Preformulation Characterization of Griffithsin, a Biopharmaceutical Candidate for HIV Prevention.

Griffithsin (GRFT) has shown potent anti-HIV activity, and it is being developed as a drug candidate for HIV prevention. Successful implementation requires thorough understanding of its preformulation characterization. In this work, preformulation assessments were conducted to characterize GRFT and identify its degradation pathways under selected conditions of temperature, light, pH, shear, ionic strength, and oxidation. Compatibility with vaginal fluid simulant, vaginal enzymes, Lactobacillus spp., and human cervicovaginal secretions was assessed. The purity, melting temperature, and HIV gp120-binding affinity of GRFT stored at 4°C and 25°C in phosphate-buffered saline (PBS) were assessed for 2 years. Chemical modifications were evaluated by intact mass analysis and peptide sequencing. Excised human ectocervical tissue permeability and localization of GRFT were evaluated. Our results demonstrated GRFT to be safe and stable under all the preformulation assessment conditions studied except oxidative stress. When GRFT was exposed to hydrogen peroxide or human cervicovaginal secretion, methionine 78 in the protein sequence underwent oxidation. GRFT did not permeate through human cervical tissue but adhered to the superficial epithelial tissue. The 2-year stability study revealed no significant change in GRFT's aggregation, degradation, melting temperature, or gp120-binding affinity despite a slow increase in oxidation over time. These studies elucidated desirable safety and bioactivity profile for GRFT, showing promise as a potential drug candidate for HIV prevention. However, susceptibility to oxidative degradation was identified. Effective protection of GRFT from oxidation is required for further development.

Novel Application of Hot Melt Extrusion for the Manufacturing of Vaginal Films Containing Microbicide Candidate Dapivirine.

Polymeric films are safe and effective and can be used for vaginal administration of microbicide drug candidates. Dapivirine (DPV), an investigational and clinically advanced antiretroviral drug, was selected as a model compound for this study. We have previously developed and clinically tested a quick-dissolving DPV film using solvent cast (SC) manufacturing technique. As an alternative to current pharmaceutical film manufacturing techniques, we investigated hot melt extrusion (HME) process in this study because it has several benefits, including its capacity as a continuous manufacturing process, lack of solvents, smaller footprint, and ease of scalability. The goal of this work was to evaluate the feasibility of using HME for DPV vaginal film manufacturing and to develop a robust manufacturing process using HME by evaluating the effect of process parameters on film quality and performance. DPV was successfully incorporated into a vaginal film using HME and maintained acceptable characteristics. Three process parameters (zone temperature, screw speed, and feed rate) had an impact on film quality and performance. Of these, the zone temperature was found to most significantly affect weight, thickness, puncture strength, and dissolution of films. Additionally, film manufacturing using HME was highly reproducible. Finally, the DPV HME film was comparable to films manufactured using SC in terms of physicochemical, biological, and safety characteristics including in vitro drug release, mechanical strength, tissue permeability, compatibility with commensal vaginal Lactobacilli, and in vitro bioactivity. These results demonstrate that HME is an effective, robust, and viable manufacturing method to produce vaginal films.

Vaginal Product Formulation Alters the Innate Antiviral Activity and Glycome of Cervicovaginal Fluids with Implications for Viral Susceptibility.

Glycosylated proteins (i.e., mucins, IgG) are important mediators of innate antiviral immunity in the vagina; however, our current knowledge of the role that glycan themselves play in genital immunity is relatively low. Herein, we evaluate the relationship between innate antiviral immunity and glycomic composition in cervicovaginal lavage fluid (CVL) collected as part of a Phase I clinical trial testing the impact of two distinct formulations of the antiretroviral drug dapivirine. Using lectin microarray technology, we discovered that formulation (hydrogel- versus film-based delivery) impacted the CVL glycome, with hydrogel formulations inducing more changes, including a loss of high-mannose. The loss of this epitope correlated to a loss of anti-HIV-1 activity. Glycoproteomic identification of high-mannose proteins revealed a cohort of antiproteases shown to be important in HIV-1 resistance, whose expression covaried with the high-mannose signature. Our data strongly suggests high-mannose as a marker for secreted proteins mediating innate antiviral immunity in vaginal fluids and that drug formulation may impact this activity as reflected in the glycome.

