RESEARCH PROPOSAL SEX- SPECIFIC METABOLISM AND EFFECTS OF DRUGS

Transcription

1 RESEARCH PROPOSAL SEX- SPECIFIC METABOLISM AND EFFECTS OF DRUGS

2 SEX- SPECIFIC METABOLISM AND EFFECTS OF DRUGS Applicants: Yvonne Bartels Charlotte Hoogstraten Krijn Reijnders Felix Tönisen Lina Wübbeke RHA Faculty of Science, Radboud University, Nijmegen, The Netherlands May 2014 First, we would like to thank the counsel from the Radboud Honours Academy (RHA) of the Faculty of Science for offering such an interesting and challenging program, and helping us to improve our skills in writing, presenting and working in a multidisciplinary group. We especially want to thank the counsel for the nice and unforgettable trip to Oxford (UK). We sincerely thank Prof. Dr. Philip Biggin, Prof. Dr. Simon Newstead, Prof. Dr. Béla Novák and Prof. Dr. Jason Schnell (Dept of Biochemistry, Oxford University, Oxford, UK) for their enthusiasm, the valuable conversations and the time and effort they put into our project, while visiting them in Oxford and afterwards. We also acknowledge Prof. Dr. Petra Thürmann (Clinical Pharmacology at the Helios Klinikum Wuppertal, Wuppertal, DE) for her advice to focus on drug efflux transporters. In addition, we like to thank Prof. Flavia Franconi (University of Sassari, Sassari, IT) for her advice. We thank Prof. Dr. Frans Russel (Radboudumc, Nijmegen, NL) for his interest, his critical view on our research proposal and his useful suggestions. Furthermore, we are grateful to Prof. Dr. David Burger and Dr. Wilbert Peters (Radboudumc, Nijmegen, NL) for their interesting and helpful conversations in October 2013, and Dr. Peter Klaren (Dept Organismal Animal Physiology, Radboud University, Nijmegen, NL) for his interest in our project and his help with the modelling part of our proposal. Our special thanks go to Prof. Dr. Gerard Martens (Donders Centre for Neuroscience, Radboud University, Nijmegen, NL), our supervisor. We thank him for the time he spent at our meetings, his involvement, enthusiasm, support, critical views and input during the process. Without his help we would not have been able to present this proposal. 1

3 ABSTRACT Sex- related differences in pharmacokinetics of the liver contribute to differences in drug efficacy and toxicity profiles in men and women. Drug metabolism in the liver and the contribution of the substrate- oxidizing enzyme cytochrome P450 (CYP450) have been well studied. However, the contributions of proteins other than CYP450 to the sex- related differences in pharmacokinetics, e.g. the drug efflux transporters, have not been described yet. The aim of our proposed research is to determine how sexual dimorphic gene expression in the liver can explain the sex- specific differences in pharmacokinetics. Our study design is unbiased because we will first perform genome- wide expression analysis (RNA sequencing and proteomics) to determine which mrnas and proteins are sex- specifically expressed in vitro in human hepatocytes from male and female. Analysis will also be performed on human hepatocytes from male and female treated with various concentrations of different hormones (growth hormone, estradiol, testosterone) and with the immunosuppressant drug cyclosporin, which is mainly metabolized in the liver. The top list of differentially expressed genes will be validated by qrt- PCR and Western blot analysis, and selected genes will be further analyzed in mice with human ectopic artificial livers (HEALs). In these humanized mice, also the plasma clearance of cyclosporin will be measured using liquid chromatography- mass spectrometry. Short hairpin RNAs will be used in vitro to downregulate the sex- specifically expressed genes in order to determine the difference in clearance efficiency between human hepatocytes from male and female. To evaluate the validity of the mouse model, we will measure the clearance of cyclosporin in male and female humans as well. Finally, based on our experimental results we will build a computational model to predict the sex- specific pharmacokinetics of cyclosporin in the liver. The knowledge about sex- specific pharmacokinetics, obtained from this research project, may lead to a reappraisal of the importance of sex- specific differences in pharmacology. 2

4 CONTENTS 1. BACKGROUND PHARMACODYNAMICS AND PHARMACOKINETICS Sex- based Differences in Pharmacokinetics DRUG METABOLISM Drug Metabolism in Phase I Cytochrome P Drug Metabolism in Phase II Transferases Drug Metabolism in Phase III Excretion CYCLOSPORIN AS AN EXAMPLE OF A DRUG WITH SEX- SPECIFIC PHARMACOKINETICS Criteria for Selecting a Drug with Sex- specific Pharmacokinetics Selection of Cyclosporin MOTIVATION HYPOTHESIS OBJECTIVES RESEARCH QUESTIONS WHICH PROTEINS, INCLUDING CYP ENZYMES AND DRUG EFFLUX TRANSPORTERS, ARE IN VITRO DIFFERENTIALLY EXPRESSED IN HUMAN HEPATOCYTES FROM MALES AND FEMALES? WHICH PROTEINS, INCLUDING CYP ENZYMES AND DRUG EFFLUX TRANSPORTERS, ARE IN VIVO DIFFERENTIALLY EXPRESSED IN HUMAN LIVERS FROM MALES AND FEMALES? HOW EFFECTIVE IS THE SEX- SPECIFIC CLEARANCE OF THE CYP3A4 SUBSTRATE CYCLOSPORIN IN ISOLATED HUMAN HEPATOCYTES AND IN HUMANS? HOW COULD A COMPUTATIONAL MODEL BE USED TO PREDICT OPTIMIZATION OF DRUG ADMINISTRATION IN MALE AND FEMALE HUMANS? RELEVANCE AND INNOVATIVE ASPECTS STUDY DESIGN AND METHODOLOGY IN VITRO ANALYSIS OF HUMAN HEPATOCYTES FROM MALES AND FEMALES Untreated Human Hepatocytes from Males and Females Hormone- treated Human Hepatocytes from Males and Females Effect of Cyclosporin on Hormone- treated Human Hepatocytes from Males and Females shrna- driven Downregulation of Top List Genes in Hormone- treated Human Hepatocytes from Males and Females IN VIVO ANALYSIS OF MALE AND FEMALE HUMANIZED MICE Engineering and Implantation of HEALs Drug Metabolism and Gene Expression in Male and Female Humanized Mice shrna- driven Downregulation of CYP3A4 in HEALs of Male and Female Humanized Mice IN VIVO CLEARANCE OF CYCLOSPORIN IN MALE AND FEMALE HUMANS COMPUTATIONAL MODEL TO OPTIMIZE DRUG ADMINISTRATION IN MALE AND FEMALE HUMANS Application of our Model STATISTICAL ANALYSIS OF THE EMPIRICAL DATA TIMETABLE BUDGET SOCIETAL RELEVANCE FEASIBILITY ETHICS REFERENCES

