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Hiroshi Yamazaki - One of the best experts on this subject based on the ideXlab platform.
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efavirenz clearances in vitro and in vivo in six cynomolgus monkeys associated with polymorphic Cytochrome P450 2C9 and simulated by individual physiologically based pharmacokinetic models
Biopharmaceutics & Drug Disposition, 2017Co-Authors: Masahiro Utoh, Tomonori Miura, Takashi Kusama, Shotaro Uehara, Makiko Shimizu, Yasuhiro Uno, Hiroshi YamazakiAbstract:Cynomolgus monkey Cytochrome P450 2C9 (formerly known as P450 2C43) variation was reportedly associated with metabolic clearance of the antiretroviral drug efavirenz in vivo (in three wild-type, one heterozygote and two homozygote animals), being unlikely in the case of human P450 2B6-dependent efavirenz clearance. In this study, the liver microsomal elimination rates of efavirenz for the same individual animals previously treated with intravenous/oral administrations of efavirenz showed significant reductions associated with the P450 2C9 p.[(I112L)] genotype (p < 0.05). Simulations of efavirenz clearance after oral administrations in individual cynomolgus monkeys were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling consisting of gut, liver and central compartments. The modeled hepatic intrinsic clearances were also significantly associated with the P450 2C9 genotypes, however, absorption rate constants or volumes of the systemic circulation were not likely determining factors for the individual efavirenz clearance variations in the six cynomolgus monkeys. This study provides important information to help simulate the clearances of efavirenz and related medicines associated with polymorphic P450 2C9 in individual cynomolgus monkeys, thereby facilitating the calculation of the fraction of liver microsomal clearance for estimating in vivo drug clearance with simplified PBPK modeling.
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in vivo individual variations in pharmacokinetics of efavirenz in cynomolgus monkeys genotyped for Cytochrome P450 2C9
Biopharmaceutics & Drug Disposition, 2016Co-Authors: Kazuhide Iwasaki, Masahiro Utoh, Shotaro Uehara, Yasuhiro Uno, Shinya Hosaka, Yusuke Kitsugi, Kanami Ikeda, Takahiro Yoshikawa, Hiroshi YamazakiAbstract:Cynomolgus monkeys are frequently used in preclinical studies for new drug development due to their evolutionary closeness to humans. An antiretroviral drug efavirenz is a typical probe substrate for human Cytochrome P450 (P450) 2B6, but is mainly metabolized by cynomolgus monkey P450 2C9. In this study, plasma concentrations of efavirenz were assessed in six cynomolgus monkeys genotyped for P450 2C9 c.334A > C (I112L) (three wild-type, one heterozygote, and two homozygotes) by high performance liquid chromatography with tandem mass spectrometry. After intravenous administration at a dose of 1.0 mg/kg, biphasic plasma elimination curves of efavirenz were seen in these cynomolgus monkeys. Plasma concentrations of primary metabolite 8-hydroxyefavirenz (1 hour after treatment, with hydrolysis by β-glucuronidase) in wild-type group mean was significantly higher (4.0-fold) than the combined heterozygous and homozygous group mean. Area under the plasma concentration-time curve values of efavirenz in the homozygous group monkey after oral administration at a dose of 2.0 mg/kg was significantly higher (2.0-fold) than the combined wild-type and heterozygous group. These results collectively indicated that P450 2C9 c.334A > C (I112L) variation was associated with efavirenz metabolic clearance in vivo. Cynomolgus P450 2C9 polymorphism might account for interindividual variations of efavirenz metabolism in cynomolgus monkeys. This article is protected by copyright. All rights reserved.
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comprehensive evaluation for substrate selectivity of cynomolgus monkey Cytochrome P450 2C9 a new efavirenz oxidase
Drug Metabolism and Disposition, 2015Co-Authors: Shinya Hosaka, Shotaro Uehara, Makiko Shimizu, Yasuhiro Uno, Norie Murayama, Masahiro Satsukawa, Kazuhide Iwasaki, Shunsuke Iwano, Hiroshi YamazakiAbstract:Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the Cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography–mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans.
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monkey liver Cytochrome P450 2C9 is involved in caffeine 7 n demethylation to form theophylline
Xenobiotica, 2013Co-Authors: Masahiro Utoh, Makiko Shimizu, Yasuhiro Uno, Shinya Hosaka, Norie Murayama, Kazuhide Iwasaki, Yui Onose, Hideki Fujino, Hiroshi YamazakiAbstract:Abstract1. Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human Cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline.2. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes.3. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4′-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably wit...
