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Introduction Dapivirine (DPV), formulated as vaginal ring, demonstrated HIV risk reduction. MTN-026 explored DPV, formulated as rectal gel, for safety, pharmacokinetics, and acceptability. Methods HIV-uninfected men and women aged 18-45 years were enrolled at United States and Thailand sites and randomized 2:1 to receive DPV 0.05% or placebo gel via rectal applicator. A single dose phase was followed by 7 observed daily doses. Plasma, and fluid and tissue from both rectum and cervix were collected at baseline and after the final dose over 72 hours for pharmacokinetics, ex-vivo HIV-1 biopsy challenge, histology, and flow cytometry. Results 28 participants were randomized; 2 terminated early; 9 were female and 19 male; 12 were white, 11 Asian, 4 black and 1 other race/ethnicity. Mean age was 28.5 and 34.2 years in the DPV and placebo arms, respectively. Thirty adverse events occurred (all Grade 1 or 2, except one unrelated Grade 3) without study arm differences. DPV rectal tissue concentrations (median [interquartile range]) 0.5-1 and 2 hours after a single dose were 256 ng/gm (below limit of quantitation [BLQ], 666) and BLQ (BLQ, 600), respectively, then BLQ (BLQ, BLQ) from 24-72 hours; concentrations following multiple doses were similar. The largest median DPV plasma concentrations were 0.33 ng/mL (0.15, 0.48) after one dose and 0.40 (0.33, 0.49) after seven doses. Conclusions The DPV rectal gel was acceptable and without safety concerns. While DPV plasma concentrations were similar to the vaginal ring, rectal tissue concentrations were well below vaginal ring tissue concentrations, suggesting need for reformulation.

Rectal microbicide (RM) researchers have increasingly been interested in using antiretroviral (ARV) drugs in the form of a sexual lubricant as a form of HIV prevention. We triangulated quantitative and qualitative assessments to evaluate participants’ acceptability of 0.05% dapivirine gel administered via two separate modalities (a rectal applicator and an artificial phallus for use as a coital simulation device) in two different randomization groups: Sequence A (Applicator/Coital Simulation Device) or Sequence B (Coital Simulation Device/Applicator) as part of a Phase I trial (N=14) among a group of men who have sex with men (MSM) randomized using a 1:1 ratio from the Microbicide Trials Network (MTN)-033 trial. Overall, participants reported favorable acceptability of the gel (n=11; 78.6%) the same or more at the end of the study compared to when they started the study. Additionally, when discussing their preferred administration modality, they noted that both methods have positive qualities but also potential areas of improvement. Our findings underscore the need to create multiple delivery methods for a future microbicide gel (i.e., with and without the need for an applicator) and highlight the importance of offering MSM choices in how biomedical HIV prevention strategies are delivered.

Objective: The antiretroviral-based dapivirine vaginal ring reduced HIV risk among women in phase III clinical trials. However, limited data exists on the impact of dapivirine on the vaginal microenvironment in adolescents. Design: A comprehensive metaproteomics approach was used to assess host proteome and microbiome changes in cervicovaginal mucus with dapivirine ring use in adolescents enrolled in the MTN-023/IPM 030 (MTN-023) trial. Methods: Participants were randomized 3 : 1 to use dapivirine or placebo vaginal rings monthly for 6 months. Cervicovaginal samples from a subset of 35 participants (8 placebo, 27 dapivirine) were analyzed. Results: Mass spectrometry analysis identified 405 human and 2467 bacterial proteins belonging to 15 unique genera. The host proteome belonged to many functional pathways primarily related to inflammation. When stratified by study treatment arm, 18 (4.4%) and 28 (6.9%) human proteins were differentially abundant (adjusted P

This study describes the acceptability of a rectal microbicide gel formulation using dapivirine (DPV) among men and women from two countries (United States and Thailand) participating in the Microbicide Trials Network-026 trial. We evaluated participants' acceptability of a rectal DPV/placebo gel as part of a Phase I trial (N = 26; 18 male, 8 female). Participants reported favorable acceptability of the study gel, with most participants reporting that they liked the gel the same (n = 14; 53.8%) or more (n = 11; 42.4%) than when they started the trial. Over half of participants noted that they would prefer the gel over condoms (n = 13; 50%) or that they liked condoms and the gel equally (n = 8; 30.8%). Side effects across products included leakage (n = 8; 30.8%), diarrhea (n = 4; 15.4%), or soiling (n = 1; 3.8%). The high acceptability of a rectal gel underscores its promise as a short-acting biomedical prevention, warranting future research for HIV prevention.Trial Registration: NCT03239483.Este estudio describe la aceptabilidad de un microbicida rectal (RM) con dapivirina (DPV) formulado como un gel por hombres y mujeres de dos países (Estados Unidos y Tailandia) que participaron como parte del Microbicide Trials Network (MTN)-026. Evaluamos la aceptabilidad de un gel rectal de DPV y un placebo como parte de un estudio de Fase I (N = 26; 18 hombres, 8 mujeres). Los participants informaron una aceptabilidad favorable sobre el gel del estudio; la mayoría de los participantes informaron que les gustó el gel igual (n = 14; 53.8%) o más (n = 11; 42.4%) que cuando comenzaron el estudio. Más de la mitad de los participantes señalaron que preferirían el gel sobre los condones (n = 13; 50%) o que les gustaban los condones y el gel por igual (n = 8; 30,8%). Los efectos de los productos incluyeron fugas (n = 8; 30,8%), diarrea (n = 4; 15,4%) o ensuciamiento (n = 1; 3,8%). La alta aceptabilidad de un gel rectal enfatiza su promesa para la prevención biomédica de acción corta y justifica futuras investigaciones para la prevención del VIH.

