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A high-throughput method for enzyme kinetic studies.

Lakshmi D Saraswat·Kimberley A Caserta·Kathy Laws·Darren Wei·Simon S Jones·Adedayo Adedoyin
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Abstract

A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 degrees C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery.

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Aryl Hydrocarbon HydroxylasesChromatography, LiquidCytochrome P-450 CYP2C9Cytochrome P-450 CYP2D6Cytochrome P-450 CYP2D6 InhibitorsCytochrome P-450 CYP3ACytochrome P-450 Enzyme InhibitorsCytochrome P-450 Enzyme SystemDextromethorphanDiclofenacDrug Evaluation, PreclinicalEnzyme InhibitorsEnzymesHalf-LifeHumansKineticsMass SpectrometryMidazolamMolecular BiologySensitivity and SpecificityTestosteroneWarfarinJournal ArticleValidation Study

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Lakshmi D Saraswat, Kimberley A Caserta, Kathy Laws, Darren Wei, Simon S Jones, Adedayo Adedoyin. 2003. A high-throughput method for enzyme kinetic studies.. https://doi.org/10.1177/1087057103257290

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