The development of rectal microbicides for HIV prevention.

Review

The development of rectal microbicides for HIV prevention Ian McGowan 1.

Introduction

University of Pittsburgh, Department of Medicine, Pittsburgh, USA

2.

The biology of HIV infection

3.

The rationale for rectal

Introduction: Individuals practicing unprotected receptive anal intercourse are at particularly high risk of HIV infection. Men who have sex with men in the developed and developing world continue to have disproportionate and increasing levels of HIV infection. The last few years have seen important progress in demonstrating the efficacy of oral antiretroviral pre-exposure prophylaxis, vaginal microbicides, and treatment as prevention, but there has also been significant progress in the development of rectal microbicides for HIV prevention. Areas covered: The purpose of this review is to summarize the status of rectal microbicide research and to identify opportunities, challenges, and future directions in this important field of HIV prevention research. The design of completed and ongoing Phase I rectal microbicide studies that include the generation of comprehensive pharmacokinetic/pharmacodynamic data may allow for more rational decisions about which rectal microbicides should be advanced to later stage development. Expert opinion: There is a strong rationale for the development of rectal microbicides for HIV prevention. Preclinical data provide supportive evidence for the feasibility of this approach, and there is significant interest in rectal microbicide development from communities at risk of HIV acquisition through unprotected receptive anal intercourse in both the developed and developing world. Demonstration of sustained safety, acceptability, and product adherence in the MTN-017 Phase II study of tenofovir 1% gel will be an important step in rectal microbicide development and will hopefully lead to Phase III effectiveness testing of this novel HIV prevention strategy.

Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by University of Maastricht on 06/15/14 For personal use only.

microbicide development 4.

Rectal microbicide formulation considerations

5.

Formulation preference studies

6.

Product distribution studies

7.

Candidate molecules for rectal microbicide development

8.

Phase I rectal microbicide studies

9.

Other ongoing rectal microbicide studies

10.

Conclusion

11.

Expert opinion

Keywords: antiretroviral, HIV infection, HIV prevention, maraviroc, mucosal immunology, rectal microbicides, tenofovir, UC781 Expert Opin. Drug Deliv. (2014) 11(1):69-82

1.

Introduction

The first cases of human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS) were recognized in 1981 among men who have sex with men (MSM) living in North American urban centers including New York, Los Angeles, and San Francisco [1]. Subsequently, cases of AIDS were recognized in subSaharan Africa [2], and the global HIV pandemic began to gather momentum. Over the last three decades, the global epidemiology of HIV/AIDS has evolved. Incidence rates in certain populations have stabilized or even fallen. However, the number of new HIV diagnoses among MSM appears to be stable or possibly increasing in most countries where data are available [3,4]. 2.

The biology of HIV infection

The specific processes underlying HIV-1 transmission are still not fully understood but are dependent on several factors that include the stage of the infection [5], the presence of other sexually transmitted diseases [6,7], and the degree to which 10.1517/17425247.2013.860132 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

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challenge experiments. These criteria are discussed in more detail below.

Article highlights. .

. . .


. .

URAI continues to be associated with high levels of HIV infection among MSM and transgender women in the developed and developing world. Use of RM may be more culturally acceptable than use of oral PrEP. Preclinical studies in NHP and mice provide proof of concept of RM as a viable HIV prevention strategy. Phase I RM development provides important PK/PD data that can inform decisions about which products should be advanced to a later stage RM development. TFV 1% gel is currently the lead RM candidate and is being evaluated in the MTN-017 Phase II study. Future Phase I studies will evaluate combination ARV RM formulations, including TFV and MVC.

This box summarizes key points contained in the article.

antiretroviral (ARV) therapy has suppressed HIV viral load in the index case [8-10]. The risks for HIV-1 acquisition for the receptive partner are about 10-fold higher than for the insertive partner [11,12]. However, these estimates are quite variable due to the factors discussed above [13]. The gastrointestinal tract is a rich source of HIV-1 target cells. Isolated lymphoid follicles, which serve as inductive sites for immune responses, are located throughout the colon [14]. The number of follicles generally increases toward the anus with the greatest numbers found in the rectum [15,16]. Antigen-presenting cells (macrophage and dendritic cells) along with effector and regulatory T cells are found within the follicles. These cells are generally activated and express HIV-1 co-receptors, CCR5 and CXCR4, as well as soluble immune mediators [17-20], thus creating the perfect environment primed for HIV-1 infection [21].

The rationale for rectal microbicide development 3.

The rationale for the development of rectal microbicides (RM) is based on several criteria: i) despite almost three decades of health education and widespread availability of condoms, significant proportions of MSM and other populations continue to practice unprotected receptive anal intercourse (URAI); ii) the majority of individuals practicing URAI use some form of sexual lubricant and so a microbicide that has lubricant-like properties would not require the type of behavioral modification associated with other forms of HIV prevention; iii) several studies have documented interest in developing RM and evaluating the potential use of RM by MSM; iv) preclinical evaluation of candidate microbicides have shown protection in colorectal explant challenge studies; v) RM have also been shown to be efficacious in non-human primate (NHP) and murine rectal challenge studies; and vi) RM have been shown to be efficacious in Phase I clinical trials that have conducted ex vivo / in vitro colorectal explant 70

