PHARMACOKINETICS AND PHARMACOGENETICS OF MYCOPHENOLIC ACID IN ASIAN RENAL TRANSPLANT PATIENTS IN SINGAPORE YAU WAI PING (B.Sc.(Pharm.)(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude to my supervisor, Assoc Prof Eli Chan, for his constant guidance and invaluable advice throughout the course of my doctoral studies. I am grateful for the many research opportunities that he has provided me. I would also like to specially thank my collaborators from the Singapore General Hospital, Assoc Prof Anantharaman Vathsala and Dr Lou Huei-Xin, for opening the door to this research project and making this thesis possible. I am grateful to the National University of Singapore (NUS) and the Department of Pharmacy for financially supporting me with the NUS Research Scholarship, NUS President’s Graduate Fellowship and Teaching Assistantship throughout the course of my doctoral studies. I gratefully acknowledge the financial support of this research project by the NUS Academic Research Funds (R-148-000-050-112 and R-148-000092-112). I would like to thank Roche Bioscience for kindly providing the two chemicals, MPAG and MPAC, for my research project. I would also like to extend my appreciation to all professors, laboratory officers, administrative staff, senior students and friends for their guidance, advice, assistance and encouragement throughout my studies. i ACKNOWLEDGEMENTS Last, but certainly not least, I would like to express my heartfelt gratitude to my parents and brother for their patience and encouragement throughout the duration of my doctoral studies. I thank them for their constant love and support. ii TABLE OF CONTENTS TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS iii SUMMARY ix LIST OF TABLES . xi LIST OF FIGURES . xviii LIST OF ABBREVIATIONS AND SYMBOLS .xxv CHAPTER 1: INTRODUCTION .1 1.1 Renal Transplantation (RTx) 2 1.1.1 Introduction 2 1.1.2 Historical perspective .2 1.1.3 Current immunosuppressive therapy .3 1.2 Mycophenolic Acid (MPA) 7 1.2.1 Introduction 7 1.2.2 Indications 7 1.2.3 Chemistry .8 1.2.4 Pharmacodynamics (PD) .9 1.2.4.1 Mechanisms of action .9 1.2.4.2 Clinical efficacy and safety of MPA in RTx 11 1.2.5 Pharmacokinetics (PK) 13 1.2.5.1 Absorption .13 1.2.5.2 Distribution .13 1.2.5.3 Metabolism .14 1.2.5.4 Excretion .15 1.2.5.5 Pharmacokinetic drug interactions 16 1.3 Therapeutic Drug Monitoring (TDM) of MPA 19 1.4 Uridine Diphosphate Glucuronosyltransferases (UGTs) 27 1.4.1 Introduction 27 1.4.2 UGT isoforms involved in metabolism of MPA to MPAG .27 1.4.3 Genetic polymorphisms of UGT1A9, 1A7, 1A8 and 1A10 and their influence on the glucuronidation and disposition of MPA 29 CHAPTER 2: HYPOTHESES AND OBJECTIVES .34 2.1 Hypotheses 35 2.2 Research Objectives 36 CHAPTER 3: ANALYTICAL METHODS AND IN VITRO STUDY .38 3.1 Reversed-Phase Ion-Pair Liquid Chromatography Assay for the Simultaneous Determination of Total MPA and Total MPAG in Human Plasma and Urine .39 iii TABLE OF CONTENTS 3.1.1 Introduction 39 3.1.2 Experimental 40 3.1.2.1 Chemicals and reagents .40 3.1.2.2 Instrumentation .40 3.1.2.3 Chromatographic conditions .41 3.1.2.4 Stock and working standard solutions 41 3.1.2.5 Sample preparation .42 3.1.2.5.1 Calibration standards of plasma samples 42 3.1.2.5.2 Calibration standards of urine samples .42 3.1.2.6 Specificity .42 3.1.2.7 Clinical samples for pharmacokinetic application 43 3.1.3 Results and Discussion 44 3.1.3.1 Chromatographic separation .44 3.1.3.1.1 Selection of the detection wavelength 45 3.1.3.1.2 Sample preparation method 46 3.1.3.1.3 Effect of pH of running buffer 47 3.1.3.1.4 Effect of acetonitrile composition of mobile phase .50 3.1.3.2 Optimal conditions and assay validation 51 3.1.3.2.1 Specificity and selectivity .53 3.1.3.2.2 Linearity 54 3.1.3.2.3 Limits of detection and quantitation .55 3.1.3.2.4 Precision and accuracy 56 3.1.3.2.5 Stability .59 3.1.3.3 Clinical application .59 3.1.4 Conclusion .60 3.2 Simple Reversed-Phase Liquid Chromatographic Assay for Simultaneous Quantification of Free MPA and Free MPAG in Human Plasma 61 3.2.1 Introduction 61 3.2.2 Experimental 62 3.2.2.1 Chemicals and reagents .62 3.2.2.2 Ultrafiltration conditions .63 3.2.2.3 Preparation of calibration standards 63 3.2.2.4 Instrumentation and chromatographic conditions .63 3.2.2.5 Specificity .64 3.2.2.6 Clinical samples for pharmacokinetic application 64 3.2.3 Results and Discussion 65 3.2.3.1 Method development 65 3.2.3.1.1 Selection of the analytical column 65 3.2.3.1.2 Sample preparation by ultrafiltration 65 3.2.3.2 Optimal conditions and assay validation 66 3.2.3.2.1 Specificity .68 3.2.3.2.2 Linearity 68 3.2.3.2.3 Limits of detection and quantitation .69 3.2.3.2.4 Precision and accuracy 70 3.2.3.2.5 Stability .70 3.2.3.3 Clinical application .71 3.2.4 Conclusion .72 iv TABLE OF CONTENTS 3.3 In Vitro Human Plasma Protein Binding Study of MPA and MPAG .73 3.3.1 Introduction 73 3.3.2 Materials and Methods .74 3.3.2.1 Chemicals and reagents .74 3.3.2.2 Sample preparation and ultrafiltration procedure .74 3.3.2.3 Sample analysis .75 3.3.2.4 Data analysis .75 3.3.3 Results 76 3.3.3.1 Human plasma protein binding of MPA .76 3.3.3.2 Human plasma protein binding of MPAG 76 3.3.3.3 Effect of MPAG on human plasma protein binding of MPA77 3.3.3.4 Effect of MPA on human plasma protein binding of MPAG77 3.3.3.5 Correlation of MPA with MPAG free fractions .78 3.3.4 Discussion 78 3.3.5 Conclusion .80 CHAPTER 4: CLINICAL PHARMACOKINETICS STUDY 82 4.1 First Dose and Multiple Dose Pharmacokinetics in Asian Renal Transplant Patients Newly Started on MMF .83 4.1.1 Introduction 83 4.1.2 Methods 84 4.1.2.1 Study design 84 4.1.2.2 Patients 84 4.1.2.3 Demographic and biochemical data collection .85 4.1.2.4 Blood sampling .85 4.1.2.5 Urine collection .85 4.1.2.6 Sample analysis .86 4.1.2.7 Pharmacokinetic analysis 86 4.1.2.7.1 Compartmental pharmacokinetic analysis 86 4.1.2.7.1.1 Pharmacokinetic model 86 4.1.2.7.1.2 Computer fitting of model . 