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Hiroshi Yamazaki - One of the best experts on this subject based on the ideXlab platform.
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Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors.
Biopharmaceutics & drug disposition, 2016Co-Authors: Shinya Hosaka, Norie Murayama, Masahiro Satsukawa, Shotaro Uehara, Makiko Shimizu, Kazuhide Iwasaki, Shunsuke Iwano, Yasuhiro Uno, Hiroshi YamazakiAbstract:Cynomolgus monkeys are widely used in drug developmental stages as non-human primate models. Previous studies used 89 compounds to investigate species differences associated with cytochrome P450 (P450 or CYP) function that reported monkey specific CYP2C76 cleared 19 chemicals, and homologous CYP2C9 and CYP2C19 metabolized 17 and 30 human CYP2C9 and/or CYP2C19 substrates/inhibitors, respectively. In the present study, 22 compounds selected from viewpoints of global drug interaction guidances and guidelines were further evaluated to seek potential substrates for monkey CYP2C8, which is highly homologous to human CYP2C8 (92%). Amodiaquine, montelukast, quercetin and rosiglitazone, known as substrates or competitive inhibitors of human CYP2C8, were metabolically depleted by recombinant monkey CYP2C8 at relatively high rates. Taken together with our reported findings of the slow eliminations of amodiaquine and montelukast by monkey CYP2C9, CYP2C19 and CYP2C76, the present results suggest that these at least four chemicals may be good marker substrates for monkey CYP2C8. Copyright © 2016 John Wiley & Sons, Ltd.
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similar substrate specificity of cynomolgus monkey cytochrome p450 2c19 to reported human p450 2c counterpart enzymes by evaluation of 89 drug clearances
Biopharmaceutics & Drug Disposition, 2015Co-Authors: Shinya Hosaka, Norie Murayama, Masahiro Satsukawa, Shotaro Uehara, Makiko Shimizu, Kazuhide Iwasaki, Shunsuke Iwano, Yasuhiro Uno, Hiroshi YamazakiAbstract:Cynomolgus monkeys are used widely in preclinical studies as non-human primate species. The amino acid sequence of cynomolgus monkey cytochrome P450 (P450 or CYP) 2C19 is reportedly highly correlated to that of human CYP2C19 (92%) and CYP2C9 (93%). In the present study, 89 commercially available compounds were screened to find potential substrates for cynomolgus monkey CYP2C19. Of 89 drugs, 34 were metabolically depleted by cynomolgus monkey CYP2C19 with relatively high rates. Among them, 30 compounds have been reported as substrates or inhibitors of, either or both, human CYP2C19 and CYP2C9. Several compounds, including loratadine, showed high selectivity to cynomolgus monkey CYP2C19, and all of these have been reported as human CYP2C19 and/or CYP2C9 substrates. In addition, cynomolgus monkey CYP2C19 formed the same loratadine metabolite as human CYP2C19, descarboethoxyloratadine. These results suggest that cynomolgus monkey CYP2C19 is generally similar to human CYP2C19 and CYP2C9 in its substrate recognition functionality.
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Comparison of cytochrome P450 2C subfamily members in terms of drug oxidation rates and substrate inhibition.
Current drug metabolism, 2012Co-Authors: Toshiro Niwa, Hiroshi YamazakiAbstract:This review focuses on identification of important active-site residues of the cytochrome P450 2C (CYP2C) subfamily in terms of substrate specificity. A meta-analysis was performed on the reported literature regarding (1) values of the Michaelis-Menten constant (K(m)), maximal velocity (V(max)), and intrinsic clearance (V(max)/K(m)) for 74 metabolic reactions of 45 substrates mediated by human CYP2C8, CYP2C9, CYP2C18, and CYP2C19 and (2) inhibition constants (K(i)) for 3 inhibitors. Although the kinetic behaviors of these CYP2C subfamily members depend on the metabolic reaction, the ratios of V(max)/K(m) values for CYP2C19/CYP2C9 and CYP2C8/CYP2C19, but not for CYP2C8/CYP2C9, were more closely correlated with K(m) values than with V(max) values, suggesting that, for many metabolic reactions, differences in affinity may be more important than differences in capacity for the substrate/reaction specificity of the CYP2C subfamily, especially for CYP2C19. In addition, it has been proposed that the residues involved in substrate recognition sites (SRS) 1, SRS 3, and/or SRS 4 are important for the metabolizing capacity and/or the substrate binding of CYP2C9 and CYP2C19. In contrast to the reasonable amount of kinetic data available, there are few reports comparing the effects of inhibitors [inhibitory constant (K(i)) or 50% inhibitory concentration (IC(50))] on metabolic reactions mediated by the CYP2C subfamily. Collectively, these findings provide insights into the contributions of CYP2C subfamily members to drug metabolism and adverse drug interactions.
