The Experts below are selected from a list of 20512803 Experts worldwide ranked by ideXlab platform
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.
Joyce A Goldstein - One of the best experts on this subject based on the ideXlab platform.
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human CYP2C8 is post transcriptionally regulated by micrornas 103 and 107 in human liver
Molecular Pharmacology, 2012Co-Authors: Shuyun Zhang, Sherry J. Coulter, Sailesh Surapureddi, Stephen S G Ferguson, Joyce A GoldsteinAbstract:The CYP2C genes are extensively regulated at the transcriptional stage. The present study shows for the first time that CYP2Cs are also regulated post-transcriptionally by microRNAs (miRNAs). By using online search engines, we found potential miRNA response elements (MREs) in the 3′-untranslated region (3′-UTR) of the CYP2C mRNAs. Among these were a MRE for the miRNAs miR-103 and miR-107 in the 3′-UTR of human CYP2C8. CYP2C8 protein levels (measured through immunoblot analyses) did not correlate with CYP2C8 mRNA levels (measured through quantitative polymerase chain reaction analyses) in human liver samples. The translation efficiency (protein/mRNA ratio) for CYP2C8 was inversely correlated with the expression of miR-103 and miR-107. When three copies of the putative MRE from CYP2C8 were inserted downstream from a luciferase expression reporter, transfection with precursors for miR-103 or miR-107 decreased luciferase activity in primary hepatocytes, whereas transfection with antisense oligonucleotides (AsOs) for miR-103/miR-107 increased luciferase activity. As expected, there was no effect of the precursors or AsOs when three copies of the putative MRE were inserted in the reverse orientation. When precursors for miR-103/miR-107 were transfected into primary human hepatocytes, CYP2C8 protein levels were decreased, whereas AsOs increased CYP2C8 protein levels. Neither precursors nor AsOs affected CYP2C8 mRNA levels, which indicated that the effect was post-transcriptional. Putative MRE motifs were also found in the 3′-UTRs of CYP2C9 and CYP2C19, which suggested that the same miRNAs could regulate translation of other members of the CYP2C family, although to a lesser degree than CYP2C8. These results clearly show that CYP2Cs are regulated post-transcriptionally by miR-103 and miR-107.
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Identification of Human CYP2C8 as a Retinoid-Related Orphan Nuclear Receptor Target Gene
The Journal of pharmacology and experimental therapeutics, 2009Co-Authors: Yuping Chen, Sherry J. Coulter, Anton M. Jetten, Joyce A GoldsteinAbstract:Retinoid-related orphan nuclear receptors (RORs) α and γ (NR1F1, -3) are highly expressed in liver, adipose tissue, thymus, and brain and are involved in many physiological processes, such as circadian rhythm and immune function. Enzymes in the cytochrome P450 2C subfamily metabolize many clinically important drugs and endogenous compounds, such as the anticancer drug paclitaxel and arachidonic acid, and are highly expressed in liver. Here, we present the first evidence that RORs regulate the transcription of human CYP2C8. Overexpression of RORα and RORγ in HepG2 cells significantly enhanced the activity of the CYP2C8 promoter but not that of the CYP2C9 or CYP2C19 promoters. Computer analyses, promoter deletion studies, gel shift assays, and mutational analysis identified an essential ROR-responsive element at -2045 base pairs in the CYP2C8 promoter that mediates ROR transactivation. Adenoviral overexpression of RORα and -γ significantly induced endogenous CYP2C8 transcripts in both HepG2 cells and human primary hepatocytes. Knockdown of endogenous RORα and -γ expression in HepG2 cells by RNA interference decreased the expression of endogenous CYP2C8 mRNA by ∼50%. These data indicate that RORs transcriptionally up-regulate CYP2C8 in human liver and, therefore, may be important modulators of the metabolism of drugs and physiologically active endogenous compounds by this enzyme in liver and possibly extrahepatic tissues where RORs are expressed.
