A high‐throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography–tandem mass spectrometry

Abstract

A sensitive and high-throughput inhibition screening liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7-hydroxycoumarin, CYP2A6; 4-hydroxytolbutamide, CYP2C9; 4'-hydroxymephenytoin, CYP2C19; α-hydroxymetoprolol, CYP2D6; and 1-hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C18 column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC50 values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13-103.37%), and inter-day (RSD < 6.20%) and intra-day (RSD < 6.13%) precision. Also, the incubation extracts of the sample were stable at room temperature (20 °C) for 48 h and for 96 h in the autosampler (4 °C). The presented method is the first HPLC-MS/MS method of this combination for simultaneous detection of the five metabolites 7-hydroxycoumarin, 4-hydroxytolbutamide, 4'-hydroxymephenytoin, α-hydroxymetoprolol and 1-hydroxymidazolam in a single-run process. It is possible that the high-quality and -throughput cocktail provides suitable information in drug discovery and screening for new drug entities.

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

“…The ratios of the IC 50 values obtained using both approaches (Table 2) were compared and ranged from 1.03 to 1.82. Because these values were within a 2-fold range of each other, they (Eagling et al, 1998;Testino and Patonay, 2003;Walsky and Obach, 2004;Zientek et al, 2008;Otten et al, 2011;Kozakai et al, 2012;Liu et al, 2015 (Dierks et al, 2001;Walsky and Obach, 2004;Otten et al, 2011;Liu et al, 2015) CYP2C9 Tolbutamide Sulfaphenazole 0.53 6 0.06 0.90 6 0.10 0.069-1.30 (Eagling et al, 1998;Shader et al, 1999;Testino and Patonay, 2003;Workman and Raynaud, 2007;Zientek et al, 2008;Otten et al, 2011;Kozakai et al, 2012;Yao et al, 2012;Qin et al, 2014;Liu et al, 2015) CYP2C19 (S)-mephenytoin Ticlopidine 3.40 6 0.32 5.81 6 0.80 0.86-4.46 (Giancarlo et al, 2001;Zientek et al, 2008;Yao et al, 2012;Qin et al, 2014;Liu et al, 2015) CYP2D6 Dextromethorphan Quinidine 0.46 6 0.02 0.84 6 0.10 0.009-0.52 (Shader et al, 1999;Testino and Patonay, 2003;Walsky and Obach, 2004;Zientek et al, 2008;Otten et al, 2011;Kozakai et al, 2012;Yao et al, 2012;Qiao et al, 2014;Qin et al, 2014;…”

“…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).…”

Comprehensive assessment of Cytochrome P450 reactions: A multiplex approach using real-time ESI-MS

“…However, results are often inconsistent implying the necessity not only to determine product formation but also to investigate the fate of the substrates [23][24][25][26][27][28]. Recent studies presented a technique that focuses on an electromembrane extraction system which can be coupled to an electrospray ionization mass spectrometer (ESI-MS) for studying drug metabolism, generating a metabolic profile and the assessment of reaction kinetics [31,32].…”

“…These conventional studies often focus on the determination of K M or IC 50 values to investigate different CYP inhibitors [23][24][25][26][27][28]. Others base their CYP activity…”

“…Most investigations so far deal with activity of CYPs and inhibition studies predominantly applying highthroughput cocktail approaches [23][24][25][26][27][28]. These conventional studies often focus on the determination of K M or IC 50 values to investigate different CYP inhibitors [23][24][25][26][27][28].…”