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。鉴定 迫切需要具有最小成瘾可能性的有效疼痛治疗的治疗靶点。这 该提案是对RFA-NS-21-029的回应，旨在建立一个多学科团队，以研究 具有最小成瘾可能性的新型疼痛治疗靶点。最近的研究表明， 疼痛涉及中枢神经系统中的小胶质细胞变化，与坏死性凋亡（程序性坏死细胞）有关 死亡）和释放促进神经炎症的细胞成分。具体来说，坏死性凋亡是一种 可由受体相互作用丝氨酸/苏氨酸蛋白激酶1（RIPK 1或 RIP 1）。在初步的RNA测序研究中，我们的数据显示RIPK 1在疼痛诱导后上调， 与疼痛处理有关的关键脑区此外，我们发现RIPK 1蛋白表达是 RIPK 1在两种小鼠神经病理性疼痛模型中上调，RIPK 1上调与神经病理性疼痛的增加有关。 小胶质细胞坏死。Necrostatin-1（Nec-1）是一种RIPK 1抑制剂，可穿过血脑屏障， 调节神经炎症的强大潜力。更重要的是，用Nec-1靶向RIPK 1可以减轻疼痛- 大鼠的相关伤害性行为。最近，我们的团队已经合成，评估和确定了一个有前途的 化合物，[11 C]CNY-06，其是放射性同位素标记的Nec-1。[11 C]CNY-06使我们能够进行正电子 发射断层扫描（PET）-CT研究，为RIPK 1成像提供了前所未有的机会， 脑中的坏死性凋亡，以表征和验证RIPK 1作为疼痛的治疗靶点。我们特别 已经确定[11 C]CNY-06在小鼠中具有优异的脑摄取和高特异性结合。使用鼠标 神经病理性疼痛模型，我们已经捕获了RIPK 1/坏死性凋亡的成像特征，并将其与 神经炎症的标志物基于这些初步数据，我们假设， RIPK 1通过Nec-1是一种安全有效的神经性疼痛治疗方法，成瘾的可能性极小。 我们将进行两个特定目标来验证这一假设。目的1：彻底表征RIPK 1表达 并在三种啮齿动物神经性疼痛模型中测试Nec-1。目的2：询问Nec-1是否会导致成瘾， 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， 使用PET-CT的新型分子成像，这项R61提案将产生关于疼痛中RIPK 1的关键数据， 严格验证我们的中心假设，即通过Nec-1抑制RIPK 1是一种安全有效的治疗方法， 痛苦这项提案不仅将提供关于Nec-1治疗疼痛的疗效和副作用的可靠数据， 还建立了一个多学科的研究团队，在翻译疼痛研究方面具有丰富的经验， 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.