The Investigation of the frequency of cytchrome P-450 2C9 (CYP2C9) and the correlation between genotype/phenotype using phenytoin

ABSTRACT M.S. Thesis THE INVESTIGATION OF THE FREQUENCY OF CYTOCHROME P4502C9 {CYP2C9) AND THE CORRELATION BETWEEN GENOTYPE/PHENOTYPE USING PHENYTOIN Ecz. Pınar GÜZELBEY Gaziantep University-Institute of Health Sciences Department of Pharmacology Supervisor: Associate Professor A. Şükrü AYNACIO?LU APRIL-2000 Cytochrome P450 (CYP2C9) is one of the enzymes identified to be polymorphic. The activation of this enzyme, which is responsible for the metabolism of phenytoin, S-warfarin, tolbutamide, fluoxetine, amitriptyline and several nonsteroidal antirheumatics including meloxicam differs interindividually and interethnically. Two important mutations which were determined to influence the activation of CYP2C9 are cysteine"! 44 (CYC2C9*2) and Ieucine359 (CYC2C9*3) amino acid variants. The aim of this study was to investigate both the mutation frequencies of CYP2C9*2 and CYP2C9*3 in Turkish individuals due to the fact that CYP2C9 polymorphism has not yet been investigated and whether there is correlation between phenotype and genotype by using phenytoin. Hundred and one Turkish individuals (30 female, 71 male) were included into the study. DNA was extracted from EDTA blood samples from each individual. The two mutation frequencies mentioned above were investigated using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The subjects in the same group were given one dose of 300 mg phenytoin orally for phenotyping and blood samples were taken 12 hours later. The concentration of mphenytoin and it's metabolite 5-(4 para-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) in the serum extracted from blood samples were analyzed by using high performance liquid cromotography (HPLC). The results of the study revealed that the distribution frequency of CYP2C9*1/*1, CYP2C9*1/*2, CYP2C9*2/2 and CYP2C9*1/*3 genotypes were 67%, 13%, 3% and 16% respectively. The frequency of CYP2C9*3/*3, the most influencing factor on CYP2C9 activity, was found to be -1%. The average phenytoin levels were determined to be 4.16 mg/L"1 for genotype CYP2C9*1/*1, 5.52 for CYP2C9*1/*2 and 5.65 for CYP2C9*1/*3. p-HPPH/phenytoin ratios for CYP2C9*1/*1, CYP2C9*1/*2, CYP2C9*2/*2, CYP2C9*1/*3 and CYP2C9*3/*3 were found to be 0.43, 0.26, 0.14, 0.21 and 0.02 respectively. On the basis of the data, it concluded that the mutation frequency of CYP2C9 does not differ from that of some European communities. It was also concluded that approximately 30% of the inter individual variations in the phenytoin level in the serum could be explained by means of genotypes, that the phenytoin level 12 hours after the application could be used in the routine phenotyping and that p-HPPH/phenytoin metabolic ratio could be one of the in vivo values. Key words: Cyctochrome P4502C9 (CYP2C9), polymorphism, phenytoin, pharmacogenetics, Turkish population. [V