Intravaginal rings (IVR) containing antiretroviral (ARV) drugs are a promising method for HIV prevention. We triangulated quantitative and qualitative assessments to evaluate the acceptability of four IVRs used continuously for 28 days as part of a Phase I trial (N=48 HIV-negative women; ages 18-45). Adherence was high throughout the trial, yet 30% of participants reported involuntary IVR expulsions followed by re-insertion. Most participants (93.6%) felt comfortable with the IVR being inside their body. Participants reported liking the IVR more (36.2%) or the same amount (55.3%) since starting the study. When given the option of choosing between the IVR and/or a male condom for HIV-prevention, most reported preferring the IVR (n=29, 63.0%), and over a quarter of the sample reported liking them equally (n=12, 26.1%). We observed no differences in IVR acceptability across the study arms. High adherence and acceptability underscores the promise of an IVR as a female-controlled, sustained mechanism for HIV prevention.

Background Vaginal rings (VR) containing antiretroviral (ARV) drugs can be utilized for prevention of human immunodeficiency virus (HIV) with potential for improved adherence compared to daily pills. Combination ARV VRs could improve efficacy. Methods MTN-027, a single-blind, randomized, placebo-controlled trial in 48 women, evaluated VRs containing MK-2048 (30 mg) and vicriviroc (VCV, 182 mg), alone or in combination, and placebo used continuously for 28 days. Safety was assessed by recording adverse events. Drug concentrations were quantified in plasma, vaginal fluid, cervical tissue, and rectal fluid. Cervical tissue was utilized for ex vivo HIV inhibition analysis. Results There was no difference in related genitourinary adverse events between treatment arms compared to placebo. VCV and MK-2048 released from single or combination VRs both achieved peak concentrations in vaginal fluids, which were substantially higher compared to plasma (200× for VCV, 30× for MK-2048) and rectal fluid. In an ex vivo challenge assay, the antiviral activity of VCV and/or MK-2048 was not correlated with tissue-associated drug concentrations. Most women (77%) were fully adherent to 28 days of continuous VR use and found the VR acceptable. Conclusions VCV and/or MK-2048 containing VRs were safe and acceptable. Both VCV and MK-2048 were quantifiable in all matrixes tested with peak compartmental drug concentrations similar for single and combination drug VRs. Tissue-associated VCV and/or MK-2048 did not correlate with inhibition of HIV infection. These data highlight the need to assess adequacy of drug dosing in the VR and measuring genital tissue drug concentrations to develop more precise concentration-response relationships.

In clinical trials evaluating HIV-1 prevention products, ex vivo exposure of mucosal tissue to HIV-1 is performed to inform drug levels needed to suppress viral infection. Understanding assay and participant variables that influence HIV-1 replication will help with assay implementation. Demographic and behavioral data were obtained from 61 healthy women aged 21–45. Paired cervical tissue (CT) and vaginal tissue (VT) biopsies were collected and treated with HIV-1(BaL) or HIV-1(JR-CSF), washed, and cultured. On days 3, 7, and/or 11, culture supernatant was collected, and viral replication was monitored by p24 ELISA. Tissue was extracted at study end, and HIV-1 relative RNA copies were determined by polymerase chain reaction. Cumulative p24 and RNA were log-transformed and analyzed using a linear mixed model, t-test, and an intraclass correlation coefficient (ICC). HIV replication was similar between CT and VT for each virus, but HIV-1(BaL) had 1.5 log(10) and 0.9 log(10) higher levels of p24 than HIV-1(JR-CSF) in CT and VT, respectively (p

Long-acting injectable antiretroviral agents are being developed for HIV-1 prevention. The MWRI-01 study was done to characterise the safety, acceptability, and pharmacokinetic and pharmacodynamic profile of long-acting rilpivirine.We did a phase 1 open-label study at the University of Pittsburgh. We enrolled healthy individuals (aged 18-45 years) who were seronegative for HIV-1. Participants were assigned alternately one intramuscular dose of either 1200 mg or 600 mg long-acting rilpivirine, beginning with the 1200 mg dose. We obtained plasma specimens, genital and rectal fluids, and tissue samples (rectal, cervical, and vaginal) before and after exposure to long-acting rilpivirine for assessment of pharmacokinetics and ex-vivo biopsy challenge with HIV-1. Our primary objective was to characterise product safety, and the analysis included all enrolled participants. This trial is registered with ClinicalTrials.gov, number NCT01656018.36 participants were enrolled into the study, of whom 24 were women and 12 men. 12 women and six men received each dose. 204 adverse events were reported among the 36 participants, of which 200 (98%) were grade 1-2. The most common adverse event was injection site reaction. All grade 3 and 4 adverse events were deemed not related to rilpivirine. Geometric mean (90% CI) concentrations in plasma of rilpivirine at day 28 post dose were 53 ng/mL (38-67) in women and 43 ng/mL (23-63) in men for the 1200 mg dose and 28 ng/mL (19-37) in women and 17 ng/mL (9-24) in men for the 600 mg dose. The tissue-to-plasma ratio for rilpivirine in rectal tissue was about two-fold higher than in vaginal and cervical tissue (1·10-1·53 vs 0·61-0·72 and 0·50-0·71, respectively). Exposure to long-acting rilpivirine suppressed viral replication significantly in rectal tissue (p0·0001), and this suppression persisted for up to 4 months. By contrast, no viral suppression was seen in cervical or vaginal tissue.Ongoing research will characterise longer term safety and acceptability of multiple injections and help ascertain whether long-acting rilpivirine should advance to assessment of efficacy in preventing HIV-1 infection.BillMelinda Gates Foundation.