5 1. BACKGROUND Currently there are a lot of chemical substances available for the production of medicines. These chemicals, the active ingredients of a medicine, are called drugs and can affect a biological system such as the human body (Thorp 2008). Drugs belong to the so- called xenobiotics, which are substances that are not familiar to the human body (Lehninger, Nelson, and Cox 2008). The study of drugs and their effects on the human body is called pharmacology. In pharmacology, the toxic and unwanted effects of drugs, which can occur when high doses are used or if the drugs are used incorrectly, are also studied. Drugs are administered to have a beneficial effect, but in general drugs have the potential to be toxic for the human body under certain circumstances. These side effects are known as adverse reactions and can be caused by a variety of mechanisms (Timbrell 1999; Thorp 2008). No matter if the effect is curative or toxic, different processes are involved in the way the chemicals affect the human body and the way the body handles them. Drugs are spread through the body by the bloodstream. Unless a drug is directly injected, it must be absorbed into the bloodstream to reach its site of action. Several membranes have to be crossed such that the drug can reach the blood for this transport. Simple diffusion, facilitated diffusion and active transport are three ways by which substances can cross cell membranes (Timbrell 1999; Thorp 2008). The drug enters the systemic circulation and can effectively be distributed through the body. Because drugs are xenobiotic, they are handled as potential toxic substances, which need to be detoxified and eliminated. This means that they are metabolized and then excreted. Enzymes change the molecular structure of the substance. The majority of xenobiotic metabolism, and thus the majority of drug metabolism, takes place in the liver. Generally there are two types of metabolic reactions, known as phase I and phase II reactions, and a phase III stage of excretion (Thorp 2008). These phases will be further explained below. Individuals get similar drug quantities, no matter if the individual is a male or female. Nevertheless, there are sex- specific differences regarding many factors, which influence the response to drugs. Furthermore, nearly all medicines that are currently prescribed have more likely been tested in male than in female laboratory animals (Schiebinger 2014). This research proposal will focus on the differences in drug metabolism between men and women. The first phase of drug metabolism is performed by the cytochrome P450- system. The second one contains conjugation reactions. These two phases and their sex- specific differences are described first. The knowledge about the differences between men and women in the third phase, excretion, is limited. Our research proposal will concern the differences in the excretion step. 1.1 PHARMACODYNAMICS AND PHARMACOKINETICS We are interested in the sex- specific differences in drug efficacy and safety between men and women. For this research field we can focus on either pharmacodynamics or pharmacokinetics. To influence the human body, drugs must interact with it. Pharmacodynamics is the study of these interactions and the effects that drugs can have on the body. Furthermore, pharmacodynamics is concerned with how drugs produce their therapeutic or adverse effects (Thorp 2008). On the other hand, pharmacokinetics describes 4

6 the way in which the body handles the drugs. The relationship between drug dosage and the concentration of the drug in certain components of the body is often used to study this (Gandhi et al. 2004). There are four major parts which belong to pharmacokinetics, namely the processes of absorption, distribution, metabolism and excretion (ADME) of drugs (Thorp 2008). We decided to focus on pharmacokinetics because there is more knowledge of and evidence for the involvement of kinetics concerning sex- specific differences: In general, pharmacokinetic differences are more numerous and consistent than disparities in pharmacodynamics. (Franconi and Campesi 2014) SEX- BASED DIFFERENCES IN PHARMACOKINETICS There are many factors in pharmacokinetics, which are different between men and women, and thus in the response to drugs. Drug absorption is the first step where differences become apparent. In females the gastric emptying time is larger than in males. This is secondarily caused by the sex hormone estradiol. The drugs stay longer in the stomach of a female and more drugs can be absorbed per taken dosage (Gandhi et al. 2004). The distribution of drugs through the body is also influenced by sex- specific characteristics of the body. Women have a higher proportion of body fat than men. Thus, for fat- soluble drugs there is a greater accumulation of lipophilic compounds in women than in men. Water- soluble compounds have a relatively lower volume of distribution. Women also have a smaller average plasma volume and the blood flow is slower. This also implicates the rate and extent of drug distribution (Gandhi et al. 2004). Sex- based differences in the metabolism of drugs seem to have a greater influence on the sex- specific variability in the pharmacokinetics than any of the other parameters of absorption or distribution, such as the higher body fat percentage in women or the differences in the gastric emptying time (Gandhi et al. 2004). We therefore focus on the differences in drug metabolism in the liver between males and females. 1.2 DRUG METABOLISM As mentioned above, the majority of drug metabolism takes place in the liver and there are three phases involved. Some drugs undergo all of the three phases, other drugs can be metabolized by only one phase and are then excreted. The drug is made more water soluble during the metabolism and can therefore be excreted more easily (Thorp 2008) DRUG METABOLISM IN PHASE I CYTOCHROME P450 In the first step of the metabolism in the liver, xenobiotics become oxidized by the CYP450 system. The CYP450 system genes are located on autosomal chromosomes. The system is called the CYP450 system because of the characteristic absorption peak at 450 nm (Anzenbacher and Anzenbacherova 2001). The CYP450 system is involved in the metabolism of a large range of drugs and other xenobiotics. This system can also be found in other tissues with a specific barrier function, e.g. heart, blood brain barrier, kidneys, placenta and intestine (Zanger and Schwab 2013). Therefore, its contribution to the metabolism of drugs is very important in pharmacology. The CYP450 system is mainly located in the microsomes, which are a part of the endoplasmic reticulum (Anzenbacher and Anzenbacherova 2001; Mode and Gustafsson 2006; Sevior, Pelkonen, and Ahokas 2012). 5

