Microbicide safety and effectiveness: an overview of recent clinical trials

Poynten, Marya,*; Brown, Joelle Mb,*; Sovero, Monicab; Millwood, Iona Ya; Kaldor, John Ma

doi: 10.1097/COH.0b013e32830b0f4c
Microbicides: Edited by John Kaldor and Melissa Robbiani

Purpose of review: This review summarizes findings from recent vaginal microbicide safety and effectiveness trials and discusses the challenges associated with undertaking these trials.

Recent findings: In safety trials, there has been a focus on the development of biomarkers of genital irritation. Recent safety studies have expanded the range of genital toxicity measures to include biomarkers of mucosal immunity, detection of haemoglobin in cervicovaginal lavage specimens and microbicide-induced vaginal flora changes. Four effectiveness trials have been stopped prematurely, two due to a lower than estimated HIV incidence rate, one as a result of an interim analysis suggesting increased risk of HIV acquisition among participants receiving active product, and one as a safety precaution. One effectiveness trial was completed and showed no reduction in HIV acquisition among participants receiving active product, and one ongoing effectiveness trial was modified by discontinuing a trial arm. Methodological challenges faced by these trials have included accurately estimating HIV incidence and pregnancy rates in trial populations, and improving adherence to and measurement of study product use.

Summary: Validated safety and surrogate efficacy endpoints and standard ways of reporting them are being pursued. Focus has shifted to antiretrovirals containing microbicides, some of which may be used independent of coitus. Research on how to improve and measure adherence should continue.

aNational Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, Australia

bDepartment of Epidemiology, School of Public Health, University of California, Los Angeles, Los Angeles, California, USA

* Co-first authors.

Correspondence to Joelle M. Brown, PhD, Assistant Professor, Department of Epidemiology, School of Public Health, University of California, Los Angeles, Los Angeles, California, CA 90024, USA Tel: +1 310 794 6205; fax: +1 310 794 2808; e-mail: joellebrown@ucla.edu

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Topical microbicides are substances that are being developed for application to the vagina or rectum to prevent or substantially reduce the acquisition of sexually transmitted HIV infections. A number of microbicides under development also have contraceptive properties, and might prevent other sexually transmitted infections. No agent has, so far, been demonstrated to be effective as a microbicide. It is estimated that approximately 60–80 different products are currently in development including several undergoing safety and effectiveness trials (Table 1) [1–20]. Several published reviews provide excellent overviews of microbicide clinical research and the development pathway of candidate products [21•,22••,23–26,27••]. The purpose of this review is to provide an update on findings and trial-related issues from candidate vaginal microbicide trials-reported since the publication of Rosenberg's review in 2006 [26].

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Results from recent clinical trials

A number of safety and effectiveness trials of candidate vaginal microbicides have been recently reported. Below, we briefly describe these studies and their findings.

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Entry/fusion inhibitors

Two phase III trials of 6% cellulose sulfate, conducted by Family Health International (FHI) and by the Contraceptive Research and Development (CONRAD) program, were closed in January 2007 when a planned interim analysis of the CONRAD trial data found more HIV infections among women randomized to cellulose sulfate gel compared with placebo. No difference was seen in the smaller FHI trial, but as a safety precaution the FHI trial was also closed. In the final analysis there were 25 new HIV infections in the CONRAD trial and 10 in the FHI trial among the women randomized to cellulose sulfate, compared with 16 and 13 in the placebo groups, respectively. These differences did not reach statistical significance in the intent-to-treat (ITT) analyses [28,29].

Past safety trials have found cellulose sulfate to be well tolerated. A recently published study of cellulose sulfate used four times daily for 14 days found that the incidence of genital epithelial disruption, candidiasis and bacterial vaginosis was low and similar to KY Jelly (Johnson & Johnson, New Brunswick, New Jersey, USA) use [30]. Rates of genital irritation were common and comparable in a 6-month trial comparing cellulose sulfate or KY Jelly used with diaphragm, and cellulose sulfate gel alone. Of 13 women with severe colposcopic findings, 12 were in the diaphragm groups. As all findings were located on the external genitalia, it was thought they might relate to insertion and removal of the diaphragm [31]. A study in HIV-positive men found mild genital irritation was more common with cellulose sulfate use (42%) than placebo (8%) [32].

