Novel Modified Erythropoietins for the Treatment of Ischemic Brain Injury

描述（由申请人提供）：EPO在体外和体内缺血性损伤模型中都显示出强大的神经保护作用。由于全身给药的促生成素通过血脑屏障（BBB）的不到1%，因此需要大剂量和多剂量促生成素才能在脑内达到有效浓度。这种给药方案可能导致红细胞压积增加，刺激血小板产生，增加微梗死和大梗死的可能性，从而严重限制甚至排除EPO在卒中中的应用。因此，降低促红细胞生成素活性及其潜在副作用的替代策略将大大提高其在脑卒中治疗中的临床应用。我们将利用两种不同的方法来克服EPO的潜在副作用和给药限制。第一种方法是利用缺乏红细胞生成活性的突变型EPO （mEPO）。我们最近成功地产生了一种新的EPO突变体，完全缺乏红细胞生成活性。重要的是，这种突变型EPO在体外和体内缺血模型中都保持了与野生型EPO相似的神经保护作用。第二种方法是使用蛋白质转导技术来增强EPO穿过血脑屏障的渗透。因此，本研究的目的是：1)在临床相关的大脑中动脉闭塞（MCAO）模型中进一步测试缺乏红细胞生成活性的突变型EPO的神经保护作用；2)确定与野生型EPO相比，将蛋白转传导结构域融合的EPO或mEPO （EPO- tat或mEPO- tat）更快地递送到大脑中是否可以转化为更低的有效剂量和更宽的有效时间窗。本文提出了以下两个具体目的：1)验证系统性给药缺乏红细胞生成活性的突变型EPO （mEPO）对局灶性缺血性损伤具有神经保护作用的假设。这些研究将确定：1)mEPO是否在不刺激血小板和红细胞压积的情况下以剂量和时间依赖的方式减少梗死体积；2) mEPO是否能改善神经预后；3) mEPO处理是否激活了与野生型EPO相同的缺血脑细胞存活通路（PI3K/Akt, ERK1/2）。2)为了验证EPO- tat或mEPO-TAT系统给药能更有效地将蛋白质递送到大脑中，并且与单独使用EPO相比，提供更宽的功效时间窗口的假设。这些研究将确定：A)与野生型EPO相比，EPO- tat或mEPO-TAT是否在较低剂量下对缺血性损伤具有相同的神经保护作用；B)与野生型EPO相比，EPO- tat或mEPO-TAT是否可以提供更宽的疗效时间窗口；C) EPO- tat或mEPO-TAT对MCAO后炎症的影响。公共卫生相关性：促红细胞生成素（EPO）是一种促进红细胞成熟的天然激素，最近被认为是缺血性卒中神经保护的有希望的候选药物。由于血脑屏障（BBB）的存在，需要大剂量和多剂量的EPO才能在脑内达到有效浓度，而血脑屏障只允许不到1%的全身给药EPO通过血脑屏障。这种给药方案可能导致红细胞和血小板生成增加，使血液更“粘”，容易凝固。我们将利用两种不同的方法来克服EPO的潜在副作用和给药限制。第一种方法是利用一种突变的EPO，它可以保护大脑免受缺血性损伤，但缺乏刺激红细胞产生的能力。因此，这种突变的EPO将使EPO治疗在中风治疗中“更安全”。第二种方法是使用蛋白质转导技术来增强EPO穿过血脑屏障的渗透。这种新方法将显著减少治疗所需的EPO量，并可能延长治疗窗口。综上所述，该项目将为EPO作为中风和其他神经系统疾病的治疗药物的应用提供新颖和安全的方法。

DESCRIPTION (provided by applicant): EPO has shown robust neuroprotective effects in both in vitro and in vivo models of ischemic injury. Since less than 1% of systemically administered EPO crosses the blood brain barrier (BBB), large and multiple doses of EPO have been required to achieve effective concentrations in the brain. Such an administration regimen of EPO may lead to increases in hematocrit and stimulate the production of platelets, increasing the likelihood of microinfarctions and macroinfarctions, thus severely limiting or even precluding the use of EPO for stroke. Accordingly, alternate strategies to reduce erythropoietic activity of EPO and its potential side effects will greatly improve its clinical applications for the treatment of stroke. We will utilize two different approaches to overcome the potential side effects and delivery limitations of EPO. The first approach is the utilization of a mutant EPO (mEPO) lacking erythropoietic activity. We have recently successfully generated a novel EPO mutant that completlely lacks erythropoietic activity. Importantly, this mutant EPO remains its neuroprotective effect in both in vitro and in vivo models of ischemia with similar efficacies as wild-type EPO. The second approach is the use of the protein transduction technique to enhance the penetration of EPO crossing the BBB. Therefore, the objectives of this proposal are 1) to further test the neuroprotective effect of mutant EPO lacking erythropoietic activity in the clinically relevant middle cerebral artery occlusion (MCAO) model, and 2) to determine whether a more rapid delivery of protein transduction domain fused EPO or mEPO (EPO-TAT or mEPO-TAT) into brain can be translated into a decreased effective dose and a wider time-window of efficacy as compared to wild-type EPO. The following two Specific Aims are proposed: 1) To test the hypothesis that systemic administration of the mutant EPO (mEPO) lacking erythropoietic activity is neuroprotective against focal ischemic injury. These studies will determine: 1) whether mEPO reduces infarct volume in dose- and time-dependent manners without stimulating platelet and hematocrit; 2) whether mEPO results in improved neurological outcomes; and 3) whether mEPO treatment activates the same cell survival pathways (PI3K/Akt, ERK1/2) in ischemic brain as wild-type EPO does. 2) To test the hypothesis that systemic administration of EPO-TAT or mEPO-TAT results in a more efficient delivery of the protein into the brain and offers a wider time window of efficacy compared to EPO alone. These studies will determine: A) whether EPO-TAT or mEPO-TAT offers same neuroprotective effects against ischemic injury at substantially lower doses as compared to the wild-type EPO, B) whether EPO-TAT or mEPO-TAT may provide a wider time window of efficacy compared to wild-type EPO and C) the effects of EPO-TAT or mEPO-TAT on inflammation following MCAO. PUBLIC HEALTH RELEVANCE: Erythropoietin (EPO) is a natural hormone for the maturation of red blood cells and has recently emerged as a promising candidate for neuroprotection in ischemic stroke. Large and multiple doses of EPO have been required to achieve effective concentrations in the brain due to the existence of the blood brain barrier (BBB), which allows only less than 1% of systemically administered EPO to cross the BBB. Such an administration regimen may lead to increased red blood cell and platelet production, making the blood more "sticky" and easily coagulated. We will utilize two different approaches to overcome the potential side effects and delivery limitations of EPO. The first approach is the utilization of a mutant EPO that protects the brain from ischemic injury, but lacks the ability to stimulate the production of red blood cell. Thus, this mutant EPO will make EPO therapy "safer" for stoke treatment. The second approach is the use of the protein transduction technique to enhance the penetration of EPO crossing the BBB. This novel approach will significantly decrease the amount of EPO required for treatment and possibly extend the treatment window. Taken together, this project will provide novel and safe approaches for the application of EPO as a therapeutic agent in stroke and in other neurological diseases.