Populations at risk of HIV infection acquired through URAI

3.1

Receptive anal intercourse (RAI) is a relatively common practice among MSM although prevalence rates are highly variable, depending on the population studied. In a recent study from Rosenberger et al. that characterized sexual behavior in 24,787 gay and bisexually identified US men, anal sex was reported by 37.2% of the respondents [22]. In contrast, in a smaller study from California 303/398 (76%) of MSM reported anal sex within the previous 12 months [23]. Unfortunately, a significant proportion of anal sex occurs without the use of condoms. The prevalence of URAI varies by the population studied, but rates of greater than 30% are commonly reported from the USA and the United Kingdom (UK) MSM [23,24]. Similar patterns of behavior have been reported for MSM across the world [4]. This includes sub-Saharan Africa, where recent studies have documented the presence of MSM; these MSM have high rates of HIV infection and little or no access to basic HIV prevention services, including the provision of condoms [3,25,26]. There is increasing recognition that anal sex is also practiced by heterosexual couples. The National Survey of Sexual Health and Behavior (NSSHB) is a study of human sexual behavior conducted in the US by the Center for Sexual Health Promotion at Indiana University in Bloomington. The study enrolled 5,865 men and women aged 14 -- 94 years in the USA. More than 20% of men aged 25 -- 49 years and women aged 20 -- 39 years reported anal sex in the last year. More than 40% of both men and women reported a lifetime experience of insertive or receptive anal intercourse [27]. A number of US studies have described linkages in young women between URAI and STIs as well as illicit drug use [28,29]. It is also apparent that women in the developing world are at risk of HIV infection through URAI as well as vaginal sex [30]. This brief survey of the epidemiology of anal sex suggests that this practice is common among men and women in both the developed and developing world. A significant proportion of anal sex is also unprotected. A safe and effective RM would therefore be a valuable addition to the HIV prevention options available to all individuals at risk of HIV infection through URAI. As oral ARV pre-exposure prophylaxis (PrEP) has become more widely available in the United States, there is increasing concern among healthcare providers that increased use of oral PrEP might be associated with reduced rates of condom use or condom migration and increased ARV resistance [31,32]. The phenomenon of condom migration has been reviewed in the context of vaginal microbicides, where it is felt that the benefits of safe and effective microbicides might outweigh the negative impact of condom migration [33]. Oral or topical

Expert Opin. Drug Deliv. (2014) 11(1)

The development of rectal microbicides for HIV prevention

PrEP associated antiretroviral resistance is another theoretical concern. Uptake of oral PrEP has been quite limited outside of clinical trials, and it is not yet possible to determine if resistance will be a significant issue. However, recent mathematical models suggest that resistance is more likely to be associated with ARV therapy rather than PrEP [34]. It is clear that data are quite limited for both of these concerns, and they will need to be revisited as rectal microbicide development advances.

Sexual lubricant use by target populations Although sexual lubricants are not intended to prevent HIV infection, their use does provide a rationale for the development of RM that may have the potential to prevent HIV infection. Lubricant use is a common practice among men and women and lubricants have been used with vaginal and anal intercourse. Carballo-Diegue´z et al. conducted a survey of rectal lubricant use among 307 Latino MSM in New York during 1995/1996. The majority of the study participants (94%) had experience of using lubricants with RAI, and 74% used a lubricant during at least 80% of all episodes of RAI. The study also demonstrated high rates of URAI (43%) with a willingness to use RM if available (92%) [35]. A second larger study conducted among 879 MSM living in San Francisco in 2002/2003 demonstrated high levels of URAI (36%), high levels of lubricant use (89%), including significant use of Nonoxynol-9 (N9) containing products (26%) [36]. The study documented variability in microbicide acceptability based on the perceived level of protection afforded by microbicides. Whereas 65% of men would consider using an RM if it was as effective as a condom, only 15% would consider using an RM if it was less effective than a condom. More recently, Clark et al. conducted a quantitative behavioral survey among 547 MSM from Lima, Peru [37]. Approximately 50% of the men reported lubricant use with RAI. Reasons for non-use of lubricants included: nonavailability of lubricant (32%), use of pre-lubricated condoms (24%), use of saliva as a lubricant (13%), or nonaffordability of lubricant (8%). URAI was common among men using lubricants (63%). In a second recent study from Latin America, Kinsler et al. used Conjoint Analysis (CJA) to explore consumer preferences among 128 MSM in Peru, Ecuador, and Brazil [38] for a potential RM. The favored product profile included the following characteristics: a unit cost of $0.30, 80% effectiveness, no side effects, use possible before sex, a gel formulation, 15 ml dosage, and provided through a prescription. Although CJA does not involve consumers trying products, it does provide an important framework for an ideal product. In this analysis, effectiveness had the greatest impact on product acceptability; participants chose 80% rather than 40% (the level of protection seen in the CAPRISA 004 study [39]), and price was also significant-$0.30 representing the approximate cost of a condom in Peru, Ecuador, or Lima.


