Pharmacokinetics and pharmacogenetics of mycophenolic acid in asian renal transplant patients in singapore

108 Table 4.7 Characteristics of Asian RTxR receiving CsA-MMF-prednisolone immunosuppression at six PK sampling days from the start of MMF therapy 121 Table 4.8 Intra- and inter-indivi

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PHARMACOKINETICS AND PHARMACOGENETICS

OF MYCOPHENOLIC ACID IN ASIAN RENAL TRANSPLANT PATIENTS IN SINGAPORE

YAU WAI PING

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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-148-000-092-112)

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

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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

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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

1.2.2 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

1.4.2 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

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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

3.2.2 Experimental 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

3.2.3.1 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

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3.3 In Vitro Human Plasma Protein Binding Study of MPA and MPAG 73

3.3.2 Materials and Methods 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

4.1.2 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

4.1.2.7 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

4.1.3.1 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

4.1.3.2.1.2 Pharmacokinetic results 123

4.1.3.2.2 Discussion 129

4.1.4 Conclusion 133

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4.2 Multiple Dose Pharmacokinetics in Stable Asian Renal Transplant Patients

receiving Chronic MMF Therapy 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

4.2.2.7 Pharmacokinetic 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-MMF-prednisolone immunosuppression 153

4.2.3.2.1 Stratification based on ethnic group 153

4.2.3.2.1.2 Steady-state pharmacokinetics 156

4.2.3.2.2 Stratification based on gender 157

4.2.3.2.2.2 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-MMF-prednisolone 201

4.3.1.3.2 Male versus female subjects receiving CsA-MMF-prednisolone 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

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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

5.3 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

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6.2 Limitations 293

6.3 Future Perspectives 294

BIBLIOGRAPHY 298

APPENDIX 1 327

APPENDIX 2 375

LIST OF PUBLICATIONS AND CONFERENCE PRESENTATIONS 378

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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 2 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

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A wide inter-subject variability in the PK parameters of MPA and MPAG in the Asian RTxR was also observed, which may in part be due to genetic factors This leads to the second part of this thesis which aims to investigate the possibility of genetic variations in the metabolic enzymes of MPA, uridine diphosphate glucuronosyltransferases (UGT), as a cause of PK variability and also as a contributing factor underlying the difference in MMF dose requirement between Asian and Western populations Various single nucleotide polymorphisms (SNPs) present in the UGT1A7, 1A8, 1A9 and 1A10 enzymes, which are mainly involved in MPA glucuronidation, were investigated The allele frequencies of most of these SNPs found in the study subjects were comparable to those reported in other Asians but somewhat different from 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 aid in the individualization

of MMF therapy to enhance efficacy and safety in Asian RTxR

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LIST OF TABLES

Table 1.1 Immunosuppressants currently used in clinical practice for renal

transplantation

5

Table 1.2 Pharmacokinetic drug interactions with MMF 17

Table 1.3 Limited sampling strategies developed based on multiple linear

regression for the estimation of the total MPA AUCss, 0-12 for

TDM in transplant recipients

22

Table 1.4 Studies investigating the UGT isoforms involved in the

metabolism of MPA to MPAG

28

Table 1.5 In vitro and in vivo effects of allelic variants of UGT1A9, 1A7,

1A8 and 1A10

31

Table 3.1 Drugs that did not show interferences to MPAG and MPA

peaks under the optimized chromatographic conditions

54

Table 3.2 Intra-day and inter-day precision (quantitation based on

absolute peak areas) of the simultaneous MPA and MPAG assay in human plasma and urine

57

Table 3.3 Accuracy of the simultaneous MPA and MPAG assay in human

plasma and urine

58

Table 3.4 Drugs that did not show interferences to MPAG, MPAC and

MPA peaks under the optimized chromatographic conditions

68

Table 3.5 Accuracy of the simultaneous free MPA and MPAG assay in

human plasma

71

Table 4.1 Differential equations describing the compartmental mass

transfer for the five-compartment drug and metabolite EHC model

88

Table 4.2 Characteristics of study subjects analyzed in the first dose study 98

Table 4.3 Parameter estimates providing the best fit of the

five-compartment drug and metabolite EHC model to the observed

plasma concentration-time data of MPA and MPAG

101

Table 4.4 Apparent clearance values and elimination half-lives of MPA

and MPAG, and apparent volume of distribution for MPA obtained based on the five-compartment drug and metabolite EHC model

103

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Table 4.5 AUC∞ values with and without EHC, and the extent of EHC for