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Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens.
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2004Co-Authors: Yoshiyuki Yamazaki, Hiroshi Yamazaki, Ken-ichi Fujita, Kazuo Nakayama, Akihiro Suzuki, Katsunori Nakamura, Tetsuya KamatakiAbstract:Abstract We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.
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formation of a dihydroxy metabolite of phenytoin in human liver microsomes cytosol roles of cytochromes p450 2c9 2c19 and 3a4
Drug Metabolism and Disposition, 2000Co-Authors: Tomoko Komatsu, Elizabeth M J Gillam, Hiroshi Yamazaki, Satoru Asahi, Peter F Guengerich, Miki Nakajima, Tsuyoshi YokoiAbstract:Formation of four oxidative metabolites from the anticonvulsant drug phenytoin (DPH) catalyzed by human liver microsomal cytochrome P450 (P450) enzymes was determined simultaneously. Under the conditions in which linearity for formation of 4′-hydroxylated DPH (4′-HPPH; main metabolite) was observed, human liver cytosol increased microsome-mediated DPH oxidation. 3′,4′-Dihydroxylated product (3′,4′-diHPPH) formation was 10 to 40% of total DPH oxidation in the presence of liver cytosol. 3′-Hydroxy DPH formation was catalyzed by only one of the human liver microsomal samples examined and 3′,4′-dihydrodiol formation could not be detected in all samples. In the presence of liver cytosol, 3′,4′-diHPPH formation activity from 100 μM 4′-HPPH was correlated with testosterone 6β-hydroxylation activity and CYP3A4 content. However, 3′,4′-diHPPH formation using 1 or 10 μM 4′-HPPH as a substrate was not correlated with contents of any P450s or marker activities. Of 10 cDNA-expressed human P450 enzymes examined, CYP2C19, CYP2C9, and CYP3A4 catalyzed 3′,4′-diHPPH formation from the primary hydroxylated metabolites (3′-hydroxy-DPH and 4′-HPPH). Fluvoxamine and anti-CYP2C antibody inhibited 3′,4′-diHPPH formation from 10 μM 4′-HPPH in a human liver sample that contained relatively high levels of CYP2C, whereas ketoconazole and anti-CYP3A antibody showed inhibitory effects on the activities in liver microsomal samples in which CYP3A4 levels were relatively high. These results suggest that CYP2C9, CYP2C19, and CYP3A4 all have catalytic activities in 3′,4′-diHPPH formation from primary hydroxylated metabolites in human liver and that the hepatic contents of these three P450 forms determine which P450 enzymes play major roles of DPH oxidation in individual humans.
Joyce A Goldstein - One of the best experts on this subject based on the ideXlab platform.
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Identification of Human CYP2C8 as a Retinoid-Related Orphan Nuclear Receptor Target Gene
The Journal of pharmacology and experimental therapeutics, 2009Co-Authors: Yuping Chen, Sherry J. Coulter, Anton M. Jetten, Joyce A GoldsteinAbstract:Retinoid-related orphan nuclear receptors (RORs) α and γ (NR1F1, -3) are highly expressed in liver, adipose tissue, thymus, and brain and are involved in many physiological processes, such as circadian rhythm and immune function. Enzymes in the cytochrome P450 2C subfamily metabolize many clinically important drugs and endogenous compounds, such as the anticancer drug paclitaxel and arachidonic acid, and are highly expressed in liver. Here, we present the first evidence that RORs regulate the transcription of human CYP2C8. Overexpression of RORα and RORγ in HepG2 cells significantly enhanced the activity of the CYP2C8 promoter but not that of the CYP2C9 or CYP2C19 promoters. Computer analyses, promoter deletion studies, gel shift assays, and mutational analysis identified an essential ROR-responsive element at -2045 base pairs in the CYP2C8 promoter that mediates ROR transactivation. Adenoviral overexpression of RORα and -γ significantly induced endogenous CYP2C8 transcripts in both HepG2 cells and human primary hepatocytes. Knockdown of endogenous RORα and -γ expression in HepG2 cells by RNA interference decreased the expression of endogenous CYP2C8 mRNA by ∼50%. These data indicate that RORs transcriptionally up-regulate CYP2C8 in human liver and, therefore, may be important modulators of the metabolism of drugs and physiologically active endogenous compounds by this enzyme in liver and possibly extrahepatic tissues where RORs are expressed.
