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The human cytochrome P450 3A4 (CYP3A4) plays a dominant role in the metabolism of numerous clinically useful drugs. Alterations in the activity or expression of this enzyme may account for a major part of the variation in drug responsiveness and toxicity. However, it is generally accepted that most of the known single nucleotide polymorphisms in the coding and 5'-flanking regions are not the main determinants for the large inter-individual variability of CYP3A4 expression and activity. We show that the allelic variation is critically involved in determining the individual total hepatic CYP3A4 mRNA level and metabolic capability. There exists a definite correlation between the total CYP3A4 mRNA level and allelic expression ratio, the relative transcript level ratio derived from the two alleles. Individuals with a low expression ratio, exhibiting a large difference of transcript level between the two alleles, revealed extremely low levels of total hepatic CYP3A4 mRNA, and thus low metabolic capability as assessed by testosterone 6beta-hydroxylation. These results present a new insight into the individualized CYP3A4-dependent pharmacotherapy and the importance of expression imbalance to human phenotypic diversity.

Background: Little is known about differences in the disposition kinetics and pharmacological effects on gastrin levels between lansoprazole and rabeprazole given in a repeated dosing scheme with respect to the polymorphic CYP2C19., Aim: To provide preliminary information that should be considered when prescribing proton-pump inhibitors (PPIs) for the treatment of acid-related diseases with reference to the CYP2C/9 genotypic status., Methods: Helicobacter pylori-negative healthy volunteers were divided into the following three groups (n = 5 each) on the basis of genotyping for CYP2C19: homozygous (hmEMs) and heterozygous extensive metabolizers (htEMs), and poor metabolizers (PMs). All received once-daily 30-mg doses of lansoprazole or 10-mg doses of rabeprazole during an 8-day course in a crossover manner., Results: The relative values for the area under the serum concentration-time curve (AUC) of lansoprazole and rabeprazole in the hmEMs, htEMs, and PMs after the final doses were 1:1.7:3.9 and 1:1.7:3.8, respectively. The relative AUCs of gastrin in the hmEMs, htEMs, and PMs were 1.6:2.6:3.1 for lansoprazole and 1.6:2.6:2.9 for rabeprazole, respectively., Conclusion: The disposition kinetic behavior of the two PPIs is co-segregated with CYP2C19. The magnitude of CYP2C19-dependent drug availability in the systemic circulation and resulting gastrin response appears to be fairly similar between the two drugs within the same CYP2C19 genotypic groups after a multiple-dosing regimen.

Objective: Genetic polymorphisms were identified in the 5'-flanking region of the human CYP2C9 gene, and their effects on the phenotype were evaluated on the basis of the luciferase reporter gene assay and the in vivo pharmacokinetics of phenytoin., Methods: Genetic polymorphisms were screened by polymerase chain reaction-single-strand conformational polymorphism analysis, following sequencing with DNA samples obtained from 50 healthy volunteers and 133 adult epileptic patients. HepG2 hepatoma cells were cotransfected with various sequence patterns of 5'-flanking region-luciferase reporter gene constructs. Pharmacokinetic parameters of phenytoin in relation to the corresponding sequence patterns were estimated by the Bayesian method, and the results were compared with in vitro activities., Results: Genetic analysis revealed the existence of 7 single nucleotide polymorphisms (SNPs). Allele frequencies of T-->C transition at position -1912 (T-1912C), C-1886G, C-1566T, G-1538A, C-1189T, G-982A, and A-162G were 0.019, 0.019, 0.077, 0.019, 0.579, 0.019, and 0.003, respectively. Some mutations occurred simultaneously, and a total of 6 sequence patterns (patterns 1-6) were observed. The luciferase reporter gene assay indicated that the presence of mutation(s) resulted in a reduction in luciferase activity of 41.4% (pattern 2) to 86.8% (pattern 5) compared with the activity of the wild-type construct. The calculated intrinsic clearance of phenytoin was also lower (up to a 40% reduction for pattern 2) when a mutation(s) was present., Conclusion: In addition to the two major mutations in the coding region (CYP2C9*2 and CYP2C9*3 ), mutations in the 5'-flanking region of the human CYP2C9 gene appear to contribute to the large interindividual variability in drug metabolism activity.

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.

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.

