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In-Vitro and In-Vivo Models of Bile Acid Metabolism and Transport

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In-Vitro and In-Vivo Models of Bile Acid Metabolism and Transport University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-15-2017 In-Vitro and In-Vivo Models of Bile Acid Metabolism and Transport Rhishikesh Thakare University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Recommended Citation Thakare, Rhishikesh, "In-Vitro and In-Vivo Models of Bile Acid Metabolism and Transport" (2017). Theses & Dissertations. 233. https://digitalcommons.unmc.edu/etd/233 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. IN-VITRO AND IN-VIVO MODELS OF BILE ACID METABOLISM AND TRANSPORT By Rhishikesh Thakare A DISSERTATION Presented to the Faculty of The Graduate College in the University of Nebraska In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Department of Pharmaceutical Sciences Under the Supervision of Professor Yazen Alnouti University of Nebraska Medical Center Omaha, Nebraska September, 2017 i ACKNOWLEDGEMENTS I would like to express my sincere gratitude to many people who have been continuously supporting and encouraging me throughout my graduate studies. First and foremost, I am deeply indebted to my mentor Dr. Yazen Alnouti for providing me the opportunity to work in his lab. Dr. Alnouti has been very instrumental in my research career. He has been a great influence in my personal and professional life and has educated me on numerous qualities that are required for becoming a good scientist. I would specially thank him for allowing me to gain hands-on experience in an industrial setting through a summer internship at Pfizer Inc, Groton. I would like to thank my committee members Dr. Rakesh Singh, Dr. Jonathan Vennerstrom, Dr. Jered Garrison for their valuable inputs, suggestions, and guidance during my training here at UNMC. I am indebted to Dr. Nagsen Gautam for nurturing my research skills and for all the suggestions and inputs during my studies. I thank the UNMC Graduate studies office and Pharmaceutical Sciences department for providing me the financial support for my graduate studies. I will cherish all the wonderful moments that I had with my friends over the last five years. I would like to especially thank my friends Prathamesh, Shrey, Pravin, Aniruddha, Shrabasti, Aditya, Rohan, Sugandha, and Kruti for all their constant support and encouragement throughout my Ph.D. program. I also thank all my friends from Omaha Cricket Club and the rest of my friends in Omaha for the wonderful company during the last five years. I would like to thank our department secretaries Elaine, Jamie, Michelle, and Katina for all the administrative help during my studies here at UNMC. Finally, I would like to thank my family including my dad- Mr. Narayan Thakare, my mom- Mrs. Jyoti Thakare, my brother- Abhishek, and my sister Mrs. Suverna for their love, prayers, support and encouragement throughout the years. I would like to thank my wife- Mrs. Rashmi for her support and commitment towards my success in graduate ii school. Finally, I appreciate all the teachers and friends from my school who played a significant role all in my life. IN-VITRO AND IN-VIVO MODELS OF BILE ACID METABOLISM AND TRANSPORT Rhishikesh Thakare University of Nebraska Medical Center, 2017 Supervisor: Yazen Alnouti, Ph.D. All biomedical research is conducted in animal models first. In addition, the Food and Drug Administration requires extrapolation from animal data to predict human responses. There are ongoing scientific and regulatory challenges translating interspecies comparisons and predictions. Metabolic pathways are a cornerstone to understanding drug metabolism and toxicities and the liver is a key organ in this process. Bile acids (BAs) play a central role in the hepatobiliary toxicities of chemicals, toxins, and biological reagents. BAs have many physiological functions including regulation of genes involved in cholesterol and glucose metabolism and BA homeostasis. However, BAs also have several pathological effects including carcinogenicity and liver toxicity. Maintenance of bile acid (BA) homeostasis is essential to achieve their physiologic functions and avoid their toxic effects. Several metabolic pathways including sulfation by sulfotransferase (SULT), glucuronidation by UDP-glucuronosyltransferases (UGTs), and oxidation by Cytochrome-P450 (CYP450) enzymes participate in the direct detoxification, enhance the elimination of BAs, and help maintain their homeostasis. In addition, influx and efflux transporters at both the sinusoidal and basolateral membranes play an important role in determining intracellular BA concentration, and therefore their hepatotoxicity. There are known species differences in BA metabolism and transporter. There are known species differences in the composition of the BA pool, the toxicity of BAs, and drug-induced