Takahiro Kubota

Genotyping of S‐mephenytoin 4′‐hydroxylation in an extended Japanese population

To assess the genotype pattern of S‐mephenytoin 4′‐hydroxylation in an extended Japanese population.

Genetic polymorphism of UDP-glucuronosyltransferase 2B7 (UGT2B7) at amino acid 268: ethnic diversity of alleles and potential clinical significance.

UGT2B7 catalyses the glucuronidation of a diverse range of drugs, environmental chemicals and endogenous compounds. Hence, coding region polymorphisms of UGT2B7 are potentially of pharmacological, toxicological and physiological significance. Two variant UGT2B7 cDNAs encoding enzymes with either His or Tyr at residue 268 have been isolated. The variants, referred to as UGT2B7*1 and UGT2B7*2, respectively, arise from a C to T transversion at nucleotide 802 of the UGT2B7 coding region. Analysis of genomic DNA from 91 unrelated Caucasians and 84 unrelated Japanese demonstrated the presence of the variant alleles encoding UGT2B7*1 and UGT2B7*2 in both populations. However, while there was an approximately equal distribution of subjects homozygous for each allele in the Caucasian population, subjects homozygous for the UGT2B7*1 allele were over 10-fold more prevalent than UGT2B7*2 homozygotes in Japanese. The frequencies of the UGT2B7*1 and UGT2B7*2 alleles were 0.511 and 0.489, respectively, in Caucasians, and 0.732 and 0.268, respectively, in Japanese. The 95% confidence intervals for the two alleles did not overlap between Caucasians and Japanese. Rates of microsomal androsterone, menthol and morphine (3-position) glucuronidation were determined for genotyped livers from Caucasian donors. Statistically significant inter-genotypic differences were not apparent for any of the three substrates. Although the UGT2B7 polymorphism characterized here is probably not associated with altered enzyme activity, the results highlight the need to consider ethnic variability in assessing the consequences of UGT polymorphisms.

Metabolism of warfarin enantiomers in Japanese patients with heart disease having different CYP2C9 and CYP2C19 genotypes

To determine whether genetic polymorphism of cytochrome P450 (CYP) 2C9 or 2C19 affects the in vivo metabolism of warfarin enantiomers.

Renormalization group estimate of the hadronic decay width of the Higgs boson

Abstract The total hadronic decay width of the Weinberg-Salam type Higgs boson is estimated in QCD for the Higgs boson mass much larger than the ordinary hadronic mass scale, by use of the operator product expansion and renormalization group equation. We give an explicit formula for the decay width in terms of quark masses including strong interaction corrections up to the next-to-leading order. A numerical analysis of the hadronic decay width of the Higgs boson is made in the six-quark model. The next-to-leading order correction is found to be significant, e.g., 30-20% of the leading term for mH of oue interest, mH ⪅ 1 TeV. Application of our scheme to the decay rates of heavy Higgs bosons of other types is also discussed.

Critical roles of residues 36 and 40 in the phenol and tertiary amine aglycone substrate selectivities of UDP-glucuronosyltransferases 1A3 and 1A4

Despite high sequence identity, UGT1A3 and UGT1A4 differ in terms of substrate selectivity. UGT1A3 glucuronidates the planar phenols 1-naphthol (1-NP) and 4-methylumbelliferone (4-MU), whereas UGT1A4 converts the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP) to quaternary ammonium glucuronides. Residues 45 to 154 (which incorporate 21 of the 35 amino acid differences) and 45 to 535 were exchanged between UGT1A3 and UGT1A4 to generate UGT1A3-4(45–535), UGT1A3-4(45–154)-3, UGT1A4-3(45–535), and UGT1A4-3(45–154)-4 hybrid proteins. Although differences in kinetic parameters were observed between the parent enzymes and chimeras, UGT1A4-3(45–535) and UGT1A4-3(45–154)-4 [but not UGT1A3-4(45–535) and UGT1A3-4(45–154)-3] retained the capacity to glucuronidate LTG and TFP. Likewise, UGT1A3-4(45–535) and UGT1A3-4(45–154)-3 retained the capacity to glucuronidate 1-NP and 4-MU, but UGT1A4-3(45–535) and UGT1A4-3(45–154)-4 exhibited low or absent activity. Within the first 44 residues, UGT1A3 and UGT1A4 differ in sequence at positions 36 and 40. “Reciprocal” mutagenesis was performed to generate the UGT1A3(I36T), UGT1A3(H40P), UGT1A4(T36I), and UGT1A4 (P40H) mutants. The T36I and P40H mutations in UGT1A4 reduced in vitro clearances for LTG and TFP glucuronidation by >90%. Conversely, the I36T and H40P mutations in UGT1A3 reduced the in vitro clearances for 1-NP and 4-MU glucuronidation by >90%. Introduction of the single H40P mutation in UGT1A3 conferred LTG and TFP glucuronidation, whereas the single T36I mutation in UGT1A4 conferred 1-NP and 4-MU glucuronidation. Thus, residues 36 and 40 of UGT1A3 and UGT1A4 are pivotal for the respective selectivities of these enzymes toward planar phenols and tertiary amines, although other regions of the proteins influence binding affinity and/or turnover.