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detection of human CYP2C8 cyp2c9 and cyp2j2 in cardiovascular tissues
Drug Metabolism and Disposition, 2007Co-Authors: Tracy C Delozier, Sherry J. Coulter, Alyce J Bradbury, Julie F Foley, Darryl C Zeldin, Grace E Kissling, Diana Dai, Elizabeth Murphy, Charles Steenbergen, Joyce A GoldsteinAbstract:The cytochrome P450 (P450) enzymes CYP2C8, CYP2C9, and CYP2J2 metabolize arachidonic acid to epoxyeicosatrienoic acids, which are known to be vital in regulation of vascular tone and cardiovascular homeostasis. Because there is limited information regarding the relative expression of these P450 enzymes in cardiovascular tissues, this study examined the expression of CYP2C8, CYP2C9, and CYP2J2 mRNA and protein in human heart, aorta, and coronary artery samples by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. CYP2J2 and CYP2C9 mRNA levels were highly variable in human hearts, whereas CYP2C8 mRNA was present in lower abundance. CYP2J2 mRNA was approximately 10 3 times higher than CYP2C9 or CYP2C8 in human heart. However, CYP2C9 mRNA was more abundant than CYP2J2 or CYP2C8 in one ischemic heart. In human aorta, mean CYP2C9 mRNA levels were ∼50 times higher than that of CYP2J2 and 5-fold higher than that of CYP2C8. In human coronary artery, mean values for CYP2C9 mRNA were ∼2-fold higher than that of CYP2J2 mRNA and 6-fold higher than that of CYP2C8 mRNA. Immunoblotting results show relatively high levels of CYP2J2 and CYP2C8 protein in human hearts, which was confirmed by immunohistochemistry. CYP2C9 protein was also detected at high levels in one ischemic heart by immunoblotting. CYP2C9 was present at higher levels than CYPJ2 in aorta and coronary artery, whereas CYP2C8 protein was below the limits of detection. The expression of CYP2J2 and CYP2C8 in human heart, and CYPC9 and CYP2J2 in aorta and coronary artery is consistent with a physiological role for these enzymes in these tissues.
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human CYP2C8 is transcriptionally regulated by the nuclear receptors constitutive androstane receptor pregnane x receptor glucocorticoid receptor and hepatic nuclear factor 4α
Molecular Pharmacology, 2005Co-Authors: Stephen S Ferguson, Edward L Lecluyse, Yuping Chen, Masahiko Negishi, Joyce A GoldsteinAbstract:Cytochrome P450 (P450) enzymes play important roles in the metabolism of endogenous and xenobiotic substrates in humans. CYP2C8 is an important member of the CYP2C subfamily, which metabolizes both endogenous compounds (i.e., arachidonic acids and retinoic acid) and xenobiotics (e.g., paclitaxel). Induction of P450 enzymes by drugs can result in tolerance as well as drug-drug interactions. CYP2C8 is the most strongly inducible member of the CYP2C subfamily in human hepatocytes, but the mechanism of induction by xenobiotics has not been delineated. To determine the mechanisms controlling the regulation of this important P450, we cloned the 5′-flanking region of CYP2C8 and investigated its transcriptional regulation by nuclear factors such as the pregnane X receptor (PXR), constitutive androstane receptor (CAR), glucocorticoid receptor (GR), and hepatic nuclear factor 4 (HNF4α) that are known to be involved in the induction of other P450 enzymes using both cell lines and primary hepatocyte models. We initially identified a distal PXR/CAR-binding site in the CYP2C8 promoter that confers inducibility of CYP2C8 via the PXR agonist/ligand rifampicin and the CAR agonist/ligand CITCO [6-(4-chlorophenyl)imidazo[2,1- b ][1,3]thiazole-5-carbaldehyde O -(3,4-dichlorobenzyl)oxime]. A glucocorticoid-responsive element was identified that mediates dexamethasone induction via the GR. We finally identified an HNF4α-binding site within the CYP2C8 basal promoter region that is cis -activated by cotransfected HNF4α. In summary, the present studies show that CAR, PXR, GR, and HNF4α can regulate CYP2C8 expression and identify specific cis -elements within the promoter that control these regulatory pathways.
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clinical relevance of genetic polymorphisms in the human cyp2c subfamily
British Journal of Clinical Pharmacology, 2001Co-Authors: Joyce A GoldsteinAbstract:The human CYP2Cs are an important subfamily of P450 enzymes that metabolize approximately 20% of clinically used drugs. There are four members of the subfamily, CYP2C8, CYP2C9, CYP2C19, and CYP2C18. Of these CYP2C8, CYP2C9, and CYP2C19 are of clinical importance. The CYP2Cs also metabolize some endogenous compounds such as arachidonic acid. Each member of this subfamily has been found to be genetically polymorphic. The most well-known of these polymorphisms is in CYP2C19. Poor metabolizers (PMs) of CYP2C19 represent approximately 3–5% of Caucasians, a similar percentage of African-Americans and 12–100% of Asian groups. The polymorphism affects metabolism of the anticonvulsant agent mephenytoin, proton pump inhibitors such as omeprazole, the anxiolytic agent diazepam, certain antidepressants, and the antimalarial drug proguanil. Toxic effects can occur in PMs exposed to diazepam, and the efficacy of some proton pump inhibitors may be greater in PMs than EMs at low doses of these drugs. A number of mutant alleles exist that can be detected by genetic testing. CYP2C9 metabolizes a wide variety of drugs including the anticoagulant warfarin, antidiabetic agents such as tolbutamide, anticonvulsants such as phenytoin, and nonsteroidal anti-inflammatory drugs. The incidence of functional polymorphisms is much lower, estimated to be 1/250 in Caucasians and lower in Asians. However, the clinical consequences of these rarer polymorphisms can be severe. Severe and life-threatening bleeding episodes have been reported in CYP2C9 PMs exposed to warfarin. Phenytoin has been reported to cause severe toxicity in PMs. New polymorphisms have been discovered in CYP2C8, which metabolizes taxol (paclitaxel). Genetic testing is available for all of the known CYP2C variant alleles.
