title: Examination of purported probes of human CYP2B6.
abstract: 7-Ethoxy-4-trifluoromethylcoumarin (7-EFC) was examined as a substrate for cytochrome P450 (P450) in microsomes from human livers and expressed in B-lymphoblastoid cells. The O-deethylation of 7-EFC to 7-hydroxy-4-trifluoromethylcoumarin (7-HFC) varied over a liver bank (n = 19) by a factor of 13 (40-507 pmol min-1 mg-1 protein). When compared with the ability of the bank of human liver samples to metabolize form-selective substrates of the P450, 7-HFC formation correlated strongly with the formation of the S-mephenytoin metabolite, nirvanol (r2 = 0.86, p < 0.0001). alpha-Napthoflavone (ANF), diethyldithiocarbamate (DDC) and chloramphenicol (CAP) inhibited the O-deethylation of 7-EFC by microsomes from human livers by greater than 60%. Orphenadrine (ORP), a reported specific CYP2B6 inhibitor, was a less potent inhibitor of 7-HFC formation by microsomes from human liver than DDC or ANF. Using microsomes from B-lymphoblastoid cells expressing specific P450s, CYP2B6 and CYP1A2 were found to produce substantial levels of 7-HFC whereas CYP2E1 and CYP2C19 produced detectable amounts of this metabolite. ORP inhibited expressed CYP2E1 and CYP2B6 mediated 7-HFC formation to a greater extent than the inhibition observed for CYP1A2. Methoxychlor and S-mephenytoin inhibited expressed CYP2B6 but not CYP1A2 mediated 7-EFC O-deethylation. Livers (n = 5) with high relative rates of 7-HFC formation displayed biphasic enzyme kinetics with the low K(m) site (average K(m) = 3.3 microM) demonstrating allosteric activation. Five livers with low relative rates of 7-HFC formation also exhibited biphasic kinetics but lacked evidence of an allosteric mechanism being involved in the low K(m) component (average K(m) = 2.4 microM). Furthermore, expressed CYP2B6 and CYP2E1 converted 7-EFC to 7-HFC with allosteric activation indicated, while CYP1A2 mediated metabolism of 7-EFC to 7-HFC best fit the classic Michaelis-Menten model. A commercially available antibody to rat CYP2B, suggested to be specific for CYP2B6, was found to cross react with all members to the CYP2 family examined including CYP2C19, which possessed a nearly identical electrophoretic mobility to that of CYP2B6 in the system examined. In total, the evidence presented indicates that multiple P450s are involved in the formation of 7-HFC from 7-EFC, therefore this does not appear to be a useful or a selective probe of CYP2B6 catalytic activity. Furthermore, the specificity of both antibody and chemical inhibitor (ORP) probes previously suggested to be specific for CYP2B6 is also questioned.
authors.affiliation: Department of Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ekins
authors.forename: M
authors.initials: M
authors.lastname: VandenBranden
authors.forename: B J
authors.initials: BJ
authors.lastname: Ring
authors.forename: S A
authors.initials: SA
authors.lastname: Wrighton
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases; D001402:B-Lymphocytes / Q000201:enzymology / Q000378:metabolism; D001571:Benzoflavones / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D002701:Chloramphenicol / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000378:metabolism; D065702:Cytochrome P-450 CYP2B6; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004050:Ditiocarb / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D015335:Molecular Probes; D009966:Orphenadrine / Q000494:pharmacology; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001571:Benzoflavones; D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D015335:Molecular Probes; C056033:7-ethoxy-4-trifluoromethylcoumarin; C084236:7-hydroxy-4-trifluoromethylcoumarin; C011512:alpha-naphthoflavone; D002701:Chloramphenicol; D003577:Cytochrome P-450 Enzyme System; D004050:Ditiocarb; D009966:Orphenadrine; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating