Study On The Effect Of CYP450 Inhibitors On The Metabolism Of TJ0711 Hydrochloride In Rats

TJ0711 hydrochloride is a novel compound synthesized by School of Pharmacy, Tongji Medical College, HUST. In our previous study, it has showed an ideal antihypertensive effect, with the blockade onα1- adrenoceptor similar to that onβ1- adrenoceptor. After oral administration of TJ0711 hydrochloride, the concentrations of the drug in feces, urine and bile of rats were fairly low, indicating that metabolism was the main process responsible for TJ0711 hydrochloride elimination. Therefore, the investigation of TJ0711 hydrochloride metabolism mediated by CYPs was very important, for it would help us to understand interindividual differences and predict potential drug- drug interactions. To identify the possible metabolic path ways of TJ0711 hydrochloride, we studied the effect of CYP450 inhibitors on the metabolism of TJ0711 hydrochloride in vitro and in vivo.We determined the enzyme kinetic parameters of TJ0711 hydrochloride in rat liver microsomes. As the structures of TJ0711 hydrochloride metabolites were still unclear, we detected the decrease of TJ0711 hydrochloride instead of the amount metabolized by CYP450. To maximize the initial velocity of enzyme reaction, the concentration of CYP450 proteins in the incubation mixture was fixed at 0.5 mg/mL and the incubation time was 5 min. The Vmax, Km and CLint values were estimated using the Michaelis-Menten model. They were 1.38μmol/min per g protein, 11.42μmol/L and 0.12 L/min per g protein, respectively,Based on the results of enzyme kinetic parameters determination, we evaluated the effect of CYPs inhibitors on the metabolism of TJ0711 hydrochloride in rat liver microsomes. Nine CYPs inhibitors were selected:α-naphthoflavone, chlorpromazine, diazepam, cimetidine, quinine, propafenone, terfenadine, zinc diethyldithiocarbamate and ketoconazole. All inhibitors were added to the incubations at final concentrations of 50μM. The results showed thatα-naphthoflavone, chlorpromazine, diazepam, propafenone, zinc diethyldithiocarbamate had negligible inhibitory effect as they inhibited the activity of CYP450 less than 22%. The IC50 values of cimetidine, quinine, terfenadine and ketoconazole were 110.79,44.15,21.77,65.11μmol/L, respectively. Lineweaver-Burk plots of TJ0711 hydrochloride showed that quinine, terfenadine and ketoconazole inhibited its metabolism through the competitive-noncompetitive mechanism, with the Ki values of 30.74,17.15,54.94μmol/L, and the Ki′values of 36.88,68.94,90.72μmol/L, respectively.For in vivo study, a sensitive and specific HPLC-FD method was established to determine the concentration of TJ0711 hydrochloride in rat plasma. Cimetidine, quinine and ketoconazole were administrated intraperitoneally by single dosing to male Sprague-Dawley rats. Compared with the control group, the main pharmacokinetic parameters of TJ0711 hydrochloride in cimetidine group showed no significant changes. In the quinine group, AUC0-t, AUC0-∞and Cmax increased significantly. In the ketoconazole group, MRT0-t increased slightly, while Cmax and t1/2z increased markedly. Meanwhile, the CLz/F in both groups showed significant decrease. From the results, we concluded that quinine and ketoconazole inhibited the biotransformation of TJ0711 hydrochloride in rats, thus increased its bioavailability.In conclusion, CYP2D and CYP3A1/2 were the responsible enzymes for the TJ0711 hydrochloride metabolism in rats, and CYP1A1/2,CYP2A,CYP2B,CYP2C,CYP2E1 were not involved in the process. However, further experiments would be carried out to understand the mechanism of TJ0711 hydrochloride metabolism.