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Charles L Crespi - One of the best experts on this subject based on the ideXlab platform.
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fluorometric high throughput screening for inhibitors of cytochrome p450
Annals of the New York Academy of Sciences, 2006Co-Authors: Vaughn P Miller, David M Stresser, Andrew P Blanchard, Stephanie D Turner, Charles L CrespiAbstract:Rapid screening for cytochrome P450 inhibitors is part of the current paradigm for avoiding development of drugs likely to give clinical pharmacokinetic drug-drug interactions and associated toxicities. We have developed microtiter plate-based, direct, fluorometric assays for the activities of the principal human drug-metabolizing enzymes, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, as well as for CYP2A6, which is an important enzyme in environmental toxicology. These assays are rapid and compatible with existing high-throughput assay instrumentation. For CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, the potency of enzyme inhibition (IC50) is consistent regardless of the probe substrate or assay method employed. In contrast, CYP3A4 inhibition for an individual inhibitor shows significant differences in potency (>300-fold) depending on the probe substrate being used. We have investigated these differences through the use of several structurally distinct fluorescent substrates for CYP3A4 and several classical substrate probes (e.g., testosterone, nifedipine, and midazolam), with a panel of known, clinically significant, CYP3A4 inhibitors. The use of multiple probe substrates appears to be needed to characterize the inhibition potential of xenobiotics for CYP3A4.
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effect of methanol ethanol dimethyl sulfoxide and acetonitrile on in vitro activities of cdna expressed human cytochromes p 450
Drug Metabolism and Disposition, 1999Co-Authors: William F Busby, Joseph M Ackermann, Charles L CrespiAbstract:The effects of methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied in vitro on nine individual, cDNAexpressed cytochrome P-450 activities (phenacetinO-deethylase for CYP1A1 and CYP1A2, coumarin 7-hydroxylase for CYP2A6, testosterone 6β-hydroxylase for CYP3A4, 7-ethoxy-4-trifluoromethylcoumarin deethylase for CYP2B6, paclitaxel 6α-hydroxylase for CYP2C8, diclofenac 4′-hydroxylase for CYP2C9,S-mephenytoin 4-hydroxylase for CYP2C19, and (±)-bufuralol 1′-hydroxylase for CYP2D6) in commercially available human lymphoblastoid microsomes. These data show that specific solvents have enzyme-selective effects on P-450 activities. Methanol did not substantially inhibit (≤10%) any of the activities at 0.3%, but did inhibit CYP1A1, CYP2B6, CYP2C9, and CYP2D6 by 12 to 26% at 1%. In contrast, 0.1% ethanol inhibited CYP1A1, CYP2B6, and CYP2C19 by 20 to 30%. Ethanol at 1% did not inhibit CYP1A2, CYP3A4, CYP2C8, and CYP2C9. DMSO inhibited CYP3A4, CYP2C19, and CYP2D6 by 15 to 25% at 0.1%. However, DMSO had little effect on CYP1A2, CYP2A6, and CYP2C8. Acetonitrile, like methanol, did not inhibit any P-450 activity at 0.3% solvent except for CYP1A1 (26%) and CYP2B6 (13%). At 1%, acetonitrile decreased activities of CYP1A1 and CYP2B6 by 40 to 60%, and inhibited CYP2A6, CYP3A4, CYP2C19, and CYP2D6 activity by 10 to 20%. Acetonitrile also increased CYP2C9 activity by 10 to 15% above control values at 1 to 3% solvent. Excluding solubility considerations, methanol and acetonitrile appear to be the most suitable solvents for the introduction of substances to cytochrome P-450 incubations for in vitro metabolism studies.
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effect of methanol ethanol dimethyl sulfoxide and acetonitrile on in vitro activities of cdna expressed human cytochromes p 450
Drug Metabolism and Disposition, 1999Co-Authors: William F Busby, Joseph M Ackermann, Charles L CrespiAbstract:The effects of methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied in vitro on nine individual, cDNAexpressed cytochrome P-450 activities (phenacetin O-deethylase for CYP1A1 and CYP1A2, coumarin 7-hydroxylase for CYP2A6, testosterone 6beta-hydroxylase for CYP3A4, 7-ethoxy-4-trifluoromethylcoumarin deethylase for CYP2B6, paclitaxel 6alpha-hydroxylase for CYP2C8, diclofenac 4'-hydroxylase for CYP2C9, S-mephenytoin 4-hydroxylase for CYP2C19, and (+/-)-bufuralol 1'-hydroxylase for CYP2D6) in commercially available human lymphoblastoid microsomes. These data show that specific solvents have enzyme-selective effects on P-450 activities. Methanol did not substantially inhibit (CYP2C9, and CYP2D6 by 12 to 26% at 1%. In contrast, 0.1% ethanol inhibited CYP1A1, CYP2B6, and CYP2C19 by 20 to 30%. Ethanol at 1% did not inhibit CYP1A2, CYP3A4, CYP2C8, and CYP2C9. DMSO inhibited CYP3A4, CYP2C19, and CYP2D6 by 15 to 25% at 0.1%. However, DMSO had little effect on CYP1A2, CYP2A6, and CYP2C8. Acetonitrile, like methanol, did not inhibit any P-450 activity at 0.3% solvent except for CYP1A1 (26%) and CYP2B6 (13%). At 1%, acetonitrile decreased activities of CYP1A1 and CYP2B6 by 40 to 60%, and inhibited CYP2A6, CYP3A4, CYP2C19, and CYP2D6 activity by 10 to 20%. Acetonitrile also increased CYP2C9 activity by 10 to 15% above control values at 1 to 3% solvent. Excluding solubility considerations, methanol and acetonitrile appear to be the most suitable solvents for the introduction of substances to cytochrome P-450 incubations for in vitro metabolism studies.
