CORE--Antiretroviral Drug Susceptibility and Drug Interactions

核心B的总体目标是为IPCP计划提供药物相互作用的药物敏感性测试和评估。BBI Biotech对HIV-1进行了多次药物敏感性分析，这为开发一种新型抗逆转录病毒药物PA457做出了贡献，该药物刚刚完成了一项成功的I期临床试验。最初，Core将筛选五种NK-1R拮抗剂(Approant、CJ-12255、CJ-96345、RP-67、580和L733060)，用于抗逆转录病毒 HIV-1ba-L在PBMC中的活性和细胞毒作用有效化合物，即那些具有可接受的抗逆转录病毒活性与细胞毒性比率的化合物，将在项目1和2以及核心B中进一步表征。为了表征抗病毒反应的广度，除了前导化合物(计划用于初步动物研究和临床试验)外，还将与由M型、O型、HIV-2主要分离株和抗药性分离株组成的25个HTV分离株进行对比测试。将针对每个分离株确定50%有效剂量(EC50)。由于大多数抗逆转录病毒疗法包含多种药物，因此将分别测试APRAPANT和其他先导化合物与不同类别抗逆转录病毒药物的药物相互作用。接受相互作用测试的药物将与HIV-1(R5和X4病毒)一起或单独在PBMC中培养。HIV p24产量的减少将在中效方程中用于计算EC50和组合指数(CI)，以指示协同或拮抗。在以下方面进行评估 体外耐药诱导时，将THP-1细胞与HIV-1、巴-L和低剂量的启动剂共同培养。药敏试验将每月进行一次，以寻找正在形成的耐药性。如果EC50增加，将对病毒被膜进行测序，以确定共受体结合区以及病毒和细胞的变化，分别发送到项目1和2进行进一步研究。为了确定在体内是否会产生显性抗性，从中国分离的SFV 在项目3中使用安非他明治疗的猴子将每隔一个月进行一次检测，并测定EC50。为了将治疗效果与患者病毒种群的可能变化联系起来，项目4中来自进入前、治疗结束和重现治疗后一个月的分离株将被检测对重现和辅助受体使用的敏感性。如果共受体的使用或药物敏感性发生变化，将在治疗前和治疗后的分离株中对包膜进行测序，以识别耐药标记。Core B提供的数据将确定阿普利康在体内和体外的抗病毒作用的广度和稳定性，并为未来的研究确定其他具有抗逆转录病毒活性的NK-1R拮抗剂的特征。

The overall goal of Core B is to provide drug susceptibility testing and evaluation for drug interactions for the IPCP program. BBI Biotech has performed numerous drug susceptibility assays for HIV-1, which have contributed to the development of a novel anti-retroviral drug, PA457, that just completed a successful Phase I clinical trial. Initially, the Core will screen five NK-1R antagonists (aprepitant, CJ-12255, CJ-96345, RP-67,580, and L733060), for anti-retroviral activity and cytotoxicity in PBMC using HIV-1 Ba-L. Effective compounds, i.e., those with acceptable ratio of antiretroviral activity to cytotoxicity, will be further characterized in Projects 1 and 2, and Core B. To characterize the breadth of the antiviral response, the lead compounds, in addition to aprepitant (planned for initial animal study and clinical trial), will be tested against a panel of 25 HTV isolates consisting of type M, type O, HIV-2 primary isolates and drug resistant isolates. The 50% Effective Dose (EC50) will be determined against each isolate. Because most antiretroviral therapies contain multiple drugs, aprepitant and the other lead compounds will each be tested for drug interactions against different classes of anti-retrovirals. Drugs to be tested for interactions will be cultured with HIV-1 (R5 and X4 viruses) in PBMC together or individually. Reduction in HIV p24 production will be used in the median-effect equation to calculate the EC50s and combination indices (CI) to indicate synergism or antagonism. To evaluate in vitro resistance induction, THP-1 cells will be cultured with HIV-1 Ba-L and low doses of aprepitant. Drug susceptibility assays will be performed once a month to look for developing resistance. If the EC50 increases, the viral envelope will be sequenced to identify changes in co-receptor binding area and virus and cells sent to Projects 1 and 2, respectively, for further study. To determine whether aprepitant resistance can develop in vivo, SFV isolated from monkeys treated with aprepitant in Project 3 will be assayed every other month and EC50 determined. To associate treatment effects with possible changes in patient viral population, isolates from pre-entry, end of treatment, and one month after aprepitant treatment from Project 4 will be assayed for susceptibility to aprepitant and co-receptor usage. If there are changes in co-receptor usage or drug susceptibility the envelope will be sequenced in pretreatment and post treatment isolates to identify resistance markers. The data provided by Core B will determine the breadth and stability of the anti-viral effects of aprepitant both in vivo and in vitro and characterize additional NK-1R antagonists with anti-retroviral activities for future studies.