FAME-04: A Phase 1 trial to assess the safety, acceptability, pharmacokinetics and pharmacodynamics of film and gel formulations of tenofovir.

INTRODUCTION: Fast-dissolving vaginal film formulations release antiretroviral drugs directly into vaginal fluid and may be as efficient at drug delivery yet more acceptable to women than gels. In this Phase 1 vaginal film study, the safety, acceptability, pharmacokinetics and pharmacodynamics of two doses of tenofovir (TFV) film and TFV 1% gel were compared to corresponding placebo formulations. METHODS: Seventy-eight healthy HIV negative women were randomized to self-insert daily vaginal film (10 mg TFV, 40 mg TFV or placebo) or 4 mL of vaginal gel (TFV 1% [40 mg] or placebo) for seven days. Grade 2 and higher adverse events (AEs) related to study product were compared across study arms using Fisher's exact test. Plasma TFV concentrations were measured before and 2 hours after last product use. Paired cervical and vaginal tissue biopsies obtained 2 hours after the last dose were measured to determine tenofovir diphosphate (TFV-DP) concentrations and exposed to HIV in an ex vivo challenge assay. Acceptability was assessed through questionnaire. RESULTS: There was only one grade 2 or higher related AE, the primary endpoint; it occurred in the placebo gel arm. AEs occurred in 90% of participants; the majority (91%) were grade 1. AEs were similar across study arms. TFV concentrations in plasma and TFV-DP concentrations in cervical and vaginal tissues were comparable between 40 mg TFV film and the TFV gel groups. There was a significant relationship between reduced viral replication and TFV-DP concentrations in cervical tissues. Film users were less likely to report product leakage than gel users (66% vs. 100%, p < 0.001). CONCLUSIONS: Films were safe and well tolerated. Furthermore, films delivered TFV to mucosal tissues at concentrations similar to gel and were sufficient to block HIV infection of genital tissue ex vivo.

Preformulation and Vaginal Film Formulation Development of Microbicide Drug Candidate CSIC for HIV prevention.

PURPOSE: 5-chloro-3-[phenylsulfonyl] indole-2-carboxamide (CSIC) is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 which has been shown to have a more desirable resistance profile than other NNRTIs in development as HIV prevention strategies. This work involves generation of preformulation data for CSIC and systematic development of a cosolvent system to effectively solubilize this hydrophobic drug candidate. This system was then applied to produce a polymeric thin film solid dosage form for vaginal administration of CSIC for use in prevention of sexual acquisition of HIV. METHODS: Extensive preformulation, formulation development, and film characterization studies were conducted. An HPLC method was developed for CSIC quantification. Preformulation tests included solubility, crystal properties, stability, and drug-excipient compatibility. Cytotoxicity was evaluated using both human epithelial and mouse macrophage cell lines. Ternary phase diagram methodology was used to identify a cosolvent system for CSIC solubility enhancement. Following preformulation evaluation, a CSIC film formulation was developed and manufactured using solvent casting technique. The developed film product was assessed for physicochemical properties, anti-HIV bioactivity, and Lactobacillus biocompatibility during 12-month stability testing period. RESULTS: Preformulation studies showed CSIC to be very stable. Due to its hydrophobicity, a cosolvent system consisting of polyethylene glycol 400, propylene glycol, and glycerin (5:2:1, w/w/w) was developed, which provided a uniform dispersion of CSIC in the film formulation. The final film product met target specifications established for vaginal microbicide application. CONCLUSIONS: The hydrophobic drug candidate CSIC was successfully formulated with high loading capacity in a vaginal film by means of a cosolvent system. The developed cosolvent strategy is applicable for incorporation of other hydrophobic drug candidates in the film platform.

The Effects of Hormones and Vaginal Microflora on the Glycome of the Female Genital Tract: Cervical-Vaginal Fluid.

