Anti-arthritic activity and therapeutic use of novel joint-homing peptides

描述（由申请人提供）： 风湿性关节炎（RA）是一种慢性衰弱性疾病，影响超过1%的美国人。女性受影响的频率约为男性的3倍。RA也是美国退伍军人的一个主要健康问题。退伍军人人口正在老龄化，在军队服役的妇女人数正在逐渐增加。此外，在战区的退伍军人面临着极端的压力和各种形式的有毒物质。这些因素都可能直接或间接地影响宿主的免疫功能，从而诱发或加重RA的发生。RA引起的疼痛、不适、工作日损失和残疾会在许多方面对退伍军人的生活和工作质量产生不利影响，并造成沉重的医疗保健和康复费用。因此，促进我们对RA发病机制和治疗的理解的研究与美国退伍军人高度相关。关节炎领域中的两个主要挑战是1）定义使关节高度倾向于受到全身性自身免疫反应攻击的机制，以及2）设计将全身施用的药物主要引导到发炎关节中（没有关节内注射）的新方法，以实现最大的治疗效果，但具有最小的副作用。我们假设，炎症关节的血管内皮的特征在于独特的分子标记物，促进致病性T细胞选择性迁移到靶器官（关节炎关节）和细胞/分子与炎症和骨损伤介质的相互作用。此外，靶向这些血管内皮细胞标志物中的一种或多种将提供用于下调关节炎症和组织损伤的新的治疗方法，而没有过度的不良反应或全身毒性。这一主张是基于我们最近在PNAS上发表的关于RA大鼠佐剂性关节炎（AA）模型中滑膜血管的研究结果，该研究是与Erkki Ruoslahti博士（Sanford-Burnham Institute，拉霍亚，CA）合作完成的。本研究的目的是通过体外和体内富集噬菌体肽展示文库克隆的创新方法来鉴定独特的关节炎关节归巢肽。用于检测组织特异性标志物的噬菌体系统的优点在于，在预测与血管内皮结合的配体时没有先验偏差。而且，与抗体不同，噬菌体展示肽与靶分子的功能结构域相互作用。这种方法是由Ruoslahti博士开创的，他提出了血管“地址分子”或“邮政编码”的概念。“他的小组在这方面检查了几个器官，但没有检查关节。我们现在已经通过鉴定两种新型9-残基肽（表示为NQR和ADK）填补了这一空白，所述肽优先归巢于发炎关节，并且与结合特异性整联蛋白的众所周知的精氨酸-甘氨酸-天冬氨酸（RGD）基序相比显示出独特的受体结合和细胞信号传导属性。此外，用NQR肽治疗关节炎大鼠可减弱AA，而ADK肽则不能。我们的研究目标是：目标1。将肽（NQR和ADK）与药物一起用于滑膜血管靶向递送到关节炎关节中，以控制炎症和骨损伤。作为概念验证，药物将 包封在脂质体中，其表面用NQR/ADK肽修饰。这些关节归巢肽将优先引导脂质体进入发炎的关节。脂质体将用聚乙二醇处理以抑制网状内皮系统对其的摄取; 目标2. 定义在与内皮细胞相互作用时由联合归巢肽触发的细胞/分子事件，并检查NQR肽的抗关节炎活性的潜在机制。我们将确定NQR/ADK肽对内皮细胞产生的致病介质的影响;内皮细胞的基因表达谱;以及白细胞的定义子集的迁移。这项研究的结果将推进我们对自身免疫性关节炎疾病过程的认识，并有助于设计新的肽导向的靶向药物递送，增加治疗RA的治疗指数。

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA) is a chronic debilitating disease affecting over 1% of Americans. Women are affected about 3 times more often than men. RA also constitutes a major health problem among the U.S. Veterans. The Veteran population is aging, and the number of women serving in the military is gradually increasing. In addition, Veterans in combat zones are exposed to extreme stress and toxic substances in different forms. All these factors may directly or indirectly influence host immunity and induction/aggravation of RA. The pain, discomfort, lost working days, and disabilities caused by RA can adversely affect Veterans' quality of life and work in many ways, as well as impose heavy cost of healthcare and rehabilitation. Therefore, studies advancing our understanding of the pathogenesis and treatment of RA are highly relevant to the U.S. Veterans. Two main challenges in the field of arthritis are 1) to define the mechanisms that render the joints highly prone to attack by a systemic autoimmune response, and 2) to devise novel ways to direct systemically-administered drugs primarily into the inflamed joints (without intra-articular injection) to achieve maximum therapeutic effect, but with minimum adverse effects. We hypothesize that the vascular endothelium of the inflamed joints is characterized by unique molecular markers that facilitate both selective migration of pathogenic T cells into the target organ (the arthritic joints) and cellular/ molecular interactions with the mediators of inflammation and bone damage. Furthermore, the targeting of one or more of these vascular endothelial cell markers would offer new therapeutic approaches for downregulating joint inflammation and tissue damage without undue adverse reactions or systemic toxicity. This proposition is based on the results of our recently published study in PNAS on the synovial vasculature in the rat adjuvant arthritis (AA) model of RA, which was done in collaboration with Dr. Erkki Ruoslahti (Sanford-Burnham Institute, La Jolla, CA). The study was aimed at identifying unique arthritic joint-homing peptides using an innovative approach of in vitro and in vivo enrichment of clones from a phage peptide-display library. The advantage of the phage system for the detection of tissue-specific markers is that there is no a priori bias in predicting the ligands that bind to the vascular endothelium. And, unlike antibodies the phage-displayed peptides interact with the functional domain of the target molecule. This approach has been pioneered by Dr. Ruoslahti, who has developed the concept of vascular "address molecules" or "zip codes." His group has examined several organs in this regard, but not the joints. We have now filled this gap by identifying two novel 9-residue peptides (denoted as NQR and ADK) that preferentially home to the inflamed joints and show unique receptor-binding and cellular-signaling attributes compared to the well-known arginine-glycine-aspartic acid (RGD) motif that binds to specific integrins. Furthermore, the treatment of arthritic rats wit NQR peptide attenuated AA, whereas ADK peptide failed to do so. The aims of our study are: Aim 1. Use the peptides (NQR and ADK) with a drug for synovial vasculature-targeted delivery into arthritic joints to control inflammation and bone damage. As a proof-of-concept, the drug will be encapsulated in liposomes, whose surface is decorated with NQR/ADK peptide. These joint-homing peptides would direct the liposomes preferentially into the inflamed joints. Liposomes will be treated with polyethylene glycol to inhibit their uptake by the reticuloendothelial system; Aim 2. Define the cellular/molecular events triggered by the joint-homing peptides upon interaction with endothelial cells and to examine the mechanisms underlying the anti-arthritic activity of NQR peptide. We will determine the effect of NQR/ADK peptide on the pathogenic mediators produced by endothelial cells; the gene expression profile of endothelial cells; and the migration of defined subsets of leukocytes. The results of this study would advance our knowledge of the disease process in autoimmune arthritis and help design novel peptide-directed, targeted drug delivery with increased therapeutic index for the treatment of RA.