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

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

i
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.

ACKNOWLEDGEMENTS

ii
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.

TABLE OF CONTENTS

iii
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

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iv
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

TABLE OF CONTENTS

v
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
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vi
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-MMF-

prednisolone 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-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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vii
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

TABLE OF CONTENTS

viii

6.2 Limitations 293

6.3 Future Perspectives 294

BIBLIOGRAPHY 298
APPENDIX 1 327
APPENDIX 2 375
LIST OF PUBLICATIONS AND CONFERENCE PRESENTATIONS 378

SUMMARY

ix
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.
SUMMARY

x
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.
LIST OF TABLES

xi
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 AUC
ss, 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
LIST OF TABLES

xii
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
LIST OF TABLES

xiii
Table 4.16
Comparison of total MPA C
0
and AUC
ss, 0–12
, normalized by
TBW-adjusted MMF dose, as well as TBW-adjusted MPA
CL
oral
, 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 C
0
and AUC
ss, 0–12
, normalized by
TBW-adjusted MMF dose, as well as TBW-adjusted MPAG

CL
oral
, 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 AUC
ss, 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 AUC

ss, 0–12
(30, 45
or 60 mg⋅h/L) according to the derived regression equations

209
Table 4.22
Distribution of individual t
max
after MMF administration

215
Table 4.23
Limited sampling strategies (LSS) for estimation of total MPA
AUC
ss, 0-12

216
Table 4.24
Predictive performance of limited sampling strategies to
estimate the total MPA AUC
ss, 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
LIST OF TABLES

xiv
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
the start of MMF therapy

328
Table A.2
Characteristics of Asian RTxR receiving SRL-MMF-
prednisolone immunosuppression at six PK sampling days from
the start of MMF therapy

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
LIST OF TABLES

xv
Table A.6
PK parameters of free MPA in Asian RTxR receiving variable
doses of MMF at six PK sampling days from the start of MMF
therapy

336
Table A.7
PK parameters of free 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 C
0
or AUC
ss, 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 CL
r
of MPA and MPAG, as well as CL
f

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 C
0
or AUC
ss, 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 CL
r
of MPA and MPAG, as well as CL
f

of MPAG, in stable Asian RTxR receiving variable steady-state
doses of MMF classified based on the immunosuppressive
regimen received

345
Table A.14
Steady-state PK parameters of total MPA and MPAG in stable
Asian RTxR receiving variable doses of MMF with
concomitant CsA and prednisolone classified based on ethnicity

346
Table A.15
Steady-state PK parameters of free MPA and MPAG in stable
Asian RTxR receiving variable doses of MMF with
concomitant CsA and prednisolone classified based on ethnicity

347
Table A.16
Metabolic ratios of total or free MPAG to MPA C
0
or AUC
ss, 0-
12

in stable Asian RTxR receiving variable steady-state doses of
MMF with concomitant CsA and prednisolone classified based
on ethnicity




348
LIST OF TABLES

xvi
Table A.17
Urinary recoveries and CL
r
of MPA and MPAG, as well as CL
f

of MPAG, in stable Asian RTxR receiving variable steady-state
doses of MMF with concomitant CsA and prednisolone
classified based on ethnicity

349
Table A.18
Steady-state PK parameters of total MPA and MPAG in stable
Asian RTxR receiving variable doses of MMF with
concomitant CsA and prednisolone classified based on gender

350
Table A.19
Steady-state PK parameters of free MPA and MPAG in stable

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

351
Table A.20
Metabolic ratios of total or free MPAG to MPA C
0
or AUC
ss, 0-
12
in stable Asian RTxR receiving variable steady-state doses of
MMF with concomitant CsA and prednisolone classified based
on gender

352
Table A.21
Urinary recoveries and CL
r
of MPA and MPAG, as well as CL
f

of MPAG, in stable Asian RTxR receiving variable steady-state
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
Table A.24
Steady-state PK parameters of MPA and MPAG in UGT1A8
genotype groups for stable Asian RTxR receiving variable
doses of MMF with concomitant CsA and prednisolone

