Since tenofovir gel was shown to reduce a woman's risk of HIV acquisition,1 there has been a renewed interest in vaginally applied microbicides for the prevention of HIV. Earlier generations of vaginal microbicides which relied on nonspecific mechanisms of action failed to demonstrate a protective effect2–6; however, the Centre for the AIDS Program of Research in South Africa (CAPRISA) trial established proof of concept that a vaginal gel containing an antiretroviral (ARV) could protect women from HIV. In the CAPRISA trial, coitally applied 1% tenofovir gel reduced the rate of HIV aquisition by 39% overall and 54% in the high adherence group. Tenofovir is just one ARV in this new class of microbicides which block specific steps in the viral life cycle; UC781, a nonnucleoside reverse transcriptase inhibitor (NNRTI), is another candidate microbicide.
Unlike tenofovir, UC781 has not been developed for the clinical care of HIV-infected individuals because of its low oral absorption. It is a tight binding NNRTI7 that binds both cell free and cell-associated HIV in vitro.8–10 A distinct advantage of UC781 over other ARVs is its potency against the virus. The inhibitory concentration of UC781 is 10 pmol/mL;11 by comparison, that of nevirapine is 100 pmol/mL. Another important feature of UC781 is its memory effect. Despite extensive washing after exposure to UC781, cells are still protected from HIV infection.10 Up to 1 week of cellular protection has been demonstrated in vitro for both cell-free virus and for cell-associated virus after a single exposure.
UC781 prevents HIV infection in rectal12 and cervical explant cultures.8 In a novel approach assessing efficacy, significant inhibition of HIV infection was observed in explant cultures after in vivo exposure to a single dose of rectally applied UC781.13 HIV-1–seronegative sexually abstinent men and women (n = 36) were randomized in a double-blind placebo-controlled trial comparing UC781 gel at 2 concentrations (0.1%, 0.25%) with placebo gel (1:1:1). Biopsies were taken after a 7-day at-home dosing schedule. Ex vivo infection of biopsies using 2 titers of HIV-1BaL showed marked suppression of p24 in tissues exposed in vivo to the higher dose of UC781; a trend toward suppression was seen with the lower 0.1% UC781 concentration.
In the first phase 1 UC781 vaginal exposure trial conducted, 48 sexually abstinent women were randomized to 1 of 4 treatment groups: placebo, 0.1%, 0.25%, and 1.0% UC781.14 Participants were exposed to 6 daily doses, and adverse events (AEs) were assessed up to 1 month after exposure. UC781 was generally well tolerated. In addition, little systemic absorption was noted. In the first completed rectal safety study of UC781, there was no difference in mucosal injury detected between UC781 and placebo groups.13
Before the current study, no human studies have attempted to describe the pharmacokinetic properties of this minimally absorbable drug, beyond looking for the presence of the drug in plasma. In addition to assessing safety and acceptability of a 1-time dose of UC781 0.1% gel for vaginal use, an important objective of this study was to quantify UC781 remaining in the vagina at variable periods postexposure to inform dosing schedules.
MATERIALS AND METHODS
This was a phase 1, 8-arm, single-site, randomized placebo-controlled trial with partially concealed group allocation comparing tolerance to and persistence of UC781 0.1% vaginal gel to placebo after a single timed exposure of different durations. The protocol was approved by the University of Pittsburgh Institutional Review Board.
A total of 60 HIV-uninfected women were enrolled from May 2007 through April 2008; they were recruited from gynecology clinics at Magee—Womens Hospital of the University of Pittsburgh Medical Center and the surrounding community. All participants provided written informed consent for screening and study procedures. Participants were 18–45 years of age and agreed to be sexually abstinent for 1 week after study product exposure. Exclusion criteria included pregnancy, hysterectomy, abnormal complete blood count with differentials or complete metabolic panel, abnormal colposcopic examination, anticipated vaginal bleeding during the first week after enrollment, and the use of antibiotics or antifungals in the 14 days preceding enrollment.
Women were randomized to 1 of 8 arms (Fig. 1) using a permuted block design with block size of 12 and a randomization ratio of UC781 to HEC placebo gel of 2:1. Type of study gel (UC781 or HEC placebo15) was double blinded. Colposcopists, but not other study staff, were blinded to duration of product exposure (0, 2, 4, or 8 hours).
