| Objective:1.To establish a reliable high performance liquid chromatography tandem mss spectrometry(HPLC-MS)for determining the plasma concentration of Warfarin in the study of human bioequivalence.2.To investigate the pharamcokinetics and bioequivalence of noval film-coated Warfarin sodium tablet and the marketed sugar-coated Warfarin sodium tablet in subjects in order to provide theoretical basis for further clinical application and approval of new drug.3.To investigate the four parameters(PT,APTT,TT and FIB)of platelet which are of great value in assessing the pharmacodynamics oftwo Warfarin dosage drugs.4.To study the effect of CYP2C9 and VKORC1 genes polymorphism on the pharmacokinetics of Warfarin for researching the reasons for the differences between individuals.Methods:1.A HPLC-MS method was established for determining the concentration of Warfarin in human plasma.The chromatographic column was Inertsil(?)ODS-3(4.6 x150mm,5??),and the mobile phase was acetonitrile:0.5%formic acid solution(60:40,V/V).The flow rate was 0.8 mL/min with the column temperature as 25?,and 5 ?L of the extracting solution was analyzed.Gradient eluted program was adopted:the concentration of acetonitrile was changed from 60?95%over the period from 5.0min to 5.5 min.The overall analytical time was 11min.The ESI ion source was used in the mass spectrometry by positive ion mode.The mass to charge ratio(m/z)of 331.1 and 324.1 were monitored for Warfarin and Glilazide as internal standard by the selective ion detection(SIM)mode,both with the fragment voltage as 110 v a.The capillary voltage wad 4000 v and the vlocity of dry gas(N2)was 11.0 L/min,with the drying temperature as 350?.The methodology validation was carried out after the Warfarin plasma samples were disposed by the liquid-liquid extraction method.2.A single-dose,open-label,randomized,2-period crossover study was performed in 24 healthy Chinese male subjects,randomly divided into two groups according to weight,administrated 2.5 mg of innovator film-coated Warfarin sodium tablets or the marketed sugar-coated Warfarin sodium tablets both are produced by Qilu Pharmaceutical company.Blood samples were collected at baseline and 0.25,0.5,0.75,1,1.5,2,4,8,12,24,48,72,120,168,240 and 360 h after dosing for investigating the pharamcokinetics of Warfarin in human plasma.Plasma was obtained and stored at-20 ? until analysis.The sensitive liquid chromatography mass spectrometry(HPLC-MS)method was used to determine Warfarin in plasma.Drug And Statistics 2.1.1 was used to calculate the pharmacokinetic parameters,and the relative bioavailability was calculated by the ratio of AUC0-360 of the test preparation with reference preparation.3.The other whole blood samples were collected at baseline and 4,24,72,120,240 and 360 h after administration these drugs for determining the four parameters(PT,APTT,TT and FIB)of platelet.Comparing the differences of these parameters between baseline and administration of drugs is to evaluate the pharmacodynamics and bioequivalence of two Warfarin dosage drugs.4.The blood(2ml)was collected from peripheral venous,and the DNA was extracted frow the leukocyte.The fluorescence in situ hybridization(FISH)method was used to detect the CYP2C9 and VKORC1 genotypes of the 24 subjects,the eluent of DNA was added to the CYP2C9 and VKORC1 gene detection kits,respectively.According to the results of CYP2C9 and VKORC1 genotypes,the subjects were divided into different groups.On the basis of the human bioequivalence test,the average pharmacokinetic parameters of each group were calculated.The statistical analysis of each two groups was counted using SPSS 17.0 software by the independent-sample t test.In consider of the effect of crystal polymorphism on bioavailability,the effect of CYP2C9 and VKORC1 gene polymorphism on the pharmacokinetics of film-coated Warfarin sodium tablet and the marketed sugar-coated Warfarin sodium tablet was