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1.  Rectal Microbicide Development 
The last few years have seen important progress in demonstrating the efficacy of oral pre-exposure prophylaxis, vaginal microbicides, and treatment as prevention as effective strategies for reducing the risk of acquiring or transmitting HIV infection. There has also been significant progress in the development of rectal microbicides. Preclinical non-human primate studies have demonstrated that antiretroviral microbicides can provide significant protection from rectal challenge with SIV or SHIV. Recent Phase 1 rectal microbicide studies have characterized the safety, acceptability, compartmental pharmacokinetics (PK), and pharmaco-dynamics (PD) of both UC781 and tenofovir gels. The tenofovir gel formulation used in vaginal studies was not well tolerated in the rectum and newer rectal-specific formulations have been developed and evaluated in Phase 1 studies. The PK/PD data generated in these Phase 1 studies may reduce the risk of advancing ineffective candidate rectal microbicides into late stage development. Tenofovir gel is currently poised to move into Phase 2 evaluation and it is possible that a Phase 2B/3 effectiveness study with this product could be initiated in the next 2–3 years.
doi:10.1007/82_2013_325
PMCID: PMC4245014  PMID: 23612991
2.  Targeted Delivery of PSC-RANTES for HIV-1 Prevention using Biodegradable Nanoparticles 
Pharmaceutical research  2008;26(3):502-511.
Purpose
Nanoparticles formulated from the biodegradable co-polymer poly(lactic-co-glycolic acid) (PLGA), were investigated as a drug delivery system to enhance tissue uptake, permeation, and targeting for PSC-RANTES anti-HIV-1 activity.
Materials and Methods
PSC-RANTES nanoparticles formulated via a double emulsion process and characterized in both in vitro and ex vivo systems to determine PSC-RANTES release rate, nanoparticle tissue permeation, and anti-HIV bioactivity.
Results
Spherical, monodisperse (PDI = 0.098 ± 0.054) PSC-RANTES nanoparticles (d = 256.58 ± 19.57 nm) with an encapsulation efficiency of 82.23 ± 8.35% were manufactured. In vitro release studies demonstrated a controlled release profile of PSC-RANTES (71.48 ± 5.25% release). PSC-RANTES nanoparticle maintained comparable anti-HIV activity with unformulated PSC-RANTES in a HeLa cell-based system with an IC50 of approximately 1pM. In an ex vivo cervical tissue model, PSC-RANTES nanoparticles displayed a fivefold increase in tissue uptake, enhanced tissue permeation, and significant localization at the basal layers of the epithelium over unformulated PSC-RANTES.
Conclusions
These results indicate that PSC-RANTES can readily be encapsulated into a PLGA nanoparticle drug delivery system, retain its anti-HIV-1 activity, and deliver PSC-RANTES to the target tissue. This is crucial for the success of this drug candidate as a topical microbicide product.
doi:10.1007/s11095-008-9765-2
PMCID: PMC4243299  PMID: 19002569
drug delivery; HIV-1 prevention; microbicide; nanoparticles; PSC-RANTES
3.  Use of Human Mucosal Tissue to Study HIV-1 Pathogenesis and Evaluate HIV-1 Prevention Modalities 
Current HIV/AIDS reports  2013;10(1):12-20.
The use of human mucosal tissue models is an important tool advancing our understanding of the specific mechanisms of sexual HIV transmission. Despite 30 years of study, major gaps remain, including how HIV-1 transverses the epithelium and the identity of the early immune targets (gate keepers). Because defining HIV-1 transmission in vivo is difficult, mucosal tissue is being used ex vivo to identify key steps in HIV-1 entry and early dissemination. Elucidating early events of HIV-1 infection will help us develop more potent and specific HIV-1 preventatives such as microbicides and vaccines. Mucosal tissue has been incorporated into testing regimens for antiretroviral drugs and monoclonal antibodies. The use of mucosal tissue recapitulates the epithelium and immune cells that would be exposed in vivo to virus and drug. This review will discuss the use of mucosal tissue to better understand HIV-1 pathogenesis and prevention modalities.
