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目的 开发一种用于检测新型重组活化凝血因子Ⅶa(rFⅦa)候选药物GEN-0828血药浓度的rFⅦa活性定量测定方法,初步探讨GEN-0828在血友病模型小鼠中的药动学特征.方法 建立GEN-0828在小鼠血浆中浓度的测定方法,并进行方法学验证.对8只FⅨ基因敲除(B-F9 KO)小鼠单次尾静脉给予GEN-082890.9 kU·kg-1.在不同时间点采集血样,采用FⅦa活性测定法测定血浆中GEN-0828浓度,使用WinNonlin软件通过非室模型分析计算药动学(PK)参数.结果 在B-F9 KO模型小鼠血浆中,在62.5～2000 U·L-1范围内GEN-0828显示出良好的线性关系(R 2=0.9988),定量下限为62.5 U·L-1;日内精密度为3.9％～5.2％、日间精密度为5.2％～11.1％,准确度为-3.5％～1.8％;选择性的RE为-15.9％～13.8％;平行性的精密度(RSD)不超过4.3％;样品稀释5～40倍范围内无稀释效应;且该方法在室温3 h,-20℃141 d,-80℃92 d稳定.GEN-0828在B-F9 KO模型小鼠中的主要药动学参数:平均消除半衰期(t1/2)为(3.73±0.15)h,曲线下面积(AUC last)为(115±23)h·kU·L-1,表观分布容积(Vz)为(3714±720)L·kg-1,总清除率(Cl)为(689±122)L·h-1·kg-1.结论 建立了一种灵敏度高、特异性好的定量分析GEN-0828血药浓度的方法,该方法可用于B型血友病模型小鼠非临床药动学研究.“,”OBJECTIVE To develop a sensitive method for the detection of GEN-0828 in plasma from hemophilia model mice and to explore the pharmacokinetic profiles of GEN-0828 in hemophilia mouse models. METHODS A quantification method for GEN-0828 in plasma was established and validated. A bonus of GEN-0828 (90.9 kU · kg-1) was injected into the tail vein of factor Ⅸ (FⅨ) knock?out (B-F9 KO) model mice. Then, the concentration of GEN-0828 in plasma was detected by activated factor Ⅶa (FⅦa) activity assay. The pharmacokinetic parameters were analyzed with WinNonlin 6.4 software for non-compartmental model analysis. RESULTS A good linearity was demonstrated from 62.5 to 2000 U·L-1 for GEN-0828 (R2=0.9988) and the lower limit was 62.5 U·L-1. The intra-day and the inter-day precisions were 3.9％-5.2％ and 5.2％-11.1％, and the accuracy was -3.5％ to 1.8％. The selective relative error (RE) was-15.9％ to 13.8％;the relative standard deviation (RSD) of parallelism was below 4.3％, and there was no dilution effect in the range of 5-40 times dilution of samples. After validation, the method was stable at room temperature for 3 h, at-20℃for 141 d, and at-80℃for 92 d. Moreover, the main pharmacokinetic parameters of GEN-0828 in B-F9 KO model mice were as follows:the terminal elimination half-life (t1/2) was (3.73 ± 0.15) h, the area under the plasma concentration-time curve from zero to the last sampling time (AUClast) was (115 ± 23) h · kU · L-1, the volume of distribution (Vz) was (3.7±0.7) L·kg-1, and the total body clearance (Cl ) was (0.69±0.12) L·h-1·kg-1. CONCLUSION A sensitive and specific quantitative method is established to determine the concentration of GEN-0828 in plasma, and may be applied in preclinical pharmacokinetic studies in hemophilia B model mice, thus facilitating fature research on this novel drug.