Recent safety studies of polynaphthalene sulfonate PRO2000/5 (Indevus Pharmaceuticals, Lexington, Massachusetts, USA) included an evaluation of inflammatory cytokine and mediator protein levels in cervicovaginal lavage (CVL) samples collected after gel application. In these studies, 0.5% PRO2000/5 did not induce a persistent inflammatory response among HIV-uninfected [33•] or HIV-infected women [2], and there were no other adverse findings compared with placebo. An assessment of virucidal activity in CVL samples after one application found 0.5% PRO2000/5 significantly inhibited HIV and HSV infection by at least 1000-fold [2]. CVL samples from these participants and others were then used to measure the activity of 0.5% PRO2000/5 and cellulose sulfate against HSV-2 infection in the presence of seminal plasma. Seminal plasma significantly interfered with anti-HSV-2 activity of both agents. The implication of these in-vitro findings is not yet clear, and will need further investigation in future trials of PRO2000/5 and other agents that are still in development [34].

PRO2000/5 is currently being evaluated in two effectiveness trials conducted by the Microbicide Trials Network (MTN) and the Microbicides Development Programme (MDP), with results expected in 2009. The MTN trial has 0.5% PRO200/5, BufferGel (ReProtect Inc., Baltimore, Maryland, USA), placebo and condom only arms. The MDP trial was designed to compare 0.5% and 2% PRO2000 with placebo. In February 2008, however, the 2% arm was discontinued after the MDP independent data monitoring committee noted that there was no more than a small chance that 2% PRO 2000 would show protection against HIV infection compared with placebo in that study [20].

Carraguard (Population Council, New York, New York, USA), a sulfated polysaccharide gel, did not appear to increase pro-inflammatory cytokine levels in HIV negative women [35•], or viral load, genital inflammation, epithelial sloughing, or microhaemorrhage among HIV-positive women [36]. A phase II trial demonstrated comparable rates of genital epithelial findings, bacterial vaginosis and candidiasis in HIV-negative women using Carraguard or placebo [37]. The evidence that Carraguard is safe and well tolerated for use up to four times a week makes all the more disappointing results from the recently reported phase III trial, which did not show evidence that precoital Carraguard protects women from HIV acquisition [38]. There were 3.3 HIV infections per 100 person-years (134 new cases) among women randomized to Carraguard, compared with 3.7 per 100 person-years (151 new cases) on placebo, a difference that did not reach statistical significance in the ITT analysis. Although 96% of participants reported using a gel with last sex, researchers utilized a staining method of verifying vaginal applicator use [39–41] and calculated that only 10% of women used the study gel 100% of the time, 20% used it on around 75% of occasions, and 30% used it on less than 25% of occasions [42]. Overall, the researchers estimated that only 44% of sex acts involved use of study gel.

In HIV-negative men, 3% w/w SPL7013, a dendrimer-based product, was reported as well tolerated and not systemically absorbed [43]. A phase I safety trial of 3% w/w SPL7013 used twice daily among sexually abstinent young women in the USA and Kenya was completed in December 2007.

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Two effectiveness studies of the surfactant 1% C31G conducted in Nigeria and Ghana [45••,46••] were prematurely discontinued on the basis of lower than anticipated HIV incidence and, therefore, insufficient power to evaluate effectiveness against HIV acquisition. In ITT analyses, the hazard ratio for HIV acquisition among women using C31G compared with placebo was 0.88 (95% CI 0.33, 2.27) in the Ghana trial [46••] and 1.7 (95% CI 0.9, 3.5) in the Nigeria trial [45••]. C31G gel is no longer being developed as a microbicide. The Invisible Condom (Laval University, Quebec City, Quebec, Canada) contains 2% sodium lauryl sulphate, an anionic surfactant. In an open label phase I study, symptoms of mild genital irritation were common (itching in 56% of women), as were erythema and discharge (27%). A rise in average vaginal pH was reported during product use. Adverse events were rare in male sexual partners [44].

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Vaginal defense enhancers

BufferGel (ReProtect, Baltimore, Maryland, USA) and Acidform (Amphora; Instead, La Jolla, California, USA) are designed to maintain an acidic vaginal pH. Recent safety studies have evaluated BufferGel and Acidform in combination with cervical barrier protection. There is biological plausibility that cervical barriers, like diaphragms, may protect women from HIV as the cervical epithelium is columnar and thinner, and thus more susceptible to pathogens than that of the vagina. The lack of an additional protective benefit of the diaphragm and lubricant gel in preventing HIV acquisition in the large scale Methods for Improving Reproductive Health in Africa (MIRA) trial was discouraging but may have been due to methodological challenges and differential condom use between study arms [47•].

