Discovery and development of BI 1265162, an ENaC inhibitor for the treatment of cystic fibrosis.

Lung selective inhibition of the endothelial sodium channel (ENaC) is a potential mutation agnostic treatment of Cystic Fibrosis (CF). We describe the discovery and development of BI 1265162, the first ENaC inhibitor devoid of the amiloride structural motif that entered clinical trials. The design of BI 1265162 focused on its suitability for inhalation via the Respimat® Soft Mist™ Inhaler and a long duration of action. A convergent and scalable route for the synthesis of BI 1265162 as dihydrogen phosphate salt is presented, that was applied to support clinical trials. A phase 2 study with BI 1265162 did not provide a clear sign of clinical benefit. Whether ENaC inhibition will be able to hold its promise for CF patients remains an open question.

Clinical and genetic characteristics of the patients with hypertension and hypokalemia carrying a novel SCNN1A mutation.

The objective of this study was to clinically and genetically characterize a pedigree with Liddle syndrome (LS). A LS pedigree comprising with one proband and seven family members was enrolled. The subjects' symptoms, laboratory results and genotypes were analyzed. Peripheral venous samples were collected from the subjects, and genomic DNA was extracted. DNA library construction and exome capture were performed on an Illumina HiSeq 4000 platform. The selected variant sites were validated using Sanger sequencing. The mutation effects were investigated using prediction tools. The proband and her paternal male family members had mild hypertension, hypokalemia and muscle weakness, including the absence of low renin and low aldosterone. Genetic analysis revealed that the proband carried a compound heterozygous mutation in SCNN1A, a novel heterozygous mutation, c.1130T > G (p.Ile377Ser) and a previously characterized polymorphism, c.1987A > G (p.Thr633Ala). The novel mutation site was inherited in an autosomal dominant manner and was predicted by in silico tools to exert a damaging effect. Alterations in the SCNN1A domain were also predicted by protein structure modeling. After six months of follow-up, treatment had significantly improved the patient's limb weakness and electrolyte levels. The novel mutation c.1130T > G of the SCNN1A gene was detected in the pedigree with LS. The clinical manifestations of the pedigree were described, which expand the phenotypic spectrum of LS. This result of this study also emphasizes the value of genetic testing for diagnosing LS.

Liddle syndrome misdiagnosed as primary aldosteronism is caused by inaccurate aldosterone-rennin detection while a novel SCNN1G mutation is discovered.

PURPOSE: Through describing the confusing misdiagnosis process of Liddle syndrome, we try to reveal the importance of accurate aldosterone-renin detection and a genetic test for Liddle syndrome. METHODS: We found a family of hypertension and hypokalaemia with the proband of a 21-year-old female who had been misdiagnosed as primary aldosteronism (PA). She presented with high aldosterone and low renin levels. Aldosterone is not suppressed in the saline infusion test and captopril challenge test. However, treatment with a standard dose of spironolactone has no blood pressure improvement effect. A heterozygous variant of SCNN1G was found with whole exome sequencing and Liddle syndrome is indicated. Treatment with amiloride was effective. We rechecked aldosterone-renin levels with two different aldosterone and renin test kits. Clinical features and the mutant gene SCNN1G of each family member were determined by the Sanger method. RESULTS: The two kits had nearly opposite results. Among those Liddle syndrome patients confirmed by a genetic test, for Test kit A all ARR were screened positive while for test kit B negative. It seems Test kit B is consistent with the diagnosis while test kit A misleads the diagnosis. A novel SCNN1G mutation, c.1729 C > T, was found in this family, which introduce a premature stop codon in the γ subunit in the epithelial Na+ channel (ENaC) and resulted in a deletion of 72 amino acids at the carboxyl end. CONCLUSION: inaccurate ARR detection might misdiagnose Liddle syndrome. A Gene test is an important method for the diagnosis of Liddle syndrome. A novel SCNN1G missense mutation, c.1729 C > T, is found in a Chinese family.

Pro-Resolving Mediator Resolvin E1 Restores Alveolar Fluid Clearance in Acute Respiratory Distress Syndrome.

ABSTRACT: Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by increased permeability of the alveolar-capillary barrier and impaired alveolar fluid clearance. Resolvin E1 (RvE1) is a specialized pro-resolving mediator derived endogenously from omega-3-polyunsaturated fatty acids. RvE1 (10 µg/kg i.v.) was injected to rats 6 h post-lipopolysaccharide (LPS) (14 mg/kg) induction. After another 3 h, alveolar fluid clearance was measured in live rats (n = 8-9). The primary Type II alveolar epithelial cell was isolated and treated by LPS (1 µg/mL) with or without RvE1 (250 nM). The expression of epithelial sodium channel (ENaC), Na+/K+-ATPase (NKA), AKT, serum- and glucocorticoid-induced kinase 1 (SGK1), and Nedd4-2 were detected. RvE1 improved survival rate (30% vs. 70%, P = 0.048), increased the clearance of alveolar fluid (13.34% vs. 18.73%, P  < 0.001), reduced lung wet-dry weight ratio (5.01 vs. 4.63, P  < 0.001), mitigated lung injury scores (13.38 vs. 7.0, P  < 0.05) and inflammation in LPS-induced ARDS in rats. RvE1 upregulated alveolar ENaC and NKA expression in vivo and in vitro. In addition, RvE1 significantly increased the expression of phosphorylated AKT, SGK1, and phosphorylated Nedd4-2 in LPS-stimulated primary alveolar type II cells. The effects of RvE1 were abrogated by blocking phosphatidylinositide3'-kinase (PI3K) and SGK1 with LY294002 and GSK650394, respectively. In summary, RvE1 upregulated ENaC and NKA expression by activating PI3K/AKT/SGK1 pathway to promote alveolar fluid clearance, suggesting that RvE1 may be a potentially effective drug for ARDS treatment.

