Microbicide excipients can greatly increase susceptibility to genital herpes transmission in the mouse.

BACKGROUND: Several active ingredients proposed as vaginal microbicides have been shown paradoxically to increase susceptibility to infection in mouse genital herpes (HSV-2) vaginal susceptibility models and in clinical trials. In addition, "inactive ingredients" (or excipients) used in topical products to formulate and deliver the active ingredient might also cause epithelial toxicities that increase viral susceptibility. However, excipients have not previously been tested in susceptibility models. METHODS: Excipients commonly used in topical products were formulated in a non-toxic vehicle (the "HEC universal placebo"), or other formulations as specified. Twelve hours after exposure to the excipient or a control treatment, mice were challenged with a vaginal dose of HSV-2, and three days later were assessed for infection by vaginal lavage culture to assess susceptibility. RESULTS: The following excipients markedly increased susceptibility to HSV-2 after a single exposure: 5% glycerol monolaurate (GML) formulated in K-Y® Warming Jelly, 5% GML as a colloidal suspension in phosphate buffered saline, K-Y Warming Jelly alone, and both of its humectant/solvent ingredients (neat propylene glycol and neat PEG-8). For excipients formulated in the HEC vehicle, 30% glycerin significantly increased susceptibility, and a trend toward increased HSV-2 susceptibility was observed after 10% glycerin, and 0.1% disodium EDTA, but not after 0.0186% disodium EDTA. The following excipients did not increase susceptibility: 10% propylene glycol, 0.18%, methylparaben plus 0.02% propylparaben, and 1% benzyl alcohol. CONCLUSIONS: As reported with other surfactants, the surfactant/emulsifier GML markedly increased susceptibility to HSV-2. Glycerin at 30% significantly increased susceptibility, and, undiluted propylene glycol and PEG-8 greatly increased susceptibility.

Vaginal transmission of cell-associated HIV-1 in the mouse is blocked by a topical, membrane-modifying agent.

Because both HIV-1 virions and HIV-infected cells are present in the semen and cervical mucus of infected individuals, HIV-1 prevention strategies must consider both cell-free and cell-associated virus. Antibodies that target HIV-1 virions have been shown to prevent vaginal transmission of cell-free virus in macaques, but since cell-associated transmission has not been reliably demonstrated in this model system, no strategies to prevent such transmission have been tested. We have employed a mouse model in which SCID mice carry human peripheral blood leukocytes (HuPBLs). In these mice, vaginal transmission of cell-associated, but not cell-free, HIV-1 transmission occurs, mediated by transepithelial migration of HIV-infected cells. Topical application of beta-cyclodextrin (beta-CD), a cholesterol-sequestering agent that interferes with cell migration and budding of virus from lipid rafts, blocks transmission of cell-associated HIV-1. The HuPBL-SCID model of vaginal HIV-1 transmission should prove useful for investigating cell-associated HIV-1 transmucosal HIV-1 transmission, as well as for screening reagents for their potential efficacy in preventing sexual HIV-1 transmission.

Vaginal microbicides: detecting toxicities in vivo that paradoxically increase pathogen transmission.

BACKGROUND: Microbicides must protect against STD pathogens without causing unacceptable toxic effects. Microbicides based on nonoxynol-9 (N9) and other detergents disrupt sperm, HSV and HIV membranes, and these agents are effective contraceptives. But paradoxically N9 fails to protect women against HIV and other STD pathogens, most likely because it causes toxic effects that increase susceptibility. The mouse HSV-2 vaginal transmission model reported here: (a) Directly tests for toxic effects that increase susceptibility to HSV-2, (b) Determines in vivo whether a microbicide can protect against HSV-2 transmission without causing toxicities that increase susceptibility, and (c) Identifies those toxic effects that best correlate with the increased HSV susceptibility. METHODS: Susceptibility was evaluated in progestin-treated mice by delivering a low-dose viral inoculum (0.1 ID50) at various times after delivering the candidate microbicide to detect whether the candidate increased the fraction of mice infected. Ten agents were tested - five detergents: nonionic (N9), cationic (benzalkonium chloride, BZK), anionic (sodium dodecylsulfate, SDS), the pair of detergents in C31G (C14AO and C16B); one surface active agent (chlorhexidine); two non-detergents (BufferGel, and sulfonated polystyrene, SPS); and HEC placebo gel (hydroxyethylcellulose). Toxic effects were evaluated by histology, uptake of a 'dead cell' dye, colposcopy, enumeration of vaginal macrophages, and measurement of inflammatory cytokines. RESULTS: A single dose of N9 protected against HSV-2 for a few minutes but then rapidly increased susceptibility, which reached maximum at 12 hours. When applied at the minimal concentration needed for brief partial protection, all five detergents caused a subsequent increase in susceptibility at 12 hours of approximately 20-30-fold. Surprisingly, colposcopy failed to detect visible signs of the N9 toxic effect that increased susceptibility at 12 hours. Toxic effects that occurred contemporaneously with increased susceptibility were rapid exfoliation and re-growth of epithelial cell layers, entry of macrophages into the vaginal lumen, and release of one or more inflammatory cytokines (Il-1beta, KC, MIP 1alpha, RANTES). The non-detergent microbicides and HEC placebo caused no significant increase in susceptibility or toxic effects. CONCLUSION: This mouse HSV-2 model provides a sensitive method to detect microbicide-induced toxicities that increase susceptibility to infection. In this model, there was no concentration at which detergents provided protection without significantly increasing susceptibility.

