Abstract

Diltiazem, a calcium channel blocker, is mainly metabolized via demethylation or deacetylation in humans. Diltiazem demethylation is catalyzed by cytochrome P450 2D6 and 3A4. Although it was previously reported that the area under the curve ratio of deacetyldiltiazem to diltiazem after oral dosing with diltiazem in rats was sevenfold higher than in humans, the molecular mechanisms underlying this species difference remain to be clarified. In the present study, we compared the diltiazem deacetylase activity in liver, intestinal, renal, and pulmonary microsome preparations of human and experimental animal tissues to identify the specific deacetylase enzyme(s) involved in deacetylation. Diltiazem deacetylase activity was detected in rat liver and small intestine microsome preparations, but not in those from human, monkey, dog, and mouse tissues. Further purification of rat liver microsome (RLM) proteins identified four carboxylesterase (Ces) enzymes (Ces1d, Ces1e, Ces1f, and Ces2a) as potential candidate deacetylases. On the basis of their tissue distribution, the Ces2a enzyme was considered to be the enzyme that was responsible for diltiazem deacetylation. Furthermore, recombinant rat Ces2a expressed in Sf21 cells displayed efficient diltiazem deacetylase activity with similar Km values as RLM. In addition, the inhibitory characteristics of various chemical inhibitors were similar between recombinant rat Ces2a and RLM. In conclusion, we determined that only rat tissues were able to catalyze diltiazem deacetylation. The characterization of Ces enzymes in animal species, as undertaken in this study, will prove useful to predict the species-specific pharmacokinetics differences between the in vivo models used for drug development.