doi:10.1007/s11904-012-0148-2
PMCID: PMC4242793  PMID: 23224426
Mucosal tissue; Sexual transmission; Sexual HIV transmission; HIV-1 infection; HIV pathogenesis; Microbicide; Vaccine; Explants; Biopsies
4.  HIV-1 Infection of Female Genital Tract Tissue for use in Prevention Studies 
Objective
Ex vivo HIV-1 challenge has been proposed as a bio-indicator of microbicide product effectiveness. The objective of this study was to establish optimal parameters for use of female genital tract tissue in this model.
Design
Ex vivo challenge involves in vivo product use, followed by tissue biopsy, and exposure of the tissue to HIV-1 in the laboratory.
Methods
Paired ectocervical and vaginal biopsies were collected from 42 women and 28 had additional biopsies from each site collected after 5% lidocaine (n=14) or chlorhexidine (n=14) treatment. Tissues were transported immediately to the laboratory and exposed to HIV-1. HIV-1 infection was followed by p24 ELISA on culture supernatants and at study end after weighing and fixing the tissue for immunohistochemistry (IHC) to detect p24 expressing cells.
Results
While both tissue types were equally infected with HIV-1 based on IHC results, ectocervical tissues had significantly higher HIV-1 replication than vaginal tissues (P < .005). Lidocaine and chlorhexidine had minimal impact on HIV-1 infection and replication. Point estimates for p24 levels were defined for 95% probability of p24-positive tissues and were 3.43 log10 for ectocervical tissue and 2.50 log10 for vaginal tissue based on weight-adjusted cumulative p24 endpoints.
Conclusions
While similar proportions of ectocervical and vaginal tissues support HIV-1 infection, higher levels of HIV-1 replication were observed in ectocervical tissues. Defining point estimates for HIV-1 infection in fresh ectocervical and vaginal tissues provides valuable information for the evaluation of HIV-1 preventative treatments during early clinical studies.
doi:10.1097/QAI.0b013e318291f331
PMCID: PMC3760971  PMID: 23514957
Microbicides; pre-exposure prophylaxis; HIV prevention; explants; mucosal tissue susceptibility; bio-indicator
5.  HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities 
PLoS ONE  2014;9(7):e101863.
Background
Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.
Methods and Findings
We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.
Conclusion
Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.
doi:10.1371/journal.pone.0101863
PMCID: PMC4108330  PMID: 25054205
6.  Development of HIV-1 Rectal-Specific Microbicides and Colonic Tissue Evaluation 
PLoS ONE  2014;9(7):e102585.
The gastrointestinal tract is structurally and functionally different from the vagina. Thus, the paradigm of topical microbicide development and evaluation has evolved to include rectal microbicides (RMs). Our interest was to create unique RM formulations to safely and effectively deliver antiretroviral drugs to mucosal tissue. RMs were designed to include those that spread and coat all surfaces of the rectum and distal colon rapidly (liquid) and those that create a deformable, erodible barrier and remain localized at the administration site (gel). Tenofovir (TFV) (1%) was formulated as an aqueous thermoreversible fluid and a carbopol-based aqueous hydrogel. Lipid-based liquid and gel formulations were prepared for UC781 (0.1%) using isopropyl myristate and GTCC (Caprylic/Capric Triglycerides), respectively. Formulations were characterized for pH, viscosity, osmolality, and drug content. Pre-clinical testing incorporated ex vivo colonic tissue obtained through surgical resections and flexible sigmoidoscopy (flex sig). As this was the first time using tissue from both sources side-by-side, the ability to replicate HIV-1 was compared. Efficacy of the RM formulations was tested by applying the products with HIV-1 directly to polarized colonic tissue and following viral replication. Safety of the formulations was determined by MTT assay and histology. All products had a neutral pH and were isoosmolar. While HIV-1BaL and HIV-1JR-CSF alone and in the presence of semen had similar replication trends between surgically resected and flex sig tissues, the magnitude of viral replication was significantly better in flex sig tissues. Both TFV and UC781 formulations protected the colonic tissue, regardless of tissue source, from HIV-1 and retained tissue viability and architecture. Our in vitro and ex vivo results show successful formulation of unique RMs. Moreover, the results of flex sig and surgically resected tissues were comparable suggesting the incorporation of both in pre-clinical testing algorithms.
