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 ~20–30-fold. Surprisingly, colposcopy failed to detect visible sign 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-1β, KC, MIP 1α, 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.JHU Woodrow Wilson Fellowship; National Institutes of Health (Program Project A1 45967

Cervicovaginal fluid and semen block the microbicidal activity of hydrogen peroxide produced by vaginal lactobacilli

BACKGROUND: H(2)O(2 )produced by vaginal lactobacilli is believed to protect against infection, and H(2)O(2)-producing lactobacilli inactivate pathogens in vitro in protein-free salt solution. However, cervicovaginal fluid (CVF) and semen have significant H(2)O(2)-blocking activity. METHODS: We measured the H(2)O(2 )concentration of CVF and the H(2)O(2)-blocking activity of CVF and semen using fluorescence and in vitro bacterial-exposure experiments. RESULTS: The mean H(2)O(2 )measured in fully aerobic CVF was 23 ± 5 μM; however, 50 μM H(2)O(2 )in salt solution showed no in vitro inactivation of HSV-2, Neisseria gonorrhoeae, Hemophilus ducreyii, or any of six BV-associated bacteria. CVF reduced 1 mM added H(2)O(2 )to an undetectable level, while semen reduced 10 mM added H(2)O(2 )to undetectable. Moreover, the addition of just 1% CVF supernatant abolished in vitro pathogen-inactivation by H(2)O(2)-producing lactobacilli. CONCLUSIONS: Given the H(2)O(2)-blocking activity of CVF and semen, it is implausible that H(2)O(2)-production by vaginal lactobacilli is a significant mechanism of protection in vivo

An interdisciplinary framework for measuring and supporting adherence in HIV prevention trials of ARV‐based vaginal rings

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138229/1/jia29158.pd

Formulating a Sulfonated Antiviral Dendrimer in a Vaginal Microbicidal Gel Having Dual Mechanisms of Action

SPL7013 is the sodium salt of a sulfonated dendrimer that has potent antiviral properties. VivaGel®, a topical gel containing 3% w/w SPL7013, has been shown to be safe and well-tolerated in human clinical studies. BufferGel® is a Carbopol®-based acidic buffering gel that enhances the natural protective action of the vagina to produce a broad-spectrum microbicidal environment. The positive attributes of both gels were combined into a combination vaginal microbicidal gel having dual mechanisms of action. A 3% w/w SPL7013 combination gel, pH 3.7, was developed and fully characterized, and was shown to have more than 2-fold greater acidic buffering capacity than BufferGel. Ultracentrifugation experiments demonstrated that SPL7013 was not sequestered or entropically trapped in the viscous gel, thereby confirming, along with viral challenge studies, that SPL7013 has sufficient mobility in the viscous gel to exert antiviral properties

Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model

BACKGROUND: Both cell-associated and cell-free HIV virions are present in semen and cervical secretions of HIV-infected individuals. Thus, topical microbicides may need to inactivate both cell-associated and cell-free HIV to prevent sexual transmission of HIV/AIDS. To determine if the mild acidity of the healthy vagina and acid buffering microbicides would prevent transmission by HIV-infected leukocytes, we measured the effect of pH on leukocyte motility, viability and intracellular pH and tested the ability of an acidic buffering microbicide (BufferGel(®)) to prevent the transmission of cell-associated HIV in a HuPBL-SCID mouse model. METHODS: Human lymphocyte, monocyte, and macrophage motilities were measured as a function of time and pH using various acidifying agents. Lymphocyte and macrophage motilities were measured using video microscopy. Monocyte motility was measured using video microscopy and chemotactic chambers. Peripheral blood mononuclear cell (PBMC) viability and intracellular pH were determined as a function of time and pH using fluorescent dyes. HuPBL-SCID mice were pretreated with BufferGel, saline, or a control gel and challenged with HIV-1-infected human PBMCs. RESULTS: Progressive motility was completely abolished in all cell types between pH 5.5 and 6.0. Concomitantly, at and below pH 5.5, the intracellular pH of PBMCs dropped precipitously to match the extracellular medium and did not recover. After acidification with hydrochloric acid to pH 4.5 for 60 min, although completely immotile, 58% of PBMCs excluded ethidium homodimer-1 (dead-cell dye). In contrast, when acidified to this pH with BufferGel, a microbicide designed to maintain vaginal acidity in the presence of semen, only 4% excluded dye at 10 min and none excluded dye after 30 min. BufferGel significantly reduced transmission of HIV-1 in HuPBL-SCID mice (1 of 12 infected) compared to saline (12 of 12 infected) and a control gel (5 of 7 infected). CONCLUSION: These results suggest that physiologic or microbicide-induced acid immobilization and killing of infected white blood cells may be effective in preventing sexual transmission of cell-associated HIV

In vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide

<p>Abstract</p> <p>Background</p> <p>Hydrogen peroxide (H₂O₂) produced by vaginal lactobacilli is generally believed to protect against bacteria associated with bacterial vaginosis (BV), and strains of lactobacilli that can produce H₂O₂are being developed as vaginal probiotics. However, evidence that led to this belief was based in part on non-physiological conditions, antioxidant-free aerobic conditions selected to maximize both production and microbicidal activity of H₂O₂. Here we used conditions more like those <it>in vivo </it>to compare the effects of physiologically plausible concentrations of H₂O₂and lactic acid on a broad range of BV-associated bacteria and vaginal lactobacilli.</p> <p>Methods</p> <p>Anaerobic cultures of seventeen species of BV-associated bacteria and four species of vaginal lactobacilli were exposed to H₂O₂, lactic acid, or acetic acid at pH 7.0 and pH 4.5. After two hours, the remaining viable bacteria were enumerated by growth on agar media plates. The effect of vaginal fluid (VF) on the microbicidal activities of H₂O₂and lactic acid was also measured.</p> <p>Results</p> <p>Physiological concentrations of H₂O₂(< 100 μM) failed to inactivate any of the BV-associated bacteria tested, even in the presence of human myeloperoxidase (MPO) that increases the microbicidal activity of H₂O₂. At 10 mM, H₂O₂inactivated all four species of vaginal lactobacilli but only one of seventeen species of BV-associated bacteria. Moreover, the addition of just 1% vaginal fluid (VF) blocked the microbicidal activity of 1 M H₂O₂. In contrast, lactic acid at physiological concentrations (55-111 mM) and pH (4.5) inactivated all the BV-associated bacteria tested, and had no detectable effect on the vaginal lactobacilli. Also, the addition of 10% VF did not block the microbicidal activity of lactic acid.</p> <p>Conclusions</p> <p>Under optimal, anaerobic growth conditions, physiological concentrations of lactic acid inactivated BV-associated bacteria without affecting vaginal lactobacilli, whereas physiological concentrations of H₂O₂produced no detectable inactivation of either BV-associated bacteria or vaginal lactobacilli. Moreover, at very high concentrations, H₂O₂was more toxic to vaginal lactobacilli than to BV-associated bacteria. On the basis of these <it>in vitro </it>observations, we conclude that lactic acid, not H₂O₂, is likely to suppress BV-associated bacteria <it>in vivo</it>.</p

SPL7013 Gel (VivaGel®) Retains Potent HIV-1 and HSV-2 Inhibitory Activity following Vaginal Administration in Humans

SPL7013 Gel (VivaGel®) is a microbicide in development for prevention of HIV and HSV. This clinical study assessed retention and duration of antiviral activity following vaginal administration of 3% SPL7013 Gel in healthy women. Participants received 5 single doses of product with ≥5 days between doses. A cervicovaginal fluid (CVF) sample was collected using a SoftCup™ pre-dose, and immediately, or 1, 3, 12 or 24 h post-dose. HIV-1 and HSV-2 antiviral activities of CVF samples were determined in cell culture assays. Antiviral activity in the presence of seminal plasma was also tested. Mass and concentration of SPL7013 in CVF samples was determined. Safety was assessed by reporting of adverse events. Statistical analysis was performed using the Wilcoxon signed-rank test with Bonferroni adjustment; p≤0.003 was significant. Eleven participants completed the study. Inhibition of HIV-1 and HSV-2 by pre-dose CVF samples was negligible. CVF samples obtained immediately after dosing almost completely inhibited (median, interquartile range) HIV-1 [96% (95,97)] and HSV-2 [86% (85,94)], and activity was maintained in all women at 3 h (HIV-1 [96% (95,98), p = 0.9]; HSV-2 [94% (91,97), p = 0.005]). At 24 h, >90% of initial HIV-1 and HSV-2 inhibition was maintained in 6/11 women. SPL7013 was recovered in CVF samples obtained at baseline (46% of 105 mg dose). At 3 and 24 h, 22 mg and 4 mg SPL7013, respectively, were recovered. More than 70% inhibition of HIV-1 and HSV-2 was observed if there was >0.5 mg SPL7013 in CVF samples. High levels of antiviral activity were retained in the presence of seminal plasma. VivaGel was well tolerated with no signs or symptoms of vaginal, vulvar or cervical irritation reported. Potent antiviral activity was observed against HIV-1 and HSV-2 immediately following vaginal administration of VivaGel, with activity maintained for at least 3 h post-dose. The data provide evidence of antiviral activity in a clinical setting, and suggest VivaGel could be administered up to 3 h before coitus

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

<p>Abstract</p> <p>Background</p> <p>Several active ingredients proposed as vaginal microbicides have been shown paradoxically to <it>increase </it>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Women and ARVâ based prevention: opportunities and challenges

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138349/1/jia29419.pd