Effects of various CYP2D6 genotypes on the steady-state plasma concentrations of risperidone and its active metabolite, 9-hydroxyrisperidone, in Japanese patients with schizophrenia.

The effects of various CYP2D6 genotypes on the steady-state plasma concentrations (Css) of risperidone and its active metabolite, 9-hydroxyrisperidone, were studied in 85 Japanese schizophrenic patients (27 men and 58 women) treated with 6 mg/d risperidone for at least 2 weeks. Plasma concentrations of risperidone and 9-hydroxyrisperidone were measured using liquid chromatography–tandem mass spectrometry. The patients had the following CYP2D6 genotypes: wild-type (wt) /wt (40 patients), CYP2D6 * 10 (* 10)/ wt (28), CYP2D6 * 5 (* 5)/ wt (8), * 10/ * 10 (5), * 5/ * 10 (3), and CYP2D6 * 4/CYP2D6 * 14 (1), respectively. The Css values of risperidone and 9-hydroxyrisperidone were corrected to the median body weight of 58 kg. The medians (ranges) of the Css of risperidone in the aforementioned genotype groups were 2.2 (0.37–35.7), 6.4 (2.1–26.5), 12.3 (4.7–39.5), 19.4 (13.4–26.4), 64.0 (41.6–68.8), and 91.8 nmol/L. Those values for risperidone-to-9-hydroxyrisperidone ratio were 0.03 (0.01–0.33), 0.06 (0.03–0.19), 0.14 (0.07–0.29), 0.28 (0.25–0.38), 0.48 (0.38–0.58), and 2.35, respectively. The Css of risperidone was significantly (P < 0.05 or P < 0.001) different among the four genotype groups (wt/wt, * 10/wt, * 5/wt, and * 10/ * 10), except between the * 5/wt and * 10/ * 10 groups. Also, the risperidone-to-9-hydroxyrisperidone ratio significantly (P < 0.005 or P < 0.001) differed among these genotype groups. No significant differences were found in the Css of 9-hydroxyrisperidone and the active moiety (the Css of risperidone plus 9-hydroxyrisperidone) among these genotype groups. This study confirms previous findings that the CYP2D6 status affects the Css of risperidone via its strong regulation of 9-hydroxylation of risperidone. However, similar active moiety of risperidone among different genotype groups suggests that the determination of the CYP2D6 genotype has little importance for clinical situations.

Effects of CYP2D6 Genotypes on Plasma Concentrations of Risperidone and Enantiomers of 9‐Hydroxyrisperidone in Japanese Patients with Schizophrenia

