SESQUITERPENES? Hana Bartikova, Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic

P76 - OLAPARIB GENOTOXICITY OBSERVED IN A NEW AND IMPROVED P21 REPORTER MOUSE Tanya Frangova, University of Dundee, Dundee, United Kingdom P77 - REGULATION OF UDP GLUCURONOSYLTRANSFERASE 2B15 AND 2B17 EXPRESSION BY MICRO-RNAS IN PROSTATE CELLS Peter Mackenzie, Flinders University, Adelaide, Australia

P65 - ANTIOXIDANT AND GST INHIBITON ACTIVITIES OF COCOA PRODUCTS AND THEIR BIOACTIVE COMPONENTS Ahmet Altay, Middle East Technical University,Department of Biological Sciences and Joint Graduate Program in Biochemistry, Ankara, Turkey

P78 - SELECTION OF REFERENCE GENES FOR IN VITRO CYP INDUCTION STUDIES IN RAT, DOG AND HUMAN HEPATOCYTES USING REAL-TIME PCR Hugues Chanteux, UCB Biopharma, Braine L'Alleud, Belgium

P66 - ASSESSMENT OF UGT ENZYME INHIBITION IN HUMAN LIVER MICROSOMES AND UGT SUPERSOMESTM IN VITRO Richard Cole, Quotient Bioresearch, Rushden, United Kingdom

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Poster Details

P79 - UDP-GLUCOSYLTRANSFERASES (UGTS) FAMILY IN THE PARASITIC NEMATODE HAEMONCHUS CONTORTUS Petra Matouskova, Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic

P91 - EXPRESSION AND FUNCTION OF DRUG TRANSPORTERS IN 3D HUMAN LUNG TISSUE MODEL Diana Feller, Humeltis Ltd., Hosszúhetény, Hungary P92 - INFLUENCE OF MECHANICAL PROPERTIES OF COLLAGEN HYDROGEL MATRICES ON METABOLIC ACTIVITY OF SANDWICH-CULTURED PRIMARY HEPATOCYTES Peter Agbekoh, University of Strathclyde, Glasgow, United Kingdom

GENOMICS / METABOLOMICS / PROTEOMICS (P80 – P81) P80 - ASSOCIATION BETWEEN HLA GENETIC POLYMORPHISM AND SEVERE CUTANEOUS ADVERSE DRUG REACTIONS ASSOCIATED WITH CO-TRIMOXAZOLE Wichittra Tassaneeyakul, Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

P93 - SKIN BIOAVAILABILITY, METABOLISM AND LOCALIZATION OF AN ANTI-ACNE ACTIVE INGREDIENT IN THE FORM OF A PRO-DRUG Carine Jacques-Jamin, Pierre Fabre Dermo-Cosmétique, Toulouse, France

P81 - DICARBONYL PROTEIN MODIFICATION: TYPE 2 DIABETES COMPLICATIONS AND METFORMIN SCAVENGING MECHANISM Serrine Lau, Dept. Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona, United States

MECHANISMS OF XENOBIOTIC TOXICITIES (P94 – P105) P94 - COMPARISON OF HEPATOTOXICITY INDUCED BY PYRROLIZIDINE ALKALOIDS AND THEIR N-OXIDES Ge Lin, School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong

HEPATOCYTES (P82 – P85) P82 - HUMAN PLURIPOTENT STEM CELL-DERIVED HEPATOCYTES WITH SUBSTANTIAL METABOLIC FUNCTIONALITY AND ADULT CHARACTERISTICS ARE HIGHLY SUITABLE FOR TOXICITY TESTING Barbara Küppers-Munther, Takara Bio Europe (formerly Cellartis), Gothenburg, Sweden

P95 - COMPARISON OF IN VITRO METHODS FOR ASSESMENT OF REACTIVITY OF ACYL GLUCURONIDE METABOLITES Ari Tolonen, Admescope Ltd., Oulu, Finland P96 - COMPARISON OF UMBILICAL CORD BLOOD SAMPLES BETWEEN URBAN AND RURAL BIRTHS FOR POLYCYCLIC AROMATIC HYDROCARBON DNA ADDUCTS Monica Valentovic, Marshall University, Huntington, West Virginia, United States

P83 - INVESTIGATION OF STABILITY AND REGENERATION OF XENOBIOTIC METABOLISM ENZYME ACTIVITIES IN PRIMARY HUMAN HEPATOCYTES DURING ISOLATION AND CULTIVATION Melanie Kießig, Charité Universitätsmedizin Berlin, Berlin, Germany

P97 - CYTOCHROME P450 - MEDIATED METABOLISM OF THE TYROSINE KINASE INHIBITOR PONATINIB TO REACTIVE ELECTROPHILES Rumen Kostov, Jacqui Wood Cancer Centre, Medical Research Institute, University of Dundee, Dundee, United Kingdom

P84 - REDUCING HUMAN DRUG OVERDOSE USING MICRORNAS Dagmara Szkolnicka, University of Edinburgh, Edinburgh, United Kingdom

P98 - CYTOTOXIC ACTIVITIES OF FOUR POLYGONUM SPECIES FROM TURKEY Gul Ozhan, Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey

P85 - UTILITY OF A MECHANISTIC MODEL TO INVESTIGATE THE UPTAKE OF ROSUVASTATIN IN HUMAN HEPATOCYTES AND TIME-DEPENDENT INHIBITION BY CYCLOSPORIN A AND CYCLOSPORIN AM1 Michael Hobbs, GlaxoSmithKline, Ware, United Kingdom

P99 - DETERMINATION OF THE ROLE OF O-ACYLGLUCURONIDE MIGRATION IN THE COVALENT BINDING OF A DRUG CANDIDATE József Pánczél, Sanofi, Frankfurt, Germany

IN SILICO (P86 – P89) P86 - IN SILICO ANALYSIS OF MICRORNA EXPRESSION PUTATIVLY REGULATED BY NUCLEAR RECEPTORS CAR, AHR AND ER Lyudmila Gulyaeva, The Institute of Molecular Biology and Biophysics/Novosibirsk State University, Novosibirsk, Russia

P100 - DNA METHYLATION PROFILES OF BISPHENOL A IN HUMAN NEUROBLASTOMA CELLS Gul Ozhan, Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Istanbul, Turkey P101 - INDUCED INTERSTITIAL PULMONARY FIBROSIS (IPF) MODEL: UNLABELED BLEOMYCIN DISTRIBUTION AND EARLY IPF MARKERS IDENTIFICATION BY MALDI IMAGING Gregory Hamm, ImaBiotech, Loos, France

P87 - IN SILICO MODELLING OF OATP1B1 INHIBITION BASED ON LIGAND STRUCTURE INFORMATION Susanne Winiwarter, AstraZeneca R&D, Mölndal, Sweden P88 - IN SILICO PREDICTION FOR BIOPHARMACEUTICS OF ORAL PIPERINE USING GASTROPLUS SOFTWARE. Chatsiri Jesadakultavee, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand

P102 - LEVODOPA AND DOPAMINE-INDUCED POSTTRANSLATIONAL MODIFICATION OF Α-SYNUCLEIN Terrence Monks, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona, United States

P89 - IN SILICO PREDICTION OF ORAL BIOAVAILABILITY Michael Lawless, Simulations Plus, Lancaster, California, United States

P103 - PROTECTION OF MALE MICE AGAINST CARBON TETRACHLORIDE-INDUCED HEPATOTOXICITY BY SOY ISOFLAVONES Yasuhiro Masubuchi, Chiba Institute of Science, Choshi, Japan

IN VITRO TECHNIQUES (P90 – P93) P90 - EVALUATION OF CYP450 AND TRANSPORTERS EXPRESSION AND ACTIVITY IN HEPARG CELL LINE UNDER DIFFERENT CONDITIONS Flavia Storelli, Geneva University Hospitals, Geneva, Switzerland

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Poster Details

P104 - ROLE OF BIOTRANSFORMATION AND FREE RADICALS IN 3,4-DICHLOROANILINE NEPHROTOXICITY IN VITRO Gary Rankin, Marshall University, Huntington, West Virginia, United States

P116 - INVESTIGATING THE MECHANISM FOR INVERSION OF CONFIGURATION OF CARBOXYLIC ACID CONTAINING DRUG CANDIDATES Fredrik Bergström, AstraZeneca R&D, Mölndal, Sweden P117 - LC-MS ANALYSIS OF THE METABOLISM OF THE DIETARY CONSTITUENT HESPERIDIN BY RAT HEPATOCYTES Khaled Omar, University of Strathclyde, Glasgow, United Kingdom

P105 - VALIDATION OF HUMAN PRECISION-CUT LIVER SLICES AS AN EX VIVO MODEL TO STUDY DRUG INDUCED CHOLESTASIS Suresh Vatakuti, Division of Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute for Pharmacy, University of Groningen, Groningen, The Netherlands

P118 - OPTIMIZING METABOLIC STABILTY VIA SOFT SPOT IDENTIFICATION (SSID) Markus Trunzer, Novartis Institutes for Biomedical Research / Metabolism and Pharmacokinetics, Basel, Switzerland

METABOLIC PROFILING (P106 – P111) P106 - A MASS BALANCE AND METABOLITE PROFILING STUDY OF LDE225 IN HEALTHY MALE SUBJECTS USING A LIGHT-LABEL APPROACH Piet Swart, DMPK, Novartis, Basel, Switzerland

P119 - PHASE 2 METABOLISM OF ACTIVE HYDROXYCLOMIPHENE METABOLITES Patrick Kroener, Dr. Margarete Fischer-Bosch-Institute Clinical Pharmacology and Universitiy of Tuebing, Stuttgart, Germany

P107 - ADME STUDIES OF [3H]-2´O-METHYLURIDINE NUCLEOSIDE IN MICE: A BUILDINGBLOCK IN SIRNA THERAPEUTICS Piet Swart, DMPK, Novartis, Basel, Switzerland

P120 - USE OF IN VIVO MODELS TO ASSESS METABOLIC LIABILITIES AND EXCRETION PATHWAYS AT EARLY STAGE IN DRUG DISCOVERY Thierry Delemonte, Novartis Institutes for Biomedical Research/ Analytical Sciences and Imaging, Basel, Switzerland

P108 - METABOLISM AND DISPOSITION OF [14C]BYL719 (ALPELISIB), AN ORAL CLASS 1 Α-SPECIFIC PI3K INHIBITOR FOR THE TREATMENT OF SOLID TUMORS IN HEALTHY MALE VOLUNTEERS Piet Swart, DMPK, Novartis, Basel, Switzerland

NON-P450 PHASE I ENZYMES (P121 – P122) P121 - DEVELOPMENT AND VALIDATION OF AN IN VITRO ASSAY TO DETERMINE THE FRACTION METABOLISED BY FMO Abhishek Srivastava, Drug Safety and Metabolism DMPK, AstraZeneca, Cambridge, United Kingdom

P109 - METABOLOMICS AS A TOOL TO INVESTIGATE MEPHEDRONE AS A DRUG OF ABUSE IN RAT HEPATOCYTES Mohammad Alwashih, University of Strathclyde, Glasgow, United Kingdom

P122 - INVOLVEMENT OF ALDEHYDE OXIDASE IN THE REDUCTION OF N-OXIDE FUNCTION Hugues Chanteux, UCB Biopharma, Braine L'Alleud, Belgium

P110 - UNDER THE SKIN: BIOMARKERS OF CUTANEOUS DEFENSES TO VACCINES USING MASS SPECTROMETRY IMAGING Gregory Hamm, ImaBiotech, Loos, France

PHARMACOGENETICS (P123 – P130) P123 - ASSOCIATION BETWEEN HUMAN TELOMERASE REVERSE TRANSCRIPTASE (HTERT) GENE VARIATIONS AND RISK OF DEVELOPING BREAST CANCER Ezgi Oztas, Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Istanbul, Turkey

P111 - USING HIGH RESOLUTION MASS SPECTROMETRY TO PREDICT SUCCESSFUL DOSING STRATEGIES FOR RADIOLABELLED CLINICAL AME STUDIES Caroline Anderson, Covance Laboratories Ltd., Harrogate, United Kingdom

P124 - ASSOCIATION BETWEEN PHOSHOLIPASE CEPSILON 1 (PLCE1) GENE VARIATIONS AND RISK OF DEVELOPING COLORECTAL CANCER Gul Ozhan, Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Istanbul, Turkey

METABOLISM (P112 – P120) P112 - CHF 6001: METABOLITE PROFILES AND IDENTIFICATION IN PLASMA, URINE, FAECES, BILE AND LIVER SAMPLES OF PXB(R) AND CD-1 MICE FOLLOWING INTRAVENOUS AND ORAL ADMINISTRATIONS Rosangela Battaglia, Accelera Srl, Nerviano Milan, Italy

P125 - ASSOCIATION OF ESR2 (RSA I AND ALU I) AND ORGANOCHLORINE PESTICIDE LEVELS WITH PROSTATE CANCER : A CASE CONTROL STUDY Basu Dev Banerjee, Department of Biochemistry, University College of Medical Sciences and GTB Hospital (Delhi University), Delhi, India

P113 - DATABASE CAPTURING AND MINING OF METABOLITE INFORMATION OF DRUG CANDIDATES: ANALYSIS OF 27 ASTRAZENECA COMPOUNDS WITH HUMAN ADME DATA IN ACD DATABASE Jessica Iegre, CVMD iMed DMPK, AstraZeneca R&D,Mölndal, Sweden

P126 - CONTRIBUTION OF CYP3A5 GENETIC POLYMORPHISM ON THE PHARMACODYNAMICS OF CLOPIDOGREL UNDER STEADY STATE CONDITION Nontaya Nakkam, Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

P114 - IDENTIFICATION OF METABOLIC PATHWAYS OF BENZIMIDAZOLE ANTHELMINTICS IN HAREBELL (CAMPANULA ROTUNDIFOLIA) Radka Podlipná, Laboratory of Plant Biotechnologies, Institute of Experimental Botany, Czech Academy of Sciences, Prague, Czech Republic

P127 - GENE ENVIRONMENT INTERACTION IN EPITHELIAL OVARIAN CANCER WITH SPECIAL REFERENCE TO ORGANOCHLORINE PESTICIDE : A CASE CONTROL STUDY Tusha Sharma, Department of Biochemistry, University College of Medical Sciences and GTB Hospital (Delhi University), Delhi, India

P115 - IMPACT OF ANTHELMINTICS IN ENVIRONMENT ON LOWER DEVELOPMENT STAGES OF HELMINTHS AND ON PLANTS Barbora Szotáková, Charles University, Faculty of Pharmacy, Hradec Králové, Czech Republic

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Poster Details

P128 - IMPACT OF CYP2C19 AND CYP2D6 GENOTYPE ON THE PHARMACOKINETICS OF ESCITALOPRAM IN HEALTHY CHINESE SUBJECTS SK Wo, School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong

P140 - THE EFFECT OF THE BLOOD SAMPLING SITE ON PK PARAMETERS AFTER OROMUCOSAL ADMINISTRATION IN DOGS Astrid Capello, WIL Research Europe, 's Hertogenbosch, The Netherlands

P129 - IMPACT OF CYP2D6 POLYMORPHISM ON STEADYSTATE PLASMA LEVELS OF RISPERIDONE AND 9HYDROXYRISPERIDONE IN THAI CHILDREN WITH AUTISTIC SPECTRUM DISORDER Chonlaphat Sukasem, Division of Pharmacogenomics and Personalized Medicine, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

RECEPTORS / NUCLEAR RECEPTORS (P141 – P145) P141 - ANTI-PROLIFERATIVE EFFECT OF NUCLEAR RECEPTOR NR0B2 IN RENAL CARCINOMA CELLS Katharina Prestin, University of Basel, Biopharmacy, Department of Pharmaceutical Science, Basel, Switzerland P142 - CHARACTERISATION OF HUMAN CAR SPLICED VARIANTS/CAR1 MUTANTS IN CAR-NULL MICE AND IN MOUSE PRIMARY HEPATOCYTES Shaohong Ding, Division of Cancer Research, Ninewells Hospital, University of Dundee, Dundee, United Kingdom

P130 - VINCRISTINE-INDUCED NEUROPATHIC PAIN IN A CYP3A5 NON-EXPRESSER WITH REDUCED CYP3A4 ACTIVITY Youssef Daali, Geneva University Hospitals, Geneva, Switzerland

P143 - EFFECTS OF CYTOTOXIC GOLD (I) COMPLEXES ON TRANSCRIPTIONAL ACTIVITY OF VARIOUS NUCLEAR RECEPTORS Katerina Kubesova, Palacky University, Olomouc, Czech Republic

PHARMACOKINETICS AND PHARMACODYNAMICS (P131 – P140) P131 - A PHARMACOKINETIC MODELING APPROACH TO PREDICT THE CONTRIBUTION OF ACTIVE METABOLITES TO HUMAN EFFICACIOUS DOSE Iain Martin, Merck, Boston, Massachusetts, United States

P144 - HEPATIC NUCLEAR RECEPTOR - ENVIRONMENTAL POLLUTION INTERACTIONS REGULATE ENERGY METABOLISM, INFLAMMATION, AND LIFESTYLE BEHAVIORS IN NON-ALCOHOLIC FATTY LIVER DISEASE Russell Prough, Department of Biochemistry & Molecular Biology, The University of Louisville School of Medicine, Louisville, Kentucky, United States

P132 - CALCITRIOL AND 20(S)-PROTOPANAXADIOL SYNERGISTICALLY INHIBIT GROWTH AND INDUCE APOPTOSIS IN PROSTATE CANCER CELLS Mohamed Ben-Eltriki, Department of Experimental Medicine, University of British Columbia and Canada and Vancouver Prostate Centre at Vancouver General Hospital, Vancouver, British Columbia, Canada

P145 - PROFILING OF ANTHOCYANIDINS AGAINST TRANSCRIPTIONAL ACTIVITIES OF STEROID AND NUCLEAR RECEPTORS Barbora Pastorkova, Department of Biochemical Sciences, Charles University, Faculty of Pharmacy, Olomouc, Czech Republic

P133 - CARBAMAZEPINE-LOADED CARBOXYMETHYL CHITOSAN NANOPARTICLES FOR DRUG DELIVERY INTO BRAIN FOLLOWING INTRANASAL ADMINISTRATION Shanshan Liu, National University of Singapore, Singapore, Singapore

TRANSPORTERS (P146 – P166) P146 - ABUNDANCE OF MEMBRANE TRANSPORTER EXPRESSION IN CELL LINES AND COMPARISON OF ABUNDANCE AND FUNCTIONAL ACTIVITY OF SELECTED TRANSPORTERS IN CELL LINES AND ISOLATED HEPATOCYTES Nikoletta Dobos, SOLVO Biotechnology, Budaörs, Hungary

P134 - CYTOCHROME P450 3A4 (CYP3A4) -HUMANISED MICE FOR THE STUDY OF ANTI-CANCER DRUG PHARMACOKINETICS Kenneth MacLeod, University of Dundee, Dundee, United Kingdom P135 - EFFECT OF HYALURONIDASES ON THE PHARMACOKINETICS OF [I-125]-INFLIXIMAB FOLLOWING SINGLE SUBCUTANEOUS ADMINISTRATION TO RATS Stephen Madden, Charles River Laboratories, Tranent, United Kingdom

P147 - CHARACTERIZATION OF STABLY TRANSFECTED HEK-293 CELLS EXPRESSING OATPS USING FLUORESCENT SUBSTANCES Dieter Runge, Primacyt Cell Culture Technology GmbH, Schwerin, Germany

P136 - OPTIONS TO BOOST EXPOSURE OF ‘PROOF OF CONCEPT’ TOOL COMPOUNDS IN RODENTS TO ACCELERATE EARLY STAGE RESEARCH Lesley Murray, Drug Metabolism and Pharmacokinetics, Genentech Inc., Campbell, California, United States

P148 - CHARACTERIZATION OF THE EXPRESSION AND FUNCTION OF ENDOGENOUS TRANSPORTERS IN HEK293 AND MDCKII CELLS Marcus Otter, University Medicine of Greifswald, Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), Greifswald, Germany

P137 - PHARMACOKINETIC STUDY OF ESCITALOPRAM AFTER ORAL ADMINISTRATION OF 10 MG ESCITALOPRAM TABLET IN HEALTHY CHINESE SUBJECTS SK Wo, School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong

P149 - CHARACTERIZATION OF TRANSPORTER ACTIVITIES IN FRESH ISOLATED PRIMARY HEPATOCYTES OF DIFFERENT SPECIES BY USING FLUORESCENT SUBSTRATES Dieter Runge, Primacyt Cell Culture Technology GmbH, Schwerin, Germany

P138 - POPULATION PHARMACOKINETICS OF LINEZOLID IN CRITICALLY ILL PATIENTS Uwe Fuhr, Department of Pharmacology, Hospital of the University of Cologne, Cologne, Germany

P150 - CHARACTERIZING HEPATO-BILIARY TRANSPORT UTILIZING THE STABLE, LONG-ENDURING CELLULAR COMPETENCY OF HµREL® CO-CULTURE TECHNOLOGY AND SINGLE-WELL DIRECT ASSAY METHOD Matthew Shipton, Hurel Corporation, Beverly Hills, California, United States

P139 - PREDICTION OF THE ORAL CLEARANCE OF ISONIAZID IN VIRTUAL SUBJECTS WITH DIFFERENT NAT2 ACETYLATOR STATUS Iain Gardner, Simcyp, Ltd., Sheffield, United Kingdom

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Poster Details

P151 - EVALUATION OF CLINICALLY RELEVANT INHIBITORS OF BSEP USING B-CLEAR® HUMAN SANDWICH-CULTURED HEPATOCYTES TO BETTER PREDICT INHIBITION AND CHOLESTASIS Chris Black, Qualyst Transporter Solutions LLC, Durham, North Carolina, United States

P163 - POTENTIAL ROLE OF BASOLATERAL EFFLUX TRANSPORTERS IN THE PREVENTION OF CHOLESTATIC HEPATOTOXICITY Chris Black, Qualyst Transporter Solutions LLC, Durham, North Carolina, United States P164 - SHOULD TOTAL PLASMA DRUG CONCENTRATION BE USED TO PREDICT TRANSPORTER MEDIATED DRUGDRUG INTERACTIONS FOR HIGHLY PROTEIN BOUND DRUGS? Yong Huang, Optivia Biotechnology Inc., Menlo Park, California, United States

P152 - FUNCTIONAL CHARACTERIZATION OF OATP2B1 USING FÖRSTER RESONANCE ENERGY TRANSFER (FRET) Paul Hagen, University Medicine of Greifswald, Department of Pharmacology, Greifswald, Germany P153 - GENETIC VARIANTS OF THE ORGANIC ANION TRANSPORTER POLYPEPTIDE 1A2 (OATP1A2) INFLUENCE THE CELLULAR UPTAKE OF DOXORUBICIN Markus Keiser, University Medicine of Greifswald, Department of Clinical Pharmacology, Greifswald, Germany

P165 - SCREENING FOR HEPATOBILIARY TRANSPORTER INHIBITION IN 3D HUMAN LIVER MICROTISSUES BY CONFOCAL IMAGING Simon Messner, InSphero AG, Schlieren, Switzerland P166 - VORTIOXETINE: IN VITRO ASSESSMENT AND CLINICAL IMPLICATIONS OF TRANSPORTER INHIBITIONBASED DRUG INTERACTIONS Liping Pan, Takeda Pharmaceutical Company, Deerfield, Illinois, United States

P154 - IN VITRO AND IN VIVO METHODS TO STUDY HEPATIC BILE SALT TRANSPORT Nikoletta Dobos, SOLVO Biotechnology, Budaörs, Hungary P155 - INFLUENCE OF ROUX-EN-Y GASTRIC BYPASS SURGERY ON DRUG TRANSPORTER EXPRESSION IN THE HUMAN SMALL INTESTINE Susanne Brueck, University Medicine of Greifswald, Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), Greifswald, Germany P156 - INTERACTION OF EMTRICITABINE WITH MATE1, OCT1, OCT2, P-GLYCOPROTEIN, BCRP AND MRP2 TRANSPORTERS Josef Reznicek, Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic P157 - INTERACTIONS OF ABACAVIR WITH NUCLEOSIDE TRANSPORTERS IN THE PLACENTA Zuzana Neumanova, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic P158 - INTERACTIONS OF SELECTED FLAVONOIDS WITH THE TRANSPORTER HOATP2B Lucie Navratilova, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Nezdenice, Czech Republic P159 - LOCALIZATION AND CHARACTERIZATION OF TRANSPORTER PROTEINS INVOLVED IN THE UPTAKE OF PULMONARY RELEVANT DRUGS Sarah Berlin, University Medicine of Greifswald, Department of Clinical Pharmacology, Greifswald, Germany P160 - LONG-TERM ADMINISTRATION OF TENOFOVIR OR EMTRICITABINE TO PREGNANT RATS; EFFECT ON ABCB1A, ABCB1B, AND ABCG2 EXPRESSION IN MATERNAL AND FETAL ORGANS Lukas Cerveny, Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic P161 - MORI CORTEX ENHANCES INTESTINAL BARRIER FUNCTION THROUGH UP-REGULATING PGLYCOPROTEIN VIA GUT MICROBIOTA- DEPENDENT AND INDEPENDENT MECHANISMS Ru Yan, University of Macau, Macau, China P162 - OVERCOMING MULTIDRUG RESISTANCE IN HUMAN BREAST CANCER CELLS Heather Wallace, University of Aberdeen, Foresterhill, United Kingdom

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Poster Information

Poster Awards Finalist Abstract Presentation Schedule Category GRADUATE/ PREDOCTORAL

Set-up Time

Poster Viewing

Tuesday, June 23 07:30 – 09:00

Tuesday, June 23 – Thursday, June 25

Authors Present

Dismantle Time

Tuesday, June 23

POSTDOCTORAL

Wednesday, June 24

Thursday, June 25 13:00 – 14:00

Poster Abstract Presentation Schedule Category Poster Session 1 June 23 Poster Session 2 June 24

Set-up Time

Poster Viewing

Tuesday, June 23 07:30 – 09:00

Tuesday, June 23 – Thursday, June 25

Authors Present

Dismantle Time

Abstracts P1 – P85 Abstracts P86 – P166

Thursday, June 25 12:00 – 13:00

OUT OF COURTESY TO THE AUTHORS, PLEASE REFRAIN FROM TAKING PHOTOGRAPHS OR VIDEO RECORDING.

Posters will be attended during designated presentation times. This is your opportunity to ask authors about their research. To obtain a copy of specific information that is being presented, contact the author directly.

Finalists Graduate / Predoctoral Award Competition Finalists Postdoctoral Fellow Award Competition

A1 – A6 A7 – A12

Absorption Analytical Bioavailability Clearance Prediction Conjugation Reactions and Enzymes Cytochrome P450 Differences in Metabolism (species, gender, age, diseases) Disposition Drug Discovery and Development Drug Interaction Enzyme Induction Enzyme Inhibition/Inactivation Extrahepatic Metabolism Gene Expression and Regulation Genomics / Metabolomics / Proteomics Hepatocytes In silico In vitro Techniques Mechanisms of Xenobiotic Toxicities Metabolic Profiling Metabolism Non-P450 Phase I Enzymes Pharmacogenetics Pharmacokinetics and Pharmacodynamics Receptors / Nuclear Receptors Transporters

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P1 – P2 P3 – P6 P7 – P9 P10 – P16 P17 – P19 P20 – P28 P29 – P32 P33 – P38 P39 – P42 P43 – P58 P59 – P63 P64 – P68 P69 P70 – P79 P80 – P81 P82 – P85 P86 – P89 P90 – P93 P94 – P105 P106 – P111 P112 – P120 P121 – P122 P123 – P130 P131 – P140 P141 – P145 P146 – P166

13th European ISSX Meeting

Poster Award Finalist Abstracts

A1 - SIMULTANEOUS QUANTIFICATION OF ESTROGENIC TAMOXIFEN METABOLITES AND SEX STEROID HORMONES IN HUMAN PLASMA VIA UHPLC-ESI-MS/MS 1 1 1 1 1&2 1&2 Janina Johänning , Jana C. Precht , Georg Heinkele , Michel Eichelbaum , Hiltrud Brauch , Matthias Schwab , 1 1 Werner Schroth , and Thomas E. Mürdter 1 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and University Tuebingen, Stuttgart, 2 Germany, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Tamoxifen is a mainstay in the treatment of estrogen-receptor (ER) positive breast cancer in pre- and postmenopausal women and acts by selective inhibition of the ER in breast tumor cells. In humans, tamoxifen is metabolized to more than 30 compounds with both, anti-estrogenic and estrogenic properties. While the effects of anti-estrogenic metabolites 4-OH-tamoxifen and endoxifen are well understood, knowledge of plasma concentrations of estrogenic metabolites, such as bisphenol (4-[1-(4-hydroxyphenyl)-2-phenylbut-1-enyl]phenol), (E)-, and (Z)-metabolite E (4-[1,2diphenylbut-1-enyl]phenol), is sparse and their potential interference with clinical effects of tamoxifen is not wellknown. Moreover, reliable data on changes in sex steroid hormone levels under tamoxifen therapy are limited. To investigate the role of estrogenic tamoxifen metabolites in tamoxifen treated patients, we developed a highly sensitive rapid resolution HPLC-positive-ESI-MS/MS quantification method for the simultaneous analysis of bisphenol, (E)-, and (Z)-metabolite E, estradiol, estrone, testosterone, androstendione and progesterone, using standard laboratory equipment. For calibration charcoal-stripped blank plasma samples were spiked with different concentrations of the analytes. Plasma samples (200 µl) were subjected to protein precipitation and the supernatant was purified by solid phase extraction. After derivatization of phenolic analytes with a pre-ionized N-methyl-nicotinic-acid active ester, all analytes were separated on a ZORBAX Eclipse plus C18 column (1.8 µm, 2.1x100 mm) with a gradient of acetonitrile in water with 0.1 % of formic acid. The analytes were detected on a triple quadrupole mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. All compounds were quantified using stable isotope labelled internal standards. Lower limits of quantification were 12, 8, and 25 pM for bisphenol, (E)-, and (Z)-metabolite E, respectively, 4 pM for estrogens, 50 pM for androgens, and 25 pM for progesterone. The method was applied to plasma samples from 100 postmenopausal breast cancer patients after 6 months of tamoxifen treatment. Notably, estrogenic tamoxifen metabolites, as their anti-estrogenic counterparts, showed a high inter-individual variability (bisphenol: 19-659 pM; (E)-metabolite E: 23-2207 pM; (Z)-metabolite E: 65-5972 pM). The analytical results for sex steroid hormone levels were as follows: Estradiol: 4-42 pM; estrone: 6-660 pM; testosterone: 53-2752 pM; androstendione: 89-4424 pM; progesterone: 20-849 pM. In the future, this method can be used to investigate the association of biotransformation of tamoxifen to estrogenic metabolites, sex steroid hormone level and clinical outcome. Supported by the Robert Bosch Foundation, Stuttgart, and the German Research Foundation, Bonn (Grants: MU 1727/2-1 and SCHR 1323/2-1).

A2 - IS THE USE OF 1-AMINOBENZOTRIAZOLE AS CYTOCHROME P450 INACTIVATOR IN RAT STUDIES APPROPRIATE? 1 2 2 2 1 Marc-Olivier Boily , Chantal Boudreau , Marie-Claude Duquet , Line Ste-Marie , François A. Leblond , Julie 2 2 1 Laterreur , Nathalie Chauret , and Vincent Pichette 1 Centre de Recherche, Hôpital Maisonneuve-Rosemont, Faculté de Médecine, Université de Montréal, Montréal, 2 Québec, Canada, Vertex Pharmaceuticals Canada, Laval, Québec, Canada 1-Aminobenzotriazole (ABT) is a well-known irreversible and non-specific cytochrome P450 inhibitor. Its pharmacokinetics properties, along with its good safety profile in rats, made it a perfect in vivo tool to understand the role of metabolism in the bioavailability or the toxicity of xenobiotics. Moreover, it was proposed that pre-treatments of rats with ABT administered orally versus intravenously could allow discrimination between intestinal and hepatic first pass metabolism (1). However, recent findings challenged the ABT rat model to address P450 metabolism effect by suggesting that ABT may affect gastric emptying and consequently the absorption process (2). Since no standard protocol is reported in the literature regarding the administration of ABT in drug-drug interaction (DDI) studies in rats, there is a need to understand the impact of varying the route of administration and the moment of administration of ABT on its effect on the metabolism and absorption of xenobiotics. To assess this question, we performed pharmacokinetic studies in untreated rats or rats pretreated with ABT given either orally 16 hours or intravenously (i.v.)

1 hour before oral dosing of metoprolol, a known P450 substrate. To evaluate the impact of ABT on hepatic and intestinal metabolism, we studied the metabolism of metoprolol in gut and liver microsomes obtained from untreated and ABT-treated rats and showed that ABT given orally or i.v inhibits similarly metoprolol intestinal and hepatic metabolism by 5-fold and 3-fold respectively. Finally, transit time studies under different ABT pre-treatments in rats were conducted to distinguish between an effect on drug absorption and metabolism. Our results indicate that ABT affects gastric emptying as we observed a 7-fold increase (p<0.001) in stomach weights when the rats were pretreated with ABT for a short period of time. This effect probably contributes significantly to the 4 hours delay in absorption of metoprolol and its metabolites in rats pretreated with ABT i.v. 1 hour before metoprolol dosing. Such delay in absorption was not seen in rats pretreated with ABT 16 hours prior to metoprolol administration or in untreated rats. In conclusion, these findings indicate that, depending on the ABT pre-treatment conditions, there could be a misinterpretation of the importance of P450 metabolism effect on the pharmacokinetic profiles of a drug in rat ABT DDI studies. Based on our results, we recommend pretreating rats with ABT 16 hours before the administration of a test compound to preserve the inhibitory effect on metabolism and avoid the effect on the gastric emptying and

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drug absorption. Finally, our results refute the assertion that administered in different routes, ABT could differentiate between intestinal and hepatic metabolism of drugs.

1) Strelevitz TJ et al. In vivo use of the P450 inactivator 1-aminobenzotriazole in the rat: varied dosing route to elucidate gut and liver contributions to first-pass and systemic clearance. J Pharm Sci. 2006. 95:1334-1341.

2) Stringer RA et al. 1-Aminobenzotriazole Modulates Oral Drug Pharmacokinetics through Cytochrome P450 Inhibition and Delay of Gastric Emptying in Rats. Drug metabolism and disposition: the biological fate of chemicals.

2014. 42:1117-1124.

A3 - PRECISION-CUT INTESTINAL SLICES AS EX VIVO MODEL TO STUDY THE UPTAKE OF BILE ACIDS BY ASBT IN THE INTESTINE 1 2 3 1 3 Ming Li , Ivan Vokral , Inge De Graaf , Marina de Jager , Bernard Evers, and Geny Groothuis 1 2 Department of Pharmacy, University of Groningen, Groningen, The Netherlands, Department of Biochemical 3 Sciences, Charles University, Hradec Králové, Czech Republic, Department of Pharmacokinetics & Drug Delivery, Groningen University Institute for Drug Exploration, Groningen, The Netherlands Introduction: The apical sodium-dependent bile acid transporter (ASBT), localized in the apical membrane of the enterocytes in the ileum, significantly participates in the enterohepatic circulation of bile acids by active uptake from the intestinal lumen. It is also considered as an important transporter involved in the uptake of drug-bile acid conjugates, designed as prodrugs to increase the oral bioavailability of drugs with low absorption. Tools to study ASBT function are traditionally based on transfected cell studies, and in vivo experiments in normal or genetically modified animals. However, cultured cell lines differ in many aspects from the conditions in vivo, while animal experiments are costly and allow only one experiment per animal. Alternatively, Precision-Cut Intestinal Slices (PCIS) are a novel ex vivo model to investigate drug metabolism, toxicity, and recently efflux transport in both human and animal intestine, allowing many experiments per animal. Moreover it can be applied to human tissue. The aim of this study is to show the application of PCIS to investigate the uptake of bile acids by ASBT in the intestine. Methods: PCIS were prepared from rat and human jejunum, ileum and colon. Taurocholic acid (TCA) and deoxycholic acid (DCA) were used as substrates of ASBT. The uptake of these bile acids into the slice was measured by a total bile acid assay. Furthermore, the toxicity of the different bile acids was characterized by the effect on intracellular ATP level of the slices well as on slice morphology. Results: The results confirmed that active uptake of TCA and DCA occurs only in ileum, but not in jejunum and colon in both rat and human and that the passive uptake is very low in all regions. In rat ileum the apparent Km and Vmax for TCA are 0.32 mM and 1.95 nmol•mg protein-1•min-1, while those in human are 0.47 mM and 3.85 nmol•mg protein-1•min-1. For DCA similar active uptake parameters were found but with a higher passive diffusion, which can be explained by its higher lipophilicity. As expected, DCA was more toxic to the ileum PCIS than TCA. Conclusion: This study shows that PCIS are a simple, reliable and fast ex vivo model to study the ASBT-mediated uptake and the toxicity of bile acids in the intestine. It clearly showed the differences among regions, species and substrates. Furthermore, our study showed for the first time the kinetic parameters of bile acid uptake in human intestinal tissue on ex vivo level. Therefore we conclude that PCIS can be used in the future to identify substrates and inhibitors of ASBT and to predict the extent of ASBT-mediated uptake in human in vivo.

A4 - INVOLVEMENT OF DIFFERENT CYTOCHROME P450 ISOFORMS IN THE SEQUENTIAL 2-STEP BIOACTIVATION OF DICLOFENAC AND THE PHYSIOLOGICAL RELEVANCE OF SUBSEQUENT DETOXIFICATION BY GLUTATHIONE S-TRANSFERASES 1 1 2 1 Michiel W. den Braver , Shalenie P. den Braver-Sewradj , Audrey Baze , Harini Venkataraman , Nico P.E. 1 2&3 1 1 Vermeulen , Lysiane Richert , J. Chris Vos , and Jan N.M. Commandeur 1 2 3 VU University Amsterdam, The Netherlands, KaLy-Cell, Plobsheim, France, Université de Franche-Comté, Besançon, France Diclofenac (DF) is a frequently prescribed non-steroidal anti-Inflammatory drug (NSAID) for the management of pain and treatment of several inflammatory disorders. Chronic exposure to DF is associated with elevated serum aminotransferase levels in 15% of the patients and a low incidence of severe idiosyncratic liver injury (IDILI). DF induced DILI is suggested to be related to bioactivation and bio-inactivation by phase I and phase II enzymes.

Bioactivation of DF is catalyzed via two steps by cytochrome P450 enzymes (P450s). First, DF is hydroxylated at the 4’- or 5- position. A secondary oxidation step results in reactive p-benzoquinone imines. These reactive metabolites are inactivated by chemical or enzymatic conjugation to GSH, which is catalyzed by the highly polymorphic glutathione S-transferases (GSTs).[1] The importance of GSTs is illustrated by a correlation of NSAID-induced IDILI and the GST M1-1 and T1-1 double-null genotype in patients.[2] The present study describes the involvement of P450 isoforms in the second oxidation step. Different P450 isoforms appeared to be involved in the sequential oxidation reactions. This finding has important implications for the predictivity of in vitro models and risk assessment in association studies. Bioinactivation of resulting p-benzoquinone imines was investigated with a complete panel of GST isoforms at GSH concentrations reflecting a liver cell under homeostasis or a cell subjected to oxidative stress. Interestingly, GSTs are shown to be active even at high, physiological relevant, GSH concentrations. Taken together, these findings implicate an important role of variability in P450s and GSTs activity in the risk for DF induced liver injury. Therefore, preliminary studies are undertaken to correlate donor specific cytotoxicity in primary human hepatocytes with differential enzyme activity.

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Acknowledgements: This project is funded by the IMI project MIP-DILI (115336).

References 1. Dragovic, S., Boerma, J. S., Vermeulen, N. P. E. & Commandeur, J. N. M. Effect of Human Glutathione STransferases on Glutathione-Dependent Inactivation of Cytochrome P450-Dependent Reactive Intermediates of Diclofenac. Chem. Res. Toxicol. 26, 1632-41 (2013).

2. Lucena, M. I. et al. Glutathione S-transferase m1 and t1 null genotypes increase susceptibility to idiosyncratic druginduced liver injury. Hepatology 48, 588–96 (2008).

A5 - THE POLYMORPHIC VARIANT P24T OF UGT1A4 AND ITS UNUSUAL CONSEQUENCES Johanna Troberg and Moshe Finel University of Helsinki, Helsinki, Finland The aim of this research was to examine the P24T single nucleotide polymorphism of UDP glucuronosyltransferase 1A4 (UGT1A4) (70C>A, *2), that occurs frequently in the population. Although this polymorphism was studied before to some degree and the results on its effects on activity were somewhat variable with only part of them reporting significant change[1,2], we reexamined it since the previous studies haven’t focused on the location of P24, which is 5 amino acids before the predicted cleavage site of the signal sequence and the starting point of the mature protein. A change in the amino acid sequence in this region might affect the cleavage of the signal sequence that leads the enzyme to the Endoplasmic Reticulum and, hence, the length of the mature protein. Moreover, this takes place very close to the active site of the enzyme. Our bioinformatic analysis of the variant suggested an effect on the signal sequence cleavage, even if the effect is rather complex. To further study this, UGT1A4 and its P24T variant were cloned and expressed in baculovirus-infected insect cells as recombinant proteins with a C-terminal His-tag. The glucuronidation activity of the UGT1A4 and UGT1A4-P24T were assayed from UGT-enriched insect membrane preparations, using two UGT1A4 substrates, dexmedetomidine and trifluoperazine. The formed glucuronides were then measured by HPLC-UV. The results showed that UGT1A4-P24T variant has a lower glucuronidation activity toward both substrates than wild-type UGT1A4. The recombinant UGTs were further purified by immobilized metal affinity chromatography (nickel column) for N-terminal amino acid sequencing. The N-terminal sequencing results of wild-type UGT1A4 and variant UGT1A4-P24T revealed a mixture of two species in the latter, as predicted by the program. In conclusion, our results revealed that the P24T variant of UGT1A4 does affect the cleavage of the signal sequence, but the effect is complex even in a recombinant system. The results also suggest an explanation for the apparent difference in the effect on activity between the native system in the human liver, to the recombinant system.

[1.] Wiener D, Fang J-L, Dossett N and Lazarus P (2004) Correlation between UDP-Glucuronosyltransferase Genotypes and 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone Glucuronidation Phenotype in Human Liver Microsomes. Cancer Research 64:1190-1196.

[2.] Ehmer U, Vogel A, Schütte JK, Krone B, Manns MP and Strassburg CP (2004) Variation of Hepatic Glucuronidation: Novel Functional Polymorphisms of the UDP-Glucuronosyltransferase UGT1A4. Hepatology 39:970977.

A6 - ABCB1, ABCC1 AND ABCG2 MODULATE TRANSPORT AND PHARMACOKINETICS OF DINACICLIB IN VITRO Daniela Cihalova, Martina Ceckova, and Frantisek Staud Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic Cyclin-dependent kinases (CDK) play an important role in cell cycle progression and their deregulation has been detected in multiple human cancers. Therefore, CDKs have become a novel attractive target and new CDK inhibitors (CDKi) are sought for to improve cancer therapy. Dinaciclib is a selective inhibitor of several CDKs and is currently in phase III clinical studies for the treatment of leukemia. Multidrug resistance (MDR) is a major obstacle in successful cancer chemotherapy and is often mediated by ATP-binding cassette (ABC) transporters. ABCB1, ABCC1 and ABCG2 actively efflux a wide range of diverse anticancer drugs, rendering the cancer cell resistant to chemotherapeutic treatment. In contrast to a number of studies evaluating pharmacological effects of dinaciclib, very little is known about the interaction of dinaciclib with drug efflux transporters. Therefore, the main objective of our study was to elucidate the effects of dinaciclib on the transport activity of ABCB1, ABCC1 and ABCG2 transporters in vitro. Employing XTT proliferation assay, we evaluated the resistance of transporter expressing MDCKII cells to dinaciclib. We found that all studied transporters, ABCB1, ABCC1 and ABCG2, confer resistance to dinaciclib.

Compared to the control parental cell line, MDCKII-ABCB1, MDCKII-ABCC1 and MDCKII-ABCG2 were 6.7-, 3.7- and 2.9-fold more resistant, respectively, indicating that dinaciclib undergoes ABC transporter-mediated efflux. The contribution of ABC transporter to the resistance was confirmed by co-incubation with ABC transporter inhibitors where the resistance was abolished. To confirm dinaciclib as a substrate of ABC transporters, we also employed monolayer transport assay in MDCKII cells transduced with human ABC transporters. In this method, we determined dinaciclib as a substrate of ABCB1 and ABCG2, but not ABCC1. We further investigated the effect of dinaciclib on the transport function of ABC transporters by accumulation assays in overexpressing MDCKII cells. We demonstrated that dinaciclib can inhibit ABCC1-mediated efflux of daunorubicin as well as ABCG2-mediated efflux of mitoxantrone,

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indicating that this drug may be able to reverse ABCC1- and ABCG2-mediated MDR. On the other hand, dinaciclib did not influence ABCB1-mediated efflux of daunorubicin. Interaction of dinaciclib with the tested transporters was also investigated in ATPase assays; dinaciclib did not stimulate the baseline activities of ABCB1, ABCG2 or ABCC1 respective ATPases. However, the interaction with the transporters was confirmed by a significant decrease of the activated ATPase activities in the inhibition assay. In conclusion, we determined dinaciclib as a substrate of ABCB1 and ABCG2 and an inhibitor of ABCC1. These findings should be taken into consideration when introducing dinaciclib into clinical therapy. We presume that ABC transporters can have a substantial effect on dinaciclib transport and may influence pharmacokinetic behavior of this drug. This study was supported by the Charles University in Prague (project SVV/2015/260-185).

A7 - DEORPHANIZATION OF HUMAN CYTOCHROME P450 4X1 1 2 2 Michal Šiller , Francis Yoshimoto , and Frederick Guengerich 1 2 Palacky University, Olomouc, Czech Republic, Vanderbilt University School of Medicine, Nashville, Tennessee, United States Cytochrome P450 (P450) 4X1 belongs to a family of orphan P450s for which the role in human body have not been well described yet. Only limited information is available about its endogenous substrates. P450 4X1 has been previously successfully expressed and purified. An endogenous cannabinoid, anandamide, was identified as a substrate for P450 4X1, which catalyzed 14,15-epoxidation (1). In this study, the approach of elucidation of unknown enzyme function followed a 21st Century paradigm, in which heterologous enzyme expression and purification lead to its use as a reagent for untargeted analysis. Purified P450 4X1 was incubated with two tissues, human liver and bovine brain, serving as sources of endogenous compounds. Two extraction protocols, ethanolic and Folch extraction, were used to obtain a broad ‘library‘of compounds. NADPH-fortified samples together with control samples were analyzed by UPLC linked to high resolution mass spectrometry (HRMS), and the data was processed with the metabolomic software XC/MS. From an ethanolic extract of bovine brain, salsolinol was identified as a possible P450 4X1 substrate. Salsolinol is a tetrahydroisoquinoline structure formed endogenously during the condesation of dopamine with acetaldehyde. It exerts various biological effects and is proposed to play an important role related to alcohol abuse (2, 3). HRMS fragmentation revealed that salsolinol is oxidized to 4-(1-aminoethyl)benzene-1,2-diol. On the basis of time-dependent incubations, the proposed reaction mechanism is multi-step and involves oxidation and CC and C-N cleavage. We also incubated purified P450 4X1 with higenamine, a natural compound with a structure similar to salsolinol, which is biologically active and is a component of certain herbal preparations (4). The reaction mechanism was identical with the one observed with salsolinol. In conclusion, our results reveal at least two novel substrates of P450 4X1. The clinical relevance of these findings is of interest, in that both compounds have multiple effects on various tissues and cell processes.

(1) Stark K, Dostalek M, Guengerich FP. Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide. FEBS J, 275 (14): 3706-3717, 2008.

(2) Quintanilla ME, Rivera-Meza M, Berrios-Cárcamo PA, Buscaglia M, Morales P, Karahanian E, Herrera-Marschitz M, Israel Y. Salsolinol, free of isosalsolinol, exerts ethanol-like motivational/sensitization effects leading to increases in ethanol intake. Alcohol, 48 (6): 551-559, 2014.

(3) Mozdzen E, Kajta M, Wasik A, Lenda T, Antkiewics-Michaluk L. Salsolinol, an endogenous compound triggers a two-phase opposing action in the central nervous system. Neurotox Res, UPDATE, 2014.

(4) Kashiwada Y, Aoshima A, Ikeshiro Y, Chen YP, Furukawa H, Itoigawa M, Fuijoka T, Mihashi K, Cosentino LM, Morris-Natschke SL, Lee KH. Anti-HIV benzylisoquinoline alkaloids and flavonoids from the leaves of Nelumbo nucifera, and structure-activity correlations with related alkaloids. Bioorg Med Chem, 13 (2): 443-448, 2005.

A8 - ACTIVITIES OF CYTOCHROMES P450 IN HUMAN SKIN EXPLANTS Nenad Manevski, Gian Camenisch, Olivier Kretz, Piet Swart, Hilmar Schiller, Kamal Kumar Balavenkatraman, and Markus Walles DMPK, Novartis, Basel, Switzerland Human skin, one of the largest organs of the body, was reported to metabolize therapeutic drugs, cosmetic ingredients, and various xenobiotics to a generally small extent [1]. Cutaneous biotransformation could be especially important for topically and transdermally applied drugs, as well as for systemically given compounds that may distribute into skin or target skin for therapeutic effect. Moreover, if reactive metabolites are formed, cutaneous drug metabolism may contribute to onset of skin adverse reactions. Despite the importance for drug development, general understanding of cutaneous drug metabolism is poor and existing experimental models lack sufficient validation. In order to address these open questions, we studied activities of cytochromes P450 (CYPs) in fresh human skin explants, an experimental model that contains all skin cell types and intact tissue architecture. To avoid enzyme inactivation and experimental artifacts, fresh human skin was collected post-surgically and transported in organ preservation medium. In our laboratories, full-thickness skin explants were prepared and incubated with specific substrates for major human CYPs. Following incubations, incubation media and skin extracts were analyzed by LCMS for the presence of specific metabolites. Results show that numerous CYPs are active in human skin, including CYP1 subfamily, CYP2B6, CYP2D6, CYP2C8, and CYP3A4. The overall level of measured CYP activities was very low, however, especially compared to human liver. Measured CYP activities were correlated with gene expression

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data. Our studies are currently extending to major and minor CYPs that are reported to be overexpressed in skin compared to liver, for example CYP4B1, CYP4X1, and CYP26 [2]. Moreover, to assess the potential for the formation of reactive skin metabolites, we are investigating the cutaneous biotransformation of benzo[a]pyrene. Taken together, results offer a novel insight into skin drug metabolism and present a usable in vitro experimental model that could be used to predict cutaneous biotransformation in vivo.

References: [1] Oesch F, Fabian E, Guth K, Landsiedel R. Xenobiotic-metabolizing enzymes in the skin of rat, mouse, pig, guinea pig, man, and in human skin models. Arch.Toxicol. 2014;88:2135-90.

[2] Baron JM, Wiederholt T, Heise R, Merk HF, Bickers DR. Expression and function of cytochrome p450-dependent enzymes in human skin cells. Curr.Med.Chem. 2008;15:2258-64.

A9 - TAB-METHYL-SEQ PROTOCOL FOR TARGETED NEXT-GENERATION SEQUENCING OF DNA (HYDROXY)METHYLATION: IMPLICATIONS FOR ADME GENE REGULATION 1 2 1 1 2 1 Maxim Ivanov , Mart Kals , Volker Lauschke , Isabel Barragan , Lili Milani , and Magnus Ingelman-Sundberg 1 2 Karolinska Institutet, Stockholm, Sweden, Estonian Genome Center, University of Tartu, Tartu, Estonia Hydroxymethylcytosine, or hmC, is a novel epigenetic modification of DNA. It is generated in vivo due to the oxidation of methylcytosine (mC) by Tet family enzymes. In contrast to mC (which is a well-established repressive epigenetic determinant), hydroxymethylcytosine was shown to correlate with active gene transcription, probably due to the different composition of hmC-binding proteins vs mC-binding ones. Thus, mC and hmC must be considered separately in hmC-rich tissues like liver, brain, small intestine and colon, otherwise the results of epigenetic analysis might be inconclusive. Bisulfite sequencing, which is the “gold standard” method for the analysis of DNA methylation, doesn’t distinguish between mC and hmC. However, the recent modification of bisulfite sequencing, called TAB-Seq (Tet1-assisted bisulfite sequencing), allows to separate the signals from mC and hmC (due to selective oxidation of mC, but not hmC, by recombinant mTet1 enzyme). However, the original TAB-Seq methodology is available only for either locus-specific or whole-genome analysis. To fill the gap between these two extremes, we developed TABMethyl-SEQ protocol, which can be considered as a fusion between TAB-Seq approach and the Agilent Methyl-SEQ platform for target enrichment of DNA prior to next-generation sequencing. We tested TAB-Methyl-SEQ protocol on 20 adult human liver samples (which are characterized by high genomic hmC content, as shown by LC-MS). To our knowledge, this is the first study, where inter-individual variability of hmC profiles was assessed with single-base resolution. Our target region includes 191 ADME genes with their 20 Kb flanking sequences (with the exception for repetitive intervals). Results of this pilot study suggest that at least in a subset of ADME genes analyzed, the local abundance of mC and hmC clearly correlates with inter-individual differences in gene expression. Importantly, these correlations wouldn’t be observed, if only the bisulfite data were available. Thus, the usage of TAB-Methyl-SEQ unlocks a previously hidden dimension in the analysis of hepatic epigenome.

A10 - IN SILICO PREDICTION OF HEPATOCELLULAR DRUG BINDING Frauke Assmus, Brian Houston, and Aleksandra Galetin Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom In vitro methods for the assessment of intracellular binding are experimentally demanding and therefore, there is a great interest in predictive in silico models. The aim of the current study was to develop a multivariate model for the prediction of the unbound fraction of drugs in hepatocytes (fu,cell), following preliminary univariate regression analysis with logP and logD7.4. The performance of models based on 3D descriptors from either lowest energy or membranebound conformations was investigated, including internal and external model validation. To this purpose, an extended database of experimental fu,cell data reported in rat and human hepatocytes was collated, comprised of structurally diverse drugs with a broad range of physicochemical properties (mlogD7.4 = -1.56 to 4.87, clogP = -0.15 to 5.81, MW = 151 to 748) and hepatocellular binding (fu,cell = 0.01-89 %). Lowest energy and smallest amphiphilic (membranebound) conformations were generated in MOE followed by calculation of 2D as well as 3D molecular descriptors in ADMET Predictor (Simulation Plus), Moloc, MOE, PgPredix, Empire and Parasurf (476 descriptors in total were considered). Reduction of the multidimensional space was performed in SIMCA by partial least squares (PLS) analysis using a subset of compounds (n = 54) for model development and a preselected set of non-redundant and physico-chemically meaningful descriptors (n = 98). Shape descriptors showed the highest sensitivity to the conformation applied; however, the consideration of membrane-bound conformations had minimal impact on the fu,cell prediction. The final PLS model highlighted the importance of lipophilicity (logD7.4, logP), charge (basic pKa, negative charge) and number of rings as significant factors governing hepatocellular drug binding. These findings were consistent and independent of the conformation used for calculation of 3D descriptors. Improved model performance in terms of explanatory (r2 = 0.71) and predictive power (q2 = 0.76 for an external test set, n = 13) was obtained compared with univariate prediction approaches based on the use of logP and logD7.4 (e.g., Austin model: r2=0.61, external q2=0.69). The developed PLS model therefore provides a promising tool for the prediction of hepatocellular binding and the estimation of unbound hepatocellular drug concentrations by modelling and simulation approaches.

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A11 - COMPARISON OF RESPIRATORY DRUG ACCUMULATION AND LYSOSOMAL SEQUESTRATION IN NR8383 AND PRIMARY HUMAN ALVEOLAR MACROPHAGES 1 2 1 1 Ayse Ufuk , Jonathan Plumb , Brian Houston , and Aleksandra Galetin 1 Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, 2 United Kingdom, University Hospital of South Manchester NHS Foundation Trust, Manchester, United Kingdom Drug accumulation in alveolar macrophages (AMs) has a number of important clinical implications including reduced efficacy of inhaled respiratory drugs and formation of foamy AMs. The latter is often associated with ‘lysosomal trapping’ of cationic amphiphilic drugs resulting from the pH difference between acidic lysosomes and cytosol. The present study compared drug accumulation and lysosomal trapping of eight respiratory drugs in the rat AM cell line NR8383 and primary human AMs from predominantly smoker donors (n=9). Drug uptake was investigated at 5 µM substrate concentration up to 10 minutes at 37 and 4°C to delineate active and passive uptake processes. The extent of lysosomal trapping was assessed at the same conditions in the presence of 20 mM ammonium chloride (NH4Cl) which abolishes cytosol-lysosome pH-gradient. Both uptake clearance (CLuptake) and cell-to-medium concentration ratio (Kp) of investigated drugs showed a wide range (>400-fold) in NR8383 and human AMs. In both systems, uptake of most drugs (n=6) was driven by an active process. The CLuptake and Kp obtained in NR8383 and human AMs showed a good agreement for the current dataset (2.9-fold bias), with fenoterol as the most pronounced outlier.

Functional and imaging data confirmed that only clarithromycin and imipramine (positive control) accumulated in the lysosomes of both systems. The reduction in Kp of both drugs in the presence of NH4Cl was up to 70%, indicating an important, but not exclusive, contribution of lysosomal trapping to their accumulation in human AMs. The Kp data obtained in the presence of NH4Cl suggest potential differences in the extent of membrane partitioning between human AMs and NR8383. The current study highlights promising application of NR8383 to assess drug accumulation and extent of lysosomal accumulation of respiratory drugs in human AMs. A12 - CAR TRANSCRIPTIONAL ACTIVITY IS UNDER THE INFLUENCE OF EGF IN PRIMARY HUMAN HEPATOCYTES. IDENTIFICATION OF CAR SPECIFIC TARGET GENES 1 1 1 2 3 4 Hugues de Boussac , Philippe Briolotti , Cedric Duret , Michael Romer , Jean-Michel Fabre , Jeanne Ramos , 1 1 Patrick Maurel , and Sabine Gerbal-Chaloin 1 2 INSERM U1183, IRMB, Hôpital Saint-Eloi, Montpellier, France, Center of Bioinformatics Center of Bioinformatics 3 Tübingen (ZBIT), University of Tübingen, Tübingen, Germany, Department of Digestive Surgery, Hospital Saint Eloi, 4 CHU, Montpellier, France, Pathological Anatomy Department, CHU, Guy de Chauliac, Montpellier, France Phenobarbital (PB), an antiepileptic drug widely used in human, has been shown to promote hepatocarcinogenesis in mice but not in human. This phenomenon is dependent on the constitutive androstane receptor (CAR), a xenosensor involved in detoxification processes in the liver, and the phenobarbital Mode Of Action (MOA) has recently been suggested to be under the influence of the EGF receptor. To date, while CAR target genes are well characterized in rodent essentially due to the KO mice models, no global transcriptomic analysis of genes modulated in a CAR dependent fashion have been performed in human. Here we aimed to investigate the role of EGF in CAR activity, to identify specific CAR target genes, and to elucidate the action of PB on CAR transcriptional activity in primary human hepatocytes (PHH). PHH were treated with CITCO, a human CAR agonist, and PB an indirect CAR activator, in the absence or the presence of EGF. We observed that EGF presence prevents CAR transcriptional activation of CYP2B6 or CYP3A4 by CITCO while the expression of PB induced genes (CYP3A4, CYP2B6) was unaffected. To determine the involvement of CAR in the CITCO and PB modulations, we used siRNA mediated depletion of the xenosensor CAR from PHH, in the presence or the absence of EGF, and analyzed gene modulations using Affymetrix microarrays. In absence of EGF where CAR can be activated, we identified 72 genes and 32 genes significantly modulated by PB and CITCO respectively. Of these genes, only 18 were modulated by CITCO in a CAR dependent manner including 4 PB-CAR dependent genes (CYP2A6, CYP2B6, CYP2B7P1 and CYP2A7). In contrast, in the presence of EGF where CAR lost its transcriptional activity, CITCO and PB modulated gene expression only in a CAR independent manner. Overally our studies suggest 1) A role for EGF in CAR transcriptional activity, 2) there are specific CAR target genes in primary human hepatocytes and 3) there is a CAR independent PB MOA on gene regulation in PHH.

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P1 - A NEW APPROACH TO ASSESS PROCESSES THAT DETERMINE HUMAN ORAL BIOAVAILABILITY USING EX VIVO PORCINE INTESTINAL TISSUE Evita van de Steeg, Joost Westerhout, Steven Erpelinck, Sieto Bosgra, and Heleen M. Wortelboer TNO, Zeist, The Netherlands In the development of pharmaceutical and nutritional products an accurate prediction of the oral fraction absorbed in humans is essential, as the bioavailability of a compound at the target site co-determines the efficacy of the active compound. Currently used animal models, in vitro (mainly cell lines) and/or in silico tools are applied pre-clinically, but their predictive value to assess oral bioavailability in humans is often insufficient. Cell-line based systems such as Caco-2 are not well suited to investigate the different processes that determine the bioavailability of specific compounds, such as intestinal metabolism, regional differences in absorption, mucus interaction, food-drug effects and/or excipient-drug effects. Human ex vivo intestinal tissue in the Ussing system has been shown a valuable alternative to address these issues. However, the availability of healthy fresh human intestinal tissue limits its practical applicability. Here, we present a new in vitro intestinal model (InTESTineTM) to evaluate these processes that determine the oral fraction absorbed using healthy porcine intestinal tissue (left over material). We have fully characterized the system and have determined the abundance of transporter proteins and metabolic enzymes, and compared these to the Caco-2 model and human intestinal tissue. We demonstrate that porcine tissue within the InTESTineTM system was well compatible with biorelevant matrices (e.g. FaSSIF and FeSSIF media). Additionally, InTESTineTM could be well applied to study the effect of intestinal wall metabolism on the fraction absorbed of compounds (e.g. testosterone, midazolam, and acetaminophen). Moreover, differences in regional (duodenum, jejunum, ileum, and colon) absorption of several compounds (e.g. antipyrine, atenolol, warfarin, verapamil) have been observed, comparable to human Ussing data. Finally, the apparent permeability (Papp) in ex vivo porcine intestinal tissue was determined for a representative set of compounds for which both Caco-2 and human Ussing Papp data were available. Porcine intestinal Papp values corresponded better to observations in human Ussing than Caco-2 Papp values did. In conclusion, viable porcine intestinal tissue mounted in the InTESTineâ&#x201E;˘ system can be applied as a reliable tool for the assessment of processes involved in human oral bioavailability.

Reference: Westerhout J, van de Steeg E, Grossouw D, Zeijdner EE, Krul CA, Verwei M, Wortelboer HM. A new approach to predict human intestinal absorption using porcine intestinal tissue and biorelevant matrices. Eur. J. Pharm. Sci. 2014, doi: 10.1016/j.ejps.2014.07.003.

P2 - SIMULATION STUDY ON CONTRIBUTIONS OF MEMBRANE PERMEABILITY, METABOLIC CLEARANCE, AND EFFLUX TRANSPORT BY P-GLYCOPROTEIN TO INTESTINAL AVAILABILITY USING TRANSLOCATION MODEL 1 1 2 3 Hirotaka Ando , Hirotaka Ando , Akihiro Hisaka , and Hiroshi Suzuki 1 2 3 Kyorin Pharmaceutical Co., Ltd., Nogi-machi, Shimotsuga-gun, Japan, Chiba University, Chiba, Japan, University of Tokyo Hospital,Tokyo, Japan Negative correlation between intrinsic clearance (CLint) and a product of absorption rate (Fa) and intestinal availability (Fg) has been reported by Kato et al(1). This simulation study aims to give theoretical considerations on the influence of membrane permeability and efflux transport by P-glycoprotein (P-gp) on the relationships between CLint and FaFg by using a newly developed Translocation model (TLM)(2). TLM is a physiologically based drug absorption model, which can analyze various non-linear phenomena during the absorption process caused by metabolism, active transport, and blood-flow limitation. TLM has one absorption site, which is translocatable along the length of the intestine, and thus location-dependent absorption properties can be considered. The translocation of a drug is defined by an arbitrary mathematical equation in TLM. By using this model, values of FaFg were simulated after random generation of the drug-related parameters; CLint [4.89-5560 mL/min/kg], membrane permeability (Papp) [8.6955.8x10^6 cm/s], and other physicochemical parameters based on information of various commercially available drugs. The relationship between FaFg and CLint was plotted on a scatter diagram. The simulation was performed with typical clinical therapeutic dose (TD, 100 mg) and microdose (MD, 100 Îźg). After the data points on the scatter diagrams were classified into several groups based on Papp and P-gp, contribution of the membrane permeability and P-gp was confirmed. The simulation suggested that the relationship between FaFg and CLint agreed with that reported by Kato et al. The relationship was rather independent to the membrane permeability at TD, but drugs with larger contribution of efflux by P-gp were indicated with slightly lower FaFg. On the other hand, drugs with high contribution ratio for P-gp and/or low permeability (Papp,Caco-2 < 15x10^6 cm/s) were indicated with a lower FaFg, resulting in poor correlation between FaFg and CLint at MD. These drugs would be suffered from significant drugdrug interactions (DDI) via P-gp inhibition. Attention should be paid to DDI via P-gp for drugs of P-gp substrates with lower permeability especially when the dose is small. These simulations need to be confirmed by clinical studies in the future. [References] 1) Kato et al., (2003) The intestinal first-pass metabolism of substrates of CYP3A4 and Pglycoprotein-quantitative analysis based on information from the literature., Drug Metab Pharmacokinet., 18(6) 365372. 2) Ando et al., (2015) A New Physiologically-Based Pharmacokinetic Model for the Prediction of Gastrointestinal Drug Absorption: Translocation Model., Drug Metab Dispos., 43(4) 590-602.