Objectives: Evidence exists to suggest that diclofenac is metabolised by CYP2C9. The present study was undertaken in order to evaluate the effect of the single CYP2C9*3 variant on drug metabolism using diclofenac as a probe drug., Methods: A single dose of diclofenac was administered orally to 12 healthy subjects in whom the genotype of CYP2C9 had been determined previously. The disposition kinetics of diclofenac were compared between homozygotes for the wild type (CYP2C9*1/*1, n = 6) and heterozygotes for the Leu359 variant (CYP2C9*1/*3, n = 6)., Results: For diclofenac, the following kinetic parameters were observed in the CYP2C9*1/*1 and CYP2C9*1/*3 subjects, respectively (mean +/- SD): apparent oral clearance (ml/kg/h) 355.8 +/- 56.9 and 484.4 +/- 155.3; area under plasma concentration time curve (microg h/ml) 2.7 +/- 0.7 and 1.9 +/- 0.6. The formation clearance of 4'-hydroxydiclofenac (ml/kg/h) was 63.6 +/- 19.1 in the CYP2C9*1/*1 subjects compared with 75.9 +/- 27.6 in the CYP2C9*1/*3 subjects. There were no significant differences in any of the kinetic parameters for either diclofenac disposition or formation clearance of 4'-hydroxydiclofenac between the two genotype groups., Conclusion: Since the disposition kinetics of diclofenac does not change in subjects with the single CYP2C9*3 mutant allele, it is suggested that the effects of CYP2C9 polymorphisms on the drug metabolism tend to be substrate specific.

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.

Aims: To evaluate the reliability of the omeprazole hydroxylation index as a marker for polymorphic CYP2C19 activity in a Japanese population of healthy young subjects (n = 78) and patients with peptic ulcer (n = 72)., Methods: Healthy subjects were administered a single dose of omeprazole (20 mg), whereas patients received 20 mg daily for at least 1 week. The ratio of the serum concentration of omeprazole to hydroxyomeprazole at 3 h postdose was determined and used as a measure of CYP2C19 activity. The CYP2C19 wild type (wt) gene and four mutant alleles associated with the poor metaboliser phenotype of (S)-mephenytoin, CYP2C19*2 in exon 5, CYP2C19*3 in exon 4, CYP2C19m4 in exon 9, and CYP2C19m3 in the initial codon were analysed., Results: In the healthy volunteer study there was complete concordance between genotype and phenotype. However, eight of the patients who had the EM genotype had a high value for their hydroxylation index, and were classified as phenotypic PMs. No CYP2C19m4 and CYP2C19m3 mutations were detected in the eight mismatched patients. They were all genotypic heterozygous EMs, elderly (> or = 65 years) and/or had hepatic disease. Therefore, impaired CYP2C19 activity combined with partial saturation of omeprazole metabolism during multiple dosing may have contributed to the discrepancy between CYP2C19 genotyping and phenotyping., Conclusion: Although omeprazole has been used instead of mephenytoin as a probe for polymorphic CYP2C19, it does not appear to be reliable enough for clinical application in Japanese patients.

Purpose: The aim of this study was to clarify the effects of genetic polymorphisms of cytochrome P450 (CYP) 2C9 and 2C19 on the metabolism of phenytoin (PHT). In addition, a population pharmacokinetic analysis was performed., Methods: The genotype of CYP2C9 (Arg144/Cys, Ile359/Leu) and CYP2C19(*1, *2 or *3) in 134 Japanese adult patients with epilepsy treated with PHT were determined, and their serum concentrations of 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) enantiomers, being major metabolites of PHT, were measured. A population pharmacokinetic analysis (NONMEM analysis) was performed to evaluate whether genetic polymorphism of CYP2C9/19 affects the clinical use of PHT by using the 336 dose-serum concentration data., Results: The mean maximal elimination rate (Vmax) was 42% lower in the heterozygote for Leu359 allele in CYP2C9, and the mean Michaelis-Menten constants (Km) in the heterozygous extensive metabolizers and the poor metabolizers of CYP2C19 were 22 and 54%, respectively, higher than those without the mutations in CYP2C9/19 genes. (R)- and (S)-p-HPPH/PHT ratios were lower in patients with mutations in CYP2C9 or CYP2C19 gene than those in patients without mutations., Conclusions: Although the hydroxylation capacity of PHT was impaired with mutations of CYP2C9/19, the impairment was greater for CYP2C9. In view of the clinical use of PHT, two important conclusions were derived from this population study. First, the serum PHT concentration in patients with the Leu359 allele in CYP2C9 would increase dramatically even at lower daily doses. Second, the patients with CYP2C19 mutations should be treated carefully at higher daily doses of PHT.