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Hiroshi Yamazaki - One of the best experts on this subject based on the ideXlab platform.
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suitable albumin concentrations for enhanced Drug Oxidation activities mediated by human liver microsomal cytochrome p450 2c9 and other forms predicted with unbound fractions and partition distribution coefficients of model substrates
Xenobiotica, 2019Co-Authors: Kanami Shimura, Norie Murayama, Saki Tanaka, Shunsuke Onozeki, Hiroshi YamazakiAbstract:Albumin has reportedly enhanced cytochrome P450 (P450)-mediated Drug Oxidation rates in human liver microsomes. Consequently, measurements of clearances and fractions metabolized could vary depending on the experimental albumin concentrations used. In this study, the Oxidation rates of diclofenac and warfarin by human liver microsomes were significantly enhanced in the presence of 0.10% (w/v) bovine serum albumin, whereas those of tolbutamide and phenytoin required 1.0% and 2.0% of albumin for significant enhancement. Values of the fractions metabolized by P450 2C9 for four substrates did not markedly change in the presence of albumin at the above-mentioned concentrations. The Oxidation rates of bupropion, omeprazole, chlorzoxazone and phenacetin in human liver microsomes were reportedly enhanced by 0.5%, 1%, 2% and 2% of albumin, respectively. Analysis of reported intrinsic clearance values and suitable albumin concentrations for the currently analyzed substrates and the reported substrates revealed an inverse correlation, with warfarin as an outlier. Suitable albumin concentrations were multivariately correlated with physicochemical properties, that is, the plasma unbound fractions, octanol-water partition coefficient and acid dissociation constant (r = 0.98, p<.0001, n = 10). Therefore, multiple physicochemical properties may be determinants of suitable albumin concentrations for substrate Oxidations in human liver microsomes.
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induction of human cytochrome p450 3a enzymes in cultured placental cells by thalidomide and relevance to bioactivation and toxicity
Journal of Toxicological Sciences, 2017Co-Authors: Norie Murayama, Kazuyuki Arata, Yasuhiro Kazuki, Norio Shibata, F. Peter Guengerich, Daisuke Satoh, T Harada, Hiroshi YamazakiAbstract:Evidence has been presented for auto-induced human cytochrome P450 3A enzyme involvement in the teratogenicity and clinical outcome of thalidomide due to Oxidation to 5-hydroxythalidomide and subsequent metabolic activation in livers. In this study, more relevant human placenta preparations and placental BeWo cells showed low but detectable P450 3A4/5 mRNA expression and Drug Oxidation activities. Human placental microsomal fractions from three subjects showed detectable midazolam 1´- and 4-hydroxylation and thalidomide 5-hydroxylation activities. Human placental BeWo cells, cultured in the recommended media, also indicated detectable midazolam 1´- and 4-hydroxylation and thalidomide 5-hydroxylation activities. To reduce any masking effects by endogenous hormones used in the recommended media, induction of P450 3A4/5 mRNA and Oxidation activities were measured in placental BeWo cells cultured with a modified medium containing 5% charcoal-stripped fetal bovine serum. Thalidomide significantly induced P450 3A4/5, 2B6, and pregnane X receptor (PXR) mRNA levels 2 to 3-fold, but rifampicin only enhanced P450 3A5 and PXR mRNA under the modified media conditions. Under these modified conditions, thalidomide also significantly induced midazolam 1´-hydroxylation and thalidomide 5-hydroxylaion activities 3-fold but not bupropion hydroxylation activity. Taken together, activation of thalidomide to 5-hydroxythalidomide with autoinduction of P450 3A enzymes in human placentas, as well as livers, is suggested in vivo.
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combining chimeric mice with humanized liver mass spectrometry and physiologically based pharmacokinetic modeling in toxicology
Chemical Research in Toxicology, 2016Co-Authors: Hiroshi Yamazaki, Makiko Shimizu, Hiroshi Suemizu, Marina Mitsui, Peter F GuengerichAbstract:Species differences exist in terms of Drug Oxidation activities, which are mediated mainly by cytochrome P450 (P450) enzymes. To overcome the problem of species extrapolation, transchromosomic mice containing a human P450 3A cluster or chimeric mice transplanted with human hepatocytes have been introduced into the human toxicology research area. In this review, Drug metabolism and disposition mediated by humanized livers in chimeric mice are summarized in terms of biliary/urinary excretions of phthalate and bisphenol A and plasma clearances of the human cocktail probe Drugs caffeine, warfarin, omeprazole, metoprolol, and midazolam. Simulation of human plasma concentrations of the teratogen thalidomide and its human metabolites is possible with a simplified physiologically based pharmacokinetic model based on data obtained in chimeric mice, in accordance with reported clinical thalidomide concentrations. In addition, in vivo nonspecific hepatic protein binding parameters of metabolically activated 14C-Drug...