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P3 - A SENSITIVE AND RAPID ANALYSIS OF ESCITALOPRAM IN HUMAN PLASMA BY UPLC/MS/MS 1 2 1 1 2 1 1 SK Wo , Benny SP Fok , Celia WS Tang , Vincent HL Lee , Brian Tomlinson , Teddy TN Lam , and Zhong Zuo 1 2 School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, Department of Medicine and Therapeutics,The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Purpose: Escitalopram, an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class, is the therapeutically active S-enantiomer of antidepressant citalopram. Due to the low dose of escitalopram (5 or 10 mg once daily), an analytical assay with high sensitivity and efficiency is essential for the quantification of escitalopram in human plasma. Methodology: UPLC/MS/MS consisted of Agilent 1290 Infinity LC-6430 triple quadrupole mass spectrometer. Chromatographic separation was achieved by Phenomenex Kinetex C18 column (50 x 2.1 mm, 2.6 μm) and 0.1% formic acid in water: acetonitrile (70:30% v/v) at 0.4 ml/min. MRM (ESI positive) was acquired at m/z 325 -> m/z 109 (escitalopram) and m/z 308 -> m/z 235 (IS, zolpidem). Liquid-liquid extraction (LLE) was conducted by vortex mixing 400 μl plasma sample (after addition of IS) with 1.5 ml methyl t-butyl ether (MTBE). After centrifugation, the organic layer was concentrated to dryness using a vacuum concentrator. The residue was then reconstituted with 200 μl of 50% acetonitrile in water. Followed by centrifugation, 2 µl supernatant was injected for analysis. Validation was conducted according to USFDA Bioanalytical Method Validation guidance. Results: Several extraction methods such as protein precipitation by either acetonitrile or methanol (with or without concentrating the sample) and LLE with dichloromethane or diethyl ether had been tried and LLE with MTBE provided high recovery and clean background.

The EIC of drug-free plasma did not indicate co-eluting interference, suggesting high specificity of the assay. The linearity of escitalopram is 0.2-30 ng/ml (free base), and LLOQ is 0.2 ng/ml (equivalent to 0.4 pg per injection), which is much lower than that from the published report (2 pg per injection). The mean extraction recovery is 89.7% and the matrix effect is negligible. The results on 3-cycle freeze (-80 °C) and thaw (ambient), auto-sampler (8 °C for 5 h) and bench (ambient for 3 h) stability tests showed that LLOQ and QC samples were all stable (94.4-106.4%). The total run time is 2 min per sample. The developed method was successfully applied to a bioequivalence study on 10 mg escitalopram tablet formulations in Chinese male subjects. Conclusion: The developed assay is highly sensitive and efficient for the quantification of escitalopram in human plasma, which provides high throughput sample analysis samples.

P4 - CONTRIBUTION OF QUANTITATIVE MASS SPECTROMETRY IMAGING (MSI) IN PHARMACEUTICAL FIELD: APPLICATION TO DRUG AND PEPTIDE ANALYSIS IN TISSUE Raphael Legouffe, Gregory Hamm, Fabien Pamelard, David Bonnel, and Jonathan Stauber ImaBiotech, Loos, France MSI permits the label-free study of several compounds of interest simultaneously on the surface of biological tissues.

Quantitative development is a key success in the application of MSI for drug discovery as it is compared to gold standard quantitative techniques (such as autoradiography or LC-MSMS). This application may play a significant role in early phases of pharmaceutical discovery to evaluate small molecule concentration, notably drugs. This presentation will describe the possibility of quantifying a drug and a peptide using MSI and will show the main advantages of this new analytical technique in pharmaceutical field. Rat were dosed with diazepam at 5 mg/Kg and sacrificed at 15 minutes post-injection according to the kinetics of the compounds. Snap frozen brain and pancreas cryosections were carried out with a Microm cryostat HM560 (Thermo Scientific) at 10µm thick and mounted on several conductive ITO glass slides before matrix automated deposition. Insulin analogue and a labeled diazepam were used to perform the quantification as internal standards. Mass spectra were acquired using a MALDI-TOF Mass Spectrometer (Bruker Daltonics) and data were generated by proprietary Quantinetix software (ImaBiotech).The quantification of a small and a large molecule, respectively the diazepam and the mouse, insulin was performed using MSI and internal standard approach. Insulin level was determined in whole pancreas tissue (approximately 200 µg/g of tissue) and the results were in agreement with previously published data (LC-MS/MS). Moreover, we have access to its content in small histological regions such as the Langerhans islets. Concerning the diazepam quantification, QMSI were used on adjacent sections and with the same dilution range. A higher amount of Diazepam (µg/g of tissue level) has been in the grey matter of the brain than in white matter which is cross-validated by pharmacokinetics data.

A good reproducibility between quantitative data has been observed with an intra and inter-variability of 10 and 20% respectively. In conclusion, this study is the first example of multiple quantitative approaches on small and large molecules by MSI. P5 - EVALUATION OF RESPONSE FACTORS OF DRUG METABOLITES ON CAPILLARY HPLC/MS WITH ELECTROSPRAY IONIZATION Joachim Blanz, Thierry Delemonte, David Pearson, Werner Gertsch, Philippe Ramstein, Jérôme Dayer, Gareth Williams, and Reiner Aichholz Novartis Institutes for Biomedical Research / Analytical Sciences and Imaging, Basel, Switzerland Drug metabolism studies are used in early phase drug discovery i) to identify metabolic soft spots, ii) to detect potentially toxic/reactive metabolites, and iii) for the early investigation of species differences in metabolism. At the discovery stage it is not only important to identity metabolites, but also paramount to esti¬mate the relative quantities of the metabolites, so that metabolic liabilities of drug candidates can be clearly identified. A major drawback of MS analysis is that electrospray ionization efficiency of a certain compound depends on its individual chemical structure,

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the mobile phase (pH, solvent) used for the chromatographic separation and electrospray conditions. In addition, ion suppression or ion enhancement due to co-elution of drug-related or matrix components is a known phenomenon.

These limitations currently require the availability of reference standards for exact quantitation of metabolites by LC/MS or the administration of radiolabelled compounds. Relative peak areas have been used in Novartis to semiquantitatively estimate the abundance of metabolites for soft spot analysis and we wanted to evaluate how reliable our estimations really are. Our poster describes the results of an investigation of 180 metabolites of 100 structurally diverse drug candidates that were synthesized in the last 10 years in Novartis to confirm chemical structures during early drug development. Using a capillary LC/MS system, the LC/MS peak areas for 26 metabolites (14%) were below 20 % of the peak area for the corresponding parent compound. Many of these metabolites derived from amide bond cleavages. The respective molecules either had a low molecular mass (below 200 Da) or were significant more polar then the respective parent. Peak areas of only 7 metabolites exceeded the peak area for the corresponding parent by more than 50 %. Thus the peak areas of 147 metabolites (82%) were within 20-150% relative to the peak areas for the corresponding parent. Our results indicate that, within the scope of early drug development, peak areas of metabolites can be used for semi-quantitative assessments that allow to correctly identify metabolic soft spots during the optimization of drug candidates.

P6 - INNOVATIVE APPROACH TO BLOOD SAMPLING USING DRIED BLOOD SPOTS. APPLICATION TO PHARMACOKINETICS AND CYTOCHROME P450 PHENOTYPING 1 1 2 1 1 Marija Bosilkovska , Julien Deglon , Aurelien Thomas , Caroline Samer , Jules Alexandre Desmeules , and Youssef 1 Daali 1 2 Geneva University Hospitals, Geneva, Switzerland, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Background: Due to its numerous advantages such as low blood volume required, the simplicity of sampling from a small finger prick, the ease of storage, shipping and sample preparation, the use of dried blood spots (DBS) has gained in popularity in the last few years over conventional whole blood or plasma sampling for PK or drug monitoring.

In order to overcome the impact that haematocrit has on the spreading of the applied drop of blood, precise knowledge of the collected volume is crucial for the determination of drug/metabolites concentrations. Material and methods: Although the collection of an accurate capillary volume using a volumetric micropipette is simpler than venous blood collection, it still needs to be conducted by trained technicians using dedicated instruments. To simplify this process a new capillary blood collection device has been developed. The prototype integrates a patented microfluidic plate (WO/2013/144743) allowing for accurate volume control and a conventional filter paper card for blood storage.The concentrations and pharmacokinetic profiles of a P-glycoprotein (P-gp) and six cytochrome P450 (CYP) probes and their metabolites obtained with the new sampling device have been compared with a conventional volumetric micropipetting method in a clinical trial including 30 volunteers who have received the Geneva cocktail for CYP and P-gp phenotyping. The cocktail was composed of: caffeine 50mg, bupropion 20 mg, flurbiprofen 10mg, omeprazole 10 mg, dextromethorphan 10mg, midazolam 1mg and fexofenadine 25mg as probes for CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A and P-gp respectively. The quantification was done using a previously validated LC/MS-MS method. Results: Concentrations obtained with the new microfluidic sampling device showed excellent correlation with conventional micropipetting concentrations with slopes values close to 1 (0.91 – 1.03) and determination coefficients R2>0.90 for all of the 13 analysed substances. Sampling could be successfully performed by the volunteers themselves with almost no previous training. Conclusion: DBS technique combined with an innovative sampling device and a sensitive analytical method can be used as a self-test for CYP and P-gp phenotyping. The use of this technique can be further enlarged to the quantification of other substances for PK studies and therapeutic drug monitoring.

P7 - KEEPING AN EYE ON MOLECULAR IMAGING: ASSESSMENT OF DRUG TOXICITY IN SMALL OCULAR STRUCTURE USING MASS SPECTROMETRY IMAGING 1 2 1 1 2 Gregory Hamm , Françoise Brignole-Baudouin , Flore Grandin , Jonathan Stauber , Christophe Baudouin 1 2 ImaBiotech, Loos, France, Institut de la Vision, Paris, France Mass spectrometry Imaging (MSI) applications to ophthalmic drug discovery have recently gained growing interest especially for preclinical studies in pharmacology or toxicology. In our study, MSI was applied to assess the distribution of Benzalkonium chloride (BAK) compound (antiglaucoma eye drops preservative) in specific areas of the eye after instillation in animal model tissues. They have been reported to cause ocular surface disorders with tear film alteration, eye irritation and to promote dry eye. The distribution of BAK compound was investigated in small specific histological regions of the eye (such as iridocorneal angle or retina regions). By this way, we can estimate efficiency of action or adverse effects of the treatment by following some molecular biomarkers localization and relative concentration. The eyes of New Zealand rabbits were instilled with different BAK solutions at 0.01%, 2 drops BID for 1 month or 5 months or 0.2% at 1 drop/day for 1 month. After sacrifice, eyes were quickly enucleated and embedded in tragacanth gum and frozen at -80°C. Serial cryosections were deposited on glass slides for Hematoxylin-Eosin staining or immunohistochemistry (IHC) and conductive glass slide for mass spectrometry imaging. Imaging experiments were performed on MALDI-TOF (Autoflex Speed, Bruker, Germany) and/or MALDI FTICR (SolariX 7T, Bruker, Germany) instruments using Multimaging Software (ImaBiotech, France) for data analysis and treatment.

High spatial resolution images were performed at cells level (30 µm). Local Drug concentration differences were observed according to histological area and position on the eye section (anterior, posterior, temporal or nasal side).

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MSI and IHC results were put side by side to correlate inflammatory areas (degradation of corneal eptithelium, or apoptosis phenomena within cornea/conjonctiva region) with BAK localization. A high accumulation of BAKs were observed at the sclerocorneal junction and near trabecular meshwork involved in aqueous humor outflow. Moreover, differential analysis was carried out to find disease state biomarkers or companion’s biomarkers of each ocular structure. Lipids and sphingolipids molecules were modulated in specific area of the eye especially in the retina’s region. Translational experiment was performed on other tissue model following the same protocol to validate BAK distribution in a real tissue. MSI offers new insight in ocular therapeutic/pharmaceutical research, especially to give a better understanding of the drug candidate migration from the front to the back of the eye to assist drug efficiency or toxicity studies for specific tissue targeting eye diseases. Multimodality approach using MSI was used to interpret ocular disorders and provide new the pharmacological insights.

P8 - PHARMACOKINETICS OF GANODERMA ACID A IN RAT PLASMA AND BRAIN DETERMINED BY A DEVELOPED UPLC-MS/MS METHOD Fangrui Cao, Bing-xin Xiao, Li Feng, Li-sha Wang, and Qi Chang Institute of Medicinal Plant Development, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China Background: Traditional Chinese medicine Ganoderma lucidum (GL) has a long history use for increasing energy, improving immunity, and promoting health and longevity. Triterpenoids are the main bioactive components of G. lucidum. Ganoderic acid A (GAA) is a representative active triterpenoid from GL, and generally exists in Ganoderma genus. GAA has been reported to exhibit antinociceptive, antioxidative and anti-cancer activities. However, the study on pharmacokinetics (PK) of GAA is limited. The present study aims to establish a sensitive and rapid UPLC-MS/MS method for studying the PK of GAA in rat plasma and brain. Methods: The plasma samples were collected via jugular vein of rats after oral and intravenous administration of GAA (20 mg/Kg). The brain micro-dialysates were collected via lateral ventricles of rats after intravenous administration of GAA (20 mg/Kg). Plasma samples were treated by protein precipitation, and micro-dialysates were directly assayed. The analytes were separated on a reversed-phase C18 column eluted with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at 0.3 ml/min. The eluate was monitored by a mass detector using MRM (m/z, 515.2-285.2) model in negative electrospray ionizations.

The method was validated using quality control samples at low, middle and high concentrations. Results: The calibration curves of GAA showed good linearity ranging from 2-5000 nmol/L (r2>0.99), with LOD and LLOQ being 0.

5 and 2 nmol/L respectively, and no significant matrix effects were observed. The intra- and inter- day precision of GAA were less than 9.99%, and accuracy were ranged from 102.3 to 115.7%. The analyte was found stable in treated samples or plasma since their concentrations were not decreased after being kept for 12 h at room temperature (20 ℃) and 7 d at -20 ℃, respectively. The extraction recoveries of GAA in rat plasma were between 92.8-97.9%. The recovery of GAA through micro-dialysis probe was 25.8%. After oral administration, GAA could be rapidly absorbed into the body with Tmax of 10 min and Cmax of 428.66 ng/ml. The PK parameters of GAA after oral and intravenous administration were estimated as t1/2 of 4.16 and 2.45 h, AUC0-∞ of 646.38 and 7026.34 ng•h/ml, Vz/F of 166.28 and 10.06 L/Kg, and Cl/F of 461.59 and 47.34 ml/(Kg•min),respectively. The PK parameters of GAA in lateral ventricles were estimated as Tmax of 15 min, Cmax of 304.55 ng/ml, t1/2 of 1.49 h, AUC0-∞ of 189.74 ng•h/ml, Vz/F of 270.68 L/Kg, and Cl/F of 1863.39 ml/(Kg•min). The absolute oral bioavailability of GAA was about 9.20%. The blood–brain barrier permeability of GAA was 2.70%. Conclusion: This is the first report on the PK of GAA in rat plasma and brain.

GAA could be rapidly absorbed into the body and distributed into the brain. This PK study seems to be useful for a further clinical study of GAA. Acknowledgments: Financial supports from the Ministry of Science and Technology of China (2012ZX09301002-001,1108).

P9 - PROTEOMIC INVESTIGATION OF SALIVA AS A SOURCE OF BIOMARKERS OF ENERGY INTAKE 1 1 2 2 2 3 Joanna Chowdry , Caroline Evans , Carmen Diaz Toledo , Roberta Re , Martin Wickham , Martin Yeoman , and 1 Bernard Corfe 1 2 Department of Oncology, University of Sheffield, Sheffield, United Kingdom, Leatherhead Food Research, Surrey, 3 United Kingdom, University of Sussex, Brighton, United Kingdom Background: Health claims made by the food industry need to be objective and substantiated in order to achieve approval by the European Food Standard Agency. Formulations aimed at modifying energy intake (EI) to manage obesity require objective measures of appetite (AP). Subjective appetite ratings are widely used though systematic literature review indicates that AP and EI don’t reliably correlate. Saliva is an alternative, less invasive biological source for global protein profiling with potential to identify novel objective measures of EI using iTRAQ (isobaric tags for absolute and relative quantification) proteomics. We previously demonstrated an increase in thioredoxin in response to a DHA-challenge, using this discovery pipeline. Methods: 34 healthy volunteers participated in a 3-way randomised crossover study to assess the effect on EI of three different pre-load challenges varying in calorific content and sensory properties, providing subjective appetite ratings, saliva samples and EI data (Kcals) pre- and post-exposure to each challenge when prompted. Samples were pooled according to ranked EI data, treatment and time-point. Pooled samples were subject to iTRAQ analyses, and ELISA for our previously identified biomarker, Thioredoxin. Results: Proteomic analysis revealed significant differences between post-prandial pooled samples, and separate relationships for fasted pooled samples. Biomarker validation using ELISA and Western Blot confirmed increases in thioredoxin corresponded to increased energy intake. Future candidate biomarkers are being selected for

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validation based on maximal differences in iTRAQ and availability of antibodies. Conclusion: Proof of concept for novel EI biomarkers has the potential to impact significantly on the food industry, speeding up the EFSA regulatory approval process. (This work was funded by University of Sheffield and Leatherhead Food Research.)

P10 - ASSESSING METABOLIC COMPETENCE IN ISOLATED HEPATOCYTES: EXPLORING THE RELATIONSHIP BETWEEN ENZYME FUNCTION AND PLASMA MEMBRANE INTEGRITY VIA SAPONIN TREATMENT Francesca Wood, David Hallifax, and Brian Houston School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom The use of human tissue for in vitro predictions of in vivo clearance is common place, yet the viability of both fresh and cryopreserved human hepatocytes is often lower than desired. Trypan Blue exclusion is a frequently used measure of viability, but reflects plasma membrane integrity rather than metabolic capability; cells with a damaged or temporarily compromised plasma membrane determined ‘non-viable’ using this method may be functionally competent [1]. The aim of this work was to investigate enzymatic function of rat hepatocytes deemed non-viable by Trypan Blue exclusion. Freshly isolated rat hepatocytes were diluted to a concentration of 1 x 106 viable cells/ml and preincubated with 0.01% saponin (a pore-forming agent) for 5 minutes. An estimate of viability was obtained using the Trypan Blue exclusion method before use. Saponin-treated and untreated (intact) rat hepatocytes of the desired cell concentration were incubated with midazolam (2.5 µM), propranolol (0.1 µM) and saquinavir (0.1 µM) in the presence and absence of exogenous NADPH (1 mM). Unbound intrinsic clearance (CLint,u) estimates were obtained from substrate depletion profiles. Treatment with 0.01% saponin rendered all cells non-viable according to the Trypan Blue exclusion method, and in the absence of exogenous NADPH, CLint,u of all substrates by these cells was negligible. Coincubation of 1 mM NADPH with saponin-treated cells and substrate restored CLint,u to levels comparable to those in intact hepatocytes. The addition of NADPH to intact cells had minor effects on CLint,u. These results confirm that damage to the cell membrane (chemical-induced pore formation) results in a marked loss of metabolic activity. Large increases in CLint,u with replenishment of NADPH indicate that Cytochrome P450 enzymes remain functional and reduction in CLint,u is principally attributable to the dissipation of internal cofactor gradients. The minimal effect of exogenous NADPH on intact cells suggests that the cell membrane presents a permeability barrier to this cofactor.

This work has important implications for assessment of viability and use of hepatocyte preparations for prediction of in vivo metabolic clearance.

1. Tran, S.-L., et al., Trypan blue dye enters viable cells incubated with the pore-forming toxin hlyii of Bacillus cereus.

Plos One, 2011. 6(9).

P11 - CYNOMOLGUS MONKEY AS A PRECLINICAL MODEL FOR THE INVESTIGATION OF HEPATIC UPTAKE OF OATP SUBSTRATES 1 2 2 1 1 1 2 Tom De Bruyn , Hugh A. Barton , Yi-An Bi , Carina Cantrill , Aleksandra Galetin , Brian Houston , Jian Lin , and 2 Rachel Kosa 1 Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, 2 United Kingdom, Pharmacokinetics, Dynamics & Metabolism, Pfizer Inc., Groton, Connecticut, United States The aim of this work was to evaluate the Cynomolgus monkey as a preclinical model to predict hepatic uptake clearance mediated by Organic Anion Transporting Polypeptide (OATP) transporters. Hepatic uptake of eight OATP substrates (rosuvastatin, pravastatin, repaglinide, fexofenadine, cerivastatin, telmisartan, pitavastatin and valsartan) was investigated in plated cynomolgus hepatocytes. Total uptake clearance values and passive diffusion were measured from initial uptake rates (up to 2 min) at a single substrate concentration (1 µM) in the absence and presence of the OATP inhibitor rifamycin SV, respectively. In vivo intrinsic hepatic clearance values (CLint,h) for these drugs were determined from systemic clearances obtained following intravenous bolus administration in female cynomolgus monkeys (n = 3) after accounting for renal excretion. Total uptake clearance values in plated monkey hepatocytes ranged from 5 to 387 µL/min/mg protein for pravastatin and telmisartan, respectively; and were comparable to in vitro data obtained under the same experimental conditions in plated human hepatocytes (ranging from 1 to 351 µL/min/mg protein). In general, there was a good agreement in the relative contribution of active transport to total uptake between cynomolgus and human data, with the exception of telmisartan and pitavastatin. In vivo CLint,h values in cynomolgus monkeys ranged from 20 to 3181 mL/min/kg for fexofenadine and cerivastatin, respectively. In vitro – in vivo extrapolation of cynomolgus total uptake clearance data resulted in good concordance of CLint,h (2.8-fold bias) with pravastatin and cerivastatin as the most pronounced outliers. Based on the current dataset, minimal species differences were observed between cynomolgus and human hepatocyte in vitro uptake data.

Additionally, extrapolation of in vitro cynomolgus total uptake data resulted in good prediction of in vivo CLint,h for most drugs, highlighting its suitability as a preclinical model for the investigation of hepatic uptake.

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P12 - DETERMINATION OF A HORSE HEPATIC MICROSOMAL SCALING FACTOR FOR PREDICTING IN VIVO METABOLIC CLEARANCE Khaled Shibany, Stuart Paine, and Sabine Totemeyer The University of Nottingham, Loughborough,United Kingdom The liver is considered as the predominant organ of metabolism for the majority of endogenous compounds and xenobiotics. This has led to the development of a set of liver-based technologies that can be used to study drug metabolism in vitro1,2. In the last three decades, in vitro metabolism has gained significant popularity in the drug discovery process. This has been primarily due to a number of factors, including the controversial ethical issues involved with studies using animals and the questionable applicability of the data to human; also there are significant financial costs associated with in vivo studies3,4. However, the in vitro data obtained using liver tissue needs to be extrapolated to predict in vivo metabolic clearance (IVIVE). This requires a knowledge of the amount of microsomal protein per gram of liver (MPPGL) or hepatocellularity per gram of liver (HPGL), and of the size the liver. Although standard values of microsomal scaling factors for different species have been determined, these values have not been determined in horses5. The aim of this study is to determine levels of MPPGL per gram of horse liver. Triplicate liver samples were used to determine values of MPPGL (n=12). Scaling factors were determined by comparing the CYP P450 content in microsomes against the CYP P450 of the frozen liver homogenate. The results of this study show that, the value of MPPGL ranged from 33 to 76 mg/gram of liver (mean= 52±17 mg/gram of liver). The result of this study indicates a consistency in scaling factor between horse and most of the other species, with the exception of dog6.

References: 1. Brandon EF., Raap CD, Meijerman I, Beijnen JH, Schellens JH. An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons. Toxicol. Appl. Pharmacol. 2003;189(3):233-246.

2. Groneberg D a., Grosse-Siestrup C, Fischer A. In Vitro Models to Study Hepatotoxicity. Toxicol. Pathol.

2002;30(3):394-399.

3. Ferdowsian HR, Beck N. Ethical and scientific considerations regarding animal testing and research. PLoS One 2011;6(9):e24059.

4. Knight A. Systematic reviews of animal experiments demonstrate poor human utility. AATEX 2008:125-130.

5. Kwon Y. Handbook of Essential Pharmacokinetics, Pharmacodynamics and Drug Metabolism for Industrial Scientists. Springer; 2001.

6. Naritomi Y, Terashita S, Kimura S, Suzuki A, Kagayama A, Sugiyama Y. Prediction of human hepatic clearance from in vivo animal experiments and in vitro metabolic studies with liver microsomes from animals and humans.

DRUG Metab. Dispos. 2001;29(10):1316-1324.

P13 - IMPACT OF AGE ON HEPATIC EXTRACTION RATIO OF DRUGS IN PAEDIATRIC POPULATION 1 2 3 Yoshiteru Kamiyama , Farzaneh Salem , and Amin Rostami-Hodjegan 1 2 Analysis & Pharmacokinetics Labs, Astellas Pharma Inc., Tsukuba-shi, Japan, Simcyp Limited (a Certara company), 3 Sheffield, United Kingdom, Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom Hepatic metabolic clearance of drugs is determined by their hepatic extraction ratio, commonly considered as an inherent attribute of drug with a fixed value. Hepatic extraction ratio consists of three age-dependent parameters [1, 2]: fraction of drug unbound in blood, hepatic intrinsic clearance of unbound drug and hepatic blood flow. Changes in the above parameters will affect absolute value of hepatic extraction ratio. For example, a rise in fraction of drug unbound in blood, for low extraction drugs (extraction ratio <0.3) increases hepatic metabolic clearance whereas for high extraction drugs (extraction ratio >0.7) this does not affect metabolic clearance. Unless the age-related physiological changes in these parameters occur in parallel, it is expected that hepatic extraction ratio of drugs varies with age. In this study, hepatic extraction ratio of midazolam and two other hypothetical drugs with ten-fold higher and lower hepatic intrinsic clearance compared to midazolam was estimated from birth to 17 years. Ontogeny of the CYP3A4 in these simulations were based on models recently published using deconvolution of in vivo systemic clearance values of probe activity [1]. Impact of “fraction of drug unbound in blood”, “hepatic intrinsic clearance of unbound drug” and “hepatic blood flow” with age were investigated on relative paediatric to adult extraction ratio.

Midazolam categorised as a low extraction ratio drug until about 7 months after birth. Hepatic extraction ratio increased with age from 0.02 in preterm neoantes to about 0.6 in healthy volunteers (n=306). A hypothetical drug with ten-fold higher hepatic intrinsic clearance than midazolam is categorised as high extraction from 4 days after birth although hepatic extraction ratio reached adult level at 8 months. For a drug with ten-fold lower hepatic intrinsic clearance compared to midazolam only 30% of adult hepatic extraction value is achieved by the first year and the drug is categorised as low extraction across all the age ranges. Hepatic extraction and therefore bioavailability of orally administered drugs were sensitive to changes in the hepatic intrinsic clearance and hepatic blood flow. Coining a drug as ‘high extraction’ cannot be universally applied at lower ages whilst if a drug was ‘low extraction’ in adults, it will be low extraction in paediatrics too.

1. Salem F, Johnson TN, Abduljalil K, Tucker GT, Rostami-Hodjegan A. A Re-evaluation and Validation of Ontogeny Functions for Cytochrome P450 1A2 and 3A4 Based on In Vivo Data. Clin Pharmacokinet 2014.

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2. Gabrielsson J., D. W. Pharmacokinetic and Pharmacodynamic Data Analysis: Concepts and Applications: Apotekarsocieteten, 2000.

P14 - LOW INTRINSIC CLEARANCE DETERMINATION USING PRIMARY HUMAN HEPATOCYTES IN MONOCULTURE COMPARED TO CO-CULTURE WITH NON-PARENCHYMAL STROMAL CELLS Petter Svanberg, Britta Bonn, Malin Darnell, Ia Hultman, and Ken Grime RIA iMed DMPK, AstraZeneca R&D, Mölndal, Sweden Drugs with low volumes of distribution, must have low clearance in order to drive a suitable elimination half-life required for a once daily drug. In Drug Discovery this requires definition of intrinsic clearance (CLint) values of less than 1µL/min/million human hepatocytes(1), which is challenging since it relates to a halflife of the drug of 12 hours in hepatocytes CLint assays. Drug metabolising enzymes (DMEs) activity decline to low levels already after 4 hours in suspended hepatocytes incubations(2). Commercially available platable multi-donor primary human hepatocytes pools cultured as a monoculture in collagenI-coated plates have shown low decline of activity for important DMEs up to 8-12 hours of incubation(3) and HµREL Corporations novel co-culture of hepatocytes with stromal cells has shown sustained activity of DMEs up to 2-3 weeks in culture(4). Both systems using plated hepatocytes can therefor supply lower CLint values than suspended hepatocytes systems due to the possibility for longer incubations, and thereby potentially provide better defined CLint and improved prediction of in vivo low clearance compounds. For comparison of the the two plated systems CLint values were defined and scaled to human in vivo clearance for a set of low turnover compounds (diazepam, disopyramide, theophylline, antipyrine, S-warfarin, metoprolol, timolol, AZ1, AZ2, AZ3 and AZ4). Changes in enzyme activity during the incubation timespan were also monitored by determination of enzyme-specific rate of metabolite formation for important drug metabolising enzymes (CYP3A, CYP2C9, CYP2D6, CYP1A2 and UGTs). Same pool of platable primary hepatocytes were tested in both systems. The results showed that both hepatocytes plated in monoculture and in co-culture can robustly produce CLint values less than 1µL/min/million human hepatocytes with sustained DME activity. When comparing the two systems, the robustness in defining low CLint values, in vivo predictability for the test compounds and differences in practicalities and cost are identifiable. From such a strength / weakness analysis, the use of each of the two systems in defining CLint for more metabolically stable compounds in Drug Discovery is proposed, with reference to suspended primary hepatocytes assays.

(1) Kenneth H. Grime, Patrick Barton, and Dermot F. McGinnity Application of In Silico, In Vitro and Preclinical Pharmacokinetic Data for the Effective and Efficient Prediction of Human Pharmacokinetics Molecular Pharmaceutics, 10 (4), pp 1191–12062, 2013 (2) Cornelia M. Smith, Christina K. Nolan, Manda A. Edwards, Jean B. Hatfield, Todd W. Stewart, Stephen S. Ferguson, Edward L. Lecluyse, Jasminder Sahi A comprehensive evaluation of metabolic activity and intrinsic clearance in suspensions and monolayer cultures of cryopreserved primary human hepatocytes Journal of Pharmaceutical Sciences, 101 (10), pp 3989–4002, 2012 (3) Petter Svanberg, Britta Bonn, Annika Janefeldt, Kajsa Kanebratt, Ia Hultman, Paul Courtney, Anshul Gupta, Ken Grime Determination of Low Intrinsic Clearance Values using Primary Human Hepatocytes and the HepaRG® Cell Line - A Comparison of Methods Poster at MDO symposium, Stuttgart, 2014.

Link: http://www.xenotechllc.com/posters/2014/intrinsic-clearance-values-az.pdf (4) Information about HµREL Corporations co-cultures, Link: http://hurelcorp.com/products-services/ P15 - PREDICTION OF PASSIVE RENAL TUBULAR REABSORPTION USING A MINIMAL PHYSIOLOGICALLYBASED MODEL 1 2 1 1 Daniel Scotcher , Christopher Jones , Amin Rostami-Hodjegan , and Aleksandra Galetin 1 Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, 2 United Kingdom, Oncology iMed, AstraZeneca, Alderley Park, United Kingdom A translational gap currently exists between in vitro data and human renal excretion clearance (CLR) in vivo. In this study, a minimal physiologically-based model was applied to predict fraction of tubular reabsorption (Freab) and nonsecretion CLR (CLR,Non-sec) using apparent permeability data (Papp) generated in Caco-2 monolayers under pH gradient conditions (6.5 vs. 7.4). CLR,Non-sec and fraction unbound in plasma (fu,p) values for 45 drugs were obtained from collated clinical studies, assuming that secretion was negligible for drugs with a ratio of total CLR/estimated glomerular filtration < 1.5. The drugs in the database represented a range of therapeutic classes and physical chemical properties; CLR values ranged from 0.02 to 145 mL/min for isoxicam and atenolol, respectively. The developed physiologically-based model was used to predict Freab in proximal tubule, loop of Henle, distal tubule and collecting duct compartments, accounting for water reabsorption and differences in tubular flow rate. The impact of correcting for microvilli-related surface area in each section of the renal tubule on the performance of the model was assessed. Assuming that glomerular filtration (CLR,filt) was the only contributing mechanism to CLR resulted in poor prediction of CLR (4-fold bias). Mechanistic model without correction for microvilli resulted in over-prediction of Freab

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(outside Âą 20% of observed Freab) for 49 % of drugs in the dataset and a 5-fold bias for predicted CLR. Correcting for microvilli-related surface area improved overall in vitro-in vivo extrapolation (IVIVE) of Freab and resulted in overall good agreement between predicted and observed CLR (2-fold bias); however, under-prediction of CLR for extensively reabsorbed drugs (Freab>0.8) was evident. Calibration of Papp data with a set of reference drugs and implications of this empirical scaling factor for Freab on IVIVE of CLR is considered. The mechanistic model proposed represents a useful addition to the IVIVE toolbox for physiologically-based predictions of renal tubular reabsorption, which can accommodate and predict the effects of the pathophysiological changes of elements affecting renal re-absorption of drugs.

P16 - UTILIZATION OF LIVER MICROSOMES FOR PREDICTION OF HEPATIC INTRINSIC CLEARANCE OF MONOAMINE OXIDASE SUBSTRATE DRUGS IN HUMANS 1 2 1 1 2 1 Yusuke Masuo , Shushi Nagamori , Kazuki Hayashi , Noritaka Nakamichi , Yoshikatsu Kanai , and Yukio Kato 1 2 Faculty of Pharmacy, Kanazawa University, Kanazawa, Japan, Division of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan Quantitative prediction of hepatic intrinsic clearance in vivo (CLint,vivo) from in vitro data is essential in the research and development of therapeutic agents. Non-CYP enzymes have recently been recognized to be involved in drug metabolism, but relatively limited information on such prediction approach has been reported for the drugs metabolized by non-CYP enzymes, compared with those metabolized by cytochrome P450 (CYP). Monoamine oxidase (MAO) is mitochondrial enzyme that is not only involved in metabolism of neurotransmitters, but also a certain drug molecules. It would be therefore, adequate to use isolated mitochondria to quantitatively predict CLint,vivo for MAO substrates. Nevertheless, human liver microsomes (HLMs) are widely and usually used to assess metabolic clearance in humans at an early phase of drug development. In addition, the major metabolic enzyme, e.g., CYP or MAO is usually not identified for drug candidate compounds at the early phase of development. It is reported that microsomal fraction exhibits comparable MAO activity with isolated mitochondria. Therefore, alternative approaches may include utilization of HLMs and appropriate scaling factor (SF) for the prediction of CLint,vivo. The purpose of the present study was to examine the feasibility of the quantitative prediction using HLMs with an aim to quantitatively estimate the risk of over/under-prediction of CLint,vivo. Three MAO substrate drugs, phenylephrine, sumatriptan and rizatriptan, as well as four CYP substrates, were selected, and their disappearance during incubation with HLMs was measured. Hepatic intrinsic clearance in vitro (CLint,vitro) was calculated from the disappearance curve, the unbound fraction during incubation and a physiological SF. The CLint,vivo was calculated according to the reported pharmacokinetic data in humans. The CLint,vitro was found to be 14-20 times lower than reported CLint,vivo values, whereas the difference between CLint,vitro and CLint,vivo was at most 2-4 times for CYP substrates, suggesting a higher in vitro-in vivo discrepancy for MAO substrates. CLint,vitro was also estimated using human liver mitochondria, but still exhibited a large discrepancy from CLint,vivo. Overall, our results indicate that hepatic intrinsic clearance of MAO substrates can be semi-quantitatively predicted from in vitro data obtained with hepatic microsomes by using appropriate reference compound(s) to compensate for the large in vitro-in vivo discrepancy. Validation of this approach using other lots of HLMs is now performed.

P17 - A NOVEL APPROACH IN MEASURING PROTEIN S-GLUTATHIONYLATION IN STRESS RESPONSE SIGNALLING 1 1 2 1 1 1 David McGarry , Wenzhang Chen , Probir Chakravarty , Douglas Lamont , Roland Wolf , and Colin Henderson 1 2 University of Dundee, Dundee, United Kingdom, Cancer Research, London Research Institute, London, United Kingdom Posttranslational modifications of protein thiols are a significant feature of redox sensing that often leads to changes in protein function. Covalent binding of the antioxidant glutathione to protein thiols (protein S-glutathionylation) is a reversible mechanism that regulates sulfhydryl homeostasis in response to perturbations in cellular oxidation and nitrosation. Although regarded as a protective mechanism, the contradictory role of glutathione in disease susceptibility and high cellular abundance of glutathione found basally makes protein S-glutathionylation an important feature of redox regulation and cell function. The aim of this study was to provide a global analysis for the identification of S-glutathionylated proteins and determine the biological consequence of such modifications in vivo. Sglutathionylated proteins were isolated from murine livers and isobarically labelled with Tandem Mass Tags (TMTs), allowing for protein identification and quantification by nLC-MS/MS. From these data, proteomic and bioinformatics analysis found that S-glutathionylation regulated a number of biological processes including oxidative stress, hypoxia, proteolysis, but also had a significant role in energy metabolism and mitochondrial pathways. We have further extended our isobaric approach to allow for cysteine labelling and subsequent identification of the modified thiol sites through the use of iodoacetamide-tagged TMTs. Furthermore, we examined the consequence of protein Sglutathionylation in response to stress in vivo. Mice nulled for glutathione S-transferase Pi (GSTP) are resistant to acetaminophen (APAP)-induced hepatotoxicity; the mechanism of which involves an ability to regenerate hepatic glutathione levels and maintain higher levels of protein S-glutathionylation in response to APAP. Using our proteomic approach, we identified a significant increase in the number of S-glutathionylated proteins related to oxidative phosphorylation, respiratory complexes, drug metabolising enzymes and mitochondrial function in Gstp1/2-/- mice in response to APAP. We identified that the stress response chaperone, HSP90, and apoptotic initiator, caspase-8, are S-glutathionylated and provide data demonstrating the biological consequence of S-glutathionylation of these proteins.

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In summary, we provide data showing an in vivo physiological role for protein S-glutathionylation and demonstrate how this important posttranslational modification regulates sulfhydryl function in response to stress and toxicity.

P18 - EFFECT OF DONOR VARIABILITY AND CULTURE CONDITIONS ON PHASE II ACTIVITY IN HUMAN HEPATOCYTES 1 1 2 1 Shalenie P. den Braver-Sewradj , Michiel W. den Braver , Audrey Baze , Nico P.E. Vermeulen , Jan N.M. 1 2&3 1 Commandeur , Lysiane Richert , J. Chris Vos 1 2 3 VU University Amsterdam, The Netherlands, KaLy-Cell, Plobsheim, France, Université de Franche-Comté, Besançon, France Primary human hepatocytes are a commonly used in vitro tool to investigate drug metabolism and resulting cellular effects. Human hepatocytes can be cultured in monolayer, however due to the ease of handling and lower costs, incubations in suspension are often preferred. Metabolism by cytochrome P450 in both culture conditions has been thoroughly investigated.[1] However, phase II metabolism also has a significant effect on toxicity as various drugs are conjugated by these enzymes. UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) are the most important phase II enzymes, and catalyze the conjugation to uridine-5’-diphospho-α-D-glucuronic acid (UDPGA) and 3’-phosphoadenosine 5’-phosphosulphate (PAPS) respectively. UGT and SULT catalyzed conjugation makes the compound more hydrophilic and is therefore generally considered to be a detoxification reaction. There are however cases known where conjugation can lead to a more active compound or a highly reactive metabolite. Moreover, phase II reactions are subjected to high interindividual variability which greatly influences metabolic fate of xenobiotics.[2] UGT activity in human hepatocytes is generally assessed using general probe substrates such as 7-hydroxycoumarin and 4-methyl-umbelliferone. UGT enzymes however exist in a superfamily, and it has been shown that not all isoforms are involved in the metabolism of such probe substrates.[3] Therefore, in the present study multiple UGT and SULT substrates are incubated under different culture conditions and using hepatocytes from different donor preparations.

Our results show substrate-specific effects of culture conditions and donor origin on UGT and SULT metabolism, indicating significant isoform-specific variability of both enzyme families. Therefore, proper characterization of primary hepatocytes is essential to understand drug-related toxicity.

Acknowledgements: this project is funded by the MIP-DILI consortium (grant agreement n° 115336).

1. Griffin SJ, Houston JB. Prediction of in vitro intrinsic clearance from hepatocytes: comparison of suspensions and monolayer cultures. Drug Metab Dispos. 2005;33(1):115–20.

2. Liu W, Ramírez J, Gamazon ER, et al. Genetic factors affecting gene transcription and catalytic activity of UDPglucuronosyltransferases in human liver. Hum Mol Genet. 2014;23(20):5558–69.

3. Uchaipichat V, Mackenzie PI, Guo X-H, et al. Human udp-glucuronosyltransferases: isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid. Drug Metab Dispos. 2004;32(4):413–23.

P19 - METABOLISM OF C-1748, 1-NTROACRIDINE ANTITUMOR AGENT, VIA HUMAN UDPGLUCURONOSYLTRANSFERASES IN PANCREATIC CANCER CELL LINES AND ITS EFFECTS ON THE MODULATION OF UGT1A9 AND UGT2B7 ACTIVITY Anna Mróz, Barbara Borowa-Mazgaj, and Zofia Mazerska Department of Pharmaceutical Technology and Biochemistry, Chemical Faculty, Gdańsk University of Technology, Gdańsk, Poland The compound 9-(2’-hydroxyethylamino)-4-methyl-1-nitroacridine, belongs to a group of 1-nitroacridine agents developed in our department, and possesses strong cytotoxic activity in several cancer cell lines. It also showed significant antitumor activity in several prostate and colon carcinoma xenografts of nude mice. It is presently accepted to I phase of clinical studies. Our laboratory demonstrated previously that C-1748 is actively metabolized by P450 into three main red-ox products in HepG2 cells. The formation of those metabolites was strongly enhanced under hypoxic conditions. These studies indicate that C-1748 can be important therapeutic drug toward solid tumors. In the present studies we investigated the conjugative metabolism of C-1748 with human liver hepatic and intestinal microsomes as well as with recombinant isoenzymes of UGT1 and UGT2 family. We also aimed to know whether the studied compound modulates enzymatic activity of selected UGT isoenzymes. First, reactions with individual recombinant UGT isoforms were carried out. All reactions products were identified by HPLC with multidiode UV-Vis and the structures of glucuronides were rigorously identified by ESI-MS. We observed the formation only one product and confirmed that C-1748 was metabolized to the glucuronide which contained glucuronic acid on the aliphatic hydroxyl group. All microsomal preparations from human liver and intestine microsomes generated the conjugated metabolite in variable amounts. However, only UGT2B7 isoenzyme had the ability to catalyze this reaction. It is recognized that UGT2B7 is expressed both in hepatic and intestinal tissues. The metabolism was also studied in pancreatic cancer cell lines: Panc-1, MiaPaCa-2 and BxPC-3. Next, glucuronidation reactions were carried out with isoform-specific substrates of UGT1A1, 1A4, 1A9-1A10 and UGT2B7: 7-ethyl-10-hydroxycamptothecin, trifluoroperazine, 7-hydroxy-4(trifluoromethyl)-coumarin and epirubicin, respectively, in the presence and in the absence of C-1748. The results showed that C-1748 inhibited the recombinant UGT1A9 activity. When UGT2B7 was exposed to different concentrations of C-1748, its activity was inhibited at high concentrations (0.1, 0.2 mM), but significant activation was observed at lower concentration (0.01 mM) of C-1748. UGT1A9 was totally inhibited in all concentrations. In

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conclusion, we demonstrated here that C-1748 underwent UGT-mediated transformations into single glucuronide and that this compound and/or its metabolite are able to modulate UGT activity. Active II phase metabolism of C-1748 in tumor patients could result in the resistance to this drug action and indicates that drug-drug interactions should be considered in the combined therapy of this compound with other antitumor therapeutic agents.

P20 - A MULTIPLEX APPROACH TO INVESTIGATE DRUG INDUCED CHANGES IN P450 ENZYME GENE EXPRESSION Jason DeLoach and Mark Schwartz HTG Molecular Diagnostics, Inc., Tucson, Arizona, United States Drug metabolizing enzymes and transporter induction can result in clinically meaningful drug interactions. Therefore, it is important to identify potential drug interactions early in the drug development process. Both p450 gene and enzyme induction studies are helpful in identifying p450-inducing compounds. Measuring gene expression has traditionally relied upon RNA extraction from treated hepatocytes followed by RT-­‐qPCR. An alternative, potentially more efficient method for measuring gene induction in this setting is the multiplex HTG Edge quantitative nuclease protection assay (HTG Edge qNPA chemistry). To measure EC50 values as well as fold-­‐changes in response to treatment, three primary hepatocyte donor lots were utilized. These hepatocytes were dosed at sixteen different concentrations for 48 hours. Expression of P450 enzymes CYP1A2 (Rifampin), CYP2B6 (Phenobarbital), and CYP3A4 (Omeprazole) were measured using both HTG Edge chemistry and qPCR. Dose response curves were generated using fold changes calculated by dividing normalized gene expression of treated samples by gene expression of vehicle control treated cells. Concentration-­‐dependent changes in gene induction were observed for CYP1A2, CYP2B6, and CYP3A4 using either assay. The HTG Edge multiplex assay provided gene expression data in triplicate using 30,000 hepatocytes per well with high precision across three P450 enzymes. Our data support the use of multiplex HTG Edge chemistry as a more efficient alternative to qRT-­‐PCR for measuring p450 gene induction in hepatocytes.

P21 - ANTIOXIDANT ACTIVITIES OF PROPOLIS AND ITS BIOACTIVE COMPONENTS, AND THEIR EFFECTS ON CYP1A1 GENE EXPRESSION IN HT-29 ADENOCARCINOMA CELL LINE Deniz Irtem Kartal, Ahmet Altay, Gülsüm Yurteri, and N. Tülün Güray Middle East Technical University, Ankara, Turkey Propolis is a resinous mixture that is collected by honeybees from plants, and is combined with beeswax and secretions from the bee’s salivary glands plus some pollen. It is a rich mixture of polyphenols, flavonoid aglycones, phenolic acids and their esters. It has been used in traditional medicine for thousands of years because of these ingredients. Propolis, just like honey, has been the subject of many studies due to its antimicrobial, antifungal, antiviral and hepatoprotective activities. The cytochrome P450 enzymes are involved in phase I xenobiotic and drug metabolism. CYP1A1 is present in high levels in human tumors demonstrated by qRT-PCR and immunohistochemical studies. In this study, Propolis collected from Datça (Mugla,Turkey) is extracted with 70% ethanol. The main phenolic contents of the propolis, caffeic acid, p-Coumaric acid and quercetin,were measured by RP-HPLC. Antioxidant activities (DPPH● and ABTS●), total phenolic (TPC) and flavanoid contents (TFC) were determined sprectrophotometrically. Cytotoxic effects of the propolis on HT-29 human colon adenocarcinoma cell lines were examined via XTT colorimetric cell proliferation assay and trypan blue dye exclusion cell viability assay. Effects of propolis extract and bioactive components on the expression of phase I detoxification enzyme, CYP1A1 in HT-29 cells were investigated bu using q-RT-PCR technique. IC50 values for DPPH● and ABTS● radicals scavenging activities of the extract were calculated as 0,042 mg/mL and 80,7 mM TE/mg extract, respectively. TPC and TFC were determined as 168,6 mg GAE/g extract and 141,8 mg QE/g extract, respectively. IC50 value for XTT assay in 48 hr incubation with the extract was evaluated as 1,16 mg/mL. Propolis extract significantly induced CYP1A1 mRNA expression with a 1,7 fold increase in HT-29 human colorectal adenocarcinoma cells in 72 h, however there was no significant modulation in 48 hr incubation with the extract. Overall, these results indicate that propolis and/or its components have regulatory activities on CYP1A1 expression and they may have a potential as a therapeutic agent in the treatment of cancer.

P22 - DEFINING SPECIES DIFFERENCES IN THE METABOLISM OF THE EGFR INHIBITOR AZD9291 USING TRANSGENIC MODELS 1 1 2 1 Lesley McLaughlin , Colin Henderson , Nico Scheer , and Roland Wolf 1 2 University of Dundee, Dundee, United Kingdom, Taconic Biosciences, Cologne, Germany Epidermal growth factor (EGFR) has been a target for anti-cancer drug development since the approval of gefitinib for non-small-cell lung carcinoma (NSCLC). As with the majority of molecularly targeted therapies the emergence of resistance is a real problem. Several pharmaceutical companies are developing EGFR inhibitors which target mutant EGFR while sparing the wild-type form. AZD9291 is a mutant EGFR inhibitor which is currently in Phase I/II trials for NSCLC. It has been reported that in rats and mice in vivo three metabolic products are formed - N-demethylation of the indole group (metabolite 27), dealkylation of the N,N,N′-trimethylethylenediamine side chain (metabolite 28) and Noxidation of the side chain; metabolite 27 appears to be the major contributor to the potency of AZD9291.

Interestingly, the half-life of AZD9291, ~3h in mice in pre-clinical work, was found to be ~50h in humans in Phase I studies (D. Cross, Personal Communication). In order to establish the basis for this difference we have used our panel

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of cytochrome P450 (P450) knockout and humanised mouse models along with recombinant human and mouse P450s to define which P450s are responsible for the metabolism of AZD9291 in mouse and man. In vitro, the major AZD9291 metabolite formed in mouse liver microsomes (MLM) was the hydroxylated product, while in human liver microsomes (HLM) the major pathway was formation of the N-demethylated metabolite. MLM were found to be 2-fold and 30-fold more efficient than HLM in generating demethylated and oxidised AZD9291, respectively. MLM generated from C57/BL6 mice nulled individually for Cyp1a, Cyp2c, Cyp2d, Cyp3a, and collectively for Cyp2c/Cyp2dCyp3a, were used to dissect which P450 sub-families were responsible for metabolite production. Deletion of Cyp3a and Cyp2d caused a 23% and 10% reduction in AZD9291 demethylation, respectively. Deletion of the Cyp2c gene cluster caused a small increase in production of this metabolite and Cyp2c/Cyp2dCyp3a triple knockout mice produced only ~12% of the levels relative to wild-type MLM. We are currently investigating the explanation for this unexpected result.

In contrast, formation of the oxidised metabolite was completely ablated in the Cyp2d knock-out microsomes and was barely detectable in liver microsomes from Cyp2c/Cyp2dCyp3a mice. Using recombinant human P450s and Spearman’s rank correlation, the enzyme with the greatest contribution to formation of demethylated AZD9291 was found to be CYP3A4, with CYP1A1, CYP2C8 and CYP2C19 also involved. In summary, oxidation of AZD9291, mediated by Cyp2d enzymes, appears to be the major pathway of disposition in mice, while the enzymes responsible for N-demethylation remain undefined. In humans, N-demethylation of AZD9291 appears to be the predominant metabolic route, carried out mainly by CYP3A4 but with contributions from other P450s, including CYP2C8.

P23 - DISCOVERY OF A HIGHLY SELECTIVE PROBE FOR HUMAN CYTOCHROME P450 3A5 Jingjing Wu, Guangbo Ge, and Ling Yang Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Human cytochrome P450 (CYP) 3A4 and CYP3A5 exhibit overlapping substrate specificity, and there is currently no specific CYP3A5 probe to differentiate the activity of CYP3A4 and CYP3A5 in biological samples. In this study, we reported Schisantherin E (SE) with a dibenzocyclooctadiene skeleton as an isoform-specific probe for CYP3A5. Phase I metabolism of SE yielded a predominant demethylated metabolite in mixed human liver microsomes (HLM) with a NADPH-generating system. Both reaction phenotyping and chemical inhibition assays revealed that CYP3A5 demonstrated a good ability to catalyze the demethylation of SE, while CYP3A4 displayed very limited activities to catalyze this biotransformation and CYP3A7 did not play a catalytic role. Kinetic characterization revealed that SE demethylation in both CYP3A5 and HLM followed the Michaelis-Menten kinetics. Intrinsic clearance values indicated that CYP3A5 contributed more than 20-fold higher than CYP3A4 on SE demethylation (10.7 μl/min/pmol CYP vs. 0.46 μl/min/pmol CYP). The CYP3A5 activities which were detected by SE demethylation as the probe reaction in fourteen individual HLM ranged from 8.68 pmol/min/mg to 158.7 pmol/min/mg with a 60.2% coefficient of variability. Strong correlations were obtained for the formation rate of demethylated SE with the content of CYP3A5 in fourteen individual HLM (r = 0.970, P < 0.0001). In addition, the results of molecular modeling also indicated that SE was a good substrate of CYP3A5 but a poor substrate of CYP3A4. In summary, SE is an attractive candidate serving as a highlyselective probe of CYP3A5, which might be used to characterize the real functions of CYP3A5 in various biological samples.

P24 - KINETIC PARAMETERS (KM AND KCAT) OF CYTOCHROMES P450: A COMPREHENSIVE SURVEY REVEALS A ROLE FOR SPECIFIC INTERACTIONS AS WELL AS LIPOPHILICITY IN SUBSTRATE AND TRANSITION STATE RECOGNITION. 1 1 2 Stephen J. Messham , Andrew G. Leach , and Nathan Kidley 1 2 Liverpool John Moores University, Liverpool, United Kingdom, Syngenta, Reading, United Kingdom A number of literature sources when taken together suggest that substrate recognition by cytochromes P450 (CYP) is more strongly influenced by the properties of the substrates than is reversible inhibition. [1,2] The kinetic parameters Km and kcat are often interpreted in a way that relates to the strength of binding to the reaction transition state and to the strength of binding of the pre-reaction reversible complex (the Michaelis-Menten complex). [3,4] All of the available literature was searched for measured values of Km and kcat for a range of CYP substrates. The CYPs involved in processing these include the human isoforms most relevant to drug metabolism (1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4) and BM3 from Bacillus megaterium. The physical properties of the substrates were calculated and compared to the observed kinetic parameters. Docking of the substrates into available crystal structures of the cytochromes P450 was also performed and the docking scores compared to the observed kinetic parameters.

Previous studies investigating the link between physical properties and both CYP inhibition and metabolism were also re-examined in order to understand the observed trends. These included metabolism in isolated microsomes, hepatocytes or in vivo.[1] Our results suggest that the property dependence of the substrate binding strength (either 1/KM or kcat/KM) for the different isoforms differs markedly and that specific molecular recognition events, as modelled by docking, often play a more important role in substrate and transition state recognition than do the physical properties of the substrates. Our results make it clear that BM3 is qualitatively and quantitatively different from the human isoforms: there is a stronger dependence of kcat/KM on physical properties and on docking scores for BM3 than is found for the other isoforms. The strongest dependency on logP for the human isoforms is found for kcat/KM in the 3A4 isoform. The same isoform is also the one for which the best rationalisation of activity by the protein structure (as assessed by docking scores) is found. Detailed data and explanations will be provided.

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References: 1. Waring, M. J. Lipophilicity in drug discovery. Expert Opin. Drug Discovery 2010, 5, 235-248.

2. Gleeson, M. P. Generation of a set of simple, interpretable ADMET rules of thumb. J Med Chem 2008, 51, 817-834.

3. Fersht, A. Structure and Mechanism in Protein Science; W. H. Freeman and Co.: New York, 1999; 4. Houk, K. N.; Leach, A. G.; Kim, S. P.; Zhang, X. Binding affinities of host-guest, protein-ligand, and proteintransition-state complexes. Angew. Chem. , Int. Ed. 2003, 42, 4872-4897.

P25 - LITTLE EFFECT OF SOLIDAGO VIRGAUREA L. EXTRACT ON PHASE I. BIOTRANSFORMATION ENZYMES IN HUMAN HEPATOCYTES AND HUMAN LIVER MICROSOMES Veronika Tománková, Jitka Ulrichová, Eva Anzenbacherová, and Pavel Anzenbacher Faculty of Medicine and Dentistry, Palacky University, Department of Medical Chemistry and Biochemistry, Olomouc, Czech Republic Solidago virgaurea L. “goldenrod” is a medicinal plant known since ancient times [1,2]. The genus Solidago is not only interesting from the viewpoint of biological effects. In addition, it contains many interesting secondary metabolites – flavonoids, phenolic acids and glucosides, polysaccharides and others [3]. Solidago virgaurea L. is known to have diuretic, antiinflammatory and newly described antitumoral, antimicrobial, sedative and hypotensive effects [1,2]. The effect on diuresis is attributed especially to flavonoids and to a phenolic ester glucoside – leiocarposide [3]. Presented study was focused on finding whether Solidago virgaurea L. extract influences phase I. biotransformation enzymes, namely cytochromes P450 (CYPs) in human hepatocytes and also in human liver microsomes. For experimental study Solidago virgaurea L. extract was used. This study was focused on properties of cytochromes P450 (CYP) in human hepatocytes, namely, on CYP protein expression and enzyme activities of CYPs. Five CYP enzyme activities with prototypical substrates were analyzed, namely, CYP1A2 (substrate phenacetin), CYP2A6 (substrate coumarin), CYP2C9 (substrate warfarin), CYP2D6 (substrate bufuralol) and CYP3A4 (substrate testosterone) were selected for measurement of enzyme activities. Enzyme activities were determined in human hepatocytes as well as in human liver microsomes. The effect of Solidago extract was however rather small. On the other hand, because of the fact that dietary supplements can interact with commonly used drugs, further studies on possible influence of Solidago virgaurea L. extract on biotransformation enzymes are needed.

[1] Kalemba D.; Constituents of the essential oil of Solidago virgaurea L.; Flavour Fragr. J., 13, 373-376 (1998) [2] Luciana D., Antonescu A., Zdrinca M., Muresan M., Vicas L., Micle O., Vicas S.; HPLC-MS analysis of flavonoids obtained from Solidago sp. (asteraceae); Analele Universitatii din Oradea, Fascicula Protectia Mediului, 19, 75-79 (2012) [3] Thiem B., Wesolowska M., Skrzypczak L., Budzianowski J.; Phenolic compounds in two Solidago L. species from in vitro culture; Acta Poloniae Pharmaceutica, 58, 277-281, (2001) Acknowledgements: We gratefully acknowledge grants no. GACR 303/12/G163 and student’s project LF_2014_014 for financial support to this work.

P26 - MEDICINAL PLANTS EPILOBIUM HIRSUTUM L . AND VISCUM ALBUM L. ALTER PROTEIN AND MRNA EXPRESSIONS OF RAT LIVER BILE ACID SYNTHESIZING CYP7A1 1 2 3 1 Tuba Culcu , Ayşe Mine Gençler Özkan , Alaatin Şen , and Orhan Adalı 1 Middle East Technical University, Department of Biological Sciences and Joint Graduate Program in Biochemistry, 2 Cankaya-Ankara, Turkey, Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Ankara, 3 Turkey, Pamukkale University, Faculty of Sciences, Department of Biology, Denizli, Turkey Epilobium hirsutum L. is known as its analgesic, anti-microbial and anti-proliferative activity, and it is used in our country as an alternative medicine. The pharmacological effect of Epilobium hirsutum L. could be explained by the presence of polyphenolics including steroids, tannins and flavonoids in the aerial parts.

Viscum album L. is a common bushy plant of the family Viscaceae, which grows as epiphyte on the branches of deciduous trees. The plant contains a great variety of compounds including lectins, viscotoxin, polysaccharides, flavonoids, phenylpropane derivates, triterpenoids and phytosterols. The use of Viscum album in the treatment of breast, lung and liver cancers and cardiovascular diseases indicates pharmaceutical importance of this plant.

In mammals, excess cholesterol is removed mainly through conversion to bile acids. Cytochromes P450s (CYPs) initiate all quantitatively significant pathways of cholesterol metabolism and bile acid biosynthesis. There are two pathways known as the classical pathway and the acidic pathway involved in bile acid synthesis. Classical pathway is initiated by CYP7A1. In this study, the possible potency of medicinal plants Epilobium hirsutum L. and Viscum album L.extracts and their major polyphenolic ingrediends, ellagic acidand o-coumarin (2-hydroxycinnamic acid) on rat liver cholesterol metabolizing CYP7A1 was investigated. The water extracts of Epilobium hirsutum L., Viscum album L., ellagic acid and coumaric acid were injected i.p. as 37.5 mg/kg, 10 mg/kg, 20mg/kg and 30mg/kg for 9 days, respectively. In vivo effects of Epilobium hirsutum L., Viscum album L., ellagic acid and coumaric acid on rat liver CYP7A1 were analyzed by determining protein and mRNA expression levels using western blotting and qPCR techniques, respectively. Protein expression of CYP7A1 decreased 60%, 86%, 70% and 76% when rats injected with Epilobium hirsutum L., Viscum album L., ellagic acid and coumaric acid, respectively. mRNA expression of CYP7A1 decreased 47% and 90% by Epilobium hirsutum L. and Viscum album L., respectively, with respect to controls and

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normalized with GAPDH expression as an internal reference. However, injection of ellagic acid and coumaric acid, caused an increase of 58% and 83%, respectively. In conclusion, bile acid synhesis catalyzed by liver CYP7A1 may be altered due to the changes in mRNA and protein expressions caused by Epilobium hirsutum L.and Viscum album L. injection to rats.

P27 - POSSIBLE MECHANISM(S) CAUSING INVERSE ENANTIOSELECTIVITY IN BUNITROLOL 4HYDROXYLATION BY CYP2D ENZYMES 1 1 2 Shizuo Narimatsu , Takahiro Matsumoto , and Kazufumi Masuda 1 2 Okayama University, Okayama, Japan, Shujitsu University, Okayama, Japan Bunitrolol (BTL) is a β-adrenoceptor blocking agent. It has an asymmetric carbon atom at the side chain, yielding enantiomers, R-BTL and S-BTL. The major metabolic pathway is known to be 4-hydroxylation of the phenyl ring, which is mainly mediated by CYP2D6 in a low substrate concentration range. We previously reported that the enantioselectivity in BTL 4-hydroxylation was different between CYP2D6 wild type having valine-374 (R-BTL>S-BTL) and its mutant having methionine-374 (S-BTL>R-BTL) [1]. In this study using AutoDock Tools (ADT, Ver. 1.5.4) and Swiss PDB viewer (Ver. 4.1), we propose a simple explanation for the inverse exnantioselectivity via possible interactions between BTL enantiomers and amino acid residues existing within the active-site cavity of the enzymes.

The crystallographic data of CYP2D6 (PDB ID, 2F9Q) was used as the wild type, and valine at position 374 was replaced with methionine. Hydrogen atoms were added to the model using the Biopolymer module of Insight II software (Molecular Simulation Inc.). A heme moiety and six peptides as substrate recognition sites (SRSs) were extracted; from 101 to 123 as SRS1, 205 to 223 as SRS2, 236 to 247 as SRS3, 296 to 311 as SRS4, 367 to 376 as SRS5 and 479 to 485 as SRS6. Energy optimization of the models was performed using Insight II/Discover and the active site models were drawn using RasMol v2.6-ucb-1.0. The structures of BTL enantiomers were made using Winmoster Ver. 3. From a number of clusters obtained, we chose four to six clusters having the mean binding energy ranging from −8.5 to −7.1 kcal/mol for each situation. Furthermore, clusters in which the distance between the oxidation site of BTL and the heme-iron is not within 6 Å were excluded. As results, the interaction between R-BTL and CYP2D6 wild type protein gave cluster ranks No. 1 and 2, having relatively high cluster numbers, in which the distance between the oxidation site of BTL and the heme iron is 3.8 to 4.8 Å. In the interaction between S-BTL and the same protein, only cluster rank No. 3, having relatively low cluster number, in which the heme iron is 3.9 Å apart from the oxidation site of the substrate was obtained as an appropriate model. A reverse tendency was observed when CYP2D6-Met374 was employed as protein. These results indicate that R-BTL is a more favorable substrate than S-BTL for CYP2D6-Val374, while S-BTL is more favorable than R-BTL for CYP2D6-Met374. Results on rat CYP2D2, showing inverse BTL enantioselectivity to that of CYP2D6 wild-type, will also be presented, and possible mechanism(s) causing the inversion of enantioselectivity will be discussed. [1] S. Narimatsu et al., Chirality, 11, 1-9 (1999).

P28 - SUBSTANTIAL INHIBITORY EFFECT OF DMSO ON IN VITRO INTRINSIC CLEARANCE MEASUREMENT – ROLE OF CYP3A4?

