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.
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