Vaginal microbicides: moving ahead after an unexpected setback

Introduction The need to develop additional HIV prevention tools, especially those that women can use, is urgent [1]. Microbicides are being developed for topical application inside the vagina or rectum to prevent infection with HIV and possibly other sexually transmitted infections. Women are often limited in their ability to abstain from sex or convince their male partners to adopt safer sex behaviours owing to social, cultural and economic gender inequalities. Furthermore, the importance of having children is a major obstacle to condom use for many women and couples, and noncontraceptive microbicides would give them an option to protect themselves from HIV while trying to conceive. Candidate microbicides Cellulose sulfate In January 2007, the Contraceptive Research and Development Program (CONRAD) and Family Health International announced that they had halted two phase 3 clinical trials of the candidate microbicide cellulose sulfate gel (Ushercell, Polydex, Canada) [2]. The independent Data Monitoring Committee for the CONRAD trial in South Africa, Benin, Uganda and India had detected a trend towards increased HIV risk associated with cellulose sulfate use, whereas that for the Family Health International trial in Nigeria had not detected any increased risk. At the time of the closures, cellulose sulfate was one of four vaginal microbicide candidates in phase 2B/3 efficacy trials for HIV prevention (Table 1) [3]. The other three candidates (Carraguard gel, PRO-2000 gel and BufferGel) are currently still in clinical development.Table 1: Phase 2b/3 microbicide trials completed, stopped, and in the field.The news about the premature closure of the cellulose sulfate trials was disappointing and unexpected. Cellulose sulfate, a polyanion, showed good activity in preclinical studies against HIV, herpes simplex virus type 1 and 2, human papillomavirus, Neisseria gonorrhoeae, Chlamydia trachomatis and Gardnerella vaginalis, and no activity against lactobacilli [4,5]. Furthermore, it was found to be safe in several safety and contraceptive efficacy trials involving 518 women (including HIV-positive women) and 48 men in the treatment arms that were conducted before the phase 3 trials were initiated [6–10]. Most studies used the sexual lubricant KY jelly (Personal Products Co, Skillman, New Jersey, USA) as the placebo gel, with gel used one to four times daily for 6 days to 6 months. No or minimal differences between the cellulose sulfate group and the placebo jelly group were found in self-reported symptoms, genital epithelial findings assessed by colposcopy, vaginal infections, proinflammatory cytokines in genital fluid and systemic safety laboratory findings. One study, however, found changes in the vaginal flora, with an increase in Escherichia coli in the cellulose sulfate group and decreases in hydrogen peroxide-producing lactobacilli in both groups [9]. The phase 3 trial of cellulose sulfate for HIV prevention was placebo-controlled, randomized and blinded [10]. The targeted sample size was 2574 HIV-negative women based on an assumed 50% effectiveness of cellulose sulfate and 4% annual HIV incidence in the control arm. A total of 66 HIV endpoints were expected and clear stopping rules were defined. When the Data Monitoring Committee met, 1333 women had been enrolled and 35 seroconversions had taken place at the African sites, where the annual HIV incidence was higher than 4%. As many before us have pointed out, the trial investigators and teams deserve praise for immediately closing the trials, recalling study gels as fast as possible, ensuring that women who tested HIV positive in the trials received good HIV-related care and transferring participants from trial services to adequate services in the community. Nonoxynol-9 Cellulose sulfate is not the first product to fail in the microbicides field; nonoxynol-9 (N-9), a nonionic surfactant, preceded it. When N-9 products were first tested for anti-HIV activity in clinical trials, they had already been used for over 25 years in spermicides and sexual lubricants (in concentrations ranging from 2 to 12%), they were cheap, and data on anti-HIV activity from in-vitro and animal models were promising [11,12]. In the 1990s, safety studies of different formulations (such as film, gel, and suppository) of N-9 were conducted [11–14]. Low-dose N-9 was considered safe and two phase 3 trials were launched. A phase 3 trial of 70 mg N-9 film showed no effect against HIV [15]. A phase 3 trial of 52.5 mg N-9 gel showed no effect against HIV at low frequency of use and an increased risk of HIV when used more than three to four times per day [16]. In-vitro studies that were conducted after these phase 3 trials had been completed confirmed that the therapeutic window of N-9 is very narrow (N-9 displays anti-HIV activity at doses that are cytotoxic for epithelial cells and lymphocytes); that N-9 cytotoxicity varies by concentration, duration of exposure and repeated exposure; and that N-9 use is associated with vaginal colonization of Escherichia coli, Enterococcus and anaerobic Gram-negative rods [11,17]. Additional studies showed that N-9 induced an inflammatory response characterized by increases in levels of cytokines, chemokines and other inflammatory mediators; recruitment of neutrophils and monocytes into the genital tract; and activation of HIV transcription in infected cells by upregulation of transcription factors NF-κB and activator protein-1 [11,18,19]. In the current scientific and regulatory environment, it is unlikely that N-9 would have progressed further than preclinical evaluation. C31G or Savvy Another surfactant, C31G or Savvy, is also no longer being developed as a vaginal microbicide. Two phase 3 trials of Savvy, in Ghana and Nigeria, were discontinued in 2005/2006 because HIV incidence at the sites was too low. Potential explanations for trial results in the direction of harm Microbicide trial results in the direction of harm can be explained in multiple ways. The most obvious