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是肌动蛋白骨架的主要调节因子，控制血管平滑肌、内皮细胞、神经元、胶质细胞、白细胞和血细胞的许多功能。其中许多与中风的病理生理有关，使ROCK成为一个独特的多效靶点，具有多种收敛和协同机制，包括脑血管舒张和降低血液粘度，改善超急性中风的侧支血流，以及急性至亚急性期的抗炎和抗水肿作用。我们和其他人在缺血性卒中模型的探索性研究中针对ROCK，绝大多数研究在多物种和卒中模型中显示出改善的组织和功能结果，并使用各种抑制剂和治疗范式。在一项对25项实验研究的系统回顾和分层荟萃分析中，ROCK抑制使梗死体积减少37%，神经学评分提高41%。法舒地尔是实验性脑卒中中最常用的ROCK抑制剂，在临床前和临床试验中，任何配方都可以立即获得法舒地尔。法舒地尔在中国和日本作为急性SAH的临床治疗标准已有20年，具有良好的安全记录。在美国和国外，有大量的全身性法舒地尔的临床试验，在健康志愿者和慢性脑梗死、冠状动脉疾病、心力衰竭和肺动脉高压等患者中进行。事实上，在一个小型的急性中风试验中，法舒地尔是有效的。然而，这些试验都没有发现急性单剂量或慢性每日剂量的任何严重不良事件。我们建议在一项使用短暂性大脑中动脉闭塞的临床前试验中证实这一前景。目的1将确定最佳剂量（Exp 1），确认对长期预后的疗效（Exp 2），测试法舒地尔是否延长了再通的治疗窗口（Exp 3），检查动脉内给药的疗效（Exp 4），并使用临床相关的MRI指标确认疗效（Exp 5）。然后，目标2将测试在有或没有tPA的血栓模型中的有效性和安全性（实验6），以及在合并症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。该建议来自一组经验丰富的调查人员，他们具有经过验证的专业知识，能够适应网络的需求。总的来说，我们已经发表了大约100篇论文，使用各种中风模型和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。除了提出的实验模型外，我们的专长还包括出血转化、tPA联合治疗和缺血性脑水肿。总之，我们热情地支持一个无偏见的多中心临床前测试平台的概念，如SPAN用于实验性脑卒中治疗。我们将全力支持这一网络。

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.