In this study, we characterized the glycome of cervical-vaginal fluid, collected with a Catamenial cup. We quantified: glycosidase levels; sialic acid and high mannose specific lectin binding; mucins, MUC1, MUC4, MUC5AC, MUC7; and albumin in the samples collected. These data were analyzed in the context of hormonal status (day of menstrual cycle, hormonal contraception use) and role, if any, of the type of the vaginal microflora present. When the Nugent score was used to stratify the subjects by microflora as normal, intermediate, or bacterial vaginosis, several important differences were observed. The activities of four of six glycosidases in the samples from women with bacterial vaginosis were significantly increased when compared to normal or intermediate women: sialidase, P = <0.001; α-galactosidase, P = 0.006; ß-galactosidase, P = 0.005; α-glucosidase, P = 0.056. Sialic acid binding sites as measured by two lectins, Maackia amurensis and Sambucus nigra binding, were significantly lower in women with BV compared to women with normal and intermediate scores (P = <0.0001 and 0.008 respectively). High mannose binding sites, a measure of innate immunity were also significantly lower in women with BV (P = <0.001). Additionally, we observed significant increases in MUC1, MUC4, MUC5AC, and MUC7 concentrations in women with BV (P = <0.001, 0.001, <0.001, 0.02 respectively). Among normal women we found that the membrane bound mucin MUC4 and the secreted MUC5AC were decreased in postmenopausal women (P = 0.02 and 0.07 respectively), while MUC7 (secreted) was decreased in women using levonorgestrel-containing IUDs (P = 0.02). The number of sialic acid binding sites was lower in the postmenopausal group (P = 0.04), but the number of high mannose binding sites, measured with Griffithsin, was not significantly different among the 6 hormonal groups. The glycosidase levels in the cervical-vaginal mucus were rather low in the groups, with exception of α-glucosidase activity that was much lower in the postmenopausal group (P<0.001). These studies present compelling evidence that the vaginal ecosystem responds to the presence of different vaginal microorganisms. These effects were so influential that it required us to remove subjects with BV for data interpretation of the impact of hormones. We also suggest that certain changes occurring in vaginal/cervical proteins are due to bacteria or their products. Therefore, the quantitation of vaginal mucins and lectin binding offers a new method to monitor bacteria-host interactions in the female reproductive tract. The data suggest that some of the changes in these components are the result of host processing, such as the increases in mucin content, while the microflora is responsible for the increases in glycosidases and the decreases in lectin binding. The methods should be considered a valid marker for insult to the female genital tract.

A Phase 1 Trial to Assess the Safety, Acceptability, Pharmacokinetics, and Pharmacodynamics of a Novel Dapivirine Vaginal Film.

BACKGROUND: Films may deliver antiretroviral drugs efficiently to mucosal tissues. In this first in-human trial of a vaginal film for delivering the nonnucleoside reverse transcriptase inhibitor dapivirine, safety, pharmacokinetics, and pharmacodynamics of film and gel formulations were compared with placebo. METHODS: Sixty-one healthy HIV-negative women were randomized to daily dapivirine (0.05%) or placebo gel, or dapivirine (1.25 mg) or placebo film for seven days. The proportion of participants experiencing grade 2 and higher adverse events related to study product were compared. Plasma dapivirine concentrations were quantified. Paired cervical and vaginal tissue biopsies obtained ∼2 hours after the last dose were measured for tissue drug concentration and exposed to HIV in an ex vivo challenge assay. RESULTS: Two grade 2 related adverse events occurred in the placebo film group. Women randomized to gel and film products had 4 log10 higher of dapivirine in cervical and vaginal tissues than plasma. Although gel and film users had comparable plasma dapivirine concentrations, tissue concentrations of dapivirine were 3-5 times higher in the gel users when compared with film users. HIV replication in the ex vivo challenge assay was significantly reduced in vaginal tissues from women randomized to dapivirine film or gel; furthermore, tissue drug concentrations were highly correlated with HIV protection. Women rated the film more comfortable with less leakage but found it more difficult to insert than gel. DISCUSSION: Both film and gel delivered dapivirine at concentrations sufficient to block HIV ex vivo. This proof-of-concept study suggests film formulations for microbicides merit further investigation.

Pharmacodynamic Activity of Dapivirine and Maraviroc Single Entity and Combination Topical Gels for HIV-1 Prevention.

