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.

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.

The structure-activity profile of mercaptobenzamides' anti-HIV activity suggests that thermodynamics of metabolism is more important than binding affinity to the target.

Mercaptobenzamide thioesters and thioethers are chemically simple HIV-1 maturation inhibitors with a unique mechanism of action, low toxicity, and a high barrier to viral resistance. A structure-activity relationship (SAR) profile based on 39 mercaptobenzamide prodrug analogs exposed divergent activity/toxicity roles for the internal and terminal amides. To probe the relationship between antiviral activity and toxicity, we generated an improved computational model for the binding of mercaptobenzamide thioesters (SAMTs) to the HIV-1 NCp7 C-terminal zinc finger, revealing the presence of a second low-energy binding orientation, hitherto undisclosed. Finally, using NMR-derived thiol-thioester exchange equilibrium constants, we propose that thermodynamics plays a role in determining the antiviral activity observed in the SAR profile.

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.

Highly Attenuated Infection With a Vpr-Deleted Molecular Clone of Human Immunodeficiency Virus-1.

A 48-year-old woman was infected with a vpr-defective human immunodeficiency virus (HIV)-1 molecular clone. Seroconversion was markedly delayed, and without treatment she had durably suppressed viremia and normal T-cell levels. Neutralizing antibody and CD8+ T-cell immune responses against HIV-1 were unremarkable. Viral sequences confirmed the source but evolved defective nef, suggesting an unknown mechanistic link to vpr. There were subtle qualitative defects in T and B cells. To our knowledge, this is the only case of human infection with a characterized defective HIV-1 molecular clone, which furthermore recapitulated live-attenuated vaccination in macaque models of HIV-1 vaccine research.

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.

Designing and developing suppository formulations for anti-HIV drug delivery.

Despite a long history of use for rectal and vaginal drug delivery, the current worldwide market for suppositories is limited primarily due to a lack of user acceptability. Therefore, virtually no rational pharmaceutical development of antiviral suppositories has been performed. However, suppositories offer several advantages over other antiviral dosage forms. Current suppository designs have integrated active pharmaceutical ingredients into existing formulation designs without optimization. As such, emerging suppository development has been focused on improving upon the existing classical design to enhance drug delivery and is poised to open suppository drug delivery to a broader range of drugs, including antiretroviral products. Thus, with continuing research into rational suppository design and development, there is significant potential for antiretroviral suppository drug delivery.

Expanding the Antiviral Spectrum of 3-Fluoro-2-(phosphonomethoxy)propyl Acyclic Nucleoside Phosphonates: Diamyl Aspartate Amidate Prodrugs.

Acyclic nucleosides containing a 3-fluoro-2-(phosphonomethoxy)propyl (FPMP) side chain are known to be moderately potent antihuman immunodeficiency virus (HIV) agents, while being completely devoid of antiviral activity against a wide range of DNA viruses. The derivatization of the phosphonic acid functionality of FPMPs with a diamyl aspartate phenoxyamidate group led to a novel generation of compounds that not only demonstrate drastically improved antiretroviral potency but also are characterized by an expanded spectrum of activity that also covers hepatitis B and herpes viruses. The best compound, the (S)-FPMPA amidate prodrug, exerts anti-HIV-1 activity in TZM-bl and peripheral blood mononuclear cells at low nanomolar concentrations and displays excellent potency against hepatitis B virus (HBV) and varicella-zoster virus (VZV). This prodrug is stable in acid and human plasma media, but it is efficiently processed in human liver microsomes with a half-life of 2 min. The (R) isomeric guanine derivative emerged as a selectively active anti-HIV and anti-HBV inhibitor, while being nontoxic to human hepatoblastoma cells. Notably, the pyrimidine containing prodrug (S)-Asp-FPMPC is the only congener within this series to demonstrate micromolar antihuman cytomegalovirus (HCMV) potency.

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.

Probing Mercaptobenzamides as HIV Inactivators via Nucleocapsid Protein†7.

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein 7 (NCp7), a zinc finger protein, plays critical roles in viral replication and maturation and is an attractive target for drug development. However, the development of drug-like molecules that inhibit NCp7 has been a significant challenge. In this study, a series of novel 2-mercaptobenzamide prodrugs were investigated for anti-HIV activity in the context of NCp7 inactivation. The molecules were synthesized from the corresponding thiosalicylic acids, and they are all crystalline solids and stable at room temperature. Derivatives with a range of amide side chains and aromatic substituents were synthesized and screened for anti-HIV activity. Wide ranges of antiviral activity were observed, with IC50 values ranging from 1 to 100 µm depending on subtle changes to the substituents on the aromatic ring and side chain. Results from these structure-activity relationships were fit to a probable mode of intracellular activation and interaction with NCp7 to explain variations in antiviral activity. Our strategy to make a series of mercaptobenzamide prodrugs represents a general new direction to make libraries that can be screened for anti-HIV activity.

Synthesis and Antiviral Evaluation of Octadecyloxyethyl Benzyl 9-[(2-Phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG), a Potent Inhibitor of Transient HPV DNA Amplification.

Human papillomavirus (HPV) high-risk genotypes such as HPV-16 and HPV-18 cause the majority of anogenital tract carcinomas, including cervical cancer, the second most common malignancy in women worldwide. Currently there are no approved antiviral agents that reduce or eliminate HPV and reverse virus-associated pathology. We synthesized and evaluated several alkoxyalkyl acyclic nucleoside phosphonate diesters and identified octadecyloxyethyl benzyl 9-[(2-phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG) as an active compound which strongly inhibited transient amplification of HPV-11, -16, and -18 origin-containing plasmid DNA in transfected cells at concentrations well below its cytotoxic concentrations. ODE-Bn-PMEG demonstrated increased uptake in human foreskin fibroblast cells and was readily converted in vitro to the active antiviral metabolite, PMEG diphosphate. The P-chiral enantiomers of ODE-Bn-PMEG were obtained and appeared to have equivalent antiviral activities against HPV. ODE-Bn-PMEG is a promising candidate for the local treatment of HPV-16 and HPV-18 and other high-risk types, an important unmet medical need.