MPA and MPAG based on the five-compartment drug and metabolite EHC model

104

Table 4.6 Parameter estimates providing the best fit of the

five-compartment drug and metabolite EHC model to the observed

plasma concentration-time data of MPA for three adult RTxR

receiving CsA-MMF-prednisone immunosuppression as reported by Shum et al

108

Table 4.7 Characteristics of Asian RTxR receiving

CsA-MMF-prednisolone immunosuppression at six PK sampling days from

the start of MMF therapy

121

Table 4.8 Intra- and inter-individual variability of PK parameters total and

free MPA and MPAG in Asian RTxR receiving variable doses

of MMF at six PK sampling days from the start of MMF therapy

128

Table 4.9 Characteristics of stable Asian RTxR classified based on the

immunosuppressive regimen received

139

Table 4.10 Characteristics of stable Asian RTxR receiving steady-state

doses of MMF with concomitant CsA and prednisolone immunosuppression classified based on ethinicity

154

Table 4.11 Characteristics of stable Asian RTxR receiving steady-state

doses of MMF with concomitant CsA and prednisolone immunosuppression classified based on gender

158

Table 4.12 Steady-state exposure parameters of total and free MPA and

MPAG in stable Asian RTxR receiving

CsA-MMF-prednisolone immunosuppression with versus without anemia,

GI side effects and/or CMV infection

170

Table 4.13 Correlation of TBW-adjusted MMF dose with hematological

results of stable Asian RTxR receiving CsA-MMF-prednisolone

immunosuppression

173

Table 4.14 Correlation of steady-state exposure parameters of total and free

MPA with hematological results of stable Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

174

Table 4.15 Correlation of steady-state exposure parameters of total and free

MPAG with hematological results of stable Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

175

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Table 4.16 Comparison of total MPA C0 and AUCss, 0–12, normalized by

TBW-adjusted MMF dose, as well as TBW-adjusted MPA

CLoral, in adult RTxR in Asian and in Western countries receiving MMF with concomitant CsA and steroid for at least 3

months

180

Table 4.17 Comparison of total MPAG C0 and AUCss, 0–12, normalized by

TBW-adjusted MMF dose, as well as TBW-adjusted MPAG

CLoral, 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 multiple dose PK study and in healthy volunteers in single dose PK studies conducted in USA

190

Table 4.20 Estimated TBW-adjusted MMF doses based on the respective

total MPA AUCss, 0–12 (30, 45 or 60 mg⋅h/L) according to the

derived regression equations

206

Table 4.21 Estimated TBW-adjusted MMF doses for male versus female

subjects based on the respective total MPA AUCss, 0–12 (30, 45

or 60 mg⋅h/L) according to the derived regression equations

209

Table 4.22 Distribution of individual tmax after MMF administration 215

Table 4.23 Limited sampling strategies (LSS) for estimation of total MPA

AUCss, 0-12

216

Table 4.24 Predictive performance of limited sampling strategies to

estimate the total MPA AUCss, 0-12

218

Table 5.1 UGT1A7, 1A8, 1A9 and 1A10 polymorphisms screened 230

Table 5.2 Primer sequences for amplification of UGT1A7, 1A8, 1A9 and

1A10 by polymerase chain reaction (PCR)

231

Table 5.3 Polymerase chain reaction conditions for amplification of

UGT1A7, 1A8, 1A9 and 1A10

232

Table 5.4 Primer sequences for DNA sequencing of UGT1A7, 1A8, 1A9

and 1A10

233

Table 5.5 Main characteristics of stable Asian RTxR in the

pharmacogenetics study classified based on the immunosuppressive regimen received

237

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Table 5.6 Comparison of variant allele frequencies of observed UGT1A7,

1A8, 1A9 and 1A10 polymorphisms in Asian RTxR in the present study and in other populations reported in literature

239

Table 5.7 Comparison of published variant allele frequencies among

populations reported in literature for UGT1A7, 1A8, 1A9 and

1A10 polymorphisms that were screened but not found in Asian

RTxR in the present study

242

Table 5.8 Comparison of observed genotype frequencies of UGT1A7,

1A8, 1A9 and 1A10 polymorphisms among the four Asian ethnic groups of the RTxR in the present study

247

Table 5.9 Haplotype analysis for UGT1A7, 1A8, 1A9 and 1A10

polymorphisms in stable Asian renal transplant patients receiving CsA-MMF-prednisolone immunosuppression

260

Table 5.10 Diplotype frequencies of UGT1A7, 1A8, 1A9 and 1A10

polymorphisms in stable Asian renal transplant patients receiving CsA-MMF-prednisolone immunosuppression

