P-hydroxylation of phenobarbital: relationship to (S)-mephenytoin hydroxylation (CYP2C19) polymorphism.

The aim of the current study was to compare the pharmacokinetics of phenobarbital (PB) in extensive metabolizers (EMs) and poor metabolizers (PMs) of S-mephenytoin. Ten healthy volunteers (5 EMs and 5 PMs) were given 30 mg PB daily for 14 days. PB and p-hydroxyphenobarbital (p-OHPB) in serum and urine were measured by high-performance liquid chromatography (HPLC). Urinary excretion (12.5% versus 7.7%) and formation clearance (29.8 versus 21.1 mL/h) of p-OHPB, one of the main metabolites of PB, were significantly lower (p < .05) in PMs than in EMs. However, area under the serum concentration-time curve (153.3 in the EMs versus 122.9 microg x h/mL in the PMs), total (210.8 versus 254.9 mL/h) and renal clearance (53.1 versus 66.1 mL/h) of PB were identical between the two groups. To compare the inducibility of CYP2C19, mephenytoin was also given prior to and on the last day of PB treatment. The urinary level of 4'-hydroxymephenytoin was analyzed by a validated gas chromatograpy/mass spectrometry (GC/MS) method. The mephenytoin hydroxylation index did not change in either EMs (1.42 versus 1.42) or PMs (341.4 versus 403.5), showing that CYP2C19 was not induced by treatment with PB. These results indicated that the p-hydroxylation pathway of PB co-segregates with the CYP2C19 metabolic polymorphism. However, the overall disposition kinetics of PB were not different between EMs and PMs, and therefore polymorphic CYP2C19 seems have no major clinical implications.

Catalytic activity of three variants (Ile, Leu, and Thr) at amino acid residue 359 in human CYP2C9 gene and simultaneous detection using single-strand conformation polymorphism analysis.

This study evaluated the catalytic activity of three variants (Ile, Leu, and Thr) at codon 359 of CYP2C9 enzymes expressed in a yeast cDNA expression system, and then established single-strand conformation polymorphism (PCR-SSCP) analysis for simultaneous detection as a screening method. Diclofenac was used for the in vitro experiment, and its hydroxy metabolite (4'-hydroxydiclofenac) was measured by HPLC. To discuss the in vivo effect of the Thr359 variant on the pharmacokinetics of phenytoin, a case report is presented. The efficiency of the SSCP method was evaluated by analyzing DNA samples from a homozygote for Ile359 and a heterozygote for Leu359 or Thr359. To evaluate the interaction between the P450 level and reductase activity, two batches of the Thr359 variant with a different P450:reductase activity ratio (1:4.0 and 1:1.4) were used. The in vitro study revealed that recombinant Ile359, Leu359, and Thr359 (2 batches) possessed a mean Km of 2.0, 16.5 and (3.8 and 2.9) micromol and Vmax of 12.4, 17.9 and (4.4 and 5.1) nmol/min/nmol P450, respectively. Although the magnitude of the change in catalytic efficiency for the Thr359 variant was close to that of the Leu359 variant, the effect of the two variants on diclofenac 4'-hydroxylation appears to be different because Leu359 variant was associated with a high Km, and Thr359 with a low Vmax. No significant differences in the kinetic data were observed between the two Thr359 enzymes, suggesting that low reductase activity in the Thr359 enzyme was not a major determinant in the present in vitro experiment. Estimated pharmacokinetic parameters of phenytoin obtained by the Bayesian method in an epileptic patient who was a heterozygote carrier for Thr359 variant were: Km = 6.45 microg/mL, Vmax = 5.77 mg/kg/d, and Vmax/Km = 0.89 L/kg/day. The Vmax/Km value in this patient was similar to the population mean value (0.90 L/kg/day) in Japanese heterozygotes for the Leu359 variant. Results for PCR-SSCP were in complete agreement with those obtained using established methods. Thus, the PCR-SSCP approach is useful for identifying these three variants of the CYP2C9 gene.

CYP2C19 polymorphism effect on phenobarbitone. Pharmacokinetics in Japanese patients with epilepsy: analysis by population pharmacokinetics.

OBJECTIVE: The aim of this study was to clarify the effect of genetic polymorphisms of CYP2C19 on the pharmacokinetics of phenobarbitone (PB) using a nonlinear mixed-effects model (NONMEM) analysis in Japanese adults with epilepsy. METHODS: A total of 144 serum PB concentrations were obtained from 74 subjects treated with both PB and phenytoin but without valproic acid. All patients were classified into three groups by CYP2C19 genotyping: G1, G2 and G3 were homozygous for the wild type of CYP2C19 (*1/*1), heterozygous extensive metabolizers (EMs), (*1/*2 or *1/*3), and poor metabolizers (PMs), (*2/*2, *2/*3), respectively. All data were analyzed using NONMEM to estimate pharmacokinetic parameters of PB with respect to the CYP2C19 genotype. RESULTS: Thirty-three patients belonged to G1 (44.6%), 35 to G2 (47.3%), and 6 to G3 (8.1%). The total clearance (CL) of PB significantly decreased by 18.8% in PMs (G3) relative to EMs (G1 and G2). The CL tended to be lower in G2 than in G1. CONCLUSION: In this study, we first demonstrated the effect of the CYP2C19 polymorphism on pharmacokinetics of PB by genotyping. The contribution of other metabolic enzymes in the metabolism of PB in humans remains to be elucidated; however, it appears that the disposition of PB is mediated in part by this enzyme. The estimated population clearance values in the three genotype groups can be used to predict the PB dose required to achieve an appropriate serum concentration in an individual patient.