3.2

3.3

Preclinical efficacy data Colorectal explant efficacy data

3.3.1

There is increasing interest in using human tissue biopsies or explants to evaluate the safety and efficacy of candidate microbicides [40]. A wide range of tissues have been used, including cervicovaginal [41], colorectal [42,43], penile [44], and foreskin tissue [45]. Colorectal and cervicovaginal explants can be harvested from surgical resection specimens or can be collected as multiple biopsies from healthy donors. With regard to the colorectal explant system, two experimental techniques have emerged. Abner et al. have described the use of a polarized tissue model in which the explant is physiologically orientated in a transwell system [43]. In contrast, Fletcher et al. have used a non-polarized system in which the colorectal biopsies are placed on a gelfoam raft [42]. Safety and efficacy can be evaluated in the presence of the candidate microbicide as well as relevant biological fluids such as semen [46]. Mucosal safety can be assessed by histology, cytokine release, or the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Antiviral efficacy is assessed by exposing the explant tissue to HIV for a defined period of time, washing the explant, and then collecting the supernatant over a 14-day period. Evidence of infection is demonstrated by quantifying HIV-1 p24 antigen levels in the explant supernatant. It is also possible to quantify accumulation of HIV-1 RNA and DNA in the explant tissue as illustrated in Figure 1. These assays have characterized the in vitro safety and efficacy of many potential RM candidates from non-specific entry inhibitors [42] to more specific non-nucleoside and nucleotide/nucleoside reverse transcriptase inhibitors, fusion inhibitors, and protease inhibitors (Table 1) [42,44,47,48]. These candidates have been typically evaluated as single entity agents, and the majority of in vitro experiments have found the formulated products to be safe (based on criteria such as the MTT assay and histopathology) and efficacious (based upon inhibition of viral replication). Two exceptions were N9, which stripped epithelium from the explants, and the original hyperosmolar formulation of TFV gel, which was associated with the induction of fractures in the explant tissue [49]. Interestingly, a reduced glycerin formulation of TFV gel did not induce these changes in colorectal explants [49]. Combinations of active pharmaceutical ingredients (APIs) are now being tested, and much like ARV therapy, they show that combinations of up to three APIs (TFV or emtricitabine (FTC) with UC781 and dapivirine) are much more potent against HIV-1 infection in colorectal tissue even against drug-resistant HIV-1 [48]. These data will inform microbicide developers as to which candidates/combinations are optimal to pursue for further development. Murine models Bone marrow liver-thymus (BLT) humanized mice are susceptible to rectal HIV infection due to the presence of human 3.3.2


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Rectal microbicide candidate

4.

HIV-1 RNA/DNA & p24

Table 1. Rectal microbicide candidates evaluated in the colorectal explant model.

transcriptase (RT-SHIV) [54]. Using the NHP model, gel formulations of an entry inhibitor, cyanovirin-N [55] and the RT inhibitors, TFV and MIV-150 [56,57], prevented rectal challenge of SHIV or RT-SHIV (Table 2). These data are informative regarding the potential of a low volume of gel to block atraumatic exposure to the infectious virus. Of note, there were correlations between plasma drug levels of TFV and protection from macaque infection [56]. To begin to address drug distribution after dosing in a more formal way, a multi-compartment pharmacokinetic study in macaques after vaginal or rectal dosing with TFV gel was done [58]. Macaques dosed with TFV gel vaginally showed rectal drug levels only 1 log10 lower than the vaginal drug levels. And both mucosal compartments were 4 -- 5 log10 higher than plasma drug levels. Similar results were found when the macaques were dosed with TFV gel rectally. These data suggest that vaginal or rectal dosing of a soluble microbicide could protect against HIV-1 regardless of the route of exposure.

108 107 106 105 104 103 102 101 100 10-1

Malnati (DNA) Li/Wong (DNA) Rouet (RNA) Drosten (RNA) HIV-1 p24


Day 4 Day 7

Day 11 Day 14

Figure 1. HIV-1 replication in colorectal explant tissue. HIV-1 RNA/DNA were measured in explant tissue and supernatant using modifications of the RNA/DNA assays previously described by Malnati, Li, Rouet, and Drosten [93-96]. HIV-1 p24 was measured by ELISA.

Cellulose acetate PRO2000 SPL7013 Vena Gel UC781 PRO2000 Dextrin sulfate Tenofovir, Nonoxynol-9 C34 T20 T1249 L’644 Tenofovir Emtricitabine UC781 TMC120 Saquinavir

Ref. [43]

[42] [49,60] [44]

[48]

[97]

CD4+ T cells, macrophages, and dendritic cells found throughout the intestines, including the rectum [50,51]. Chateau et al. have recently demonstrated that BLT mice treated with rectal tenofovir (TFV) 1% gel were significantly protected from infection with HIV-1JRCSF and HIV-1THRO [52]. These observations not only support the further development of TFV gel as an RM but also provide an important platform for testing the efficacy of future novel RM. Non-human primate models NHP infection models use two different challenge schemes that involve either a multiple low-dose or single high-dose challenge with simian immunodeficiency virus (SIV) or a chimeric SIV/HIV construct (SHIV) [53]. Typically, the virus used to evaluate microbicides is a SIV modified to express the HIV-1 envelope (SHIV) or the HIV-1 reverse 3.3.3

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Rectal microbicide formulation considerations

Formulation of APIs for rectal use will likely be in a liquid or semi-solid dosage form to cover areas that are at most risk for HIV-1 exposure [59]. Preclinical testing of formulated APIs adds additional complexity because pH, osmolality, and viscosity of the product will impact the results. For instance, the polymers used in the formulation may enhance toxicity or efficacy due to smothering individual cells or nonspecifically binding HIV-1. These results may be overinterpreted by inexperienced researchers. Therefore, it is critical to include the vehicle control, the same formulation but without the API, in all assays to accommodate the impact the formulation may have on the testing results. As with unformulated APIs, testing algorithms have been developed [60]. Typically, the testing done with the formulations is to ensure the APIs activity has not been impaired and the formulation is safe. Mucosal tissues are used for testing the formulations but are polarized, keeping the apical surface at the liquid/air interface [43,60,61]. The formulation, with or without HIV-1, can be applied to the apical surface recapitulating the use by a person. Using this testing algorithm, it was recently reported that the exposure to TFV 1% gel was associated with epithelial fracture and sloughing in polarized mucosal tissue and that this effect might be related to the hyperosmolar nature of this formulation [60]. The gel was subsequently reformulated to reduce the glycerin content and thus reduce the osmolality [49]. The reduced glycerin (RG)TFV 1% gel showed improved epithelial retention in polarized rectal and ectocervical tissue explants. These data support the clinical trial results (discussed below) that showed that gastrointestinal adverse events (AEs) were significantly more common with the rectal use of the original TFV 1% gel than with the use of RG gel formulation. The preclinical



Table 2. Non-human primate rectal microbicide challenge studies.


