An In Vitro, High Throughput, Seven CYP Cocktail Inhibition Assay for the Evaluation of New Chemical Entities Using LC-MS/MS

Abstract

A validated method for the simultaneous characterization of xenobiotic compound-mediated inhibition of seven major cytochrome P450 (CYP) enzymes in pooled human liver microsomes through the use of specific CYP probe substrates (cocktail assay) with low protein content, and a rapid, three minute LC-MS/MS analytical method is described. The specific CYP substrates used in this cocktail assay included phenacetin (CYP1A2), bupropion (CYP2B6), amodiaquine (CYP2C8), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4/5). The LC-MS method incorporated the aforementioned seven CYP substrates along with their respective major metabolites, and one internal standard, labetalol. In a cross-validation analysis, the concentrations of each CYP probe substrate in the assay had minimal effect (i.e., inhibition or activation) on the other CYP activities. Furthermore, the assay conditions for the multiple probe substrate, ie., cocktail assay, were validated against the single probe substrate assay using 18 compounds with known CYP inhibition liabilities and 10 proprietary compounds. The inhibitory constant (Ki) determined with this cocktail assay was highly correlated (R(2) ≥ 0.77 for each individual probe substrate) with that of the single probe substrate assay for all 27 CYP inhibitors. This seven CYP inhibition cocktail assay has increased the efficiency to assess compounds for inhibition of the major CYP isoforms in a high throughput, drug discovery setting.

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;…”

“…IC 50 values are extrinsic constants and depend on experimental conditions, but K i values are intrinsic constants. Therefore, researchers sometimes estimate K i from IC 50 values (Otten et al, 2011). Assuming competitive inhibition, K i values can be calculated using the ChengPrusoff equation (K i = IC 50 /2 when substrate concentration is %K m ; and K i = IC 50 /1.1 when the substrate concentration is K m /10).…”

“…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.…”

Identification of Selective ERRγ Inverse Agonists

“…Aliquots of the supernatant were injected onto an LC-MS/MS system. The CYP-mediated activities in the presence of inhibitors were expressed as percentages of the corresponding control values [19].…”