Masahiro Tohkin

Comprehensive UGT1A1 Genotyping in a Japanese Population by Pyrosequencing

Glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), is important in the detoxification and enhanced elimination of a large number of endogenous and exogenous substrates. The human UGT1A gene complex contains at least nine variations of exon 1, common exons 2–5, and a single exon 1 splices to exons 2–5 (1).nnOf the UGT1A isoforms, UGT1A1 is primarily responsible for the glucuronidation of bilirubin in the human liver and can also conjugate phenols, anthraquinones, flavonoids, and a variety of therapeutic drugs and their metabolites (e.g., SN-38, an active irinotecan metabolite) (2)(3). Several functional polymorphisms in UGT1A1 are associated with reduced bilirubin glucuronidation activity and cause hyperbilirubinemia (Gilbert and Crigler–Najjar syndromes).nnUGT1A1 TATA box variants [A(TA)6TAA>A(TA)5/7/8TAA] are associated with enhanced [(TA)5] or reduced [(TA)7/8] UGT1A1 transcription (4). Among them, the (TA)6 and (TA)7 repeats have been reported in Asians. The variant (TA)7 is associated with reduced glucuronidation of SN-38 and bilirubin, as well as the pathogenesis of Gilbert syndrome (5). In addition, a T-to-G substitution at nucleotide −3279 (A of the translational start codon in GenBank accession no. AF297093.1 is nucleotide number 1) in the UGT1A1 phenobarbital-responsive enhancer module reduces transcriptional activity (6).nnThe most common nonsynonymous single-nucleotide polymorphism (SNP) (211G>A) that causes an amino acid alteration (glycine to arginine at codon 71) is found in Asian populations at frequencies of 13–23% (7)(8). The 686C>A (P229Q) variation in the Taiwanese has a frequency of 2.8% (8). Also associated with Gilbert syndrome are 211G>A (G71R) and 686C>A (P229Q). Rare in Japanese and Taiwanese patients is 1456T>G (Y486D), which is associated with the more severe type II Crigler–Najjar syndrome (8)(9). Our previous study demonstrated that …

CYP3A4*16 and CYP3A4*18 Alleles Found in East Asians Exhibit Differential Catalytic Activities for Seven CYP3A4 Substrate Drugs

CYP3A4, the major form of cytochrome P450 (P450) expressed in the adult human liver, is involved in the metabolism of approximately 50% of commonly prescribed drugs. Several genetic polymorphisms in CYP3A4 are known to affect its catalytic activity and to contribute in part to interindividual differences in the pharmacokinetics and pharmacodynamics of CYP3A4 substrate drugs. In this study, catalytic activities of the two alleles found in East Asians, CYP3A4*16 (T185S) and CYP3A4*18 (L293P), were assessed using the following seven substrates: midazolam, carbamazepine, atorvastatin, paclitaxel, docetaxel, irinotecan, and terfenadine. The holoprotein levels of CYP3A4.16 and CYP3A4.18 were significantly higher and lower, respectively, than that of CYP3A4.1 when expressed in Sf21 insect cell microsomes together with human NADPH-P450 reductase. CYP3A4.16 exhibited intrinsic clearances (Vmax/Km) that were lowered considerably (by 84–60%) for metabolism of midazolam, carbamazepine, atorvastatin, paclitaxel, and irinotecan compared with CYP3A4.1 due to increased Km with or without decreased Vmax values, whereas no apparent decrease in intrinsic clearance was observed for docetaxel. On the other hand, Km values for CYP3A4.18 were comparable to those for CYP3A4.1 for all substrates except terfenadine; but Vmax values were lower for midazolam, paclitaxel, docetaxel, and irinotecan, resulting in partially reduced intrinsic clearance values (by 34–52%). These results demonstrated that the impacts of both alleles on CYP3A4 catalytic activities depend on the substrates used. Thus, to evaluate the influences of both alleles on the pharmacokinetics of CYP3A4-metabolized drugs and their drug-drug interactions, substrate drug-dependent characteristics should be considered for each drug.

Specific HLA types are associated with antiepileptic drug-induced Stevens-Johnson syndrome and toxic epidermal necrolysis in Japanese subjects.