Hugues Chanteux, Maria Rosa, Julia Lopes, Nathalie Latour, Melanie Golding, Robert Barnaby, Sylvie Dell'aiera, Claude Delatour, and Anna-Lena Ungell UCB Biopharma, Braine L'Alleud, Belgium Dimethylsulfoxide (DMSO) is the organic solvent of choice in drug discovery to solubilize chemical libraries that will be tested in high throughput screening in ADME and pharmacology. However, DMSO is known to inhibit some CYP450s even though the extent of inhibition (less than 40% at 0.5% DMSO) is acceptable for metabolic stability screening where discrimination between high, moderate and low turn-over compounds is required. Within the framework of a drug discovery project in the lead optimization stage, we first discovered that one compound (tested at 0.5µM) demonstrated, in human hepatocytes, a 10-fold higher in vitro intrinsic clearance (CLint) in presence of 1% acetonitrile (ACN) compared to 0.5% DMSO. The effect of DMSO was confirmed by measuring the CLint in presence of different concentration of DMSO (0.5% to 0.025%). At the lowest concentration tested (0.025%), DMSO still showed some inhibition (35%) compared to ACN. In order to better characterize this unexpected finding, other compounds from the same chemical series were selected in order to check whether DMSO would affect systematically the CLint measurement. To this aim, CLint was measured by parent drug disappearance in human hepatocytes (1 million cells/mL) and in human liver microsomes (1mg/mL). The results showed that a significant DMSO effect (>5-fold difference with ACN) was observed with other compounds (not all of them) as well as in another in vitro test system (human liver microsomes). Thereafter, the CLint of two compounds showing a major effect of DMSO (~10-fold) and being also fairly soluble, was also measured in human hepatocytes containing no organic solvent and clearly demonstrated the inhibitory effect of DMSO as the CLint measured in absence of organic solvent was similar to the one obtained with 1% ACN. In addition, the involvement of CYP3A4 in metabolic clearance of these compounds was assessed in hepatocytes treated with azamulin (3µM), a specific CYP3A4 inhibitor. The results showed that all the compounds demonstrating a major effect of DMSO have also major CYP3A4 involvement (>70%) in their metabolic clearance. For the sake of comparison, the effect of DMSO on the CLint of different compounds known to be metabolized partially or totally by CYP3A4 (midazolam, felodipine, simvastatin, cerivastatin, quinidine, sildenafil, nifedipine, omeprazole, pimozide and zolpidem) was also measured. As expected, DMSO (0.5%) has only a slight effect (less than 30%) on the CLint of all marketed drug, except pimozide which demonstrated also a significant effect

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of DMSO (>6-fold decrease compared to 1% ACN). Overall, the data clearly demonstrated that DMSO had a significant inhibitory effect on the metabolic clearance of a series of compounds belonging to the same chemical family. This effect is compound specific as no correlation have been identified so far, with chemical structure or other molecular descriptors. Currently, the only hypothesis is linked to the involvement of CYP3A4 and further investigations are warranted to decipher the mechanisms responsible for this substantial inhibition.

P29 - APPLICATION OF A MOUSE LINE HUMANISED FOR HPXR/CAR/CYP3A4/CYP3A7/CYP2D6/CYP2C9 TO INVESTIGATE THE REVERSIBLE AND TIME-DEPENDENT INHIBITION OF MIDAZOLAM 1’-HYDROXYLATION 1 2 2 1 1 1 Yury Kapelyukh , Nico Scheer , Anja Rode , De Lin , Colin Henderson , and Roland Wolf 1 2 University of Dundee, Dundee, United Kingdom, Taconic Biosciences, Cologne, Germany Accurate prediction of clearance and the magnitude of potential drug-drug interactions in patients are important objectives in drug development and use. Both extrapolation from in vitro data and interspecies scaling using pharmacokinetics of drug candidates in preclinical species demonstrate a similar level of predictive accuracy. The latter approach is affected by differences between rodents and man in catalytic efficacy and/or substrate specificity of drug metabolising enzymes. For example, the mouse Cyp2c cytochrome P450 subfamily plays a major role in midazolam 1’-hydroxylation in mice. However more than 90% of midazolam in humans is eliminated by CYP3A4.

Murine Cyp2c enzymes have a much lower sensitivity than CYP3A4 to the CYP3A4-specific inhibitor ketoconazole.

This can result in an underestimation of the capacity of new chemical entities to inhibit CYP3A4 if assessment is carried out in mice. In order to develop a more predictive model for estimating drug/drug interactions in man we have now generated a complex humanised mouse line where all the murine Cyp3a, Cyp2d and Cyp2c genes (except Cyp2c44) have been replaced by human CYP3A4/3A7, CYP2D6 and CYP2C9 respectively. The impact of nonCYP3A4 enzymes on midazolam 1’-hydroxylation in this humanised mouse line was estimated by studying ketoconazole inhibition kinetics. The Km values for midazolam 1’-hydroxylation and inhibition type and parameters of interaction with ketoconazole were determined for human CYP3A4 and murine Cyp3a, Cyp2c and non-Cyp3a enzymes using liver microsomes from human donors and Cyp2c, Cyp3a and Cyp2c/Cyp2d/Cyp3a KO mice, respectively. The generated kinetic parameters were used to establish the relative contribution of the different murine or human P-450s to midazolam hydroxylation in wild type, hPXR/CAR/CYP3A4/3A7 and hPXR/CAR/CYP3A4/CYP2D6/CYP2C9 mouse liver microsomes by analysis of the dependency of the reaction rate on substrate and inhibitor concentrations as a sum of the two corresponding individual kinetic components. Vmax values of each component were calculated by simultaneous non-linear regression. For wild type and hPXR/hCAR/CYP3A4/3A7 mice the contribution of Cyp2c enzymes to midazolam 1’-hydroxylation was estimated to be ~30%. For microsomes from hPXR/CAR/CYP3A4/CYP2D6/CYP2C9 mice a single enzyme non-competitive inhibition was statistically a better fit compared to the two components system, suggesting a minimal contribution of the non-CYP3A4 component to midazolam 1’-hydroxylation in this model These data were in good agreement with the markedly increased sensitivity of microsomes from hPXR/hCAR/CYP3A4/CYP2D6/CYP2C9 mice to the timedependent inhibitor erythromycin, and illustrate the utility of this model as a powerful adjunct to existing approaches for predicting drug/drug interactions in man.

P30 - DIFFERENCES OF BETAHISTINE PHARMACOKINETICS AND METABOLISM IN DOG, CAT AND MAN Matthias Olbrich Abbott Laboratories, Hannover, Germany Betahistine is a histamine analog and has been used for over 40 years in the symptomatic treatment of Ménière’s disease and vestibular vertigo. There are limited data available about pharmacokinetics and metabolism of betahistine. Pharmacokinetic profiles in dog, cat and man revealed strong differences in betahistine exposure following administration of the same dose. Betahistine undergoes a fast absorption and nearly complete metabolism by monoamine oxidases (MAO) in man. In vitro permeation assays demonstrated good passive diffusion across cell monolayers with no involvement of transporters. Absorption is assumed to be comparable between species.

Consequently, exposure differences could be related to differences in metabolism in dog, cat and man. Betahistine metabolism was studied in incubations with human recombinant expressed MAO-A or MAO-B enzymes, male human, dog or cat hepatocytes, intestinal homogenate and blood. Decrease of betahistine as well as appearance of the major metabolite 2-pyridyl acetic acid was monitored. Data suggest that betahistine is metabolised by both human MAO-A and MAO B however there is a species specific contribution of MAO-A and MAO-B to the metabolism of betahistine. In man and dog betahistine was metabolized by MAO-A in the intestine and by MAO-B in the liver. In the cat betahistine was nearly exclusively metabolized by MAO-B in both liver and intestine. Minimal metabolism of betahistine was observed in blood of the two animal species and man. The appearance of the major metabolite 2-pyridylacetic acid was observed in incubations with liver. Only minimal levels of 2-pyridylacetic acid were observed in incubationswith recombinant expressed enzymes, intestine homogenate or blood. Most likely this is due to the absence of the enzyme responsible for conversion of the intermediate aldehyde metabolite to the acid metabolite. Summarizing our results, it is suggested that the differences in exposure, observed in the two animal species and in man, are mainly due to different metabolism of betahistine in dog, cat and man.

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P31 - FORMATION OF THE CARBOXYLIC ACID METABOLITE OF AZD5069 Anja Ekdahl, Anna-Pia Palmgren, Richard A. Thompson, and Ken Grime RIA iMed DMPK, AstraZeneca R&D, Mölndal, Sweden AZD5069 is a selective and reversible oral chemokine receptor 2 (CXCR2) antagonist in development for the treatment of inflammatory disease. AZD5069 is an azetidinesulfonamide compound with an aliphatic side chain containing a terminal alcohol. The major circulating metabolite of AZD5069 in clinical studies was found to be the carboxylic acid of the aliphatic alcohol. The pre-cursor aldehyde was also found to be a circulating metabolite. In in vitro incubations of AZD5069 in human hepatocytes the carboxylic acid was detected as a minor metabolite. Here we present in vitro data on the formation mechanisms of the carboxylic acid metabolite of AZD5069 in order to understand the high exposure levels of the metabolite in humans. We used a close analogue of AZD5069 for the investigation and the metabolites were detected using LC-MS. The aldehyde and carboxylic acid were found in incubations in human hepatocytes but neither of them was found in incubations using dog and rat hepatocytes.

Heterologously expressed human CYP2C9 was capable of forming the aldehyde and the acid metabolite and aldehyde oxidase (AO) was capable of forming the acid from the corresponding aldehyde, but not the aldehyde from the alcohol. A surprising limitation of the available in vitro drug metabolising systems was that in human liver microsomes (HLM) the aldehyde but not the acid metabolite was formed, despite the presence of functional CYP2C9.

CYPs 2C8, 3A4 and 3A5 were also found to metabolise AZD5069 and its analogue, but not to the aldehyde or acid metabolites. Interestingly a cocktail of these CYPs allowed formation of the aldehyde and acid metabolites but when CYP2C9 was co-incubated with HLM neither aldehyde nor carboxylic acid were formed. These studies demonstrate that CYPs can metabolise the aliphatic alcohol to the aldehyde and to the acid, but do not explain why the acid is not formed in HLM. However since AO was shown to oxidise the aldehyde to the acid, species differences in AO activity could explain the unexpected high plasma levels in humans of the carboxylic acid of AZD5069.

P32 - IN-SITU METABOLITE PROFILING OF REMODELED ARTERIES IN PULMONARY ARTERIAL HYPERTENSION USING INNOVATIVE MASS SPECTROMETRY IMAGING TOOLS 1 2 2 2 2 2 Sébastien J. Dumas , Gregory Hamm , Raphael Legouffe , David Bonnel , Gaël Picard de Muller Fabien Pamelard , 1 1 Marc Humbert , and Peter Dorfmüller 1 2 INSERM UMR-S 999, Univ Paris-Sud, LabEx LERMIT, Le Plessis Robinson, France, ImaBiotech, Loos, France Introduction: Pulmonary arterial hypertension (PAH) is associated to increased pulmonary arterial pressure, resulting in right heart failure and death. Understanding vascular remodeling pathways is a challenge for discovering novel therapeutic and diagnostic tools. Direct in-situ metabolite profiling of pulmonary arteries using mass spectrometry imaging (MSI) combined with the MultimagingTM analysis tool could be a pertinent strategy to better understand PAH pathways. Aims: 1) In-situ detection and quantification of metabolites in pulmonary arteries in order to determine the molecular signature of vascular remodeling. 2) Assessment of MultimagingTM in the context of multiple comparisons of complex tissues. Methods: Human frozen lung samples from the tissue bank of the French Referral Center for PAH were cut in 10µm-thick sections and mounted onto ITO conductive slides. Analysis was performed with a SolariX FTICR 7.0T Mass Spectrometer (Bruker Daltonics) at a 30 µm spatial resolution combined to the MultimagingTM analysis tool, allowing selection of areas containing vessels using contrast ions, and automated analysis of metabolites. Results: Several metabolites showed differential levels between healthy and PAH samples with some m/z signals remaining to be characterized. Typical molecular signatures of the different types of vascular lesions were highlighted. For example, cADP (Cyclic ADP) ribose (key mediator of hypoxic pulmonary vasoconstriction) and ADP ribose, two metabolites belonging to the same pathway, were overexpressed in PAH lesions. Conclusion: MSI combined with MultimagingTM allowed the discovery of molecular signature of vascular remodeling in PAH. This strategy could open new avenues in understanding complex diseases on the basis of multiple tissue comparison.

P33 - APPLICATION OF A MULTI-COMPARTMENT PERMEABILITY LIMITED LUNG MODEL TO PREDICT LUNG CONCENTRATIONS OF ANTI-TUBERCULOSIS DRUGS IN VIRTUAL HUMAN SUBJECTS 1 1 1 1 1 2 2 Gaohua Lu , Iain Gardner , Janak Wedagedera , Ben Small , Lisa Almond , Klaus Romero , Debra Hanna , and 1 Masoud Jamei 1 2 Simcyp, Ltd., Sheffield, United Kingdom, Critical Path Institute, Tucson, Arizona, United States Tuberculosis (TB) remains a major global health problem. According to WHO reports, an estimated 8.6 million people developed TB and 1.3 million died from the disease in 2012 [1]. Current therapies for pulmonary TB use combinations of orally dosed drugs that need to achieve adequate concentrations in the lungs of infected individuals to achieve therapeutic benefit. The ability to predict lung concentration of anti-TB drugs primarily from in vitro experiments would be of great benefit in screening new drug candidates and designing appropriate dosing regimens for novel TB drugs.

As a first step in this process the aim of this study was to develop a physiologically-based pharmacokinetic (PBPK) model to predict the concentration of different anti-TB drugs in the human lung. A multi-compartment PBPK model where the lung airways and lobes were represented by a total of 7 compartments was developed and implemented in the Simcyp simulator (V14 R1). Each of the 7 compartments was sub-divided into compartments representing the pulmonary capillary blood, pulmonary tissue mass, epithelial lining fluid and alveolar air. The PBPK model allows the possibility to account for both passive and active movement of drugs between the sub-compartments. For each of the drugs simulated in the PBPK model (Rifampicin, Pyrazinamide, Ethambutol and Isoniazid) the movement of drug

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within the compartments of the PBPK model was predicted using in vitro permeability data in Calu-3 or Caco-2 cell lines together with in vitro-in vivo extrapolation procedures. The concentrations predicted to occur in the lung epithelial lining fluid (ELF) and the ELF:plasma ratio were compared to reported measured values from human clinical studies [2-6]. The simulated ELF:plasma ratio in virtual subjects dosed with rifampicin (simulated 0.10-0.17, observed 0.030.45) and isoniazid (simulated 0.93 to 1.1, observed 1.2 +/-1.9 in fast acetyators and 3.2+/- 8.1 in slow acetylators) showed overlap with the reported clinical data. For ethambutol the simulated ELF:plasma ratio were of a similar magnitude to (0.4-3.5) the observed clinical values (0.8-1.3) but showed marked inter-individual variability. The simulated ELF:plasma ratio for pyrazinamide was significantly underpredicted (simulated 0.88 to 0.93, observed 1122). Sensitivity analyses were conducted and showed that altering the pH of the epithelial lining fluid in the lung PBPK model or incorporating the effect of an efflux transporter between the lung tissue and the ELF resulted in pyrazinamide ELF:plasma concentration ratios that matched those observed clinically. The described lung PBPK model has shown some utility in predicting the lung pharmacokinetics of anti-TB drugs and testing of the model with a wider range of compounds is underway. For some compounds assuming purely passive diffusion between the plasma and ELF results in underprediction of the lung fluid concentrations and for these compounds additional processes (eg drug transporters) may be involved.

References [1] World-Health-Organization (2013) http://www.who.int/tb/publications/global_report/en/.

[2] Goutelle et al (2009) Antimicrob Agents Chemother 53:2974-2981.

[3] Conte et al. (1999) Antimicrob Agents Chemother 43:1329-1333.

[4] Conte et al (2001) Antimicrob Agents Chemother 45:2891-2896.

[5] Conte et al (2002) Antimicrob Agents Chemother 46:2358-2364.

[6] Katiyar et al (2008) J Postgrad Med 54:245-246.

P34 - DICLOFENAC: BIOCONCENTRATION, TISSUE DISTRIBUTION AND METABOLISM IN FISH Andrew McEwen, Adam Woods, Claire Henson, Lucy Gilhooly, and Stuart Wood Quotient Bioresearch Limited, Rushden, United Kingdom Diclofenac (2-(2,6-dichloranilino) phenylacetic acid) is a nonsteroidal anti-inflammatory drug that, due to human activities, has become widely distributed in the environment. Veterinary use of diclofenac in cattle has been linked to the dramatic decline of vulture populations in the Indian subcontinent, whilst therapeutic use in man has resulted in diclofenac appearing in the aquatic environment. The reportedly high octanol-water partition coefficient (log Kow 4.5) has raised concerns that the drug could bioaccumulate in fish thus presenting a risk to both fish-eating birds and man.

A bioconcentration study has been performed in rainbow trout using radiolabelled diclofenac (14C) according to the standard OECD guideline 305. The study procedures were subjected to ethical review. Water samples were taken from the fish tanks on a daily basis and the radioactive concentration determined using liquid scintillation counting.

Fish were sampled on five occasions during the uptake phase and on four occasions during the depuration phase.

Selected fish were homogenised and total radioactive concentrations determined by combustion-liquid scintillation analysis, whilst additional fish were taken to investigate the distribution of radioactivity between edible and non-edible portions of the fish. This enabled concentrations of radioactivity to be determined for specific organs (liver, kidney and gastrointestinal tract) that are potential target sites for diclofenac toxicity. Fish carcasses were subjected to wholebody autoradiography using procedures based on the work of Ullberg (Acta. Radiol. Suppl. 118, 22-31, 1954) and sections obtained using a Leica CM3600 microtome (Leica Microsystems). Distribution of radioactivity was determined and quantified using a Fuji FLA-5100 image analyser and associated Tina and SeeScan Software. Tissue concentrations of radioactivity were determined using a standard curve produced using calibrated standards. The use of a radiolabelled test compound also enabled distribution in target organs to be investigated at the cellular level using microautoradiographic techniques.

P35 - NOVEL MULTIPLE ASSAY SYSTEM FOR HEPATOCELLULAR DRUG DISPOSITION 1 2 Ryosuke Takahashi and Katsuhiro Kanda 1 2 Central Research Laboratory, Hitachi, Ltd., Saitama, Japan, Hitachi High-Technologies Corporation, Tokyo, Japan Better prediction of drug disposition in human is critical for efficient new drug development. Therefore, it is needed to establish the methodology to comprehend the entire image of pharmacokinetics of drug in liver. The purpose of this study is to develop the novel multiple assay system for hepatocellular drug disposition, which is desired to be available for evaluating in vitro uptake, biliary excretion and basal efflux disposition in serial procedure. We started the sandwich culture using rat fresh hepatocytes. After 5 days culture, hepatocytes were rinsed and preincubated in 37°C, 5% CO2 incubator with Hanks’ balanced salt solution (HBSS) containing Ca2+ and Mg2+. Then, hepatocytes were incubated with dosing solution including 5 (and 6)-carboxy-2'-7'- dichlorofluorescein (CDF) or Rhodamine 123 in the CO2 incubator. After incubation, the dosing solution was aspirated from the cells, and uptake process was stopped by washing the cells with ice-cold HBSS. Subsequently, three different sequences were performed in parallel. The first one is the method to disrupt the tight junctions comprising bile canalicular network at 37°C (seq. 1). The second is that to maintain the network at 37°C (seq. 2). And the third is that to maintain the network at 4°C (seq. 3). The supernatant fractions were collected from each sequence. Finally, cells were lysed with 1% Triton X-100 in DW and cell lysate was collected. The samples were analyzed for relative luminescence unit (RLU) of CDF and Rhodamine 123 by microplate

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reader. The disposition rates of each fraction were calculated using the obtained fluorescent data. The disposition rates of basal efflux by diffusion, those by transporter-mediated, biliary excretion, and residual cellular fraction of CDF and Rhodamine 123 were 38.2% and 11.0%, 26.6% and 12.1%, 18.6% and 4.9%, and, 16.7% and 72.0%, respectively. CDF was revealed to be likely to excrete extracellularly whereas Rhodamine 123 was to remain intracellularly. CDF showed relatively higher biliary excretion rate rather than Rhodamine 123. These results reflected the agent properties. In conclusion, we have developed the novel multiple assay system for hepatocellular drug disposition. The system is available for not only evaluating in vitro uptake, biliary excretion and basal efflux clearance in serial procedure, but also distinguishing diffusion and transporter-mediated efflux as well. We next illustrate the application with human hepatocyte by using real drug in order to estimate the better prediction in human.

P36 - PROTEIN BINDING IN COMBINATION WITH PH, METALS AND INTERACTING DRUGS CAN AUGMENT THE DISPOSITION OF RALTEGRAVIR Darren Moss, Marco Siccardi, and Andrew Owen University of Liverpool, Liverpool, United Kingdom Introduction: Raltegravir (RAL) is an antiretroviral integrase inhibitor which exhibits high inter- and intra-patient variability in oral absorption (400mg bid). Variability is not fully characterised and this makes predicting pharmacokinetics difficult. RAL binds to magnesium and exhibits pH-dependent cell membrane permeation. RAL also shows moderate plasma protein binding and its effect on these mechanisms is not understood. Therefore, we hypothesized that RAL disposition may be effected by protein binding, which may need to be considered when interpreting in vitro cellular permeation data. Methods. RAL membrane permeation was investigated using the PAMPA model, which are free of drug transporters and which models the intestinal surface. RAL (100µM) permeation was measured (5hr, 22˚C) with and without magnesium chloride (10mM) and 0.1% human serum albumin (HSA). The impact of human serum on permeation of RAL through Caco-2 cell monolayers was investigated as a model of transporter-expressing intestinal epithelium. The pH of the apical chamber was either 5 or 7.4 and the basolateral chamber was maintained at pH 7.4. The apical chamber contained either no protein, 5% (v/v) human serum (HS), 10% (v/v) HS or 0.1% (w/v) HSA. RAL (10µM) permeability was determined in the apical to basolateral (A-to-B) direction at 60 minutes. Results are given as apparent permeability (Papp, cm/s). Parallel Caco-2 monolayer experiments were performed in the presence of 0.1% HSA to investigate the effects of various potentially interacting drugs (efavirenz, ritonavir, verapamil, furosemide, montelukast, MK571, cepheranthine, omeprazole, minocycline, dipyridamole, 20 µM) on RAL Papp. Permeation of RAL was determined in both the A-to-B and B-to-A directions and were used to determine the efflux ratio (B-to-A Papp/A-to-B Papp). Mannitol was used to confirm Caco-2 monolayer integrity. Results. RAL PAMPA permeation was significantly lower (43% reduction, p<0.01) in the presence of magnesium but was not influenced by HSA. In Caco-2 experiments, addition of 10% HS at pH5 caused a significantly altered RAL A-to-B permeation (24% reduction, p<0.01). Protein addition caused no change in RAL permeability at pH 7.4. RAL permeability was significantly higher at pH 5 compared to pH 7.4 under all protein conditions (p<0.01).

Permeation of raltegravir through Caco-2 monolayers was modestly altered in the A-to-B direction by furosemide (17% increase, p=0.02) and minocycline (29% decrease, p<0.01), and was modestly altered in the B-to-A direction by omeprazole (16% increase, p=0.04), minocycline (26% decrease, p=0.02) and dipyridamole (37% decrease, p<0.01).

RAL efflux ratio was decreased by MK571 (25% decrease, p=0.04) and dipyridamole (47% decrease, p<0.01).

Discussion. RAL cellular permeation is altered in the presence of magnesium and by altering pH, most likely due to the formation of charged and metal-complexed drug. Human serum did not alter the effects of magnesium, but did alter the effects of pH, on RAL membrane permeation. MK571 (an MRP inhibitor) and dipyridamole (a nucleoside transporter inhibitor) both reduce the RAL efflux ratio using a Caco-2 cell monolayer, suggesting a possible role in RAL PK for transporters affected by these drugs. Protein concentration should be considered when interpreting such in vitro data for PH-sensitive drugs.

P37 - RODENT PHARMACOKINETICS AND IN VITRO ADME PROPERTIES OF IV3086, AN ORALLY AVAILABLE AND BRAIN PENETRANT NURR1/RXR ACTIVATOR Bruno Bournique, Didier Bressac, Emmanuel Hardillier, and Olivier Lacombe INVENTIVA, Daix, France Introduction: IV3086 is a Nurr1/RXR activator developed by INVENTIVA as disease modifier approach to treat Parkinson’s Disease. IV3086 has shown to be significantly neuroprotective at low oral doses in the mouse MPTP model. Method: Wistar or SD rats and C57BL/6JRj mice were administered the compound orally in 1% methylcellulose / 0.1% Poloxamer 188 or IV as a 3 min infusion in saline containing 2% Cremophor EL. 4 h constant rate IV infusion was performed in jugular vein cannulated rats; blood and brain were collected to determine Kp (brain/plasma concentration ratio). Rats were also placed in metabolic cages to collect urine and feces. Protein binding was analyzed by equilibrium dialysis using plasma and brain homogenate. Metabolic stability was assessed in mouse and human liver microsomes. CYP inhibitions were performed by co-incubation of IV3086 with cocktails of substrates. CYP induction was evaluated using plated human hepatocytes. Permeability was studied on PAMPA (hexadecane, pH 7.4), Caco-2, MDCK-hMDR1 and MDCK-WT layers. In vitro and in vivo samples were analyzed by LC-MS/MS. Results: After IV administration the plasma profile of IV3086 seemed biphasic with a Vd of 0.5-2.0 L/kg.

Clearance was 9.0 mL/min/kg, i.e. ~11% of liver blood flow in both species and was not dose dependent (1.0 - 10 mg/kg). After oral administration, bioavailability was 45% in mice and 60% in rats. In the mouse, oral Cmax increased

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linearly and more than proportionally to the dose (300 - 21000 ng/mL at 1.0 - 30 mg/kg). A similar relationship was observed for oral AUC, but only from 1 to 10 mg/kg. Tmax increased with the dose from 0.25 h to 1 h. In rats, exposures were dose proportional at 75 and 250 mg/kg (Cmax 9400 and 27200 ng/mL, respectively). Half-life was short: 1 h in the mouse and 3 h in the rat. Brain Kp was 0.5 and Kpu,u 0.33 (fu plasma 0.003, fu brain 0.002). The molecule was a weak human P-gp substrate as measured by the net efflux value of 2.9 in MDCK MDR1/WT cells.

Permeability on Caco-2 was moderate (3.5 10-6 cm/s) with no efflux, while it was high on PAMPA (24 10-6 cm/s). In rats, 40% to 90% of the dose was recovered as parent compound in the 0-24 h feces. In liver microsomes, Clint values were similar with and without NADPH, suggesting either a non-CYP metabolism or a physico-chemical issue.

No or weak CYP inhibition was observed on CYP1A2, 2A6, 2B6, 2C9, 2D6 and 3A4/5. At 1 and 10 µM IV3086, CYP2C8 was inhibited by 27% and 74%, respectively. No CYP1A2 mRNA induction was detected in human hepatocytes, and CYP3A4 mRNA induction was 20% of rifampicin effect. Conclusion: IV3086 rodent PK shows good oral bioavailability, low clearance and good brain penetration. Half-life was short and free fraction very low. Further investigations are needed regarding CYP450s and P-gp involvement.

P38 - WHAT DATA CAN A 14C CLINICAL STUDY DELIVER? A DISPOSITION DASHBOARD: INNOVATIVE AND INTEGRATED 14C STUDY DESIGNS TO UNDERSTAND DRUG ABSORPTION AND DISPOSITION IN HUMAN SUBJECTS Iain Shaw, Lloyd Stevens, Vanessa Zann, and Helen Walker Quotient Clinical Ltd., Nottingham, United Kingdom Introduction: Utilising 14C radiolabelled drug to obtain definitive mass balance data and to characterize metabolites for regulatory submissions is long established as a necessary part of the drug development program.

However, studies in man can be used to elucidate greater information about drug behaviour and can help build a far more comprehensive understanding of drug pharmacokinetics and metabolism.

This presentation will demonstrate, through published examples, how a comprehensive understanding of drug absorption and disposition can be achieved from administration of 14C radiolabelled drug to human subjects, thereby providing information on fraction absorbed, absolute bioavailability, routes and rates of elimination, clearance, distribution volumes as well asmetabolic disposition. Materials and Methods: Data are collated from 14C radiolabelled studies conducted by Quotient Clinical in the last 10 years. Using a ‘Human Drug Disposition Dashboard’ (Figure 1) to offer a visual portrayal of the complete picture from dosing through to elimination we will describe the data generated by published examples of various different 14C human study designs to generate a complete picture of drug absorption and disposition. See Figure 1. Human Drug Disposition Dashboard. Data discussed will include outcomes from conventional human mass balance and metabolism (ADME) studies, intravenous microtracer (ivMT) studies, integrated ivMT/ADME and oral and intravenous tracer studies. Results: The data generated from various 14C study designs can be used to compile the ‘Human Drug Disposition Dashboard. The complexity and utility of the completed dashboard in each individual case will be driven by the particular drug product being investigated and its behaviour in human subjects. Use in supporting regulatory submission may require a different level of complexity compared to a dashboard used to support drug formulation development. Conclusion: Human studies involving the dosing of 14C radiolabelled drug product have the capability to deliver a thorough understanding of drug absorption and disposition.

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P39 - NOVEL PHTHALOCYANINES AND TETRAPYRIDOPORPHYRAZINES IN PHOTODYNAMIC THERAPY AND VASCULAR-TARGETED PHOTODYNAMIC THERAPY OF CANCER – IN VITRO STUDY. Miloslav Macháček, Petra Brázdová, Adéla Jedličková, Petr Zimčík, Veronika Nováková, and Tomáš ŠImůnek Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic Photodynamic Therapy (PDT) and Vascular-Targeted Photodynamic Therapy (VTP) are two similar modalities of localized non metastatic solid tumors treatment using the same fundamental elements – photosensitizer (PS), molecular oxygen and activating light. In PDT, PSs require time to be taken up by tumor cells before the irradiation.

Red light, that has the highest permeability in tissues, is absorbed and excited state of PS passes back to ground state by emitting photon and forming reactive oxygen species (ROS). In PDT, subcellular structures of cancer cells are the primary targets of generated ROS. In contrast, no pre incubation period is applied in VTP and light activation starts immediately after administration of PS which is localized in tumor vasculature. ROS cause damage of endothelium surface that leads to vascular injury and deprivation of nutrients and oxygen to the tumor. PSs involved in this study are highly hydrophilic non aggregating phthalocyanines and their aza-analogues from the group of tetra-3,4pyridoporphyrazines, with absorption in near infra-red area (around 780 nm). Their photodynamic effect was studied on several malignant and nonmalignant cell lines – human cervical carcinoma (HeLa), melanoma (SK MEL 28) and colorectal carcinoma (HCT 116) cells in PDT or human immortalized endothelial cell line (EA.Hy926) and human primary endothelial cells (HUVEC) in VTP protocol respectively. Dark toxicity (toxicity without activating light) was also established on HeLa, EA.hy926 and 3T3 (mouse nonmalignant fibroblast cell line). All the examined compounds were efficient photosensitizers after irradiation with red light (λ > 570 nm, 12.4 mW/cm2, 11.2 J/cm2) reaching EC50 values at nano molar concentration, whereas without the activating light they showed very low inherent toxicity – TC50 values in hundreds of micro molar concentrations. Fluorescence microscopy using specific probes for different organelles showed predominant localization of photosensitizers inside lysosomes. Subcellular localization of PSs determines mode of cellular demise – therefore damage to lysosomal membrane allowed spreading of PSs throughout cytoplasm leading to quick cell death with necrotic appearance and activation of executioner caspases without previous activation of initial caspases probably by non-caspase proteases following the lysosomal damage. ROS production was measured using H2DCFDA probe and occurred only during irradiation itself; no significant secondary ROS production was detected. Involvement of autophagy was also investigated. The study was supported by the Grant Agency of Charles University grant no. 1916214 and the Czech Science Foundation grant no. 13-27761-S. P40 - ON-LINE ELECTROCHEMISTRY/MASS SPECTROMETRY (EC/MS) – A POWERFUL TOOL FOR THE PREDICTION OF DRUG OXIDATIVE METABOLISM REACTIONS AND THEIR MOLECULAR MECHANISM. Agnieszka Potęga, Kamila Wilczewska, and Zofia Mazerska Department of Pharmaceutical Technology and Biochemistry, Chemical Faculty, Gdańsk University of Technology, Gdańsk, Poland Drug metabolites can be physiologically inactive, some can express either therapeutic activity or toxic properties.

Thus, a solid understanding of metabolic pathway of drug candidate is a crucial point in drug discovery and development. Electrochemistry (EC) in an on-line combination with mass spectrometry (MS) creates a powerful platform for a quick metabolic profiling and/or reconfirmation of potential drug metabolites, which is often sufficient for decision making. This analytical technique allows to simulate various oxidation and reduction processes occurring in living organisms. EC/MS improves the conventional metabolism studies, because it helps to overcome many of the laborious tasks related to isolation and detection of metabolic products formed in vivo (urine, plasma, etc.) or in vitro (liver microsomes). Moreover, it delivers the redox fingerprint of a (drug) molecule, allowing to predict a metabolic profile, in a very short time. The aim of the present study is to demonstrate the potential of an on-line EC/MS system for the prediction of oxidative metabolism of the selected antitumor C-1311 (5-diethylaminoethylamino-8hydroxyimidazoacridinone) and C-1305 (5-dimethylaminopropylamino-8-hydroxytriazoloacridinone) drug candidates, developed in our laboratory (1). An analytical cell equipped with a glassy carbon (GC) working electrode and a Pd/H2 reference electrode was used for the oxidation of drug compounds. The cell potential, applied using a ROXY EC System (Antec, The Netherlands), was ramped from -1.5 to 2.5 V during the experiments. The outlet of the electrochemical cell was connected directly to the electrospray source of a Q-TOF mass spectrometer (Agilent Technologies, USA). 10 μM solutions in 50% methanol and 50% 10 mM aqueous formic acid were pumped through the electrochemical cell at a flow rate of 10 μL/min. Structural analogues of C-1311 and C-1305, 2-hydroxyacridinone and antitumor agent ellipticine, were choosen as model compounds. In addition, comparative study with liver cell microsomes was carried out (HPLC/MS analysis). The results of our study indicate that some products, which were generated in the EC reactor cell and on-line identified by MS, were also detected as phase I metabolites in the conventional enzymatic approach (e.g. N-dealkylated metabolites of C-1311 and C-1305, hydroxylated species).

Moreover, additional products were found only during electrochemical simulation (e.g. derivatives of quinone). They are suspected to be reactive intermediates of enzymatic reactions, which were unable to be discovered in reaction medium. To conclude, the oxidation pathway was successfully simulated under electrochemical conditions for all studied compounds. The obtained results allowed to define new possibilities of metabolic transformations of the selected antitumor agents and confirmed the known metabolic pathways. Therefore, it was clearly shown that purely instrumental approach, based on electrochemical conversion, is a feasible alternative to classical microsomal studies.

Overall, EC/MS can be a versatile and user friendly tool in predicting human responses during drug discovery and

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development when applied complementary to established in vitro or in vivo approaches (project No 2012/07/D/NZ7/03395).

(1) Kuśmierczyk H, Chołody WM, Łukowicz J, Radzikowski C, Konopa J Experimental antitumor activity and toxicity of selected triazolo- and imidazoacridinones. Arch Immunol Ther Exper 42: 415 – 423, 1994.

P41 - SMALL MOLECULE TARGETING OF THE PYRUVATE DEHYDROGENASE COMPLEX (PDC)/PYRUVATE DEHYDROGENASE KINASE (PDK) AXIS Peter Stacpoole, University of Florida, Gainesville, Florida, United States The mitochondrial pyruvate dehydrogenase complex (PDC) irreversibly decarboxylates pyruvate to acetyl CoA, thereby linking glycolysis to the tricarboxylic acid (TCA) cycle and defining a critical step in cellular bioenergetics.

Rapid regulation of the PDC is primarily by reversible phosphorylation which, in humans, occurs by tissue-specific expression of four isoforms of pyruvate dehydrogenase kinase (PDK 1-4) and two isoforms of pyruvate dehydrogenase phosphatase (PDP1 and 2). Inhibition of PDC activity has been causally associated with many disorders, including 1) congenital and acquired lactic acidosis, 2) diabetes and other insulin resistant states, 3) cerebrovascular and cardiovascular disorders, 4) neurodegenerative diseases, 5) pulmonary arterial hypertension and 6) cancer. The most common underlying mechanism accounting for PDC inhibition in these conditions is posttranscriptional up-regulation of one or more PDK isoforms, leading to phosphorylation of the E1α subunit of PDC. Such perturbations of the PDC/PDK axis induce a “glycolytic shift,” whereby affected cells favor ATP production by glycolysis or (in some tissues) fatty acid oxidation over mitochondrial glucose oxidation. For the above reasons, the PDC/PDK axis has long been a therapeutic target. Dichloroacetate (DCA) is the prototypic xenobiotic inhibitor of PDK, thereby maintaining PDC in its unphosphorylated, catalytically active, form. The amino-terminal domain of PDK contains a small binding pocket for both the natural inhibitor pyruvate and DCA, as well as sites for binding by other synthetic PDK inhibitors. DCA, certain other halogenated short chain fatty acids and pyruvate are most active against the ubiquitously expressed PDK2 isoform, but are weak inhibitors, with IC50s in the high micromolar-low millimolar range. The first orally active PDK inhibitors not based on α-dihalogenated carbonyl compounds were halogenated propionamides (Novartis) and anilide tertiary carbinols (AstraZeneca) that bound to the lipoyl group site of PDC. These compounds achieved micromolar IC50 values, but never reached clinical trials. Advances in the understanding of tumor metabolism have placed mitochondria, and particularly the PDC/PDK axis, at the center of cancer bioenergetics, proliferation and survival. Accordingly, many new PDK inhibitors have been synthesized as putative anti-cancer drugs. So far, most of these contain DCA, either coupled to standard chemotherapeutics or to molecules designed to enhance mitochondrial bioavailability of DCA. Here, I summarize the current state of small molecule inhibitors of PDKs and their therapeutic potentials in the chronic treatment of acquired and congenital disorders of the PDC. P42 - THE EFFECT OF CHRYSIN ON LYMPHANGIOGENESIS IN VITRO 1 1 2 Orawin Prangsaengtong , Sirivan Athikomkulchai , and Keiichi Koizumi 1 2 Department of Biopharmacy, Faculty of Pharmacy, Srinakharinwirot University, Nakornayok, Thailand, Department of Kampo Diagnostics, Institute of Natural Medicine, University of Toyama, Toyama, Japan Lymphangiogenesis refers to the formation of lymphatic vessels from preexisting lymphatic vessels. Lymphatic system plays an important role in tissue-fluid homeostasis, immunosurveillance, and absorption of dietary fat. In adult tissue, the growth induction new lymphatic vessel can promotes inflammation and wound healing. Moreover, tumorlymphangiogenesis is an important process to promote cancer growth and cancer metastasis via lymphatic system[1].

Recently, we successfully in establishing a rat lymphatic endothelial cells (TR-LE) from the thoracic duct of transgenic rat harboring a temperature-sensitive simian virus 40 (SV40) large T antigen[2]. TR-LE cells shown the property of endothelial cells by formation thick cord network on Matrigel, a substrate derived from mouse tumor which rich in extracellular matrix proteins and growth factors[3]. This cells were used as a model of lymphatic endothelial cells in this study. Chrysin, 5,7-dihydroxyflavone (Molecular Formula: C15H10O4, Molecular Weight: 254.2375 g/mol), is a naturally occurring flavone extracted from plants, fruits, honey, and propolis. Chrysin has the multiple bioactivities such as anti-diabetic, anti-estrogenic, anti-inflammatory, anti-allergic, anti-oxidant, anti-bacterial and anti-tumor activities. In vitro and in vivo models have shown that chrysin inhibits cancer growth and metastasis by induction of apoptosis, alteration of cell cycle inhibit cell invasion and inhibition of angiogenesis through the modulation of multiple cell signaling pathways[4]. However, the information between chrysin and lymphangiogenesis is not currently available. This study investigated the effect of chrysin that extracted from Thai propolis on lymphangiognesis in vitro.

Propolis was collected from a hive of Apis mellifera from Chiangmai province, Thailand[5]. First, we had tried to find out the nontoxic doses of chrysin on TR-LE cells by using proliferation assay and then a maximal nontoxic dose at 25 micromolar chrysin was selected to perform cord formation assay to see cord formation ability of TR-LE cells on Matrigel. After exposing to this compound, we found that chrysin at nontoxic dose suppressed cord formation of TRLE cells at 4 hours and significantly inhibited cord formation on Matrigel at 6 hours of incubation by 57.25% (*p<0.05) when compared with control group. In conclusion, we report, for the first time that chrysin inhibited lymphangiogenesis in vitro model. This finding may provide a natural compound for anti-lymphangiogenesis that involved in cancer therapy.

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References: 1. Tammela T and Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise. Cell. 2010 Feb 19;140(4):460-76.

2. Matsuo M, Koizumi K, Yamada S, Tomi M, Takahashi R, Ueda M, Terasaki T, Obinata M, Hosoya K, Ohtani O, Saiki I. Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats. Cell Tissue Res. 2006 Dec;326(3):749-58.

3. Kleinman HK, Martin GR. Matrigel: basement membrane matrix with biological activity. Semin Cancer Biol. 2005 Oct;15(5):378-86.

4. Kasala ER, Bodduluru LN, Madana RM, V AK, Gogoi R, Barua CC. Chemopreventive and therapeutic potential of chrysin in cancer: mechanistic perspectives Toxicol Lett. 2015 Mar 4;233(2):214-25.

5. Athikomkulchai S., Awale S., Ruangrungsi N., Ruchirawat S., Kadota S. Chemical constituents of Thai propolis.

Fitoterapia 2013. 88: 96-100.

P43 - ‘TIME-DEPENDENT’ INHIBITION OF HEPATIC UPTAKE TRANSPORTERS IN HEPATOCYTES AND IMPLICATIONS ON THE ASSESSMENT OF TRANSPORTER-MEDIATED DRUG-DRUG INTERACTIONS Michiharu Kageyama, Brian Houston, and Aleksandra Galetin Center for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom Recently, time-dependent increase in OATP1B1 inhibition potency has been reported for cyclosporine in transfected cell lines following pre-incubation step. In this study, we investigated the effect of different pre-incubation and coincubation conditions on the inhibition of active uptake of pitavastatin, clinically relevant OATP1B1 probe. The inhibitory potential of cyclosporine, rifampicin and clarithromycin was investigated in plated rat hepatocytes; 1h preincubation was performed either with the buffer or in the presence of an inhibitor. Following the washing step, uptake of pitavastatin (0.5 µM) was investigated ±co-incubation with the inhibitor. Pitavastatin cellular concentrations under different conditions were quantified using LC-MS/MS. Pre-incubation followed by the co-incubation step resulted in the most pronounced increase in cyclosporine and rifampicin potency compared to control (up to 3-fold). However, the estimated potency in rat hepatocytes in pre-incubation conditions (IC50 of 2.1±0.3 and 21.2±6.1 µM for cyclosporine and rifampicin, respectively) was more than 10-fold lower compared to data in transfected cell lines for these inhibitors. Pre-incubation step showed no effect on clarithromycin inhibition potency in rat hepatocytes. This study is the first to report time-dependent increase in potency of OATP inhibitors in rat hepatocytes for cyclosporine and rifampicin. Reduced potency observed relative to transfected cell lines is likely due to species differences.

Confirmation of the ‘time-dependent’ effect in human hepatocytes is currently ongoing.

P44 - DIRECT AND CYTOKINE-MEDIATED EFFECTS OF ALBUMIN-FUSED HUMAN GROWTH HORMONE, TV1106, ON CYP ENZYME EXPRESSION IN HUMAN HEPATOCYTES IN VITRO 1 1 2 2 2 1 Maciej Czerwiński , Paul Bolliger , Victor Piryatinsky , Hussein Hallak , Yousif Sahly , Immaculate Amunom , and 1 David Buckley 1 2 XenoTech LLC, Lenexa, Kansas, United States, Teva Pharmaceutical Industries Ltd., Netanya, Israel Some therapeutic proteins can evoke a cytokine response that may change expression of drug-metabolizing enzymes and lead to pharmacokinetic interactions with small molecule drugs. TV-1106 is an albumin-fused human growth hormone (GH) being developed as a long-acting treatment of GH deficiency. The GH cytokine response and its effects on drug metabolism were investigated in vitro and in vivo and indicated a potential for clinically relevant drugdrug interactions (DDI), but the DDI potential of TV-1106 has not been examined. Cytokine release assay is a risk identification tool that assesses changes in the levels of cytokines in vitro. Translation of in vitro results into effects of plurality of cytokines, which may be affected by a therapeutic protein, is poorly understood, but they may be examined in cultured human hepatocytes. In this study, human blood was treated with bacterial lipopolysaccharide, recombinant human growth hormone (rhGH) or TV-1106. While treatment of blood with rhGH resulted only in modest increases of cytokines, TV-1106 caused smaller increases of 6 cytokines (up to 4.3-fold increase in IL-1β) and a 50% reduction in IFN-γ, IL-10 and IL-12p70. Treatment of hepatocytes with TV-1106 had little or no effects on CYP1A2, CYP2C19 or CYP3A4 mRNA expression or enzyme activity. Treatment of hepatocytes with plasma from rhGH-treated blood reduced activity of the enzymes by up to 50% of the control, but the plasma from TV-1106-treated blood had little or no effect on CYP activities. The results indicated that the fusion of GH with albumin made TV-1106 an unlikely perpetrator of CYP-facilitated, direct or cytokine-mediated DDI.

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P45 - EFFECT OF PARACETAMOL ON THE PHARMACOKINETICS AND TISSUE DISTRIBUTION OF SUNITINIB IN FEMALE MICE 1 1 2 3 3 Ming Hui Liew , Evelyn Chee , Adeline Lim , Eduardo L. Mariño , and Ignacio Segarra 1 Department of Pharmaceutical Technology, School of Pharmacy, International Medical University, Kuala Lumpur, 2 Malaysia, Department of Human Biology, School of Medicine, International Medical University, Kuala Lumpur, 3 Malaysia, Clinical Pharmacy and Pharmacotherapy Unit, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Barcelona, Spain Sunitinib (SU-11248) is a chemotherapeutic agent approved for the treatment of advanced renal cell carcinoma (RCC), pancreatic neuroendocrine tumors and GIST refractory to imatinib. It is a multi-targeted tyrosine kinase receptor with also targets the VEGF receptors, the c-KIT receptor and the PDGF receptors. It is largely metabolized by the CYP3A4 isoenzyme and is an ABCB1B and ABCG2 efflux transporters substrate. Hypothesis: There is a pharmacokinetic drug-drug interaction with paracetamol (a pain management drug widely used by cancer patients) at plasma and tissue level. Methods: Paracetamol, 200 mg/kg (study group) or vehicle (control group) was administered PO to female ICR mice followed 30 min later by 60 mg/kg sunitinib PO. Sunitinib plasma, kidney, liver and brain concentration at predetermined time points were measured using a validated HPLC method [1, 2] and the pharmacokinetic parameters estimated using non-compartmental techniques. Results: Sunitinib plasma AUCo→∞ remained similar in the control (23.82 ± 0.39 µg·h/ml) and the study (25.68 ± 1.61 µg·h/ml) groups (p = 0.188) as well as Cmax (2.11 ± 0.61 µg/ml, 2.32 ± 1.06 µg/ml in control and study groups respectively, p = 0.871) which was delayed in the study group (Tmax = 2 h versus 0.5 h in the control group). The half-life decreased from 9.2 h (control group) to 6.5 h (study group) as well as the MRT (13.1 h and 10.0 h in control versus study group). Lastly, Cl/F decreased 11% and V/F was 32% lower. Sunitinib tissue AUCo→∞ after paracetamol coadministration decreased 20.3% in kidney, 9% in liver and 45% in brain, (p < 0.001 in all of them). In addition, Cmax in kidney and liver were halved (p<0.001 in both) and was achieved later in all tissues. MRT remained similar except in brain tissue that was 30% shorter. These results suggest a pharmacokinetic interaction between sunitinib and paracetamol which is not evident after plasma pharmacokinetic analysis only. Paracetamol seem to affect fundamentally the tissue uptake efficiency: the tissue-toplasma AUC ratio decreased from 14.6 ±1.5 to 10.8 ± 1.9 in kidney (p<0.05), from 2.3 ± 0.1 to 1.2 ± 0.1 in brain (p<0.05) and from 17.8 ± 1.2 to 15.5 ± 2.7 in liver (n.s.). Conclusions: Paracetamol decreased sunitinib tissue penetration but did not alter its plasma pharmacokinetics. This interaction may be unnoticed in plasma and may have clinical consequences in patients undergoing treatment for brain- or tissue- localized tumors if the appropriate pain management drug is not selected due to the diminished tissue distribution of sunitinib.

Financial support: International Medical University B1/06Res(09).

[1] Sunitinib tissue distribution changes after coadministration with ketoconazole in mice. Chee EL, Lim AY, Modamio P, Fernandez-Lastra C, Segarra I. Eur J Drug Metab Pharmacokin (Feb 2015).

[2] Histopathology and biochemistry analysis of the interaction between sunitinib and paracetamol in mice. Lim AY, Segarra I, Chakravarthi S, Akram S, Judson JP. BMC Pharmacol 10:14 (2010).

P46 - EFFECTS OF METRONIDAZOLE AND TINIDAZOLE ON THE HEPATIC CYP EXPRESSION Kiyomi Ito, Yumiko Endo, Hidehiko Kibayashi, and Toshiyuki Kudo Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan Blood levels of S-warfarin have been reported to be increased by concomitant administration of metronidazole (MTZ), an antiprotozoal agent. Previously, we found that MTZ does not inhibit the CYP2C9-mediated hydroxylation of Swarfarin by human liver microsomes. In the present study, for the purpose of elucidating the mechanism of this interaction and identifying other possible drug-drug interactions, we conducted an in vitro study with the primary cultured human hepatocytes on the ability of MTZ and tinidazole (TNZ), another nitroimidazole compound, to reduce the expression of various cytochrome P450 (CYP) isozymes as well as nuclear receptors that regulate the expression of these enzymes. The cryopreserved human hepatocytes were cultured with or without MTZ (20, 100, or 500 μM) or TNZ (40 or 400 μM) for 3 days and the mRNA levels of CYP isozymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4) as well as nuclear receptors (CAR and PXR) were determined by real-time RT-PCR. More than 80% viability was confirmed by MTT assay with the hepatocytes cultured under the same condition. Although lotto-lot variability existed, the mRNA levels of all the CYP isozymes investigated except for CYP2C19 were decreased

by MTZ or TNZ treatment in a concentration dependent manner. The mRNA levels of nuclear receptors were also decreased by MTZ or TNZ treatment, with a greater reduction observed for CAR expression. The average reduction in the mRNA levels of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A4, CAR, and PXR were 17%, 78%, 71%, 50%, 31%, 65%, 73%, and 34%, respectively, with the treatment of 100 μM MTZ, which is close to the reported maximum plasma unbound concentration when 250 mg MTZ was taken orally twice daily for 7 days. The corresponding reduction was 18%, 64%, 65%, 57%, 37%, 59%, 61%, and 43%, respectively, with the treatment of 40 μM TNZ. Our findings suggest that the reported interaction between MTZ and S-warfarin may be associated with the MTZ-induced down-regulation of CYP2C9 and CAR which regulates CYP2C9 expression. We also found the possibility that the reduction of CYP levels could be a common effect of nitroimidazole derivatives. Whether the metabolic activities of CYP2B6 and CYP3A4 were altered with MTZ or TNZ treatment are currently under investigation.

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P47 - EVALUATION OF THE DRUG-DRUG INTERACTION BETWEEN ZIDOVUDINE AND PROBENECID BY USING A MECHANISTIC KIDNEY MODEL (MECH KIM) NESTED WITHIN A FULL PHYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODEL Hilary Kim Crewe, Sibylle Neuhoff, and Karen Rowland Yeo Simcyp, Ltd., Sheffield, United Kingdom Introduction: Zidovudine (AZT) undergoes glucuronidation by UGT2B7 (65-75% of a dose) and is renally cleared (1520%). As active tubular secretion contributes to the renal elimination, drug interactions of transporter-mediated uptake in the kidney should be considered in parallel to metabolic drug-drug interactions (DDIs). The aim was to build a mechanistic PBPK model describing the metabolic and renal components of zidovudine elimination and to investigate the effect of probenecid on UGT2B7-mediated metabolism of zidovudine and Organic Anion Transporter 1 (OAT1) uptake in the kidney. Methods: In vitro information on the permeability, metabolism and transporter kinetics for OAT1 of zidovudine were combined with physicochemical data in a PBPK model (Simcyp Population-based Simulator Version 14), which included a permeability-limited model for the kidney (Mech KiM). In addition to hepatic and renal metabolism, renal secretion and reabsorption described by optimised transporter kinetics were accounted for to recover the observed renal clearance. Similarly, a fullPBPK model was also used for probenecid incorporating in vitro Ki data relating to inhibition of UGT2B7 and OAT1. Results: The simulated concentration-time profiles of zidovudine were consistent with observed data across 4 independent studies.The predicted increase in exposure of zidovudine following co-administration of probenecid was consistent with observed data; values were 1.97 (trial range: 1.65-2.18) and 2.20, respectively [1]. Corresponding predicted and observed decreases in renal clearance of zidovudine were 0.62 (trial range: 0.45-0.76) and 0.80, respectively. Conclusion: Incorporation of Mech KiM within a full PBPK model for zidovudine allowed simultaneous assessment of metabolism and transporter interactions with probenecid.

Inhibition of UGT2B7-mediated metabolism appears to be a more significant determinant of the DDI than inhibition of renal clearance.

[1] De Miranda P, Good SS, Yarchoan R, Thomas RV, Blum MR, Myers CE and Broder S (1989). Alteration of zidovudine pharmacokinetics by probenecid in patients with AIDS or AIDS-related complex; Clin Pharmacol Ther 46: 494-500.

P48 - EVALUATION OF THE IN VITRO BINDING AFFINITY OF PHOTOTOXIC CHEMICALS TO SYNTHETIC MELANIN Jelle Reinen, Leticia Rubio, Mira Wenker, Saskia Krebbers, and Beppy Van De Waart WIL Research Europe, 's Hertogenbosch, Netherlands Chemical-induced phototoxicity is an adverse reaction that is caused by interaction of photoactivated chemicals with biomolecules, such as lipids, proteins and DNA present in skin and eyes. This phototoxic effect is initiated after exposure of topically or systemically administered chemicals to sunlight. Chemicals which absorb UVB (wavelengths between 280-300 nm), UVA (320-400 nm) or visible light and show partitioning in the skin therefore need to be tested for their phototoxic potential (ICH S10 Guideline). Melanin is a naturally occurring pigment that colors the hair, skin, and eyes and binding of chemicals to melanin is one mechanism by which tissue retention and/or accumulation may occur. Since the turnover of melanin in the body is very low, a long-term retention of chemicals with high melanin affinity may take place in melanin-containing tissues and a phototoxic tissue reaction is more likely to develop. Binding of chemicals to melanin has been reported to correlate with ocular toxicity, ototoxicity, pigment disturbances of skin and hair, and carcinogenicity. As melanin-rich areas of the body are extensively exposed to light, binding of phototoxic chemicals to melanin potentially increases their phototoxicity. For in vivo phototoxicity studies animal models with both pigmented and non-pigmented animals are available. Although non-pigmented skin tends to be more sensitive than pigmented skin for detecting phototoxicity, pigmented skin should be considered for chemicals that bind significantly to melanin (ICH S10 Guideline). Therefore, to aid species selection in order to obtain an accurate risk assessment melanin binding needs to be investigated for phototoxic chemicals. In the present study we have evaluated the melanin binding affinity of a selection of structurally diverse chemicals that have been associated with phototoxic effects. First, the melanin binding affinity of 20 phototoxic chemicals was screened at two substrate concentrations (100 and 1 ÂľM) in order to identify high, moderate and low affinity melanin binders. Based upon this screening compounds were selected for determination of the melanin binding parameters. Subsequently, the time-dependent and melanin concentration-dependent binding of the selected phototoxicants was investigated and the effect of organic solvents upon the melanin binding affinity of the selected chemicals was tested. It is concluded that we can successfully evaluate the binding affinity of phototoxic chemicals to synthetic melanin in vitro. The presented research strategy can be used as a valuable tool during the photosafety evaluation of chemicals.

P49 - HERB-DRUG INTERACTION MEDIATED HEPATOTOXICITY OF TRIPTOLIDE: ROLES OF CYP AND EFFLUX TRANSPORTER Hua Li, Xiaomei Zhuang, Linglei Kong, and Lin Chen Beijing Institute of Pharmacology and Toxicology, Beijing, China Triptolide is the major active ingredient of Tripterygium wilfordii Hook F. and has been widely used to treat autoimmune and inflammatory diseases. However, it has a narrow therapeutic window with the dose-limiting

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hepatotoxicity being reported in the clinic. Triptolide is a dual substrate of CYP3A and P-gp and the toxicity may be associated with its hepatic metabolism and canalicular efflux. In this study, the roles of CYP3A and P-gp in the triptolide hepatic disposition and toxicity were investigated in vitro and in vivo with enzyme and transporter modulators and small interference RNA (siRNA). In sandwich-cultured rat hepatocytes (SCRH), the biliary clearance of triptolide was reduced 73.7% upon treatment of tariquidar (P-gp inhibitor, 5 mM) and increased 346% with phenobarbital (CYP3A and P-gp inducer, 1.0 mM), respectively. When CYP3A activity was blocked by the enzyme inhibitors 1aminobenzotriazole and ketoconazole, the triptolide-induced hepatotoxicity was increased by 2 or 1.8-fold, respectively. Data analysis indicated that CYP3A and P-gp likely contributed equally toward the detoxification of triptolide in the SCRH model and modulation of CYP3A and P-gp would result in an additive or synergistic effect on the hepatotoxicity. In the in vivo experiment, pretreatment with mdr1a-1 siRNA resulted in 62% of silencing efficiency in P-gp protein expression of BALB/C mice liver, and the systemic and hepatic exposures of triptolide were increased up to 1.5 and 2.4-fold of the control. Concomitant with tariquidar also caused 1.4 and 1.8-fold increases in the plasma and liver concentrations. The aggravated hepatotoxicity was evidenced with the remarkably lifted levels of serum biomarkers and pathological changes in liver. The toxicological indicators（AST, ALT, MDA, SOD and Bcl-2/Bax) were quantitatively correlated to the exposure increase in the presence of CYP3A and P-gp modulators. The results indicated that CYP3A and P-gp based herb-drug interaction may enhance triptolide hepatotoxicity. The dose of the drug needs to be closely monitored in the clinic when it is concomitant with CYP3A and P-gp inhibitors or substrates.

P50 - IN VITRO ASSESSMENT OF THE EFFECT OF GLUCURONIDES ON METABOLIC ENZYMES AND OATP1B1 1 2 2 2 1 Rebecca Sullivan , Pradeep Sharma , Helen Rollison , Katherine Fenner , and Aleksandra Galetin , 1 Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, 2 United Kingdom, Drug Safety and Metabolism DMPK, AstraZeneca, Cambridge, United Kingdom The latest US FDA guidance recommends investigation of the effect of metabolites on CYP450 enzymes if present at ≥25% of parent systemic exposure. Some glucuronide metabolites have been reported to inhibit metabolic enzymes and/or uptake transporters e.g., OATP1B1 and CYP2C8 (gemfibrozil glucuronide). The in vitro inhibitory effect of glucuronides on enzymes and transporters has not been characterised and their potency in relation to parent compounds is unknown. This study investigated the inhibitory potential of 10 glucuronides and selected parent compounds against OATP1B1 using estradiol 17β glucuronide (0.02 µM) and pitavastatin (1 µM) as probes. The inhibitory potential was assessed in stably transfected HEK-293 cells ± 30 minute pre-incubation with inhibitor prior to addition of probe substrate. Additionally, the effect on CYP3A4, CYP2C8, and UGT1A1 was investigated in human liver microsomes ± a 30 minute pre-incubation to assess any potential time-dependent inhibitory effect. Different metabolic pathways of repaglinide were used as markers of CYP3A4, CYP2C8 and UGT1A1 activity, enabling simultaneous investigation of the effects of glucuronides by monitoring the formation of corresponding probe metabolites. OATP1B1 inhibition was observed for 8/10 glucuronides with IC50 ranging 1.7 - 55 µM for telmisartan and mefenamic glucuronides, respectively, with estradiol 17β glucuronide. Comparable values were obtained with pitavastatin. In the case of repaglinide, diclofenac and gemfibrozil, the OATP1B1 inhibitory potency of parent compounds was comparable to glucuronides. In contrast, ezetimibe glucuronide was 4-fold more potent than its parent and the opposite trend was seen in the case of telmisartan. No trend was observed in the effect of preincubation on glucuronide or parent OATP1B1 inhibitory potency. Of the metabolic enzymes investigated, CYP2C8 inhibition was observed for all glucuronides, whereas the inhibitory effect on CYP3A4 and UGT1A1 was marginal. The CYP2C8 IC50 ranged from 5 – 57 µM for gemfibrozil and diclofenac glucuronides, respectively. Time-dependent inhibition was observed only for clopidogrel and gemfibrozil glucuronides resulting in up to 8-fold more potent CYP2C8 inhibition. Glucuronides caused equal or more potent CYP2C8 inhibition than their corresponding parent compounds with the exception of mefenamic glucuronide. No time-dependent inhibition was observed for parent compounds. In conclusion, glucuronides were found to inhibit both CYP2C8 and OATP1B1 in vitro causing similar or more potent inhibition than their parent compounds and thus may contribute to clinical drug-drug interactions.

P51 - IN VITRO ASSESSMENT OF THE PHARMACOKINETIC DRUG-DRUG INTERACTION POTENTIAL OF RASAGILINE AND ITS MAJOR METABOLITE AMINOINDAN Gregory Loewen, Lydia Vermeer, David Buckley, Rebecca Campbell, Andrew Mandracchia, Chase McCoy, Brian Ogilvie, and Julie Scheinkoenig XenoTech LLC, Lenexa, Kansas, United States Rasagiline mesylate is the active pharmaceutical ingredient of the anti-Parkinson’s drug Azilect, marketed by Teva Neuroscience, Inc. Rasagiline (R-N-2-Propynyl-1-indanamine) is a second generation, selective and irreversible inhibitor of monoamine oxidase (MAO)-B. Prior to FDA approval in 2006, the metabolism of rasagiline to its major metabolite aminoindan by CYP1A2 was characterized in vitro by traditional reaction phenotyping approaches and in a follow-up clinical study where the AUC of rasagiline (2 mg/day) increased by 83% when co-dosed with the strong CYP1A2 inhibitor ciprofloxacin (500 mg b.i.d.). Additionally, the potential for rasagiline to cause direct and/or metabolism-dependent inhibition of cytochrome P450 (CYP450) enzymes was evaluated; rasagiline did not inhibit any of the CYP450 enzymes tested (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5 and CYP4A11). In the current study, we further evaluated rasagiline and its metabolite aminoindan for the potential to be the victim or perpetrator of pharmacokinetic based drug-drug interactions. The experimental procedures were based

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on the recommendations and scientific principles described in the FDA DDI draft guidance for industry (2012), the EMA guideline on the investigation of drug interactions (2013) and in the 2014 Japanese MHLW DDI draft guidance.

Rasagiline and aminoindan were tested for the potential to cause induction of CYP450 enzymes (CYP1A2, CYP2B6, CYP3A4/5) in human hepatocytes and for direct and/or metabolism-dependent inhibition of CYP450 enzymes in human liver microsomes (rasagiline, CYP2B6, CYP2C8 and CYP3A4/5; aminoindan, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5) . Furthermore, the potential of rasagiline and aminoindan to be substrates (P-gp and BCRP) or inhibitors (P-gp, BCRP, OATP1B1, OATP1B3, OCT2, OAT1, OAT3, MATE1 and MATE2-K) of drug transporters was evaluated in vitro. With the exception of the previously mentioned CYP1A2 interaction, based on the results of the in vitro drug-drug interaction studies rasagiline and its metabolite aminoindan are not expected to be the victim or perpetrators of clinical pharmacokinetic based drug-drug interactions.

P52 - IN VITRO STUDY OF HERBAL CONSTITUENT INHIBITION ON HUMAN LIVER MICROSOMAL MORPHINE GLUCURONIDATION: A PREDICTION OF METABOLIC DRUG-DRUG INTERACTION ARISING FROM ANDROGRAPHOLIDE INHIBITION Verawan Uchaipichat Khon Kaen University, Khon Kaen, Thailand Accumulating evidences showed that silymarin flavonolignans, andrographolide and curcuminoids can inhibit UGT2B7 activities in vitro. Inhibitions of morphine clearance via glucuronidation pathway by these herbal constituents are potentially occurred and may consequently alter its analgesic efficacy or side effect profile. This study aims to (i) investigate the kinetics of morphine 3- and 6-glucuronidation by using pooled human liver microsomes, (ii) investigate the inhibitory effect of herbal constituents including major silymarin flavonolignans (viz., silybin and silychristine), andrographolide and curcuminoids on morphine 3- and 6-glucuronidation in vitro, and (ii) predict the magnitude of metabolic drug-drug interaction arising from andrographolide inhibition by using in vitro and in vivo extrapolation.

Morphine 3- and 6-glucuronide formations in the incubation without bovine serum albumin (BSA) were quantified by using the method which modified from previous studies (1,2). For inhibition study, morphine at S50 concentration was employed, while herbal constituents were used as concentration range of 1-1000 µM. Inhibitory data was presented as IC50 value. The inhibition type and Ki value were further investigated for andrographolide inhibition. Morphine 3and 6-glucuronidation kinetics exhibited Hill’s equation with the same degree of negative cooperative kinetic (n=0.91).

The respective S50 and Vmax values were 8.6 mM and 4398 pmol/min.mg protein, and 7.3 mM and 540 pmol/min.mg protein. The Clint for morphine 3-glucuronide formation was about 7-fold higher than those from morphine 6glucuronidation pathway. Using morphine concentration of 8 mM, andrographolide and curcuminoids showed markedly inhibition on morphine 3- and 6- glucuronidation with IC50 values of 50 and 87 µM, and 96 and 111 µM, respectively. However, morphine 3- and 6- glucuronidation were moderately inhibited by silybin and silychristine with the respective IC50 values of 583 and 862 µM, and 3257 and 3504 µM. Andrographolide inhibition of morphine 3and 6-glucuronidation was competitive. The Ki values for inhibition of morphine 3- and 6- glucuronide formation by andrographolide were 7.1 ± 0.02 µM and 9.5 ± 3.02 µM, respectively. Based on total inhibitor concentration in plasma at a dose of 60 mg andrographolide daily, the predicted AUC increase for morphine when coadministered with andrographolide was 15%. Although only a minor drug-drug interaction was predicted, magnitude of inhibition would be underestimated due to the absence of BSA in the in vitro condition. For more accurate prediction, in vitro study for andrographolide inhibition on morphine 3- and 6-glucuronide formation in the presence of BSA should be further investigated.