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Drug interactions of diclofenac and its oxidative metabolite with human liver microsomal cytochrome p450 1a2 dependent Drug Oxidation
Xenobiotica, 2014Co-Authors: Katsuhiro Ohyama, Norie Murayama, Makiko Shimizu, Hiroshi YamazakiAbstract:1. The purpose of this study was to investigate the inhibitory effects of diclofenac on human cytochrome P450 1A2-, 2C19- and 3A4-mediated Drug Oxidations and to evaluate the Drug interaction potential of diclofenac and 4'-hydroxydiclofenac. 2. Diclofenac was converted to 4'-hydroxydiclofenac by recombinantly expressed human P450 1A2 with Km and Vmax values of 33 µM and 0.20 min(-1), respectively. Diclofenac and 4'-hydroxydiclofenac suppressed flurbiprofen 4'-hydroxylation by P450 2C9 strongly and moderately, respectively; however, they did not affect P450 2C19-dependent S-mephenytoin hydroxylation or P450 3A4-dependent midazolam hydroxylation. 3. Although the caffeine 3-N-demethylation activity of liver microsomal P450 1A2 was inhibited by simultaneous incubation with diclofenac, the riluzole N-hydroxylation activities of recombinant P450 1A2 and human liver microsomes were inhibited after preincubation with diclofenac or 4'-hydroxydiclofenac for 20 min in the presence of NADPH. Using the inhibition constant (37 µM) of diclofenac on caffeine 3-N-demethylation and the reported 95th percentiles of maximum plasma concentration (10.5 µM) after an oral dose of diclofenac, the in vivo estimated increase in area under the plasma concentration-time curve was 29%. 4. These results suggest that diclofenac could inhibit Drug clearance to a clinically important degree that depends on P450 1A2. Clinically relevant Drug interactions in vivo with diclofenac are likely to be invoked via human P450 1A2 function in addition to those caused by the effect of diclofenac on P450 2C9.
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the cyp3a4 intron 6 c t polymorphism cyp3a4 22 is associated with reduced cyp3a4 protein level and function in human liver microsomes
Journal of Toxicological Sciences, 2013Co-Authors: Maho Okubo, Norie Murayama, Makiko Shimizu, Tsutomu Shimada, Peter F Guengerich, Hiroshi YamazakiAbstract:Effects of the CYP3A4 intron 6 C>T (CYP3A4*22) polymorphism, which has recently been reported to have a critical role in vivo, were investigated by measuring CYP3A4 protein expression levels and CYP3A4-dependent Drug Oxidation activities in individual human liver microsomes in vitro. Prior to protein analysis, analysis of DNA samples indicated that 36 Caucasian subjects were genotyped as CYP3A4*1/*1 and five subjects were CYP3A4*1/*22, with a CYP3A4*22 allelic frequency of 6.1%. No CYP3A4*22 alleles were found in the Japanese samples (106 alleles). Individual differences in CYP2D6-dependent dextromethorphan O-demethylation activities in liver microsomes from Caucasians were not affected by either the CYP3A4*1/*22 or CYP3A5*1/*3 genotype. Liver microsomes genotyped as CYP3A4*1/*22 (n = 4) showed significantly lower CYP3A-dependent dextromethorphan N-demethylation, midazolam 1'-hydroxylation, and testosterone 6β-hydroxylation activities, as well as lower expression levels of CYP3A protein (28% of control), compared with those of the CYP3A4*1/*1 group (n = 19). The other polymorphism, CYP3A5*1/*3, did not show these differences (n = 4). The CYP3A4*22 polymorphism was associated with reduced CYP3A4 protein expression levels and resulted in decreased CYP3A4-dependent activities in human livers. The present results suggest an important role of low expression of CYP3A4 protein associated with the CYP3A4*22 allele in the individual differences in Drug clearance.
Norie Murayama - One of the best experts on this subject based on the ideXlab platform.