90 4.1.2.7.1.3 Computer simulation of model 92 4.1.2.7.2 Non-compartmental pharmacokinetic analysis .93 4.1.2.8 Statistical analysis .94 4.1.3 Results and Discussion 96 4.1.3.1 First dose study by pharmacokinetic modeling 96 4.1.3.1.1 Results .96 4.1.3.1.1.1 Patient demographics . 96 4.1.3.1.1.2 Model fitting 99 4.1.3.1.1.3 Model simulation . 108 4.1.3.1.2 Discussion .111 4.1.3.2 Multiple dose study by non-compartmental pharmacokinetic analysis 118 4.1.3.2.1 Results .118 4.1.3.2.1.1 Patient demographics . 118 4.1.3.2.1.2 Pharmacokinetic results . 123 4.1.3.2.2 Discussion .129 4.1.4 Conclusion .133 v TABLE OF CONTENTS 4.2 Multiple Dose Pharmacokinetics in Stable Asian Renal Transplant Patients receiving Chronic MMF Therapy .134 4.2.1 Introduction 134 4.2.2 Methods 135 4.2.2.1 Study design 135 4.2.2.2 Patients 135 4.2.2.3 Demographic and biochemical data collection .135 4.2.2.4 Blood sampling .136 4.2.2.5 Urine collection .136 4.2.2.6 Sample analysis .136 4.2.2.7 Pharmacokinetic analysis 136 4.2.2.8 Statistical analysis .136 4.2.3 Results 137 4.2.3.1 All stable subjects recruited 137 4.2.3.1.1 Patient demographics 137 4.2.3.1.2 Steady-state pharmacokinetics 138 4.2.3.2 Subgroup analyses of stable subjects receiving CsA-MMFprednisolone immunosuppression .153 4.2.3.2.1 Stratification based on ethnic group .153 4.2.3.2.1.1 Patient demographics . 153 4.2.3.2.1.2 Steady-state pharmacokinetics . 156 4.2.3.2.2 Stratification based on gender .157 4.2.3.2.2.1 Patient demographics . 157 4.2.3.2.2.2 Steady-state pharmacokinetics . 159 4.2.3.2.3 Effect of kidney graft function on pharmacokinetics of MPA and MPAG .161 4.2.3.2.4 Pharmacokinetic-pharmacodynamic relationships .169 4.2.4 Discussion 177 4.2.5 Conclusion .197 4.3 Applications: Proposed Dosing Strategies for MMF 200 4.3.1 Proposed Optimal Dose of MMF .200 4.3.1.1 Introduction .200 4.3.1.2 Methods .201 4.3.1.2.1 Patients and pharmacokinetic data 201 4.3.1.2.2 Statistical analysis .201 4.3.1.3 Results .201 4.3.1.3.1 All stable subjects receiving CsA-MMFprednisolone 201 4.3.1.3.2 Male versus female subjects receiving CsA-MMFprednisolone 206 4.3.1.4 Discussion .209 4.3.1.5 Conclusion 211 4.3.2 Limited Sampling Strategy (LSS) for Therapeutic Drug Monitoring (TDM) of MPA 212 4.3.2.1 Introduction .212 4.3.2.2 Methods .213 4.3.2.2.1 Patients and pharmacokinetic data 213 vi TABLE OF CONTENTS 4.3.2.2.2 Development and validation of limited sampling strategies .213 4.3.2.3 Results .214 4.3.2.3.1 Concentration-time profiles 214 4.3.2.3.2 Development of limited sampling strategies .215 4.3.2.3.3 Validation of limited sampling strategies .217 4.3.2.4 Discussion .220 4.3.2.5 Conclusion 223 CHAPTER 5: CLINICAL PHARMACOGENETICS STUDY 225 5.1 Introduction .226 5.2 Methods .227 5.2.1 Study design .227 5.2.2 Patients .227 5.2.3 Pharmacokinetic data .227 5.2.4 Pharmacogenetic analysis 228 5.2.4.1 Blood sampling and genomic DNA extraction .228 5.2.4.2 Genotyping of UGT1A7, 1A8, 1A9 and 1A10 polymorphisms 228 5.2.4.3 Polymerase chain reaction (PCR) amplification .228 5.2.4.4 Purification of PCR amplified products and DNA sequencing .229 5.2.4.5 Statistical analysis .234 5.3 Results .236 5.3.1 Stable Asian renal transplant patients receiving chronic MMF therapy 236 5.3.1.1 Patient demographics 236 5.3.1.2 Allele frequencies of UGT1A7, 1A8, 1A9 and 1A10 polymorphisms 238 5.3.1.3 Linkage disequilibrium (LD) analysis of UGT1A7, 1A8, 1A9 and 1A10 polymorphisms .243 5.3.1.4 Genotype frequencies of UGT1A7, 1A8, 1A9 and 1A10 polymorphisms 245 5.3.2 Subgroup analyses of stable Asian renal transplant patients receiving CsA-MMF-prednisolone immunosuppression .248 5.3.2.1 Impact of UGT1A7, 1A8, 1A9 and 1A10 polymorphisms on the steady-state PK of MPA and MPAG 249 5.3.2.2 Haplotype analysis of UGT1A7, 1A8, 1A9 and 1A10 polymorphisms and impact of haplotypes and diplotypes on the steady-state PK of MPA and MPAG 259 5.3.2.3 Contribution of genetic, demographic and clinical variables to inter-individual variability of steady-state PK of MPA and MPAG .268 5.4 Discussion .279 5.5 Conclusion 289 CHAPTER 6: CONCLUDING REMARKS .291 6.1 Summary and Contribution .292 vii TABLE OF CONTENTS 6.2 Limitations 293 6.3 Future Perspectives .294 BIBLIOGRAPHY 298 APPENDIX 327 APPENDIX 375 LIST OF PUBLICATIONS AND CONFERENCE PRESENTATIONS .378 viii SUMMARY SUMMARY This thesis is a clinical study on the pharmacokinetics (PK) and pharmacogenetics of mycophenolic acid (MPA) in Asian renal transplant recipients (RTxR) in Singapore. MPA is the active entity of its ester prodrug, mycophenolate mofetil (MMF), which is a potent immunosuppressant approved for the prophylaxis of organ rejection in patients receiving renal, cardiac or hepatic transplants. In view of the limited PK data of MPA in the Asian population, the first part of this thesis aims to evaluate the PK of MPA in local Asian RTxR. Reversed-phase liquid chromatographic assays were developed for the quantification of total and free MPA and its glucuronide metabolite (MPAG) in human plasma and urine, which were applied to the clinical PK studies. The acute and steady-state PK of MPA and MPAG were characterized in Asian RTxR receiving immunosuppressive therapy consisting of MMF and prednisolone, in combination with cyclosporine, tacrolimus or sirolimus. In the local Asian population, the body weight-adjusted MPA oral clearance showed tendency to be lower than the Western population; hence, Asian patients may require a lower MMF dose. The observed correlation between drug exposure and body weight-adjusted MMF dose suggested that MMF may be dosed based on body weight, rather than the recommended standard fixed dose of g/day, so as to reduce the potential complications of excessive immunosuppression. An empiric MMF dose