Effect of rociverine on P450-dependent monooxygenases and its N-deethylation metabolism in rat liver microsomes

Rociverine [2-(diethylamino)-1-methylethyl cis-1-hydroxy [bicyclohexyl]-2-carboxylate] citrate (ROC) is an antispasmodic agent therapeutically active in humans at doses of 0.5-1 mg/kg. This study investigated the effect of acute administration of the drug on hepatic microsomal cytochrome P450 (P450)-catalysed drug metabolism. Only high doses (> or = 100 mg/kg) of ROC were able to induce in rats the hepatic microsomal pentoxyresorufin O-depenthylase (PROD) and 16 beta-testosterone hydroxylase activities both associated with P4502B1/2 and the erythromycin N-dimethylase (ErD) and 2 beta-testosterone hydroxylase activities both dependent on P4503A1/2. However, at 100 and 200 mg/kg of ROC, the 16 beta-testosterone hydroxylase and PROD were the most induced activities, suggesting that P4502B1/2 are the isoforms most sensitive to ROC induction. Accordingly, ROC treatment enhanced, in a dose-dependent manner, the amount of P4502B1/2 and 3A1/2 in microsomes as assayed by western blotting. The northern blot analysis of ROC-treated rat liver showed that the P4502B1/2 induction appears to be regulated at the mRNA level as in the induction by phenobarbital (PB). The oxidative metabolism of ROC with hepatic microsomes from control or PB- and ROC-induced rats resulted in a N-deethyl ROC derivative (major metabolite) and an unknown minor ROC derivative. The kinetic parameters for the N-deethylation of ROC were studied with purified P4502B1 and with microsomes from control or rats treated with various inducers (phenobarbital, ethanol, beta-naphthoflavone, dexamethasone and rociverine). It was found that phenobarbital-, dexamethasone- and rociverine-induced microsomes deethylated ROC with a Vmax about five times higher than that (0.9 nmol/min/mg protein) of control microsomes, although with a similar affinity (Km approximately 0.3 mM). In a reconstituted system, the purified P4502B1 metabolized ROC with a high deethylation rate (22 nmol/min/nmol P450). Moreover, the ROC deethylation was inhibited by compounds such as hexobarbital, metyrapone and triacetyloleandomicin, selective inhibitors for P4502B and/or P4503A enzymes. On the other hand ROC, when added in vitro, inhibited the 16 beta- and 2 beta-testosterone hydroxylases and the PROD and ErD activities. Taken together, these results indicate that the ROC-inducible P4502B and P4503A are involved in ROC deethylation. In conclusion, it has been demonstrated that ROC is a weak phenobarbital-like inducer of P450, probably able at high and reiterated doses to alter its own metabolism, at least in the rat liver.