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Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies

Curcio, Raphaele
Published in Analytical and Bioanalytical Chemistry. 2010, vol. 398, p. 2163-2171
Collection Open Access - Licence nationale Springer
Abstract An automated in-capillary assay requiring very small quantities of reagents was developed for performing in vitro cytochrome P450 (CYP450) drug metabolism studies. The approach is based on the following: (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sandwich mode, within a capillary; (ii) mixing the reagents by diffusion across the interfaces between the injected solutions; (iii) collection of the capillary content at the end of the in-capillary assay; and (iv) off-line analysis of the incubation mixture by ultrahigh pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After optimizing the injection sequence of the reagents, the in-capillary approach was applied to the quantitative determination of the kinetics of drug metabolism reactions catalyzed by three CYP450 isozymes involved in human drug metabolism: CYP1A2, CYP2D6, and CYP3A4. It was demonstrated that this in-capillary method was able to provide similar kinetic parameters for CYP450 activity (e.g., Michaelis constants and turnover values) as the classical in vitro method, with a drastic reduction of reagent consumption.
Keywords Drug metabolismDrug-drug interactionsCYP450In-capillary enzymatic assaysCYP450 kinetics
PMID: 20835864
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CURCIO, Raphaele et al. Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies. In: Analytical and Bioanalytical Chemistry, 2010, vol. 398, p. 2163-2171. https://archive-ouverte.unige.ch/unige:12152

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Deposited on : 2010-10-19

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