Genital epithelial barrier function is conserved by intravaginal rings releasing etonogestrel and ethinyl estradiol.

The injectable progestin depot-medroxyprogesterone acetate (DMPA) is a popular contraceptive choice in sub-Saharan Africa although mouse models indicate it weakens genital epithelial integrity and barrier function and increases susceptibility to genital infection. The intravaginal ring NuvaRing® is another contraceptive option that like DMPA suppresses hypothalamic pituitary ovarian (HPO) axis function with local release of progestin (etonogestrel) and estrogen (ethinyl estradiol). As we previously reported that treating mice with DMPA and estrogen averts the loss of genital epithelial integrity and barrier function induced by DMPA alone, in the current investigation we compared genital levels of the cell-cell adhesion molecule desmoglein-1 (DSG1) and genital epithelial permeability in rhesus macaques (RM) treated with DMPA or a NuvaRing®re-sized for RM (N-IVR). While these studies demonstrated comparable inhibition of the HPO axis with DMPA or N-IVR, DMPA induced significantly lower genital DSG1 levels and greater tissue permeability to intravaginally administered low molecular mass molecules. By identifying greater compromise of genital epithelial integrity and barrier function in RM administered DMPA vs. N-IVR, our results add to the growing body of evidence that indicate DMPA weakens a fundamental mechanism of anti-pathogen host defense in the female genital tract.

Loss of genital epithelial barrier function is greater with depot-medroxyprogesterone acetate than intravaginal rings that release etonogestrel and ethinyl estradiol.

The injectable progestin depot-medroxyprogesterone acetate (DMPA) is a popular contraceptive choice in sub-Saharan Africa although mouse models indicate it weakens genital epithelial integrity and barrier function and increases susceptibility to genital infection. The intravaginal ring NuvaRing® is another contraceptive option that like DMPA suppresses hypothalamic pituitary ovarian (HPO) axis function with local release of progestin (etonogestrel) and estrogen (ethinyl estradiol). As we previously reported that treating mice with DMPA and estrogen averts the loss of genital epithelial integrity and barrier function induced by DMPA alone, in the current investigation we compared genital levels of the cell-cell adhesion molecule desmoglein-1 (DSG1) and genital epithelial permeability in rhesus macaques (RM) treated with DMPA or a NuvaRing®re-sized for RM (N-IVR). While these studies demonstrated comparable inhibition of the HPO axis with DMPA or N-IVR, DMPA induced significantly lower genital DSG1 levels and greater tissue permeability to intravaginally administered low molecular mass molecules. By identifying greater compromise of genital epithelial integrity and barrier function in RM administered DMPA vs. N-IVR, our results add to the growing body of evidence that indicate DMPA weakens a fundamental mechanism of anti-pathogen host defense in the female genital tract.

Preclinical Considerations for Long-acting Delivery of Tenofovir Alafenamide from Subdermal Implants for HIV Pre-exposure Prophylaxis.

PURPOSE: Long-acting formulations of the potent antiretroviral prodrug tenofovir alafenamide (TAF) hold potential as biomedical HIV prevention modalities. Here, we present a rigorous comparison of three animal models, C57BL/6 J mice, beagle dogs, and merino sheep for evaluating TAF implant pharmacokinetics (PKs). METHODS: Implants delivering TAF over a wide range of controlled release rates were tested in vitro and in mice and dogs. Our existing PK model, supported by an intravenous (IV) dosing dog study, was adapted to analyze mechanistic aspects underlying implant TAF delivery. RESULTS: TAF in vitro release in the 0.13 to 9.8 mg d-1 range with zero order kinetics were attained. Implants with equivalent fabrication parameters released TAF in mice and sheep at rates that were not statistically different, but were 3 times higher in dogs. When two implants were placed in the same subcutaneous pocket, a two-week creep to Cmax was observed in dogs for systemic drug and metabolite concentrations, but not in mice. Co-modeling IV and TAF implant PK data in dogs led to an apparent TAF bioavailability of 9.6 in the single implant groups (compared to the IV group), but only 1.5 when two implants were placed in the same subcutaneous pocket. CONCLUSIONS: Based on the current results, we recommend using mice and sheep, with macaques as a complementary species, for preclinical TAF implant evaluation with the caveat that our observations may be specific to the implant technology used here. Our report provides fundamental, translatable insights into multispecies TAF delivery via long-acting implants.

