SELECTIVITY AND KINETICS OF INACTIVATION OF RABBIT HEPATIC CYTOCHROMES P450 2B4 AND 2B5 BY N-ARALKYLATED DERIVATIVES OF 1-AMINOBENZOTRIAZOLE

The kinetics of mechanism-based inactivation of phenobarbital-inducibl e rabbit hepatic cytochromes P450 2B4 and 2B5 by N-benzyl-(BBT) and N- alpha-methylbenzyl (alpha MB) 1-aminobenzotriazole were investigated u sing reconstituted P450 2B4, a stable heterologous expression system, and hepatic microsomes. Low micromolar concentrations of the 1-aminobe nzotriazole derivatives caused reversible inhibition as well as rapid inactivation of reconstituted P450 2B4 and recombinant P450 2B4 and 2B 5. In contrast, even at a 1000-fold higher concentration, aminobenzotr iazole inactivated the expressed P450 2B enzymes less rapidly. Preincu bation of phenobarbital-induced hepatic microsomes with BET and alpha MB resulted in concentration-dependent decreases in marker activities of P450 2B4 and 2B5, benzyloxyresorufin O-debenzylase and androstenedi one 15 alpha-hydroxylase, respectively. BET caused the inactivation of P450 2B4 and 2B5 in hepatic microsomes with apparent K-I values of 1. 9 and 2.4 mu M and maximal rate constants of 0.29 and 0.18 min(-1), re spectively, alpha MB inactivated both P450 2B enzymes with similar K-I values (similar to 7 mu M) and maximal rate constants only slightly h igher for 2B4 compared with 2B5 (0.68 vs. 0.55 min(-1)). Similar P450 2B selectivity of BBT and alpha MB in both hepatic microsomes and the stable expression system further validates this new expression system and the use of the selective markers identified for 2B4 and 2B5 in hep atic microsomes. The results also provide a mechanistic basis for the high potency of the N-aralkylated 1-aminobenzotriazole derivatives in vivo and suggest that treatments that inactivate 2B4 will also lead to 2B5 inactivation.