Rectal microbicides are needed for individuals who engage in unprotected anal intercourse (UAI) [1–5]. The rectal compartment is highly vulnerable to HIV transmission with a conservative estimate of a 10–20 fold increased risk of HIV transmission associated with anal [6,7] compared with vaginal intercourse [8,9]; these data do not reflect the increased risk when non-HIV sexually transmitted infections are present. Women in the developed [10,11] and developing world [12–14] engage in anal intercourse at epidemiologically significant levels. It is, therefore, assumed that vaginal microbicides, once available, will also be used rectally and it will be important to know whether a microbicide that is safe in the vagina will also be safe in the rectal compartment. This is a lesson first learned with nonoxynol-9. Men who have sex with men (MSM) continue to engage in UAI [1,2] and this behaviour fuels the HIV epidemic in the developed world. Recently, epidemiological and anthropological studies have suggested that MSM associated UAI also occurs in the developing world [15,16•,17,18,19•] with predictable acquisition of HIV infection [20•].
Rectal safety of vaginal microbicides and sexual lubricants
As women may well use vaginal microbicides as lubricants to facilitate anal intercourse, often during the same sexual episode, it is important to determine the rectal safety of these products during the microbicide development process and, preferably, before large scale effectiveness studies are undertaken. Commercially available sexual lubricants are widely used by MSM as well as women and may not necessarily have an appropriate safety profile for anal intercourse [21,22••]. Further safety studies are indicated for this group of over-the-counter (OTC) lubricant products.
Rectal safety of vaginal microbicides
The first rectal safety studies evaluated a vaginal formulation of nonoxynol-9 in MSM. Tabet et al.  described mild rectal histological changes seen in participants receiving up to 6 weeks of nonoxynol-9 or placebo gel use. In contrast, marked epithelial exfoliation was seen after a single exposure to nonoxynol-9 in studies conducted by Phillips et al. [24,25] using rectal lavage and histology as endpoints. Epithelial reconstitution can occur within 1–8 h after exposure to nonoxynol-9 [25,26]. In the Tabet study samples were collected up to 12 h after nonoxynol-9 exposure but after only 15 min in the Phillips study. The implication of these studies is that rectal safety should be assessed after acute (within 1 h) and chronic (at least 7 days) product exposure.
There is increasing concern that repeated mucosal exposure to vaginal microbicides or rectal microbicides could induce subtle immunological changes in the vaginal or rectal mucosa, potentially increasing the risk of HIV transmission . As a consequence, it will be necessary to develop more sensitive, immunological safety biomarkers. Over time and with trial experience, these new indices may be reduced to the most predictive ones, but it is increasingly clear that the current panel of endoscopic appearance and histology may be inadequate.
A recent study, HPTN-056, investigated the biological variability of potential intestinal safety biomarkers [28•]. To assess stability and intra/inter-subject variability of specified indices, colorectal biopsies were collected at 15 and 30 cm from the anal verge from 16 participants on three occasions over a 4-week period in the absence of any microbicide exposure. Tissue was evaluated for biological variability of a broad range of parameters including histology, mucosal cytokine gene expression, rectal immunoglobulins, and mucosal T-cell phenotype. The study demonstrated that tissue from both sites was essentially equivalent and that the most stable parameters included mucosal cytokine gene expression and T-cell phenotype. The study also demonstrated that histological quantification of specific cell types was far more variable than using a pre-established qualitative score. Importantly, in contrast to the vaginal microbicides field, endoscopic appearance of the rectal mucosa was not a study parameter, given the experience in gastroenterology that the subtle endoscopic changes may have no clinical or histological significance.
The first microbicide product to undergo phase I rectal safety assessment with this broader range of safety biomarkers is the vaginally formulated microbicide, non-nucleoside reverse transcriptase inhibitor UC-781 . In the rectal safety study, following baseline blood and mucosal assessments, the participants received a single rectal dose of UC-781 gel with a flexible sigmoidoscopy 30 min later to assess acute mucosal responses . After a minimum of a 1-week recovery period, seven daily doses of UC-781 were administered followed by a final mucosal assessment. A unique feature of this study was the evaluation of intestinal tissue explants, exposed to UC-781 in vivo, to resist HIV infection in vitro . This design feature allows for preliminary assessment of microbicide efficacy as well as safety, before potentially proceeding to much larger clinical effectiveness studies.
Rectal safety of sexual lubricants
Sexual lubricants are widely used by MSM  and have even been considered as potential rectal microbicides themselves because some products appear to have in-vitro efficacy against HIV . Unfortunately, sexual lubricants may also have the capacity to induce rectal damage. This situation has been well documented for nonoxynol-9 containing lubricants but may also be seen with other commercially available products that do not contain nonoxynol-9 . Studies conducted in 2001–2003 among MSM in San Francisco documented that 26–67% had used nonoxynol-9 containing products, often in the absence of condoms [34,35]. There is a clear need for wider evaluation of the safety profile of sexual lubricants as well as increased health education around this issue.
