Investigation of 5'-Norcarbocyclic Nucleoside Analogues as Antiprotozoal and Antibacterial Agents.

Carbocyclic nucleosides have long played a role in antiviral, antiparasitic, and antibacterial therapies. Recent results from our laboratories from two structurally related scaffolds have shown promising activity against both Mycobacterium tuberculosis and several parasitic strains. As a result, a small structure activity relationship study was designed to further probe their activity and potential. Their synthesis and the results of the subsequent biological activity are reported herein.

Novel 5'-Norcarbocyclic Pyrimidine Derivatives as Antibacterial Agents.

A series of novel 5'-norcarbocyclic derivatives of 5-alkoxymethyl or 5-alkyltriazolyl-methyl uracil were synthesized and the activity of the compounds evaluated against both Gram-positive and Gram-negative bacteria. The growth of Mycobacterium smegmatis was completely inhibited by the most active compounds at a MIC99 of 67 µg/mL (mc²155) and a MIC99 of 6.7⁻67 µg/mL (VKPM Ac 1339). Several compounds also showed the ability to inhibit the growth of attenuated strains of Mycobacterium tuberculosis ATCC 25177 (MIC99 28⁻61 µg/mL) and Mycobacterium bovis ATCC 35737 (MIC99 50⁻60 µg/mL), as well as two virulent strains of M. tuberculosis; a laboratory strain H37Rv (MIC99 20⁻50 µg/mL) and a clinical strain with multiple drug resistance MS-115 (MIC99 20⁻50 µg/mL). Transmission electron microscopy (TEM) evaluation of M. tuberculosis H37Rv bacterial cells treated with one of the compounds demonstrated destruction of the bacterial cell wall, suggesting that the mechanism of action for these compounds may be related to their interactions with bacteria cell walls.

Pharmacokinetic and Pharmacodynamic Evaluation following Vaginal Application of IQB3002, a Dual-Chamber Microbicide Gel Containing the Nonnucleoside Reverse Transcriptase Inhibitor IQP-0528 in Rhesus Macaques.

We evaluated the in vivo pharmacokinetics and used a complementary ex vivo coculture assay to determine the pharmacodynamics of IQB3002 gel containing 1% IQP-0528, a nonnucleoside reverse transcriptase inhibitor (NNRTI), in rhesus macaques (RM). The gel (1.5 ml) was applied vaginally to 6 simian-human immunodeficiency (SHIV)-positive female RM. Blood, vaginal fluids, and rectal fluids were collected at 0, 1, 2, and 4 h. RM were euthanized at 4 h, and vaginal, cervical, rectal, and regional lymph node tissues were harvested. Anti-human immunodeficiency virus (HIV) activity was evaluated ex vivo by coculturing fresh or frozen vaginal tissues with activated human peripheral blood mononuclear cells (PBMCs) and measuring the p24 levels for 10 days after an HIV-1Ba-L challenge. The median levels of IQP-0528, determined using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) methods, were between 10(4) and 10(5) ng/g in vaginal and cervical tissue, between 10(3) and 10(4) ng/g in rectal tissues, and between 10(5) and 10(7) ng/ml in vaginal fluids over the 4-h period. The vaginal tissues protected the cocultured PBMCs from HIV-1 infection ex vivo, with a viral inhibition range of 81 to 100% in fresh and frozen tissues that were proximal, medial, and distal relative to the cervix. No viral inhibition was detected in untreated baseline tissues. Collectively, the median drug levels observed were 5 to 7 logs higher than the in vitro 50% effective concentration (EC50) range (0.21 ng/ml to 1.29 ng/ml), suggesting that 1.5 ml of the gel delivers IQP-0528 throughout the RM vaginal compartment at levels that are highly inhibitory to HIV-1. Importantly, antiviral activity was observed in both fresh and frozen vaginal tissues, broadening the scope of the ex vivo coculture model for future NNRTI efficacy studies.

Engineering soya bean seeds as a scalable platform to produce cyanovirin-N, a non-ARV microbicide against HIV.

There is an urgent need to provide effective anti-HIV microbicides to resource-poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant cyanovirin-N (rCV-N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV-N was isolated with a yield of 350 µg/g of dry seed weight. The observed amino acid sequence of rCV-N matched the expected sequence of native CV-N, as did the mass of rCV-N (11 009 Da). Purified rCV-N from soya is active in anti-HIV assays with an EC50 of 0.82-2.7 nM (compared to 0.45-1.8 nM for E. coli-produced CV-N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV-N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti-HIV microbicide development.

Synthesis and Biological Evaluation of 5'-O-Dicarboxylic Fatty Acyl Monoester Derivatives of Anti-HIV Nucleoside Reverse Transcriptase Inhibitors.

A number of 5'-O-dicarboxylic fatty acyl monoester derivatives of 3'-azido-3'-deoxythymidine (zidovudine, AZT), 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T), and 3'-fluoro-3'-deoxythymidine (alovudine, FLT) were synthesized to improve the lipophilicity and potentially the cellular delivery of parent polar 2', 3'-dideoxynucleoside (ddN) analogues. The compounds were evaluated for their anti-HIV activity. Three different fatty acids with varying chain length of suberic acid (octanedioic acid), sebacic acid (decanedioic acid), and dodecanedioic acid were used for the conjugation with the nucleosides. The compounds were evaluated for anti-HIV activity and cytotoxicity. All dicarboxylic ester conjugates of nucleosides exhibited significantly higher anti-HIV activity than that of the corresponding parent nucleoside analogs. Among all the tested conjugates, 5'-O-suberate derivative of AZT (EC50 = 0.10 nM) was found to be the most potent compound and showed 80-fold higher anti-HIV activity than AZT without any significant toxicity (TC50 > 500 nM).

Novel preclinical models of topical PrEP pharmacodynamics provide rationale for combination of drugs with complementary properties.

BACKGROUND: The limited success of recent HIV topical pre-exposure prophylaxis clinical trials highlights the need for more predictive models of drug efficacy that better simulate what may happen during sexual exposure. To address this gap, we developed complementary in vitro models to evaluate the ability of drugs to retain anti-HIV activity if cells were washed with seminal plasma (simulating what may happen following exposure to ejaculate), and to protect drug-naive T cells (representing newly recruited immune cells) co-cultured with explants that had been pretreated with drug. We focused on tenofovir disoproxil fumarate (TDF), the non-nucleoside reverse transcriptase inhibitors dapivirine (DPV) and IQP-0528, and the entry inhibitors maraviroc (MVC) and the D-peptide chol-PIE-12 trimer (PIE12). Studies were extended to macaques and the ability of cervical biopsies obtained from animals treated with an intravaginal ring formulation of IQP-0528 to protect ex vivo co-cultured T cells was determined. The antiviral activity of cervicovaginal lavage samples against a primary Clade C isolate was also measured and correlated with drug levels. RESULTS: Cells exposed to TDF were equally protected from HIV whether or not the drug-treated cells were washed with medium or seminal plasma prior to challenge. In contrast, several-fold higher concentrations of NNRTIs and entry inhibitors were needed to attain similar levels of HIV inhibition following a wash with seminal plasma. Conversely, the NNRTIs and PIE12, but not TDF or MVC, were effectively transferred from ex vivo treated explants and protected co-cultured T cells. Biopsies obtained from IQP-0528 ring-treated macaques also protected co-cultured T cells with viral inhibition ranging from 42-72%. Antiviral activity correlated with the concentration of drug recovered. Combinations of TDF with IQP-0528 protected in both in vitro models. CONCLUSIONS: Together, these models suggest that intracellularly retained drugs such as TDF may protect resident immune cells following coitus but sustained delivery may be required to protect immune cells subsequently recruited into the genital tract. Sustained delivery may also be critical for NNRTIs, which are rapidly transported out of cells and could be lost following sexual intercourse. An ideal approach may be a combination of drugs with complementary bioavailability profiles formulated for sustained delivery.

Synthesis and anti-HIV activities of symmetrical dicarboxylate esters of dinucleoside reverse transcriptase inhibitors.

Three nucleoside analogues, 3'-fluoro-2',3'-dideoxythymidine (FLT), 3'-azido-2',3'-dideoxythymidine (AZT), and 2',3'-dideoxy-3'-thiacytidine (3TC) were conjugated with three different dicarboxylic acids to afford the long chain dicarboxylate esters of nucleosides. In general, dinucleoside ester conjugates of FLT and 3TC with long chain dicarboxylic acids exhibited higher anti-HIV activity than their parent nucleosides. Dodecanoate and tetradecanoate dinucleoside ester derivatives of FLT were found to be the most potent compounds with EC(50) values of 0.8-1.0 nM and 3-4 nM against HIV-1(US/92/727) and HIV-1(IIIB) cells, respectively. The anti-HIV activity of the 3TC conjugates containing long chain dicarboxylate diester (EC(50)=3-60 nM) was improved by 1.5-66 fold when compared to 3TC (EC(50)=90-200 nM). This study reveals that the symmetrical ester conjugation of dicarboxylic acids with a number of nucleosides results in conjugates with improved anti-HIV profile.

Designing Dual Compartment HIV Prevention Products: Women's Sensory Perceptions and Experiences of Suppositories for Rectal and Vaginal Use.

Dual compartment suppositories are being developed to prevent HIV and other sexually transmitted infections. Such products, for use in the rectum, the vagina, or both, could have a significant public health impact by decreasing global incidence of these diseases. In this study, 16 women each used two rheologically distinct suppositories in their vagina and rectum. User Sensory Perception and Experience (USPE) scales assessed sensory experiences during sexual activity to understand whether, and how, women perceive formulation properties in the vagina and rectum. Qualitative data from individual in-depth interviews captured women's descriptions and comparisons of the experiences. Significant differences and large Cohen's d effect sizes between vaginal and rectal experiences of suppository-A were found for three scales: Application (APP): Product Awareness, SEX: Initial Penetration; and SEX: Effortful. Qualitative data provided user experience details that credibly align with these score differences. Near significant differences and large effect sizes were found for two additional scales: SEX: Perceived Wetness with suppository-A and SEX: Messiness with suppository-B. In addition, other scale scores showed medium-to-large effect sizes that correspond to hypothesized sensations associated with biophysical properties of the suppositories. Statistical significance combined with large effect sizes and qualitative data accurately represent the hypothesized perceptibility of suppository properties and identifies performance characteristics relevant to acceptability and adherence; together these data provide discernment of factors that can guide the development of dual compartment products. The Clinical Trial Registration number: NCT02744261.

Examining the Safety, Pharmacokinetics, and Pharmacodynamics of a Rectally Administered IQP-0528 Gel for HIV Pre-Exposure Prophylaxis: A First-In-Human Study.

A lubricating microbicide gel designed for rectal and vaginal use would provide a behaviorally congruent strategy to enhance pre-exposure prophylaxis adherence and reduce HIV infection risk. In this study, we report the first-in-human evaluation of such a gel containing 1% IQP-0528, an investigational antiretroviral. Seven HIV-1-negative participants received one 10 mL rectal dose of radiolabeled 1% IQP-0528 gel. We assessed safety; IQP-0528 pharmacokinetics in plasma, and rectal and vaginal tissue; ex vivo local pharmacodynamics (PD); and colorectal distribution. The 1% gel was determined to be safe with one mild event attributed to study product and no effects on rectal tissue histology. All concentrations measured in plasma and vaginal tissue were below the limit of quantitation. Median IQP-0528 concentrations in rectal tissue exceeded the in vitro EC95 against HIV-1 (0.07 ng/mg) by 3-5 h of dosing and remained above this concentration for at least 24 h, despite a 3-log reduction in concentration over this duration of time. Rectal tissue PD-assessed by ex vivo HIV challenge-demonstrated significant p24 antigen reduction 3-5 h postdose compared with baseline (p = .05), but not 24-26 h postdose (p = .75). Single-photon emission computed tomography/computed tomography imaging revealed that product distribution was localized to the rectosigmoid. The IQP-0528 gel possesses desirable features for a topical microbicide including: local safety with no systemic absorption, delivery of locally high IQP-0528 concentrations, and significant reductions in ex vivo HIV infectivity. However, the gel is limited by its rapid clearance and inability to penetrate vaginal tissues following rectal dosing. Clinical Trial Registration number: NCT03082690.

The Role of Volume in the Perceptibility of Topical Vaginal Formulations: User Sensory Perceptions and Experiences of Heterosexual Couples During Vaginal Sex.

Users' sensory perceptions and experiences (USPEs; perceptibility) of drug formulations can critically impact product adoption and adherence, especially when products rely on appropriate user behaviors (timing of administration, dosing measurement) for effectiveness. The use of topical gel formulations for effective antihuman immunodeficiency virus/sexually transmitted infection (HIV/STI) vaginal microbicides has been associated with messiness and other use-associated challenges, resulting in low adherence. Nonetheless, such formulations remain attractive due to good pharmacokinetics and resulting pharmacodynamics through their volume and surface contact for drug delivery into luminal fluids and mucosa. Consequently, advocates and scientists continue to pursue topical forms [semisolid (e.g., gel, suppository); solid (e.g., film)] to deliver select drugs and offer user choice in HIV/STI prevention. The current data build on previously validated USPE scales evaluating perceptibility of gels with various biophysical/rheological properties. Specifically, increased formulation parameter space adds a new set of properties inherent in quick-dissolving film. We compared film, a product adding no discernable volume to the vaginal environment, to 2 and 3.5 mL hydroxyethyl cellulose gel to consider the impact of volume on user experience. We also examined the USPE scales for evaluation of male sexual partners' experiences. The original USPE scales functioned as expected. Additionally, six new USPE scales were identified in this enhanced parameter space. Significant differences were noted between USPEs in pairwise comparisons, with largest differences between film and high-volume gel. Product developers and behavioral scientists can use these scales to design products, optimizing user experience and maximizing adherence and delivery of efficacious anti-HIV/STI pharmaceuticals. They can be extended to evaluation of additional formulations, devices, and compartments, as well as single- and multipurpose pharmaceuticals. In broader contexts, USPEs could be of value in evaluating formulations and devices to prevent/treat other diseases (e.g., ophthalmologic, dermatologic). Steadfast attention should be given to patient experience, and, where applicable, experiences of partners and/or caregivers.

Effects of gel volume on pharmacokinetics for vaginal and rectal applications of combination DuoGel-IQB4012, a dual chamber-dual drug HIV microbicide gel, in pigtailed macaques.

This study evaluated effects of differing gel volumes on pharmacokinetics (PK). IQB4012, a gel containing the non-nucleoside reverse transcriptase inhibitor IQP-0528 and tenofovir (TFV), was applied to the pigtailed macaque vagina and rectum. Vaginal gel volumes (1% loading of both drugs) were 0.5 or 1.5 ml; following wash-out, 1 or 4 ml of gel were then applied rectally. Blood, vaginal, and rectal fluids were collected at 0, 2, 4, and 24 h. Vaginal and rectal tissue biopsies were collected at 4 and 24 h. There were no statistically significant differences in concentrations for either drug between gel volumes within compartments at matched time points. After vaginal gel application, median IQP-0528 concentrations were ~ 104-105 ng/g, 105-106 ng/ml, and 103-105 ng/ml in vaginal tissues, vaginal fluids, and rectal fluids, respectively (over 24 h). Median vaginal TFV concentrations were 1-2 logs lower than IQP-0528 levels at matched time points. After rectal gel application, median IQP-0528 and TFV concentrations in rectal fluids were ~ 103-105 ng/ml and ~ 102-103 ng/ml, respectively. Concentrations of both drugs sampled in rectal tissues were low (~ 101-103 ng/g). For 1 ml gel, half of sampled rectal tissues had undetectable concentrations of either drug, and over half of sampled rectal fluids had undetectable TFV concentrations. These results indicate differences in drug delivery between the vaginal and rectal compartments, and that smaller vaginal gel volumes may not significantly compromise microbicide PK and prophylactic potential. However, effects of rectal gel volume on PK for both drugs were less definitive.

Lubricant Provides Poor Rectal Mucosal HIV Coverage.