261

Table 5.11 Determinants of the steady-state PK parameters of MPA and

MPAG in stable Asian RTxR receiving

CsA-MMF-prednisolone immunosuppression

271

Table 5.12 Multiple linear regression models for estimation of the

steady-state PK parameters of MPA and MPAG in stable Asian RTxR

receiving CsA-MMF-prednisolone immunosuppression

274

Table A.1 Characteristics of Asian RTxR receiving

TAC-MMF-prednisolone immunosuppression at six PK sampling days from

328

Table A.2 Characteristics of Asian RTxR receiving

SRL-MMF-prednisolone immunosuppression at six PK sampling days from

330

Table A.3 Characteristics of Asian RTxR receiving MMF-prednisolone

immunosuppression at six PK sampling days from the start of

MMF therapy

332

Table A.4 PK parameters of total MPA in Asian RTxR receiving variable

doses of MMF at six PK sampling days from the start of MMF

therapy

334

Table A.5 PK parameters of total MPAG in Asian RTxR receiving

variable doses of MMF at six PK sampling days from the start

of MMF therapy

335

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Table A.6 PK parameters of free MPA in Asian RTxR receiving variable

therapy

336

Table A.7 PK parameters of free MPAG in Asian RTxR receiving variable

therapy

338

Table A.8 Metabolic ratios of total or free MPAG to MPA C0 or AUCss,

0-12 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 MMF at

six PK sampling days from the start of MMF therapy

341

Table A.10 Steady-state PK parameters of total MPA and MPAG in stable

Asian RTxR receiving variable doses of MMF classified based

on the immunosuppressive regimen received

342

Table A.11 Steady-state PK parameters of free MPA and MPAG in stable

Asian RTxR receiving variable doses of MMF classified based

on the immunosuppressive regimen received

343

Table A.12 Metabolic ratios of total or free MPAG to MPA C0 or AUCss,

0-12 in stable Asian RTxR receiving variable steady-state doses of

MMF classified based on the immunosuppressive regimen received

344

Table A.13 Urinary recoveries and CLr of MPA and MPAG, as well as CLf

of MPAG, in stable Asian RTxR receiving variable steady-state

doses of MMF classified based on the immunosuppressive regimen received

345

Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone classified based on ethnicity

346

Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone classified based on ethnicity

347

MMF with concomitant CsA and prednisolone classified based

on ethnicity

348

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doses of MMF with concomitant CsA and prednisolone classified based on ethnicity

349

Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone classified based on gender

350

Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone classified based on gender

351

MMF with concomitant CsA and prednisolone classified based

on gender

352

doses of MMF with concomitant CsA and prednisolone classified based on gender

352

Table A.22 Steady-state PK parameters of MPA and MPAG in UGT1A7

genotype groups for stable Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone

353

Table A.23 Steady-state PK parameters of MPA and MPAG in officially

named UGT1A7 genotype groups a for stable Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone

356

358

Table A.25 Steady-state PK parameters of MPA and MPAG in officially

named UGT1A8 genotype groups a for stable Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone

360

promoter region genotype groups for stable Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone

362

intronic region genotype groups for stable Asian RTxR receiving variable doses of MMF with concomitant CsA and prednisolone

365

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368

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

370

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

373

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LIST OF FIGURES

Figure 1.1 Chemical structures of (A) MMF (B) MPA and (C)

MPA-glucuronide (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 acetonitrile composition of mobile phase on the

qualitative retention of MPA, MPAG and endogenous plasma interferences

51

Figure 3.3 Representative chromatograms showing the simultaneous

analysis of MPA and MPAG in human plasma: (A) blank pooled human plasma; (B) blank pooled human plasma spiked with MPA (10 mg/L) and MPAG (200 mg/L); (C) plasma sample from a stable RTxR under chronic immunosuppressive therapy with MMF obtained 1 h after MMF administration (MPA: 8.97 mg/L, MPAG: 111 mg/L); (D) plasma sample from a RTxR under immunosuppressive therapy without MMF

52

analysis of MPA and MPAG in human urine: (A) blank human urine; (B) blank human urine spiked with MPA (25 mg/L) and MPAG (250 mg/L); (C) 12-h urine sample (collected at the time the evening dose of MMF was administered to the time the next morning dose was administered) from a stable RTxR under chronic immunosuppressive therapy with MMF (MPA: 17.0 mg/L, MPAG: 565 mg/L)

53

Figure 3.5 PK profiles of MPA and MPAG of an individual stable RTxR

under chronic immunosuppressive therapy, receiving 500 mg MMF BD

60

analysis of free MPA and MPAG in human plasma: (A) blank ultrafiltrate; (B) blank ultrafiltrate spiked with MPA (0.05 mg/L), MPAG (10 mg/L) and MPAC (15 mg/L); (C) ultrafiltrate from the plasma sample of a RTxR under immunosuppressive therapy with MMF obtained 0.5 h after MMF administration (free MPA: 0.0844 mg/L, free MPAG: 13.2 mg/L)

67

Figure 3.7 PK profiles of both free and total MPA and MPAG of an

individual stable RTxR under chronic immunosuppressive therapy, receiving 500 mg MMF (CellCept®) twice daily