An optimized Factor H-Fc fusion protein against multidrug-resistant Neisseria gonorrhoeae.

Novel therapeutics against the global threat of multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococci evade killing by complement by binding factor H (FH), a key inhibitor of the alternative pathway. FH comprises 20 short consensus repeat (SCR) domains organized as a single chain. Gonococci bind FH through domains 6 and 7, and C-terminal domains 18 through 20. Previously, we showed that a chimeric protein comprising (from the N- to C-terminus) FH domains 18-20 (containing a point mutation in domain 19 to prevent lysis of host cells) fused to human IgG1 Fc (called FH*/Fc1) killed gonococci in a complement-dependent manner and reduced the duration and bacterial burden in the mouse vaginal colonization model of gonorrhea. Considering the N. gonorrhoeae-binding FH domains 18-20 are C-terminal in native FH, we reasoned that positioning Fc N-terminal to FH* (Fc1/FH*) would improve binding and bactericidal activity. Although both molecules bound gonococci similarly, Fc1/FH* displayed a 5-fold lower IC50 (the concentration required for 50% killing in complement-dependent bactericidal assays) than FH*/Fc1. To further increase complement activation, we replaced human IgG1 Fc in Fc1/FH* with Fc from human IgG3, the most potent complement-activating IgG subclass, to obtain Fc3/FH*. Bactericidal activity was further increased ~2.3-fold in Fc3/FH* compared to Fc1/FH*. Fc3/FH* killed (defined by <50% survival) 45/45 (100%) diverse PorB1B-expessing gonococci, but only 2/15 PorB1A-expressing isolates, in a complement-dependent manner. Decreased Fc3/FH* binding accounted for the limited activity against PorB1A strains. Fc3/FH* was efficacious against all four tested PorB1B gonococcal strains in the mouse vaginal colonization model when administered at a dose of 5 µg intravaginally, daily. Furthermore, Fc3/FH* retained bactericidal activity when reconstituted following lyophilization or spray-drying, suggesting feasibility for formulation into intravaginal rings. In conclusion, Fc3/FH* represents a promising prophylactic immunotherapeutic against multidrug-resistant gonococci.

At the Crossroads of Life and Death: The Proteins That Influence Cell Fate Decisions.

When a cell is damaged, it must decide how to respond. As a consequence of a variety of stresses, cells can induce well-regulated programmes such as senescence, a persistent proliferative arrest that limits their replication. Alternatively, regulated programmed cell death can be induced to remove the irreversibly damaged cells in a controlled manner. These programmes are mainly triggered and controlled by the tumour suppressor protein p53 and its complex network of effectors, but how it decides between these wildly different responses is not fully understood. This review focuses on the key proteins involved both in the regulation and induction of apoptosis and senescence to examine the key events that determine cell fate following damage. Furthermore, we examine how the regulation and activity of these proteins are altered during the progression of many chronic diseases, including cancer.

Fundamental aspects of long-acting tenofovir alafenamide delivery from subdermal implants for HIV prophylaxis.

Global efforts aimed at preventing human immunodeficiency virus type one (HIV-1) infection in vulnerable populations appear to be stalling, limiting our ability to control the epidemic. Long-acting, controlled drug administration from subdermal implants holds significant potential by reducing the compliance burden associated with frequent dosing. We, and others, are exploring the development of complementary subdermal implant technologies delivering the potent prodrug, tenofovir alafenamide (TAF). The current report addresses knowledge gaps in the preclinical pharmacology of long-acting, subdermal TAF delivery using several mouse models. Systemic drug disposition during TAF implant dosing was explained by a multi-compartment pharmacokinetic (PK) model. Imaging mass spectrometry was employed to characterize the spatial distribution of TAF and its principal five metabolites in local tissues surrounding the implant. Humanized mouse studies determined the effective TAF dose for preventing vaginal and rectal HIV-1 acquisition. Our results represent an important step in the development of a safe and effective TAF implant for HIV-1 prevention.

CAPRISA 018: a phase I/II clinical trial study protocol to assess the safety, acceptability, tolerability and pharmacokinetics of a sustained-release tenofovir alafenamide subdermal implant for HIV prevention in women.

INTRODUCTION: Young African women bear a disproportionately high risk for HIV acquisition. HIV technologies that empower women to protect themselves are needed. Safe, potent antiretroviral agents such as tenofovir alafenamide (TAF), formulated as long-acting subdermal implants, offer an innovative solution. METHODS AND ANALYSIS: CAPRISA 018 is a phase I/II trial to evaluate the safety, acceptability, tolerability and pharmacokinetics (PKs) of a TAF free base subdermal silicone implant containing 110 mg of TAF with an anticipated 0.25 mg/day release rate.The phase I trial (n=60) will assess the safety of one implant inserted in six participants (Group 1), followed by dose escalation components (Groups 2 and 3) assessing the safety, tolerability and PK of one to four TAF 110 mg implants releasing between 0.25 mg and 1 mg daily in 54 healthy women at low risk for HIV infection. Data from this phase I trial will be used to determine the dosing, implant location and implant replacement interval for the phase II trial.The phase II component (Group 4) will assess extended safety, PK, tolerability and acceptability of the implant in 490 at risk women, randomised in a 1:1 ratio to the TAF implant and placebo tablet or to the placebo implant and an oral pre-exposure prophylaxis tablet. Safety will be assessed by calculating the percentage change in creatinine clearance from baseline at weeks 4, 12, 24, 36, 72, 96 and 120, compared with the percentage change in the control group. ETHICS AND DISSEMINATION: The South African Health Products Regulatory Authority and the University of KwaZulu-Natal's Biomedical Research Ethics Committee have approved the trial. Results will be disseminated through open access peer reviewed publications, conference presentations, public stakeholder engagement and upload of data into the clinical trials registry. TRIAL REGISTRATION NUMBER: PACTR201809520959443.

Drug-releasing vaginal rings for HIV/STI and pregnancy prevention: a review of recent advances and clinical applications.

INTRODUCTION: Adolescent girls and young women (AGYW), as well as pre- and post-menopausal women globally would benefit from expanded choice to address their sexual and reproductive health (SRH) needs related to Human Immunodeficiency Virus (HIV), sexually transmitted infections (STIs) and pregnancy prevention. Lack of adequate preventative vaccines for HIV/STIs reinforces public health prioritization for options women may use to mitigate risk for infectious disease and unplanned pregnancy. Drug releasing intravaginal rings (IVRs) represent one such technology that has garnered attention based on the modality's success as a pre-exposure prophylaxis (PrEP) delivery option in HIV risk reduction. AREAS COVERED: This article provides a synopsis of three IVR technologies in active clinical development for prevention of HIV, STI, and unintended pregnancy demonstrating advancements in terms of compatibility with a wide range of drug types with a focus on dapivirine-based silicone rings (International Partnership for Microbicides (IPM), tenofovir-based polyurethane rings (Conrad), and pod-based rings (Oak Crest Institute of Science)). EXPERT OPINION: The goals of IVR research are to reduce burdens of HIV/STIs and unplanned pregnancies. Through the evolution of IVR technologies, the potential exists to trigger integration of health-care services through formulation of products with multiple indications.

Tenofovir Alafenamide for HIV Prevention: Review of the Proceedings from the Gates Foundation Long-Acting TAF Product Development Meeting.

The ability to successfully develop a safe and effective vaccine for the prevention of HIV infection has proven challenging. Consequently, alternative approaches to HIV infection prevention have been pursued, and there have been a number of successes with differing levels of efficacy. At present, only two oral preexposure prophylaxis (PrEP) products are available, Truvada and Descovy. Descovy is a newer product not yet indicated in individuals at risk of HIV-1 infection from receptive vaginal sex, because it still needs to be evaluated in this population. A topical dapivirine vaginal ring is currently under regulatory review, and a long-acting (LA) injectable cabotegravir product shows strong promise. Although demonstrably effective, daily oral PrEP presents adherence challenges for many users, particularly adolescent girls and young women, key target populations. This limitation has triggered development efforts in LA HIV prevention options. This article reviews efforts supported by the Bill & Melinda Gates Foundation, as well as similar work by other groups, to identify and develop optimal LA HIV prevention products. Specifically, this article is a summary review of a meeting convened by the foundation in early 2020 that focused on the development of LA products designed for extended delivery of tenofovir alafenamide (TAF) for HIV prevention. The review broadly serves as technical guidance for preclinical development of LA HIV prevention products. The meeting examined the technical feasibility of multiple delivery technologies, in vivo pharmacokinetics, and safety of subcutaneous (SC) delivery of TAF in animal models. Ultimately, the foundation concluded that there are technologies available for long-term delivery of TAF. However, because of potentially limited efficacy and possible toxicity issues with SC delivery, the foundation will not continue investing in the development of LA, SC delivery of TAF products for HIV prevention.

