anti-HIV Microbicide Research Articles

Quantitative analysis of microbicide concentrations in fluids, gels and tissues using confocal Raman spectroscopy.

Topical vaginal anti-HIV microbicides are an important focus in female-based strategies to prevent the sexual transmission of HIV. Understanding microbicide pharmacokinetics is essential to development, characterization and implementation of efficacious microbicide drug delivery formulations. Current methods to measure drug concentrations in tissue (e.g., LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry) are highly sensitive, but destructive and complex. This project explored the use of confocal Raman spectroscopy to detect microbicide drugs and to measure their local concentrations in fluids, drug delivery gels, and tissues. We evaluated three candidate microbicide drugs: tenofovir, Dapivirine and IQP-0528. Measurements were performed in freshly excised porcine buccal tissue specimens, gel vehicles and fluids using two Horiba Raman microscopes, one of which is confocal. Characteristic spectral peak calibrations for each drug were obtained using serial dilutions in the three matrices. These specific Raman bands demonstrated strong linear concentration dependences in the matrices and were characterized with respect to their unique vibrational signatures. At least one specific Raman feature was identified for each drug as a marker band for detection in tissue. Sensitivity of detection was evaluated in the three matrices. A specific peak was also identified for tenofovir diphosphate, the anti-HIV bioactive product of tenofovir after phosphorylation in host cells. Z-scans of drug concentrations vs. depth in excised tissue specimens, incubated under layers of tenofovir solution in a Transwell assay, showed decreasing concentration with depth from the surface into the tissue. Time-dependent concentration profiles were obtained from tissue samples incubated in the Transwell assay, for times ranging 30 minutes - 6 hours. Calibrations and measurements from tissue permeation studies for tenofovir showed good correlation with gold standard LC-MS/MS data. These results demonstrate that confocal Raman spectroscopy holds promise as a tool for practical, minimally invasive, label-free measurement of microbicide drug concentrations in fluids, gels and tissues.

Development and in vitro evaluation of a vaginal microbicide gel formulation for UAMC01398, a novel diaryltriazine NNRTI against HIV-1

Diaryltriazines (DATAs) constitute a class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are being investigated for use as anti-HIV microbicides. The aim of the present study was (1) to assess the biopharmaceutical properties of the DATA series, (2) to select the lead candidate as vaginal microbicide and (3) to develop and evaluate gel formulations of the lead candidate. First, the vaginal tissue permeation potential of the different DATAs was screened by performing permeability and solubility measurements. To obtain a suitable formulation of the lead microbicide candidate, several hydroxyethylcellulose-based gels were assessed for their cellular toxicity, stability and ability to enable UAMC01398 epithelial permeation. Also, attention was given to appropriate preservative selection. Because of its favourable in vitro activity, safety and biopharmaceutical profile, UAMC01398 was chosen as the lead microbicide candidate among the DATA series. Formulating UAMC01398 as a vaginal gel did not affect its anti-HIV activity. Safe and chemically stable gel formulations of UAMC01398 (0.02%) included a non-solubilizing gel and a gel containing sulfobutyl ether-β-cyclodextrin (SBE-βCD, 5%) as solubilizing excipient. Inclusion of SBE-βCD in the gel formulation resulted in enhanced microbicide flux across HEC-1A epithelial cell layers, to an extent that could not be achieved by simply increasing the dose of UAMC01398. The applied rational (pre)formulation approach resulted in the development of aqueous-based gel formulations that are appropriate for further in vivo investigation of the anti-HIV microbicide potential of the novel NNRTI UAMC01398.

“Set it and Forget it”: Women’s Perceptions and Opinions of Long-Acting Topical Vaginal Gels

Women's initial understandings and anticipated acceptability of long-acting vaginal gels as potential anti-HIV microbicides was investigated by exploring the perceptibility variables associated with prototype formulations. Four focus groups with 29 women, aged 18-45, were conducted to consider gel prototypes with varied physicochemical and rheological properties. Participants responded favorably to the concept of long-acting vaginal gels as microbicides. Distinctions in understandings and stated needs regarding product dosing, characteristics, and effectiveness offer valuable insights into product design. Long-acting vaginal gels capable of protecting against HIV/STIs will be a viable option among potential users, with dosing frequency being an important factor in willingness to use.