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clinical relevance of genetic polymorphisms in the human cyp2c subfamily
British Journal of Clinical Pharmacology, 2001Co-Authors: Joyce A GoldsteinAbstract:The human CYP2Cs are an important subfamily of P450 enzymes that metabolize approximately 20% of clinically used drugs. There are four members of the subfamily, CYP2C8, CYP2C9, CYP2C19, and CYP2C18. Of these CYP2C8, CYP2C9, and CYP2C19 are of clinical importance. The CYP2Cs also metabolize some endogenous compounds such as arachidonic acid. Each member of this subfamily has been found to be genetically polymorphic. The most well-known of these polymorphisms is in CYP2C19. Poor metabolizers (PMs) of CYP2C19 represent approximately 3–5% of Caucasians, a similar percentage of African-Americans and 12–100% of Asian groups. The polymorphism affects metabolism of the anticonvulsant agent mephenytoin, proton pump inhibitors such as omeprazole, the anxiolytic agent diazepam, certain antidepressants, and the antimalarial drug proguanil. Toxic effects can occur in PMs exposed to diazepam, and the efficacy of some proton pump inhibitors may be greater in PMs than EMs at low doses of these drugs. A number of mutant alleles exist that can be detected by genetic testing. CYP2C9 metabolizes a wide variety of drugs including the anticoagulant warfarin, antidiabetic agents such as tolbutamide, anticonvulsants such as phenytoin, and nonsteroidal anti-inflammatory drugs. The incidence of functional polymorphisms is much lower, estimated to be 1/250 in Caucasians and lower in Asians. However, the clinical consequences of these rarer polymorphisms can be severe. Severe and life-threatening bleeding episodes have been reported in CYP2C9 PMs exposed to warfarin. Phenytoin has been reported to cause severe toxicity in PMs. New polymorphisms have been discovered in CYP2C8, which metabolizes taxol (paclitaxel). Genetic testing is available for all of the known CYP2C variant alleles.
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Gene structure of CYP2C8 and extrahepatic distribution of the human CYP2Cs.
Journal of biochemical and molecular toxicology, 1999Co-Authors: Theresa S. Klose, Joyce Blaisdell, Joyce A GoldsteinAbstract:Extrahepatic tissue distribution of the mRNAs for the four human CYP2Cs (2C8, 2C9, 2C18, and 2C19) was examined in kidney, testes, adrenal gland, prostate, brain, uterus, mammary gland, ovary, lung, and duodenum. CYP2C mRNAs were detected by RT-PCR using specific primers for each individual CYP2C. CYP2C8 mRNA was detected in the kidney, adrenal gland, brain, uterus, mammary gland, ovary, and duodenum. CYP2C9 mRNA was detected in the kidney, testes, adrenal gland, prostate, ovary, and duodenum. CYP2C18 mRNA was found only in the brain, uterus, mammary gland, kidney, and duodenum and CYP2C19 mRNA was found only in the duodenum. Immunoblot analysis of small intestinal microsomes detected both 2C9 and 2C19 proteins. In addition, genomic clones for CYP2C8 were sequenced, and long-distance PCR was performed to determine the complete gene structure. CYP2C8 spanned a 31 kb region. Comparative analysis of the 2.4 kb upstream region of CYP2C8 with CYP2C9 revealed two previously unidentified transcription factors sites, C/EBP and HPF-1, and the latter might be involved in hepatic expression. Although CYP2C8 has been shown to be phenobarbital inducible, neither a barbiturate-responsive regulatory sequence (a Barbie box) nor a phenobarbital-responsive enhancer module (PBREM) was found within the upstream region analyzed.
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Human CYP2C19 is a major omeprazole 5-hydroxylase, as demonstrated with recombinant cytochrome P450 enzymes.