7 The CYP450 system is a mono- oxygenase enzyme and therefore it adds one oxygen atom to the substrate. The functional group of this protein is a heme- group. The energy that is necessary for the oxygenation comes from NAD(P)H. The biotransformation of the lipophilic substrates results in a more polar form of the drug. The drug becomes more water soluble and thus it is easier to clear such metabolites from the body (Sevior, Pelkonen, and Ahokas 2012; Zanger and Schwab 2013). There are a lot of human isoforms of the CYP450 system. These isoforms are grouped into several families and subfamilies based on their degrees of nucleotide sequence identities. There are 18 different families and 44 subfamilies (Sevior, Pelkonen, and Ahokas 2012; Zanger and Schwab 2013). A lot of these isoforms are only involved in the synthesis of steroid hormones, prostaglandins, bile acids and other endogenous compounds. Only three families of the CYP450 system (the CYP1, CYP2 and CYP3 families) are drug metabolizing enzymes. Because of this, these three families are the most important ones in pharmacokinetics (Sevior, Pelkonen, and Ahokas 2012). The CYP3A4 isoform displays the highest level of expression, is involved in the metabolism of more than fifty per cent of the existing drugs and has a high substrate capacity (Anzenbacher and Anzenbacherova 2001) SEX- SPECIFIC DIFFERENCES AND REGULATORY MECHANISMS IN PHASE I The sex- specific differences in the regulation of the CYP3A4 isoform are not clear yet. Research on various regulatory factors has shown their possible contribution to the sex- specific expression of CYP3A4. An important hormone for the regulation of the human CYP450 system is the growth hormone (GH). This hormone is produced by somatotrophic cells in the hypothalamus and is secreted by the lateral wings of the anterior pituitary gland into the bloodstream. It is a hormone consisting of 191 amino acids and acts as a stimulator for longitudinal bone growth. In addition, it induces diverse effects on cell growth, differentiation and metabolism (Waxman and O Connor 2006). In the neonatal period the secretion pattern of GH becomes imprinted. This imprinting is due to the action of the gonadal hormones. GH is secreted continuously in females, but in males it is released every 3,5 hours followed by GH- free intervals (Wiwi and Waxman 2004; Waxman and O Connor 2006). Therefore the plasma levels of GH differ in men and women. The key factor of the sex- specific expression of CYP3A4 might be the hepatocyte nuclear factor HNFα. The continuous secretion pattern of GH in females upregulates HNF4α activation of the promoter of CYP3A4. This contributes to the sex- specific expression levels of CYP3A4 (Figure 1. In addition to the sex- dependent expression, there are also several single- nucleotide polymorphisms (SNPs) known in the CYP3A4 gene. However, these SNPs do not contribute to the sex- specific variability of CYP3A4 expression (Thangavel, Boopathi, and Shapiro 2011). 6

8 Figure 1: The influence of the sex- specific growth hormone release pattern on the HNF4 pathway leading to transcription of CYP- genes (Waxman and O Connor 2006). STAT5b might be important for the sex- dependent effect of GH in the liver. It is a signal transducer that mediates many of the transcriptional responses of GH and certain other hormones. Phosphorylation of this transducer is needed for translocation from cytosol into the nucleus, where it is required for transcriptional activities. This phosphorylation seems much more efficient in male livers than female livers due to GH pulses instead of continuous release. Every incoming GH pulse rapidly translocates the phosphorylated STAT5b to the nucleus. STAT5b is deactivated via dephosphorylation and returns to the cytosol in the inactive form. The near- continuous plasma GH pattern in females is responsible for the lower nuclear STAT5b activity (Waxman et al. 1995; Tannenbaum et al. 2001). High levels of the nuclear phosphorylated STAT5b protein can generally be found in male but not female livers (Waxman et al. 1995). Studies of GH pattern- dependent sex- specific differences in the activation of liver STAT5b suggest that STAT5b may be an important intracellular mediator of the transcriptional effects of GH on sex- specific liver genes (Gebert, Park, and Waxman 1997; Ji, Frank, and Messina 2002) DRUG METABOLISM IN PHASE II TRANSFERASES During the second step in the metabolism of drugs, biotransformation by enzymes occurs. The purpose of phase II reactions is to perform conjugation reactions, which means that ionized or other functional endogenous molecules attach to the compound. Several enzymes called transferases perform this reaction. These reactions occur when the drug contains a group suitable for combination with an endogenous compound (e.g. glutathione, glycine, sulfate, glucuronic acid). The chemical groups on the drug molecule usually associated with these reactions are - OH, - COOH, - NH 2 and SH (Timbrell 1999). Example reactions include glucuronidation, sulfation, methylation, acetylation, and glutathione and amino acid conjugation. In general, these conjugates are most of the time more polar and more hydrophilic than the parent compounds. This facilitates excretion via 7

9 bile and urine, and decreases the pharmacological activity (Jancova, Anzenbacher, and Anzenbacherova 2010). Phase II enzymes have attracted much less attention in clinical pharmacology than cytochrome P450, because drug interactions involving these enzymes are relatively rare. Nevertheless, the reduced metabolizing capacity of the phase II enzymes can lead to manifestations of the toxic effect of clinical drugs. Although phase II reactions are generally detoxifying, the conjugates formed may also mediate adverse effects. Individual differences in metabolic response exist for both phase I and phase II enzymes. Both external factors, such as smoking, medication, diet and effects of the environment, and internal factors, such as age, diseases and genetics, are known to have an impact on phase II enzymes (Jancova, Anzenbacher, and Anzenbacherova 2010). UDP- glucuronosyltransferases (UGTs) are an example of such transferases. UGTs are the key enzymes in the process called glucuronidation. The formation of these conjugates is the most important detoxification pathway of the second phase in the metabolism of drugs. Approximately forty to seventy percent of all drugs are metabolized through glucuronidation reactions metabolized by UGTs in phase II. These enzymes are responsible for the metabolism of many xenobiotics and endogenous compounds (Jancova, Anzenbacher, and Anzenbacherova 2010). UGTs are membrane- bound enzymes which catalyze the formation of a chemical bond between a nucleophilic O-, N-, S- or C- atom with uridine- 5 - diphospho- α- D- glucuronic acid (UDPGA). The glucuronic acid is in the α- configuration at the C1 atom when bound to the coenzyme. The transfer occurs with an inversion of configuration. This reaction leads to the formation of β- D- glucuronides. A typical conjugation reaction is illustrated in the following equation: R- OH + UDPGA + glucuronyltransferase - - > R- O- glucuronide + UDP The expression of UDP- glucuronosyltransferase enzymes is sex specific in mice (Nicolson, Mellor, and Roberts 2010). It is not exactly clear whether only this type of transferase has a significant sex- related expression. More research on this topic is needed (Buckley and Klaassen 2007; Nicolson, Mellor, and Roberts 2010) SEX- SPECIFIC DIFFERENCES AND REGULATORY MECHANISMS IN PHASE II Although the transferase reactions performed by the UGTs are sex specific in mice, it is difficult to extrapolate these results to humans. Furthermore, there are no indications in the literature that phase II is sex specific in humans. There are a lot of different reactions involved in phase II and the mechanism of the probable sex- specific regulation and genes involved remains unclear. Therefore, phase II is too understudied at the moment to be amenable to a sex- specific study. However, in a genome- wide approach (see section 6.1.1) indications for a sex specificity of phase II may be found DRUG METABOLISM IN PHASE III EXCRETION The third step of drug metabolism involves the excretion of phase I and II metabolites which is done by drug efflux transporters. They remove a wide range of structurally and functionally distinct molecules from the cells against a concentration gradient. The 8

