Hydroxylation of Phenytoin (PHT) and the Cytochrome P450 (CYP) 2C Subfamily

Abstract

Purpose: Four members of the CYP2C subfamily, CYP2C8, CYP2C9, CYP2C18, and CYP2C19, have been identified in humans, and a number of allelic variants of the CYP2C9, CYP2C18. and CYP2C19 genes associated with metabolic polymorphisms have been reported (Pharmacogenetics) 1994;4:285–99). CYP2C9 is a major enzyme responsible for the formation of 5–(4‐hydroxyphenyl)‐5‐phenylhydantoin (p‐HPPH), a major hydroxylation metabolite of PHT. However, we recently reported that CYP2C19 contributes to the stereoselective hydroxylation of PHT (Br J Clin Pharmacol 1997;43:431–5). To clarify the relation between hydroxylation of PHT and the CYP2C subfamily, we examined their stereoselective para‐hydroxylation properties by using cDNAs expressing CYP2C8, 9, 18, or 19. In addition, the allelic linkage among members of the CYP2C subfamily was evaluated. Methods: PHT (200 μM) and microsomes from yeast expressing human CYP2C8, 9, 18, or 19 (200 pmol of P450/ml) were incubated in a reaction buffer (0.1M Kpi [pH 7.41 and 0.1 mM EDTA 2 Na) with the addition of a NADPH‐generating system at 37°C for 60 min. The PHT metabolites, (R)‐ and (S)‐pHPPH, were analyzed by reverse‐phase high‐performance liquid chromatography. Blood samples were obtained from 175 adult Japanese patients with epilepsy who were treated at the Department of Neuropsychiatry of Kyushu University Hospital, and genomic DNA was isolated from peripheral lymphocytes with an extraction kit. The CYP2C19*1 (wild‐type) gene and two mutant alleles, CYP2C19*2 (G to A in exon 5) and CYP2C*3 (G to A in exon 4), and the CYP2C18wt (wild‐type) gene and two mutant alleles, CYP2C18ml (T to A in exon 2) and CYP2C18m2 (T to C in 5′‐flanking region), were identified by the polymerase chain reaction (PCR) and restriction fragment‐length polymorphism (RFLP) analysis by using specific primers and restriction enzymes. Results: The mean formation rates of (R)‐pHPPH by CYP2C19 and by CYP2C18 were 0.37 and 3.05 pmol/h/pmol P450, respectively. The rates of (S)‐pHPPH formation by CYP2C19, by CYP2C18, and by CYP2C9 were 0.69, 2.84, and 0.68 pmol/h/pmo1 P450, respectively. (R)‐ and (S)‐pHPPH formation by CYP2C8 and (R)‐pHPPH formation by CYP2C9 were not detected; stereoselective hydroxylation was suspected for CYP2C9. The genotyping results for CYP2C18 were completely consistent with those for CYP2C19: CYP2C19*1/*1 (n = 75; 42.98). *2/*2 (n = 11; 6.3%), and *3/*3 (n = 4; 2.3%) homozygotes and *1/*2 (n = 54; 30.9%), *1/*3 (n = 25; 14.38). and *2/*3 (n = 6; 3.4%) heterozygotes were homozygous for CYP2C18wt/wt/m2/m2 and m1/m1 and heterozygous for wt/m2, wt/ml and m2/m1, respectively. Conclusions: Not only CYP2C9 but also CYP2C18 and CYP2C19 contributed to the formation of p‐HPPH. The stereoselective differences in the formation of p‐HPPH among the CYP2C subfamily were elucidated. Because the CYP2C18 and CYP2C19 gene mutations were linked, the poor metabolizer (PM) phenotype of CYP2C19 might also be the PM phenotype of CYP2C18. Evidence collected in vivo and in vitro suggested that CYP2C9 might become a key enzyme when PHT is given to patients with the PM phenotype of CYP2C19.