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comparative studies on the catalytic roles of Cytochrome P450 2C9 and its cys and leu variants in the oxidation of warfarin flurbiprofen and diclofenac by human liver microsomes
Biochemical Pharmacology, 1998Co-Authors: Hiroshi Yamazaki, Joyce A Goldstein, Kiyoshi Inoue, Koji Chiba, Naoki Ozawa, Takao Kawai, Yasuhiko Suzuki, Peter F Guengerich, Tsutomu ShimadaAbstract:Abstract S -Warfarin 7-hydroxylation, S -flurbiprofen 4′-hydroxylation, and diclofenac 4′-hydroxylation activities were determined in liver microsomes of 30 humans of which 19 were wild-type (Arg144 · Ile359), 8 were heterozygous Cys (Cys144 · Ile359), and 3 were heterozygous Leu (Arg144 · Leu359) allelic variants of the Cytochrome P450 2C9 ( CYP 2 C 9) gene. All of the human samples examined contained P450 protein(s) immunoreactive with anti-CYP2C9 antibodies in liver microsomes. Individuals with the Cys144 allele of CYP 2 C 9 had similar, but slightly lower, activities for the oxidations of these substrates than those of wild-type CYP 2 C 9. One of the three human samples heterozygous for the Leu359 allele had very low V max and high K m values for the oxidation of three substrates examined, while the other two individuals gave kinetic parameters comparable to those seen in the wild-type and Cys144 CYP 2 C 9. Reverse transcriptase-polymerase chain reaction analysis, however, showed that all of the three human samples with the heterozygous Leu359 variant were found to express both Ile359 and Leu359 variants at relatively similar extents in liver RNA of three humans. These results suggest that the Cys144 variant of CYP2C9 catalyzes the CYP2C9 substrates at rates comparative to, but slightly lower than, those of wild-type CYP2C9, while the Leu359-allelic variant has slower rates for the oxidation of these drug substrates. Activities for the oxidation of these CYP2C9 substrates in humans with heterozygous Leu359 allele is likely to be dependent on the levels of expression of each of the wild- and Leu-variants in the livers. However, one of the humans with a heterozygous Leu allele was found to have very low activities towards the oxidation of CYP2C9 substrates. The basis of this defect in catalytic functions towards CYP2C9 substrates is unknown.
Shufeng Zhou - One of the best experts on this subject based on the ideXlab platform.
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clinical drugs undergoing polymorphic metabolism by human Cytochrome P450 2C9 and the implication in drug development
Current Medicinal Chemistry, 2011Co-Authors: Zhi Wei Zhou, Shufeng ZhouAbstract:CYP2C9 metabolizes more than 100 clinically used drugs including phenytoin, S-warfarin, tolbutamide, glipizide, diclofenac, and losartan with varying contributions. CYP2C9 is considered one of the most important CYPs, with substrate specificity typical of many new chemical entities (i.e. lipophilic bases). A large interindividual variation has been identified for the CYP2C9 activity and for the clinical response to the therapeutics metabolised by the enzyme. So far, at least 33 variants of CYP2C9 (*2 through to *34) have been identified. CYP2C9 is one of the clinically significant drug metabolising enzymes that demonstrates genetic variants with significant phenotype and clinical outcomes. This review updates our current knowledge on the polymorphic metabolism of drugs by CYP2C9 and discusses its implications in drug development. The authors have searched through computer-based literatures by full text search in Medline (via Pubmed), ScienceDirect, Genetics Abstracts (CSA), SCOPUS, Chemical Abstracts, Current Contents Connect (ISI), Cochrance Library, CINAHL (EBSCO), CrossRef Search and Embase (all from inception to October 31 2010). A comprehensive literature search has identified 32 drugs that are subject to CYP2C9-mediated polymorphic metabolism. Drugs that are subject to polymorphic metabolism with clinical significance include nine nonsteroidal anti-inflammatory agents, six sulfonylurea antidiabetic drugs and, most critically, three oral coumarin anticoagulants. Polymorphisms in CYP2C9 have the potential to affect the clearance and clinical response of CYP2C9 substrate drugs with low therapeutic indices such as warfarin, phenytoin, and certain antidiabetic drugs. Warfarin has served as a model drug of how pharmacogenetics can be employed to achieve maximum efficacy and minimum toxicity. Minimizing interindividual variability in drug exposure due to CYP2C9 polymorphisms is an important goal in drug development and discovery.