of 12 mg/kg/dose for Asian patients on MMF with concomitant CsA was proposed. In addition, with regards to clinical toxicity, free MPA levels were demonstrated to better correlate with adverse effects such as anemia, as compared to total MPA levels. This finding provides evidence to suggest that therapeutic monitoring of free MPA, rather than total MPA, may be of greater clinical value to ensure the safe use of MMF. ix MPAG 61.6 (34.5–97.8) 50.7 (19.6–92.1) 55.6 (26.7–97.8) 65.3 (19.6–96.1) 53.4 (45.8–64.2) 54.8 (34.5–97.4) 60.0 (19.6–97.8) 53.8 (26.7–92.1) 60.3 (19.6–97.8) 61.7 (45.8–96.1) MPA and MPAG 63.6 (35.5–99.2) 52.1 (19.8–93.3) 56.1 (27.7–99.2) 67.5 (19.8–98.9) 53.9 (46.5–65.0) 55.3 (35.5–99.2) 62.7 (19.8–98.9) 56.1 (27.7–93.3) 63.3 (19.8–99.2) 62.7 (46.5–98.9) 0.214 (0.046–1.849) 0.178 (0.032–0.609) 0.178 (0.059–0.656) 0.265 (0.032–1.849) 0.155 (0.069–0.704) 0.297 (0.059–0.656) 0.179 (0.032–1.849) 0.145 (0.094–0.435) 0.233 (0.032–1.849) 0.181 (0.069–0.704) 0.715 (0.170–1.740) 0.450 (0.130–2.020) 0.700 (0.130–1.570) 0.700 (0.210–2.020) 0.630 (0.400–0.970) 0.410 (0.170–1.570) 0.705 (0.130–2.020) 0.500 (0.130–1.340) 0.645 (0.170–2.020) 0.825 (0.400–1.540) 0.335 (0.080–2.720) 0.358 (0.041–1.383) 0.288 (0.103–1.383) 0.461 (0.041–2.720) 0.241 (0.124–1.696) 0.594 (0.103–1.058) 0.335 (0.041–2.720) 0.288 (0.196–0.880) 0.382 (0.041–2.720) 0.346 (0.124–1.696) 0.0434 (0.0106– 0.1267) 0.0279 (0.0058– 0.1654) 0.0436 (0.0058– 0.0972) 0.0432 (0.0073– 0.1654) 0.0351 (0.0240– 0.0613) 0.0274 (0.0106– 0.0972) 0.0434 (0.0058–0.1654) 0.0424 (0.0058– 0.0715) 0.0352 (0.0073– 0.1654) 0.0453 (0.0240– 0.1267) 0.186 (0.063–0.522) 0.134 (0.037–0.373) 0.163 (0.094–0.330) 0.188 (0.037–0.522) 0.141 (0.077–0.279) 0.152 (0.107–0.330) 0.169 (0.037–0.522) 0.151 (0.094–0.373) 0.172 (0.037–0.522) 0.187 (0.077–0.318) 33.5 (10.9–99.9) 30.0 (4.7–69.6) 34.3 (7.9–58.5) 32.9 (4.7–99.9) 23.8 (15.8–43.7) 32.9 (12.4–58.5) 32.4 (4.7–99.9) 33.4 (7.9–54.8) 32.1 (4.7–99.9) 32.9 (15.8–87.1) CLr (× 10-2) (L/h), normalized by TBW (kg) MPA MPAG CLur (L/h), normalized by TBW (kg) MPA MPAG CLf (L/h), normalized by TBW (kg) MPAG CLuf (L/h), normalized by TBW (kg) MPAG wt, wild-type; m, mutation. All data are expressed as median (range). * Significantly different among the three genotype groups (p < 0.05, Kruskal-Wallis test), with groups being significantly different as indicated (post-hoc multiple comparisons at the experimentwise 0.01 level). ** Significantly different between wt/m and m/m (p < 0.05). a APPENDIX 364 364 Table A.27. Steady-state PK parameters of MPA and MPAG in UGT1A9 intronic region genotype groups for stable Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone a Parameter I143C>T Total MPA C0 (mg/L), normalized by MMF dose (mg/kg) Cmax (mg/L), normalized by MMF dose (mg/kg) tmax (h) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) CLoral (L/h), normalized by TBW (kg) Total MPAG C0 (mg/L), normalized by MMF dose (mg/kg) Cmax (mg/L), normalized by MMF dose (mg/kg) tmax (h) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) CLoral (× 10-1) (L/h), normalized by TBW (kg) Free MPA C0 (× 10-3) (mg/L), normalized by MMF dose (mg/kg) Cmax (× 10-2) (mg/L), normalized by MMF dose (mg/kg) tmax (h) -2 AUCss, 0-12 (× 10 ) (mg⋅h/L), normalized by MMF dose (mg/kg) fu (%) CLu (L/h), normalized by TBW (kg) I152G>A b wt/m (n = 17) wt/wt (n = 47) wt/m (n = 2) wt/wt (n = 31) 0.176 (0.065–0.747) 1.35 (0.32–4.25) 0.750 (0.467–6.017) 4.17 (2.04–9.14) 0.177 (0.081–0.362) 0.164 (0.099–0.230) 1.32 (0.76–1.88) 0.508 (0.483–0.533) 3.52 (2.83–4.21) 0.218 (0.176–0.261) 0.186 (0.065–0.747) 1.18 (0.32–4.25) 0.77 (0.47–6.02) 4.14 (2.04–9.14) 0.178 (0.081–0.362) 0.176 (0.079–0.362) 1.62 (0.76–2.83) 0.53 (0.48–1.98) 4.50 (2.83–7.49) 0.164 (0.099–0.261) 5.8 (1.8–19.5) 11.9 (5.3–27.6) 2.02 (1.00–6.33) 103 (47–284) 0.112 (0.040–0.242) 12.2 (9.3–15.2) 15.2 (12.8–17.6) 1.79 (1.50–2.08) 161 (129–193) 0.074 (0.059–0.089) 6.29 (2.37–19.52) 10.2 (5.3–27.6) 2.00 (1.02–6.33) 99 (47–284) 0.116 (0.040–0.242) 5.68 (1.81–15.17) 12.1 (6.6–24.8) 2.03 (1.00–5.93) 110 (49–233) 0.104 (0.049–0.236) 0.650 (0.237–4.525) 0.856 (0.164–2.109) 0.750 (0.467–6.017) 2.11 (1.08–6.46) 0.508 (0.284–1.770) 35.0 (11.4–68.1) 0.779 (0.702–0.856) 0.903 (0.795–1.012) 0.508 (0.483–0.533) 2.15 (1.92–2.37) 0.622 (0.563–0.680) 34.8 (31.2–38.4) 0.769 (0.237–4.525) 0.764 (0.164–2.109) 0.75 (0.47–6.02) 2.11 (1.08–6.46) 0.564 (0.336–1.770) 35.0 (11.4–68.1) 0.702 (0.344–1.810) 0.883 (0.651–1.851) 0.60 (0.48–1.98) 2.13 (1.39–4.81) 0.446 (0.284–1.195) 34.7 (15.4–53.3) m/m (n = 1) wt/wt (n = 9) I219T>A/I313A>C wt/m (n = 26) m/m (n = 14) 0.150 0.150 (0.079–0.276) 1.18 (0.76–2.45) 1.00 (0.48–1.67) 4.07 (2.83–6.01) 0.181 (0.123–0.261) 0.185 (0.065–0.747) 0.30 (0.39–3.53) 0.69 (0.47–6.02) 4.38 (2.08–8.85) 0.169 (0.084–0.356) 0.187 (0.120–0.370) 1.51 (0.32–4.25) 0.63 (0.48–2.00) 4.16 (2.04–9.14) 0.178 (0.081–0.362) 7.77 (2.37–15.17) 12.8 (5.7–24.8) 2.05 (1.00–5.93) 129 (47–233) 0.089 (0.049–0.242) 5.78 (1.81–19.52) 12.4 (6.6–27.6) 2.03 (1.05–6.33) 109 (49–284) 0.105 (0.040–0.236) 6.14 (2.38–8.24) 9.3 (5.3–19.9) 1.58 (1.02–6.10) 88 (51–176) 0.130 (0.065–0.224) 0.702 (0.406–1.810) 0.836 (0.368–1.851) 1.00 (0.48–1.50) 2.03 (1.27–3.89) 0.494 (0.336–1.195) 36.5 (19.0–58.2) 0.709 (0.322–4.525) 0.800 (0.258–2.109) 1.00 (0.47–6.02) 2.09 (1.08–6.46) 0.522 (0.284–1.527) 35.3 (11.4–68.1) 0.696 (0.237–1.962) 0.921 (0.164–1.964) 0.61 (0.48–1.60) 2.28 (1.14–3.62) 0.491 (0.357–1.770) 32.6 (20.4–64.6) 0.93 1.67 4.10 0.180 7.77 14.8 2.17 139 0.083 0.570 0.479 1.25 2.03 0.495 36.5 365 365 Free MPAG C0 (mg/L), normalized by MMF dose (mg/kg) Cmax (mg/L), normalized by MMF dose (mg/kg) tmax (h) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) fu (%) CLu (L/h), normalized by TBW (kg) Metabolic ratios Total MPAG to Total MPA C0 Total MPAG to Total MPA AUCss, 0-12 Free MPAG to Free