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.

Synergistic activity profile of carbosilane dendrimer G2-STE16 in combination with other dendrimers and antiretrovirals as topical anti-HIV-1 microbicide

Polyanionic carbosilane dendrimers represent opportunities to develop new anti-HIV microbicides. Dendrimers and antiretrovirals (ARVs) acting at different stages of HIV replication have been proposed as compounds to decrease new HIV infections. Thus, we determined the potential use of our G2-STE16 carbosilane dendrimer in combination with other carbosilane dendrimers and ARVs for the use as topical microbicide against HIV-1. We showed that these combinations obtained 100% inhibition and displayed a synergistic profile against different HIV-1 isolates in our model of TZM.bl cells. Our results also showed their potent activity in the presence of an acidic vaginal or seminal fluid environment and did not activate an inflammatory response. This study is the first step toward exploring the use of different anionic carbosilane dendrimers in combination and toward making a safe microbicide. Therefore, our results support further studies on dendrimer/dendrimer or dendrimer/ARV combinations as topical anti-HIV-1 microbicide. From the Clinical EditorThis paper describes the first steps toward the use of anionic carbosilane dendrimers in combination with antivirals to address HIV-1, paving the way to further studies on dendrimer/dendrimer or dendrimer/ARV combinations as topical anti-HIV-1 microbicides.

Gravity-driven thin film flow of an ellis fluid

The thin film lubrication approximation has been studied extensively for moving contact lines of Newtonian fluids. However, many industrial and biological applications of the thin film equation involve shear-thinning fluids, which often also exhibit a Newtonian plateau at low shear. This study presents new numerical simulations of the three-dimensional (i.e. two-dimensional spreading), constant-volume, gravity-driven, free surface flow of an Ellis fluid. The numerical solution was validated with a new similarity solution, compared to previous experiments, and then used in a parametric study. The parametric study centered around rheological data for an example biological application of thin film flow: topical drug delivery of anti-HIV microbicide formulations, e.g. hydroxyethylcellulose (HEC) polymer solutions. The parametric study evaluated how spreading length and front velocity saturation depend on Ellis parameters. A lower concentration polymer solution with smaller zero shear viscosity (η0), τ1/2, and λ values spread further. However, when comparing any two fluids with any possible combinations of Ellis parameters, the impact of changing one parameter on spreading length depends on the direction and magnitude of changes in the other two parameters. In addition, the isolated effect of the shear-thinning parameter, λ, on the front velocity saturation depended on τ1/2. This study highlighted the relative effects of the individual Ellis parameters, and showed that the shear rates in this flow were in both the shear-thinning and plateau regions of rheological behavior, emphasizing the importance of characterizing the full range of shear-rates in rheological measurements. The validated numerical model and parametric study provides a useful tool for future steps to optimize flow of a fluid with rheological behavior well-described by the Ellis constitutive model, in a range of industrial and biological applications.

(4-Benzyloxy-Phenylmethylene)-Bis-Diisopentanoyl Phloroglucinol: Evaluation of Anti-HIV-1 Properties and Use as HIV-1 Microbicide Candidate