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polymorphisms of human Cytochrome P450 2C9 and the functional relevance
Toxicology, 2010Co-Authors: Shufeng Zhou, Zhi Wei Zhou, Min HuangAbstract:Human Cytochrome P450 2C9 (CYP2C9) accounts for ∼20% of hepatic total CYP content and metabolizes ~15% clinical drugs such as phenytoin, S-warfarin, tolbutamide, losartan, and many nonsteroidal anti-inflammatory agents (NSAIDs). CYP2C9 is highly polymorphic, with at least 33 variants of CYP2C9 (*1B through *34) being identified so far. CYP2C9*2 is frequent among Caucasians with ~1% of the population being homozygous carriers and 22% are heterozygous. The corresponding figures for the CYP2C9*3 allele are 0.4% and 15%, respectively. There are a number of clinical studies addressing the impact of CYP2C9 polymorphisms on the clearance and/or therapeutic response of therapeutic drugs. These studies have highlighted the importance of the CYP2C9*2 and *3 alleles as a determining factor for drug clearance and drug response. The CYP2C9 polymorphisms are relevant for the efficacy and adverse effects of numerous NSAIDs, sulfonylurea antidiabetic drugs and, most critically, oral anticoagulants belonging to the class of vitamin K epoxide reductase inhibitors. Warfarin has served as a practical example of how pharmacogenetics can be utilized to achieve maximum efficacy and minimum toxicity. For many of these drugs, a clear gene-dose and gene-effect relationship has been observed in patients. In this regard, CYP2C9 alleles can be considered as a useful biomarker in monitoring drug response and adverse effects. Genetic testing of CYP2C9 is expected to play a role in predicting drug clearance and conducting individualized pharmacotherapy. However, prospective clinical studies with large samples are warranted to establish gene-dose and gene-effect relationships for CYP2C9 and its substrate drugs.
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new insights into the structural features and functional relevance of human Cytochrome P450 2C9 part i
Current Drug Metabolism, 2009Co-Authors: Zhi Wei Zhou, Li Ping Yang, Ming Q Wei, Shufeng ZhouAbstract:Part I of this article published in the same issue of Current Drug Metabolism discussed the substrate specificity, inhibitor selectivity and structure-activity relationship (SAR) of human CYP2C9. The features of CYP2C9 pharmacophore and SAR models have been elaborated. Part II of this article will address the homology models of CYP2C9, data from site-directed mutagenesis studies, and crystal structural features of CYP2C9. The heteroactivation of CYP2C9 and its interactions with other CYPs will also be discussed. A number of ligand-based and homology models of CYP2C9 have been reported and this has provided insights into the binding of ligands to the active site of CYP2C9. Site-directed mutagenesis studies have revealed that a number of residues (e.g. R97, F110, F114, R132, R144, D293, F476 and A477) play an important role in ligand binding and determination of substrate specificity. The resolved crystal structures of CYP2C9 have confirmed the importance of these residues in substrate recognition and ligand orientation. Currently, there are three X-ray structures of the human CYP2C9 in Protein Database (PDB): one ligand-free protein (1OG2), and two in complex with Swarfarin (1OG5) or flurbiprofen (1R9O). The published structures of 1OG2 and 1OG5 differ in comparison with 1R9O in residues 30—53 of N-termini, residues 97—121 of B/C-loops, and residues 196—233 of helix F and F/G-loops. CYP2C9 is a two-domain protein with typical fold characteristics of the CYPs. The B-C loop forms part of the active site and contributes to substrate specificity. In the structures of CYP2C9 without ligand bound or with bound S-warfarin, residues 101-106 in the B-C loop form helix B. In addition, residues 212—222 in the F—G loop form helices F and G, which was not observed in rabbit CYP2C5 and bacterial CYPs. In the 1OG2 and 1OG5 structures, the heme is stabilized by hydrogen bonds between the propionates and the side chains of W120, R124, H368 and R433. In addition, R97 forms hydrogen bonds to the propionates as well as the carbonyl oxygen atoms of V113 and P367. CYP2C9 is activated by dapsone and its analogues and R-lansoprazole in a stereo-specific and substrate-dependent manner, probably through binding to the active site and inducing positive cooperativity. Further studies are needed to investigate the molecular determinants for ligand-CYP2C9 interactions.
