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RNA水平。与Telaprevir孵育24,48,72和120小时后的IC50分别确定为0.574,0.488,0.21和0.139μM,表明抑制效应随时间增加。在2％FBS存在下使用48小时孵育进行三次独立实验后,Telaprevir的平均IC50确定为0.354±0.035μM,平均IC90为0.830±0.190μM[1]。 Telaprevir(VX-950)是丙型肝炎病毒(HCV)NS3-4A丝氨酸蛋白酶的有效选择性肽模拟抑制剂,Telaprevir在基因型1b HCV复制子细胞(IC50 = 354 nM)和人体中均表现出优异的抗病毒活性。胎儿肝细胞感染基因型1a HCV阳性患者血清(IC50 = 280 nM)[2]。",{"key":228,"value":229},"体内研究","以10或25 mg\u002Fkg给予Telaprevir(VX-950)的小鼠血清SEAP活性降低约5倍,其平均值(±SEM)为18.7±8.3％或18.4±5.4％分别与给药载体(100±28％)进行比较。这些数据表明Telaprevir能够抑制小鼠肝脏中的HCV NS3-4A丝氨酸蛋白酶活性,并阻断这些小鼠的切割和随后SEAP分泌到血液循环中[2]。",{"key":231,"value":232},"细胞实验","进行HCV复制子细胞中Telaprevir（VX-950）或IFN-α的IC50，IC90，CC50的测定。简言之，将每孔1×104个复制子细胞接种在96孔板中。第二天，将复制子细胞在37℃下孵育指定的时间，用抗病毒剂在DMEM加2％FBS和0.5％DMSO中连续稀释。使用RNeasy-96试剂盒提取总细胞RNA，并使用定量RT-PCR（QRT-PCR）测定法测定HCV RNA的拷贝数。每个基准点代表细胞培养中五次重复的平均值。使用基于四唑（MTS）的细胞活力测定，在相同的实验设置下测量Telaprevir的细胞毒性。对于用人肝细胞系的细胞毒性测定，使用每孔1×104个亲本Huh-7细胞或每孔4×104个HepG2细胞。为了确定Telaprevir对静息PBMC的细胞毒性，将每孔1×10 5个细胞与Telaprevir在RPMI-1640培养基（无血清）中孵育48小时，并通过基于MTS的测定确定细胞活力。为了确定VX-950对增殖的PBMC的细胞毒性，将RPMI-1640培养基中每孔1×10 5个细胞加入到96孔板中，该板用抗人CD3抗体预涂覆。将细胞与Telaprevir和抗人CD28抗体在37℃温育72小时，并在第48和72小时之间通过[3H]胸苷更新确定细胞生长[1]。",{"key":234,"value":235},"动物实验","小鼠[2]通过尾静脉向5只6周龄SCID小鼠（每组6只动物）注射109只IFU \u002F小鼠重组腺病毒Ad-WT-HCVpro-SEAP。每组小鼠口服给予以下剂量之一的Telaprevir（VX-950）：10,25,75,150或300mg \u002F kg。第一次Telaprevir剂量在腺病毒注射前2小时给予，第二剂量在注射后10小时给予。另外一组10只小鼠单独给予载体。在注射后24小时收集血清样品，并将每个Telaprevir给药组中的SEAP活性与载体组的SEAP活性进行比较。大鼠和狗[2]在大鼠和狗中评估Telaprevir（VX-950）的静脉内和口服药代动力学。给予一组体重为250-300g的3只雄性Sprague-Dawley大鼠静脉推注剂量为0.95mg \u002F kg的Telaprevir。在给药前和给药后0.083,0.167,0.25,0.5,1,1.5,2,3,4,6和8小时，将系列血液样品收集在肝素化管中。给一组3只雄性比格犬（8至12kg）静脉推注剂量为3.5mg \u002F kg Telaprevir的10％乙醇，40％聚乙二醇400和50％D5W。在给药前和给药后0.083,0.167,0.25,0.5,1,1.5,2,4,6,8,12和24小时，将系列血液样品收集在肝素化管中。对于大鼠和狗的口服研究，Telaprevir配制成聚乙烯吡咯烷酮（PVP）K-30加2％十二烷基硫酸钠，然后以口服强饲法给药。一组3只雄性Sprague-Dawley大鼠（250至300g）口服给予40mg \u002F kg VX-950，并且给予一组4只雄性比格犬（10.9至12.0kg）口服剂量9.6mg \u002F kg VX-950。在两次口服研究中，在给药前和给药后0.25,0.5,1,1.5,2,3,4,6,8,12和24小时取血样。在静脉内和口服研究中，通过离心获得血浆样品并储存在-70℃直至分析。通过手性液相色谱然后串联质谱（LC \u002F MS \u002F MS）方法分析来自静脉内研究的样品，并使用非手性LC \u002F MS \u002F MS方法分析来自口服研究的样品。",{"key":237,"value":238},"参考文献","[1].\nLin K, et al. 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