Highly synergistic drug combination prevents vaginal HIV infection in humanized mice.

The HIV-1 epidemic remains an urgent global health concern. Young women are disproportionately at risk of acquiring the virus. A range of highly effective, female-controlled, discrete vaginal products therefore is needed to help curb the epidemic. Oral tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are effective in HIV-1 pre-exposure prophylaxis (PrEP) and form a promising basis for a vaginal product. Here, we evaluate TDF and FTC in combination with the broadly neutralizing antibody VRC01-N using a highly reproducible humanized mouse model. The agents were vaginally dosed individually and in combination, and the efficacy of HIV-1 prevention was analyzed using the established, rigorous median-effect model. Surprisingly, the triple combination showed a high degree of synergism, unprecedented for in vivo HIV-1 PrEP, leading to a possible fivefold dose reduction for some of the agents. Vaginal administration of the TDF-FTC-VRC01-N combination holds significant promise for HIV-1 PrEP.

Multispecies Evaluation of a Long-Acting Tenofovir Alafenamide Subdermal Implant for HIV Prophylaxis.

New HIV-1 infection rates far outpace the targets set by global health organizations, despite important progress in curbing the progression of the epidemic. Long-acting (LA) formulations delivering antiretroviral (ARV) agents for HIV-1 pre-exposure prophylaxis (PrEP) hold significant promise, potentially facilitating adherence due to reduced dosing frequency compared to oral regimens. We have developed a subdermal implant delivering the potent ARV drug tenofovir alafenamide that could provide protection from HIV-1 infection for 6 months, or longer. Implants from the same lot were investigated in mice and sheep for local safety and pharmacokinetics (PKs). Ours is the first report using these animal models to evaluate subdermal implants for HIV-1 PrEP. The devices appeared safe, and the plasma PKs as well as the drug and metabolite concentrations in dermal tissue adjacent to the implants were studied and contrasted in two models spanning the extremes of the body weight spectrum. Drug and drug metabolite concentrations in dermal tissue are key in assessing local exposure and any toxicity related to the active agent. Based on our analysis, both animal models were shown to hold significant promise in LA product development.

Safety and pharmacokinetics of single, dual, and triple antiretroviral drug formulations delivered by pod-intravaginal rings designed for HIV-1 prevention: A Phase I trial.