1) Uchaipichat V, Raungrut P, Chau N, Janchawee B, Evans AM and Miners JO. Effects of ketamine on human UDPglucuronosyltransferases in vitro predict potential drug-drug interactions arising from ketamine inhibition of codeine and morphine glucuronidation. Drug Metab Dispos, 2011; 39(8): 1324-8.

2) Chau N, Elliot DJ, Lewis BC, Burns K, Johnston MR, Mackenzie PI and Miners JO. Morphine glucuronidation and glucosidation represent complementary metabolic pathways that are both catalyzed by UDP-Glucuronosyltransferase 2B7: Kinetic, Inhibition, and Molecular Modeling Studies. JPET, 2014; 349: 126-37.

P53 - IN-VITRO EVALUATION OF DDI WITH COBICISTAT AND RITONAVIR USING HEPARG CELL LINE 1 2 1 1 1 Flavia Storelli , Christel Bruggmann , Fabienne Doffey-Lazeyras , Caroline Samer , Jules Alexandre Desmeules , 1 and Youssef Daali 1 2 Geneva University Hospitals, Geneva, Switzerland, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Cobicistat (Tybost®) is a new pharmacoenhancer which has recently been approved by the EMEA as a boosting agent for the HIV protease inhibitors atazanavir and darunavir. As opposed to ritonavir, a protease inhibitor used as a boosting agent in HIV treatment combinations, cobicistat does not show any antiviral activity. Also, previous inhibition studies on human hepatic microsomal fractions showed a more specific inhibition of CYP3A4 of cobicistat versus ritonavir and a lack of induction potential on CYP3A4. The aim of this work was to investigate CYP450 inhibition/induction potential of major CYPs by cobicistat and ritonavir in HepaRG. On this purpose, low densityseeded confluent HepaRG cells were differentiated in a culture medium containing 1.5% DSMO and plated in 24-well plates. At day 12 after plating, cobicistat or ritonavir both at various concentrations ranging from 0 to 50 µM were incubated with a probe cocktail composed of midazolam 5 µM (CYP3A4), S-mephenytoin 50 µM (CYP2C19), bupropion 50 µM (CYP2B6) and flurbiprofen 10 µM (CYP2C9). Metabolite production after 3 hours of incubation at

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37°C was quantified by LC-MS/MS and IC50 were calculated by linear regression. For induction assay, cobicistat or rifampicin for positive control at various concentrations ranging from 0 to 50 µM were incubated with HepaRG cells for 72 hours in a medium composed of 0.5% DMSO and deprived of FBS. DMSO concentration was decreased from 1.5 to 0.5 % 24 hours before addition of the inducers. CYP3A4 activity was assessed by incubation of midazolam 5 µM for 3 hours at 37°C and quantification of metabolite production by LC-MS/MS. Cobicistat and ritonavir showed similar inhibition of CYP 3A4 with IC50 of 0.107 µM and 0.105 µM respectively. IC50 values of CYP 2B6, 1A2, 2C19 and 2C9 were lower for cobicistat than for ritonavir, thus suggesting that cobicistat may be a more potent inhibitor of these CYP450 than ritonavir. Considering CYP3A4 induction, cobicistat did not seem to induce CYP 3A4. Rifampicin, which was used as a positive control, showed induction of CYP 3A4 up to 2.5 fold. This work has showed the suitability of the HepaRG cell line to study inhibition drug interactions in vitro. Induction assays however have still to be optimized to reach greater levels of induction.

P54 - POTENTIAL INTERACTION OF ANXIOLYTIC DRUG, BUSPIRONE, WITH HUMAN ORGANIC CATION TRANSPORTER 1 1 2 2 1 1 Chutima Srimaroeng , Promsuk Jutabha , Naohiko Anzai , Atcharaporn Ontawong , Metee Jinakote , and Chaliya 1 Jaiyen 1 2 Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, Department of Pharmacology and Toxicology, Dokkyo Medical University, School of Medicine, Tochigi, Japan Buspirone is an anxiolytic agent that has been used for the short-term management of anxiety disorders. It can be metabolized by cytochrome P450 3A4 (CYP3A4) and can be produced several metabolites, including hydroxylated derivatives and 1-(2-pyrimidinyl)-piperazine (1-PP). Buspirone was shown to be a partial agonist of 5hydroxytryptamine 1A (5-HT1A) receptor and has a moderate affinity for brain dopamine D2 receptor, whereas 1-PP, its pharmacologically active metabolite, was shown to be an antagonist of alpha2-adrenergic receptor. Organic cation transport system has extremely high impact on the systemic levels of cationic drugs and plays a crucial role on these drug excretion. Currently, human organic cation transporter 1 (hOCT1) has revealed to be highly expressed in the liver and plays important role for cellular uptake of several cationic drugs into the hepatocytes, resulting in exertion of their pharmacologic actions, including antidiabetic metformin and anticancer oxaliplatin, imatinib, irinitecan, and paclitaxel.

Recent study had shown that the transport of typical hOCT1 substrate, 1-methyl-4-phenyl pyridinium (MPP+), was interfered by several anti-depressant and anti-psychotic drugs, including trimipramine, desipramine and fluoxetine.

However, the effect of anxiolytic buspirone on the transport of hOCT1 substrates remains unknown. Therefore, the objective of this study was to examine whether buspirone and its major derivative, 1-PP, interact with the human organic cation transporter 1 (hOCT1). The uptake of MPP+ into the second segment of the renal proximal tubule (S2) cells that stably expressing human OCT1 (S2hOCT1) in the presence or absence of either buspirone or 1-PP were determined. The effect of buspirone on the uptake of cationic fluorescence 4-(4-(dimethylamino)styryl)-nmethylpyridinium (ASP+) into human hepatocellular carcinoma cells (HepG2) mediated by hOCT1 was also assessed.

The results showed that buspirone was able to inhibit hOCT1 mediated MPP+ uptake in stable S2hOCT1 cells in a dose-dependent manner. The estimated half maximal inhibitory concentration (IC50) was approximately 132.0 ± 12.9 uM. In contrast, 1-PP had no interaction with hOCT1 in S2hOCT1 cells. Similar to overexpressing hOCT1 cells, buspirone, but not 1-PP, inhibited ASP+ mediated by hOCT1 transport in HepG2 cells in a dose-dependent manner with IC50 of 31.1 ± 0.8 uM. The inhibitory effect of buspirone on ASP+ transport in HepG2 cells was not by a decrease of cell viability as indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Hence, the present study indicates that buspirone moderately interacts with hOCT1 in both overexpressing hOCT1 and HepG2 cells. Nevertheless, further investigation is needed to examine the potential inhibitory effect of buspirone on the exertion of pharmacological cationic drugs.

P55 - PREDICTION OF IN VIVO DRUG-DRUG INTERACTIONS BETWEEN DRONEDARONE AND RIVAROXABAN BASED ON IN VITRO INHIBITION STUDIES Yanjun Hong, Eleanor Jing Yi Cheong, and Eric Chan National University of Singapore, Singapore, Singapore Dronedarone is an anti-arrhythmic agent approved in 2009 for the treatment of atrial fibrillation (AF). Previous inhouse studies have established that dronedarone and its main metabolite, N-desbutyldronedarone (NDBD) can cause mechanism-based inactivation (MBI) and reversible inhibition of CYP3A4 and CYP2J2 with testosterone and astemizole as probe substrates, respectively. Rivaroxaban, a new anti-coagulant approved in 2011, is a safer alternative to warfarin. Clinical therapy involving the co-administration of rivaroxaban and dronedarone is likely in patients with AF. Given that CYP3A4 and CYP2J2 are responsible for approximately 32% of rivaroxaban elimination, the MBI and reversible inhibition of CYP3A4 and CYP2J2 by dronedarone may augment exposure to rivaroxaban during co-administration, resulting in potential drug-drug interactions (DDIs) which may lead to hematologic toxicities.

Given the absence of prior clinical data, this study thus aims to predict in vivo DDIs between rivaroxaban and dronedarone using a mechanistic static model based on in vitro inhibition data. Kinetic parameters for competitive inhibition (Ki) and MBI (KI and kinact) were obtained based on in vitro studies using recombinant P450 enzymes, with rivaroxaban as the probe substrate. A mechanistic static model was fitted with the inhibition data to predict the increase in systemic exposure of rivaroxaban. The KI and kinact describing the inactivation of CYP3A4 by dronedarone were determined to be 0.302 µM and 0.056 min-1 whereas the corresponding values for NDBD were

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0.876 µM and 0.047 min-1. The calculated inactivation kinetic constants, KI and kinact, describing inactivation of CYP2J2 were 0.031 µM and 0.021 min-1 for dronedarone and 0.037 µM and 0.025 min-1 for NDBD. By comparing the kinact/KI ratio, the potency of CYP2J2 inactivation by dronedarone and NDBD is considerably greater than the potency of CYP3A4 inactivation. The inhibition constant Ki for CYP3A4 was determined to be 0.641 µM and 1.030 µM for dronedarone and NDBD respectively. As for CYP2J2, the calculated Ki was 0.927 µM for dronedarone and 2.531 µM for NDBD. The fold change in rivaroxaban exposure was calculated to be 2.30 and 2.58 respectively in the absence and presence of intestinal inhibition, which translates into a moderate risk of DDI. In conclusion, hepatic oxidative biotransformation of rivaroxaban by CYP3A4 and CYP2J2 is likely to be impaired by dronedarone through the simultaneous mechanisms of MBI and reversible inhibition. Risks associated with the concomitant use of both agents in the management of AF have to be recognized. However, the possibility of P-gp inhibition by dronedarone and the involvement of inhibitory metabolites cannot be simulated by the mechanistic static model and future research will necessitate the use of physiologically-based pharmacokinetic modeling in order to refine the DDI prediction.

P56 - SILENSOMES™: A NEW IN VITRO MODEL FOR HUMAN CYTOCHROMES P450 PHENOTYPING ASSAYS 1 1 1 2 3 Yannick Parmentier , Fabrice Caradec , Corinne Pothier , Belkacem Bouaita , Fabrice Guillet , and Christophe 2 Chesné 1 2 Institut de Recherches Servier, Croissy-sur-Seine, France, R&D, Biopredic International, Saint Gregoire, 3 France, EUROSAFE, Saint Gregoire, France Pharmacokinetic drug-drug interaction can significantly impact drug safety and efficacy. Prediction of this drug-drug interaction risk is a requisite in the development plan of a new drug candidate to the submission of the registration dossier. In vitro identification and measurement of the contribution of the major cytochrome P450 enzymes involved in the human metabolism of a new drug candidate, also called “CYP phenotyping assay”, allows predicting the impact of another co-administered drug (perpetrator) on the pharmacokinetics of the new chemical entity (victim). Up to now, a battery of in vitro tests (recommended by the regulatory agencies) is required for this CYP phenotyping assay. Each of these tests suffers from numerous inconvenients: no direct quantitative measurement of the contribution of each CYP in the metabolism of a drug, biologic system not fully representative of the liver situation (eg human recombinant CYP450), lack of specificity (eg antibody anti-CYP450), too specific conditions of use (eg chemical competitive CYP specific inhibitors). A new in vitro model, called, Silensomes™ has been developed to encounter the disadvantages of the current methodologies. Silensomes™ correspond to human pooled liver microsomes chemically desactivated for one specific CYP450. CYP3A4 Silensomes™ were chosen as a proof of concept of this new model. Following their preparation, the cryopreserved batches of desactivated-CYP3A4 and control pooled liver microsomes were incubated with CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 specific substrates to measure their respective activities. Results showed that CYP3A4 Silensomes™ abolished CYP3A4 activities of testosterone, nifedipine and midazolam to more than 80 % with no impact on the other CYP450 activities tested showing the high specificity and potency of the inhibition. Silensome™ CYP3A4 desactivation was preserved after storage at -80°C. Moreover, CYP3A4 contribution measured for known multiCYP substrates, following their incubation with CYP3A4 Silensomes™ were similar to the fm values observed in vivo or in vitro. These results showed that the CYP3A4 Silensomes™ model responds to the different criteria of specificity, potency, stability and predictability to ensure a good extrapolation of the risk of pharmacokinetic drug-drug interaction. Moreover, this new “ready to use” in vitro model is very easy to handle and can be easily fully automated. This approach will be extended to all the human major CYP450 in order to provide a complete kit ready to use for the CYP450 phenotyping assay. It is therefore the model of choice for a rapid and robust determination of the CYP contribution all along the development plan of a new chemical entity.

P57 - THE LACK OF UTILITY OF PHARMACOLOGICAL INTERFERENCE FOR THE STUDY OF PROTEIN DEGRADATION 1 1 1 1 2 1 Christina Chan , Neill Liptrott , Philip Martin , Marco Siccard , Lisa Almond , and Andrew Owen 1 2 University of Liverpool, Liverpool, United Kingdom, Simcyp Limited (a Certara company), Sheffield, United Kingdom Physiologically-based pharmacokinetic (PBPK) modelling can be used to predict the magnitude and time-course of drug-drug interactions (DDIs). There are currently a wealth of data within the literature on the induction of pharmacologically-relevant metabolising enzymes and transporter proteins. However, the accuracy PBPK DDI prediction at the molecular level is also dependent upon the rate of degradation (kdeg) of these proteins. Protein synthesis inhibitors are commonly used for estimating kdeg but a thorough investigation of the cytotoxicity of such agents in relation to protein synthesis inhibition has not been conducted. This study aimed to identify concentrations of single inhibitors or combinations that provide optimal protein synthesis inhibition at non-cytotoxic concentrations for use in estimation of Kdeg. Actinomycin D, cycloheximide, emetine and puromycin were assessed individually and in 2, 3 and 4 drug combinations for protein synthesis inhibition (IC50) and cytotoxicity (CC50). Two-drug combinations were assessed for synergy by the fixed-ratio isobologram method. Protein synthesis inhibition was determined by leucine incorporation assay and cytotoxicity was assessed by standard MTT assay in HepG2 cells. A range of toxicity assays including CellTiter Glo, GSH-Glo glutathione and Trypan Blue exclusion were carried out for 3 and 4 drug combinations to confirm the robustness of MTT assays as a measure of cytotoxicity. IC50 and CC50 were calculated using non-linear regression with Graphpad Prism. The IC50 for the four individual protein synthesis inhibitors actinomycin D, cycloheximide, emetine and puromycin were calculated from concentration-response curves as 0.032

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± 0.008, 5.8 ± 2.6, 2.1 ± 1.5 and 2.8 ± 0.66 µM, respectively. The CC50 values were found to be 0.10 ± 0.007, 0.43 ± 0.23, 0.072 ± 0.011 and 1.2 ±0.1 µM, respectively. Two-drug combinations of actinomycin D/cycloheximide and actinomycin D/emetine were found to be synergistic for protein synthesis inhibition and antagonistic for cytotoxicity at 5:1 and 4:2 ratios. However, the CC50 values for these drug pairs alone and in combination were again lower than corresponding IC50 values. The 4-drug combination at sub-cytotoxic concentrations of each inhibitor (CC10) provided only 24% protein synthesis inhibition, which was insufficient for subsequent degradation studies. Therefore, no single, 2, 3 or 4 combinations of these protein synthesis inhibitors provided sufficient protein synthesis inhibition without also causing cytotoxicity. The inhibitors investigated generally exhibited cytotoxicity with lower concentrations than those required for inhibition of protein synthesis. Identification of single agent or combinations of inhibitors for accurate determination of kdeg was not possible in the absence of other effects on cell function. These data indicate that methodologies avoiding the use of chemical inhibitors may be preferred for estimating kdeg.

P58 - TOWARDS QUANTITATIVE PREDICTION OF DISEASE-DRUG INTERACTIONS USING A PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELLING APPROACH: SUPPRESSION OF CYP1A2 BY IL-6 1 1 1 2 1 Krishna Machavaram , Lisa Almond , Masoud Jamei , Amin Rostami-Hodjegan , and Iain Gardner 1 2 Simcyp, Ltd., Sheffield, United Kingdom, Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Manchester, United Kingdom It is known that circulating levels of pro-inflammatory cytokines (e.g. Interleukin [IL]-6) are elevated in patients with heart failure (1) and an inverse relationship between cytokine levels and cytochrome P450 (CYP) activity is observed (2). Clinical studies have indicated that a decrease in CYP1A2 activity in congestive heart failure (CHF) patients (2, 3).

Exposure of hepatocytes to IL-6 in vitro leads to decreased expression/activity of CYP1A2 (4). The aim of this work was to investigate the role of a physiologically based pharmacokinetic (PBPK) model to predict the impact of systemic IL-6 levels on the disposition of CYP1A2 probe substrates (caffeine and theophylline) in patients with heart failure. The PBPK model included a semi-mechanistic suppression model (Simcyp v14.1, Sheffield, UK) (5) that combined in vitro suppression data with a range of steady-state levels of IL-6 (5 to 1000 pg/mL) to quantitatively predict the diseasedrug interaction via IL-6 mediated suppression of CYP1A2. The characteristics of the virtual subjects were matched to participants of the clinical trials in terms of numbers, age, and sex. Using steady-state plasma IL-6 levels of 500 pg/mL, the predicted median caffeine metabolic ratio was comparable with the observed data (0.33 vs. 0.36) from one study. Although 500 pg/mL is higher than the observed mean IL-6 concentrations, it is within the reported range for CHF patients. The same assumptions were then applied to a different group of CHF patients administered with theophylline. Simulations showed a decrease in mean total clearance of theophylline that was reasonably comparable with the clinical data (7-50% vs. 25-69%). The impact of other potential contributing factors such as high variability in circulating levels of IL-6 in CHF patients, significant inter-donor variability in the in vitro data and the role of cytokines other than IL-6 warrant further investigation. However, these findings support the application of PBPK models, that have been validated with independent clinical data, for the quantitative prediction as well as mechanistic understanding of disease-drug interactions via suppression of CYPs by elevated levels of IL-6.

REFERENCES 1. Matsumori A, Yamada T, Suzuki H, Matoba Y, Sasayama S. Increased circulating cytokines in patients with myocarditis and cardiomyopathy. Br Heart J. 72(6):561-6, 1994.

2. Frye RF, Schneider VM, Frye CS, Feldman AM. Plasma levels of TNF-alpha and IL-6 are inversely related to cytochrome P450-dependent drug metabolism in patients with congestive heart failure. J Card Fail. 8(5):315-9, 2002.

3. Jeong CS, Hwang SC, Jones DW, Ryu HS, Sohn K, Sands CD. Theophylline disposition in Korean patients with congestive heart failure. Ann Pharmacother. 28(3):396-40, 1994.

4. Dickmann LJ, Patel SK, Rock DA, Wienkers LC, Slatter JG. Effects of interleukin-6 (IL-6) and an anti-IL-6 monoclonal antibody on drug-metabolizing enzymes in human hepatocyte culture. Drug Metab Dispos. 39(8):1415-22, 2011.

5. Machavaram KK, Almond LM, Rostami-Hodjegan A, Gardner I, Jamei M, Tay S, Wong S, Joshi A, Kenny JR. A physiologically based pharmacokinetic modeling approach to predict disease-drug interactions: suppression of CYP3A

by IL-6. Clin Pharmacol Ther. 94(2):260-8, 2013.

P59 - EVALUATION OF CYTOCHROME P450 ENZYME INDUCTION IN VITRO: TREND ANALYSIS AND CORRELATION OF ENZYME ACTIVITY DATA WITH MRNA EXPRESSION David Wilkinson, Sarah Andrews, Emma Alexander, Gang Luo, Anthony Glazier, and John Kendrick Covance Laboratories Ltd., Harrogate, United Kingdom The induction of drug metabolising enzymes, particularly cytochromes P450 (CYP), can have significant pharmacological and toxicological consequences. The use of monolayer cultures of human hepatocytes is established as the definitive model for the prediction of CYP induction in vitro, with earlier difficulties in the procurement of fresh human liver largely ameliorated through the commercial availability of plateable cryopreserved cells. Covance has been performing in vitro enzyme induction studies for many years and, as such, a significant amount of data on the effects of positive control inducers in hepatocyte cultures has been generated. In this presentation, noteworthy trends in Covance historical data generated using freshly isolated and commercially sourced

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cryopreserved hepatocytes will be discussed, including the effect of donor demographics and the impact of basal enzyme activity on the extent of induction elicited. In addition, recent FDA/EMA regulatory guidance has advocated the use of CYP mRNA expression as the preferred endpoint for the quantification of induction [1, 2]. Through the use of RT-PCR and Taqman® methodology, the effects of prototypical inducers on CYP1A2, CYP2B6 and CYP3A4 mRNA expression in hepatocytes from multiple human donors will be presented, together with the corresponding enzyme activity data from the same cells. The extent of induction elicited using both endpoints will be directly compared and discussed in relation to regulatory guidance.

1. FDA Draft Guidance for Industry. Drug Interaction Studies – Study Design, Data Analysis, Implications for Incubation, and Labelling Recommendations (2012).

2. The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (2012).

P60 - EVALUATION OF DIFFERENT NORMALISATION METHODS TO PREDICT CYP3A4 INDUCTION IN SIX FULLY CHARACTERIZED CRYOPRESERVED HUMAN HEPATOCYTE PREPARATIONS AND HEPARG CELLS Helene Vermet and Xavier Boulenc Sanofi, Montpellier, France Prediction of drug-drug interaction due to CYP3A4 over-expression is important because this CYP isoform is involved in the metabolism of more than 50 % of small marketed drugs and can lead to sub-therapeutic concentrations of the affected drug, resulting in therapeutic failure. It is therefore mandatory to understand and predict a new compound’s potential for CYP3A4 induction. Using in vitro data obtained in human hepatocytes, the risk of induction at a given therapeutic dose is estimated through the principle of in vitro – in vivo extrapolation (IVIVE), also called "bottom-up" approach. Recently, several methods were proposed in the literature. In particular, an approach that consists in considering a scaling factor for a given hepatocytes batch to bridge the gap between in vitro and clinical situation has been taken over by health authorities and reported in the guidelines. In this approach, the value of this factor is determined by comparing in vitro and clinical data for a set of well-known inducers through a statistical analysis for each hepatocyte batch. The purpose of our work was to challenge the relevance of this calibration factor determined with a set of 15 well-known inducers or with Rifampicin only. The approaches were evaluated using different values for hepatic inducer concentration: unbound (with Fup) or total (without Fup) Cmax. These investigations were conducted with six human hepatocytes batches as well as with an established cell line HepaRG. Consistently with previous authors, our data show that the absence of calibration factor leads to a clear overestimation of the clinical effects of the inducer when using standard IVIVE technics. The use of d-factor calculated without Fup is the best method to avoid underestimation. More interestingly, our results suggest that the "d" factor value determined with Rifampicin alone provide similar accuracy in prediction than with a set of well-known clinical inducers. In conclusion, these findings not only show that a calibration factor was mandatory for clinical predictions but they also suggest that the calculation of this factor for a given cell batch requested the use of Rifampicin only, and not the use of a large set of fully characterized CYP3A4 clinical inducers. For HepaRG established cell line, preliminary findings obtained with three thawing cycle of only one batch suggests that a calibration factor does not lead to a reliability improvement of the predictions. Additional investigations with different batches are needed to confirm or not these results.

P61 - NUCLEAR RECEPTOR-MEDIATED CHANGES IN GENE EXPRESSION IN A HUMAN HEPATOCYTE MICROPATTERNED CO-CULTURE SYSTEM FOLLOWING TREATMENT WITH HEPATOTOXIC COMPOUNDS 1 1 2 2 2 1 Kelly Rose , Kristina Wolf , Okechukwu Ukairo , Amanda Moore JeanneMarie Gaffney , Melvin Andersen , Edward 1 LeCluyse 1 2 The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina, United States, Hepregen Corporation, Medford, Massachusetts, United States The current suite of in vitro ToxCast assays may not represent complex biochemical and multi-cellular responses observed in vivo with adequate fidelity. We recently have shown that a novel hepatic culture model in which cryopreserved primary human hepatocytes are seeded onto micropatterned 96-well plates and co-cultured with murine embryonic fibroblasts (MPCC; HepatoPac®) retains key biochemical functions of the liver in vivo, including metabolic capacity. However, the retention of functional nuclear receptor pathways in this model has not yet been determined. Human MPCCs were treated with 17 compounds, including prototypical nuclear receptor activating compounds rifampin (RIF), phenobarbital (PB) and GW7647, along with additional compounds from the ToxCast datasets (e.g. PFOA and haloperidol), using an 8 point concentration range (0-100 µM). Changes in the expression of eight different genes, including a house-keeping gene (ACOX1, CYP4A11, HMGCS2, CYP1A2, CYP2B6, CYP2C9, CYP3A4, HPRT), were determined following 24 and 72 hr exposures. Results show concentration-dependent induction of the majority of target genes by the prototypical inducers. However, we found that hepatocytes in micropatterned co-cultures are more sensitive to induction by some compounds, such as RIF and CITCO, compared to the standard sandwich-culture model. A minimum three-fold induction was observed in at least one target gene at one time point following treatment with each compound, except for the negative control caffeine. A 50% suppression of at least one target gene at a single time point also was observed for the majority of compounds tested. Human MPCCs appear to be a sensitive, robust, and reproducible model for the measurement of changes in nuclear receptormediated gene expression.

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P62 - THE EFFECT OF CYP1A2 INDUCTION ON MELATONIN PHARMACOKINETICS IN HEALTHY THAI SUBJECTS 1 1 1 1 1 Sirimas Kanjanawart , Nuttawut Jenjirattithigarn , Thachanan Kongpan , Nontaya Nakkam , Katcharin Phunikom , 2 1 Jeffrey Roy Johns , and Wichittra Tassaneeyakul 1 2 Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand Melatonin is a pineal hormone which controls sleep pattern. Exogenous melatonin has been used for sleep disorders, antioxidant and adjuvant to chemotherapy treatments as an oncostatic agent. Melatonin is mainly metabolized by CYP1A2. Several reports documented increased in oral bioavailability of melatonin after co-administration of the strong CYP1A2 inhibitor fluoxamine. On the other hand, omeprazole is known as CYP1A2 inducer and the role of CYP1A2 inducer on melatonin is still unclear. This study aims to investigate the effect of omeprazole, CYP1A2 inducer on melatonin pharmacokinetics. A single oral dose 20 mg melatonin capsule was administered to 25 healthy subjects under fasting condition in two phase trials, before (non-induction) and after 60 mg omeprazole twice a day for seven days (induction). The paraxanthine and caffeine ratio in saliva after administration of 150 mg caffeine beverage for 6 hours was used as an index of CYP1A2 activity before and after induction. Blood samples were collected at 0, 0.5, 0.75, 1.0, 1.25, 1.5, 2, 3, 4, 5, 6 and 8 hours after melatonin administration. Melatonin concentrations in plasma and caffeine concentrations in saliva were determined using high performance liquid chromatography. Pharmacokinetic parameters of melatonin in both phases were calculated via non-compartmental model. After administration of omeprazole, the mean paraxantine and caffeine ratio was increase from 0.62 to 1.02. The mean (SD) Cmax and AUC0-inf for non-induction phase were 55.49 (48.26) ng/ml and 67.1(52.7) hr*ng/ml, while after induction phase both Cmax and AUC0-inf were reduced to 26.83 (34.83) ng/mL and 30.86 (28.36) hr*ng/ml. Co-administration of omeprazole with melatonin resulted in significant reduce the systemic exposure of melatonin (53 % for Cmax and 54% for AUC0-inf). The geometric mean ratios of Cmax and AUC0-inf with 90% CI of melatonin (induction phase/noninduction phase) were 0.44 (0.32 -0.54) and 0.58 (0.36 - 0.53). This result suggests that the induction of CYP1A2 may reduce systemic exposure of melatonin. This study was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the High Education Commission, through the Health cluster (SHeP-GMS), Faculty of Medicine, Khon Kaen University, Thailand.

P63 - THE EFFECT OF PROTOTYPICAL INDUCERS ON MRNA EXPRESSION OF CYP1A1, CYP1A2, CYP2B1, CYP3A1 AND UGT1A1 IN CRYOPRESERVED RAT HEPATOCYTES David Stresser, Duan Wang, and George Zhang Corning Gentest Contract Research Services, Woburn, Massachusetts, United States Cytochrome P450 isoforms 1A1, 1A2, 2B1 are 3A1 well known to be inducible in rat liver. Prototypical inducers of these isoforms include ß-naphthoflavone (BNF) for CYP1A1/2, phenobarbital (PB) for CYP2B1 and dexamethasone (DEX) and pregnenolone 16α-carbonitrile (PCN) for CYP3A1. The P450 induction response elicited by drug candidates in preclinical species is often used to help explain or anticipate lower drug exposures in animals upon repeat dosing. In addition, induction elicited through constitutive androstane receptor (typified by PB treatment) is associated with a proliferative response and can trigger safety concerns. Often, induction information is gathered by evaluating mRNA or enzyme activity ex vivo using liver tissue obtained from a toxicity study. Unfortunately, this information may be gathered too late to impact medicinal chemistry efforts. In this study, cryopreserved hepatocytes obtained from three lots of male, Sprague-Dawley rat hepatocytes (each lot pooled from 5-8 animals) were plated and treated with 25 μM BNF, 1000 μM PB, 10 μM DEX and 10 μM PCN along with solvent vehicle controls. Cells were treated for 48 h, with a media change after the first 24 h. The mRNA relative expression levels were determined for CYP1A1, CYP1A2, CYP2B1, CYP3A1 and UGT1A1 using Taqman® Real Time RT PCR. Significant induction of CYP1A1 and CYP1A2 mRNA expression was observed with BNF treatment, with mean induction of 179 ± 108 and 76 ± 50 fold, respectively whereas other inducers caused no or minimal induction. Significant induction of CYP2B1 mRNA expression was observed with PB treatment, with a mean induction of 1576 ± 717-fold. DEX and PCN also caused clear, but much lower induction of CYP2B1 with mean fold induction of 6.9 ± 4.3 and 3.5 ± 3.6, respectively, whereas BNF did not induce. Substantial induction of CYP3A1 mRNA expression was observed with DEX and PCN treatments, with mean induction of 587 ± 205 and 338 ± 89, respectively. In addition, PB also caused robust induction of CYP3A1 with a mean fold induction of 192 ± 15-fold; BNF induced at a relatively low level of 6.7 ± 3.1 -fold. The overlapping induction response of CYP2B1 and CYP3A1 elicited by PB, DEX and PCN are consistent with nuclear receptor crosstalk between inducers of these enzymes, although PB was much more selective as an inducer of CYP2B1. No induction of UGT1A1 was observed for any of the inducers tested (e.g. < 1.5-fold). Instead, BNF caused a decrease in mRNA expression of UGT1A1 with a mean decrease of 69%. Global CV of fold-induction response was relatively high (0.4), although the very large dynamic range may compensate. These data demonstrate the applicability of a time and cost-saving in vitro model to evaluate induction potential of major, inducible rat P450 isoforms. We suggest that model could be used to explain or anticipate findings of lower exposures in rats upon repeat dosing, as a result of autoinduction.

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P64 - ACTIVITY OF ANTIOXIDANT ENZYMES IN HUMAN COLORECTAL ADENOCARCINOMA CELL LINES: CAN IT BE MODIFIED BY SELECTED SESQUITERPENES? 1 2 1 1 1 Hana Bartikova , Veronika Hanusova , Petra Matouskova , Barbora Szotakova , and Lenka Skalova 1 Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Hradec Králové, Czech 2 Republic, Department of Medical Biology and Genetics, Faculty of Medicine, Charles University, Hradec Králové, Czech Republic As the cancer represents one of the major causes of death all over the world, there is increased interest in the natural substances which are the source of new anticancer agents. Most biologically active natural products are secondary metabolites with sesquiterpenes being an important group. Sesquiterpenes, defined as 15-carbon compounds from 3 isoprenoid units, possess many biological activities including the effect against cancer. Besides other things, anticancer activity of sesquiterpenes may lie in impairment of antioxidant defence system in cancer cells. In the current study, we focused on the influence of sesquiterpenes on the activity of antioxidant enzymes in human colorectal adenocarcinoma cell lines HT29 and Caco2. We tested Myrica rubra essential oil (mixture of sesquiterpenes) and its 2 substantial sesquiterpene components -humulene (2,6,6,9-tetramethyl-1,4-8cycloundecatriene) and trans-nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol), which showed the most profound antiproliferative effect on used cancer cell lines. Both cell lines were incubated with Myrica rubra essential oil, transnerolidol or -humulene at concentration 25 and 50 µg/ml for 48 hours. Control group was treated with corresponding amount of DMSO (0.1%) in the absence of sesquiterpenes. Specific activity of selected antioxidant enzymes was assayed in cytosolic fraction from cell homogenate by spectrophotometric methods based on detection of rising product or diminishing substrate/cofactor. The attention was focused on glutathione S-transferase, glutathione peroxidase, glutathione reductase, catalase and superoxide dismutase. In HT29 cell lines, only catalase was affected – the activity was lowered by trans-nerolidol (25 µg/ml). On the contrary, in Caco2 trans-nerolidol at concentration 50 µg/ml increased the activity of glutathione reductase and superoxide dismutase. Moreover, superoxide dismutase activity was elevated also by lower concentration of trans-nerolidol and by Myrica rubra essential oil (50 µg/ml). Our results showed that decrease in catalase activity in cancer cells can contribute to anticancer effect of trans-nerolidol.

However, as this effect was not predominantly occurring for tested sesquiterpenes, it suggests that sesquiterpenes may impair antioxidant defence of cancer cells rather by different mechanism, e.g. reactive oxygen species production. This would also explain increased activity of some antioxidant enzymes which can be considered as the adaptation to excessive amount of reactive oxygen species. This project was supported by Czech Science Foundation (GACR) - Centre of Drug-Dietary Supplements Interactions and Nutrigenetics (Grant No. P303/12/G163).

P65 - ANTIOXIDANT AND GST INHIBITON ACTIVITIES OF COCOA PRODUCTS AND THEIR BIOACTIVE COMPONENTS 1 2 1 Ahmet Altay , Gülçin Sağdıçoğlu Celep , and Faruk Tahsin Bozoğlu 1 2 Middle East Technical University, Ankara, Turkey, Gazi Universit, Ankara, Turkey In recent years, it has been reported with a number of clinical studies that regular consumption of fresh fruits and vegetables in daily diet is correlated with lower health risks. This benefical effects are usually attributed to particularly polyphenols found in certain food sources. Polyphenols are set of bioactive compounds having benefits mainly on cardivascular health and flavonoids belong to the greatest group among them with notable specific action on arterial walls. In the literature flavanoids are mostly mentioned regarding to their antixidant and enzyme modulatory activities.

Lipid peroxidation is chain reaction that can cause deleterious effects on vasculo-endothelial systems causing atherosclerosis and related diseases. Glutathione S-transferases (GSTs) are a group of antioxidant enzymes that catalyze the conjugation reaction of reduced GSH with compounds containing an electrophilic center. In certain types of cancers such as breast cancer, GSTs activities increase significantly and inhibition of GSTs activitiy is considered to be a potential therapeutic target. In this research, processed (natural and alkaline cocoa cake) and non-processed (natural and alkaline cocoa) samples are selected as high flavonoid containing food sources. The antioxidant activities of both cocoa samples and their products are investigated using DPPH● radical scavenging and microsomal lipid peroxidation inhibition methods. Their total phenolic contents are also determined spectrophotometrically. GSTs inhibition activities of the cocoa samples are investigated and their IC50 values are determined. The main component of flavonoid ingradients of cocoa samples, (-)-epicatechin and (+)-catechin are measured analitically by HPLC. DPPH● radical scavenging activities of non-processed (natural and alkaline cocoa) and processed (natural and alkaline cocoa cake) samples were determined as 0.047, 0.064, 1.24, 2,034 with an IC50 (mg/ml) values, respectively. Total phenolic contents (mg GAE/g extract) were determined as 252 ,122, 7.37, 3.78, respectively. IC50 (µg/ml) values for GST inhibition activities of samples were determined as 27.75, 58.45, 399 and 836, respectively.

Epicatechin concentrations (mg EC/g extract) for non-processed and processed cocoa samples were determined as 7.87, 2.504, 0.257, 0.0525, respectively. Catechin concentrations (mg C/g extract) were 1.423, 2.709, 0.053, 0,0968, respectively. As a conclusion, cocoa samples particularly non processed natural cocoa samples are considered as potential GST inhibitors regarding to their low IC50 datas.

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P66 - ASSESSMENT OF UGT ENZYME INHIBITION IN HUMAN LIVER MICROSOMES AND UGT SUPERSOMESTM IN VITRO Vicky Birks, Jenny Lewis, Sebastian Lopez, Stephanie Geoffroy, Carina Cantrill, and Richard Cole Quotient Bioresearch, Rushden, United Kingdom Metabolism by UDP-glucuronosyltransferase enzymes (UGTs) represents a major Phase II conjugation pathway in humans. These enzymes catalyse the addition of glucuronic acid to xenobiotics and endogenous compounds, making them more polar and hence more readily excreted. In recent years there has been an increased interest in drug-drug interactions (DDIs) involving UGTs. This has led to regulatory authorities requiring an assessment of UGT inhibition potential of a candidate drug if glucuronidation is a predominant pathway of elimination. In response, the aim of this study was to establish methods for the in vitro measurement of UGT enzyme activities in human liver microsomes (HLM) and UGT SupersomesTM using standard metabolic substrates at Quotient Bioresearch. The following 6 UGTs were investigated using both human liver microsomes and SupersomesTM: UGT1A1, 1A4, 1A6, 1A9, 2B7 and 2B17.

An additional 7 UGTs were investigated using SupersomesTM only: 1A3, 1A7, 1A8, 1A10, 2B4, 2B10 and 2B15. The use of 96-well plates for both incubations and analysis of metabolite concentrations using internal standards (as relevant) via radio-HPLC or robust LC-MS/MS methods allowed the rapid screening for the potential of compounds to act as UGT inhibitors. Optimal incubation conditions with respect to protein concentration and time were first determined to provide linearity of substrate metabolism. This was followed by the detection of inhibition and generation of IC50 values for selected positive control inhibitors. Greater than 50% inhibition of UGT activity was elicited by the control inhibitors in all UGTs assessed. Where possible, selective inhibitors were used, viz. hecogenin (UGT1A4; IC50 HLM: 0.141 µM, SupersomesTM: 0.433 µM), niflumic acid (UGT1A9; IC50 HLM: 0.202 µM, SupersomesTM: 0.0554 µM) and fluconazole (UGT2B7; IC50 HLM: 2493 µM, SupersomesTM: 1937 µM). For the remaining UGTs, the non-selective inhibitors troglitazone and bisphenol A (BPA) (UGT2B4 only) were used, providing IC50 values ranging between 0.285 – 42.3 µM and 6.03 µM for troglitazone and BPA, respectively. In conclusion, robust methods to assess DDI risk in 13 individual UGTs have been developed at Quotient Bioresearch (Rushden) Ltd. These assays will be used in the future assessment of the potential of candidate drugs to inhibit UGTs and hence risk of clinical DDIs. P67 - IN VITRO ASSESSMENT OF DIRECT AND TIME-DEPENDENT INHIBITORY EFFECTS ON MAJOR HUMAN CYTOCHROME P450 ENZYMES BY SPASMOLYTICS Dominik Dahlinger, Sebastian Frechen, Sevinc Aslan, and Uwe Fuhr Department of Pharmacology, Hospital of the University of Cologne, Cologne, Germany Approximately one in six adults in the United States and Europe suffer from overactive bladder syndrome (OAB).

Anticholinergic agents are considered as the pharmacotherapy of choice, with seven drugs being approved for the German market. Yet, only limited information on the drug-drug interaction potential of these agents exists. Therefore, we examined inhibition of the seven major cytochrome P450 (CYP) enzymes by darifenacin, fesoterodin, oxybutinin, propiverin, solifenacin, tolterodin, trospium chloride. To assess direct inhibition, an in vitro cocktail of seven specific, clinically relevant model substrates was incubated with pooled human liver microsomes for 10 minutes. For all experiments, the major metabolites of the probe substrates were simultaneously analysed using a validated liquid chromatography - tandem mass spectrometry method and enzyme kinetics were estimated by determining IC50 values. These IC50 values were then converted to an inhibition constant Ki using the Cheng-Prusoff equation (1).

Subsequently, to assess time-dependent inhibition, a single concentration equivalent to the IC25, determined in the previous direct inhibition experiments, was used to examine TDI of the test compounds. After a 30 minute preincubation with NADPH, a TDI was identified by a decrease in enzyme activity. In this study, 49 IC50 experiments were conducted. In 18 out of these 49 cases, point estimates for the IC50 values were smaller than 100µM. In these cases CYP2D6 and CYP3A4 were most frequently inhibited (5 cases for CYP2D6; 5 cases for CYP3A4). The strongest inhibition was observed for darifenacin, propiverin and tolterodin on CYP2D6 with the calculated Ki in the lower micromolar range (associated 95% confidence intervals): darifenacin 0.0053 µM (0.0032 µM-0.0075 µM); propiverin 1.9µM (1.4µM-2.3µM) and tolterodin 2.7µM (1.7µM-3.6µM). The assessment of the time-dependent inhibitory potential of the spasmolytics most notably resulted in a decrease of CYP2B6 and CYP2D6 activty for oxybutynin (32% and 25% respectively) and a 28% decrease of CYP3A4 activity for darifenacin. These screening experiments suggest that in particular darifenacin, propiverin and tolterodin may have clinically relevant inhibitory effects on especially CYP2D6 in humans. To characterize the potential clinical impact of these interactions and recommend dosage modifications, further in vitro data is currently gathered, allowing for physiologically based pharmacokinetic modeling.

Reference: (1) Cheng Y, Prusoff WH. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099-108.

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P68 - REVERSIBLE INHIBITION AND MECHANISM-BASED INACTIVATION OF HUMAN CYP2J2 BY DRONEDARONE AND AMIODARONE Aneesh Karkhanis, Hui Yuan Lam, and Eric Chan National University of Singapore, Singapore, Singapore Dronedarone and amiodarone are anti-arrhythmic drugs (AADs) that have been reported to cause hepatotoxicity(1) and worsen cardiac failure condition(2). Dronedarone and amiodarone are metabolized to main circulatory metabolites N-desbutyldronedarone (NDBD) and N-desethylamiodarone (NDEA) respectively by CYP3A4 (Figure 1). Reports suggest that dronedarone(3) and amiodarone(4) are competitive and mechanism-based inactivators of CYP3A4.

CYP2J2, a predominantly cardiac CYP450 enzyme, plays a central role in maintaining cardiac homeostasis via metabolism of arachidonic acid to epoxyeicosatrienoic acids (EETs)(5). Both in-house and literature data(5) reported that AADs are substrates of CYP2J2. Since CYP3A4 and CYP2J2 demonstrate similar substrate specificity, we hypothesize that dronedarone, amiodarone and their main metabolites may also inhibit CYP2J2. Multiple probe substrate [astemizole (0.3-1.2 µM)] and test inhibitor concentrations (0-2 µM) were used to evaluate the reversible inhibition of recombinant CYP2J2. Time-, concentration- and NADPH-dependent inhibition experiments testing multiple inhibitor concentrations (0-10 µM) were performed to determine mechanism-based inactivation (MBI) of CYP2J2. Inactivation parameters (KI and Kinact) and partition ratio (r) were calculated to determine the potency and efficiency of MBI. Spectral difference scanning and dialysis experiments were performed to ascertain the formation of metabolite-intermediate (MI) complex. We observed that dronedarone, amiodarone, NDBD are strong reversible inhibitors and mechanism-based inactivators of CYP2J2 while NDEA showed no significant inhibitory potential.

Dronedarone was found to be more potent than amiodarone and NDBD with regards to both reversible inhibition (Ki: 0.029, 0.070, 0.364 µM respectively) and irreversible MBI of CYP2J2 (Kinact/KI: 675.4, 68.5, 49.1 mM-1 min-1 respectively). Partition ratio calculation confirmed that dronedarone (r: 3.3) inactivates CYP2J2 more efficiently compared to amiodarone (r: 20.7) and NDBD (r: 21.7). In terms of reversible inhibition, competitive kinetics was observed for dronedarone while mix-mode inhibition kinetics was determined for amiodarone and NDBD. Regarding MBI, none of the AADs and their main metabolites displayed MI complex formation with CYP2J2. In conclusion, we report for the first time that dronedarone, amiodarone and NDBD cause reversible inhibition and MBI of CYP2J2. This in turn fuels an impetus for scientists to investigate clinical drug-drug interactions and cardiac failure exacerbations associated with AADs via the respective inactivation of hepatic and cardiac CYP2J2.

1. Felser A, Blum K, Lindinger PW, Bouitbir J, Krähenbühl S. Mechanisms of hepatocellular toxicity associated with dronedarone--a comparison to amiodarone. Toxicol Sci 2013;131(2):480-90.

2. Køber L, Torp-Pedersen C, McMurray JJ, Gøtzsche O, Lévy S, Crijns H, et al. Increased mortality after dronedarone therapy for severe heart failure. N Engl J Med 2008;358(25):2678-87.

3. Hong Y, Hng Yeo RH, Chan ECY. Inhibitiory effects of dronedarone and its N-desbutyl metabolite on human CYP3A4 and CYP3A5 activities. In: 19th North American/29th Japanese ISSX meeting. San Fransisco, USA; October 2014.

4. Ohyama K, Nakajima M, Suzuki M, Shimada N, Yamazaki H, Yokoi T. Inhibitory effects of amiodarone and its Ndeethylated metabolite on human cytochrome P450 activities: prediction of in vivo drug interactions. Br J Clin Pharmacol 2000;49(3):244-53.

5. Xu M, Ju W, Hao H, Wang G, Li P. Cytochrome P450 2J2: distribution, function, regulation, genetic polymorphisms and clinical significance. Drug Metab Rev 2013;45(3):311-52.

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P69 - IN VITRO ASSESSMENT OF SKIN METABOLISM: COMPARISON OF METHODS AND IMPLICATIONS FOR WIDER APPLICATION Katie Plant, Rachel Upcott Gill, Victoria Hare, David Higton, Caroline Bauch, Phil Butler, and Clive Dilworth Cyprotex, Macclesfield, United Kingdom The largest organ in the human body, skin has an important role in providing a barrier to drugs and chemicals.

However it also acts as a route of entry for delivering drugs and chemicals into systemic circulation through absorption either directly or following bioactivation. The relative expression of xenobiotic-metabolising enzymes in skin compared with liver is much lower, especially for phase I cytochrome P450s. However it does express a range of other phase I enzymes such as flavin-containing monoxygenases, alcohol and aldehyde dehydrogenases, aldo-keto reductases and phase II enzymes, glutathione transferases, N-acetyl transferases and UDP-glucuronosyltransferases, which have been reported to have an increased expression profile in activated keratinocytes. First-pass metabolism in the skin should be evaluated for all drugs or chemicals with a topical route of administration. This is of importance due to the possibility of drugs or chemicals acquiring a sensitising potential following oxidative activation in the skin. With the practical limitations of using freshly excised human skin and other reconstructed 3D systems being relatively expensive there is a requirement for a simple in vitro system to assess skin metabolism. Skin S9 presents a low-cost option for an early assessment and despite low enzyme expression and activity levels could offer a more relevant option compared with liver S9. We have evaluated a panel of 12 different substrates including Phase I and Phase II markers alongside known sensitisers requiring activation in human skin S9 (derived from epidermis of abdominal skin, pooled from 3 donors), human liver S9 and monolayer-culture of human keratinocyte cell line, HaCaT cells.

Compounds were incubated in both S9 sources (1 mg/mL), with a mixture of cofactors NADPH, UDPGA and PAPS, for up to 120 mins (0, 10, 20, 40, 60, 120), and HaCaT cells for 24 hr (0, 1, 2, 4, 8, 24 hrs). At each incubation time point, supernatant was removed into pre-chilled acetonitrile to precipitate the protein. Substrate depletion was monitored by LC-MS/MS and metabolite identification performed using a Waters Xevo G2-S Q-Tof UPLC MS/MS platform. Despite observing a lack of parent depletion in skin S9 and the wide difference in levels of metabolism between skin S9 and liver S9, these systems still have potential for improving methods for identifying metabolic routes and predicting skin sensitisation. Addition of a relevant metabolic-activation system to increase the basal metabolic levels of HaCaT cells and thus keratinocyte activation may help to improve the prediction of skin sensitising potential and distinguish between irritants and sensitisers.

P70 - AHR, PXR, NRF2 AND GR INDUCE THE EXPRESSION OF THE HUMAN GLUTATHIONE S-TRANSFERASE ALPHA 1 (HGSTA1) IN HEPG2 CELLS María Del Carmen Martínez-Guzmán, Emmanuel González-Barbosa, Alejandro Mejía-García, and Guillermo Elizondo Department of Cell Biology, CINVESTAV-IPN, México City, México The glutathione S transferases (GSTs) are a superfamily of isoenzymes which play an important role on xenobiotics and endobiotics detoxification, cellular protection from oxidative stress, and modulation of signaling pathways [1].

Human GSTA1 (hGSTA1), a cytosolic isoform, is the most abundant GST in the liver, and is involved in the metabolism of carcinogenic compounds, chemotherapeutic agents and lipid peroxidation products [2]. In rodents, GSTA1 gene expression is under several xenobiotic/endobiotic sensors (XE-sensors) such as Pregnenolone X Receptor (PXR), Aryl Hydrocarbon Receptor (AhR), Constitutive Androstane Receptor (CAR) and the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) [3]. However, the information about hGSTA1 gene regulation is limited. Previous studies have shown that PXR, AhR and Nrf2 ligands increase hGSTA1 mRNA levels, suggesting the involved of these transcription factors in hGSTA1 expression [4]. Therefore, the aim of the present study was to better understand the regulation of hGSTA1 gene expression by XE-sensors. In order to identify XE-sensor responsive elements in hGSTA1 gene promoter an in silico analysis was performed. Putative elements for AhR, PXR, Glucocorticoid Receptor (GR), and Nrf2 were identified. Then, HepG2 cells were treated with rifampicin (RFP), β-naphthoflavone (β-NF), oltipraz (OPZ) and dexamethasone (DEX), ligands of PXR, AhR, Nrf2 and GR, respectively, and hGSTA1 mRNA levels were determined by qPCR. All treatments induced hGSTA1 expression, been DEX the stronger inducer followed by RFP<βNF<OPZ. The latter effects were inhibited by Actinomycin D indicating that the observed inductions were at transcriptional level. Transactivation assays were carried out through the transfection of HepG2 cells with a PGL4 reporter vector containing hGSTA1 gene promoter. Data indicated that RIF, β-NF, OPZ, and DEX treatment transactivated the hGSTA1 gene promoter. Western blotting revealed that the induction on hGSTA1 mRNA levels also results in increased levels of hGSTA1 protein. Furthermore, a spectrophotometric assay of conjugation of GSH to CDNB shows an increase in the total hGST activity. In conclusion, the present data showed that activation of several XE-sensors results in the induction of hGSTA1. In particular, we demonstrated for the first time that hGSTA1 is under GR regulation. (This work was supported by Consejo Nacional de Ciencia y Tecnología CO NAC yT, proje ct 153377).

1.

Laborde, E. 2010. Glutathione transferases as mediators of signaling pathways involved in cell proliferation and cell death. Cell Death and Differentiation. 17, 1373–1380.

2.

Hayes, J.D., Flanagan, J.U., and Jowsey, I.R. 2005. Glutathione transferases. Annu Rev Pharmacol Toxicol.

45, 51-88

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3.

Coles, B.F., and Kadlubar, F.F. 2005. Human alpha class glutathione S-transferases: genetic polymorphism, expression, and susceptibility to disease. Methods Enzymol. 401, 9-42.

4.

Aleksunes, L.M., and Klaassen, C.D. 2012. Coordinated Regulation of Hepatic Phase I and II DrugMetabolizing Genes and Transporters using AhR-, CAR-, PXR-, PPARalpha-, and Nrf2-Null Mice. Drug Metab Dispos 40, 1366-1379.

P71 - ARYL HYDROCARBON RECEPTOR (AHR) UP-REGULATES UBCM4 EXPRESSION IN THE MOUSE BRAIN Emmanuel González-Barbosa, Alejandro Mejía-García, and Guillermo Elizondo Department of Cell Biology, CINVESTAV-IPN, México City, México The aryl hydrocarbon receptor (AhR) is a transcription factor that regulates the expression of several genes in a ligand-dependent manner. Besides its role in xenobiotic detoxification, it has been determined that AhR modulates several cell processes such as cell proliferation and cell differentiation, among others [1]. Previous studies indicate that the AhR is also involved in the regulation of several genes encoding for ubiquitin enzymes (UBEs) [2]. The ubiquitin-proteasome system (UPS) is the main specific mechanism for degradation of nuclear and cytoplasmic proteins. It participates in a wide variety of regulatory pathways, from cell cycle control to immune system homeostasis [3]. Despite its importance, little is known about the transcriptional regulation of the UBEs. UbcM4 is an ubiquitinconjugating enzyme (E2), which interacts with several E3s such as Parkin. When interacting with Parkin promotes the degradation of proteins avoiding its accumulation and dopaminergic cell death in the midbrain region, specifically in the substantia nigra [4]. The aim of this study was to determine whether AhR mediates UbcM4 gene and protein expression in mouse brain. To achieve this, we first evaluated the AhR expression in the olfactory bulb, midbrain, hippocampus, striatum, cerebral cortex, brain stem and medulla oblongata regions of C57BL/6 Wild type (WT) mice by Real-time PCR. Results show that AhR is present in all the brain regions analyzed. We then treated WT and Ahr-null mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) an AhR ligand, and mRNA levels of UbcM4 were determined.

When wild-type mice were treated with TCDD increased UbcM4 levels were observed. In contrast, TCDD fails to induce its expression in AhR-null mice. Western blot analysis also shows an up-regulation of UbcM4 protein levels after TCDD treatment. Finally, chromatin immunoprecipitation (ChIP) studies revealed that the AhR interacts with the promoter region of the UbcM4 gene. The present data suggest that AhR plays an important role in the modulation of UbcM4, which, along with Parkin has an important role in the UPS-mediated degradation of proteins in the substantia nigra. (This work was supported by Consejo Nacional de Ciencia y Tecnología [CONACyT], Project 153377).

[1] Ma C., Marlowe J. L., Puga A. The aryl hydrocarbon receptor at the crossroads of multiple signaling pathways.

EXS. 2009; 99: 231-57.

[2] Reyes-Hernández O. D., Mejía-García A., Sánchez-Ocampo E. M., Cabañas-Cortes M. A., Ramírez P., ChávezGonzález L., González F. J., Elizondo G. Ube2l3 gene expression is modulated by activation of the aryl hydrocarbon receptor: implications for p53 ubiquitination. Biochem Pharmacol. 2010 Sep 15; 80(6): 932-40.

[3] Wang J. and Maldonado M. A. The Ubiquitin-Proteasome System and Its Role in Inflammatory and Autoimmune Diseases. Cell Mol Immunol. 2006 Aug; 3(4): 255-61.

[4] Morris L. G. T., Veeriah S. and Chan T. A. Genetic determinants at the interface of cancer and neurodegenerative disease. Oncogene. 2010 Jun 17; 29(24): 3453-64.

P72 - DEFINING THE REGULATORY NETWORKS THAT CONTROL THE IN VIVO EXPRESSION OF HUMAN CYTOCHROME P450 CYP2B6 USING A NOVEL LACZ REPORTER MOUSE MODEL 1 1 2 1 1 Aileen McLaren , Rita Moreno , Nico Scheer , Colin Henderson , and Roland Wolf 1 2 University of Dundee, Dundee, United Kingdom, Taconic Biosciences, Cologne, Germany Human cytochrome P450 CYP2B6 plays a major role in the metabolism certain important drugs such as cyclophosphamide. The expression of this protein is subject to marked individuality due to nuclear receptor-mediated regulation and genetic polymorphism. Increased interest in a reliable method to measure its induction has been stimulated by the need of routine assessment during drug and chemical safety testing, including FDA Guidance. Here we report on the development of a number of novel transgenic mouse lines that carry a LacZ reporter gene expressed under the control of the human CYP2B6 promoter, and either humanised or deleted for the nuclear receptors CAR (constitutive androstane receptor) or PXR (pregnane X receptor). Phenobarbital (PB) treatment of CYP2B6LacZ mice carrying murine CAR or PXR markedly increased reporter gene expression in the periportal and midzonal regions of the liver, a pattern which was confirmed by immunohistochemistry. A number of compounds which exert their effects through CAR or PXR were used to treat reporter mice carrying either endogenous CAR and PXR, or humanised for both nuclear receptors. Whereas 1,4-dichlorobenzene (DCB) or PB induced hepatic reporter expression with either murine or human nuclear receptors, with greater activity towards the human receptor, cyproterone acetate (CPA) resulted in LacZ staining only in the presence of mCAR/mPXR, and rifampicin (RIF) treatment induced reporter expression exclusively in mice humanised for CAR and PXR. Further analysis with CYP2B6LacZ reporter mice lacking either CAR or CAR and PXR determined that RIF was dependent on PXR, and DCB on CAR, for their ability to induce reporter expression. When CYP2B6LacZ/hCAR/hPXR reporter mice were treated with the anti-convulsant medications valproic acid, phenytoin and carbamazepine, reporter expression was observed for the latter two drugs but not for valproic acid. These provide powerful models allow us to measure the factors which affect CYP2B6 expression by

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analysing β-galactosidase activity. They also provide a powerful tool to study the in vivo regulation of gene expression by human nuclear receptors, as well as to investigate species differences in drug and chemical effects, these interactions being of central importance in drug/drug interactions in man.

This work was supported by the EU Innovative Medicine Initiative Joint Undertaking (115001) MARCAR project; http://www.imi-marcar.eu.

P73 - DETERMINATION OF CELLULAR SIGNALING PATHWAYS MODULATED BY EXPOSURE OF HUMAN HEPATIC CELLS TO CYLINDROSPERMOPSIN Antoine Huguet, Hélène Quenault, Yannick Blanchard, and Valérie Fessard ANSES, Fougeres, France Cylindrospermopsin (CYN) is a cyanotoxin produced by several freshwater cyanobacteria species. Due to eutrophization, these microbes are able to massively proliferate in the environment. The particularity of CYN is that released fraction in water during these blooms can represents up to 98% of the total produced. Therefore, humans are exposed to this toxin through drinking water and consumption of contaminated food. Experiments performed on rodents revealed that CYN induces damage largely restricted to the liver. In consequence, this toxin is recognized as a potential public health risk. Some studies reported that CYN irreversibly inhibits eukaryotic protein synthesis, but also induced DNA fragmentation through an increase of reactive oxygen species. However, others CYN-induced effects are probably contributors to the toxic process, and therefore information concerning the mechanisms of action producing this toxicity is needed. As liver appears as the main target organ of the toxin, the objective of this study was to investigate the cellular signaling pathways modulated by exposure of human hepatic cells to CYN. We used differentiated HepaRG cells as a human hepatic cell model. By using a non-targeted approach with microarrays, we studied the transcriptomic profile of differentiated HepaRG cells exposed for 24 h to a sub-toxic concentration of CYN (0.8 µM). After extraction of total RNA, nucleic acids were labeled with Cyanine 3 or Cyanine 5 and hybridized on miccroarrays. Thereafter raw data were normalized and filtered on threshold intensity. Compared with the control, only genes with expression level above 2 or below 0.5 with a P value less than 0.05 were selected. Using these selected genes, an analysis of biological functions modulated by exposure to CYN was performed and based on the gene ontology. The results indicated that 1061 genes had their expression increased and 1055 genes had their expression decreased significantly. Analyze of the biological and molecular processes related to the overexpressed genes indicated that exposure to CYN resulted in an up-regulation of RNA maturation, such as splicing, and translation.

However, from the down-regulated genes cluster, there were more biological processes which were mainly related to mitosis and metabolism of various components such as lipids, alcohols and organic acids. Our results on modulated functions and individual genes expression were consistent and indicated that exposure to CYN resulted in a strong deregulation of cell cycle and hepatic metabolism.

P74 - DIRECT TRANSCRIPTIONAL REGULATION OF CYTOCHROME P450, CYP2C8, BY PPARΑ IN HUMAN LIVER 1 1 1 2 1 1 Maria Thomas , Britta Klumpp , Stefan Winter , Thomas S. Weiss , Matthias Schwab , and Uli Zanger 1 2 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany, Surgery, University of Regensburg, Regensburg, Germany CYP2C8 is an enzyme whose importance in the metabolism of retinoic acid and arachidonic acid has been recognized for many years, but which has only recently been associated with the metabolism of more than 60 clinically used drugs, including mainly antimalarials and antidiabetics. However predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently it has been demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis, directly regulates the transcription of cytochrome P450, 3A4 (1). Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status (2) and clinical outcome of simvastatin (3) showed significant associations with protein level and enzyme activity (amodiaquine N-desethylation) of CYP2C8 by univariate statistical analysis.

Furthermore, siRNA-mediated knock-down of PPARα in primary human hepatocytes (PHH) resulted in up to ~60% and ~50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150% and >100%, respectively. Interestingly, ligand-mediated activation of PPARβ/δ with GW501516 led to a significant downregulation of CYP2C8 mRNA and activity. Combination of PPARα induction following PPARβ/δ knock-down resulted in a further increase of CYP2C8 mRNA expression compared to induction alone, suggesting that PPARα and PPARβ/δ play antagonistic roles in regulating CYP2C8. Chromatin immunoprecipitation scanning assay in primary human hepatocytes identified two regions of in vivo occupation for PPARα. Electromobility shift assay (EMSA) identified two direct binding repeats of PPARα, which appeared to have been previously characterized as regulatory elements for constitutive androstane receptor, CAR and pregnane X receptor (PXR) (4). Validation of the functional activity of these elements using luciferase reporter gene assays is currently in progress. Our data suggest significant, potentially direct regulation of CYP2C8 by PPARα. This might be important in view of the increased attention to CYP2C8-mediated adverse drug reactions and warrants further investigation.

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(1) Thomas M, Burk O, Klumpp B, Kandel BA, Damm G, Weiss TS, Klein K, Schwab M, Zanger UM. Direct Transcriptional Regulation of Human Hepatic Cytochrome P450 3A4 (CYP3A4) by Peroxisome Proliferator-Activated Receptor Alpha (PPARα). Molecular Pharmacology 2013 83(3):709-18 (2) Klein K., Thomas M., Winter S., Nuessler A.K., Niemi M., Schwab M., Zanger U.M. PPARα is a Novel Genetic Determinant of CYP3A4 In Vitro and In Vivo. Clinical Pharmacology & Therapeutics 2012 (91):1044-1052 (3) de Keyser CE, Becker ML, Uitterlinden AG, Hofman A, Lous JJ, Elens L, Visser LE, van Schaik RH, Stricker BH. Genetic variation in the PPARA gene is associated with simvastatin-mediated cholesterol reduction in the Rotterdam Study. Pharmacogenomics. 2013 Aug; 14(11):1295-304.

(4) Ferguson SS, Chen Y, LeCluyse EL, Negishi M, Goldstein JA. Human CYP2C8 is transcriptionally regulated by the nuclear receptors constitutive androstane receptor, pregnane X receptor, glucocorticoid receptor, and hepatic nuclear factor 4alpha. Mol Pharmacol. 2005 Sep;68(3):747-57.

Supported by the Robert Bosch Foundation, Stuttgart, Germany.

P75 - NEW METHODOLOGICAL APPROACHES FOR DISTINGUISHING DIRECT AND INDIRECT CONSTITUTIVE ANDROSTANE RECEPTOR (CAR) ACTIVATORS 1 1 1 2 1 Alejandro Carazo , Lucie Navratilova , Tomas Smutny , Karel Berka , and Petr Pavek 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Králové, Czech 2 Republic, Palacky University, Olomouc, Czech Republic The constitutive androstane receptor (CAR, NR1I3) is the critical transcriptional regulator of key xenobioticmetabolizing enzymes such as cytochrome P450 CYP3A4, CYP2C9 and specially, CYP2B6. CAR can be activate either via direct ligand binding to CAR ligand binding domain (LBD) or indirectly via inhibition of EGFR signaling with subsequent dephosphorylation of CAR at Thr38. The CITCO 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5carbaldehyde O-(3,4-dichlorobenzyl)oxime) is the prototype direct CAR ligand and phenobarbital is the classic indirect CAR activator. In our work, we introduce methods that can demonstrate direct interaction with the CAR ligand binding pocket. Further, we studied whether the tested compounds exert their activity through the EGFR signaling, phenobarbital-like activation. We tested the flavonoids chrysin, balcalein and galangin, which have been reported to be activators of CAR and interacting compounds with EGFR signaling. Employing a TR-FRET coactivator assay with recombinant human CAR and gene reporter-based CAR assembly assay, a consistent activation of CAR with flavonoids and phenobarbital was not observed. It was determined, however, that galangin, chrysin, and baicalin may repress EGFR-Tyr1068 autophosphorylation after EGF treatment. These data suggest that flavonoids chrysin, galangin and balcalein are not direct human CAR agonists and that they may interfere with EGFR signaling. This study thus demonstrates the application of new approaches for the testing of the direct CAR interaction of both natural and synthetic ligands. This research has been supported by the Czech Scientific Agency GACR303/12/G163.

P76 - OLAPARIB GENOTOXICITY OBSERVED IN A NEW AND IMPROVED P21 REPORTER MOUSE Tanya Frangova, Colin Henderson, Roland Wolf, and Michael McMahon University of Dundee, Dundee, United Kingdom Genomic instability is one of the most pervasive liabilities of tumour cells. To exploit this liability in the clinic many drugs that target the DNA damage response (DDR) have been developed and are in clinical trial. The first successful such drug, the PARP inhibitor olaparib, has recently been licensed for use. However, our understanding of the DDR is founded largely on in vitro studies and the phenomenon is underexplored in vivo. It is essential that we reduce this knowledge gap if we are to optimally use drugs targeting the DDR. To enable the necessary in vivo studies to be performed we have created a new and improved DDR reporter mouse line based on the DDR-responsive p21 gene.