72

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Figure 3.8 Free fraction of MPAG at various concentrations of total MPAG

spiked in human plasma

76

Figure 3.9 Free fraction of MPA at various concentrations of total MPA

spiked in human plasma, in the absence (control) or presence of

a fixed concentration of total MPAG (100 mg/L) in human plasma

77

Figure 3.10 Correlation of MPA free fraction with MPAG free fraction 78

Figure 4.1 A five-compartment drug and metabolite EHC model describing

the PK of MPA and MPAG after oral administration

87

Figure 4.2 Best fit of model to the observed plasma concentration-time data

of total MPA and total MPAG for two typical subjects after oral administration of the first dose of MMF in combination with (A) concomitant prednisolone and CsA, and (B) concomitant prednisolone without CsA, respectively

100

Figure 4.3 Correlations of extent of EHC for (A) MPA and (B) MPAG with

TBW-adjusted CsA daily dose

105

Figure 4.4 Best fit of model to the observed plasma concentration-time data

of total MPA after an oral dose of MMF in three adult RTxR receiving CsA-MMF-prednisone for at least five days, demonstrating (A) a lag time in absorption, (B) a complex absorption process and (C) a markedly significant EHC process, respectively

106

Figure 4.5 Influence of T bile on plasma concentration-time profiles of total

MPA and total MPAG for two typical subjects after oral administration of the first dose of MMF in combination with (A) concomitant prednisolone and CsA, and (B) concomitant prednisolone without CsA, respectively

109

Figure 4.6 Influence of τgall on plasma concentration-time profiles of total

MPA and total MPAG for two typical subjects after oral administration of the first dose of MMF in combination with (A) concomitant prednisolone and CsA, and (B) concomitant prednisolone without CsA, respectively

110

Figure 4.7 Box plot of free MPA C0 normalized by TBW-adjusted MMF

dose for early and late post-Tx patients receiving prednisolone immunosuppression over the six study days

CsA-MMF-124

Figure 4.8 Box plots of (A) free MPA AUCss, 0-12 normalized by

TBW-adjusted MMF, (B) TBW-TBW-adjusted MPA CLu and (C) MPA fu

for late post-Tx patients receiving CsA-MMF-prednisolone immunosuppression (n = 6) over the six study days

125

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Figure 4.9 Box plots of (A) total MPAG Cmax, (B) total MPAG AUCss, 0-12,

(C) free MPAG Cmax and (D) free MPAG AUCss, 0-12, normalized

by TBW-adjusted MMF dose for late post-Tx patients receiving CsA-MMF-prednisolone immunosuppression (n = 6) over the six study days

126

Figure 4.10 Box plots of metabolic ratios of (A) total MPAG to MPA AUCss,

0-12 and (B) free MPAG to MPA AUCss, 0-12 for late post-Tx patients receiving CsA-MMF-Prednisolone immunosuppression (n = 6) over the six study days

127

Figure 4.11 Individual plasma concentration-time profiles of total MPA for

stable Asian RTxR receiving chronic dosing of (A) prednisolone (n = 53), (B) TAC-MMF-prednisolone (n = 9), (C) SRL-MMF-prednisolone (n = 3) or (D) MMF-prednisolone immunosuppression (n = 2)

CsA-MMF-141

Figure 4.12 Individual plasma concentration-time profiles of total MPAG for

CsA-MMF-142

Figure 4.13 Individual plasma concentration-time profiles of free MPA for

CsA-MMF-143

Figure 4.14 Individual plasma concentration-time profiles of free MPAG for

CsA-MMF-144

Figure 4.15 Average plasma concentration-time profiles of total MPA and

MPAG for stable Asian RTxR receiving chronic dosing of (A) CsA-MMF-prednisolone immunosuppression (n = 53), (B) TAC-MMF-prednisolone immunosuppression (n = 9), (C) SRL-MMF-prednisolone immunosuppression (n = 3) and (D) MMF-prednisolone immunosuppression (n = 2)

145

Figure 4.16 Average plasma concentration-time profiles of free MPA and

MPAG for stable Asian RTxR receiving chronic dosing of (A) CsA-MMF-prednisolone immunosuppression (n = 53), (B) TAC-MMF-prednisolone immunosuppression (n = 9), (C) SRL-MMF-prednisolone immunosuppression (n = 3) and (D) MMF-prednisolone immunosuppression (n = 2)

146

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Figure 4.17 Box plots of (A) total and (B) free MPA C0, before (left) and

after (right) normalization by TBW-adjusted MMF dose for

stable Asian RTxR receiving variable doses of MMF classified based on the immunosuppressive regimen received

148

Figure 4.18 Box plot of free MPA AUCss, 0-12 normalized by TBW-adjusted

MMF dose for stable Asian RTxR receiving variable doses of MMF classified based on the immunosuppressive regimen received