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suitable albumin concentrations for enhanced Drug Oxidation activities mediated by human liver microsomal cytochrome p450 2c9 and other forms predicted with unbound fractions and partition distribution coefficients of model substrates
Xenobiotica, 2019Co-Authors: Kanami Shimura, Norie Murayama, Saki Tanaka, Shunsuke Onozeki, Hiroshi YamazakiAbstract:Albumin has reportedly enhanced cytochrome P450 (P450)-mediated Drug Oxidation rates in human liver microsomes. Consequently, measurements of clearances and fractions metabolized could vary depending on the experimental albumin concentrations used. In this study, the Oxidation rates of diclofenac and warfarin by human liver microsomes were significantly enhanced in the presence of 0.10% (w/v) bovine serum albumin, whereas those of tolbutamide and phenytoin required 1.0% and 2.0% of albumin for significant enhancement. Values of the fractions metabolized by P450 2C9 for four substrates did not markedly change in the presence of albumin at the above-mentioned concentrations. The Oxidation rates of bupropion, omeprazole, chlorzoxazone and phenacetin in human liver microsomes were reportedly enhanced by 0.5%, 1%, 2% and 2% of albumin, respectively. Analysis of reported intrinsic clearance values and suitable albumin concentrations for the currently analyzed substrates and the reported substrates revealed an inverse correlation, with warfarin as an outlier. Suitable albumin concentrations were multivariately correlated with physicochemical properties, that is, the plasma unbound fractions, octanol-water partition coefficient and acid dissociation constant (r = 0.98, p<.0001, n = 10). Therefore, multiple physicochemical properties may be determinants of suitable albumin concentrations for substrate Oxidations in human liver microsomes.
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induction of human cytochrome p450 3a enzymes in cultured placental cells by thalidomide and relevance to bioactivation and toxicity
Journal of Toxicological Sciences, 2017Co-Authors: Norie Murayama, Kazuyuki Arata, Yasuhiro Kazuki, Norio Shibata, F. Peter Guengerich, Daisuke Satoh, T Harada, Hiroshi YamazakiAbstract:Evidence has been presented for auto-induced human cytochrome P450 3A enzyme involvement in the teratogenicity and clinical outcome of thalidomide due to Oxidation to 5-hydroxythalidomide and subsequent metabolic activation in livers. In this study, more relevant human placenta preparations and placental BeWo cells showed low but detectable P450 3A4/5 mRNA expression and Drug Oxidation activities. Human placental microsomal fractions from three subjects showed detectable midazolam 1´- and 4-hydroxylation and thalidomide 5-hydroxylation activities. Human placental BeWo cells, cultured in the recommended media, also indicated detectable midazolam 1´- and 4-hydroxylation and thalidomide 5-hydroxylation activities. To reduce any masking effects by endogenous hormones used in the recommended media, induction of P450 3A4/5 mRNA and Oxidation activities were measured in placental BeWo cells cultured with a modified medium containing 5% charcoal-stripped fetal bovine serum. Thalidomide significantly induced P450 3A4/5, 2B6, and pregnane X receptor (PXR) mRNA levels 2 to 3-fold, but rifampicin only enhanced P450 3A5 and PXR mRNA under the modified media conditions. Under these modified conditions, thalidomide also significantly induced midazolam 1´-hydroxylation and thalidomide 5-hydroxylaion activities 3-fold but not bupropion hydroxylation activity. Taken together, activation of thalidomide to 5-hydroxythalidomide with autoinduction of P450 3A enzymes in human placentas, as well as livers, is suggested in vivo.
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Drug interactions of diclofenac and its oxidative metabolite with human liver microsomal cytochrome p450 1a2 dependent Drug Oxidation
Xenobiotica, 2014Co-Authors: Katsuhiro Ohyama, Norie Murayama, Makiko Shimizu, Hiroshi YamazakiAbstract:1. The purpose of this study was to investigate the inhibitory effects of diclofenac on human cytochrome P450 1A2-, 2C19- and 3A4-mediated Drug Oxidations and to evaluate the Drug interaction potential of diclofenac and 4'-hydroxydiclofenac. 2. Diclofenac was converted to 4'-hydroxydiclofenac by recombinantly expressed human P450 1A2 with Km and Vmax values of 33 µM and 0.20 min(-1), respectively. Diclofenac and 4'-hydroxydiclofenac suppressed flurbiprofen 4'-hydroxylation by P450 2C9 strongly and moderately, respectively; however, they did not affect P450 2C19-dependent S-mephenytoin hydroxylation or P450 3A4-dependent midazolam hydroxylation. 3. Although the caffeine 3-N-demethylation activity of liver microsomal P450 1A2 was inhibited by simultaneous incubation with diclofenac, the riluzole N-hydroxylation activities of recombinant P450 1A2 and human liver microsomes were inhibited after preincubation with diclofenac or 4'-hydroxydiclofenac for 20 min in the presence of NADPH. Using the inhibition constant (37 µM) of diclofenac on caffeine 3-N-demethylation and the reported 95th percentiles of maximum plasma concentration (10.5 µM) after an oral dose of diclofenac, the in vivo estimated increase in area under the plasma concentration-time curve was 29%. 4. These results suggest that diclofenac could inhibit Drug clearance to a clinically important degree that depends on P450 1A2. Clinically relevant Drug interactions in vivo with diclofenac are likely to be invoked via human P450 1A2 function in addition to those caused by the effect of diclofenac on P450 2C9.