The MWRI-01 study characterized the safety, acceptability, pharmacokinetic (PK), and pharmacodynamic (PD) profile of rilpivirine (RPV) long acting (LA) in a model of preexposure prophylaxis (PrEP). Prospective, open-label Phase 1 study. The safety and acceptability of three repeated doses of RPV LA were monitored. Blood, tissue (rectal, cervical, and vaginal), and biological fluids (vaginal and endocervical) were collected at baseline and at 1- to 2-month intervals throughout the study for PK and PD assessment. Eight women and four men received three intramuscular doses of 1,200 mg of RPV LA given 8 weeks apart. There were a total of 195 adverse events (AEs) reported, of which 138 (70.8%) were Grade 1 and 55 (28.2%) were Grade 2. The most common AE was injection site pain. Geometric mean (90% confidence interval) plasma RPV concentrations at 56 days after the first and third doses were 39 (33-45) ng/mL (female)/29 (17-40) ng/mL (male) and 59 (45-62) ng/mL (female)/40 (30-51) ng/mL (male), respectively. Exposure to RPV LA was associated with significant inhibition of HIV-1

Breastfeeding (BF) women are an important population for biomedical HIV prevention strategies, but they are rarely included in trials. The 25-mg dapivirine vaginal ring (VR) reduced women’s risk of sexually transmitted HIV infection in two phase 3 trials conducted in Africa. We conducted a phase 1, open-label study (MTN-029/IPM 039) of dapivirine VR use among lactating women in Pittsburgh, PA, and Birmingham, AL, USA. MTN-029/IPM 039 enrolled 16 healthy adult women who had already weaned their infants but were still able to express breast milk. Women were instructed to use the VR continuously for 14 days and provided milk, plasma, and cervicovaginal fluid (CVF) samples for pharmacological analysis. No infants were exposed to the drug, but infant dosage was estimated according to FDA guidance. Adverse events (AEs) were collected at all contacts. The study was completed with 100% participant retention. Median dapivirine concentrations were 676 pg/ml in breast milk, 327 pg/ml in plasma (milk/plasma ratio ∼2.0), and 36.25 ng/mg in CVF. Six participants experienced 10 total AEs, none of which required VR discontinuation. The estimated mean daily infant dosage was 74.3 ng/kg/day. In this first study of dapivirine exposure during lactation, dapivirine VR use was associated with lower concentrations of detectable dapivirine in milk and plasma than in CVF samples and a favorable safety profile. Estimated daily levels of infant dapivirine exposure were also low. Additional studies are needed to evaluate longer periods of dapivirine VR use among BF mother-infant pairs living in regions with higher incidence of sexually transmitted HIV infection. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT02808949","term_id":"NCT02808949"}}NCT02808949.)

A long-acting injectable form of rilpivirine (RPV) is being evaluated in clinical trials for the prevention of HIV infection. Preclinical testing was undertaken to define RPV pharmacokinetic (PK) and pharmacodynamic (PD) activities in ectocervical and colonic tissue treated in vitro . Tenfold dilutions of RPV were added to the basolateral medium of polarized ectocervical and colonic explant tissues. To half the explants, HIV-1 BaL was applied to the apical tissue surface. After culture overnight, all the explants were washed and the RPV in the explants not exposed to HIV was quantified using a validated liquid chromatography-mass spectrometry assay. For efficacy, explants exposed to HIV remained in culture, and supernatants were collected to assess viral replication using a p24 enzyme-linked immunosorbent assay. The data were log 10 transformed, and PK/PD correlations were determined using GraphPad Prism and SigmaPlot software. The application of RPV to the basolateral medium at 10 μM and 1 μM was effective in protecting ectocervical and colonic tissues, respectively, from HIV infection. When the RPV in paired ectocervical and colonic explant tissues was quantified, significant inverse linear correlations ( P < 0.001) between p24 and RPV concentrations were obtained; more viral replication was noted at lower drug levels. Using a maximum effect model, RPV concentrations of 271 nM in ectocervical tissue and 45 nM in colonic tissue were needed to achieve a 90% effective concentration (EC 90 ). These data demonstrate that RPV can suppress HIV infection in mucosal tissue but that higher levels of RPV are needed in female genital tract tissue than in gastrointestinal tract tissue for protection.

Background Films may deliver antiretroviral drugs efficiently to mucosal tissues. In this first in-human trial of a vaginal film for delivering the nonnucleoside reverse transcriptase inhibitor dapivirine, safety, pharmacokinetics, and pharmacodynamics of film and gel formulations were compared with placebo. Methods Sixty-one healthy HIV-negative women were randomized to daily dapivirine (0.05%) or placebo gel, or dapivirine (1.25 mg) or placebo film for seven days. The proportion of participants experiencing grade 2 and higher adverse events related to study product were compared. Plasma dapivirine concentrations were quantified. Paired cervical and vaginal tissue biopsies obtained ∼2 hours after the last dose were measured for tissue drug concentration and exposed to HIV in an ex vivo challenge assay. Results Two grade 2 related adverse events occurred in the placebo film group. Women randomized to gel and film products had 4 log10 higher of dapivirine in cervical and vaginal tissues than plasma. Although gel and film users had comparable plasma dapivirine concentrations, tissue concentrations of dapivirine were 3-5 times higher in the gel users when compared with film users. HIV replication in the ex vivo challenge assay was significantly reduced in vaginal tissues from women randomized to dapivirine film or gel; furthermore, tissue drug concentrations were highly correlated with HIV protection. Women rated the film more comfortable with less leakage but found it more difficult to insert than gel. Discussion Both film and gel delivered dapivirine at concentrations sufficient to block HIV ex vivo. This proof-of-concept study suggests film formulations for microbicides merit further investigation.

