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Hepatic Microsomal Bile Acid Biotransformation Studies Carried out So Far Seem to Be Restricted by Concentrating Only on CYP3A-Mediated Bile Acid Biotransformation

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Hepatic Microsomal Bile Acid Biotransformation Studies Carried out So Far Seem to Be Restricted by Concentrating Only on CYP3A-Mediated Bile Acid Biotransformation HEPATIC MICROSOMAL BILE ACID BIOTRANSFORMATION: IDENTIFICATION OF METABOLITES AND CYTOCHROME P450 ENZYMES INVOLVED by Anand K. Deo B. Pharm., University of Pune, India, 2000 M. Pharm. Sci., University of Mumbai, 2002 M.Sc., The University of British Columbia, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (PHARMACEUTICAL SCIENCES) THE UNIVERSITY OF BRITISH COLUMBIA Vancouver February 2009 © ANAND K. DEO, 2009 ABSTRACT Bile acids are end-products of cholesterol metabolism and essential for absorption of dietary lipids in the body. Impaired bile flow leads to hepatic bile acid accumulation and liver damage. Hepatic microsomal oxidation offers a potential mechanism for efficient elimination of bile acids. The present study investigated the cytochrome P450 (P450)-mediated hepatic microsomal biotransformation profiles of lithocholic acid, cholic acid and chenodeoxycholic acid using a liquid chromatography-mass spectrometry (LCIMS) based assay. Incubation of lithocholic acid with rat hepatic microsomes resulted in the formation of a major 6J3-hydroxylated metabolite, murideoxycholic acid, followed by isolithocholic acid and 3- ketocholanoic acid. Ursodeoxycholic acid, hyodeoxycholic acid and 6-ketolithocholic acid were identified as minor metabolites. Studies using P450-specific antibodies, chemical inducers, and rat recombinant enzymes showed that formation of murideoxycholic acid and 3-ketocholanoic acid were mediated by CYP3A2 and CYP2C 11. Similar metabolite profiles were obtained by incubation of lithocholic acid with mouse hepatic microsomes generating murideoxycholic acid as the major metabolite. Studies using P450 inducers and chemical inhibitors suggested the involvement of murine CYP3A in murideoxycholic acid and 3-ketocholanoic acid formation, and CYP1A, CYP2B and CYP3A enzymes in ursodeoxycholic acid, hyodeoxycholic acid and 6-ketolithocholic acid formation by mouse liver microsomes. Biotransformation of lithocholic acid by human hepatic microsomes generated 3-ketocholanoic acid as the major metabolite, and hyodeoxycholic acid, ursodeoxycholic acid, 6-ketolithocholic acid and murideoxycholic acid, as minor metabolites. Studies with chemical inhibitors and human recombinant enzymes demonstrated that CYP3A4 catalyzed the formation of all five metabolites. The biotransformation of cholic acid and chenodeoxycholic acid by human hepatic microsomes revealed the formation of a single cholic acid metabolite, 3-dehydrocholic acid. 11 Chenodeoxycholic acid biotransformation generated 7ct-hydroxy-3 -oxo-5 3-cholan-24-oic acid as the major metabolite followed by ‘y-muricholic acid, 7-ketolithocholic acid and cholic acid, respectively. CYP3A4 was found to be the major enzyme involved in the biotransformation of cholie acid and chenodeoxycholic acid in human liver microsomes. A comparison of metabolite profiles demonstrated the dominant role of human CYP3A4 in the oxidation of bile acids at the C-3 position. In contrast, 63-hydroxy1ation catalyzed by multiple P450 (CYP1A, CYP2B, CYP2C and CYP3A) enzymes was the preferred biotransformation pathway in rodent liver microsomes. 111 TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xiii ACKNOWLEDGEMENTS xiv DEDICATION xv CO-AUTHORSHIP STATEMENT xvi 1. 1 1.1 Bile Acids: Structure And Physicochemical Properties 2 1.2 Bile Acid Biosynthesis: Steps Involved 3 1.2.1 Biosynthesis of primary bile acids 4 1.2.2 Biosynthesis of secondary bile acids 8 1.3 Biliary Bile Acids: Composition And Physiological Relevance 9 1.4 Bile Acids: Enterohepatic Circulation 10 1.5 Bile Acid Toxicity 12 1.6 Cholestasis And Bile Acid Toxicity 13 1.7 Nuclear Receptors In Bile Acid Regulation 18 1.8 Bile Acid Biotransformation By Conjugation 22 1.9 Bile Acid Biotransformation By P450 Enzymes 24 1.10 Rationale 28 1.11 Hypotheses 31 1.1 1.1 Specific research objectives 32 1.12 References 50 iv 2. BIOTRANSFORMATION OF LITHOCHOLIC ACID BY RAT HEPATIC MICROSOMES: METABOLITE ANALYSIS BY LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY 67 2.1 Summary 68 2.2 Introduction 69 2.3 Materials And Methods 71 2.3.1 Chemicals and reagents 71 2.3.2 Animal treatment and preparation of hepatic microsomes 72 2.3.3 Lithocholic acid biotransformation assay 72 2.3.4 Antibody inhibition 74 2.3.5 Analytical methods 74 2.3.6 Data analysis and calculation of enzyme kinetic parameters 75 2.3.7 Statistical analysis 76 2.4 Results 76 2.4.1 Biotransformation of lithocholic acid and metabolite identification 76 2.4.2 Kinetic analysis of hepatic microsomal metabolite formation 