Inhibition and metabolite complexation of rat hepatic microsomal cytochrome p450 by tricyclic antidepressants

Abstract

Administration of imipramine (IMIP) and other tricyclic antidepressants to humans and experimental animals has been associated with inhibition of hepatic cytochrome P450 (P450)-mediated drug oxidation. This study investigated the capacity of several structurally related tricyclic antidepressants to inhibit musomal P450 activity in vitro. It was found that IMIP, desipramine (DES), amitriptyline (AMIT) and nortriptyline (NOR) were poor inhibitors of P450 activity unless they were preincubated with musomes and NADPH prior to transfer to flasks containing substrate. Thus, subsequent experiments characterized the time-dependent intensification of inhibition produced by the drugs. Preincubation of the N-methylaminoalkyi agents DES and NOR (200 μM) with NADPH-supplemented musomes for 30 min led to an approximate 30% decrease in spectrally apparent P450 content; the N, N-dimethylaminoalkyl drugs IMIP and AMIT did not significantly decrease apparent P450 content. Analysis of optical difference spectra of musomes during NADPH-mediated metabolism of these drugs revealed a prominent increase in absorbance at 454 nm with DES and NOR but not IMIP or AMIT. Monospecific antibodies to the male-specific P450 2C11 and, to a lesser extent, P450 3A2 were effective in preventing the formation of the DES metabolite 454 nm-Soret peak. In addition, the 454 nm absorbance was not produced by the incubation of DES with NADPH-fortified hepatic musomes from adult female or immature male rats. Studies with the steroid substrate testosterone, which undergoes P450-specific positional hydroxylation, indicated that P450 2C11-mediated 2α- and 16α-hydroxylation were most susceptible to the time-dependent intensification of inhibition produced by DES (8.5 and 7.0 min preincubation required for loss of 50% activity, respectively) and NOR (4.0 and 4.0 min for loss of 50% of both activities). The 6β- (P450 3A2) and 7α-hydroxylase (P450 2A1) pathways were somewhat less susceptible to inhibition than 2α- and 16α-hydroxylation. These findings suggest that DES and NOR form a metabolite intermediate (MI)-complex, characterized by a Soret region absorbance maximum near 454 nm in the optical difference spectrum, with musomal P450 in male rat liver in vitro. Studies with the steroid substrate testosterone as well as immunoinhibition experiments are consistent with the proposition that this MI complex forms principally with the male-specific enzymes P450 2C11 and 3A2. Although a human orthologue of P450 2C11 has not yet been identified, P450s of the 3A subfamily are quantitatively important enzymes in human liver. MI complexation of such enzymes could be a feasible underlying mechanism for certain clinically important drug interactions involving tricyclic antidepressants.