Rectal microbicide candidate

Model

Findings

Ref.

1% cyanovirin-N gel (n = 5) 2% cyanovirin-N gel (n = 5) Placebo gel (n = 4) Virus control (n = 4)

Cynomolgus macaques SHIV89.6P Single viral challenge 20 min after gel application

All 1 and 2% cyanovirin-N gel recipients protected. All virus control and placebo recipients infected

[55]

1% TFV gel (n = 12) Placebo gel (n = 4) Virus control (n = 4)

Indian rhesus macaques SIVmac251/32H Single viral challenge 2 h before and 15 min to 2 h after gel exposure

6/9 macaques receiving TFV gel up to 2 h before virus exposure were protected. All untreated and 3/4 placebo gel recipients were infected

[56]

MIV-150/ carageenan gel (n = 18) Placebo gel (n = 6)

Chinese rhesus macaques SHIV-RT Single viral challenge 30 min to 4 h after gel exposure

SHIV-RT TCID50 103: 8/8 macaques receiving MIV-150/ carageenan gel protected. SHIV-RT TCID50 104: 2/4 receiving MIV-150/carageenan gel protected. All placebo gel recipients infected.

[98]

MIV-150/ zinc acetate/ carageenan gel (n = 14) Placebo gel (n = 6)

Chinese and Indian rhesus macaques SHIV-RT Single viral challenge 8 to 24 h after gel exposure

5/7 macaques exposed to virus 24 h and 4/7 macaques exposed to virus 8 h after receiving MIV-150/zinc acetate/carageenan gel were infected. 5/6 placebo recipients infected

[99]

testing of formulations is thus important to ensure that those products that move into clinical trials are safe as well as effective. 5.

Formulation preference studies

It is not known what volume of a microbicide gel will be needed to provide protection from HIV infection associated with URAI. The majority of vaginal microbicide studies have used approximately 4 ml of study product. However, since the vaginal compartment is a relatively enclosed anatomical space and the rectum a potentially much larger space, it has been assumed that a greater volume of gel would be needed in the rectal versus vaginal compartments. This study, undertaken by Carballo-Diegue´z et al., was conducted among HIV-uninfected MSM in Boston, Massachusetts and was designed to explore the acceptability of escalating volumes of placebo gel in MSM practicing anal sex [62]. The placebo gel used in the study was FemGlide, an isosmolar vaginal lubricant (Trumbull, Connecticut, USA). The gel was administered using a syringe; each of the 18 participants received sequentially 5, 20, 35, and 50 ml of study product. The participant then used the study product at the highest volume that he had rated as acceptable during receptive anal intercourse (RAI). Up to 35 ml of FemGlide was found to be acceptable to study participants. Beyond this volume, there were increasing reports of leaking and messiness, which were associated with poor product acceptability. In a second study from the same group, again conducted in Boston Massachusetts, USA, 77 MSM were enrolled in a

crossover study where they were asked to use either a placebo gel or a suppository [63]. FemGlide was administered at a volume of 35 ml using an accordion-type applicator, and the suppository was manufactured using two polyethylene glycol bases (PEG 1450 and PEG 300) (JE Apothecary, Inc., Brookline, Massachusetts, USA). Participants were asked to insert the products up to 2 h before anal intercourse on three separate occasions. Rectal gel was preferred over the suppository by both the recipient (75 vs 25%, p < 0.001) and the partner (71 vs 29%, p < 0.01). Although these data suggest a clear preference for gel over suppository, the authors did comment that the suppository used in this study was quite large (8 g and 2.5 inches) and they felt that a smaller suppository might have been more acceptable. In a recent study, Pines et al. evaluated a gel, suppository, and enema in 117 HIV-uninfected males (79%) and females (21%) [64]. The study was conducted in Los Angeles, California, USA, and participants received an isosmolar gel (4 ml of Pre-Seed; Spokane, Washington, USA), an isotonic enema (125 ml of Normosol-R; Hospira Inc., Lake Forest, Illinois, USA), and a suppository commonly used for the treatment of haemorrhoids (Tucks; McNEIL-PPC Inc., Skillman, New Jersey, USA). The study products were administered in a randomized sequence and participants were asked to use each product 3 -- 5 times over a 2-week period before moving on to the next product. During follow-up, 75, 76, and 66% of participants reported using the enema, applicator, and suppository, respectively. There was no difference in the frequency of anorectal symptoms or AEs by product


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and, based on computer-assisted self-interviews (CASI), the applicator was the most acceptable product overall. Galea et al. conducted qualitative interviews among 140 Peruvian MSM to determine whether rectal douches might be an acceptable formulation for RM delivery. Only 27% of the study participants had a history of rectal douching, but 80% reported that they would use an RM formulated as a douche [65]. Leyva et al. have studied the safety, acceptability, and distribution of isoosmolar, hyperosmolar, and hypoosmolar enemas and found that isoosmolar enemas had the greatest proximal colonic distribution, greater colon tissue concentrations of 99mTc-DTPA, and equivalent acceptability in comparison to the other enema formulations [66]. These data suggest that an enema/douche formulation should be further evaluated as a potential RM. Morrow et al. have argued for more sophisticated analysis of consumer formulation preference for vaginal microbicides [67] and integration of these data into product development [68]. It will be important to evaluate this approach in the context of RM development. The negative results of the VOICE Phase IIB study of vaginal TFV 1% gel was largely due to product non-adherence [69]. The extent to which product characteristics, including formulation, influenced patterns of non-adherence in the VOICE study is not clear but is currently under evaluation. At this point in time, completed RM studies have only involved single or 7-day exposure to the study product, and so adherence has not been a major focus of attention. However, given the major negative impact of product non-adherence on microbicide effectiveness, the MTN-017 study, in which participants will receive 8 weeks of product exposure, will scrutinize product adherence in greater detail. In addition to self-reported adherence behavior, pharmacokinetic (PK) samples from study participants will be evaluated in real time to provide objective evidence of product use and, more importantly, to focus counseling messages on improving product adherence. 6.

