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.
Footnotes
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The support for this project by Senter-Novem/BTS (#BTS00091) and Merck Research Laboratories (Drug Metabolism Department) is kindly acknowledged.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.106.012245.
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ABBREVIATIONS: P450, cytochrome P450; BTFC, 7-benzyloxy-4-trifluoromethylcoumarin; DEX, dexamethasone; EAD, enzyme affinity detection; FIA, flow injection analysis; HRS, high resolution screening; IPA, isopropyl alcohol; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MeCN, acetonitrile; MeOH, methanol; β-NF, β-naphthoflavone; PB, phenobarbital; PEG6000, polyethyleneglycol 6000; S/N, signal-to-noise ratio.
- Received July 26, 2006.
- Accepted January 23, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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