An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV.

Short Communication: HIV Controller T Cells Effectively Inhibit Viral Replication in Alveolar Macrophages.

Macrophages are targets of HIV-1 infection, and control of viral replication within these cells may be an important component of a T-cell-based vaccine. Although several studies have analyzed the ability of CD8+ T cells to inhibit viral replication in monocyte-derived macrophages, the effect of T cells on HIV-1-infected tissue macrophages is less clear. We demonstrate here that both CD4+ and CD8+ T-cell effectors from HIV controllers are capable of suppressing viral replication in bronchoalveolar lavage-derived alveolar macrophages. These findings have implications for HIV-1 vaccine and eradication strategies.

Safety and Pharmacokinetics of Quick-Dissolving Polymeric Vaginal Films Delivering the Antiretroviral IQP-0528 for Preexposure Prophylaxis.

For human immunodeficiency virus (HIV) prevention, microbicides or drugs delivered as quick-dissolving films may be more acceptable to women than gels because of their compact size, minimal waste, lack of an applicator, and easier storage and transport. This has the potential to improve adherence to promising products for preexposure prophylaxis. Vaginal films containing IQP-0528, a nonnucleoside reverse transcriptase inhibitor, were evaluated for their pharmacokinetics in pigtailed macaques. Polymeric films (22 by 44 by 0.1 mm; providing 75% of a human dose) containing IQP-0528 (1.5%, wt/wt) with and without poly(lactic-co-glycolic acid) (PLGA) nanoparticle encapsulation were inserted vaginally into pigtailed macaques in a crossover study design (n = 6). With unencapsulated drug, the median (range) vaginal fluid concentrations of IQP-0528 were 160.97 (2.73 to 2,104), 181.79 (1.86 to 15,800), and 484.50 (8.26 to 4,045) µg/ml at 1, 4, and 24 h after film application, respectively. Median vaginal tissue IQP-0528 concentrations at 24 h were 3.10 (0.03 to 222.58) µg/g. The values were similar at locations proximal, medial, and distal to the cervix. The IQP-0528 nanoparticle-formulated films delivered IQP-0528 in vaginal tissue and secretions at levels similar to those obtained with the unencapsulated formulation. A single application of either formulation did not disturb the vaginal microflora or the pH (7.24 ± 0.84 [mean ± standard deviation]). The high mucosal IQP-0528 levels delivered by both vaginal film formulations were between 1 and 5 log higher than the in vitro 90% inhibitory concentration (IC90) of 0.146 µg/ml. The excellent coverage and high mucosal levels of IQP-0528, well above the IC90, suggest that the films may be protective and warrant further evaluation in a vaginal repeated low dose simian-human immunodeficiency virus (SHIV) transmission study in macaques and clinically in women.

Systematic evaluation of methyl ester bioisosteres in the context of developing alkenyldiarylmethanes (ADAMs) as non-nucleoside reverse transcriptase inhibitors (NNRTIs) for anti-HIV-1 chemotherapy.

The alkenyldiarylmethanes (ADAMs) are a class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) targeting HIV-1. Four chemically and metabolically stabilized ADAMs incorporating N-methoxyimidoyl halide replacements of the methyl esters of the lead compound were previously reported. In this study, twenty-five new ADAMs were synthesized in order to investigate the biological consequences of installing nine different methyl ester bioisosteres at three different locations. Attempts to define a universal rank order of methyl ester bioisosteres and discover the 'best' one in terms of inhibitory activity versus HIV-1 reverse transcriptase (RT) led to the realization that the potencies are critically dependent on the surrounding structure at each location, and therefore the definition of universal rank order is impossible. This investigation produced several new non-nucleoside reverse transcriptase inhibitors in which all three of the three methyl esters of the lead compound were replaced by methyl ester bioisosteres, resulting in compounds that are more potent as HIV-1 RT inhibitors and antiviral agents than the lead compound itself and are expected to also be more metabolically stable than the lead compound.

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.

The Promise of Intravaginal Rings for Prevention: User Perceptions of Biomechanical Properties and Implications for Prevention Product Development.

Intravaginal rings (IVRs) are currently under investigation as devices for the delivery of agents to protect against the sexual transmission of HIV and STIs, as well as pregnancy. To assist product developers in creating highly acceptable rings, we sought to identify characteristics that intravaginal ring users consider when making decisions about ring use or non-use. We conducted four semi-structured focus groups with 21 women (aged 18-45) who reported using an IVR in the past 12 months. Participants manipulated four prototype rings in their hands, discussed ring materials, dimensionality, and "behavior," and shared perceptions and appraisals. Five salient ring characteristics were identified: 1) appearance of the rings' surfaces, 2) tactile sensations of the cylinder material, 3) materials properties, 4) diameter of the cylinder, and 5) ring circumference. Pliability (or flexibility) was generally considered the most important mechanical property. Several ring properties (e.g., porousness, dimensionality) were associated with perceptions of efficacy. Women also revealed user behaviors that may impact the effectiveness of certain drugs, such as removing, rinsing and re-inserting the ring while bathing, and removing the ring during sexual encounters. As product developers explore IVRs as prevention delivery systems, it is critical to balance product materials and dimensions with use parameters to optimize drug delivery and the user experience. It is also critical to consider how user behaviors (e.g., removing the ring) might impact drug delivery.

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.