doi:10.1371/journal.pone.0102585
PMCID: PMC4099179  PMID: 25025306
8.  Mucosal Escherichia coli Bactericidal Activity and Immune Mediators Are Associated With HIV-1 Seroconversion in Women Participating in the HPTN 035 Trial 
The Journal of Infectious Diseases  2012;206(12):1931-1935.
The mucosal environment may impact the risk for human immunodeficiency virus type 1 (HIV-1) acquisition. Immune mediators were measured in vaginal fluid collected from HPTN 035 participants who acquired HIV-1 and from those who remained HIV-1 negative (controls). Mediator concentrations were similar in samples obtained before as compared to after HIV-1 acquisition in the 8 seroconverters. Compared with controls, seroconverters were more likely to have detectable levels of HβD-2 (odds ratio [OR], 2.39; P = .005) and greater Escherichia coli bactericidal activity (OR, 1.22; P = .01) prior to seroconversion. E. coli bactericidal activity remained significant in a multivariable analysis (P = .02) and may be a biomarker for HIV-1 acquisition.
doi:10.1093/infdis/jis555
PMCID: PMC3502373  PMID: 22966121
9.  Safety and Efficacy of tenofovir / IQP-0528 combination gels – a dual compartment microbicide for HIV-1 prevention 
Antiviral research  2012;96(2):221-225.
Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor and IQP-0528 is a nonnucleoside reverse transcriptase inhibitor that also blocks virus entry. TFV and IQP-0528 alone have shown antiviral activity as microbicide gels. Because combination therapy will likely be more potent than mono-therapy, these drugs have been chosen to make a combination microbicide gel containing 2.5% TFV/1% IQP-0528. Safety and efficacy testing was done to evaluate five prototype combination gels. The gels retained TZM-bl cell and ectocervical and colorectal tissue viability. Further, the epithelium of the ectocervical and colorectal tissue remained intact after a 24 hour exposure. The ED50 calculated from the formulations for IQP-0528 was ~32 nM and for TFV was ~59 nM and their inhibitory activity was not affected by semen. The ED50 of TFV in the combination gels was ~100-fold lower than when calculated for the drug substance alone reflecting the activity of the more potent IQP-0528. When ectocervical and colorectal tissue were treated with the combination gels, HIV-1 p24 release was reduced by ≥1 log10 and ≥2 log10, respectively. Immunohistochemistry for the ectocervical tissues treated with combination gels showed no HIV-1 infected cells at study end. With the increased realization of receptive anal intercourse among heterosexual couples often in conjunction with vaginal intercourse, having a safe and effective microbicide for both mucosal sites is critical. The safety and efficacy profiles of the gels were similar for ectocervical and colorectal tissues suggesting these gels have the potential for dual compartment use.
doi:10.1016/j.antiviral.2012.08.004
PMCID: PMC3501576  PMID: 22940075
HIV prevention; combination microbicide; rectal microbicide; pyrimidinedione; topical gel
10.  Extracts from Acacia catechu suppress HIV-1 replication by inhibiting the activities of the viral protease and Tat 
Virology Journal  2013;10:309.
Background
Acacia catechu (Mimosa family) stem bark extracts have been used traditionally as a dietary supplement as well as a folk medicine given its reported anti-inflammatory, immunomodulatory, hepatoprotective, antioxidant, anti-microbial and anti-tumor activities. The present study was undertaken to evaluate the anti-HIV-1 activity of the extracts from stem bark of A. catechu.
