Multicenter preclinical trial of rho-kinase inhibitor fasudil in acute focal cerebral ischemia and reperfusion

在这项临床前试验中，我们建议在急性局灶性脑缺血再灌注模型中使用法舒地尔靶向Rho相关蛋白激酶(ROCK)。ROCK是肌动蛋白细胞骨架的主要调节者，控制着血管平滑肌、内皮、神经元、神经胶质细胞、白细胞和血细胞的多种功能。其中许多与中风的病理生理学有关，使ROCK成为具有多种汇聚和协同机制的独特的多效性靶点，包括脑血管扩张和降低血液粘度，改善超急性中风的侧支循环，以及在急性到亚急性阶段的抗炎和抗水肿作用。我们和其他人在探索性研究中针对缺血性中风模型中的ROCK进行了研究，其中绝大多数研究显示，在多个物种和中风模型中，使用各种抑制剂和治疗范例，组织和功能结果都有所改善。在一项对25项实验研究的系统回顾和分层荟萃分析中，岩石抑制使脑梗塞体积减少了37%，神经学评分提高了41%。法舒地尔是实验性中风中最常用的岩石抑制剂，在临床前和临床试验中的任何配方中都可以立即获得专利。二十年来，法舒地尔一直是中国和日本治疗急性蛛网膜下腔出血的临床标准，并有明确的安全记录。在美国和国外，系统法舒地尔已经在健康志愿者以及慢性脑梗塞、冠状动脉疾病、心力衰竭和肺动脉高压患者中进行了大量临床试验。事实上，在一项治疗急性中风的小型试验中，法舒地尔是有效的。然而，这些试验中没有一项显示出任何严重的不良反应，无论是急性单剂量还是慢性每日剂量。我们建议在一项使用暂时性大脑中动脉闭塞的临床前试验中证实这一有希望的特征。目的1确定最佳剂量(Exp 1)，确定远期疗效(Exp 2)，测试Fasudil是否延长再通治疗窗口(Exp 3)，检查动脉内给药的疗效(Exp 4)，并使用临床相关的MRI指标(Exp 5)确认疗效。然后，AIM 2将在有或没有tPA的血栓模型中测试有效性和安全性(实验6)，以及在合并高血压和糖尿病以及联合治疗(实验7)和永久性缺血(实验8)中的安全性。这项提议来自一群经验丰富的调查人员，他们拥有成熟的专业知识，能够适应网络的需求。我们总共发表了约100篇手稿，使用了各种中风模型以及神经认知和组织读数，适用于小鼠和大鼠的急性(72小时)和慢性(长达2个月)评估。除了建议的实验模型外，我们的专业知识还包括出血性转化、tPA联合治疗和缺血性脑水肿。总而言之，我们热情地支持一个不偏不倚的多中心临床前测试平台的概念，例如用于实验性中风治疗的SPAN。我们完全致力于支持这个网络。

In this preclinical trial, we propose to target rho-associated protein kinase (ROCK) using fasudil in models of acute focal cerebral ischemia followed by reperfusion. ROCK is a major regulator of actin cytoskeleton controlling numerous functions in vascular smooth muscle, endothelium, neurons, glia, leukocytes and blood cells. Many of these are relevant for the pathophysiology of stroke, making ROCK a unique pleiotropic target with multiple converging and synergistic mechanisms, including cerebrovasodilation and reduced blood viscosity improving collateral flow in hyperacute stroke, and anti-inflammatory and anti-edema effects in acute to subacute stages. We and others have targeted ROCK in models of ischemic stroke in exploratory studies, vast majority of which showed improved tissue and functional outcomes, in multiple species and stroke models, and using various inhibitors and therapeutic paradigms. In a systematic review and stratified metaanalysis of 25 experimental studies, ROCK inhibition reduced infarct volume by 37% and improved neurological scores by 41%. Fasudil is the most commonly used ROCK inhibitor in experimental stroke and is off-patent to be procured in any formulation for both preclinical and clinical trials without delay. Fasudil has been the clinical standard of care for two decades in acute SAH in China and Japan, with a clean safety record. There have been numerous clinical trials of systemic fasudil in both US and abroad in healthy volunteers, and in patients with chronic cerebral infarcts, coronary artery disease, heart failure, and pulmonary hypertension among others. Indeed, in a small trial in acute stroke fasudil was efficacious. Nevertheless, none of these trials revealed any serious adverse events either with acute single doses or chronic daily dosing. We propose to confirm this promising profile in a preclinical trial using transient middle cerebral artery occlusion. Aim 1 will establish the optimal dose (Exp 1), confirm efficacy on long-term outcome (Exp 2), test whether fasudil extends the therapeutic window for recanalization (Exp 3), examine the efficacy of intraarterial delivery (Exp 4), and confirm efficacy using clinically-relevant MRI indices (Exp 5). Aim 2 will then test efficacy and safety in a clot model with or without tPA (Exp 6), and safety in comorbid hypertension and diabetes as well as combination treatments (Exp 7) and in permanent ischemia (Exp 8). The proposal comes from an experienced group of investigators with proven expertise, capable of adjusting to the needs of the network. Collectively, we have published ~100 manuscripts using various stroke models and neurocognitive and tissue readouts suitable for both acute (<72h) and chronic (up to 2 months) assessments in mice and rats. Besides the proposed experimental models, our expertise covers hemorrhagic transformation, combination treatment with tPA and ischemic brain edema. Altogether, we passionately support the concept of an unbiased multicenter preclinical testing platform such as SPAN for experimental stroke therapeutics. We are fully committed to support this network.