HIV-1 transmission occurs mostly through sexual contact with infected individual. HIV crosses vaginal mucosa and HIV-1 can infect dendritic cells, macrophages and T lymphocytes in the submucosa and then target circulating CD4+ T cells. In the developing world, women have limited freedom to choose sexual situations or to insist on condom use. In such scenario, microbicide would prove an effective prevention tool. Therefore, the development of an anti-HIV microbicide is extremely important. The recent trend in the development of new microbicide candidates includes the utilization of FDA-approved drugs for HIV therapy that target the early stages of the HIV life cycle, including entry inhibitors and reverse transcriptase (RT) inhibitors. We have evaluated 4-BPMDP, a potent reverse transcriptase inhibitor, for its spectrum of anti-HIV-1 activity in hPBMCs. 4-BPMDP significantly inhibited several strains of HIV-1 including X4, R5 and X4R5 dual tropic viruses. It also inhibited replication of subtype C R5 tropic clinical isolates at low micromolar concentrations. Further we investigated 4-BPMDP as microbicide candidate. 4-BPMDP is nontoxic to vaginal epithelial cells as evident from cytokine analysis. In dual chamber model system of cervical epithelial cells, 4-BPMDP moderately inhibited transmission of HIV-1NL4-3. Our data indicate that 4-BPMDP displays potent anti-HIV-1 activity and could serve as tools for the identification of novel anti-HIV targets. However further chemical modification would be needed to enhance its HIV-1 microbicidal properties.

An engineered HIV-1 gp41 trimeric coiled coil with increased stability and anti-HIV-1 activity: implication for developing anti-HIV microbicides

We previously constructed a trimeric coiled coil, N28Fd, based on the N-heptad repeat (NHR) sequence of HIV-1 gp41, as a promising HIV-1 entry inhibitor. Here, we attempted to engineer a stabilized trimeric coiled coil, ccN28Fd, by adding interchain disulphide bonds at the N terminus of N28Fd to improve its biophysical properties and anti-HIV-1 efficacy. Molecular biology techniques were applied to engineer ccN28Fd. Circular dichroism and sedimentation velocity analysis were used to determine its secondary structure and thermostability and polymeric states, respectively. The anti-HIV-1 activity was assessed by p24 or luciferase expression. Its cytotoxicity was evaluated by XTT assay. At low pH, ccN28Fd and N28Fd were in trimeric and monomeric conformation, respectively. ccN28Fd showed higher thermostability and much stronger antiviral activity against HIV-1 IIIB (X4) and Bal (R5) strains than N28Fd. ccN28Fd was effective in inhibiting infection by a broad spectrum of primary HIV-1 isolates and enfuvirtide-resistant strains and blocking cell-to-cell HIV-1 transmission. A combination of ccN28Fd with tenofovir, a nucleoside reverse transcriptase inhibitor-based microbicide, exhibited potent synergistic anti-HIV-1 activity. ccN28Fd was highly resistant to digestion by proteinase K at pH 7.2 and pepsin at pH 1.5, and its anti-HIV-1 activity was not significantly affected by the presence of hydroxyethyl cellulose gel, seminal fluid or vaginal fluid simulant. It possessed no significant in vitro cytotoxicity. The engineered ccN28Fd maintains high stability in a low pH environment and exhibits potent and broad anti-HIV-1 activity, suggesting good potential for development as an effective and safe vaginal microbicide to prevent HIV sexual transmission.

3-Hydroxyphthalic Anhydride-Modified Human Serum Albumin as a Microbicide Candidate Against HIV Type 1 Entry by Targeting Both Viral Envelope Glycoprotein gp120 and Cellular Receptor CD4

We previously reported that 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) as an anti-HIV microbicide could potently inhibit infection by a broad spectrum of HIV-1 strains; however, its mechanism of action is still elusive. Here, we aimed to identify the target(s) of HP-HSA. HIV-1 envelope glycoprotein (Env)-mediated cell-cell fusion assays were conducted using noninfectious CHO-WT cells or infectious HIV-1IIIB-infected H9 cells as effector cells and MT-2 as target cells. The cell-to-cell transmission and single-round HIV-1 infection assays were performed by measuring luciferase activity. Binding of HP-HSA to CD4 or gp120 was determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, while binding of HP-HSA to the coreceptor CXCR4 or CCR5 was detected by cell-based ELISA. HP-HSA strongly inhibited HIV-1 Env-mediated cell-cell fusion and blocked infection by HIV-1 pseudoviruses bearing Env of HIV-1HXB2 (X4 strain) or HIV-1SF162 (R5 strain). HP-HSA was also effective in blocking HIV-1BaL transmission from infected to uninfected cells. HP-HSA could strongly bind to HIV-1 Env gp120 and cellular receptor CD4. These results suggest that HP-HSA inhibits HIV-1 entry into the target cell by interacting with viral Env gp120 and/or the cellular CD4 receptor, making it a promising microbicide candidate for preventing HIV-1 sexual transmission.