Broadly neutralizing monoclonal antibodies (nAbs) specific for HIV are being investigated for use in HIV prevention. Due to their ability to inhibit HIV attachment to and entry into target cells, nAbs may be suitable for use as topical HIV microbicides. As such, they would present an alternative intervention for individuals who may not benefit from using antiretroviral-based products for HIV prevention. We theorize that nAbs can inhibit viral transmission through mucosal tissue, thus reducing the incidence of HIV infection. The efficacy of the PG9, PG16, VRC01, and 4E10 antibodies was evaluated in an ex vivo human model of mucosal HIV transmission. nAbs reduced HIV transmission, causing 1.5- to 2-log 10 reductions in HIV replication in ectocervical tissues and ≈3-log 10 reductions in HIV replication in colonic tissues over 21 days. These antibodies demonstrated greater potency in colonic tissues, with a 50-fold higher dose being required to reduce transmission in ectocervical tissues. Importantly, nAbs retained their potency and reduced viral transmission in the presence of whole semen. No changes in tissue viability or immune activation were observed in colonic or ectocervical tissue after nAb exposure. Our data suggest that topically applied nAbs are safe and effective against HIV infection of mucosal tissue and support further development of nAbs as a topical microbicide that could be used for anal as well as vaginal protection.

BACKGROUND: Postmenopausal women have unique sociobiological human immunodeficiency virus (HIV) risks. We evaluated the safety, pharmacokinetics, and acceptability of a microbicide dapivirine (DPV) vaginal ring (VR) versus placebo in postmenopausal women. METHODS: We enrolled 96 HIV-negative postmenopausal US women in a phase 2a double-blind, randomized (3:1) trial of monthly VRs containing 25 mg DPV or placebo used continuously for 12 weeks. We assessed safety by adverse events (AEs). DPV concentrations were quantified in plasma and vaginal fluid. Steady-state concentrations were analyzed at 4, 8, and 12 weeks using repeated measures ANOVA. We assessed acceptability by self-report. RESULTS: We found no differences in the proportion of women with related grade 2 or higher reproductive system AEs (DPV: 6/72 (8%), placebo: 3/24 (13%), P = .68) or grade 3 or higher AEs (DPV: 4/72 (6%), placebo: 0/24 (0%), P = .57). In the DPV arm, 2/72 (3%) declined to resume product use due to AEs. Median DPV concentrations in plasma (262.0 pg/mL at week 12) and vaginal fluid (40.6 ng/mg at week 12) were constant over 12 weeks and exceeded the in vitro 50% effective concentration by 5000-fold in vaginal fluid by week 4. VR acceptability was high; 84/93 (90%) “very much liked or liked” the VR. CONCLUSIONS: DPV VRs were safe, well tolerated, and acceptable in postmenopausal women. Plasma concentrations were comparable to published data on DPV use in reproductive-age women (median plasma concentration: 264 pg/mL). Given the reassuring safety and pharmacokinetic data, the DPV VR is promising for preexposure prophylaxis in postmenopausal women. CLINICAL TRIALS REGISTRATION: NCT02010593.

Background Vaginal rings (VRs) are a promising approach for sustained delivery of antiretroviral (ARV) medication to prevent human immunodeficiency virus (HIV) infection in women. Combination ARV VRs could increase efficacy. Methods MTN-028, a phase 1 trial in 19 HIV-uninfected women, evaluated 2 VRs containing vicriviroc (VCV) and MK-2048. Participants were randomized 2:1 to a low-dose (VCV, 91 mg; MK-2048, 10 mg) or original-dose (VCV, 182 mg; MK-2048, 30 mg) ring used for 28 days. Safety was assessed by documenting adverse events (AEs). Drug concentrations were evaluated in plasma, cervicovaginal fluid (CVF), and cervical tissue samples. Results All AEs reported were grade 1 or 2, with no statistically significant differences in related genitourinary AEs or grade ≥2 AEs observed between arms (P = >.99). VCV/MK-2048 concentrations rose rapidly, with higher plasma area under the concentration-time curve (AUC) in the original-dose arm (geometric mean ratio, 3.29 for VCV and 1.49 for MK-2048) and similar AUCs across arms for CVF samples. Cervical tissue concentrations were higher in the original-dose arm (geometric mean ratio, 7.94 for VCV and 6.45 for MK-2048), with greater drug released based on residual drug levels. Plasma and CVF concentrations for both drugs fell rapidly after ring removal. Conclusions In this first study evaluating 2 doses of a combination VCV/MK-2048 VR, both rings were found to be safe and well tolerated. VCV and MK-2048 were detectable in plasma, CVF, and cervical tissue samples, and drug release and plasma drug exposure were higher for the original-dose than for the low-dose ring.

© 2018 Hughes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Cryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol. Methods and findings To find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1C/ min) with 10% dimethylsulfoxide (designated “cryopreservation”) and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol (“vitrification”). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59–84%]) than before (50% [38–62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones. Conclusions Cryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.

Background Evidence is lacking regarding whether vaginal pre-exposure prophylaxis with topical tenofovir (TFV) reduces the risk of rectal HIV acquisition. Setting Bronx, NY. Methods MTN-014 was a phase 1, cross-over, randomized sequence trial comparing the cross-compartment pharmacokinetics and pharmacodynamics of daily TFV reduced-glycerin 1% gel after 14 days each of rectal and vaginal application, with directly observed dosing and a 6-week washout period between phases. Results Fourteen HIV-uninfected women enrolled; 91% of doses were observed and 13 women completed all study procedures. TFV and TFV diphosphate (TFV-DP) were detected in most samples collected from the dosing compartment. After vaginal dosing, TFV was detected in 10/14 samples of rectal fluid (RF) (median 4.4 ng/sponge) and 1/13 rectal tissue samples (0.2 ng/mg); TFV-DP was detected in 2/13 rectal tissue samples at 59.8 and 76.5 fmol/mg. After rectal dosing, TFV was detected in 9/14 samples of vaginal fluid (median 1.1 ng/swab) and in 6/14 vaginal tissue samples (median below limit of quantification); TFV-DP was detected in 3/14 vaginal tissue samples at 17.3, 87.6, and 77.1 fmol/mg. Neither cervicovaginal lavage fluid nor RF collected 24 hours after rectal or vaginal dosing resulted in a statistically significant suppression of viral replication. Conclusions In this study of 14 days each of vaginal and rectal application of TFV reduced-glycerin 1% gel, we found only a small degree of cross-compartment distribution of TFV in RF and vaginal fluids and no pharmacodynamic activity in ex vivo testing. Although high TFV concentrations in the dosing compartment may be protective, low cross-compartment tissue concentrations are not likely to be protective.

