| Objectives: Imrecoxib is a compound of completely new structure, which is a moderately selective inhibitor of cyclooxygenase(COX)-2 developed in China. It has a potential therapeutic effect on acute and chronic inflammation. It has been reported that CYP 3A and CYP 2D both played a role in the metabolism of imrecoxib in rat liver microsomes in vitro, while in the investigations with recombinant human CYPs, CYP 2C9, CYP 2D6 and CYP 3A were all involved in the metabolism of imrecoxib. The results obtained from above studies both in vitro are not consistent. So far, it is still unclear which CYP isoforms take a part in the metabolism of imrecoxib in vivo and it will be explored in the following experiments.Methods: Chromatography conditions: The samples were separated on a chromatographic column of Diamonsil C18(150 mm x 4.6 mm, 5 μm) and the column temperature was set at 30 ℃. A mobile phase consisting of acetonitrile-water-formic acid(86:15:0.1, V/V/V) was used at a flow rate of 1.0 m L·min-1. The injected volume was 10 μL and tolbutamide was selected as the internal standard.Mass spectrometry conditions: 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 k V and the ion source temperature was set at 550 ℃; The curtain gas pressure was fixed at 40 psi and the collision gas pressure was at 6 psi; Nitrogen was used as GS1(pressure was 55 psi) and GS2(pressure was 55 psi); The declustering potential(DP) and collision energy(CE) values of imrecoxib were 100 V and 45 e V, respectively; The DP and CE values of tolbutamide were 22 V and 32 e V, respectively; Quantification was performed using multiple reaction monitoring(MRM) of the transitions of m/z: 370.1â†'236.2 for imrecoxib and m/z: 270.1â†'155.2 for tolbutamide, respectively.Animals treatment: Eighty rats were randomly divided into four groups(n=19). Four groups of rats were pretreated with distilled water, amiodarone(40 mg·kg-1, inhibiter of CYP 2C9), paroxetine(2 mg·kg-1, inhibitor of CYP 2D6) and ketoconzole(20 mg·kg-1, inhibitor of CYP 3A) for consecutive 7 days, respectively. On day 8, imrecoxib was administered orally at a dose of 20 mg·kg-1 to all rats. Blood samples(approximate 0.5 m L) from plexus venous at fundus oculiwere collected pre-dose and at 10 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h and 24 h post dose. All blood samples were centrifuged at 6500×g for 5 min. Then 200 μL plasma samples were transferred to another clean tube and stored frozen at –40 ℃ for LC/MS/MS analysis.Sample preparation: For the sample preparation, the plasma(200 μL) was added to a 5 m L tube containing 20 μL tolbutamide(500 ng·m L-1) as the internal standard, vortexed for 10 s, then 1 m L ethyl acetate was added and mixed for 2 min. The mixture was centrifuged for 10 min at 4500×g and the upper organic layer was transferred and evaporated to dryness under the stream of nitrogen. The dry residue was reconstituted with 400 μL mobile phase. After mixing for 1 min, the mixture was centrifuged for 5 min at 6500×g, then a 10 μL aliquot of supernatant was injected into LC-MS/MS system.Data analysis and statistical analysisAll the data was analyzed by using DAS 2.0 software and standard methods were used to calculate the pharmacokinetic parameters. Statistical analysis was performed on SPSS 13.0 software. Difference in parameters of two groups was analyzed by using the Student’s t-test or nonparametric test. A P value less than 0.05 was deemed significant.Results: The retention times of imrecoxib and