Product distribution studies

Increasingly, sophisticated imaging technology is being used to track semen and microbicide surrogates to understand more about their distribution and persistence following rectal administration. Craig Hendrix and his colleagues at Johns Hopkins University (JHU), Baltimore, Maryland, USA have developed this area of prevention research with a number of innovative studies. In their first study, Hendrix et al. administered hydroxy ethyl cellulose (HEC)-based microbicide surrogate and a viscosity-matched semen surrogate to three participants [70]. Each participant underwent seven phases of evaluation wherein the impact of variables such as enema administration, semen and microbicide surrogate, and simulation of anal intercourse on product distribution were evaluated. Both the microbicide and semen surrogates were labeled with gadolinium-DTPA (as a magnetic resonance imaging (MRI) 74

contrast agent) and 99mTechnetium-sulfur colloid (for single photon emission computed tomography (SPECT) imaging). MRI studies conducted at the end of one and four hours following product administration demonstrated presence of signal throughout the rectum with the highest signal being seen in the dependent/posterior aspect of the rectum (participants remained supine throughout the procedure). In contrast, SPECT imaging demonstrated more proximal product migration. Four hours post product exposure 4/16 studies demonstrated signal in the descending colon, two of which reached the splenic flexure. An example of SPECT/CT imaging is shown in Figure 2. In a second study, the JHU team evaluated the distribution of cell-free and cell-associated HIV surrogates in the colon after simulated RAI in six men [71]. In this study, autologous leukocytes, collected from study participants were radiolabeled with 111Indium-oxine as the cell associated surrogate and sulfur colloid particles were labeled with 99mTechnetium-sulfur as the cell-free surrogate. Both products were mixed with 3 ml of autologous semen and delivered to the rectum via a coital dynamic simulator (CDS) after 5 min of simulated RAI. SPECT/CT images were acquired at 1, 4, 8, and 24 h post dosing. In this study, cell-associated and cellfree surrogates generally co-localized, were largely confined to the rectosigmoid colon, and their signal persisted for up to 24 h in the absence of defecation. Furthermore, cell-free and cell-associated surrogates were found in tissue biopsies collected from the study participants. In the third and most recently published study, the group further evaluated microbicide and semen surrogate colonic distribution in eight participants using the methodological approach described above [72]. In this study, gel distribution was limited to the rectosigmoid in 84% (26/31) of the participants although 16% (5/31) participants showed proximal spread to descending colon. Comparison of Dmax, DCmax, and Dave across different phases of the study did not generate any significant differences suggesting that microbicide and semen surrogates were generally co-localized. Collectively, these highly innovative studies suggest that the rectosigmoid is the area of highest viral exposure following RAI and that the RM also appears to co-localize in this area. Obviously, these data need to be interpreted with some degree of caution; the participants are undergoing simulated RAI and receiving microbicide and semen surrogates. Actual semen and specific microbicide products may have different patterns of distribution.

Candidate molecules for rectal microbicide development

7.

UC781 UC781 is a thiocarboxanilide non-nucleoside RT inhibitor (NNRTI) that was being developed as both a vaginal and rectal microbicide. UC781 has reduced activity against NNRTI resistant HIV-1 [73] but remains active at concentrations of 7.1



nucleoside kinases, whose activity and availability are dependent upon the activation state of the cell. Consequently, it is unknown how long it will take lymphocytes, dendritic cells, and monocyte/macrophages in mucosal surfaces to convert TFV to its active diphosphorylated antiviral metabolite. It is possible that the absolute rate of TFV activation will depend upon a wide range of factors including the presence or absence of mucosal inflammation. TFV is probably the most widely studied ARV for HIV prevention [78].

S

Maraviroc The majority of available PrEP PK data relate to TFV and TDF/FTC. However, the CCR5 antagonist, maraviroc (MVC) (Selzentry, ViiV Healthcare, North Carolina, USA [79]), is also being considered as both an oral (HPTN-069 study) and a topical agent for HIV prevention, as a vaginal ring (MTN-013) and an RM in the Combination HIV Antiretroviral Rectal Microbicide (CHARM) Program. Brown et al. conducted a PK study in 12 HIV-negative men who received 300 mg of MVC twice daily for 8 days [80]. Rectal tissue MVC area under the curve (AUC) was 7.5- to 26-fold higher than blood plasma. In a second study in women, MVC AUC in cervicovaginal fluid following oral dosing was only 1.9- to 2.5-fold higher than blood plasma [81]. MVC has the potential to become an important drug for HIV prevention since greater than 95% of new HIV infections are transmitted by CCR5 (R5) tropic viruses [82,83]. One note of caution was raised by a recent NHP study conducted by the Centers for Disease Control and Prevention (CDC), Atlanta, USA in which 10 macaques received oral MVC (44 mg/kg) and were challenged rectally with SHIV162p3 [84]. Despite similar PK profiles to human PK studies of MVC, including high concentrations of MVC in rectal secretions (median = 2,329 ng/ml vs median = 451 ng/ml in blood), 5/6 macaques receiving MVC and 3/4 control macaques were infected with SHIV162p3. One possible explanation for these NHP data is that there are eight amino acid differences between the macaque and human CCR5 receptor. Although both receptors bind MVC, disassociation of the MVC from the macaque CCR5 receptor was 10-fold faster than from the human CCR5 receptor [85]. Additional studies, including explant challenge, will be required to further assess the utility of MVC as a PrEP agent.


