Novel Modified Erythropoietins for the Treatment of Ischemic Brain Injury

描述(由申请人提供)：EPO在体外和体内的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EPO只有不到1%穿过血脑屏障(BBB)，因此需要大剂量和多次剂量的EPO才能在大脑中达到有效浓度。这种EPO给药方案可能会导致红细胞压积增加，刺激血小板的产生，增加微梗塞和大梗塞的可能性，从而严重限制甚至排除EPO在卒中中的应用。因此，减少EPO的红细胞生成活性及其潜在副作用的替代策略将极大地改善其在中风治疗中的临床应用。我们将利用两种不同的方法来克服促红细胞生成素的潜在副作用和传递限制。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完全缺乏红细胞生成活性的新型EPO突变体。重要的是，这种突变的EPO在体外和体内的缺血模型中都保持了它的神经保护作用，与野生型EPO的效果相似。第二种途径是利用蛋白质转导技术增强EPO对血脑屏障的穿透能力。因此，这项建议的目的是：1)在临床相关的大脑中动脉阻塞(MCAO)模型中进一步测试缺乏红细胞生成活性的突变体EPO的神经保护作用；2)确定与野生型EPO相比，更快地将蛋白质转导结构域融合的EPO或mEPO(EPO-TAT或mEPO-TAT)转移到脑内是否可以转化为更低的有效剂量和更宽的有效时间窗。提出了以下两个具体目标：1)验证全身应用缺乏红细胞生成活性的突变型EPO(MEPO)对局灶性脑缺血损伤具有神经保护作用的假说。这些研究将确定：1)mEPO是否以剂量和时间依赖的方式减少梗塞体积，而不刺激血小板和红细胞压积；2)mEPO是否能改善神经功能；以及3)mEPO治疗是否激活与野生型EPO相同的缺血脑细胞生存通路(PI3K/Akt，ERK1/2)。2)检验假设，与单独使用EPO-TAT相比，全身应用EPO-TAT或mEPO-TAT可更有效地将蛋白质输送到大脑，并提供更宽的有效时间窗口。这些研究将确定：A)与野生型EPO相比，EPO-TAT或mEPO-TAT是否在低得多的剂量下提供了相同的抗缺血损伤的神经保护作用；B)与野生型EPO相比，EPO-TAT或mEPO-TAT是否提供了更宽的有效时间窗口；以及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，最近被认为是治疗缺血性中风的一种有前途的神经保护候选药物。由于血脑屏障(BBB)的存在，需要大剂量和多次剂量的EPO才能在脑内达到有效浓度，这只允许不到1%的全身给药的EPO通过血脑屏障。这样的给药方案可能会导致红细胞和血小板的生成增加，使血液更“粘性”，更容易凝结。我们将利用两种不同的方法来克服促红细胞生成素的潜在副作用和传递限制。第一种方法是利用突变的EPO来保护大脑免受缺血损伤，但缺乏刺激红细胞产生的能力。因此，这种突变的促红细胞生成素将使促红细胞生成素疗法在中风治疗中“更安全”。第二种途径是利用蛋白质转导技术增强EPO对血脑屏障的穿透能力。这种新的方法将显著减少治疗所需的促红细胞生成素的量，并可能延长治疗窗口。综上所述，该项目将为EPO作为治疗药物在中风和其他神经疾病中的应用提供新的和安全的方法。

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.