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genetic polymorphism of the human Cytochrome P450 2C9 gene and its clinical significance
Current Drug Metabolism, 2009Co-Authors: Bo Wang, Jing Wang, Shuiqing Huang, Shufeng ZhouAbstract:Human Cytochrome P450 2C9 (CYP2C9) accounts for approximately 20% of total hepatic CYP content and metabolizes approximately 15% clinically used drugs including S-warfarin, tolbutamide, phenytoin, losartan, diclofenac, and celecoxib. To date, there are at least 33 variants of CYP2C9 (*1B through to *34) being identified. CYP2C9*2 and CYP2C9*3 differ from the wild-type CYP2C9*1 by a single point mutation: CYP2C9*2 is characterised by a 430C>T exchange in exon 3 resulting in an Arg144Cys amino acid substitution, whereas CYP2C9*3 shows an exchange of 1075A>C in exon 7 causing an Ile359Leu substitution in the catalytic site of the enzyme. CYP2C9*2 is frequent among Caucasians with approximately 1% of the population being homozygous carriers and 22% heterozygous. The corresponding figures for the CYP2C9*3 allele are 0.4% and 15%, respectively. Worldwide, a number of other variants have also to be considered. The CYP2C9 polymorphisms are relevant for the efficacy and adverse effects of numerous nonsteroidal anti-inflammatory agents, sulfonylurea antidiabetic drugs and, most critically, oral anticoagulants belonging to the class of vitamin K epoxide reductase inhibitors. Numerous clinical studies have shown that the CYP2C9 polymorphism should be considered in warfarin therapy and practical algorithms how to consider it in therapy are available. These studies have highlighted the importance of the CYP2C9*2 and *3 alleles. Warfarin has served as a practical example of how pharmacogenetics can be utilized to achieve maximum efficacy and minimum toxicity. Polymorphisms in CYP2C9 have the potential to affect the toxicity of CYP2C9 drugs with somewhat lower therapeutic indices such as warfarin, phenytoin, and certain antidiabetic drugs. CYP2C9 is one of the clinically significant drug metabolising enzymes that demonstrates genetic variants with significant phenotype and clinical outcomes. Genetic testing of CYP2C9 is expected to have a role in predicting drug clearance and implementing individualized pharmacotherapy. Prospective clinical studies with large samples are required to establish gene-dose and gene-effect relatiohsips for CYP2C9.
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substrates inducers inhibitors and structure activity relationships of human Cytochrome P450 2C9 and implications in drug development
Current Medicinal Chemistry, 2009Co-Authors: Shufeng Zhou, Zhi Wei Zhou, Li Ping Yang, Jianping CaiAbstract:Cytochrome P450 2C9 (CYP2C9) is one of the most abundant CYP enzymes in the human liver. CYP2C9 metabolizes more than 100 therapeutic drugs, including tolbutamide, glyburide, diclofenac, celecoxib, torasemide, phenytoin losartan, and S-warfarin). Some natural and herbal compounds are also metabolized by CYP2C9, probably leading to the formation of toxic metabolites. CYP2C9 also plays a role in the metabolism of several endogenous compounds such as steroids, melatonin, retinoids and arachidonic acid. Many CYP2C9 substrates are weak acids, but CYP2C9 also has the capacity to metabolise neutral, highly lipophilic compounds. A number of ligand-based and homology models of CYP2C9 have been reported and this has provided insights into the binding of ligands to the active site of CYP2C9. Data from the site-directed mutagenesis studies have revealed that a number of residues (e.g. Arg97, Phe110, Val113, Phe114, Arg144, Ser286, Asn289, Asp293 and Phe476) play an important role in ligand binding and determination of substrate specificity. The resolved crystal structures of CYP2C9 have confirmed the importance of these residues in substrate recognition and ligand orientation. CYP2C9 is activated by dapsone and its analogues and R-lansoprazole in a stereo-specific and substrate-dependent manner, probably through binding to the active site and inducing positive cooperativity. CYP2C9 is subject to induction by rifampin, phenobarbital, and dexamethasone, indicating the involvement of pregnane X receptor, constitutive androstane receptor and glucocorticoid receptor in the regulation of CYP2C9. A number of compounds have been found to inhibit CYP2C9 and this may provide an explanation for some clinically important drug interactions. Tienilic acid, suprofen and silybin are mechanism-based inhibitors of CYP2C9. Given the critical role of CYP2C9 in drug metabolism and the presence of polymorphisms, it is important to identify drug candidates as potential substrates, inducer or inhibitors of CYP2C9 in drug development and drug discovery scientists should develop drugs with minimal interactions with this enzyme. Further studies are warranted to explore the molecular determinants for ligand-CYP2C9 binding and the structure-activity relationships.
Yasuhiro Uno - One of the best experts on this subject based on the ideXlab platform.