explanation is that the candidate microbicide increases women's vulnerability to HIV. However, results in the direction of harm could also be caused by differences between study arms in nonadherence with study products and/or condom use, particularly if the candidate microbicide is coitally dependent and less efficacious than condoms. For example, if a candidate microbicide gel is more acceptable to women than the placebo gel, gel use may be higher and condom use lower in the microbicide arm compared with the placebo arm. Finally, the placebo could be more protective than the candidate microbicide or trial results could be explained by statistical chance. The last two explanations are unlikely and the remainder of this review will, therefore, focus on the first two. Increased vulnerability: are pieces of the safety puzzle missing? After vaginal exposure, HIV is capable of establishing infection through multiple pathways involving a variety of target cells and receptors [5]. The relative importance of each pathway is not yet clear. The virus can cross the epithelium by infecting epithelial cells, by transcytosis through epithelial cells, by epithelial transmigration of infected donor cells, via uptake by Langerhans cells, and by direct entry through epithelial disruptions. Crossing the epithelium is likely easier when there are fewer layers of epithelial cells, such as the one layer of columnar epithelium in the cervix as opposed to the multilayer squamous epithelium in the vagina [20]. In the submucosa, the virus can infect CD4 T cells, dendritic cells and macrophages. It can use the gp120 receptor in conjunction with the CXCR4 or CCR5 coreceptor, but it can also attach to mannose-binding C-type lectins such as DC-SIGN. The ideal microbicide should be able to cope with this variety of HIV infection mechanisms but should also be able to block HIV replication and release once HIV is intracellular; this may require a combination of several active ingredients in one formulation [21]. Microbicide-induced enhancement of HIV entry Cellulose sulfate is a polyanion. Polyanion acitivity was first identified in experiments using CXCR4 viruses, in which they were able to bind to positively charged regions of gp120, thereby inactivating cell-free virus [4,22,23]. However, recent studies showed that most sexually transmitted HIV-1 strains use the CCR5 coreceptor for transmission, and these CCR5 viruses have significantly lower levels of exposed positive charges on their gp120 [24]. The current understanding is that polyanions are most effective against CCR5 viruses following conformational change induced by CD4 binding, which exposes the basic charge regions in gp120 (Fig. 1). Therefore, to be effective against CCR5 transmission, polyanions must be present at the site of virus–target cell interaction within mucosal tissue. Even then, such protection could be circumvented by the rapid transport of virus from mucosal sites by migratory dendritic cells [21]. The degree of tissue penetration required to block viral attachment and fusion is not known. However, current polyanion candidates were selected on the basis of undetectable mucosal absorption to minimize systemic side effects, with cellulose sulfate (2000 kDa) being the largest in its class.Fig. 1: Current understanding of polyanion mechanism of action. X4, CXCR4 coreceptor; R5, CCR5 coreceptor.While these new insights may explain why cellulose sulfate lacked efficacy, they do not explain a potential increased susceptibility to infection. One of the ways in which a candidate microbicide could increase a women's vulnerability to HIV is by increasing HIV binding to target cells, increasing HIV entry in permissive cells and/or facilitating cell-associated transepithelial infection. To rule out these possibilities, the US Food and Drug Administration (FDA) recommends cell-based antiviral activity and cytotoxicity assays using a variety of cell lines (laboratory cell lines, peripheral blood mononuclear cells, primary macrophages and dendritic cells) and different laboratory and clinical virus isolates (including both cell-associated and cell-free systems, and CXCR4 and CCR5 viruses) under conditions likely to be encountered in real life (e.g., in the presence of semen and vaginal secretions) [25]. In the case of cellulose sulfate, the FDA-recommended assays did not give cause for concern, but CONRAD is considering additional nonclinical studies to further investigate potential mechanisms of harm [26]. Ex-vivo human explant studies and in-vivo animal models are increasingly being used to compare the efficacy and safety of different candidate microbicides, and new in-vitro, ex-vivo and in-vivo models continue to be developed [25]. For example, recent human cervical explant studies suggest that the smaller polyanion candidate PRO-2000 (5 kDa) can inhibit both localized infection and dissemination pathways [23]. The most relevant model of human efficacy is likely to be the macaque model of vaginal CCR5 transmission. Recently, two compounds (PSC-RANTES and CMPD-167) have demonstrated good protection against CCR5-utilizing SHIV162P3 in this model [27,28]. Some argue that all candidate microbicides that are currently in phase 3 trials should first have been evaluated in this model [29]. The problem with newer models is that their utility and reproducibility in different hands and under different study conditions have not yet been fully explored, and results have not yet been correlated with clinical trial results. The experiences with N-9 and cellulose sulfate may provide an opportunity to validate these models further. Microbicide-induced changes in genital epithelial integrity and/or permeability Repeated exposure of the cervicovaginal mucosa to a candidate microbicide may lead to changes in epithelial integrity and/or permeability, which could facilitate pathogen transmission instead of preventing it. In the microbicides field, in-vivo safety studies typically include rabbit and/or primate vaginal irritation models, employing histology, colposcopy and inflammatory