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PHARMACOKINETICS, LIMITED SAMPLING STRATEGIES, AND PHARMACOGENETICS OF MYCOPHENOLIC ACID IN THORACIC TRANSPLANT RECIPIENTS by LILLIAN S.L. TING 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) March, 2009. © Lillian S.L. Ting, 2009 ABSTRACT Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil, is an immunosuppressive agent known to exhibit wide inter-patient pharmacokinetic variability. The metabolism and transport of MPA and the phenolic (MPAG) and acyl (AcMPAG) glucuronides are mediated by UDP-glucuronosyltransferases (UGTs) and multidrug resistance-associated protein 2 (MRP2/ABCC2), respectively. Increasing evidence supports monitoring MPA area-under-the-concentration-time-curve; however, it is impractical and costly to implement. The objectives of this clinical study were to characterize MPA pharmacokinetics, develop MPA limited sampling strategies for estimating MPA exposure, and assess contribution of UGT and ABCC2 genetics to MPA pharmacokinetics and clinical outcomes in thoracic transplant recipients. Seventy thoracic (36 lung, 34 heart) transplant recipients were recruited. Eleven blood samples were obtained over a 12-hour dosing period at steady state. Plasma concentrations of MPA, MPAG, AcMPAG, and free MPA were measured by a high performance liquid chromatography-ultraviolet detection method, and conventional dose- normalized pharmacokinetic parameters were determined via non-compartmental methods. Limited sampling strategies were developed in 64 subjects by stepwise multiple regression analysis using the index group data, and tested in the validation group to determine bias and precision. Genetic polymorphisms in UGT and ABCC2 were determined by sequencing and their contributions to pharmacokinetic variability were investigated in 68 thoracic transplant recipients using multivariate analysis. Significantly lower MPA pharmacokinetic exposure was observed in lung transplant recipients (compared with heart), and in patients taking cyclosporine ii (compared with tacrolimus). There was wide inter-patient variability of MPA, MPAG and AcMPAG pharmacokinetics. The best limited sampling strategies for both transplant populations were developed from the lung transplant group data; equations (Log C1.5, C2) and (Log C0, Log C1.5) were the best candidates for the heart and lung transplant population, respectively. For both transplant groups, UGT2B7 variants 802T (*2a) and - 138A (*2g) were associated with increased AcMPAG AUC (~3-fold) and AcMPAG/MPA (~10-fold) in multivariate analyses, respectively, and high AcMPAG exposure and metabolic ratio were associated with poor clinical outcomes. UGT2B7 is a promising gene candidate that may influence MPA pharmacokinetics clinically; however, larger clinical pharmacogenetic studies in thoracic transplant subpopulations are warranted to corroborate the role of AcMPAG and UGT2B7 variants in optimizing mycophenolate therapy. iii TABLE OF CONTENTS Abstract............................................................................................................................... ii Table of Contents............................................................................................................... iv List of Tables .................................................................................................................... vii List of Figures.................................................................................................................... ix List of Abbreviations .......................................................................................................... x Acknowledgements..........................................................................................................xiii Co-authorship Statement.................................................................................................. xiv 1. Introduction................................................................................................................. 1 1.1. Heart and Lung Transplantation ......................................................................... 1 1.2. Maintenance Immunosuppressive Agents .......................................................... 3 1.2.1. Cyclosporine ............................................................................................... 4 1.2.2. Tacrolimus .................................................................................................. 5 1.2.3. Sirolimus..................................................................................................... 6 1.2.4. Corticosteroids ............................................................................................ 8 1.3. Mycophenolate Mofetil....................................................................................... 9 1.3.1. Mycophenolic acid pharmacokinetics and variability .............................. 10 1.3.2. Drug interactions with other immunosuppressive agents ......................... 12 1.3.3. Mycophenolic acid therapeutic drug monitoring...................................... 13 1.3.4. Limited sampling strategies of mycophenolic acid .................................. 17 1.4. UDP-glucuronosyltransferases ......................................................................... 20 1.4.1. Role of UGTs in mycophenolic acid metabolism..................................... 22 1.4.2. UDP-glucuronosyltransferase genetic polymorphisms............................. 23 1.5. Multidrug Resistance-Associated Protein 2...................................................... 25 1.5.1. Role of ABCC2 in mycophenolic acid disposition................................... 26 1.5.2. Multidrug resistance-associated protein 2 genetic polymorphisms.......... 27 1.6. Pharmacogenetics of Mycophenolic Acid ........................................................ 29 1.6.1. Clinical impact of UDP-glucuronosyltransferase genetic polymorphisms on MPA metabolism ................................................................................. 30 1.6.2. Clinical impact of multidrug resistance-associated protein 2 genetic polymorphisms in MPA disposition ......................................................... 36 1.6.3. Prospectus ................................................................................................. 38 1.7. Hypothesis......................................................................................................... 39 1.8. Objectives ......................................................................................................... 40 1.9. Tables................................................................................................................ 41 1.10. Figures............................................................................................................... 56 1.11. References......................................................................................................... 58 iv 2. Pharmacokinetics of Mycophenolic Acid and its Phenolic-Glucuronide and Acyl- Glucuronide Metabolites in Stable Thoracic Transplant Recipients ................................ 81 2.1. Introduction....................................................................................................... 82 2.2. Materials and Methods...................................................................................... 84 2.2.1. Subjects..................................................................................................... 84 2.2.2. Study protocol........................................................................................... 84 2.2.3. Chemicals.................................................................................................. 86 2.2.4. Plasma concentrations of MPA, MPAG and AcMPAG ........................... 86 2.2.5. Free MPA analysis.................................................................................... 88 2.2.6. Urine concentrations of MPA, MPAG and AcMPAG.............................. 89 2.2.7. Pharmacokinetic parameters assessment .................................................. 92 2.2.8. Statistical analyses.................................................................................... 93 2.3. Results............................................................................................................... 93 2.3.1. Patient characteristics................................................................................ 93 2.3.2. MPA pharmacokinetics............................................................................. 94 2.3.3. Urine data.................................................................................................. 96 2.4. Discussion......................................................................................................... 96 2.5. Conclusions..................................................................................................... 104 2.6. Tables.............................................................................................................. 106 2.7. Figures............................................................................................................. 122 2.8. References....................................................................................................... 131 3. Limited Sampling Strategy for Predicting Mycophenolic Acid Area-Under-the-