Given the rising HIV incidence in men who have sex with men (MSM) despite repeatedly proven effectiveness of oral HIV pre-exposure prophylaxis, behaviorally congruent periodic dosing strategies, such as dosing microbicides as lubricants, are now in demand. Rectal microbicide gel studies largely administer gels using vaginal applicators, which have not been well received and do not mimic lubricant use. We compared rectal gel manually dosed as lubricant with applicator dosing in five healthy, HIV-negative MSM who received 10 or 3.5 ml of 99mTc-DTPA-radiolabeled hydroxyethyl cellulose universal placebo gel intrarectally. After washout, participants received 10 ml of radiolabeled Wet® Original® lubricant to apply to the anus with fingers and/or a phallus in a manner typical of sexual lubricant use with a partner, followed by simulated receptive anal intercourse. Single-photon emission computed tomography with transmission computed tomography was performed 4 h after each gel administration. Manual dosing was associated with more variable rectosigmoid distribution, 4.4-15.3 cm from the anorectal junction, compared with more uniform distribution, 5.9-7.4 and 5.3-7.6 cm after 10 and 3.5 ml applicator dosing, respectively. A significantly smaller fraction of the initial 10 ml dose was retained within the colon after manual dosing, 3.4%, compared with 94.9% and 88.4% after 10 and 3.5 ml applicator dosing, respectively (both p < .001). Manual dosing of a sexual lubricant delivered a small, variable fraction of the dose with variable rectosigmoid distribution compared with applicator dosing. These results raise concern that dosing a rectal microbicide gel as a sexual lubricant may not provide adequate or predictable mucosal coverage for HIV protection.

Current Concepts for the IND-Directed Development of Microbicide Products to Prevent the Sexual Transmission of HIV.

In the absence of an approved and effective vaccine, topical microbicides have become the strategy of choice to provide women with the ability to prevent the sexual transmission of HIV. Topical microbicides are chemical and physical agents specifically developed and formulated for use in either the vaginal or rectal environment to prevent the sexual transmission of infectious organisms. Although a microbicide product will have many of the same properties as other anti-infective therapeutic agents, the microbicide development pathway has significant differences which reflect the complex biological environment in which the products must act. These challenges to the development of an effective microbicide are reflected in the recently released FDA Guidance document which defines the microbicide development algorithm and includes the evaluation of preclinical efficacy and toxicity, and safety and toxicology, and indicates the necessity of testing of the active pharmaceutical product as well as an optimal formulation for delivery of the microbicide product. The microbicide development algorithm requires evaluation of the potential microbicidal agent and final formulated product in assays which mimic the microenvironment of the vagina and rectum during the sexual transmission of HIV, including the evaluation of activity and cytotoxicity in the appropriate biological matrices, toxicity testing against normal vaginal flora and at standard vaginal pH, testing in ectocervical and colorectal explant tissue, and irritation studies to vaginal, rectal and penile tissue. Herein, we discuss currently accepted practices required for the development of a successful microbicide product which will prevent virus transmission in the vaginal and rectal vaults.

An Intravaginal Ring for the Simultaneous Delivery of an HIV-1 Maturation Inhibitor and Reverse-Transcriptase Inhibitor for Prophylaxis of HIV Transmission.

Nucleocapsid 7 (NCp7) inhibitors have been investigated extensively for their role in impeding the function of HIV-1 replication machinery and their ability to directly inactivate the virus. A class of NCp7 zinc finger inhibitors, S-acyl-2-mercaptobenzamide thioesters (SAMTs), was investigated for topical drug delivery. SAMTs are inherently unstable because of their hydrolytically labile thioester bond, thus requiring formulation approaches that can lend stability. We describe the delivery of N-[2-(3,4,5-trimethoxybenzoylthio)benzoyl]-ß-alaninamide (SAMT-10), as a single agent antiretroviral (ARV) therapeutic and in combination with the HIV-1 reverse-transcriptase inhibitor pyrimidinedione IQP-0528, from a hydrophobic polyether urethane (PEU) intravaginal ring (IVR) for a month. The physicochemical stability of the ARV-loaded IVRs was confirmed after 3 months at 40°C/75% relative humidity. In vitro, 25 ± 3 mg/IVR of SAMT-10 and 86 ± 13 mg/IVR of IQP-0528 were released. No degradation of the hydrolytically labile SAMT-10 was observed within the matrix. The combination of ARVs had synergistic antiviral activity when tested in in vitro cell-based assays. Toxicological evaluations performed on an organotypic EpiVaginal(™) tissue model demonstrated a lack of formulation toxicity. Overall, SAMT-10 and IQP-0528 were formulated in a stable PEU IVR for sustained release. Our findings support the need for further preclinical evaluation. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3426-3439, 2015.

