Examination of purported probes of human CYP2B6

Pharmacogenetics. 1997 Jun;7(3):165-79. doi: 10.1097/00008571-199706000-00001.


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.

MeSH terms

  • Aryl Hydrocarbon Hydroxylases*
  • B-Lymphocytes / enzymology
  • B-Lymphocytes / metabolism
  • Benzoflavones / pharmacology
  • Blotting, Western
  • Cell Line
  • Chloramphenicol / pharmacology
  • Chromatography, High Pressure Liquid
  • Coumarins / metabolism
  • Cytochrome P-450 CYP2B6
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / metabolism*
  • Ditiocarb / pharmacology
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Microsomes / enzymology
  • Microsomes / metabolism
  • Microsomes, Liver / enzymology
  • Microsomes, Liver / metabolism
  • Molecular Probes*
  • Orphenadrine / pharmacology
  • Oxidoreductases, N-Demethylating / antagonists & inhibitors
  • Oxidoreductases, N-Demethylating / metabolism*
  • Substrate Specificity


  • Benzoflavones
  • Coumarins
  • Cytochrome P-450 Enzyme Inhibitors
  • Molecular Probes
  • 7-ethoxy-4-trifluoromethylcoumarin
  • 7-hydroxy-4-trifluoromethylcoumarin
  • alpha-naphthoflavone
  • Chloramphenicol
  • Cytochrome P-450 Enzyme System
  • Ditiocarb
  • Orphenadrine
  • Aryl Hydrocarbon Hydroxylases
  • CYP2B6 protein, human
  • Cytochrome P-450 CYP2B6
  • Oxidoreductases, N-Demethylating