Methods
The aqueous and 50% ethanolic extracts of A. catechu stem bark were prepared and 50% ethanolic extract was further fractioned by successively partitioning with petroleum ether, chloroform and n-butanol. All the extracts and fractions were evaluated for cytotoxicity and anti-HIV-1 activity using different in vitro assays. The active n-butanol fraction was evaluated for its inhibition against HIV-1 reverse transcriptase, integrase, protease, pro-viral genome integration and viral Tat protein mediated transactivation. The effect of n-butanol fraction on the induction of pro-inflammatory cytokines secretion in Vk2/E6E7 cells and transepithelial resistance in Caco-2 and HEC-1A cells was investigated.
Results
The aqueous and 50% ethanolic extracts of A. catechu showed IC50 values of 1.8 ± 0.18 μg/ml and 3.6 ± 0.31 μg/ml, respectively in cell-free virus based assay using TZM-bl cells and HIV-1NL4.3 (X-4 tropic). In the above assay, n-butanol fraction exhibited anti-HIV-1 activity with an IC50 of 1.7 ± 0.12 μg/ml. The n-butanol fraction showed a dose-dependent inhibition against HIV-1NL4.3 infection of the peripheral blood lymphocytes and against HIV-1BaL(R-5-tropic) as well as two different primary viral isolates of HIV-1 infection of TZM-bl cells. The n-butanol fraction demonstrates a potent inhibitory activity against the viral protease (IC50 = 12.9 μg/ml), but not reverse transcriptase or integrase. Further, in Alu-PCR no effect on viral integration was observed. The n-butanol fraction interfered with the Tat-mediated Long Terminal Repeat transactivation in TZM-bl cells, mRNA quantitation (qRT-PCR) and electrophoretic mobility shift assay (EMSA). The n-butanol fraction did not cause an enhanced secretion of pro-inflammatory cytokines in Vk2/E6E7 cells. Additionally, no adverse effects were observed to the monolayer formed by the Caco-2 and HEC-1A epithelial cells.
Conclusions
The results presented here show a potential anti-HIV-1 activity of A. catechu mediated by the inhibition of the functions of the viral protein and Tat.
doi:10.1186/1743-422X-10-309
PMCID: PMC3819669  PMID: 25228267
11.  Reformulated tenofovir gel for use as a dual compartment microbicide 
Objectives
Coital use of 1% tenofovir gel was shown to be modestly effective at preventing HIV transmission when applied vaginally in the CAPRISA 004 trial. Because the gel is hyperosmolar, which would reduce the integrity of the epithelium and induce fluid movement into the lumen, rectal use may not be acceptable. This study evaluated the pre-clinical safety and efficacy of a reformulated (reduced osmolality) tenofovir gel product.
Methods
Reduced glycerine (RG)-tenofovir gel was compared with the original tenofovir gel for physiochemical characteristics, product safety and anti-HIV-1 activity.
Results
The formulations were similar in all characteristics except for osmolality and spreadability/firmness. The RG-tenofovir gel had a 73% lower osmolality, a 29.6% increase in spreadability and a 27% decrease in firmness as compared with the original tenofovir gel. When applied to epithelial cell monolayers, tenofovir gel showed a transient reduction in the transepithelial resistance while the RG-tenofovir gel did not. Both gels retained ectocervical and colorectal explant viability. However, tenofovir gel treatment resulted in epithelial stripping that was absent after RG-tenofovir gel treatment of the polarized explants. Anti-HIV-1 activity was confirmed by lack of HIV-1 infection in polarized explants treated with either gel as compared with the control explants.
