title: Further characterization of the expression in liver and catalytic activity of CYP2B6.
abstract: Previous studies in this laboratory have determined the lack of specificity of several antibody and substrate probes of CYP2B6. The goals of the current study were to examine the expression of CYP2B6 in a bank of human liver microsome (HLM) samples using a new specific monoclonal antibody (MAb 49-10-20) and to further characterize the substrate specificity of CYP2B6. A 100-fold variability in expression of immunodetectable CYP2B6 was demonstrated in a bank of 19 HLM samples (0.7 pmol/mg protein to 71. 1 pmol/mg protein) using MAb 49-10-20. CYP2B6 levels were found to significantly (P < .0001) correlate with S-mephenytoin N-demethylation to nirvanol (r2 = 0.89), 7-hydroxy-4-trifluoromethylcoumarin formation (r2 = 0.81) and several markers of CYP3A levels and activity. The relationships between nirvanol formation and CYP3A levels or activity were found to depend on two HLM samples. Km (apparent) values were generated for benzyloxyresorufin O-deethylation (1.3 microM), benzphetamine N-demethylation (93.4 microM), 3-cyano 7-ethoxycoumarin O-deethylation (71.3 microM), midazolam 1'-hydroxylation (46.1 microM) and 4-chloromethyl-7-ethoxycoumarin O-deethylation (33.7 microM) using expressed CYP2B6. Testosterone 16beta-hydroxylation by expressed CYP2B6 resulted in atypical kinetics characteristic of substrate activation. The data best fit the Hill equation with a Km (apparent) of 50.5 microM and an n of 1.3 (n = number of sites bound by activator). In conclusion, the highly specific MAb 49-10-20 was used to provide further confirmation that S-mephenytoin N-demethylation to nirvanol is a CYP2B6 selective probe. Finally, some, but not all substrates of CYP2B6 demonstrate autoactivation.
authors.affiliation: Department of Drug Disposition, Lilly Research Laboratories, Eli Lilly and Co., Lilly Corporate Center, Indianapolis, Indiana, 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: J S
authors.initials: JS
authors.lastname: Gillespie
authors.forename: T J
authors.initials: TJ
authors.lastname: Yang
authors.forename: H V
authors.initials: HV
authors.lastname: Gelboin
authors.forename: S A
authors.initials: SA
authors.lastname: Wrighton
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D065702:Cytochrome P-450 CYP2B6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D008099:Liver / Q000201:enzymology*; D008874:Midazolam / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D008874:Midazolam