Study participants received a single dose of UC781 0.1% vaginal gel or placebo gel at the study site and remained in the study area for the duration of the randomly assigned product exposure time. A cervicovaginal lavage (CVL) was used to remove all visible study products at the participant's designated time: 0, 2, 4, or 8 hours after product administration. Cervical and vaginal fluid samples were then collected using sterile Dacron swabs.
Participants returned for 3 follow-up visits as follows: 1 to 2 days, 1 week, and 1 month after exposure. At these visits, participants completed a medical history, pelvic exam, and laboratory evaluations including complete blood count with differential and complete metabolic panel. Colposcopy was performed at 1–2 days after exposure and at subsequent visits only if indicated by the evidence of deep epithelial disruption or other suspicious finding. Deep epithelial disruption was defined as a lesion that penetrated into and exposed the subepithelial tissue and possible blood vessels. If bleeding from the lesion was present, the disruption was to be recorded as deep, even if it appeared that only a minimal amount or tissue had been lost. Findings observed by colposcopy alone were not collected as AEs.
Local and systemic safety was assessed by eliciting AEs through history, physical exam, and laboratory evaluation. AEs were defined and graded according to the Division of AIDS Table for Grading Severity of Adult and Pediatric AEs, Verison 1.0. The number of AEs by body system and relationship to study product was tabulated; individual participants contributed once to the calculation of event rates.
Persistence of UC781 was determined by measuring UC781 levels in CVL and vaginal fluid swabs. CVL sample aliquots were mixed with 2 volumes of acetonitrile and vortexed vigorously. The suspension was centrifuged at 13000g for 20 minutes to remove precipitated proteins and other particulates and a defined aliquot of the clarified supernatant was removed and dried in vacuo. The dried residue was dissolved in 100 μL of acetonitrile and quantified for UC781 content by high performance liquid chromatography. Vaginal swab UC781 levels were directly measured; a median value of 100 μL per swab sample was used to calculate the quantity per μL. The lower limit of detection for CVL samples was 25 pmol/mL and 500 pmol/mL for the vaginal fluid swab. CVL and vaginal fluid swab drug levels were measured for both active and placebo arms. There were three positive CVL samples in the placebo group with quantities of drug ranging from 92 to 427 pmol/mL. Systemic absorption of UC781 was determined by measuring UC781 in plasma samples by liquid chromatography essentially as previously described.16
Participant opinions on product acceptability were collected via questionnaires immediately after product exposure and at the final visit using a Likert scale format.
Sample size was based on the assumption that for a given arm, if the true rate of a given toxicity endpoint was 5%, 9 women would provide 93% power to exclude toxicity endpoint rates greater than 45% given that the probability of observing zero or 1 event is less than 0.05 when the true rate is 45%. Four and 9 women per arm would assure that a 95% confidence interval for the difference between the placebo and active gel toxicity rates would have an upper limit no more than 26% when the true toxicity rates for placebo and active gel were both 5%. Student t test, Mann–Whitney U, Fisher exact, and χ2 for linear trend tests were used to compare results between groups.
One hundred seventy-five screening visits were conducted and 60 women enrolled. Forty women were randomized to the UC781 group and 20 to the placebo group. Of 300 scheduled visits, 298 were completed. Figure 1 outlines the disposition of study participants.
As described in Table 1, there was no difference with respect to demographic characteristics, sexual behavior, and familiarity with vaginal products and microbicides between study arms. In total, 87% of participants reported at least 1 AE during the course of study follow-up. Only 4 AEs were deemed either related or probably related to study product; these included vulvar pruritis, yellow vaginal discharge, external genital tingling, and external genital itching. The placebo and UC781 groups had a similar distribution of mild and moderate genitourinary events (Table 2). Twenty-three colposcopic findings were noted in 18 women, 5 in the placebo group and 13 in the UC781 group. These included erythema, petechiae, peeling, and 3 reports of edema. All findings were superficial and none required follow-up colposcopy. There was no change in cytokines pre- and postexposure between the UC781 and placebo groups. In the UC781 group, there was a decrease in the pro-inflammatory cytokines, IL-1 and IL-12, and an increase in IL-6 after product exposure. In the placebo group, there was a statistically significant decreased in IL-6 pre and postexposure (Table 3).