It has been shown that risperidone (+)‐9‐hydroxylation is enantioselectively catalyzed by the polymorphic CYP2D6 in human liver. This study aimed to examine the effect of CYP2D6 genotype on (+)‐9‐hydroxylation of risperidone in schizophrenic patients. Subjects were 38 Japanese schizophrenic inpatients receiving 6 mg/day of risperidone. Plasma concentrations of risperidone and (+)‐ and (−)‐9‐hydroxyrisperidone at steady state were quantified using LC/MS/MS and HPLC with α1 acid‐AGP chiral column, respectively. The CYP2D6*5(*5) and *10 alleles were identified using polymerase chain reaction (PCR) methods. Twenty patients had no mutated allele, 14 had one mutated allele, and 4 had two mutated alleles. There were significant differences in the steady‐state plasma concentrations of risperidone (ANOVA; p < 0.0001) among the three genotype groups, while the CYP2D6 genotype did not affect the steady‐state plasma concentrations of (+)‐9‐hydroxyrisperidone (p = 0.314) or (−)‐9‐hydroxyrisperidone (p = 0.957). The concentration ratio of risperidone to 9‐hydroxyrisperidone was strongly dependent on the CYP2D6 genotypes. This study suggests that CYP2D6 activity strongly influences the steady‐state plasma concentrations of risperidone and risperidone/9‐hydroxyrisperidone concentration ratios but is unlikely to determine enantioselectivity in the steady‐state plasma concentrations of 9‐hydroxyrisperidone in the clinical situation.

Effects of genetic defects in the CYP2C19 gene on the N-demethylation of imipramine, and clinical outcome of imipramine therapy

Abstract  The relationship between the genetic polymorphism of S‐mephenytoin 4′‐hydroxylation catalyzed by CYP2C19 and the N‐demethylation of imipramine was examined in 10 Japanese depressed patients. Five patients, who were poor metabolizers of S‐mephenytoin, were determined to be either homozygous for a mutation in exon 5 or heterozygous for mutations in exon 4 and exon 5 of the CYP2C19 gene. In contrast, five patients, who were extensive metabolizers, had no mutations. The demethylation index (the desipramine/imipramine ratio) was significantly lower in patients with genetic defects. Plasma levels of imipramine and 2‐hydroxyimipramine normalized by the daily dose (mg) per weight (kg) were significantly higher in patients with genetic defects. This suggests that the N‐demethylation of imipramine is impaired in patients with genetic defects in the CYP2C19 gene, and that genotype determination may be useful in preventing side effects induced by unexpectedly elevated levels of imipramine.

Frequent occurrence of CYP2D6*10 duplication allele in a Japanese population.

Allele-specific long-polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (RFLP) and haplotype analysis using XbaI and EcoRI were used to determine whether gene duplication of CYP2D6*10 exists in a Japanese population of 162 healthy subjects. Based on the results of PCR and haplotype analysis, the frequencies of CYP2D6*1X2, CYP2D6*2X2 and CYP2D6*10X2 in the Japanese population were estimated to be 0.3, 0.3 and 0.6%, respectively. The results suggest that duplicated alleles of CYP2D6*10 exist in the Japanese population and that it may be one of the factors affecting the capacity of Japanese to metabolize various CYP2D6 substrate drugs.

Frequency distribution of thiopurine S-methyltransferase activity in red blood cells of a healthy Japanese population.

Thiopurine S-methyltransferase (TPMT), which exhibits a genetic polymorphism, plays an important role in the metabolism of thiopurine drugs such as mercaptopurine, thioguanine, and azathioprine. To determine the frequency distribution of TPMT activity in 157 Japanese subjects with different TPMT genotypes, ie, TPMT*1/*1 and TPMT*1/*3, the authors measured levels of 6-methylmercaptopurine formed from 6-mercaptopurine in red blood cells lysates by HPLC. The TPMT activities in our Japanese subjects ranged from 11.0 to 42.6 pmol/h/mgHb. Although the mean value of TPMT activities in 6 subjects with TPMT*1/*3C (20.3 ± 8.1 pmol/h/mgHb) was 25% lower than that in 151 subjects with TPMT*1/*1 (27.0 ± 5.1 pmol/h/mgHb), there was overlap. The ranges of TPMT activity in subjects with TPMT*1/*1 and those with TPMT*1/*3C were similar. The median values in TPMT*1/*3C and TPMT*1/*1 individuals were 20.1 (11.0–31.2) and 26.8 pmol/h/mgHb (15.7–42.7), respectively (Mann-Whitney U-test: median difference 6.7 pmol/h/mgHb, 95% CI 0–25.5, P < 0.05). This observation may have relevance for the use of 6-mercaptopurine and azathioprine as therapeutic agents in Japanese patients.