Drug metabolism and disposition: the biological fate of chemicals, 1996Co-Authors: W G Karam, Joyce A Goldstein, J. M. Lasker, Burhan I GhanayemAbstract:Omeprazole (OP) is a potent antiulcer drug that is metabolized by liver cytochrome P450 (P450) enzymes. However, the identities of the P450 isoforms responsible for its metabolism have been controversial. 5-Hydroxyomeprazole (5OH-OP) formation cosegregates with the polymorphism of (S)-mephenytoin 4'-hydroxylation in humans, which is now known to be mediated by CYP2C19. Previous in vitro studies have indicated that liver microsomal 50H-OP formation correlates with both (S)-mephenytoin 4'-hydroxylase and CYP3A content. Inhibitor and CYP2C antibody studies also suggested that both enzymes may be involved in the 5-hydroxylation of OP, whereas CYP3A appears to be the predominant enzyme involved in OP sulfone (OP-S) formation. The present studies assessed the contribution of various CYP2C and CYP3A4 enzymes to OP metabolism by using recombinant human enzymes. CYP2C19, CYP2C8, CYP2C18, and CYP2C9 formed a single metabolite with an HPLC retention time identical to that of 5OH-OP. The turnover number for CYP2C19 was 13.4 +/- 1.4 nmol/min/nmol of P450, whereas those for CYP2C8, CYP2C18, and CYP2C9 were 2.2 +/- 0.1, 1.5 +/- 0.1, and approximately equal to 0.5 nmol/min/nmol of P450, respectively. Recombinant human CYP3A4 formed 5OH-OP and OP-S with turnover numbers of 5.7 +/- 1.1 and 7.4 +/- 0.9 nmol/min/nmol of P450, respectively, and formed a minor unidentified metabolite. CYP2C19 had a substantially lower KM for 5OH-OP formation than did CYP3A4, CYP2C8, or CYP2C18. Antibody to CYP2C proteins inhibited approximately equal to 70% of OP 5-hydroxylation at low substrate concentrations, comparable to those that may be encountered at therapeutically relevant doses, whereas antibody to CYP3A4 inhibited approximately equal to 30% of the activity. At high substrate concentrations, the contributions of the two enzymes to OP hydroxylation were roughly comparable (40-50%). In contrast, OP-S formation was completely inhibited by antibody to CYP3A4 proteins. The present study provides the first direct confirmation, using human recombinant P450 enzymes and selective antibody inhibition, that CYP2C19 is a major high affinity OP 5-hydroxylase and CYP3A4 is a low affinity OP-hydroxylating enzyme. The current work also shows, for the first time, that other CYP2C enzymes (CYP2C8, CYP2C9, and CYP2C18) may contribute to OP hydroxylation at high substrate concentrations. In contrast, OP-S was formed principally by CYP3A4.
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Biochemistry and molecular biology of the human CYP2C subfamily.
Pharmacogenetics, 1994Co-Authors: Joyce A Goldstein, Sonia M.f. De MoraisAbstract:The cytochromes P450 (CYP) are a superfamily of hemoproteins which metabolize foreign chemicals as well as a number of endogenous compounds such as steroids. The human CYP2C subfamily appears to principally metabolize a number of clinically used drugs. Four members of this subfamily have been identified in humans: CYP2C8, CYP2C9, CYP2C18, and CYP2C19. CYP2C9 is important in the metabolism certain of therapeutically used drugs including the anticoagulant drug warfarin and a number of nonsteroidal antiinflammatory drugs. A number of allelic variants of CYP2C9 exist in humans, but the effects of these allelic variants on metabolism in vivo remain to be determined. A well-characterized genetic polymorphism occurs in the 2C subfamily which is associated with the metabolism of the anticonvulsant drug mephenytoin. In population studies, individuals can be segregated into extensive and poor metabolizers of mephenytoin. Poor metabolizers are unable to 4'-hydroxylate the S-enantiomer of mephenytoin. There are marked interracial variations in the frequency of the poor metabolizer phenotype which represents 3-5% of Caucasians, but 18-23% of Oriental populations. The mechanism of this polymorphism has been recently elucidated. The enzyme responsible for S-mephenytoin metabolism has been shown to be CYP2C19, and two defects in the CYP2C19 gene have been described in poor metabolizers. The first defect, CYP2C19m1, consists of the creation of an aberrant splice site in exon 5. This defect accounts for approximately 75-85% of Caucasian and Japanese poor metabolizers. A second defect, CYP2C19m2, has been found only in Oriental populations and accounts for the remaining 25% of poor metabolizers in Japanese populations. The availability of genotyping tests for this polymorphism will enhance the assessment of the role of this pathway in clinical studies.
Ichiro Ieiri - One of the best experts on this subject based on the ideXlab platform.