Introduction Fast‐dissolving vaginal film formulations release antiretroviral drugs directly into vaginal fluid and may be as efficient at drug delivery yet more acceptable to women than gels. In this Phase 1 vaginal film study, the safety, acceptability, pharmacokinetics and pharmacodynamics of two doses of tenofovir (TFV) film and TFV 1% gel were compared to corresponding placebo formulations. Methods Seventy‐eight healthy HIV negative women were randomized to self‐insert daily vaginal film (10 mg TFV, 40 mg TFV or placebo) or 4 mL of vaginal gel (TFV 1% [40 mg] or placebo) for seven days. Grade 2 and higher adverse events (AEs) related to study product were compared across study arms using Fisher's exact test. Plasma TFV concentrations were measured before and 2 hours after last product use. Paired cervical and vaginal tissue biopsies obtained 2 hours after the last dose were measured to determine tenofovir diphosphate (TFV‐DP) concentrations and exposed to HIV in an ex vivo challenge assay. Acceptability was assessed through questionnaire. Results There was only one grade 2 or higher related AE, the primary endpoint; it occurred in the placebo gel arm. AEs occurred in 90% of participants; the majority (91%) were grade 1. AEs were similar across study arms. TFV concentrations in plasma and TFV‐DP concentrations in cervical and vaginal tissues were comparable between 40 mg TFV film and the TFV gel groups. There was a significant relationship between reduced viral replication and TFV‐DP concentrations in cervical tissues. Film users were less likely to report product leakage than gel users (66% vs. 100%, p

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 ≤ 400 mmol/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 = 10 nM). Over 12 month 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.

Problem To explore the impact of race and geographic region on biomarkers of HIV risk and vaginal health, differences in soluble immune mediators were measured in US versus African and US white versus US black women at enrollment into a phase 2 microbicide trial. Methods Levels of soluble mucosal immune mediators and inhibitory activity against E. coli, which may serve as biomarkers of risk for HIV and other genital tract infections, were quantified in cervicovaginal lavage (CVL) collected from HIV-uninfected women in the United States (n = 73) and Africa (n = 73). Differences between groups were analyzed with multivariable logistic regression models for dichotomous variables and linear regression models for continuous variables. Results Secretory leukocyte protease inhibitor, lactoferrin, human beta defensins, interleukin (IL)-8, and interferon-gamma-induced protein-10 were significantly higher in US compared to African women in multivariable analysis, but only IL-1β was significantly different between US white and black women. E. coli inhibitory activity did not differ among groups in adjusted analyses. Conclusion Differences in soluble mucosal immunity between US and African women may play an important role in women's risk for HIV and other genital tract infections and response to prevention strategies including vaginal microbicides and should be considered in future studies.

EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV.Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations.No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection.Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission.

To prevent the global health burdens of human immunodeficiency virus [HIV] and unintended/mistimed pregnancies, we developed an intravaginal ring [IVR] that delivers tenofovir [TFV] at ~10mg/day alone or with levonorgestrel [LNG] at ~20μg/day for 90 days. We present safety, pharmacokinetics, pharmacodynamics, acceptability and drug release data in healthy women. CONRAD A13-128 was a randomized, placebo controlled phase I study. We screened 86 women; 51 were randomized to TFV, TFV/LNG or placebo IVR [2:2:1] and 50 completed all visits, using the IVR for approximately 15 days. We assessed safety by adverse events, colposcopy, vaginal microbiota, epithelial integrity, mucosal histology and immune cell numbers and phenotype, cervicovaginal [CV] cytokines and antimicrobial proteins and changes in systemic laboratory measurements, and LNG and TFV pharmacokinetics in multiple compartments. TFV pharmacodynamic activity was measured by evaluating CV fluid [CVF] and tissue for antiviral activity using in vitro models. LNG pharmacodynamic assessments were timed based on peak urinary luteinizing hormone levels. All IVRs were safe with no significant colposcopic, mucosal, immune and microbiota changes and were acceptable. Among TFV containing IVR users, median and mean CV aspirate TFV concentrations remained above 100,000 ng/mL 4 hours post IVR insertion and mean TFV-diphosphate [DP] concentrations in vaginal tissue remained above 1,000 fmol/mg even 3 days post IVR removal. CVF of women using TFV-containing IVRs completely inhibited [94-100%] HIV infection in vitro. TFV/LNG IVR users had mean serum LNG concentrations exceeding 300 pg/mL within 1 hour, remaining high throughout IVR use. All LNG IVR users had a cervical mucus Insler score

To develop polymeric films containing dual combinations of anti-HIV drug candidate tenofovir, maraviroc and dapivirine for vaginal application as topical microbicides.A solvent casting method was used to manufacture the films. Solid phase solubility was used to identify potential polymers for use in the film formulation. Physical and chemical properties (such as water content, puncture strength and in vitro release) and product stability were determined. The bioactivity of the film products against HIV was assessed using the TZM-bl assay and a cervical explant model.Polymers identified from the solid phase solubility study maintained tenofovir and maraviroc in an amorphous state and prevented drug crystallization. Three combination film products were developed using cellulose polymers and polyvinyl alcohol. The residual water content in all films was10% (w/w). All films delivered the active agents with release of50% of film drug content within 30 min. Stability testing confirmed that the combination film products were stable for 12 months at ambient temperature and 6 months under stressed conditions. Antiviral activity was confirmed in TZM-bl and cervical explant models.Polymeric films can be used as a stable dosage form for the delivery of antiretroviral combinations as microbicides.

