Development of a Neutrophil Degranulation Inhibitor to Treat ARDS

项目摘要 急性呼吸窘迫综合征(ARDS)是肺部医学中的一个严重问题，占 美国重症监护病房(ICU)入院人数的10%，每年总计超过200,000名患者 每个患者的治疗费用通常超过7万美元。数十年的临床试验都未能确定有效 ARDS的药物治疗，而死亡率保持在30%以上。因此，有一个危急的，未得到满足的临床 需要成功的药物策略来治疗ARDS。中性粒细胞在肺部起着至关重要的作用。 损伤导致急性呼吸窘迫综合征，包括新冠肺炎，通过细胞外释放活性氧 种类(ROS)、颗粒成分和中性粒细胞胞外陷阱(Net)。科学基础 我们创新疗法的基础是抑制中性粒细胞脱颗粒也能阻止ROS的释放 和Nets，使脱颗粒成为治疗目标。我们产生了一种重组蛋白，去颗粒蛋白-23 (SNAP-23脱颗粒抑制剂，DGN-23)，含有SNAP-23的SNARE基序和一个细胞 通透性多肽。DGN-23在体外和体内迅速进入人中性粒细胞，通过以下方式抑制脱颗粒 50%至80%，防止ROS释放，并减少净形成。重要的是，体外研究表明 DGN-23不损害中性粒细胞吞噬、颗粒与吞噬小体融合或内部细菌杀灭 吞噬小体和体内研究表明，DGN-23在肺损伤开始时给药可抑制急性 3只大鼠肺损伤模型，无明显毒性反应。因此，体外和体内数据提供了强有力的支持 我们的新治疗策略是抑制中性粒细胞胞吐，以减轻导致ARDS的肺损伤。 下一步是开发制造能力和质量控制，从实验室转移到 将重组蛋白分级为药用级药物及无毒疗效验证 在临床相关的情况下。这一过渡将通过两个目标实现。目标1：确定特征 DGN-23的生产特性和脱靶/副作用。这个目标将决定纯度，存在 在DGN-23的多个生产批次中，细菌污染物、产量、效力的再现性和稳定性。 将确定细胞毒性和对循环免疫细胞的脱靶效应。器官和细胞的定位 DGN-23将在进行目标2所述实验的动物的所有主要器官中被检测。 2：在临床相关条件下确定DGN-23的治疗效果。DGN-23的能力 预防急性肺损伤和提高死亡率在初始损伤后的不同时间给药 将在H1N1流感病毒诱导的小鼠急性肺损伤模型中确定。提高了存活率和 血液含氧量的改善将成为继续开发的结果。在此结束时 工作，这个第一阶段的项目将确定一种候选药物，具有最佳的疗效、产量和 稳定性，以及在第二阶段项目中推进高级临床前研究的效力。

Project Summary Acute respiratory distress syndrome (ARDS) is a critical problem in pulmonary medicine, accounting for 10% of intensive care unit (ICU) admissions and totaling over 200,000 patients/year in the U.S. Current treatment cost typically exceeds $70,000 per patient. Decades of clinical trials failed to identify effective pharmacologic therapy for ARDS, while mortality remains above 30%. Thus, there is a critical, unmet clinical need for successful pharmacologic strategies to treat ARDS. Neutrophils play an essential role in the lung injury leading to ARDS, including that due to COVID-19, through extracellular release of reactive oxygen species (ROS), granule constituents, and neutrophil extracellular traps (NETs). The scientific foundation underlying our innovative therapy is that inhibition of neutrophil degranulation also prevents release of ROS and NETs, making degranulation a therapeutic target. We generated a recombinant protein, degranin-23 (degranulation inhibitor of SNAP-23, DGN-23) containing a SNARE motif from SNAP-23 and a cell permeability peptide. DGN-23 rapidly enters human neutrophils in vitro and in vivo, inhibits degranulation by 50% to 80%, prevents priming of ROS release, and reduces NET formation. Importantly, in vitro studies show DGN-23 does not impair neutrophil phagocytosis, granule fusion with phagosomes, or bacterial killing within phagosomes, and in vivo studies show that DGN-23 administration at the initiation of lung injury inhibits acute lung injury in 3 rodent models, without obvious toxicity. Thus, in vitro and in vivo data provide strong support for our novel therapeutic strategy that inhibition of neutrophil exocytosis attenuates lung injury leading to ARDS. The next steps are development of manufacturing capability and quality control to move from a laboratory grade recombinant protein to a pharmaceutical grade drug and validation of therapeutic effect without toxicity under clinically relevant conditions. This transition will be accomplished by two Aims. Aim 1: Characterize production characteristics and off-target/side effects of DGN-23. This aim will determine purity, presence of bacterial contaminants, yield, reproducibility of potency, and stability in multiple production runs of DGN-23. Cell toxicity and off target effects on circulating immune cells will be determined. Organ and cell localization of DGN-23 will be determined in all major organs from animals undergoing experiments described in Aim 2. Aim 2: Determine therapeutic efficacy of DGN-23 under clinically relevant conditions. The ability of DGN-23 to prevent acute lung injury and improve mortality when administered at various times after the initiating injury will be determined in a mouse model of H1N1 influenza virus-induced acute lung injury. Improved survival and improved blood oxygenation will serve as outcomes to proceed with development. At the conclusion of this work, this Phase 1 project will have identified a drug candidate with an optimal combination of efficacy, yield, stability, and potency to advance to advanced pre-clinical studies in a Phase 2 project.