Development of three parallel cytochrome P450 enzyme affinity detection systems coupled on-line to gradient high-performance liquid chromatography

J. Kool, S.M. van Liempd, H. van Rossum, D.A. van Elswijk, H. Irth, J.N.M. Commandeur, N.P.E. Vermeulen

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

A high resolution screening (HRS) technology is described, in which gradient high-performance liquid chromatography (HPLC) is connected on-line to three parallel placed bioaffinity detection systems containing mammalian cytochromes P450 (P450s). The three so-called enzyme affinity detection (EAD) systems contained, respectively, liver microsomes from rats induced by β-naphthoflavone (CYP1A activity), phenobarbital (CYP2B activity), and dexamethasone (CYP3A activity). Each P450-EAD system was optimized for enzyme, substrate, and organic modifier (isopropyl alcohol, methanol, and acetonitrile) in flow injection analysis mode. Characteristic P450 ligands were used to validate the P450-EAD systems. IC50 values of the ligands were measured and found to be similar to those obtained with conventional microtiter plate reader assays. Detection limits (n = 3; signal-to-noise ratio = 3) of potent inhibitors ranged from 1 to 3 pmol for CYP1A activity, 4 to 17 pmol for CYP2B activity, and 4 to 15 pmol for CYP3A activity. The three optimized P450-EAD systems were subsequently coupled to gradient HPLC and used to screen compound mixtures for individual ligands. Finally, to increase analysis efficiency, a HRS system was constructed in which all three P450-EAD systems were coupled on-line and in parallel to gradient HPLC. The triple parallelized P450-EAD system was shown to enable rapid profiling of individual components in complex mixtures for inhibitory activity to three different P450s. Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics.
Original languageEnglish
Pages (from-to)640-8
JournalDrug Metabolism and Disposition
Volume35
Issue number4
DOIs
Publication statusPublished - 2007

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