High-Throughput, 384-Well, LC-MS/MS CYP Inhibition Assay Using Automation, Cassette-Analysis Technique and Streamlined Data Analysis

Jason       S. Halladay; Erlie Marie       Delarosa; Daniel      Tran; Leslie       Wang; Susan       Wong; S.       Cyrus Khojasteh

Abstract

Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using wellknown HLM-based MS probes. We provide consistently robust IC50 values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC50 values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)- warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC50 results for five major CYP isoforms using our method compared to values reported in the literature.

Keywords: Automation, Cytochrome P450, drug-drug interactions, high-throughput, human liver microsomes, inhibition, in vitro, new chemical entities (NCEs), mass spectrometry (MS), clinical trials