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Preclinical Evaluation of Synthetic -2 RANTES as a Candidate Vaginal Microbicide To Target CCR5
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".....In conclusion, the excellent safety profile of -2 RANTES provides strong support for the advancement of -2 RANTES as a vaginal microbicide. The ideal formulation for any vaginal microbicide requires a balance between minimizing the toxicity of the microbicide to the cervicovaginal mucosa and maintaining maximal activity against HIV-1. This report on the preclinical evaluation of -2 RANTES demonstrates that -2 RANTES is favorable for topical vaginal application for prevention of the sexual transmission of R5 tropic strains of HIV-1...."
Antimicrobial Agents and Chemotherapy, April 2006, p. 1497-1509, Vol. 50, No. 4
Tina M. Kish-Catalone, Wuyuan Lu, Robert C. Gallo, and Anthony L. DeVico*
Division of Basic Science, Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201
ABSTRACT
A potential strategy that can be used to combat the worldwide AIDS epidemic is the development of a vaginal microbicide that prevents the sexual transmission of human immunodeficiency virus type 1 (HIV-1). Certain CC chemokines, including RANTES, MIP-1, and MIP-1B, might facilitate the development of such microbicides since they potently suppress HIV-1 infection by binding to CCR5, the viral coreceptor used by most sexually transmitted strains of HIV-1 to enter host cells. In this study, we evaluated whether a CCR5-specific fragment of RANTES that lacks two N-terminal residues (-2 RANTES) and possesses especially potent HIV-1 suppressive activity has toxicity profiles conducive to the advancement of testing in candidate microbicide formulations. Analyses were carried out with a synthetic version of the chemokine, which was formulated with either Novasomes 7474, a nonphospholipid liposome, or methylcellulose gel. Dialysis studies demonstrated that the formulated -2 RANTES was released from both vehicles and retained anti-HIV-1 activity.
Preclinical toxicity studies carried out with Swiss Webster mouse and New Zealand White rabbit vaginal irritation models demonstrated minimal inflammation and minimal adverse changes in cervicovaginal tissue integrity after short-term (10 min) and long-term (24 h) exposure to formulations containing up to 1 mg/ml of -2 RANTES. Similarly, no toxicity was observed with formulations of bioactive murine RANTES in the Swiss Webster mouse vaginal irritation model. Overall, these preclinical studies suggest that -2 RANTES is suitable for further testing as a candidate anti-HIV-1 microbicide.
Background
Heterosexual contact has emerged as the predominant mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide, accounting for more than 90% of all adolescent and adult HIV-1 infections (27). Consequently, half of all individuals currently infected with HIV-1 are female (22). In the absence of an effective prophylactic anti-HIV vaccine, other preventative methods are urgently needed to control the spread of the virus (6, 13). Vaginal microbicides may provide an alternative, female-controlled method of protection against the sexual transmission of HIV-1. The ideal vaginal microbicide should be effective, safe, acceptable, affordable, colorless, odorless, stable, easy to store and use, available in contraceptive and noncontraceptive formulations, and available without a prescription (6). However, the immediate priority is the development of a microbicide that effectively blocks HIV-1 infection in a manner that is nontoxic to the genital tract.
Early attempts to develop vaginal microbicides focused on agents with nonspecific mechanisms of action, such as nonionic surfactant, nonoxynol-9 (N-9). N-9 has previously been demonstrated to possess in vitro activity against several sexually transmitted disease pathogens, including HIV-1 (24, 31). However, high concentrations and/or frequent exposures to these agents resulted in genital irritation and ulceration in clinical trials, which increased the risk for HIV-1 infection (35, 39). For these reasons, attention has turned toward the development of candidate microbicides with specific biological mechanisms of action.
In order to initiate replication, HIV-1 uses the host cell CD4 receptor for attachment and a coreceptor for viral entry. The vast majority of sexually transmitted HIV-1 strains use the CCR5 coreceptor, which naturally functions as a chemokine receptor (42, 47-49). Consequently, strong natural resistance to infection is associated with the genetic loss of CCR5 expression (14, 20, 23, 36). Notably, there are no apparent health consequences associated with this phenotype (14, 15, 20, 23, 25, 33, 36, 50). Taken together, these findings suggest that sexual transmission of HIV-1 infection may be safely blocked by agents that interfere with the coreceptor function of CCR5.