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the cyp3a4 intron 6 c t polymorphism cyp3a4 22 is associated with reduced cyp3a4 protein level and function in human liver microsomes
Journal of Toxicological Sciences, 2013Co-Authors: Maho Okubo, Norie Murayama, Makiko Shimizu, Tsutomu Shimada, Peter F Guengerich, Hiroshi YamazakiAbstract:Effects of the CYP3A4 intron 6 C>T (CYP3A4*22) polymorphism, which has recently been reported to have a critical role in vivo, were investigated by measuring CYP3A4 protein expression levels and CYP3A4-dependent Drug Oxidation activities in individual human liver microsomes in vitro. Prior to protein analysis, analysis of DNA samples indicated that 36 Caucasian subjects were genotyped as CYP3A4*1/*1 and five subjects were CYP3A4*1/*22, with a CYP3A4*22 allelic frequency of 6.1%. No CYP3A4*22 alleles were found in the Japanese samples (106 alleles). Individual differences in CYP2D6-dependent dextromethorphan O-demethylation activities in liver microsomes from Caucasians were not affected by either the CYP3A4*1/*22 or CYP3A5*1/*3 genotype. Liver microsomes genotyped as CYP3A4*1/*22 (n = 4) showed significantly lower CYP3A-dependent dextromethorphan N-demethylation, midazolam 1'-hydroxylation, and testosterone 6β-hydroxylation activities, as well as lower expression levels of CYP3A protein (28% of control), compared with those of the CYP3A4*1/*1 group (n = 19). The other polymorphism, CYP3A5*1/*3, did not show these differences (n = 4). The CYP3A4*22 polymorphism was associated with reduced CYP3A4 protein expression levels and resulted in decreased CYP3A4-dependent activities in human livers. The present results suggest an important role of low expression of CYP3A4 protein associated with the CYP3A4*22 allele in the individual differences in Drug clearance.
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monkey liver cytochrome p450 2c19 is involved in r and s warfarin 7 hydroxylation
Biochemical Pharmacology, 2012Co-Authors: Yoshio Hosoi, Norie Murayama, Makiko Shimizu, Masahiro Utoh, Yasuhiro Uno, Hideki Fujino, Mitsunori Shukuya, Kazuhide Iwasaki, Hiroshi YamazakiAbstract:Abstract Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R - and S -warfarin are model substrates for stereoselective Oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R - and S -warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R - and S -warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S -warfarin 7-hydroxylation. R -Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S -warfarin 7-hydroxylation activities were not correlated with the four marker Drug Oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R - and S -warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R - and S -warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V max and low K m values for R -warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S -warfarin 7-hydroxylation with V max and V max / K m values comparable to those for recombinant human P450 2C9. R -warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R - and S -warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S -warfarin 7-hydroxylation.
Makiko Shimizu - One of the best experts on this subject based on the ideXlab platform.
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combining chimeric mice with humanized liver mass spectrometry and physiologically based pharmacokinetic modeling in toxicology
Chemical Research in Toxicology, 2016Co-Authors: Hiroshi Yamazaki, Makiko Shimizu, Hiroshi Suemizu, Marina Mitsui, Peter F GuengerichAbstract:Species differences exist in terms of Drug Oxidation activities, which are mediated mainly by cytochrome P450 (P450) enzymes. To overcome the problem of species extrapolation, transchromosomic mice containing a human P450 3A cluster or chimeric mice transplanted with human hepatocytes have been introduced into the human toxicology research area. In this review, Drug metabolism and disposition mediated by humanized livers in chimeric mice are summarized in terms of biliary/urinary excretions of phthalate and bisphenol A and plasma clearances of the human cocktail probe Drugs caffeine, warfarin, omeprazole, metoprolol, and midazolam. Simulation of human plasma concentrations of the teratogen thalidomide and its human metabolites is possible with a simplified physiologically based pharmacokinetic model based on data obtained in chimeric mice, in accordance with reported clinical thalidomide concentrations. In addition, in vivo nonspecific hepatic protein binding parameters of metabolically activated 14C-Drug...
