Development of three parallel cytochrome P450 enzyme affinity detection systems coupled on-line to gradient high-performance liquid chromatography.
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 beta-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. IC(50) 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.
Kool
Jeroen
J
Vrije Universiteit, Department of Pharmacochemistry, LACDR-Division of Molecular Toxicology, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
van Liempd
Sebastiaan M
SM
van Rossum
Huub
H
van Elswijk
Danny A
DA
Irth
Hubertus
H
Commandeur
Jan N M
JN
Vermeulen
Nico P E
NP
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017022:Flow Injection Analysis; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D010078:Oxazines / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013997:Time Factors; D019324:beta-Naphthoflavone / Q000494:pharmacology
C419147:7-benzyloxy-4-trifluoromethylcoumarin; D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D010078:Oxazines; D019324:beta-Naphthoflavone; D003907:Dexamethasone; C044943:pentoxyresorufin; D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1; D051544:Cytochrome P-450 CYP3A; D010634:Phenobarbital