Human rElafin Inhibits HIV-1 Replication in Its Natural Target Cells

Trappin-2/elafin is a novel innate immune factor that belongs to the serine protease inhibitor family and has known antibacterial, antifungal, and antiviral properties. In this study, we further investigated the anti-HIV activity of elafin using different cellular models and both X4– and R5–HIV-1 laboratory strains. We compared the antiviral activity of human recombinant elafin (rElafin) with three well-known antiretroviral drugs, AZT, tenofovir, and enfuvirtide. We have found that when the virus is pre-incubated with rElafin prior to the infection of the cells, HIV-1 replication is significantly inhibited. In target T cells and human peripheral blood mononuclear cells, maximal inhibition was achieved using submicromolar concentrations, and rElafin was found to be as potent as enfuvirtide, showing its potential for therapeutic application. We also show data on the mechanism of the antiviral activity of rElafin. We have demonstrated that rElafin neither binds to CD4, CXCR4, or CCR5 host cell receptors, nor to the viral glycoproteins gp120 and gp41. Furthermore, in our cell-to-cell fusion assays, in contrast to enfuvirtide, rElafin failed to block cell fusion. Altogether our results indicate that rElafin interferes with HIV replication at the early steps of its cycle but with a different mechanism of action than enfuvirtide. This study provides the first experimental evidence that elafin inhibits HIV replication in its natural target cells; therefore, elafin might have potential for its development as a new anti-HIV drug or microbicide.

Chemically modified bovine beta-lactoglobulin inhibits human papillomavirus infection

Previous studies have shown that 3-hydroxyphthalic anhydride-modified bovine beta-lactoglobulin is a promising anti-HIV microbicide candidate. Here we found that this chemically modified protein, designated JB01, exhibited highly potent antiviral activity against infection by human papillomaviruses (HPV), including HPV6, HPV16 and HPV18. Its anti-HPV activity was correlated with the percentage of modified lysine and arginine residues in JB01. This modified milk protein had no cytotoxicity at the concentration of 1 mg/ml, and it is highly stable at room temperature and 37 °C for at least 12 weeks. These results suggest that JB01 has good potential to be developed as an effective, safe and inexpensive antiviral agent for treatment and prevention of HPV infection.

Homeostatic properties of Lactobacillus jensenii engineered as a live vaginal anti-HIV microbicide

