Validation of SPX-101 as a candidate for the treatment of cystic fibrosis

上皮Na+通道(ENaC)是许多上皮细胞吸收Na+的限速步骤。在……里面 囊性纤维化(CF)患者的呼吸道，ENaC异常活跃导致不成比例 Na+的吸收和呼吸道表面液体(ASL)的耗竭。ENAC过度活跃对 粘液淤积和呼吸道感染发生率增加，经常导致死亡 有耐心的。尽管之前做出了努力，但目前还没有治疗ENaC异常活动的现有疗法。 阿龙。利用蛋白质组学筛选，我们已经确定SPLunc1蛋白是一种有效的变构抑制物 ENaC与胞外ENaC结合，减少质膜中ENaC的数量 并随后限制ENaC的活性。我们还鉴定了ENaC抑制结构域 并合成了与该结构域对应的小肽，称为SPX-101。这 多肽强效抑制CF24小时培养的ENaC和防止ASL高吸收 在单剂注射后。SPX-101在中性粒细胞弹性蛋白酶存在的情况下继续发挥作用，这是 在CF呼吸道中含量非常丰富，表明这种多肽可能在治疗上有益于 慢性阻塞性肺疾病的治疗我们还证明了SPX-101可以提高小鼠的存活率。 肺上皮细胞特异性高表达ENaC的转基因小鼠模型(βENaC-TG) SPX-101可以通过雾化吸入有效地输送到肺部。值得注意的是，SPX-101没有 静脉给药可致大鼠利尿或高钾血症。此外，雾化了 SPX-101仍然隔离在肺内，给肺1%的剂量到达 大鼠的体循环。 为了促进治疗CF的SPX-101的开发，我们正在寻求这一阶段的SBIR 授予：(I)测量SPX-101对大鼠、犬和绵羊支气管上皮细胞的影响 证明这些物种与毒物学和翻译研究的相关性，以及(2)界定 在CFTR抑制剂中，SPX-101的最大有效剂量和完全增强剂量依赖效应。 对绵羊气管粘液流速有抑制作用。拟议工作的完成将导致 第二阶段申请，目标是完成毒理学研究和人体临床试验 这是一种治疗CF的新疗法。

The Epithelial Na+ Channel (ENaC) is the rate limiting step for Na+ absorption across many epithelia. In the airways of cystic fibrosis (CF) patients, ENaC is abnormally hyperactive leading to disproportionate Na+ absorption and a depletion of airway surface liquid (ASL) volume. ENaC hyperactivity contributes to mucus stasis and increased incidence of airway infections that frequently lead to the death of the patient. Despite previous efforts, there are no existing therapies to treat abnormal ENaC activity in the lung. Using a proteomic screen, we have identified the protein SPLUNC1 as a potent allosteric inhibitor of ENaC that binds extra-cellularly to ENaC, reducing the amount of ENaC in the plasma membrane and subsequently limiting ENaC activity. We have also identified the ENaC-inhibitory domain of SPLUNC1, and have synthesized small peptide that corresponds to this domain, called SPX-101. This peptide robustly inhibits ENaC and prevents ASL hyperabsorption in CF airway cultures for 24 h following a single dose. SPX-101 continues to function in the presence of neutrophil elastase, which is highly abundant in CF airways, suggesting that this peptide may be therapeutically beneficial in the treatment of CF lung disease. We have also demonstrated that SPX-101 improves survival in a transgenic mouse model of CF that specifically overexpresses ENaC in the lung epithelia (βENaC-Tg) and that SPX-101 can be effectively delivered to the lung via nebulization. Notably, SPX-101 does not induce diuresis or hyperkalemia when delivered by intravenous infusion in rats. Furthermore, nebulized SPX-101 remains sequestered within the lung and <1% of the dose administered to the lung reaches systemic circulation in rats. To advance the development of a SPX-101 for the treatment of CF we are seeking this Phase I SBIR grant to: (i) measure the effect of SPX-101 in rat, canine and ovine bronchial epithelial cells to demonstrate the relevance of these species for toxicological and translational studies, and (ii) define the maximal effective dose and fully power the dose dependent effects of SPX-101 in a CFTR inhibitor- induced inhibition of tracheal mucus velocity in sheep. Completion of the proposed work will lead to a Phase II application that will be aimed at completion of toxicology studies and human clinical trials of this novel therapy for CF.