cytokine assays to detect local toxic effects [25]. More recently, the rabbit vaginal irritation model has been refined [30], and in-vivo models have been expanded to correlate toxic effects with increased susceptibility to infection with a variety of sexually transmitted pathogens [11,19,31]. Genital epithelial findings and clinical signs of inflammation have always been important safety endpoints in clinical trials of candidate microbicides [32]. Attempts have been made to standardize the diagnosis and documentation of genital findings across trials, trial sites and trial clinicians [33,34]. In the case of cellulose sulfate, no evidence was found for visible genital irritation in any of the safety trials. However, microtrauma that is invisible to the naked eye was not evaluated in these trials but may also play a role in facilitating HIV transmission, but this was not evaluated in these trials [35]. Studies suggest that 12–45% of women not using any experimental vaginal products have visible genital epithelial findings upon pelvic examination [36]. Sexual intercourse and several factors that are common in women of reproductive age (such as increased parity, tampon use, traditional vaginal product use and vaginal infections) are correlated with such findings. Given these high background rates, the contribution of experimental vaginal products to the prevalence of genital epithelial findings is difficult to assess. Furthermore, placebo products (especially gels) may also cause genital epithelial findings. The clinical significance of many of these findings, including their potential significance in heterosexual HIV transmission, remains unknown. For all of these reasons, validated biomarkers of genital irritation could be useful. For example, microtrauma might be detected by measuring haemoglobin and red blood cells in cervicovaginal lavage specimens [35]. Epithelial breaches of sufficient size to allow red blood cells out would be of sufficient size to allow the entry of HIV and possibly even HIV-infected cells. Furthermore, the upper genital tract cannot be assessed by colposcopy. These new techniques, however, are currently limited by the absence of clear normative values, intersubject variability and lack of standardization. Ultimately, studies are needed to determine the relationships between visual genital findings and these types of biomarker in the absence of experimental product use, as well as their relationships with HIV acquisition. Experiments and studies with N-9 have shown that timing of safety measurements is important: for example, rectal application of N-9 leads to rapid exfoliation of sheets of epithelial cells (within 15 min of application) and regeneration of the epithelium takes place within hours [37]. Susceptibility to infection is increased immediately after the rapid exfoliation but also during the regeneration of the epithelium. Nonclinical safety experiments, and possibly also early safety trials in humans, should, therefore, include acute as well as chronic safety measurements. Microbicide-induced genital inflammation Repeated exposure of the cervicovaginal mucosa to a candidate microbicide may also lead to a subclinical inflammatory reaction, increased activation and/or dendritic and cells at mucosal chemokines and cytokines, which cells and and (such as other and of cells could be as CD4 cells are also target cells for The between proinflammatory and inflammation measurements were in microbicide trials but correlate with clinical signs and self-reported of genital inflammation were in one phase 1 trial of cellulose sulfate with no evidence for [9]. However, of inflammation and activation over and cytokine measurements are not typically out in microbicide trials and were not in any of the cellulose sulfate trials. concentrations in cervicovaginal lavage are difficult to different types of cytokine are at different time and after repeated exposure Furthermore, the cytokine at most mucosal under conditions has not been fully and cytokine levels may be by use of vaginal variability and other factors between different are and the clinical or significance of changes in or groups of cytokines is not known. It therefore, be to develop assays that the of candidate microbicides on mucosal as a opposed to measuring levels of and that protective and A understanding of mucosal to candidate microbicides is not important from a safety but could also be used to thereby mucosal and microbicide Microbicide-induced vaginal changes The last of to the effect of candidate microbicides on the cervical and vaginal The and of cervical is not in phase 1 safety trials for from However, the effect of candidate microbicides on lactobacilli is assessed in and in clinical trials using in the vaginal have been for cellulose sulfate, and Most of these changes were considered or and the activity against vaginal lactobacilli acceptable if the product sufficient [25]. However, the evidence for and as risk factors for HIV in women is and even changes of the vaginal may in with high HIV prevalence It is not clear levels of vaginal changes are acceptable in different and more of vaginal should be Microbicide trial that may explain results in the direction of harm No validated endpoints for the activity of microbicides currently Therefore, the primary of microbicide efficacy trials must be HIV incidence [32]. of a vaginal microbicide the of of women at risk of HIV infection through heterosexual The higher the HIV incidence in a trial the lower the required sample For reasons, trials are typically conducted in that a of HIV each For reasons, efficacy trials must the effect of the potential microbicide over and a of already HIV prevention that include HIV and condom and treatment of sexually transmitted and vaginal such an effect may be particularly for microbicides that have a lower expected efficacy than condoms. Another microbicide trial is that a placebo not Even a placebo typically not the active of the candidate it may or HIV transmission. 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