10 transporters are located in the liver, on the luminal side of the blood- brain barrier, blood- testis barrier and placenta (Wang, Siahaan, and Soltero 2005). Efflux transporters can be divided into three major groups: P- glycoproteins (P- gp) multidrug resistance- associated proteins (MRP) breast cancer- resistant proteins (BCRP) Most relevant for this research project is the P- gp drug efflux transporter because one of its substrates is the drug cyclosporin that will be central in our project (see below under 1.3). Therefore, only the P- gp is described in detail P- GLYCOPROTEIN Cells can become multidrug resistant via P- gp. It is a multidrug resistance protein (MDR) and belongs to the ATP- binding cassette (ABC) transporter family. MDRs are ATP- dependent efflux transporters, which have a broad spectrum of substrate specificity. Most of the compounds that are excreted by these efflux transporters are hydrophilic, positively charged or neutral and with a planar structure (Wang, Siahaan, and Soltero 2005). The structure of a P- gp consists of two homologous halves, each of which consists of six transmembrane helices. Furthermore, it has a hydrophilic intracellular nucleotide binding domain, which is characteristic for ABC- transporters. The nucleotide binding sites provide ATPase activity. The hydrolysis of ATP provides energy, which is needed for the translocation function of the protein (Wang, Siahaan, and Soltero 2005) (Figure 2). Figure 2: Conformational changes of ATP- binding cassette exporters (ABC exporters) (Martinez & Falson 2014). P- gp has been proposed to facilitate excretion by two different mechanisms: hydrophobic vacuum cleaner or the flippase mechanism. The first one binds the substrates directly within the plasma membranes and pumps them out of the cells. The second mechanism contains a substrate- binding site in the inner leaflet of the plasma membrane bilayer. The 9

11 substrates are then flipped by the P- gp to the outer leaflet, from which they diffuse into the extracellular space (Wang, Siahaan, and Soltero 2005). All P- gp substrates are amphipathic, which means they have lipophilic as well as hydrophilic properties. This may have to do with the mechanism used by the P- gp. Only amphipathic molecules would have the proper membrane insertion properties, because the substrate first has to insert to the inner leaflet before being flipped to the outer leaflet (Schinkel and Jonker 2003). High levels of P- gp expression have been found in tissues such as the liver, kidney, gastrointestinal tract, the blood- brain and blood- testis barriers, as well as the adrenal glands. P- gp has been shown to be predominantly located on the apical surface of the epithelial (or endothelial) cells with a specific barrier function (Wang, Siahaan, and Soltero 2005). The expression of this exporter protein is polarized in the excretory organs and this protein has the ability to transport a wide diversity of chemicals. This indicates that P- gp may play an important role in protecting cells and tissues from toxins and toxic metabolites. This is done by actively excreting these toxic agents into the bile, urine and intestinal lumen (Nicolson et al. 2010) SEX- SPECIFIC DIFFERENCES AND REGULATORY MECHANISMS IN PHASE III It has been shown that the expression of drug efflux transporters varies among individuals. This can be due to age and sex- differences, genetic polymorphisms or prior exposure to drugs, food and environmental compounds (Wang, Siahaan, and Soltero 2005). During the third phase in the metabolism of drugs, metabolites leave the liver, a process that is facilitated by drug efflux transporters in the apical membrane. These transporters, mainly P- gp and MRP are usually co- expressed and co- induced with CYP3A in the liver and intestine. It appears that these may be regulated by steroid and xenobiotic receptor (SXR) or pregnane activated receptor (PXR) (Johnson, Charman, and Porter 2003). These nuclear receptors function as a sensor for the presence of toxic substances and in response upregulate the expression of proteins involved in clearance and detoxification. The exact mechanism of the regulation of these receptors and other factors that may influence this third phase are still obscure (Xu, Li, and Kong 2005). 1.3 CYCLOSPORIN AS AN EXAMPLE OF A DRUG WITH SEX- SPECIFIC PHARMACOKINETICS In this paragraph the selection of cyclosporin, which is central in this research proposal, is described. In the first step selection criteria were chosen CRITERIA FOR SELECTING A DRUG WITH SEX- SPECIFIC PHARMACOKINETICS The fact that the drug is mainly eliminated through the liver, rather than e.g. kidneys, is an important criterion. Furthermore, in this research proposal the sex- specific expression of the cytochrome P450 isoform CYP3A4 will be analyzed. Therefore, it is necessary that the drug is mainly metabolized by this isoform. Additionally, the chosen drug must be a substrate of one of the major drug efflux transporters, such as P- gp. The third criterion is that there has to be evidence for sex- specific pharmacokinetics of the drug in the literature. Furthermore, it is important that the chosen drug is not a pro- drug. Pro- drugs have to be biotransformed in the liver to become activated, which results in two forms, active and inactive, to be present 10