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 (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.
Laurence S Kaminsky - One of the best experts on this subject based on the ideXlab platform.
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human extrahepatic cytochromes p450 function in xenobiotic metabolism and tissue selective chemical toxicity in the respiratory and gastrointestinal tracts
Annual Review of Pharmacology and Toxicology, 2003Co-Authors: Laurence S KaminskyAbstract:Cytochrome P450 (CYP) enzymes in extrahepatic tissues often play a dominant role in target tissue metabolic activation of xenobiotic compounds. They may also determine drug efficacy and influence the tissue burden of foreign chemicals or bioavailability of therapeutic agents. This review focuses on xenobiotic-metabolizing CYPs of the human respiratory and gastrointestinal tracts, including the lung, trachea, nasal respiratory and olfactory mucosa, esophagus, stomach, small intestine, and colon. Many CYPs are expressed in one or more of these organs, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2S1, CYP3A4, CYP3A5, and CYP4B1. Of particular interest are the preferential expression of certain CYPs in the respiratory tract and the regional differences in CYP expression profile in different parts of the gastrointestinal tract. Current research activities on the characterization of CYP expression, function, and regulation in the...
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human extrahepatic cytochromes p450 function in xenobiotic metabolism and tissue selective chemical toxicity in the respiratory and gastrointestinal tracts
Annual Review of Pharmacology and Toxicology, 2003Co-Authors: Laurence S KaminskyAbstract:Cytochrome P450 (CYP) enzymes in extrahepatic tissues often play a dominant role in target tissue metabolic activation of xenobiotic compounds. They may also determine drug efficacy and influence the tissue burden of foreign chemicals or bioavailability of therapeutic agents. This review focuses on xenobiotic-metabolizing CYPs of the human respiratory and gastrointestinal tracts, including the lung, trachea, nasal respiratory and olfactory mucosa, esophagus, stomach, small intestine, and colon. Many CYPs are expressed in one or more of these organs, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2S1, CYP3A4, CYP3A5, and CYP4B1. Of particular interest are the preferential expression of certain CYPs in the respiratory tract and the regional differences in CYP expression profile in different parts of the gastrointestinal tract. Current research activities on the characterization of CYP expression, function, and regulation in these tissues, as well as future research needs, are discussed.
Anne M Filppula - 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.
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reevaluation of the microsomal metabolism of montelukast major contribution by CYP2C8 at clinically relevant concentrations
Drug Metabolism and Disposition, 2011Co-Authors: Anne M Filppula, Pertti J. Neuvonen, Jouko Laitila, Janne T. BackmanAbstract:According to published in vitro studies, cytochrome P450 3A4 catalyzes montelukast 21-hydroxylation (M5 formation), whereas CYP2C9 catalyzes 36-hydroxylation (M6), the primary step in the main metabolic pathway of montelukast. However, montelukast is a selective competitive CYP2C8 inhibitor, and our recent in vivo studies suggest that CYP2C8 is involved in its metabolism. We therefore reevaluated the contributions of different cytochrome P450 (P450) enzymes, particularly that of CYP2C8, to the hepatic microsomal metabolism of montelukast using clinically relevant substrate concentrations in vitro. The effects of P450 isoform inhibitors on montelukast metabolism were examined using pooled human liver microsomes, and montelukast oxidations by human recombinant CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were investigated. The results verified the central role of CYP3A4 in M5 formation. The CYP2C8 inhibitors gemfibrozil 1-O-β glucuronide and trimethoprim inhibited the depletion of 0.02 μM montelukast and formation of M6 from 0.05 μM montelukast more potently than did the CYP2C9 inhibitor sulfaphenazole. Likewise, recombinant CYP2C8 catalyzed montelukast depletion and M6 formation at a 6 times higher intrinsic clearance than did CYP2C9, whereas other P450 isoforms produced no M6. On the basis of depletion of 0.02 μM montelukast, CYP2C8 was estimated to account for 72% of the oxidative metabolism of montelukast in vivo, with a 16% contribution for CYP3A4 and 12% for CYP2C9. Moreover, CYP2C8 catalyzed the further metabolism of M6 more actively than did any other P450. In conclusion, CYP2C8 plays a major role in the main metabolic pathway of montelukast at clinically relevant montelukast concentrations in vitro.
