FURTHER CHARACTERIZATION OF THE EXPRESSION IN LIVER AND CATALYTIC ACTIVITY OF CYP2B6

Previous studies in this laboratory have determined the lack of specif icity 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 spe cificity of CYP2B6. A 100-fold variability in expression of immunodeta ctable CYP2B6 was demonstrated in a bank of 19 HLM samples (0.7 pmol/m g protein to 71.1 pmol/mg protein) using MAb 49-10-20. CYP2B6 levels w ere found to significantly (P < .0001) correlate with S-mephenytoin N- demethylation to nirvanol (r(2) = -0.89), 7-hydroxy-4-trifluoromethylc oumarin formation (r(2) = 0.81) and several markers of CYP3A levels an d activity. The relationships between nirvanol formation and CYP3A lev els or activity were found to depend on two HLM samples. K-m(apparent) values were generated for benzyloxyresorufin O-deethylation (1.3 mu M ), benzphetamine N-demethylation (93.4 mu M), 3-cyano 7-ethoxycoumarin O-deethylation (71.3 mu M), midazolam 1'-hydroxylation (46.1 mu M) an d 4-chloromethyl-7-ethoxycoumarin O-deethylation (33.7 mu M) using exp ressed CYP2B6. Testosterone 16 beta-hydroxylation by expressed CYP2B6 resulted in atypical kinetics characteristic of substrate activation. The data best fit the Hill equation with a K-m (apparent) of 50.5 mu M and an n of 1.3 (n = number of sites bound by activator). In conclusi on, the highly specific MAb 49-10-20 was used to provide further confi rmation that S-mephenytoin N-demethylation to nirvanol is a CYP2B6 sel ective probe. Finally, some, but not all substrates of CYP2B6 demonstr ate autoactivation.