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efavirenz clearances in vitro and in vivo in six cynomolgus monkeys associated with polymorphic Cytochrome P450 2C9 and simulated by individual physiologically based pharmacokinetic models
Biopharmaceutics & Drug Disposition, 2017Co-Authors: Masahiro Utoh, Tomonori Miura, Takashi Kusama, Shotaro Uehara, Makiko Shimizu, Yasuhiro Uno, Hiroshi YamazakiAbstract:Cynomolgus monkey Cytochrome P450 2C9 (formerly known as P450 2C43) variation was reportedly associated with metabolic clearance of the antiretroviral drug efavirenz in vivo (in three wild-type, one heterozygote and two homozygote animals), being unlikely in the case of human P450 2B6-dependent efavirenz clearance. In this study, the liver microsomal elimination rates of efavirenz for the same individual animals previously treated with intravenous/oral administrations of efavirenz showed significant reductions associated with the P450 2C9 p.[(I112L)] genotype (p < 0.05). Simulations of efavirenz clearance after oral administrations in individual cynomolgus monkeys were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling consisting of gut, liver and central compartments. The modeled hepatic intrinsic clearances were also significantly associated with the P450 2C9 genotypes, however, absorption rate constants or volumes of the systemic circulation were not likely determining factors for the individual efavirenz clearance variations in the six cynomolgus monkeys. This study provides important information to help simulate the clearances of efavirenz and related medicines associated with polymorphic P450 2C9 in individual cynomolgus monkeys, thereby facilitating the calculation of the fraction of liver microsomal clearance for estimating in vivo drug clearance with simplified PBPK modeling.
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in vivo individual variations in pharmacokinetics of efavirenz in cynomolgus monkeys genotyped for Cytochrome P450 2C9
Biopharmaceutics & Drug Disposition, 2016Co-Authors: Kazuhide Iwasaki, Masahiro Utoh, Shotaro Uehara, Yasuhiro Uno, Shinya Hosaka, Yusuke Kitsugi, Kanami Ikeda, Takahiro Yoshikawa, Hiroshi YamazakiAbstract:Cynomolgus monkeys are frequently used in preclinical studies for new drug development due to their evolutionary closeness to humans. An antiretroviral drug efavirenz is a typical probe substrate for human Cytochrome P450 (P450) 2B6, but is mainly metabolized by cynomolgus monkey P450 2C9. In this study, plasma concentrations of efavirenz were assessed in six cynomolgus monkeys genotyped for P450 2C9 c.334A > C (I112L) (three wild-type, one heterozygote, and two homozygotes) by high performance liquid chromatography with tandem mass spectrometry. After intravenous administration at a dose of 1.0 mg/kg, biphasic plasma elimination curves of efavirenz were seen in these cynomolgus monkeys. Plasma concentrations of primary metabolite 8-hydroxyefavirenz (1 hour after treatment, with hydrolysis by β-glucuronidase) in wild-type group mean was significantly higher (4.0-fold) than the combined heterozygous and homozygous group mean. Area under the plasma concentration-time curve values of efavirenz in the homozygous group monkey after oral administration at a dose of 2.0 mg/kg was significantly higher (2.0-fold) than the combined wild-type and heterozygous group. These results collectively indicated that P450 2C9 c.334A > C (I112L) variation was associated with efavirenz metabolic clearance in vivo. Cynomolgus P450 2C9 polymorphism might account for interindividual variations of efavirenz metabolism in cynomolgus monkeys. This article is protected by copyright. All rights reserved.
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comprehensive evaluation for substrate selectivity of cynomolgus monkey Cytochrome P450 2C9 a new efavirenz oxidase
Drug Metabolism and Disposition, 2015Co-Authors: Shinya Hosaka, Shotaro Uehara, Makiko Shimizu, Yasuhiro Uno, Norie Murayama, Masahiro Satsukawa, Kazuhide Iwasaki, Shunsuke Iwano, Hiroshi YamazakiAbstract:Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the Cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography–mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans.
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monkey liver Cytochrome P450 2C9 is involved in caffeine 7 n demethylation to form theophylline
Xenobiotica, 2013Co-Authors: Masahiro Utoh, Makiko Shimizu, Yasuhiro Uno, Shinya Hosaka, Norie Murayama, Kazuhide Iwasaki, Yui Onose, Hideki Fujino, Hiroshi YamazakiAbstract:Abstract1. Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human Cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline.2. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes.3. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4′-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably wit...
Bruno H. Stricker - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome P450 2C9 2 and 3 polymorphisms and the dose and effect of sulfonylurea in type ii diabetes mellitus
Clinical Pharmacology & Therapeutics, 2008Co-Authors: Matthijs L Becker, Ron H.n. Van Schaik, Albert Hofman, Loes E. Visser, Paul H Trienekens, Bruno H. StrickerAbstract:Sulfonylurea hypoglycemics are mainly metabolized by the Cytochrome P450 2C9 (CYP2C9) enzyme. The CYP2C9*2 and *3 polymorphisms encode proteins with less enzymatic activity and are correlated with elevated serum levels of sulfonylurea, as demonstrated in healthy volunteers. In this study, the effect of these variants is described for patients with diabetes mellitus treated with sulfonylurea. Associations between CYP2C9 polymorphisms, prescribed doses of sulfonylurea, and change in glucose levels after the start of sulfonylurea therapy were assessed in all patients with incident diabetes mellitus starting on sulfonylurea therapy in the Rotterdam Study, a population-based cohort study of 7,983 elderly people. In CYP2C9*3 allele carriers using tolbutamide, the prescribed dose was lower compared to patients with the wild-type CYP2C9 genotype. No differences in the prescribed dose were found in tolbutamide users with the CYP2C9*1/*2 or CYP2C9*2/*2 genotype compared to wild-type patients or in patients using other sulfonylurea. In CYP2C9*3 allele carriers, the mean decrease in fasting serum glucose levels after the start of tolbutamide therapy was larger than in patients with the wild-type genotype, although not statistically significant. Patients with diabetes mellitus who are carriers of a CYP2C9*3 allele require lower doses of tolbutamide to regulate their serum glucose levels compared to patients with the wild-type genotype.
