title: Development of an in vitro cytochrome P450 cocktail inhibition assay for assessing the inhibition risk of drugs of abuse.
abstract: Drugs of abuse are not tested for cytochrome P450 (CYP) inhibition potential before distribution. Therefore, a cocktail assay should be developed for testing the inhibition potential for all relevant CYPs. The following CYP test substrates and selective inhibitors were incubated in pooled human liver microsomes: phenacetin (alpha-naphthoflavone for CYP1A2), coumarin (tranylcypromine, CYP2A6), bupropion (sertraline, CYP2B6), amodiaquine (trimethoprim, CYP2C8), diclofenac (sulfaphenazole, CYP2C9), omeprazole (fluconazole, CYP2C19), dextromethorphan (quinidine, CYP2D6), chlorzoxazone (clomethiazole, CYP2E1), testosterone (verapamil, CYP3A). Samples were analyzed after protein precipitation using a Thermo Fisher Q-Exactive LC-high-resolution-MS/MS. The IC50 values were calculated by plotting the concentration of the formed metabolite, relative to the control sample, over the logarithm of the inhibitor concentration. They were determined either for single substrate or the cocktail incubation. Unfortunately, the cocktail assay had to be split because of interferences during incubation caused by substrates or metabolites, but the mixture of both incubates could be analyzed in one analytical run. The IC50 values determined in the single substrate or both cocktail incubations were comparable among themselves and with published data. In conclusion, the new inhibition cocktail assay was reproducible and applicable for testing the inhibition potential of drugs of abuse as exemplified for 2,5-dimethoxy-4-iodo-amfetamine (DOI).
authors.affiliation: Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, D-66421 Homburg (Saar), Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Dinger
authors.affiliation: Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, D-66421 Homburg (Saar), Germany.
authors.forename: Markus R
authors.initials: MR
authors.lastname: Meyer
authors.affiliation: Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, D-66421 Homburg (Saar), Germany. Electronic address: hans.maurer@uks.eu.
authors.forename: Hans H
authors.initials: HH
authors.lastname: Maurer
mesh_terms: D001681:Biological Assay / Q000379:methods*; D001711:Biotransformation; D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000633:toxicity*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D015203:Reproducibility of Results; D018570:Risk Assessment; D019966:Substance-Related Disorders / Q000150:complications / Q000201:enzymology*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System
keywords: CYP inhibition; Cocktail assay; Drugs of abuse; LC-HR–MS/MS