PURPOSE: Dapivirine (DPV), a non-nucleoside reverse transcriptase inhibitor, and maraviroc (MVC), a CCR5 antagonist, were formulated into aqueous gels designed to prevent mucosal HIV transmission. METHODS: 0.05% DPV, 0.1% MVC, 0.05% DPV/0.1% MVC and placebo gels were evaluated for pH, viscosity, osmolality, and in vitro release. In vitro assays and mucosal tissues were used to evaluate anti-HIV activity. Viability (Lactobacilli only) and epithelial integrity in cell lines and mucosal tissues defined safety. RESULTS: The gels were acidic and viscous. DPV gel had an osmolality of 893 mOsm/kg while the other gels had an osmolality of <100 mOsm/kg. MVC release was similar from the single and combination gels (~5 µg/cm(2)/min(1/2)), while DPV release was 10-fold less from the single as compared to the combination gel (0.4331 µg/cm(2)/min(1/2)). Titrations of the gels showed 10-fold more drug was needed to protect ectocervical than colonic tissue. The combination gel showed ~10- and 100-fold improved activity as compared to DPV and MVC gel, respectively. All gels were safe. CONCLUSIONS: The DPV/MVC gel showed a benefit blocking HIV infection of mucosal tissue compared to the single entity gels. Combination products with drugs affecting unique steps in the viral replication cycle would be advantageous for HIV prevention.

Impact of bacterial vaginosis, as assessed by nugent criteria and hormonal status on glycosidases and lectin binding in cervicovaginal lavage samples.

The objective of this study was to evaluate the impact of hormonal status and bacterial vaginosis (BV) on the glycosidases present and glycosylation changes as assessed by lectin binding to cervicovaginal lavage constituents. Frozen cervicovaginal lavage samples from a completed study examining the impact of reproductive hormones on the physicochemical properties of vaginal fluid were utilized for the present study. In the parent study, 165 women were characterized as having BV, intermediate or normal microflora using the Nugent criteria. The presence of glycosidases in the samples was determined using quantitative 4-methyl-umbelliferone based assays, and glycosylation was assessed using enzyme linked lectin assays (ELLA). Women with BV had elevated sialidase, α-galactosidase, ß-galactosidase and α-glucosidase activities compared to intermediate or normal women (P<0.001, 0.003, 0.006 and 0.042 respectively). The amount of sialic acid (Sambucus nigra, P = 0.003) and high mannose (griffithsin, P<0.001) were reduced, as evaluated by lectin binding, in women with BV. When the data were stratified according to hormonal status, α-glucosidase and griffithsin binding were decreased among postmenopausal women (P<0.02) when compared to premenopausal groups. These data suggest that both hormonal status and BV impact the glycosidases and lectin binding sites present in vaginal fluid. The sialidases present at increased levels in women with BV likely reduce the number of sialic acid binding sites. Other enzymes likely reduce griffithsin binding. The alterations in the glycosidase content, high mannose and sialic acid binding sites in the cervicovaginal fluid associated with bacterial vaginosis may impact susceptibility to viruses, such as HIV, that utilize glycans as a portal of entry.

Assessing the potential of the Woman's Condom for vaginal drug delivery.

BACKGROUND: The Woman's Condom is a new female condom that uses a dissolvable polyvinyl alcohol capsule to simplify vaginal insertion. This preclinical study assessed the feasibility to incorporate an antiviral drug, UC781, into the Woman's Condom capsule, offering a unique drug delivery platform. STUDY DESIGN: UC781 capsules were fabricated using methods from the development of the Woman's Condom capsules as well as those used in vaginal film development. Capsules were characterized to evaluate physical/chemical attributes, Lactobacillus compatibility, in vitro safety and bioactivity, and condom compatibility. RESULTS: Two UC781 capsule platforms were assessed. Capsule masses (mg; mean±SD) for platforms 1 and 2 were 116.50±18.22 and 93.80±8.49, respectively. Thicknesses were 0.0034±0.0004 in and 0.0033±0.0004 in. Disintegration times were 11±3 s and 5±1 s. Puncture strengths were 21.72±3.30 N and 4.02±0.83 N. Water content measured 6.98±1.17% and 7.04±1.92%. UC781 content was 0.59±0.05 mg and 0.77±0.11 mg. Both platforms retained in vitro bioactivity and were nontoxic to TZM-bl cells and Lactobacillus. Short-term storage of UC781 capsules with the Woman's Condom pouch did not decrease condom mechanical integrity. CONCLUSIONS: UC781 was loaded into a polymeric capsule similar to that of the Woman's Condom product. This study highlights the potential use of the Woman's Condom as a platform for vaginal delivery of drugs relevant to sexual/reproductive health, including those for short- or long-acting HIV prevention. IMPLICATIONS: We determined the proof-of-concept feasibility of incorporation of an HIV-preventative microbicide into the Woman's Condom capsule. This study highlights various in vitro physical and chemical evaluations as well as bioactivity and safety assessments necessary for vaginal product development related to female sexual and reproductive health.