SPX-101 Is a Novel Epithelial Sodium Channel-targeted Therapeutic for Cystic Fibrosis That Restores Mucus Transport.

RATIONALE: Cystic fibrosis (CF) lung disease is caused by the loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) combined with hyperactivation of the epithelial sodium channel (ENaC). In the lung, ENaC is responsible for movement of sodium. Hyperactivation of ENaC, which creates an osmotic gradient that pulls fluid out of the airway, contributes to reduced airway hydration, causing mucus dehydration, decreased mucociliary clearance, and recurrent acute bacterial infections. ENaC represents a therapeutic target to treat all patients with CF independent of their underlying CFTR mutation. OBJECTIVES: To investigate the in vitro and in vivo efficacy of SPX-101, a peptide mimetic of the natural regulation of ENaC activity by short palate, lung, and nasal epithelial clone 1, known as SPLUNC1. METHODS: ENaC internalization by SPX-101 in primary human bronchial epithelial cells from healthy and CF donors was assessed by surface biotinylation and subsequent Western blot analysis. SPX-101's in vivo therapeutic effect was assessed by survival of ß-ENaC-transgenic mice, mucus transport in these mice, and mucus transport in a sheep model of CF. MEASUREMENTS AND MAIN RESULTS: SPX-101 binds selectively to ENaC and promotes internalization of the α-, ß-, and γ-subunits. Removing ENaC from the membrane with SPX-101 causes a significant decrease in amiloride-sensitive current. The peptide increases survival of ß-ENaC-transgenic mice to greater than 90% with once-daily dosing by inhalation. SPX-101 increased mucus transport in the ß-ENaC mouse model as well as the sheep model of CF. CONCLUSIONS: These data demonstrate that SPX-101 promotes durable reduction of ENaC membrane concentration, leading to significant improvements in mucus transport.

ß1-Na(+),K(+)-ATPase gene therapy upregulates tight junctions to rescue lipopolysaccharide-induced acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with diverse disorders and characterized by disruption of the alveolar-capillary barrier, leakage of edema fluid into the lung, and substantial inflammation leading to acute respiratory failure. Gene therapy is a potentially powerful approach to treat ALI/ARDS through repair of alveolar epithelial function. Herein, we show that delivery of a plasmid expressing ß1-subunit of the Na(+),K(+)-ATPase (ß1-Na(+),K(+)-ATPase) alone or in combination with epithelial sodium channel (ENaC) α1-subunit using electroporation not only protected from subsequent lipopolysaccharide (LPS)-mediated lung injury, but also treated injured lungs. However, transfer of α1-subunit of ENaC (α1-ENaC) alone only provided protection benefit rather than treatment benefit although alveolar fluid clearance had been remarkably enhanced. Gene transfer of ß1-Na(+),K(+)-ATPase, but not α1-ENaC, not only enhanced expression of tight junction protein zona occludins-1 (ZO-1) and occludin both in cultured cells and in mouse lungs, but also reduced pre-existing increase of lung permeability in vivo. These results demonstrate that gene transfer of ß1-Na(+),K(+)-ATPase upregulates tight junction formation and therefore treats lungs with existing injury, whereas delivery of α1-ENaC only maintains pre-existing tight junction but not for generation. This indicates that the restoration of epithelial/endothelial barrier function may provide better treatment of ALI/ARDS.

[Na+ Transport in the lungs: differential impact of ENaC in the airways and alveoli]. / Transport du Na+ dans les poumons : impact différentiel du canal ENaC dans les voies aériennes et les alvéoles.

Na+ transport by airway epithelial cells, in conjunction with Cl- secretion is crucial for maintaining an adequate level of airway surface liquid (ASL) for an effective mucociliary clearance by the ciliated airway epithelial cells. It is also an important mechanism for lung liquid absorption at birth and oedema absorption during an acute respiratory distress syndrome (ARDS). The epithelial Na+ channel (ENaC) is the channel mostly involved in this process. The consequences of an imbalance in ENaC activity in the airways and in the distal lung are different. Experimental over expression of ENaC in the airways leads to a decrease in mucociliary clearance and inflammation similar to cystic fibrosis and chronic bronchitis. However, bacterial and viral pathogens, as well as pro-inflammatory cytokines present during lung infection downregulate ENaC expression and activity in airway and alveolar epithelial cells. ENaC downregulation by pathogens or inflammatory products could participate in the modulation of the severity of ARDS. Pharmacological strategies that modulate ENaC expression or activity could be important in the treatment of different lung diseases since it is actively involved in the lung innate defence mechanisms.