Effect of glucuronides on metabolic enzymes and active hepatic uptake: in vitro assessment and prediction of drug-drug interaction risk A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Medical and Human Sciences 2015 Rebecca Alice Sullivan Manchester Pharmacy School 1 Contents CONTENTS ................................................................................................... 2 LIST OF FIGURES ........................................................................................ 6 LIST OF TABLES ........................................................................................ 12 ABSTRACT ................................................................................................. 16 DECLARATION ........................................................................................... 17 COPYRIGHT STATEMENT ......................................................................... 17 LIST OF ABBREVIATIONS......................................................................... 18 ACKNOWLEDGEMENTS ........................................................................... 20 THE AUTHOR ............................................................................................. 21 CHAPTER 1 INTRODUCTION .................................................................... 22 1.1 Overview and relevance of drug-drug interactions and glucuronide metabolites . 22 1.2 Metabolising enzymes and drug-drug interactions .................................................... 22 1.2.1 Cytochrome P450 enzymes ...................................................................................... 23 1.2.2 Uridine di-phospho-glucuronosyltransferase enzymes ............................................. 24 1.2.3 Classes of drug-drug interactions - enzyme induction and inhibition ....................... 25 1. 3 Transporter membrane proteins – importance to drug-drug interactions .............. 27 1.3.1 The OATP1B1 transporter ........................................................................................ 28 1.3.2 The role of OATP1B1 in drug-drug interactions ........................................................ 29 1.3.3 In vitro investigation of inhibition of OATP1B1 .......................................................... 30 1.4 Investigation of drug-drug interactions in vitro .......................................................... 33 1.4.1 Investigating enzyme inhibition in vitro ..................................................................... 33 1.4.2 In vitro systems for investigation of metabolic drug-drug interactions ...................... 34 1.4.3 Investigation of inhibition of OATP1B1 in vitro .......................................................... 35 1.4.4 In vitro systems for investigation of OATP1B1 inhibition .......................................... 35 1.4.5 In vitro systems suitable for investigation of metabolising enzymes and OATP1B1 inhibition ............................................................................................................................. 36 1.4.6 Quantification of drug-drug interactions in vitro ........................................................ 36 1.4.7 Prediction of drug-drug interactions .......................................................................... 37 2 1.5 The contribution of metabolites to metabolic drug-drug interactions ..................... 40 1.6 The contribution of metabolites to transporter drug-drug interactions ................... 42 1.7 The contribution of glucuronide metabolites to drug-drug interactions ................. 42 1.7.1 Inhibition of metabolising enzymes by glucuronides ................................................. 43 1.7.2 Glucuronides and parent drugs inhibiting the OATP1B1 transporter ....................... 49 1.8 Project Aims ................................................................................................................... 52 1.9 Compounds selected for investigation ........................................................................ 53 1.9.1 Gemfibrozil acyl-β-D-glucuronide.............................................................................. 53 1.9.2 Clopidogrel acyl-β-D-glucuronide.............................................................................. 54 1.9.3 Raloxifene-4’-glucuronide ......................................................................................... 54 1.9.4 Ezetimibe phenoxy-β-D-glucuronide ......................................................................... 55 1.9.5 Mefenamic acid acyl-β-D-glucuronide ...................................................................... 55 1.9.6 Mycophenolic acid β-D-glucuronide .......................................................................... 56 1.9.7 Repaglinide acyl-β-D-glucuronide ............................................................................. 56 1.9.8 Diclofenac acyl-β-D-glucuronide ............................................................................... 57 1.9.9 Telmisartan acyl-β-D-glucuronide ............................................................................. 57 1.9.10 Raltegravir β-D-glucuronide .................................................................................... 58 CHAPTER 2 IN VITRO ASSESSMENT OF INHIBITION OF METABOLISING ENZYMES BY GLUCURONIDE METABOLITES ........... 59 2.1 Introduction .................................................................................................................... 59 2.2 Aims................................................................................................................................. 60 2.3 Methods .......................................................................................................................... 60 2.3.1 Selection of repaglinide as a probe substrate ........................................................... 60 2.3.2 Reagents ................................................................................................................... 62 2.3.3 Assessment of CYP2C8 and CYP3A4 inhibition by repaglinide glucuronide ........... 62 2.3.4 Assessment of CYP2C8, CYP3A4 and UGT1A1 inhibition by reference inhibitors . 63 2.3.5 Assessment of CYP2C8, CYP3A4 and UGT1A1 inhibition by glucuronides ............ 64 2.3.6 Analysis of inhibitor concentrations and glucuronide stability ................................... 65 2.3.7 LC/MS-MS Analysis of samples ................................................................................ 65 2.3.8 Data analysis ............................................................................................................. 66 2.3.9 Correction of IC50 data for nonspecific binding ......................................................... 66 2.4 Results ............................................................................................................................ 68 2.4.1 Inhibition of CYP2C8 and CYP3A4 by repaglinide glucuronide ............................... 68 2.4.2 Inhibition studies in human liver microsomes – reference inhibitors ........................ 69 2.4.3 CYP2C8, CYP3A4 and UGT1A1 inhibition by glucuronide metabolites ................... 71 2.4.4 CYP2C8 and CYP3A4 inhibition studies in human liver microsomes – P450 co- factors ................................................................................................................................. 76 2.4.5 Comparison of the enzyme inhibitory potency between glucuronides and parent drugs .................................................................................................................................. 80 2.4.6 Impact of pre-incubation on P450 and UGT inhibition .............................................. 82 2.4.7 Impact of co-factor selection on enzyme inhibitory potential .................................... 83 2.4.8 Monitoring of inhibitor concentrations ....................................................................... 86 3 2.5 Discussion ...................................................................................................................... 89 2.5.1 Inhibitory effects of reference inhibitors on CYP2C8, CYP3A4 and UGT1A1 .......... 89 2.5.2 Inhibition of CYP2C8, CYP3A4 and UGT1A1 by glucuronides – combined co-factor conditions ........................................................................................................................... 90 2.5.3 IC50 experiments using P450 only co-factors – impact of co-factor conditions on the assessment of the inhibitory potential of glucuronides ...................................................... 92 2.5.4 Comparison of CYP2C8 and CYP3A4 inhibitory potential between glucuronides and parent drugs ....................................................................................................................... 93 2.6.5 Comparison of glucuronide and parent compound P450 inhibitory potency ............ 94 2.5.6 Investigation of time-dependent enzyme inhibition ................................................... 95 2.5.7 Conclusion ................................................................................................................ 96 CHAPTER 3 IN VITRO INVESTIGATION OF OATP1B1 INHIBITION BY GLUCURONIDES ........................................................................................ 97 3.1 Introduction .................................................................................................................... 97 3.2 Aims................................................................................................................................