Simultaneous determination of multiple CYP inhibition constants using a cocktail-probe approach.

To identify cytochrome P450 (CYP) drug-drug interaction (DDI) potential of a new chemical entity, the use of a specific clinically relevant probe substrate in the presence of a test compound is common place. In early discovery of new chemical entities, a balance of rigor, the ability to predict clinical DDI, and throughput is desired in an in vitro assay. This chapter describes a high-throughput CYP-mediated DDI assay method that balances these characteristics. The method utilizes a cassette approach using a cocktail of five selective probe substrates for the major clinically relevant CYPs involved in drug interactions. CYP1A2, 2C9, 2C19, 2D6, and 3A activities are assessed with liquid chromatography/tandem mass spectrometry (LC-MS/MS) quantification of metabolite formation. The method also outlines specific inhibitors to evaluate dynamic range and as a positive control. The benefits and needs for caution of this method are noted and discussed.