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Drug interactions of diclofenac and its oxidative metabolite with human liver microsomal cytochrome p450 1a2 dependent Drug Oxidation
Xenobiotica, 2014Co-Authors: Katsuhiro Ohyama, Norie Murayama, Makiko Shimizu, Hiroshi YamazakiAbstract:1. The purpose of this study was to investigate the inhibitory effects of diclofenac on human cytochrome P450 1A2-, 2C19- and 3A4-mediated Drug Oxidations and to evaluate the Drug interaction potential of diclofenac and 4'-hydroxydiclofenac. 2. Diclofenac was converted to 4'-hydroxydiclofenac by recombinantly expressed human P450 1A2 with Km and Vmax values of 33 µM and 0.20 min(-1), respectively. Diclofenac and 4'-hydroxydiclofenac suppressed flurbiprofen 4'-hydroxylation by P450 2C9 strongly and moderately, respectively; however, they did not affect P450 2C19-dependent S-mephenytoin hydroxylation or P450 3A4-dependent midazolam hydroxylation. 3. Although the caffeine 3-N-demethylation activity of liver microsomal P450 1A2 was inhibited by simultaneous incubation with diclofenac, the riluzole N-hydroxylation activities of recombinant P450 1A2 and human liver microsomes were inhibited after preincubation with diclofenac or 4'-hydroxydiclofenac for 20 min in the presence of NADPH. Using the inhibition constant (37 µM) of diclofenac on caffeine 3-N-demethylation and the reported 95th percentiles of maximum plasma concentration (10.5 µM) after an oral dose of diclofenac, the in vivo estimated increase in area under the plasma concentration-time curve was 29%. 4. These results suggest that diclofenac could inhibit Drug clearance to a clinically important degree that depends on P450 1A2. Clinically relevant Drug interactions in vivo with diclofenac are likely to be invoked via human P450 1A2 function in addition to those caused by the effect of diclofenac on P450 2C9.
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the cyp3a4 intron 6 c t polymorphism cyp3a4 22 is associated with reduced cyp3a4 protein level and function in human liver microsomes
Journal of Toxicological Sciences, 2013Co-Authors: Maho Okubo, Norie Murayama, Makiko Shimizu, Tsutomu Shimada, Peter F Guengerich, Hiroshi YamazakiAbstract:Effects of the CYP3A4 intron 6 C>T (CYP3A4*22) polymorphism, which has recently been reported to have a critical role in vivo, were investigated by measuring CYP3A4 protein expression levels and CYP3A4-dependent Drug Oxidation activities in individual human liver microsomes in vitro. Prior to protein analysis, analysis of DNA samples indicated that 36 Caucasian subjects were genotyped as CYP3A4*1/*1 and five subjects were CYP3A4*1/*22, with a CYP3A4*22 allelic frequency of 6.1%. No CYP3A4*22 alleles were found in the Japanese samples (106 alleles). Individual differences in CYP2D6-dependent dextromethorphan O-demethylation activities in liver microsomes from Caucasians were not affected by either the CYP3A4*1/*22 or CYP3A5*1/*3 genotype. Liver microsomes genotyped as CYP3A4*1/*22 (n = 4) showed significantly lower CYP3A-dependent dextromethorphan N-demethylation, midazolam 1'-hydroxylation, and testosterone 6β-hydroxylation activities, as well as lower expression levels of CYP3A protein (28% of control), compared with those of the CYP3A4*1/*1 group (n = 19). The other polymorphism, CYP3A5*1/*3, did not show these differences (n = 4). The CYP3A4*22 polymorphism was associated with reduced CYP3A4 protein expression levels and resulted in decreased CYP3A4-dependent activities in human livers. The present results suggest an important role of low expression of CYP3A4 protein associated with the CYP3A4*22 allele in the individual differences in Drug clearance.
