Inhibiting RIPK1 with Necrostatin-1 for Safe and Effective Pain Treatment

慢性疼痛影响着数百万美国人，每年花费超过6350亿美元。身份识别 迫切需要以最小的成瘾可能性有效地治疗疼痛的治疗目标。这 建议是对RFA-NS-21-029的回应，即建立一个多学科小组，以评估 具有最小成瘾潜力的新的疼痛治疗靶点。最近的研究报告说，神经病态 疼痛涉及中枢神经系统的小胶质细胞改变，与坏死性下垂(程序性坏死细胞)有关 死亡)和促进神经炎症的细胞成分的释放。具体地说，坏死性下垂是一种 受体相互作用的丝氨酸/苏氨酸蛋白激酶1(RIPK1或RIPK1)介导的坏死性细胞死亡 RIP1)。在初步的RNA序列研究中，我们的数据显示RIPK1在诱导疼痛后上调，在 与疼痛处理有关的关键脑区。此外，我们还发现RIPK1蛋白的表达是 在两个小鼠神经病理性疼痛模型中表达上调，并且RIPK1上调与 小胶质细胞坏死性下垂。NEC-1(NEC-1)是一种RIPK1抑制剂，它可以穿越血脑屏障，具有 有很强的调节神经炎症的潜力。更重要的是，靶向RIPK1的NEC-1减轻了疼痛- 大鼠的相关伤害性行为。最近，我们的团队合成、评估并确定了一个有希望的 化合物，[11C]CNY-06，是一种标记NEC-1的放射性同位素。[11C]CNY-06允许我们进行正电子 发射断层扫描(PET)-CT研究为RIPK1和RIPK1成像提供了前所未有的机会 大脑中的坏死性下垂，以表征和验证RIPK1作为治疗疼痛的靶点。具体来说，我们 已经证实[11C]CNY-06在小鼠体内具有良好的脑摄取和高特异性结合。使用鼠标 我们建立了神经病理性疼痛模型，捕捉了RIPK1/坏死性下垂的影像特征，并与 已确定的神经炎症标志物。根据这些初步数据，我们假设目标是 RIPK1至NEC-1是一种安全有效的神经病理性疼痛治疗方法，成瘾可能性最小。 我们将实现两个具体目标来检验这一假说。目的1：深入研究RIPK1的表达特征 并在三种啮齿动物神经病理性疼痛模型上测试NEC-1。目标2：询问NEC-1是否会导致成瘾或是否 它增强了其他药物的成瘾潜力。结合疼痛机制研究、行为测试和 使用PET-CT的新分子成像，这项R61提案将通过以下方式生成关于RIPK1在疼痛中的关键数据 严格检验我们的中心假设，即通过NEC-1抑制RIPK1是安全有效的治疗方法 疼痛。这项提案不仅将为NEC-1治疗疼痛的疗效和副作用提供强有力的数据， 而且还建立了一支在转化性疼痛研究方面拥有丰富经验的多学科研究团队 随后的U19团队研究阶段。

Chronic pain affects millions of Americans and costs more than $635 billion annually. Identification of therapeutic targets for effective pain treatment with minimal potential for addiction is urgently warranted. This proposal is in response to RFA-NS-21-029 to develop a multidisciplinary team for the feasibility and validity of a novel therapeutic target of pain with minimal potential of addiction. Recent studies have reported that neuropathic pain involves microglial changes in central nervous system, linked to necroptosis (programmed necrotic cell death) and release of cellular components that facilitate neuroinflammation. Specifically, necroptosis is a type of necrotic cell death that can be mediated by the receptor-interacting serine/threonine-protein kinase 1 (RIPK1 or RIP1). In preliminary RNA Seq studies, our data showed that RIPK1 was upregulated after induction of pain, in key brain regions implicated in pain processing. Moreover, we found that RIPK1 protein expression was upregulated in two mouse neuropathic pain models and that RIPK1 upregulation was associated with increased microglial necroptosis. Necrostatin-1 (Nec-1) is a RIPK1 inhibitor which crosses the blood-brain barrier, with strong potential of modulating neuroinflammation. More importantly, targeting RIPK1 with Nec-1 attenuated pain- related nociceptive behaviors in rats. Recently, our team has synthesized, evaluated and identified a promising compound, [11C]CNY-06, which is a radioisotope labeled Nec-1. [11C]CNY-06 allows us to perform positron emission tomography (PET) -CT studies which provides unprecedented opportunities to image RIPK1 and necroptosis in the brain, to characterize and validate RIPK1 as a therapeutic target for pain. Specifically, we have established that [11C]CNY-06 has excellent brain uptake and high specific binding in mice. Using a mouse neuropathic pain model we have captured imaging features of RIPK1/necroptosis, and compared them with established markers of neuroinflammation. Based on these preliminary data, we hypothesize that targeting RIPK1 through Nec-1 is a safe and effective treatment for neuropathic pain, with minimal potential for addiction. We will carry out two Specific Aims to test this hypothesis. Aim 1: To thoroughly characterize RIPK1 expression and to test Nec-1 in three rodent neuropathic pain models. Aim 2: To interrogate if Nec-1 leads to addiction or if it enhances other medication’s addiction potential. Combining pain mechanistic studies, behavioral testing, and novel molecular imaging using PET-CT, this R61 proposal will generate pivotal data regarding RIPK1 in pain, by rigorously testing our central hypothesis that inhibiting RIPK1 through Nec-1 is a safe and effective treatment for pain. This proposal will not only provide robust data on the efficacy and side effects of Nec-1 for pain treatment, but also establish a multidisciplinary research team with extensive experience in translational pain research for the subsequent U19 Team Research phase.