Using viral 2A technology we have expressed two separate reporter molecules, luciferase and LacZ, from a single endogenous p21 locus. Both reporters rise-and-fall concomitantly with p21 protein levels. This enabled us to not only to follow DNA damage non-invasively in live mice (luciferase) but also to map DNA damage at single-cell resolution (LacZ ). Constitutive p21 expression was observed in a number of tissues in particularly in the bronchiolar epithelium, the gastro-intestinal tract, myocardiocytes and certain subpopulations of neurons. Exposure of the mice to ionising radiation (IR) induced the p21 reporter in essentially all tissues . The combination of olaparib with IR is currently being trialled in cancer patients in the hope that the former sensitises tumours to the DNA damaging effects of the latter. We found that olaparib pretreatment markedly increased IR-induced DNA damage signalling in the kidney and gastrointestinal tract. These data suggest that when applying this combination treatment in the clinic, there is the risk of increased toxicity to normal tissues.

P77 - REGULATION OF UDP GLUCURONOSYLTRANSFERASE 2B15 AND 2B17 EXPRESSION BY MICRORNAS IN PROSTATE CELLS Dhilushi Wijayakumara, Dong Gui Hu, and Peter Mackenzie Flinders University, Adelaide, Australia The UDP glucuronosyltransferases, UGT2B15 and UGT2B17 play a major role in the inactivation of the male hormone, testosterone and the more active derivative, dihydrotestosterone. Hence their altered expression in the prostate has the potential to impact on androgen signalling, androgen-dependent prostate function and the development of prostate cancer. As microRNAs negatively regulate gene expression by modulating mRNA stability

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and translatability, we sought to determine whether UGT2B15 and UGT2B17 expression is modulated by microRNAs in the prostate cell line, LNCaP. Using bioinformatic databases, we identified a putative miR-376c target site in the 3’untranslated regions (UTR) of both UGT2B15 and UGT2B17 mRNAs and a putative miR-331-5p target site in the UGT2B15 3’-UTR. Transfection experiments with LNCaP cells demonstrated that MiR-376c reduced both UGT2B15 and UGT2B17 mRNA levels and associated glucuronidation activities, as well as the activities of luciferase reporters containing UGT2B15 or UGT2B17 3’-UTRs. In contrast, miR-331-5p was more effective in reducing UGT2B15 mRNA levels and the activity of the reporter harbouring the UGT2B15 3’-UTR. This microRNA-mediated repression was significantly abrogated by mutating the miR-376c or miR-331-5p binding sites in the UGT 3’-UTRs. In support for a role for miR-376c in regulating UGT2B15 and UGT2B17 expression in prostate cells, an inverse correlation in the levels of this microRNA and the mRNAs of UGT2B15 and UGT2B17 was observed in prostate cancer cell lines versus normal prostate tissue. Overall, these data indicate that the expression of UGT2B15 and UGT2B17 is negatively regulated by the binding of miR-376c and/or miR-331-5p to sites within the UGT2B15 and UGT2B17 3’-UTRs in prostate cells.

P78 - SELECTION OF REFERENCE GENES FOR IN VITRO CYP INDUCTION STUDIES IN RAT, DOG AND HUMAN HEPATOCYTES USING REAL-TIME PCR Brigitte Gerin, Catherine Vandenplas, and Hugues Chanteux UCB Biopharma, Braine L'Alleud, Belgium Identification of the potential of new chemical entities to induce CYP450 is an important activity in drug development in order to assess the risk of drug-drug interaction. To this aim, quantification of mRNA is the gold-standard end point for in vitro CYP induction studies. Reference genes are frequently used to normalize mRNA between different samples.

However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances as it has been demonstrated for the well-known reference genes ß-actin and glyceraldehyde 3phosphate dehydrogenase. Thus, the selection of reference genes is critical for gene expression studies. The aim of the present study was to select 3 different reference genes for rat, dog and human which showed stable expression under the experimental conditions of a classical in vitro CYP induction using plated hepatocytes. To this aim, the monolayers of hepatocytes from rat, dog and human were treated for 48h with vehicle or ß-naphtoflavone, phenobarbital and rifampicin (dexamethasone for rat) as known reference inducers for CYP1A, CYP2B and CYP3A, respectively. At the end of treatment, the hepatocytes were collected and total RNA was extracted using the RNeasy Plus Mini Kit (RNeasy Micro kit for dog hepatocytes) from Qiagen and further reverse-transcribed into cDNA. PCR was then applied on cDNA from selected treated and non-treated samples to detect expression changes of minimum 9 putative reference genes for each species. The amplification of the reference genes was then statistically analyzed using geNorm algorithm (qBase+ from Biogazelle). This analysis allowed the selection of the 3 most stable reference genes for each species, i.e. B2m, Gapdh and Sdha for rat, TBP, SDHA and PGK1 for dog and HPRT, HMBS and RPL41 for human. Thereafter, PCR was performed on the same samples to measure the relative gene expression of the CYP1A, CYP2B and CYP3A. For each species, results were normalized using the geometric mean of 3 selected reference genes and demonstrated induction of CYP expression in line with the known effect of reference inducers. In conclusion, the present study identified 3 references genes per species that demonstrated stable expression within the frame of in vitro CYP induction studies and that were used successfully to normalize the expression of CYP450 genes.

P79 - UDP-GLUCOSYLTRANSFERASES (UGTS) FAMILY IN THE PARASITIC NEMATODE HAEMONCHUS CONTORTUS 1 1 2 1 Petra Matouskova , Lenka Lecová , Barbora SzotákováIvan Vokral , and Lenka Skálová 1 Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Hradec Králové, Czech 2 Republic, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic Haemonchus contortus, a hematophagous gastrointestinal parasite of sheep, has a great impact on livestock production worldwide. The prevalence of anthelmintic resistance has dramatically increased and has evolved into a serious problem facing small ruminant production in many countries. Resistant parasites have several mechanisms that protect them from the effects of anthelmintics. Highly effective mechanisms of resistance are differences in anthelmintics metabolism and their detoxification. Parasitic nematodes are armed with a large number of inducible metabolizing enzymes and transporters. An important role in the inactivation and excretion of anthelmintics plays glucuronidation or glucosidation, performed by enzymes from the family of UDP-glucuronosyl/glucosyl transferases (UGTs). In the genome of H. contortus more than 40 UGT genes were identified, which displays great variety in this family of enzymes. Previously, we have analysed biotransformation of benzimidazole anthelmintic - albendazole in adults of two H. contortus strains. The multi-resistant (WR) strain formed significantly more glucose conjugates of albendazole than the drug-susceptible (ISE) strain [1]. In this project we have selected several UGT candidates based on preliminary RNA-seq analysis of WR and ISE H. contortus strains and quantified their constitutive expression in adults. Generally our qPCR experiments were in accordance with the RNA-seq analysis and confirmed observed differences. However, those differences were quite small and did not fully reflect higher amount of glucose conjugates of albendazole in the WR strain. We propose that higher resistance of WR strain to benzimidazole anthelmintic can be

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partially caused by inducibility of UGT expression, rather than higher constitutive expression. Therefore, we have analysed inducible changes of selected UGTs upon albendazole treatment.

1. Vokral, I.; Jirasko, R.; Stuchlikova, L.; Bartikova, H.; Szotakova, B.; Lamka, J.; Varady, M.; Skalova, L., Biotransformation of albendazole and activities of selected detoxification enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics. Veterinary Parasitology, 196 (3-4), 373-381, 2013.

This study was supported by the Charles University in Prague (Research Project SVV 260 186).

P80 - ASSOCIATION BETWEEN HLA GENETIC POLYMORPHISM AND SEVERE CUTANEOUS ADVERSE DRUG REACTIONS ASSOCIATED WITH CO-TRIMOXAZOLE 1 1 2 3 Wichittra Tassaneeyakul , Thachanan Kongpan , Surakameth Mahasirimongkol , Parinya Konyoung , Pansu 1 1 2 1 Chumworathayi , Sirimas Kanjanawart , Nuanjun Wichukchinda , and Suda Vannaprasaht 1 2 Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, Department of 3 Medical Sciences, Ministry of Public Health, Bangkok, Thailand, Pharmacy Unit, Udon Thani Hospital, Udon Thani, Thailand Co-trimoxazole is a sulfonamide antibiotic which effective for treatment of several infections and for prophylaxis of Pneumocystis jiroveci pneumonia. This drug is the most common culprit reported drug for Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) in Thailand. Recently, severe cutaneous reactions induced by several drugs have been reported to be associated with Human leukocyte antigens (HLA). This study aims to identify HLA alleles that may be used as genetic markers for prediction of SJS/TEN-induced by co-trimoxazole. We enrolled 47 Thai patients with co-trimoxazole induced SJS/TEN and 91 co-trimoxazole tolerant patients. The HLA classes I and II were genotyped by the reverse sequence-specific oligonucleotide probes method. HLA genotyping of the SJS/TEN and tolerant control patients revealed that the frequencies of 4 alleles including HLA-B*15:02, HLA-C*06:02, HLA-C08:01 and HLA-DRB1*15:01 were significantly higher in the co-trimoxazole-induced SJS/TEN group compared with those observed in the controls. Moderate to strong associations between co-trimoxazole-induced SJS/TEN and HLA alleles including HLA-B*15:02, HLA-C*06:02, HLA-C*08:01 and HLA-DRB1*15:01 were found in our study population.

Whether these HLA alleles are a valid genetic markers for screening of severe cutaneous advers drug reactions associated with co-trimoxazole in other ethnics need to be investigated further.

P81 - DICARBONYL PROTEIN MODIFICATION: TYPE 2 DIABETES COMPLICATIONS AND METFORMIN SCAVENGING MECHANISM 1 1 1 2 2 2 2 Serrine Lau , Owen Kinsky , Tiffanie Hargraves , Tarun Anumol , Neil Jacobsen , Jixun Dai , Shane Snyder , and 1 George Tsaprailis 1 Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona, United 2 States, Department of Chemical and Environmental Engineering, University of Arizona, Tucson, Arizona, United States Reactive dicarbonyls, such as methylglyoxal (MG), are elevated in type-2 diabetes mellitus (T2DM) patients, and the ability of dicarbonyls to covalently modify proteins contributes to a number of diabetic complications. Protein-MG adducts may drive retinopathy, neuropathy, and many other common diabetic complications. The irreversible modification of R residues by dicarbonyls causes a net loss of positive charge, most commonly via hydroimidazolone formation. We identified the fibrinolytic system protein plasminogen (Pg) as a sensitive target of MG adduction, and which has functional consequences. Thus, molecular modeling indicated that modification of R561 at the cleavage site of Pg would impact enzymatic activation due to drastically altered energy of interaction. The current work has identified, via 2D gel and subsequent in-gel digestion and Orbitrap mass spectrometry analysis, the most sensitive sites of MG-R modification as R504 and R530. The functional significance of Pg adduction by MG was further tested in vitro. Pg activation was significantly delayed via modification with MG, as determined by cleavage of a chromogenic substrate (S-2251) by plasmin, the product of Pg activation. The findings indicate that MG-modification of Pg may disrupt the fibrinolytic cascade sufficient to represent an underlying mechanism of vascular complications in diabetics.

The T2DM first-line drug metformin (MF) significantly reduces diabetes-related endpoints and mortality more effectively than other glucose-lowering medications. We have examined whether, in addition to its ability to reduce hepatic gluconeogenesis, MF directly scavenges dicarbonyls as an additional mechanism to reduce T2DM complications. We synthesized a MF/MG cyclized product (183 mw) and characterized the product by 13C, 1H and HMBC NMR (Bruker DRX-600), X-ray diffraction analysis (Bruker APEX-II CCD) as well as ESI-MS/MS mass spectrometry (MH+, 184 m/z; Agilent 6490). Using an LC-MS-based multiple reaction monitoring analysis we measured MF and the imidazolinone product (IMZ) in human urine with nM sensitivity of detection. The IMZ was detected in all MF-treated T2DM subjects analyzed to date. Subjects examined that have not taken metformin show no IMZ peak, as anticipated. Quantitation of IMZ in a cohort of >90 MF-treated subjects is ongoing, utilizing specific gravity normalization. The data reveal that urine from every T2DM patient treated with MF contains this IMZ product as a result of direct reaction with MG, and increased levels of MF correlate with elevations in IMZ. In addition to lowering hepatic gluconeogenesis, MF may play a role in scavenging the highly reactive MG. The role of the IMZ in the reduction of diabetic complications warrants further study. (DK090958, ABRC1115, T32ES007091, P30ES006694).

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P82 - HUMAN PLURIPOTENT STEM CELL-DERIVED HEPATOCYTES WITH SUBSTANTIAL METABOLIC FUNCTIONALITY AND ADULT CHARACTERISTICS ARE HIGHLY SUITABLE FOR TOXICITY TESTING 1 2 1 2 2 Barbara Küppers-Munther , Annika Asplund , Mariska Van Giezen , Nidal Ghosheh , Jane Synnergren , and 1 Josefina Edsbagge 1 2 Takara Bio Europe (formerly Cellartis), Gothenburg, Sweden, University of Skövde, Skövde, Sweden Human pluripotent stem cells (hPSC) derived hepatocytes have the potential to serve as predictive human in vitro model systems for drug discovery and toxicity testing provided that they have relevant hepatic functions. However, up to now, the functionality of hPSC-derived hepatocytes has been insufficient for applications demanding high expression of multiple drug metabolizing enzymes. In order to obtain more functional hepatocytes from human induced pluripotent stem cells (hiPSC), we have developed a novel, highly robust 2D differentiation protocol. The resulting hiPSC-derived hepatocytes have substantial CYP1A, 2C9, 2C19, 2D6, and 3A4 enzyme activities and important adult hepatic features, such as low expression of fetal genes (e.g., CYP3A7 and alpha-fetoprotein) and high expression of adult genes (e.g., CYP2C9, 2C19, and 3A4). In addition, we will present results on toxicity testing using these novel hiPSC-derived hepatocytes with various read-outs, e.g. High Content Analysis and cell viability.

Importantly, we can generate homogenous hepatocyte cultures from multiple hPSC lines which show diverse CYP activity profiles reflecting inter-individual variation present in the population. These developments now provide a platform for an inexhaustible source of functional human hepatocytes suitable for toxicity testing and drug metabolism studies from different genetic backgrounds.

P83 - INVESTIGATION OF STABILITY AND REGENERATION OF XENOBIOTIC METABOLISM ENZYME ACTIVITIES IN PRIMARY HUMAN HEPATOCYTES DURING ISOLATION AND CULTIVATION. Melanie Kießig, Daniel Seehofer, and Georg Damm Charité Universitätsmedizin Berlin, Berlin, Germany The use of primary human hepatocytes (PHH) is still considered as the gold standard in in vitro testing of drug metabolism and hepatotoxicity. PHH are a well-balanced liver model regarding complexity and relevance, but especially the activity of xenobiotic metabolism enzymes such as Cytochrome P450 monooxigenases (CYP450) are known to decrease quickly during culture. The main disadvantage of this model is the limited availability of PHH due to tissue scarcity. Therefore optimization of cell isolation and cell culture conditions are perquisite for the utilization of limited resources. We hypothesize that especially PHH with insufficient energy resources (e.g. glycogen or lipids) are more sensitive to isolation stress resulting in loss of viability, function, and regeneration capacity when used in cell culture. Thus aim of the present study was to investigate the influence of glucose and insulin supplementation (GIS) during cell isolation on viability as well as stability and regeneration of xenobiotic metabolism enzyme activity and other hepatic functions. Therefore we simultaneously isolated PHH from the same donor with and without GIS using a two-step collagenase perfusion technique. Yield and viability were quantified after isolation. Cells were cultured for three days and xenobiotic enzyme activity (fluorescence-based CYP450 and Phase II assays), ammonia detoxification (urea synthesis), fibrinogen (ELISA) and albumin (ELISA) production were studied. Results were correlated with donor data. In case of a high donor Body Mass Index (BMI >30) our data indicated no significant effect of GIS on viability and hepatic functions. In case of a low or normal donor BMI we observed a positive effect of GIS on the viability of freshly isolated cells with varying improvements. In general CYP450 activities were decreasing during cultivation time of three days. CYP450 profiles strongly varied between donors and in some donors initial activity was very low. Phase II enzyme activities were more stable during cultivation and in some cases increased during the regeneration phase.

In some cases GIS was able to stabilize or increase selective CYP450 activities as well as phase II activities during cultivation in some of the donors with low or normal BMI. Other hepatic functions were usually not affected by GIS, but were slightly increased in one donor. We observed no significant correlation with other known donor anamnesis data (i.e. age, or diagnosis). Our results suggest that the effect of GIS depends on intracellular lipid content related to donor BMI. We conclude that in case of a low intracellular glycogen content before, during and after cell isolation the only alternative available energy source are stored lipids. Therefore we think GIS has the potential to stabilize energy resources in cells with little or no lipid content. Whether this results in better viability and functionality during culture needs further investigation with additional donors.

P84 - REDUCING HUMAN DRUG OVERDOSE USING MICRORNAS Dagmara Szkolnicka, Baltasar Lucendo-Villarin, Stuart Forbes, and David Hay University of Edinburgh, Edinburgh, United Kingdom Despite major progress in the knowledge and management of human liver injury, there are approximately 2000 cases per year of acute liver failure (ALF) in the United States (1,2,3). Acetaminophen (APAP) overdose is a major factor in ALF and accounts for approximately 50% of cases (4,5). Although current medical treatments for APAP overdose such as gastric lavage, activated charcoal ingestion, or N-acetyl cysteine (NAC) administration are successful, these treatments may cause side effects, including the inhibition of key metabolic functions of the liver (6,7), as well as negatively affect gastrointestinal or central nervous system (8). In the absence of notable effect and at the onset of severe liver decompensation and failure, liver transplant is the only effective approach to treat patients. While highly successful, this is not an off the shelf or scalable alternative. Therefore, other more specific treatments must be developed to improve patient outcome. Our study centres on the major metabolic cell type of the liver, the hepatocyte.

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In particular we were interested in the hepatocyte damage following drug overdose. While a number of hepatocyte models exist, their drawbacks outweigh their advantages, resulting in poor performance. To circumvent those issues, we employed pluripotent stem cell derived models which are scalable, renewable and from defined genetics. As microRNAs have been recently demonstrated to play an important role in the regulation of drug metabolism, we hypothesised that microRNAs could control enzymatic network promoting non-toxic drug metabolism and clearance, thereby reducing the compound cytotoxicity. Our experiments focussed on human APAP toxicity following overdose, with a view to identifying non-coding microRNAs which could reduce or attenuate this process. We identified a novel of antimicroRNA against miR-324-5p which not only reduced drug cytotoxicity(p = 0.03), but also reduced the depletion of glutathione (p = 0.018), the major cause of liver failure in vivo. In conclusion, we believe that these studies represent an important advance in the field, demonstrating the power of stem cell derived systems to model human biology and identify new targets which could lead to clinical translation in the future.

References: 1. Hoofnagle, JH., Carithers, RL., Shapiro C., et al (1995) Fulminant hepatic failure: summary of a workshop.

Hepatology. 21: 240-252.

2. Polson, JP., and Lee,WM.(2005) AASLD position paper: The management of acute liver failure. Hepatology. 41: 1179-97.

3. Fontana, RJ (2008) Acute liver failure including acetaminophen overdose. Medical Clinics of North America.

97(4):761-794.

4. Nourjah, P., Ahmad, SR., Karwoski, C., and Willy, M (2006) Estimates of acetaminophen(paracetamol) – associated overdoses in the United States. Pharmacoepidemiology and Drug Safety. 15(6): 398-405.

5. Bari K., and Fontana, RJ (2014) Acetaminophen overdose: what practitioners need to know. Clinical Liver Disease.

4(1): 17-21.

6. Makin, AJ., Wendon, J., and Williams, R (1995) A 7-year experience of severe acetaminophen-induced hepatotoxicity (1987-1993). Gastroenterology. 109: 1907-1916.

7. Kozer, E., and Koren, G. (2001) Management of paracetamol overdose: current controversies. Drug Safety. 24(7): 503-512.

8. Chun, LJ., Tong, MJ., Busuttil, RW., and Hiatt, JR (2009) Acetaminophen hepatotoxicity and acute liver failure.

Journal of Clinical Gastroenterology. 43(4).

P85 - UTILITY OF A MECHANISTIC MODEL TO INVESTIGATE THE UPTAKE OF ROSUVASTATIN IN HUMAN HEPATOCYTES AND TIME-DEPENDENT INHIBITION BY CYCLOSPORIN A AND CYCLOSPORIN AM1.

1 1 2 2 Michael Hobbs , Jackie Bloomer , Simon Mackay , and Mary Grant 1 2 GlaxoSmithKline, Ware, United Kingdom, University of Strathclyde, Glasgow, United Kingdom Rosuvastatin disposition is mediated by the transporters, organic anion transporting polypeptide (OATP) -1B1, -1B3 and sodium taurocholate co-transporting polypeptide (NTCP) and cyclosporin A (CsA) and its metabolite cyclosporin AM1(AM1) are potent inhibitors of these transporters [1]. An increase in potency has been observed with CsA in HEK293 cells over-expressing OATP1B1, when it was pre-incubated with certain statins [2]. In this study, the uptake kinetic parameters (Vmax, Km,u, CLactive,u, Pdiff,u and Fucell) of rosuvastatin into plated cryopreserved adult human hepatocytes from three donors (HU8116, HU8119 and HU1411) have been determined using a mechanistic model [3].

In addition, the nature of the inhibition of this uptake was determined using CsA and AM1. Plated hepatocytes were incubated in triplicate (at 37°C) using 6 concentrations of rosuvastatin over 8 time points, so that steady state was achieved. The uptake of a single concentration of rosuvastatin (1 µM) over 3 minutes was determined with 10 concentrations of CsA or AM1. CsA and AM1 were either co-incubated (i.e. added at exactly the same time) with rosuvastatin or pre-incubated with the hepatocytes (approximately 40 minutes) prior to addition of rosuvastatin. The mean (± standard error of the mean) parameter values for rosuvastatin were, Vmax 25.8 ± 12.8 pmol/min/106 cells, Km,u 3.66 ± 1.82 µM, CLactive,u 8.58 ± 2.31, Pdiff,u 0.90 ± 0.32 µL/min/106 cells and Fucell 0.17 ± 0.05. Both CsA and AM1 were potent inhibitors of rosuvastatin uptake in cryopreserved adult human hepatocytes from all three donors. The mean IC50 values for co-incubation and pre-incubation with CsA were, 0.45 and 0.32 µM, respectively and for AM1 were, 0.75 and 0.31 µM, respectively. There does not appear to be any statistical difference in the IC50 values between co-incubation and pre-incubation with either CsA or AM1. In conclusion, the uptake kinetic parameters of rosuvastatin in adult human hepatocytes from three donors were determined at steady state kinetics using a mechanistic model. CsA and AM1 were both potent inhibitors of rosuvastatin uptake in adult human hepatocyte preparations and there did not appear to be a time-dependent shift in the IC50 values with either CsA or AM1.

References: 1. Ho RH, Tirona RG, Leake BF, Glaeser H, Lee W, Lemke CJ, Wang Y and Kim RB (2006). Drug and Bile Acid Transporters in Rosuvastatin Hepatic Uptake: Function, Expression, and Pharmacogenetics. Gastroenterology, 130: 1793–1806.

2. Izumi S, Nozaki Y, Maeda K, Komori T, Takenaka O, Kusuhara H and Sugiyama Y (2015). Investigation of the Impact of Substrate Selection on In Vitro Organic Anion Transporting Polypeptide 1B1 Inhibition Profiles for the Prediction of Drug-Drug Interactions. Drug Metab. Dispos., 43: 235–247.

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3. Ménochet K, Kenworthy KE, Houston B, and Galetin A (2012) Use of Mechanistic Modeling to Assess Interindividual Variability and Interspecies Differences in Active Uptake in Human and Rat Hepatocytes. Drug Metab.

Dispos. 40 (9): 1744-1756.

P86 - IN SILICO ANALYSIS OF MICRORNA EXPRESSION PUTATIVLY REGULATED BY NUCLEAR RECEPTORS CAR, AHR AND ER Lyudmila Gulyaeva, Semyon Kolmykov, and Sergey Nechkin The Institute of Molecular Biology and Biophysics/Novosibirsk State University, Novosibirsk, Russia MicroRNAs play important role in post-transcriptional regulation of numerous genes, in particular the cytochrome P450. The effects caused by xenobiotics can be attributed to changes in the miRNA expression profile, thus leading to changes in gene regulation, which can explain the noxious effects that these chemicals have on human health. In our study we used in silico methods, trying to find out whether the DDT and BP affect the expression of miRNAs through the activation of the nuclear receptors (NRs): CAR (DDT) and AhR (BP), as well as estrogen receptor ER1 and ER2.

To find miRNAs that might be expressed due to CAR activity, we searched for a PB-responsive enhancer module (PBREM) in promoters of known rat genes and miRNAs. Promoter regions were extracted from the rat genome (rn5), 10000 nt upstream from the transcription start site of a gene (as annotated by the UCSC refGene track for rn5) and 10000 nt upstream from the start of a precursor miRNA sequence (miRBase v21). We searched for NR1, NR2 binding sites motif that is known to be found within PBREM in the extracted promoter regions allowing not more than 4 mismatches, on both positive and negative strands. The motif sequence is TGTACT.{3,4}TGACCT.{4,20}TCAACT.{3,4}TGACAC. The “{3,4}” designates any sequence of length 3 to 4 nt.

TGTACT.{3,4}TGACCT corresponds to NR1, TCAACT.{3,4}TGACAC corresponds to NR2. NR1 and NR2 binding sites had to be delimited by any base spacer of 4 to 20 nt. Rat genes that contain putative PBREM in their promoter were overlapped with annotated miRNAs (miRBase v21) to make a list of putative intragenic miRNAs (rno-mir-208a, rno-mir-3577, rno-mir-193b, rno-mir-1843a, rno-mir-190a-2, rno-mir-190a-1). No intergenic miRNAs that have PBREM in the promoter were found. We extracted a list of rat genes with DREs in the promoter from “Sun et al. 2004” [1] and overlapped them with the miRNA annotation (miRBase v21) to make a list of putative AhR induced miRNAs (rno-mir336, rno-mir-207, rno-mir-208b, rno-mir-3573, rno-mir-326, rno-mir-483, rno-mir-126a). A comprehensive list of human ER sites was found in “Chin-Yo Lin et al. 2007” [2]. Using the tool liftOver (UCSC Genome Browser utility tool) we identified the corresponding homologous regions in rat (rn5). We then searched for the presence of consensus EREs (AGGTCA.{1,10}TGACCT) with up to two mutations. Genes that have promoters containing ERE binding sites found were selected. Only one of these genes had a single annotated miRNA (rno-mir-6327), which was selected as potentially induced by ER. None of the ERE sites was found in the 10 000 bp upstream region of intergenic miRNAs (miRBase v21). This study showed that expression of microRNA could vary under the xenobiotic induction through NRs activation. The mechanism of regulation of miRNA under the exposure of xenobiotics warrants further experimental investigation. This work was supported by RFBR (grant №15-03-01700).

References: 1. Sun YV et al. Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences.

Nucleic Acids Res. 2004; 32(15):4512-23 2. Lin CY et al. Whole-genome cartography of estrogen receptor alpha binding sites. PLoS Genet. 2007; 3(6):e87.

P87 - IN SILICO MODELLING OF OATP1B1 INHIBITION BASED ON LIGAND STRUCTURE INFORMATION 1 2 Susanne Winiwarter and Ismael Zamora 1 2 AstraZeneca R&D, Mölndal, Sweden, Lead Molecular Design, Sant Cugat del Vallès, Spain The influence of transporter proteins for the uptake of compounds into different tissues is increasingly recognized, as is the potential for drug-drug interactions as a consequence. Thus, understanding the possibility of a drug to be a substrate or an inhibitor of a transporter protein is of key importance in the design of safe and effective therapeutics.

We show here the development of a ligand based in silico model for inhibition of OATP1B1. This organic anion transporter protein enables active transport of amphiphilic drugs into hepatocytes which results in clearance from the blood while leading to increased intrahepatic concentration.1 Some drugs, for instance many statins, utilize this transporter to be taken up into the liver to reach a sufficient concentration at their target.2 Consequently, new pharmaceutics likely to be co-medicated with such drugs need to avoid any interaction with this transporter.

Especially, drug discovery projects in the cardiovascular area, where statins are considered standard of care, benefit from better understanding the structure-transporter activity relationship early on. The presented model is based on a structural overlay of six known OATP1B1 inhibitors using the shape similarity software ROCS.3 For each of the six compounds the conformation that fitted best with all other compounds was used. The resulting ‘transportophor’ was trained with almost 150 compounds with literature OATP1B1 inhibition data.4 For each compound the conformation that was most similar to one of the six known inhibitors was selected and the similarity score calculated. Compounds with an inhibition <40% were considered as inactive whereas compounds with an inhibition >60% seen as active in this dataset. Using this definition and the similarity scores for each compound a similarity threshold to distinguish actives from inactives could be defined. We subjected in house compounds for which OATP1B1 inhibition had been measured to the same procedure and used the established similarity threshold to distinguish between likely inhibitors and non-inhibitors. Projects can use this model to estimate inhibition likelihood for their compounds based on the

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similarity to the ‘transportophor’. Additionally, the structural overlay to the most similar known inhibitor gives information on which part of the structure of a new compound may give raise to the potential interaction, which will be useful in drug design. The model is available to drug projects at AZ using a web-interface.

References: 1. Hilgendorf et al DMD 2007, 35: 1333-1340 2. Elsby et al Clin.Pharmacol.Ther. 2012, 92: 584-598 3. ROCS, OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com 4. Karlgren et al PharmRes 2012, 29: 411-426 P88 - IN SILICO PREDICTION FOR BIOPHARMACEUTICS OF ORAL PIPERINE USING GASTROPLUS SOFTWARE. 1 1 2 3 Chatsiri Jesadakultavee , Korbtham Sathirakul , Pleumchitt Rojanapanthu , and Ariya Khunvichai 1 2 Faculty of Pharmacy, Mahidol University, Bangkok, Thailand, Drug Discovery and Development Center, Thammasat 3 University, Bangkok, Thailand, Medica Innova, Bangkok, Thailand The aims of this study were to predict and develop piperine absorption model in human by using in vitro, in vivo and/or in silico estimates parameters for input in GastroPlus software. In addition models were built to demonstrate the involvement of the CYP enzyme in the metabolism of piperine. The simulation for absorption and plasma concentration-time profiles after oral administration of piperine in human were performed based on compartmental model integrated with physicochemical properties and biopharmaceutical properties such as solubility, permeability, molecular weight, diffusion coefficient, particle size and particle density. Seven human oral absorption models, all predicted input, clearance input scaling from mice, clearance input scaling from rat, PK value input from fitting parameters, input from fitting PK parameters plus predicted Vmax and Km, input from fitting PK parameters plus CL from well-stirred model and input from fitting PK parameters plus CL from parallel tube model, were established, respectively. Predicted plasma concentration time profiles were compared with references which obtained from literature. The best described absorption model was from integrated input from fitting PK parameters while other models were overestimate or underestimate in elimination phase. The predicted AUC of all models except model that calculated CL from well stirred and parallel tube model were within 2-fold error. In addition, prediction of dose escalation absorption trend to show linear pharmacokinetics. Parameter sensitivity analysis suggested that solubility and permeability of piperine were only small impact on percent absorption and percent bioavailability. Base on the result, the models were well describe absorption phase meanwhile elimination phase needed for more precise inputs.

Thus metabolism pathway of piperine should be further investigated.

P89 - IN SILICO PREDICTION OF ORAL BIOAVAILABILITY Michael Lawless, John DiBella, Michael B. Bolger, Robert D. Clark, Eva Huehn, Marvin Waldman, Jinhua Zhang, and Viera Lukacova Simulations Plus, Lancaster, California, United States Oral bioavailability is an important pharmacokinetic property that can determine the fate of a compound in clinical trials. Thus, predicting oral bioavailability prior to first-in-human dosing is highly desirable. We created a database of 62 drugs including their percent oral bioavailability (F%) and dose. The major clearance of each compound is mediated by cytochrome P450 (CYP) enzymes. The F% values varied from 3% (fluphenazine) to 99% (diazepam, galantamine, glimepiride, indomethacin, and tamsulosin) with an average of 60%. Our strategy for F% prediction involves integration of artificial neural network ensemble (ANNE) model predictions based on 2D molecular structures with physiologically based pharmacokinetic (PBPK) modeling. Predicted aqueous and biorelevant solubility, pKa, logD, gastrointestinal permeability, fraction unbound in human plasma, blood to plasma concentration ratio, and Michaelis-Menten Km and Vmax parameters for five major CYP isoforms (1A2, 2C9, 2C19, 2D6, and 3A4) were used as inputs for the PBPK models. A hierarchical set of models was used to determine CYP metabolism. First, classification models predicted whether each compound was a substrate for each of five major CYPs. Next, sites of metabolism were predicted for those compounds that were projected to be substrates. Finally, Km and Vmax predictions were made for each predicted site of metabolism. Oral bioavailability for each drug was computed using a PBPK model for a 35-year-old American male. The tissue:plasma partition coefficients for the PBPK models were calculated using the Lukacova method (1). Absorption of the compound was simulated with the Advanced Compartmental Absorption and Transit™ (ACAT™) model in humans under fasted conditions. CYP metabolism in gut and liver was accounted for utilizing built-in enzyme expression levels in gut and liver along with predicted Km and Vmax values. All molecules were correctly predicted to be substrates of the CYPs associated with their major clearance pathways. Furthermore, these pathways had the highest predicted CYP intrinsic clearance in 42 of the 62 molecules. Overall, 68% of the molecules were predicted within 2-fold of their reported oral bioavailability, indicating that this methodology can be used to prioritize compounds for development.

(1) V. Lukacova, N. Parrott, T. Lave, G. Fraczkiewicz, M. Bolger, W. Woltosz. General approach to calculation of tissue:plasma partition coefficients for physiologically based pharmacokinetic (PBPK) modeling, AAPS National meeting, Atlanta, November 15-20, 2008.

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P90 - EVALUATION OF CYP450 AND TRANSPORTERS EXPRESSION AND ACTIVITY IN HEPARG CELL LINE UNDER DIFFERENT CONDITIONS Flavia Storelli, Christel Bruggmann, Fabienne Doffey-Lazeyras, Caroline Samer, Jules Alexandre Desmeules, and Youssef Daali Geneva University Hospitals, Geneva, Switzerland HepaRG is a newly developed human hepatoma cell line which is able to differentiate to both hepatocyte-like and biliary-like cells after reaching confluence. When differentiated, HepaRG cells have shown good drug-metabolizing properties compared to primary human hepatocytes. Previous works have shown that confluent HepaRG cells start to differentiate when adding 2% DMSO in the culture medium. However, DMSO is well known to induce cell death. In order to optimize CYP450 activity while decreasing cell apoptosis, we tested different culture conditions for differentiation by varying DMSO concentration from 0 to 2% and by adding growth factors EGF and HGF at concentrations of 10 ng/ml. CYP activity was assessed using a cocktail approach by LC-MS/MS. Expression of several CYP450, phase II enzymes UGT1A1 and UGT2B7 and various uptake and efflux transporters was assessed with Nanostring® technology. The culture media tested were all compared to differentiation medium MIL720 from Biopredic®. The higher DMSO concentration, the more differentiated cells were observed under microscopy.

However, cell viability was significantly decreased when adding DMSO up to 2%. Considering CYP450 activity, DMSO increased significantly the activity of CYP3A4, 2B6 and 1A2. The addition of growth factors EGF and HGF was found to have a negative impact on cell differentiation and thus CYP activity, but significantly improved cell viability.

There was a good correlation between CYP activity and CYP expression (P<0.05), except for CYP1A2. In all conditions tested, CYP 2D6 showed a weak activity and expression levels were undetectable. UGT1A1 and UGT2B7 transcripts were found at appreciable levels and were influenced by DMSO concentration. Considering hepatic transporters, the same profile regarding influence of DMSO was observed. Efflux transporters MRP2, MRP3 and MDR1 (P-gp) levels were high, whereas BSEP, BCRP and MRP1 levels were low. Considering hepatic uptake transporters, OCT1 was largely expressed. OATP1B1, 2B1, OCT3, OAT2 expression was found in acceptable levels.

On the contrary, NTCP and OATP1B3 transcripts were undetectable. Differentiation medium containing 1.5% showed similar viability compared to MIL720 from Biopredic used as reference, with slightly lower CYP450 activities. This medium was thus chosen for further metabolism experiments on the HepaRG cell line.

P91 - EXPRESSION AND FUNCTION OF DRUG TRANSPORTERS IN 3D HUMAN LUNG TISSUE MODEL 1 1 1 1 2 1 Diana Feller , Monika Avdicevic , Edit Kiss , Judit Rapp , Peter Krajcsi , and Judit E. Pongracz 1 2 Humeltis Ltd., Hosszúhetény, Hungary, SOLVO Biotechnology, Budaörs, Hungary Lung cancers are the most common and are amongst the deadliest malignant diseases worldwide. While the various subtypes have different molecular and clinical characteristics, they still frequently share similar treatment approaches.

The similarity in treatment stems from the fact that the majority of patients are presented at advanced or even metastatic stage of the disease where surgical resection is not an option. Additionally, specific biological therapies are only applicable in a small fraction of cases therefore combination chemotherapy is frequently used in an attempt to prolong survival and to slow down inevitable disease progression. Unfortunately, pre-existing or acquired multidrug resistance makes chemotherapy ineffective. Multidrug resistance (MDR) often originates from activation of drug transporters that pump out chemotherapeutic drugs from cells. Drug transporter compositions are tissue microenvironment-dependent making the expression and functional analysis of transporters extremely difficult.

In our study mRNA, protein levels and functional analysis of four drug transporters, namely, ABCB1, ABCG2 SLC22A3 and SLCO4C1 were tested in human non-small cell lung cancer (NSCLC) subtypes, adenocarcinoma and squamous cell lung carcinoma cell lines. The effect of microenvironmental stimuli on drug transporter expression and function, 2 and 3 dimensional co-cultures were set up using primary human lung cell types including epithelial, endothelial and fibroblast cells. Significant differences have been identified in drug transporter expression between adenocarcinoma and squamous cell carcinoma cell lines as well as primary NSCLC subtypes. Additionally, traditional 2D monocultures of primary human lung cell types expressed drug transporters at very low levels that did not change significantly even in 3D co-cultures when the aggregates consisted only of epithelial cells and fibroblasts. However, incorporation of endothelial cells into the aggregates triggered a drastic change in the drug transporter expression pattern making it greatly similar to the drug transporter pattern of the primary healthy human lung.

Modeling the human lung in vitro using a 3D co-culture aggregate is not only a proper tool for analyzing drug transporter expression, but combining the carefully selected tissue model with MultiDrugQuant Kit (by Solvo Biotechnology) makes the setup suitable for analysis of drug transporter function at similar to in vivo conditions.

Based on the above results new therapeutic agents can be tested more reliably in our newly developed in vitro model system.

P92 - INFLUENCE OF MECHANICAL PROPERTIES OF COLLAGEN HYDROGEL MATRICES ON METABOLIC ACTIVITY OF SANDWICH-CULTURED PRIMARY HEPATOCYTES Peter Agbekoh, Catherine Henderson, Philip Riches, and Mary Grant University of Strathclyde, Glasgow, United Kingdom Sandwich-cultured primary hepatocytes are regarded as a good in vitro model for drug metabolism studies. Despite the widespread use, the system is not ideal and further investigations are required to optimise the system to better

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reflect in vivo conditions and lead to reproduction of in vivo functionality. The type and properties of the extracellular matrix used in the in vitro system are known to significantly influence the characteristics of the sandwich-cultured hepatocytes including their metabolic activity. This study was aimed at investigating the effect of mechanical properties (stiffness and hydraulic permeability) of collagen hydrogel matrices on metabolism of 7-hydroxycoumarin (7-HC) by sandwich-cultured primary adult rat hepatocytes. Primary hepatocytes were isolated from adult Sprague Dawley and maintained on collagen gel matrices with varying collagen content (0.1%, 0.3% and 0.45% w/v collagen).

Hepatocytes were seeded at 1.5 x 105 cells/cm2 and extent of metabolism of 7-HC assessed at 48h, 72h and 7d. The mechanical properties of collagen gels significantly influenced drug metabolic activity of the cells with levels of 7-HC glucuronide and 7-HC sulphate significantly (p<0.05) higher in primary hepatocytes maintained on 0.1% w/v collagen gels metabolic compared to hepatocytes maintained on 0.3% and 0.45% w/v collagen gels. Mechanical measurements indicated that stiffness and hydraulic permeability of collagen gels were inversely correlated, with stiffness and hydraulic permeability values increasing and decreasing respectively, with increasing collagen content of gels. Stiffness increased from 0.70 ± 0.08 kPa to 1.30 ± 0.09 kPa for 0.1% w/v to 0.45% w/v collagen gels, respectively. Furthermore, morphological features exhibited by hepatocytes in culture also demonstrated that primary hepatocytes displayed a comparative preference for the least stiff/ more permeable gel matrix. The formation of bile canaliculi was more extensive and retained throughout the 7 day culture period on the 0.1% w/v gels compared to 0.3% and 0.45% w/v gel matrices. Data from this study indicate that changes in mechanical properties of the extracellular matrix must be very important considerations during optimisation of sandwich culture systems for xenobiotic metabolism studies.

P93 - SKIN BIOAVAILABILITY, METABOLISM AND LOCALIZATION OF AN ANTI-ACNE ACTIVE INGREDIENT IN THE FORM OF A PRO-DRUG 1 2 1 1 1 Carine Jacques-Jamin , Nicolas Desbenoit , Corinne JeanJean ,Cecile Viode , Sylvie Daunes-Marion , Daniel 1 2 1 Redoulès , Gilles Frache , and Sandrine Bessou-Touya 1 2 Pierre Fabre Dermo-Cosmétique, Toulouse, France, Luxembourg Institute of Science and Technology, Sanem, Luxembourg Acne is a chronic inflammatory disease of the pilosebaceous follicle progressing by eruptions until spontaneous extinction. It involves several phases in its origin. The main ones are hyper-seborrhea and the latter depends mainly on the proliferation of the germ of Propionibacterium acnes (P.acnes) leading to inflammatory acne lesions.

We proposed to take advantage of the enzymatic activity of P. acnes to release both antibacterial and antiinflammatory active ingredients, from a unique lipase activated diol-fatty acid pro-drug molecule.

The potential release of the fatty acid part, i.e linolenic acid, and the diol part from the diol-fatty acid pro-drug, was initially checked in tubo using a recombinant lipase from P. acnes. In this study, we investigated its metabolism by using an ex vivo skin organ culture, as close as possible of the in use conditions. For the kinetic study of the pro-drug metabolism, experiments were carried out with an emulsion containing 1% of radiolabeled parent molecule (14C), topically applied in a finite dose during 24 hours. Radiolabeled linolenic acid was followed and quantified using radioHPLC. To localize the pro-drug and the linolenic acid, as metabolite, TOF-SIMS surface imaging experiments were performed. Following the skin permeation, skin samples were punched and frozen under liquid nitrogen. The frozen samples were sectioned into slices and analysed by TOF-SIMS. Following topical application of the pro-drug emulsion, 5.5% of the applied dose was recovered into the stratum corneum and 5.1% of the applied dose was located into the skin and the receptor fluid. The results confirmed the detection of free radiolabeled linolenic acid in the ex vivo tissue model which resulted from the topically applied prodrug, biotransformed after endogenous enzymatic activity. As the enzyme availability increased in the skin, the proportion of the metabolite increased.

Moreover, our results demonstrated a reservoir effect of the parent compound as a potential source ready to be activated by lipases not only of endogenous origin but also of P.acnes origins The TOF-SIMS analyses permitted to detect the pro-drug and the linolenic acid into the stratum corneum, the epidermis, and pilosebaceous unit according to the results obtained during the penetration study.

To conclude, using different analyses, either quantitative or qualitative, we confirmed the proof of concept of pro-drug targeting and releasing active ingredients after topical application, using ex-vivo skin models. Consequently, we supported the innovative potential of a selective and local action for an anti-acne treatment by targeting the infected pilosebaceous follicule and releasing both anti-inflammatory anti-P.acnes activities.

Key words: diffusion, ex vivo skin model, metabolism, acne, prodrug P94 - COMPARISON OF HEPATOTOXICITY INDUCED BY PYRROLIZIDINE ALKALOIDS AND THEIR N-OXIDES 1 2 1 Mengbi Yang , Jianqing Ruan , and Ge Lin , 1 2 School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, Soochow University, Suzhou, China Pyrrolizidine alkaloids (PAs) are natural toxins presented in various plants and also contaminated in a variety of foodstuffs. Intake of PA-containing plants, herbs, and PA-contaminated foodstuffs causes numerous cases of liver injury, especially hepatic sinusoidal obstruction syndrome (HSOS) worldwide. There are two types of toxic PAs, namely retronecine-type and otonecine-type. Both types of toxic PAs elicit hepatotoxicity via metabolic activation in the liver to reactive metabolites, which form metabolite-derived protein adducts (prrole-protein adducts) leading to

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toxicity. Retronecine-type PAs also has their corresponding N-oxides, and both forms co-exist naturally with the content of N-oxides less, equal or even higher than the parent PAs. However, the hepatotoxicity of PA N-oxides remains largely unknown. The present study aims to compare the hepatotoxicity induced by retronecine-type PAs and their corresponding N-oxides. Male ICR mice were orally dosed with alkaloid extract of Gynura segetum (containing retronecine-type PAs (seneciphylline and senecionine) only) or Gynura pseudochina (containing the corresponding PA N-oxides (>92% of total amount of PAs/N-oxides) at the dosage of 0.167 mmol (total PAs or PA N-oxides)/kg/day for 3 consecutive days. After treatment with PA N-oxides, the corresponding PAs with comparable concentrations of their N-oxides were found in the blood. In both treated groups, remarkable hepatotoxicity was evidenced by the observation of hepatic hemorrhagic necrosis and ascites. Serum ALT activity was significantly elevated in both PA-treated groups (158.7±2.7 IU/L) and PA N-oxide-treated groups (95.2±15.2 IU/L). Immunostaining demonstrated significant damage of endothelial cells in central vein and sinusoids in the liver, which corresponded to characteristic lesion of clinical HSOS. The sinusoids sinusoidal damage was found much severe in PA-treated mice compared to PA N-oxide-treated mice. Pyrrole-protein adducts were detected in the serum and liver of both treated groups while absent in control groups. Significantly higher pyrrole-protein adduct levels were found in PA-treated group (15.5±3.3 nmol/g in liver and 3.5±0.8 umol/L in serum) compared with PA N-oxides-treated group (9.4±0.7 nmol/g in liver and 2.0±0.3 umol/L in serum). All the results suggested a similar toxicity mechanism but lower hepatotoxic potency of PA N-oxides. In summary, the present study investigated the hepatotoxicity of PA N-oxides and directly compared with the hepatotoxicity induced by their parent PAs for the first time. PA N-oxide intoxication was revealed to be due to metabolic conversion into the corresponding PA firstly followed by a same metabolism-induced toxicity mechanism.

Furthermore, hepatotoxic potency of PA N-oxide was found to be significantly lower than that of the parent PA. [The present studies were supported by Research Grant Council of Hong Kong (GRF Grant, Project No.: 471310 and 469712) and CUHK (Direct Grants: 4054134, 4054047 and 2041744].

P95 - COMPARISON OF IN VITRO METHODS FOR ASSESMENT OF REACTIVITY OF ACYL GLUCURONIDE METABOLITES 1 1 2 1 Juho Hokkanen , Sanna-Mari Aatsinki , Toni Lassila , and Ari Tolonen 1 2 Admescope Ltd., Oulu, Finland, University of Oulu, Department of Chemistry, Oulu, Finland Reactive acyl glucuronide conjugate (AGs) metabolites can be formed in the presence of carboxylic acid moiety in a parent drug or a phase I metabolite. These conjugates can mediate adverse drug reactions (ADRs) and drug induced liver toxicity (DILI) by binding to cellular enzymes and proteins, either via transacylation reaction or via glycation that is intermediated by acyl migration to aldehyde isomer [1]. In literature, the rate of acyl migration reaction is suggested as a measure of reactivity and further the toxicity of acyl glucuronide metabolites [2 - 3], although no thorough validation of the method has been published. The aim of this work was to compare several in vitro methods for assessment of the acyl-glucuronide conjugate caused toxicity, using test battery of 13 compounds with known in vivo toxicity (diclofenac, furosemide, gemfibrozil, ibuprofen, indomethacin, isoxepac, montelukast, naproxen, repaglinide, telmisartan, tolmetin, valproic acid, and zomepirac). The assays were based on a) acyl migration rate, measured by taking account the half-life of 1-O-β-AG produced with liver microsomal incubation and simultaneous formation of isomeric AGs (to eliminate the effect of hydrolysis), b) total amount of acyl glucuronide formation in HepaRG cells, and c-d) in vitro cytotoxicity and mitochondrial toxicity experiments with HepaRG cells. The results were used also in combination of the maximum daily in vivo dose levels of these compounds. Of the used methods, the rate of acyl migration showed clearly the best correlation to the classification of the test compounds based on their known toxicity profile. The observed total formation of acyl glucuronide, cytotoxicity, or mitochondrial toxicity did not have correlation to the toxicity classification of the test compounds, with or without combination to their maximum daily dose.

[1] Sophie L. Regan, James L. Maggs, Thomas G. Hammond, Craig Lambert, Dominic P. Williams, P. Kevin Park.

Acyl glucuronides: The good, the bad, and the ugly. Biopharmaceutics & Drug Disposition (2010), 31: 367 - 395.

[2] Zdhesheng Chen, Tom G. Holt, James V. Pivhichny, Kwan Leung. A simple in vitro model to study the stability of acylglucuronides. Journal of Pharmacological and Toxicological Methods (2007), 55: 91 - 95.

[3] Ryoko Sawamura, Noriko Okudaira, Kengo Watanabe, Takahiro Murai, Yshomasa Kobayashi, Masaya Tachibana, Takashi Ohnuki, Kayoko Masuda, Hidehito Honma, Atsushi Kurihara, Osamu Okazaki. Predictability of idiosyncratic drug toxicity risk for carboxylic acid containing drugs based on the chemical stability of acyl glucuronide. Drug Metabolism and Disposition (2010), 38: 1857 - 1864.

P96 - COMPARISON OF UMBILICAL CORD BLOOD SAMPLES BETWEEN URBAN AND RURAL BIRTHS FOR POLYCYCLIC AROMATIC HYDROCARBON DNA ADDUCTS Monica Valentovic, Jesse Cottrell, John Ball, Jason Childress, Lawrence Harbrecht, and Brenda Mitchell Marshall University, Huntington, West Virginia, United States Polycyclic aromatic hydrocarbons (PAHs) are bioactive compounds that are biotransformed by cytochrome p450 enzymes. The reactive metabolites are known to bind covalently to DNA and form PAH-DNA adducts. There is a paucity of data comparing umbilical cord blood levels of PAH-DNA adducts between the rural Appalachian area and their urban counterparts. The overall goal of this study was to isolate and quantitate PAH-DNA adducts from umbilical cord blood of children from urban and rural areas of West Virginia. This study may provide better understanding of the causes of increased health disparities individuals in urban and rural West Virginia. A comparative

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cross-sectional study was conducted on 172 babies born at Cabell Huntington Hospital. The babies were divided as 79 rural and 93 urban. Rural and urban locations were based on Rural-Urban Commuting Area Codes. Cord blood was collected at the time of delivery, between April 2013-February 2014. Maternal use of tobacco was recorded for each patient.. However, umbilical cord blood cotinine levels were measured as a biomarker for cigarette smoke exposure of the fetus. Umbilical cord blood was processed to isolate DNA. DNA and potential PAH-DNA adducts were hydrolyzed and prepared for HPLC analysis. The concentration of DNA retrieved per sample was recorded using NanoDrop UV spectroscopy. High-performance liquid chromatography (HPLC) analysis was then initiated for measurement of benzo(a)pyrene tetrol each sample. Benzo(a)pyrene tetrols were confirmed in patients that were positive for cotinine. PAH-DNA adducts were detected in both rural and urban umbilical cord samples. DNA levels were similar between urban and rural umbilical cord samples (p>0.05). Our studies showed that newborn babies have sufficiently high enough exposure to PAH that is biotransformed to detectable PAH-DNA adducts. Further evaluations are needed to determine whether the PAH-DNA adducts can be attributed to a specific source for urban and rural newborns.

P97 - CYTOCHROME P450 - MEDIATED METABOLISM OF THE TYROSINE KINASE INHIBITOR PONATINIB TO REACTIVE ELECTROPHILES Rumen Kostov, De Lin, Jeffrey TJ Huang, Sheila Sharp, Colin Henderson, and Roland Wolf Jacqui Wood Cancer Centre, Medical Research Institute, University of Dundee, Dundee, United Kingdom Ponatinib (Iclusig) is a tyrosine-kinase inhibitor whose primary target is BCR-ABL tyrosine-kinase. Ponatinib, whose disposition is mediated by the cytochrome P450 system, is used for the treatment of imatinib-resistant chronic myeloid leukemia (CML) and Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL). Ponatinib can cause severe arterial and venous thrombosis and occlusions, pancreatitis and hepatotoxicity. The mechanisms of toxicity however, remain unknown. In order to study the pathways of ponatinib disposition we have investigated its metabolism by a panel of purified recombinant human CYP450 enzymes (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYPC8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP2E1). Ponatinib was metabolised down multiple pathways and in agreement with published data, cytochrome P450 CYP3A4 played a predominant role in the production of the major N-demethylation product, with the hydroxylation reaction catalyzed principally by CYP1A2.

Interestingly, on the addition of glutathione to the incubation medium a glutathione conjugate was formed which paralleled the disappearance of the hydroxylation product (PonaOH-276-II). This suggested that the initial reaction involved the generation of an epoxide. We also found that addition of mouse or human liver cytosol or recombinant mouse glutathione S-transferase Gstp1 to the incubation medium catalysed the formation of a glutathione conjugate in presence of CYP1A2. The formation of reactive metabolites and their trapping by glutathione indicates that the electrophilic intermediates formed primarily by CYP1A2 may be sufficiently reactive to bind to the cysteine groups of proteins in vivo and could contribute to the mechanism of ponatinib toxicity.

P98 - CYTOTOXIC ACTIVITIES OF FOUR POLYGONUM SPECIES FROM TURKEY 1 2 3 4 Gul Ozhan , Tugba Yilmaz-Ozden , Nina Taher Nasabi , and Mine Kocyigit 1 2 Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey, Department of 3 Biochemistry, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey, Department of Pharmacology, Institute of 4 Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey Polygonum L. (Polygonaceae) species are used in traditional folk medicine in many countries [1, 2]. In Turkey, some Polygonum species are consumed as a popular vegetable and its regional name is “Madımak”. Four Polygonum species, namely P. aviculare L., P. patulum Beib. subsp. pulchellum (Lois.) Leblebici, P. lapathifolium L. and P. istanbulicum Keskin (endemic) were chosen for this study. The plants were collected from Istanbul, Turkey. The aerial parts of the plants were dried and extracted with three different solvents (ethanol, methanol and chloroform). The obtained extracts were screened for their cytotoxic activity against normal rat kidney epitelial (NRK-52E) and Henrietta Lacks (HeLa) cervical cancer cell lines using the MTT assay [3]. The results showed that, all the ethanol extracts possessed selective cytotoxic activity against HeLa cells and the most potent activity was obtained with P. lapathifolium (IC50 8.70 µg/mL) while they were not cytotoxic on NRK-52E cells. The methanol extracts of the plants displayed non-selective cytotoxic activity. Methanol extract of P. lapathifolium showed the most potent cytotoxic activity against both normal (IC50 4.81 µg/mL) and cancer (IC50 8.65 µg/mL) cells. Chloroform extracts of the tested plants did not display cytotoxicity against HeLa cancer cell lines whereas showed cytotoxic activity against normal cell lines. In this study among the tested species, P. patulum subsp. pulchellum and P. istanbulicum extracts have been screened against NRK-52E and HeLa cell lines for the first time. The observations from this study suggest that these Polygonum species can act as a natural source for drug discovery and cancer studies.

1. Jia W, Gao W, Tang L. Antidiabetic herbal drugs officially approved in China. Phytotherapy Research, 17:11271134, 2003.

2. Tuttolomondo T, Licata M, Leto C, Savo V, Bonsangue G, Letizia Gargano M, Venturella G, La Bella S. Ethnobotanical investigation on wild medicinal plants in the Monti Sicani Regional Park (Sicily, Italy). Journal of Ethnopharmacology, 153:568-586, 2014.

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3. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. The Journal of Immunological Methods, 65:55-63, 1983.

P99 - DETERMINATION OF THE ROLE OF O-ACYL-GLUCURONIDE MIGRATION IN THE COVALENT BINDING OF A DRUG CANDIDATE József Pánczél, Anzar Siddiqui, Jens Riedel, Markus Kohlmann, Angela Dudda, and Gert Ulrich Kürzel Sanofi, Frankfurt, Germany Idiosyncratic adverse drug reactions have been associated with covalent binding of drug molecules. Thus, several in vitro systems have been proposed to study and rank order drug candidates for their extent of covalent binding either in human liver microsomes or in human hepatocytes. Acylglucuronides are of particular interest due to their potential to form unstable glucuronides resulting in acylmigration and potential formation of reactive species. Here we describe the investigation of the covalent binding potential of a drug candidate forming an acylglucuronide as main metabolite in human but to a significant lower amount in animals. The in vitro metabolism of 3H labeled drug candidate called Cpd X (R-CH2-COOH, 5 M, 3H la be l in the „R” moiety) has been studied in liver microsomes and cryopreserved hepatocytes of human and animal species. A low metabolic turnover was found with no significant covalent binding with and without cofactor NADPH in liver microsomes. The main metabolic pathways in human hepatocytes were the O-acyl glucuronidation with subsequent glucuronide migration (9-13%, in sum). Only in human hepatocytes, a slight potential of covalent binding (75-96 pmol / 2x106 cells) was observed. The O-acyl-glucuronide metabolites were the sole metabolites suspected for covalent binding potential, as their abundancies were far below 5% in animal hepatocytes and the other human metabolites were observed at least in one in-vitro animal species with similar or higher abundances, as seen in human hepatocytes. In order to confirm the relation between the slight, but distinct covalent binding and the O-acyl glucuronide formation and - migration, the 3H- Cpd X was incubated in liver microsomes of human, dog and rat with or without addition of NADPH, and with or without addition of activated glucuronic acid (UDPGA) for 0h, 2h and 6h in sevenfolds replicates. The metabolic profiles of these incubations, particular the formation of O-acyl-glucuronides, were compared to the corresponding quantity of covalent binding to protein pellets in these incubations. The slight potential of covalent binding has been confirmed in liver microsomes of human and dog (~27 pmol eq/mg) after addition of UDPGA in 6h incubation compared to 2h incubations (~14 pmol eq/mg). There was no significant difference in the amounts of O-acylglucuronide metabolites found after 2 and 6 hours incubations. Nevertheless the migration of the glucuronic acid was pronounced (t1/2 O-acyl migration: 2.6h, at pH: 7.4, determined by LC-MS) after 6h incubation. The 6h incubations in liver microsomes of human and dog without UDPGA showed definitely lower covalent bindings (~8-15 pmol eq/mg) than they were obtained in the presence of UDPGA. These slight but distinct differences in the amounts of covalent binding in liver microsomes with or without cofactors suggested that the O-acyl glucuronide metabolites of Cpd X were the root cause of binding to the protein pellets after O-acyl migration, supposingly by glycosylation.

P100 - DNA METHYLATION PROFILES OF BISPHENOL A IN HUMAN NEUROBLASTOMA CELLS Gul Ozhan, Mine Senyildiz, and Sibel Ozden Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Istanbul, Turkey Bisphenol A (BPA), as synthetic monomer used in industry in the production of polycarbonate plastic and epoxy resins, has endocrine disruptor properties and high risk on human health. Continuous release of free BPA into food, beverages, and the environment have resulted in a widespread human exposure to this chemical. Defining the hazard identification under the risk assessment of chemicals is an important step in the elucidation of the mechanism of toxicity. Recently, showing the role of endocrine effects of environmental chemicals on the changes in gene expression may be associated with epigenetic mechanisms are emerging as an alternative way (Kundakovic and Champagne, 2011: Rezg et al., 2014). Therefore, epigenetic events such as DNA methylation and histone modifications may play an important role in the toxicity of endocrine disrupting chemicals such as BPA. The aim of this study is to investigate the effects of BPA on global DNA methylation, CpG promoter DNA methylation and gene expressions in human neuroblastoma cell line (SH-SY5Y). 5-hydroxymethylcytosine (5-hmC), oxidation product of 5methylcytosine (5-mC), is considered as a new epigenetic DNA modification with relevant roles in cell homeostasis regulating DNA demethylation and transcription. For DNA methylation analysis, cells were treated with BPA at 0, 100 nM, 1 and 10 M conce ntra tions for 48 a nd 96 hours . IC50 va lue of BP A wa s de te rm ine d a s 183 a nd 129 µM in S HSY5Y cells by MTT and neutral red uptake test. We observed that alterations on the global levels of 5-mC and 5-hmC levels after 48 and 96 hours BPA exposure. To better understand the epigenetic effects of BPA in SH-SY5Y cells, CpG island promoter methylation and expression profile of key tumor-suppressor genes is currently analysed. It is expected that results from this study will contribute to better understanding of key molecular events in the toxicity of endocrine disrupting chemicals for risk assessment process.

References: Kundakovic, M., Champagne, F.A. 2011. Epigenetic perspective on the developmental effects of bisphenol A. Brain Behav. Immun., 25, 1084–1093.

Rezg, R., El-Fazaa, S., Gharbi, N., Mornagui, B. 2014. Bisphenol A and human chronic diseases: Current evidences, possible mechanisms, and future perspectives. Environment International, 64, 83–90.

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P101 - INDUCED INTERSTITIAL PULMONARY FIBROSIS (IPF) MODEL: UNLABELED BLEOMYCIN DISTRIBUTION AND EARLY IPF MARKERS IDENTIFICATION BY MALDI IMAGING 1 2 1 1 1 1 David Bonnel , Mary McElroy , Emeline Falaux , Gaël Picard de Muller , Fabien Pamelard , Gregory Hamm , 2 1 Stephen Madden , and Jonathan Stauber 1 2 ImaBiotech, Loos, France, Charles River Laboratories, Discovery Research Services, Edinburgh, United Kingdom Interstitial Pulmonary Fibrosis (IPF) is a chronic and progressive lung disease. It is currently believed that fibrosis is caused by aberrant alveolar epithelial cell activation and repair leading to fibroblastic/myofibroblastic foci, accumulation of extracellular matrix and irreversible destruction of the lung tissue. Combined with classical histological staining, Mass Spectrometry Imaging (MSI) was used to improve the understanding of the Bleomycin IPF rat model and to identify several potential early biomarkers of this pathology. Rats were administered seven doses of Bleomycin delivered to the lungs at 1 mg/kg. Control animals received seven doses of saline. Both Bleomycin and saline were administered by oropharyngeal aspiration route. Three treated animals were followed for 7 days; three control and three treated animals were followed for 22 days. Several lung fresh sections were prepared and analyzed by mass spectrometry imaging (MALDI-FTICR, SolariX 7T, Bruker, Germany). Data treatment and statistical analysis were performed using Multimaging software (ImaBiotech, France). We describe a process which combines MSI and classical staining approach directly on tissue to follow the distribution of molecules implicated in fibrosis, and allows a better understanding of lung damage and repair. Indeed most of the identified biomarkers were located in extracellular medium and in the plasma membrane, as some upregulated lipids (included LPA18:0 and LPA16:0) and novel lipids not previously associated with fibrosis. All these lipids biomarkers were characterized on-tissue section using MS/MS experiments (CID mode) and high mass accuracy measurement (below ppm level). These lipids are known to be involved in cell signaling, chemotaxis or membrane stability, which might be associated with disregulated alveolar epithelial repair and/or fibrosis. Combination of MSI and histological staining provides information regarding molecules distribution and identification in the tissue by studying their co-distribution and by comparing their relative abundance at active sites of fibrosis. Here we identified a number of biomarkers which may be useful diagnostic and/or therapeutic targets and therefore useful tools to help understand the efficacy and safety of novel treatments in IPF. Correlation between disease related alterations and molecules localization provides reliable information about disease mechanism taking place within tissue.

P102 - LEVODOPA AND DOPAMINE-INDUCED POST-TRANSLATIONAL MODIFICATION OF Α-SYNUCLEIN Terrence Monks, Aram Cholanians, and Serrine Lau Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona, United States α-Synuclein (α-Syn) plays a central role in synucleinopathies and comprises the major component of aggregates present in Lewy Bodies (LB), a major hallmark of Parkinson‘s disease (PD). Moreover, dopamine (DA) mediated αSyn post-translational modifications (PTM) play an important role in the etiology of the disease, although the consequences of such PTMs remains the subject of debate. The DA precursor, levodopa (L-DOPA), remains the drug of choice for the treatment of PD. We therefore compared the effects of L-DOPA and DA on α-Syn aggregation.