MPA C0 Free MPAG to Free MPA AUCss, 0-12 Urinary recovery (% dose) MPA MPAG MPA and MPAG CLr (× 10-2) (L/h), normalized by TBW (kg) MPA MPAG CLur (L/h), normalized by TBW (kg) MPA MPAG 0.92 (0.21–3.82) 2.08 (0.82–6.12) 1.97 (0.75–6.07) 16.9 (5.9–60.5) 16.3 (11.7–29.0) 0.0678 (0.0189–0.1930) 1.79 (1.31–2.26) 2.88 (2.09–3.68) 1.52 (1.50–1.53) 26.2 (20.5–31.8) 16.2 (15.8–16.5) 0.0460 (0.0360–0.0559) 1.01 (0.37–3.28) 2.08 (1.09–6.12) 2.00 (0.75–6.07) 17.5 (7.9–60.5) 18.4 (12.2–29.0) 0.0654 (0.0189–0.1457) 0.89 (0.21–3.82) 2.09 (0.82–5.27) 1.55 (1.25–2.42) 15.8 (5.9–53.1) 15.7 (11.7–28.6) 0.0726 (0.0216–0.1930) 1.28 29.4 (6.2–137.4) 24.1 (8.7–77.5) 1610 (195–4980) 806 (282–2450) 79.9 (66.1–93.8) 45.8 (45.8–45.9) 2380 (1530–3220) 1200 (1070–1340) 29.0 (6.2–137.4) 23.7 (8.7–77.5) 1450 (195–4980) 802 (282–2450) 31.0 (10.3–113.7) 27.0 (10.8–71.6) 1520 (416–3610) 810 (387–1360) 1.02 (0.27–11.78) 59.4 (19.6–97.8) 60.3 (19.8–99.2) 1.01 (0.83–1.20) 80.6 (69.2–92.1) 81.6 (70.0–93.3) 1.04 (0.34–11.78) 60.2 (34.5–97.8) 63.4 (35.5–99.2) 0.83 (0.27–3.57) 50.7 (19.6–92.1) 52.1 (19.8–93.3) 0.183 (0.032–1.849) 0.700 (0.130–2.020) 0.229 (0.145–0.313) 0.615 (0.410–0.820) 0.265 (0.046–1.849) 0.710 (0.170–1.740) 0.139 (0.032–0.609) 0.600 (0.130–2.020) 0.435 0.358 (0.041–2.720) 0.0424 (0.0058–0.1654) 0.359 (0.257–0.461) 0.0382 (0.0249–0.0515) 0.385 (0.080–2.720) 0.0405 (0.0106–0.1267) 0.304 (0.041–1.383) 0.0436 (0.0058–0.1654) 0.880 2.43 2.17 22.2 16.0 0.0517 51.7 33.8 2250 1090 2.41 53.8 56.3 0.450 0.0278 1.28 (0.39–3.82) 2.09 (1.29–5.27) 1.53 (1.25–2.42) 20.5 (8.9–53.1) 16.0 (11.7–22.8) 0.0559 (0.0216–0.1287) 0.95 (0.21–3.28) 2.15 (0.82–6.12) 1.98 (1.45–6.07) 17.2 (5.9–60.5) 16.3 (12.2–29.0) 0.0666 (0.0189–0.1930) 0.98 (0.37–1.86) 1.93 (1.09–3.38) 1.56 (0.75–2.07) 16.4 (7.9–28.8) 17.0 (12.2–26.1) 0.0705 (0.0398–0.1457) 39.1 (18.3–113.7) 33.8 (13.2–71.6) 1530 (747–3220) 927 (550–1360) 30.8 (6.2–137.4) 27.3 (8.7–77.5) 1520 (195–4550) 871 (282–2450) 27.9 (16.7–53.2) 21.1 (15.3–50.5) 1320 (237–4980) 753 (332–1490) 0.83 (0.50–2.41) 53.8 (26.7–92.1) 56.1 (27.7–93.3) 1.32 (0.27–11.78) 60.3 (19.6–97.8) 63.3 (19.8–99.2) 0.89 (0.54–3.97) 61.7 (45.8–96.1) 62.7 (46.5–98.9) 0.145 (0.094–0.435) 0.500 (0.130–1.340) 0.233 (0.032–1.849) 0.645 (0.170–2.020) 0.181 (0.069–0.704) 0.825 (0.400–1.540) 0.288 (0.196–0.880) 0.0424 (0.0058–0.0715) 0.382 (0.041–2.720) 0.0352 (0.0073–0.1654) 0.346 (0.124–1.696) 0.0453 (0.0240–0.1267) 366 366 CLf (L/h), normalized by TBW (kg) MPAG CLuf (L/h), normalized by TBW (kg) MPAG 0.159 (0.037–0.522) 0.281 (0.188–0.373) 0.198 (0.063–0.522) 0.126 (0.037–0.373) 0.151 0.151 (0.094–0.373) 0.172 (0.037–0.522) 0.187 (0.077–0.318) 31.3 (4.7–99.9) 44.1 (33.4–54.8) 33.5 (10.9–99.9) 30.0 (4.7–69.6) 30.5 33.4 (7.9–54.8) 32.1 (4.7–99.9) 32.9 (15.8–87.1) wt, wild-type; m, mutation. All data are expressed as median (range). Results are the same as for UGT1A7 756G>A as UGT1A7 756G>A and UGT1A9 I152G>A were in complete LD (D' = 1, r2 = 1). a b 367 367 Table A.28. Steady-state PK parameters of MPA and MPAG in UGT1A10 genotype groups for stable Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone a Parameter Total MPA C0 (mg/L), normalized by MMF dose (mg/kg) Cmax (mg/L), normalized by MMF dose (mg/kg) tmax (h) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) CLoral (L/h), normalized by TBW (kg) Total MPAG C0 (mg/L), normalized by MMF dose (mg/kg) Cmax (mg/L), normalized by MMF dose (mg/kg) tmax (h) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) CLoral (× 10-1) (L/h), normalized by TBW (kg) Free MPA C0 (× 10-3) (mg/L), normalized by MMF dose (mg/kg) Cmax (× 10-2) (mg/L), normalized by MMF dose (mg/kg) tmax (h) AUCss, 0-12 (× 10-2) (mg⋅h/L), normalized by MMF dose (mg/kg) fu (%) CLu (L/h), normalized by TBW (kg) wt/wt (n = 48) wt/m (n = 1) wt/wt (n = 37) wt/m (n = 12) 0.175 (0.065–0.747) 1.35 (0.32–4.25) 0.742 (0.467–6.017) 4.16 (2.04–9.14) 0.178 (0.081–0.362) 0.346 1.73 0.500 6.14 0.120 0.186 (0.065–0.747) 1.18 (0.32–4.25) 0.767 (0.467–6.017) 4.17 (2.04–9.14) 0.177 (0.081–0.362) 0.174 (0.099–0.362) 1.61 (1.06–2.27) 0.567 (0.483–1.983) 4.29 (2.83–6.14) 0.173 (0.120–0.261) 6.14 (1.81–19.52) 12.0 (5.3–27.6) 3.76 11.4 6.00 (2.37–19.52) 11.9 (5.3–27.6) 6.96 (1.81–14.70) 12.4 (6.6–24.8) 2.02 (1.00–6.33) 106 (47–284) 0.108 (0.040–0.242) 2.00 108 0.106 2.00 (1.02–6.33) 99 (47–284) 0.116 (0.040–0.242) 2.04 (1.00–5.93) 120 (49–233) 0.096 (0.049–0.236) 0.68 (0.24–4.52) 0.846 (0.164–2.11) 1.75 1.46 0.702 (0.237–4.525) 0.785 (0.164–2.109) 0.625 (0.344–1.810) 1.010 (0.654–1.851) * 0.742 (0.467–6.017) 2.08 (1.08–6.46) 0.500 4.81 1.00 (0.47–6.02) 2.13 (1.08–6.46) 0.57 (0.48–1.98) 1.99 (1.39–4.81) 0.515 (0.284–1.770) 35.5 (11.4–68.1) 0.783 15.4 0.523 (0.284–1.770) 34.7 (11.4–68.1) 0.434 (0.340–1.195) 37.2 (15.4–53.3) 0.96 (0.21–3.82) 2.09 (0.82–6.12) 1.17 3.46 0.92 (0.37–3.28) 1.97 (1.09–6.12) 1.21 (0.21–3.82) 2.31 (0.82–5.27) APPENDIX Free MPAG C0 (mg/L), normalized by MMF dose (mg/kg) Cmax (mg/L), normalized by MMF dose (mg/kg) 693C>T b 605C>T 368 368 tmax (h) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) fu (%) CLu (L/h), normalized by TBW (kg) 1.94 (0.75–6.07) 17.2 (5.9–60.5) 16.2 (11.7–29.0) 0.0666 (0.0189–0.1930) 1.58 30.8 28.6 0.0372 1.98 (0.75–6.07) 16.9 (7.9–60.5) 17.0 (12.2–29.0) 0.0678 (0.0189–0.1457) 1.58 (1.25–2.42) 21.3 (5.9–53.1) 15.6 (11.7–28.6) 0.0537 (0.0216–0.1930) Metabolic ratios Total MPAG to Total MPA C0 Total MPAG to Total MPA AUCss, 0-12 Free MPAG to Free MPA C0 Free MPAG to Free MPA AUCss, 0-12 30.0 (6.2–137.4) 25.5 (8.7–77.5) 1520 (195–4980) 814 (282–2450) 10.9 17.5 670 640 29.5 (6.2–137.4) 23.7 (8.7–77.5) 1440 (195–4980) 781 (282–2450) 30.2 (10.3–113.7) 27.4 (10.8–71.6) 1530 (480–3610) 981 (387–1360) Urinary recovery (% dose) MPA MPAG MPA and MPAG 1.03 (0.34–11.78) 59.9 (26.7–97.8) 62.5 (27.7–99.2) 0.267 19.6 19.8 1.02 (0.34–11.78) 60.2 (34.5–97.8) 63.4 (35.5–99.2) 0.98 (0.27–2.77) 50.3 (19.6–92.1) 51.5 (19.8–93.3) 0.192 (0.046–1.849) 0.700 (0.130–2.020) 0.0320 0.210 0.244 (0.046–1.849) 0.710 (0.170–1.740) 0.159 (0.032–0.609) 0.440 (0.130–2.020) 0.361 (0.080–2.720) 0.0428 (0.0058–0.1654) 0.0410 0.0073 0.364 (0.080–2.720) 0.0432 (0.0106–0.1267) 0.335 (0.041–1.383) 0.0338 (0.0058–0.1654) CLf (L/h), normalized by TBW (kg) MPAG 0.169 (0.063–0.522) 0.0365 0.184 (0.063–0.522) 0.130 (0.037–0.373) CLuf (L/h), normalized by TBW (kg) MPAG 33.1 (7.9–99.9) 4.65 33.4 (10.9–99.9) 27.9 (4.7–69.6) CLr (× 10-2) (L/h), normalized by TBW (kg) MPA MPAG CLur (L/h), normalized by TBW (kg) MPA MPAG APPENDIX wt, wild-type; m, mutation. a All data are expressed as median (range). b Results are the same as for UGT1A8 765A>G as UGT1A8 765A>G and UGT1A10 693C>T were in complete LD (D' = 1, r2 = 1). * Significantly different between wt/wt and wt/m (p < 0.05). 369 369 Table A.29. Steady-state PK parameters of MPA and MPAG in the five most common UGT1A7 to 1A10 haplotype groups for stable Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone a Parameter Haplotype Non-carriers Carriers (n = 20) (n = 29) Haplotype Non-carriers Carriers (n =33 ) (n = 16) Haplotype Non-carriers Carriers (n = 35) (n = 14) Haplotype Non-carriers Carriers (n = 38) (n = 11) Haplotype Non-carriers Carriers (n = 42) (n = 7) 0.144 (0.067–0.306) 0.194 (0.065–0.747) 0.188 (0.065–0.747) 0.143 (0.067–0.306) 0.176 (0.067–0.370) 0.191 (0.065–0.747) 0.180 (0.065–0.747) 0.176 (0.104–0.362) 0.187 (0.065–0.747) 0.138 (0.067–0.257) Cmax (mg/L), normalized by MMF dose (mg/kg) 1.11 (0.32–2.45) 1.52 (0.36–4.25) 1.56 (0.36–4.25) 1.23 (0.32–3.56) 1.44 (0.32–4.25) 1.12 (0.39–3.53) 1.23 (0.32–4.25) 1.61 (1.06–2.27) 1.49 (0.32–4.25) 0.80 (0.56–1.35) * tmax (h) 1.02 (0.48–2.00) 0.60 (0.47–6.02) 0.60 (0.47–6.02) 1.00 (0.50–2.00) 0.62 (0.48–2.00) 1.02 (0.47–6.02) 0.759 (0.467–6.017) 0.600 (0.500–1.983) 0.61 (0.47–6.02) 1.47 (0.48–1.57) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) 3.65 (2.51–6.01) 4.27 (2.04–9.14) * 4.21 (2.04–8.85) 3.65 (2.51–9.14) 4.19 (2.04–9.14) 3.97 (2.08–8.85) 4.16 (2.04–9.14) 4.50 (3.26–6.14) 4.18 (2.04–9.14) 3.52 (2.60–5.85) CLoral (L/h), normalized by TBW (kg) 0.203 (0.123–0.295) 0.173 (0.081–0.362) * 0.176 (0.084–0.362) 0.203 (0.081–0.295) 0.176 (0.081–0.362) 0.186 (0.084–0.356) 0.178 (0.081–0.362) 0.164 (0.120–0.227) 0.177 (0.081–0.362) 0.210 (0.126–0.284) 7.03 (2.37–19.52) 5.68 (1.81–18.13) 5.74 (1.81–18.13) 6.14 (2.44–19.52) 6.29 (1.81–19.52) 5.73 (2.37–18.13) 6.14 (2.37–19.52) 5.68 (1.81–14.70) 5.79 (1.81–18.13) 9.20 (4.11–19.52) Cmax (mg/L), normalized by MMF dose (mg/kg) 13.6 (5.3–27.6) 10.9 (6.6–24.4) 11.9 (5.7–24.8) 13.6 (5.3–27.6) 12.0 (5.3–27.6) 11.1 (5.7–24.8) 11.9 (5.3–27.6) 12.1 (6.6–24.8) 11.6 (5.3–24.8) 15.7 (7.6–27.6) tmax (h) 2.00 (1.00–6.33) 2.02 (1.02–6.10) 2.03 (1.00–6.10) 1.95 (1.05–6.33) 2.00 (1.02–6.33) 2.08 (1.00–6.07) 2.00 (1.02–6.33) 2.05 (1.00–5.93) 2.02 (1.00–6.10) 2.00 (1.50–6.33) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) 128 (47–284) 93 (49–266) 103 (47–266) 119 (51–284) 108 (49–284) 106 (47–266) 101 (47–284) 110 (49–233) 101 (47–266) 137 (71–284) CLoral (× 10-1) (L/h), normalized by TBW (kg) 0.089 (0.040–0.242) 0.123 (0.043–0.236) 0.112 (0.043–0.242) 0.096 (0.040–0.224) 0.113 (0.040–0.236) 0.108 (0.043–0.242) 0.114 (0.040–0.242) 0.104 (0.049–0.236) 0.114 (0.043–0.242) 0.084 (0.040–0.161) Total MPA C0 (mg/L), normalized by MMF dose (mg/kg) Total MPAG C0 (mg/L), normalized by MMF dose (mg/kg) 370 370 Free MPA C0 (× 10-3) (mg/L), normalized by MMF dose (mg/kg) 0.635 (0.344–1.810) 0.783 (0.237–4.525) 0.783 (0.322–4.525) 0.606 (0.237–1.857) 0.617 (0.237–1.962) 0.890 (0.387–4.525) 0.735 (0.237–4.525) 0.440 (0.344–1.810) 0.776 (0.237–4.525) 0.557 (0.322–1.165) 0.725 (0.164–1.851) 0.900 (0.258–2.109) 0.869 (0.258–2.109) 0.775 (0.164–1.354) 0.856 (0.164–1.964) 0.866 (0.258–2.109) 0.79 (0.16–2.11) 1.01 (0.65–1.85) 0.890 (0.164–2.109) 0.676 (0.324–0.764) * tmax (h) 1.00 (0.48–1.57) 0.62 (0.47–6.02) 0.617 (0.467–6.017) 0.867 (0.500–1.567) 0.62 (0.48–2.00) 1.02 (0.47–6.02) 0.875 (0.467–6.017) 0.600 (0.500–1.983) 0.66 (0.47–6.02) 1.47 (0.48–2.00) AUCss, 0-12 (× 10-2) (mg⋅h/L), normalized by MMF dose (mg/kg) 2.02 (1.27–3.89) 2.26 1.08–6.46) 2.13 (1.08–6.46) 2.08 (1.14–3.27) 2.11 (1.14–4.81) 2.15 (1.08–6.46) 2.12 (1.08–6.46) 2.06 (1.39–4.81) 2.08 (1.08–6.46) 2.26 (1.64–3.20) 0.560 (0.336–1.230) 0.502 (0.284–1.770) 0.498 (0.284–1.770) 0.569 (0.357–1.230) 0.502 (0.284–1.770) 0.551 (0.336–1.527) 0.540 (0.284–1.770) 0.428 (0.340–1.195) 0.500 (0.284–1.770) 0.575 (0.349–1.230) 36.5 (19.0–58.2) 32.8 (11.4–68.1) 34.7 (11.4–68.1) 35.5 (22.6–64.6) 35.0 (15.4–64.6) 34.6 (11.4–68.1) 34.8 (11.4–68.1) 36.0 (15.4–53.3) 35.5 (11.4–68.1) 32.8 (23.1–45.1) 1.26 (0.39–3.82) 0.92 (0.21–3.28) 1.01 (0.21–3.82) 1.03 (0.37–2.96) 1.04 (0.21–2.96) 0.85 (0.39–3.82) 0.96 (0.37–3.28) 1.17 (0.21–3.82) 0.90 (0.21–3.82) 1.40 (0.68–2.96) Cmax (mg/L), normalized by MMF dose (mg/kg) 2.15 (1.27–5.27) 1.97 (0.82–6.12) 1.97 (0.82–6.12) 2.16 (1.09–5.08) 2.09 (0.82–5.08) 1.99 (1.21–6.12) 2.03 (1.09–6.12) 2.50 (0.82–5.27) 2.09 (0.82–6.12) 2.95 (1.27–5.08) tmax (h) 1.95 (1.25–2.42) 1.70 (0.75–6.07) 1.70 (0.75–6.07) 1.95 (1.50–2.07) 1.65 (0.75–2.25) 2.03 (1.25–6.07) 1.98 (0.75–6.07) 1.58 (1.25–2.42) 1.63 (0.75–6.07) 2.00 (1.52–2.05) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) 19.0 (8.9–53.1) 16.9 (5.9–60.5) 16.9 (5.9–60.5) 17.6 (7.9–46.4) 17.7 (5.9–46.4) 16.0 (8.9–60.5) 17.2 (7.9–60.5) 22.2 (5.9–53.1) 16.6 (5.9–60.5) 25.1 (11.5–46.4) fu (%) 16.4 (11.7–23.4) 16.0 (12.2–29.0) 16.0 (11.7–29.0) 16.3 (12.2–23.7) 16.1 (12.2–28.6) 18.7 (11.7–29.0) 16.7 (12.2–29.0) 15.5 (11.7–28.6) 16.0 (11.7–29.0) 18.4 (13.0–22.5) 0.0606 (0.0216–0.1287) 0.0678 (0.0189–0.1930) 0.0678 (0.0189–0.1930) 0.0650 (0.0247–0.1457) 0.0647 (0.0247–0.1930) 0.0723 (0.0189–0.1287) 0.0666 (0.0189–0.1457) 