Targeting toll like receptor 4 (TLR4) and TLR2 to resolve EAE-associated paralysis, pain and cognitive deficits: efficacy of a clinically-relevant blood brain barrier permeable TLR4/TLR2 antagonist

项目总结 多发性硬化症(MS)是一种男性和女性的终生衰弱疾病。症状包括 运动功能丧失、神经性疼痛、认知障碍和社交障碍。MS IS 此外，还与循环和中枢(脊髓和脑)PRO水平的升高有关。 炎性细胞因子和抗炎细胞因子水平降低，表明 免疫和神经胶质过程导致慢性炎症。这种持续的炎症在 脱髓鞘、慢性神经胶质细胞激活和神经元死亡是该病的特征。 使用抗炎策略显著地靶向抑制脊髓神经炎 改善实验性自身免疫性脑脊髓炎(EAE)的症状，这是MS的大鼠模型，展望未来 对于翻译，需要的不是我们目前鞘内注射的方法，而是一种 通过临床相关的、口服的、血脑屏障可穿透的小分子药物有效治疗EAE/MS的手段 针对EAE/MS病理的分子，由神经炎症驱动。 我们已经发现了这样一个小分子，并对其进行了广泛的表征。这种药物(非阿片类药物 纳曲酮的异构体((+)-纳曲酮)正迅速向FDA申请研发新药 状态。这是Toll样受体4(TLR4)和TLR2的选择性拮抗剂。因为它不能结合经典的阿片类药物 受体，(+)-纳曲酮不干扰阿片类药物的止痛效果或 阿片受体系统。以TLR2和TLR4为目标源于大量文献，该文献展示了 这些受体在神经炎症过程和EAE/MS症状中的重要性将在这里进行研究。 一系列免费的行为和免疫组织化学研究建议在男性和 将探索(+)-纳曲酮抑制EAE诱导的(A)麻痹/瘫痪的能力的女性，(B) 神经性疼痛，(C)认知障碍，(D)社会交往障碍，以及(E)改善 生存期，当(+)-纳曲酮系统地跨天给药时，比较早期和晚期的剂量 东非航空公司的时间通道。IHC的研究将分析早期和晚期(+)后收集的脑和脊髓组织。 纳曲酮治疗(映射到行为学研究)以确定(+)-纳曲酮是否抑制神经胶质细胞 正如预测的那样，激活、神经细胞死亡和脱髓鞘，以及刺激重新髓鞘形成。 这些研究创造了两个相辅相成的目标。目标1探索一种积极干预的新方法 EAE导致的运动功能障碍、神经病理性疼痛、社交和认知障碍，以及因 通过全身应用(+)-纳曲酮靶向TLR4/TLR2。目标2过渡到对以下内容的初步探索 潜在的病理生理学机制，重点是TLR4/TLR2阻断对血管内皮细胞的影响 脱髓鞘和重新髓鞘形成、神经细胞死亡和神经炎症。这些研究提供了一个 深入研究TLR4/TLR2在EAE主要MS相关症状中的作用。

PROJECT SUMMARY Multiple sclerosis (MS) is a life-long, debilitating disease in both males and females. Symptoms include loss of motor function, neuropathic pain, cognitive impairments, and impaired social interaction. MS is furthermore associated with elevations in circulating and central (spinal cord and brain) levels of pro- inflammatory cytokines and decreased levels of anti-inflammatory cytokines, suggesting a dysregulation of immune and glial processes resulting in chronic inflammation. This ongoing inflammation is important in demyelination, chronic glial activation, and neuronal death characteristic of the disease. Targeted suppression of spinal cord neuroinflammation using anti-inflammatory strategies dramatically improves symptoms of experimental autoimmune encephalomyelitis (EAE), a rat model of MS. Looking forward toward translation, what is needed is not our current approaches that are injected intrathecally, but rather a means to effectively treat EAE/MS via a clinically relevant, orally available, blood-brain barrier permeable small molecule that targets EAE/MS pathology driven by neuroinflammation. We have discovered, and extensively characterized, such a small molecule. This drug (the non-opioid (+)-isomer of naltrexone; (+)-naltrexone) is rapidly moving toward FDA application for Investigational New Drug status. This is a selective antagonist at toll-like receptor 4 (TLR4) and TLR2. As it fails to bind classical opioid receptors, (+)-naltrexone does not interfere with the efficacy of opioids for pain control or normal functioning of opioid receptor systems. Targeting TLR2 and TLR4 arises from an extensive literature demonstrating the importance of these receptors in the neuroinflammatory processes and EAE/MS symptoms to be studied here. A complimentary series of behavioral and immunohistochemistry studies are proposed in males and females which will explore the ability of (+)-naltrexone to suppress EAE-induced (a) paresis/paralysis, (b) neuropathic pain, (c) cognitive impairment, and (d) social interaction impairment, as well as (e) improve survival, when (+)-naltrexone is systemically administered across days, comparing dosing early vs. late in the EAE timecourse. The IHC studies will analyze brain and spinal cord tissues collected after early vs. late (+)- naltrexone treatment (mapping onto the behavioral studies) to define whether (+)-naltrexone suppresses glial activation, neuronal cell death, and demyelination, as well as stimulates remyelination, as predicted. These studies create two complimentary Aims. Aim 1 explores a novel means of positively intervening in EAE-induced motor dysfunction, neuropathic pain, deficits in social interaction and cognition, and loss of life by targeting TLR4/TLR2 by systemic administration of (+)-naltrexone. Aim 2 transitions to an initial exploration of potential mechanisms underlying pathophysiology, focusing on the impact of TLR4/TLR2 blockade on demyelination and remyelination, neuronal cell death, and neuroinflammation. These studies provide a thorough investigation of the role of TLR4/TLR2 in major MS-relevant symptoms of EAE.