Core B: HIV Antiretroviral Drug Susceptibility and Drug Interactions (Director: Mark Manak)
The overall goal of Core B is to provide drug susceptibility testing and evaluation of drug interaction for the four projects included in this proposal. SeraCare BioServices 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 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 anti-retroviral 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 HIV 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 anti-retroviral therapies contain multiple drugs, aprepitant and the other lead compounds will each be tested for drug interactions with representatives of different classes of anti-retrovirals. Compounds/drugs to be tested for interactions will be cultured with HIV-1 (R5 and X4 viruses will be used) 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, additive effects, and antagonism. To evaluate in vitro resistance that may occur with extended use of aprepitant, THP-1 cells will be cultured with HIV-1 Ba-L and low doses of aprepitant. Virus and cells will used in drug susceptibility assays 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 SIV isolated from monkeys treated with aprepitant in project 3 will be assayed every other month and EC50 determined. To associate treatment effects in the clinical trial with possible changes in patient viral population, isolates from pre-entry, end of treatment, and one month after 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/in vitro and characterize additional NK-1R antagonists with anti-retroviral activities for future studies.