tolbutamide were 2.27 min and 2.26 min, respectively. The calibration curve was Y = 0.016 X + 0.072(r=0.9997), and the linear range was 10~400 ng·m L-1. The absolute recoveries of imrecoxib at the concentrations of 20 ng·m L-1, 80 ng·m L-1 and 320 ng·m L-1 were 61.65%, 65.33% and 72.57%, respectively and the absolute recovery of tolbutamide was 51.64%. The relative recoveries of quality control samples at above three concentrations were 102.2%, 98.71% and 97.01%, respectively. The RSD values of intra-day and inter-day precision were all less than 8%. Plasma samples were stable when stored at –40℃ for a month or under freeze-thaw cycles for three times and the processed samples had a good stability within 12 hours at 4℃.Pharmacokinetic parameters of imrecoxib in rats treated with distilled water were as follows: AUC0-t: 1125.1 ± 457.6 mg·h·L-1, AUC0-∞: 1331.3 ± 592.6 mg·h·L-1, t1/2: 7.4 ± 3.8 h, Tmax: 1.5 ± 0.6 h, CL: 0.02 ± 0.01 L·h-1·kg-1, V: 0.17 ± 0.07 L·kg-1, Cmax: 162.2 ± 53.0 mg·L-1; Pharmacokinetic parameters of imrecoxib in rats treated with amiodarone were as follows: AUC0-t: 1814.8 ± 693.4 mg·h·L-1, AUC0-∞: 2091.6 ± 887.1 mg·h·L-1, t1/2: 7.8 ± 4.5 h, Tmax: 1.7 ± 0.6 h, CL: 0.01 ± 0.01 L·h-1·kg-1, V: 0.11 ± 0.05 L·kg-1, Cmax: 268.2 ± 115.7 mg·L-1; Pharmacokinetic parameters of imrecoxib in rats treated with paroxetine were as follows: AUC0-t: 1485.5 ± 480.3 mg·h·L-1, AUC0-∞: 1730.4 ± 606.5 mg·h·L-1, t1/2: 7.3 ± 5.2 h, Tmax: 1.5 ± 0.7 h, CL: 0.01 ± 0.01 L·h-1·kg-1, V: 0.12 ± 0.06 L·kg-1, Cmax: 192.1 ± 70.6 mg·L-1; Pharmacokinetic parameters of imrecoxib in rats treated with ketoconzole were as follows: AUC0-t: 1191.9 ± 626.7 mg·h·L-1, AUC0-∞: 1391.0 ± 692.0 mg·h·L-1, t1/2: 8.0 ± 4.2 h, Tmax: 1.5 ± 0.7 h, CL: 0.02 ± 0.01 L·h-1·kg-1, V: 0.19 ± 0.11 L·kg-1, Cmax: 159.0 ± 70.5 mg·L-1. Compared with the control group, there was a significant increase in AUC0-24 h, AUC0-∞ and Cmax and a marked decrease in CL and V of the group treated with amiodarone; At the same time, increased AUC0-24 h, AUC0-∞, and Cmax and reduced CL in the group pretreated with paroxetine were also found. On the contrary, no apparent difference was observed on the pharmacokinetic parameters of imrecoxib betweengroup treated with ketoconzole andcontrol group.Conclusions: The LC-MS/MS method has a good performance in terms of accuracy, sensitivity and precision. It is suitable to determine the plasma concentration of imrecoxib in rats.Compared with control group, it was observed that there was an obvious increase in the exposure and a marked reduction in CL of imrecoxib in the group pretreated with amiodarone, which suggested the metabolism of imrecoxib slowed down when the activity of CYP 2C9 was inhibited by amiodarone. It was concluded that CYP 2C9 was, as expected from in vitro studies with recombinant human CYP isoforms, responsible for the metabolism of imrecoxib. The metabolism of imrecoxib in rats treated with paroxetine was slower than control group followedwith increased AUC and decreased CL. The results demonstrated that CYP 2D6 had the metabolic capacity of imrecoxib in vivo. There was no obvious variation on the pharmacokinetic parameters of imrecoxib between control group and the group treated with ketoconzole. So inhibiting the activity of CYP 3A in rats did not increase the exposure or decrease the elimination of imrecoxib, which indicated that CYP 3A may have no influence on the metabolism of imrecoxib in rats. |
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