Mikko Niemi - One of the best experts on this subject based on the ideXlab platform.
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an automated cocktail method for in vitro assessment of direct and time dependent inhibition of nine major cytochrome p450 enzymes application to establishing cyp2c8 inhibitor selectivity
European Journal of Pharmaceutical Sciences, 2021Co-Authors: Helina Kahma, Laura Aurinsalo, Mikko Neuvonen, Jani Katajamaki, Marienoelle Paludetto, Jenni Viinamaki, Terhi Launiainen, Anne M Filppula, Aleksi Tornio, Mikko NiemiAbstract:Abstract We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-β-glucuronide and clopidogrel acyl-β-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-β-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-β-glucuronide was a strong (>90% inhibition) and selective (
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an automated cocktail method for in vitro assessment of direct and time dependent inhibition of nine major cytochrome p450 enzymes application to establishing cyp2c8 inhibitor selectivity
European Journal of Pharmaceutical Sciences, 2021Co-Authors: Helina Kahma, Laura Aurinsalo, Mikko Neuvonen, Jani Katajamaki, Marienoelle Paludetto, Jenni Viinamaki, Terhi Launiainen, Anne M Filppula, Aleksi Tornio, Mikko NiemiAbstract:We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-β-glucuronide and clopidogrel acyl-β-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-β-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-β-glucuronide was a strong (>90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 μM, while the selectivity of clopidogrel acyl-β-D-glucuronide was limited at concentrations above its IC80 for CYP2C8. The time-dependent IC50 values of these glucuronides for CYP2C8 were 8.1 and 38 µM, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting time-dependent inhibition. Moreover, gemfibrozil 1-O-β-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies.
Zeruesenay Desta - One of the best experts on this subject based on the ideXlab platform.
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in vitro analysis and quantitative prediction of efavirenz inhibition of eight cytochrome p450 cyp enzymes major effects on cyps 2b6 2c8 2c9 and 2c19
Drug Metabolism and Pharmacokinetics, 2013Co-Authors: Zeruesenay DestaAbstract:In order to quantitatively predict drug interactions associated with efavirenz-based anti-HIV therapy, we evaluated reversible and time-dependent inhibitions of efavirenz on eight cytochrome P450 (CYP) enzymes in vitro. The present study showed that efavirenz was a potent competitive inhibitor of CYP2B6 (average K(i) = 1.68 µM in HLMs and K(i) = 1.38 µM in expressed CYP2B6) and CYP2C8 (K(i) = 4.78 µM in pooled HLMs and K(i) = 4.80 µM in HLMs with CYP2C8*3/*3 genotype). Efavirenz was a moderate inhibitor of CYP2C9 (K(i) = 19.46 µM) and CYP2C19 (K(i) = 21.31 µM); and a weak inhibitor of CYP3A (K(i) = 40.33 µM). No appreciable inhibition was observed on CYP1A2, CYP2A6 or CYP2D6. No time-dependent inhibition of the CYPs by efavirenz was observed in this study. Quantitative predictions showed that single dose of efavirenz may substantially slow the elimination of drugs predominantly cleared by CYP2B6, CYP2C19 or by both enzymes and may also lower the area under the plasma concentration time curve (AUC) of active metabolites of some pro-drugs (e.g., clopidogrel and proguanil) by up to 30%. Depending on substrates, chronic administration of efavirenz may increase the AUC of CYP2C8 and CYP2C9 substrates about 3.5-4.4-fold and 1.7-2.0-fold at steady state.
