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

描述（由申请人提供）：本R21探索性/发展性转化研究项目旨在测试小分子坏死他汀-1 （nec1）作为治疗肌萎缩性侧索硬化症（ALS）的潜在神经保护剂。肌萎缩性侧索硬化症是一种无法治愈的致命瘫痪疾病。超氧化物歧化酶-1 （SOD1）突变导致家族性ALS，嵌合或条件突变小鼠的研究表明，非神经元细胞在突变型SOD1相关的神经变性中起重要作用。我们和其他人发现，野生型原代脊髓运动神经元（MNs）被表达sod1的突变星形胶质细胞或其条件培养基通过程序性细胞死亡选择性杀死。现在，我们证明了Nec-1，一种激酶受体相互作用蛋白1 （RIP1）的变构抑制剂，可以防止这种突变的星形胶质细胞诱导的MN死亡。因此，为了确定Nec-1的细胞靶标，特异性靶(SA)-1将确定Nec-1在ALS MN/星形胶质细胞共培养模型系统中提供的保护是否与MN和/或星形胶质细胞中RIP1的抑制有关。Nec-1是否能像保护MN细胞体的神经过程一样有效也将被研究。我们还发现，通过计算机模拟，预测Nec-1可以穿过血脑屏障，这增加了Nec-1在体内作为ALS治疗剂使用的可能性。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及其无活性结构类似物Nec-1在适当的慢性体内使用的溶解度和稳定性，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定义的Nec-1给药的最佳条件，SA-3将通过一套全面的行为和形态学研究来确定Nec-1在转基因突变SOD1 ALS小鼠模型中的神经保护效力。我们预计，到本项目结束时，Nec-1的临床前适用性和有效性将得到评估，这可能导致ALS的治疗开发项目。后者将包括临床前监管研究，作为未来临床试验的先决条件的一部分。

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.