BACKGROUND Surrogate markers of HIV-1 pre-exposure prophylaxis and microbicide efficacy are needed. One potential surrogate is the antiviral activity in cervicovaginal lavage (CVL) after exposure to candidate products. We measured CVL antiviral activity in women using oral or vaginal tenofovir-based pre-exposure prophylaxis and correlated activity with drug and immune mediator levels. METHODS Inhibitory activity against HIV-1 and herpes simplex virus (HSV)-2 and concentrations of interleukin (IL)-1β, IL-6, IL-8, interferon-γ, induced protein 10 (IP-10), macrophage inflammatory protein (MIP)-1α, MIP-3a, lactoferrin, secretory leukocyte protease inhibitor, and defensins were measured in CVL obtained from 60 women at baseline and after 6 weeks of a randomized sequence of oral and topical tenofovir. CVL tenofovir concentrations were measured by mass spectrometry. RESULTS The number of women with CVL anti-HIV activity ≥ 90% increased significantly from 5.0% at baseline to 89.1% after daily use of 1% tenofovir gel (relative risk = 17.85, P < 0.001), but there was no increase after daily oral tenofovir. The CVL anti-HIV activity correlated with drug levels (Spearman correlation coefficient 0.64 after tenofovir gel; P < 0.001) but not with the concentrations of mucosal immune mediators. No increase in CVL anti-HSV activity was observed after either drug regimen, an observation consistent with the higher concentrations of tenofovir needed to inhibit HSV-2 infection. The CVL anti-HSV activity correlated with lactoferrin, defensins, IP-10, IL-8, and detectable levels of MIP-1α but not with drug levels. CONCLUSIONS CVL may provide a surrogate for local but not systemic drug efficacy and a tool to better understand mucosal factors that modulate antiviral activity in genital tract secretions.

The ex vivo challenge assay is being increasingly used as an efficacy endpoint during early human clinical trials of HIV prevention treatments. There is no standard methodology for the ex vivo challenge assay, although the use of different data collection methods and analytical parameters may impact results and reduce the comparability of findings between trials. In this analysis, we describe the impact of data imputation methods, kit type, testing schedule and tissue type on variability, statistical power, and ex vivo HIV growth kinetics. Data were p24 antigen (pg/ml) measurements collected from clinical trials of candidate microbicides where rectal (n = 502), cervical (n = 88), and vaginal (n = 110) tissues were challenged with HIV-1BaL ex vivo. Imputation of missing data using a nonlinear mixed effect model was found to provide an improved fit compared to imputation using half the limit of detection. The rectal virus growth period was found to be earlier and of a relatively shorter duration than the growth period for cervical and vaginal tissue types. On average, only four rectal tissue challenge assays in each treatment and control group would be needed to find a one log difference in p24 to be significant (alpha = 0.05), but a larger sample size was predicted to be needed for either cervical (n = 21) or vaginal (n = 10) tissue comparisons. Overall, the results indicated that improvements could be made in the design and analysis of the ex vivo challenge assay to provide a more standardized and powerful assay to compare efficacy of microbicide products.

Several microbicides, including nonoxynol-9 (N-9) and cellulose sulfate (CS), looked promising during early trials but failed in efficacy trials. We aimed to identify Phase I mucosal safety endpoints that might explain that failure. In a blinded, randomized, parallel trial, 60 healthy premenopausal sexually abstinent women applied Universal HEC placebo, 6% CS or 4% N-9 gel twice daily for 13½ days. Endpoints included immune biomarkers in cervicovaginal lavage (CVL) and endocervical cytobrushes, inflammatory infiltrates in vaginal biopsies, epithelial integrity by naked eye, colposcopy, and histology, CVL anti-HIV activity, vaginal microflora, pH, and adverse events. Twenty women enrolled per group. Soluble/cellular markers were similar with CS and placebo, except secretory leukocyte protease inhibitor (SLPI) levels decreased in CVL, and CD3(+) and CD45(+) cells increased in biopsies after CS use. Increases in interleukin (IL)-8, IL-1, IL-1RA, and myeloperoxidase (MPO) and decreases in SLPI were significant with N-9. CVL anti-HIV activity was significantly higher during CS use compared to N-9 or placebo. CS users tended to have a higher prevalence of intermediate Nugent score, Escherichia coli, and Enterococcus and fewer gram-negative rods. Most Nugent scores diagnostic for bacterial vaginosis were in N-9 users. All cases of histological inflammation or deep epithelial disruption occurred in N-9 users. While the surfactant N-9 showed obvious biochemical and histological signs of inflammation, more subtle changes, including depression of SLPI, tissue influx of CD45(+) and CD3(+) cells, and subclinical microflora shifts were associated with CS use and may help to explain the clinical failure of nonsurfactant microbicides.

OBJECTIVE Ex vivo HIV-1 challenge has been proposed as a bioindicator of microbicide product effectiveness. The objective of this study was to establish optimal parameters for use of female genital tract tissue in this model. DESIGN Ex vivo challenge involves in vivo product use, followed by tissue biopsy, and exposure of the tissue to HIV-1 in the laboratory. METHODS Paired ectocervical and vaginal biopsies were collected from 42 women, and 28 women had additional biopsies from each site collected after 5% lidocaine (n = 14) or chlorhexidine (n = 14) treatment. Tissues were transported immediately to the laboratory and exposed to HIV-1. HIV-1 infection was followed by p24 enzyme-linked immunosorbent assay on culture supernatants and at study end after weighing and fixing the tissue for immunohistochemistry to detect p24 expressing cells. RESULTS Although both tissue types were equally infected with HIV-1 based on the immunohistochemistry results, ectocervical tissues had significantly higher HIV-1 replication than vaginal tissues (P < 0.005). Lidocaine and chlorhexidine had minimal impact on HIV-1 infection and replication. Point estimates for p24 levels were defined for 95% probability of p24-positive tissues and were 3.43 log10 for ectocervical tissue and 2.50 log10 for vaginal tissue based on the weight-adjusted cumulative p24 end points. CONCLUSIONS Although similar proportions of ectocervical and vaginal tissues support HIV-1 infection, higher levels of HIV-1 replication were observed in ectocervical tissues. Defining point estimates for HIV-1 infection in fresh ectocervical and vaginal tissues provides valuable information for the evaluation of HIV-1 preventative treatments during early clinical studies.