149

Figure 4.19 Box plot of total MPA CLoral for stable Asian RTxR receiving

variable doses of MMF classified based on the immunosuppressive regimen received

149

Figure 4.20 Box plots of MPA CLu (A) before and (B) after normalization by

TBW for stable Asian RTxR receiving variable doses of MMF classified based on the immunosuppressive regimen received

150

Figure 4.21 Box plots of (A) total and (B) free MPAG tmax for stable Asian

RTxR receiving variable doses of MMF classified based on the immunosuppressive regimen received

150

Figure 4.22 Box plots of metabolic ratios of (A) total MPAG to MPA C0 and

(B) free MPAG to MPA C0 for stable Asian RTxR receiving variable doses of MMF classified based on the immunosuppressive regimen received

151

Figure 4.23 Histograms showing inter-individual variability of total and free

normalization, and MPA and MPAG CLoral and CLu before and after normalization by TBW, in Asian RTxR receiving CsA-MMF-prednisolone (n = 53)

152

Figure 4.24 Box plots of metabolic ratio of free MPAG to free MPA C0 for

the four ethnic groups

156

Figure 4.25 Box plots of (A) total and (B) free MPA Cmax, normalized by

TBW-adjusted MMF dose, in Asian RTxR receiving MMF-prednisolone immunosuppression classified based on gender

CsA-160

Figure 4.26 Box plots of (A) total MPA AUCss, 0-12 normalized by

TBW-adjusted MMF dose and (B) TBW-TBW-adjusted total MPA CLoral in Asian RTxR receiving CsA-MMF-prednisolone immunosuppression classified based on gender

160

Figure 4.27 Box plots of TBW-adjusted MPAG (A) CLf and (B) CLuf in

Asian RTxR receiving CsA-MMF-prednisolone immunosuppression classified based on gender

161

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Figure 4.28 Relationship between MPA plasma clearance or MPA AUCss,

0-12, normalized by TBW-adjusted MMF dose, and calculated creatinine clearance

163

Figure 4.29 Relationship between MPAG plasma clearance or MPAG

AUCss, 0-12, normalized by TBW-adjusted MMF dose, and calculated creatinine clearance

164

Figure 4.30 Relationship between MPA or MPAG renal clearance and

calculated creatinine clearance

165

Figure 4.31 Relationship between MPA or MPAG fu and calculated

creatinine clearance or serum albumin or serum urea

167

Figure 4.32 Relationship between serum albumin or serum urea and

calculated creatinine clearance

168

Figure 4.33 Relationship between MPA and MPAG fu 168

Figure 4.34 Relationship between MPAG formation clearance and calculated

creatinine clearance

168

Figure 4.35 Relationship between total or free MPA AUCss, 0-12 and

hemoglobin (Hb), hematocrit (Hct) or red blood cell (RBC) count

176

Figure 4.36 Correlation of total MPA AUCss, 0–12 with TBW-adjusted MMF

dose, (A) before and (B) after omission of outliers

203

Figure 4.37 Correlation of total MPA C0 with TBW-adjusted MMF dose, (A)

before and (B) after omission of outliers

204

Figure 4.38 Box plots of (A) total MPA C0 and (B) total MPA AUCss, 0–12

against TBW-adjusted MMF dose range

205

Figure 4.39 Correlation of total MPA AUCss, 0–12 with TBW-adjusted MMF

dose, (A) before and (B) after omission of outliers, for male versus female subjects

208

Figure 4.40 Bland-Altman plots testing agreement between the full measured

total MPA AUCss, 0-12 and the estimated total MPA AUCss, 0-12

derived from the limited sampling strategies Models 22 and 33

219

Figure 5.1 Relative positions of the observed UGT1A7, 1A8, 1A9 and

1A10 polymorphisms in the study population on the UGT1A gene

238

Figure 5.2 Pairwise linkage disequilibrium analysis for UGT1A7, 1A8, 1A9

and 1A10 polymorphisms

243

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TBW-adjusted MMF dose, according to UGT1A9 331C>T genotypes in Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

-440T>C/-251

Figure 5.4 Box plot of free MPA Cmax, normalized by TBW-adjusted MMF

dose, according to UGT1A8 765A>G/UGT1A10 693C>T genotypes in Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

251

Figure 5.5 Box plots of metabolic ratios of (A) total MPAG to MPA C0, (B)

total MPAG to MPA AUCss, 0–12, (C) free MPAG to MPA C0 and (D) free MPAG to MPA AUCss, 0–12, according to UGT1A9 -440T>C/-331C>T genotypes in Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

254

Figure 5.6 Box plots of metabolic ratios of (A) total MPAG to MPA C0 and

(B) total MPAG to MPA AUCss, 0–12 according to UGT1A9 I143C>T genotypes in all Asian RTxR (Chinese, Malay, Indian,