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the risk of myocardial infarction in patients with reduced activity of Cytochrome P450 2C9
Pharmacogenetics and Genomics, 2007Co-Authors: Loes E. Visser, Ron H.n. Van Schaik, Albert Hofman, Cornelia M Van Duijn, A H J Danser, Jacqueline C M Witteman, Andre G Uitterlinden, Huibert A P Pols, Bruno H. StrickerAbstract:Objective The aim of the present follow-up study was to investigate whether the enzyme activity of the human Cytochrome P450 (CYP) 2C9 isoenzyme is associated with myocardial infarction. Methods We investigated whether the variant alleles CYP2C9*2 and CYP2C9*3 or the use of CYP2C9 substrates or inhibitors was associated with an increased risk of myocardial infarction in 2210 men and 3534 women from the Rotterdam Study, a prospective population-based cohort study of individuals aged 55 years or older. Results In women, the use of CYP2C9 substrates or inhibitors was significantly associated with incident myocardial infarction with a hazard ratio of 2.48 (95% confidence interval: 1.55-3.96). Within the group of female carriers of a variant allele, the use of CYP2C9 substrates or inhibitors was associated with a fourfold increased risk of myocardial infarction (hazard ratio 3.86, 95% confidence interval: 1.93-7.75), as compared with non-use. Neither the use of CYP2C9 inhibitors or substrates nor the variant CYP2C9 alleles were associated with an increased risk of myocardial infarction in men. Conclusions Drugs that are metabolized by CYP2C9 increase the risk of myocardial infarction in women. This risk was even higher in women with allelic variants of CYP2C9 with reduced enzyme activity.
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allelic variants of Cytochrome P450 2C9 modify the interaction between nonsteroidal anti inflammatory drugs and coumarin anticoagulants
Clinical Pharmacology & Therapeutics, 2005Co-Authors: Loes E. Visser, Ron H.n. Van Schaik, Albert Hofman, Peter A. G. M. De Smet, Martin H Van Vliet, Arnold G Vulto, Cornelia M Van Duijn, Paul H Trienekens, Bruno H. StrickerAbstract:INTRODUCTION: Cytochrome P450 (CYP) plays a key role in the metabolism of coumarin anticoagulants and nonsteroidal anti-inflammatory drugs (NSAIDs). Because CYP2C9 is a genetically polymorphic enzyme, genetic variability could play an important role in the potential interaction between NSAIDs and coumarins. We investigated whether NSAIDs were associated with overanticoagulation during therapy with coumarins and evaluated the effect of the CYP2C9 polymorphisms on this potential interaction. METHODS: We conducted a population-based cohort study among patients of an anticoagulation clinic who were treated with acenocoumarol or phenprocoumon between April 1, 1991, and May 31, 2003, and whose CYP2C9 status was known. Patients were followed up until an international normalized ratio (INR) of 6.0 or greater was reached or until the end of treatment, death, or the end of the study. Proportional hazards regression analysis was used to estimate the risk of an INR of 6.0 or greater in relation to concomitant use of a coumarin anticoagulant and NSAIDs after adjustment for several potentially confounding factors. To study effect modification by CYP2C9 genotype, stratified analyses were performed for wild-type patients and patients with a variant genotype. RESULTS: Of the 973 patients in the cohort, 415 had an INR of 6.0 or greater. Several NSAIDs increased the risk of overanticoagulation. The risk of overanticoagulation was 2.98 (95% confidence interval, 1.09-7.02) in coumarin-treated patients taking NSAIDs with a CYP2C9*2 allele and 10.8 (95% confidence interval, 2.57-34.6) in those with a CYP2C9*3 allele. CONCLUSIONS: Several NSAIDs were associated with overanticoagulation. For NSAIDs that are known CYP2C9 substrates, this risk was modified by allelic variants of CYP2C9. More frequent INR monitoring of patients taking NSAIDs is warranted.
