Examination of purported probes of human CYP2B6.
Ekins, Sean; VandenBranden, Mark; Ring, Barbara J.; Wrighton, Steven A.
7(3):165-179, June 1997.
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7-Ethoxy-4-trifluoromethyIcoumarin (7-EFC) was examined as a substrate for cytochrome P450 (P450) in microsomes from human livers and expressed in B-lymphoblastoid cells. The Odeethylation of 7-EFC to 7-hydroxy-4-trifluoromethyIcoumarin (7-HFC) varied over a liver bank (n=19) by a factor of 13 (40-507 pmol min-'mg-' 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). a-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 m site (average m=3.3 HM) 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 m component (average m=2.4 JUM). 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 of 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.
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