In a phase I study, the diaphragm used with BufferGel, Acidform, or KY Jelly was well tolerated. Mild genital irritation and moderate colposcopic findings were slightly more frequent in the BufferGel and Acidform groups compared with KY Jelly [48]. A contraceptive effectiveness trial of BufferGel with diaphragm demonstrated no significant difference in colposcopic findings or genital symptoms compared with the control arm [13]. The control for this study was the diaphragm used with nonoxynol-9 (N-9), which causes genital epithelial disruption with frequent use [49]. In a study of the BufferGel Duet (an oval shaped device containing 10 g of BufferGel that acts as an applicator and a cervical barrier), seven events of abdominal or genital pain/tenderness were reported in 29 women. Colposcopic findings were common (79% of women), with superficial peeling of the cervicovaginal epithelium being the most common finding (62% of women) [50]. BufferGel is currently being evaluated in phase II/IIB effectiveness trial.

A study of the safety of 10% and 20% diluted lime juice [51] found significantly more burning and dryness and an increase in the chemotactic cytokines IL-8 (CXCL8) and MIP-1β (CCL4) among women using 20% lime juice, confirming earlier findings of increased toxicity at higher concentrations [52]. In an important example of the need to integrate clinical and preclinical findings, an evaluation of the virucidal activity and cytotoxicity of lime juice showed that concentrations lower than 50% are unlikely to inactivate HIV, but do lead to significant toxicity as measured by cervicovaginal and penile tissue models, as well as in clinical studies [53].

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Highly active antiretroviral drugs

Antiretroviral (ARV)-based agents represent the new generation of candidate microbicides. One potential advantage of these agents is that they can be used independent of coitus [25]. If the concern regarding selection of drug-resistant HIV variants with subtherapeutic drug levels is confirmed, it may be overcome by the use of combinations of microbicides with complementary mechanisms of action [22••].

Tenofovir, a nucleoside reverse transcriptase inhibitor (NRTI), was reported to be well tolerated in a phase I study among women using gel concentrations of 0.3% and 1% [54]. In addition, in a 6-month phase II trial of daily and precoital use of 1% tenofovir gel among 200 HIV-negative women, tenofovir was reported to be safe. Adherence and acceptability were high overall [55]. One percent tenofovir gel is currently being evaluated for safety and effectiveness in a phase II/IIb study.

Three concentrations of the nNRTI, TMC120 (dapivirine) were reported to be safe and well tolerated in a phase I study among HIV-negative and HIV-positive women [56•]. After 7 days of use, three-quarters of women had detectable plasma levels albeit very low. The implication for nNRTI resistance with such low levels is unclear. Another phase I study of TMC120, used twice daily for 42 days at three dose levels reported this agent to be safe and well tolerated in HIV-negative women [57].

UC-781, another nNRTI, is currently being developed as both a vaginal and rectal microbicide. Dose levels of 0.1%, 0.25%, and 1.0% UC-781 gel were well tolerated in 48 sexually abstinent women. Urogenital irritation, generally mild, was common in all UC-781 treatment groups. Two participants in the 1% group had very low, detectable plasma levels of UC-781 [58•].

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Other product classes

Praneem (Panacea Biotec, New Delhi, India) polyherbal formulations containing purified extracts of Azadirachta indica (neem tree) have an uncertain mechanism, but show activity against HIV in vitro [59]. A Praneem pessary was well tolerated in a phase I study in HIV negative women [60]. A phase II study of 6 months precoital use in HIV-uninfected women found adverse events related to product use more frequent among Praneem tablet users, with transient genital discomfort the most common event [61].

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Issues in microbicide trials

A number of challenges have surfaced during the implementation of recently reported microbicide trials. Below we comment on challenges that relate to safety endpoints, HIV incidence, adherence and pregnancy.

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Safety endpoints

Genital epithelial findings identified on visual inspection have been the traditional endpoints of toxicity in clinical microbicide trials and the subject of guidelines for standardized interpretation [62–64]. Van de Wijgert et al. [65] provided additional data on the normal background rates of genital findings. In a cohort of 165 healthy women followed for a year, genital findings with intact epithelium were common at screening but genital findings with disrupted epithelium were infrequent, occurring in less than 3% of women. More general toxicity tables for the grading of female and male genital and rectal findings in microbicide studies have recently been developed [66].

Efforts are underway to identify biomarkers of genital tract inflammation, in addition, which might serve as more objective safety endpoints. There is growing evidence that microbicide use may induce immunological changes in the vaginal mucosa, which may in turn facilitate the transmission or acquisition of HIV [67]. The role of individual cytokines in assessing the genital epithelial safety of microbicides is yet to be established [67]. Bioassays or panels predictive of mucosal function as a whole, rather than individual immune mediators, may have more utility [27••]. Understanding the normal variation of these biomarkers within and across individuals, as well as the effect of hormonal status, menses, sexual activity, semen, vaginal products, and other factors on the expression of these biomarkers is critical [65], as these factors may also impact on a product's range of potential virucidal activity.