Lactation studies are necessary evaluations of medications for reproductive-age women. We evaluated pharmacokinetics (PK), pharmacodynamics, safety, and adherence profiles associated with 7 days of 1% tenofovir (TFV) vaginal gel use during lactation. Tenofovir levels (maternal/infant serum, milk) and anti-HIV activity (milk), adverse events (AEs), and adherence were measured for 17 HIV-1-seronegative breast-feeding mother-infant pairs. Tenofovir use was well-tolerated and detected at low levels in maternal serum, milk, and infant serum but demonstrated no anti-HIV activity in milk.

Non-antiretroviral microbicide candidates were previously explored as a female-controlled method of preventing sexual transmission of HIV. These products contained non-HIV specific active compounds that were ultimately found to disrupt the vaginal epithelium, cause increased immune activation in the female genital tract, disturb vaginal flora, and/or cause other irritation that precluded their use as vaginal microbicides. Due to the failure of these first-generation candidates, there was a shift in focus to developing HIV pre-exposure prophylaxis and microbicides containing small-molecule antiretrovirals. Even with the limited success of the antiretroviral-based microbicides in clinical evaluations and no commercially available products, there has been significant progress in microbicide research. The lessons learned from previous trials have given rise to more rigorous preclinical evaluation that aims to be better at predicting microbicide efficacy and safety and to novel formulation and delivery technologies. These advances have resulted in renewed interest in developing non-antiretroviral-based microbicides, such as broadly neutralizing antibodies (for example, VRC01) and anti-viral proteins (for example, Griffithsin), as options for persons not wanting to use antiretroviral drugs, and for their potential to prevent multiple sexually transmitted infections.

Background: The ex vivo challenge assay is a bio-indicator of drug efficacy and was utilized in this randomized, placebo controlled trial as one of the exploratory endpoints. Fresh and cryopreserved tissues were evaluated for human immunodeficiency virus (HIV) infection and pharmacokinetic (PK)/pharmacodynamic (PD) relationships. Methods: HIV-negative women used vaginal rings containing 25 mg dapivirine (DPV)/100 mg maraviroc (MVC) (n = 12), DPV only (n = 12), MVC only (n = 12), or placebo (n = 12) for 28 days. Blood plasma, cervicovaginal fluid (CVF), and cervical biopsies were collected for drug quantification and the ex vivo challenge assay; half (fresh) were exposed immediately to HIV while the other half were cryopreserved, thawed, then exposed to HIV. HIV replication was monitored by p24 enzyme-linked immunosorbent assay from culture supernatant. Data were log-transformed and analyzed by linear least squared regression, nonlinear Emax dose–response model and Satterthwaite t test. Results: HIV replication was greater in fresh compared to cryopreserved tissue (P = 0.04). DPV was detected in all compartments, while MVC was consistently detected only in CVF. Significant negative correlations between p24 and DPV levels were observed in fresh cervical tissue (P = 0.01) and CVF (P = 0.03), but not plasma. CVF MVC levels showed a significant negative correlation with p24 levels (P = 0.03); drug levels in plasma and tissue were not correlated with HIV suppression. p24 levels from cryopreserved tissue did not correlate to either drug from any compartment. Conclusion: Fresh tissue replicated HIV to greater levels and defined PK/PD relationships while cryopreserved tissue did not. The ex vivo challenge assay using fresh tissue could prioritize drugs being considered for HIV prevention.

BackgroundUnderstanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible.Methods and findingsTo find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes.Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension.ConclusionsLeukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

Author(s): Hughes, Sean M; Shu, Zhiquan; Levy, Claire N; Ferre, April L; Hartig, Heather; Fang, Cifeng; Lentz, Gretchen; Fialkow, Michael; Kirby, Anna C; Adams Waldorf, Kristina M; Veazey, Ronald S; Germann, Anja; von Briesen, Hagen; McElrath, M Juliana; Dezzutti, Charlene S; Sinclair, Elizabeth; Baker, Chris AR; Shacklett, Barbara L; Gao, Dayong; Hladik, Florian | Abstract: BackgroundUnderstanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible.Methods and findingsTo find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension.ConclusionsLeukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

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 24 h exposure. The ED50 calculated from the formulations for IQP-0528 was ∼32 nM and for TFV was ∼59 nM and their inhibitory activity was not affected by semen. The ED50 of TFV in the combination gels was ∼100-fold lower than when calculated for the drug substance alone reflecting the activity of the more potent IQP-0528. When ectocervical and colorectal tissue were treated with the combination gels, HIV-1 p24 release was reduced by ⩾1 log10 and ⩾2 log10, respectively. Immunohistochemistry for the ectocervical tissues treated with combination gels showed no HIV-1 infected cells at study end. With the increased realization of receptive anal intercourse among heterosexual couples often in conjunction with vaginal intercourse, having a safe and effective microbicide for both mucosal sites is critical. The safety and efficacy profiles of the gels were similar for ectocervical and colorectal tissues suggesting these gels have the potential for dual compartment use.