studied separately.Results:1.The endogenous impurity in plasma did not interference the determination of Warfarin and Gliclazide as internal standard,indicating that the new established HPLC-MS method was high specificity.Linearity of Warfarin was achieved over a plasma concentration range from 5 to 600 ng·mL-1(r2>0.99),and the lower limit of quantification(LLOQ)was 5.0 ng·mL-1.The results of the intra-and inter-day accuracy and precision investigated by analyzing the low,middle and high(10,50,500 ng·mL-1)QC samples were wthin ± 15%,achieving the determination requirements.The extraction recovery of Warfarin was about 60%,and the matrix effect was 93.5?100.2%,indicating that the plasma matrix did not influence the plasma concentration of Warfarin.Stability studies include post-preparative(12h and 24h),freeze-thaw(one and two cycles),long term freeze(1?7?28 and 63d)stability.And the results of them were stable.The established HPLC-MS method was sensitive,accurate and suitable for analyzing the Warfarin in plasma samples for human bioequivalence study.2.The main pharmacokinetic parameters of the film-coated Warfarin sodium tablet(T)and the marketed sugar-coated Warfarin sodium tablet(R)were as follows:t1/2(103.533±18.862)h and(105.801±21.302)h,respectively;Tmax(0.719±0.496)h and(1.323±0.813)h;Cmax(347.766± 74.803)ng·mL-1 and(322.867±75.691)ng·mL-1;AUC0?360(16024.169±3713.939)ng·mL-1·h and(15586.606±3476.975)ng·mL-1·h;AUC0??(17335.649±4089.072)ng·mL-1·h and(16911.987±3911.233)ng·mL-1·h.The bioavailability of film-coated Warfarin sodium tablet(T)relative to the sugar-coated Warfarin sodium tablet(R)was 103.3%a±12.1%.The 90%confidence interval of ln(Cmax)and ln(AUC0?360)of film-coated Warfarin sodium tablet(T)relative to the sugar-coated Warfarin sodium tablet(R)were 98.9%?118.4%and 98.7%?106.5%,respectively,which both were within 80%?125%,indicating that the former was bio equivalent to the later.3.After 24 healthy Chinese male subjects were administrated 2.5 mg of film-coated Warfarin sodium tablets or the marketed sugar-coated Warfarin sodium tablets,some of four parameters(PT,APTT,TT and FIB)of platelet changed considerably including the APTT-S and the TT-S.The other parameters merely changed within the 4h.And then after 8h,all the parameters recovered to the normal level.These results revealed that these two dosage drugs were bioequivalence in the pharmacodynamics.4.The results of the CYP2C9 and VKORC1 genotype of 24 subjects were as follow:wild type CYP2C9(AA)as 22 subjects,heterozygous mutation type CYP2C9(CA)as 3 subjects,homozygous mutation type VKORC1(AA)as 21 subjects,heterozygous mutation type VKORC1(GA)as 3 subjects.For the film-coated Warfarin sodium tablet,the pharmacokinetics parameters of subjects who have different CYP2C9 and VKORC1 genotype shows no significant difference among them.But for the sugar-coated Warfarin tablet,Tmawas different considerably between the different CYP2C9 and VKORC1 genotype,which may be caused by the coating material.The other pharmacokinetics parameters were slightly different between the different CYP2C9 and VKORC1 gengtype.Conclusion:1.The film-coated Warfarin sodium tablet was bioequivalent to the sugar-coated Warfarin tablet because there were slight differences in pharmacokinetics between them.However,Because of the material for coating,the Tmax was dramatically between these two dosage drugs after being examined by nonparametric tests.And the bioavailability of film-coated Warfarin sodium tablet was higher than the sugar-coated Warfarin tablet.2.The film-coated Warfarin sodium tablet was bioequivalent to the sugar-coated Warfarin tablet in pharmacodynamics.And there are no adverse drug reactions in this experiment.3.CYP2C9 and VKORC1 genes polymorphism has slight effects on the pharmacokinetics and bioequivalence between the film-and sugar-coated Warfarin sodium tables... |
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