Nanosystem Formulations for Rectal Microbicides: a Call for More Research

Keywords::

• antiretroviral drugs
• HIV/AIDS
• nanomedicine
• nanotechnology
• prevention
• rectal drug delivery
• sexually transmitted diseases
• VivaGel^®

HIV/AIDS is undoubtedly one of the main global health concerns of modern days. Latest numbers estimate that around 33.3 million individuals were living with HIV/AIDS by 2009, while 2.6 million new infections occurred during the same year [1]. Even if antiretroviral treatment provide an indispensable tool in the management of the disease, the infection is still incurable and prevention is considered a cornerstone strategy. In particular, prevention of sexual transmission is paramount. In this context, microbicides were proposed as products intended for vaginal or rectal administration around the time of sexual intercourse in order to prevent HIV transmission (and, possibly, other sexually transmitted pathogens) [2]. After frustrating results of different vaginal microbicide products, proof-of-concept was finally achieved in a Phase IIb clinical trial reported in 2010 for a vaginal gel containing 1% tenofovir, in which partial protection against the acquisition of HIV infection was found [3]. A lot of effort is now being focused on further confirmatory clinical studies for vaginal microbicides, either containing tenofovir or other antiretroviral drugs.

In the case of rectal microbicides, advances have been slower, but important preclinical and clinical data regarding preclinical effectiveness, and clinical safety and acceptability has been obtained in recent years [4,5]. It is widely recognized that these microbicides are of extreme importance, not only for men who have sex with men, but also for women who are receptive to anal intercourse (RAI), since rectal microbicides will empower users of self-protection without depending on condom use by partners [5]. Moreover, the risk of HIV rectal transmission can be estimated approximately 20-fold higher than for vaginal transmission [6,7]. Several factors may justify this fact, namely particular anatomical and physiological features.

One important aspect of microbicides development is related with product design. Choosing and formulating adequate systems for delivering microbicide drugs is regarded as increasingly important in achieving successful protection. Since both the vagina and colorectum present particular features, ‘one-size fits all‘ may not be applicable even if highly desirable. Nonetheless, hydrophilic gels have been regarded as the gold standard for both types of microbicides but other more complex dosage forms have also been proposed (e.g., vaginal rings and films). With the advent and recent achievements of nanomedicine, nanotechnology-based systems have been advocated as potentially advantageous for developing next-generation microbicides [8,9]. However, efforts have been mostly focused on nanosystems for vaginal administration, while research on those intended for rectal administration have been nearly non-existant.

Challenges in formulating rectal microbicides

The colorectal mucosa is covered by a single layer of columnar epithelial cells. This barrier is easily damageable (e.g., during RAI itself), providing a direct pathway to the underlying lamina propria, where HIV-susceptible cells (T cells, macrophages and dendritic cells) and lymphoid nodules are present [10]. Lubricants, which are often used during RAI [11], can also significantly damage the mucosa [12]; however, this practice may have a positive side since it can assure that rectal microbicides will be well accepted and used consistently, therefore contributing to adequate efficacy. When formulating rectal microbicides, the focus on safety cannot be too much. Past vaginal microbicides trials provided valuable lessons on how these products can damage and increase susceptibility to HIV infection [2]. The composition of the mucosal fluid present in the rectum and descending colon is in line with fluids covering other mucosal tissues, presenting pH values near neutrality in healthy individuals [13]. Comprising a thin sheet around 125 µm, the rectal mucosal fluid possesses protective and lubricant functions, posing as a barrier to the diffusion of particles (including virions and therapeutic nanosystems) to the underlying mucosa [14]. Damaging this film, for example, by using rectal products, may lead to enhanced HIV transmission and altered diffusion of drugs/nanosystems.

The continuum of the rectum towards the colon raises issues about product spreading: too little could compromise effectiveness, while too much can lead to safety issues. Nonetheless, much larger quantities of a product are expected to be necessary than for vaginal microbicides in order to outdistance and outlast the virus. A recent study indicates that quantities of up to 35 ml of a gel were considered acceptable by men who have sex with men [15]. Drug absorption by the rectal route is generally inline with absorption by oral administration. Therefore, systemic exposure and even toxicity are important issues when delivering molecules that are well absorbed.

A couple of questions requiring further insight may help solving these problems:

▪	Which are the boundaries for viral infection in the lower GI tract?
▪	Are there any particular regions of the lower GI tract that are more prone than others to infection by HIV?
▪	If so, is it feasible to specifically target them?

One recent pilot study in men engaging in RAI showed that nanoparticles made of ^99mTe-sulfur colloid (mean size 100 nm; range 10–500 nm) could migrate as far as the splenic flexure after simulated rectal ejaculation. However, in most cases (75%), the distribution was confined to the rectosigmoid colon [16]. A further study by the same researchers confirmed that distribution of these particles was mostly limited to this last region. Moreover, they used radio-labeled lymphocytes in the semen simulant as cell-associated HIV surrogates and observed that the distribution of these cells was also limited to the rectosigmoid colon up to 24 h [17]. Although these results should be analyzed with caution, mainly due to the use of HIV-particle substitutes and the low number of studied individuals, they provide valuable insight as to the distribution of both viral particles and nanotechnology-based microbicides.