Eurasian) (left) and in the Chinese ethnic group (right) receiving

CsA-MMF-prednisolone immunosuppression

255

total MPAG to MPA AUCss, 0–12, (C) free MPAG to MPA C0 and (D) free MPAG to MPA AUCss, 0–12, according to UGT1A7 -57T>G/33C>A/622T>C genotypes in Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

256

total MPAG to MPA AUCss, 0–12, (C) free MPAG to MPA C0 and (D) free MPAG to MPA AUCss, 0–12, according to UGT1A7 genotypes in Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

257

Figure 5.9 Box plots of (A) total MPA AUCss, 0-12 normalized by

TBW-adjusted MMF dose, (B) TBW-TBW-adjusted total MPA CLoral, and metabolic ratios of (C) total MPAG to MPA C0 and (B) total MPAG to MPA AUCss, 0–12, in the absence or presence of haplotype H1 in Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

263

TBW-adjusted MMF dose, and metabolic ratios of (C) total MPAG to MPA C0 and (D) free MPAG to MPA C0 in the absence or presence of haplotype H5 in Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

264

Figure 5.11 Box plot of TBW-adjusted MPAG CLf in the absence or

presence of haplotype H2 in Asian RTxR receiving prednisolone immunosuppression

CsA-MMF-265

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Figure 5.12 Box plot of total MPA Cmax normalized by TBW-adjusted MMF

dose according to UGT1A7 to 1A10 diplotypes in Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

266

total MPAG to MPA AUCss, 0–12, (C) free MPAG to MPA C0 and (D) free MPAG to MPA AUCss, 0–12 according to UGT1A7 to 1A10 diplotypes in Asian RTxR receiving CsA-MMF-prednisolone immunosuppression

267

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 1 regions

376

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 1 region

376

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 1 and intron 1 regions

377

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

377

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LIST OF ABBREVIATIONS AND SYMBOLS

Area under the plasma concentration-time curve at steady-state from 0-12 h AUCss, 0-12

Aspartate aminotransferase AST

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Gastrointestional GI

Hour(s) h

Intravenous IV

Minute(s) min

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Peak time tmax

Sirolimus SRL

Tacrolimus TAC

Ultraviolet UV

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CHAPTER 1 : INTRODUCTION

INTRODUCTION

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1.1 Renal Transplantation (RTx)

1.1.1 Introduction

In renal failure, two therapeutic options are available, namely renal dialysis and RTx Due to the higher quality of life [1-3], improved cost-effectiveness [2] and reduced long-term mortality rates [4] as compared with dialysis, RTx, in particular live-donor transplantation, is the optimal treatment for renal failure in patients across all age groups [5]

In Singapore, the first successful deceased-donor and live-donor RTx was performed in 1970 and 1977, respectively, in Singapore General Hospital (SGH) [6]

By 1999, 1000 RTx had been performed in Singapore [7] Currently, RTx programmes are locally available in SGH and National University 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 donor RTx were performed in Singapore [9]

live-1.1.2 Historical perspective

The first experimental kidney transplantation was preformed between dogs in Vienna in 1902 [10] And in 1906, the first transplants in humans were attempted using a sheep and a pig kidney, respectively, but these were unsuccessful [10] The first successful clinical organ transplantation occurred in 1954 when live-donor RTx was performed between identical twins at the Peter Bent Brigham Hospital in Boston [11]

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Subsequently, from 1955 to 1962, attempts were made to perform RTx beyond identical twins [12,13] using sublethal total body irradiation and cortisone to suppress organ rejection [14] However, not many of these transplants were successful [14] and the observed life-threatening side effects of total body irradiation prompted the search and development of other immunosuppressants [10] In 1959, the anti-cancer agent, 6-mercaptopurine, was demonstrated to suppress the immune response in rabbits [15,16] Azathioprine, a less toxic derivative of 6-mercaptopurine, was later synthesized and used concomitantly with corticosteroids in RTxR since 1962 [17] In the early 1980s, cyclosporine (CsA) [10,14] and the first monocloncal antibody against T lymphocytes, OKT3 [10], were introduced into clinic practice to prevent organ rejection in transplantations Since then, many other anti-lymphocyte antibodies and immunosuppressive drugs, such as tacrolimus (TAC), mycophenolate mofetil (MMF) and sirolimus (SRL), have been developed for clinical use [10]

1.1.3 Current immunosuppressive therapy

Today, a wide array of immunosuppressants is used in different combinations for induction immunosuppression, maintenance immunosuppression and treatment of acute rejection episodes (Table 1.1)

During the early post-transplantation (post-Tx) period, initial induction with antibody-based immunosuppressive therapy is started with high-dose corticosteroids This is followed by a maintenance immunosuppressive regimen comprising of a few immunosuppressants, typically triple therapy involving a primary immunosuppressant (calcineurin inhibitor: CsA or TAC), an adjunctive agent (azathioprine or MMF or SRL) and a corticosteroid (prednisolone) [18] When graft stabilization is achieved,