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
Table A.26
Steady-state PK parameters of MPA and MPAG in UGT1A9
promoter region genotype groups for stable Asian RTxR
receiving variable doses of MMF with concomitant CsA and

prednisolone

362
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
365
LIST OF TABLES

xvii
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

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

LIST OF FIGURES

xviii
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
Figure 3.4
Representative chromatograms showing the simultaneous
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
Figure 3.6
Representative chromatograms showing the simultaneous
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
LIST OF FIGURES

xix
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
τ
gall
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 C
0
normalized by TBW-adjusted MMF
dose for early and late post-Tx patients receiving CsA-MMF-
prednisolone immunosuppression over the six study days

124
Figure 4.8
Box plots of (A) free MPA AUC
ss, 0-12
normalized by TBW-
adjusted MMF, (B) TBW-adjusted MPA CL

u
and (C) MPA f
u

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

125
LIST OF FIGURES

xx
Figure 4.9
Box plots of (A) total MPAG C
max
, (B) total MPAG AUC
ss, 0-12
,
(C) free MPAG C
max
and (D) free MPAG AUC
ss, 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 AUC
ss,

0-12
and (B) free MPAG to MPA AUC
ss, 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) CsA-MMF-
prednisolone (n = 53), (B) TAC-MMF-prednisolone (n = 9), (C)
SRL-MMF-prednisolone (n = 3) or (D) MMF-prednisolone
immunosuppression (n = 2)

141
Figure 4.12
Individual plasma concentration-time profiles of total MPAG for
stable Asian RTxR receiving chronic dosing of (A) CsA-MMF-
prednisolone (n = 53), (B) TAC-MMF-prednisolone (n = 9), (C)
SRL-MMF-prednisolone (n = 3) or (D) MMF-prednisolone
immunosuppression (n = 2)

142
Figure 4.13
Individual plasma concentration-time profiles of free MPA for
stable Asian RTxR receiving chronic dosing of (A) CsA-MMF-
prednisolone (n = 53), (B) TAC-MMF-prednisolone (n = 9), (C)
SRL-MMF-prednisolone (n = 3) or (D) MMF-prednisolone
immunosuppression (n = 2)

143
Figure 4.14 Individual plasma concentration-time profiles of free MPAG for
stable Asian RTxR receiving chronic dosing of (A) CsA-MMF-
prednisolone (n = 53), (B) TAC-MMF-prednisolone (n = 9), (C)
SRL-MMF-prednisolone (n = 3) or (D) MMF-prednisolone
immunosuppression (n = 2)

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
LIST OF FIGURES

xxi
Figure 4.17
Box plots of (A) total and (B) free MPA C
0
, 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 AUC
ss, 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 CL
oral
for stable Asian RTxR receiving
variable doses of MMF classified based on the
immunosuppressive regimen received

149
Figure 4.20
Box plots of MPA CL
u

(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 t
max
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 C
0
and
(B) free MPAG to MPA C
0
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
MPA and MPAG AUC
ss, 0-12
before and after dose
normalization, and MPA and MPAG CL

oral
and CL
u
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 C
0
for
the four ethnic groups

156
Figure 4.25
Box plots of (A) total and (B) free MPA C
max
, normalized by
TBW-adjusted MMF dose, in Asian RTxR receiving CsA-
MMF-prednisolone immunosuppression classified based on
gender

160
Figure 4.26
Box plots of (A) total MPA AUC
ss, 0-12
normalized by TBW-
adjusted MMF dose and (B) TBW-adjusted total MPA CL
oral

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

160
Figure 4.27
Box plots of TBW-adjusted MPAG (A) CL
f
and (B) CLu
f
in
Asian RTxR receiving CsA-MMF-prednisolone
immunosuppression classified based on gender