Another important issue when considering rectal microbicides is to guarantee the local availability of the active molecule in sufficient concentrations and for long enough in order to inhibit viral infection. Additional significant aspects include the development of specific rectal applicators and product acceptability and affordability [5].

Nanotechnology-based rectal microbicides

Nanosystems for microbicide development encompass those with intrinsic antiviral activity (due to their surface chemistry, nanosystems interact directly with the virus and inactivate it), and those designed as antiretroviral drug carriers. In the case of the former, the main advantage of nanosizing comprises the exponential increase of the available surface area for interaction with the virus; as for antiretroviral drug nanocarriers, general advantages include the possibility of labile drug protection, modified drug release, penetration of epithelial cell linings, intracellular drug delivery, drug targeting and enhanced tissue retention [8,9]. Modulation of mucoadhesion according to specific needs is also achievable [18]. However, investigation on nanosystems for rectal microbicide development has been scarce.

The only exception has been VivaGel^® (Starpharma Pty Ltd., Melbourne, Australia), a carbomer gel containing the antiviral dendrimer SPL7013. SPL7013 is constituted by polylysine branches terminally derivatized with naphthalene disulfonate groups and attached to a central core of benzhydrylamine amide [19]. Due to its nanosized dimensions, the large surface area exposing the naphthalene disulfonate groups increases the probability of interaction with the virus. Even if most of the studies that have been conducted so far focused on vaginal use, some data has been reported on its rectal use. In vitro studies using a 5% SPL7013 carbomer gel showed that the formulation had reduced toxicity in the Caco-2 colorectal cell line. The cytotoxicity was mainly associated to the excipients of the gel rather than SPL7013 itself. Moreover, the integrity of Caco-2 monolayers was not damaged by the formulation. In addition, the dendrimer-containing gel prevented the transfer of HIV-1 from Caco-2 cells to peripheral blood mononuclear cells [20]. A further study by this group showed that the 5% SPL7013 gel induced epithelial shedding in human colorectal explants but without damaging the lamina propria. Even if these observations raise some concern, the formulation was still able to reduce over 85% the viral infection of explants [21]. In an in vivo animal study, the safety of a 3% SPL7013 gel was evaluated in pigtailed macaques upon three-times daily rectal application for four days. The choice for testing a reduced concentration gel (3 vs 5%) was based on previous observations of toxicity of the 5% concentration upon vaginal administration in the same model. During the study no significant differences in terms of pH, normal microflora and epithelial shedding were observed as compared to the placebo gel. Overall, the dendrimer-containing gel was well tolerated even if long-term safety studies are necessary in order to confirm these results [22].

In the particular case of microbicide drug nanocarriers, to the best of our knowledge, no major studies have been conducted so far. Our group has recently reported on polycaprolactone-based nanoparticles for delivering dapivirine, a potent antiretroviral drug. In vitro results showed that the encapsulation of dapivirine into nanoparticles was able to reduce its toxicity to Caco-2 cells, depending on the surface properties of the nanosystem. Moreover, drug-loaded nanoparticles were successfully used to increase the intracellular levels of drug in macrophages and dendritic cells, which play an important role in HIV mucosal transmission. Dapivirine-loaded nanoparticles were also able to inhibit cell infection by HIV-1 in different experimental settings. Overall, our results showed that differently engineered nanoparticles were able to modulate the selectivity index of dapivirine [23].

What is needed to move forward?

Advocacy for rectal microbicides has been increasing over the last years [101]. Although always a few steps behind vaginal microbicides, efforts have been made in order to close the gap and move forward in the field. Apart from Vivagel, research on nanotechnology-based systems for rectal microbicide development is still in its infancy. Future groundbreaking work includes both basic and applied research on rectal microbicides. Important questions on rectal HIV transmission for microbicide development remain to be answered, namely:

▪	What is the ‘anatomical range‘ for colorectal transmission and are there any particular areas more susceptible than others?
▪	What is the influence of changes to the colorectal microenvironment on viral transmission?
▪	Which important physiological alterations during and after RAI can influence microbicide design?

While waiting for answers, more investigation on nanosystems designed to specifically meet the particularities of the colorectal environment needs to be engaged. In particular, studies on the interaction of nanosystems with the mucosa, colorectal fluid and content (e.g., stool), and semen are in order. Moreover, it is necessary to develop appropriate vehicles for the administration of these nanosystems.

Financial & competing interests disclosure

J das Neves acknowledges financial support from Fundação para a Ciência e a Tecnologia, Portugal (grant SFRH/BD/43393/2008). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.