Use of the murine model of cervicovaginal toxicity to examine factors that adversely affect epithelial integrity and inflammation following microbicide application
Authors:   , Lozenski Karissa , Krebs Fred C 
Published: July 2024

The human immunodeficiency virus type 1 (HIV-1) pandemic is a current global health issue that is showing no signs of slowing it's spread. 33 million people are infected with HIV-1, approximately 22 million of whom are localized to sub-Saharan Africa. Within the HIV-1+ population, over 50% those afflicted are women. Women are also the leading population for the development of new infections. The involvement of women in the current epidemic has created a great need for female controlled prevention methods that will provide women with the tools necessary to protect themselves. The answer to this demand is a microbicide, which is a topical product that can be applied before intercourse to provide protection against HIV-1 infection. During the past 10 years of microbicide development, a number of candidate microbicides have failed to provide protection in clinical trials of microbicide efficacy. In addition, two of these trials indicated a greater risk of HIV-1 infection associated with product use. Specifically, in the case of nonoxynol-9 (N-9), the risk of infection in the treatment group was approximately 50% higher than what was observed in the placebo group. As a consequence of these failures, researchers are now questioning the stringency of preclinical development strategies. Our laboratory has worked to develop a small animal model of cervicovaginal toxicity that could be used to predict clinical microbicide safety. Initial studies involving this model system strongly suggested that murine microbicide toxicity is predictive of clinical toxicity. In the present studies, this model system was utilized in multiple exposure studies focusing on the effects of multiple daily applications of N-9. These studies were performed in response to clinical results that indicated a greater risk of HIV-1 infection associated with increase frequency of N-9 use. Experiments showed that N-9-induced damage is not cumulative and that tissue actually being to become tolerant to N-9. In addition, immunological studies identified pro- inflammatory cytokines that are released in response to this damage, and demonstrated immune cell recruitment as a result of N-9 application. These effects provide a plausible explanation for the increased risk of HIV-1 infection that was observed in clinical studies. In addition to N-9, a candidate microbicide was examined in this animal model system. NB325, a polybiguanide molecule characterized by activity against HIV-1 and low in vitro cytotoxicity, was examined using the murine model of microbicide toxicity to assess the potential safety of both unformulated and formulated compound. Unformulated NB325 (1%) had little impact on vaginal epithelial integrity and no effect on cervical tissues. Interestingly, NB325 formulation increased its toxicity slightly compared to the neat compound, suggesting that retention of a compound as a function of its formulation may be a factor that decreases its safety. Experiments were also performed to seek parallels between in vitro assays of microbicide cytotoxicity and in vivo indications of product toxicity. Specifically, these studies focused on multiple exposures to N-9. After multiple, daily applications of N-9, cell lines of human cervical epithelial origin became increasingly sensitive to N-9 with each additional exposure. These in vitro results were in sharp contrast to the results of the animal model experiments, which indicated the emergence of N-9 tolerance. The levels of cytokine expression and release from N-9-exposed cells also differed from the levels and timing of cytokine release and expression in the murine model experiments. These results strongly advocate the use of a small animal model, such as the murine model of microbicide toxicity, in the course of pre-clinical microbicide development and the judicious use of in vitro experiments to predict compound safety.