AIMnThis preliminary study investigated genomic biomarkers for Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), related to three antiepileptic drugs, zonisamide, phenobarbital and phenytoin.nnnPATIENTS & METHODSnHLA class I and HLA-DRB1 loci were genotyped for Japanese patients with zonisamide-, phenobarbital- or phenytoin-induced SJS/TEN (n = 12, 8 and 9, respectively) and for healthy Japanese volunteers (n = 2878).nnnRESULTSnCarrier frequencies of HLA-A*02:07 in patients with zonisamide-induced SJS/TEN and in the general Japanese population were 41.7 and 6.81%, respectively. Carrier frequencies of HLA-B*51:01 in patients with phenobarbital- and phenytoin-induced SJS/TEN and in controls were 75.0, 55.6 and 15.2%, respectively. HLA-A*02:07 and HLA-B*51:01, in a dominant model, were significantly associated with zonisamide- and phenobarbital-induced SJS/TEN, respectively (Pc = 0.0176 and 0.0042, respectively).nnnCONCLUSIONnOur data suggest that HLA-A*02:07 and HLA-B*51:01 are potential biomarkers for zonisamide- and phenobarbital-induced SJS/TEN, respectively, in Japanese individuals.

The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A.

Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein-protein interactions with GR.

Absence of ethnic differences in the pharmacokinetics of moxifloxacin, simvastatin, and meloxicam among three East Asian populations and Caucasians

Aim To examine whether strict control of clinical trial conditions could reduce apparent differences of pharmacokinetic (PK) parameters among ethnic groups. Methods Open‐label, single dose PK studies of moxifloxacin, simvastatin and meloxicam were conducted in healthy male subjects from three East Asian populations (Japanese, Chinese and Koreans) and one Caucasian population as a control. These three drugs were selected because differences in PK parameters have been reported, even though the backgrounds of these East Asian populations are similar. Moxifloxacin (400 mg) was administered orally to 20 subjects, and plasma and urine levels of moxifloxacin and its metabolite (M2) were measured. Simvastatin (20 mg) was given to 40 subjects, and plasma levels of simvastatin and simvastatin acid were measured. Meloxicam (7.5 mg) was given to 30 subjects and its plasma concentration was determined. Intrinsic factors (polymorphism of UGT1A1 for moxifloxacin, SLCO1B1 for simvastatin, and CYP2C9 for meloxicam) were also examined. Results AUCinf values for moxifloxacin, simvastatin and meloxicam showed no significant differences among the East Asian groups. Cmax values of moxifloxacin and simvastatin, but not meloxicam, showed significant differences. There were no significant differences of data for M2 or simvastatin acid. Genetic analysis identified significant differences in the frequencies of relevant polymorphisms, but these differences did not affect the PK parameters observed. Conclusions Although there were some differences in PK parameters among the three East Asian groups, the present study performed under strictly controlled conditions did not reproduce the major ethnic differences observed in previous studies.

cDNA cloning and expression of a novel CYP3A from the Syrian hamster, CYP3A31

A clone, encoding a cytochrome P450 protein consisting of 501 amino acids, was isolated from a cDNA library constructed from mRNA of Syrian hamster liver. The deduced amino acid sequence of this clone showed a high homology (65 to 81%) with other mammalian CYP3As and hence, this novel isozyme was named CYP3A31. By Northern blotting, using an oligonucleotide specific to CYP3A31, the mRNA for this isozyme was shown to be expressed constitutively in liver and induced by treatment with phenobarbital but repressed by 3-methylcholanthrene or dexamethasone treatments. The increase in mRNA expression by phenobarbital and decrease by dexamethasone corresponded to changes in CYP3A protein as analysed by Western blotting. These indicate that CYP3A31 might constitute one of the major CYP3A isozymes in the hamster.

Cross-Classification of Human Urinary Lipidome by Sex, Age, and Body Mass Index

Technological advancements in past decades have led to the development of integrative analytical approaches to lipidomics, such as liquid chromatography-mass spectrometry (LC/MS), and information about biogenic lipids is rapidly accumulating. Although several cohort-based studies have been conducted on the composition of urinary lipidome, the data on urinary lipids cross-classified by sex, age, and body mass index (BMI) are insufficient to screen for various abnormalities. To promote the development of urinary lipid metabolome-based diagnostic assay, we analyzed 60 urine samples from healthy white adults (young (c.a., 30 years) and old (c.a., 60 years) men/women) using LC/MS. Women had a higher urinary concentration of omega-3 12-lipoxygenase (LOX)-generated oxylipins with anti-inflammatory activity compared to men. In addition, young women showed increased abundance of poly-unsaturated fatty acids (PUFAs) and cytochrome P450 (P450)-produced oxylipins with anti-hypertensive activity compared with young men, whereas elderly women exhibited higher concentration of 5-LOX-generated anti-inflammatory oxylipins than elderly men. There were no significant differences in urinary oxylipin levels between young and old subjects or between subjects with low and high BMI. Our findings suggest that sex, but neither ages nor BMI could be a confounding factor for measuring the composition of urinary lipid metabolites in the healthy population. The information showed contribute to the development of reliable biomarker findings from urine.