161
LIST OF FIGURES

xxii
Figure 4.28
Relationship between MPA plasma clearance or MPA AUC
ss, 0-
12
, normalized by TBW-adjusted MMF dose, and calculated
creatinine clearance

163
Figure 4.29
Relationship between MPAG plasma clearance or MPAG
AUC
ss, 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 f
u
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 f
u

168
Figure 4.34 Relationship between MPAG formation clearance and calculated
creatinine clearance

168

Figure 4.35
Relationship between total or free MPA AUC
ss, 0-12
and
hemoglobin (Hb), hematocrit (Hct) or red blood cell (RBC)
count

176
Figure 4.36
Correlation of total MPA AUC
ss, 0–12
with TBW-adjusted MMF
dose, (A) before and (B) after omission of outliers

203
Figure 4.37 Correlation of total MPA C
0
with TBW-adjusted MMF dose, (A)
before and (B) after omission of outliers

204
Figure 4.38
Box plots of (A) total MPA C
0
and (B) total MPA AUC
ss, 0–12

against TBW-adjusted MMF dose range

205

Figure 4.39
Correlation of total MPA AUC
ss, 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 AUC
ss, 0-12
and the estimated total MPA AUC
ss, 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
LIST OF FIGURES

xxiii
Figure 5.3
Box plots of (A) total and (B) free MPA C
max
, normalized by
TBW-adjusted MMF dose, according to UGT1A9 -440T>C/-
331C>T genotypes in Asian RTxR receiving CsA-MMF-
prednisolone immunosuppression

251
Figure 5.4
Box plot of free MPA C
max
, 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 C
0
, (B)
total MPAG to MPA AUC
ss, 0–12
, (C) free MPAG to MPA C
0
and
(D) free MPAG to MPA AUC
ss, 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 C
0
and
(B) total MPAG to MPA AUC
ss, 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
Figure 5.7
Box plots of metabolic ratios of (A) total MPAG to MPA C
0
, (B)
total MPAG to MPA AUC
ss, 0–12
, (C) free MPAG to MPA C
0
and
(D) free MPAG to MPA AUC
ss, 0–12
, according to UGT1A7 -
57T>G/33C>A/622T>C genotypes in Asian RTxR receiving

CsA-MMF-prednisolone immunosuppression

256
Figure 5.8
Box plots of metabolic ratios of (A) total MPAG to MPA C
0
, (B)
total MPAG to MPA AUC
ss, 0–12
, (C) free MPAG to MPA C
0
and
(D) free MPAG to MPA AUC
ss, 0–12
, according to UGT1A7
genotypes in Asian RTxR receiving CsA-MMF-prednisolone
immunosuppression

257
Figure 5.9
Box plots of (A) total MPA AUC
ss, 0-12
normalized by TBW-
adjusted MMF dose, (B) TBW-adjusted total MPA CL
oral
, and
metabolic ratios of (C) total MPAG to MPA C
0
and (B) total
MPAG to MPA AUC

ss, 0–12
, in the absence or presence of
haplotype H1 in Asian RTxR receiving CsA-MMF-prednisolone
immunosuppression

263
Figure 5.10
Box plots of (A) total and (B) free MPA C
max
, normalized by
TBW-adjusted MMF dose, and metabolic ratios of (C) total
MPAG to MPA C
0
and (D) free MPAG to MPA C
0
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 CL
f
in the absence or
presence of haplotype H2 in Asian RTxR receiving CsA-MMF-
prednisolone immunosuppression
265
LIST OF FIGURES

xxiv
Figure 5.12

Box plot of total MPA C
max
normalized by TBW-adjusted MMF
dose according to UGT1A7 to 1A10 diplotypes in Asian RTxR
receiving CsA-MMF-prednisolone immunosuppression

266
Figure 5.13
Box plots of metabolic ratios of (A) total MPAG to MPA C
0
, (B)
total MPAG to MPA AUC
ss, 0–12
, (C) free MPAG to MPA C
0
and
(D) free MPAG to MPA AUC
ss, 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
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 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
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
377