The rational design and development of a dual chamber vaginal/rectal microbicide gel formulation for HIV prevention.

The DuoGel™ was developed for safe and effective dual chamber administration of antiretroviral drugs to reduce the high incidence of HIV transmission during receptive vaginal and anal intercourse. The DuoGel™s containing IQP-0528, a non-nucleoside reverse transcriptase inhibitor (NNRTI), were formulated from GRAS excipients approved for vaginal and rectal administration. The DuoGel™s were evaluated based upon quantitative physicochemical and biological evaluations defined by a Target Product Profile (TPP) acceptable for vaginal and rectal application. From the two primary TPP characteristics defined to accommodate safe rectal administration three DuoGel™ formulations (IQB3000, IQB3001, and IQB3002) were developed at pH 6.00 and osmolality ⩽400mmol/kg. The DuoGel™s displayed no in vitro cellular or bacterial toxicity and no loss in viability in ectocervical and colorectal tissue. IQB3000 was removed from consideration due to reduced NNRTI delivery (∼65% reduction) and IQB3001 was removed due to increase spread resulting in leakage. IQB3002 containing IQP-0528 was defined as our lead DuoGel™ formulation, possessing potent activity against HIV-1 (EC50=10nM). Over 12month stability evaluations, IQB3002 maintained formulation stability. This study has identified a lead DuoGel™ formulation that will safely deliver IQP-0528 to prevent sexual HIV-1 transmission in the vagina and rectum.

In vitro and ex vivo evaluations on transdermal delivery of the HIV inhibitor IQP-0410.

The aim of this study was to investigate the physicochemical and in vitro/ex vivo characteristics of the pyrmidinedione IQP-0410 formulated into transdermal films. IQP-0410 is a potent therapeutic anti-HIV nonnucleoside reverse transcriptase inhibitor that would be subjected to extensive first pass metabolism, through conventional oral administration. Therefore, IQP-0410 was formulated into ethyl cellulose/HPMC-based transdermal films via solvent casting. In mano evaluations were performed to evaluate gross physical characteristics. In vitro release studies were performed in both Franz cells and USP-4 dissolution vessels. Ex vivo release and permeability assays were performed on human epidermal tissue models, and the permeated IQP-0410 was collected for in vitro HIV-1 efficacy assays in CEM-SS cells and PBMCs. Film formulation D3 resulted in pliable, strong transdermal films that were loaded with 2% (w/w) IQP-0410. Composed of 60% (w/w) ethyl cellulose and 20% (w/w) HPMC, the films contained < 1.2% (w/w) of water and were hygroscopic resulting in significant swelling under humid conditions. The water permeable nature of the film resulted in complete in vitro dissolution and drug release in 26 hours. When applied to ex vivo epidermal tissues, the films were non-toxic to the tissue and also were non-toxic to HIV target cells used in the in vitro efficacy assays. Over a 3 day application, the films delivered IQP-0410 through the skin tissue at a zero-order rate of 0.94 ± 0.06 µg/cm(2)/hr with 134 ± 14.7 µM collected in the basal media. The delivered IQP-0410 resulted in in vitro EC50 values against HIV-1 of 2.56 ± 0.40 nM (CEM-SS) and 0.58 ± 0.03 nM (PBMC). The film formulation demonstrated no significant deviation from target values when packaged in foil pouches under standard and accelerated environmental conditions. It was concluded that the transdermal film formulation was a potentially viable method of administering IQP-0410 that warrants further development.