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Impact of Genetic Polymorphisms in CYP2C9 and CYP2C19 on the Pharmacokinetics of Clinically Used Drugs
Drug metabolism and pharmacokinetics, 2012Co-Authors: Takeshi Hirota, Shunsuke Eguchi, Ichiro IeiriAbstract:Human cytochrome P450 (CYP) is a superfamily of hemoproteins which oxidize a number of endogenous compounds and xenobiotics. The human CYP2C subfamily consists of four members: CYP2C8, CYP2C9, CYP2C18 and CYP2C19. CYP2C9 and CYP2C19 are important drug-metabolizing enzymes and together metabolize approximately 20% of therapeutically used drugs. Forty-two allelic variants for CYP2C9 and 34 for CYP2C19 have been reported. The frequencies of these variants show marked inter-ethnic variation. The functional consequences of genetic polymorphisms have been examined, and many studies have shown the clinical importance of these polymorphisms. Current evidence suggests that taking the genetically determined metabolic capacity of CYP2C9 and CYP2C19 into account has the potential to improve individual risk/benefit relationships. However, more prospective studies with clinical endpoints are needed before the paradigm of "personalized medicine" based on the variants can be established. This review summarizes the currently available important information on this topic.
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the effects of genetic polymorphisms of cyp2c9 and cyp2c19 on phenytoin metabolism in japanese adult patients with epilepsy studies in stereoselective hydroxylation and population pharmacokinetics
Epilepsia, 1998Co-Authors: Kohsuke Mamiya, Junta Shimamoto, Hideaki Ninomiya, Jun Imai, Kenji Otsubo, Ichiro Ieiri, Eiji Yukawa, Hiroaki Yamada, Shun HiguchiAbstract:Summary: 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 (Arg'44/Cys, Ile359/Leu) and CYP2C19 (*l, *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 @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 (K,) 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 (5')-pHPPHPHT ratios were lower in patients with mutations in CYP2C9 or CYP2C19 gene than those in patients without rnutations. 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.
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genetic polymorphism of cytochrome p450s cyp2c19 and cyp2c9 in a japanese population
Therapeutic Drug Monitoring, 1998Co-Authors: Miyuki Kimura, Kohsuke Mamiya, Ichiro Ieiri, Akinori Urae, Shun HiguchiAbstract:Genotypings of two mutations (*2 and *3) in CYP2C19 and the amino acid variants (Arg144/Cys, Tyr358/Cys, Ile359/Leu, and Gly417/Asp) in CYP2C9 were carried out in 140 unrelated Japanese subjects. Thirty-three subjects (23.6%) were genotypically identified as poor metabolizers of CYP2C19, and the allele frequencies of the CYP2C19*2 and CYP2C19*3 were 0.35 and 0.11, respectively. The authors' findings are in agreement with the 18% to 23% prevalence of poor metabolizers in the Japanese populations previously phenotyped. In CYP2C9, all subjects were homozygous (CYP2C9*1) for Arg144, Tyr358, Ile359, and Gly417, except for five subjects (3.6%) who were heterozygous for the Leu359 (CYP2C9*3). The frequencies of Arg144, Tyr358, Ile359, Leu359, and Gly417 variants were 1.0, 1.0, 0.982, 0.018, and 1.0, respectively. The low frequency of the Cys144 allele (CYP2C9*2) in the Japanese population is different from the frequency recently found in British subjects (allele frequency, 0.125 to 0.192). The results suggest that the known interindividual variations in the CYP2C9 sequence among Japanese subjects is small, and that Ile359/Leu is one possible site showing interracial polymorphism.