Conclusions
Reducing the osmolality of the tenofovir gel resulted in improved epithelial integrity, which suggests better safety upon rectal use. The improved gel safety did not compromise drug release or anti-HIV-1 activity. These data support the use of this gel as a dual compartment microbicide.
doi:10.1093/jac/dks173
PMCID: PMC3417689  PMID: 22581908
HIV prevention; rectal microbicide; formulation; preclinical testing; safety
12.  Characterization of UC781-Tenofovir Combination Gel Products for HIV-1 Infection Prevention in an Ex Vivo Ectocervical Model 
HIV continues to be a problem worldwide. Topical vaginal microbicides represent one option being evaluated to stop the spread of HIV. With drug candidates that have a specific action against HIV now being studied, it is important that, when appropriate and based on the mechanism of action, the drug permeates the tissue so that it can be delivered to specific targets which reside there. Novel formulations of the nucleotide reverse transcriptase inhibitor tenofovir (TFV) and the nonnucleoside reverse transcriptase inhibitor UC781 have been developed and evaluated here. Gels with three distinct rheological properties were prepared. The three gels released both UC781 and TFV under in vitro conditions at concentrations equal to or above the reported 50% effective concentrations (EC50s). The drug concentrations in ectocervical tissues were well in excess of the reported EC50s. The gels maintain ectocervical viability and prevent infection of ectocervical explants after a HIV-1 challenge. This study successfully demonstrates the feasibility of using this novel combination of antiretroviral agents in an aqueous gel as an HIV infection preventative.
doi:10.1128/AAC.06284-11
PMCID: PMC3370776  PMID: 22430977
13.  Sublimable C5A Delivery Provides Sustained and Prolonged Anti-HIV Microbicidal Activities 
We have identified a short amphipathic helical peptide, called C5A, which exhibits potent microbicidal activities in vitro and which offers protection from vaginal HIV transmission in vivo in a humanized mouse model. However, there are many obstacles to overcome before C5A can be considered a true microbicidal candidate. First, it must be stabilized against enzymatic degradation in a continuously warm and moist environment. Second, it must be delivered in a controlled manner to achieve long-term and coitally independent efficacy. We demonstrate in this in vitro study that the combination of two matrices with different subliming properties ((hexamethylcyclotrisiloxane [HMCS] and cyclododecane [CDD]) containing 10% labile C5A yielded the best results in terms of controlled release and preserved anti-HIV activity of the peptide when pre-exposed to cell-free medium or cell culture at body temperature for up to 2 months.
doi:10.1128/AAC.00186-12
PMCID: PMC3370808  PMID: 22430971
14.  Is Wetter Better? An Evaluation of Over-the-Counter Personal Lubricants for Safety and Anti-HIV-1 Activity 
PLoS ONE  2012;7(11):e48328.
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.
doi:10.1371/journal.pone.0048328
PMCID: PMC3492332  PMID: 23144863
15.  Sustained Local Delivery of Structurally Diverse HIV-1 Microbicides Released from Sublimation Enthalpy Controlled Matrices 
Pharmaceutical Research  2012;29(11):3156-3168.
ABSTRACT
Purpose
Use of coital-dependent products to prevent HIV-1 transmission has resulted in mixed success. We hypothesize that incorporation of antiviral drug candidates into a novel controlled delivery system will prolong their activity, making their use coital independent, thus increasing their chance of prophylactic success.
Methods
Tenofovir, emtricitabine, and C5A peptide HIV microbicides were mechanically incorporated into matrices comprising a series of subliming solids. Matrix sublimation rates and drug release rates were measured in three in vitro and one in vivo environments intended to model human vaginal interior. Antiviral activity studies evaluating matrix incorporated microbicides were performed using in vitro cell cultures and human ectocervical explants.
Results
Drug release rates were identical to matrix sublimation rates, and were zero order. Differences in matrix material sublimation enthalpies determined drug release and matrix erosion rates in a thermodynamically definable manner, in vitro and in vivo. Durations of release ranging from several days to several months were readily achieved. Prolonged duration of anti HIV-1 activity was shown for matrix incorporated microbicides, using ectocervical explant and cell culture models of HIV-1 infection.