Studying the effects of reproductive hormones and bacterial vaginosis on the glycome of lavage samples from the cervicovaginal cavity.

The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depo-medroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system.

Vaginal Microbicide Film Combinations of Two Reverse Transcriptase Inhibitors, EFdA and CSIC, for the Prevention of HIV-1 Sexual Transmission.

PURPOSE: EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV. METHODS: Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations. RESULTS: No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection. CONCLUSIONS: Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission.

The effect of menopause on the innate antiviral activity of cervicovaginal lavage.

OBJECTIVE: Reproductive hormones are known to impact innate mucosal immune function of the lower genital tract. Our objectives were to determine the effect of hormonal status on intrinsic antiviral (herpes simplex virus [HSV]-1, HSV-2, and human immunodeficiency virus [HIV]-1) activity of cervicovaginal lavage (CVL). STUDY DESIGN: CVL was collected from 165 asymptomatic women including postmenopausal women (n = 29); women not on contraception in days 1-14 (n = 26) or days 15-28 (n = 27) of the menstrual cycle; and women using the levonorgestrel intrauterine device (n = 28), depot medroxyprogesterone acetate (n = 28), or combined oral contraceptives (n = 27). The anti-HSV-1/-2 and the anti-HIV-1 activity of the CVL were measured using plaque assays and the Jurkat-Tat-CCR5 assay, respectively. RESULTS: CVL from all of the groups had modest antiviral activity. Anti-HIV-1 activity was decreased in CVL from postmenopausal women when compared to premenopausal women (11% vs 34%, P = .002). However, there was no difference in anti-HIV-1 activity among premenopausal women regardless of phase of menstrual cycle or contraceptive use. Anti-HIV-1 activity was associated with the protein content of the CVL (r = 0.44, P < .001). There was no difference in anti-HSV-1 or -2 activity by hormonal group. CONCLUSION: Menopause is associated with decreased innate HIV-1 activity in the lower genital tract, suggesting that factors in the vaginal fluid could play a role in increased susceptibility of HIV-1 infection in postmenopausal women. Hormonal contraceptive use, menopause, and phase of menstrual cycle did not have a measurable impact on the intrinsic anti-HSV-1 or -2 activity.

Formulation and characterization of polymeric films containing combinations of antiretrovirals (ARVs) for HIV prevention.

PURPOSE: To develop polymeric films containing dual combinations of anti-HIV drug candidate tenofovir, maraviroc and dapivirine for vaginal application as topical microbicides. METHODS: A solvent casting method was used to manufacture the films. Solid phase solubility was used to identify potential polymers for use in the film formulation. Physical and chemical properties (such as water content, puncture strength and in vitro release) and product stability were determined. The bioactivity of the film products against HIV was assessed using the TZM-bl assay and a cervical explant model. RESULTS: Polymers identified from the solid phase solubility study maintained tenofovir and maraviroc in an amorphous state and prevented drug crystallization. Three combination film products were developed using cellulose polymers and polyvinyl alcohol. The residual water content in all films was <10% (w/w). All films delivered the active agents with release of >50% of film drug content within 30 min. Stability testing confirmed that the combination film products were stable for 12 months at ambient temperature and 6 months under stressed conditions. Antiviral activity was confirmed in TZM-bl and cervical explant models. CONCLUSIONS: Polymeric films can be used as a stable dosage form for the delivery of antiretroviral combinations as microbicides.

The effects of reproductive hormones on the physical properties of cervicovaginal fluid.

