| Part 1 The Effects of Amiodarone on the Pharmacokinetic of Ticagrelor in ratsObjective: To study the pharmacokinetics of reversible anti-platelet aggregation drug-Ticagrelor in rats and effect of amiodarone on the metabolism of TicagrelorMethods: The concentrations were determined by LC-MS/MS with a Diamonsil C18 chromatographic column(150 mm × 4.6 mm, 5 μm); Dtetctor: API 4000+ type- three weight four grade rod mass spectrometer; the mobile phase was consisted of acetonitrile-10 m M Ammonium acetate solution(adjust the p H to 3.14 with formic acid)(80:20,V/V),with a flow rate of 1 m L·min-1 and at the temperature of 30 ℃, The injection volume was 10 μl and Ibuprofen was selected as internal standard.Rats were random Ly divided into test group and control group with 8 in each group. Each rat in test group was given amiodarone intragastricaly(60 mg/kg) and ticagrelor(18 mg/kg) at the first day, then 2-6 days given amiodarone intragastricaly(20 mg/kg) and ticagrelor(9 mg/kg), twice a day. On the 7th day, each rat in test group was given amiodarone intragastricaly(20 mg/kg) and ticagrelor(9 mg/kg), the blood samples were collected from epicanthal folds at different time after administration.Each rat in control group was given ticagrelor(18 mg/kg) intragastricaly at the first day,then 2-6 days given ticagrelor(9 mg/kg) intragastricaly and equal volume of distilled water, twice a day. On the 7th day, Each rat in control group was given ticagrelor(9 mg/kg) intragastricaly and equal volume of distilled water, the blood samples were collected at different time after giving medicine. 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:Methodology part: The calibrationcurve was Y=0.0075X﹣0.053(r=0.9982), and the linear range was 10.0~500.0 ng/m L of ticagrelor. The absolute recoveries of Ticagrelor at the concentrations of 25 ng/m L, 100 ng/m L and 400 ng/m L were 93.43%, 92.27% and 93.61%, respectively and the absolute recovery of ibuprofen was 91.38%. The method recoveries of Ticagrelor at the concentrations of 25 ng/m L, 100 ng/m L and 400 ng/m L were 101.65%, 94.46% and 92.77%. The RSD of intra-day and inter-day precision were all less than 10%. Plasma samples were stable after stored at-40°C for 5 days or freeze-thaw for three times and had a good stability within 24 hours at room temperature.The main pharmacokinetic parameters of test group and control group were: AUC0-36(2637.29 ± 1059.26)mg·h·L-1 and(3806.35 ± 1604.03)mg·h·L-1;AUC0-∞(2913.53 ± 1319.84)mg·h·L-1 and(4478.14 ± 2054.48)mg·h·L-1;Tmax(1.19 ± 0.26)h and(1.31 ± 0.37)h; V(44.02 ± 22.17)L·kg-1 and(35.98 ± 18.85)L·kg-1;CL(3.54±1.21)L·h-1·kg-1 and(2.45 ± 1.12)L·h-1·kg-1.There was a significant difference in Cmax between test group and control group(P<0.05), CL and V of t1/2 and AUC0-t, AUC0-∞, Tmax have no significant difference(P>0.05).Conclusion: The LC-MS/MS method was found to detect the concentration of Ticagrelor in rats plasma, The method is simple, sensitive and reproducible, and is able to meet the dynamic studies. The results showed that after the combination of Amiodarone, Ticagrelor has slowed metabolism and clearance rate decreased, and area under the curve increased. Part 2 The Effects of Amiodarone on the Pharmacodynamics of Ticagrelor in ratsObjective: To study the platelet aggregation inhibitory rate of Amiodarone on Ticagrelor.Methods: Use sterile capillary pipette to puncture rat blood in the eye canthus, and anticoagulant is 3.8% sodium citrate(blood: anticoagulant = 4: 1), centrifuged 10 min with 700r/min, That portion of the supernatant is platelet-rich plasma(PRP), centrifuge the rest 3min with 3000r/min, That portion of the supernatant is platelet-poor plasma(PRP). Platelet number is 4.0* 10/mm=I in the PRP. According to Bom Nephelometry, put the turbidity tube with 225 μL PRP and one small magnet into platelet aggregation instrument. After calibrated with PPP, add inducer ADP 50 μL with stirring to induce aggregation.The end concentration of ADP is 5 μg/m L, measure the maximum platelets aggregate rate before and after the combination of within 5 min, Formula is Platelet aggregation inhibition rate=(Platelet aggregation rate before giving medicine- Platelet aggregation rate after giving medicine)/ Platelet aggregation rate before medicine. By comparing the change of combination therapy ticagrelor rats in vivo inhibition of platelet aggregation rate, we could obtain the Pharmacodynamics of the Effects of Amiodarone on Ticagrelor.Take comparison between two groups, 16 healthy male SD rats, clean, were random Ly divided into a control group and the test group, get the eye canthus blood before medicine. To give Ticagrelor only to the control group from 1 to 14 days, give Ticagrelor and Amiodarone to the test group from 1 to 14 days, and get the eye canthus blood after medicine in the 7th and 14 th day.Conclusion: The platelet aggregation inhibition rate of Ticagrelor combined with Amiodarone has no significance.After the combination with Amiodarone, Ticagrelor in rats platelet aggregation inhibition rate has no significant difference, the pharmacodynamics of amiodarone for ticagrelor in rats had no significant difference. |
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