BACKGROUND: Intravaginal rings (IVRs) for HIV pre-exposure prophylaxis (PrEP) theoretically overcome some adherence concerns associated with frequent dosing that can occur with oral or vaginal film/gel regimens. An innovative pod-IVR, composed of an elastomer scaffold that can hold up to 10 polymer-coated drug cores (or "pods"), is distinct from other IVR designs as drug release from each pod can be controlled independently. A pod-IVR has been developed for the delivery of tenofovir (TFV) disoproxil fumarate (TDF) in combination with emtricitabine (FTC), as daily oral TDF-FTC is the only Food and Drug Administration (FDA)-approved regimen for HIV PrEP. A triple combination IVR building on this platform and delivering TDF-FTC along with the antiretroviral (ARV) agent maraviroc (MVC) also is under development. METHODOLOGY AND FINDINGS: This pilot Phase I trial conducted between June 23, 2015, and July 15, 2016, evaluated the safety, pharmacokinetics (PKs), and acceptability of pod-IVRs delivering 3 different ARV regimens: 1) TDF only, 2) TDF-FTC, and 3) TDF-FTC-MVC over 7 d. The crossover, open-label portion of the trial (N = 6) consisted of 7 d of continuous TDF pod-IVR use, a wash-out phase, and 7 d of continuous TDF-FTC pod-IVR use. After a 3-mo pause to evaluate safety and PK of the TDF and TDF-FTC pod-IVRs, TDF-FTC-MVC pod-IVRs (N = 6) were evaluated over 7 d of continuous use. Safety was assessed by adverse events (AEs), colposcopy, and culture-independent analysis of the vaginal microbiome (VMB). Drug and drug metabolite concentrations in plasma, cervicovaginal fluids (CVFs), cervicovaginal lavages (CVLs), and vaginal tissue (VT) biopsies were determined via liquid chromatographic-tandem mass spectrometry (LC-MS/MS). Perceptibility and acceptability were assessed by surveys and interviews. Median participant age was as follows: TDF/TDF-FTC group, 26 y (range 24-35 y), 2 White, 2 Hispanic, and 2 African American; TDF-FTC-MVC group, 24.5 y (range 21-41 y), 3 White, 1 Hispanic, and 2 African American. Reported acceptability was high for all 3 products, and pod-IVR use was confirmed by residual drug levels in used IVRs. There were no serious adverse events (SAEs) during the study. There were 26 AEs reported during TDF/TDF-FTC IVR use (itching, discharge, discomfort), with no differences between TDF alone or in combination with FTC observed. In the TDF-FTC-MVC IVR group, there were 12 AEs (itching, discharge, discomfort) during IVR use regardless of attribution to study product. No epithelial disruption/thinning was seen by colposcopy, and no systematic VMB shifts were observed. Median (IQR) tenofovir diphosphate (TFV-DP) tissue concentrations of 303 (277-938) fmol/10(6) cells (TDF), 289 (110-603) fmol/10(6) cells (TDF-FTC), and 302 (177.1-823.8) fmol/10(6) cells (TDF-FTC-MVC) were sustained for 7 d, exceeding theoretical target concentrations for vaginal HIV prevention. The study's main limitations include the small sample size, short duration (7 d versus 28 d), and the lack of FTC triphosphate measurements in VT biopsies. CONCLUSIONS: An innovative pod-IVR delivery device with 3 different formulations delivering different regimens of ARV drugs vaginally appeared to be safe and acceptable and provided drug concentrations in CVFs and tissues exceeding concentrations achieved by highly protective oral dosing, suggesting that efficacy for vaginal HIV PrEP is achievable. These results show that an alternate, more adherence-independent, longer-acting prevention device based on the only FDA-approved PrEP combination regimen can be advanced to safety and efficacy testing. TRIAL REGISTRATION: ClinicalTrials.gov NCT02431273.

Phase I trial of pod-intravaginal rings delivering antiretroviral agents for HIV-1 prevention: Rectal drug exposure from vaginal dosing with tenofovir disoproxil fumarate, emtricitabine, and maraviroc.

BACKGROUND: Intravaginal rings (IVRs) can deliver antiretroviral (ARV) agents for HIV pre-exposure prophylaxis (PrEP), theoretically overcoming adherence concerns associated with frequent dosing. However, topical vaginal ARV drug delivery has not simultaneously led to sufficient rectal drug exposure to likely protect from HIV infection as a result of receptive anal intercourse (RAI). Unprotected RAI has a higher risk of infection per sex act and, for women, also can be associated with vaginal exposure during a single sexual encounter, especially in higher-risk subsets of women. The physiologically inflamed, activated, immune-cell dense colorectal mucosa is increasingly appreciated as the sexual compartment with highly significant risk; this risk is increased in the setting of co-infections. Ex vivo studies have shown that colorectal tissue and rectal fluid concentrations correlated with HIV protection. Given these important results, efforts to document colorectal compartment ARV drug concentration from pod-IVR delivery was assessed to determine if vaginal application could provide protective ARV levels in both compartments. METHODOLOGY/PRINCIPAL FINDINGS: A crossover clinical trial (N = 6) evaluated 7 d of continuous TDF pod-IVR use, a wash-out phase, followed by 7 d with a TDF-FTC pod-IVR. A subsequent clinical trial (N = 6) consisted of 7 d of continuous TDF-FTC-MVC pod-IVR use. Rectal fluids were collected on Day 7 at IVR removal in all three ARV-exposures (two Phase 1 trials) and drug concentrations quantified by LC-MS/MS. Median rectal fluid concentrations of TFV, the hydrolysis product of the prodrug TDF, were between 0.66 ng mg-1 (TDF pod-IVR group) and 1.11 ng mg-1 (TDF-FTC pod-IVR group), but below the analytical lower limit of quantitation in 5/6 samples in the TDF-FTC-MVC pod-IVR group. Unexpectedly, median FTC (TDF-FTC pod-IVR, 20.3 ng mg-1; TDF-FTC-MVC pod-IVR, 0.18 ng mg-1), and MVC rectal fluid concentrations (0.84 ng mg-1) were quantifiable and higher than their respective in vitro EC50 values in most samples. Due to participant burden in these exploratory trials, rectal fluid was used as a surrogate for rectal tissue, where drug concentrations are expected to be higher. CONCLUSIONS/SIGNIFICANCE: The concentrations of FTC and MVC in rectal fluids obtained in two exploratory clinical trials of IVRs delivering ARV combinations exceeded levels associated with in vitro efficacy in HIV inhibition. Unexpectedly, MVC appeared to depress the distribution of TFV and FTC into the rectal lumen. Here we show that vaginal delivery of ARV combinations may provide adherence and coitally independent dual-compartment protection from HIV infection during both vaginal and receptive anal intercourse.

