UNIVERSITI TEKNOLOGI MARA
A PHARMACOGENETIC STUDY OF
CYTOCHROME P450 2C9 (CYP2C9) AND VITAMIN K EPOXIDE REDUCTASE COMPLEX 1 (VKORCl)
IMMACULATE MBONGO LANGMIA
Thesis submitted in fulfillment of the requirements for the degree of
Master of Science
Faculty of Pharmacy
May 2011
A B S T R A C T
A fast and reliable genotyping method for detection of single nucleotide polymorphisms (SNPs) of VKORCl was developed. PCR methods used for detection of CYP2C9 (CYP2C9*1 and CYP2C9*3) SNPs were previously developed in house. The optimized nested allele specific polymerase chain reaction (AS-PCR) method was used to investigate the influence of VKORCl and CYP2C9 in warfarin therapy. Genotype frequencies among warfarin patients were explored and specific clinical parameters of the patients was correlated with VKORCl and CYP2C9 genotypes. A total of 163 (50% of Malay and the remaining Chinese) warfarin patients were recruited after written informed consent. The patients were monitored for a period of six month and their medical records reviewed. Four SNPs including 2 for VKORCl (G-1639A (rs9923231) in the 5 flanking region and C1173T (rs9934438) in exon 2) and 2 for CYP2C9 (CYP2C9*! and CYP2C9*3) were examined in this study. For VKORCl-1639G/A (rs992323i;, three genotypes were detected, A-1639A, G-1629A and G-1639G. For VKORCl-1173C/T (rs9934438) three genotypes were also detected, T1173T, C1173T and C1173C respectively. The variant alleles A-1639A and T1173T were the most frequent. When grouped into the different races, the two genotypes were higher in the Chinese patients than the Malay. The frequency of the A-1639A genotype was 76.5% in the Chinese in contrast to 53.7% in the Malay. The T1173T genotype existed in 76.5 per 100 Chinese patients compared to 45.3 per 100 Malay patients. CYP2C9*1/*1 was detected in 93.3%
of the patients and the remaining were having CYP2C9*l/*3 (6.7%). Following analysis results, variants of VKORCl and CYP2C9 were associated with variability in warfarin dose. Patients with the T1173T genotype required a mean dose of 3.03 mg (SD 1.1) opposed to 4.4 mg (SD 1.4) and 5.6 mg (SD 2.5) for patients with the C1173T and C1173C respectively. Patients with the CYP2C9*l/*3 required significantly lower dose to achieve therapeutic INR levels. Patients with the wild type genotype required 3.5 mg (SD 1.4) of warfarin compared to 2.5 (SD 1.1) mg for patients with CYP2C9*l/*3 to maintain the same INR level of 2.0 to 4.0. This clearly demands for individualized warfarin therapy in the Malaysian population and it can be achieved by genotyping of CYP2C9 and VKORCl. There was a poor relationship between INR values and the amount of doses given to the patients. In terms of environmental factors, analysis results showed that age was a significant predictor but weight and height were not. There was no significant difference in dose between male (3.6 mg SD 1.29) and female (3.4 mg SD 1.54). Patients with the variant genotype (A-1639A and T1173T) experienced the highest bleeding rate. Thirty five (35) patients experienced bleeding during the treatment period.
Key words: Pharmacogenetics, CYP2C9, VKORCl, Genotype, Individualized therapy.
ACKNOWLEDGEMENTS
Thanks to God Almighty who gave me the grace throughout the entire studies. Glory and adoration be unto Him for provision, protection and favor. He was my shield, my comfort and my hope. During difficult moments He gave me assurance and His everlasting peace kept me going till the end of this studies. Blessed be His Holy name forever and ever.
I wish to express my deepest gratitude to my supervisor Professor Mohd Zaki Salleh, I'm grateful for his kindness, advice and fatherly support when I needed them most during the course of this study. I respect him for his unlimited diligence in achieving goals and his pursuit of excellence in everything. Indeed he has been a father, full of love and wisdom.
I count it a privilege for being under his my supervision.
I wish to extend my deep and sincere appreciation to my co-supervisor Assoc. Prof. Dr.
Teh Lek Kek for her consistence support throughout this study. Her wide knowledge, logical advice and persistence encouragement towards a timely and realistic achievement have been of great value for me.
I also wish to appreciate the Dean of Faculty of Pharmacy Prof. Dr. Aishah Adam for her support and encouragement.
My heart felt thanks to Assoc. Prof. Dr. Roziah Mohd Janor for her guidance and contribution in the statistical analysis of this study.
TABLE OF CONTENTS
Page TITLE PAGE
AUTHOR'S DECLARATION ii
ABSTRACT iii ACKNOWLEGEMENTS iv
TABLE OF CONTENTS vi LIST OF TABLES x LIST OF FIGURES xiii LIST OF PALTE xiv LIST OF ABREVIATIONS xv
CHAPTER 1: INTRODUCTION TO PHARMACOGENETICS 1
1.1 Pharmacogenetics 1 1.2 Statement of research problems 3
1.3 Objectives 4 CHAPTER 2: LITERATURE REVIEW 5
2.1 Warfarin 5 2.1.1 Structure of warfarin 6
2.1.2 Pharmacogenetics of warfarin 7 2.1.3 Pharmacodynamics of warfarin and the vitamin K cycle 8
2.1.4 Pharmacokinetics of warfarin 11 2.1.4.1 Absorption and transportation 11
2.1.4.2 Metabolism and Excretion 11
CHAPTER 1
AN INTRODUCTION TO PHARMACOGENETICS
1.1 Pharmacogenetics
The ultimate aim of drug therapy is to achieve therapeutic efficacy with little or no adverse effect. This can be acquired by prescribing the right drug with the right dosage to the right patient and at the right time (Jon et al, 1999). Although non- genetic factors such as age, sex, height and disease or drug interaction may affect drug response, most often clinicians feel challenged when patients with similar non-genetic factors return to the hospital with different outcomes from the same drug (Wendell, 2008). For example, two patients (Mr. A and Mr. B) with similar clinical and environmental parameters, suffering from the same disease were prescribed 5 mg of warfarin and discharged home. Two days later, Mr. B returns to the hospital with severe bleeding while Mr. A responded well. We cannot help but wonder why a drug that is therapeutic in one person may be toxic or even ineffective in another. The cause of this variability lies in inheritance (Wendell, 2008). Genetic make-up of an individual patient is a significant factor that influences both the efficacy of a drug and the probability of the occurrence of adverse drug reactions (ADR) (Takayashi 2003).
Genetic variation in genes that code for drug transporters, metabolizing enzymes and drug targets (receptors) greatly affects the efficacy of most drugs resulting in ADR.
