| Objective To pass the clinical trial phase Ⅰ and establish a rapid, sensitive and accurate HPLC-MS/MS method for determination of Entecavir in human plasma. To research the relative bioavailability between Entecavir Dispersible Tablets developed by Jiangsu Wuzhong, Suzhou Pharmaceutical Pharmaceutical Group Co., Ltd. and Entecavir Tablets developed by Sino-American Shanghai Squibb Pharmaceuticals Ltd. and evaluate the bioequivalence of Entecavir test.To reference experimental basis on approval of new drugs and safe, rational use of drugs.Methods This trial introduce an open, randomized, two-period crossover trial design.Twenty-four Chinese healthy volunteers were classified into two groups randomized,each of which includes twelve males. A liquor of blood were taken from upper limb vein before and 10 min, 20 min, 30 min, 45 min, 1h, 1.5h, 2h, 4h, 6h, 8h, 12 h, 24 h, 48 h, 72 h, 96 h,120h after single dose orally taken Entecavir Dispersible Tablets test or reference, transferred to calparine tube and centrifuged at 2000 rpm for 10 minutes after 30 min, plasma was collected and stored at-30℃ for analysis. The drug washout period is 28 days, that is the volunteers need to take pills on the first and twenty-ninth days. To repeat the test after the twenty-ninth day. Abacavir with Rob as an internal standard, HPLC-MS/MS methods were used to determine the plasma concentrations of Entecavir. The pharmacokinetic parameters of Entecavir were calculated with Win Nonlin 6.1. The results Cmax、AUC0-tn and AUC0-∞ after logarithmic conversion were implemented with analysis of variance, two-sided t test, 90% confidence interval, as well as non-parametric test of Tmax to evaluate the bioequivalence of Entecavir test and reference and calculated the mean and standard deviation.Results The research had chosen 24 Chinese males to be volunteers,and eventually completed all the clinical trial. All the volunteers took medicine under the supervision of researchers, and completed all requirements including exsanguinations, vital signs examination and laboratory tests with good compliance. In the research of HPLC-MS/MS methods for determination of Entecavir, impurities does not interfere with the plasma sample measured, which was not affected by matrix effects. The extraction recovery of Entecavir in the plasma sample was in the interval of 67.97%-70.89%. The linear range of the standard curve changed in the range of 0.05μg·L-1-20μg·L-1 with good linear relationship. The accuracy of low, medium, high level in batch and out of batch were all under 15.0%. The liquid storage kept stable for 20 days at 5℃. The plasma sample measured stabled in sampler for 24 hours, the same at room temperature, repeated freezing and thawing at-20℃ for three times, at-20℃ for three months. Detection Methodology was be in line with the requirement of biological sample analysis. In the process of research, there was no serious adverse reactions. Few Parameters appeared mildly adnormal in biochemical tests and blood routine test and urine routine test, which returned to normal without any treatments. The plasma concentration- time data of all the 24 volunteers had beeninvolved in the Pharmacokinetics and bioequivalence analysis. The plasma concentration of Entecavir had good linear relationship in the range of 0.05μg·L-1-20μg·L-1(r=0.9998). Minimum quantitative concentration was 0.05μg·L-1. RSD was under 15% in and out of the day. The main pharmacokinetics parameters of Entecavir test and reference were as following: t1/2 was 62.35±30.26 h and 60.48±26.54 h, Tmax was 0.63±0.21 h wad 0.62±0.40 h, Cmax was 10.51±3.11μg·L-1 and 10.25±2.98μg·L-1, AUC0-tn was 28.91±6.63μg·h·L-1 and 28.58±5.73μg·h·L-1, AUC0-￥ was 35.22±9.50μg·h·L-1 and 34.61±7.90μg·h·L-1. The relative bioavailability of Entecavir Dispersible Tablets F0-tn and F0-∞ was 101.25±12.02% and 102.99±20.40%. The bioequivalence was calculated with Win Nonlin 6.1. After logarithmic conversion, the AUC0-tn,AUC0- ∞ and Cmax of the pharma test andreference was analyzed by variance. The results showed that there was no big difference among the pharma, the administration order and cycle time. 90% confidence interval of AUC0-tn was 95.98%-105.22%,which of AUC0- ∞ was 94.13%-108.39%, and which of Cmax was 90.49%-116.85%. All of them were in the range of 80%-125%. So the pharma test and reference of Entecavir was bioequivalent.Conclusion 1.The assay was proved to be sensitive, accurate and convenient, and it is suitable for Entecavir pharmacokinetics studies after single oral doses of Entecavir Dispersible Tablets. 2. The relative bioavailabilities of Entecavir in Entecavir Dispersible Tablets were 102.99±20.40%. 3. Win Nonlin 6.1 analysis showed that no significant difference existed between the main pharmacokinetics parameters as AUC, Cmax and Tmax of Entecavir test and reference, as well as periods, but in dividual statistical differences of existed. Further of t-test analysis showed that the hypothesis of bioequivalence were consistent with the fact among individuals, periods and preparations. Entecavir test and reference were bioequivalence. |
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