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monkey liver cytochrome p450 2c19 is involved in r and s warfarin 7 hydroxylation
Biochemical Pharmacology, 2012Co-Authors: Yoshio Hosoi, Norie Murayama, Makiko Shimizu, Masahiro Utoh, Yasuhiro Uno, Hideki Fujino, Mitsunori Shukuya, Kazuhide Iwasaki, Hiroshi YamazakiAbstract:Abstract Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R - and S -warfarin are model substrates for stereoselective Oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R - and S -warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R - and S -warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S -warfarin 7-hydroxylation. R -Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S -warfarin 7-hydroxylation activities were not correlated with the four marker Drug Oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R - and S -warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R - and S -warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V max and low K m values for R -warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S -warfarin 7-hydroxylation with V max and V max / K m values comparable to those for recombinant human P450 2C9. R -warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R - and S -warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S -warfarin 7-hydroxylation.
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cytochrome p450 depedent Drug Oxidation activity of liver microsomes from microminipigs a possible new animal model for humans in non clinical studies
Drug Metabolism and Pharmacokinetics, 2009Co-Authors: Norie Murayama, Makiko Shimizu, Katsuhiro Ohyama, Naoki Kaneko, Kana Horiuchi, Katzuhiko Ito, Hiroshi YamazakiAbstract:Summary: Small minipigs (Bland name, Micromini Pig; registered as a novel variety of pig in the Japanese Ministry of Agriculture, Forestry and Fisheries) were developed with the aim of non-clinical pharmacological/toxicological use. They were principally mated with
Ulrich M Zanger - One of the best experts on this subject based on the ideXlab platform.
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cyp2d6 genotyping strategy based on gene copy number determination by taqman real time pcr
Human Mutation, 2003Co-Authors: Elke Schaeffeler, Matthias Schwab, Michel Eichelbaum, Ulrich M ZangerAbstract:The genetic polymorphism of the cytochrome P450 monooxygenase, CYP2D6, comprises at least 43 alleles giving rise to distinct Drug metabolism phenotypes termed ultrarapid, extensive, intermediate, and poor metabolizers. As a consequence, Drug side effects or lack of Drug effect may occur if standard doses are applied. Genetic prediction of Drug Oxidation phenotype as a basis for dose selection requires analysis of single nucleotide polymorphisms and of alleles with duplicated or deleted genes. Here we developed a novel method to determine the CYP2D6 gene dose per genome. A TaqMan real-time PCR assay to specifically amplify genomic CYP2D6 was established by using a specific set of amplification primers and probe, located in exon 9, which effectively prevent amplification of CYP2D7 and CYP2D8 pseudogenes. Quantitative CYP2D6 amplification data were normalized to albumin as an internal reference gene which was coamplified simultaneously in a single-tube biplex assay. The assay was validated with a selection of previously genotyped DNA samples containing none, one, two, or three CYP2D6 gene copies. The results were highly reproducible and closely matched the number of genes with no overlap between the groups. Analysis of DNA samples comprising all major alleles and genotypes revealed high sensitivity and specificity of the assay, as demonstrated by agreement of the determined gene dose with the presence of CYP2D6(*)2 x 2 (gene duplication) and CYP2D6(*) 5 (gene deletion) alleles. The predictability of the new strategy was systematically evaluated. The semiautomatic TaqMan assay allows high sample throughput and will be useful for pharmacogenetic studies and in the clinical setting.
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elucidation of the genetic basis of the common intermediate metabolizer phenotype for Drug Oxidation by cyp2d6
Pharmacogenetics, 2000Co-Authors: Sebastian Raimundo, Matthias Schwab, Michel Eichelbaum, Joachim Fischer, Ernstulrich Griese, Ulrich M ZangerAbstract:A subgroup of 10-15% of Caucasians are termed phenotypical 'intermediate metabolizers' of Drug substrates of CYP2D6 because they have severely impaired yet residual in-vivo function of this cytochrome P450. Genotyping based on the currently known CYP2D6 alleles does not predict this phenotype satisfactorily. A systematic sequencing strategy through 1.6 kb of the CYP2D6 5'-flanking sequence revealed six mutations of which three were exclusively associated with the functional CYP2D6*2 allele (-1496 C to G; -652 C to T; and -590 G to A), two were associated with the nonfunctional *4 and with the functional *10-alleles (-1338 C to T and -912 G to A) and one (-1147 A to G) was seen in all *2, *4 and *10-alleles investigated. The -1496 C to G mutation was found to be polymorphic within CYP2D6*2 alleles. In a family study, the wild-type CYP2D6 *2[-1496 C] and the novel variant [-1496 G] allele co-segregated with lower and higher CYP2D6 in-vivo function, respectively, as shown by phenotyping using sparteine as probe Drug. In a representative population sample selected for genotypes comprising one CYP2D6*2 and one non-functional allele, the median urinary metabolic ratio (MRs) for sparteine Oxidation was 4.4-fold reduced in individuals with the variant allele (*2[-1496 G], MRs = 0.53, n = 27) compared with individuals lacking the mutation (*2[-1496 C], MRs = 2.33, n = 12; P < 0.0001). The mutation -1496 C to G has an estimated frequency of approximately 20% in the general population and allows establishment of a genotype for the identification of over 60% of intermediate metabolizers in Caucasian populations.
