Tina Kish-Catalone

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

ABSTRACT 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.

Structure-activity relationships of polybiguanides with activity against human immunodeficiency virus type 1

Previous investigations showing that polydisperse biguanide (PDBG) molecules have activity against human immunodeficiency virus type 1 (HIV-1) also suggested a relationship between PDBG biologic activity and the lengths of hydrocarbon linkers surrounding the positively charged biguanide unit. To better define structure-activity relationships, PDBG molecules with select linker lengths were evaluated for cytotoxicity, anti-HIV-1 activity, and in vivo toxicity. Results of the in vitro experiments demonstrated that increases in linker length (and, therefore, increases in compound lipophilicity) were generally associated with increases in cytotoxicity and antiviral activity against HIV-1. However, a relationship between linker length asymmetry and in vitro therapeutic index (TI) suggested structural specificity in the mechanism of action against HIV-1. Polyethylene hexamethylene biguanide (PEHMB; biguanide units spaced between alternating ethylene and hexamethylene linkers) was found to have the highest in vitro TI (CC₅₀/IC₅₀) among the compounds examined. Recent improvements in PEHMB synthesis and purification have yielded preparations of PEHMB with in vitro TI values of 266 and 7000 against HIV-1 strains BaL and IIIB, respectively. The minimal toxicity of PEHMB relative to polyhexamethylene biguanide (PHMB; biguanide units alternating with hexamethylene linkers) in a murine model of cervicovaginal microbicide toxicity was consistent with considerable differences in cytotoxicity between PEHMB and PHMB observed during in vitro experiments. These structure-activity investigations increase our understanding of PDBG molecules as agents with activity against HIV-1 and provide the foundation for further preclinical studies of PEHMB and other biguanide-based compounds as antiviral and microbicidal agents.

Decreased cervical epithelial sensitivity to nonoxynol-9 (N-9) after four daily applications in a murine model of topical vaginal microbicide safety

BackgroundThe disappointing clinical failures of five topical vaginal microbicides have provided new insights into factors that impact microbicide safety and efficacy. Specifically, the greater risk for human immunodeficiency virus type 1 (HIV-1) acquisition associated with multiple uses of a nonoxynol-9 (N-9)-containing product has highlighted the importance of application frequency as a variable during pre-clinical microbicide development, particularly in animal model studies.MethodsTo evaluate an association between application frequency and N-9 toxicity, experiments were performed using a mouse model of cervicovaginal microbicide safety. In this model system, changes in cervical and vaginal epithelial integrity, cytokine release, and immune cell infiltration were assessed after single and multiple exposures to N-9.ResultsAfter the initial application of N-9 (aqueous, 1%), considerable damage to the cervical epithelium (but not the vaginal epithelium) was observed as early as 10 min post-exposure and up to 8 h post-exposure. Subsequent daily exposures (up to 4 days) were characterized by diminished cervical toxicity relative to single exposures of like duration. Levels of pro-inflammatory cytokines released into the cervicovaginal lumen and the degree of CD14-positive immune cell infiltration proximal to the cervical epithelium were also dependent on the number of N-9 exposures.ConclusionsRather than causing cumulative cervical epithelial damage, repeated applications of N-9 were characterized by decreased sensitivity to N-9-associated toxicity and lower levels of immune cell recruitment. These results provide new insights into the failure of N-9-based microbicides and illustrate the importance of considering multiple exposure protocols in pre-clinical microbicide development strategies.

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 Anti-HIV-1 Polybiguanide Vaginal Microbicides in a Murine Model of Toxicity and Inflammation

The use of vaginal microbicides has gained support as a strategy for the protection of women against HIV-1 and other sexually transmitted disease (STD) pathogens. The preclinical Swiss Webster murine model has been developed to specifically measure cervicovaginal tissue integrity and inflammation following application of candidate vaginal microbicides, when potential exposure to an STD pathogen may occur. This model demonstrates both mechanistic and temporal differences in inflammatory responses following microbicide exposure. Currently, specific markers of inflammation, including pro-inflammatory cytokines, are being evaluated in the cervicovaginal mucosa. Safety profiles of polybiguanides (PBGs), which demonstrated significant in vitro efficacy against HIV-1, are being assessed in vivo. Intravaginal application of PEHMB (1%) resulted in little or no cervicovaginal toxicity after shortor long-term exposure. Collectively, these studies support the Swiss Webster model as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides. Furthermore, these results strongly support further development of polybiguanide derivatives as vaginal microbicidal agents. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

CONRAD Testing Algorithm: Microbicidal Compounds Screened For Cytotoxicity and Activity Against HIV-1

Cytotoxicity and virucidal/antiviral testing conducted for the CONRAD Program identifies compounds that may be developed as microbicides used to reduce or eliminate the risk of HIV-1 sexual transmission. Preliminary testing begins with a cytotoxicity screen (CTS) to assess the impact of each compound on cell viability. Activity against infectious HIV-1 was measured using viral binding/entry inhibition (VBI) assays, in which each compound was evaluated for the ability to inhibit binding or entry events between target cells and HIV-1 strains IIIB (X4 phenotype) or BaL (R5 phenotype). Finally, compounds have been screened to determine their ability to interfere with cell-to-cell (CTC) HIV-1 transmission. In all assays, dextran sulfate was used as a control because of its minimal cytotoxicity and potent anti-HIV-1 activity. Out of 477 compounds tested since May 2001, 77 compounds tested using this algorithm were shown to have high selectivity indices (little or no cytotoxicity and consistently high activity in all three viral assays). These endeavors will greatly facilitate the search for compounds and formulations that can be used globally as topical microbicides. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005