Analytical approaches to determine cytochrome P450 inhibitory potential of new chemical entities in drug discovery

The use of a cassette incubation of probe substrates with human liver microsomes (HLM) - also known as the 'cocktail' approach - is becoming a widely accepted approach to determine the interaction of new chemical entities (NCEs) with cytochrome P450 enzymes (CYP450) in early drug discovery. This article describes two LC-MS/MS-based analytical methods used at the high-throughput (HT) stage and late discovery (LD) stage for analysis of 'cocktail' incubates to analyze the probe metabolites 1'-hydroxymidazolam (CYP3A4), 4'-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), 1'-hydroxytacrine (CYP1A2) and 4'-hydroxymephenytoin (CYP2C19). The analytical methods are advantageous over currently reported methods due to their sensitivity, shorter analyses times (<2 min/sample for the HT method and 4 min/sample for the LD method) and their ability to monitor a unique set of clinically relevant probe metabolites from a biological incubate containing low microsomal protein (0.1mg/mL). The analytical methods employ the same mobile phase, acetonitrile and 0.1% formic acid, under similar LC-MS/MS conditions. In the HT method, the chromatographic method consists of a short robust step-gradient where the probe metabolites are simultaneously and quickly eluted to enhance throughput. The probe metabolites are chromatographically resolved in the LD stage by utilizing a true linear gradient to obtain optimal peak separation. The IC50 data generated by both analytical methods using single incubations versus cocktail incubations for various test compounds are in good agreement (correlation coefficient (r2)>or=0.98). The scientist conducting the analysis is provided with a choice of method selection depending on the stage of the test compound and on whether throughput or minimizing interference from other co-eluting metabolites is the most important criterion.