OBJECTIVE: The purpose of this study was to determine the impact of contraception, menopause, and vaginal flora on the physical and biochemical properties of cervicovaginal fluid (CVF). STUDY DESIGN: Vaginal swabs, CVF, and cervicovaginal lavage (CVL) were collected from a total of 165 healthy asymptomatic women including: postmenopausal women (n = 29), women in the proliferative (n = 26) or follicular (n = 27) phase, and women using the levonogestrel intrauterine device (n = 28), depomedroxyprogesterone acetate (n = 28) or combined oral contraceptives (n = 27). Vaginal smears were evaluated using the Nugent score. The osmolality, viscosity, density, and pH of CVL samples were measured. RESULTS: CVL from postmenopausal women and women with abnormal vaginal flora was less viscous and had higher pH than premenopausal women and women with normal flora, respectively. Women using hormonal contraceptives had more viscous CVL as compared with premenopausal women not using hormonal contraceptives, but this increase in viscosity was mitigated in the presence of bacterial vaginosis. Women using depomedroxyprogesterone acetate had less total protein in the CVL as compared with women using the levonogestrel intrauterine device, and had similar protein content when compared with postmenopausal women. CONCLUSION: The differences in CVL protein content between depomedroxyprogesterone acetate and levonogestrel intrauterine device suggest that type of progesterone and route of delivery impact the vaginal environment. Contraceptive hormone users had more viscous CVL than women not using contraceptives. However, the presence of bacterial vaginosis impacted both the pH and viscosity (regardless of hormonal contraceptive use), demonstrating that vaginal flora has a greater impact on the physical properties of CVF than reproductive hormones.

Evaluation of PD 404,182 as an anti-HIV and anti-herpes simplex virus microbicide.

PD 404,182 (PD) is a synthetic compound that was found to compromise HIV integrity via interaction with a nonenvelope protein viral structural component (A. M. Chamoun et al., Antimicrob. Agents Chemother. 56:672-681, 2012). The present study evaluates the potential of PD as an anti-HIV microbicide and establishes PD's virucidal activity toward another pathogen, herpes simplex virus (HSV). We show that the anti-HIV-1 50% inhibitory concentration (IC50) of PD, when diluted in seminal plasma, is ∼1 µM, similar to the IC50 determined in cell culture growth medium, and that PD retains full anti-HIV-1 activity after incubation in cervical fluid at 37°C for at least 24 h. In addition, PD is nontoxic toward vaginal commensal Lactobacillus species (50% cytotoxic concentration [CC50], >300 µM), freshly activated human peripheral blood mononuclear cells (CC50, ∼200 µM), and primary CD4(+) T cells, macrophages, and dendritic cells (CC50, >300 µM). PD also exhibited high stability in pH-adjusted Dulbecco's phosphate-buffered saline with little to no activity loss after 8 weeks at pH 4 and 42°C, indicating suitability for formulation for transportation and storage in developing countries. Finally, for the first time, we show that PD inactivates herpes simplex virus 1 (HSV-1) and HSV-2 at submicromolar concentrations. Due to the prevalence of HSV infection, the ability of PD to inactivate HSV may provide an additional incentive for use as a microbicide. The ability of PD to inactivate both HIV-1 and HSV, combined with its low toxicity and high stability, warrants additional studies for the evaluation of PD's microbicidal candidacy in animals and humans.

Is wetter better? An evaluation of over-the-counter personal lubricants for safety and anti-HIV-1 activity.