0.0516 (0.0216–0.1930) 0.0690 (0.0189–0.1930) 0.0455 (0.0247–0.0997) Cmax (× 10-2) (mg/L), normalized by MMF dose (mg/kg) fu (%) CLu (L/h), normalized by TBW (kg) Free MPAG C0 (mg/L), normalized by MMF dose (mg/kg) CLu (L/h), normalized by TBW (kg) 371 371 Metabolic ratios Total MPAG to Total MPA C0 40.4 (18.3–137.4) 27.4 (6.2–85.0) * 27.9 (6.2–113.7) 31.1 (18.5–137.4) 29.5 (10.3–137.4) 33.3 (6.2–113.7) 30.0 (6.2–137.4) 29.4 (10.3–113.7) 28.5 (6.2–113.7) 67.4 (25.6–137.4) * Total MPAG to Total MPA AUCss, 0-12 30.5 (13.2–71.6) 20.7 (8.7–77.5) * 24.1 (8.7–77.5) 25.5 (15.3–66.7) 23.7 (10.8–66.7) 26.9 (8.7–77.5) 23.9 (8.7–77.5) 27.0 (10.8–71.6) 23.9 (8.7–77.5) 29.8 (20.4–66.7) Free MPAG to Free MPA C0 1580 (686–4550) 1320 (195–4980) 1320 (195–4980) 1570 (686–4550) 1530 (237–4980) 1170 (195–3760) 1450 (195–4980) 1530 (480–3610) 1380 (195–4980) 1900 (1280–4550) * Free MPAG to Free MPA AUCss, 0-12 1020 (545–1900) 731 (282–2450) 806 (282–2450) 916 (479–1900) 819 (332–1900) 806 (282–2450) 792 (282–2450) 927 (387–1360) 804 (282–2450) 1030 (598–1900) Urinary recovery (% dose) MPA 0.93 (0.34–2.94) 1.02 (0.27–11.8) 0.93 (0.27–11.78) 1.03 (0.34–2.94) 0.93 (0.27–3.97) 1.21 (0.50–11.78) 1.03 (0.34–11.78) 0.93 (0.27–2.77) 0.91 (0.27–11.78) 1.68 (0.34–2.94) MPAG 57.6 (26.7–97.8) 60.4 (19.6–96.1) 54.1 (19.6–94.6) 66.6 (34.5–97.8) * 60.2 (19.6–97.4) 57.5 (26.7–97.8) 61.6 (34.5–97.8) 49.9 (19.6–85.7) * 60.0 (19.6–97.8) 54.8 (34.5–97.4) MPA and MPAG 58.3 (27.7–99.2) 63.4 (19.8–98.9) 57.0 (19.8–98.7) 68.3 (35.5–99.2) 61.9 (19.8–99.2) 60.2 (27.7–98.7) 63.6 (35.5–99.2) 51.0 (19.8–86.2) * 62.7 (19.8–98.9) 55.3 (35.5–99.2) 0.201 (0.032–1.849) 0.181 (0.059–0.656) 0.179 (0.032–0.704) 0.260 (0.046–1.849) 0.255 (0.046–1.849) 0.139 (0.032–0.609) 0.179 (0.032–1.849) 0.297 (0.059–0.656) CLr (× 10-2) (L/h), normalized by TBW (kg) MPA 0.179 0.201 (0.059–0.656) (0.032–1.849) MPAG 0.600 (0.130–1.570) 0.700 (0.210–2.020) 0.630 (0.130–2.020) 0.805 (0.170–1.570) 0.700 (0.170–2.020) 0.680 (0.130–1.740) 0.715 (0.170–1.740) 0.380 (0.130–2.020) 0.705 (0.130–2.020) 0.410 (0.170–1.570) CLur (L/h), normalized by TBW (kg) MPA 0.297 (0.103–1.383) 0.385 (0.041–2.720) 0.358 (0.041–2.720) 0.374 (0.103–1.383) 0.313 (0.041–1.696) 0.361 (0.080–2.720) 0.375 (0.080–2.720) 0.313 (0.041–1.383) 0.335 (0.041–2.720) 0.594 (0.103–1.058) 0.0405 (0.0073–0.1654) 0.0385 (0.0058–0.1654) 0.0503 (0.0106–0.1267) 0.0432 (0.0073–0.1654) 0.0405 (0.0058–0.0866) 0.0434 (0.0106–0.1267) 0.0251 (0.0058–0.1654) 0.0434 (0.0058–0.1654) 0.0274 (0.0106–0.0972) 0.159 (0.037–0.522) 0.149 (0.037–0.522) 0.218 (0.107–0.330) * 0.152 (0.037–0.373) 0.174 (0.063–0.522) 0.186 (0.063–0.522) 0.126 (0.037–0.271) 0.169 (0.037–0.522) 0.152 (0.107–0.330) 31.3 (4.6–99.9) 30.7 (4.6–99.9) 34.7 (12.3–87.1) 32.9 (4.6–87.1) 32.8 (7.9–99.9) 33.5 (10.9–99.9) 25.8 (4.6–69.6) 32.4 (4.6–99.9) 32.9 (12.3–58.5) MPAG 0.0430 (0.0058–0.0972) CLf (L/h), normalized by TBW (kg) MPAG 0.175 (0.094–0.373) CLuf (L/h), normalized by TBW (kg) MPAG 33.9 (7.9–58.5) a All data are expressed as median (range). * Significantly different between non-carriers and carriers (p < 0.05). 372 372 Table A.30. Steady-state PK parameters of MPA and MPAG in the five most common UGT1A7 to 1A10 diplotype groups for stable Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone a Parameter H1/H1 (n = 7) H1/H2 (n = 5) H1/H3 (n = 7) H1/H4 (n = 6) H2/H5 (n = 5) Total MPA C0 (mg/L), normalized by MMF dose (mg/kg) Cmax (mg/L), normalized by MMF dose (mg/kg) * tmax (h) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) CLoral (L/h), normalized by TBW (kg) 0.188 (0.126–0.370) 1.70 (0.36–4.25) *H2/H5 0.52 (0.48–1.60) 4.19 (2.04–6.85) 0.176 (0.108–0.362) 0.149 (0.120–0.296) 1.44 (1.18–3.56) 0.73 (0.50–1.05) 3.85 (3.13–9.14) 0.192 (0.081–0.236) 0.207 (0.065–0.747) 0.85 (0.39–3.53) 1.18 (0.47–6.02) 3.86 (2.08–8.85) 0.191 (0.084–0.356) 0.248 (0.104–0.362) 1.61 (1.26–2.27) 0.61 (0.50–1.98) 5.14 (3.57–6.14) 0.144 (0.120–0.207) 0.138 (0.067–0.257) 0.80 (0.56–1.35) *H1/H1 1.47 (0.62–1.57) 3.38 (2.60–5.85) 0.219 (0.126–0.284) Total MPAG C0 (mg/L), normalized by MMF dose (mg/kg) Cmax (mg/L), normalized by MMF dose (mg/kg) tmax (h) AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) CLoral (× 10-1) (L/h), normalized by TBW (kg) 6.31 (2.38–6.97) 10.9 (7.2–17.0) 1.52 (1.02–6.10) 99 (59–145) 0.116 (0.079–0.195) 6.00 (3.49–8.24) 9.3 (7.7–19.9) 2.02 (1.52–2.08) 92 (65–176) 0.125 (0.065–0.177) 7.49 (3.53–18.13) 9.6 (6.6–24.4) 2.27 (1.97–6.07) 93 (62–266) 0.123 (0.043–0.184) 4.96 (1.81–9.70) 10.9 (6.6–17.3) 2.04 (1.98–2.25) 103 (49–170) 0.111 (0.067–0.236) 9.98 (4.22–19.52) 17.1 (7.6–27.6) 1.98 (1.50–6.33) 144 (71–284) 0.079 (0.040–0.161) Free MPA C0 (× 10-3) (mg/L), normalized by MMF dose (mg/kg) Cmax (× 10-2) (mg/L), normalized by MMF dose (mg/kg) tmax (h) AUCss, 0-12 (× 10-2) (mg⋅h/L), normalized by MMF dose (mg/kg) fu (%) CLu (L/h), normalized by TBW (kg) 0.990 (0.335–1.962) 0.953 (0.663–1.964) 0.52 (0.48–1.60) 2.46 (1.68–3.62) 0.459 (0.359–1.770) 30.1 (20.4–43.9) 0.595 (0.237–1.857) 0.941 (0.785–1.354) 0.73 (0.50–1.05) 2.44 (1.14–3.27) 0.607 (0.357–0.672) 30.3 (22.6–64.6) 0.940 (0.387–4.525) 0.520 (0.258–2.109) 1.18 (0.47–6.02) 2.02 (1.08–6.46) 0.580 (0.413–1.527) 36.5 (11.4–68.1) 0.625 (0.366–1.748) 0.986 (0.767–1.463) 0.61 (0.50–1.98) 2.16 (1.39–4.81) 0.421 (0.340–0.784) 34.2 (15.4–53.3) 0.650 (0.452–1.165) 0.686 (0.559–0.764) 1.47 (0.62–1.57) 2.44 (1.91–3.20) 0.575 (0.521–1.230) 30.2 (23.1–38.8) 1.04 (0.47–1.67) 2.08 (1.39–3.26) 1.10 (0.75–2.03) **H1/H3 18.1 (10.4–25.8) 17.7 (12.2–26.1) 0.0631 (0.0445–0.1105) 0.80 (0.37–1.86) 2.13 (1.09–3.38) 1.65 (1.52–2.07) 17.7 (7.9–28.8) 15.0 (12.2–23.7) 0.0647 (0.0398– 1.76 (0.58–3.28) 2.54 (1.21–6.12) 2.12 (1.53–6.07) **H1/H1 26.2 (10.4–60.5) 20.1 (13.1–29.0) 0.0438 (0.0189–0.1099) 1.01 (0.21–1.28) 2.10 (0.82–3.46) 1.84 (1.52–2.25) 18.1 (5.9–30.8) 14.9 (12.2–28.6) 