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.
Ieiri
I
I
Department of Hospital Pharmacy, Faculty of Medicine, Tottori University, Yonago, Japan.
Tainaka
H
H
Morita
T
T
Hadama
A
A
Mamiya
K
K
Hayashibara
M
M
Ninomiya
H
H
Ohmori
S
S
Kitada
M
M
Tashiro
N
N
Higuchi
S
S
Otsubo
K
K
D000328:Adult; D000483:Alleles; D019943:Amino Acid Substitution; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D002384:Catalysis; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004008:Diclofenac / Q000378:metabolism; D004827:Epilepsy / Q000201:enzymology / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D007527:Isoenzymes; D011994:Recombinant Proteins; D007532:Isoleucine; D004008:Diclofenac; D013912:Threonine; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; D007930:Leucine