Multivalent benzoboroxole functionalized polymers as gp120 glycan targeted microbicide entry inhibitors.

Microbicides are women-controlled prophylactics for sexually transmitted infections. The most important class of microbicides target HIV-1 and contain antiviral agents formulated for topical vaginal delivery. Identification of new viral entry inhibitors that target the HIV-1 envelope is important because they can inactivate HIV-1 in the vaginal lumen before virions can come in contact with CD4+ cells in the vaginal mucosa. Carbohydrate binding agents (CBAs) demonstrate the ability to act as entry inhibitors due to their ability to bind to glycans and prevent gp120 binding to CD4+ cells. However, as proteins they present significant challenges in regard to economical production and formulation for resource-poor environments. We have synthesized water-soluble polymer CBAs that contain multiple benzoboroxole moieties. A benzoboroxole-functionalized monomer was synthesized and incorporated into linear oligomers with 2-hydroxypropylmethacrylamide (HPMAm) at different feed ratios using free radical polymerization. The benzoboroxole small molecule analogue demonstrated weak affinity for HIV-1BaL gp120 by SPR; however, the 25 mol % functionalized benzoboroxole oligomer demonstrated a 10-fold decrease in the K(D) for gp120, suggesting an increased avidity for the multivalent polymer construct. High molecular weight polymers functionalized with 25, 50, and 75 mol % benzoboroxole were synthesized and tested for their ability to neutralize HIV-1 entry for two HIV-1 clades and both R5 and X4 coreceptor tropism. All three polymers demonstrated activity against all viral strains tested with EC(50)s that decrease from 15000 nM (1500 microg mL(-1)) for the 25 mol % functionalized polymers to 11 nM (1 microg mL(-1)) for the 75 mol % benzoboroxole-functionalized polymers. These polymers exhibited minimal cytotoxicity after 24 h exposure to a human vaginal cell line.

Transport theory for HIV diffusion through in vivo distributions of topical microbicide gels.

Topical microbicide products are being developed for the prevention of sexually transmitted infections. These include vaginally-applied gels that deliver anti-HIV molecules. Gels may also provide partial barriers that slow virion diffusion from semen to vulnerable epithelium, increasing the time during which anti-HIV molecules can act. To explore the barrier function of microbicide gels, we developed a deterministic mathematical model for HIV diffusion through realistic gel distributions. We applied the model to experimental data for in vivo coating distributions of two vaginal gels in women. Time required for a threshold number of virions to reach the tissue surface was used as a metric for comparing different scenarios. Results delineated how time to threshold increased with increasing gel layer thickness and with decreasing diffusion coefficient. We note that for gel layers with average thickness > approximately 100 microm, the fractional area coated, rather than the gel layer thickness, was the primary determinant of time to threshold. For gel layers < approximately 100 microm, time to threshold was brief, regardless of fractional area coated. Application of the model to vaginal coating data showed little difference in time to threshold between the two gels tested. However, the protocol after gel application (i.e., with or without simulated coitus) had a much more significant effect. This study suggests that gel distribution in layers of thickness >100 microm and fractional area coated >0.8 is critical in determining the ability of the gel to serve as a barrier to HIV diffusion.

Dilution of microbicide gels with vaginal fluid and semen simulants: effect on rheological properties and coating flow.

Microbicides are agents applied topically to the vagina to prevent HIV transmission. Microbicide products formulated as semi-solid dosage forms or "gels" coat vulnerable tissue to deliver active ingredients. Effective microbicide delivery vehicles must have appropriate rheological properties to ensure appropriate deployment in vivo. Microbicide products become diluted by fluids in the vagina after application; dilution affects vehicle rheological properties and mechanics of vaginal distribution, thus affecting efficacy. To simulate the changes that might occur after application, this study analyzed the effects of small dilutions (10-30%) with vaginal fluid and semen simulants on three semi-solid vaginal formulations: a cellulose lubricant (KY Jelly), a polyacrylic acid moisturizer (Replens), and a carrageenan prototype microbicide (Carraguard). Rheological behavior was characterized using cone-and-plate rheometry. Data were fitted to either the power-law, Carreau, or Herschel-Bulkley model. Rheological parameters from these fits were input to models of coating flow due squeezing, and the simulated area coated output from these models was used to compare the responses of the different formulations to the two diluents for varying degrees of dilution. There were differences in the responses of the three materials to dilution. Even small dilutions altered the rank order of vaginal coating rates compared to the undiluted formulations.

Semi-solid gels function as physical barriers to human immunodeficiency virus transport in vitro.

Vaginal gels may act as physical barriers to HIV during sexual transmission. However, the extent and significance of this effect are not well understood. During male-to-female sexual transmission of HIV, semen containing infectious HIV is present within the lower female reproductive tract. In cases where a topical gel has previously been applied to the vaginal epithelium, virions must move through gel layers before reaching vulnerable tissue. This additional barrier could affect the functioning of anti-HIV microbicide gels and placebos. To better understand HIV transport in gels, we: (1) quantified diffusion coefficients of HIV virions within semi-solid delivery vehicles; and (2) tested the barrier functioning of thin gel layers in a Transwell system. Two gels used as placebos in microbicides clinical trials, hydroxyethyl cellulose (HEC) and methylcellulose (MC), were found to hinder HIV transport in vitro. The diffusion coefficients for HIV virions in undiluted HEC and MC were 4±2 x 10⁻¹² and 7±1 x 10⁻¹² cm²/s, respectively. These are almost 10,000 times lower than the diffusion coefficient for HIV in water. Substantial gel dilution (80%:diluent/gel, v/v) was required before diffusion coefficients rose to even two orders of magnitude lower than those in water. In the Transwell system, gel layers of approximately 150-µm thickness reduced HIV transport. There was a log reduction in the amount of HIV that had breached the Transwell membrane after 0-, 4-, and 8-h incubations. The ability of a gel to function as a physical barrier to HIV transport from semen to tissue will also depend on its distribution over the epithelium and effects of dilution by vaginal fluids or semen. Results here can serve as a baseline for future design of products that act as barriers to HIV transmission. The potential barrier function of placebo gels should be considered in the design and interpretation of microbicides clinical trials.

Distribution of extracellular matrix proteins type I collagen, type IV collagen, fibronectin, and laminin in mouse folliculogenesis.

The extracellular matrix (ECM) plays a prominent role in ovarian function by participating in processes such as cell migration, proliferation, growth, and development. Although some of these signaling processes have been characterized in the mouse, the relative quantity and distribution of ECM proteins within developing follicles of the ovary have not been characterized. This study uses immunohistochemistry and real-time PCR to characterize the ECM components type I collagen, type IV collagen, fibronectin, and laminin in the mouse ovary according to follicle stage and cellular compartment. Collagen I was present throughout the ovary, with higher concentrations in the ovarian surface epithelium and follicular compartments. Collagen IV was abundant in the theca cell compartment with low-level expression in the stroma and granulosa cells. The distribution of collagen was consistent throughout follicle maturation. Fibronectin staining in the stroma and theca cell compartment increased throughout follicle development, while staining in the granulosa cell compartment decreased. Heavy staining was also observed in the follicular fluid of antral follicles. Laminin was localized primarily to the theca cell compartment, with a defined ring at the exterior of the follicular granulosa cells marking the basement membrane. Low levels of laminin were also apparent in the stroma and granulosa cell compartment. Taken together, the ECM content of the mouse ovary changes during follicular development and reveals a distinct spatial and temporal pattern. This understanding of ECM composition and distribution can be used in the basic studies of ECM function during follicle development, and could aid in the development of in vitro systems for follicle growth.