Identification of the human and animal hepatic cytochromes P450 involved in clonazepam metabolism

Abstract

This report characterizes the cytochrome P450 isozyme involved in clonazepam metabolism. This study was undertaken using a library of liver microsomal fractions prepared from untreated rabbits or those treated with drugs known to specifically induce various cytochrome P450 isozymes (ie P450 2B4 by phenobarbital, P450 1A1 and P450 1A2 by 3-methylcholanthrene and beta-naphthoflavone, P450 2E1 by acetone and ethyl alcohol, and P450 3A6 by erythromycin). Only microsomes obtained from phenobarbital-treated rabbits exhibited a type II binding spectrum upon addition of clonazepam (Ks(app) = 31.4 +/- 3.8 microM) and significantly metabolized clonazepam to 7-aminoclonazepam. Benzphetamine, which is a known substrate for P450 2B1 was also extensively metabolized by microsomes prepared from phenobarbital treated rabbits. This indicates that the same isozyme (P450 2B subfamily) was involved in the biotransformation of both substrates. Experiments performed on 14 human liver microsomal preparations showed a wide interindividual variability (from 1-4) and a good correlation (r = 0.70) between benzphetamine and clonazepam metabolism. Since P450 3A4 (nf25) was involved in benzphetamine metabolism, clonazepam was probably nitroreduced by the same isozyme. An oligonucleotide specific for the P450 3A4 gene subfamily was synthetized and used for hybridization on total RNA from human liver samples. Two transcripts of 2.2 and 3.0 kb were detected and the level of the 2.2 kb mRNA expression was significantly correlated (r = 0.61) with the intensity of clonazepam nitroreduction by the corresponding microsome batches.