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assessment of the predictive power of genotypes for the in vivo catalytic function of cyp2d6 in a german population
Pharmacogenetics, 1998Co-Authors: Ernstulrich Griese, Ulrich M Zanger, Ulrich Brudermanns, Andrea Gaedigk, Gerd Mikus, Klaus Morike, Thomas Stuven, Michel EichelbaumAbstract:The polymorphic cytochrome P450 CYP2D6 catalyses the biotransformation of at least 40 Drugs. The CYP2D6 genetic polymorphism is responsible for pronounced interindividual differences in plasma concentrations and, hence, in Drug action and side-effects after administration of the same dose. Provided there is a close relationship between CYP2D6 genotypes and catalytic function, genotyping could be used in the clinical setting for individualization of Drug dose. In the present study, we evaluated the relationship between the in-vivo enzyme activity and 35 different genotypes in order to determine whether genotyping can be used to predict a person's metabolic capacity for CYP2D6-catalysed Drug Oxidation using sparteine as a probe Drug. One hundred and ninety-five Caucasian individuals were genotyped for seven nonfunctional (CYP2D6 x 3, x 4, x 5, x 6, x 7, x 8, x 16) and eight functional alleles (CYP2D6 x 1, x 2, x 2 x 2, x 2B, x 2B x 2, x 9, x 10, x 17). The metabolic ratio distribution for sparteine showed trimodality, with 15 poor metabolizers, 21 intermediate metabolizers, and 1.59 extensive and ultrarapid metabolizers. All poor metabolizers were unambiguously identified as carriers of two nonfunctional alleles. In contrast, the most frequent functional genotypes extensively overlapped and, with few exceptions, genotype was not a useful predictor of function. Gene dose effects among homozygotes and heterozygotes of the major functional alleles were not significant and could not explain the wide variations. Only a minor fraction of phenotypical ultrarapid metabolizers, arbitrarily defined as individuals with a metabolic ratio < 0.2, could be identified as carriers of three functional gene copies, including duplicated CYP2D6 x 2 x 2 alleles. Similarly, only a minor fraction of the intermediate metabolizers had predictive genotypes involving alleles coding for enzyme with impaired function. Thus, genotyping correctly identifies poor metabolizers, but quantitative prediction of Drug metabolism capacity among extensive metabolizers is not possible.
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molecular mechanisms of genetic polymorphisms of Drug metabolism
Annual Review of Pharmacology and Toxicology, 1997Co-Authors: Urs A Meyer, Ulrich M ZangerAbstract:One of the major causes of interindividual variation of Drug effects is genetic variation of Drug metabolism. Genetic polymorphisms of Drug-metabolizing enzymes give rise to distinct subgroups in the population that differ in their ability to perform certain Drug biotransformation reactions. Polymorphisms are generated by mutations in the genes for these enzymes, which cause decreased, increased, or absent enzyme expression or activity by multiple molecular mechanisms. Moreover, the variant alleles exist in the population at relatively high frequency. Genetic polymorphisms have been described for most Drug metabolizing enzymes. The molecular mechanisms of three polymorphisms are reviewed here. The acetylation polymorphism concerns the metabolism of a variety of arylamine and hydrazine Drugs, as well as carcinogens by the cytosolic N-acetyltransferase NAT2. Seven mutations of the NAT2 gene that occur singly or in combination define numerous alleles associated with decreased function. The debrisoquine-sparteine polymorphism of Drug Oxidation affects the metabolism of more than 40 Drugs. The poor metabolizer phenotype is caused by several “loss of function” alleles of the cytochrome P450 CYP2D6gene. On the other hand, “ultrarapid” metabolizers are caused by duplication or amplification of an active CYP2D6gene. Intermediate metabolizers are often heterozygotes or carry alleles with mutations that decrease enzyme activity only moderately. The mephenytoin polymorphism affects the metabolism of mephenytoin and several other Drugs. Two mutant alleles of CYP2C19have so far been identified to cause this polymorphism.