Anti-PEG antibodies alter the mobility and biodistribution of densely PEGylated nanoparticles in mucus.

UNLABELLED: Antibodies that specifically bind polyethylene glycol (PEG) can lead to rapid elimination of PEGylated therapeutics from the systemic circulation. We have recently shown that virus-binding IgG can immobilize viruses in mucus via multiple low-affinity crosslinks between IgG and mucins. However, it remains unclear whether anti-PEG antibodies in mucus may also alter the penetration and consequently biodistribution of PEGylated nanoparticles delivered to mucosal surfaces. We found that both anti-PEG IgG and IgM can readily bind nanoparticles that were densely coated with PEG polymer to minimize adhesive interactions with mucus constituents. Addition of anti-PEG IgG and IgM into mouse cervicovaginal mucus resulted in extensive trapping of mucus-penetrating PEGylated nanoparticles, with the fraction of mobile particles reduced from over 95% to only 34% and 7% with anti-PEG IgG and IgM, respectively. Surprisingly, we did not observe significant agglutination induced by either antibody, suggesting that particle immobilization is caused by adhesive crosslinks between mucin fibers and IgG or IgM bound to individual nanoparticles. Importantly, addition of corresponding control antibodies did not slow the PEGylated nanoparticles, confirming anti-PEG antibodies specifically bound to and trapped the PEGylated nanoparticles. Finally, we showed that trapped PEGylated nanoparticles remained largely in the luminal mucus layer of the mouse vagina even when delivered in hypotonic formulations that caused untrapped particles to be drawn by the flow of water (advection) through mucus all the way to the epithelial surface. These results underscore the potential importance of elucidating mucosal anti-PEG immune responses for PEGylated therapeutics and biomaterials applied to mucosal surfaces. STATEMENT OF SIGNIFICANCE: PEG, generally considered a 'stealth' polymer, is broadly used to improve the circulation times and therapeutic efficacy of nanomedicines. Nevertheless, there is increasing scientific evidence that demonstrates both animals and humans can generate PEG-specific antibodies. Here, we show that anti-PEG IgG and IgM can specifically immobilize otherwise freely diffusing PEG-coated nanoparticles in fresh vaginal mucus gel ex vivo by crosslinking nanoparticles to the mucin mesh, and consequently prevent PEG-coated nanoparticles from accessing the vaginal epithelium in vivo. Given the increasing use of PEG coatings to enhance nanoparticle penetration of mucosal barriers, our findings demonstrate that anti-PEG immunity may be a potential concern not only for systemic drug delivery but also for mucosal drug delivery.

Enhanced vaginal drug delivery through the use of hypotonic formulations that induce fluid uptake.

Mucosal epithelia use osmotic gradients for fluid absorption and secretion. We hypothesized that administration of hypotonic solutions would induce fluid uptake that could be advantageous for rapidly delivering drugs through mucus to the vaginal epithelium. We found that hypotonic formulations markedly increased the rate at which small molecule drugs and mucoinert nanoparticles (mucus-penetrating particles, or MPP), but not conventional mucoadhesive nanoparticles (CP), reached the vaginal epithelial surface in vivo in mice. Additionally, hypotonic formulations greatly enhanced drug and MPP delivery to the entire epithelial surface, including deep into the vaginal folds (rugae) that drugs or MPP in isotonic formulations failed to reach efficiently. However, hypotonic formulations caused unencapsulated "free" drugs to be drawn through the epithelium, reducing vaginal retention. In contrast, hypotonic formulations caused MPP to accumulate rapidly and uniformly on vaginal surfaces, ideally positioned for localized sustained drug delivery. Using a mouse model of vaginal genital herpes (HSV-2) infection, we found that hypotonic delivery of free drug led to improved immediate protection, but diminished longer-term protection. In contrast, as we previously demonstrated, hypotonic delivery of drug via MPP led to better long-term retention and protection in the vagina. Importantly, we demonstrate that slightly hypotonic formulations provided rapid and uniform delivery of MPP to the entire vaginal surface, thus enabling formulations with minimal risk of epithelial toxicity. Hypotonic formulations for vaginal drug delivery via MPP may significantly improve prevention and treatment of reproductive tract diseases and disorders.

Mucus-penetrating nanoparticles for vaginal drug delivery protect against herpes simplex virus.

Incomplete coverage and short duration of action limit the effectiveness of vaginally administered drugs, including microbicides, for preventing sexually transmitted infections. We investigated vaginal distribution, retention, and safety of nanoparticles with surfaces modified to enhance transport through mucus. We show that mucus-penetrating particles (MPPs) provide uniform distribution over the vaginal epithelium, whereas conventional nanoparticles (CPs) that are mucoadhesive are aggregated by mouse vaginal mucus, leading to poor distribution. Moreover, when delivered hypotonically, MPPs were transported advectively (versus diffusively) through mucus deep into vaginal folds (rugae) within minutes. By penetrating into the deepest mucus layers, more MPPs were retained in the vaginal tract after 6 hours compared to CPs. After 24 hours, when delivered in a conventional vaginal gel, patches of a model drug remained on the vaginal epithelium, whereas the epithelium was coated with drug delivered by MPPs. We then developed MPPs composed of acyclovir monophosphate (ACVp). When administered before vaginal herpes simplex virus 2 challenge, ACVp-MPPs protected 53% of mice compared to only 16% protected by soluble drug. Overall, MPPs improved vaginal drug distribution and retention, provided more effective protection against vaginal viral challenge than soluble drug, and were nontoxic when administered daily for 1 week.