Development of human thioredoxin as a mucolytic therapy for Cystic Fibrosis

描述(由申请人提供)：阻塞性肺疾病的有效治疗，特别是囊性纤维化(CF)，是一个重要的未得到满足的医疗需求。这类疾病的特点是粘液增厚，导致肺功能受损。这种粘液屏障主要由交联的蛋白质链(粘蛋白)与持续肺部炎症和感染所释放的核酸复合而成。CF是一种常染色体隐性遗传病，在美国约有30,000名患者，全世界约有70,000名患者。目前，旨在增加粘液流动性和促进清除的治疗方法包括使用高渗盐水吸入稀薄分泌物和DNA酶(商标为Pulmozyme)来消化粘液核酸。此外，一半的CF患者服用四种不同的药物，而约四分之一的患者服用六种以上的药物，包括短效β-受体激动剂、粘液溶解药物、抗生素、吸入和鼻腔注射。 皮质类固醇。因此，迫切需要通过开发与现有疗法和给药装置兼容的配方和给药方案来减少治疗负担，并在更大比例的CF患者中有效。OrPro Treateutics，Inc.正在开发硫氧还蛋白，这是一种新型的粘液溶解蛋白药物，具有高效和安全的特点，可以专门破坏交联粘蛋白之间的硫醇连接。还原的重组人硫氧还蛋白-1(RhTrx)在实验室研究中已被证明是一种高效的人CF痰液化剂。Trx是一种小的、稳定的非糖基化氧化还原蛋白，适合在一系列原核和真核表达宿主中制造，不需要哺乳动物细胞表达。我们的总体目标是建立一种重组人Trx的配方策略，以支持该分子作为一种新的、更有效的囊性纤维化粘液溶解疗法的发展。这种疗法的一个关键组成部分是一种药物配方，它可以实现保持蛋白质处于稳定的化学还原状态(对其粘液溶解和抗蛋白酶功能至关重要)的目标，并促进与现代高效气雾剂输送系统的兼容性，从而将CF患者的治疗负担降至最低。对于这一阶段的应用，我们计划专注于实验室规模的纯化生产 蛋白质(天然的重组人Trx对照和单个氨基酸突变体r(Cys)hTrx，具有提高安全性和有效性的潜力)，这些蛋白质的物理特性顺序 评估其化学性质和相对活性，并进行初步配方前研究，以评估硫氧还蛋白是否适合通过一系列雾化装置给药。如果实现了该项目的人工智能，将建立对二硫化物减少用于粘液溶解药物开发的概念的关键验证，从而实现第二阶段项目，其目标是重点开发特定配方，以支持临床前研究，从而导致使用改进的、患者友好的输送装置的潜在更安全和更有效的治疗方案的人体试验。 与公共卫生相关：囊性纤维化(CF)是高加索人群中最常见的遗传性致命疾病，在美国有30,000多人受到影响，目前仍有安全有效的治疗需求尚未得到满足。我们的总体目标是开发一种新的、改进的治疗CF的粘液破坏(粘液溶解)疗法，该疗法是高效的，并且不会给CF患者带来过多的治疗负担。我们将通过开发吸入性人类硫氧还蛋白的最佳配方来实现这一点，这是一种具有新作用机制的重组蛋白粘液溶解物质，与目前的治疗方法相比，它有可能使更广泛的CF患者受益。

DESCRIPTION (provided by applicant): Effective management of obstructive pulmonary diseases, especially Cystic Fibrosis (CF), represents a significant unmet medical need. Such diseases are characterized by thickened mucus that contributes to impaired lung function. This mucus barrier is composed primarily of cross-linked chains of proteins (mucins) complexed with nucleic acids released as a consequence of persistent lung inflammation and infection. In the case of CF, an autosomal recessive disease affecting approximately 30,000 patients in the US and 70,000 world-wide, current therapies designed to increase the fluidity of mucus and facilitate clearance involve the use of hypertonic saline inhalation to thin secretions and DNase enzymes (dornase alfa, trademarked as Pulmozyme") to digest mucus nucleic acids. In addition, half of patients with CF take four different medications, while about a quarter take upwards of six drugs including short-acting beta agonists, mucolytics, antibiotics, and inhaled and intranasal corticosteroids. There is consequently a great need to reduce treatment burden by developing formulations and delivery regimes that are compatible with existing therapies and delivery devices and that are effective across a broader percentage of CF patients. OrPro Therapeutics, Inc. is developing thioredoxins, a new class of mucolytic protein drugs with high efficacy and safety which specifically disrupt the thiol linkages between crosslinked mucin proteins. Reduced recombinant human thioredoxin-1 (rhTrx) has been demonstrated to be a highly potent liquefier of human CF sputum in laboratory studies. Trx is a small, stable non-glycosylated redox protein suitable for manufacturing in a range of prokaryotic and eukaryotic expression hosts and does not require mammalian cell expression. Our overall goal is to establish a formulation strategy for rhTrx that will support development of this molecule as a new and more efficacious mucolytic therapy for cystic fibrosis. A key component of such a therapy is a drug formulation that accomplishes the objectives of maintaining the protein in a stable, chemically reduced state (essential for its mucolytic and anti-protease functions) as well as facilitating compatibility wit modern, highly efficient aerosol delivery systems that minimize the treatment burden on CF patients. For this Phase I application, we plan to focus on laboratory-scale production of purified proteins (both native rhTrx control and a single amino-acid mutant variant, r(Cys)hTrx, having the potential to enhance safety and efficacy), physical characterization of these proteins in order to assess their chemical properties and relative activities, and initial preformulation studies to evaluate the suitability of thioredoxin for delivery via a range of nebulization devices. If the Ais of this project are achieved, key validation of the concept of disulfide reduction for mucolytic drug development will be established, enabling a Phase II project with the goal of focused development of specific formulations that will support preclinical studies leading to human trials of potentially safer and more efficacious treatment regimens that utilize improved, patient-friendly delivery devices. PUBLIC HEALTH RELEVANCE: There remains a critical unmet need for safe and effective treatment for Cystic Fibrosis (CF), the most common inherited lethal disease in Caucasian populations affecting more than 30,000 individuals in the United States. Our overall goal is to develop a new and improved mucus-disrupting (mucolytic) therapy for CF that is highly efficacious and which does not impose excessive treatment burdens on CF patients. We will accomplish this through development of optimal formulations of inhaled human thioredoxin, a recombinant protein mucolytic with a novel mechanism of action that has the potential to benefit a broader range of CF patients than current therapies.