12 in the blood. This makes it more difficult to measure the concentration of the active compound in the blood SELECTION OF CYCLOSPORIN There are several drugs which are common substrates of CYP3A4 and P- gp, e.g., cyclosporin, colchicine, dexamethasone, doxorubicin, erythromycin, imatinib, nelfinavir, verapamil and vinblastine (Kim 2002; B. Chen et al. 2011; Department of Medicine at Indiana University 2013; Fruit et al. 2013; Zanger and Schwab 2013). These drugs are immunosuppressants, anticancer agents, anti- inflammatory agents, antiarrhythmic agents, anti- retroviral agents and antibiotics. We do not want to choose an anticancer agent, because cancer patients are often co- administered with other drugs. The pharmacokinetics of co- administered drugs is more complex due to possible drug- drug interactions. We selected the immunosuppressive drug cyclosporin. It is often used for suppressing the immune reaction of patients with grafted organs and as a standard therapy after the transplantation of organs (Barbarino et al. 2013; Fruit et al. 2013). Cyclosporin is mainly eliminated and excreted through the liver to the bile. Other ways of excretion do not contribute significantly to the pharmacokinetics of cyclosporin (Fahr 1993). The decrease of the concentration of cyclosporin and its metabolites in the blood is therefore representative for its clearance by the liver. Furthermore, the pharmacokinetics of cyclosporin is sex specific (Fruit et al. 2013; Zanger and Schwab 2013). Cyclosporin is metabolized into approximately 25 metabolites. The three major metabolites AM1, AM9 and AM4N are formed by CYP3A4. AM1 and AM9 are hydroxylated products of cyclosporin, AM4N is a N- demethylated product (Akhlaghi et al. 2012). Furthermore, cyclosporin is a substrate of the drug efflux transporter and is excreted from the hepatocytes to the bile (Fricker et al. 1996; Hebert 1997; Lown et al. 1997). 1.4 MOTIVATION The efficiency and effectiveness of drugs are strongly influenced by sex- specific differences. Therefore, it is definitely time to perform sex- specific research in all steps of drug development. Female sex appears to be a potential risk factor for adverse drug effects, likely because mainly male laboratory animals are studied during drug development (Franconi and Campesi 2014). Medical research is mainly performed in male animals. From 1995 until 2005, there is a 79 per cent preponderance of whole- animal studies for preclinical trials performed only on male laboratory animals (Mogil and Chanda 2005). In phase III human trials on the other hand, women have to be included since 1993 (required by US law). But how can women safely be included, if the drugs are never tested in female laboratory animals first? How can we ignore half of the population in basic biomedical research? Not using female animals can cause greater health risks for women (Schiebinger 2014) and is likely the reason why women have a fold greater risk than men to react with adverse reactions after drug intake (Gandhi et al. 2004). The reason for not using female laboratory animals is simple: avoiding higher costs. The development of a drug costs billions of dollars. When using female animals their reproductive status and ovarian cycle have to be taken into account. But a recent study reveals that no problematic variation is produced through the female animals hormonal cycle (Prendergast, Onishi, and Zucker 2014). There will be less failure and the use of drugs 11

13 will be optimized, if the drugs are tested on female laboratory animals as well (Becker et al. 2005). The importance of studying sex- based differences in pharmacology is evident. From October 2014 onwards, the largest source of funding for medical research in the world, the National Institutes of Health (USA), will strictly only fund research projects, which balance their use of female and male animals (Clayton and Collins 2014). Even more importantly, the efficacy of medicines in men and women and potential adverse reactions are obviously influenced by sex- specific differences (Gandhi et al. 2004). Genetic variation in CYP enzymes and drug efflux transporters is homogeneously distributed in the human population and not expected to be sex specific. Therefore, in this research proposal physiological, non- genetic sex- specific differences will be explored. Since we do not know enough about how the body interacts with a drug, understanding these interactions will optimize the specificity and use of drugs. The results of our research will greatly contribute to our understanding of sex- based differences in pharmacology and are therefore of great importance. 2. HYPOTHESIS In recent research it has been shown that differences in sex have a significant influence on the clearance of cyclosporin by the liver (Fruit et al. 2013). Cyclosporin is mainly metabolized by the CYP3A4 isoform of the cytochrome P450 system in the liver. Women appear to have a more rapid CYP3A4- dependent clearance of certain drugs, including cyclosporin (Waxman and O Connor 2006). We therefore expect to find a strong relation between the differences in the expression of CYP enzymes, e.g. CYP3A4, and the difference in the clearance of cyclosporin. Nevertheless, we expect that the sexual dimorphic clearance of cyclosporin cannot only be explained by the sex- specific expression of CYP3A4. Therefore, by applying a genome- wide approach we expect to find other sex- specifically expressed genes. Besides CYP3A4, we anticipate to find at least P- gp in a top list of the most significant sex- specifically expressed genes, because it is co- expressed and co- induced with CYP3A4 (Xu, Li, and Kong 2005). We thus expect that the expression of CYP3A4 as well as the expression of other genes, e.g. P- gp, is influenced by sex. With our research project we aim to test this hypothesis and determine the statistical correlation of the sexual dimorphic expression patterns of CYP3A4 and the top list genes with the sex- specific clearance of cyclosporin. 3. OBJECTIVES The overall objective of this research proposal is to highlight the importance of considering sex- specific differences in future pharmacokinetic studies and reduce sex- specific adverse drug effects. The factors responsible for the sex- specific differences in pharmacokinetics will be explored. Furthermore, a computational model that will allow prediction of the sex- specificity of phase III in the liver will be developed. Missing parameters in our model will be measured experimentally, including the sex- specific regulation of the drug efflux transporters. 12