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immunosuppression is usually reduced for long-term therapy to diminish the risks of opportunistic infection and malignancy associated with immunosuppression, while balancing the control of organ rejection [18] The reduction of immunosuppression may be carried out by (i) dose reduction or elimination of corticosteroid, (ii) dose minimization or withdrawal of calcineurin inhibitor, or (iii) substitution of calcineurin inhibitor with SRL in patients who are particularly sensitive to the nephrotoxicity of calcineurin inhibitors [18,19]

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CHAPTER 1: INTRODUCTION

Table 1.1 Immunosuppressants currently used in clinical practice for renal transplantation [18,20-22]

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 T-cell activation

Maintenance immunosuppression

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

Calcineurin inhibitors Cyclosporine (Sandimmune®,

Neoral®)

Binds to cyclophilin, resulting in cyclophilin-cyclosporine complex which inhibits calcineurin phosphatase and T-cell activation

(FKBP12), resulting in tacrolimus complex which inhibits calcineurin phosphatase and T-cell activation

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CHAPTER 1: INTRODUCTION

Antiproliferative agents Azathioprine (Imuran®) Blocks the de novo and salvage pathways

of purine synthesis and inhibits DNA replication

enzyme in the de novo synthesis of

guanosine nucleotides

Anti-lymphocyte

monoclonal antibodies

Muromonab-CD3 (OKT3®) Binds to T-cell-receptor associated CD3

complex, resulting in initial activation and release of cytokines, followed by

depletion and functional alteration of T cells

monocytes, macrophages and natural killer cells, causing cell lysis and prolonged depletion

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1.2 Mycophenolic Acid (MPA)

1.2.1 Introduction

MPA, first discovered in 1893 [23] and isolated in 1896 [24], is the fermentation

product of Penicillium [24-27] MPA was originally developed in the 1960s and

1970s as a potential antimicrobial [28-30], antineoplastic [29,31-34] and antipsoriatic drug [35-38] before its development as an immunosuppressant

Currently, two formulations of MPA are commercially available The first is the 2-morpholinoethyl ester prodrug of MPA, MMF (CellCept®, Roche Pharmaceuticals, Basel, Switzerland) which has been available since 1995 and the second is the enteric-coated mycophenolate sodium (sodium salt of MPA) (EC-MPS, Myfortic®, Novartis Pharma AG, Basel, Switzerland) which is a new formulation approved for use only in

2004 For the studies performed in this thesis, MMF, was used and hence would be the focus of this thesis Details on EC-MPS would therefore not be further discussed

1.2.2 Indications

MMF is approved by the US Food and Drug Administration (FDA) for the prophylaxis of organ rejection in patients receiving allogeneic renal, cardiac or hepatic transplants It is used in combination with a calcineurin inhibitor (CsA [39-41]

or TAC [42-45]) or mTOR inhibitor (SRL) [46-50], and corticosteroids

Other non-FDA-approved therapeutic uses of MMF have also been reported in literature These include its use in the prophylaxis of graft-versus-host disease [51,52], and in autoimmune [53-61] and inflammatory diseases [62-65]

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1.2.3 Chemistry

The chemical name of MMF is

2-morpholinoethyl-(E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl)-4-methyl-4-hexenoate and its empirical formula is

C23H31NO7 (molecular weight: 433.5) (Fig 1.1A) [66,67] MMF is a white to white crystalline powder with melting point around 93°C to 94°C [66] Its pKa values are 5.6 for the morpholino group and 8.5 for the phenolic group [67] Its solubility is low in water but increases at acidic pH Its apparent partition coefficients, namely log

off-P (n-octanol/buffer pH 2) and log off-P (n-octanol/buffer pH 7.4), are 0.0085 and 238, respectively [66]

MMF was developed as a prodrug to enhance the oral bioavailability of MPA [68] The chemical name of MPA is 6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl)-4-methyl-4-hexenoic acid and its empirical formula is C17H20O6 (molecular weight: 320.3) [66] It is formed as needles from hot water and has a melting point of 141°C [66] It is a weak dibasic acid (Fig 1.1B), with pKa values of 4.5 for the aliphatic carboxylic acid group [66] and 8.5 for the phenolic group [67] Its apparent partition coefficients, namely log P (n-octanol/buffer pH 2) and log P (n-octanol/buffer pH 7.4), are 570 and 1.6, respectively [66]

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Figure 1.1 Chemical structures of (A) MMF (B) MPA and (C) MPA-glucuronide

