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
Autor: | Mikko Niemi, Mikko Neuvonen, Janne T. Backman, Terhi Launiainen, Anne M. Filppula, Jani Katajamäki, Helinä Kahma, Aleksi Tornio, Laura Aurinsalo, Jenni Viinamäki, Marie-Noëlle Paludetto |
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Přispěvatelé: | INDIVIDRUG - Individualized Drug Therapy, HUSLAB, Medicum, Department of Clinical Pharmacology, University of Helsinki, Faculty of Medicine, Research Programs Unit, Department of Diagnostics and Therapeutics, Janne Backman / Principal Investigator, Clinicum |
Rok vydání: | 2020 |
Předmět: |
Time-dependent inhibition
Furafylline CYP3A4 CYP2B6 Metabolite Pharmaceutical Science VIVO EXTRAPOLATION Cytochrome P450 02 engineering and technology HUMAN LIVER-MICROSOMES Pharmacology METABOLISM 030226 pharmacology & pharmacy Cytochrome P-450 CYP2C8 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Cytochrome P-450 Enzyme System Tandem Mass Spectrometry medicine Gemfibrozil Cytochrome P-450 Enzyme Inhibitors Humans ASSAY Drug Interactions DRUG-DRUG INTERACTIONS Metabolic interactions Substrate cocktail MECHANISM-BASED INHIBITION Cytochrome P-450 CYP2C9 Drug metabolism GEMFIBROZIL Chemistry CYP1A2 021001 nanoscience & nanotechnology Cytochrome P-450 CYP2C8 Inhibitors Tienilic acid 317 Pharmacy UNSATURATED FATTY-ACIDS Microsomes Liver Ritonavir INACTIVATION 3111 Biomedicine 0210 nano-technology 1-O-beta-glucuronide medicine.drug |
Zdroj: | European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 162 |
ISSN: | 1879-0720 |
Popis: | 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-beta-glucuronide and clopidogrel acyl-beta-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-beta-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-beta-glucuronide was a strong (90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 mu M, while the selectivity of clopidogrel acyl-beta-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 mu M, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting timedependent inhibition. Moreover, gemfibrozil 1-O-beta-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies. |
Databáze: | OpenAIRE |
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