Timothy S. Tracy - One of the best experts on this subject based on the ideXlab platform.
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Functional Analysis of Phenylalanine Residues in the Active Site of Cytochrome P450 2C9
Biochemistry, 2008Co-Authors: Carrie M. Mosher, Timothy S. Tracy, Matthew A. Hummel, Allan E. RettieAbstract:The two published crystal structures of Cytochrome P450 2C9, complexed with (S)-warfarin or flurbiprofen, implicate a cluster of three active site phenylalanine residues (F100, F114, F476) in ligand binding. However, these three residues appear to interact differently with these two ligands based on the static crystal structures. To elucidate the importance of CYP2C9’s active site phenylalanines on substrate binding, orientation, and catalytic turnover, a series of leucine and tryptophan mutants were constructed and their interactions with (S)-warfarin and (S)-flurbiprofen examined. The F100→L mutation had minor effects on substrate binding and metabolism of each substrate. In contrast, the F114L and F476L mutants exhibited substantially reduced (S)-warfarin metabolism and altered hydroxy metabolite profiles but only modestly decreased nonsteroidal antiinflammatory drug (NSAID) turnover while maintaining product regioselectivity. The F114→W and F476→W mutations also had opposing effects on (S)-warfarin ve...
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use of simple docking methods to screen a virtual library for heteroactivators of Cytochrome P450 2C9
Journal of Medicinal Chemistry, 2007Co-Authors: Charles W Locuson, Peter M Gannett, Robyn Ayscue, Timothy S. TracyAbstract:Several laboratories have demonstrated that activation of drug metabolism by P450s may occur via a mechanism that resembles allosterism from an enzyme kinetic standpoint. Because the effector drug binding site may be located in the same P450 binding pocket where the drug substrate is located, the ability to find and characterize novel effectors (aka heteroactivators) will prove to be important in probing the mechanism of activation. We have used analogues of the prototypical CYP2C9 heteroactivator dapsone to validate a simple docking method that can be used to predict heteroactivators based on ligand binding location in a P450 crystal structure. As proof of concept for the described docking method, a protocol was developed to discover potential heteroactivators from a virtual chemical library through efficient sorting of >40 000 compounds. One of the top-scoring compounds identified was verified to be a CYP2C9 heteroactivator in vitro, and it possessed activity similar to dapsone.
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activation of Cytochrome P450 2C9 mediated metabolism mechanistic evidence in support of kinetic observations
Archives of Biochemistry and Biophysics, 2003Co-Authors: Matthew J Hutzler, Larry C Wienkers, Jan L Wahlstrom, Timothy J Carlson, Timothy S. TracyAbstract:Studies were designed to investigate the possible mechanisms associated with the kinetic observation of CYP2C9 activation by dapsone and its phase I metabolite, N-hydroxydapsone. Kinetic studies suggested that dapsone activated CYP2C9-mediated flurbiprofen 4(')-hydroxylation by decreasing the K(m) (alpha=0.2) and increasing the V(max) (beta=1.9). Interestingly, N-hydroxydapsone also activated flurbiprofen 4(')-hydroxylation by increasing V(max) (beta=1.5) but had no effect on K(m) (alpha=0.98). To study the effects of these modulators on the binding affinity of flurbiprofen, spectral binding studies were performed. In the presence of dapsone, the spectral binding constant (K(s)) for flurbiprofen was reduced from 14.1 to 2.1 microM, while in the presence of N-hydroxydapsone, the K(s) remained unchanged (14.0 microM), which suggests that dapsone causes an increase in the affinity of flurbiprofen for CYP2C9, whereas N-hydroxydapsone does not. Additionally, stoichiometry measurements under activation conditions in the presence of dapsone resulted in a doubling of both NADPH and oxygen consumption for flurbiprofen 4(')-hydroxylation, with an overall increase in metabolite formation and a decrease in formation of peroxide and excess water. Interestingly, the presence of N-hydroxydapsone generally caused the same effects on stoichiometry as those of flurbiprofen 4(')-hydroxylation but failed to reduce excess water formation, which suggests that, while N-hydroxydapsone activates CYP2C9, it does so less efficiently and possibly through a mechanism different from that of dapsone.