Because lubricants may decrease trauma during coitus, it is hypothesized that they could aid in the prevention of HIV acquisition. Therefore, safety and anti-HIV-1 activity of over-the-counter (OTC) aqueous- (n = 10), lipid- (n = 2), and silicone-based (n = 2) products were tested. The rheological properties of the lipid-based lubricants precluded testing with the exception of explant safety testing. Six aqueous-based gels were hyperosmolar, two were nearly iso-osmolar, and two were hypo-osmolar. Evaluation of the panel of products showed Gynol II (a spermicidal gel containing 2% nonoxynol-9), KY Jelly, and Replens were toxic to Lactobacillus. Two nearly iso-osmolar aqueous- and both silicone-based gels were not toxic toward epithelial cell lines or ectocervical or colorectal explant tissues. Hyperosmolar lubricants demonstrated reduction of tissue viability and epithelial fracture/sloughing while the nearly iso-osmolar and silicon-based lubricants showed no significant changes in tissue viability or epithelial modifications. While most of the lubricants had no measurable anti-HIV-1 activity, three lubricants which retained cell viability did demonstrate modest anti-HIV-1 activity in vitro. To determine if this would result in protection of mucosal tissue or conversely determine if the epithelial damage associated with the hyperosmolar lubricants increased HIV-1 infection ex vivo, ectocervical tissue was exposed to selected lubricants and then challenged with HIV-1. None of the lubricants that had a moderate to high therapeutic index protected the mucosal tissue. These results show hyperosmolar lubricant gels were associated with cellular toxicity and epithelial damage while showing no anti-viral activity. The two iso-osmolar lubricants, Good Clean Love and PRÉ, and both silicone-based lubricants, Female Condom 2 lubricant and Wet Platinum, were the safest in our testing algorithm.

The inhibitory activity of typified propolis against Enterococcus species.

Propolis, a natural bee product widely used for its antimicrobial activity, was tested against isolates of Enterococcus from humans, pig-tailed macaques, isolates of refractory endodontic treatment cases, and isolates from Lactobacillus-containing food supplements. Typification of the propolis was performed by high-performance liquid chromatography (HPLC) by which prenylated compounds, cinnamic acid derivatives, and flavonoids were detected as the main constituents. Minimum inhibitory concentrations (MIC) were determined using the agar dilution method. All human and animal Enterococcus isolates demonstrated MIC values of 1600 microg/mL. Enterococcal species of human and animal origin were inhibited by propolis. Particularly, human isolates of E. faecium and E. faecalis of refractory endodontic treatment cases were susceptible to propolis of Brazilian origin.

The safety, persistence, and acceptability of an antiretroviral microbicide candidate UC781.

OBJECTIVE: : To evaluate the persistence and acceptability of a minimally absorbed vaginal gel antiretroviral designed to block the acquisition of HIV. METHODS: : Sixty healthy women aged 18-45 participated in a phase-1 randomized placebo-controlled trial of a vaginal gel containing the nonnucleoside reverse transcriptase inhibitor UC781. Women underwent a single timed exposure ranging from 0 to 8 hours and were followed for 35 days. Safety was assessed by symptoms, physical exam, laboratory evaluation, and colposcopy. Persistence was assessed by drug levels in cervicovaginal lavage (CVL) and vaginal swab specimens. RESULTS: : The participants' mean age was 26 years; 77% were white. The most frequently reported adverse events were genitourinary; however, the placebo and UC781 arms had a similar distribution of mild and moderate genitourinary events. All colposcopic findings were superficial. Measuring systemic UC781 levels in the plasma revealed that 2 (5%) women in the UC781 gel group had detectable UC781; however, the amount was below the limits of quantification (2.5 ng/mL) in both participants. UC781 was detected in 37 of 40 CVL samples obtained 1-2 days after drug exposure and initial CVL; the median level of UC781 was 4965 pmol/mL, significantly higher than the known IC50 of 10 pmol/mL. Eighty percent of participants reported that they would use the product if it were found to be protective against HIV. CONCLUSION: : In this population of HIV-uninfected women, the gel was well tolerated and acceptable. Active levels of drug were detected in CVL and vaginal swab specimens at 1-2 days at concentrations supporting the role for daily dosing.

Degradation of naturally occurring and engineered antimicrobial peptides by proteases.

We hypothesized that current antimicrobial peptides should be susceptible to proteolytic digestion. The antimicrobial peptides: Griffithinsin, RC-101, LL-37, LSA-5, PSC-RANTES and DJ007 were degraded by commercially available proteases. Two different species of anaerobic vaginal flora, Prevotella bivia and Porphyromonas asaccharolytica also degraded the materials. Griffithsin was resistant to digestion by 8 of the 9 proteases and the bacteria while LL-37 was the most sensitive to protease digestion. These data suggests most of the molecules may not survive for very long in the proteolytic rich environments in which they are intended to function.