0.0664 (0.0372–0.1930) 1.52 (0.71–2.96) 3.21 (1.27–5.08) 2.00 (1.52–2.05) 32.5 (11.5–46.4) 18.4 (16.1–22.5) 0.0352 (0.0247– Free MPAG C0 (mg/L), normalized by MMF dose (mg/kg) Cmax (mg/L), normalized by MMF dose (mg/kg) tmax (h) ** AUCss, 0-12 (mg⋅h/L), normalized by MMF dose (mg/kg) fu (%) CLu (L/h), normalized by TBW (kg) 373 373 0.1457) 0.0997) Metabolic ratios Total MPAG to Total MPA C0 Total MPAG to Total MPA AUCss, 0-12 Free MPAG to Free MPA C0 Free MPAG to Free MPA AUCss, 0-12 27.9 (16.7–53.2) 23.7 (16.2–35.1) 1320 (237–4980) 781 (332–1490) 29.0 (26.5–47.6) 20.7 (15.3–50.5) 1520 (716–1980) 687 (479–1360) 49.3 (6.2–85.0) 30.0 (8.7–77.5) 1450 (195–3760) 937 (282–2450) 24.3 (10.3–54.3) 19.1 (10.8–32.0) 1320 (480–1930) 723 (387–1080) 75.9 (30.7–137.4) 41.2 (21.0–66.7) 1900 (1580–4550) 1080 (602–1900) Urinary recovery (% dose) MPA *** MPAG MPA and MPAG 0.88 (0.54–3.97) 53.4 (45.8–64.2) 53.9 (46.5–65.0) 1.02 (0.70–2.76) 87.0 (63.8–96.1) 87.7 (65.9–98.9) 3.53 (0.55–11.78) ***H1/H4 65.3 (48.7–94.6) 68.8 (49.3–98.7) 0.59 (0.27–1.49) ***H1/H3 53.4 (19.6–85.7) 54.4 (19.8–86.2) 1.04 (0.34–2.94) 54.8 (34.5–97.4) 55.3 (35.5–99.2) 0.155 (0.069–0.704) 0.63 (0.40–0.97) 0.183 (0.083–0.477) 1.02 (0.42–1.54) 0.757 (0.046–1.849) 0.66 (0.26–1.74) 0.105 (0.032–0.201) 0.54 (0.21–2.02) 0.297 (0.059–0.656) 0.36 (0.17–1.57) 0.24 (0.12–1.70) 0.0351 (0.0240–0.0613) 0.50 (0.21–0.72) 0.0523 (0.0254– 0.1267) 1.29 (0.08–2.72) 0.0279 (0.0129–0.0866) 0.27 (0.04–0.40) 0.0365 (0.0073–0.1654) 0.24 (0.10–1.06) 0.0191 (0.0106– 0.0972) CLf (L/h), normalized by TBW (kg) MPAG 0.141 (0.077–0.279) 0.210 (0.109–0.318) 0.199 (0.063–0.522) 0.116 (0.037–0.271) 0.152 (0.107–0.330) CLuf (L/h), normalized by TBW (kg) MPAG 23.8 (15.8–43.7) 34.6 (30.6–87.1) 31.3 (10.9–99.9) 25.5 (4.7–69.6) 20.6 (12.4–58.5) CLr (× 10-2) (L/h), normalized by TBW (kg) MPA MPAG CLur (L/h), normalized by TBW (kg) MPA MPAG a All data are expressed as median (range). * Significantly different among the five diplotype groups (p < 0.05, Kruskal-Wallis test), with groups being significantly different as indicated (post-hoc multiple comparisons at the experimentwise 0.15 level). ** Significantly different among the five diplotype groups (p < 0.05, Kruskal-Wallis test), with groups being significantly different as indicated (post-hoc multiple comparisons at the experimentwise 0.01 level). *** Significantly different among the five diplotype groups (p < 0.05, Kruskal-Wallis test), with groups being significantly different as indicated (post-hoc multiple comparisons at the experimentwise 0.025 level). 374 374 APPENDIX APPENDIX APPENDIX 375 APPENDIX M 10.0 kb – 8.0 kb – 6.0 kb – 5.0 kb – 4.0 kb – 3.0 kb – 2.0 kb – 1.5 kb – – 1295 bp 1.0 kb – 0.5 kb – Figure A.1. Photograph of agarose gel (stained with ethidium bromide) showing PCR products (1295 bp; position 98246–99540 in reference sequence for human UGT1 gene: GenBank accession number AF297093) to confirm specific PCR amplification of UGT1A7 promoter and exon regions. bp, base pairs; kb, kilo base pairs; M, DNA ladder marker. M 1517 bp – 1200 bp – 1,000 bp – 900 bp – 800 bp – 700 bp – 600 bp – 500, 517 bp – – 1033 bp 400 bp – 300 bp – 200 bp – 100 bp – Figure A.2. Photograph of agarose gel (stained with ethidium bromide) showing PCR products (1033 bp; position 34175–35207 in reference sequence for human UGT1 gene: GenBank accession number AF297093) to confirm specific PCR amplification of UGT1A8 exon region. bp, base pairs; M, DNA ladder marker. 376 APPENDIX (A) M 10.0 kb – 8.0 kb – 6.0 kb – 5.0 kb – 4.0 kb – 3.0 kb – – 2302 bp 2.0 kb – 1.5 kb – 1.0 kb – 0.5 kb – (B) M 10.0 kb – 8.0 kb – 6.0 kb – 5.0 kb – 4.0 kb – 3.0 kb – 2.0 kb – 1.5 kb – – 1464 bp 1.0 kb – 0.5 kb – Figure A.3. Photographs of agarose gel (stained with ethidium bromide) showing PCR products ((A): 2302 bp; position 86309–88610 in reference sequence for human UGT1 gene: GenBank accession number AF297093; (B): 1464 bp; position 88271– 89734) to confirm specific PCR amplification of (A) UGT1A9 promoter region and (B) UGT1A9 exon and intron regions. bp, base pairs; kb, kilo base pairs; M, DNA ladder marker. M 1517 bp – 1200 bp – 1,000 bp – 900 bp – 800 bp – 700 bp – 600 bp – 500, 517 bp – 400 bp – 300 bp – 200 bp – – (i) 657 bp – (ii) 416 bp 100 bp – Figure A.4. Photograph of agarose gel (stained with ethidium bromide) showing PCR products ((i) 657 bp; position 53042–53698 in reference sequence for human UGT1 gene: GenBank accession number AF297093; (ii) 416 bp; position 53659 - 54074) to confirm specific PCR amplifications of two different regions of UGT1A10 exon 1. bp, base pairs; M, DNA ladder marker. 377 LIST OF PUBLICATIONS PRESENTATIONS AND CONFERENCE LIST OF PUBLICATIONS AND CONFERENCE PRESENTATIONS 378 Publications and Conference Presentations The following have been published or presented in advance of this thesis. Publications 1) Yau WP, Vathsala A, Lou HX, Chan E. Is a standard fixed dose of mycophenolate mofetil ideal for all patients? Nephrol Dial Transplant 2007; 22(12): 3638- 3645. 2) Yau WP, Vathsala A, Lou HX, Zhou SF, Chan E. Simple reversed-phase liquid chromatographic assay for simultaneous quantification of free mycophenolic acid and its glucuronide metabolite in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2007; 846(1-2): 313-318. 3) Yau WP, Vathsala A, Lou HX, Chan E. Simple reversed-phase ion-pair liquid chromatography assay for the simultaneous determination of mycophenolic acid and its glucuronide metabolite in human plasma and urine. J Chromatogr B Analyt Technol Biomed Life Sci 2004; 805(1): 101-112. Conference Presentations 1) Yau WP, Lou HX, Vathsala A, Zhou SF, Chan E. Therapeutic Drug Monitoring of Mycophenolic Acid in Renal Transplant Recipients in Singapore. Poster presentation at the NUS Science Faculty Graduate Congress 2005 (21 Sep 2005, NUS, Singapore). 