PROJECT SUMMARY Osteoarthritis (OA), the gradual erosion of articular cartilage which covers the ends of bones in diathroidial joints, is a debilitating disease that affects over 32.5 million U.S. adults. There is no cure for OA, and the two most common intra-articular treatments, corticosteroids and hyaluronic acid, are for pain relief and increased joint range of motion. Thus, there is an urgent, unmet need for disease-modifying OA drugs (DMOADs) that would actually inhibit disease progression. Punicalagin (PCG) has been identified as a promising DMOAD candidate. Our long-term objective is to create a PLGA-based, in situ forming microparticle system capable of ultralong, intra-articular PCG delivery. We envision semiannual injections that could be given prophylactically to patients at high risk of developing OA or to slow disease progression in patients with early-stage OA. PCG is the major polyphenol present in pomegranate. It plays regulatory roles in multiple signaling pathways involved in the inflammatory process and also inactivates MMP-13, the enzyme primarily responsible for destruction of cartilage collagen in OA. The approach follows an established workflow for designing an intra- articular drug delivery system to treat OA, starting with characterization of PCG as a DMOAD, proceeding to optimization of the delivery system formulation (PCG-containing PLGA solution emulsified into sesame oil), and then moving to a study of the system’s safety. Specific Aim 1 is to determine the potential for PCG to inhibit production of IL-1β, TNF-α, and IL-6 (important OA cytokines), as well as MMP-13 and ADAMTS-5 (key catabolic enzymes in OA) by human chondrocytes, synoviocytes, osteoblasts and macrophages. Cultures stimulated with the alarmin S100A8 to induce inflammation will be treated with PCG and analyzed two days later using ELISA. Aim 2 is to optimize emulsion formulation with respect to chondrocyte survival, in vitro PCG release kinetics, and microparticle size. Variables include PLGA MW, PLGA concentration, solvent, and polymer:oil ratio, which are expected to influence microparticle size and structure, porosity, and the rate of solvent diffusion. Some microparticles will be formed in dialysis bags and maintained at 37 °C to monitor drug release. Additional aliquots of emulsion will be used to assess their cytotoxicity to a chondrocyte cell line and to characterize the size and morphology of microparticles formed upon injection into water. Specific Aim 3 is to evaluate the potential for the optimal, drug-free microparticle system to stimulate production of inflammatory mediators in vitro and to be tolerated in vivo within a joint. Biocompatibility of the optimal emulsion will be evaluated in cultures of human synoviocytes, chondrocytes, osteoblasts, and macrophages based on production of proinflammatory cytokines and eicosanoids quantified using multiplex ELISA and LC-MS/MS. The safety of the emulsion with respect to the intra-articular route ...