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

Ichiro Ieiri, Hitoshi Tainaka, Toshihiro Morita, Atsuko Hadama, Kohsuke Mamiya, Masakazu Hayashibara, Hideaki Ninomiya, Shigeru Ohmori, Mitsukazu Kitada, Nobutada Tashiro, Shun Higuchi, Kenji Otsubo

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Abstract

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 hatches 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 K(m) of 2.0, 16.5 and (3.8 and 2.9) μmol and V(max) 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 K(m), and Thr359 with a low V(max). 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: K(m) = 6.45 μug/mL, V(max) = 577 mg/kg/d, and V(max)/K(m) = 0.89 L/kg/day. The V(max)/K(m) value in this patient was similar 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.

Original languageEnglish
Pages (from-to)237-244
Number of pages8
JournalTherapeutic drug monitoring
Volume22
Issue number3
DOIs
Publication statusPublished - Jun 1 2000
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmacology (medical)

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