Western blot analysis of L-DOPA and DA-modified α-Syn revealed that both compounds stimulated α-Syn aggregation, although the pattern of the PTMs differed. MALDI-TOF analysis of DA-modified α-Syn revealed an addition of 4.5 kDa to the α-Syn monomer, and up to 9 kDa to the α-Syn dimer, suggesting poly-DA interactions with α-Syn, crosslinking of protein, and aggregation of α-Syn. Since their are no cysteine residues in α-Syn, modification by DA primarily involves either oxidation of methionine or adduction of amino groups in lysine (K). We have previously shown that quinones preferentially form adducts with K-rich proteins, and in particular with proteins expressing KXK or XKKX motifs. α-Syn is a K-rich protein (10.7%K; 15/140 AAs) and contains 6 KXK/XKKX motifs. Acetylating α-Syn -K residues with either acetylsalicylic acid or acetic anhydride, and peptides containing the KXK or XKKX motifs substantially ameliorated DA-induced α-Syn aggregation. Under physiological conditions, L-DOPA and DA oxidation occur in the presence of GSH, and L-DOPAQ and DQ are scavenged by GSH to form thiol conjugates of the mercapturic acid pathway. At least one of these thiols (5-Cys-DA) is elevated in patients with advanced PD and both 5-Cys-DA and 5-NACys-DA are present in human striatum. Carbon at position 5 is the primary site of thiol adduction (C5:C2 = 4:1) leaving the remaining carbon as a reactive center for electrophilic addition to α-Syn, since C6 is the site of attack by the side-chain nitrogen to form aminochrome. This greatly limits the potential for post-adduction chemistry and reduces the complexity of the subsequent MS analysis. Mono-L-DOPA and DA conjugates oxidize and generate α-Syn adducts in a pH-dependent manner similar to DA, but fail to cause insoluble α-Syn aggregates. The findings reveal that scavenging of L-DOPA and DA quinones by cysteine and/or GSH has profound effects on their interaction with α-Syn, and on subsequent aggregation reactions. Thiol modulation of α-Syn PTMs and of α-Syn aggregation suggests potential interventions to alleviate α-Syn -dependent cytotoxicity. (P30ES006694 & T32 ES007091).

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P103 - PROTECTION OF MALE MICE AGAINST CARBON TETRACHLORIDE-INDUCED HEPATOTOXICITY BY SOY ISOFLAVONES Yasuhiro Masubuchi, Yuma Hayashi, and Miri Nomura Chiba Institute of Science, Choshi, Japan It has been reported that female mice are less susceptible to hepatotoxicity induced by acetaminophen and carbon tetrachloride (CCl4). These findings suggest that endogenous estrogens can act as a protective factor in the pathogenesis of drug-induced liver injury. Soy isoflavones such as genistein are one of the major members of phytoestrogens, which are expected to exert effects similar to estrogens on various tissues including the liver. In the present study, we investigated the effects of genistein and daidzein on CCl4-induced hepatotoxicity in male mice, and characteristically compared to the resistance of female mice to the hepatotoxicity. Male and female CD-1 mice were given CCl4 intraperitoneally (1.0 mmol/kg), and were sacrificed 3, 8 and 24 hr after dosing. Some male mice were also given isoflavone aglycons intraperitoneally (10 mg/kg; genistein, daidzein, and isoflavone aglycon mixture from soybean), which were pre- (16 hr before) and coadministered with CCl4. Hepatotoxicity was assessed by serum leakage of alanine aminotransferase (ALT) and histochemical analysis. Hepatic expression of cytokines and other inflammatory mediators were assayed by real-time RT-PCR. Serum ALT leakage observed at 24 hr after the treatment of male mice with CCl4 was much higher than female mice. The data on ALT leakage were supported by the hematoxylin/eosin staining of liver sections. Both genistein and daidzein attenuated the CCl4 hepatotoxicity, but daidzein, an inactive analogue of genistein for inhibition of tyrosine kinase activity, was less effective than genistein.

On the other hand, isoflavone aglycon mixture was ineffective. It is thus suggested that genistein is a major hepatoprotective constituent in soy isoflavone aglycons, and some other component in the aglycon mixture may antagonize the action of genistein. Treatment of male mice with CCl4 caused an increase in hepatic expression of heme oxygenase-1, tumor necrosis factor-α and interleukin-6 prior to the development of hepatotoxicity. The expression of all of these genes was suppressed by genistein, which suggests that genistein exerts its protective effects through the potent antioxidant and/or anti-inflammatory activities. This mode of action may be different from those of the resistance of female mice to the CCl4 hepatotoxicity, which does not accompany the suppression of cytokine expression in female liver. In conclusion, soy isoflavones attenuate CCl4-induced hepatotoxicity, but there may be a mechanistic difference in protective actions between the endogenous estrogens involved in the gender difference and exogenously supplied phytoestrogens.

P104 - ROLE OF BIOTRANSFORMATION AND FREE RADICALS IN 3,4-DICHLOROANILINE NEPHROTOXICITY IN VITRO Gary Rankin, Christopher Racine, Dianne Anestis, and Monica Valentovic Marshall University, Huntington, West Virginia, United States 3,4-Dichloroaniline (3,4-DCA) is used in the manufacture of dyes, pesticides and numerous chemical intermediates.

3,4-DCA is also a metabolite of several pesticides and has been identified as a priority environmental pollutant. 3,4DCA induces several toxicities, including nephrotoxicity in vivo and in vitro (1). However, it isn’t clear if 3,4-DCA is bioactivated to nephrotoxic metabolites by the kidney. This study was designed to test the hypotheses that the kidney bioactivates 3,4-DCA to a nephrotoxic metabolite and that free radicals play a role in 3,4-DCA nephrotoxicity in vitro.

Isolated renal cortical cells (IRCC; 3 mL 4.0 X 106 cells/ml) from male Fisher 344 rats were treated with DMSO (vehicle control), 1.0 mM 3,4-DCA, a pretreatment, or pretreatment plus 1.0 mM 3,4-DCA, followed by incubation for 2 hr at 37oC. The pretreatments used in this study were: antioxidants [ascorbate (2.0 mM), glutathione (1.0 mM), and αtocopherol (1.0 mM)]; cytochrome P450 (CYP) inhibitors [piperonyl butoxide (0.1 mM) or metyrapone (1.0 mM)]; a cyclooxygenase inhibitor [indomethacin (1.0 mM)]; a peroxidase inhibitor [mercaptosuccinate (1.0mM)]; and a flavin monooxygenase (FMO) inhibitor [methimazole (1.0 mM)]. Cytotoxicity was determined using lactate dehydrogenase (LDH) release analysis. LDH release was significantly increased from control levels (~18-20% release of total LDH) by 3,4-DCA treatment (~32-37% release of total LDH). The FMO inhibitor methimazole and the peroxidase inhibitor mercaptosuccinate did not significantly reduce 3,4-DCA cytotoxicity, suggesting that FMO or peroxidase-mediated bioactivation does not play a role in 3,4-DCA cytotoxicity. Also, the CYP inhibitor metyrapone did not attenuate 3,4DCA cytotoxicity, indicating that CYPs inhibited by metyrapone do not contribute to 3,4-DCA nephrotoxicity. However, 3,4-DCA cytotoxicity was significantly reduced by pretreatment with the antioxidants ascorbate (26 ± 0.4% release of total LDH) or glutathione (25 ± 0.6% release of total LDH), the CYP inhibitor piperonyl butoxide (27 ± 1.2% release of total LDH), and the cyclooxygenase inhibitor indomethacin (29 ± 1.0% release of total LDH). These results suggest that multiple enzyme systems, including cyclooxygenase and piperonyl butoxide-inhibited CPYs, may play a role in the bioactivation of 3,4-DCA to cytotoxic metabolites, and that free radicals play a role in the in vitro renal biotransformation of 3,4-DCA and/or its mechanism of inducing cell death. (Supported in part by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence.)

1. Valentovic, M.A., Yahia, T., Ball, J.G., Hong, S.K., Brown, P.I., Rankin, G.O. 3,4-Dichloroaniline acute toxicity in male Fischer 344 rats. Toxicology 124; 125-134, 1997.

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P105 - VALIDATION OF HUMAN PRECISION-CUT LIVER SLICES AS AN EX VIVO MODEL TO STUDY DRUG INDUCED CHOLESTASIS 1 2 1 Suresh Vatakuti, Peter Olinga , Jeroen Pennings , and Geny Groothuis 1 Division of Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute for Pharmacy, University of 2 Groningen, Groningen, The Netherlands, National Institute for Public Health and the Environment, Bilthoven, The Netherlands Introduction: Hepatotoxicity is one of the major reasons for withdrawal of drugs from the market due to the fact that the liver is the major organ involved in drug metabolism and thus can generate toxic metabolites. There is a need to screen the molecules for drug-induced hepatotoxicity in human at an earlier stage. Transcriptomics is a technique widely used to screen molecules for toxicity and to unravel the possible mechanisms of toxicity. The aim of this study was to validate human precision-cut liver slices (PCLS) as an ex vivo model to predict drug-induced cholestasis and identify the possible mechanisms of cholestasis toxicity using gene expression profiles. Methods: Five hepatotoxicants, which are known to induce cholestasis (alpha-naphthylisothiocyanate, chlorpromazine, cyclosporine, ethinylestradiol and methyltestosterone) were used at concentrations inducing low (<20%) and medium (30-50%) toxicity, based on ATP content and/or or LDH leakage. Human PCLS were incubated with the drugs in the presence of a non-toxic concentration (60 µM) of a bile acid mixture (mimicking the portal vein composition) as model for the bile acid induced cholestasis. Transcriptomics analysis was performed using Illumina bead arrays. Significantly regulated genes (FC ≤ or ≥ 1.5 and adjusted p-value ≤ 0.05) were identified using LIMMA and pathway analysis of regulated genes was performed using IPA pathway analysis software. Results: Dose dependent increase in the number of regulated genes was observed. Regulated genes include bile acid transporters ABCB11 (BSEP) and ABCB4 (MDR3), and the cholesterol transporters ABCG5 and ABCG8. Pathway analysis revealed that signaling pathways such as FXR-, LXR-, PXR- and VDR-mediated responses, which are known to play a role in cholestasis, are significantly affected by all cholestatic compounds. Other significant affected pathways include unfolded protein response and protein ubiquitination implicating the role of endoplasmic reticulum stress in bile acid induced cholestasis. This is in line with a classification analysis, performed on array data of human PCLS exposed to cholestatic and necrotic compounds to identify classifier genes which can discriminate cholestasis inducing drugs from necrosis inducing drugs where the classifier genes identified are also related to ER stress. NRF2-mediated oxidative stress response is also affected indicating the adaptive response to ROS mediated cell death caused by accumulation of bile acids.

Conclusion: This study shows that human PCLS incubated in the presence of a physiological bile acid mixture correctly reflect the pathways affected in drug-induced cholestasis. These pathways are clearly affected for all of the tested cholestatic drugs including alpha-naphthylisothiocyanate, which is proved to be cholestatic so far only in animals. In the future this human PCLS model can be used to identify cholestatic adverse drug reaction of new chemical entities.

P106 - A MASS BALANCE AND METABOLITE PROFILING STUDY OF LDE225 IN HEALTHY MALE SUBJECTS USING A LIGHT-LABEL APPROACH 1 2 1 1 1 2 Piet Swart , Ad Roffel , Frederic Lozac'h , Arnold Demailly , Ulrike Glaenzel , Sjoerd van Marle , Ewoud van 2 1 Hoogdalem , and Markus Zollinger 1 2 DMPK, Novartis, Basel, Switzerland, PRA Health Sciences, Groningen, The Netherlands LDE225 (sonidegib) is a potent, selective, oral inhibitor of Smo, developed for the treatment of human malignancies involving the Hedgehog (Hh) pathway. Animal studies showed that LDE225-related radioactivity was mainly eliminated into feces following iv and po administration, and that renal excretion was minor. The current study investigated the PK and ADME properties of LDE225 after administration of a single oral dose of 800 mg / 74 kBq 14C-LDE225 to healthy male subjects. Prestudy dosimetry indicated an expected effective whole body dose of 0.63 mSv after the administration of 2.5 MBq 14C-LDE225 (ICRP 62 Category IIa). However, it was decided to administer a dose of 74 kBq, based on the variable half-lifes for LDE225 and metabolites as observed in humans, and the uncertainty about the half-life of total drug-related 14C in humans. For this dose, no formal dosimetry calculation was generated, since the effective whole body dose in humans was assumed to be below 0.1 mSv based on ICRP 30. Analysis of 14C was conducted largely using AMS. Study subjects were housed in the Clinical Unit until Day 22 after drug intake, followed by 24-h visits at 1 to 2-week intervals until Day 99. The radiolabeled drug substance and capsules were manufactured by Novartis. Chemical and radiochemical purity were ≥98%. Six subjects aged 22-40 years were enrolled and completed the study. The rate of absorption of total 14C was moderate to high (median Tmax 2 hours), but the extent of absorption was low (6-7% of dose). Total radioactivity and LDE225 showed similar terminal T1/2 in plasma, with mean values of ~14 and ~13 days, respectively. AUCinf of LDE225 accounted for 34.9% of the AUCinf of total radioactivity in plasma. The median Tmax of the major circulating metabolite (LGE899) was 60 h. Its AUCinf accounted for 15.1% of the AUCinf of total radioactivity in plasma and for 44.8% of the AUCinf of LDE225. The elimination of absorbed LDE225 occurred predominantly by metabolism. Metabolite profiles in plasma showed a total of 17 metabolites, and confirmed the major contribution of LDE225 and LGE899 to total radioactivity. The acylglucuronide accounted for most of the radioactivity in urine (0.9% of the dose). Unchanged LDE225 was not detected in urine. The morpholine oxygenation product M31 was the most prominent metabolite in feces (0.75% of the dose), followed by the morpholine dehydrogenation product M50 and the pyridine oxidation product M43. Overall, the metabolic pathways that contributed to the total metabolic clearance of LDE225 were quantified as approximately 50% oxidation in the morpholine part, 25% amide hydrolysis, and 10% oxidation in pyridine part. Fecal excretion of

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radioactivity up to infinity was 93.4% of the dose; urinary excretion 1.95%. Post-hoc dosimetry indicated an effective whole body dose of 0.043 mSv after intake of 74 kBq of 14C-LDE225. In conclusion, the mass balance and metabolite profile of LDE225 were successfully characterized in healthy males, using a light label approach and AMS. The radiation burden calculated after the study confirmed the categorization of the study as ICRP 62 Category I. P107 - ADME STUDIES OF [3H]-2´O-METHYLURIDINE NUCLEOSIDE IN MICE: A BUILDINGBLOCK IN SIRNA THERAPEUTICS Piet Swart, Frederic Lozac'h, Francois Natt, Juerg Hunziker, Thomas Faller, Esther van de Kerkhof, and Jesper Christensen DMPK, Novartis, Basel, Switzerland The chemical modification 2´-O-methyl of nucleosides is often used to increase siRNA stability towards nuclease activities. However, the fate of modified nucleosides remains unclear. In this study, the ADME properties of [3H]-2´-Omethyluridine was studied in CD-mice, following intravenous administration of 1 mg/kg. 2´-O-methyluridine and its metabolites were quantified in plasma by liquid chromatography and off line radioactivity detection. Tissue distribution was determined using whole body autoradiography, and metabolite profiles were generated in individual plasma time points, excreta pools and selected tissues by high-performance liquid chromatography (HPLC) with radioactivity detection. The chemical structures of the 2´-O-methyluridine metabolites were characterized by LC-MS/MS in the presence of Na+ and Li+ ions and by comparison with reference compounds. The radioactivity was rapidly and widely distributed throughout the body, and remained detectable in all tissues investigated throughout the observation period (48 hours). After an initial rapid decline, the concentrations of total radiolabeled components in plasma declined with a half-life of 21 h, while the half-life for [3H]-2´-O-methyluridine was about 5 hours. The parent compound represented a minor component of the radioactivity in plasma (5.9% of AUClast). Metabolism was the major route of elimination of 2´-O-methyluridine, with the O-demethylation and N-dealkylations being the major metabolism pathways, ending up with the endogenous compounds uridine, cytidine, uracil and cytosine. Following the 48 hour study period the majority of the radioactivity, was excreted via the urine (~ 60%) with ~ 2% in the feces, while the carcass still contained ~40 % of the radioactive dose. In contrast to the metabolite profiles in urine and feces, metabolite profiles in tissues (spleen, lung, small intestine, liver, kidney and heart) showed that most of the radioactivity could be attributed to uridine and uracil. Our studies show that the modified uridine is rapidly excreted and that the metabolic degradation ends up with endogenous existing material. Assuming similar metabolite pathways in human and with human tolerating relative high oral doses of uridine or uracil, it is unlikely that that the degradation products will cause safety concerns at the intended siRNA doses.

P108 - METABOLISM AND DISPOSITION OF [14C]BYL719 (ALPELISIB), AN ORAL CLASS 1 Α-SPECIFIC PI3K INHIBITOR FOR THE TREATMENT OF SOLID TUMORS IN HEALTHY MALE VOLUNTEERS Piet Swart, Lars Blumenstein, Ulrike Glaenzel, Yi Jin, Arnold Demailly, Annamaria Jakab, Regine Hansen, and Anuradha Metha DMPK, Novartis, Basel, Switzerland The disposition and biotransformation of 14C-BYL719 were investigated in four healthy male subjects after a single oral dose of 400 mg. Blood, plasma and urine collected over 7 days, and feces over 9 days, were analyzed for total radioactivity. BYL719 was quantified in plasma by liquid chromatography coupled to tandem mass spectrometry (LCMS/MS), and metabolite profiles were generated in individual plasma time points and excreta pools by highperformance liquid chromatography (HPLC) with radioactivity detection. The chemical structures of BYL719 metabolites were characterized by LC-MS/MS and by comparison with reference compounds. BYL719 was safe and well tolerated in this study population. BYL719 absorption was approximately 56% of the administered dose, reaching mean plasma Cmax values of 1240 ng/mL (BYL719) and 1850 ng-eq/mL (total radioactivity) at 2 hours. Thereafter, BYL719 in plasma, blood and plasma total radioactivity concentrations declined with mean apparent half-lives of 13.7, 11.1 and 18.0 hours, respectively. Metabolism was found to contribute to the elimination of BYL719, with the major pharmacologically inactive metabolite BZG791 being formed by amide hydrolysis. Other minor metabolic pathways included C-hydroxylation of BYL719, de-amination with formation of an aldehyde, and further C-hydroxylation of major metabolite BZG791. The subjects were mainly exposed to BYL719 and BZG791, which combined accounted for 94.6% of 14C-AUC0-12h. The formation of BZG719 was subsequently investigated with a series of in vitro incubations, which concluded that it is likely formed by a combination of enzymatic hydrolysis, with a small possibility of chemical/microbial hydrolysis in the stomach/gut. After 9 days, the mean balance of total radioactivity excretion was almost complete (94.2% of dose), with 80.7% recovered in feces and 13.5% recovered in urine.

P109 - METABOLOMICS AS A TOOL TO INVESTIGATE MEPHEDRONE AS A DRUG OF ABUSE IN RAT HEPATOCYTES Mohammad Alwashih, David Watson, Mary Grant, and Catherine Henderson University of Strathclyde, Glasgow, United Kingdom Metabolomics approach was used to investigate the role of (±)-4-Methylmethcathinone hydrochloride [(±)mephedrone, 4-MMC] a synthetic “legal high” in hepatotoxicity. An in vitro incubation of 4-MMC and a blank with Sprague-Dawley rat hepatocytes, reaction mixture of the drug and the blank with the hepatocyte solution at 0, 30 and

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120 min was analysed on a zwitterionic hydrophilic interaction (ZICp-HILIC) column using LC–MS(Khreit et al., 2013).

MzMatch software was used to convert Ms out-put to identifiable metabolites, and metabolite intensities were transformed to approximate to normality. Principal component analysis (PCA) and Orthogonal partial least square discriminant analysis (OPLS-DA) were applied to the results by using SIMCA P software, and finally a univariate analysis t-test was used to confirm the significance of metabolite changes. The results showed significant up regulation of metabolites in the pentose phosphate pathway, the Krebs cycle, bile acids pathway and fatty acid levels.

The most marked elevations in metabolites were in the levels of indole lactate and phenylalanine acetate which are metabolites of amino acids required for the biosynthesis of serotonin and dopamine respectively. There was significant decrease in diacylglycerol levels. A previous paper observed effects of 4-MMC on on the redox pathways in cells which may relate to some of the currently observed changes (den Hollander et al. 2014). This study showed the metabolomics effect of mephedrone on rat hepatocytes for the first time.

1. KHREIT, O. I., GRANT, M. H., ZHANG, T., HENDERSON, C., WATSON, D. G. & SUTCLIFFE, O. B. 2013.

Elucidation of the Phase I and Phase II metabolic pathways of (+/-)-4'-methylmethcathinone (4-MMC) and (+/-)-4'(trifluoromethyl)methcathinone (4-TFMMC) in rat liver hepatocytes using LC-MS and LC-MS(2). J Pharm Biomed Anal, 72, 177-85.

2. den Hollander, B.; Sundström, M.; Pelander, A.; Ojanperä, I.; Mervaala, E.; Korpi, E. R.; Kankuri, E., Keto Amphetamine Toxicity—Focus on the Redox Reactivity of the Cathinone Designer Drug Mephedrone. Toxicological Sciences 2014, 141 (1), 120-131.

P110 - UNDER THE SKIN: BIOMARKERS OF CUTANEOUS DEFENSES TO VACCINES USING MASS SPECTROMETRY IMAGING 1 2 1 3 2 4 Juliette Masure , Hélène Perrin , Gregory Hamm , Maxence Wisztorski , Mélody Dufossé , Charlotte Primard , 3 3 Isabelle Fournier , and Michel Salzet 1 2 3 ImaBiotech, Loos, France, INSERM U1135‐Cimi‐Paris, Paris, France, PRISM Laboratory INSERM U 1192, Lille 4 University, Lille, France, Adjuvatis, Lyon, France The better understanding of skin immunological process regarding to vaccines has a great impact on human health and therapies against infectious diseases. To address these issues, the combination of mass spectrometry imaging and histology provides useful and precise information about exogenous compounds’ targeting skin substructures (at the micrometer scale), their amount, their impact on endogenous molecules and their potential adverse effects (inflammatory or toxicity response). This study takes place in the French National Research Agency (ANR) project ASIO (Advanced Skin Immunology by Omics Analysis). ASIO will holistically dissect key elements (skin cellular functions, gene expression, molecules including proteins, lipids and metabolites) of skin cell communication and behavior to define biomarkers of immune defenses. Human skin explants were used and Influenza vaccines administrated following two modes, TC (trans cutaneous) vs ID (intra-dermal). Snap frozen skin biopsies were sectioned and thaw mounted on ITO glass slide. DHB (40 mg/mL in MeOH/W+0.1% TFA) and 9AA (10 mg/mL in MeOH) were respectively used as MALDI matrices for positive and negative detection mode. Two matrix applications were used, the classical way with SunCollect sprayer device (SunChrom). Mass spectrometric images were performed in positive or negative detection mode using a SolariX MALDI-FTICR 7.0T Mass Spectrometer (Bruker Daltonics) at low spatial resolution (30 µm). Data were generated and statistically analyzed using SpectraViewer (CEA-LIST, France) and Multimaging (Imabiotech, France) software. In this study, a multimodal approach to focus on various molecular classes (small metabolites & lipids) and using different polarities was used to describe skin immunological response to vaccine. Statistical tests were used to measure up or down regulation of metabolite profile from both conditions (treated TD vs treated ID vs control). The comparison of metabolites signal at high spatial resolution, 25 µm, was performed to differentiate substructures of the skin such as epidermis, dermis, and sebaceous gland but especially inside the hair follicle and in surrounding area. By this way, several molecules have been identified such as carnitine and its related species which are linked to keratinocytes or some phospholipids modulated by the treatment. For a high degree of confidence, MS/MS experiment (SORI-CID) was performed on tissue section to finely characterize all targets as well as high mass accuracy measurement (below ppm level). On that basis, immunohistochemical (IHC) staining was used on adjacent skin section to label several cells involved in skin defense mechanism. Notably, we are able to follow Langerhans cells & dermal dendritic cells surrounding the hair duct using respectively CD1a or CD209 labeling. These cells have specific behaviors in regards to danger signal (in our case the vaccine) based on molecular changes. MS images were then correlated with IHC results to establish a relationship between cells type and molecular species function. In conclusion, the combination of MSI and Histology provides a global approach: gives information not only on target molecule skin penetration/fixation, but also on potential toxicity/inflammatory or disease state by using specific biomarkers.The correlation between immunohistological features and molecular species’ localization provides important information about skin immunological response.

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P111 - USING HIGH RESOLUTION MASS SPECTROMETRY TO PREDICT SUCCESSFUL DOSING STRATEGIES FOR RADIOLABELLED CLINICAL AME STUDIES 1 2 1 1 1 2 Caroline Anderson , Yan Qin , Lindsay Corfield , Catherine Holdsworth , John Kendrick , and Svein Olaf Hustvedt 1 2 Covance Laboratories Ltd., Harrogate, United Kingdom, BASF, Lysaker, Norway Understanding the metabolism of a drug is essential to make informed decisions regarding the appropriate radioactive dose to be used in the human AME study. The requirements of the ICH M3[1] and the FDA MIST[2] guidelines imply that the administered radioactivity must be sufficient that metabolites formed at >10% of the total administered dose may be characterised. Data from the analysis of plasma samples taken from non-radiolabelled pre clinical, and first in human PK studies, can be used to support better informed decisions regarding the appropriate radioactive dose.

Plasma samples from preclinical species and human subjects were analysed using high resolution mass spectrometry. Relative proportions of the parent compound and detected metabolites were determined in the appropriate extracted ion chromatograms. The LC-MS data from all species were initially assessed for potential unique and disproportion human metabolites; with the results used for safety assessment. The number and nature of metabolites in human plasma samples taken at steady state was then further investigated, so that in advance of the radiolabelled clinical AME study, predictions of the likely concentrations of radioactivity in human plasma could be made; based on the following four considerations: (a) the non-radiolabelled parent compound PK data; (b) the specific activity of the proposed clinical dose formulation; (c) the number and proportions of metabolites as indicated from the cold metabolite profiles and (d) sample preparation and concentration techniques. As a consequence, the initial proposed clinical dose was doubled from 108 μCi (0.5 mSv), as suggested by Public Health England based upon dosimetry estimates, to 200 µCi (1 mSv). This increased dose level ensured that there was sufficient radioactivity present in the resulting human plasma samples, to generate suitable radio-HPLC profiling data (using fraction collection with offline radio-detection), and that it was possible to quantify and identify metabolites formed at less than 10% of the total drug related radioactivity without the need to employ other techniques (i.e. Accelerator Mass Spectrometry). Therefore characterising the metabolism of a compound at an early stage in its development can be used to ensure that the objectives of the radiolabelled human AME study can be met, and the regulatory requirements satisfied.

References: [1] Guidance on Non-Clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals M3. ICH Harmonised Tripartite Guideline.

[2 ] Guidance for Industry, Safety Testing of Drug Metabolites, U.S. Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research (CDER), P112 - CHF 6001: METABOLITE PROFILES AND IDENTIFICATION IN PLASMA, URINE, FAECES, BILE AND LIVER SAMPLES OF PXB(R) AND CD-1 MICE FOLLOWING INTRAVENOUS AND ORAL ADMINISTRATIONS 1 2 2 2 1 Valentina Cenacchi , Rosangela Battaglia , Flavio Cinato , Daniele Pezzetta , Giandomenico Brogin , and Paola 1 Puccini 1 2 Chiesi Farmaceutici Spa, Parma, Italy, Accelera SRL, Nerviano Milan, Italy Purpose: To investigate the metabolite profiles and to identify the metabolites of CHF 6001 in plasma, urine, faeces, bile and liver samples from PXB(R) mice, in comparison with CD-1 mice, after intravenous (IV) and oral (PO) administration of the drug. PXB(R) mice are chimeric animals with humanized liver (Phoenixbio Co, Ltd, Hiroshima, Japan) that are generated from urokinase-type plasminogen activator/severe combined immunodeficiency mice trasplanted with human hepatocytes (1). In these mice approximately 80% of the hepatocytes are human. The expression levels and metabolic activities of P450 and non-P450 enzymes in livers of PXB(R) mice with a high replacement index (RI) are similar to those of humans and human-specific metabolites are formed in PXB(R) mice (2).

PXB(R) mice could be a good model for predicting drug metabolism in human. Methods: CHF 6001 was administered intravenously and orally at the doses of 1 mg/kg and 50 mg/kg, respectively. The metabolite profiles were determined

by LC-UV-MS and the metabolites formed were identified by LC-MS/MS (accurate mass). Results: After IV and PO administrations to PXB(R) and CD-1 mice, CHF 6001 was found mainly unchanged in plasma and metabolized through phase I and phase II reactions in urine, faeces and bile. In plasma only CHF 6001 was detected in small amounts. In urine, CHF 6001 was not detected; only polar metabolites were present. In faeces, CHF 6001 accounted for 18-39% of total drug-related material after IV treatment and for 81-87% after PO dosing. No CHF 5956 or its derivatives were detected in faeces. In bile, CHF 6001 was detected in traces (<1% of total drug-related material) after both administration routes. The major metabolite, derived from CHF6001 through conjugation with glucuronic acid and pyridine contraction (3) accounted for 39-49% of total drug-related material. In liver extracts CHF 6001, when detected, was present in trace levels. Conclusions: After IV and PO administrations of CHF 6001 to PXB(R) and CD-1 mice, the metabolite profiles in the PXB(R) mice were similar to those in the CD-1 mice from a qualitative point of view, with some differences from a quantitative point of view. However, no strain-specific metabolite was observed, hence no human specific metabolites are expected. In addition previous metabolic profiles observed in rats (3) are in agreement with those obtained in mice.

1.Tateno et al (2004). Near completely humanized liver in mice shows human –type metabolic response to drugs. Am, J, Pathol. 165:901-912.

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2. Inoue T.et al (2009). Prediction of human disposition toward S-3H-warfarin using chimeric mice with humanized liver. Drug Metab. Pharmacokinet 24:153-160.

3. Cenacchi et al (March 2015). In vitro and in vivo metabolism of CHF6001, a selective phosphodieserase (PDE4) inhibitor. Xenobiotica, Early on line: 1-18.

(R) Registered P113 - DATABASE CAPTURING AND MINING OF METABOLITE INFORMATION OF DRUG CANDIDATES: ANALYSIS OF 27 ASTRAZENECA COMPOUNDS WITH HUMAN ADME DATA IN ACD DATABASE Jessica Iegre, Martin A. Hayes, Richard A. Thompson, Lars Weidolf, and Emre M. Isin CVMD iMed DMPK, AstraZeneca R&D,Mölndal, Sweden As part of the drug discovery and development process, it is of critical importance to understand the human metabolism of candidate drugs prior to clinical studies (1). In vitro and preclinical in vivo cross species comparison experiments are conducted to build an understanding of potential human circulating metabolites and to increase confidence in the expected biotransformation pathways of the drug candidates in human. The workflow used in AstraZeneca R&D from early discovery to development to obtain metabolite identification (Met-ID) information will be described. Within AstraZeneca, all Met-ID information generated for compounds, are captured in a global Met-ID database using ACD software (ChemFolderEnterprise) (2). In this study, a database mining exercise was performed on 27 AstraZeneca drugs/drug candidates in order to understand the predictive quality of our in vitro and pre-clinical in vivo experiments with respect to human circulating metabolites. For the 27 compounds analysed, the Met-ID information derived from in vitro and in vivo ADME studies utilizing radiolabeled compounds, was extracted from the ACD database. An evaluation of the predictability of human circulating metabolites from the most commonly used in vitro systems and matrices (human and rat hepatocytes, rat plasma) will be presented. Furthermore, a structural analysis was carried out in order to understand which human circulating metabolites are missed in pre-clinical experiments. The reverse relationship was also investigated, where metabolites observed in human hepatocytes and rat plasma but not seen in circulation in human were extracted. The results of the structural analysis will be discussed.

(1) Emre M. Isin, Charles S. Elmore, Göran N. Nilsson, Richard A. Thomson, Lars Weidolf (2012) Use of Radiolabeled Compounds in Drug Metabolism and Pharmacokinetic Studies. Chem Res Toxicol 25: 532-542 (2) ACD/Spectrum DB Enterprise, version S20E41, Advanced Chemistry Development, Inc., Toronto, ON, Canada, www.acdlabs.com, 2014.

P114 - IDENTIFICATION OF METABOLIC PATHWAYS OF BENZIMIDAZOLE ANTHELMINTICS IN HAREBELL (CAMPANULA ROTUNDIFOLIA) 1 2 3 2 2 2 Radka Podlipná , Lucie Stuchlíková , Robert Jirásko , František Pavlík , Lenka Skálová , and Barbora Szotáková 1 Laboratory of Plant Biotechnologies, Institute of Experimental Botany, Czech Academy of Sciences, Prague, Czech 2 Republic, Department of Biochemical Sciences, Faculty of Pharmacy in, Charles University, Hradec Králové, Czech 3 Republic, Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic Benzimidazole anthelmintics (BNZ), the drugs against parasitic worms, are widely used in human as well as veterinary medicine. Usefulness of the anthelmintic drugs is uncontested, but at the same time they represent a risk to ecosystems. Anthelmintics administered to animals enter into environment primarily through their excretion in faeces or urine. Following excretion, these substances may persist in the environment and impact non-target organisms.

There is a significant risk that free living helminths and other invertebrates can be affected by anthelmintics found in the environment. As plants are able to detoxify xenobiotics, including drugs, via biotransformation, they represent a possible tool for detoxification of anthelmintics in ecosystems surrounding pastures. For this reasons, the study of metabolic pathways of BNZ in plants is very important. The aim of our work was to identify metabolic pathways of BNZ (flubendazole, albendazole) in harebell (Campanula rotundifolia), common meadow plant. In vitro culture was achieved from primary callus rising on stem segments of harebell seedlings. Suspension cultures derived from this callus were grown in the dark at 24 °C. The suspensions were incubated with BNZ (10 µM) for 6, 24, 42, 66, 90 and 114 h. Before analysis, homogenized suspensions were subjected to liquid–liquid extraction. The samples were analysed by UHPLC/MS (QqTOF) in positive-ion mode. The results showed that enzymatic systems of harebell are able to biotransform BNZ via first and second phase biotransformation steps. Based on obtained results, schemes of metabolic pathway of BNZ in Campanula rotundifolia were proposed. This project was supported by Czech Science Foundation, grant No. 15-05325S.

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P115 - IMPACT OF ANTHELMINTICS IN ENVIRONMENT ON LOWER DEVELOPMENT STAGES OF HELMINTHS AND ON PLANTS 1 1 2 2 2 3 Barbora Szotáková , Jiří Lamka , Lenka Lecová , Lukáš Prchal , Lenka Skálová , and Radka Podlipná 1 Department of Pharmacology and Toxicology Charles University, Faculty of Pharmacy, Hradec Králové, Czech 2 Republic, Department of Biochemical Sciences, Faculty of Pharmacy in, Charles University, Hradec Králové, Czech 3 Republic, Laboratory of Plant Biotechnologies, Institute of Experimental Botany, Czech Academy of Sciences, Prague, Czech Republic Pharmaceuticals from human and veterinary medication are a new class of micro-pollutants that possess a serious threat to environment. Anthelmintics administered to animals enter into environment and may persist there and impact dung fauna and dung decomposition which represents a key factor for herbage yields in agricultural grasslands.

Moreover, exposure of lower development stages of helminths to low concentrations of anthelmintics in the manure and soil may also encourage the development of drug-resistant strains of helminths. Albendazole (ABZ) is a benzimidazole anthelmintic widely used in veterinary medicine against parasitic worms. In host, ABZ is oxidized to anthelmintically active ABZ sulphoxide (ABZ.SO) and both substances are excreted in faeces of animals. The aim of our work was to find out the kinetics of ABZ and ABZ.SO elimination in faeces from treated animals and to evaluate the effect of these anthelmintics on lower development stages of Barber’s pole worm (Haemonchus contortus), on germination of mustard (Sinapis alba) seeds, and on suspension cell cultures of harebell (Campanula rotundifolia).

Texel lambs were treated with ABZ (10 mg/kg) and faeces were collected (0-72 h). Concentrations of ABZ and metabolites were measured by HPLC/MS. The highest concentrations of ABZ (10 µg/g) and ABZ.SO (5 µg/g) in faeces were found 12 h after ABZ administration. H. contortus eggs were isolated from faeces of infected Texel lambs.

Obtained eggs were subsequently pre-incubated with ABZ or ABZ.SO for 24 h at 8°C. Micro-agar larval development tests was than performed. After 7 days the numbers of unhatched eggs and L1 – L3 larvae were counted. No impact of ABZ or ABZ.SO pre-incubation was observed. Seeds of white mustard germinated in Petri dishes on filtration paper moistened with cultivation medium supplemented with different concentrations of ABZ or ABZ.SO. The seeds sprouted in dark at 24°C. The length of roots was measured after 3 days. This test did not show any significant difference in roots length between control and treated plants. The phytotoxicity of monitored compounds was also tested on suspension cell cultures of harebell using test of viability. No significant difference in viability between control and treated plants was observed. Taken together, no effect of low ABZ or ABZ.SO concentrations on H. contortus hatching, mustard germination, and viability of harebell cell suspensions was found. This project was supported by Czech Science Foundation, grant No. 15-05325S. P116 - INVESTIGATING THE MECHANISM FOR INVERSION OF CONFIGURATION OF CARBOXYLIC ACID CONTAINING DRUG CANDIDATES Fredrik Bergström, Andreas Dannhorn, and Emre M. Isin CVMD iMed DMPK, AstraZeneca R&D, Mölndal, Sweden Considering, the number of drugs containing a carboxylic acid moiety on the market, biotransformation of drugs/drug candidates possessing this moiety is of interest from pharmaceutical industry point of view. One particular biotransformation pathway of interest is the acyl-CoA mediated inversion of configuration which can render the drug inactive, hence there is a general interest in increasing the understanding of mechanisms of and enzymes involved in this biotransformation reaction. In this work, we investigated the mechanism of inversion of configuration in a novel series of carboxylic acid containing drug candidates where inversion at the alpha carbon is a major in vitro metabolic pathway in human cryopreserved hepatocytes. The compounds were studied in human liver microsomal incubations with and without addition of CoA. LC-MS/MS-TOF and LC-MS/MS was used for identification and quantification of drug candidate, intermediates, and resulting metabolites. The inversion of configuration for the studied compounds occurs via the formation of acyl-CoA conjugates followed by inversion of the CoA-conjugates and subsequent hydrolysis to yield the inverted parent drug candidate. The acyl-CoA formation, which is the initial step, was studied using lauric acid, as a competitive inhibitor for Long-chain Coenzyme A ligases. The inhibition was investigated and the IC50 value of lauric acid was determined to be ~100 µM. The acyl-CoA inversion step of the drug candidates is likely to be mediated by a mechanism similar to the inversion of branched-chain-fatty acid CoA conjugates. For ibuprofenyl-CoA conjugates this is reported to occur via an enol intermediate and to be mediated by the dibasic enzyme alpha-methylacyl-CoA racemase (AMACR)[1]. The hydrolysis of the formed CoA-conjugates was studied using diisopropylfluorophosphate (DFP), which is an irreversible inhibitor for a broad range of esterases[2]. Upon addition of DFP, we observed a concentration dependent inhibition of the hydrolysis of acyl-CoA conjugates (of parent and inverted parent drug candidate) that reached a maximum at a DFP concentration of about 100 µM. Our data indicate that the hydrolysis is enzymatically driven, and these findings are in line with earlier observations on hydrolysis of ibuprofenoyl-CoA by acyl-CoA thioesterases[3]. We performed kinetic experiments where the rate of formation of acyl-CoA conjugates as well as the appearance of inverted compound was monitored. These experiments indicate that formation of acyl-CoA conjugates is the rate limiting step with a maximum activity at a compound concentration of ~75µM. We will present the results of our mechanistic studies on the inversion of configuration of novel carboxylic acid containing drug candidates.

1. M.D. Lloyd et al, alpha-Methylacyl-CoA racemase (AMACR): Metabolic enzyme, drug metabolizer and cancer marker P504S, Prog. Lipid Res., 52, 220-230, 2013.

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2. T. Minagawa et al, Species Differences in Hydrolysis of Isocarbacyclin Methyl ester (TEI-9090) By Blood Esterases, Biochem. Pharmacol., 49(10), 1361-1365, 1995.

3. X. Qu et al, Hydrolysis of ibuprofenoyl-CoA and other 2-APA-CoA esters by human acyl-CoA thioesterases-1 and -2 and their possible role in the chiral inversion of profens, Biochem. Pharmacol., 86, 1621-1625, 2013.

P117 - LC-MS ANALYSIS OF THE METABOLISM OF THE DIETARY CONSTITUENT HESPERIDIN BY RAT HEPATOCYTES Khaled Omar, Mary Grant, Catherine Henderson, and David Watson University of Strathclyde, Glasgow, United Kingdom Hesperidin (4’-methoxy-7-O-rutinosyl-3’,5-dihydroxyflavanone), is a naturally occurring flavanone glycoside predominant in citrus fruits [1]. Dietary hesperidin is hydrolyzed by gut microflora to give hesperetin (4’-methoxy-3’,5,7 trihydroxyflavanone), the aglycone form of hesperidin [2]. In recent years, there have been an increasing number of reports on the physiological functions or pharmacological effects of hesperidin including antioxidant, anti-cancer, antiallergenic, anti-inflammatory, neuroprotective, antihypotensive, vasodilator properties and antimicrobial activities.

[3], [4], [5]. In the present study, we observed eleven metabolites of hesperidin formed in vitro by incubating hesperidin with rat hepatocytes. The most abundant metabolite was the sulphate of hesperidin followed by hesperidin glucuronide and glutathione conjugation as a phase III metabolite. A wide range of novel hesperidin metabolites have been detected due to aromatic hydroxylation, O-demethylation, desaturation, desaturation+S-GSH, hydroxylation, methylation and glycine conjugation. These were detected and reported for the first time, also this is the first study of hesperidin metabolism in vitro using rat hepatocytes. The hepatocytes completely lacked the ability to convert hesperidin to its aglycone. Conclusions: There has been extensive interest in the health benefits of dietary phenolics over nearly two decades, much of the benefits are still unproven. The occurrence of glutathione conjugation seems to be commonplace in phenolic compounds which suggests their health benefits might not be that clear cut [6, 7].

References: 1. Yang, C.Y., et al., Determination of hesperetin and its conjugate metabolites in serum and urine (vol 10, pg 143, 2002). Journal of Food and Drug Analysis, 2002. 10(4): p. 284-284.

2. Haidari, F., et al., Orange Juice and Hesperetin Supplementation to Hyperuricemic Rats Alter Oxidative Stress Markers and Xanthine Oxidoreductase Activity. Journal of Clinical Biochemistry and Nutrition, 2009. 45(3): p. 285-291.

3. Cho, J., Antioxidant and neuroprotective effects of hesperidin and its aglycone hesperetin. Archives of Pharmacal Research, 2006. 29(8): p. 699-706.

4. Lee, K.-H., et al., The inhibitory effect of hesperidin on tumor cell invasiveness occurs via suppression of activator protein 1 and nuclear factor-kappaB in human hepatocellular carcinoma cells. Toxicology Letters, 2010. 194(1-2): p.

42-49.

5. Garg, A., et al., Chemistry and pharmacology of the Citrus bioflavonoid hesperidin. Phytotherapy Research, 2001.

15(8): p. 655-669.

6. Omar, K., et al., The complex degradation and metabolism of quercetin in rat hepatocyte incubations. Xenobiotica, 2014. 44(12): p. 1074-1082.

7. Omar, K., et al., The abundant dietary constituent ferulic acid forms a wide range of metabolites including a glutathione adduct when incubated with rat hepatocytes. Xenobiotica, 2014. 44(5): p. 432-437.

P118 - OPTIMIZING METABOLIC STABILTY VIA SOFT SPOT IDENTIFICATION (SSID) Markus Trunzer, Alfred G. Zimmerlin, and Bernard Faller Novartis Institutes for Biomedical Research / Metabolism and Pharmacokinetics, Basel, Switzerland The optimization for metabolic stability of a structural series is a key factor in Drug Discovery as it may dictate systemic exposure. In early stages, metabolic stability is typically determined by the disappearance of test article (TA) in microsomal incubations analyzed by liquid-chromatography-mass-spectrometry (LC-MS). No information about metabolites or the metabolic soft spot is provided. MS-technologies like quadrupole-time-of-flight (Q-ToF) or Orbitrap, which allow high resolution mass spectrometry in MS and MSMS mode, can identify drug related material in complex mixtures like microsomal incubations in addition to monitoring of the test article disappearance. Harvesting this data from metabolic stability incubations can be used for the structure elucidation of metabolites. However, data mining and interpretation of the large datasets remains the bottleneck in this process. We used MassMetaSite to help data interpretation and created a fully automated work-flow - the metabolic soft spot identification (SSID) approach – which is presented here. This additional knowledge enables a better understanding of metabolism and a more rational design of follow-up compounds.

P119 - PHASE 2 METABOLISM OF ACTIVE HYDROXYCLOMIPHENE METABOLITES 1 1 1 1 1&2 1 Patrick Kroener , Georg Heinkele , Boian Ganchev , Kathrin Klein , Matthias Schwab , and Thomas E. Mürdter 1 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and University Tuebingen, Stuttgart, 2 Germany, Department of Clinical Pharmacology University Hospital Tübingen, Tübingen Clomiphene citrate which is administered as a mixture of cis- and trans-isomers (Fig.) is the first line therapy for the treatment of infertility in women affected by the polycystic ovary syndrome. However, treatment outcome is variable

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and approximately ¼ of patients do not benefit from clomiphene treatment. Recently, the polymorphic cytochrome P450 (CYP) 2D6 was identified to be the major enzyme involved in 4-hydroxylation of trans-clomiphene. The resulting metabolites trans-4-hydroxyclomiphene and trans-4-hydroxydesethylclomiphene showed an approximately 100-fold higher potency in inhibiting estradiol mediated activation of the estrogen receptor compared to the parent compound [1]. Variability in plasma concentrations of these active metabolites could be explained by CYP2D6 pharmacogenetics only partially. Besides the formation of active metabolites, their plasma concentrations are influenced by their clearance via glucuronidation. Here, we characterized trans- and cis-4-hydroxyclomiphene with respect to their antiestrogenic activity in the estrogen response element reporter assay and their glucuronidation in human liver microsomes and supersomes expressing UDP-glucuronosyl transferase (UGT) isoforms. In addition, we included another CYP2D6 dependent metabolite, trans-3-hydroxyclomiphene in the investigations. The anti-estrogenic activity of trans-3-hydroxyclomiphene was more than tenfold lower compared to trans-4-hydroxyclomiphene (of 2.6 nM versus 34.5 nM). This finding is in agreement with previous data and correlates to the respective hydroxylated tamoxifen metabolites (IC50 values of 7 and 94 nM for trans-4-hydroxytamoxifen and trans-3-hydroxytamoxifen, respectively [2]).

First, the kinetic profile of the glucuronidation of the separate isomers was analyzed by incubation with a pool of human liver microsome from 150 donors representing the Caucasian population. The highest intrinsic clearance was observed for cis-4-hydroxyclomiphene (551 µL*mg-1*min-1) followed by trans-3-hydroxyclomiphene (188 µL*mg1*min-1), cis-3-hydroxyclomiphene (11 µL*mg-1*min-1). The active trans-4-hydroxyclomiphene showed the lowest intrinsic clearance (8 µL*mg-1*min-1). Trans-4-hydroxyclomiphene was mainly glucuronidated by the UGTs 1A8, 1A9, 2B7, 1A3 and 1A10. Of note, two of these isoforms are located extra-hepatic. In contrary, cis-4-hydroxyclomiphene was glucuronidated only by hepatic UGTs (2B15, 1A3, 1A9, 1A1 and 2B7). Whereas both, UGT 1A3 and 2B7 contributed to the glucuronidation of cis-3-hydoxyclomiphene, trans-3-hydroxyclomiphene was conjugated exclusively

by UGT2B7. Interestingly, the respective hydroxylated tamoxifen metabolites showed a comparable pattern of UGT isoforms [3]. In summary, we could show that the more potent trans-4-hydroxyclomiphene is glucuronidated by various UGT isoforms; therefore, an influence of genetics on its clearance is rather unlikely.

[1] Mürdter TE, Kerb R, Turpeinen M, Schroth W, Ganchev B, Böhmer GM, Igel S, Schaeffeler E, Genetic polymorphism of cytochrome P450 2D6 determines oestrogen receptor activity of the major infertility drug clomiphene via its active metabolites, Human Molecular Genetics, 2012, 21(5), 1145-1154.

[2] Mürdter TE, Schroth W, Bacchus-Gerybadze L, Winter S, Heinkele G, Simon W, Fasching PA, Fehm T, Activity levels of tamoxifen metabolites at the estrogen receptor ant the impact of genetic polymorphisms of phase I and II enzymes on their concentration levels in plasma, Clinical Pharmacology & Therapeutics, 2011, 89(5), 708-711.

[3] Lazarus P, Sun D, Potential role of UGT pharmacogenetics in cancer treatment and prevention: focus on tamoxifen and aromatase inhibitors, Drug Metabolism Reviews, 2010, 42(1), 182-194 P120 - USE OF IN VIVO MODELS TO ASSESS METABOLIC LIABILITIES AND EXCRETION PATHWAYS AT EARLY STAGE IN DRUG DISCOVERY Thierry Delemonte, Joachim Blanz, Jérôme Dayer, Werner Gertsch, Claude Hager, Ralf Endres, Ernst Gassmann, and Sandrine Desrayaud Novartis Institutes for Biomedical Research / Analytical Sciences and Imaging, Basel, Switzerland Metabolism-related liabilities continue to be a major challenge in drug discovery and development. Consequently, assessment of the main metabolic pathways at an early stage of drug discovery allows rationalization of the selection

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of lead candidates and enables compound optimization. This ultimately has an impact on the development towards high quality drugs. The use of in vivo metabolism studies for early compound optimization is advantageous because of different aspects. Biotransformation occurs in many tissues, with the liver being the most important organ. In addition, extra-hepatic metabolism occurs in kidney, lungs and intestine and may contribute significantly to the metabolic fate of a drug candidate. Thus in vivo studies give an integrated picture of the metabolic fate (phase I and phase II) and provide additional information on the extent of direct elimination of unchanged drug (phase III). For this purpose we apply two animal models at early stage of drug development for the investigation of unlabeled parent compounds.

First, the bile-duct cannulated (BDC) rat model is frequently used for early profiling of chemotypes or the identification of major metabolic pathways of selected drug candidates. It is also used to uncover disconnects between intrinsic clearance as measured in metabolic stability assays in vitro and in vivo clearance and half-life. Second, the noninvasive bile collection (NIBCo) model in dogs is used for cross-species comparison of metabolism, preferably when disconnects between in vivo PK of rat and dog are observed and which cannot be explained by in vitro data. The poster describes the experimental set-up of both models, and how they can be used for assessment of metabolic liabilities and excretion pathways. For this purpose a selected drug candidate was administered to a BDC rat and NIBCo dogs respectively. The results obtained with both animal models propose species specific metabolites and a certain extent of direct excretion of unchanged parent compound.

P121 - DEVELOPMENT AND VALIDATION OF AN IN VITRO ASSAY TO DETERMINE THE FRACTION METABOLISED BY FMO Abhishek Srivastava and Barry Jones Drug Safety and Metabolism DMPK, AstraZeneca, Cambridge, United Kingdom It is important to clearly understand the contribution of different metabolic enzymes involved in the clearance of new compounds. A precise prediction of routes of metabolism can help to evaluate risk associated with inter-patient variability in metabolism (due to enzyme polymorphism) or clinically significant drug-drug interactions at an early phase of drug development. The role of flavin containing monooxygenases (FMO) on drug disposition has been insufficiently explored partly due to overlapping substrate specificity with cytochrome P450 (CYP) enzymes and unavailability of a robust assay to differentiate their relative contributions. In this study, we discuss development and validation of an in vitro assay for estimating the fraction metabolised (fm) by FMO enzymes using human liver microsomes (HLM). Here, we used benzydamine as a probe substrate for FMO activity and monitored the formation of its N-oxide metabolite at different experimental conditions. We evaluated previously described conditions for thermal and chemical inactivation1 of FMO activity in HLM and derived conditions exhibiting maximum inactivation of FMO. For further validation we studied the effect of the above thermal and chemical inactivation conditions on CYP activity.

The optimized conditions used in this assay are highly selective/specific to FMO enzymes and show minimal interference with the major CYP isoforms. In conclusion, this study provides a robust method to estimate the fraction metabolised by FMO and could potentially allow to distinguish between contribution by CYP and FMO enzymes on the metabolism of discovery/development compounds.

Reference: 1. StoÈrmer E, Roots I & BrockmoÈ ller J (2000) Benzydamine N-oxidation as an index reaction reflecting FMO activity in human liver microsomes and impact of FMO3 polymorphisms on enzyme activity. Br J Clin Pharmacol, 50, 553-56.

P122 - INVOLVEMENT OF ALDEHYDE OXIDASE IN THE REDUCTION OF N-OXIDE FUNCTION Maria Rosa, Julia Lopes, Jean-Marie Nicolas, Sylvie Dell'aiera, Claude Delatour, Anna-Lena Ungell, and Hugues Chanteux UCB Biopharma, Braine L'Alleud, Belgium Identifying the major enzyme(s) involved in the metabolism of a NCE (new chemical entity) is critical in drug development in order to predict the risk for potential drug-drug interactions in the clinic if the NCE is co administered with drugs being inducers or inhibitors of these enzymes. The aim of the present work was to identify the major enzyme(s) involved in the major metabolic pathway of a novel NCE, which is the reduction of its N oxide function.

For that purpose, the compound was incubated with several human liver fractions (S9, cytosol, hepatocytes) and the formation of the reduced metabolite was monitored by LC-MS/MS. Preliminary assays showed that the formation of the reduced metabolite (normalized per gram of liver) was 10-fold lower in human liver S9 fractions compared with human hepatocytes suspensions. In S9 liver fractions, NADPH did not play a role in the formation of the reduced metabolite, ruling out a major involvement of NADPH-dependent enzymes (CYP450 and FMO). In order to assess a possible involvement of xanthine oxidase (XO) and aldehyde oxidase (AO), in the reduction of N oxide, specific electron donors (2 hydroxypyrimidine (2 mM) for AO and xanthine (1 mM) for XO) were added to incubations with S9 and cytosol fractions. The formation of the reduced metabolite was only observed in presence of 2 hydroxypyrimidine, suggesting a significant role of AO in this reaction. Incubations with specific inhibitors of AO (menadione (100 µM), raloxifene (0.5 µM) and hydralazine (25µM)) confirmed the major involvement of this enzyme. Also, potential formation of the reduced metabolite by non enzymatic reaction was checked and confirmed that the reduction was primarily catalyzed by liver enzymes. In conclusion, the present results clearly demonstrated that AO plays a major role in the

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reduction of the N-oxide function present on our NCE and that specific incubation conditions are required to catalyze this reaction when liver fractions (S9 and cytosol) are used.

P123 - ASSOCIATION BETWEEN HUMAN TELOMERASE REVERSE TRANSCRIPTASE (HTERT) GENE VARIATIONS AND RISK OF DEVELOPING BREAST CANCER 1 2 2 1 Ezgi Oztas , Halil Kara , Cihan Uras , and Gul Ozhan 1 2 Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Istanbul, Turkey, Acibadem University, Faculty of Medicine, Department of General Surgery, Istanbul, Turkey Despite the reduction of deaths from cancers through development of early detection tests, improvements in treatment, changes in the age distribution of the population, changes of personal behaviors as a result of awareness, breast cancer is still a major health problem. Breast cancer is most common cancer and second leading cause of cancer death in women (1). Several genetic and environmental factors are involved in breast cancer pathogenesis, but its exact etiology is complicated and is not clearly identified (2). The structure and integrity of telomeres are pivotal for genome stability and telomere length is maintained by the expression of the telomerase enzyme. Human Telomerase Reverse Transcriptase (hTERT) gene is one of the main functional subunits of the telomerase. Several recent studies have provided evidence that hTERT gene variants may have important role in cancer development (3-4). Therefore, in this case-control study, we aimed to evaluate the influence of three single-nucleotide polymorphisms (SNPs) (rs2736100, rs2736098 and rs2853669) of hTERT gene on susceptibility to breast cancer. For the SNPs, we genotyped in 100 patients with breast cancer and 110 healthy controls in Turkish women. In conclusion; hTERT rs2736098 was slightly associated with breast cancer risk (OR=1.09; P=0.05) while rs2736100 and rs2853669 did not significantly differ. The findings are the first results of hTERT allele distributions in the Turkish population and may contribute to understanding of breast cancer development. Nevertheless, further large population studies are needed to understand the role of the hTERT polymorphisms and haplotypes in the development of breast cancer.

1. Independent U.K. Panel on Breast Cancer Screening (2012) The benefits and harms of breast cancer screening: An independent review. Lancet 380(9855):1778-1786.

2. Pharoah PD, Antoniou AC, Easton DF and Ponder BA (2008) Polygenes, risk prediction, and targeted prevention of breast cancer. N Engl J Med 358:2796-2803.

3. Vodenicharov MD and Wellinger RJ (2007) The cell division cycle puts up with unprotected telomeres: Cell cycle regulated telomere uncapping as a means to achieve telomere homeostasis. Cell Cycle 6(10):1161-1167.

P124 - ASSOCIATION BETWEEN PHOSHOLIPASE C-EPSILON 1 (PLCE1) GENE VARIATIONS AND RISK OF DEVELOPING COLORECTAL CANCER 1 1 1 2 Gul Ozhan , Ezgi Oztas , Merve Arıcı , and Hakan Teoman Yanar 2 Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Istanbul, Turkey, Istanbul University, Faculty of Medicine, Department of General Surgery, Istanbul, Turkey Colorectal cancer (CRC), is one of the common complex diseases, is the fourth most common cause of cancer-related mortality in the world and causes nearly one million deaths worldwide each year (1). It is given rise by genetic and environmental factors such as an unbalanced diet, nutritional deficiencies, environmental exposure to carcinogens, and infectious agents such as viruses and bacteria might contribute to carcinogenesis of CRC. Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation, and they may have a role to an individual’s susceptibility to cancer. Phosholipase C-epsilon 1 (PLCE1) is one of the phospholipase family of enzymes and controls cellular responses which lead to cell growth, differentiation and gene expression (2). In the study, it was aimed to determine whether the PLCE1 polymorphisms are associated with CRC development and progression. We evaluated PLCE1 rs2274223 and rs3765524 genotypes in 110 patients with CRC and 130 healthy controls in Turkish population. In conclusion, rs2274223 was significantly associated with CRC risk (OR=2.018; 95% CI=1.012-3.981; p=0.041) while rs3765524 did not significantly differ (OR=0.959; 95%CI=0.543-1.694; p=0.888). We believe that the findings are the first results of PLCE1 allele distributions in the Turkish population and provide an understanding of aetiology in CRC. 1. Fan C, et al (2007) Case-only study of interactions between metabolic enzymes and smoking in colorectal cancer.

BMC Cancer 7:115.

2. Wang X, et al (2012) Phospholipase C epsilon plays a suppressive role in incidence of colorectal. Med Oncol 29:1051.

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P125 - ASSOCIATION OF ESR2 (RSA I AND ALU I) AND ORGANOCHLORINE PESTICIDE LEVELS WITH PROSTATE CANCER : A CASE CONTROL STUDY 1 1 2 2 Basu Dev Banerjee , Tusha Sharma , Nivedita Sharma , and Sanjay Gupta 1 Department of Biochemistry, University College of Medical Sciences and GTB Hospital (Delhi University), Delhi, 2 India, Department of Surgery, University College of Medical Sciences and GTB Hospital (Delhi University), Delhi, India Prostate cancer (PC) is a common disease worldwide with a higher incidence rate in developed countries. The incidence rate in the Asian and Central or Eastern European countries is increasing. It accounts for about 14% (about 90,350 cases) of the total new cancer cases and 6% (about 2,58,400 cases) of the total cancer deaths for males. The heterogeneous expression of androgen receptor, ESR1 (stromal) and ESR2 receptors (epithelial cells) occurs in prostate cancer. Apart from steroid hormone there are several environmental factors which are associated with the pathogenesis of prostate cancer. Organochlorine pesticides (OCPs), potent endocrine disrupters, are found to be associated with several cancers such as prostate, breast, bladder, etc. ESR2 (ESRβ) is a polymorphic supergene family involved in the pathogenesis of hormone mediated cancer. The present study was carried out in PC subjects and healthy control subjects (n=50 in each group) with an aim to (i) determine the role of ESR 2 RsaI and ESR 2 AluI polymorphism and its implication on the OCP detoxification or bioaccumulation which may increase the risk of PC in humans and (ii) identify the “gene-environment interaction” specifically between gene polymorphism in estrogen receptor and blood OCP levels. ESR 2 RsaI and ESR 2 AluI gene polymorphism was analysed by using multiplex PCR. OCPs (α,β,γ,total-HCH, endosulfan I & II, aldrin, dielrin, heptachlor, p’p’-DDT, p’p’ DDE and p’p’DDD) levels in whole blood were estimated by gas chromatography equipped with electron capture detector. For gene environment interaction one way analysis of variance followed by tukey’s test and student –t test was performed for multiple comparisons. The results demonstrated the blood levels of β, γ, total -HCH and p,p’-DDE were found to be significantly (p<0.001) high in PC cases as compared to controls. The frequency of wild variant of ESR2 RsaI was higher in cases than controls. The frequency of hetero and mutant variant of ESR2 AluI was also higher in cases as compared to controls but no significantly difference was observed in cases as compared to controls. A significant interaction was found between Endosulfan- II and ESR2-RsaI genotype and α-HCH and ESR2-AluI genotype in controls. This is the first report indicating the correlation of different polymorphic variant of ESR2 with the level of pesticides and tried to assess the role of gene environmental interaction in the etiology of prostate cancer. The present study also suggests that the presence of wild variant has the protective effect from the disease progression and indicate that “gene-environment interaction” may play a key role in decreasing the risk for PC in individuals who are genetically not susceptible due to presence of wild variant of ESR 2 RsaI and ESR2 AluI during their routine encounter with exposure to OCPs. However, further such studies are required with larger sample size to explore the exact role of gene-environment interaction and risk of developing prostate cancer in different ethnic population with reference to other xenobiotics as environmental pollutant.

P126 - CONTRIBUTION OF CYP3A5 GENETIC POLYMORPHISM ON THE PHARMACODYNAMICS OF CLOPIDOGREL UNDER STEADY STATE CONDITION 1 1 2 1 Nontaya Nakkam , Sirimas Kanjanawart , Somsak Tiamkao , and Wichittra Tassaneeyakul 1 2 Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, Division of Neurology, Department of Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand Clopidogrel is an antiplatelet drug that commonly used for treatment and prevention of atherothrombotic events in patients who have cardiovascular and cerebrovascular diseases. This drug is a prodrug which requires bioactivation by several drug metabolizing enzymes for conversion into an active thiol metabolite and subsequently act by inhibiting the binding of adenosine 5-diphosphate (ADP) to its platelet P2Y12 receptor. Recent data demonstrated that genetic polymorphism of these drug metabolizing enzymes may affect the antiplatelet aggregation response of clopidogrel.

Apart from CYP2C19, CYP3A5 is one of the major enzyme involve in the bioactiavation of clopidogrel to its active metablite, however, the impact of CYP3A5 genetic polymorphism on clopidogrel response is less clear. Thus, the study aims to evaluate the impact of CYP3A5 on the pharmacodynamics of clopidogrel under steady state condition.

Thirty-five male healthy subjects were treated with clopidogrel (75 mg/day) for 7 days and blood samples were collected at pre-dose, 1, 2, 3, 4, 8, 12 and 24 hr after the last dose administration. The platelet aggregation was determined using the whole blood impedance aggregometry and VerifyNow®P2Y12 assay. Genotyping for CYP3A5 was performed by custom Taqman® SNP genotyping method and CYP2C19 were performed by PCR-RFLP method.

The areas under the time-antiplatelet effect curves (AUEC0-24hr), maximal antiplatelet effect (Emax), and minimal antiplatelet effect (Emin) of clopidogrel obtained from both whole blood impedance aggregometry method and VerifyNow®P2Y12 assay were significance different among subjects with three different CYP2C19 genotypes.

However, these three pharmacodymamic parameters of clopidogrel were not significantly different among subjects with different CYP3A5 genotypes. Among the subjects who are heterozygous of CYP2C19*2 allele, the AUEC0-24hr and Emin of clopidogrel observed from the CYP3A5*3/*3 carriers measured by whole blood impedance aggregometry was significantly lower than those of the CYP3A5*1/*3 carriers. Although the Emax of clopidogrel observed from the CYP3A5*3/*3 carriers measured by whole blood impedance aggregometry was apparently lower but it was not significantly different. Interestingly, all three pharmacodynamics parameters for cloipidogrel response measured by VerifyNow® P2Y12 assay were not significantly different among the heterozygous CYP2C19*2 subjects who carried different CYP3A5 genotypes. It is clearly that CYP2C19 genetic polymorphism play a major role in the

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pharmacodynamic response of clopidogrel after administration of 75 mg once daily under steady state condition while CYP3A5 genetic polymorphism may be pronounced in the subjects who carried loss-functional allele of CYP2C19.

Moreover, the assay systems used for assessment antiplatelet aggregation effect of clopidogrel may influence the impact of study factor, thus more than one platelet functional methods should be employed.

