Novel PA1-Inhibitors of Neutrophil Proteinases

说明(申请人提供)：中性粒细胞弹性蛋白酶和组织蛋白酶G是储存在中性粒细胞初级颗粒内的丝氨酸蛋白酶。中性粒细胞在炎症部位的激活和脱颗粒导致这些蛋白酶的释放，在这些酶遇到抑制物时，它们在特定的炎症条件下调节其活性。这些蛋白水解酶与多种疾病的发病机制有关。与中性粒细胞重新聚集相关的炎性疾病的一个例子是慢性阻塞性肺疾病(COPD)。慢性阻塞性肺疾病是一种进展缓慢且无法治愈的呼吸道疾病，其特征是肺功能逐渐丧失。据估计，目前有2090万美国人受到慢性阻塞性肺病的影响，其中1830万人患有慢性支气管炎，270万人患有肺气肿。我们的第一阶段研究包括开发两个合理设计的纤溶酶原激活物抑制物1(PAI-1)突变体。PAL-1的正常靶点是纤溶酶原激活物tPA和uPA。PAL-1的基因已经改变，产生了具有改变的蛋白酶特异性的突变。这两种新的突变体抑制剂现在针对中性粒细胞弹性蛋白酶和组织蛋白酶G。由于PAL-1的独特性质，这些突变体实际上表现出优于天然抑制剂阿尔法酮蛋白水解酶抑制剂和抗糜蛋白酶的性质。具体地说，在肝素和DNA等多阴离子表面的存在下，这些突变体既能使细胞内吞作用失活，又能促进中性粒细胞蛋白酶的降解，这些表面能隔离这些非常基本的酶。编码这些突变体的DNA已经克隆到大肠杆菌中，以便在非常高的水平表达，在我们的第二阶段研究中，一种正在申请专利的PAL-1纯化方法将被放大。第二阶段的提案扩展和扩大了我们的研究，包括：改善用于治疗的突变体的性质，在一些动物模型中进一步表征突变体与商业上可获得的和内源性抑制剂相比的性能，开发灵敏和特异的中性粒细胞弹性蛋白酶和组织蛋白酶G免疫分析方法，以及使用快速申请专利的纯化过程来检查PAL-1突变体的大规模生产的研究。破坏性肺部疾病是美国发病率的主要原因。我们相信这些突变可以开发成治疗各种炎症性疾病的治疗剂。

DESCRIPTION (provided by applicant): Neutrophil elastase and cathepsin G are serine proteases stored within the primary granules of neutrophils. The activation and degranulation of neutrophils at inflammatory sites result in the release of these proteases where they encounter inhibitors that regulate their activity in certain inflammatory conditions. These proteases have been implicated in the pathogenesis of a variety of diseases. One example of an inflammatory disease associated with neutrophil recruitment is Chronic Obstructive Pulmonary Disease (COPD). COPD is a slowly progressive and incurable disease of the airways, characterized by a gradual loss of lung function. An estimated 20.9 million Americans are currently affected by COPD; of which 18.3 million suffer from chronic bronchitis and 2.7 million suffer from emphysema. Our Phase I study involved the development of two rationally designed mutants of Plasminogen Activator Inhibitor One (PAI-1). The normal targets for PAl-1 are the plasminogen activators tPA and uPA. The gene for PAl-1 has been altered to produce mutants with altered protease specificity. These two new mutant inhibitors now target neutrophil elastase and cathepsin G. Due to the unique properties of PAl-1, these mutants actually exhibit properties superior to the natural inhibitors Alpha One Proteinase Inhibitor and Antichymotrypsin. Specifically, these mutants both inactivate and facilitate the cellular endocytosis and degradation of the neutrophil proteinases in the presence of polyanionic surfaces such as heparin and DNA, which sequester these very basic enzymes. The DNA that codes for these mutants has been cloned into a E. coli bacterial construct for expression at very high levels, and a patent pending method for the purification of PAl-1 will be scaled up in our Phase II study. The Phase II proposal extends and expands our studies to include: improving the properties of the mutants for therapeutic use, further characterization of the performance of the mutants compared with commercially available and endogenous inhibitors in a number of animal models, development of sensitive and specific immunoassays for neutrophil elastase and cathepsin G and, a study to examine the large scale production of the PAl-1 mutants using a rapid patent pending purification process. Destructive lung diseases are a major cause of morbidity in the U.S. We believe that these mutants can be developed into therapeutic agents for the treatment of a variety of inflammatory diseases.