Novel analytical methodologies for the characterization and determination of cytochrome P450 enzymes

The increased flux of compounds into drug discovery due to combinatorial chemistry and high-throughput screening techniques has placed an increased demand for speed and efficiency on the supporting analytical methodologies used for evaluating cytochrome P450 (CYP) metabolism. This dissertation describes novel analytical methodologies that have been developed for the characterization and determination of CYP enzymes.; A method for the determination of testosterone and its metabolite, 6β-hydroxytestosterone, in liver microsomal incubates employing gas chromatography with selected ion monitoring mass spectrometric detection (GC/SIM/MS) was developed. The method did not require derivatization and testosterone and its metabolites were separated on a HP-5MS fused-silica capillary column in less than 15 min. The assay has been used to characterize the CYP3A metabolic activity of multiple preparations of human, rat, and dog liver microsomes.; A method was also developed for the high-throughput inhibition screening of the major human cytochrome P450 (CYP) using an in vitro substrate cocktail and liquid chromatography-tandem mass spectrometry (LC/MS/MS). The concentrations of the substrate metabolites in incubated microsomal samples were determined by LC/MS/MS in a single assay. IC50s (μM) of model CYP inhibitors determined using the substrate cocktail were in good agreement with those obtained with individual substrates and with previously reported values in the literature.; A novel LC/MS method was developed for the study of CYP induction in rat liver microsomes. As reported previously in the literature, the primary CYP enzymes determined by LC/MS to be induced by β-napthaflavone were CYP1A, CYP2B by phenobarbital, CYP2E1 by isoniazid, and CYP3A by dexamethasone. Levels of induction of other microsomal proteins (epoxide hydrolase, glutathione s-transferase, and cytochrome b₅) were able to be determined by LC/MS simultaneously with CYP enzymes.; Finally, preliminary experiments to investigate the utility of capillary electrophoresis (CE) for the separation of CYP enzymes were conducted. Capillary coating, either by addition of a modifier to the run buffer or chemical modification of the capillary itself, was necessary to achieve reproducible results. Experiments with capillary isoelectric focusing (CIEF) demonstrated that this technique may be successful if a non-ionic detergent is used to prevent protein aggregation during the CIEF process.