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comprehensive in vitro analysis of voriconazole inhibition of eight cytochrome p450 cyp enzymes major effect on cyps 2b6 2c9 2c19 and 3a
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: Seong Wook Jeong, Phuong D Nguyen, Zeruesenay DestaAbstract:Voriconazole is an effective antifungal drug, but adverse drug-drug interactions associated with its use are of major clinical concern. To identify the mechanisms of these interactions, we tested the inhibitory potency of voriconazole with eight human cytochrome P450 (CYP) enzymes. Isoform-specific probes were incubated with human liver microsomes (HLMs) (or expressed CYPs) and cofactors in the absence and the presence of voriconazole. Preincubation experiments were performed to test mechanism-based inactivation. In pilot experiments, voriconazole showed inhibition of CYP2B6, CYP2C9, CYP2C19, and CYP3A (half-maximal [50%] inhibitory concentrations, <6 microM); its effect on CYP1A2, CYP2A6, CYP2C8, and CYP2D6 was marginal (<25% inhibition at 100 microM voriconazole). Further detailed experiments with HLMs showed that voriconazole is a potent competitive inhibitor of CYP2B6 (K(i) < 0.5), CYP2C9 (K(i) = 2.79 microM), and CYP2C19 (K(i) = 5.1 microM). The inhibition of CYP3A by voriconazole was explained by noncompetitive (K(i) = 2.97 microM) and competitive (K(i) = 0.66 microM) modes of inhibition. Prediction of the in vivo interaction of voriconazole from these in vitro data suggests that voriconazole would substantially increase the exposure of drugs metabolized by CYP2B6, CYP2C9, CYP2C19, and CYP3A. Clinicians should be aware of these interactions and monitor patients for adverse effects or failure of therapy.
Yasuhiro Uno - One of the best experts on this subject based on the ideXlab platform.
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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 UnoAbstract: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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comprehensive evaluation for substrate selectivity of cynomolgus monkey cytochrome p450 2c9 a new efavirenz oxidase
Drug Metabolism and Disposition, 2015Co-Authors: Shinya Hosaka, Masahiro Satsukawa, Shotaro Uehara, Kazuhide Iwasaki, Shunsuke Iwano, Yasuhiro UnoAbstract: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.
Guangji Wang - One of the best experts on this subject based on the ideXlab platform.
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a comprehensive assay for nine major cytochrome p450 enzymes activities with 16 probe reactions on human liver microsomes by a single lc ms ms run to support reliable in vitro inhibitory drug drug interaction evaluation
Xenobiotica, 2015Co-Authors: Ying Peng, Yunxi Zhong, Hua Sang, Fengyi Zhang, Huanhuan Qi, Hui Wu, Xueyuan Zhang, Yu Wang, Guangji WangAbstract:Abstract1. A comprehensive method for the simultaneous characterization of xenobiotic compound inhibition of nine major CYP enzymes in human liver microsomes was established by using 16 CYP-catalyzed reactions of 14 probe substrates with three cocktail incubation sets and a single LC/MS/MS analysis.2. The three cocktail subgroups were developed to minimize the effects of organic solvents, polyunsaturated fatty acids and mutual substrate interactions: Group I was composed of tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), testosterone (CYP3A4), dextromethorphan (CYP2D6); Group II was composed of nifedipine (CYP3A4), midazolam (CYP3A4), coumarin (CYP2A6), bupropion (CYP2B6), diclofenac (CYP2C9); Group III was composed of phenacetin (CYP1A2), chlorzoxazone (CYP2E1), omeprazole (CYP2C19 and CYP3A4), paclitaxel (CYP2C8), (+)-bufuralol (CYP2D6). In the case of CYP2C9, CYP2C19, CYP2D6 and CYP3A4, multiple probe substrates were used due to the phenomenon of multiple substrate-binding pockets and substrate-dependen...
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A comprehensive assay for nine major cytochrome P450 enzymes activities with 16 probe reactions on human liver microsomes by a single LC/MS/MS run to support reliable in vitro inhibitory drug–drug interaction evaluation
2015Co-Authors: Ying Peng, Hua Sang, Fengyi Zhang, Guangji Wang, Xueyuan Zhang, Yu Wang, Jianguo Sun, Yunxi ZhongAbstract:1. A comprehensive method for the simultaneous characterization of xenobiotic compound inhibition of nine major CYP enzymes in human liver microsomes was established by using 16 CYP-catalyzed reactions of 14 probe substrates with three cocktail incubation sets and a single LC/MS/MS analysis.2. The three cocktail subgroups were developed to minimize the effects of organic solvents, polyunsaturated fatty acids and mutual substrate interactions: Group I was composed of tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), testosterone (CYP3A4), dextromethorphan (CYP2D6); Group II was composed of nifedipine (CYP3A4), midazolam (CYP3A4), coumarin (CYP2A6), bupropion (CYP2B6), diclofenac (CYP2C9); Group III was composed of phenacetin (CYP1A2), chlorzoxazone (CYP2E1), omeprazole (CYP2C19 and CYP3A4), paclitaxel (CYP2C8), (+)-bufuralol (CYP2D6). In the case of CYP2C9, CYP2C19, CYP2D6 and CYP3A4, multiple probe substrates were used due to the phenomenon of multiple substrate-binding pockets and substrate-dependent inhibition. All probe metabolites were simultaneously analyzed with a polarity switching mode in a single LC/MS/MS run.3. This method was validated against the single probe substrate assay using 12 well-characterized CYP inhibitors and two new entities (GT0918, MDV3100). The IC50 values of each inhibitor in the cocktail agreed well with that of the individual probe drug as well as with values reported in previous literatures.