Development of rectal specific microbicides
The development of a safe, effective, and acceptable rectal microbicide will require significant formative research in the areas of formulation science, preclinical evaluation, product distribution, compartmental pharmacokinetics, mucosal safety, and acceptability before moving towards effectiveness studies. There seems little doubt that MSM would use such products. MSM commonly use sexual lubricants to facilitate anal intercourse , are interested in participating in rectal microbicide trials , and would use products if commercially available [35,37].
Formulation is a pivotal component of rectal microbicide development, especially when one considers the dynamic compartmental differences between the lower intestine/rectum and the vagina. It will be a challenge to identify a single agent that will perform optimally in both compartments. Microbicides could be formulated as gels, suppositories, or douches . To date, the majority of rectal formulation work has focused on a microbicide gel formulation. Carballo-Dieguez et al. [39••] recently conducted a rectal microbicide volume escalation study in which participants were asked to insert increasing volumes of a rectal microbicide surrogate (5–50 ml of Femglide; Trumbull, Connecticut, USA) on three consecutive days. When an unacceptable volume was encountered the participants then evaluated the previously tolerated volume during anal intercourse. Using this approach, the investigators were able to demonstrate volume acceptability of up to 35 ml in couples engaging in anal intercourse [39••]. A practical and likely clinically relevant problem is that the majority of gel formulations are hyper-osmolar. It is known that hyper-osmolar products can induce epithelial damage [22••], probably an undesirable characteristic for rectal microbicides. Rectal douches might be another useful vehicle for microbicide delivery. In one US study 53% of HIV-negative and 96% of HIV-positive men douched prior to rectal sex [40•]. Again, this approach might not be safe if the douche is hyper or hypo-osmolar. If enema results turn out to be similar to published gel results (studies underway), an iso-osmolar enema/douche could be safer and potentially deliver a microbicide product to the left side of the colon.
There are limited preclinical data evaluating microbicide safety in the rectal compartment and the majority of data focus on nonoxynol-9. The nonoxynol-9 data have provided important insights concerning the intestinal mucosal response to microbicide-induced injury. With this factor as an agreed concern, the rectal injury model still needs tighter evidence linking epithelia and histological focal injury with actual increased HIV infection. In the future, murine, nonhuman primate and human explant models may provide this needed link.
Phillips et al.  demonstrated that rectal application of nonoxynol-9 in mice resulted in rapid exfoliation of intestinal epithelium within 10 min of product exposure. The changes were transient and histological examination of the intestinal biopsy samples collected at 1 h after nonoxynol-9 exposure appeared normal. The study also demonstrated a nonoxynol-9 dose-dependent increase in murine susceptibility to anorectal herpes simplex infection. Increasingly sophisticated humanized mouse models are providing exciting opportunities to screen microbicides for rectal safety and efficacy [41,42].
Preclinical evaluation of candidate microbicides for safety and efficacy has been conducted using human intestinal explants [43,44]. In these models, intestinal tissue explants are collected using endoscopy or harvested from surgical resection specimens. The explants can be exposed to product and evaluated for toxicity using histological techniques and/or the MTT assay (an assay that measures cellular mitochondrial toxicity as an index of product induced damage ). One limitation of intestinal explant safety assessment is that explants undergo profound architectural deterioration within 24 h of collection; consequently, any meaningful histological assessment of toxicity can only be conducted within this period.
Intestinal explants have also been used to demonstrate the efficacy of antiretroviral microbicide candidates such as tenofovir, UC-781, and TMC-120, alone and in combination. In these studies, intestinal tissue is exposed to the product in vitro and then virus is added to the model. Explants are then cultured for up to 2 weeks with repeated measurement of HIV-1 p24 in the culture supernatant to indicate the presence or absence of productive HIV infection . Despite the limitations of the system, explants can provide important preliminary safety and efficacy data on a microbicide candidate before moving into more expensive animal models.
Nonhuman primate studies
Dramatic intestinal epithelial exfoliation has been documented in macaques rectally exposed to nonoxynol-9 . Other candidate microbicides, however, including Buffergel, Savvy, and VivaGel appeared to be safe in the macaque rectal model [46–48].
Despite concerns about the feasibility of developing a safe and effective rectal microbicide, two macaque studies evaluating cyanovirin and tenofovir have demonstrated significant protection from rectal challenge with SIV/SHIV. In 2003, Tsai et al.  reported that adult male cynomolgus macaques that received a 2 ml dose of 1 or 2% cyanovirin gel 20 min before rectal exposure to SHIV89.6P were completely protected from infection. In contrast, all the animals receiving placebo or virus alone were infected. In the second study, Cranage et al.  exposed Indian rhesus macaques to rectal challenge with SIVmac251/32H. The macaques given tenofovir per rectum up to 2 h prior to virus challenge were protected from infection (n = 6) or had modified virus outcomes (n = 2) while all untreated macaques and three of four macaques given placebo gel were infected.
