Biologic Evaluation of Liposome-Encapsulated Hemoglobin

描述(申请人提供)：严重出血是创伤受害者的主要死亡原因，全球约有300万人死亡。通过及时提供安全和通用的复苏液体，大部分死亡是可以预防的。尽管全血或红细胞(RBC)仍然是首选的输液，但安全血液的供应不足仍然是一个问题。尽管在抗击与输血有关的感染和确保受过教育的献血者群体方面持乐观态度，但血液的有限保质期和交叉配型的需要经常导致血液库存短缺。这些问题刺激了人们对安全、免疫耐受、货架稳定和有效的民用应急和军事应用氧气载体的研究。以氦-移动珠蛋白为基础的氧气载体增加了氧气的携带和心脏的前负荷。我们正在开发脂质体包裹的血红蛋白或LEH，作为一种接近模拟红细胞的替代氧载体。与其他化学修饰但游离的血红蛋白产品相比，包裹的血红蛋白被认为对血流动力学有更好的控制。使用新的配方和先进的加工技术，我们克服了低包封率、氧化不稳定性、成本和放大的障碍。该产品(称为NeoLEH)在血液中循环较长时间，并改善大脑新陈代谢。临床前数据显示，与红细胞(7.5 5m)相比，LEH(275 Nm)较小的尺寸导致更有效的氧气输送到大脑，这反过来又改善了大脑的氧气和能量代谢。在这项拟议的工作中，我们将检验我们的总体假设，即NeoLEH可以改善存活率和脑代谢，并在失血性休克中作为一种长循环和无毒的氧气载体。其具体目的是评估：1)40%失血大鼠模型的存活和脑代谢，2)NeoLEH的药代动力学和生物分布，以及再注射的影响，3)NeoLEH对40%失血动物模型的急性毒性作用，4)NeoLEH复苏和缺血再灌注(I/R)损伤。我们提出了一个为期4年的项目来测试NeoLEH的有效性和安全性。我们将研究接受NeoLEH复苏的大鼠在失血40%后24小时的存活情况。林格氏乳酸盐、失血和HEXTEND将用作控制液。脑氧、葡萄糖和能量代谢的改善将通过正电子发射断层扫描和磁共振成像进行评估。将通过监测放射性标记的99mTC-NeoLEH的血液和器官水平来评估NeoLEH的循环持久性和生物分布。为了测试NeoLEH的安全性，将研究NeoLEH输注对肝脏吞噬能力和补体介导的血小板活化的急性影响。最后，我们将通过监测肠道完整性、中性粒细胞隔离等来评估NeoLEH输注对缺血再灌注现象是否有任何有益的影响。拟议的研究结果将对NeoLEH的临床发展至关重要。NeoLEH的目标是在因手术或创伤而导致的急性失血中复苏，它将同时服务于民用和军用需求。 公共卫生相关性：失血性休克的输血实践将受益于充分和安全地获得通用的红细胞替代品。本研究涉及一种新型脂质体包裹的血红蛋白作为氧气载体的研究。该产品名为NeoLEH，将在失血性休克的大鼠模型中进行评估。

DESCRIPTION (provided by applicant): Severe hemorrhage is the leading cause of death among trauma victims, and is responsible for approximately 3 million deaths worldwide. Much of the mortality may be prevented by the timely availability of a safe and uni- versal resuscitation fluid. While whole blood or red blood cells (RBCs) remain the preferred transfusion fluids, inadequate availability of safe blood remains a problem. Despite the optimism in combating transfusion-related infections and ensuring an educated donor population, the limited shelf-life of blood and the need to cross- match frequently cause blood inventory shortages. These issues have stimulated a search for safe, immune- tolerant, shelf-stable and efficacious oxygen carrier for both civilian emergency and military applications. He- moglobin based oxygen carriers increase both oxygen-carriage and cardiac preload. We are developing liposome-encapsulated hemoglobin or LEH as a substitute oxygen carrier closely mimick- ing the RBCs. Encapsulated hemoglobin is believed to have better control over hemodynamics as compared to other chemically-modified, but free hemoglobin products. Using a novel formulation and advanced processing technologies, we have overcome the barriers of low encapsulation, oxidative instability, expense and scale-up. The product (called NeoLEH) circulates in blood for a prolonged time, and improves cerebral metabolism. Pre- liminary data demonstrate that the smaller size of LEH (275 nm) as compared to the RBCs (7.5 5m) results in more efficient oxygen delivery to the brain, which in turn improves cerebral oxygen and energy metabolism. In the proposed work, we will test our overall hypothesis that NeoLEH improves survival and cerebral metabol- ism, and serves as a long-circulating and non-toxic oxygen carrier in hemorrhagic shock. The specific aims are to assess: 1) Survival and cerebral metabolism in a rat model of 40% hemorrhage, 2) Pharmacokinetics and biodistribution of NeoLEH, and the effect of reinjection, 3) Acute toxic effects of NeoLEH in the animal model of 40% hemorrhage, and 4) NeoLEH resuscitation and ischemia-reperfusion (I/R) injury. We propose a 4-y project for testing efficacy and safety of NeoLEH. We will study 24 h survival of rats receiv- ing NeoLEH resuscitation after 40% blood loss. Ringer's lactate, shed blood and Hextend will be used as con- trol fluids. Improvement in cerebral oxygen, glucose and energy metabolism will be evaluated by positron emission tomography and magnetic resonance imaging. Circulation persistence and biodistribution of NeoLEH will be assessed by monitoring the blood and organ levels of radiolabeled 99mTc-NeoLEH. In order to test the safety of NeoLEH, acute effects of NeoLEH infusion on phagocytic capacity of liver and complement-mediated platelet activation will be studied. Lastly, we will assess if NeoLEH infusion has any salutary effects in ische- mia-reperfusion phenomenon by monitoring intestinal integrity, neutrophil sequestration, etc. The results of the proposed investigations will be critical for the clinical development of NeoLEH. Targeted towards resuscitation in acute blood loss due to surgery or trauma, NeoLEH will serve civilian as well as military needs alike. PUBLIC HEALTH RELEVANCE: Transfusion practices in hemorrhagic shock would benefit from adequate and safe availability of a universal substitute for red blood cells. This research pertains to the development of a novel liposome-encapsulated hemoglobin preparation as an oxygen carrier. The product, called NeoLEH, will be evaluated in a rat model of hemorrhagic shock.