78 2.4.3 Effect of P450 inducers on lithocholic acid biotransformation 80 2.4.4 Antibody inhibition studies 80 2.4.5 Biotransformation studies with recombinant P450 enzymes 81 2.5 Discussion 82 2.6 References 99 3. IDENTIFICATION OF HUMAN HEPATIC CYTOCHROME P450 ENZYMES INVOLVED IN THE BIOTRANSFORMATION OF CHOLIC AND CHENODEOXYCHOLIC ACID 104 3.1 Summary 105 3.2 Introduction 106 3.3 Materials And Methods 108 3.3.1 Chemicals and reagents 108 V 3.3.2 Cholic acid and chenodeoxycholic acid biotransformation assays 109 3.3.3 Analytical methods 110 3.3.4 Data analysis and calculation of enzyme kinetic parameters 111 3.4 Results 112 3.4.1 Biotransformation and kinetic analysis of cholic acid metabolites 112 3.4.2 Biotransformation and kinetic analysis of chenodeoxycholic acid metabolites 113 3.4.3 Biotransformation studies with human recombinant P450 enzymes 114 3.5 Discussion 116 3.6 References 130 4. 3-KETOCHOLANOIC ACID IS TifE MAJOR CYP3A4 MEDIATED LITHOCHOLIC ACID METABOLITE IN HUMAN HEPATIC 133 4.1 Summary 134 4.2 Introduction 135 4.3 Materials And Methods 137 4.3.1 Chemicals and Reagents 137 4.3.2 Lithocholic acid biotransformation assay 138 4.3.3 Analytical methods 139 4.3.4 Data analysis and calculation of enzyme kinetic parameters 139 4.3.5 Chemical inhibition studies 140 4.3.6 Statistical analysis 141 4.4 Results 141 4.4.1 Lithocholic acid biotransformation by human liver microsomes 141 4.4.2 Lithocholic acid biotransformation by human recombinant P450 enzymes 142 4.4.3 Chemical inhibition studies 143 4.4.4 Lithocholic acid biotransformation in human hepatic microsomes with varying CYP3A4 levels 143 4.5 Discussion 144 4.6 References 158 vi 5. HEPATIC MICROSOMAL P450 MEDIATED BIOTRANSFORMATION OF LITHOCHOLIC ACID BY MOUSE HIPi4.TIC 1’II1SO1VIES 162 5.1 Summary 163 5.2 Introduction 164 5.3 Materials And Methods 165 5.3.1 Chemicals and reagents 165 5.3.2 Animal treatment and preparation of microsomes 166 5.3.3 Lithocholic acid biotransformation assay 166 5.3.4 Chemical inhibition studies 166 5.3.5 Statistical analysis 167 5.4 Results 167 5.4.1 Hepatic biotransformation of lithocholic acid by mouse hepatic microsomes 167 5.4.2 Effect of P450 inducers in lithocholic acid biotransformation 167 5.4.3 Chemical inhibition studies 168 5.5 Discussion 170 5.8 References 178 6. ENE1J4.IJ DISCIJSSIO1i 181 6.1 Overview 182 6.2 Cholic Acid Biotransformation By Human Hepatic Microsomes 183 6.3 Chenodeoxycholic Acid Biotransformation By Human Hepatic Microsomes 183 6.4 Comparison of Lithocholic Acid Metabolite Formation Patterns By Rat, Human And Mouse Liver Microsomes 184 6.5 Limitations 188 6.6 Overall Significance of Thesis Research 190 6.7 Future Studies 191 6.8 References 200 vii ITIA £OZJ )CIGtsJaJcI’r LIST OF TABLES Table 1.1 Oxidative metabolites of bile acids 33 Table 1.2 Different P450 enzymes and their levels in human and rat hepatic microsomes 34 Table 2.1 Kinetic parameters of lithocholic acid metabolite formation by rat hepatic microsomes 88 Table 2.2 Effect of sex and treatment with P450 inducers on lithocholie acid metabolite formation by rat hepatic microsomes 89 Table 4.1 Kinetic parameters of lithocholic acid metabolite formation by human hepatic microsomes 149 Table 4.2 Kinetic parameters of lithocholic acid metabolite formation by recombinant CYP3A4 150 Table 4.3 Rate of formation of lithocholic acid metabolites in human liver microsomes with varying CYP3A4 activities 151 Effect of treatment with P450 inducers on lithocholic acid metabolite Table 5 1 formation by mouse hepatic microsomes 174 Table 6.1 Regio-selective oxidation of lithocholic acid by human, rat and mouse hepatic microsomes 195 Table 6.2 Comparison of P450 enzymes involved in lithocholic acid metabolite formation in human and rodent hepatic microsomes 196 ix LIST OF FIGURES Figure 1.1 Cholesterol and bile acid structure 35 Figure 1.2 Chemical structures of unconjugated and conjugated bile acids 36 Figure 1.3 Bile acid levels in human, rat and mouse liver 37 Figure 1.4 Neutral and acidic pathways in bile acid formation 38 Figure 1.5 Bile acid levels in human, rat and mouse bile 39 Figure 1.6 Enterohepatic circulation of bile acids 40 Figure 1.7 Amphipathic nature of bile acids 41 Figure 1.8 Obstruction to the flow of bile acids from the liver to the intestine is termed as cholestasis 42 Figure 1.9 Comparison of bile acid levels in livers of patients with end-stage cholestasis and normal humans 43 Figure 1.10 Bile acid regulation: A complex process involving various receptors, enzymes, and transporters 44 Figure 1.11 Regulation of bile acids by nuclear receptors 45 Figure 1.12 PXR and CAR cross-talk mediated metabolism of lithocholic acid 46 Figure 1.13 Major hepatic P450 enzymes involved in drug metabolism 47 Figure 1.14 Biotransformation of bile acids 48 Figure 2.1 General bile acid structure showing positions available for hydroxylation 90 Figure 2.2 Representative
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