Therapeutic Targeting of the Class IB PI3-Kinase Gamma for Treatment of Acute Ischemic Stroke

描述（由申请方提供）：IB类PI 3-激酶γ治疗急性缺血性卒中的治疗靶点。重组tPA仍然是FDA批准的唯一一种治疗缺血性卒中的最有效的干预措施。然而，tPA的临床应用受到限制，因为其治疗窗窄且出血性转化（HT）的风险增加。在该提案中，我们假设单独使用AS 605240或与tPA联合使用治疗性抑制PI 3 Kg可防止缺血和tPA诱导的脑损伤和HT，并增加tPA治疗缺血性卒中的治疗窗。我们想法的分子基础是PI 3 Kg的基因缺失不仅减少了中风引起的BBB破坏和组织梗死，而且几乎完全阻止了中风后6小时给予tPA引起的脑出血。我们的数据是有希望的，并建议在缺血性中风后的tPA诱导的HT和脑损伤的PI 3 Kg的关键作用。此外，我们的初步数据表明，AS 605240抑制PI 3 Kg可显着防止脑损伤，并减少大鼠栓塞性中风后缺血脑中的纤维蛋白沉积/血栓形成。这些令人兴奋的初步数据表明，PI 3 Kg可能是治疗急性缺血性卒中的一个有前途的治疗靶点。在本申请中，我们建议开发一个 通过AS 605240治疗性靶向PI 3 Kg治疗急性缺血性卒中的新卒中疗法，并研究AS 605240介导的有益作用的分子机制。我们现在提出三个目标来研究这种新的中风疗法。在目标1中，我们将确定大鼠局灶性栓塞性卒中后6小时单独使用和与tPA联合使用AS 605240的影响。我们将确定AS 605240单药治疗和与tPA联合治疗急性卒中的最佳剂量和延长的治疗时间窗。在卒中后72小时检查脑梗死、神经功能缺损、脑出血和动物死亡率。在目标2中，我们将定义AS 605240单独或与tPA联合治疗急性卒中的有益作用的机制。将通过关键BB B蛋白的空间和时间分析以及与脑血管上NF-κB、CD 147、MMP-9和/或MMP-3的活化/表达变化的潜在相关性来评估BB B损伤。通过分析脑血管壁内和周围的纤维蛋白沉积以及缺血性脑中血栓形成前因子（派-1、组织因子-TF）的表达，评估卒中后缺血性脑中的血栓形成。在目标3中，我们将评估栓塞性卒中后6小时单独使用AS 605240和与tPA联合使用的长期结局。将对动物进行为期5周的一系列行为测试。最后，取出脑以测量病变大小、纤维蛋白沉积/血栓形成、脑血管密度/效力以及它们与促血管生成因子（VEGF、Ang-1）和促血栓形成因子（派-1、组织因子-TF）表达的相关性。如果成功的话，这些提出的实验应该提供关于IB类PI 3 Kg信号传导如何促进缺血和tPA诱导的HT和脑损伤的新见解，并可能最终产生治疗急性缺血性卒中和最小化tPA对缺血性神经血管损伤的不良影响的新疗法，从而改善缺血性卒中后的长期结局。

DESCRIPTION (provided by applicant): Therapeutic targeting of the class IB PI3-kinase gamma for treatment of acute ischemic stroke. Recombinant tPA remains the only FDA approved and the most beneficial proven intervention for treatment of ischemic stroke. However, clinical use of tPA is limited due to its narrow therapeutic window and increased risk of hemorrhagic transformation (HT). In this proposal, we hypothesize that therapeutic inhibition of PI3Kg with AS605240, alone or in combination with tPA, protects against the ischemia- and tPA-induced brain injury and HT and increases therapeutic window for tPA treatment of ischemic stroke. The molecular basis for our idea is that genetic deletion of PI3Kg not only reduces stroke-induced BBB disruption and tissue infarction but also almost completely blocked cerebral hemorrhage induced by tPA administered at 6 hours after stroke. Our data are promising and suggest a critical role of PI3Kg in tPA-induced HT and brain injury after ischemic stroke. Further, our pilot data show that inhibition of PI3Kg with AS605240 significantly protects against brain injury and reduces fibrin deposition/thrombosis in the ischemic brain after embolic stroke in rats. These exciting preliminary data suggest that PI3Kg may represent a promising therapeutic target for treating acute ischemic stroke. In this application, we propose to develop a new stroke therapy by therapeutic targeting of PI3Kg with AS605240 for treatment of acute ischemic stroke and to investigate molecular mechanisms underlying the AS605240-mediated beneficial effects. We now propose three aims to investigate this new stroke therapy. In Aim 1, we will determine the effects of AS605240, alone and in combination with tPA, treated at 6 hrs after focal embolic stroke in rats. We will identify the optimal dosage and the extended therapeutic time window for AS605240 alone and in combination with tPA for treatment of acute stroke. Brain infarction, neurological deficits, brain hemorrhage, and animal mortality will be examined 72h after stroke. In Aim 2, we will define the mechanisms that underlie the beneficial effects of AS605240 alone or in combination with tPA in the treatment of acute stroke. BBB damage will be assessed by the spatial and temporal analysis of key BBB proteins and potential correlation with changes in activation/expression of NF-κB, CD147, MMP-9 and/or MMP-3 on cerebral vessels. Thrombus formation in the ischemic brain after stroke will be assessed by analyzing fibrin deposition within and around the cerebral vessel wall and the expression of prothrombotic factors (PAI-1, tissue factor-TF) in the ischemic brain. In Aim 3, we will assess long term outcomes of AS605240 alone and in combination with tPA treated at 6 hrs after embolic stroke. Animals will be followed over 5 weeks with a battery of behavioral tests. At the end, brains are removed for measurements of lesion size, fibrin deposition/thrombosis, cerebral vascular density/potency, and their correlation with the expression of proangiogenic factors (VEGF, Ang-1) and prothrombotic factors (PAI-1, tissue factor-TF). If successful, these proposed experiments should provide new insights of how the class IB PI3Kg signaling contributes to the ischemia- and tPA-induced HT and brain injury and may ultimately yield new therapies to treat acute ischemic stroke and to minimize the adverse effect of tPA on ischemic neurovascular damage, leading to improved long term outcomes after ischemic stroke.