Imaging studies including MRI have been used to define vaginal microbicide product distribution . For rectal microbicides, Hendrix et al. [52••], have used MRI, single photon emission tomography (SPECT), computed tomography, and endoscopic pharmacokinetic sampling to carefully characterize the distribution of a semen simulant and microbicide candidate in participants undergoing simulated anal intercourse with an artificial phallus. The semen simulant was a 1: 1 combination of hydroxy-ethylcellulose gel (K-Y Jelly; Johnson & Johnson, New Brunswick, New Jersey, USA) and saline designed to have the same viscosity of coagulated semen. K-Y Jelly was used as the microbicide surrogate. Over a 4-h period, the semen surrogate introduced into the rectum after simulated anal intercourse showed significant retrograde movement into the descending colon. In 12% of SPECT studies, the semen simulant migrated as far as the splenic flexure, approximately 60 cm from the anal verge. This observation has led to skepticism about the feasibility of delivering an adequate volume of microbicide to protect regions potentially at risk of HIV transmission. Such pessimism is probably premature as gastroenterologists routinely prescribe topical products (gels, enemas, foams, and suppositories) that are able to deliver 5′ aminosalicylic acid (5′ASA) products to the left side of the colon in patients with ulcerative colitis [53,54]. Hopefully, pharmacological experience with the design and delivery of 5′ASA products can be used in the development of rectal microbicides.
A critical issue in prevention research is the determination of whether antiretroviral prophylaxis should be given orally, topically, or using both approaches. Varying degrees of protection against rectal viral challenge has been achieved by both routes [50,55,56•] but further studies are needed to document whether compartmental pharmacokinetics vary by route of administration and whether either route alone or both together are needed to achieve tissue and intracellular concentrations of drug that correlate with protection.
The design of phase I rectal safety studies of rectal specific formulations will be similar to the assessment of rectal safety of vaginal microbicides outlined above. Given the differences in the compartments' structure, however, histology, baseline inflammation and constitutive physiology, safety parameters will need to include many of the indices used in vaginal microbicides trials with the addition of assays that may better assess the vulnerable rectal linings. As well, rectal microbicides acceptability may be better characterized in participants who regularly engage in anal intercourse.
The current generation of rectal safety studies has used vaginal applicators or syringe-like devices to deliver test product to the rectal compartment. Clearly, this approach is suboptimal. Vaginal applicators are not designed for rectal insertion and have not been found to be acceptable in rectal safety studies . Carballo-Dieguez et al.  recently conducted a comprehensive interview-based qualitative assessment of the design requirements for a rectal applicator. This assessment has led to the production of a prototype model that will hopefully be evaluated in future rectal safety studies.
The last decade of vaginal microbicide development has generated significant experience in the logistical challenges associated with conducting microbicide effectiveness studies. Key issues are identifying populations with an adequate background annual rate of HIV seroincidence (usually considered to be about 3%), maintaining high adherence rates to study product, and avoiding interruptions in administration of study product due to events such as unplanned pregnancy. These lessons can be directly transferred to the design of future rectal microbicide effectiveness studies. Identifying suitable populations for these studies will be critical. Fortunately, HIV vaccine and antiretroviral pre-exposure prophylaxis studies have already identified populations of MSM at risk of HIV acquisition secondary to UAI. It is also important to include women who engage in anal intercourse in rectal microbicide development. It would be extremely challenging, however, to identify women with the required seroincidence rates who have a history of UAI.
Breban et al. have published a mathematical model that explores the impact of introducing rectal microbicides into a high-risk sexual zone for HIV transmission. The setting they chose was a ‘virtual’ bathhouse. Bathhouses are venues commonly found in Europe and North America where MSM congregate and often have sex with multiple partners [59,60]. In this model, the authors varied microbicide effectiveness and frequency of use by the bathhouse clients and assessed the reduction of new, secondary infections. After making appropriate adjustments for condom use, it was possible to demonstrate that if a product that was thought to be only 30% effective but was used by 30–50% of the clients, a rectal microbicide could prevent HIV dissemination within the bathhouse environment . Based on the macaque data, it should be possible to develop products with greater than 30% effectiveness especially those products that incorporate antiretroviral compounds.
A significant component of the increased interest in rectal microbicide development has been driven by the advocacy community. The International Rectal Microbicides Advocates (IRMA) group has lobbied extensively for increased funding support for rectal microbicide research while producing their own field research on lubricant use and preference as well as holding monthly educational teleconferences. IRMA recently published an overview of the rectal microbicides field, Less Silence, More Science , distributed at the 2008 International Microbicide Conference in Delhi, India.
Future priorities in rectal microbicides research are to optimize the design of phase I safety studies, to develop rectal specific formulations, and to define the operational requirements needed to evaluate rectal specific microbicides in effectiveness studies. These studies may not occur for another 5 years but the preparatory work needs to start now.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 602).
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