Protection Efficacy of C5A Against Vaginal and Rectal HIV Challenges in Humanized Mice.

INTRODUCTION: In the absence of a vaccine, there is an urgent need for the identification of effective agents that prevent HIV transmission in uninfected individuals. Non-vaccine Biomedical Prevention (nBP) methods, such as topical or systemic pre-exposure prophylaxis (PrEP), are promising strategies to slow down the spread of AIDS. METHODS: In this study, we investigated the microbicidal efficacy of the viral membrane-disrupting amphipathic SWLRDIWDWICEVLSDFK peptide called C5A. We chose the bone marrow/liver/thymus (BLT) humanized mouse model as vaginal and rectal HIV transmission models. RESULTS: We found that the topical administration of C5A offers complete protection against vaginal and rectal HIV challenges in humanized mice. After demonstrating that C5A blocks genital HIV transmission in humanized mice, we examined the molecular requirements for its microbicidal property. We found that the removal of four amino acids on either end of C5A does not diminish its microbicidal efficacy. However, the removal of four amino acids at both the ends, abolishes its capacity to prevent vaginal or rectal HIV transmission, suggesting that the length of the peptide is a critical parameter for the microbicidal activity of C5A. Moreover, we demonstrated that the amphipathicity of the helical peptide as well as its hydrophobic surface represents key factors for the microbicidal activity of C5A in humanized mice. CONCLUSION: With its noncellular cytotoxic activity, its property of neutralizing both HSV and HIV, and its unique mechanism of action that disrupts the stability of the viral membrane, C5A represents an attractive multipurpose microbicidal candidate to be combined with other anti-HIV agents including antiretrovirals.

Novel multipurpose pod-intravaginal ring for the prevention of HIV, HSV, and unintended pregnancy: Pharmacokinetic evaluation in a macaque model.

Globally, women bear an uneven burden for sexual HIV acquisition. Results from two clinical trials evaluating intravaginal rings (IVRs) delivering the antiretroviral agent dapivirine have shown that protection from HIV infection can be achieved with this modality, but high adherence is essential. Multipurpose prevention technologies (MPTs) can potentially increase product adherence by offering protection against multiple vaginally transmitted infections and unintended pregnancy. Here we describe a coitally independent, long-acting pod-IVR MPT that could potentially prevent HIV and HSV infection as well as unintended pregnancy. The pharmacokinetics of MPT pod-IVRs delivering tenofovir alafenamide hemifumarate (TAF2) to prevent HIV, acyclovir (ACV) to prevent HSV, and etonogestrel (ENG) in combination with ethinyl estradiol (EE), FDA-approved hormonal contraceptives, were evaluated in pigtailed macaques (N = 6) over 35 days. Pod IVRs were exchanged at 14 days with the only modification being lower ENG release rates in the second IVR. Plasma progesterone was monitored weekly to determine the effect of ENG/EE on menstrual cycle. The mean in vivo release rates (mg d-1) for the two formulations over 30 days ranged as follows: TAF2 0.35-0.40; ACV 0.56-0.70; EE 0.03-0.08; ENG (high releasing) 0.63; and ENG (low releasing) 0.05. Mean peak progesterone levels were 4.4 ± 1.8 ng mL-1 prior to IVR insertion and 0.075 ± 0.064 ng mL-1 for 5 weeks after insertion, suggesting that systemic EE/ENG levels were sufficient to suppress menstruation. The TAF2 and ACV release rates and resulting vaginal tissue drug concentrations (medians: TFV, 2.4 ng mg-1; ACV, 0.2 ng mg-1) may be sufficient to protect against HIV and HSV infection, respectively. This proof of principle study demonstrates that MPT-pod IVRs could serve as a potent biomedical prevention tool to protect women's sexual and reproductive health and may increase adherence to HIV PrEP even among younger high-risk populations.

