ROLE OF CYP3A4 IN HUMAN HEPATIC DILTIAZEM N-DEMETHYLATION - INHIBITION OF CYP3A4 ACTIVITY BY OXIDIZED DILTIAZEM METABOLITES

The antihypertensive agent diltiazem (DTZ) impairs hepatic drug metabo lism by inhibition of cytochrome P450 (CYP). The accumulation of DTZ m etabolites in serum occurs during prolonged therapy and leads to decre ased DTZ elimination. Thus, DTZ metabolites may contribute to CYP inhi bition. This study assessed the role of human CYPs in microsomal DTZ o xidation and the capacity of DTZ metabolites to inhibit specific CYP a ctivities. DTZ N-demethylation varied 10-fold in microsomal fractions from 17 livers (0.33-3.31 nmol/mg of protein/min). DTZ oxidation was c orrelated with testosterone GP-hydroxylation (r = 0.82) and, to a less er extent, tolbutamide hydroxylation (r = 0.59) but not with activitie s mediated by CYP1A2 or CYP2E1. CYP3A4 in lymphoblastoid cell microsom es catalyzed DTZ N-demethylation but CYP2C8 and CYP2C9 were also activ e (similar to 20% and 10% of the activity supported by CYP3A4); seven other CYPs produced little or no N-desmethyl DTZ from DTZ. The CYP3A4 inhibitors ketoconazole and troleandomycin decreased microsomal DTZ ox idation, but inhibitors or substrates of CYP2C, CYP2D and CYP2E1 produ ced no inhibition. Some inhibition was produced by alpha-naphthoflavon e, a chemical that inhibits CYP1As and also interacts with CYP3A4. In further experiments, the capacities of DTZ and three metabolites to mo dulate human CYP 1A2, 2E1, 2C9 and 3A4 activities were evaluated in vi tro. DTZ and its N-desmethyl and N,N-didesmethyl metabolites selective ly inhibited CYP3A4 activity, whereas O-desmethyl DTZ was not inhibito ry. The IC50 value of DTZ against GYP3A4-mediated testosterone 6 beta- hydroxylation (substrate concentration, 50 mu M) was 120 mu M The N-de smethyl (IC50 = 11 mu M) and N,N-didesmethyl (IC50 = 0.6 mu M) metabol ites were 11 and 200 times, respectively, more potent. From kinetic st udies, N-desmethyl DTZ and N,N-didesmethyl DTZ were potent competitive inhibitors of CYP3A4 (K-i = similar to 2 and 0.1 mu M, respectively). CYP3A4 inhibition was enhanced when DTZ and N-desmethyl DTZ underwent biotransformation in NADPH-supplemented hepatic microsomes in vitro, supporting the contention that inhibitory metabolites may be generated in situ. These findings suggest that N-demethylated metabolites of DT Z may contribute to CYP3A4 inhibition in vivo, especially under condit ions in which N-desmethyl DTZ accumulates, such as during prolonged DT Z therapy.