Peter F Guengerich - One of the best experts on this subject based on the ideXlab platform.
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combining chimeric mice with humanized liver mass spectrometry and physiologically based pharmacokinetic modeling in toxicology
Chemical Research in Toxicology, 2016Co-Authors: Hiroshi Yamazaki, Makiko Shimizu, Hiroshi Suemizu, Marina Mitsui, Peter F GuengerichAbstract:Species differences exist in terms of Drug Oxidation activities, which are mediated mainly by cytochrome P450 (P450) enzymes. To overcome the problem of species extrapolation, transchromosomic mice containing a human P450 3A cluster or chimeric mice transplanted with human hepatocytes have been introduced into the human toxicology research area. In this review, Drug metabolism and disposition mediated by humanized livers in chimeric mice are summarized in terms of biliary/urinary excretions of phthalate and bisphenol A and plasma clearances of the human cocktail probe Drugs caffeine, warfarin, omeprazole, metoprolol, and midazolam. Simulation of human plasma concentrations of the teratogen thalidomide and its human metabolites is possible with a simplified physiologically based pharmacokinetic model based on data obtained in chimeric mice, in accordance with reported clinical thalidomide concentrations. In addition, in vivo nonspecific hepatic protein binding parameters of metabolically activated 14C-Drug...
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the cyp3a4 intron 6 c t polymorphism cyp3a4 22 is associated with reduced cyp3a4 protein level and function in human liver microsomes
Journal of Toxicological Sciences, 2013Co-Authors: Maho Okubo, Norie Murayama, Makiko Shimizu, Tsutomu Shimada, Peter F Guengerich, Hiroshi YamazakiAbstract:Effects of the CYP3A4 intron 6 C>T (CYP3A4*22) polymorphism, which has recently been reported to have a critical role in vivo, were investigated by measuring CYP3A4 protein expression levels and CYP3A4-dependent Drug Oxidation activities in individual human liver microsomes in vitro. Prior to protein analysis, analysis of DNA samples indicated that 36 Caucasian subjects were genotyped as CYP3A4*1/*1 and five subjects were CYP3A4*1/*22, with a CYP3A4*22 allelic frequency of 6.1%. No CYP3A4*22 alleles were found in the Japanese samples (106 alleles). Individual differences in CYP2D6-dependent dextromethorphan O-demethylation activities in liver microsomes from Caucasians were not affected by either the CYP3A4*1/*22 or CYP3A5*1/*3 genotype. Liver microsomes genotyped as CYP3A4*1/*22 (n = 4) showed significantly lower CYP3A-dependent dextromethorphan N-demethylation, midazolam 1'-hydroxylation, and testosterone 6β-hydroxylation activities, as well as lower expression levels of CYP3A protein (28% of control), compared with those of the CYP3A4*1/*1 group (n = 19). The other polymorphism, CYP3A5*1/*3, did not show these differences (n = 4). The CYP3A4*22 polymorphism was associated with reduced CYP3A4 protein expression levels and resulted in decreased CYP3A4-dependent activities in human livers. The present results suggest an important role of low expression of CYP3A4 protein associated with the CYP3A4*22 allele in the individual differences in Drug clearance.
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kinetics and thermodynamics of ligand binding by cytochrome p450 3a4
Journal of Biological Chemistry, 2006Co-Authors: Emre M Isin, Peter F GuengerichAbstract:Abstract Cytochrome P450 (P450) 3A4, the major catalyst involved in human Drug Oxidation, displays substrate- and reaction-dependent homotropic and heterotropic cooperative behavior. Although several models have been proposed, these mainly rely on steady-state kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and α-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system, and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity.
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roles of nadph p450 reductase and apo and holo cytochrome b5 on xenobiotic Oxidations catalyzed by 12 recombinant human cytochrome p450s expressed in membranes of escherichia coli
Protein Expression and Purification, 2002Co-Authors: Hiroshi Yamazaki, Mami Nakamura, Tomoko Komatsu, Naoya Hatanaka, Satoru Asahi, Peter F Guengerich, Tsutomu ShimadaAbstract:Drug Oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent Drug Oxidations.