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studies of flurbiprofen 4 hydroxylation additional evidence suggesting the sole involvement of Cytochrome P450 2C9
Biochemical Pharmacology, 1996Co-Authors: Timothy S. Tracy, Steven A Wrighton, Frank J Gonzalez, Christina Marra, Kenneth R. KorzekwaAbstract:Abstract Flurbiprofen, a non-steroidal anti-inflammatory drug (NSAID), is metabolized by both oxidation via the Cytochrome P450 system and by glucuronidation. The major oxidative pathway in flurbiprofen metabolism is to a 4′-hydroxy metabolite, and recently we demonstrated that Cytochrome P450 2C9 and its R144C variant were involved in this process (Tracy et al., Biochem Pharmacol 49 : 1269–1275, 1995). Using complementary DNA (cDNA)-expressed cell systems, it has been demonstrated that at physiological concentrations of flurbiprofen there is a lack of involvement of P450s 1A2, 2C8, 2E1, and 3A4. In evaluating flurbiprofen as a potential probe for Cytochrome P450 2C9, it is important to assess the involvement of additional P450s in this process. To this end, further studies were undertaken using specific inhibitors of P450 2C9 and P450 cDNA-expressed microsomes for P450 1A1, 2A6, 2B6, 2C19, and 2D6 to assess their potential involvement. We observed the inhibition of ( R )- and ( S )-flurbiprofen 4′-hydroxylation by an inhibitor of P450 2C9, sulfaphenazole ( K i = 0.07 and 0.06 μ M, respectively), and the NSAID piroxicam ( K i = 10 and 7 μ M, respectively). Furthermore, using microsomes from a lymphoblastoid cell line, we found that P450s 1A1, 2A6, 2B6, 2C19, and 2D6 were not involved in flurbiprofen hydroxylation at physiological concentrations of flurbiprofen. This finding is particularly important due to the sequence homology and potential substrate overlap of P450 2C9 and 2C19. These studies then provide additional evidence to suggest that P450 2C9 may be the only isoform involved to any substantial degree in flurbiprofen 4′-hydroxylation, and thus this reaction is useful as an in vitro probe for this particularly Cytochrome P450 isoform and may be useful as an in vivo probe.
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role of Cytochrome P450 2C9 and an allelic variant in the 4 hydroxylation of r and s flurbiprofen
Biochemical Pharmacology, 1995Co-Authors: Timothy S. Tracy, Brady W Rosenbluth, Steven A Wrighton, Frank J Gonzalez, Kenneth R. KorzekwaAbstract:Flurbiprofen is a chiral non-steroidal anti-inflammatory drug used in the treatment of pain or inflammation. The primary routes of biotransformation for (R)- and (S)-flurbiprofen are oxidation (presumably Cytochrome P450) and conjugation. To date, the specific Cytochrome P450 (P450) involved in the oxidative metabolism of this compound (specifically 4'-hydroxylation) has not been elucidated. Experiments were conducted to characterize the kinetic parameters (Km and Vmax) for the 4'-hydroxylation of (R)- and (S)-flurbiprofen in human liver microsomes, to determine if enantiomeric interactions occur when both enantiomers are present, and to identify the specific P450 form(s) involved in this reaction. In human liver microsomes, the Km and Vmax (mean +/- SD) for (R)-4'-hydroxy-flurbiprofen formation were 3.1 +/- 0.8 microM and 305 +/- 168 pmol.min-1.mg protein)-1, respectively. In comparison, the Km and Vmax (mean +/- SD) for (S)-4'-hydroxy-flurbiprofen formation were 1.9 +/- 0.4 microM and 343 +/- 196 pmol.min-1.mg protein-1, respectively. Enantiomeric interaction studies revealed a decrease in Km and Vmax for both enantiomers and an apparent loss of stereoselectivity. Racemic-warfarin, tolbutamide, alpha-naphthoflavone and erythromycin were studied as potential inhibitors of this process. The estimated Ki values for the inhibition of (R)- and (S)-4'-hydroxy-flurbiprofen formation by racemic-warfarin were 2.2 and 4.7 microM. This reaction was also inhibited by tolbutamide. In contrast, erythromycin and alpha-naphthoflavone had no appreciable effect on 4'-hydroxy-flurbiprofen formation. cDNA-expression of individual forms was used to determine which P450 was involved in 4'-hydroxy-flurbiprofen formation. P450 2C9 and an allelic variant (R144C) readily catalyzed the formation of 4'-hydroxy-flurbiprofen. P450 1A2 was also active albeit with a turnover rate 1/140th that of P450 2C9R144C (P450s 2C8, 2E1 and 3A4 were not active toward either enantiomer). The results of these studies indicate that the enantiomers of flurbiprofen may exhibit stereoselectivity with respect to enzyme affinity but have roughly equal maximum formation velocities. Additionally, these two enantiomers may compete for the enzyme resulting in lower maximum velocities for both enantiomers. Finally, of those P450 forms examined, only P450 2C9 and an allelic variant catalyzed the 4'-hydroxylation of both (R)- and (S)-flurbiprofen.