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Hydroxylation of Phenytoin (PHT) and the Cytochrome P450 (CYP) 2C Subfamily
Epilepsia, 1998Co-Authors: Kohsuke Mamiya, Hideaki Ninomiya, Jun Imai, Shun Higuchi, Ichiro Ieiri, Sayaka Miyahar, Nobutada Tashiro, Hiroaki YamadaAbstract:Purpose: Four members of the CYP2C subfamily, CYP2C8, CYP2C9, CYP2C18, and CYP2C19, have been identified in humans, and a number of allelic variants of the CYP2C9, CYP2C18. and CYP2C19 genes associated with metabolic polymorphisms have been reported (Pharmacogenetics) 1994;4:285–99). CYP2C9 is a major enzyme responsible for the formation of 5–(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), a major hydroxylation metabolite of PHT. However, we recently reported that CYP2C19 contributes to the stereoselective hydroxylation of PHT (Br J Clin Pharmacol 1997;43:431–5). To clarify the relation between hydroxylation of PHT and the CYP2C subfamily, we examined their stereoselective para-hydroxylation properties by using cDNAs expressing CYP2C8, 9, 18, or 19. In addition, the allelic linkage among members of the CYP2C subfamily was evaluated. Methods: PHT (200 μM) and microsomes from yeast expressing human CYP2C8, 9, 18, or 19 (200 pmol of P450/ml) were incubated in a reaction buffer (0.1M Kpi [pH 7.41 and 0.1 mM EDTA 2 Na) with the addition of a NADPH-generating system at 37°C for 60 min. The PHT metabolites, (R)- and (S)-pHPPH, were analyzed by reverse-phase high-performance liquid chromatography. Blood samples were obtained from 175 adult Japanese patients with epilepsy who were treated at the Department of Neuropsychiatry of Kyushu University Hospital, and genomic DNA was isolated from peripheral lymphocytes with an extraction kit. The CYP2C19*1 (wild-type) gene and two mutant alleles, CYP2C19*2 (G to A in exon 5) and CYP2C*3 (G to A in exon 4), and the CYP2C18wt (wild-type) gene and two mutant alleles, CYP2C18ml (T to A in exon 2) and CYP2C18m2 (T to C in 5′-flanking region), were identified by the polymerase chain reaction (PCR) and restriction fragment-length polymorphism (RFLP) analysis by using specific primers and restriction enzymes. Results: The mean formation rates of (R)-pHPPH by CYP2C19 and by CYP2C18 were 0.37 and 3.05 pmol/h/pmol P450, respectively. The rates of (S)-pHPPH formation by CYP2C19, by CYP2C18, and by CYP2C9 were 0.69, 2.84, and 0.68 pmol/h/pmo1 P450, respectively. (R)- and (S)-pHPPH formation by CYP2C8 and (R)-pHPPH formation by CYP2C9 were not detected; stereoselective hydroxylation was suspected for CYP2C9. The genotyping results for CYP2C18 were completely consistent with those for CYP2C19: CYP2C19*1/*1 (n = 75; 42.98). *2/*2 (n = 11; 6.3%), and *3/*3 (n = 4; 2.3%) homozygotes and *1/*2 (n = 54; 30.9%), *1/*3 (n = 25; 14.38). and *2/*3 (n = 6; 3.4%) heterozygotes were homozygous for CYP2C18wt/wt/m2/m2 and m1/m1 and heterozygous for wt/m2, wt/ml and m2/m1, respectively. Conclusions: Not only CYP2C9 but also CYP2C18 and CYP2C19 contributed to the formation of p-HPPH. The stereoselective differences in the formation of p-HPPH among the CYP2C subfamily were elucidated. Because the CYP2C18 and CYP2C19 gene mutations were linked, the poor metabolizer (PM) phenotype of CYP2C19 might also be the PM phenotype of CYP2C18. Evidence collected in vivo and in vitro suggested that CYP2C9 might become a key enzyme when PHT is given to patients with the PM phenotype of CYP2C19.
Moon Kyo In - One of the best experts on this subject based on the ideXlab platform.
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AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes
Molecules, 2017Co-Authors: Soon-sang Kwon, Tae Yeon Kong, Jae Chul Cheong, Moon Kyo InAbstract:AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.
Soon-sang Kwon - One of the best experts on this subject based on the ideXlab platform.
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AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes
Molecules, 2017Co-Authors: Soon-sang Kwon, Tae Yeon Kong, Jae Chul Cheong, Moon Kyo InAbstract:AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.
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Inhibitory Effects of Aschantin on Cytochrome P450 and Uridine 5′-diphospho-glucuronosyltransferase Enzyme Activities in Human Liver Microsomes
MDPI AG, 2016Co-Authors: Soon-sang Kwon, Ju-hyun Kim, Hyeon-uk Jeong, Yong Yeon Cho, Hye Suk LeeAbstract:Aschantin is a bioactive neolignan found in Magnolia flos with antiplasmodial, Ca2+-antagonistic, platelet activating factor-antagonistic, and chemopreventive activities. We investigated its inhibitory effects on the activities of eight major human cytochrome P450 (CYP) and uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes of human liver microsomes to determine if mechanistic aschantin–enzyme interactions were evident. Aschantin potently inhibited CYP2C8-mediated amodiaquine N-de-ethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. Aschantin at 100 µM negligibly inhibited CYP1A2-mediated phenacetin O-de-ethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, and CYP2D6-mediated bufuralol 1′-hydroxylation. At 200 µM, it weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively, but did not show inhibition against UGT1A3, UGT1A4, or UGT2B7 up to 200 µM. These in vitro results indicate that aschantin should be examined in terms of potential interactions with pharmacokinetic drugs in vivo. It exhibited potent mechanism-based inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4