Conclusion
Subliming solid matrices show promise as a delivery system providing multi month intravaginal release of a wide range of HIV-1 microbicides.
doi:10.1007/s11095-012-0811-8
PMCID: PMC3473190  PMID: 22736232
enthalpically controlled release; HIV-1; intravaginal delivery; prolonged antiviral effect; subliming solid matrix
17.  Vaginal Microbicide Gel for Delivery of IQP-0528, a Pyrimidinedione Analog with a Dual Mechanism of Action against HIV-1▿ †  
Pyrimidinediones, a novel class of compounds, have previously been shown to possess antiviral activity at nanomolar concentrations. One member of this class of compounds, IQP-0528, was selected as the lead molecule for formulation development owing to its stability at physiologically relevant conditions, wide therapeutic window, and antiviral activity in the nanomolar range. Here, we report the development of two vaginal gels—3.0% hydroxyethyl cellulose (HEC) formulation and a 0.65% Carbopol formulation—for the sustained delivery of IQP-0528. Stability studies under accelerated conditions confirmed the chemical stability of IQP-0528 and mechanical stability of the gel formulation for 3 months. In vitro release studies revealed that diffusion-controlled release of IQP-0528 occurred over 6 h, with an initial lag time of approximately 1 h. Based on the drug release profile, the 3.0% HEC gel was selected as the lead formulation for safety and activity evaluations. The in vitro and ex vivo safety evaluations showed no significant loss in cell viability or significant inflammatory response after treatment with a 3.0% HEC gel containing 0.25% IQP-0528. In an in vitro HIV-1 entry inhibition assay, the lead formulation showed an 50% effective concentration of 0.14 μg/ml for gel in culture media, which corresponds to ∼0.001 μM IQP-0528. The antiviral activity was further confirmed by using polarized cervical explants, in which the formulation showed complete protection against HIV infection. In summary, these results are encouraging and warrant further evaluation of IQP-0528 gel formulations in in vivo models, as well as the development of alternative formulations for the delivery of IQP-0528 as a microbicide.
doi:10.1128/AAC.01368-10
PMCID: PMC3067148  PMID: 21245437
18.  Performance of Swabs, Lavage, and Diluents to Quantify Biomarkers of Female Genital Tract Soluble Mucosal Mediators 
PLoS ONE  2011;6(8):e23136.
Background
Measurement of immune mediators and antimicrobial activity in female genital tract secretions may provide biomarkers predictive of risk for HIV-1 acquisition and surrogate markers of microbicide safety. However, optimal methods for sample collection do not exist. This study compared collection methods.
Methods
Secretions were collected from 48 women (24 with bacterial vaginosis [BV]) using vaginal and endocervical Dacron and flocked swabs. Cervicovaginal lavage (CVL) was collected with 10 mL of Normosol-R (n = 20), saline (n = 14), or water (n = 14). The concentration of gluconate in Normosol-R CVL was determined to estimate the dilution factor. Cytokine and antimicrobial mediators were measured by Luminex or ELISA and corrected for protein content. Endogenous anti-HIV-1 and anti-E. coli activity were measured by TZM-bl assay or E. coli growth.
Results
Higher concentrations of protein were recovered by CVL, despite a 10-fold dilution of secretions, as compared to swab eluents. After protein correction, endocervical swabs recovered the highest mediator levels regardless of BV status. Endocervical and vaginal flocked swabs recovered significantly higher levels of anti-HIV-1 and anti-E. coli activity than Dacron swabs (P<0.001). BV had a significant effect on CVL mediator recovery. Normosol-R tended to recover higher levels of most mediators among women with BV, whereas saline or water tended to recover higher levels among women without BV. Saline recovered the highest levels of anti-HIV-1 activity regardless of BV status.
Conclusions
Endocervical swabs and CVL collected with saline provide the best recovery of most mediators and would be the optimal sampling method(s) for clinical trials.
doi:10.1371/journal.pone.0023136
PMCID: PMC3155537  PMID: 21858008
19.  Rectal microbicides: clinically relevant approach to the design of rectal specific placebo formulations 
Background
The objective of this study is to identify the critical formulation parameters controlling distribution and function for the rectal administration of microbicides in humans. Four placebo formulations were designed with a wide range of hydrophilic characteristics (aqueous to lipid) and rheological properties (Newtonian, shear thinning, thermal sensitive and thixotropic). Aqueous formulations using typical polymers to control viscosity were iso-osmotic and buffered to pH 7. Lipid formulations were developed from lipid solvent/lipid gelling agent binary mixtures. Testing included pharmaceutical function and stability as well as in vitro and in vivo toxicity.