BackgroundVaginal probiotics are investigated as a binary strategy for prevention of bacterial vaginosis and HIV. We applied an innovative experimental model using primary and immortalized human cervical and vaginal epithelial cells to assess the functional properties of Lactobacillus jensenii, a predominant constituent of the healthy vaginal microbiome, engineered to express the HIV-1 entry inhibitor modified cyanovirin-N (mCV-N). In this model bacteria colonize the epithelial cells over a period of 24-72 h. Staurosporine and the Toll-like receptor 2/6 ligand macrophage-activating lipopeptide-2 (MALP-2) serve as positive controls for apoptosis and proinflammatory activation, respectively. In 24-hour intervals, the colonized epithelium is assessed microscopically, supernatants are collected for measurement of soluble immunoinflammatory mediators and production of CV-N, and cells are lysed for assessment of: 1) apoptosis by cleaved versus total caspase-3 assay; 2) NF-κB activation by a luciferase reporter assay; or 3) epithelia-associated colony forming units (CFU) in Brucella agar.ResultsWild type (WT) L. jensenii 1153 consistently colonized cervical and vaginal cells in the absence of epithelial damage and apoptosis. The bioengineered derivatives expressing mCV-N or control plasmids showed the same stable colonization pattern, which was reproducible between technologists and bacterial batches (CFU coefficient of variation <10% within and between experiments and epithelial cell types). MALP-2 activated NF-κB and caused fold-increased levels of proinflammatory mediators with clinically established significance in the cervicovaginal environment (IL-1α, IL-1β, IL-6, TNF-α, IL-8, RANTES, MIP-3α, and ICAM-1), measured by a multiplex electrochemiluminescence assay. At the same time levels of protective anti-inflammatory mediators interleukin 1 receptor antagonist (IL-1RA) and secretory leukocyte protease inhibitor (SLPI), both measured by ELISA, remained constant (IL-1RA) or moderately increased (SLPI). Similarly to MALP-2, colonization by L. jensenii WT activated NF-κB; however, unlike the synthetic TLR2/6 ligand, the live microorganisms did not induce significant changes in the secreted levels across all inflammation-associated proteins. The mCV-N production and function were confirmed by western blot and a HIV-1 gp120 binding assay, respectively. The bioengineered lactobacilli expressed mCV-N with anti-HIV activity preserved in the epithelial cell context and caused no significant immunoinflammatory changes as compared to the WT L. jensenii.ConclusionsThese results highlight the translational value of the colonization model and justify further clinical investigation of the homeostatic and anti-HIV effectiveness of the L. jensenii derivates.

In search of the optimal delivery method for anti-HIV microbicides: are intravaginal rings the way forward?

Microbicides are urgently needed to help stem the HIV epidemic [1]. With time and resources at a premium, donors, researchers and regulators agree that only the most promising candidates should be ...

MiniCD4 microbicide prevents HIV infection of human mucosal explants and vaginal transmission of SHIV(162P3) in cynomolgus macaques.

In complement to an effective vaccine, development of potent anti-HIV microbicides remains an important priority. We have previously shown that the miniCD4 M48U1, a functional mimetic of sCD4 presented on a 27 amino-acid stable scaffold, inhibits a broad range of HIV-1 isolates at sub-nanomolar concentrations in cellular models. Here, we report that M48U1 inhibits efficiently HIV-1Ba-L in human mucosal explants of cervical and colorectal tissues. In vivo efficacy of M48U1 was evaluated in nonhuman primate (NHP) model of mucosal challenge with SHIV162P3 after assessing pharmacokinetics and pharmacodynamics of a miniCD4 gel formulation in sexually matured female cynomolgus macaques. Among 12 females, half were treated with hydroxyethylcellulose-based gel (control), the other half received the same gel containing 3 mg/g of M48U1, one hour before vaginal route challenge with 10 AID50 of SHIV162P3. All control animals were infected with a peak plasma viral load of 105–106 viral RNA (vRNA) copies per mL. In animals treated with miniCD4, 5 out of 6 were fully protected from acquisition of infection, as assessed by qRT-PCR for vRNA detection in plasma, qPCR for viral DNA detection in PBMC and lymph node cells. The only infected animal in this group had a delayed peak of viremia of one week. These results demonstrate that M48U1 miniCD4 acts in vivo as a potent entry inhibitor, which may be considered in microbicide developments.

A modified SILCS contraceptive diaphragm for long-term controlled release of the HIV microbicide dapivirine

BackgroundThere is considerable interest in developing new multipurpose prevention technologies to address women's reproductive health needs. This study describes an innovative barrier contraceptive device — based on the SILCS diaphragm — that also provides long-term controlled release of the lead candidate anti-HIV microbicide dapivirine. Study DesignDiaphragm devices comprising various dapivirine-loaded polymer spring cores overmolded with a nonmedicated silicone elastomer sheath were fabricated by injection molding processes. In vitro release testing, thermal analysis and mechanical characterization were performed on the devices. ResultsA diaphragm device containing a polyoxymethylene spring core loaded with 10% w/w dapivirine provided continuous and controlled release of dapivirine over a 6-month period, with a mean in vitro daily release rate of 174 mcg/day. The mechanical properties of the new diaphragm were closely matched to the SILCS diaphragm. ConclusionsThe study demonstrates proof of concept for a dapivirine-releasing diaphragm with daily release quantities potentially capable of preventing HIV transmission. In discontinuous clinical use, release of dapivirine may be readily extended over 1 or more years.

