View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ScholarBank@NUS PHARMACODYNAMICS, PHARMACOKINETICS AND PHARMACOGENETICS OF DOXORUBICIN IN SINGAPOREAN BREAST CANCER PATIENTS Fan Lu A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I wish to express my thanks to my supervisors, Dr. Goh Boon Cher, Dr Lee Soo Chin and Associate Professor Lee How Sung, for providing me with the opportunity to pursue research in this field. I am grateful for their kind supports, guidance and scientific enthusiasm during my five years’ training. Through their patience and persistence, my postgraduate experience was immeasurably enriched. Special appreciation goes to Associate Professor Lee How Sung. She has always been ready with advice, encouragement and direction from the time I started to work with her in the Department of Pharmacology. I would like to thank Prof. Philip Moore for his support to my application for a part time study. I would like to acknowledge excellent advice and suggestions from my Ph.D. qualification examination committee members, A/Prof. Theresa, Tan May Chin, A/Prof. Paul Ho, and A/Prof. Boelsterli, Urs Alex. My thesis is the result of much more than individual effort. Thanks are also due to many scientific contributions and assistance of our many lab colleagues, collaborators, as well as friends. I would like to thank: Ms Khoo Yok Moi, Dr Wang Ling Zhi, Mr Guo Jia Yi, Ms Yap Hui Ling, Ms Jamsine Lim, Ms Norita Sukri and Dr Tham Lai Sam for sharing their knowledge, demonstrating bio-analytical techniques and providing their help and encouragement. I also would like to thank: Dr Rorbet Lim, Dr Ross Soo, Dr Yong Wei Peng, Dr Wong Chiung Ing, Dr Lim Siew Eng, Dr Benjamin Chuah and Dr Benjamin Mow from the National University Hospital, for I their kind help and advice in my whole study, especially in the preparation of presentation for American Society of Clinical Oncology annual meeting (2007). Last but not least, I would like to thank my family for their continued support and encouragement. II TABLE OF CONTENTS ACKNOWLEDGEMENTS I TABLE OF CONTENTS III SUMMARY VIII LIST OF TABLES XI LIST OF FIGURES XIII LIST OF ABBREVIATIONS XV LIST OF PUBLICATIONS AND PRESENTATIONS XVIII CHAPTER 1 INTRODUCTION 1 CHAPTER 2 LITERATURE REVIEW 9 2.1 Introduction of doxorubicin 10 2.2 Molecular structure of doxorubicin 10 2.3 Main mechanisms of anticancer cytotoxicity action 11 2.4 The clinical use of doxorubicin for breast cancer 16 2.4.1 Doxorubicin as a single agent for breast cancer 17 2.4.1.1 Efficacy of doxorubicin 17 2.4.1.2 Doxorubicin dose-response relationship 18 2.4.1.3 Dose limiting toxicities of doxorubicin 18 2.4.2 Chemotherapy with doxorubicin and docetaxel for breast cancer 20 2.5 Doxorubicin clinical pharmacokinetics 25 2.5.1 Distribution 25 2.5.2 Metabolism 27 2.5.2.1 Metabolic pathway of doxorubicin 27 2.5.2.2 Major metabolite of doxorubicinol 29 III 2.5.3 Elimination 31 2.6 Inter-patient variations in the PK and PD of doxorubicin 32 2.6.1 Inter-individual variations in PK 32 2.6.2 Inter-individual variations in PD 33 2.7 Reported PK-PD correlations 34 2.8 Inter-ethnic variations in the PD of doxorubicin 35 2.9 The pharmacogenetics of doxorubicin 36 2.9.1 The early studies on the pharmacogenetics of doxorubicin 36 2.9.2 Metabolism of doxorubicin to doxorubicinol 37 2.9.3 Human CBR1 substrates and CBR1 expression 41 2.9.4 Human CBR3 substrates and CBR3 expression 42 2.9.5 Functional alleles on human CBR1 and CBR3 43 2.9.6 Inter-ethnic variations of functional genetic variants on CBR1 46 and CBR3 2.10 Objectives 48 CHAPTER 3 MATERIAL AND METHODS 50 3.1 Clinical trial design and the patient recruitment 51 3.2 Efficacy and toxicity assessment 54 3.3 Chromatographic analysis of doxorubicin and doxorubicinol 57 3.3.1 Reagents and standards 57 3.3.2 Standard solutions, calibration and quality control samples 57 3.3.3 Sample collections and sample preparations 58 3.3.4 Chromatographic separation (HPLC) 59 3.3.5 Method validation 59 3.3.5.1 Chromatography 59 IV 3.3.5.2 Linearity 61 3.3.5.3 Precision and accuracy 63 3.3.5.4 Recovery 64 3.3.5.5 Stability 66 3.4 Pharmacokinetic analysis 69 3.5 Genetic variants on CBR1 and CBR3 71 3.5.1 Genomic DNA isolation 71 3.5.2 PCR amplification and purification 71 3.5.3 DNA sequencing 74 3.6 Cancer-free Asian subject population assay 75 3.7 Tumour tissue transcription analyses on CBR1 and CBR3 75 3.8 Statistical analysis 76 CHAPTER 4 RESULTS AND DISCUSSION 80 4.1 Patient characteristics 81 4.1.1 Characteristics of patients before the first cycle of doxorubicin 81 treatment 4.1.2 Comparison of patient characteristics between the two treatment 84 Arms 4.2 Efficacy 88 4.2.1 Anticancer