As expected, UC781 was detected in the plasma of only 2 (5%) women in the UC781 gel group, and the concentration was below the limits of quantification (2.5 ng/mL) in both participants. None of the participants in the placebo group had detectable drug in the plasma.
The primary objective of this study, however, was to determine the persistence of UC781 drug in the vaginal compartment. One day after exposure, UC781 was still detected in 93% of CVL samples and 42% of vaginal swab samples in relatively large concentrations (Table 4).
For both CVL and vaginal swab samples, the longer the duration of exposure, the lesser the drug was recovered immediately after exposure. Although the amount of UC781 recovered from the 0-hour CVL specimen was significantly greater than that of the later postexposure samples (P < 0.001), there were no statistically significant differences in drug recovered among the 2-hour, 4-hour, and 8-hour postexposure samples (P > 0.5). For vaginal samples, the only significant difference in the amount of UC781 recovered was between the 0-hour and 8-hour samples (P = 0.004).
Although only 20% of participants had ever been prescribed a vaginal product, the majority of participants reported that the product was comfortable to insert and denied problems with odor or leakage. The majority (80%) also reported that they would buy the product if it was proven to be effective and they perceived themselves at risk for HIV. Women expressed a variety of preferences for drug formulation and drug-dosing schedules.
This study demonstrated that a single timed exposure of 0.1% UC781 gel applied vaginally is well tolerated among HIV-seronegative women. The rate of AEs, including that of genitourinary irritation, was similar between UC781 and placebo arms and similar to rates of AEs published in other microbicide trials. In addition, colposcopic findings were reassuring. The correlation between specific colposcopic findings and an increased risk of HIV acquisition has not been demonstrated, however, epithelial disruption is the most concerning finding, as gross findings of epithelial disruption were associated with HIV acquisition in participants exposed to N-9.5 None of the participants in this study experienced deep epithelial disruption; all colposcopic findings of peeling were noted to be superficial. Furthermore, in the UC781 group, there was a decrease in the pro-inflammatory cytokines, IL-1 and IL-12 (−0.54 ± 0.71 pg/mL, −0.26 ± 0.65 pg/mL, respectively) and an increased in IL-6 (0.08 ± 0.44 pg/mL), after product exposure. Only the changes in IL-1 and IL-12 reached statistical significance. There was no association between length of exposure to UC781 and the amount of change in cytokine levels (P > 0.05). This study supports the findings from the first published safety study of UC78114 which reported that urogenital irritation was common in all treatment groups, but it was notably mild and transient.
The most unique aspect of this study was the pharmacokinetic design. To date, pharmacokinetic studies of candidate microbicides have largely been limited to measuring systemic absorption of the drug. To our knowledge, this is among the first published vaginal pharmacokinetic studies of a minimally absorbable drug.16,17 The challenges for determining persistence of this product in the vagina are obvious. Given that little systemic absorption of UC781 was expected and a tissue assay for quantifying UC781 does not exist, persistence had to be measured in nontraditional ways, namely determining CVL and vaginal fluid swab concentrations at various time points.
The results from this study suggest that UC781 persists in the vaginal fluid at levels that could block cell-free HIV for more than a day. Although 2 CVL samples were uninterpretable, likely due to interference of blood with the assay, 38 of 40 samples had detectable levels of UC781, 3 logs greater that the IC50. At 24 hours, even after the extensive vaginal washing during the CVL collection procedure 24 hours earlier, 93% of the second lavage samples had quantities of drug that were 500 times the IC50 concentration. The vaginal swab sample quantification confirmed the persistence of high quantity of drug, particularly considering that these samples were collected after a thorough lavage. The proportion of vaginal fluid swabs with quantifiable UC781 was less than that of CVL, but it should be noted that the limit of detection differed between the 2. Taken together, results of these assays from these sample types suggest that UC781 could be dosed a day before sexual exposure.
The persistence of drug in the vaginal lumen is particularly important considering that an important advantage of UC781 over tenofovir is its ability to bind cell-free virus. Unlike tenofovir, which requires intracellular metabolic activation, UC781 is able to inactivate isolated HIV-1 and cell-associated virus. UC781 is hydrophobic and passes through the HIV core membrane easily; once inside the virion, it is able to access to reverse transcriptase enzyme and inhibit initial HIV infection and replication should integration of the virion into the target cells occur. Whether declining levels in the lumen are attributable to absorption or loss is impossible to determine from this study.
The limited systemic absorption assessed in this trial supports findings observed in multiple animal experiments which demonstrated that UC781 does not readily distribute beyond the local mucosa and that systemic exposure is minimal after vaginal dosing.18 Limitations of this study include the single-dose exposure and the lack of a tissue assay which would accurately quantify the level of UC781 in HIV target cells in the female genital tract.
Significant absorption of a microbicide not only has the potential to expose healthy individuals to systemic toxic effects of the active agent or its metabolites, but also raises the concern about selecting for resistant virus. Drugs such as tenofovir have been shown to distribute systemically in human and animal models at concentrations that could be assoicated with antiviral activity though the potential for the development of resistance is unknown.19 These concerns are minimized with UC781 as its absorption is minimal. Furthermore, unlike first-generation NNRTIs that only require 1 mutation before viral resistance occurs, the virus usually undergoes at least 2 mutations before becoming resistant to UC781.20
Ultimately, limited systemic absorption coupled with high vaginal concentrations demonstrated by this study underscore the tremendous potential of UC781 for use as a topical microbicide. The presence of high concentrations of UC781 in CVL samples up to 24 hours after exposure suggests a window of protection after a single application and suggests that a daily dosing regimen is adequate to achieve luminal concentrations of drug that may confer protection. It should be noted that the reduction in luminal concentrations of drug overtime does not necessarily reflect absorption but may in fact be due to leakage. Sexual activity has been shown to impact the persistence of at least 1 vaginal microbicide in CVL21 and was not considered in the design of this study. Describing the vaginal pharmacokinetics and biologic activity of UC781 in sexually active participants is a reasonable next step in a thorough evaluation of this product. In addition, working toward a tissue drug assay would contribute substantially to informing dosing regimens.
Finally, in addition to being well tolerated, a single application of UC781 was acceptable to participants. A majority of participants reported that they would use the product were it shown to be protective against HIV. Importantly, only 9% of participants from the UC781 group reported that gel leakage would prevent future gel use. In addition, women expressed interest in a variety of formulations supporting plans to study film preparations. Prototype and clinical formulation for the vaginal films has been developed and has been tested for safety in pigtailed macqaques but not yet in humans.
UC781 is early in the drug development process, but given its safety profile and efficacy in vitro, it merits further investigation. However, when tenofovir was found to reduce the incidence of infections in the CAPRISA 004 study, the financial resources at CONRAD, the entity, which holds the licensing rights for both tenofovir and UC781 gel product, were prioritized to tenofovir gel development. As tenofovir gel and the dapivirine ring continue through the development pathway, the future role of UC781 and other NNRTIs as microbicide products will be clarified. A product like UC781 could offer complementary advantages to the first generation of ARV-based microbicides like tenovofir including decreased systemic absoprtion and its increased potency relative to tenofovir. In colorectal explants, the combination of NRTI-NNRTIs was more effective at preventing cellular and tissue infection than either agent alone.22 UC781 has in vitro and in vivo properties that make it a promising microbicide agent to add to the tool box of HIV prevention products.
1. Abdool Karim Q, Abdool Karim SS, Frohlich JA, et al.. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science. 2010;329:1168–1174.
2. Feldblum PJ, Adeiga A, Bakare R, et al.. SAVVY vaginal gel (C31G) for prevention of HIV infection: a randomized controlled trial in Nigeria. PLoS One. 2008;3:e1474.
3. Peterson L, Nanda K, Opoku BK, et al.. SAVVY (C31G) gel for prevention of HIV infection in women: a phase 3, double-blind, randomized, placebo-controlled trial in Ghana. PLoS One. 2007;2:e1312.
4. Skoler-Karpoff S, Ramjee G, Ahmed K, et al.. Efficacy of Carraguard for prevention of HIV infection in women in South Africa: a randomised, double-blind, placebo-controlled trial. Lancet. 2008;372:1977–1987.
