Multipurpose Prevention Approaches with Antiretroviral-Based Formulations

We compared the preclinical safety and efficacy of tenofovir (TFV) 1% gel with that of MZC gel [containing 50 μM MIV-150, 14 mM Zn(O2CCH3)2(H2O)2, and 3% carrageenan] through a series of in vitro, ex vivo, and in vivo assays. The two gels showed good antiviral therapeutic indexes (50% cytotoxic concentration/50% effective concentration ratios; range, >25 to 800). MZC showed greater anti-simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) activity than TFV 1% gel in rhesus macaque vaginal explants. MZC protected mice from vaginal herpes simplex virus 2 (HSV-2) challenge (P < 0.0001), but the TFV 1% gel did not.

T he most recent lead microbicide formulations, tenofovir (TFV) 1% gel and the dapivirine intravaginal ring (IVR), have been investigated as means to prevent primarily human immunodeficiency virus (HIV) acquisition. Recent clinical trials like CAPRISA 004 (clinical trial registration number NCT00441298) (1,2), conducted by the Centre for the AIDS Programme of Research in South Africa, and the VOICE trial (Vaginal and Oral Interventions to Control the Epidemic; clinical trial registration number NCT00705679) (3) have investigated the effectiveness of TFV 1% gel to prevent HIV acquisition. CAPRISA 004 was a study comparing TFV 1% vaginal gel with a placebo when used before and after sex while the VOICE trial looked at daily use of an oral tablet (TFV or Truvada) or a vaginal gel (TFV 1% gel). CAPRISA 004 and the VOICE trial showed a reduction in herpes simplex virus 2 (HSV-2) acquisition that correlated with the use of TFV vaginal gel. This was surprising since TFV has modest anti-HSV activity in cell-based assays (4), ex vivo explants (4), and murine models (4,5). Herein, we explored the preclinical safety and efficacy of a dual compartment multipurpose prevention technology (MPT) microbicide gel (MZC, which contains 50 M MIV-150, 14 mM zinc acetate dihydrate, and 3% carrageenan [CG]) targeting HIV, HSV, and human papillomavirus (HPV) (6) in a sideby-side comparison with the TFV 1% gel. A recently completed phase 3 trial (FACTS 001; clinical trial registration number NCT01386294) of TFV 1% gel was designed that followed the CAPRISA 004 dosing strategy but had a larger number of participants. The gel was not proven to be effective and poor user adherence to the dosing regimen likely contributed to the outcome of the trial. A reduced glycerin version of this gel is currently in a phase 2 trial as a potential rectal microbicide (7).
MZC, TFV 1%, and 3% CG gels were formulated as previously described (1,6). MZC, like TFV 1% gel, is a clear and semisolid formulation. However, based on physicochemical properties, the formulations differ mainly in pH (TFV 1%, 5.0; MZC, 6.9) and osmolality (TFV 1% gel, 3,358 mosmol/kg; MZC gel, 447 mosmol/kg). The MZC gel contains only 0.002% of the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 versus 1% of the NRTI TFV in the 1% gel. Antiviral activity against HIV-1 was tested using the standardized TZM-bl-based assay (6) or the peripheral blood mononu-clear cell (PBMC)-based assay (6). Briefly, TZM-bl cells (1.5 ϫ 10 5 /ml) or activated PBMCs (2 ϫ 10 6 /ml) were treated for 1 h with dilutions of gels (triplicates) before adding 100 focus forming units (FFU) or one hundred 50% tissue culture infective doses (TCID 50 ) of virus, respectively. TZM-bl cells were incubated for 72 h before staining with 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal) to count FFU. The supernatant was replaced for PBMCs with fresh stimulation medium on days 1 and 4 postinfection. The p24 level in the supernatant was determined on day 7 after infection by p24 enzyme-linked immunosorbent assay (ELISA) (ZeptoMetrix, Buffalo, NY). The 50% cytotoxic concentration (CC 50 values) of each gel formulation was estimated using XTT and CyQuant by running the antiviral assay in the absence of virus (6). The 50% effective concentrations (EC 50 s) were calculated based on gel dilution factor in order to compare the efficacies of the two gels, each containing a different active pharmaceutical ingredient (API). By comparing the EC 50 s based on gel dilution, we observed how MZC with only 0.002% of MIV-150 can achieve better or similar antiviral activity than TFV gel containing 1% of the