hepatotoxicity related to BAs. These species differences can prevent extrapolation of toxicity profiles of xenobiotics between species causing a serious disconnect between preclinical safety findings in rodent and canine animal models and safety finding at clinical stages. In this thesis, we compare the metabolic profile of representative BAs between several species including humans, chimpanzee, monkeys, minipigs, hamster, rabbits, dogs, rats, and mice. The metabolic profile was characterized by the identification of BA metabolites and by quantifying the kinetics of their formation in hepatocyte S9 in-vitro system. The relative contributions of individual metabolic pathways were determined. LC-MS/MS was used for the qualitative and quantitative analysis of BAs and their metabolites. A mixture of stable-isotope labeled 2 ( H4) and unlabeled BAs were used to facilitate the identification of all minor and major metabolites. Major species differences were found in the metabolism of BAs. Amidation with taurine and glycine was the major pathway in all species. Sulfation was predominant in humans, whereas oxidation and glucuronidation were predominant in rodents and dogs, respectively. Glucuronidation and amidation of BAs are exclusive, where glucuronidation only takes place for unamidated BAs. In vitro-In vivo extrapolation (IVIVE) is performed to establish the correlation of the in vitro results and the bile acid profile in vivo. These results explain, at least in part, the dissociation between preclinical toxicity data in various in vitro and in vivo models and toxicities observed in humans. Furthermore, more relevant species are suggested based on the similarity to the human BA metabolism. In addition, we also screened different bile acids as a potential biomarker for transporter activity using cynomolgus monkey as preclinical model. This resulted in identification of key bile acid sulfates as biomarker for transporter mediated drug-drug interactions. TABLE OF CONTENTS ACKNOWLEDGEMENTS i ABSTRACT iii TABLE OF CONTENTS V LIST OF FIGURES IX LIST OF TABLES XII LIST OF ABBREVIATIONS XIV CHAPTER 1: SPECIES DIFFERENCES IN BILE ACID METABOLISM 1 1.1 Introduction 1 1.2 Materials and Method 13 1.2.1 Chemicals and Reagents 13 1.2.2 Hepatocyte S9 fractions Incubation Condition 14 1.2.3 Identification of Metabolites Generated by Hepatocyte S9 fractions 15 1.2.4 Biosynthesis and NMR-based Quantification of UDCA Metabolites 16 1.2.5 BAs Quantification by LC-MS/MS 18 1.2.6 Calculation of BA Indices 19 1.3 Results 20 1.3.1 BA Profiles in Plasma 20 1.3.1 BA Profiles in Urine 21 1.3.3 Identification of Ursodeoxycholic acid (UDCA) Metabolites Generated by Hepatocyte S9 fractions 23 1.3.4 Biosynthesis and NMR-quantification of UDCA Metabolites 27 1.3.5 Species Differences of BA Metabolism by Hepatocyte S9 fractions 28 1.4 Discussion 31 vi 1.4.1 In-vivo BA Profile 31 1.4.2 Literature Summary of BA Profiles in Different Species 36 1.4.3 Biosynthesis of UDCA Metabolites and Quantification Using NMR 38 1.4.4 Optimization of Hepatocyte S9 Incubation Conditions 41 1.4.5 Species Differences in BA Metabolism by Hepatocyte S9 fractions 42 1.4.6 In-vivo and In-vitro Correlation 46 1.5 Conclusions 46 1.6 References 74 CHAPTER 2: BILE ACIDS AS POTENTIAL NOVEL BIOMARKERS FOR ORGANIC ANION-TRANSPORTING POLYPEPTIDE 91 2.1 Introduction 91 2.2 Materials and Methods 93 2.2.1 Chemicals and Reagents 93 2.2.2 Animal Handling, Dosing, Plasma Draws, and Urine Collection 94 2.2.3 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis 96 2.2.4 Determination of Uptake Clearance in the Presence of Cynomolgus Monkey Plated Primary Hepatocytes 98 2.2.5 Pharmacokinetics Analysis 99 2.2.6 Statistical Analysis of RIF Dose Response 100 2.3 Results 101 2.3.1 Pharmacokinetics of RIF in Cynomolgus Monkeys 101 2.3.2 Impact of RIF on 2H4-Pitavastatin Pharmacokinetics 101 2.3.3 Profiling of Plasma BAs at Different Doses of RIF 102 2.3.4 Assessment of RIF Dose Response 103 2.3.5 Impact of RIF on BA Renal Clearance 104 2.3.6 Incubation of GCDCA-S, GDCA-S, DCA-S, TDCA-S, TCA and Pitavastatin with Cynomolgus Monkey Plated Hepatocytes 105 vii 2.4 Discussion and Conclusion 106 2.5 References 138 CHAPTER 3: QUANTITATIVE ANALYSIS OF ENDOGENOUS COMPOUNDS 142 3.1 Introduction 142 3.2 Quantitative Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis 143 3.3 Approaches for Quantification of Endogenous Analytes 146 3.3.1 Method of Background Subtraction 146 3.3.2 Method of Standard Addition 148 3.3.3 Surrogate Matrices 150 3.3.4 Surrogate Analytes 152 3.4 Regulatory Guidelines 154 3.5 Conclusions 155 3.6 References 169 CHAPTER 4: SIMULTANEOUS LC-MS/MS ANALYSIS OF EICOSANOIDS AND RELATED METABOLITES IN HUMAN SERUM, SPUTUM, AND BALF 177 4.1 Introduction 177 4.2 Experimental 179 4.2.1 Chemicals and Reagents 179 4.2.2 Instrumentation 180 4.2.3 Liquid chromatographic and Mass spectrometric Conditions 180 4.2.4.
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