Cervicovaginal Safety of the Formulated, Biguanide-Based Human Immunodeficiency Virus Type 1 (HIV-1) Inhibitor NB325 in a Murine Model

Vaginal microbicides that reduce or eliminate the risk of HIV-1 sexual transmission must do so safely without adversely affecting the integrity of the cervicovaginal epithelium. The present studies were performed to assess the safety of the biguanide-based antiviral compound NB325 in a formulation suitable for topical application. Experiments were performed using a mouse model of cervicovaginal microbicide application, which was previously shown to be predictive of topical agent toxicity revealed in microbicide clinical trials. Mice were exposed vaginally to unformulated NB325 or NB325 formulated in the hydroxyethyl cellulose “universal placebo.” Following exposures to formulated 1% NB325 for 10 min to 24 h, the vaginal and cervical epithelia were generally intact, although some areas of minimal vaginal epithelial damage were noted. Although formulated NB325 appeared generally safe for application in these studies, the low but observable level of toxicity suggests the need for improvements in the compound and/or formulation

Preclinical Evaluation of Synthetic −2 RANTES as a Candidate Vaginal Microbicide To Target CCR5

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-1α, and MIP-1β, 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

Mouse Model of Cervicovaginal Toxicity and Inflammation for Preclinical Evaluation of Topical Vaginal Microbicides

Clinical trials evaluating the efficacy of nonoxynol-9 (N-9) as a topical microbicide concluded that N-9 offers no in vivo protection against human immunodeficiency virus type 1 (HIV-1) infection, despite demonstrated in vitro inactivation of HIV-1 by N-9. These trials emphasize the need for better model systems to determine candidate microbicide effectiveness and safety in a preclinical setting. To that end, time-dependent in vitro cytotoxicity, as well as in vivo toxicity and inflammation, associated with N-9 exposure were characterized with the goal of validating a mouse model of microbicide toxicity. In vitro studies using submerged cell cultures indicated that human cervical epithelial cells were inherently more sensitive to N-9-mediated damage than human vaginal epithelial cells. These results correlated with in vivo findings obtained by using Swiss Webster mice in which intravaginal inoculation of 1% N-9 or Conceptrol gel (containing 4% N-9) resulted in selective and acute disruption of the cervical columnar epithelial cells 2 h postapplication accompanied by intense inflammatory infiltrates within the lamina propria. Although damage to the cervical epithelium was apparent out to 8 h postapplication, these tissues resembled control tissue by 24 h postapplication. In contrast, minimal damage and infiltration were associated with both short- and long-term exposure of the vaginal mucosa to either N-9 or Conceptrol. These analyses were extended to examine the relative toxicity of polyethylene hexamethylene biguanide (PEHMB), a polybiguanide compound under evaluation as a candidate topical microbicide. In similar studies, in vivo exposure to 1% PEHMB caused minimal damage and inflammation of the genital mucosa, a finding consistent with the demonstration that PEHMB was >350-fold less cytotoxic than N-9 in vitro. Collectively, these studies highlight the murine model of toxicity as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides

Prolonged exposure to the candidate microbicide C31G differentially reduces cellular sensitivity to agent re-exposure

Comparative assays of in vitro cytotoxicity using nonoxynol-9 (N-9) and the candidate microbicides C31G and sodium dodecyl sulfate (SDS) demonstrated that these agents, which are, respectively, characterized as nonionic, amphoteric, and anionic surfactants, differed in their concentration-dependent effects on cell viability, especially after prolonged exposure. We hypothesized that differences in cellular sensitivity may have been due, in part, to cellular changes induced by long-term exposure to each agent. To examine this possibility, HeLa cells were exposed to N-9, C31G, or SDS for extended periods of time and subsequently reassessed for sensitivity to each of these agents. Following 10 continuous days of C31G exposure, HeLa cells were less sensitive to a subsequent C31G exposure compared to cells that had not undergone long-term C31G treatment. Interestingly, long-term C31G exposure also changed subsequent sensitivity to N-9 but not SDS. In contrast, prolonged exposure to either N-9 or SDS did not reduce sensitivity to re-exposure. The effect of long-term C31G exposure was both concentration-dependent and transient, as treated cells reverted to pre-exposure sensitivity in a time-dependent manner following the cessation of C31G exposure. Lipid analyses of cells exposed to C31G for extended durations revealed altered phospholipid profiles relative to C31G-naïve cells. Experiments examining the individual components of C31G demonstrated the involvement of the amine oxide moiety in reductions in cellular sensitivity. These studies, which provide new information concerning the cytotoxicity of surfactant microbicides, suggest that cervicovaginal epithelial cells may have greater in vivo tolerance for products containing C31G through unique interactions between C31G and components of the cellular membranes

