Role of seminal plasma in the anti-HIV-1 activity of candidate microbicides

Abstract

Background: Evaluation of microbicides for prevention of HIV-1 infection in macaque models for vaginal infection has indicated that the concentrations of active compounds needed for protection by far exceed levels sufficient for complete inhibition of infection in vitro. These experiments were done in the absence of seminal plasma (SP), a vehicle for sexual transmission of the virus. To gain insight into the possible effect of SP on the performance of selected microbicides, their anti-HIV-1 activity in the presence, and absence of SP, was determined.

Methods: The inhibitory activity of compounds against the X4 virus, HIV-1 IIIB, and the R5 virus, HIV-1 BaL was determined using TZM-bl indicator cells and quantitated by measuring beta-galactosidase induced by infection. The virucidal properties of cellulose acetate 1,2-benzene-dicarboxylate (CAP), the only microbicide provided in water insoluble, micronized form, in the presence of SP was measured.

Results: The HIV-1 inhibitory activity of the polymeric microbicides, poly(naphthalene sulfonate), cellulose sulfate, carrageenan, CAP (in soluble form) and polystyrene sulfonate, respectively, was considerably (range approximately 4 to approximately 73-fold) diminished in the presence of SP (33.3%). Formulations of micronized CAP, providing an acidic buffering system even in the presence of an SP volume excess, effectively inactivated HIV-1 infectivity.

Conclusion: The data presented here suggest that the in vivo efficacy of polymeric microbicides, acting as HIV-1 entry inhibitors, might become at least partly compromised by the inevitable presence of SP. These possible disadvantages could be overcome by combining the respective polymers with acidic pH buffering systems (built-in for formulations of micronized CAP) or with other anti-HIV-1 compounds, the activity of which is not affected by SP, e.g. reverse transcriptase and zinc finger inhibitors.

Figure 1

Inhibition of HIV-1 IIIB infection by negatively charged polymers in the presence (33.3%; v/v), and absence of seminal plasma, respectively. TZM-bl indicator cells and β-galactosidase readout were used for quantitative analysis of HIV-1 infection.

Figure 2

pH changes caused by addition of seminal plasma to 180 mg Aquateric. Increasing volumes of seminal plasma were added to 180 mg Aquateric, and pH was measured. Aquateric is a micronized form of CAP and consists of ≈ 67% CAP and ≈ 33% poloxamer + distilled acetylated monoglycerides. CAP formulations have been designed to contain 180 mg Aquateric per gram of formulation.

Figure 3

Dissolution rate of CAP as a function of pH. Data are derived from J. Spitael and R. Klinget: Solubility and dissolution rate of enteric polymers, Acta Pharmaceutica technologica 1979; S7:163–168 [56].

Figure 4

CAP solubility in seminal plasma-Aquateric formulation mixtures. Increasing volumes of seminal plasma were added to one ml of formulation 1 (see Methods section). The pH was measured; the mixtures were centrifuged to pellet most of Aquateric and CAP in the supernatant fluids was quantitated [25].

Figure 5

HIV-1 BaL inactivation in seminal plasma-Aquateric formulation mixtures. Increasing volumes of seminal plasma were added to aliquots of formulation 2 (see Methods section). After 5 min at 37°C, the samples were cooled in ice, and residual infectivity was measured. The decrease in extent of virus inactivation reflects both dilutions of the Aquateric formulation by seminal plasma and a concomitant pH increase. Samples diluted ≤ 8-fold were cytotoxic due to low pH, and the percentage of virus inactivation could not be determined.