Highly Miniaturized Formats for In Vitro Drug Metabolism Assays Using Vivid ® Fluorescent Substrates and Recombinant Human Cytochrome P450 Enzymes

®fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid ® fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors .I C 50 values obtained for the modifiers in 96- and 1536well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall exam