An algorithm for the preclinical development of anti-HIV topical microbicides.

Throughout the world, and especially in countries comprising the developing world, women are now bearing the brunt of the HIV pandemic, with over 50% living with HIV infection primarily contracted through sexual transmission in monogamous relationships. Thus, effective chemical or physical means of preventing HIV transmission are urgently needed and in the absence of an approved and effective vaccine, microbicides have become the strategy of choice to provide women with the ability to prevent HIV transmission from their infected partners. Topical microbicides include agents specifically developed and formulated for use in either the vaginal or rectal environment to prevent the sexual transmission of infectious organisms, including pathogenic viruses, bacteria and fungi. Although a microbicide product will have many of the same properties as other anti-infective therapeutic agents and would be similarly developed through a defined preclinical program leading to human clinical trials, microbicide development bears its own challenges related to appropriate and informative preclinical investigation, formulation and delivery, and the complex biological environment in which the product must act, as well as the requirement to develop a product that is acceptable to the user. Following years of microbicide development and a series of unsuccessful human clinical trials, a preclinical microbicide development algorithm has been continuously evolving as greater understanding of the required properties of a successful microbicide are defined through laboratory and clinical experience. Herein, we discuss currently accepted practices required for the development of a successful microbicide product which will prevent cell-free and cell-associated virus transmission in the vaginal and rectal vaults.

Factors Important to the Prioritization and Development of Successful Topical Microbicides for HIV-1.

Significant advancements in topical microbicide development have occurred since the prevention strategy was first described as a means to inhibit the sexual transmission of HIV-1. The lack of clinical efficacy of the first generation microbicide products has focused development attention on specific antiretroviral agents, and these agents have proven partially successful in human clinical trials. With greater understanding of vaginal and rectal virus infection, replication, and dissemination, better microbicide products and delivery strategies should result in products with enhanced potency. However, a variety of development gaps exist which relate to product dosing, formulation and delivery, and pharmacokinetics and pharmacodynamics which must be better understood in order to prioritize microbicide products for clinical development. In vitro, ex vivo, and in vivo models must be optimized with regard to these development gaps in order to put the right product at the right place, at the right time, and at the right concentration for effective inhibition of virus transmission. As the microbicide field continues to evolve, we must harness the knowledge gained from unsuccessful and successful clinical trials and development programs to continuously enhance our preclinical development algorithms.

Safety and efficacy of tenofovir/IQP-0528 combination gels - a dual compartment microbicide for HIV-1 prevention.

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor and IQP-0528 is a non-nucleoside 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 24h exposure. The ED(50) calculated from the formulations for IQP-0528 was ~32nM and for TFV was ~59nM and their inhibitory activity was not affected by semen. The ED(50) 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 ≥1log(10) and ≥2log(10), 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.

Lactobacillus proteins are associated with the bactericidal activity against E. coli of female genital tract secretions.

BACKGROUND: Female genital tract secretions are bactericidal for Escherichia (E.) coli ex vivo. However, the intersubject variability and molecules that contribute to this activity have not been defined. METHODS: The bactericidal activity and concentration of immune mediators in cervicovaginal lavage (CVL) collected from 99 healthy women were determined. RESULTS: CVL reduced the number of E. coli colonies by 68% [-26, 100] (median [range]). CVL were active against laboratory and clinical isolates of E. coli, but were inactive against Lactobacillus species. Bactericidal activity correlated with the concentration of protein recovered (p<0.001), but not with cytokines, chemokines or antimicrobial peptides. Four CVL with>90% inhibitory activity (active) and two with<30% activity were subjected to MS/MS proteomic analysis. 215 proteins were identified and six were found exclusively in active samples. Four of these corresponded to Lactobacillus crispatus or jensenii proteins. Moreover, culture supernatants from Lactobacillus jensenii were bactericidal for E. coli. CONCLUSION: Both host and commensal microbiota proteins contribute to mucosal defense. Identification of these proteins will facilitate the development of strategies to maintain a healthy vaginal microbiome and prevent colonization with pathogenic bacteria such as E. coli that increase the risk for urinary tract infections, preterm labor and perinatal infection.