Development and validation of a HPLC method for the assay of dapivirine in cell-based and tissue permeability experiments

Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is being currently used for the development of potential anti-HIV microbicide formulations and delivery systems. A new high-performance liquid chromatography (HPLC) method with UV detection was developed for the assay of this drug in different biological matrices, namely cell lysates, receptor media from permeability experiments and homogenates of mucosal tissues. The method used a reversed-phase C18 column with a mobile phase composed of trifluoroacetic acid solution (0.1%, v/v) and acetonitrile in a gradient mode. Injection volume was 50μL and the flow rate 1mL/min. The total run time was 12min and UV detection was performed at 290nm for dapivirine and the internal standard (IS) diphenylamine. A Box-Behnken experimental design was used to study different experimental variables of the method, namely the ratio of the mobile phase components and the gradient time, and their influence in responses such as the retention factor, tailing factor, and theoretical plates for dapivirine and the IS, as well as the peak resolution between both compounds. The optimized method was further validated and its usefulness assessed for in vitro and ex vivo experiments using dapivirine or dapivirine-loaded nanoparticles. The method showed to be selective, linear, accurate and precise in the range of 0.02–1.5μg/mL. Other chromatographic parameters, namely carry-over, lower limit of quantification (0.02μg/mL), limit of detection (0.006μg/mL), recovery (equal or higher than 90.7%), and sample stability at different storage conditions, were also determined and found adequate for the intended purposes. The method was successfully used for cell uptake assays and permeability studies across cell monolayers and pig genital mucosal tissues. Overall, the proposed method provides a simple, versatile and reliable way for studying the behavior of dapivirine in different biological matrices and assessing its potential as an anti-HIV microbicide drug.

In vivooptical imaging of human vaginal gel thickness distributions with a probe-based, dual-modality instrument

ABSTRACT. We used a probe-based dual-modality optical imaging instrument to measure in vivo coating thickness distributions of a gel distributed along the vaginal lumen, in a clinical study. The gel was a surrogate for one delivering an anti-HIV topical microbicide. Imaging data from Fourier-domain multiplexed low-coherence interferometry (mLCI) and fluorimetric measurements were compared to assess the feasibility and accuracy of mLCI in measuring in vivo gel coating thickness distributions. In each study session, 3.5mL of Replens gel was inserted to the vaginal fornix while the participant was supine. The participant either: 1. remained supine (10 or 60min); or 2. sat up (1min), stood up (1min), sat down (1min) and returned to the supine position; net elapsed time was 10 or 60min after which the gel distribution was imaged. Local coating thickness distributions were qualitatively and quantitatively similar. Here mLCI did not accurately measure thicker gel coatings (&gt;0.8 mm), a limitation not seen with fluorimetry. However, mLCI is capable of measuring in vivo microbicide gel distributions with resolution on the order of 10μm, without the need for exogenous contrast agents, and can accurately capture relevant summary coating measures in good agreement with fluorimetry.

Antiviral and Immunological Effects of Tenofovir Microbicide in Vaginal Herpes Simplex Virus 2 Infection

