On-going research in our lab, which is supported by the parent grant, has begun to clarify the critical role played by activated mast cells (MC) in the development of diabetic foot ulcers (DFU). More specifically, we found that in the skin from patients with diabetes (DM) the number of activated MC is significantly higher than healthy controls and correlates with dermal inflammatory cells and dermal inflammatory cytokines, as well as with circulating levels of inflammatory markers such as IL6 and TNF?. Furthermore, we were able to demonstrate in a murine model of diabetic wound healing that the number of degranulated MC was significantly higher in the skin of streptozotocin-induced diabetic (STZ-DM) mice. Treatment with the MC stabilizer disodium cromoglycate (DSCG) significantly accelerated wound healing, accompanied with significantly reduced amount of degranulated MC. In addition, DSCG considerably reduced the M1 macrophage phenotype, both pre- and post-wounding in this animal model. Thus, our preclinical proof-of-concept results strongly support that the inhibition of MC degranulation is a viable approach for treating DFU. MC degranulation is controlled by an elevated level of cytosolic calcium that is mediated by the stored operated calcium (SOC), and to a lesser extent, by the receptor potential canonical (TRPC) channels. The best characterized SOC channel is the calcium selective orai, also known as calcium-release activated calcium (CRAC) channel that is expressed not only by MC but also by T cells as well as vascular smooth muscle cells. Activation of MC stimulates the opening of the orai channels for calcium influx. Small molecule orai/CRAC channel blockers have been shown to potently inhibit MC degranulation and T-cell activation. In this revised proposal, we plan to evaluate the effects of calcium channel blockers in our mouse model of diabetic wound healing. Our main hypothesis is that calcium channel blockers are more efficacious in improving diabetic wound healing. In the first specific aim, we will focus on the synthesis of the prototype calcium channel blockers and formulation development for topical drug administration. The synthetic process for each compound will require three chemical steps from available reagents and standard purification technics such as column separation and recrystallization. Because of the intrinsic hydrophobic properties of the prototype compounds, we will also employ solubilizing enhancers to formulate the drugs for topical delivery in in vivo efficacy studies. In the second specific aim, we will test the efficacy of developed prototypes in animal models of diabetic wound healing. For this, we will employ the streptozotocin-induced diabetic (STZ-DM) wild-type (WT) C57BL/6J mouse model that was used in the parent application to evaluate its ability to improve wound healing either used prophylactically before the wound creation or applied topically post wounding. We will also compare the efficacy of this treatment to the only commercially available MC stabilizer, DSCG.
The main aims of this revision are to synthesize a prototype mast cell stabilizer that is a calcium channel blocker, complete formulation development for topical drug administration and test its efficacy in animal models of diabetic wound healing. We will also compare the efficacy of this treatment to the only commercially available mast cell stabilizer, disodium cromoglycate (DSCG).