HIV continues to be a problem worldwide. Topical vaginal microbicides represent one option being evaluated to stop the spread of HIV. With drug candidates that have a specific action against HIV now being studied, it is important that, when appropriate and based on the mechanism of action, the drug permeates the tissue so that it can be delivered to specific targets which reside there. Novel formulations of the nucleotide reverse transcriptase inhibitor tenofovir (TFV) and the nonnucleoside reverse transcriptase inhibitor UC781 have been developed and evaluated here. Gels with three distinct rheological properties were prepared. The three gels released both UC781 and TFV under in vitro conditions at concentrations equal to or above the reported 50% effective concentrations (EC 50 s). The drug concentrations in ectocervical tissues were well in excess of the reported EC 50 s. The gels maintain ectocervical viability and prevent infection of ectocervical explants after a HIV-1 challenge. This study successfully demonstrates the feasibility of using this novel combination of antiretroviral agents in an aqueous gel as an HIV infection preventative.

Objectives: Coital use of 1% tenofovir gel was shown to be modestly effective at preventing HIV transmission when applied vaginally in the CAPRISA 004 trial. Because the gel is hyperosmolar, which would reduce the integrity of the epithelium and induce fluid movement into the lumen, rectal use may not be acceptable. This study evaluated the pre-clinical safety and efficacy of a reformulated (reduced osmolality) tenofovir gel product. Methods: Reduced glycerine (RG)-tenofovir gel was compared with the original tenofovir gel for physiochemical characteristics, product safety and anti-HIV-1 activity. Results: The formulations were similar in all characteristics except for osmolality and spreadability/firmness. The RG-tenofovir gel had a 73% lower osmolality, a 29.6% increase in spreadability and a 27% decrease in firmness as compared with the original tenofovir gel. When applied to epithelial cell monolayers, tenofovir gel showed a transient reduction in the transepithelial resistance while the RG-tenofovir gel did not. Both gels retained ectocervical and colorectal explant viability. However, tenofovir gel treatment resulted in epithelial stripping that was absent after RG-tenofovir gel treatment of the polarized explants. Anti-HIV-1 activity was confirmed by lack of HIV-1 infection in polarized explants treated with either gel as compared with the control explants. Conclusions: Reducing the osmolality of the tenofovir gel resulted in improved epithelial integrity, which suggests better safety upon rectal use. The improved gel safety did not compromise drug release or anti-HIV-1 activity. These data support the use of this gel as a dual compartment microbicide.

Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is a potent and promising anti-HIV molecule. It is currently being investigated for use as a vaginal microbicide in two-dosage forms, a semi-solid gel and a silicone elastomer ring. Quick-dissolving films are promising and attractive dosage forms that may provide an alternative platform for the vaginal delivery of microbicide drug candidates. Vaginal films may provide advantages such as discreet use, no product leakage during use, lack of requirement for an applicator for insertion, rapid drug release, and minimal packaging and reduced wastage. Within this study, the in vitro bioactivity of dapivirine as compared with the non-nucleoside reverse transcriptase inhibitor UC781 was further established, and a quick dissolve film was developed for vaginal application of dapivirine for prevention of HIV infection. The developed film was characterized with respect to its physical and chemical attributes including water content, mechanical strength, drug release profile, permeability, compatibility with lactobacilli, and bioactivity. The anti-HIV activity of the formulated dapivirine film was confirmed in in vitro and ex vivo models. Importantly, the physical and chemical properties of the film as well as its bioactivity were maintained for a period of 18 months. In conclusion, a vaginal film containing dapivirine was developed and characterized. The film was shown to prevent HIV-1 infection in vitro and ex vivo and have acceptable characteristics which make this film a promising candidate for testing as vaginal microbicide.

Pyrimidinediones, a novel class of compounds, have previously been shown to possess antiviral activity at nanomolar concentrations. One member of this class of compounds, IQP-0528, was selected as the lead molecule for formulation development owing to its stability at physiologically relevant conditions, wide therapeutic window, and antiviral activity in the nanomolar range. Here, we report the development of two vaginal gels—3.0% hydroxyethyl cellulose (HEC) formulation and a 0.65% Carbopol formulation—for the sustained delivery of IQP-0528. Stability studies under accelerated conditions confirmed the chemical stability of IQP-0528 and mechanical stability of the gel formulation for 3 months. In vitro release studies revealed that diffusion-controlled release of IQP-0528 occurred over 6 h, with an initial lag time of approximately 1 h. Based on the drug release profile, the 3.0% HEC gel was selected as the lead formulation for safety and activity evaluations. The in vitro and ex vivo safety evaluations showed no significant loss in cell viability or significant inflammatory response after treatment with a 3.0% HEC gel containing 0.25% IQP-0528. In an in vitro HIV-1 entry inhibition assay, the lead formulation showed an 50% effective concentration of 0.14 g/ml for gel in culture media, which corresponds to 0.001 M IQP-0528. The antiviral activity was further confirmed by using polarized cervical explants, in which the formulation showed complete protection against HIV infection. In summary, these results are encouraging and warrant further evaluation of IQP-0528 gel formulations in in vivo models, as well as the development of alternative formulations for the delivery of IQP-0528 as a microbicide. The need for prophylactic strategies to prevent heterosexual transmission of human immunodeficiency virus (HIV) was recognized more than 20 years ago (30). Since then researchers from various fields have actively been developing technologies to combat the rapid spread of HIV. These strategies include pre-exposure prophylactics (28, 41) and microbicides (14, 31) designed to topically deliver single or combination antiviral agents. Currently, microbicide delivery systems under development include gels, rings, films, and suppositories. Of these systems, vaginal gels remain the preferred choice for the first line of microbicide product development. This can be attributed to the ease of vaginal gel development, the extensive work reported in the literature using this dosage form, and the existence of several vaginal semisolid products in the market. Microbicide administration via the vaginal route is advantageous since it allows local, noninvasive delivery of antiviral agents and enhanced biodistribution of drugs facilitated by the rich underlying blood supply (37, 40). Furthermore, vaginal gels can be self-administered, which is critical in the development of woman-controlled preventive strategies such as vaginal microbicides (37).