Prevention of vaginal and rectal HIV transmission by antiretroviral combinations in humanized mice.

With more than 7,000 new HIV infections daily worldwide, there is an urgent need for non-vaccine biomedical prevention (nBP) strategies that are safe, effective, and acceptable. Clinical trials have demonstrated that pre-exposure prophylaxis (PrEP) with antiretrovirals (ARVs) can be effective at preventing HIV infection. In contrast, other trials using the same ARVs failed to show consistent efficacy. Topical (vaginal and rectal) dosing is a promising regimen for HIV PrEP as it leads to low systematic drug exposure. A series of titration studies were carried out in bone marrow/liver/thymus (BLT) mice aimed at determining the adequate drug concentrations applied vaginally or rectally that offer protection against rectal or vaginal HIV challenge. The dose-response relationship of these agents was measured and showed that topical tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) can offer 100% protection against rectal or vaginal HIV challenges. From the challenge data, EC50 values of 4.6 µM for TDF and 0.6 µM for FTC for HIV vaginal administration and 6.1 µM TDF and 0.18 µM for FTC for rectal administration were obtained. These findings suggest that the BLT mouse model is highly suitable for studying the dose-response relationship in single and combination ARV studies of vaginal or rectal HIV exposure. Application of this sensitive HIV infection model to more complex binary and ternary ARV combinations, particularly where agents have different mechanisms of action, should allow selection of optimal ARV combinations to be advanced into pre-clinical and clinical development as nBP products.

An intravaginal ring for real-time evaluation of adherence to therapy.

Two recent Phase III clinical trials to investigate an intravaginal ring for preventing HIV infection demonstrated that adherence to prescribed device use was a primary driver of efficacy. Surrogate methods for determining adherence in the studies were limited in their inability to monitor temporal patterns of use and allow deconvolution of the effects of adherence and device efficacy on HIV infection rates. To address this issue, we have developed functionality in an intravaginal ring to continuously monitor when the device is being used and maintain a log of adherence that can be accessed by clinicians after it is removed. An electronic module fabricated with common, inexpensive electronic components was encapsulated in a silicone intravaginal ring. The device uses temperature as a surrogate measure of periods of device insertion and removal, and stores a record of the data for subsequent retrieval. The adherence-monitoring intravaginal ring accurately recorded the device status over 33 simulated IN-OUT cycles and more than 1000 measurement cycles in vitro. Following initial in vitro testing in a temperature-controlled chamber, the device was evaluated in vivo in sheep using a predetermined insertion/removal pattern to simulate intravaginal ring use. After insertion into the vaginal cavity of a sheep, the logged data correctly indicated the device status over 29 hours of continuous measurement including three cycles of insertion and removal. The device described here is a promising, low-cost method for real-time adherence assessment in clinical trials involving medicated intravaginal rings or other intravaginal devices.

Pharmacokinetics and Preliminary Safety of Pod-Intravaginal Rings Delivering the Monoclonal Antibody VRC01-N for HIV Prophylaxis in a Macaque Model.

