Evaluation Of The Effects Of Imrecoxib On Cytochrome P450 Activity By Probe Drug

Part 1 An Evaluation of the Effects of Imrecoxib on CYP3A Enzyme by Probe DrugObjective:To develop an HPLC method for the determination of midazolam, the probe drug of CYP3A. By comparing the pharmacokinetic parameters of midazolam in rats after single or combined administration of imrecoxib to investigate whether imrecoxib induces or inhibits the activity of CYP3A enzyme.Methods:The HPLC condition was as following:sample was separated by Diamonsil C18chromatographic column (250 mm×4.6 mm,5.0μm) at the temperature of 30℃, the mobile phase was consisted of acetonitrile-water (56:44,V/V) with a flow rate of 1.0 mL·min-1. The determination wavelength was set at 225 nm and the injected volume was 20μL. Diazepam was selected as internal stanstard. Forty rats were randomly divided into test group and control group with 20 rats in each group. Each rat in test group was given 20 mg·kg-1 of imrecoxib intragastricaly and each rat in control group was given equal volum of distilled water, twice daily for 7 days. On day 8, each rat recieved a single dose of midazolam (10 mg·kg-1) intravenously. The blood samples were collected from epicanthal folds at different time after medication. After centrifugation (6525×g),200μL plasma sample was added into 5 mL centrifuge tube,20μL internal standard solution and 1 mL acetonitrile was added to precipitate protein.Then the samples were vortexed for 3 min and centrifugated(6525×g) for 2 min,950 μL supernatant was dropped and added into 1.5 mL centrifuge tube and blowed by nitrogen under 40℃ The residue was dissolved by 100μL mobile phase. Then 20μL sample was injected to analysis. The pharmacokinetic parameters of the two groups were calculated by DAS 2.1.1 and the statistic analysis was processed by SPSS 13.1.Results:The main pharmacokinetic parameters of test group and control group were obtained as follows:AUC0-twere (3.42±0.91) mg·h·mL-1 and (3.83±0.84) mg·h·mL-1; AUC0-∞ were (3.34±0.88) mg·h·mL-1 and (3.77±0.86) mg·h·mL-1;ti1/2 were (0.37±0.07) h and (0.35±0.08) h; Cmax were (11.85±4.45) mg·L-1 and (12.36±3.48) mg·L-1; V were (1.62±0.45) L·kg-1 and (1.37±0.39) L·kg-1; CL were (3.14±0.85) L·h-1·kg-1 and (2.74±0.66) L·h-1·kg-1, respectively. There was no significant difference between the pharmacokinetic parameters of test group and control group (P>0.05)Conclusions:An HPLC method was established for the determination of midazolam in the plasma of rats. The results showed no significant difference on the pharmacokinetic parameters of midazolam between administration singlely or coadministered with imrecoxib in rats, imrecoxib dosen’t influence the pharmacokinetics of midazolam in rats by intravenous administration, indicating that the activity of CYP3A enzyme was not influenced by imrecoxib.Part 2 An Evaluation of the Effects of Imrecoxib on C YP2C9 Enzyme by Probe DrugObjective:To develop an HPLC method of determinating the concentration of tolbutamide and evaluate the effects of imrecoxib on CYP2C9 enzyme in rats by probe drug.Methods:The concentrations of tolbutamide were determined by Diamonsil C18 chromatographic column (250 mm×4.6 mm,5μm) at the temperature of 30℃, the mobile phase was consisted of acetonitrile-water-acetic acid-triethylamine (50:50:0.4:0.5,V/V/V/V) with a flow rate of 1.0 mL·min-1. The determination wavelength was set at 230 nm and the injected volume was 20μL. Carbamazepine was selected as internal stanstard. Rats were randomly divided into test group and control group with 18 rats in each group. Each rat in test group was given imrecoxib intragastricaly (20 mg·kg-1), and each rat in control group was given equal volume of distilled water, twice daily for 7 days. On day 8, each rat was given tolbutamide intragastricaly (50 mg·kg-1), The blood samples were collected from epicanthal folds at different time after medication. After centrifugation (6525×g),200μL plasma sample was added into 5 mL centrifuge tube,20 μL carbamazepine standard solution and 1 mL dichloromethane was added to precipitate protein,3 min vortexed and 2 min centrifugated,950μL supernatant was