The anti-HIV microbicide, tenofovir (TFV) gel, has been shown to decrease HIV-1 acquisition by 39% and reduce herpes simplex virus 2 (HSV-2) transmission by 51%. We evaluated the effect of a 1% TFV gel on genital HSV-2 infection in a mouse vaginal challenge model. In vitro plaque assays and luminex multiplex bead analysis were used, respectively, to measure postinfection vaginal viral shedding (day 1) and cytokine secretion (day 2). To further investigate the anti-HSV-2 properties, we evaluated the direct antiviral effect of TFV and the oral prodrug tenofovir disoproxil fumerate (TDF) in cell culture. Compared to placebo-treated mice, TFV-treated mice had significantly lower clinical scores, developed later genital lesions, and showed reduced vaginal viral shedding. Furthermore, the levels of IFN-γ, IL-2, TNF-α, and other cytokines were altered in the vaginal fluid following topical tenofovir treatment and subsequent HSV-2 challenge. Finally, we found that both TFV and TDF inhibited HSV-2 infection in vitro; TDF showed a 50-fold greater potency than TFV. In conclusion, we confirmed that the microbicide TFV had direct anti-HSV-2 effects in a murine vaginal challenge model. Therefore, this model would be suitable for evaluating present and future microbicide candidates. Furthermore, the present study warrants further investigation of TDF in microbicides.

Transient spreading and swelling behavior of a gel deploying an anti-HIV topical microbicide

Drug delivery of topical microbicidal molecules against HIV offers promise as a modality to prevent sexual transmission of the virus. Success of any microbicide product depends, in an interactive way, upon its drug (the microbicide active pharmaceutical ingredient, API) and its delivery system (e.g. a gel, film or intravaginal ring). There is a widespread agreement that more effective drug delivery vehicles, as well as better APIs, must be developed to improve the efficacy of microbicide products. Non-Newtonian gels are primary microbicide vehicles, but those to date have been created with limited understanding of how their properties govern their spreading and retention in the vagina, which, in turn, govern successful drug delivery. Here, we apply fundamental fluid mechanical and physicochemical transport theory to help better understand how successful microbicide API delivery depends upon properties of a gel and the vaginal environment. We address several critical components of this complex process, including: elastohydrodynamic flow of the bolus of a non-Newtonian fluid; and mass transfer due to inhomogeneous dilution of the gel by vaginal fluid contacting it along a moving boundary (the locally deforming vaginal epithelial surface). Local dilution of gel alters local rheological properties. We evaluated this experimentally, delineating the way that constitutive parameters of a shear-thinning gel are modified by dilution. We supplement the Reynolds lubrication equation with a mass conservation equation to model diluting fluid movement across the moving vaginal epithelial surface and into the gel bolus. This is a physicochemically complex phenomenon that is not well understood. We implement a boundary flux model based upon the elevated hydrodynamic pressures in the cells. Results show that this model produces fluxes that lie within the range of mean values that have been reported. Further experimental characterization of the vaginal wall is required for a more precise set of parameters and a more sophisticated theoretical treatment of epithelium.

In vitro profiling of the vaginal permeation potential of anti-HIV microbicides and the influence of formulation excipients

In the search for an effective anti-HIV microbicidal gel, limited drug penetration into the vaginal submucosa is a possible reason for failed protection against HIV transmission. To address this issue in early development, we here describe a simple in vitro strategy to predict the tissue permeation potential of vaginally applied drugs, based on solubility, permeability and flux assessment. We demonstrated this approach for four model microbicides (tenofovir, darunavir, saquinavir mesylate and dapivirine) and additionally examined the influence of formulation excipients on the permeation potential. When formulated in an aqueous-based HEC gel, high flux values across an HEC-1A cell layer were reached by tenofovir, as a result of its high aqueous solubility. In contrast, saquinavir and dapivirine fluxes remained low due to poor permeability and solubility, respectively. These low fluxes suggest limited in vivo tissue penetration, possibly leading to lack of efficacy. Dapivirine fluxes, however, could be enhanced up to 30-fold, by including formulation excipients such as polyethylene glycol 1000 (20%) or cyclodextrins (5%) in the HEC gels. Alternative formulations, i.e. emulsions or silicone elastomer gels, were less effective in flux enhancement compared to cyclodextrin-HEC gels. In conclusion, implementing the proposed solubility and permeability profiling in early microbicide development may contribute to the successful selection of promising microbicide candidates and appropriate formulations.