Novel Therapeutics for Interstitial Lung Disease: Modified, Water-Soluble Caveolin-1 Scaffolding Domain Peptides with Improved Characteristics for Drug Development

摘要我们的长期目标是改进治疗许多毁灭性疾病的有限选择。 它们共同构成间质性肺疾病(ILD)，其中最常见的是硬皮病和 特发性肺纤维化。由于ILD影响到那些试图保持活跃的中年后期人群， 治疗将迅速增长。小窝蛋白-1是治疗纤维化疾病的一个有前景的靶点。纤维化症患者 小窝蛋白-1缺乏对细胞和小鼠模型的影响可被一种与其活性相当的多肽抑制 站点(Caveolin-1支架结构域，CSD)。然而，CSD缺乏合适的药物药理性质 发展。为了克服这个问题，我们开发了CSD的新的修改版本。我们首先划分了CSD 分成三个亚区，发现它们都能抑制博莱霉素诱导的肺纤维化。为了改善制药行业- 生物学特性，然后我们修改了CSD和每个分区，使其具有水溶性，并防止蛋白质- 解体。这种修饰增强了细胞对所有四种修饰多肽的摄取，并增强了它们对 体外抑制几种纯化的蛋白水解酶。到目前为止，我们只在体内测试了四种修饰多肽中的一种， 对博莱霉素诱导的肺纤维化有显著的抑制作用。这些初步研究有力地证明了 支持我们的建议，从四个候选化合物中确定一个先导化合物，然后评估其Thera- Putic Index(毒性剂量与有益剂量的比率)。我们的研究和文献也表明，我们的 多肽将比FDA批准的重磅炸弹药物九替达尼更有效，副作用更少 (品牌名称Ofev)。总而言之，要继续药物开发，我们必须确定一种先导化合物。到期 鉴于它们独特的药理和功能特点，我们必须将我们的四个 修饰的多肽。1)使用两个模型系统选择先导化合物：全身博莱霉素治疗和 异硫氰酸荧光素(FITC)处理。我们将选择一种先导化合物，然后展示它的 通过将其与对照多肽(混杂铅)、9tedanib和吡非尼酮进行比较来确定其特异性和活性 (据报道，FDA批准的治疗ILD的药物Esbriet会影响小窝蛋白-1的水平)。多肽将是 已交付S.C.在治疗方案中，从诱导纤维化后7天开始。主要读数将为 肺功能和纤维化标记物的定量、微血管渗漏和组织形态。成功 将被定义为由先导化合物抑制50%的对肺功能的有害影响 和&gt；75%的有害影响纤维化和微血管渗漏。2)确定治疗指标 铅化合物的含量。将确定先导化合物的有益作用的剂量依赖关系 使用高于和低于我们当前标准剂量的剂量。它的毒性将通过一次治疗进行评估 最大耐受剂量实验使用我们当前标准剂量的1X、5X、25X和125X。我们会 如果治疗指数为50，则认为这些研究是成功的。总而言之，这些研究将提供 一种新型的先导化合物，在抑制ILD和安全性方面都符合我们的成功标准。

Abstract Our long-term goal is to improve on the limited choices for treating the many devastating diseases that together constitute the Interstitial Lung Diseases (ILD), the most prevalent of which are scleroderma and Idiopathic Pulmonary Fibrosis. As ILD affects those in late middle age trying to remain active, the market for a treatment will grow rapidly. Caveolin-1 is a promising therapeutic target in fibrotic diseases. The profibrotic effects of caveolin-1 deficiency in cells and in mouse models is suppressed by a peptide equivalent to its active site (caveolin-1 scaffolding domain, CSD). However, CSD lacks suitable pharmacologic properties for drug development. To overcome this problem, we developed novel, modified versions of CSD. We first divided CSD into three subregions and found they all suppressed bleomycin-induced lung fibrosis. To improve the pharma- cological properties, we then modified CSD and each subregion to be water soluble and protected from proteo- lysis. This modification enhanced the uptake by cells of all four modified peptides and increased their ability to inhibit several purified kinases in vitro. We have so far tested only one of the four modified peptides in vivo and it was outstandingly active in inhibiting bleomycin-induced lung fibrosis. These initial studies justify and strongly support our proposal to identify a Lead Compound from among the four candidates, then evaluate its Thera- peutic Index (ratio between toxic and beneficial doses). Our studies and the literature also suggest that our peptides will be more effective and have fewer side effects than the FDA-approved blockbuster drug nintedanib (brand name Ofev). In summary, to proceed with drug development we must identify a Lead Compound. Due to their distinct pharmacological and functional features, we must do a side-by-side comparison of our four modified peptides. 1) Select a Lead Compound using two model systems: Systemic Bleomycin Treatment and Fluorescein Isothiocyanate (FITC) Treatment. We will choose a Lead Compound, then demonstrate its specificity and activity by comparing it to a control peptide (scrambled Lead), nintedanib, and pirfenidone (brand name Esbriet, an FDA-approved drug for ILD reported to affect caveolin-1 levels). Peptides will be delivered s.c. in a Therapeutic Protocol, beginning 7 days after fibrosis is induced. Primary Readouts will be lung function and quantification of fibrosis markers, microvascular leakage, and tissue morphology. Success will be defined as the suppression by the Lead Compound of >50% of the deleterious effect on lung function and >75% of the deleterious effect on fibrosis and microvascular leakage. 2) Determine the Therapeutic Index of the Lead Compound. The dose-dependence of the Lead Compound’s beneficial effects will be determined using doses above and below our current standard dose. Its toxicity will be evaluated in a Single-Treatment Maximum Tolerated Dose Experiment using 1X, 5X, 25X, and 125X our current standard dose. We will consider these studies to be a success if the Therapeutic Index is >50. In summary, these studies will provide a novel Lead Compound that meets our Criteria for Success, both in terms of suppression of ILD and of safety.