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more dependent on this pathway than other cell types that can utilize the salvage pathway, which is not blocked by MPA [71] (Fig 1.2) Moreover, the type II isoform

of IMPDH, which is expressed in activated lymphocytes, is approximately five times more sensitive to MPA than the type I isoform of IMPDH, which is expressed in most cell types [69] Hence, MPA has a more potent cytostatic effect on lymphocytes than

on other cell types [71] Thus, MPA selectively inhibits T and B lymphocyte proliferation, thereby inhibiting cell-mediated immune responses and antibody formation [72] This is the principal mechanism of action of MPA

DNA

synthesis

RNA synthesis Glycoprotein synthesis

Salvage pathway Guanosine

PNP Guanine HGPRTase

PRPP

IMPDH

x MPA

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 ATP, adenosine triphosphate; dGDP,

deoxyguanosine diphosphate; dGTP, deoxyguanosine triphosphate; DNA, deoxyribonucleic acid; GDP, guanosine diphosphate; GMP, guanosine monophosphate; GTP, guanosine triphosphate; HGPRTase, hypoxanthine guanine phosphoribosyltransferase; IMP, inosine monophosphate; IMPDH, inosine monophosphate dehydrogenase; PNP, purine nucleoside phosphorylase; PRPP, 5-phosphoribosyl-1-pyrophosphate; RNA, ribonucleic acid; Ribose-5P, ribose-5-phosphate; XMP, xanthine monophosphate

There are other proposed mechanisms by which MPA prevents organ rejection These include the induction of apoptosis of activated T lymphocytes [73,74], the inhibition of glycosylation and expression of adhesion molecules resulting in reduced recruitment of lymphocytes and monocytes to sites of inflammation, including

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rejection sites [75-78], as well as the suppression of nitric oxide (NO) production by the depletion of a cofactor of inducible NO synthase, tetrahydrobiopterin, thereby inhibiting the generation of tissue-damaging peroxynitrite at rejection sites [79]

1.2.4.2 Clinical efficacy and safety of MPA in RTx

Three randomized, double-blind, multi-centre clinical trials carried out in America, Australia, Canada and Europe have demonstrated that MMF given at either 2 g/day or

3 g/day in two divided doses, administered in combination with CsA and corticosteroids, was superior to placebo [39] or azathioprine [40,41] in reducing the incidence of acute allograft rejection in adult Caucasian RTxR In terms of safety, all three studies showed that the lower dose of 2 g/day was better tolerated as the incidence of adverse effects, mainly gastrointestinal (GI) disturbances (diarrhoea, abdominal pain and nausea), hematological side effects (anemia, leukopenia and thrombocytopenia) and opportunistic infections (mainly tissue-invasive cytomegalovirus (CMV) infections), was less [39-41] Due to the overall better safety profile, the recommended starting dose of MMF for the prophylaxis of organ rejection

in adult Caucasian RTxR is 2 g/day [39-41] However, for adult African American RTxR who are known to be at higher risk for rejection as compared to non-African Americans, the larger dose of 3 g/day MMF was recommended as it was significantly more effective in reducing the incidence of biopsy-proven acute rejection than 2 g/day MMF or azathioprine [80]

Based on the recommended MMF dose of 2 g/day [39-41], a randomized clinical study carried out in Korea also confirmed the benefit of this MMF dose, administered with CsA and corticosteroids, in reducing the incidence of acute allograft rejection in

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Asians after live-donor RTx as compared to a dual regimen (CsA and corticosteroids) without MMF [81] The efficacy of 2 g/day MMF over azathioprine was also demonstrated in a randomized, single-centre controlled trial conducted locally in Singapore on Asian RTxR receiving concomitant CsA and prednisolone but the dose

of MMF in terms of mg/kg body weight was found to be higher in patients with

leukopenia [82] In Chinese RTx population, MMF dosed at 1.5 g/day was shown to

be comparable in efficacy to the standard dose of 2 g/day [83,84] and this lower dose was associated with reduced occurrence of adverse effects [83] An even lower MMF dose of 1 g/day was also demonstrated to significantly improve graft survival after RTx in Taiwanese patients receiving concomitant CsA and prednisolone [85]

Besides the improved efficacy of including MMF to CsA-based immunosuppression, the addition of 2 g/day MMF to a combination of TAC and prednisone also demonstrated significantly reduced incidence of acute renal allograft rejection in a randomized clinical trial [42] The efficacy of 2 g/day MMF over azathioprine in TAC-based immunosuppression was similarly proven with good safety profile in RTxR [43] In comparison with TAC-SRL [86,87] or CsA-SRL [86] combinations, MMF co-administered with TAC showed comparable patient and graft survival [86,87], comparable [87] or reduced [86] acute rejection rates, and significantly improved renal function and cardiovascular risk profile [86,87]

MMF is also being used clinically in SRL-based immunosuppression as calcineurin inhibitor-free regimens When MMF was administered in combination with SRL and corticosteroids in RTx, efficacy in terms of patient survival, graft

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