efficacy 88 4.2.2 Comparison of patient’s anticancer efficacies between the two 89 treatment arms 4.2.3 Discussion 90 4.3 Toxicities 91 4.3.1 Hematologic toxicities 91 V 4.3.1.1 All patients’ hematologic toxicities 91 4.3.1.2 Comparison of patient’s hematologic toxicities between 92 two treatment arms 4.3.2 Non-hematological toxicities 94 4.3.3 Discussion 95 4.4 Clinical pharmacokinetics of doxorubicin 98 4.4.1 Comparison of pharmacokinetic parameters between the two 98 treatment arms 4.4.2 Clinical pharmacokinetics of doxorubicin 100 4.4.3 Discussion 102 4.5 Pharmacokinetic-pharmacodynamic relationships 104 4.5.1 Relationship of doxorubicin efficacy with PK 104 4.5.2 Relationship of hematologic toxicities with PK 108 4.5.3 Discussion 111 4.6 Doxorubicin pharmacogenetics on human CBR1 and CBR3 114 4.6.1 Human CBR1 and CBR3 genetic variants in Singaporean breast 114 cancer patients 4.6.1.1 Identified genetic variants on CBR1 and CBR3 114 4.6.1.2 Allelic frequencies of CBR1 and CBR3 genetic variants 118 4.6.1.3 Linkage disequilibrium 121 4.6.2 Influence of common CBR3 variants on the PK, PD of 122 doxorubicin and the tumour tissue CBR3 expression 4.6.2.1 CBR3 C4Y (11G>A) variant 122 4.6.2.1.1 PK correlations 122 4.6.2.1.2 Hematologic toxicity correlations 122 VI 4.6.2.1.3 Efficacy correlation 123 4.6.2.1.4 Tumour tissue CBR3 expression 123 4.6.2.2 CBR3 730G>A (V244M) variant 128 4.6.3 Influence of common CBR1 variants on the PK of doxorubicin 131 4.6.4 Correlation of leukocyte toxicity 132 4.6.5 Discussion 133 4.7 Inter-ethnic variations on the PD of doxorubicin 138 4.7.1 Inter-ethnic variations on hematologic toxicities 138 4.7.2 Inter-ethnic comparisons of CBR3 genotype 142 4.7.3 Genotype-phenotype correlation after stratifying for ethnicity 147 4.7.4 Discussion 151 CHAPTER 5 CONCLUSIONS 153 CHAPTER 6 FUTURE WORKS 160 REFERENCES 165 APPENDIX – Clinical trial protocol 176 VII SUMMARY Doxorubicin, as one of the most efficacious cytotoxic anticancer drugs, has been clinically used for more than thirty years. Despite its long history of use, the efficacy of treatment and the severity of myelosuppression after chemotherapy vary greatly from patient to patient, thus posing a major obstacle for clinical treatment of patients with doxorubicin. Understanding the sources of this pharmacodynamic variability, especially in terms of pharmacokinetic variability, potentially enhances the prospect of predicting toxicity, and individualising dosing for optimal outcome. Therefore, in the studies we have focused on investigation of the associations of the pharmacodynamics of doxorubicin (doxorubicin-induced hematologic toxicities and the tumour response) with the pharmacokinetic parameters and the genetic variants in CBR1 and CBR3 which encode the two main metabolizing enzymes that extensively reduce doxorubicin to doxorubicinol, the less hematologic and tumoural active metabolite. In this clinical study, ninety-nine female breast cancer patients (64 Chinese, 26 Malays, 7 Indians and 2 other ethnic origins) received the first-line doxorubicin at 75mg/m2 per dose every 3 weeks. Intra-tumoral CBR1 and CBR3 expressions had been investigated before the patients received chemotherapy. Pharmacokinetic data, toxicities, and tumour reductions were evaluated after the first cycle of doxorubicin treatment. Comprehensive sequencing of all coding regions, including the splice-site junctions of CBR1 and CBR3, was performed in the breast cancer patients. The allele frequencies of the important variants identified in the breast cancer patients were also examined in larger cancer-free Asian groups. VIII We found that the patient’s body surface area was not associated with the pharmacokinetics and pharmacodynamics of doxorubicin. However, the wide inter- patient variations observed in leukocyte suppression at nadir may be correlated with the plasma concentration ratios of doxorubicinol to doxorubicin at 1 hr after doxorubicin administration and the non-synonymous coding region variant in CBR3, C4Y (11G>A); whereas the doxorubicin-induced tumour reduction may be related to the pharmacokinetics parameter, K21, (the rate constant of peripheral tissue compartment to central blood compartment) and the genetic variant, CBR3, C4Y (11G>A). The most influential variant of CBR3 (11G>A) appeared to be associated with lower doxorubicinol AUC and lower doxorubicinol AUC/doxorubicin AUC metabolic ratios, suggesting that patients with the G allele may have greater catalytic conversion of doxorubicin to the less active metabolite, doxorubicinol. Consistently, patients with the G allele experienced significantly less leukocyte suppression at nadir and less tumour reduction.