P127 - GENE ENVIRONMENT INTERACTION IN EPITHELIAL OVARIAN CANCER WITH SPECIAL REFERENCE TO ORGANOCHLORINE PESTICIDE : A CASE CONTROL STUDY 1 1 2 1 1 3 Tusha Sharma , Basu Dev Banerjee , Kiran Guleria , Rafat Ahmed , Ashok Kumar Tripathi , and Vinod Kumar Arora 1 Department of Biochemistry, University College of Medical Sciences and GTB Hospital (Delhi University), Delhi, 2 India, Department of Obstetrics and Gynaecology, University College of Medical Sciences and GTB Hospital (Delhi 3 University), Delhi, India, Department of Pathology, University College of Medical Sciences and GTB Hospital (Delhi University), Delhi, India Organochlorine pesticides (OCPs) belongs to the class of hydrocarbons characterize by its cyclic structure. Worldwide particularly in developing countries these compounds are widely used for pest control in agriculture and public health program. Due to their persistence nature OCP gets accumulated in the food chain and cause possible adverse health effects. OCPs, potent endocrine disrupters, are found to be associated with hormone mediated disorders like reproductive health including preterm birth, Intra uterine growth retardation, birth defect like hypospadias, and several cancers such as prostate, breast, bladder, etc. Epithelial ovarian cancer (EOC) is also one of the hormone dependant cancer and contributes to a large portion of mortality and morbidity among all cancer occurring in women. Glutathione S-transferase (GST) is a polymorphic supergene family involved in the detoxification of numerous environmental toxins including OCPs. The present study was carried out in EOC cases and healthy control subjects (n=60 in each group respectively) with an aim to determine the role of GSTM1 and GSTT1 polymorphism and its implication on the OCP detoxification or bioaccumulation which may increase the risk of EOC in human. This study was also designed to identify the “gene-environment interaction” specifically between gene polymorphism in xenobiotic metabolizing genetic enzyme(s) and blood OCP levels. GSTM1/GSTT1 gene polymorphism was analysed by using multiplex PCR. OCPs levels in whole blood were estimated by Gas chromatography equipped with electron capture detector. The results demonstrated a significant (p<0.05) increase in frequency of GSTM1-/GSTT1- (null) genotype in EOC cases without interfering the distribution of other GSTT1/GSTM1 genotypes. The blood levels of β-HCH, endosulfan I, p’p’-DDT, p’p’DDE and heptachlor were found significantly high (p<0.05) in cases of epithelial ovarian cancer as compare to control.

A significant association was also observed between higher levels of β-HCH and heptachlor and EOC with odds ratio of 2.76 and 2.97 respectively which may indicates the plausible role of OCPs with the pathogenesis of EOC. Multiple regression analysis revealed a significant interaction between β-HCH and GSTM1-genotype (p<0.05) as well as in βHCH and GSTT1- genotype (p<0.05) respectively. These findings indicate that “gene-environment interaction” may play a key role in increasing the risk for EOC in individuals who are genetically more susceptible due to presence of GSTM1-/GSTT1- (null) deletion during their routine encounter with exposure to OCPs. However, it remains independent of exposure with reference to other GSTT1/GSTM1 genotypes.

P128 - IMPACT OF CYP2C19 AND CYP2D6 GENOTYPE ON THE PHARMACOKINETICS OF ESCITALOPRAM IN HEALTHY CHINESE SUBJECTS 1 2 1 1 2 1 1 SK Wo , Benny SP Fok , Celia WS Tang , Vincent HL Lee , Brian Tomlinson , Teddy TN Lam , and Zhong Zuo 1 2 School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Purpose: As an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class, escitalopram is mainly metabolized in liver via CYP2C19, CYP3A4 and CYP2D6 to S-demethylcitalopram. Although CYP2C19 polymophism was reported to affects its plasma concentrations in Caucasian, the related information was barely investigated in Chinese populations [1]. The current study aims to explore the potential pharmacogenetic factor that may affect the pharmacokinetic parameters of escitalopram in Chinese subjects. Methodology: Pharmacokinetics studies of a single oral Lexapro tablet 10 mg under fasting condition were conducted in thirteen healthy male subjects. The plasma concentration of escitalopram was determined by a developed UPLC/MS/MS method. Pharmacokinetic parameters of escitalopram were calculated by non-compartmental model from the plasma concentration vs time profiles. The polymorphism of CYP2C19 and CYP2D6 of all subjects were also genotyped. Statistical analysis using Student’s ttest was conducted to evaluate the effect of genotyping on Cmax and AUC0-96h of escitalopram. Results: Comparing Cmax and AUC0-96h of escitalopram from subjects with CYP2D6 C188T C allele vs. those without C allele, these parameters from subjects with C allele (Cmax of 12.9 +/- 2.6 ng/ml, AUC0-96h of 387.0 +/- 160.9 ng.h/ml, n = 6) were found to be significantly lower (p <0.05) than those without C allele (Cmax of 17.3 +/- 3.9 ng/ml, AUC0-96h of 572.1 +/- 117.2 ng.h/ml, n = 7). Similarly, both Cmax and AUC0-96h of escitalopram obtained from subjects with CYP2D6 G4268C G allele (Cmax of 12.3 +/- 2.5 ng/ml, AUC0-96h of 325.3 +/- 83.8 ng.h/ml, n = 4) were significantly lower (p <0.05 for Cmax and p <0.01 for AUC) than those without G allele (Cmax of 16.6 +/- 3.8 ng/ml, AUC0-96h of 558.4 +/137.4 ng.h/ml, n = 9). On the other hand, difference in Cmax and AUC0-96h was not observed between CYP2C19*2 rs4244285 GG (n = 7) and AG (n = 6). Due to limited subjects expressed in different variants of CYP2C19*3 rs4986893, CYP2D6*5, CYP2D6 G1846A/T, CYP2D6 G1934A and CYP2D6 C2938T, no relevant comparison could be conducted. Conclusion: CYP2C19 and CYP2D6 genotype in thirteen healthy Chinese subjects were evaluated and correlated with the Cmax and AUC0-96h of escitalopram after its oral administration. The results suggest that the

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genotype of CYP2D6 may have significant impact on the pharmacokinetics of escitalopram in Chinese subjects.

Future study using a larger sample size could provide would warrant further verification.

Reference: [1] Noehr-Jensen et al. Eur. J. Clin. Pharmacol. (2009) 65:887-894.

P129 - IMPACT OF CYP2D6 POLYMORPHISM ON STEADY-STATE PLASMA LEVELS OF RISPERIDONE AND 9HYDROXYRISPERIDONE IN THAI CHILDREN WITH AUTISTIC SPECTRUM DISORDER Chonlaphat Sukasem Division of Pharmacogenomics and Personalized Medicine, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Objective: To investigate the influence of genetic polymorphisms in CYP2D6 genes on plasma concentrations of risperidone and its metabolite in Thai children and adolescents with autism spectrum disorder. Methods: All 97 autism spectrum disorder patients included in this study had been receiving risperidone at least for 1 month. CYP2D6 genotypes were determined by real-time PCR-based allelic discrimination for CYP2D6*4,*5,*10, and *41 alleles.

Plasma concentrations of risperidone and 9-hydroxyrisperidone were quantified using LC/MS/MS. Results: Among the 97 patients, the most important null alleles detected were CYP2D6*4 (splice defect) and CYP2D6*5 (gene deletion), while the most common allele was CYP2D6*10 (55.9%). Presence of the CYP2D6*10 (100T/T) allele was significantly associated with higher risperidone levels (P = 0.01) and risperidone/9-hydroxyrisperidone ratio (P = 0.01), as compared to patients with the wild type allele. Plasma levels of risperidone were significantly increased in individuals with CYP2D6*5/*10 (P=0.02), CYP2D6*10/*10 (P=0.04), and CYP2D6*10/*41 (P=0.04). No significant influence was found between CYP2D6 polymorphisms, plasma concentrations of 9-hydroxyrisperidone and the total active moiety.

Conclusions: This is the first study to investigate the effects of CYP2D6 genetic polymorphisms on the plasma concentrations of risperidone in Thai children with ASD. The findings indicate that the CYP2D6*10 allele affects the plasma concentrations of risperidone and the risperidone/9-hydroxyrisperidone ratio, and that genetic screening for the CYP2D6 polymorphisms might help reduce unexpected adverse events due to the higher plasma concentration of risperidone.

P130 - VINCRISTINE-INDUCED NEUROPATHIC PAIN IN A CYP3A5 NON-EXPRESSER WITH REDUCED CYP3A4 ACTIVITY Marija Bosilkovska, Kuntheavy Ing Lorenzini, Jules Alexandre Desmeules, Youssef Daali, and Monica Escher Geneva University Hospitals, Geneva, Switzerland Background: Vincristine is metabolised by CYP3A5 and CYP3A4 isoforms with CYP3A5 contributing to 75% of vincristine intrinsic clearance. Vincristine is a substrate of the P-glycoprotein (P-gp) transporter. An increase in vincristine neurotoxicity in CYP3A5 non-expressers has been observed [1]. The severity of neuropathy was found to be inversely correlated to vincristine metabolite concentrations. However a clear correlation between genetic polymorphisms and vincristine toxicity has not been established. Case presentation: We report the case of a 21-year old African male patient who received vincristine 2 mg on three occasions (on 14.10, 30.10 and 06.11.14) for the treatment of pre-B acute lymphoblastic leukemia. Six weeks after the last vincristine dose the patient complained of bilateral severe burning pain in the toes and allodynia, suggestive of neuropathic pain. The patient was genotyped for CYP3A5 using a real-time PCR method as well as for ABCB1 (coding for P-gp) G2677T/A and C3435T SNPs. The results showed that the patient presented a CYP3A5*3/*3 polymorphism indicating that he did not express CYP3A5 enzyme. He was a homozygous â&#x20AC;&#x2DC;wild typeâ&#x20AC;&#x2122; carrier for ABCB1 SNPs. Furthermore, CYP activity was evaluated using the Geneva phenotyping cocktail including midazolam as a probe for CYP3A4 [2]. The patient had a decreased CYP3A activity which could not be explained by the concomitant medication. Similarly he had no CYP3A inhibitor in his medication at the time he received vincristine. Conclusion: The lack of CYP3A5 expression together with decreased CYP3A4 activity probably led to a decrease in vincristine clearance and to an increase in its plasma concentrations. It is a likely explanation for the occurrence of neurotoxicity in our patient despite the low doses of vincristine he received. In patients treated with vincristine, CYP phenotyping and genotyping could be crucial in preventing serious side effects.

[1] Egbelakin, A., et al., Increased risk of vincristine neurotoxicity associated with low CYP3A5 expression genotype in children with acute lymphoblastic leukemia. Pediatr Blood Cancer, 2011. 56(3): p. 361-7.

[2] Bosilkovska, M., et al., Geneva cocktail for cytochrome p450 and p-glycoprotein activity assessment using dried blood spots. Clin Pharmacol Ther, 2014. 96(3): p. 349-59.

P131 - A PHARMACOKINETIC MODELING APPROACH TO PREDICT THE CONTRIBUTION OF ACTIVE METABOLITES TO HUMAN EFFICACIOUS DOSE Susan Hill and Iain Martin Merck, Boston, Massachusetts, United States During preclinical evaluation, an oncology candidate compound was found to elicit tumor regression in a mouse xenograft model. Analysis of plasma samples from these studies revealed the presence of significant levels of circulating metabolites whose identities were confirmed by comparison to authentic standards using LC/MS. These

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metabolites were found to have similar in vitro potency at the target to the parent compound and were therefore thought likely to contribute towards the observed in vivo efficacy. In this poster we present a pharmacokinetic modeling tool that was developed for the program team to estimate if the predicted human dose for this compound was feasible in terms of size, formulation and likely safety margins. Additionally, the model could be used to aid the design of preclinical safety studies in that it facilitated species and dose selection to achieve appropriate exposure multiples not only for the parent but also for the major metabolites. The model was developed based on the assumption of an AUC target for efficacy. The mouse unbound plasma AUCs of the active metabolites at the efficacious dose were normalized by their in vitro potency compared to the parent. These AUCs were added to that of the parent to yield a total efficacious unbound AUC expressed in terms of parent ‘equivalents’ which was used as the target exposure for predictions of the human efficacious dose. In vitro and preclinical pharmacokinetic studies afforded predictions of the pharmacokinetics of the parent compound and the two major active metabolites in man including the relative metabolic pathway flux to each of the active metabolites. These predictions were combined with the relative in vitro potencies of parent compound and the active metabolites in man to parameterize a pharmacokinetic model and estimate the human dose required to achieve the target compound “equivalents” exposure. The model readily allowed interrogation of various “what if” scenarios and was applied to several molecules over the course of the program. A description of the model and its output including examples of how this information was used are presented.

P132 - CALCITRIOL AND 20(S)-PROTOPANAXADIOL SYNERGISTICALLY INHIBIT GROWTH AND INDUCE APOPTOSIS IN PROSTATE CANCER CELLS 1&2 2 2 Mohamed Ben-Eltriki , Subrata Deb , and Emma Guns 1 Department of Experimental Medicine, University of British Columbia, Vancouver, British Columbia, 2 Canada, Vancouver Prostate Centre at Vancouver General Hospital, Vancouver, British Columbia, Canada The potential role of calcitriol, the dihydroxylated metabolite of vitamin D3, and 20(S)-protopanaxadiol (aPPD), the aglycone of the protopanaxadiol family of ginsenosides, in prevention/treatment of prostate cancer (PCa) has gained much attention in recent years (Bikle, 2014, Deb et al., 2014, Musende et al., 2010). In the present study, we evaluated the anticancer effects of calcitriol and aPPD, either alone or in combination, in two well-characterized human PCa cell lines: androgen dependent non-metastatic LNCaP cells and androgen independent metastatic C4-2 cells. We hypothesized that combining calcitriol at clinically relevant concentrations with aPPD would significantly sensitize their individual anti-cancer efficacies. The effects of the treatments on PCa cell viability and proliferation rates were evaluated by MTS and Brdu assays, respectively. We assessed the synergism between calcitriol and aPPD action using the constant ratio combination design. Combination Indices (CI) and Dose Reduction Indices (DRI) were subsequently estimated using the Calcusyn software (Biosoft, Cambridge, UK) based on the median effect principle. We also assessed the potential mechanisms of pharmacodynamic interaction by measuring the following protein markers using Western blot: prostate cancer specific protein, androgen receptor, vitamin D receptors (VDR), and markers that are known to control cell cycle (cyclin D1 and cdk2) and apoptosis (Bcl-2, Bax, and caspase-3). Our results show that addition of 10nM calcitriol to aPPD significantly lowered its IC50 values from 41-53 µM to 13-23 µM, in LNCaP and C4-2 prostate cancer cells. The cell proliferation rate was significantly lower for combinations compared to the cells treated with aPPD alone. Similarly, aPPD significantly upregulated VDR expression, while calcitriol further enhances the ability of aPPD to induce BAX and inhibit cdk2 levels. Thus, the interaction between aPPD and calcitriol in growth inhibition and apoptosis appears to be synergistic in nature. In conclusion, calcitriol sensitizes PCa cells to aPPD-mediated anticancer effects by enhancing its ability to induce apoptosis and reduce cell proliferation. The associated increase in VDR expression may be mechanistically associated with this sensitization effect. Our findings suggest that there is a potential clinical value in combining aPPD with calcitriol for the treatment of human PCa and this approach could limit calcitriol toxicity by facilitating the use of lower calcitriol concentrations.

References: Bikle DD. (2014). Vitamin D and cancer: the promise not yet fulfilled. Endocrine, 46, 29-38.

Deb S, Chin MY, Adomat H, Guns ES. (2014). Ginsenoside-mediated blockade of 1alpha,25-dihydroxyvitamin D3 inactivation in human liver and intestine in vitro. J Steroid Biochem Mol Biol, 141, 94-103.

Musende AG, Eberding A, Jia W, Ramsay E, Bally MB, Guns ET (2010). Rh2 or its aglycone aPPD in combination with docetaxel for treatment of prostate cancer. Prostate, 70, 1437-47.

P133 - CARBAMAZEPINE-LOADED CARBOXYMETHYL CHITOSAN NANOPARTICLES FOR DRUG DELIVERY INTO BRAIN FOLLOWING INTRANASAL ADMINISTRATION Shanshan Liu and Chi Lui Ho National University of Singapore, Singapore, Singapore OBJECTIVE: Epilepsy has been defined as a common and diverse set of chronic neurological disorders which generally cannot be cured but its symptoms can be controlled with antiepileptic drugs. However, with the presence of the blood-brain barrier, the entry of most drugs for treatment of epilepsy is restricted. To overcome that problem, our study aims to increase the drug concentration in the brain by using carboxymethyl chitosan modified carbamazepine nanoparticles and intranasal administration to bypass the blood-brain barrier thus to enhance the treatment efficacy of antiepileptic drugs. METHODS: To achieve that objective, a carboymethyl chitosan derivative was synthesized and characterized using FT-IR, 1H NMR and 13C NMR. Then the carbamazpine-loaded carboxymethyl chitosan

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nanoparticles (CBZ- NP) were prepared by modified emulsion evaporation method and evaluated in vitro. After oral and intranasal administration of CBZ-NP, the concentration of CBZ in plasma and brain of mice were analyzed by high performance liquid choromatography (HPLC). RESULTS: Results up to date indicate that when the CBZ-NP was administered orally, the CBZ brain to plasma exposure in area under curve was found to be around 40% (AUCs in brain and plasma after oral administration were 302.80 and 705.62 min*μg/mL respectively). The value is lower than the reported value of 100% (1:1 brain to plasma concentration ratio) in humans and 75% in mice. On the other hand, when the CBZ-NP was administered intranasally, the CBZ brain to plasma exposure in AUC was 146% (AUCs in brain and plasma after intranasal administration were 55.55 and 37.15 min*μg/mL respectively). CONCLUSION: The results so far have proved our hypothesis that CBZ can be preferentially delivered to the brain when the carboxymethyl chitosan nanoparticles of carbamazepine were administered intranasally.

P134 - CYTOCHROME P450 3A4 (CYP3A4) -HUMANISED MICE FOR THE STUDY OF ANTI-CANCER DRUG PHARMACOKINETICS 1 1 2 1 Kenneth MacLeod , Colin Henderson , Nico Scheer , and Roland Wolf 1 2 University of Dundee, Dundee, United Kingdom, Taconic Biosciences, Cologne, Germany Most of the targeted small molecule anti-cancer therapies approved for clinical use within the last 15 years exhibit high inter- and intra-patient variability in exposure, as determined through their quantification in circulating blood. In almost all cases the influence of this variability on therapeutic outcome is unclear. Europeans Medicines Agency (EMA) and Food and Drug Administration (FDA) reports state that the vast majority of targeted agents are metabolised, to a greater or lesser extent, by CYP3A4. As CYP3A4 levels are known to vary >100-fold between individuals, it is possible that CYP3A4 phenotype is a critical mediator of circulating drug concentration and, thereby, the beneficial and adverse outcomes of therapy. We have utilised two novel mouse lines to examine the contribution of CYP3A4 to the disposition of targeted anti-cancer agents. In the first line, Cyp3aKO, a targeted deletion of seven of the eight murine Cyp3a genes has rendered this animal functionally null for Cyp3a activity. In the second line, hCYP3A4/3A7, a large human genomic region containing CYP3A4 and its foetal form, CYP3A7, has been inserted at the nulled Cyp3a locus.

CYP3A4 is expressed at a low basal level in the liver of this humanised line, requiring transcriptional induction through the chemically-mediated activation of its upstream regulator, pregnane X receptor (Pxr). After an extensive characterisation of these mouse lines and their response to Pxr activation, we determined the in vitro metabolic stability of 27 targeted anti-cancer agents in liver microsomal preparations. This allowed the identification of those compounds which showed the greatest degree of interaction with CYP3A4. The EGFR-inhibitor, erlotinib, had a halflife of 39 minutes with 3aKO liver microsomes and 23 minutes with h3A4 liver microsomes. The multi-kinase inhibitor, regorafenib, had a half-life of 53 minutes with 3aKO liver microsomes and 12 minutes with h3A4 liver microsomes.

These compounds were prioritised for in vivo pharmacokinetic study. Relative to 3aKO mice, the maximum plasma concentration (Cmax) of erlotinib (at a dose of 100 mg/kg) in h3A4 was decreased by 39% (from 13.7 ± 7.2 to 8.3 ± 1.1 µg/mL). The area under the curve (AUC) was decreased by 64% (89.5 ± 50.4 to 32.2 ± 4.2 hr*µg/mL) and the clearance (CL) was increased 2.3-fold (1345 ± 592 to 3136 ± 430 mL/hr/kg). For regorafenib (at a dose of 25 mg/kg), Cmax decreased by 65% (26.1 ± 8.0 to 9.2 ± 3.6 µg/mL), AUC decreased by 82% (170.9 ± 29.1 to 30.9 ± 10.7 hr*µg/mL) and CL increased 5.8-fold (148 ± 26 to 864 ± 259 mL/hr/kg) in h3A4, relative to 3aKO. In conclusion, Cyp3a null and CYP3A4-humanised mice constitute a platform for the study of anti-cancer drug metabolism and pharmacokinetics and may be of utility in defining the role of CYP3A4 in inter- and intra-patient variability of exposure.

P135 - EFFECT OF HYALURONIDASES ON THE PHARMACOKINETICS OF [I-125]-INFLIXIMAB FOLLOWING SINGLE SUBCUTANEOUS ADMINISTRATION TO RATS 1 1 1 2 1 Stephen Madden , Karen Stevenson , Marco Bottacini , Luciano Messina , and Ross Hoey 1 2 Charles River Laboratories, Tranent, United Kingdom, Fidia Farmaceutici s.p.a., Abano Terme, Italy Hyaluronidases are a family of enzymes that degrades hyaluronic acid and they are used as adjuvant in pharmaceutical products in order to increase dispersion and absorption of subcutaneously injected drugs. Using a host strain of bacteria Generally Regarded As Safe (GRAS) and non pathogenic, Fidia Farmaceutici’s R&D has developed rHyal_Sk, a new, soluble recombinant bacterial hyaluronidase having the same effectiveness as native hyaluronidase, but with a considerably better cost, safety and purity profile. The purpose of this study was to investigate the effect of the co-administration of rHyal_Sk on the pharmacokinetic profile of [I-125]-labelled Infliximab®. Infliximab® is a monoclonal antibody against tumour necrosis factor alpha (TNF-α) used to treat autoimmune diseases. This product has been employed in the past in early investigations in rats as a probe drug to test the hypothesis that improvements in the pharmacokinetics can be achieved with hyaluronidase as a “spreading” adjuvant. Three groups of Sprague Dawley female rats were administered with a subcutaneous dose of [I-125]Infliximab® alone, [I-125]-Infliximab® in combination with rHyal_Sk or Hylenex®, a recombinant human hyaluronidase currently marked as tissue permeability enhancer. A group of rats was also dosed with the labelled Infliximab® by the intravenous route. With the co-administration of rHyal_Sk, the Tmax of radioactivity in blood decreased from 48 hours ([I-125]-Infliximab® administered alone) to 6 hours post dose, indicating that Cmax is achieved quicker. The Tmax was achieved much earlier than with the co-administration of Hylenex® (24 h post dose). The study confirmed that hyaluronidases have a favourable effect in terms of absorption after subcutaneous administration, with an increase in exposure, both as Cmax and AUC. In this study, rHyal_Sk showed a superior effect compared to Hylenex®, both in terms of a quicker absorption and higher exposure.

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P136 - OPTIONS TO BOOST EXPOSURE OF ‘PROOF OF CONCEPT’ TOOL COMPOUNDS IN RODENTS TO ACCELERATE EARLY STAGE RESEARCH Lesley Murray, Kang-Jye Chou, Gauri Deshmukh, Hank La, Justin Ly, Kirsten Messick, and Cornelis Hop Drug Metabolism and Pharmacokinetics, Genentech Inc., Campbell, California, United States With recent increased exploration of novel targets for small molecule inhibitors (SMI), there is increasing need for early in vivo proof of concept (POC) studies, prior to investing a large amount of resources. Often there is still a high level of chemistry support at this stage to provide potent and selective compounds with sufficiently high exposure in rodent models to expect a pharmacodynamic (PD) effect. Inducible knockdown of target in mice provides a valuable tool for target validation, as well as informing whether any on-target toxicity is expected. Unfortunately, not all projects benefit from having access to an ‘inducible KO’ mouse model sufficiently early in discovery. An alternative strategy is to artificially boost exposure of potent and selective tool compounds with less than ideal PK properties to allow assessment of PD effect, efficacy and safety, without excessive use of resources. Techniques that have been used at Genentech and that will be discussed with examples include: •Dosing by non-oral route, e.g. IP or SC dosing to bypass gut metabolism and absorption issues •Enabling formulation for poorly soluble compounds, e.g. self-emulsifying drug delivery systems (SEDDs) and amorphous dispersion used for lipophilic or highly crystalline compounds •Inhibition of in vivo metabolism to decrease clearance •Aminobenzotriazole (ABT) to inhibit CYP-mediated metabolism •Use of HRN or HRN-nude mice which lack hepatic p450 metabolism •Inhibition of efflux transport in gastointestinal tract - inhibit Pgp transporter in GI tract – Cremaphor in SEDDs or Cyclosporin. Consider for poorly soluble compounds that are good Pgp substrates •Dosing tool compounds in place of drinking water by adding to Medidrop or Medigel – aims for more sustained exposure and avoidance of sharp peak to trough variation in plasma concentration, and reduction of stress caused by long term BID oral gavage •Exposure of a tool compound in brain can be increased by pre-dosing with a Pgp inhibitor such as Elacridar to determine whether brain entry is required for efficacy e.g. for pain drug candidate, or whether high brain concentrations are tolerated.

This arsenal of techniques to boost exposure in rodents can allow a shorter timeline to validate a novel small molecule drug target, without using up precious drug discovery resources to identify the perfect drug-like in vivo tool compound.

P137 - PHARMACOKINETIC STUDY OF ESCITALOPRAM AFTER ORAL ADMINISTRATION OF 10 MG ESCITALOPRAM TABLET IN HEALTHY CHINESE SUBJECTS 1 2 1 1 2 1 1 SK Wo , Benny SP Fok , Celia WS Tang , Vincent HL Lee , Brian Tomlinson , Teddy TN Lam , and Zhong Zuo 1 2 School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Purpose: Escitalopram, an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class, is the therapeutically active S-enantiomer of antidepressant citalopram. The pharmacokinetics of escitalopram in Chinese subjects is very limited. The present study aims to determine the pharmacokinetics of escitalopram in Chinese subjects after a single oral dose of 10 mg escitalopram tablet. Methodology: Protocol was approved from the local ethics committee prior to the study. This study is a bioequivalence study in which a single dose, two-treatment, twoperiod, two-sequence crossover design was conducted. Healthy male adults were administered with a single oral dose of 10 mg escitalopram from either the generic formulation or reference formulation (Lexapro tablet 10 mg) after an overnight fast of 10 h. Venous blood samples were collected at pre-dose (0 h) and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12, 24, 48, 72 and 96 h post-dose. Quantification of escitalopram in plasma was conducted using a validated UPLC/MS/MS method. Pharmacokinetic parameters of escitalopram were calculated by non-compartmental model from the plasma concentration vs time profiles. Results: Totally 14 male adults completed the study. The generic drug was bioequivalent to the branded product with similar pharmacokinetics parameters. The Cmax (ng/ml) obtained from 14 Chinese subjects administered with 10 mg Lexapro tablet was 15.7 +/- 4.1 ng/ml, occurred at Tmax of 3.4 +/- 1.2 h with elimination half-life (t1/2) of 35.4 +/- 11.0 h The corresponding AUC0-96h and AUC0-inf were 483.8 +/- 157.8 and 586.9 +/- 225.2 ng.h/ml, respectively. Comparing these parameters with those reported in the literature for other ethnic groups, the mean Cmax of escitalopram obtained from Chinese subjects were similar to those (12-15 ng/ml) obtained from Caucasian [1] and Brazilian [2]. The mean AUC0-inf obtained from Chinese subjects was slightly higher than Brazilian (437 ng.h/ml) [2] and between Caucasian CYP2C19 poor metabolizers (713 ng.h/ml) and extensive metabolizers (351 ng.h/ml)[1]. Conclusion: The pharmacokinetics of escitalopram in healthy Chinese subjects administered with 10 mg escitalopram was investigated and the results were compared with different ethnic groups. Further study on the phenotyping and genotyping of escitalopram and its metabolite would be required to better understanding of the metabolism of this medication in Chinese subjects.

Reference: [1] Noehr-Jensen et al. Eur. J. Clin. Pharmacol. (2009) 65:887-894. [2] Mendes et al. Int. J. Clin.

Pharmacol. Thera. (2010) 48:554-562.

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P138 - POPULATION PHARMACOKINETICS OF LINEZOLID IN CRITICALLY ILL PATIENTS 1 2 3 1 2 2 Max Taubert , Michael Zoller , Barbara Maier , Sebastian Frechen , Uwe Fuhr, Christina Scharf , Lorenz Frey , and 3 Michael Vogeser 1 2 Department of Pharmacology, Hospital of the University of Cologne, Cologne, Germany, Department of 3 Anesthesiology, Hospital of the Ludwig-Maximilians-University of Munich, Munich, Germany, Institute of Laboratory Medicine, Hospital of the Ludwig-Maximilians-University of Munich, Munich, Germany Linezolid ((S)-N-({3-[3-fluoro-4-(morpholin-4-yl)phenyl]-2-oxo-1,3-oxazolidin-5-yl}methyl)acetamide) is an antimicrobial agent for the treatment of severe infections including those with Gram-positive multi-resistant pathogens. Little is known on pharmacokinetic properties of linezolid and respective sources of variability in critically ill patients. Our objective was to develop a population pharmacokinetic model in this special group of patients and to identify and quantify the influence of covariates. The investigated data set was obtained from 53 intensive care patients (19 women, 18 on Continuous Renal Replacement Therapy [CRRT], 16 with Acute Respiratory Distress Syndrome [ARDS], 15 with lung transplantation, 8 with liver transplantation, 16 with sepsis, age 58 years, weight 75 kg, APACHE-II score 28, SOFA score 12 [medians]). Linezolid doses of 600 mg were administered intravenously or, in a few cases, orally every 12 hours for 4 days. Multiple serum samples per day were taken and linezolid was quantified by a liquid chromatography / tandem mass spectrometry method. Based on this data we developed a population pharmacokinetic model and investigated the effect of covariates (including demographics, laboratory values, intensive care scores) using NONMEM. A two-compartment model with first-order elimination kinetics was most suitable to describe the data. The central and peripheral volume of distribution were 19 (15, 26) L and 25 L respectively, while the elimination clearance was 7.6 (4.4, 12) L/h (median and interquartile range [where applicable] of individual estimates).

For oral administration, complete bioavailability and an absorption constant of 1.7 /h was found.

The body weight was linked allometrically to the central volume of distribution and to elimination clearance. An increase in the SOFA score was linked to a decrease of clearance. The need for CRRT was linked to a 38% higher apparent central volume of distribution and patients suffering from ARDS had a clearance increased by 79%. The identified covariates explained 28 % and 52 % of the observed between-subject variance in clearance and central volume of distribution, respectively. Our analysis shows that individual pharmacokinetic parameters of linezolid very much depend on body weight and various parameters describing the severity of disease. The model allows the prediction of individual concentrations required for optimization of linezolid dosing.

P139 - PREDICTION OF THE ORAL CLEARANCE OF ISONIAZID IN VIRTUAL SUBJECTS WITH DIFFERENT NAT-2 ACETYLATOR STATUS 1 1 2 2 1 1 1 Iain Gardner , Ben Small , Klaus Romero , Debra Hanna , Dave Hermann , Masoud Jamei , and Lisa Almond 1 2 Simcyp, Ltd., Sheffield, United Kingdom, Critical Path Institute, Tucson, Arizona, United States N-acetyltransferase-2 (NAT-2) is the predominant drug metabolising enzyme responsible for the metabolism of isoniazid. NAT-2 is polymorphic with evidence for 3 phenotypes in South African populations (slow, intermediate and fast acetylators). The aim of this study was to develop a scaling strategy for cytosolic enzymes that incorporates in vitro metabolism data with information on relative abundance in conjunction with the appropriate phenotype frequencies and demographics to simulate the range of isoniazid clearances in a virtual South African Population. An advantage of developing a semi-mechanistic PBPK model for isoniazid is that it readily enables in silico prediction of isoniazid clearance in other populations with differing NAT-2 phenotype distributions. Information relating to demographics and bodyweights in South African populations were collected from publically available databases. A meta-analysis was carried out for NAT-2 phenotype frequencies in South African subjects where mean values were weighted for the size of study populations. Clinical data were collected to provide oral and renal (CLR = 2.76 L/h) clearance values for isoniazid [1-5]. The in vitro intrinsic clearance for the cytosolic metabolism of isoniazid (3.13 ÂľL/min/mg) was back calculated from the average oral clearance (CLpo = 26.3 L/h) in fast acetylator subjects reported

by Peloquin et al.[3], using a retrograde approach. Relative abundances of the enzyme in fast and slow acetylators were calculated based on data reported by Weber and Hein [6] and Werely et al., [7]. The pharmacokinetics of isoniazid in South Africans were simulated in a virtual population (n=1000 individuals), with demographics matching those represented in available clinical studies, using the Simcyp Simulator (V14R1). Simulated plasma concentrationtime profiles and distribution of CLpo values were compared to observed. The meta-analysis of available data indicated that the frequency of fast, intermediate and slow acetylator subjects in a South African population was 0.236, 0.425 and 0.359, respectively. The relative activity in slow acetylators was calculated to be 0.25 of that in fast acetylators. The activity in intermediate acetylators was assumed to be 0.61. Using the phenotype frequencies obtained from the meta-analysis and phenotype-specific relative abundances, the distribution of CLpo values in a South African population were well recovered when fast, slow and intermediate phenotypes were considered. Using data for only fast and slow acetylator phenotypes did not recover the observed data as well. In conclusion using information on NAT-2 metabolism in fast acetylators together with information on the frequency of intermediate and slow acetylator phenotypes and relative activity of NAT-2 across phenotypes it was possible to simulate the plasma concentration-time profile and distribution of clearances of isoniazid within a South African population.

[1] Agrawal S et al (2002). Int J Pharmaceutics 233:169-177 [2] McIlleron H et al (2006) Antimicrob Agents Chemo 50:1170-1177 [3] Peloquin C et al (1999) Int J Tuberc Lung Dis 3:703-710.

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[4] Israili Z et al (1987) J Clin Pharmacol 27:78-83 [5] Schall et al (1995) Arzneim-Forsch/Drug Res 45: 1236-1239 [6] Weber Wand Hein D(1979) Clinical Pharmacokinetics 4:401-422.

[7] Werely CJ (2012) Pharmacogenetics of Arylamine N-acetyltransferase genes in South African populations. PhD Thesis, Stellenbosch University P140 - THE EFFECT OF THE BLOOD SAMPLING SITE ON PK PARAMETERS AFTER OROMUCOSAL ADMINISTRATION IN DOGS 1 1 2 1 3 Astrid Capello , Mira Wenker , Lauri Vuorilehto , Harry Emmen , and Jarmo S. Salonen 1 2 WIL Research Europe, 's Hertogenbosch, Netherlands, University of Turku, Department of Pharmacology, Drug 3 Development and Therapeutics, Bioanalytical Laboratory, Turku, Finland, R&D, Chemistry and Safety Sciences, Orion Corporation ORION PHARMA, Espoo, Finland Commonly, drugs are administered orally, but not all compounds are suited for oral administration due to poor absorption or extensive first pass metabolism. Such compounds are generally given intravenously or intramuscularly.

To find a less invasive route of administration, the interest in other dosing routes like oromucosal (buccal), sublingual and nasal is increasing. However, when using these routes of administration, consideration should be given to the site of blood sampling, in relation to the measured plasma concentration. These data are essential for a proper pharmacokinetic evaluation, but are not always taken in consideration in the design of pharmacological studies.

In this study 9 male healthy beagle dogs were dosed intravenously, intragastricly (oral gavage) and oromucosally with 125 Âľg/m2 dexmedetomidine. Blood was sampled from the jugular vein with all dosing routes. In addition, in 6 dogs, blood was also sampled from the cephalic vein following oromucosal administration. Dexmedetomidine concentrations were determined by a qualified LC-MS/MS method. The pharmacokinetic parameters were calculated using noncompartmental analysis.Following intravenous administration, dexmedetomidine was a highly distributed and highly cleared compound, which was also rapidly eliminated. After intragastric gavage administration, all dexmedetomidine concentrations were below the limit of quantification, suggesting negligible bioavailability by this route, as expected. After oromucosal administration, blood samples taken from the jugular vein displayed higher and more variable plasma concentrations compared with sampling from the cephalic vein. The differences between the two sampling sites were highest within the first 4 hours after sampling. Furthermore, a difference was noted in the concentrations measured from the different jugular veins, most likely caused by the site of the administration in the mouth. The plasma exposure measured after sampling from the jugular vein was, in terms of Cmax and AUC, respectively 4.1-fold and 3.2-fold higher compared with the sampling from the cephalic vein. In some of the dogs, the estimated (apparent) systemic bioavailability clearly exceeded 100%. These results demonstrate that after oromucosal administration sampling from the jugular vein leads to a harsh overestimation of the systemic exposure and bioavailability of dexmedetomidine compared with sampling from the cephalic vein. In order not to overestimate the exposure after oromucosal administration, a blood sampling site where the absorbed compound has passed the heart prior to blood sampling must be chosen.

P141 - ANTI-PROLIFERATIVE EFFECT OF NUCLEAR RECEPTOR NR0B2 IN RENAL CARCINOMA CELLS 1 3 3 3 1 4 Katharina Prestin , Janine Hussner , Tamara Isenegger , Daniel Gliesche , Kerstin BĂśttcher , Uwe Zimmermann , 3 and Henriette E. Meyer Zu Schwabedissen 1 University of Basel, Biopharmacy, Department of Pharmaceutical Science, Basel, Switzerland 2 2 Maria Olbert , University Medicine of Greifswald, Department of Pharmacology, Center of Drug Absorption and 3 Transport (C_DAT), Greifswald, Germany, University of Basel, Biopharmacy, Department of Pharmaceutical Science, 4 Basel, Switzerland, University Medicine of Greifswald, Department of Urology, Greifswald, Germany Members of the transcription factor family of nuclear receptors (NR) are assumed to regulate the expression of target genes which play an important role in drug metabolism, transport and cellular signaling pathways. The orphan and structurally unique receptor small heterodimer partner 1 SHP1 (NR0B2) is not only known for its modulation of drug response, but has also been reported to be involved in carcinogenesis. Indeed, previous studies show that NR0B2 is down-regulated in human hepatocellular carcinoma, suggesting that NR0B2 acts as a tumor suppressor via inhibition of cellular growth and activation of apoptosis [1]. The aim of our study was to elucidate whether NR0B2 may also play a role in other tumor entities.

Performing quantitative real-time RTPCR and Western Blot for mRNA and protein expression analysis revealed that NR0B2 expression was significantly lower in tumor samples of human renal cell carcinoma compared to nonmalignant transformed tissue, suggesting that mechanisms of reduced NR0B2 expression might also play a role in this tumor entity. To test the impact of NR0B2 on proliferation of human kidney cells, we performed an adenoviral transfer of NR0B2 in a renal carcinoma cell line (RCC-EW) thereby modulating the expression in those cells. For validation of adenoviral transfer of NR0B2, Western blot analysis and immunofluorescence staining of infected RCC-EW cells were performed, showing that an increasing amount of adenoviral load resulted in an increasing expression of NR0B2.

Finally the impact of heterologous expression of NR0B2 on cell cycle progression and proliferation was characterized.

Monitoring fluorescence intensity of resazurin turnover in RCC-EW cells revealed no significant differences in metabolic activity in presence of NR0B2. However, there was a significant decrease of cellular proliferation in cells overexpressing the nuclear receptor. Interestingly, inhibition of proliferation by heterologous NR0B2 was more pronounced than treatment with the commonly used chemotherapeutics sunitinib or temsirolimus. Furthermore flow

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cytometry analysis showed that heterologous overexpression of NR0B2 significantly reduced the amount of cells passing G1 phase while on the other hand more cells in S/G2 phase were detected. Taken together, we show that NR0B2 is down-regulated in renal cell carcinoma in vivo. In addition, our data also suggest that increased expression of NR0B2 diminishes cellular proliferation of kidney tumor cells in vitro, supporting its regulatory role in renal cell cancer progression. Future studies have to specify the mechanism responsible for diminished proliferation of renal carcinoma cells and the shift of cell cycle phases after overexpression of NR0B2.

P142 - CHARACTERISATION OF HUMAN CAR SPLICED VARIANTS/CAR1 MUTANTS IN CAR-NULL MICE AND IN MOUSE PRIMARY HEPATOCYTES 1 1 1 1 2 1 Shaohong Ding , Michael McMahon , Lourdes Acosta Jimenez , Julia Carr , Nico Scheer , Colin Henderson , and 1 Roland Wolf 1 2 Division of Cancer Research, Ninewells Hospital, University of Dundee, Dundee, United Kingdom, Taconic Biosciences, Cologne, Germany The constitutive androstane receptor (CAR) is a xenobiotic sensor that regulates the expression of drug-metabolising enzymes. It also impacts upon other liver functions such as gluconeogenesis and may promote tumour growth. For all these reasons, there is great interest in academia and industry in developing systems to identify activators of human CAR (hCAR), and to study their mode-of-action. We now report a new and improved system for assaying hCAR activity in vivo. Using tail vein injection of recombinant adenoviral particles, we successfully reconstituted the livers of mCAR-/- animals with hCAR isoforms. Strikingly, hCAR1, the reference isoform, is expressed predominantly in the nucleus of hepatocytes in control animals, whereas hCAR2 and hCAR3, are retained in the cytoplasm. We validated the model by demonstrating that the prototypic indirect activator of CAR, phenobarbital (PB), and the direct hCARbinding ligand CITCO, both activated hCAR1 and drove transcription of Cyp2b10 and Cyp3a11. Neither hCAR2 nor hCAR3 were activated by PB, CITCO, or a third agent, DEHP, in our in vivo assay. These data conflict with previous work using artificial CAR trans-activation assays that suggested CITCO could stimulate all three hCAR isoforms, and that DEHP could stimulate hCAR2. In order to increase the throughput of this CAR activation assay, we successfully reconstituted mCAR-/- hepatocytes with hCAR isoforms. Finally, we have used this system to explore how PB activates hCAR1 by expressing mutant forms of hCAR in the mCAR-/- background. We demonstrate that the T38D mutant form of hCAR1 cannot respond to either indirect (PB) or direct acting (CITCO) CAR activators, suggesting that T38 plays a more fundamental role in CAR regulation than just responding to indirect activators through EGFR. Our reconstitution system provides a powerful approach to identify chemicals that can activate hCAR, whether direct or indirect, and to dissect the underlying signalling mechanisms.

P143 - EFFECTS OF CYTOTOXIC GOLD (I) COMPLEXES ON TRANSCRIPTIONAL ACTIVITY OF VARIOUS NUCLEAR RECEPTORS Katerina Kubesova, Zdenek Travnicek, and Zdenek Dvorak Palacky University, Olomouc, Czech Republic The development of new metal-based anticancer and anti-inflammatory agents is a very important direction of research. The interest in this field was unambiguously triggered by medicinal success of cisplatin, as an antineoplastic drug. However, the negative side effects of cisplatin have resulted in an extensive search for more favourable compounds with the aim to improve the anticancer activity and/or decrease the side effects compared to cisplatin.

Therefore platinum complexes were later followed by coordination compounds of other transition metals (e.g. copper, gold, zinc, ruthenium, etc.) [1], however, no such compounds have been approved for clinical use as anticancer drugs to date. On the other hand, clinical use of gold complexes is known, in particular, for their application in the treatment of rheumatoid arthritis. In this work, three gold (I) mixed-ligand complexes with the general formula [Au(Ln)(PPh3)] (13) involving triphenylphosphine (PPh3) and a deprotonated form of O-substituted derivatives of 9-deazahypoxanthine (Ln), i.e. 6-isopropyloxy-9-deazapurine (HL1); 6-phenethyloxy-9-deazapurine (HL2) and 6-benzyloxy-9-deazapurine (HL3) [2] were studied for their in vitro cytotoxic effects and influence on the transcriptional activity of several nuclear receptors in cell-based models. The complexes were screened for their in vitro antitumour activity against human cancer cell lines HepG2 (hepatocarcinoma), 22Rv1 (prostate carcinoma) and LS180 (colon adenocarcinoma) in range from 0.01 µM to 50 µM by the MTT assay. The results showed cytotoxic activity with low micromolar IC50 values in all the tested cell lines. The best results were achieved for the complex 3 against 22Rv1 and LS180, with the IC50 values in the range of 4.2 – 6.3 µM. Then, we tested the effects of these complexes (non-toxic concentrations) on the transcriptional activity (agonist and antagonist mode) in nuclear receptors by a gene reporter assay. Nuclear receptors play a crucial role in the regulation of drug metabolism, energy metabolism, immunity and other physiological functions. We used a stably transfected gene reporter cell lines for assessment of transcriptional activity of aryl hydrocarbon receptor (AhR) [3], androgen receptor (AR) and thyroid receptor (TR) and transiently transfected gene reporter cell line for assessment of transcription activity pregnane X receptor (PXR). The obtained results of the investigated Au(I) complexes displayed an activity of the aryl hydrocarbon receptor, thyroide receptor, androgen receptor and pregnane X receptor. The results suggested here might be of toxicological and clinical importance.

REFERENCES [1] Gielen M, Tiekink ERT (2005) Metallotherapeutic Drugs and Metal-based Diagnostic Agents: The Use of Metals in Medicine. London: John Wiley and Sons, Ltd., Chichester, England.

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[2] Vančo J, Gáliková J, Hošek J, Dvořák Z, Paráková L, Trávníček Z (2014) PLOS One 9(10): e109901.

[3] Novotna A, Pavek P, Dvorak Z (2013) Environ. Sci. Technol. 10.1021: es2029334.

P144 - HEPATIC NUCLEAR RECEPTOR - ENVIRONMENTAL POLLUTION INTERACTIONS REGULATE ENERGY METABOLISM, INFLAMMATION, AND LIFESTYLE BEHAVIORS IN NON-ALCOHOLIC FATTY LIVER DISEASE 1 2 3 3 3 Russell Prough , Banrida Wahlang , K. Cameron Falkner , Ming Song , and Matt Cave 1 Department of Biochemistry & Molecular Biology, The University of Louisville School of Medicine, Louisville, 2 Kentucky, United States, Department of Pharmacology & Toxicology and Division of Gastroenterology/Medicine, The 3 University of Louisville School of Medicine, Louisville, Kentucky, United States, Division of Gastroenterology/Medicine, The University of Louisville School of Medicine, Louisville, Kentucky, United States Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of US adults and dosedependently associated with non-alcoholic fatty liver disease in epidemiologic studies. PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism. We recently demonstrated that exposure to the commercial PCB mixture, Aroclor 1260, worsened non-alcoholic steatohepatitis (NASH) in a diet-induced obesity mouse model and activated nuclear receptors, including the pregnane-xenobiotic receptor (PXR) and constitutive androstane receptor (CAR). In this study, we further characterized the role of these receptors in PCB induced-NASH. C57Bl/6 (wildtype), PXR-/- and CAR-/- mice were exposed to Aroclor 1260 (20 mg/kg) and fed a high fat diet (42% kCal fat) for 12 weeks. Serum, liver and adipose samples were taken for immunohistochemistry, RT-PCR, lipid and adipocytokine measurements. In PXR-/- and CAR-/- mice, Aroclor 1260 exposure resulted in steatohepatitis with increased basal hepatic TNFα and IL-6 expression. PXR-/- mice had increased % body fat and liver to body weight ratio regardless of exposure while PXR-/- mice exposed to Aroclor 1260 showed increased hepatic gluconeogenic and lipogenic gene expression. CAR and PXR activation appeared to decrease hepatic lipogenic (FAS, SCD-1) and gluconeogenic (PEPCK-1, Glucose-6-phosphatase) gene expression. The knockout groups also demonstrated increased basal mTOR1 activity while Aroclor 1260 exposure increased AMPKα activity. Intriguingly, Aroclor 1260 exposure resulted in decreased serum insulin levels and HOMA-IR, an index for insulin resistance, in all groups. Thus, PXR and CAR participate in hepatic energy metabolism and appear to play an important protective role in Aroclor 1260-induced liver injury. Supported by National Institutes of Environmental Health Science grant ES021375.

P145 - PROFILING OF ANTHOCYANIDINS AGAINST TRANSCRIPTIONAL ACTIVITIES OF STEROID AND NUCLEAR RECEPTORS 1 2 Barbora Pastorkova and Zdenek Dvorak 1 Department of Biochemical Sciences, Charles University, Faculty of Pharmacy, Olomouc, Czech 2 Republic, Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Olomouc, Czech Republic The anthocyanidins are plant pigments. They represent aglycon (sugar-free) backbones of anthocyanins, belonging to the phenolic compounds known as flavonoids. They are important antioxidants and possess antitumor and antimutagenic activities [1]. The anthocyanidins are constituents of common diet and can cause food-drug interaction; pharmacokinetic and toxicokinetic interactions between food and drugs that change their activity. Food-drug interactions proceed either by inhibition or induction of drug metabolizing enzymes. These enzymes are transcriptionally regulated by steroid and nuclear receptors [2]. In presented work, the effect of the most common anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin and peonidin) on the transcriptional activity of steroid and nuclear receptors was studied. It has already been shown that pelargonidin slightly induces the aryl hydrocarbon receptor (AhR) that regulates xenobiotic-metabolizing enzymes, such as cytochrome P450 [3]. It was examined other nuclear and steroid receptors: glucocorticoid receptor (GR), androgen receptor (AR) and thyroid receptor (TR) in a stably transfected luciferase gene reporter cell lines HeLa (cervical cancer cells), 22RV1 (human prostate carcinoma cells), HepG2 (human Caucasian hepatocellular carcinoma cells), respectively. Activity of pregnane X receptor (PXR) and vitamin D receptor (VDR) in transiently transfected luciferase gene reporter cell line LS180 (colorectal adenocarcinoma cells) was also investigated. First, the cytotoxicity of the anthocyanidins with increasing concentrations was assessed and the range of concentration was set. The cells were incubated for 24 h with tested compounds with increasing concentrations (10 nM - 50 µM) in reporter gene assay. Model agonists were used as a positive controls and vehicle (DMSO) as a negative control. Experiments were performed in agonist and antagonist mode. In the antagonist mode, the cells were treated with increasing concentrations of substances in the presence of model agonist. It was found that PXR is inhibited by malvidin (IC50 39 μM) and pelargonidin (IC50 53 μM) in the antagonist mode. Moreover, the inhibition of VDR activity was observed by malvidin (IC50 43 μM) and pelargonidin (IC50 38 μM).

References: [1] Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R. Analysis and biological activities of anthocyanins.

Phytochemistry. 2003; 64(5):923-33.

[2] Kamenickova A, Anzenbacherova E, Pavek P, Soshilov AA, Denison MS, Zapletalova M, Anzenbacher P, Dvorak Z. Effects of anthocyanins on the AhR-CYP1A1 signaling pathway in human hepatocytes and human cancer cell lines.

Toxicol Lett. 2013; 221(1):1-8.

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[3] Kamenickova A, Anzenbacherova E, Pavek P, Soshilov AA, Denison MS, AnzenbacherP, Dvorak Z. Pelargonidin activates the AhR and induces CYP1A1 in primary humanhepatocytes and human cancer cell lines HepG2 and LS174T. Toxicol Lett. 2013; 218(3):253-9.

P146 - ABUNDANCE OF MEMBRANE TRANSPORTER EXPRESSION IN CELL LINES AND COMPARISON OF ABUNDANCE AND FUNCTIONAL ACTIVITY OF SELECTED TRANSPORTERS IN CELL LINES AND ISOLATED HEPATOCYTES 1 2 1 3 Berend Oosterhuis , John Keogh , Remi Magnan , Yann Courbebaisse 1 2 3 SOLVO Biotechnology, Budaörs, Hungary, JPK Consulting, Hitchin, United Kingdom, Bertin Pharma, Orléans, France The major human hepatic transporters BSEP, OCT1, MATE1, OATP1B1, OATP1B1*1A, OATP1B3, MDR1, MRP2 and BCRP, and the renal transporters OAT1, OAT3, OCT2, MATE1 and MATE 2K transporters were measured in overexpressing cell lines or vesicles using quantitative targeted absolute proteomics (QTAP). In addition, the abundance of selected transporters was measured in human hepatocytes. [1] The relative abundance of these transporters was compared with literature values for native tissues. Transport rates of standard substrate(s) and of protein abundance were measured in paired samples of overexpressing cell lines and isolated human hepatocytes, to investigate the relationship between transporter abundance and functional activity in vitro, and to compare hepatocyte uptake with that in over-expressing cell lines. Results indicate that transporter abundance in over-expressing cell lines or vesicles is typically greater than observed in hepatocytes, although this is transporter-dependant. For example, OATP1B1 and OATP1B3 abundances in cell lines are approximately 77 and 3 times the average levels measured in hepatocytes[2]. Transport rates of the tested substrate(s) are also significantly different between hepatocytes and over-expressing cells. The correlation between protein abundance in hepatocytes and cell lines and probe substrate uptake is discussed.

Bibliography: [1] T. T. Ohtsuki S, Uchida Y, Kubo Y, “Novel Applications of LC / MS-based Membrane Protein Quantification for Transporter Research and Drug Development Sumio Ohtsuki,” J Pharm Sci, 2011.

[2] O. Schaefer, S. Ohtsuki, H. Kawakami, T. Inoue, S. Liehner, A. Saito, A. Sakamoto, N. Ishiguro, T. Matsumaru, T. Terasaki, and T. Ebner, “Absolute quantification and differential expression of drug transporters, cytochrome P450 enzymes, and UDP-glucuronosyltransferases in cultured primary human hepatocytes.,” Drug Metab. Dispos., vol. 40, no. 1, pp. 93–103, Jan. 2012.

P147 - CHARACTERIZATION OF STABLY TRANSFECTED HEK-293 CELLS EXPRESSING OATPS USING FLUORESCENT SUBSTANCES 1 1 2 1 1 Jia Jia , Claudia Garve , Markus Keiser , Dieter Runge , and Anett Ullrich 1 2 Primacyt Cell Culture Technology GmbH, Schwerin, Germany, University Medicine of Greifswald, Department of Clinical Pharmacology, Greifswald, Germany Membrane transporters are major variables for disposition, efficacy and safety of many drugs. Transport proteins can be divided into uptake and ATP-dependent efflux transporters. Organic anion transporting polypeptides (OATPs, gene family: SLCO) belong to the uptake transporters and are important for the absorption, distribution and excretion of drugs. Therefore, we developed a cell platform using stably transfected cells expressing pharmacologic relevant OATPs. Traditionally, the function of cells has been characterized using radiolabeled substrates, which causes potential health risks and requires a specific equipment and an isotope laboratory facility. To overcome these issues, we wanted to analyse the uptake function of OATPs with fluorescent substrates, which can be performed in normal labs. Three fluorescent substances (Fluorescein methotrexate (FMTX); Fluorescein; Rhodamine 123) were analyzed with stably transfected HEK-293 cells expressing OATP1A2, 1B1, 1B3 and 2B1. HEK-OATP1B3 showed an uptake of FMTX (Km = 1.5 ± 0.4 µmol/l, Vmax = 22.8 ± 1.7 pmol/mg × min), which could be inhibited by rifampicin (IC50 = 0.37 µmol/l) efficiently. FMTX was also a substrate for HEK-OATP1B1 cells with a Km value of 4.7 ± 1.3 µmol/l and a Vmax value of 59.1 ± 4.7 pmol/mg × min. This uptake was inhibited by rifampicin with an IC50 value of 0.69 µmol/l. HEKOATP1B1 showed an affinity to fluorescein (Km = 18.2 ± 6.7 µmol/l, Vmax = 147.9 ± 23.8 pmol/mg × min), which could be inhibited by rifampicin (IC50 = 1.05 µmol/l). HEK-OATP1A2 showed an uptake of Rhodamine 123 (Km = 3.8 ± 1.3 µmol/l, Vmax = 577.7 ± 86.6 pmol/mg × min). This uptake was inhibited by rifampicin (IC50 = 35.4 µmol/l).

However, OATP2B1 stably transfected HEK-293 cells showed no specific transporter-mediated uptake of FMTX, Fluorescein and Rhodamine 123. Thus, the transport function of stably transfected HEK-293 cells expressing OATP1A2, OATP1B1 and OATP1B3 can be characterized with fluorescent substances. Having demonstrated that transporter activities in stably transfected HEK cells can be analyzed with fluorescent compounds we are currently adopting these methods for the use with primary hepatocyte cultures.

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P148 - CHARACTERIZATION OF THE EXPRESSION AND FUNCTION OF ENDOGENOUS TRANSPORTERS IN HEK293 AND MDCKII CELLS Marcus Otter, Paul-Ole Eriksson, Markus Keiser, and Stefan Oswald University Medicine of Greifswald, Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), Greifswald, Germany BACKGROUND: Drug transporters are known to be important determinants in the pharmacokinetics and efficacy of many drugs. In order to characterize the affinity or inhibitory properties of drugs to transporter proteins in preclinical drug development, transporter-overexpressing cellular models based on MDCKII and HEK293 cells are widely used and recommended by current guidelines by the medical authorities. In this study, we investigated the endogenous (“background”) transporter expression in frequently used HEK293 and MDCKII cells. Furthermore, we examined the functional consequences of endogenous ABCB1 in cellular uptake studies. METHODS: The following cell lines have been investigated: HEK293 wild-type cells or stably transfected with control vector, OATP1A2, OATP1B1, OATP1B3 or OATP2B1; MDCKII wild-type cells or stably transfected with control vector or ABCB1. The mRNA expression of 5 human ABC and 16 SLC transporters (HEK293 cells) and 16 canine transporters (MDCKII cells) were determined by validated TaqMan® gene expression assays. Protein abundance was quantified by LC-MS/MS-based targeted proteomics. The functional study was performed with talinolol as probe substrate of OATP1A2 and ABCB1 using wildtype, stably transfected vector control and OATP1A2-overexpressing HEK293 cells. Intracellular accumulation after 5 min and 30 min incubation of [3H]-talinolol was measured by liquid scintillation counting after cell lysis. RESULTS: The observed gene expression and protein abundance data were mostly not correlated. Several transporters could be identified as endogenous transporters in both cell lines. In HEK293 cells, the endogenous ABCB1 protein content was considerably lower in vector control-, OATP1A2-, OATP1B1-, OATP1B3- and OATP2B1-transfected cells compared to wild-type cells. On the contrary, markedly higher expression has been observed for endogenous ABCC2 in HEKOATP1B1 and for endogenous ABCC3 in MDCK-ABCB1. Time-adjusted influx rate of talinolol in wild-type, vector control and OATP1A2 cells was significantly decreased after 30 min in comparison to 5 min incubation. Higher protein abundance of endogenous ABCB1 in wild-type cells resulted by trend in reduced intracellular accumulation of talinolol compared to vector-transfected cells. CONCLUSION: There are markedly differences in expression pattern of endogenous transporters in the investigated HEK293 and MDCKII cells. These endogenous transporters may affect drug transport and the estimated transporter affinity to the focused transporter protein.

P149 - CHARACTERIZATION OF TRANSPORTER ACTIVITIES IN FRESH ISOLATED PRIMARY HEPATOCYTES OF DIFFERENT SPECIES BY USING FLUORESCENT SUBSTRATES Anett Ullrich, Jia Jia, and Dieter Runge Primacyt Cell Culture Technology GmbH, Schwerin, Germany Primary mammalian hepatocytes are used for several in vitro applications like testing of drug metabolism, toxicity and transporter assays. A comprehensive characterisation of plated primary hepatocytes is of high interest for researchers involved in ADME studies. Beside CYP inducibility we have therefore focused our work on transporter activities in fresh isolated human and Cynomolgus monkey hepatocytes. Previous studies have shown that radiolabeled substances can be used to characterize transporter activities in fresh and cryopreserved hepatocytes. Our current work is focused on the use of fluorescent substances in assays than can be performed in all standard laboratories.

Human and monkey hepatocytes were incubated in serum free media. After cell isolation a time and concentration dependent uptake of FMTX (Fluoresceine Methotrexate) was measured in presence or absence of Rifampicin as inhibitor. FMTX was evaluated as a suitable transporter substrate for primary hepatocytes. Hepatocytes from both species showed a saturable time-dependent uptake of FMTX within 30 minutes. The intracellular accumulation of FMTX was inhibited by Rifampicin to approx. 70 %. In addition, a concentration-dependent uptake assay for FMTX was performed for 2 and 5 min. Here, the substance transport was impaired by Rifampicin only to a minor extent.

Other fluorescent substances like Fluoresceine did not show a transporter-mediated uptake. In conclusion, fluorescent compounds may be used as an unhazardous alternative to radiolabeled chemicals for investigating transporter activities in primary hepatocytes.

P150 - CHARACTERIZING HEPATO-BILIARY TRANSPORT UTILIZING THE STABLE, LONG-ENDURING CELLULAR COMPETENCY OF HµREL® CO-CULTURE TECHNOLOGY AND SINGLE-WELL DIRECT ASSAY METHOD Eric Novik, Matthew Shipton, Amit Parekh, Cheul Cho, Eric Pludwinski, and Anil Shrirao Hurel Corporation, Beverly Hills, California, United States Accurately predicting the pharmacokinetic and toxicological properties of drugs as early as possible in the development process can materially reduce the number of late-stage pre-clinical and clinical failures, the very expensive investment write-offs associated with such late-stage failures and, hence, the total, fully burdened cost of securing regulatory approval for a new drug. As such, there is an accelerating drive to identify in vitro models that offer greater translational relevance in predicting human physiological responses to xenobiotics. Current systems provide in vivo data obtained using whole animals or in vitro using microsomes, isolated hepatocytes, and cell lines such as HepG2. Isolated hepatocytes or their enzymes comprise the basis of most in vitro DMPK and toxicity assays used to predict human hepatic outcomes. Because hepato-specific functions are typically lost in the first hours or days of

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culture in many in vitro hepatic models, such models are of limited use in instances where meaningfully predictive simulation of a compound’s properties require that the in vitro model maintain such function stably over longer time periods, such as where compounds clear slowly, where metabolites are generated over extended periods of time, where membrane polarization is required for biliary canalicular formation, or where cellular conditions may cumulate after repeated dosing, such as in cholestatic toxicities. To address this limitation, HµREL has developed and extensively characterized, in the human and several pre-clinical species, a long-enduring, stable primary hepatocyte co-culture model. This long-term model has been shown to more accurately predict intrinsic clearance, metabolite generation, and toxicity profiles. Here we report the ability of this human and rat model to assess biliary efflux mediated by a number of key canalicular transporters when assayed utilizing Hurel’s single-well direct measurement method. The results show that all compounds tested were taken up into the cell and subsequently secreted into the bile canaliculus in the 20 minute incubation period. Uptake rates of the compounds were measured in pmol/min/mg protein for Taurocholic Acid, Digoxin, Rosuvastatin, Estradiol-Glucuronide and Pravastatin. The uptake rates were 41, 1.34, 7.62, 1.29 and 0.4 respectively. In conclusion, utilizing Hurel’s single-well, direct measurement method, cryopreserved hepatocytes plated in HµREL’s co-culture model can assay biliary excretion mediated by a number of important canalicular transporters. In addition, the direct measurement of biliary and cellular content drawn from the same single well via LC/MS analysis may offer an improvement over current methods of assessing biliary excretion.

P151 - EVALUATION OF CLINICALLY RELEVANT INHIBITORS OF BSEP USING B-CLEAR® HUMAN SANDWICH-CULTURED HEPATOCYTES TO BETTER PREDICT INHIBITION AND CHOLESTASIS 1 1 2 2 2 1 Lydia Vermeer , Caleb Isringhausen , Kimberly Freeman , Chris Black , Kenneth Brouwer , and Gregory Loewen 1 2 XenoTech LLC, Lenexa, Kansas, United States, Qualyst Transporter Solutions LLC, Durham, North Carolina, United States Drug induced liver injury (DILI) has caused many drugs to be withdrawn from the market. A hypothesized mechanism behind DILI is cholestasis due to inhibition of the bile salt efflux pump (BSEP), an efflux transporter expressed in the canalicular membrane of hepatocytes. BSEP is a major route of efflux for bile acids and inhibition of this transporter can be measured in vitro through the use of systems such as transfected vesicles. It is important to note though, that many xenobiotics which have been shown to cause DILI in vivo have not been identified as BSEP inhibitors in currently employed in vitro assays. While inhibition of BSEP has been implicated as a mechanism of drug-induced cholestasis and hepatotoxicity, studies have also demonstrated that many xenobiotics can lead to changes in bile acid disposition through inhibition of both uptake and efflux in hepatocytes. It is hypothesized that an in vitro system which better models in vivo transporter expression and functionality will better predict both inhibition and the potential for hepatotoxicity. To study this, B-CLEAR® human sandwich-cultured hepatocytes were used to screen a panel of known BSEP inhibitors clinically demonstrated to cause hepatotoxicity. Changes in uptake, efflux, and intracellular concentrations of taurocholic acid ([3H]-TCA) were investigated. Following this, a range of concentrations of compounds that demonstrated inhibition were studied to better understand how uptake and efflux into the hepatocytes and bile caniculi were affected. Toxicity studies are currently being carried out utilizing the MTT, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and urea formation assays to better correlate inhibition of uptake and efflux and hepatotoxicity. Inhibition results for cyclosporine A (CspA) indicate that compared to solvent control treatment the intracellular concentration (ICC) of TCA decreased over 8-fold in the presence of 10 µM CspA. The biliary excretion index (BEI), a measure of retained TCA in the bile caniculi, decreased almost 6-fold, and the Kp ratio decreased 9-fold. These results indicate inhibition of uptake by CspA in the human hepatocytes. Typically, a higher ICC and Kp values may indicate inhibition of TCA efflux, and a high potential for toxicity, while a lower ICC and Kp values may indicate inhibition of uptake. Additional compounds will be discussed. Utilizing the B CLEAR® sandwichcultured hepatocytes, we evaluated changes in intracellular concentration, BEI, Kp, and the uptake and efflux of TCA which are not captured by other in vitro systems currently in use. Overall, these data demonstrate the complexity of transporter inhibition and the impact many xenobiotics may have on hepatic uptake and efflux, and clearance from the body. The use of the B-CLEAR® sandwich-cultured hepatocyte system more closely models in vivo conditions encountered in the clinic, which could lead to more accurate results regarding BSEP inhibition and hepatotoxicity.