Results
The aqueous fluid placebo, based on poloxamer, was fluid at room temperature, thickened and became shear thinning at 37°C. The aqueous gel placebo used carbopol as the gelling agent, was shear thinning at room temperature and showed a typical decrease in viscosity with an increase in temperature. The lipid fluid placebo, myristyl myristate in isopropyl myristate, was relatively thin and temperature independent. The lipid gel placebo, glyceryl stearate and PEG-75 stearate in caprylic/capric triglycerides, was also shear thinning at both room temperature and 37°C but with significant time dependency or thixotropy. All formulations showed no rectal irritation in rabbits and were non-toxic using an ex vivo rectal explant model.
Conclusions
Four placebo formulations ranging from fluid to gel in aqueous and lipid formats with a range of rheological properties were developed, tested, scaled-up, manufactured under cGMP conditions and enrolled in a formal stability program. Clinical testing of these formulations as placebos will serve as the basis for further microbicide formulation development with drug-containing products.
doi:10.1186/1742-6405-8-12
PMCID: PMC3061893  PMID: 21385339
20.  Multisite Comparison of Anti-Human Immunodeficiency Virus Microbicide Activity in Explant Assays Using a Novel Endpoint Analysis ▿ †  
Journal of Clinical Microbiology  2009;47(11):3530-3539.
Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development.
doi:10.1128/JCM.00673-09
PMCID: PMC2772583  PMID: 19726602
21.  In Vitro and Ex Vivo Testing of Tenofovir Shows It Is Effective As an HIV-1 Microbicide 
PLoS ONE  2010;5(2):e9310.
Background
Tenofovir gel has entered into clinical trials for use as a topical microbicide to prevent HIV-1 infection but has no published data regarding pre-clinical testing using in vitro and ex vivo models. To validate our findings with on-going clinical trial results, we evaluated topical tenofovir gel for safety and efficacy. We also modeled systemic application of tenofovir for efficacy.
Methods and Findings
Formulation assessment of tenofovir gel included osmolality, viscosity, in vitro release, and permeability testing. Safety was evaluated by measuring the effect on the viability of vaginal flora, PBMCs, epithelial cells, and ectocervical and colorectal explant tissues. For efficacy testing, PBMCs were cultured with tenofovir or vehicle control gels and HIV-1 representing subtypes A, B, and C. Additionally, polarized ectocervical and colorectal explant cultures were treated apically with either gel. Tenofovir was added basolaterally to simulate systemic application. All tissues were challenged with HIV-1 applied apically. Infection was assessed by measuring p24 by ELISA on collected supernatants and immunohistochemistry for ectocervical explants. Formulation testing showed the tenofovir and vehicle control gels were >10 times isosmolar. Permeability through ectocervical tissue was variable but in all cases the receptor compartment drug concentration reached levels that inhibit HIV-1 infection in vitro. The gels were non-toxic toward vaginal flora, PBMCs, or epithelial cells. A transient reduction in epithelial monolayer integrity and epithelial fracture for ectocervical and colorectal explants was noted and likely due to the hyperosmolar nature of the formulation. Tenofovir gel prevented HIV-1 infection of PBMCs regardless of HIV-1 subtype. Topical and systemic tenofovir were effective at preventing HIV-1 infection of explant cultures.
Conclusions
These studies provide a mechanism for pre-clinical prediction of safety and efficacy of formulated microbicides. Tenofovir was effective against HIV-1 infection in our algorithm. These data support the use of tenofovir for pre-exposure prophylaxis.
doi:10.1371/journal.pone.0009310
PMCID: PMC2824823  PMID: 20174579
22.  Cervical Shedding of Human T Cell Lymphotropic Virus Type I Is Associated with Cervicitis 
The Journal of infectious diseases  2002;186(11):1669-1672.