7.3

I

Figure 2. SPECT/CT images of RM distribution. Images were captured 2 h after rectal administration of a placebo gel. Single-photon emission computerized tomography (SPECT) was performed using 111Indium-Diethylene Triamine Pentacaetic Acid (DTPA). Signal intensity from the sagittal plane with the greatest signal intensity was used to create a full thickness image, not a single plane. Images provided courtesy of Dr. Craig Hendrix, Professor of Medicine, Johns Hopkins University, Baltimore, Maryland, USA.

25 µM, which is below the levels delivered by current UC781 formulations. Combinations of cellulose acetate 1, 2-benzenedicarboxylate (CAP) and UC781 have been shown to provide synergistic inhibition of HIV replication [74]. Preclinical evaluation of UC781 was unremarkable [75], and explant studies have demonstrated inhibition of HIV-1 replication in cervical tissue and prevented migration of infected cells from the explant [76]. Unfortunately, the clinical development of UC781 as a microbicide was suspended in 2010 despite supportive safety data from completed Phase I studies.

8.

Tenofovir TFV (Gilead Sciences Inc., Foster City, CA, USA) is an adenosine nucleoside monophosphate (nucleotide) analog belonging to the class of acyclic phosphonomethylether nucleosides. The prodrug of TFV, Tenofovir disoproxil fumarate (TDF) is licensed for the treatment of chronic HIV infection (Viread) and TDF/emtricitabine (Truvada) has recently been licensed for a HIV prevention indication [77]. Activation of TFV is dependent upon anabolic phosphorylation by intracellular 7.2

Phase I rectal microbicide studies

RMP-01 In the Rectal Microbicide Program (RMP)-01 study, 36 HIV-1 seronegative, sexually-abstinent men and women were enrolled in Los Angeles, CA and randomized into a double-blind, placebo-controlled trial comparing UC781 gel at two concentrations (0.10%, 0.25%) with a placebo gel (1:1:1) [86]. Safety and acceptability were primary study endpoints. Changes in colorectal mucosal safety biomarkers and UC781 plasma drug levels were secondary endpoints. 8.1


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Ex vivo explant infectibility with HIV-1 was an ancillary study endpoint. Samples were collected at enrollment after a single rectal dose of study product and after seven daily doses. The majority of AEs were mild. Product acceptability was high, including likelihood of future use. No changes in mucosal safety biomarkers were identified. Plasma levels of UC781 were not detected. Ex vivo infection of biopsies using two titers of HIV-1BaL showed marked suppression of HIV-1 p24 in tissues exposed in vivo to 0.25% UC781. Ideally, the product would have been advanced into Phase II development, but the FDA Investigational New Drug (IND) sponsor (CONRAD; http://www.conrad.org/) terminated the UC781 development program. RMP-02/MTN-006 The RMP-02/MTN-006 study enrolled 18 participants from Pittsburgh, PA and Los Angeles, CA. All participants received a single 300 mg dose of TDF and were then randomized 2:1 to receive a single, then seven daily doses of TFV 1% gel or the HEC placebo gel [87]. Safety endpoints included clinical AEs and mucosal safety biomarkers. Participants were assessed at enrollment, after single doses of oral TFV and study gel, and after seven daily doses of study gel. Blood and colonic biopsies were collected for PK analysis and ex vivo challenge with HIV-1. No serious AEs were reported. However, AEs, especially gastrointestinal AEs, were significantly increased with 7-day use of the TFV 1% gel. Only 25% of participants liked the TFV gel; however, likelihood of use, if the product was somewhat protective, was high (75%). No significant mucosal injury was detected. Tissue TFV diphosphate (TFV-DP) Cmax 30-min after single rectal exposure to TFV gel was 112-times greater than single oral-exposure to TDF. Tissue 7-day exposure to TFV gel resulted in 5-times greater levels of TFV-DP than single rectal-exposure to TFV gel. Seven-day exposure to rectal TFV was associated with significant suppression of explant infection. Increased AEs suggested that the vaginal formulation of TFV 1% gel used rectally was not entirely safe nor fully acceptable, suggesting a need for improved formulations. 8.2

MTN-007 The MTN-007 study was designed to assess the safety, adherence, and acceptability of a new RG formulation of TFV 1% gel [88]. Participants were randomized 1:1:1:1 to receive the RG formulation of TFV 1% gel, a HEC placebo gel, a 2% (N9) gel, or no treatment, respectively. Each gel was administered as a single dose followed by 7 daily doses. Mucosal safety evaluation included histology, fecal calprotectin, epithelial sloughing, cytokine expression (mRNA and protein), microarrays, flow cytometry of mucosal T cell phenotype, and rectal microflora. Acceptability and adherence were determined by computer-administered questionnaires and interactive telephone responses, respectively. Sixty-five participants (45 men and 20 women) were recruited into the study. There were no significant differences between the numbers 8.3

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of ‡ grade 2 adverse events across the arms of the study. Likelihood of future product use (acceptability) was 87% (RG-TFV 1% gel), 93% (HEC placebo gel), and 63% (N9 gel). Fecal calprotectin, rectal microflora, and epithelial sloughing did not differ by treatment arms during the study. Suggestive evidence of differences was seen in histology, mucosal gene expression, protein expression, and T cell phenotype. These changes were mostly confined to comparisons between the N9 gel and other study arms. One exception was the microarray data, where the majority of changes in mucosal transcriptome were seen in the TFV arm of the study. The biological significance of these microarray changes and whether they represent a safety signal are currently uncertain. Additional proteomic and immunohistochemical testing of rectal samples from MTN-007 is ongoing and may clarify this situation. Overall, the RG-TFV 1% gel was considered safe and well tolerated rectally and has now been advanced to Phase II development (MTN-017). 9.