API. MZC gel was generally more potent than TFV 1% gel in blocking HIV-1 infection in TZM-bl or PBMC with the clear exception of one multidrug-resistant (MDR) strain [OL-1/4(II)d4] containing 2 mutations (K101E and Y181I) associated with decreased susceptibility of viruses to NNRTI (Table 1). Similarly, TFV 1% gel showed an increase in EC 50 s for two strains (71361-1 and 56252-1) containing the 65R amino acid change associated with HIV resistance to TFV. Although NNRTIs are known to select resistant viruses rapidly, MIV-150 seems to select resistance at a slower pace compared to other NNRTIs and requires two or more mutations in a single genome to decrease HIV susceptibility (8). Additionally, it is important to mention that resistance development in topically applied antiretrovirals is not yet fully understood.
Cell-based assays are excellent tools for screening potential microbicides, for testing antiviral properties against a variety of isolates/MDR strains, and for monitoring the stability of formulations. However, the testing of a lead formulation in the explant model allows for assessment of preclinical safety and efficacy in a more relevant HIV target cell and architectural context. We tested MZC and TFV 1% gel in our ex vivo rhesus macaque (RM) vaginal explant model using cell-free virus inoculum and also cell-associated virus.
Similar results were observed with cumulative analysis (not shown).
We have previously shown that the combination of CG and zinc acetate (as in the MZC formulation) results in antiviral synergy (in vitro and in vivo) against HSV-2 (11). We explored the anti-HSV-2 activity of MZC and of TFV 1% gels in a murine model. Depo-Provera-treated BALB/c mice were dosed with 10 l of test gel intravaginally 1 h prior to HSV-2 infection plus 1 h after HSV-2 infection to mimic the BAT24 dosing strategy used in the CAPRISA 004 and FACTS 001 trials (BAT24 refers to one dose of gel before sex and a second dose of gel as soon as possible after sex and no more than two doses in a 24-hour period) (12). Mice were challenged with 10 l of HSV-2 G (5 ϫ 10 3 PFU/mouse) and were examined and scored daily for 21 days as previously described (11). Despite being tested in a less-stringent murine model (lower virus inoculum compare to previous evaluation in this model [6,11]), the TFV 1% gel did not protect mice from HSV-2 infection while the MZC gel protected 100% of the animals (Fig. 2).
A possible explanation for these divergent results (compared to CAPRISA 004 results in humans) is that TFV phosphorylation and/or TFV uptake may be less efficient in mice than in human cells. In fact, subtherapeutic (below a lower limit of quantification [LLOQ] of Ͻ100 ng/g) TFV diphosphate (TFV-DP) levels were found in murine cervicovaginal tissue, and even TFV-only levels were low (median, 5400 ng/g) as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (13).
MZC's potent in vitro and in vivo anti-HPV activities make this formulation a very appealing MPT candidate targeting three noncurable viral sexually transmitted infections (STIs) (6). However, poor adherence in clinical trials is an important issue that has overshadowed the success of microbicide gels in the HIV preexposure prophylaxis field. In light of this, the MZC combination may be explored not only as a gel (with potential for a rectal microbicide) but also as an IVR that incorporates levonorgestrel (LNG) to prevent unintended pregnancy (14) (a similar approach is being tested in a phase 1 trial with a TFV ϩ LNG IVR [Clinical-Trials registration no. NCT02235662] [15]). Importantly, the results shown in this paper provide information about API levels that need to be released from alternative delivery systems (e.g., IVR) in order to be safe and achieve protection against HIV infection. Adding a contraceptive, targeting more than one STI, and providing different choices for drug delivery may increase demand/uptake as well as the efficiencies of delivery and access. The MZC combination is a promising MPT that was successfully evaluated in a phase 1 trial (Population Council 558; clinical trial registration number NCT02033109), and the results shown herein support moving forward with its clinical evaluation.

FUNDING INFORMATION
The United States Agency for International Development (USAID) provided funding under award GPO-A-00-04-00019.