Human T cell lymphotropic virus type I (HTLV-I) is sexually transmitted. The purpose of this study was to determine the prevalence and risk factors for cervical shedding of HTLV-I DNA among Peruvian sex workers. HTLV tax DNA was detected in cervical specimens from 43 (68%) of 63 HTLV-I–infected sex workers and in samples obtained during 113 (52%) of 216 clinic visits between 1993 and 1997. Detection of HTLV DNA was associated with the presence of ≥30 polymorphonuclear cells (PMNs) within cervical mucus per 100×microscopic field (odds ratio [OR], 4.3, 95% confidence interval [CI], 1.8–10.1) and with the presence of cervical secretions (OR, 2.0; 95% CI 1.2–3.4). Hormonal contraceptive use (OR 1.7; 95% CI, 0.8–3.6) and concomitant cervical infection by Chlamydia trachomatis (OR, 1.5; 95% CI, 0.3–4.3) or Neisseria gonorrhoeae (OR, 1.1; 95% CI, 0.6–3.7) were not significantly associated with HTLV-I shedding. Our results suggest that cervicitis may increase cervical HTLV-I shedding and the sexual transmission of this virus.
doi:10.1086/345364
PMCID: PMC2675941  PMID: 12447745
23.  Biological and Technical Variables Affecting Immunoassay Recovery of Cytokines from Human Serum and Simulated Vaginal Fluid: A Multicenter Study 
Analytical Chemistry  2008;80(12):4741-4751.
The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1β and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1β and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal−Wallis analysis of variance with Dunn’s multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1β determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.
doi:10.1021/ac702628q
PMCID: PMC2646866  PMID: 18484740
24.  Can a Topical Microbicide Prevent Rectal HIV Transmission? 
PLoS Medicine  2008;5(8):e167.
Florian Hladik and Charlene Dezzutti discuss a macaque study of whether rectal simian immunodeficiency virus transmission can be prevented by topical pre-exposure application of tenofovir gel.
doi:10.1371/journal.pmed.0050167
PMCID: PMC2494564  PMID: 18684009
25.  Effect of Topical Microbicides on Infectious Human Immunodeficiency Virus Type 1 Binding to Epithelial Cells▿  
Topical microbicides (cellulose acetate 1,2 benzene dicarboxylate [CAP], PRO 2000, SPL7013, and UC781) are being investigated to reduce the sexual transmission of human immunodeficiency virus type 1 (HIV-1). These products were shown to prevent the transfer of infectious HIV-1 from urogenital and colorectal epithelial cell lines to peripheral blood mononuclear cells. However, it was unclear if the topical microbicides rendered the virus noninfectious and/or reduced the binding to the epithelial cells. To test this, epithelial cells were cultured with HIV-1 in the presence or absence of topical microbicides or their placebos. The cells were washed, RNA lysates were made, and real-time PCR was performed for HIV-1. PRO 2000 and SPL7013 significantly (P < 0.0001) reduced the amount of bound HIV-1 to the colorectal epithelial cell line across clades A, B, C, and CRF01-AE. While none of the products reduced the binding of HIV-1 clades A and C to the urogenital cell line, CAP, PRO 2000, and SPL7013 significantly (P ≤ 0.002) reduced the binding of clades B and CRF01-AE. In general, PRO 2000 and SPL7013 placebos significantly (P < 0.0001) reduced the amount of bound HIV-1 but were less than the active products. UC781, its placebo, and hydroxyethyl cellulose (placebo for CAP) minimally affected the amount of bound HIV-1. These results suggest that rendering HIV-1 noninfectious may not correlate to the amount of HIV-1 bound to epithelial cells and possible shedding into mucosal secretions. Therefore, functional virological assays in addition to measuring viral RNA should be included when clinically evaluating topical microbicide use by infected persons.
doi:10.1128/AAC.01358-06
PMCID: PMC1891390  PMID: 17404008

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