Other potential safety endpoints include the detection of haemoglobin and red blood cells in CVL specimens, as epithelial disruption sufficient for microhaemorrhage may allow transmission of HIV or HIV-infected cells [27••,49]. Bacterial vaginosis, vaginal pH and candidiasis are recognized from observational research to be associated with increased risk of HIV transmission. However, there is no consensus as to what rate of microbicide-induced vaginal flora changes would preclude progression to further clinical trials [27••].

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HIV incidence

Due to the lack of reliable and validated surrogate markers, HIV acquisition is the only endpoint that can be used to assess effectiveness. Effectiveness trials are typically conducted among populations with an HIV incidence of at least 2.5% [68] and several thousands of women are required to give statistically meaningful results [21•]. Accurately estimating HIV incidence in trial populations before trial implementation is often difficult [69]. Site preparedness activities have provided important information on HIV incidence, accrual and retention rates and attrition due to pregnancy. In one such preparedness study at four potential trial sites in Africa [70•], participant retention was high (86–97%) and condom use low (28%) among 958 women followed for 12 months. HIV incidence overall was 3.8 per 100 person-years, but varied between1.3 and 6.0 per 100 person-years between sites. The lower than expected HIV incidence at some sites highlights the need to be conservative in estimates for sample size in effectiveness trials [70•].

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Despite varied and rigorous attempts to counsel participants to use condoms and study gel with each act of intercourse, most of the recently reported phase III trials had higher than anticipated levels of nonadherence. Imperfect adherence reduces product effectiveness, making it more difficult to assess efficacy [71], highlighting the importance of defining and accurately measuring adherence, determining barriers to and strategies to enhance adherence, and employing techniques to account for imperfect adherence at the time of data analysis. Trials of microbicides have until now mostly relied on self-reported adherence estimates, which are influenced by recall and social desirability biases [21•]. More objective measures are being developed [21•] to corroborate self-reports of risk behavior and product use, including biomarkers of exposure to semen [27••,72] such as prostate specific antigen and Y-chromosome DNA testing and an assay to evaluate whether applicators have been exposed to the vagina [39–41]. With many of the newer ARV-based microbicides, adherence can be monitored by measuring ARV levels in genital tract tissues [27••], but the utility of this method will depend on factors such as product half-life and timing of last dose and study visit. Because of the effect of nonadherence on effect estimates from both ITT and per-protocol analyses [73–76], analytic methods such as structural nested modeling, which preserves randomization while allowing for arbitrarily complex (i.e. time-varying) adherence patterns [74,77•,78,79], offer a robust alternative for analyzing trial data with imperfect adherence. Research on how to improve and measure adherence should continue [67], and power calculations should be conservative with respect to anticipated rates of nonadherence [80].

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Pregnancy during trials is an important issue because women who become pregnant are required to stop product use due to a lack of data on fetal safety. This time ‘off-product’ can have a major impact on the statistical power of the study [21•]. Very high rates of pregnancy were seen in the cellulose sulfate and C31G trials ranging from 23–60 per 100 person-years corresponding to 5–10% of time off-product [28,29,45••,46••,81]. High pregnancy rates (20.2 per 100 person-years) also occurred in a site preparedness study at four African sites [70•]. Although the pregnancy rates in trials may be somewhat exaggerated due to increased frequency of testing resulting in the detection of subclinical pregnancies [81], pregnancies are unavoidable as women in trials are in their peak reproductive years. Importantly, the women most likely to become pregnant, (18–25 year olds), are those at higher risk of acquiring HIV infection [70•].

The provision of contraception both during and after trials is an important ethical consideration [70•]. How to successfully sustain posttrial contraception and fund its provision are questions that need to be addressed. Further research on reproductive toxicity of candidate microbicides may provide the basis for defining conditions under which pregnant women can remain in a phase II/III trial [69,71,82].

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Despite the numerous challenges faced in their development, efforts to identify safe, acceptable and effective microbicides must continue. Researchers are addressing issues related to toxicity measurement, validation of endpoints and nonadherence. These efforts have the ultimate aim of providing a much needed method of prevention of HIV infection.

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References and recommended reading

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Papers of particular interest, published within the annual period of review, have been highlighted as:

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• of special interest

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•• of outstanding interest

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Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 601–602).

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effectiveness trials; HIV prevention; microbicide; safety trials

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