added into 1.5 mL centrifuge tube and blowed by nitrogen in 40℃ water, residue was dissolved by 100μL mobile phase. Then 20 uL sample was injected to analysis.The pharmacokinetic parameters of the two groups were calculated by DAS 2.1.1 and the statistic analysis was processed by SPSS 13.1.Results:The main pharmacokinetic parameters of test group and control group:AUC0-t were (766.25±145.49) mg·h·mL-1 and (1015.51±194.80) mg·h·mL-1; AUC0-∞ were (811.96±172.82) mg·h·mL-1 and (1077.64±254.16) mg·h·mL-1; Tmaxwere (0.99±0.68) hand (1.38±0.62) h; V were (0.54±0.11) L·kg-1 and (0.25±0.09) L·kg-1; CL were (0.06±0.01) L·h-1·kg-1 and (0.05±0.01) L·h-1·kg-1. There were significant differences of the AUC0-t, AUC0-∞, Tmax, CL and V between test group and control group (P<0.05)Conclusion:An HPLC method was developed for determinating of the concentration of tolbutamide in rat plasma. The AUC0-t and AUCo-∞, of tolbutamide in test group decreased significantly compared with that of the control group, while the CL of tolbutamide increased apparently. It indicated that imrecoxib could induce the activity of CYP2C9 enzyme and increase the metableolism of tolbutamide.Part 3 An Evaluation of the Effects of Imrecoxib on CYP2D6 Enzyme by Probe DrugObjective:To develop an LC-MS/MS method of determinating the concentration of dextromethorphan. The pharmacokinetic of probe drug in rats before and after combined with imrecoxib was compared to determine whether imrecoxib induces or inhibits the activity of CYP2D6 enzyme.Methods:The plasma concentrations of dextromethorphan were determined by LC-MS/MS. The concentrations of dextromethorphan were determined by Symmetry C18chromatographic column (2.1 mm* 100 mm,3.5 μm), at the temperature of 40℃, the mobile phase was consisted of A:water(1‰ formic acid)-B:acetonitrile(0～3 min:60%â†'90%B,3～3.5 min:90%â†'60%B; 3.5～5 min:60%B;) with a flow rate of 0.4 mL·min-1. The mass spectrometer was operated in the positive electrospray ionization mode with an electrospray ionization interface (ESI); the source voltage was maintained at 5.5 kV and the ion source temperature was set at 550℃; The curtain gas (CUR) pressure was fixed at 10 psi and the collision gas (CAD) pressure was at 4 psi; Nitrogen was used as GS1 (pressure was 55 psi) and GS2 (pressure was 55 psi); The DP and CE values of dextromethorphan were 95 and 37, respectively; The DP and CE values of sulfamethyloxazole were 73 and 22, respectively; Quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 272.3â†'212.8 for dextromethorphan and m/z 254.2â†'155.8 for sulfamethyloxazole, respectively. Rats were randomly divided into test group and control group with 15 rats in each group. The test group was given imrecoxib (20 mg·kg-1), the control group was administrated distilled water intragastrically, two times a day, for consecutive 8 d. Control group and test group were given dextromethorphan (12 mg·kg-1) on the eighth day immediately after gavage. The blood samples were collected from epicanthal folds at different time after medication. The blood samples were collected from epicanthal folds at different time after medication. After centrifugation (6525xg×g),200μL plasma sample was added into 5 mL centrifuge tube,20μL internal standard solution and 1 mL ethyl acetate was added to precipitate protein,3 min vortexed and 2 min centrifugated (6525×g),950μL supernatant was added into 1.5mL centrifuge tube and blowed by nitrogen in 40℃ water, residue was dissolved by 300 μL mobile phase. Then 10μL sample was injected to analysis. The pharmacokinetic parameters of the two groups were calculated and compared.Results:The main pharmacokinetic parameters of test group and control group:t1/2were (5.48±2.93) h and (7.23±2.16) h. There was a significant difference between the t1/2 of test group and control group (P<0.05).Conclusion:The study developed an LC-MS/MS method for the determination of dextromethorphan in rat plasma. The t1/2 of dextromethorphan test group increased significantly compared with that of control group, which indicated that imrecoxib can induce the activity of CYP2D6 enzyme and decrease the metableolism of dextromethorphan.