14 4. RESEARCH QUESTIONS Because of the great lack of knowledge about the sex- specific differences between men and women in pharmacokinetics, we raise the following main question: To what extent do CYP enzymes and drug efflux transporters in the liver contribute to the sex- specific differences in pharmacokinetics? To answer the main question, a number of aspects have to be considered. Therefore, we split this question into four subquestions. 4.1 WHICH PROTEINS, INCLUDING CYP ENZYMES AND DRUG EFFLUX TRANSPORTERS, ARE IN VITRO DIFFERENTIALLY EXPRESSED IN HUMAN HEPATOCYTES FROM MALES AND FEMALES? First, an unbiased genome- wide study will be performed to detect sex- specific mrna and protein expression patterns in the liver, which are expected to include CYP enzymes and drug efflux transporters. In order to investigate this, RNA- sequencing and proteomics of human hepatocytes from males and females will be performed. The top list of differentially expressed mrnas and proteins will be validated by quantitative reverse transcriptase polymerase chain reaction (qrt- PCR) and Western blot analysis, respectively. One enzyme that will be differentially expressed in males and females is CYP3A4. We also expect a difference in the expression of P- gp between males and females. Therefore, these proteins will be included in the to- be- validated list, and their mrna and protein expression levels will be measured in human hepatocytes from males and females by using qrt- PCR and Western blot analysis, respectively. The methodologies mentioned above will be used in untreated as well as hormone- and cyclosporin- treated hepatocytes. Next, the mrna expression levels of CYP3A4, P- gp and other sex- specifically expressed genes derived from the top list will be downregulated by using short- hairpin (sh) RNA. This will be done to determine the efficiency of human hepatocytes from males and females with respect to drug clearance by sex- specifically expressed genes. 4.2 WHICH PROTEINS, INCLUDING CYP ENZYMES AND DRUG EFFLUX TRANSPORTERS, ARE IN VIVO DIFFERENTIALLY EXPRESSED IN HUMAN LIVERS FROM MALES AND FEMALES? Human ectopic artificial livers (HEALs) will be engineered and implanted into mice, and analyzed for sex- specific differences in mrna and protein expression by RNA- sequencing and proteomics as described above. Liver biopsies are needed to measure mrna expression levels. Again, the to- be- validated list will include CYP3A4 and P- gp, and their mrna and protein levels will be determined by qrt- PCR and Western blot analysis, respectively. Downregulating the mrna expression level of top list genes in male and female HEALs by using shrna, will give insights into the efficiency of hepatocytes in males and females with respect to drug clearance by sex- specifically expressed genes. 4.3 HOW EFFECTIVE IS THE SEX- SPECIFIC CLEARANCE OF THE CYP3A4 SUBSTRATE CYCLOSPORIN IN ISOLATED HUMAN HEPATOCYTES AND IN HUMANS? To answer this question, the concentrations of cyclosporin and its major metabolites will be measured in vitro in human hepatocytes from males and females. Furthermore, concentrations of cyclosporin and its metabolites will be measured over time in the blood of 13

15 male and female humanized mice (HEALs) and humans. To determine these concentrations, liquid chromatography- mass spectrometry (LC- MS) will be used. 4.4 HOW COULD A COMPUTATIONAL MODEL BE USED TO PREDICT OPTIMIZATION OF DRUG ADMINISTRATION IN MALE AND FEMALE HUMANS? To predict optimal drug dosing for men and women, a computational model will be created. Therefore, rate constants between compartments, which are involved in drug metabolism (blood, liver and bile), will be determined by using the results of the cyclosporin concentration measurements in vitro (measured under 4.1). This model will give us insights into the differences in drug metabolism between the two sexes and will also help to determine the optimal drug dose for men and women. 5. RELEVANCE AND INNOVATIVE ASPECTS Surprisingly little is known about sex- specific differences in metabolism and effects of drugs, and the reasons for these differences at the molecular level. This research proposal is unique and innovative in that for the first time we approach these topics in an integrated and comprehensive way, using up- to- date research approaches and methodology. If there are differences in the expression of drug efflux transporters between males and females, this could lead to different medication or dosing between males and females. As a result, side effects could be reduced and drug efficiency and efficacy could be increased. Some researchers even go as far as to claim that it might save us from life- threatening errors (Schiebinger 2014). A sex- specific difference in drug efflux transporters could help us with our current understanding of drug metabolism, which is of course an important factor for our health. Most drugs are now overprescribed for women and create an unnecessary risk for women by causing side effects and decreased efficiency. Also, if the mechanism of drug efflux transporters is completely understood, this could help us to improve the pharmacokinetics of new drugs. If we know there is a sex- related difference, this could help with the development of new drugs. It is also a step forward in the direction of personalized medicine. Furthermore, our research could lead to a better understanding of the pharmacokinetics of drugs in general. Understanding the pharmacokinetics of a drug is essential knowledge for the medical and biological world. This includes understanding of why and how sex- specific differences are caused. 6. STUDY DESIGN AND METHODOLOGY In this research we will focus on the sex- specific metabolism of drugs. First, in vitro experiments will be performed with human hepatocytes from males and females. HEALs will be made from human hepatocytes from males and females, so that they can be used in mouse models. Furthermore, the clearance of cyclosporin from blood from the humanized mice and from human subjects will be measured. Finally, using the experimental in vitro results, a computational approach will be applied to predict the optimization of drug administration for men and women. 14

16 6.1 IN VITRO ANALYSIS OF HUMAN HEPATOCYTES FROM MALES AND FEMALES This section will focus on in vitro studies on human hepatocytes from males and females. These hepatocytes will be obtained from Life Technologies TM - Gibco (Human Suspension Hepatocytes, Metabolism Qualified, Male/Female, (9-12 x 10 6 cells); catalog number: HMCS1L, HMCS2L). First of all, we will describe the cells that will be used as controls. The second part is focusing on human hepatocytes from males and females, which will be cultured in the presence of sex hormones and the growth hormone to identify whether these affect the expression and regulation of metabolizing genes. This will be done, because sex hormones and growth hormone patterns are one of the major differences in the regulation of processes between men and women. In the last part of this section, we will discuss this hormone treatment in combination with the use of cyclosporin. The aim of this experiment is to reveal the genome- wide expression levels of enzymes in the hepatocytes, including the CYP enzymes and P- gp. The intra- and extracellular concentrations of cyclosporin and its major metabolites will also be measured. We expect to find sex- specific differences in the expression of CYP enzymes and P- gp, but it is likely that these differences are not the only differences that can be found at the molecular level between human hepatocytes from males and females. Therefore, a genome- wide detection of differences in mrna and protein expression between the sexes is needed. Furthermore, a back- up plan will be used in which specifically the expression of CYP3A4 and P- gp is measured, because these proteins are the most likely ones to have a sex- specific influence on drug metabolism UNTREATED HUMAN HEPATOCYTES FROM MALES AND FEMALES Human hepatocytes from males and females will be cultured in a medium that is optimal for hepatocytes. Because this experiment will deal with untreated cells, neither the sex hormones estradiol, testosterone and growth hormone nor cyclosporin will be added to these hepatocytes. If the cells lose their sex specificity in the absence of hormones, this experiment will thus monitor the time it will take to reach mrna and protein expression levels in human hepatocytes from males that are comparable with human hepatocytes from females GENOME- WIDE STUDY TO DETECT SEX- SPECIFIC MRNA AND PROTEIN EXPRESSION PATTERNS The quantitative profiling of genome- wide mrna expression, also called transcriptomics, will be performed by RNA- sequencing. Quantitative protein expression profiling will be performed to determine the expression of proteins in human hepatocytes from males and females on a large- scale. Quantitative mass spectrometry can be used to determine the quantities in which the enzymes are produced. qrt- PCR and Western blot analysis will be used to validate the RNA- sequencing and proteomics results of the potential sex- specifically expressed mrnas and proteins, respectively. To interpret our RNA- sequencing and proteomics data, bioinformatics analysis will be used. In this study, known reference points like the sex- specific expression profile of CYP enzymes will be used as controls. This experiment will thus give us a top list of sex- specifically expressed genes. The mrna and protein levels of the hepatocytes will be measured in the absence of the substrate cyclosporin and important hormones. This genome- wide study will be performed in 15

