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Drug Metabolism and Disposition Fast Forward
First published on January 24, 2007; DOI: 10.1124/dmd.106.012245

0090-9556/07/3504-640-648$20.00
DMD 35:640-648, 2007

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Development of Three Parallel Cytochrome P450 Enzyme Affinity Detection Systems Coupled On-line to Gradient High-Performance Liquid Chromatography

Jeroen Kool, Sebastiaan M. van Liempd, Huub van Rossum, Danny A. van Elswijk, Hubertus Irth, Jan N. M. Commandeur, and Nico P. E. Vermeulen

Leiden/Amsterdam Center for Drug Research-Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, The Netherlands (J.K., S.M.v.L., H.v.R., J.N.M.C., N.P.E.V.); and Kiadis B.V., Groningen, The Netherlands (H.I., D.A.v.E.)

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 ß-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.

Address correspondence to: Dr. Nico P. E. Vermeulen, Vrije Universiteit, Department of Pharmacochemistry, LACDR-Division of Molecular Toxicology, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. E-mail: npe.vermeulen{at}few.vu.nl






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