Drug synergy of tenofovir and nanoparticle-based antiretrovirals for HIV prophylaxis

Abstract

Background: The use of drug combinations has revolutionized the treatment of HIV but there is no equivalent combination product that exists for prevention, particularly for topical HIV prevention. Strategies to combine chemically incompatible agents may facilitate the discovery of unique drug-drug activities, particularly unexplored combination drug synergy. We fabricated two types of nanoparticles, each loaded with a single antiretroviral (ARV) that acts on a specific step of the viral replication cycle. Here we show unique combination drug activities mediated by our polymeric delivery systems when combined with free tenofovir (TFV).

Methodology/principal findings: Biodegradable poly(lactide-co-glycolide) nanoparticles loaded with efavirenz (NP-EFV) or saquinavir (NP-SQV) were individually prepared by emulsion or nanoprecipitation techniques. Nanoparticles had reproducible size (d ∼200 nm) and zeta potential (-25 mV). The drug loading of the nanoparticles was approximately 7% (w/w). NP-EFV and NP-SQV were nontoxic to TZM-bl cells and ectocervical explants. Both NP-EFV and NP-SQV exhibited potent protection against HIV-1 BaL infection in vitro. The HIV inhibitory effect of nanoparticle formulated ARVs showed up to a 50-fold reduction in the 50% inhibitory concentration (IC50) compared to free drug. To quantify the activity arising from delivery of drug combinations, we calculated combination indices (CI) according to the median-effect principle. NP-EFV combined with free TFV demonstrated strong synergistic effects (CI50 = 0.07) at a 1∶50 ratio of IC50 values and additive effects (CI50 = 1.05) at a 1∶1 ratio of IC50 values. TFV combined with NP-SQV at a 1∶1 ratio of IC50 values also showed strong synergy (CI50 = 0.07).

Conclusions: ARVs with different physicochemical properties can be encapsulated individually into nanoparticles to potently inhibit HIV. Our findings demonstrate for the first time that combining TFV with either NP-EFV or NP-SQV results in pronounced combination drug effects, and emphasize the potential of nanoparticles for the realization of unique drug-drug activities.

Figure 1. Schematic diagram for combination effect analysis.

Drug combinations were analyzed for their ability to affect dose reduction and synergy. First, individual drugs, either free or encapsulated, were used to create dose response curves using the TZM-bl assay. These curves were fit to a sigmoid curve using nonlinear least squares regression to estimate drug IC₅₀ and the Hill slope. Next, combinations of drugs at their equipotency ratio (1∶1 ratios of IC₅₀ values) were used to create similar curves using the TZM-bl assay. These were also fit to a sigmoid curve using nonlinear least squares regression to estimate the IC₅₀ and Hill slope of the combination. Finally, comparison of IC₅₀ values combinations were used to estimate dose reduction. The median-effect analysis was performed to measure combination effects.

Figure 2. Properties of PLGA nanoparticles loaded with efavirenz or saquinavir.

(A) Scanning electron photomicrographs (magnification, 15,000×) of nanoparticles encapsulated with antiretroviral drugs efavirenz (NP-EFV) or saquinavir (NP-SQV). (B) Fourier transform infrared spectroscopy (FTIR) confirmation of the antiretroviral drugs loaded into PLGA nanoparticles. Insets show characteristic frequencies of SQV and EFV and the PLGA polymer (Vehicle Control). (C) HPLC chromatograms of vehicle control (black), SQV (blue) and EFV (red) nanoparticles showing the detection of SQV and EFV only in ARV loaded nanoparticles. No drug peak was detected in the vehicle control nanoparticles. (D) Cumulative release of EFV and SQV from nanoparticles in a vaginal fluid simulant (VFS) showing the release of SQV and EFV over 24 h.

Figure 3. PLGA nanoparticles loaded with EFV or SQV show low cytotoxity.

Viability of TZM-bl cells measured by the CellTiter-Blue™ (Promega) viability assay demonstrating non-toxic concentrations (>80% viability) of efavirenz nanoparticles (NP-EFV) and saquinavir nanoparticles (NP-SQV) at ≤1,000 µg of polymer/mL (≤48 µM EFV and ≤26 µM SQV). Vehicle control nanoparticles at the concentrations tested showed no reduction of viability (100%±8%), indicating non-cytotoxicity of PLGA polymer. Negative control = media only, Positive control = DMSO. *Vehicle control for NP-SQV was measured at 10,000 µg of polymer/mL.

Figure 4. Ectocervical explants confirm the safety of NP-ARVs.

Viability of explants from two macaques was assessed at 18–24 h after application using an MTT assay and histology. (A) A circular tissue punch of macaque ectocervical explants was inserted through a transwell membrane and placed to assure exposure to products (luminal epithelium up). (B) Viability of explants measured by the MTT assay demonstrating viability of explant tissue exposed to nanoparticles loaded with efavirenz (NP-EFV, n = 1) or saquinavir (NP-SQV, n = 1). Tissue viability was similar to media control (untreated explants, n = 3) while the toxicity control (nonoxynol-9 (N-9) treated explant, n = 1) showed significant reduction in tissue viability. (C) Histological photographs of macaque ectocervical explants (hematoxylin and eosin stain; magnification, ×100) show no visual changes following exposure to both NP-EFV and NP-SQV treated explants, and the destruction of the epithelial layer of an N-9-treated explant.

Figure 5. NP-ARVs alone and in combination with free TFV show potent antiviral activity.

Tenofovir (TFV) alone and in combination with efavirenz (EFV) or saquinavir (SQV) was investigated in TZM-bl cells. Data represent mean values obtained from triplicate infections. The dose-response curves show antiviral activity of free EFV, free SQV, EFV and SQV loaded nanoparticles (NP-EFV and NP-SQV) alone (A and C) or in combination with free TFV (B and D). At a 1∶11 molar ratio (1∶1 ratio of IC₅₀ values of free EFV and free TFV, the antiviral activity of free TFV combined with NP-EFV showed a 3-fold reduction in the IC₅₀ value compared to the free drug combinations (B). The antiviral activity of free TFV combined with free SQV or NP-SQV was tested at a 1∶1 ratio of IC₅₀ values (1∶5 TFV∶SQV and 1∶3 TFV∶NP-SQV molar ratio). Free TFV combined with NP-SQV showed a 20-fold reduction in the IC₅₀ value compared to the free drug combinations (D).

Figure 6. TFV combined with NP-EFV or NP-SQV showed strong synergism.

Combination effects of free tenofovir (TFV) with efavirenz (EFV) or saquinavir (SQV) were quantified using the TZM-bl infectivity assay and the HIV-1 BaL isolate. The combination index (CI) was determined as described by Chou and Talalay. CI<1, = 1, and >1 indicate synergistic, additive, and antagonistic effects, respectively. The red line at CI = 1 represents the additive effect. (A) Combination of free TFV with free EFV or with nanoparticles loaded with EFV (NP-EFV) at a 1∶11 EFV∶TFV molar ratio demonstrated strong synergism, with the CI at 50% inhibition (CI₅₀) of 0.01 and 0.07, respectively. (B) Combination of free TFV with free EFV or with NP-EFV at a 1∶600 NP-EFV∶TFV molar ratio demonstrated synergism and addition, with the CI₅₀ of 0.58 and 1.05, respectively. (C) Combination of free TFV with free SQV at a 1∶5 TFV∶SQV molar ratio showed an additive effect (CI₅₀ = 1.04) while free TFV combined with nanoparticles loaded with SQV (NP-SQV) at a 1∶3 TFV∶NP-SQV molar ratio showed a synergistic effect (CI₅₀ = 0.07).