PROJECT 2 The demonstration of safety is a central component in the development of topical non-vaccine biomedical prevention (nBP) candidates for the prevention of sexual HIV transmission. When orally administered FDA- approved drugs are reformulated for topical delivery to the vagina, safety studies have involved general toxicology and irritation/inflammation in one or more animal species, along with a rudimentary assessment of the effect on vaginal microflora, usually in macaques. These established studies do not adequately address all important safety aspects of antiretroviral (ARV) combinations administered to the vagina, particularly those relating to increased risk of HIV acquisition through product use that could counteract the drugs'protective effects. The objective of Project 2 is to assess the safety/local toxicity of intravaginal rings (IVRs) delivering ARV combinations formulated in Core B.
In Aim 1, nine initial ARV IVR formulations will be screened ex vivo in a high-throughput vaginal epithelial cell (VEC) culture system to measure the impact of drug combinations and dosing on: physiology and health;innate immune stimulation;apoptosis and inflammation;and commensal microorganism viability and health using VECs colonized with complex vaginal microbiota. Preliminary safety also will be established in a first round of mouse and sheep studies. Results from these safety and efficacy (Project 3) outcomes will be used to select three ARV IVR candidates for more in-depth, second round of safety evaluation under Aim 2 in a sheep model using advanced, in vivo, high-resolution imaging together with traditional histology and colposcopy. The local toxicity of these IVRs will be assessed further in Aim 3 using pig-tailed macaques by measuring the upregulation of inflammatory cytokines in the vaginal epithelium and the impact of the IVRs on commensal microflora. The latter will be achieved using an innovative combination of molecular techniques to obtain culture-independent microbial community composition and microscopic techniques to characterize the community structure in the vaginal tract and on the surface of the IVRs with a special emphasis on microbial biofilms. An analogous assessment of local toxicity will be applied in a (pre- Phase I) Exploratory Clinical Trial in women using our lead candidate IVR triple ARV combination (Aim 3). Successful completion of these safety studies will provide the necessary data for selection of a lead candidate and will form a cornerstone of the IND-enabling activities in the transition of this formulation to post-IPCP clinical studies.