P152 - FUNCTIONAL CHARACTERIZATION OF OATP2B1 USING FÖRSTER RESONANCE ENERGY TRANSFER (FRET) Paul Hagen, Gabriele Jedlitschky, Werner Siegmund, and Markus Grube University Medicine of Greifswald, Department of Pharmacology, Greifswald, Germany Introduction: Organic anion-transporting polypeptides represent a family of multispecific uptake transporters for a wide range of drugs as well as endogenous compounds like statins and steroid-conjugates. While little is known about the exact molecular transport mechanism, it is assumed that the transmembranal transfer of OATP substrates involves a rocker-switch-type movement of the protein. Under this assumption the transmembrane domains of the protein should alter their relative orientation to each other during the transport process. FRET (Förster resonance energy transfer) microscopy is a suitable technology to investigate such conformational changes. We therefore modified OATP2B1 by introducing FRET donor and acceptor dyes to characterize this transporter.

Methods and Results: OATP2B1 was modified by fusing the FRET donor ECFP to the C-terminus of the protein. In addition, the FRET acceptor EYFP was added to the N-terminus or the third intracellular loop of the protein and the fusion proteins were subsequently expressed in HeLa cells. The constructs were inserted at least in part into the

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plasma membrane. Intramolecular FRET was measured by confocal laser scanning microscopy using the acceptor photobleaching as well as the sensitized emission method. In dependence of the acceptor position, different FRETefficiencies were detected: While the N-terminal EYFP construct demonstrated a FRET efficiency of 7 %, the FRET efficiency of the construct with the EYFP in the intracellular loop was 16 % indicating a closer spatial proximity of this loop to the C-terminal domain compared to the N-terminus. Additionally, the FRET efficiency of the latter construct was measured in the presence and absence of the OATP2B1 substrate Estrone-3-sulfate (10 µM) revealing a significant lower FRET-efficiency (14 %) in the presence of the substrate.

Conclusions: Intramolecular FRET can be used to investigate the molecular conformation of OATPs and may be useful tool to characterize drug transporter interactions.

P153 - GENETIC VARIANTS OF THE ORGANIC ANION TRANSPORTER POLYPEPTIDE 1A2 (OATP1A2) INFLUENCE THE CELLULAR UPTAKE OF DOXORUBICIN Markus Keiser, Katharina Wille, Marcus Otter, Sandra Bien-Möller, Susanne Brueck, Werner Siegmund, and Stefan Oswald University Medicine of Greifswald, Department of Clinical Pharmacology, Greifswald, Germany BACKGROUND: Doxorubicin is a frequently used antineoplastic drug to treat solid tumors like nephroblastoma (Wilms´ tumor) or brochial carcinomas as well as haematological malignancies such as acute lymphoblastic leukemia and acute myeloblasic leukemia. The clinical efficacy of doxorubicin is limited by its poorly predictable cardiotoxicity the reasons of which remain so far unknown. Previous studies showed that doxorubicin is a substrate of the organic anion transporter polypeptide (OATP) 1A2. Thus, it was the aim of this study to investigate whether clinically relevant genetic variants of OATP1A2 affect the cellular accumulation of doxorubicin which might be an explanation for the observed inter-subject variability in terms of efficacy and cardiac toxicity. METHODS: HEK293-cells were transfected with the organic anion transporting polypeptides OATP1A2*1, OATP1A2*2 and OATP1A2*3. Expression rates of OATP1A2*1, *2 and *3 were quantified by mass spectrometry-based targeted proteomics using a validated LCMS/MS method. Cellular uptake of doxorubicin was measured by UV-HPLC and competition of doxorubicin with the cellular uptake of [3H]-estrone-3-sulfate (E1S) by OATP1A2 was determined using liquid scintillation counting. Toxic effects of doxorubicin on transfected cell lines were measured using a resazurin assay. Expression of OATP1A2 and other clinically relevant transporters in human heart, pulmonary macrophages and blood monocytes was quantified by real-time RT-PCR and targeted proteomics. RESULTS: OATP1A2-mediated transport of [3H]-E1S was efficiently inhibited by doxorubicin with an IC50-value of 3.5 (1.6; 7.8) µmol/l. Doxorubicin showed a similar affinity to OATP1A2*1 and the genetic variants *2 and *3 (Km = 7.5±3.3; 5.8±5.9; 5.9±4.9 µmol/l). After normalizing transport capacity (Vmax) of doxorubicin to the OATP1A2 protein amount in transfected cells, genetic variants *2 and *3 showed a significantly reduced transport of doxorubicin compared to the wild-type protein (Vmax = 90.6±13.1 vs.

26.3±7.9 (*2) and 53.8±13.2 (*3) pmol/mg × min). After 72 h incubation time, doxorubicin had the most pronounced cytotoxic effect on OATP1A2*1 transfected cells which could be diminished in all transfected cell lines in the presence of the OATP1A2 inhibitor naringin. OATP1A2 was found to be expressed in human broncho-epithelial cells, pulmonary macrophages, and blood monocytes but not in human heart. CONCLUSION: Genetic variants of OATP1A2 affect the OATP1A2-mediated cellular uptake transport of doxorubicin. Genetic OATP1A2 polymorphisms cannot account for cardiac toxicity as it is not expressed in human heart. However, distribution of doxorubicin in tissue that shows considerable OATP1A2 expression such as pulmonary tissue and white blood cells may be influenced by these genetic polymorphisms and in turn may result in differences in drug response and toxicity.

P154 - IN VITRO AND IN VIVO METHODS TO STUDY HEPATIC BILE SALT TRANSPORT Berend Oosterhuis, Beata Toth, Peter Trampus, Mirabella Sike, Franciska Erdo, Marton Jani, Remi Magnan, Peter Krajcsi SOLVO Biotechnology, Budaörs, Hungary Intro: Biliary transport of bile salts is essential for detoxification, digestion and cholesterol catabolism. Inhibition of biliary bile salt transport by drugs may lead to cholestasis, intrahepatic accumulation of toxic bile salts and eventually hepatotoxicity. In vitro and in vivo preclinical data on inhibition of hepatic bile salt transport may be useful to predict and prevent potential hepatotoxicity in humans. Aim: Develop and optimize a toolbox of in vitro and in vivo methods to assess inhibition of hepatic bile salt transport by drugs. Methods: Membrane vesicles were used to study the inhibition of Bsep mediated bile salt transport. Stable transfectants expressing rat Ntcp and / or rat Bsep were used to study transcellular bile salt transport. Biliary cannulation of rats was used as an in vivo model. Results: Cyclosporine A (CsA) is a cholestatic drug that is thought to mediate inhibition of bile salt transport by inhibiting Bsep. IC50 values of CsA mediated inhibition of taurocholate (TC) transport in the VT assay using Bsep expressing membranes and in a vectorial transport assay in MDCKII-Ntcp/Bsep cells were in acceptable correlation. We utilized 4 models employing bile duct cannulated rats to study inhibition of biliary transport of TC by CsA such as measurement of tracer radiolabelled TC or measurement of endogenous TC in the bile upon bolus administration or infusion of CsA. Measurement of biliary transport of endogenous taurocholate upon CsA infusion was the only model that showed an inhibition of cumulative TC excretion. In the other models inhibition of CsA on biliary TC excretion was transient.

Conclusion: In vitro and in vivo tools to study inhibition of hepatic bile salt transport by drugs have been developed.

The toolset includes the first stable double transfectant cellular model co-expressing Ntcp and Bsep and in vivo

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models utilizing cannulated rats. The in vitro tools allow for mechanistic studies and generation of kinetic parameters.

Measurement of endogenous TC levels is the more appropriate approach in vivo.

P155 - INFLUENCE OF ROUX-EN-Y GASTRIC BYPASS SURGERY ON DRUG TRANSPORTER EXPRESSION IN THE HUMAN SMALL INTESTINE 1 1 2 3 3 2 Susanne Brueck , Diana Busch , Sierk Haenisch , Kaja Ludwig , Joern Bernhardt , Ingolf Cascorbi , Werner 1 1 Siegmund , and Stefan Oswald 1 University Medicine of Greifswald, Department of Clinical Pharmacology, Center of Drug Absorption and Transport 2 (C_DAT), Greifswald, Germany, University Hospital Schleswig-Holstein, Campus Kiel, Institute of Experimental and 3 Clinical Pharmacology, Kiel, Germany, Klinikum Südstadt Rostock, Department of Surgery, Rostock, Germany Background: Roux-en-Y gastric bypass surgery is the most frequently used bariatric surgery method to reduce body weight in morbid obese patients. In this procedure the stomach and upper intestine are circumvented, which leads to decreased intestinal absorption of nutrients and subsequently to a loss of weight. Pharmacokinetics of drugs may be influenced in patients after gastric surgery due to several reasons such as decreased intestinal surface area and altered volume of distribution. It remains unknown whether the regulation of intestinal transporter proteins contributes to this phenomenon. Thus, it was the aim of our study to investigate the impact of Roux-en-Y gastric bypass surgery on drug transporter expression in the human small intestine. Methods: Intestinal tissue samples (~ 2-5 mg) were taken before (duodenum), during (jejunum) and one year after (jejunum) surgery. Protein abundance of clinically relevant intestinal transporters and villin-1 as enterocyte-specific marker were quantified via LC-MS/MS-based targeted proteomics. In parallel, gene expression analysis was conducted using TaqMan® real-time RT-PCR assays. Statistical analysis was performed with Mann-Whitney and Wilcoxon test. Results: With exception of OATP1A2, all investigated uptake and efflux transporters could be detected in all intestinal samples on mRNA level. Reliable protein data could be assessed for ABCB1, ABCC2, ABCG2 and PEPT1, whereas protein contents of ABCC3, ASBT, OATP1A2, OATP2B1, OCT1 and OCT3 were below theirs limits of quantification which may be explained by the small size of biopsies. As expected from previous expression analysis, the protein abundance of ABCB1, ABCC2, ABCG2 and PEPT1 was significantly higher in the jejunum compared to duodenal tissue. One year after surgery, the transporter protein content in originally more distal parts of jejunum tended to adapt to the expression pattern that has been initially observed in the duodenum. This down-regulation was found to be significant for ABCC2, ABCG2 and PEPT1.

The observed changes in gene expression and protein content were correlated to intestinal miRNA expression.

Conclusion: Gene expression and protein abundance of several drug transporters is markedly affected by Roux-en-Y gastric surgery. These compensatory changes may result in differences in intestinal drug absorption.

P156 - INTERACTION OF EMTRICITABINE WITH MATE1, OCT1, OCT2, P-GLYCOPROTEIN, BCRP AND MRP2 TRANSPORTERS Josef Reznicek, Martina Ceckova, Zuzana Neumanova, Lukas Cerveny, and Frantisek Staud Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic Emtricitabine, chemically named 5-fluoro-1-(2R,5S)-[2(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine is a nucleoside reverse transcriptase inhibitor. It constitutes an important component of combination antiretroviral therapy (cART) used in treatment of HIV positive patients including pregnant women. Pharmacokinetic behavior of emtricitabine and its transfer across the placenta could be largely affected by membrane expressed drug transporters. Interactions of emtricitabine with these drug transporters could also lead to clinically relevant drug-drug interactions with other antiretrovirals used in cART. We therefore focused on describing interactions of emtricitabine with P-glycoprotein (ABCB1), BCRP (ABCG2), MRP2 (ABCC2), MATE1 (SLC47A1), OCT1 (SLC22A1) and/or OCT2 (SLC22A2) transporters, which are abundantly expressed in many tissues and physiological barriers including placenta and influence pharmacokinetics of various drugs and their drug-drug interactions. Transfected MDCK cells stably expressing P-gp, BCRP, MRP2, MATE1, OCT1 and OCT2 were used for in vitro accumulation assays and transcellular transport experiments. Moreover, we used method of dually perfused rat term placenta in closed-circuit arrangements to assess the contribution of drug transporters in transplacental transfer of emtricitabine. Employing conventional bi-directional (concentration gradient) transport experiments and the concentration equilibrium method in MDCK cells, we did not notice any contribution of ABCB1, ABCG2, ABCC2, OCT1 or OCT2 in transcellular transport of emtricitabine. However, in MATE1 expressing cells, transcellular transport of emtricitabine was significantly increased and its intracellular accumulation was correspondingly significantly reduced. Further experiments in MATE1 cells reveal significantly reduced transcellular transport of emtricitabine and significantly increased intracellular accumulation in presence of ritonavir or cimetidine, known MATE1 inhibitors. The basolateral-to-apical transport of emtricitabine across the MATE1 expressing cellular monolayers was significantly reduced at low temperature and with increasing pH in apical compartment. Moreover, emtricitabine was able to significantly reduce uptake of ASP+, the fluorescent substrate of OCT1, OCT2 and MATE1, into relevant OCT1-, OCT2- and MATE1-overexpressing cells.

Based on our results, we conclude that emtricitabine is transported by MATE1 and shows inhibitory potency to OCT1 and OCT2, but it is not a substrate of examined ABC or OCT transporters. Nevertheless, the impact of our findings for the pharmacokinetic behavior of emtricitabine and the risk of DDI on OCT1, OCT2 and MATE1 transporters in vivo remains to be further elucidated. The study was supported by the Grant Agency of the Charles University in Prague (GAUK 1148213/C/2013) and GACR 303/13-31118P.

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P157 - INTERACTIONS OF ABACAVIR WITH NUCLEOSIDE TRANSPORTERS IN THE PLACENTA Zuzana Neumanova, Lukas Cerveny, Martina Ceckova, and Frantisek Staud Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic Abacavir, a nucleoside reverse transcriptase inhibitor, is used during gestation in order to prevent mother-to-child transmission of HIV. Its transplacental passage was found to be sufficient to provide adequate prevention; however the exact mechanism of abacavir transport across the placenta has not been fully elucidated yet. Nucleoside-derived drugs are known to be transported by secondary active Na+ dependent concentrative nucleoside transporters (CNTs) and/or bidirectional equilibrative nucleoside transporters (ENTs) mediating facilitated diffusion of their substrates. The aim of our study was to detect whether transplacental transport of abacavir can be mediated by placental CNTs and/or ENTs. For this purpose we employed in vitro accumulation assays in human syncytiotrophoblast-derived BeWo cell line and in situ method of dually perfused rat term placenta. Accumulation studies revealed that abacavir uptake was sodium-dependent and was decreased by the specific ENT1 inhibitor NBMPR (100 nM) suggesting involvement of CNTs and ENT1 in its transcellular transport in BeWo cells. The method of dually perfused rat term placenta was used to investigate the effects of NBMPR and sodium depletion on abacavir transplacental transport on the organ level.

NBMPR (100 nM) reduced abacavir clearance in both materno-fetal and feto-maternal directions confirming the role of rat ENT1 in abacavir transplacental passage. Conversely to accumulation studies in BeWo cells, Na+-dependency in abacavir transport was not detected. In summary we suggest that nucleoside transporters, most probably ENT1, may be involved in the penetration of abacavir across the placenta from mother to fetus. Nevertheless, additional experiments are needed to profoundly elucidate this issue. The study was supported by the Czech Science Foundation (GACR P303/120850) and by the Charles University in Prague (project SVV/2015/260 185).

P158 - INTERACTIONS OF SELECTED FLAVONOIDS WITH THE TRANSPORTER HOATP2B Lucie Navratilova, Jana Mandikova, and Frantisek Trejtnar Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic Flavonoids are plant-specific secondary metabolites and represent one of the largest groups of secondary metabolites. The main dietary sources are fruits, vegetables, grains, honey, they are also contained in beverages (tea, wine, juices).1 It was shown that some flavonoids can interact with the transporters from the family organic anion transporting polypeptide (OATPs).1 OATPs significantly contribute to the intestinal drug absorption.2 One of the most expressed transporters from this family in human intestine is OATP2B1.3 Interactions of flavonoids with concomitantly administered drugs at OATP2B1 level may lead to a change in absorption, pharmacokinetics and safety profile of the drugs transported by this carrier. The aim of this study was to assess the potential inhibition of hOATP2B1 by natural compounds from the group of flavonoids (quercetin, myricetin, galangin, pinobanksin, pinocembrin, chrysin, fisetin) in vitro. MDCK II cells transiently transfected with hOATP2B1 were used as the experimental model in the study. The inhibition of the hOATP2B1-mediated [3H]-estrone 3 sulfate uptake was observed, quercetin served as a positive control. All mentioned flavonoids showed the inhibition of the hOATP2B1. However, the found inhibitory potency towards the hOATP2B1 was significantly different. Galangin, chrysin and fisetin were the most potent inhibitors with IC50 of 15.0 µM, 18.2 µM and 18.5 µM, respectively. Myricetin and pinobanksin exhibited IC50 more than one order of magnitude higher. According to the obtained results, these natural compounds could potentially affect in varying degrees uptake of the drug substrates transported by human OATP2B1 (e.g. statins) into the enterocytes. Therefore, the food-drug interactions in humans based on this interaction cannot be excluded. The study was supported by grant of the Czech Science Foundation (GACR) no. GBP303/12/G163 and Charles University in Prague (SVV 260185 and PRVOUK P40).

References: 1. Passamonti S. et al.: Bioavailability of flavonoids: a review of their membrane transport and the function of bilitranslocase in animal and plant organisms. Curr Drug Metab., 2009, 10(4):369-394 2. Tamai I., Nakanishi T.: OATP transporter-mediated drug absorption and interaction. Curr Opin Pharmacol., 2013, 13:859-863 3. Tamai I.: Oral drug delivery utilizing intestinal OATP transporters. Adv Drug Deliv Rev., 2012, 64(6):508-514 P159 - LOCALIZATION AND CHARACTERIZATION OF TRANSPORTER PROTEINS INVOLVED IN THE UPTAKE OF PULMONARY RELEVANT DRUGS 1 1 1 1 1 2 Sarah Berlin , Markus Grube , Andrea Hubeny , Stefan Oswald , Markus Keiser , Ralf Ewert , and Werner 1 Siegmund 1 2 University Medicine of Greifswald, Department of Clinical Pharmacology, Greifswald, Germany, University Medicine of Greifswald, Department of Internal Medicine, Pulmonary Diseases, Greifswald, Germany Background: Members of the organic anion transporting polypeptide family (OATPs) are important uptake transporters in pharmacokinetically relevant organs like liver or intestine. In contrast, the expression and pharmacological impact of OATPs in peripheral compartments such as the pulmonary/bronchoalveolar system is less well understood. Since the respiratory tract is an important target structure for numerous drugs including antibiotics, bronchodilators or anti-

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inflammatory compounds, we studied the expression and localization of OATPs in this target structure as well as their functional interaction with pulmonary active drugs. Methods: mRNA expression of OATP-transporters was measured in bronchial and nasal biopsies (BEC, NEC) obtained from eight healthy volunteers (4 female, 4 male, 22-37 years) using the TaqManTM array card real-time PCR technology. Transporter localization was analyzed in paraffinembedded tissue samples by immunofluorescence microscopy. Functional analyses were carried out as competition assays measuring the uptake of OATP standard substrates into OATP-overexpressing MDCKII cells in the presence and absence of the test compounds. Results: On mRNA levels significant amounts of OATP1A2, OATP1B3, OATP2A1, OATP2B1, OATP3A1, OATP4A1 and OATP4C1 were detected. Among those, immunofluorescence staining indicated a localization of OATP1A2 und OATP2B1 within the alveolar wall (pneumocytes type I) and, additionally for OATP2B1, in the bronchial epithelium. Subsequently, the interaction of OATP1A2 and OATP2B1 with pulmonary relevant drugs was investigated. Thereby, a significant inhibition of the estrone-3-sulfate uptake by more than 50 % was detected for roxithromycin, telithromycin, levofloxacin, moxifloxacin, rifampicin, ipratropium bromide, tiotropium bromide, prednisolone, cortisone, hydrocortisone, beclometasone and beclometasone dipropionate (each 100 µM). For the most potent inhibitors telithromycin and ipratropium bromide, half maximal inhibitory concentrations (IC50) of 0.4 µM and 4.7 µM, respectively for OATP1A2 and of 9.8 µM for telithromycin and OATP2B1 were determined, indicating a potential relevance of these transporters for the pulmonary distribution of the mentioned compounds. Conclusion: We were able to demonstrate a significant expression of several OATPs in the pulmonary system including OATP1A2 and OATP2B1. These transporters are located in vascular structures (OATP1A2) and the bronchial epithelium (OATP2B1). Their interactions with certain pulmonary relevant drugs indicate a possible role in drug distribution in the bronchoalveolar system and need to be investigated further.

P160 - LONG-TERM ADMINISTRATION OF TENOFOVIR OR EMTRICITABINE TO PREGNANT RATS; EFFECT ON ABCB1A, ABCB1B, AND ABCG2 EXPRESSION IN MATERNAL AND FETAL ORGANS Lukas Cerveny, Zuzana Neumanova, Sara Karbanova, Ivana Havlova, and Frantisek Staud Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic Nucleotide/nucleoside reverse transcriptase inhibitors tenofovir and emtricitabine are very effective and well tolerated antiretrovirals representing current backbone of the therapeutic combination regimens for prevention of perinatal HIV transmission. Effects of chronic therapy on expression of ATP-binding cassette (ABC) transporters, important pharmacokinetic determinants, in maternal or fetal tissues have not been investigated to date. The aim of our study was to determine whether tenofovir or emtricitabine administered in long-term fashion affect expression of two widely expressed ABC transporters, ABCB1 and ABCG2, in maternal and fetal biological barriers. To investigate this issue we treated pregnant Wistar rats with i.m. injection of tenofovir (2.25 mg/kg), emtricitabine (3.5 mg/kg), or saline for ten days (from 12th to 21st gestation day). On the 22nd gestation day the placenta and maternal/fetal brain, kidneys, intestine, and liver were collected and relative expression of Abcb1a, Abcb1b, and Abcg2 mRNA was assessed. As we routinely weighed the fetuses and their placentas, placenta-to-birth weight ratio, an indicator of physiological programming of the fetus, was calculated. We first evaluated expression profile of Abcb1a, Abcb1b, and Abcg2 in the organs of animals exposed to saline; Abcb1a and/or Abcb1b showed ontogenic expression in the brain, liver, and kidneys. Subsequently it was demonstrated that administration of tenofovir or emtricitabine did not alter expression of Abcb1a, Abcb1b, and Abcg2 in the organs analyzed. Placenta-to-birth weight ratio in tenofovir group of animals was significantly increased, suggesting tenofovir might cause developmental reprogramming of the fetus. In conclusion we bring the evidence that long term treatment with tenofovir/emtricitabine covering half of gestation might not affect expression of the tested transporters in the placenta and both maternal and fetal organs. Our data thus broadens knowledge on safety profile of tenofovir and emtricitabine use in pregnancy, however, needs to be verified in humans.

This research was financially supported by the Czech Science Foundation (GACR P303/12/0850). We thank Gilead Sciences, Inc. (333 Lakeside Drive Foster City, California 94404, USA) for providing tenofovir and emtricitabine.

P161 - MORI CORTEX ENHANCES INTESTINAL BARRIER FUNCTION THROUGH UP-REGULATING PGLYCOPROTEIN VIA GUT MICROBIOTA- DEPENDENT AND INDEPENDENT MECHANISMS Wanghui Jing, Xuejiao Gao, and Ru Yan University of Macau, Macau, China Background: P-glycoprotein (P-gp) constitutes an important part in intestinal barrier function. Dysregulation of P-gp has been implicated in inflammatory bowel diseases. Mori Cortex, the root bark of Morus alba L., has been used for alleviation of refractory colitis in folk medicine. The present study aimed to evaluate the regulatory effects of Mori Cortex on intestinal P-gp in vivo and in vitro and unravel the underlying mechanism. Methods: Mori Cortex extract (MCE) was prepared with 70% aqueous ethanol and the chemical profiling was characterized by tandem mass spectrometry [1]. Male SD rats (n=6 each group) received either drinking water (normal group), 5% dextran sulfate sodium (DSS) in drinking water (UC group) for 7 days or DSS stimulation plus 3-week MCE intervention (p.o.

0.5g/kg/day) (MCE group). Animals were sacrificed on the last day and colon and serum were collected to measure Pgp expression, MPO activity and cytokines levels. Pooled fecal bacteria suspension was prepared for each group and cultured overnight to obtain the supernatant (CS). The short- (up to 24-h) and long- (7-day) term effects of MCE and major components on P-gp were examined in Caco-2 cells using western blot and rhodamine-123 transport. The involvement of gut microbiota in P-gp regulation by MCE was assessed by incubating the CS from each group with

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Caco-2 cells. Results: DSS stimulation resulted in increased MPO activity and pro- and anti-inflammatory mediator levels in rats. MCE intervention reversed the elevation of MPO and pro-inflammatory cytokines but enhanced the increase of anti-inflammatory cytokines. P-gp expression showed a significant decrease in colitic colon, which was prevented by MCE intervention. In Caco-2 cells, short-term MCE treatment resulted in a time-dependent inhibition of P-gp expression and correspondingly, an enhanced accumulation of Rho-123. In contrast, long-term MCE treatment up-regulated P-gp expression and activity. Distinct regulatory effects on P-gp by the major components in short- and long- term treatments may account for the biphasic direct effects of MCE. More interestingly, CS from colitic rats down-regulated P-gp expression in Caco-2 cells while CS from normal group showed no effects. This down-regulation could be reversed by co-incubation of cells with CS from MCE-treated colitic rats or pretreatment of Caco-2 cells with MCE. Conclusion: Mori Cortex enhanced intestinal barrier function in DSS-induced colitic rats by up-regulating P-gp.

The mechanisms involve a direct effect as well as gut microciota-mediated pathway. (Supported by Science and Technology Development fund of Macao SAR (FDCT043/2011/A2) and University of Macau (MYRG2015-00220ICMS-QRCM)).

Reference: [1] Wanghui Jing, Ru Yan* and Yitao Wang. A practical strategy for chemical profiling of herbal medicines using ultra-high performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry: a case study of Mori Cortex. Anal. Methods 2015;7:443-457.

P162 - OVERCOMING MULTIDRUG RESISTANCE IN HUMAN BREAST CANCER CELLS Heather Wallace, Andrew Irvine, Aidan Seeley, Tara Schmitz, Laurent Trembleau, Andrew McEwan, Wael Houssen, and Marcel Jaspars University of Aberdeen, Foresterhill, United Kingdom A major factor in unsuccessful cancer chemotherapy is the development of multidrug resistance (MDR), the primary mechanism by which cancers develop resistance to several chemotherapeutic drugs. Heterogenic cancer tumours contain both drug-sensitive and drug-resistant cells, with drug-sensitive cells killed by early stages of chemotherapy leaving a higher population of drug-resistant cells. Patellamides have shown promise in the resensitisation of MDR cells1 but progressed no further following NCI60 screens2. It is hoped biosynthetic manipulation of patellamides will yield invaluable therapeutics, termed patellamide-like compounds (PLCs). This project aimed to establish the cytotoxic profile of a new patellamide peptide family in drug-resistant breast adenocarcinoma cell line (MDA-MB-231 TAX30) to assess whether patellamide-like structures are potentially resistance modifiers. MDA-MB-231 TAX30 cells were less sensitive to docetaxel than the parent cell line with an IC50 value of 112 +/- 1 M (m e a n +/- SEM, n=18). The Phase III P-glycoprotein inhibitor, zosuquidar, was used as a positive response modifier. Treatment with zosquidar and docetaxel showed a significant shift in the dose response curve to the left and a decrease in docetaxel IC50 values Six PLCs (MBC-536-541) were available and all six were tested initially for effects on cell growth alone as zosuquidar was found to be toxic at concentrations above 15 M. P LCs we re te s te d up to conce ntra tions of 40 M a nd little toxicity was observed. Initial experiments with PLCs showed promising results. In conclusion, while zosuquidar is effective in terms of counteracting P-glycoprotein linked drug resistance but the drug is toxic at relatively low concentrations. The PLCs with no obvious toxicity alone may offer a realistic alternative.

1. WILLIAMS, A.B. and JACOBS, R.S., 1993. A marine natural product, patellamide D, reverses multidrug resistance in a human leukemic cell line. Cancer letters, 71(1-3), pp. 97-102.

2. RASHID, M.A., GUSTAFSON, K.R., CARDELLINA, J.H.,2nd and BOYD, M.R., 1995. Patellamide F, A new cytotoxic cyclic peptide from the colonial ascidian Lissoclinum patella. Journal of natural products, 58(4), pp. 594-597.

P163 - POTENTIAL ROLE OF BASOLATERAL EFFLUX TRANSPORTERS IN THE PREVENTION OF CHOLESTATIC HEPATOTOXICITY 1 1 1 1 1 2 Kenneth Brouwer , Jonathan Jackson , Kimberly Freeman , Robert St. Claire , Weslyn Friley , Jeffrey Edwards, and 1 Chris Black 1 2 Qualyst Transporter Solutions LLC, Durham, North Carolina, United States, Intercept Pharma, New York, New York, United States In vivo, concentrations of bile acids are very highly regulated through synthesis, metabolism and transport mechanisms. Previous experiments have demonstrated the potential of compounds to alter the hepatobiliary disposition of bile acids through inhibition of their hepatic uptake and efflux. The net effect of a compound on these processes determines the acute change in the intracellular concentration of bile acids. Acute changes in the intracellular concentrations of bile acids through inhibitory processes, may lead to chronic effects through multiple pathways responsible for maintaining bile acid homeostasis. Synthesis, metabolism and expression of transporter proteins are regulated by multiple nuclear regulatory factors. We evaluated the hepatobiliary disposition of d8taurocholate (TCA) and regulatory pathways for the synthesis, metabolism and transport of bile acids following chronic exposure to a model bile acid (CDCA). Transporter Certified™, cryopreserved human hepatocytes (N=3 donors) and B-CLEAR® technology were used to evaluate the effects of CDCA exposure at 0.1, 0.3, 1, 3.16, 10, 31.6, and 100 µM for 72 hours. The gene expression of various transporters, synthetic enzymes, and regulatory factors was determined using the Qiagen RNeasy kit, followed by quantitative PCR. Relative-fold mRNA content was determined for each

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treatment relative to a solvent control. Following a 72 hour exposure to CDCA, the total accumulation and intracellular concentration of TCA decreased to 29.6 ± 2.1 and 26.3 ± 7.5 % of control, respectively compared to vehicle treatment. Dose dependent decreases (>200X) in CYP7A1 (bile acid synthesis) and HMG CoA Synthase A (cholesterol synthesis) were observed. No change in NTCP (bile acid uptake) was observed. BSEP (canalicular efflux of bile acids) expression increased (5X) in a dose dependent fashion. The expression of basolateral bile acid efflux transporters MRP3 or MRP4 was not altered. However the expression of OSTα and OSTβ (basolateral efflux transporters) increased by 10X and 100X, respectively as the CDCA exposure increased. The nuclear regulatory factors FGF19 and SHP were also increased by 1000X and 5X, respectively. In response to elevated intracellular concentrations of bile acids, hepatocytes decreased the synthesis of bile acids, and increased both the basolateral and canalicular efflux pathways for bile acids. Induction of OSTα/β mediated basolateral efflux may be an important pathway for elimination of bile acids. Inhibition of OSTα/β mediated basolateral efflux in addition to BSEP mediated canalicular efflux inhibition may be critical in the development of cholestatic hepatotoxicity.

P164 - SHOULD TOTAL PLASMA DRUG CONCENTRATION BE USED TO PREDICT TRANSPORTER MEDIATED DRUG-DRUG INTERACTIONS FOR HIGHLY PROTEIN BOUND DRUGS? Mirza Jahic, Chien-Ming Li, Jason Baik, Wenjie Jiang, Xuexiang Zhang, and Yong Huang Optivia Biotechnology Inc., Menlo Park, California, United States Despite the significant progress made in the drug transporter field, predicting clinical Drug-Drug Interactions (DDIs) based on in vitro transporter studies still remains a major challenge. Specifically, the extent of DDIs in terms of changes in Cmax and AUC of victim drugs is often significantly underestimated using in vitro transporter inhibition constants (Ki/IC50), as exemplified by the well-studied OATP mediated DDIs between statins and rifampicin, and the recently reported dolutegravir-metformin interaction. Using OATP1B1 and OCT2 as examples, this talk will discuss the effect of protein binding on "in vivo" transporter inhibition and present a "binding equilibrium shift" hypothesis to explain our experimental findings. Dose-dependent OCT2 and OATP1B1 inhibitions by various drugs with both high and low plasma protein binding, were assessed in protein-free HBSS and protein-rich solutions including HBSS with 4% albumin, 100% human serum and 100% bovine serum. Strikingly, for highly protein bound drugs, unbound fraction (fu) adjusted IC50 values determined in protein-rich solutions were significantly lower than that obtained from assays using protein-free HBSS. For example, dolutegravir, a HIV integrase inhibitor with >99% protein binding in the human, was significantly more potent in inhibiting OCT2 mediated metformin transport in serum than in HBSS, with fu adjusted IC50 values of 87nM and 536nM respectively. In addition, the fu adjusted permeability of dolutegravir tested in serum was also >5x higher than that assessed in HBSS. On the contrary, the fu adjusted IC50 for low protein bound Cimetidine (in vivo protein binding ~20%) was independent on assay matrix. These data suggest that for highly protein bound drugs, the "actual" free drug concentrations in presence of transporters and other drug binding membrane proteins could be substantially higher than the calculated free concentration based on fu measured in vitro, as high affinity binding to cell membrane proteins could effectively change the equilibrium of nonspecific binding between drug and serum proteins. Lastly, the talk will present a partial PBPK model incorporating total plasma drug concentration and in vitro transporter IC50s obtained with human serum, demonstrating improved IVIVC of dolutegravir and rifampicin associated DDIs. These studies, for the first time to our best knowledge, showed that for highly protein bound drugs, conventional approach based on unbound drug concentration may lead to underestimation of in vivo transporter inhibition. As such, we may consider conducting in vitro transporter studies in human serum and using total plasma drug concentration for modeling and prediction of transporter mediated DDIs. P165 - SCREENING FOR HEPATOBILIARY TRANSPORTER INHIBITION IN 3D HUMAN LIVER MICROTISSUES

BY CONFOCAL IMAGING 1 1 2 2 2 2 Stefan Letzsch , Karin Boettcher , Wolfgang Moritz , Zoe Weydert , Simon Messner , and Jens M. Kelm 1 2 PerkinElmer Cellular Technologies Germany GmbH, Hamburg, Germany, InSphero AG, Schlieren, Switzerland A functional impairment of hepatobiliary transporters such as bile salt export pump (BSEP) and multidrug resistanceassociated protein 2 (MRP2) are strongly associated with an increased risk of liver injury. Currently mostly artificial models, such as BSEP expressing membrane vesicles, are used for studying efflux transporter function. However, they lack the integration of the functional complexity of the natural 3-dimensional (3D) liver environment. The presence of BSEP and MRP2 on the apical membrane towards the bile canaliculi indicates that 3D human liver microtissues might be a useful tool to study the effects of efflux-inhibiting compounds in a complex 3D environment. Since the 3D liver microtissues are viable and functional over several weeks, long-term studies of efflux transporters are possible.

However, the complex 3D structure of the spheroids poses challenges for cellular high-content analysis (HCA). Here we describe the use of confocal imaging to assess BSEP-mediated efflux of cholyl-lysyl-fluorescein (CLF) and MRP2mediated efflux of 5-chloromethylfluorescein diacetate (CMFDA) into the bile canaliculi. Automated analysis of bile canaliculi area was achieved by analysis of maximum intensity projection images of the 60 µm stacks. The HCA analysis of 3D human liver microtissues revealed a fluorescent bile canaliculi area after CLF and CMFDA exposure.

Fluorescent CLF area was decreased with BSEP inhibitor Sitaxentan, whereas fluorescent CMF area was unaffected by this compound. Cytochalasin, an F-actin inhibitor, induced a drop in fluorescent bile canaliculi area of both CLF and CMF. In support to the confocal microscopy, the fluorescence of 5(and 6)-carboxy-2’, 7’-dichlorofluorescin diacetate (CDFDA) exposed human liver microtissues increased over time in the supernatant. The secretion of fluorescent CDF was inhibited by cyclosporine A and accompanied by an accumulation of intracellular fluorescent CDF. The technique

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presented herein utilizes a 384-well plate, containing a single microtissue per well. This allows automated measurement and image processing in a high-throughput setting. These results indicate that 3D human liver microtissues are a useful tool to study hepatobiliary transporter activity in a complex organotypic in vitro liver model system.

P166 - VORTIOXETINE: IN VITRO ASSESSMENT AND CLINICAL IMPLICATIONS OF TRANSPORTER INHIBITION-BASED DRUG INTERACTIONS 1 1 2 Liping Pan , Grace Chen , and Christina Sveigaard 1 2 Takeda Pharmaceutical Company, Deerfield, Illinois, United States, Department of Discovery, ADME, H. Lundbeck A/S, Valby, Denmark BACKGROUND: The evaluation of drug interactions during drug development and its impact on regulatory approval have dramatically changed over the past decade. These changes have evolved based on our increased knowledge of drug metabolizing enzymes and drug transporters, and the development of tools to study drug interactions. Recently, the FDA, EMA, and PMDA revised the Guidance for Industry on the investigation of drug interactions, specially emphasizing transporter-based drug interactions. To fullfil Takeda’s postmarketing commitment as specified in the FDA’s vortioxetine approvable letter, the potential inhibitory effects of vortioxetine and its major metabolite Lu AA34443 on the major transporters were evaluated in vitro. METHODS: To evaluate the inhibitory potential of vortioxetine and Lu AA34443 on the activities of BCRP, BSEP, MATE1, MATE2-K, MDR1, OAT1, OAT3, OATP1B1, OATP1B3, OCT1 and/or OCT2, cell- or membrane-based assay systems with radiolabeled substrates were employed.

The concentrations of radiolabeled substrates were measured by liquid scintillation spectrometry. Efflux ratios or uptake activities of each transporter were determined in the absence or presence of vortioxetine or Lu AA34443. The IC50 values were calculated and the clinical implications of the in vitro findings were assessed. RESULTS: Vortioxetine inhibited MDR1, MATE1, MATE2-K, OCT1, and OCT2 activities with respective IC50 values of 4.14, 21.7, 43.7, 1.93, and 12.3 µmol/L. The major metabolite Lu AA34443 inhibited MATE1 and OCT1 activities with respective IC50 values of 18.5 and 9.18 µmol/L. For the other transporters examined, vortioxetine and Lu AA34443 showed little or no inhibition up to the highest concentration tested, with IC50 values >30 µmol/L, except for BCRP for which the IC50 value of vortioxetine was >3 µmol/L, the highest concentration tested in this study. The likelihood of a projected in vivo interaction is based on the Cmax/IC50 ratio. Following multiple oral QD doses of vortioxetine at 20 mg, the highest label approved dose, the mean steady-state Cmax values of vortioxetine and its metabolite Lu AA34443 are 0.11 and 0.10 µmol/L, respectively. All estimated Cmax/IC50 ratios of MDR1, MATE1, MATE2-K, OCT1, and/or OCT2 for vortioxetine or Lu AA34443 were <0.1, indicating that neither vortioxetine nor Lu AA34443 would significantly inhibit the activities of the transporters examined at systemic level in humans at clinically relevant Cmax. Based on the Decision Trees of the FDA, EMA, and PMDA Drug Interaction Guidance and up-to-date clinical findings, no additional in vivo transporter-based drug interaction study will be needed. CONCLUSION: Vortioxetine, a serotonin transporter inhibitor, up to 20 mg QD is unlikely to have any clinically significant effects on the pharmacokinetics of comedications, in which some of these transporters play an important role. The current research also addressed FDA’s postmarketing requirement.

121

13th European ISSX Meeting Aatsinki, Sanna-Mari, P95 Adalı, Orhan, P26 Agbekoh, Peter, P92 Ahmed, Rafat, P127 Aichholz, Reiner, P5 Alexander, Emma, P59 Almond, Lisa, P33, P57, P58, P139 Altay, Ahmet, P21, P65 Alwashih, Mohammad, P109 Amaral, Margareta, S14 Amberntsson, Sara, SC1.2 Amunom, Immaculate, P44 Andersen, Melvin, P61 Anderson, Caroline, P111 Andersson, Tommy, S4 Ando, Hirotaka, P2 Andrews, Sarah, P59 Anestis, Dianne, P104 Anumol, Tarun, P81 Anzai, Naohiko, P54 Anzenbacher, Pavel, P25 Anzenbacherová, Eva, P25 Arıcı, Merve, P124 Armstrong, Martin, S17 Arora, Vinod Kumar, P127 Aslan, Sevinc, P67 Asplund, Annika, P82 Assmus, Frauke, A10 Athikomkulchai, Sirivan, P42 Avdicevic, Monika, P91 Baik, Jason, P164 Balavenkatraman, Kamal Kumar, A8 Ball, John, P96 Ballard, Peter, S40 Banerjee, Basu Dev, P125, P127 Barnaby, Robert, P28 Barragan, Isabel, A9 Bartikova, Hana, P64 Barton, Hugh A., P11 Battaglia, Rosangela, P112 Bauch, Caroline, P69 Baudouin, Christophe, P7 Baze, Audrey, A4, P18 Ben-Eltriki, Mohamed, P132 Bergström, Fredrik, P116 Berka, Karel, P75 Berlin, Sarah, P159 Bernhardt, Joern, P155 Bessou-Touya, Sandrine, P93 Bi, Yi-An, P11 Bickmore, Wendy, S37 Bien-Möller, Sandra, P153 Birks, Vicky, P66 Black, Chris, P151, P163 Blanchard, Yannick, P73 Blanz, Joachim, P5, P120 Bloomer, Jackie, P85 Blumenstein, Lars, P108 Boettcher, Karin, P165 Boily, Marc-Olivier, A2 Bolger, Michael B., P89 Bolliger, Paul, P44

Author Index Bonn, Britta, P14 Bonnel, David, P4, P32, P101 Booth-Genthe, Catherine, S41 Borowa-Mazgaj, Barbara, P19 Bosgra, Sieto, P1 Bosilkovska, Marija, P6, P130 Bottacini, Marco, P135 Böttcher, Kerstin, P141 Bouaita, Belkacem, P56 Boudreau, Chantal, A2 Boulenc, Xavier, P60 Bournique, Bruno, P37 Bozoğlu, Faruk Tahsin, P65 Brauch, Hiltrud, A1 Brázdová, Petra, P39 Bressac, Didier, P37 Brignole-baudouin, Françoise, P7 Briolotti, Philippe, A12 Brogin, Giandomenico, P112 Brouwer, Kenneth, P151, P163 Brueck, Susanne, P153, P155 Bruggmann, Christel, P53, P90 Buckley, David, P44, P51 Burgess, Karl, SC4.4 Busch, Diana, P155 Butler, Phil, P69 Camenisch, Gian, A8 Campbell, Rebecca, P51 Cantrill, Carina, P11, P66 Cao, Fangrui, P8 Capello, Astrid, P140 Caradec, Fabrice, P56 Carazo, Alejandro, P75 Carr, Julia, P142 Cascorbi, Ingolf, P155 Cave, Matt, P144 Ceckova, Martina, A6, P156, P157 Cenacchi, Valentina, P112 Cerveny, Lukas, P156, P157, P160 Chakravarty, Probir, P17 Chan, Christina, P57 Chan, Eric, S31, P55, P68 Chang, Qi, P8 Chanteux, Hugues, P28, P78, P122 Chauret, Nathalie, A2 Chee, Evelyn, P45 Chen, Grace, P166 Chen, Lin, P49 Chen, Wenzhang, P17 Cheong, Eleanor Jing Yi, P55 Chesné, Christophe, P56 Childress, Jason, P96 Cho, Cheul, P150 Cholanians, Aram, P102 Chou, Kang-Jye, P136 Chowdry, Joanna, P9 Christensen, Jesper, P107 Chumworathayi, Pansu, P80 Cihalova, Daniela, A6 Cinato, Flavio, P112 Clark, Robert D., P89

122

Cohen, Philip, S1 Cole, Richard, P66 Commandeur, Jan N.M., A4, P18 Corfe, Bernard, S33, P9 Corfield, Lindsay, P111 Cottrell, Jesse, P96 Courbebaisse, Yann, P146 Crewe, Hilary Kim, P47 Culcu, Tuba, P26 Czerwiński, Maciej, P44 Daali, Youssef, P6, P53, P90, P130 Dahlinger, Dominik, P67 Dai, Jixun, P81 Damm, Georg, P83 Danhof, Meindert, S6 Dannhorn, Andreas, P116 Darnell, Malin, P14 Daunes-Marion, Sylvie, P93 Dayer, Jérôme, P5, P120 de Boussac, Hugues, A12 De Bruyn, Tom, P11 de Graaf, Inge, A3 De Jager, Marina, A3 Deb, Subrata, P132 Deglon, Julien, P6 Delatour, Claude, P28, P122 Delemonte, Thierry, P5, P120 Dell'aiera, Sylvie, P28, P122 DeLoach, Jason, P20 Demailly, Arnold, P106, P108 den Braver, Michiel W., A4, P18 den Braver-Sewradj, Shalenie P., A4, P18 Denning, Chris, SC2.2 Desbenoit, Nicolas, P93 Deshmukh, Gauri, P136 Desmeules, Jules Alexandre, P6, P53, P90, P130 Desrayaud, Sandrine, P120 Diaz Toledo, Carmen, P9 DiBella, John, P89 Dickie, Anthony, SC1.1 Dilworth, Clive, P69 Ding, Shaohong, P142 Dobos, Nikoletta, P146, P154 Doffey-Lazeyras, Fabienne, P53, P90 Dorfmüller, Peter, P32 Dudda, Angela, P99 Dufossé, Mélody, P110 Dumas, Sébastien J., P32 Duquet, Marie-claude, A2 Duret, Cedric, A12 Dvorak, Zdenek, P143, P145 Edsbagge, Josefina, P82 Edwards, Jeffrey, P163 Eichelbaum, Michel, A1 Ekdahl, Anja, P31 Elizondo, Guillermo, P70, P71 Emmen, Harry, P140 Endo, Yumiko, P46 Endres, Ralf, P120

13th European ISSX Meeting Erdo, Franciska, P154 Eriksson, Paul-Ole, P148 Erpelinck, Steven, P1 Escher, Monica, P130 Evans, Caroline, P9 Evers, Bernard, A3 Ewert, Ralf, P159 Fabre, Jean-Michel, A12 Falaux, Emeline, P101 Falkner, K. Cameron, P144 Faller, Bernard, P118 Faller, Thomas, P107 Feller, Diana, P91 Feng, Li, P8 Fenner, Katherine, P50 Fessard, Valérie, P73 Finel, Moshe, A5 Fok, Benny SP, P3, P128, P137 Forbes, Stuart, P84 Fournier, Isabelle, P110 Frache, Gilles, P93 Frangova, Tanya, P76 Frechen, Sebastian, P67, P138 Freeman, Kimberly, P151, P163 Frey, Lorenz, P138 Friley, Weslyn, P163 Fromm, Martin, S21 Fuhr, Uwe, P67, P138 Funk, Christoph, S19 Gaffney, Jeannemarie, P61 Galetin, Aleksandra, S8, A10, A11, P11, P15, P43, P50 Ganchev, Boian, P119 Gao, Xuejiao, P161 Gardner, Iain, P33, P58, P139 Garve, Claudia, P147 Gassmann, Ernst, P120 Ge, Guangbo, P23 Gençler öZkan, Ayşe Mine, P26 Geoffroy, Stephanie, P66 Gerbal-Chaloin, Sabine, A12 Gerin, Brigitte, P78 Gertsch, Werner, P5, P120 Gertz, Michael, SC3.1 Ghosheh, Nidal, P82 Gil Berglund, Eva, S18 Gilhooly, Lucy, P34 Glaenzel, Ulrike, P106, P108 Glatt, Hansruedi, S48 Glazier, Anthony, P59 Gliesche, Daniel, P141 Golding, Melanie, P28 Goldring, Chris, SC2.1 González-Barbosa, Emmanuel, P70, P71 Grandin, Flore, P7 Grant, Mary, P85, P92, P109, P117 Grime, Ken, P14, P31 Groothuis, Geny, A3, P105 Grube, Markus, P152, P159 Guengerich, Frederick, A7 Guillet, Fabrice, P56

Author Index Guleria, Kiran, P127 Gulyaeva, Lyudmila, P86 Guns, Emma, P132 Gupta, Sanjay, P125 Güray, N. Tülün, P21

Isenegger, Tamara, P141 Isin, Emre M., P113, P116 Isringhausen, Caleb, P151 Ito, Kiyomi, P46 Ivanov, Maxim, A9

Haenisch, Sierk, P155 Hagen, Paul, P152 Hager, Claude, P120 Hallak, Hussein, P44 Hallifax, David, P10 Hamm, Gregory, P4, P7, P32, P101, P110 Hanna, Debra, P33, P139 Hansen, Regine, P108 Hanusova, Veronika, P64 Harbrecht, Lawrence, P96 Hardillier, Emmanuel, P37 Hare, Victoria, P69 Hargraves, Tiffanie, P81 Havlova, Ivana, P160 Hay, David, S2, P84 Hayashi, Kazuki, P16 Hayashi, Yuma, P103 Hayes, John D., S22 Hayes, Martin A., P113 Heinkele, Georg, A1, P119 Henderson, Catherine, P92, P109, P117 Henderson, Colin, S26, P17, P22, P29, P72, P76, P97, P134, P142 Henson, Claire, P34 Hermann, Dave, P139 Higton, David, P69 Hill, Susan, P131 Hisaka, Akihiro, P2 Ho, Chi Lui, P133 Hobbs, Michael, P85 Hoey, Ross, P135 Hokkanen, Juho, P95 Holdsworth, Catherine, P111 Hong, Yanjun, P55 Hop, Cornelis, P136 Houssen, Wael, P162 Houston, Brian, A10, A11, P10, P11, P43 Hu, Dong Gui, P77 Huang, Jeffrey T.J., P97 Huang, Yong, P164 Hubeny, Andrea, P159 Huehn, Eva, P89 Huguet, Antoine, P73 Hultman, Ia, P14 Humbert, Marc, P32 Hunziker, Juerg, P107 Hussner, Janine, P141 Hustvedt, Svein Olaf, P111

Jackson, Jonathan, P163 Jacob, Claus, S24 Jacobsen, Neil, P81 Jacques-Jamin, Carine, P93 Jahic, Mirza, P164 Jaiyen, Chaliya, P54 Jakab, Annamaria, P108 Jamei, Masoud, P33, P58, P139 Jani, Marton, P154 Jaspars, Marcel, P162 Jeanjean, Corinne, P93 Jedličková, Adéla, P39 Jedlitschky, Gabriele, P152 Jenjirattithigarn, Nuttawut, P62 Jenkins, Rosalind, SC4.3 Jesadakultavee, Chatsiri, P88 Jia, Jia, P147, P149 Jiang, Wenjie, P164 Jimenez, Lourdes Acosta, P142 Jin, Yi, P108 Jinakote, Metee, P54 Jing, Wanghui, P161 Jirásko, Robert, P114 Johänning, Janina, A1 Johns, Jeffrey Roy, P62 Jones, Barry, P121 Jones, Christopher, P15 Jutabha, Promsuk, P54

Iegre, Jessica, P113 Ing Lorenzini, Kuntheavy, P130 Ingelman-Sundberg, Magnus, S27, A9 Irtem Kartal, Deniz, P21 Irvine, Andrew, P162

123

Kageyama, Michiharu, P43 Kals, Mart, A9 Kamiyama, Yoshiteru, P13 Kanai, Yoshikatsu, P16 Kanda, Katsuhiro, P35 Kanjanawart, Sirimas, P62, P80, P126 Kapelyukh, Yury, P29 Kara, Halil, P123 Karbanova, Sara, P160 Karkhanis, Aneesh, P68 Kato, Yukio, P16 Keiser, Markus, P147, P148, P153, P159 Kelm, Jens M., P165 Kendrick, John, P59, P111 Keogh, John, P146 Khunvichai, Ariya, P88 Kibayashi, Hidehiko, P46 Kidley, Nathan, P24 Kießig, Melanie, P83 Kinsky, Owen, P81 Kiss, Edit, P91 Klein, Kathrin, P119 Klingmueller, Ursula, S11 Klumpp, Britta, P74 Kocyigit, Mine, P98 Kohlmann, Markus, P99 Koizumi, Keiichi, P42 Kolmykov, Semyon, P86

13th European ISSX Meeting Kong, Linglei, P49 Kongpan, Thachanan, P62, P80 Konyoung, Parinya, P80 Kosa, Rachel, P11 Kostov, Rumen, P97 Krajcsi, Peter, P91, P154 Krebbers, Saskia, P48 Kretz, Olivier, A8 Kroener, Patrick, P119 Kubesova, Katerina, P143 Kudo, Toshiyuki, P46 Küppers-Munther, Barbara, P82 Kürzel, Gert Ulrich, P99 La, Hank, P136 Lacombe, Olivier, P37 Lam, Hui Yuan, P68 Lam, Teddy TN, P3, P128, P137 Lamka, Jiří, P115 Lamont, Douglas, P17 Lassila, Toni, P95 Laterreur, Julie, A2 Latour, Nathalie, P28 Lau, Serrine, P81, P102 Lauschke, Volker, A9 Lawless, Michael, P89 Leach, Andrew G., P24 Leblond, François A., A2 LeCluyse, Edward, P61 Lecová, Lenka, P79, P115 Lee, Vincent HL, P3, P128, P137 Legouffe, Raphael, P4, P32 Letzsch, Stefan, P165 Lewis, Jenny, P66 Li, Chien-Ming, P164 Li, Hua, P49 Li, Linzhong, S9 Li, Ming, A3 Liew, Ming Hui, P45 Lim, Adeline, P45 Lin, De, P29, P97 Lin, Ge, P94 Lin, Jian, P11 Liptrott, Neill, P57 Liu, Shanshan, P133 Loewen, Gregory, P51, P151 Lopes, Julia, P28, P122 Lopez, Sebastian, P66 Lozac'h, Frederic, P106, P107 Lu, Gaohua, P33 Lucendo - Villarin, Baltasar, P84 Ludwig, Kaja, P155 Lukacova, Viera, P89 Luo, Gang, P59 Ly, Justin, P136 Macháček, Miloslav, P39 Machavaram, Krishna, P58 Mackay, Simon, P85 Mackenzie, Peter, P77 MacLeod, Kenneth, P134 Madden, Stephen, P101, P135 Magnan, Remi, P146, P154 Mahasirimongkol, Surakameth, P80

Author Index Maier, Barbara, P138 Mandikova, Jana, P158 Mandracchia, Andrew, P51 Manevski, Nenad, A8 Mariño, Eduardo L., P45 Marowsky, Anne, S46 Martin, Iain, P131 Martin, Philip, P57 Martínez-Guzmán, María del Carmen, P70 Masubuchi, Yasuhiro, P103 Masuda, Kazufumi, P27 Masuo, Yusuke, P16 Masure, Juliette, P110 Matouskova, Petra, P64, P79 Matsson, Pär, S20 Matsumoto, Takahiro, P27 Maurel, Patrick, A12 Mazerska, Zofia, P19, P40 McElroy, Mary, P101 McCoy, Chase, P51 McEwen, Andrew, P34, P162 McGarry, David, P17 McLaren, Aileen, P72 McLaughlin, Lesley, P22 McMahon, Michael, P76, P142 Mejía-García, Alejandro, P70, P71 Messham, Stephen J., P24 Messick, Kirsten, P136 Messina, Luciano, P135 Messner, Simon, P165 Metha, Anuradha, P108 Meyer Zu Schwabedissen, Henriette E., P141 Milani, Lili, A9 Miners, John, S47 Mitchell, Brenda, P96 Moggs, Jonathan, S34 Monks, Terrence, P102 Moore, Amanda, P61 Moreno, Rita, P72 Morgan, Edward, S52 Moritz, Wolfgang, P165 Mortishire-Smith, Russell, SC1.3 Moss, Darren, P36 Mróz, Anna, P19 Mürdter, Thomas E., A1, P119 Murray, Lesley, P136 Nagamori, Shushi, P16 Nakamichi, Noritaka, P16 Nakkam, Nontaya, P62, P126 Narimatsu, Shizuo, P27 Natt, Francois, P107 Navratilova, Lucie, P75, P158 Nechkin, Sergey, P86 Neuhoff, Sibylle, P47 Neumanova, Zuzana, P156, P157, P160 Nicolas, Jean-Marie, P122 Nomura, Miri, P103 Nováková, Veronika, P39 Novik, Eric, P150

124

Ogilvie, Brian, P51 Ogungbero, Kay, SC3.3 Olbert, Maria, P141 Olbrich, Matthias, P30 Olinga, Peter, P105 Omar, Khaled, P117 Ontawong, Atcharaporn, P54 Oosterhuis, Berend, P146, P154 Oswald, Stefan, P148, P155, P159 Otter, Marcus, P148, P153 Owen, Andrew, P36, P57 Ozden, Sibel, P100 Ozhan, Gul, P98, P100, P123, P124 Oztas, Ezgi, P123, P124 Paine, Stuart, P12 Palmgren, Anna-Pia, P31 Pamelard, Fabien, P4, P32, P101 Pan, Liping, P166 Pánczél, József, P99 Parekh, Amit, P150 Park, Kevin, S38 Parmentier, Yannick, P56 Pastorkova, Barbora, P145 Pavek, Petr, P75 Pavlík, František, P114 Pearson, David, P5 Pennie, Bill, S44 Pennings, Jeroen, P105 Perrin, Hélène, P110 Pettersson, Sven, S32 Pezzetta, Daniele, P112 Phunikom, Katcharin, P62 Picard de Muller, Gaël, P32, P101 Pichette, Vincent, A2 Pirmohamed, Munir, S15 Piquette-Miller, Micheline, S51 Piryatinsky, Victor, P44 Pitt, Andy, SC4.1 Plant, Katie, P69 Pludwinski, Eric, P150 Plumb, Jonathan, A11 Podlipná, Radka, P114, P115 Polak, Sebastian, S13 Pongracz, Judit E., P91 Potęga, Agnieszka, P40 Pothier, Corinne, P56 Prangsaengtong, Orawin, P42 Prchal, Lukáš, P115 Precht, Jana C., A1 Prestin, Katharina, P141 Primard, Charlotte, P110 Prough, Russell, P144 Puccini, Paola, P112 Qin, Yan, P111 Quenault, Hélène, P73 Racine, Christopher, P104 Ramos, Jeanne, A12 Ramstein, Philippe, P5 Rankin, Gary, P104

13th European ISSX Meeting Rapp, Judit, P91 Rasmussen, Henrik, S49 Re, Roberta, P9 Redoulès, Daniel, P93 Reinen, Jelle, P48 Reznicek, Josef, P156 Richert, Lysiane, A4, P18 Riches, Philip, P92 Riedel, Jens, P99 Rietjens, Ivonne, S45 Roberts, Ruth, S43 Rode, Anja, P29 Roffel, Ad, P106 Rojanapanthu, Pleumchitt, P88 Rollison, Helen, P50 Romer, Michael, A12 Romero, Klaus, P33, P139 Rosa, Maria, P28, P122 Rose, Kelly, P61 Rostami-Hodjegan, Amin , P13, P15, P58 Rowland Yeo, Karen, P47 Ruan, Jianqing, P94 Rubio, Leticia, P48 Runge, Dieter, P147, P149 Sağdıçoğlu Celep, Gülçin, P65 Sahly, Yousif, P44 Salem, Farzaneh, P13 Salonen, Jarmo S., P140 Salzet, Michel, P110 Samer, Caroline, P6, P53, P90 Sathirakul, Korbtham, P88 Scharf, Christina, P138 Scheer, Nico, P22, P29, P72, P134, P142 Scheinkoenig, Julie, P51 Schiller, Hilmar, A8 Schinkel, Alfred, S28 Schmitz, Tara, P162 Schroth, Werner, A1 Schwab, Matthias, S16, A1, P74, P119 Schwartz, Mark, P20 Scotcher, Daniel, P15 Seehofer, Daniel, P83 Seeley, Aidan, P162 Segarra, Ignacio, P45 Şen, Alaatin, P26 Senyildiz, Mine, P100 Sharma, Nivedita, P125 Sharma, Pradeep, P50 Sharma, Tusha, P125, P127 Sharp, Sheila, P97 Shaw, Iain, P38 Shibany, Khaled, P12 Shipton, Matthew, P150 Shoffner, Mann, P61 Shrirao, Anil, P150 Siccardi, Marco, P36, P57 Siddiqui, Anzar, P99 Siegmund, Werner, P152, P153, P155, P159 Sike, Mirabella, P154 Šiller, Michal, A7

Author Index Sitia, Roberto, S25 ŠImůnek, Tomáš, P39 Skálová, Lenka, P64, P79, P114, P115 Small, Ben, P33, P139 Smutny, Tomas, P75 Snyder, Shane, P81 Song, Ming, P144 Srimaroeng, Chutima, P54 Srivastava, Abhishek, P121 St. Claire, Robert, P163 Stacpoole, Peter, P41 Stauber, Jonathan, P4, P7, P101 Staud, Frantisek, A6, P156, P157, P160 Ste-Marie, Line, A2 Stevens, Lloyd, P38 Stevenson, Karen, P135 Storelli, Flavia, P53, P90 Stresser, David, P63 Stuchlíková, Lucie, P114 Sukasem, Chonlaphat, P129 Sullivan, Rebecca, P50 Suzuki, Hiroshi, P2 Svanberg, Petter, P14 Sveigaard, Christina , P166 Swart, Piet, A8, P106, P107, P108 Synnergren, Jane, P82 Szkolnicka, Dagmara, P84 Szotáková, Barbora, P64, P79, P114, P115 Taher Nasabi, Nina, P98 Takahashi, Ryosuke, P35 Tang, Celia WS, P3, P128, P137 Tassaneeyakul, Wichittra, P62, P80, P126 Taubert, Max, P138 Tew, Kenneth, S23 Thomas, Aurelien, P6 Thomas, Maria, P74 Thompson, Richard A., P31, P113 Thomson, John, S35 Tiamkao, Somsak, P126 Tolonen, Ari, P95 Tománková, Veronika, P25 Tomlinson, Brian, P3, P128, P137 Totemeyer, Sabine, P12 Toth, Beata, P154 Trampus, Peter, P154 Travnicek, Zdenek, P143 Trejtnar, Frantisek, P158 Trembleau, Laurent, P162 Tripathi, Ashok Kumar, P127 Troberg, Johanna, A5 Trunzer, Markus, P118 Tsaprailis, George, P81 Uchaipichat, Verawan, P52 Ufuk, Ayse, A11 Ukairo, Okechukwu, P61 Ullrich, Anett, P147, P149

125

Ulrichová, Jitka, P25 Ungell, Anna-Lena, P28, P122 Upcott Gill, Rachel, P69 Uras, Cihan, P123 Valentovic, Monica, P96, P104 Vallier, Ludovic, S3 van de Kerkhof, Esther, P107 van de Steeg, Evita, P1 van de Waart, Beppy, P48 van de Water, Bob, S10 Van Giezen, Mariska, P82 Van Hoogdalem, Ewoud, P106 Van Marle, Sjoerd, P106 Vandenplas, Catherine, P78 Vannaprasaht, Suda, P80 Vatakuti, Suresh, P105 Venkataraman, Harini, A4 Vermeer, Lydia, P51, P151 Vermet, Helene, P60 Vermeulen, Nico P.E., A4, P18 Viode, Cecile, P93 Vogeser, Michael, P138 Vokral, Ivan, A3, P79 Vos, Chris J., A4, P18 Vuorilehto, Lauri, P140 Wahlang, Banrida, P144 Waldman, Marvin, P89 Walker, Helen, P38 Wallace, Heather, P162 Walles, Markus, A8 Wang, Duan, P63 Wang, Li-sha, P8 Watkins, Paul, S12 Watson, David, P109, P117 Wedagedera, Janak, P33 Weidolf, Lars, P113 Weiss, Thomas S., P74 Wenker, Mira, P48, P140 Westerhout, Joost, P1 Weydert, Zoe, P165 Wichukchinda, Nuanjun, P80 Wickham, Martin, P9 Wijayakumara, Dhilushi, P77 Wilczewska, Kamila, P40 Wilkinson, David, P59 Wille, Katharina, P153 Williams, Gareth, P5 Wilson, Ian, SC1.4 Winiwarter, Susanne, P87 Winter, Stefan, P74 Wisztorski, Maxence, P110 Wo, SK, P3, P128, P137 Wolf, Kristina, P61 Wolf, Roland, S42, P17, P22, P29, P72, P76, P97, P134, P142 Wood, Francesca, P10 Wood, Stuart, P34 Woods, Adam, P34 Wortelboer, Heleen M., P1 Wright, Matt, SC2.4 Wu, Jingjing, P23

13th European ISSX Meeting

Author Index

Xiao, Bing-xin, P8 Yan, Ru, P161 Yanar, Hakan Teoman, P124 Yang, Ling, P23 Yang, Mengbi, P94 Yeo, Karen Rowland, SC3.2 Yeoman, Martin, P9 Yilmaz-Ozden, Tugba, P98 Yoshimoto, Francis, A7 Yurteri, Gülsüm, P21 Zamora, Ismael, P87 Zanger, Ulrich, S50, P74 Zann, Vanessa, P38 Zhang, George, P63 Zhang, Jinhua, P89 Zhang, Xuexiang, P164 Zhuang, Xiaomei, P49 Zimčík, Petr, P39 Zimmerlin, Alfred G., P118 Zimmermann, Uwe, P141 Zink, Daniele, SC2.3 Zoller, Michael, P138 Zollinger, Markus, P106 Zuo, Zhong, P3, P128, P13

126

13th European ISSX Meeting

Notes

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13th European ISSX Meeting

Notes

128