Application of rat hepatocyte culture to predict in vivo metabolic auto-induction: Studies with DFP, a cyclooxygenase-2 inhibitor

The drug candidate DFP [5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonylp henyl)-2(5H)-furanone] is a selective cyclooxygenase-2 inhibitor under eval uation for analgesic and anti-inflammatory therapy. The in vitro metabolic pathways (rat microsomes) involve hydroxylation of the isopropyl side chain at either of two positions, the methyl or the methine, thus producing a hy droxylated metabolite (DFHP) or a dealkylated metabolite (DFH). DFH formati on was the major pathway. Using hepatic microsomes from rats treated with a gents that induce specific CYP isozymes, it was shown that the dexamethason e-inducible rat CYP3A isozyme(s) play a major role in DFH formation. The ro les of CYP3A1 and -3A2 were confirmed with genetically engineered rat CYP e nzymes. The potential for induction of rat CYP3A by DFP was evaluated by in cubating DFP in rat hepatocyte cultures and measuring the CYP3A levels. Bot h CYP3A immunoreactive protein and enzyme activity were induced in a dose-d ependent manner. The induction was confirmed in vivo by dosing rats with DF P at 100 mg/kg for 4 days. Microsomes prepared from the excised livers show ed that DFP gave similar to 55% of the induction observed with dexamethason e, as determined by Western blot. In vitro metabolic auto-induction of DFP was assessed by measuring the metabolism of DFP in hepatocytes treated with DFP. DFH formation was significantly enhanced in the DFP-treated cells. In vivo, treating rats with DFP at doses of 10 to 100 mg/(kg.day) for 13 week s indicated that DFP induced its own metabolism. The C-max and plasma drug area under the curve values during the thirteenth week were significantly l ower than that on the first day, and the effect was dose-dependent.