17 collaboration with one of the leading experts in proteomics in The Netherlands: Prof. Dr. Albert Heck and his colleagues from the Department of Biomolecular Mass Spectrometry and Proteomics, University Utrecht QRT- PCR AND MASS SPECTROMETRY OF CYTOCHROME P450 AND P- GLYCOPROTEINS As described in section , an unbiased genome- wide study will be performed, such that it can be ensured that all the relevant factors are taken into account. However, it is already known that the CYP450 system is an important sex- specific factor in drug metabolism. Furthermore, we expect a sex- specific difference in the expression of P- gp. Therefore, as a back- up plan in case the genome- wide analysis will not give additional differentially expressed mrnas/proteins, also some specific experiments will be performed. The mrna expression levels of CYP3A4 and P- gp will be measured by qrt- PCR. The protein expression levels will be determined using quantitative mass spectrometry. This experiment will also reveal the time it takes for the hepatocytes to reach expression levels, which are the same for males and females HORMONE- TREATED HUMAN HEPATOCYTES FROM MALES AND FEMALES In this experiment, a number of concentrations of various hormones, which are expected to have a sex- specific influence, are added to the cells. The growth hormone, estradiol and testosterone are expected to be the most important sex- specific signaling molecules. The hormone concentrations that are added will be different for men and women, because the hormone patterns of men and women are different. The male cells will get the male hormone pattern and the female cells will get the female hormone pattern. Furthermore, an experiment will be done in which human hepatocytes derived from men will get the female hormone pattern and vice versa. This will show the importance of genetic differences compared to hormonal differences. No cyclosporin will be added to the cells in these measurements. Therefore two variables are taken into account in these experiments: sex and hormones. To determine if the hormones have a significant effect by down- or upregulating the genes that are involved in cyclosporin metabolism, the mrna and protein expression levels after adding growth hormone, estradiol and testosterone will be measured separately. After that, combinations of the different hormones will be added, so that can be determined what the most important regulatory factors are. First, a genome- wide approach will be applied in which all the down- and upregulated genes will be detected. After that it will be clear which proteins are produced by the cells in the presence of hormones that are sex specific. Furthermore, an experiment will be done in which the expression levels of cytochrome P450 and the P- gp are measured in the presence of hormones GENOME- WIDE STUDY TO DETECT SEX- SPECIFIC MRNA AND PROTEIN EXPRESSION PATTERNS To determine which mrnas and proteins are expressed in the absence (see section ) and presence of hormones, RNA- sequencing and proteomics will be used, respectively (Nikolov, Schmidt, and Urlaub 2012; Anon 2014). To validate the top list results, qrt- PCR and Western blot analysis will be used. 16

18 QRT- PCR AND MASS SPECTROMETRY OF CYTOCHROME P450 AND P- GLYCOPROTEINS Again, as a back- up plan, the mrna and protein expression of CYP3A4 and P- gp will be quantitatively measured before and after hormones are added. qrt- PCR will be used to measure the mrna expression levels. The expression level of the proteins will be determined by quantitative mass spectrometry (see section ) EFFECT OF CYCLOSPORIN ON HORMONE- TREATED HUMAN HEPATOCYTES FROM MALES AND FEMALES The goal of these experiments is to determine which factors cause the sex- specific difference in the clearance of cyclosporin. Important factors of the drug metabolism will already be detected in the sections and 6.1.2, but adding cyclosporin could induce the production of additional proteins. Therefore, sex- specific measurements will be done in which cyclosporin is added. In these measurements also the hormones will be added, to keep the environment of the cells as close as possible to the natural environment. There are three variables used in these experiments: sex, hormones and cyclosporin. In this approach again genome- wide and specific experiments will be performed GENOME- WIDE STUDY TO DETECT SEX- SPECIFIC MRNA AND PROTEIN EXPRESSION PATTERNS After the mrna and protein expression profiles are measured as described in section 6.1.2, cyclosporin will be added. Then the expression levels will again be measured. This will make clear if the expression of the proteins is induced by the cyclosporin QRT- PCR AND MASS SPECTROMETRY OF CYTOCHROME P450 AND P- GLYCOPROTEINS The mrna and protein expression levels of the CYP450 and P- gp are known (section ). These measurements will be done again, after the addition of cyclosporin. It can again be determined if the expression of these protein is induced by the cyclosporin INTRA- AND EXTRACELLULAR CONCENTRATION OF CYCLOSPORIN AND METABOLITES Furthermore, the intra- and extracellular concentrations of cyclosporin and its major metabolites (AM1, AM9 and AM4N) will be measured. These values are needed to determine the parameters that can be used in our computational model. To measure the difference between the intra- and extracellular concentrations, we will separate the cells from the extracellular medium by centrifugation. By using liquid chromatography and mass spectrometry (LC- MS) the intra- and extracellular concentrations of cyclosporin and its major metabolites can be measured (Falck et al. 2007). This procedure can be seen in figure 3. 17

