Pre-clinical testing of necrostatin as a potential small molecule for the treatme

描述(申请人提供)：这个R21转译研究的探索性/发展项目旨在测试小分子NEC-1(NEC-1)作为治疗肌萎缩侧索硬化症(ALS)的潜在神经保护剂的作用。ALS是一种无法治愈的致命瘫痪疾病。超氧化物歧化酶-1(SOD1)基因突变导致家族性ALS，对嵌合或条件突变小鼠的研究表明，非神经细胞在突变的SOD1相关神经变性中发挥重要作用。我们和其他人发现，野生型初级脊髓运动神经元(MN)可以被表达SOD1的突变型星形胶质细胞或其条件培养液通过程序性细胞死亡选择性地杀死。现在，我们证明了NEC-1，一种变构的激酶受体相互作用蛋白-1(RIP1)的抑制剂，可以保护这种突变型星形胶质细胞诱导的MN死亡。因此，为了确定NEC-1的细胞靶点，特异性靶点(SA)-1将确定NEC-1在我们的ALS MN/星形胶质细胞共培养模型系统中提供的保护是否与抑制MNS和/或星形胶质细胞内的RIP1有关。NEC-1是否像它们的神经突起一样有效地保护MN细胞体也将被检验。我们还发现，通过计算机模拟，NEC-1被预测可以跨越血脑屏障，增加了NEC-1可能在体内用作ALS治疗剂的可能性。因此，在我们测试这种可能性的最初步骤中，SA-2将评估NEC-1及其非活性结构类似物NEC-1ia在适合体内慢性使用的载体中的溶解度和稳定性，以及它们在野生型小鼠中的中枢神经系统渗透、药代动力学、给药途径和耐受性。然后，基于SA-2中定义的NEC-1的最佳给药条件，SA-3将通过一系列全面的行为和形态学研究来确定NEC-1在ALS转基因突变SOD1小鼠模型中的神经保护作用。我们预计，到本项目结束时，NEC-1的临床前适宜性和有效性将得到评估，这可能导致ALS的治疗开发项目。后者将包括临床前调控研究，作为未来临床试验的先决条件。 公共卫生相关性：肌萎缩侧索硬化症(ALS)是一种无法治愈的致命性瘫痪疾病，炎症是越来越多人认识到的对疾病过程的贡献。我们在一个培养皿中发现，一种名为Necrostatin的小分子可以阻止特定炎症细胞对导致ALS瘫痪的神经细胞的有害影响。在这里，我们建议确定使用这种小分子作为活的有机体的适宜性，并在ALS的实验模型中证明它是否具有保护性。

DESCRIPTION (provided by applicant): This R21 exploratory/developmental project in translational research aims to test the small-molecule necrostatin-1 (Nec-1) as a potential neuroprotective agent for the treatment of amyotrophic lateral sclerosis (ALS), an incurable fatal paralytic disorder. Mutations in superoxide dismutase-1 (SOD1) cause familial ALS and studies in chimeric or conditional mutant mice indicate that non-neuronal cells play an important role in mutant SOD1-related neurodegeneration. We and others find that wild-type primary spinal motor neurons (MNs) are selectively killed by mutant SOD1-expressing astrocytes or their conditioned medium by programmed cell death. Now, we show that Nec-1, an allosteric inhibitor of the kinase receptor interacting protein-1 (RIP1), protects against this mutant astrocyte-induced MN death. Thus, to define the cellular target of Nec- 1, specific aim (SA)-1 will determine whether the protection afforded by Nec-1 in our MN/astrocyte co-culture model system of ALS is linked to an inhibition of RIP1 within MNs and/or within astrocytes. Whether Nec-1 protects MN cell bodies as potently as their nerve processes will also be examined. We also find, by in silico modeling, that Nec-1 is predicted to cross the blood-brain barrier raising the possibility that Nec-1 may be usable in vivo as a therapeutic agent for ALS. Thus, in our initial steps toward testing this possibility, SA-2 will assess Nec-1 and its inactive structural analogue Nec-1ia solubility and stability in suitable vehicles for chronic in vivo use, and their central nervous system penetration, pharmacokinetics, routes of administration and tolerability in wild- type mice. Then, based on the optimal conditions of Nec-1 administration defined in SA-2, SA-3 will determine the neuroprotective potency of Nec-1 in the transgenic mutant SOD1 mouse model of ALS using a comprehensive set of behavioral and morphological investigations. We anticipate that, by the end of this project, the preclinical suitability and effectiveness of Nec-1 will have been evaluated, which may lead into a therapeutic development project for ALS. The latter will include preclinical regulatory studies as part of the prerequisites for future clinical trials. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is an incurable fatal paralytic disease in which inflammation is an increasingly recognized contributor to the disease process. We have found in a dish that a small molecule called necrostatin blocks the deleterious effects of specific inflammatory cells on the nerve cells responsible for ALS paralysis. Herein, we propose to determine the suitability of using this small molecule is a living organism and to demonstrate whether it is protective in an experimental model of ALS.