Received for publication August 11, 2006.
Revised January 12, 2007.
Accepted for publication January 23, 2007.

Development of three parallel Cytochrome P450 enzyme affinity detection systems coupled on-line to gradient HPLC

Abstract

A High Resolution Screening (HRS) technology is described, in which gradient HPLC is connected on-line to three parallel placed bioaffinity detection systems containing mammalian Cytochrome P450's (CYP's). The three so-called Enzyme Affinity Detection (EAD) systems contained respectively liver microsomes from rats induced by alpha-naphthoflavone (CYP1A-activity), phenobarbital (CYP2B-activity) and dexamethasone (CYP3A-activity). Each CYP EAD system was optimized for enzyme, substrate and organic modifier (isopropylalcohol, methanol and acetonitrile) in flow injection analysis (FIA) mode. Characteristic CYP ligands were used to validate the CYP 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; S/N = 3) of potent inhibitors ranged from 1 - 3 pmol for CYP1A activity, 4 - 17 pmol for CYP2B activity and 4 - 15 pmol for CYP3A activity. The three optimized CYP EAD systems were subsequently coupled to gradient HPLC and used to screen compound mixtures for individual ligands. In order to increase analysis efficiency, finally a HRS system was constructed in which all three CYP EAD systems were coupled on-line and in parallel to gradient HPLC. The triple parallelized CYP EAD system was shown to enable rapid profiling of individual components in complex mixtures for inhibitory activity to three different CYP's.

Key words: analytical pharmacology/toxicology, CYP inhibition, cytochrome P450, drug analysis, drug-drug interactions, high throughput screening, HPLC