19 Figure 3: Determination of the intra- and extracellular concentrations of cyclosporin and its metabolites. To determine if one or both of the metabolizing enzymes CYP3A4 and P- gp are saturated at a certain cyclosporin concentration, experiments with different doses of cyclosporin will be done. By using the methods described above we will determine which protein will be saturated earlier, indicating which of the phases gives the limiting step SHRNA- DRIVEN DOWNREGULATION OF TOP LIST GENES IN HORMONE- TREATED HUMAN HEPATOCYTES FROM MALES AND FEMALES Virus- mediated transfer will be used to introduce shrna into a cell to silence the expression of a certain gene via RNA interference. In our experiments this methodology will be used for the downregulation of the top list genes. The antisense shrna will be transferred into human hepatocytes from males and females. Then, the cells will be treated with cyclosporin and differences in clearance due to the downregulation will be detected. The goal is to determine whether male and female cells show any differences in clearance efficiency at a certain level of downregulation. As a back- up plan, if the genome- wide approach does not identify other sex- specifically expressed genes that differ in expression between human hepatocytes from males and females, this methodology could also be used to manipulate the levels of CYP3A4 and P- gp to study the effects of their downregulation. 6.2 IN VIVO ANALYSIS OF MALE AND FEMALE HUMANIZED MICE After investigating the sex- specific differences in in vitro cell lines, we also have to measure these differences in in vivo conditions. Therefore, we will use a humanized mouse model. Because of the implantation of tissue- engineered human ectopic artificial livers (HEALs), this mouse model exhibits humanized liver functions persistent for weeks (A.A. Chen et al. 2011). They display human drug metabolism and the synthesis of human proteins, which makes them just perfect to predict the sex- specific differences in human drug metabolism. By using these humanized mouse models with male and female HEALs in male and female mice, respectively, the sex- specific differences in the mrna and protein expression of CYP3A4 and P- gp and in the clearance of cyclosporin can be determined for humans ENGINEERING AND IMPLANTATION OF HEALS HEALs will be generated as described in Chen et al. (2011). Hereby the function of cryopreserved primary human hepatocytes will be stabilized in polymeric scaffolds through 18

20 juxtacrine and paracrine signals (Chen et al. 2011). To establish the sex- specific differences, human hepatocytes from males and females will be used to create male and female HEALs. Before these HEALs are implanted, the mrna and protein expression levels of CYP3A4 and P- gp will be quantitatively measured for the first time. This will also be done by using qrt- PCR and mass spectrometry. In a following step the HEALs have to be implanted in the subcutaneous or peritoneal cavity of mice. They can be efficiently established in immunocompetent mice with normal liver function. A group of male mice will become implanted with a male HEAL and the same number of female mice will become implanted with a female HEAL (Figure 4). Figure 4: Schematic depicting the fabrication, implantation, and utility of HEALs for humanizing mice (A.A. Chen et al. 2011) DRUG METABOLISM AND GENE EXPRESSION IN MALE AND FEMALE HUMANIZED MICE In this experiment a genome- wide and a specific approach, in which CYP450 and P- gp will be studied, will also be performed. The methods used will be the same as described in and After a time of conditioning of the HEALs in the body of the mice models, a liver biopsy will be done to measure the mrna expression of P- gp and CYP3A4 by qrt- PCR for the next time. This measurement will show if there occur differences in expression due to implantation in the mice and the resulting change of the environment from in vitro cells. Afterwards the mice will be daily injected intravenously (i.v.) with cyclosporin and the clearance will be measured over time by taking blood samples. Using LC- MS, the plasma concentration of cyclosporin and its metabolites can be determined (Falck et al. 2007). Also the hormone cycle of the mice will be taken into account by measuring the blood concentrations of the growth hormone and estradiol. After investigating the differences in clearance, a second liver biopsy will be done to measure the mrna expression of CYP3A4 and P- gp by qrt- PCR for the last time. This measurement is needed to determine whether differences in expression occur due to the administration of cyclosporin SHRNA- DRIVEN DOWNREGULATION OF CYP3A4 IN HEALS OF MALE AND FEMALE HUMANIZED MICE As described under , shrnas can be used to downregulate CYP3A4 and other sex- specifically expressed genes. This will also be done in vivo using the male and female humanized mouse models with male and female HEALs. Again the mice will be daily injected i.v. with cyclosporin and any differences in clearance due to the downregulation will be detected with the same methods as described under

21 6.3 IN VIVO CLEARANCE OF CYCLOSPORIN IN MALE AND FEMALE HUMANS The extrapolation of the data to humans, which we will get from the in vitro experiments and the mouse models, is an important aspect of this research project. To evaluate the empirical data, we will test to what extent the HEAL mouse model is representative for humans. Therefore, the sex- specific clearance of cyclosporin in human subjects will be compared to the clearance of cyclosporin in the HEAL mouse model. A single dose of cyclosporin will be administered to male and female HEAL mice, and to healthy male and female subjects. Male and female subjects will get the same dose. At several time intervals following the administration of cyclosporin, intravenous blood samples will be collected and stored for analysis. The plasma concentrations of cyclosporin and its metabolites will be determined using LC- MS (Falck et al. 2007). The averaged concentrations from male and female subjects will be plotted against time. These results can be used to determine if the data from the mouse models can be extrapolated to humans. 6.4 COMPUTATIONAL MODEL TO OPTIMIZE DRUG ADMINISTRATION IN MALE AND FEMALE HUMANS In this part of the research, a computational model will be generated and used to predict optimization of the drug administration process. If the differences in drug metabolism between men and women are known, calculations can be made to gain specific values for the rate constants. These can be used to predict the most optimal drug doses for men and women. Our model consists of three compartments: the blood, the hepatocytes and the bile. This is illustrated in Figure 5. Figure 5: Model of drug metabolism in the liver. K1, k2 and k3 represent the different rate values for the various steps. K1 is a constant rate value, and k2 and k3 are Michaelis- Menten rate values. The process of drug metabolism by cytochrome P450 takes place in the liver and drug efflux transporters take care of the excretion to the bile. If we know the difference in concentration between the blood and the bile and the intra- and extracellular concentrations (see section ), we can understand what happens in the liver. Therefore we want to measure these values. With the results we can determine the parameters. The determined values can be filled in into the equations that can be found in figure 6. With these equations the optimal dose can be predicted for men as well as for women. 20