Toxicity, inflammation, and anti-human immunodeficiency virus type 1 activity following exposure to chemical moieties of C31G

C31G, which has potent activity against the human immunodeficiency virus type 1 (HIV-1) and an established record of safety in animal studies and human trials, is a microbicidal agent comprised of a buffered equimolar mixture of two amphoteric, surface-active agents: an alkyl amine oxide (C14AO) and an alkyl betaine (C16B). Studies of long-term in vitro exposure to C31G and its constituents have suggested that the components of C31G may contribute differentially to its toxicity and efficacy. In the present studies, in vitro assays of cytotoxicity and anti-HIV-1 activity demonstrated that C16B was slightly less cytotoxic compared to either C31G or C14AO, whereas the anti-HIV-1 activities of C31G and its individual constituents were similar. In the murine model of cervicovaginal microbicide toxicity, in vivo exposure to C14AO resulted in severe cervical inflammation followed by a delayed disruption of the columnar epithelium. In contrast, exposure to C16B caused severe cervical epithelial disruption and a secondary, less intense inflammatory response. These results demonstrate that (i) there are both mechanistic and temporal differences in toxicity associated with the components of C31G not necessarily predicted by in vitro assessments of cytotoxicity and (ii) contributions of each component to the anti-HIV-1 activity of C31G appear to be equal. In addition, these findings indicate that direct and indirect mechanisms of in vivo toxicity can be observed as separate but interrelated events. These results provide further insight into the activity of C31G, as well as mechanisms potentially associated with microbicide toxicity

Human β-Defensins Suppress Human Immunodeficiency Virus Infection: Potential Role in Mucosal Protection

β-Defensins are small (3 to 5 kDa in size) secreted antimicrobial and antiviral proteins that are components of innate immunity. β-Defensins are secreted by epithelial cells, and they are expressed at high levels in several mucosae, including the mouth, where the concentration of these proteins can reach 100 μg/ml. Because of these properties, we wondered whether they could be part of the defenses that lower oral transmission of human immunodeficiency virus (HIV) compared to other mucosal sites. Our data show that select β-defensins, especially human β-defensin 2 (hBD2) and hBD3, inhibit R5 and X4 HIV infection in a dose-dependent manner at doses that are compatible with or below those measured in the oral cavity. We observed that β-defensin treatment inhibited accumulation of early products of reverse transcription, as detected by PCR. We could not, however, detect any reproducible inhibition of env-mediated fusion, and we did not observe any modulation of HIV coreceptors following treatment with hBD1 and hBD2, in both resting and phytohemagglutinin-activated cells. Our data instead suggest that, besides a direct inactivation of HIV virions, hBD2 inhibits HIV replication in the intracellular environment. Therefore, we speculate that β-defensins mediate a novel antiretroviral mechanism that contributes to prevention of oral HIV transmission in the oral cavity. Immunohistochemical data on hBD2 expression in oral mucosal tissue shows that hBD2 is constitutively expressed, forming a barrier layer across the epithelium in healthy subjects, while in HIV-positive subjects levels of hBD2 expression are dramatically diminished. This may predispose HIV-positive subjects to increased incidence of oral complications associated with HIV infection