Nanoparticle-Based ARV Drug Combinations for Synergistic Inhibition of Cell-Free and Cell-Cell HIV Transmission.

Nanocarrier-based drug delivery systems are playing an emerging role in human immunodeficiency virus (HIV) chemoprophylaxis and treatment due to their ability to alter the pharmacokinetics and improve the therapeutic index of various antiretroviral (ARV) drug compounds used alone and in combination. Although several nanocarriers have been described for combination delivery of ARV drugs, measurement of drug-drug activities facilitated by the use of these nanotechnology platforms has not been fully investigated for topical prevention. Here, we show that physicochemically diverse ARV drugs can be encapsulated within polymeric nanoparticles to deliver multidrug combinations that provide potent HIV chemoprophylaxis in relevant models of cell-free, cell-cell, and mucosal tissue infection. In contrast to existing approaches that coformulate ARV drug combinations together in a single nanocarrier, we prepared single-drug-loaded nanoparticles that were subsequently combined upon administration. ARV drug-nanoparticles were prepared using emulsion-solvent evaporation techniques to incorporate maraviroc (MVC), etravirine (ETR), and raltegravir (RAL) into poly(lactic-co-glycolic acid) (PLGA) nanoparticles. We compared the antiviral potency of the free and formulated drug combinations for all pairwise and triple drug combinations against both cell-free and cell-associated HIV-1 infection in vitro. The efficacy of ARV-drug nanoparticle combinations was also assessed in a macaque cervicovaginal explant model using a chimeric simian-human immunodeficiency virus (SHIV) containing the reverse transcriptase (RT) of HIV-1. We observed that our ARV-NPs maintained potent HIV inhibition and were more effective when used in combinations. In particular, ARV-NP combinations involving ETR-NP exhibited significantly higher antiviral potency and dose-reduction against both cell-free and cell-associated HIV-1 BaL infection in vitro. Furthermore, ARV-NP combinations that showed large dose-reduction were identified to be synergistic, whereas the equivalent free-drug combinations were observed to be strictly additive. Higher intracellular drug concentration was measured for cells dosed with the triple ARV-NP combination compared to the equivalent unformulated drugs. Finally, as a first step toward evaluating challenge studies in animal models, we also show that our ARV-NP combinations inhibit RT-SHIV virus propagation in macaque cervicovaginal tissue and block virus transmission by migratory cells emigrating from the tissue. Our results demonstrate that ARV-NP combinations control HIV-1 transmission more efficiently than free-drug combinations. These studies provide a rationale to better understand the role of nanocarrier systems in facilitating multidrug effects in relevant cells and tissues associated with HIV infection.

Polyanionic (i.e., polysulfonate) dendrimers can inhibit the replication of human immunodeficiency virus by interfering with both virus adsorption and later steps (reverse transcriptase/integrase) in the virus replicative cycle.

Polyanionic dendrimers were synthesized and evaluated for their antiviral effects. Phenyldicarboxylic acid (BRI6195) and naphthyldisulfonic acid (BRI2923) dendrimers were found to inhibit the replication of human immunodeficiency virus type 1 (HIV-1; strain III(B)) in MT-4 cells at a EC(50) of 0.1 and 0.3 microg/ml, respectively. The dendrimers were not toxic to MT-4 cells up to the highest concentrations tested (250 microg/ml). These compounds were also effective against various other HIV-1 strains, including clinical isolates, HIV-2 strains, simian immunodeficiency virus (SIV, strain MAC(251)), and HIV-1 strains that were resistant to reverse transcriptase inhibitors. HIV strains containing mutations in the envelope glycoprotein gp120 (engendering resistance to known adsorption inhibitors) displayed reduced sensitivity to the dendrimers. The compounds inhibited the binding of wild-type virus and recombinant virus (containing wild-type gp120) to MT-4 cells at concentrations comparable to those that inhibited the replication of HIV-1(III(B)) in these cells. Cellular uptake studies indicated that BRI2923, but not BRI6195, permeates into MT-4 and CEM cells. Accordingly, the naphtyldisulfonic acid dendrimer (BRI2923) proved able to inhibit later steps of the replication cycle of HIV, i.e., reverse transcriptase and integrase. NL4.3 strains resistant to BRI2923 were selected after passage of the virus in the presence of increasing concentrations of BRI2923. The virus mutants showed 15-fold reduced sensitivity to BRI2923 and cross-resistance to known adsorption inhibitors. However, these virus mutants were not cross-resistant to reverse transcriptase inhibitors or protease inhibitors. We identified several mutations in the envelope glycoprotein gp120 gene (i.e., V2, V3, and C3, V4, and C4 regions) of the BRI2923-resistant NL4.3 strains that were not present in the wild-type NL4.3 strain, whereas no mutations were found in the reverse transcriptase or integrase genes.