A growing body of evidence suggests that the natural chemokine ligands for CCR5, which include RANTES, MIP-1, and MIP-B, may provide a means to block mucosal CCR5 use by HIV-1 (37). These chemokines suppress HIV-1 entry by competitively blocking HIV-CCR5 interactions and by down-regulating cell surface CCR5 (1, 3, 19, 29, 41, 46). A number of clinical studies have shown that an inborn capacity to produce high levels of HIV-suppressive CCR5 ligands is associated with persistent seronegative status, despite repeated exposure to HIV-1 (14, 20, 23, 25, 33, 36, 50). In agreement, primate vaccine studies have correlated increased chemokine production with protection from virus challenge (4).
Among CCR5 ligands, a truncated fragment of RANTES (termed -2 RANTES) exhibits qualities that are particularly conducive to microbicide development. Under natural conditions this fragment is generated by a dipeptidyl peptidase, CD26, which removes two N-terminal residues from the chemokine (21, 30, 32, 38). Although unprocessed RANTES binds to several chemokine receptors, -2 RANTES is a dedicated CCR5 ligand (40). This specificity appears to render -2 RANTES a more potent HIV-1 suppressive agent than the unprocessed chemokine (38). However, the N-terminal truncation also abrogates the ability of RANTES to stimulate calcium mobilization in monocytes and to chemoattract monocytes and eosinophils in vitro (21, 30, 32, 38). This reduced capacity to activate receptors could minimize the likelihood that -2 RANTES would cause local inflammation or other adverse side effects if it was used in a topical microbicide formulation.
In this study, standardized animal models were used to examine the safety and toxicity profiles of topical microbicide formulations containing -2 RANTES. Formulation vehicles providing sustained contact of the chemokine to the mucosal surface were selected. One formulation vehicle, methylcellulose, has previously been used for vaginal delivery of neutralizing antibodies (43) and has demonstrated excellent safety profiles in phase I trials (9). The second vehicle of choice, Novasomes 7474, a nonphospholipid liposome, has previously been tested as a vaginal vehicle in phase I trials and was shown to have acceptable safety profiles (Novavax investigational new drug permit 57,550). Here we report that both formulations release antiviral concentrations of -2 RANTES without resulting in detectable levels of inflammation or toxicity. These results indicate that microbicide formulations containing CCR5 ligands may represent candidate vaginal microbicides for the prevention of HIV-1 transmission.
DISCUSSION
The development of topical microbicides has gained increasing attention as a means of containing sexually transmitted disease epidemics, including the HIV and AIDS epidemic. Over thirty candidate compounds are being evaluated for their potential use as topical microbicides (Alliance for Microbicide Development [www.microbicide.org]). Each class of compounds demands specific types of analyses to assess potential safety and efficacy. In the case of biological antiviral molecules formulated in carrier vehicles, it is necessary to evaluate whether a candidate compound is nontoxic, whether it is released from the vehicle in an antiviral form and concentrations, and whether biological activity causes inflammation or irritation which could subsequently promote infection.
In this study, we evaluated whether -2 RANTES formulated in a carrier vehicle warrants testing for efficacy as a vaginal anti-HIV-1 microbicide. Since RANTES is a chemoattractant cytokine that plays a role in inflammatory responses, our analytical models were selected and configured to determine whether the test formulations caused significant inflammation or cell infiltration of vaginal tissues. The nonionic surfactant N-9 was used as a positive control in these experiments for severe cervicovaginal toxicity. N-9 has repeatedly been demonstrated to cause gross disturbances in the cervicovaginal mucosa, subsequently increasing the risk of acquiring HIV-1 infection through sexual intercourse (28, 34, 35, 39).
Four of our analytical parameters indicated that vehicles deliver and release -2 RANTES in an active form and without evidence of toxicity. First, concentrations of -2 RANTES up to 2,000-fold higher than what is necessary for blocking HIV-1 infection in vitro (1 mg/ml) were nontoxic, as determined in MTS cytotoxicity assays (Fig. 1). Thus, it was reasonable to expect that relatively high concentrations of -2 RANTES can be incorporated into carrier vehicles with a minimal risk of causing general toxic effects in vivo.