2) Yau WP, Vathsala A, Lou HX, Zhou SF, Chan E. Effect of Gender on the Pharmacokinetics of Mycophenolic Acid and its Metabolite in Renal Transplant Patients in Singapore receiving Concomitant Cyclosporine. Oral presentation at the Inter-varsity Symposium of the 17th Singapore Pharmacy Congress 2005 (1 Jul 2005, NUS, Singapore). 3) Yau WP, Lou HX, Vathsala A, Zhou SF, Chan E. Is a Fixed Dosing Regimen of Mycophenolate Mofetil Ideal for All Patients? Poster presentation at the American Transplant Congress 2005 (21–25 May 2005, Washington State Convention and Trade Center, Seattle, Washington, USA). Am J Transplant 2005; (Suppl 11): 323 (Abstract 655). 4) Yau WP, Lou HX, Vathsala A, Sy J, Zhou SF, Chan E. Pharmacokinetics of Mycophenolic Acid in Renal Transplant Recipients on Chronic Dosing. Oral presentation at The Society of Transplantation (Singapore) Annual Scientific Meeting 2004 (13 Nov 2004, The Fullerton Hotel, Singapore). 5) Lou HX, Yau WP, Vathsala A, Sy J, Zhou SF, Chan E. Pharmacokinetics of Mycophenolic Acid in Renal Transplant Recipients in Singapore. Poster presentation at the SingHealth Scientific Meeting 2004 (15–17 Oct 2004, Shangrila Hotel, Singapore). 379 [...]... in adult RTxR in Asian and in Western countries receiving MMF with concomitant CsA and steroid for at least 3 months 181 Table 4.18 Comparison of renal mechanism of MPA inferred in stable Asian RTxR in the present multiple dose PK study and in healthy volunteers in single dose PK studies conducted in USA 189 Table 4.19 Comparison of renal mechanism of MPAG inferred in stable Asian RTxR in the present... activation Maintenance immunosuppression Methylprednisolone (Solumedrol®) Inhibit expression of cytokines, namely interleukin-1, -2, -3, -6, tumor necrosis factor- and interferon- , resulting in the inhibition of all stages of T-cell activation Induction immunosuppression and treatment of acute rejection Cyclosporine (Sandimmune®, Neoral®) Binds to cyclophilin, resulting in cyclophilin-cyclosporine complex... nephrotoxicity of calcineurin inhibitors [18,19] 4 CHAPTER 1: INTRODUCTION Table 1.1 Immunosuppressants currently used in clinical practice for renal transplantation [18,20-22] Drug Mechanism of action Use Corticosteroids Prednisolone and its prodrug, Prednisone Inhibit expression of cytokines, namely interleukin-1, -2, -3, -6, tumor necrosis factor- and interferon- , resulting in the inhibition of all stages of. .. MPAG in Asian RTxR receiving variable doses of MMF at six PK sampling days from the start of MMF therapy 338 Table A.8 Metabolic ratios of total or free MPAG to MPA C0 or AUCss, 012 in Asian RTxR receiving variable doses of MMF at six PK sampling days from the start of MMF therapy 340 Table A.9 Urinary recoveries and CLr of MPA and MPAG, as well as CLf of MPAG, in Asian RTxR receiving variable doses of. .. Hospital (NUH), with the former being the major RTx centre in Singapore According to data from the Singapore Renal Registry, there were 874 prevalent renal transplant recipients (RTxR) as of end 2000 [8] Of these, 59.8% of the transplants were performed in SGH, 13.8% in NUH and 25.3% at overseas centres [8] In 2005, 43 deceased-donor and 53 livedonor RTx were performed in Singapore [9] 1.1.2 Historical... those reported in Caucasians and African Americans Some SNPs were found to influence the PK of MPA and MPAG These findings suggested the ethnic diversity of polymorphisms in the UGT1A7 to 1A10 metabolic enzymes and their likely impact on the PK of MPA and MPAG in Asian patients receiving MMF therapy Together with the PK results and other non-genetic patient factors, these pharmacogenetics findings may potentially... which inhibits calcineurin phosphatase and Tcell activation Maintenance immunosuppression Tacrolimus (Prograf®) Binds to FK506-binding protein 12 (FKBP12), resulting in FKBP12tacrolimus complex which inhibits calcineurin phosphatase and T-cell activation Maintenance immunosuppression Sirolimus (Rapamune®) and its derivative, Everolimus (Certican®) Bind to FKBP12, resulting in FKBP12sirolimus and FKBP12-everolimus... structures of (A) MMF (B) MPA and (C) MPAglucuronide (MPAG) 9 Figure 1.2 Schematic representation of the de novo and salvage pathways of guanosine nucleotide biosynthesis, showing the mechanism of action of MPA by inhibition of the de novo pathway 10 Figure 3.1 The influence of pH of running buffer on the qualitative retention of MPA, MPAG and endogenous plasma interferences 50 Figure 3.2 The influence of. .. which inhibit mTOR and interleukin-2 driven T-cell proliferation Maintenance immunosuppression Calcineurin inhibitors Mammalian target of rapamycin (mTOR) inhibitors 5 5 CHAPTER 1: INTRODUCTION General class CHAPTER 1: INTRODUCTION Azathioprine (Imuran®) Maintenance immunosuppression Mycophenolate: mycophenolate Selective, uncompetitive, reversible mofetil (CellCept®), mycophenolate inhibitor of inosine... immunosuppression and treatment of acute rejection Anti-interleukin-2 receptor monoclonal antibodies Basiliximab (Simulect®); Daclizumab (Zenapax®) Binds to and blocks CD25 (interleukin-2 receptor chain) on activated T cells, resulting in depletion of these cells and inhibition of interleukin-2 induced T-cell activation Induction immunosuppression Anti-lymphocyte monoclonal antibodies 6 6 CHAPTER 1: INTRODUCTION . PHARMACOKINETICS AND PHARMACOGENETICS OF MYCOPHENOLIC ACID IN ASIAN RENAL TRANSPLANT PATIENTS IN SINGAPORE YAU WAI PING (B.Sc.(Pharm.)(Hons.), NUS). thesis is a clinical study on the pharmacokinetics (PK) and pharmacogenetics of mycophenolic acid (MPA) in Asian renal transplant recipients (RTxR) in Singapore. MPA is the active entity of its ester. binding of MPA 76 3.3.3.2 Human plasma protein binding of MPAG 76 3.3.3.3 Effect of MPAG on human plasma protein binding of MPA77 3.3.3.4 Effect of MPA on human plasma protein binding of MPAG77 3.3.3.5