Evaluation of WLBU2 peptide and 3-O-octyl-sn-glycerol lipid as active ingredients for a topical microbicide formulation targeting Chlamydia trachomatis

Abstract

Topical microbicides for prevention of sexually transmitted diseases (STDs) would be especially useful for women who are not able to persuade their partner(s) to take precautions. Many topical microbicides are in various stages of development, based on a variety of active ingredients. We investigated the in vitro activity of an engineered antimicrobial peptide (WLBU2) and a lipid (3-O-octyl-sn-glycerol [3-OG]) which could potentially be used as active ingredients in such a product. Using commercially available cytotoxicity reagents [Alamar Blue, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH)], we first determined the toxicity of WLBU2 and 3-OG to the host cells in our assay procedure and excluded toxic concentrations from further testing. To determine activity against Chlamydia trachomatis, we used an assay previously developed by our laboratory in which chlamydial elementary bodies (EBs) were exposed to microbicides prior to contact with epithelial cells: the minimum (microbi)cidal concentration (MCC) assay. To further simulate conditions of transmission, we carried out the same assay in the presence of a simulated vaginal fluid, a simulated seminal fluid, human serum albumin, and a range of pH values which might be found in the human vagina at the time of exposure. Last, we tested WLBU2 and 3-OG in combination to determine if adding them together resulted in synergistic activity. We found that WLBU2 and 3-OG both have excellent activity in vitro against C. trachomatis and significantly more activity when added together. The simulated fluids reduced activity, but the synergy seen is good evidence that they would be effective when combined in a microbicide formulation.

FIG. 1.

The activity of WLBU2 (50 to 0.1 μM) against C. trachomatis serovar L2 in our standard MCC assay after 5 and 120 min of exposure. Ten 2-fold dilutions of the negative and positive inhibition controls (penicillin G and polymyxin B, respectively) were also run in this assay. *, the highest test concentrations for penicillin G and polymyxin B were 2.69 mM and 0.72 mM, respectively. Percent inhibition of inclusion formation was calculated as a percentage based on the number of inclusions in the no-drug control. Each test was performed in triplicate, and the results are reported as the average of results from those three tests. Standard deviations (SD) of the results are indicated on the graph with error bars.

FIG. 2.

The activity of 3-OG (25 to 0.05 mM) against C. trachomatis serovar L2 in our standard MCC assay after 5 and 120 min of exposure. The remainder of the assay conditions are described in the legend for Fig. 1.

FIG. 3.

Activity of WLBU2 (60 to 3.75 μM) against C. trachomatis serovar D in our simulated environment assay. The conditions for the standard MCC assay were followed except for the addition of 50% VFS, 50% SFS, or 5% HSA. Percent inhibition was calculated as for Fig. 1 but based on a no-drug control with the same simulated environment. Experiments were performed in triplicate, and results that are statistically different from those for SPG alone (Tukey's test; P ≤ 0.05) are indicated with an asterisk.

FIG. 4.

Activity of WLBU2 (60 to 3.75 μM) against C. trachomatis serovar E in our simulated environment assay. The remainder of the assay conditions are described in the legend for Fig. 3.

FIG. 5.

Activity of WLBU2 (60 to 3.75 μM) against C. trachomatis serovar L2 in our simulated environment assay. The remainder of the assay conditions are described in the legend for Fig. 3.

FIG. 6.

Activities of 1.88 mM 3-OG against C. trachomatis serovars D, E, and L2 in the presence of plain SPG, HSA, VFS, and SFS for 120 min of exposure. Percent inhibition and SD were calculated as for Fig. 1 but based on a no-drug control in the same simulated environment. An asterisk indicates that the activity of 3-OG is significantly different from the activity in the presence of plain SPG (Tukey's test; P ≤ 0.05).

FIG. 7.

Activities of 60 μM WLBU2 against C. trachomatis serovars D, E, and L2 in our pH assay. The conditions for the standard MCC assay were followed except that they were carried out in the presence of SPG at various pHs (4, 5.5, 7, and 8). Percent inhibition and SD were calculated as for Fig. 1 but based on a no-drug control with the same pH. An asterisk indicates that the activity is significantly different against serovar D or E than the activity against L2 (Tukey's test; P ≤ 0.05).