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.

SPX-101 is stable in and retains function after exposure to cystic fibrosis sputum.

BACKGROUND: In healthy lungs, epithelial sodium channel (ENaC) is regulated by short, palate, lung, and nasal clone 1 (SPLUNC1). In cystic fibrosis (CF), ENaC is hyperactivated in part due to a loss of SPLUNC1 function. We have developed SPX-101 to replace the lost function of SPLUNC1 in the CF lung. METHODS: Expression of SPLUNC1 was determined in sputum from healthy and CF donors. Stability of SPLUNC1, S18 (the ENaC regulatory domain of SPLUNC1), and SPX-101 was determined in sputum from CF donors and towards neutrophil elastase. Activity of SPX-101 after exposure to CF sputum was determined in airway epithelial cells from CF donors and in the ßENaC transgenic mouse model. RESULTS: SPLUNC1 protein expression is significantly reduced in CF as compared to healthy sputum. SPLUNC1 is rapidly degraded in CF sputum as well as by a number of individual proteases known to be found in the sputum. SPX-101, but not S18, is stable in CF sputum. Finally, SPX-101 retains its ability to internalize ENaC, regulate airway surface liquid height, and increase survival of ßENaC mice after exposure to CF sputum. CONCLUSIONS: Our results demonstrate that SPX-101, but not SPLUNC1 or S18, is stable in CF sputum. These results support the therapeutic development of SPX-101 for the treatment of cystic fibrosis.

Signal transducer and activator of transcription 3 is involved in cell growth and survival of human rhabdomyosarcoma and osteosarcoma cells.

BACKGROUND: Stat3 has been classified as a proto-oncogene and constitutive Stat3 signaling appears to be involved in oncogenesis of human cancers. However, whether constitutive Stat3 signaling plays a role in the survival and growth of osteosarcomas, rhabdomyosarcomas, and soft-tissue sarcomas is still unclear. METHODS: To examine whether Stat3 is activated in osteosarcomas, rhabdomyosarcomas and other soft-tissue sarcomas we analyzed sarcoma tissue microarray slides and sarcoma cell lines using immunohistochemistry and Western blot analysis, respectively, with a phospho-specific Stat3 antibody. To examine whether the activated Stat3 pathway is important for sarcoma cell growth and survival, adenovirus-mediated expression of a dominant-negative Stat3 (Y705F) and a small molecule inhibitor (termed STA-21) were used to inhibit constitutive Stat3 signaling in human sarcoma cell lines expressing elevated levels of Stat3 phosphorylation. Cell viability was determined by MTT assays and induction of apoptosis was analyzed by western blotting using antibodies that specifically recognize cleaved caspases-3, 8, and 9. RESULTS: Stat3 phosphorylation is elevated in 19% (21/113) of osteosarcoma, 27% (17/64) of rhabdomyosarcoma, and 15% (22/151) of other soft-tissue sarcoma tissues as well as in sarcoma cell lines. Expression of the dominant-negative Stat3 and treatment of STA-21 inhibited cell viability and growth and induced apoptosis through caspases 3, 8 and 9 pathways in human sarcoma cell lines expressing elevated levels of phosphorylated Stat3. CONCLUSION: This study demonstrates that Stat3 phosphorylation is elevated in human rhabdomyosarcoma, osteosarcomas and soft-tissue sarcomas. Furthermore, the activated Stat3 pathway is important for cell growth and survival of human sarcoma cells.

Functional ultrasound imaging for assessment of extracellular matrix scaffolds used for liver organoid formation.

A method of 3D functional ultrasound imaging has been developed to enable non-destructive assessment of extracellular matrix scaffolds that have been prepared by decellularization protocols and are intended for recellularization to create organoids. A major challenge in organ decellularization is retaining patent micro-vascular structures crucial for nutrient access and functionality of organoids. The imaging method described here provides statistical distributions of flow rates throughout the tissue volumes, 3D vessel network architecture visualization, characterization of microvessel volumes and sizes, and delineation of matrix from vascular circuits. The imaging protocol was tested on matrix scaffolds that are tissue-specific, but not species-specific, matrix extracts, prepared by a process that preserved >98% of the collagens, collagen-associated matrix components, and matrix-bound growth factors and cytokines. Image-derived data are discussed with respect to assessment of scaffolds followed by proof-of-concept studies in organoid establishment using Hep3B, a human hepatoblast-like cell line. Histology showed that the cells attached to scaffolds with patent vasculature within minutes, achieved engraftment at near 100%, expressed liver-specific functions within 24 h, and yielded evidence of proliferation and increasing differentiation of cells throughout the two weeks of culture studies. This imaging method should prove valuable in analyses of such matrix scaffolds.