High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions

• DOI: 10.1124/dmd.115.065987
• Publication Date: 2015-08-19T02:48:50Z
• Authors: Guannan Li; Ke Huang; Dejan Nikolic; Richard B. van Br...
• Keywords: Binding Sites; Herb-Drug Interactions; High-Throughput Screening Assays; Isoenzymes; 03 medical and health sciences; 0302 clinical medicine; Cytochrome P-450 Enzyme System; Tandem Mass Spectrometry; Glycyrrhiza; Microsomes, Liver; Cytochrome P-450 Enzyme Inhibitors; Humans; Drug Interactions; Chromatography, High Pressure Liquid

Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry-based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography-tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example.