5. Van Damme L, Ramjee G, Alary M, et al.. Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet. 2002;360:971–977.
6. Van Damme L, Govinden R, Mirembe FM, et al.. Lack of effectiveness of cellulose sulfate gel for the prevention of vaginal HIV transmission. N Engl J Med 2008;359:463–472.
7. Barnard J, Borkow G, Parniak MA. The thiocarboxanilide nonnucleoside UC781 is a tight-binding inhibitor of HIV-1 reverse transcriptase. Biochemistry. 1997;36:7786–7792.
8. Zussman A, Lara L, Lara HH, et al.. Blocking of cell-free and cell-associated HIV-1 transmission through human cervix organ culture with UC781. AIDS. 2003;17:653–661.
9. Fletcher P, Kiselyeva Y, Wallace G, et al.. The nonnucleoside reverse transcriptase inhibitor UC-781 inhibits human immunodeficiency virus type 1 infection of human cervical tissue and dissemination by migratory cells. J Virol. 2005;79:11179–11186.
10. Borkow G, Barnard J, Nguyen TM, et al.. Chemical barriers to human immunodeficiency virus type 1 (HIV-1) infection: retrovirucidal activity of UC781, a thiocarboxanilide nonnucleoside inhibitor of HIV-1 reverse transcriptase. J Virol. 1997;71:3023–3030.
11. Motakis D, Parniak MA. A tight-binding mode of inhibition is essential for anti-human immunodeficiency virus type 1 virucidal activity of nonnucleoside reverse transcriptase inhibitors. Antimicrob Agents Chemother. 2002;46:1851–1856.
12. Fletcher PS, Elliott J, Grivel JC, et al.. Ex vivo culture of human colorectal tissue for the evaluation of candidate microbicides. AIDS. 2006;20:1237–1245.
13. Anton PA, Saunders T, Elliott J, et al.. First phase 1 double-blind, placebo-controlled, randomized rectal microbicide trial using UC781 gel with a novel index of ex vivo efficacy. PLoS One. 2011;6:e23243.
14. Schwartz JL, Kovalevsky G, Lai JJ, et al.. A randomized six-day safety study of an antiretroviral microbicide candidate UC781, a non-nucleoside reverse transcriptase inhibitor. Sex Transm Dis. 2008;35:414–419.
15. Schwartz JL, Ballagh SA, Kwok C, et al.. Fourteen-day safety and acceptability study of the universal placebo gel. Contraception. 2007;75:136–141.
16. Lacey CJ, Wright A, Weber JN, et al.. Direct measurement of in-vivo vaginal microbicide levels of PRO 2000 achieved in a human safety study. AIDS. 2006;20:1027–1030.
17. Nel AM, Coplan P, Smythe SC, et al.. Pharmacokinetic assessment of dapivirine vaginal microbicide gel in healthy, HIV-negative women. AIDS Res Hum Retroviruses. 2011;26:1181–1190.
18. Patton DL, Sweeney YT, Balkus JE, et al.. Preclinical safety assessments of UC781 anti-human immunodeficiency virus topical microbicide formulations. Antimicrob Agents Chemother. 2007;51:1608–1615.
19. Mayer KH, Maslankowski LA, Gai F, et al.. Safety and tolerability of tenofovir vaginal gel in abstinent and sexually active HIV-infected and uninfected women. AIDS. 2006;20:543–551.
20. Buckheit RW Jr, Snow MJ, Fliakas-Boltz V, et al.. Highly potent oxathiin carboxanilide derivatives with efficacy against nonnucleoside reverse transcriptase inhibitor-resistant human immunodeficiency virus isolates. Antimicrob Agents Chemother. 1997;41:831–837.
21. Keller MJ, Mesquita PM, Torres NM, et al.. Postcoital bioavailability and antiviral activity of 0.5% PRO 2000 gel: implications for future microbicide clinical trials. PLoS One. 2010;5:e8781.
22. Herrera C, Cranage M, McGowan I, et al.. Reverse transcriptase inhibitors as potential colorectal microbicides. Antimicrob Agents Chemother. 2009;53:1797–1807.