Other ongoing rectal microbicide studies

Project gel Project Gel is a study that attempts to evaluate RM safety, adherence, and acceptability in young ethnic minority MSM in Boston, MA; Pittsburgh, PA; and San Juan, PR. The study design has two stages: A clinical and behavioral evaluation (Stage 1A) with an acceptability and adherence trial (Stage 1B), followed by a Phase I randomized, double-blind, multisite, placebo-controlled safety trial (Stage 2). The first 120 eligible participants who complete Stage 1A and report URAI in the previous 3 months will continue on to Stage 1B. During Stage 1B, participants will be given condoms and a placebo gel to use during RAI. Over a 3 month period, they will report the frequency of product use and be interviewed about the acceptability of the product. The first 24 participants who complete Stage 1B will be eligible to participate in Stage 2, where they will be randomized to receive an actual microbicide RG-TFV 1% gel) or matched placebo. Project Gel was completed in Q3 2013, and it is hoped that data from this study will provide unique insights into the acceptability, safety, and adherence of RM in young MSM. 9.1

CHARM Program The goal of the CHARM Program is to develop and evaluate a combination ARV rectal specific microbicide. TFV and MVC are the two lead compounds and the ultimate goal is to develop a TFV/MVC combination product. Two Phase I studies, CHARM-01 and CHARM-02, will be completed in Q4 2013. CHARM-01 will assess the safety, acceptability, and PK/pharmacodynamic (PD) profile of three TFV gel formulations: the original TFV 1% gel used in vaginal microbicide studies, the RG-TFV 1% gel, and a rectal specific TFV 1% gel. CHARM-02 will evaluate the safety, PK, and distribution of the same three gels. Similar techniques have been used to characterize the distribution of semen surrogates and 9.2



microbicide products in the presence and absence of simulated RAI [71,72]. Collectively, these studies will provide unique data on the influence of formulation characteristics, including osmolality, and product safety, PK/PD, and distribution. A CHARM-03 study is in development and will evaluate the safety, acceptability, and PK/PF profile of both oral MVC and a rectal specific gel formulation of MVC. MTN-014 The MTN-014 study will explore the PK profile of a RGTFV 1% gel in women after rectal and vaginal administration. Nuttall et al. have previously demonstrated that when TFV gel is administered vaginally in macaques, TFV can be detected in the rectal compartment. The converse is true when TFV gel is administered rectally [58]. Clearly, these NHP data have significant implications for microbicide development. Demonstration that a single compartment gel delivery could provide dual compartment protection from HIV infection would significantly increase enthusiasm for this type of product. Rectal fluid samples were collected in a subset of women enrolled in the MTN-001 study that characterized the PK profile of TFV following oral, vaginal, and oral/vaginal combination administration [89]. In the MTN-001 study, the median concentration of TFV in rectal fluid was almost 6 times higher than after oral administration of TFV. The purpose of the MTN-014 is to build upon these NHP and human data sets and provide tissue PK to further determine whether dual compartment PK exposure also occurs in humans. A unique feature of the study is that gel administration will be directly observed, circumventing any problems associated with non-adherence to study product. It is hoped that the MTN-014 study will start in Q4 2013.


9.3

MTN-017 The MTN-017 study is a Phase II evaluation of the RG-TFV 1% gel. The study will enroll 186 MSM and transgender women in the US, Peru, Thailand, and South Africa. Participants will receive the TFV gel daily for 8 weeks, TFV gel before and after sex for 8 weeks, and oral Truvada for 8 weeks. In addition to collection of generic safety and acceptability data, a subset of participants in the US and Thailand will undergo intensive evaluation of rectal tissue for PK, PD, and mucosal safety. The MTN-017 study started enrollment in the US in October 2013. 9.4

10.

Conclusion

The contemporary global epidemiology of HIV infection demonstrates that there is an important need to develop novel interventions to prevent or at least significantly reduce HIV infections in individuals at risk through URAI. The majority of RAI is facilitated by the use of sexual lubricants. Development of an RM that could be used like a sexual lubricant is likely to be culturally and socially acceptable, which may increase rates of product adherence by at-risk populations.

The first RM studies evaluated the rectal safety of vaginal microbicides, but subsequent studies are evaluating rectal specific products. NHP studies have provided proof of concept that insertion of an ARV gel prior to rectal viral challenge can reduce rates of infection and similar observations have been seen in humanized mouse models. Importantly, antiviral efficacy has also been seen in the ex vivo / in vitro explant challenge system. It is not clear whether these latter observations will translate into effectiveness in Phase IIB/III studies, but they certainly increase optimism in RM as a novel HIV prevention strategy. As a consequence, development of a TFV 1% gel RM has now progressed to Phase II evaluation. High rates of HIV infection associated with URAI impact communities within both the developed and developing world. This observation raises the possibility that development of a safe and effective RM may have a commercial value within the developed world and may encourage more direct engagement in RM development by pharmaceutical companies. 11.

