Direct and metabolism‐dependent cytochrome P450 inhibition assays for evaluating drug–drug interactions

Abstract

We developed methods for evaluating the ntial inhibition of human cytochrome P450 (CYP) enzymes, including CYP1A2, CYP2A6, CYP2B6, CYP2 C9, CYP2 C19, CYP2D6, CYP2E1 and CYP3A4, using pooled human liver microsomes (HLMs). The CYP inhibition assay used substrate cocktail sets [set A: phenacetin for CYP1A2, coumarin for CYP2A6, (S)-(+)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4; set B: bupropion for CYP2B6, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1, and testosterone for CYP3A4] with quantitation by liquid chromatography-tandem mass spectrometry. A direct inhibition assay was performed with the substrate cocktails without β-nicotinamide adenine dinucleotide phosphate (NADPH) pre-incubation, and a metabolism-dependent inhibition (MDI) assay was performed after 30 min of pre-incubation with NADPH in HLMs. MDI was identified based on the half-maximal inhibitory concentration (IC(50)) shifts. The IC(50) values of the direct inhibitors determined using the probe substrate cocktails were in good agreement with previously reported values. Eight metabolism-dependent inhibitors including furafylline, 8-methoxypsoralen, tienilic acid, ticlopidine, fluoxetine, paroxetine, disulfiram and verapamil against CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, respectively, resulted in significant IC(50) shifts (≥2.5-fold) after pre-incubation. Thus, these CYP inhibition assays are considered to be useful tools for evaluating both direct inhibition and MDI at an early stage of the drug discovery and development process.

Expression of hepatic cytochrome P450s and UDP-glucuronosyltransferases in PXR and CAR double humanized mice treated with rifampicin

“…7-Penthoxyresorufin is commonly used as a probe substrate for the CYP2B (Burke et al, 1994), but is also metabolized by CYP1A2 (Kobayashi et al, 2002). Chlorzoxazone hydroxylation to 6-hydroxy chlorzoxazone is mediated by the CYP2E1 isoform (Lee and Kim, 2013). Midazolam is predominantly metabolized to 1 0 -hydroxy midazolam by CYP3A (Turpeinen et al, 2007).…”

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

“…To expedite these assays, several cocktail approaches, also known as n-in-one assays, have been developed to test for inhibition of several P450 isoforms simultaneously. Most of these assays test for inhibition of five to eight P450 isoforms and use a wide variety of experimental conditions and probe substrates (Dierks et al, 2001;Testino and Patonay, 2003;He et al, 2007;Li et al, 2007;Smith et al, 2007;Workman and Raynaud, 2007;Zientek et al, 2008;Alden et al, 2010;Otten et al, 2011;Yao et al, 2012;Kozakai et al, 2012;Lee and Kim, 2013;Qiao et al, 2014;Qin et al, 2014;Liu et al, 2015). Although a few approaches have claimed using 9 or 10 substrates to evaluate nine isoforms, they actually carry out separate incubations of subsets of probe substrates to avoid P450 interactions before pooling the mixtures immediately prior to a quantitative analysis step, use P450 substrates which are not recommended by the FDA, and/or use long incubation times of up to 60 minutes (Kim et al, 2005;Turpeinen et al, 2005;Tolonen et al, 2007;Dinger et al, 2014).…”

“…Simultaneously assessing the inhibition of nine P450 isoforms can significantly reduce the cost and time needed for the evaluation of drug-drug and drug-botanical interactions. Our in vitro high-throughput cocktail approach optimized enzyme protein concentration, minimized probe substrate interactions, minimized solvent effects, and used a fast and sensitive ultrahigh pressure liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS) quantitative assay (Chauret et al, 1998;Busby et al, 1999;Yuan et al, 2002;Turpeinen et al, 2005;Jia and Liu, 2007;Smith et al, 2007;Otten et al, 2011;Kozakai et al, 2012;Lee and Kim, 2013;Spaggiari et al, 2014). After validating the new assay using nine known P450 inhibitors, an extract of the botanical dietary supplement licorice (Glycyrrhiza glabra) was evaluated for P450 inhibition.…”

Human microsomal cyttrochrome P450-mediated reduction of oxysophocarpine, an active and highly toxic constituent derived from Sophora flavescens species, and its intestinal absorption and metabolism in rat

“…CYP3A4/5 and CYP2D6 are among the main drug-metabolizing enzymes in humans that are responsible for the metabolism of more than 50% of marketed drugs [19,21]. Drugs metabolized by CYPs are prone to drug-drug interactions, thereby modifying their response [18,32]. Metabolic inhibition is also involved in many drug-drug interactions; in some instances, the drug interactions can be lifethreatening for the human body.…”