The broadly neutralizing antibody (bNAb) VRC01, capable of neutralizing 91% of known human immunodeficiency virus type 1 (HIV-1) isolates in vitro, is a promising candidate microbicide for preventing sexual HIV infection when administered topically to the vagina; however, accessibility to antibody-based prophylactic treatment by target populations in sub-Saharan Africa and other underdeveloped regions may be limited by the high cost of conventionally produced antibodies and the limited capacity to manufacture such antibodies. Intravaginal rings of the pod design (pod-IVRs) delivering Nicotiana-manufactured VRC01 (VRC01-N) over a range of release rates have been developed. The pharmacokinetics and preliminary safety of VRC01-N pod-IVRs were evaluated in a rhesus macaque model. The devices sustained VRC01-N release for up to 21 days at controlled rates, with mean steady-state VRC01-N levels in vaginal fluids in the range of 102 to 103 µg g-1 being correlated with in vitro release rates. No adverse safety indications were observed. These findings indicate that pod-IVRs are promising devices for the delivery of the candidate topical microbicide VRC01-N against HIV-1 infection and merit further preclinical evaluation.

Topical Delivery of Tenofovir Disoproxil Fumarate and Emtricitabine from Pod-Intravaginal Rings Protects Macaques from Multiple SHIV Exposures.

Topical preexposure prophylaxis (PrEP) against HIV has been marginally successful in recent clinical trials with low adherence rates being a primary factor for failure. Controlled, sustained release of antiretroviral (ARV) drugs may help overcome these low adherence rates if the product is protective for extended periods of time. The oral combination of tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) is currently the only FDA-approved ARV drug for HIV PrEP. A novel pod-intravaginal ring (IVR) delivering TDF and FTC at independently controlled rates was evaluated for efficacy at preventing SHIV162p3 infection in a rigorous, repeat low-dose vaginal exposure model using normally cycling female pigtailed macaques. Six macaques received pod-IVRs containing TDF (65 mg) and FTC (68 mg) every two weeks, and weekly vaginal exposures to 50 TCID50 of SHIV162p3 began one week after the first pod-IVR insertion. All pod-IVR-treated macaques were fully protected throughout the study (P = 0.0002, Log-rank test), whereas all control animals became infected with a median of 4 exposures to infection. The topical, sustained release of TDF and FTC from the pod-IVR maintained protective drug levels in macaques over four months of virus exposures. This novel and versatile delivery system has the capacity to deliver and maintain protective levels of multiple drugs and the protection observed here warrants clinical evaluation of this pod-IVR design.

Combination Pod-Intravaginal Ring Delivers Antiretroviral Agents for HIV Prophylaxis: Pharmacokinetic Evaluation in an Ovine Model.

Preexposure prophylaxis (PrEP) against HIV using oral regimens based on the nucleoside reverse transcriptase inhibitor tenofovir disoproxil fumarate (TDF) has been effective to various degrees in multiple clinical trials, and the CCR5 receptor antagonist maraviroc (MVC) holds potential for complementary efficacy. The effectiveness of HIV PrEP is highly dependent on adherence. Incorporation of the TDF-MVC combination into intravaginal rings (IVRs) for sustained mucosal delivery could increase product adherence and efficacy compared with oral and vaginal gel formulations. A novel pod-IVR technology capable of delivering multiple drugs is described. The pharmacokinetics and preliminary local safety characteristics of a novel pod-IVR delivering a combination of TDF and MVC were evaluated in the ovine model. The device exhibited sustained release at controlled rates over the 28-day study and maintained steady-state drug levels in cervicovaginal fluids (CVFs). Dilution of CVFs during lavage sample collection was measured by ion chromatography using an inert tracer, allowing corrected drug concentrations to be measured for the first time. Median, steady-state drug levels in vaginal tissue homogenate were as follows: for tenofovir (TFV; in vivo hydrolysis product of TDF), 7.3 × 10(2) ng g(-1) (interquartile range [IQR], 3.0 × 10(2), 4.0 × 10(3)); for TFV diphosphate (TFV-DP; active metabolite of TFV), 1.8 × 10(4) fmol g(-1) (IQR, 1.5 × 10(4), 4.8 × 10(4)); and for MVC, 8.2 × 10(2) ng g(-1) (IQR, 4.7 × 10(2), 2.0 × 10(3)). No adverse events were observed. These findings, together with previous pod-IVR studies, have allowed several lead candidates to advance into clinical evaluation.