Expert opinion

RM research has benefited from many of the lessons learned during several decades of vaginal microbicide research. Apart from the HIVNET-008 study that evaluated N9 gel as a potential RM [90,91], subsequent research has focused exclusively on the development of ARV microbicide formulations. The design of Phase I studies has been optimized to generate safety, acceptability, and PK/PD data that will hopefully reduce the risk of advancing unsuitable products into later stage development. Human studies have been complemented by preclinical safety and efficacy evaluations in explant, murine, and NHP models. In addition, responding to concerns about product acceptability, microbicide formulations have been optimized for rectal use. It is clear that there is an urgent need to provide individuals practicing URAI with other options for HIV prevention. The iPrEx study demonstrated that use of Truvada for oral PrEP significantly reduced HIV acquisition rates in MSM and transgender women [92]. This was a major milestone in HIV prevention research; however, the overall effect size was modest (44%; 95% CI: 15 -- 63%), and following licensure of a HIV prevention indication uptake of oral PrEP in the US has been slow, suggesting that other options are also needed. The CAPRISA 004 study provided proof of concept that use of an ARV microbicide could significantly reduce HIV acquisition rates in women [39]. However, like the iPrEx study, the effect size was modest (39%; 95% CI: 6 -- 60%), and the subsequent VOICE study did not demonstrate the effectiveness of daily TFV 1% gel use in a similar population [69]. Preliminary data suggest that the VOICE results were driven by significant levels of non-adherence to product use. Not surprisingly, if a product is not used, it will not be effective. A key issue for rectal microbicide development is whether individuals at risk of HIV infection from URAI will use a


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rectal gel. Sexual lubricants are commonly used to facilitate RAI, and so it is hoped that RM might be perceived as a culturally acceptable intervention for HIV prevention. The MTN-017 study will provide important data on the levels of RM use, including real time PK monitoring of product use. A critical component of the MTN-017 study is that participants will be provided with details of the PK monitoring, and it is hoped that this intervention may increase product adherence. The future of RM research is probably contingent upon the results of the MTN-017 study. If PK data from the MTN-017 study suggest very low levels of product adherence, despite intensive counseling and PK monitoring, it is unlikely that a daily RM will advance into Phase IIB/III effectiveness testing. It is possible that an alternative formulation such as a suppository, fast dissolving tablet, or enema product that could be given once prior to URAI might circumvent adherence problems associated with daily product use and allow this type of topical product to move towards effectiveness testing. The MTN-007 study has generated intriguing data that demonstrate that even 1 week’s rectal exposure to TFV gel results in quite significant changes in the mucosal transcriptome. These changes may result from the high concentrations of ARV drugs that can be delivered by rectal administration. The biological significance of these changes is uncertain. Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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The MTN-017 study, in which participants will receive 8 weeks of daily TFV gel, will provide critical data about the biological effects of more prolonged rectal exposure to potent ARV agents. If these transcriptomic changes persist or increase in magnitude, there may be a rationale for the exploration of microbicide candidates that could be given before and perhaps after episodes of URAI. It is clear that there is an ongoing need for safe and effective interventions for HIV prevention. Individuals practicing URAI are at high risk of HIV infection and this provides a strong rationale for the ongoing development of an ARV RM.

Declaration of interest I McGowan is the Co-Principal Investigator of the Microbicide Trials Network which is supported by the Division of AIDS, at the National Institute of Allergy and Infectious Disease, NIH grant number 3UM1AI068633. McGowan also acknowledges support from the Integrated Preclinical/Clinical Program for HIV Topical Microbicides at DAIDS for the CHARM program (Grant number: 3U19AI082637) and from the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Institute of Mental Health for support of Project Gel (5R01HD059533). The views expressed by the author do not necessarily reflect the views of the NIH.

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Affiliation Ian McGowan University of Pittsburgh, Department of Medicine, Pittsburgh, USA E-mail: [email protected]

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Non-Antiretroviral Microbicides for HIV Prevention.

Beyond HIV microbicides: multipurpose prevention technology products.

Nanotech-derived topical microbicides for HIV prevention: the road to clinical development.

Development of HIV-1 rectal-specific microbicides and colonic tissue evaluation.

Topical microbicides and HIV prevention in the female genital tract.

Precise engineering of dapivirine-loaded nanoparticles for the development of anti-HIV vaginal microbicides.

Formulation and delivery of anti-HIV rectal microbicides: advances and challenges.

Microbicides for the Treatment of Sexually Transmitted HIV Infections.

rectal microbicide gel formulation for HIV prevention.

Development of a protocol for predicting bacterial resistance to microbicides.

Preparing for rectal microbicides: sociocultural factors affecting product uptake among potential South American users.

Combining biomedical preventions for HIV: Vaccines with pre-exposure prophylaxis, microbicides or other HIV preventions.

Historical development of vaginal microbicides to prevent sexual transmission of HIV in women: from past failures to future hopes.

Modified silicone elastomer vaginal gels for sustained release of antiretroviral HIV microbicides.

HIV transmission biology: translation for HIV prevention.

Efficacy of rectal misoprostol for prevention of postpartum hemorrhage.

Prevention of vaginal and rectal HIV transmission by antiretroviral combinations in humanized mice.

Semen enhances HIV infectivity and impairs the antiviral efficacy of microbicides.

The context of HIV prevention.

Prevention and management of recurrent rectal cancer.

Interest in using rectal microbicides among Australian gay men is associated with perceived HIV vulnerability and engaging in condomless sex with casual partners: results from a national survey.

Emergent HIV technology: urban Tanzanian women's narratives of medical research, microbicides and sexuality.

AIDS.

Antiretroviral Therapy for the Prevention of HIV-1 Transmission.