Second, analyses in the Swiss Webster mouse vaginal toxicity model demonstrated that -2 RANTES, either alone (Fig. 2 and 3) or in a formulation with methylcellulose or Novasomes 7474 (Fig. 7), causes no morphological damage to the cervical or vaginal epithelium at concentrations up to 1 mg/ml. Furthermore, neither vehicle alone caused detectable damage, in accordance with the findings of clinical trials that used these compounds as placebo controls (Novavax investigational new drug permit 57,550) (9). It is particularly noteworthy that no damage was evident between 30 min and 4 h after microbicide application, which corresponds to the period when the maximum risk of potential exposure to HIV-1 is likely to occur. Previous studies with this model have also demonstrated that these are the time points when the greatest amount of tissue toxicity and inflammation occurred following 1% N-9 application. Immunohistochemical analyses of murine CD45-positive cells further demonstrated that up to 1 mg/ml -2 RANTES (Fig. 2 and 3) caused minimal infiltration of cells over the 24-h time course of the experiment. Collectively, these findings suggest that vaginal application of -2 RANTES is unlikely to cause recruitment of potential host cells to HIV-1-exposed areas of the vaginal epithelium or to induce major changes in tissue architecture that might promote mucosal HIV-1 infection. It is reasonable to use these findings to predict the safety of -2 RANTES in humans, since studies demonstrate that the human chemokine is biologically active in mice (7, 26).
Analyses of 2 ƒÊg/ml of full-length murine RANTES in the Swiss Webster mouse model revealed signs of more significant cell infiltrate in the animals compared to that observed with human -2 RANTES. However, the murine chemokine did not result in gross visible signs of inflammation or morphological damage to the cervicovaginal mucosa (Fig. 4). These differences could be due to the higher activity of the murine chemokine in the mouse or to the generally lower chemotactic activity of -2 RANTES compared with that of the unprocessed form (38). Recent studies in our laboratory demonstrated that the vaginal application of formulated -2 RANTES was nontoxic to the cervicovaginal epithelium of the cynomolgus macaque model (T. M. Kish-Catalone et al., unpublished data).
Third, toxicity studies with the standardized New Zealand White rabbit vaginal irritation model emphasize that minimal tissue damage resulted following 10 consecutive daily applications of the -2 RANTES formulations. Specifically, the Novasomes 7474 formulations containing 50 ƒÊg/ml or 500 ƒÊg/ml of -2 RANTES received an acceptable vaginal irritation rating, according to established scoring methodologies for assessment of the toxicities of vaginal products. Notably, this model system is a required in vivo assay necessary for all candidate vaginal microbicides advancing into clinical trials.
Fourth, the dialysis experiments demonstrated that -2 RANTES was released from formulation vehicles with retention of antiviral activity. However, more -2 RANTES was released from methylcellulose than from Novasomes 7474. These experiments were not expected to predict precisely how much -2 RANTES would be released in the cervicovaginal environment in situ, since many factors (e.g., pH, temperature, enzymatic activities, and the presence of vaginal microflora and semen) are not represented. However, the system allowed us to compare different formulations for chemokine release. In general, the vehicles impeded the dialysis of -2 RANTES into the buffer compared to that for -2 RANTES in PBS (Fig. 5). Between the two vehicles, more chemokine was released from methylcellulose than from Novasomes 7474. The physical and/or chemical basis for this difference is unclear. An important consideration is that methylcellulose is a mixture of heterogeneous polymers, with the smallest having a molecular weight of 90,000. Therefore, a minor portion of the vehicle might have passed through the dialysis bag along with the chemokine. Such an effect could explain why methylcellulose appeared to release more -2 RANTES than the corresponding Novasomes 7474 formulations. Notably, our control assays determined that methylcellulose did not interfere in the ELISA or inhibit HIV infection when it was tested alone (data not shown). Therefore, we do not believe that the quantitative assays were influenced by methylcellulose contamination. However, the antiviral potency of the -2 RANTES released from methylcellulose was significantly reduced compared to that of the -2 RANTES released from PBS or Novasomes 7474 (Fig. 6). These results collectively emphasize the importance of formulation design in the development of a vaginal microbicide based on biological antiviral molecules.
In conclusion, the excellent safety profile of -2 RANTES provides strong support for the advancement of -2 RANTES as a vaginal microbicide. The ideal formulation for any vaginal microbicide requires a balance between minimizing the toxicity of the microbicide to the cervicovaginal mucosa and maintaining maximal activity against HIV-1. This report on the preclinical evaluation of -2 RANTES demonstrates that -2 RANTES is favorable for topical vaginal application for prevention of the sexual transmission of R5 tropic strains of HIV-1.
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