N-Aralkylated derivatives of 1-aminobenzotriazole as isozyme-selective, mechanism-based inhibitors of guinea pig hepatic cytochrome P-450 dependent monooxygenase activity

Abstract

The mechanism-based inactivation of hepatic cytochrome P-450 by the suicide inhibitor 1-aminobenzotriazole and two of its derivatives, N-benzyl-1-aminobenzotriazole and N-α-memylbenzyl-1-aminobenzotriazole, was investigated in microsomes from untreated, phenobarbital-induced, and β-naphthoflavone-induced guinea pigs. Microsomal 7-ethoxyresorufin O-deethylase, 7-pentoxyresorufm O-dealkylase, and benzphetamine N-demethylase activities, and cytochrome P-450 content were determined following incubation with 1-aminobenzotriazole and its analogues. The loss of hepatic cytochrome P-450 content and monooxygenase activity was dependent on inhibitor concentration and required NADPH. N-Benzyl-1-aminobenzotriazole and N-α-methylbenzyl-1-aminobenzotriazole were more potent inhibitors of monooxygenase activity than the parent compound in microsomes from untreated and phenobarbital-induced guinea pigs. In microsomes from phenobarbital-induced guinea pigs, N-α-methylbenzyl-1-aminobenzotriazole (10 μM) was highly selective for the inactivation of the major cytochrome P-450 isozyme catalyzing 7-pentoxyresorufin O-dealkylation (the guinea pig ortholog of P-450IIB1) compared with those isozymes catalyzing 7-ethoxyresorufin O-deethylation or benzphetamine N-demethylation (88 ± 3% loss of activity vs. 35 ± 11 and 13 ± 7%, respectively). N-Benzyl-1-aminobenzotriazole was also selective for the inactivation of 7-pentoxyresorufm O-dealkylase activity, but to a lesser degree (56 ± 6 vs. 31 ± 8 and 21 ± 8%, respectively). In hepatic microsomes from untreated guinea pigs, the two N-substituted analogues were selective for the inhibition of 7-pentoxyresorufin O-dealkylation compared with benzphetamine N-demethylation, but not 7-ethoxyresorufin O-deethylation. The spectrally assayed loss of cytochrome P-450 caused by 1-aminobenzotriazole paralleled the inhibition of enzyme activity in all three treatment groups; however, the loss of cytochrome P-450 caused by N-benzyl-1-aminobenzotriazole and N-α-methylbenzyl-1-aminobenzotriazole was never greater than 45% even when monooxygenase activity was virtually 100% inhibited. In general, N-benzyl-1-aminobenzotriazole and N-α-methylbenzyl-1-aminobenzotriazole were more potent inhibitors of cytochrome P-450-dependent monooxygenase activity in hepatic microsomes from untreated compared with induced guinea pigs (for example, 100 μM N-benzyl-1-aminobenzotriazole inhibited 93 ± 3, 81 ± 1, and 61 ± 7% of the 7-ethoxyresorufin O-deethylase activity in hepatic microsomes from untreated, phenobarbital-induced, and β-naphthoflavone-induced guinea pigs, respectively). These latter data are consistent with the facile inactivation of guinea pig P-450IA1 but not P-450IA2 by N-benzyl-1-aminobenzotriazole.Key words: cytochrome P-450, guinea pig, isozyme selective, suicide inhibitors.