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

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 SPLUNC 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 SPLUNC and have synthesized small peptide that corresponds to this domain called SPX This peptide robustly inhibits ENaC and prevents ASL hyperabsorption in CF airway cultures for h following a single dose SPX 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 improves survival in a transgenic mouse model of CF that specifically overexpresses ENaC in the lung epithelia ENaC Tg and that SPX can be effectively delivered to the lung via nebulization Notably SPX does not induce diuresis or hyperkalemia when delivered by intravenous infusion in rats Furthermore nebulized SPX remains sequestered within the lung and andlt of the dose administered to the lung reaches systemic circulation in rats To advance the development of a SPX for the treatment of CF we are seeking this Phase I SBIR grant to i measure the effect of SPX 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 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 The epithelial sodium channel ENaC is a molecular target for cystic fibrosis CF When the cystic fibrosis transmembrane conductance regulator protein is inactive due to genetic mutation the body loses the ability to secrete chloride ions into the fluid that lines the airways Since salt in this fluid controls the total volume of the fluid decreases and the mucus in the airways becomes dehydrated The loss of salt due to reduced chloride secretion is made worse by ENaC which removes sodium from the airway fluid and further dehydrates the lung ENaC inhibition can counteract the loss of fluid and accumulation of viscous dehydrated mucus that blocks small airways and provides a nutrient source for bacterial growth We have identified a novel peptide agent that inhibits ENaC activity and may provide a valuable new approach for treatment We propose to test this novel peptide known as SPX for the treatment of CF