Mechanisms of Neuropeptides Action in Diabetes

 描述（由申请人提供）：在我们实验室正在进行的研究，这是由父母资助，已经开始澄清激活的肥大细胞（MC）中发挥的关键作用， 糖尿病足溃疡（DFU）的发展。更具体地说，我们发现，在糖尿病（DM）患者的皮肤中，活化MC的数量显著高于健康对照，并与真皮炎性细胞和真皮炎性细胞因子以及炎症标志物（如IL 6和TNFα）的循环水平相关。此外，我们能够证明在糖尿病伤口愈合的小鼠模型中，在链脲佐菌素诱导的糖尿病（STZ-DM）小鼠的皮肤中，脱颗粒MC的数量显著较高。用MC稳定剂色甘酸二钠（DSCG）处理显著加速伤口愈合，伴随着脱颗粒MC的量显著减少。此外，DSCG大大减少了M1巨噬细胞表型，在这个动物模型中的创伤前和创伤后。因此，我们的临床前概念验证结果强烈支持抑制MC脱粒是治疗DFU的可行方法。MC脱粒是由升高水平的胞质钙，这是由储存的操作钙（SOC）介导的，并在较小程度上，由受体电位典型（TRPC）通道控制。最佳表征的SOC通道是钙选择性奥赖，也称为钙释放激活的钙（CRAC）通道，其不仅由MC表达，而且由T细胞以及血管平滑肌细胞表达。MC的激活刺激奥赖通道的开放以供钙内流。小分子奥赖/CRAC通道阻断剂已显示出有效抑制MC脱粒和T细胞活化。在这个修订的建议，我们计划评估钙通道阻滞剂在我们的糖尿病伤口愈合的小鼠模型的影响。我们的主要假设是钙通道阻滞剂在改善糖尿病伤口愈合方面更有效。在第一个具体的目标，我们将集中在原型钙通道阻滞剂的合成和局部给药的制剂开发。每种化合物的合成过程需要三个化学步骤，从现有的试剂和标准的纯化技术，如柱分离和重结晶。由于原型化合物的固有疏水性质，我们还将采用增溶增强剂来配制用于在体内功效研究中局部递送的药物。在第二个具体目标中，我们将在糖尿病伤口愈合的动物模型中测试开发的原型的功效。为此，我们将采用在母公司申请中使用的链脲霉素诱导的糖尿病（STZ-DM）野生型（WT）C57 BL/6 J小鼠模型，以评价其在伤口形成前局部使用或在伤口后局部应用时改善伤口愈合的能力。我们还将比较这种治疗与唯一市售MC稳定剂DSCG的疗效。

 DESCRIPTION (provided by applicant): 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 TNFα. 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.