Molecular cloning, heterologous expression, and characterization of a novel member of CYP2A in the Syrian hamster

The cDNA clone coding for a novel cytochrome P-450 2A subfamily member (CYP2A16) was isolated from a Syrian hamster liver cDNA library. The deduced amino acid sequence of CYP2A16 showed more than 90% identity with those of rat CYP2A3 and mouse CYP2A4/5. The catalytic activity of CYP2A16 was determined by transient expression of its cDNA in transfected COS7 cells and CYP2A16 was found to have the testosterone 2 beta-, 15 alpha-, and 15 beta-hydroxylases, coumarin 7-hydroxylase, and ethoxycoumarin O-deethylase activities. These enzymatic characteristics of CYP2A16 are different from those of other Syrian hamster CYP2A subfamily members, CYP2A8 and CYP2A9. Northern blot analysis showed that CYP2A16 was expressed in kidney and lung while most of the other CYP2A subfamily members have been reported to be expressed in liver and olfactory. These observations indicated that the Syrian hamster CYP2A16 had unique properties compared with those of other CYP2A subfamily members.

Lack of ethnic differences of moxifloxacin and metabolite pharmacokinetics in East Asian men

This study was designed to investigate ethnic differences in the pharmacokinetics (PKs) of moxifloxacin and its metabolites, M1 (sulfo conjugate) and M2 (acyl-glucuronate), among Japanese, Chinese, and Korean populations, following oral administration. We used a population PK modeling approach using data from a clinical study involving 79 healthy male volunteers. A comprehensive population PK model considering the PK mechanism of moxifloxacin and its metabolites was newly built. The structures of the final model were two-compartment for moxifloxacin and one-compartment for M1 and M2, with first-order absorption with lag time for all three compounds. The formation of M1 and M2 from moxifloxacin via a first-pass effect and subsequent metabolic clearance in the system were also modeled. Lean body mass on the central volume of distribution (Vc) and estimated glomerular filtration rate on renal clearance (CLr) were identified as covariates of PKs of moxifloxacin. Additionally, bioavailability was slightly higher in Koreans, whereas CLr, non-renal clearance (CLnr), and Vc were slightly lower. Regarding M1 and M2, body surface area on CLr of M2 and UGT1A1*6 on F of M2 were modeled. Korean ethnicity was observed to influence CLnr of M2, F of M2, and the metabolic clearance of moxifloxacin to M2. However, the exposure levels of moxifloxacin, M1, and M2 in Koreans were comparable to those in Japanese and Chinese because the effects of Korean ethnicity on some PK parameters were counterbalanced. These results suggest that PKs for moxifloxacin and its metabolites among East Asian populations are essentially similar.

Ethnic Difference in the Pharmacodynamics‐efficacy Relationship of Dipeptidyl Peptidase‐4 Inhibitors Between Japanese and non‐Japanese Patients: A Systematic Review

A systematic review of the differences in the efficacy of dipeptidyl peptidase‐4 (DPP‐4) inhibitors between Japanese and non‐Japanese subjects was conducted. We searched for randomized controlled trials in patients with type 2 diabetes mellitus (T2DM) that studied the intervention of a DPP‐4 inhibitor once‐daily vs. placebo, as monotherapy or as add‐on therapy. Data regarding placebo‐corrected HbA1c reduction and trough DPP‐4 inhibition rate after ≥12 weeks treatment were extracted. In the 12 eligible studies, linear regression analysis revealed that the hemoglobin A1c (HbA1c) reduction at each DPP‐4 inhibition level was larger in studies involving Japanese patients than in studies involving non‐Japanese patients, with statistical significance between the two groups (P < 0.0001). Sensitivity analysis excluding studies of add‐on therapies supported the robustness of the result. Our study indicated that DPP‐4 inhibitors show greater efficacy in Japanese patients than in non‐Japanese patients, which may be an important consideration in the global development strategy of new diabetic medications.