Investigations into the Fate of Inhaled Salmon Calcitonin at the Respiratory Epithelial Barrier

Purpose The fate of inhaled salmon calcitonin (sCT) at the respiratory epithelial barrier was studied with particular emphasis on enzymatic degradation by trypsin, chymotrypsin, and neutrophil elastase. Degradation of sCT was assessed by HPLC in cell homogenate, supernatant and intact monolayers of human respiratory epithelial cells (hBEpC, Calu-3, 16HBE14o-, A549) and Caco-2 as comparison at 37A degrees C for 2 h. Breakdown of sCT by trypsin, chymotrypsin and neutrophil elastase was investigated. The presence of enzymes in cell supernatant and homogenate was studied by immunoblot and enzyme activity by model substrate assay. Transport studies across Calu-3 monolayers were performed. sCT concentration remained unchanged over 2 h, when incubated in supernatant or with cell monolayers, independent of cell type studied. When cell homogenates were used, sCT concentrations were reduced to varying extents. sCT was degraded when incubated with enzymes alone. Western blot revealed abundance of all proteinases in cell homogenates and weaker expression in supernatants. Transport studies indicated net-absorptive sCT translocation; presence of bacitracin resulted in increased amount of sCT in receiver compartments. Epithelial proteases play a role in the disposition of sCT after pulmonary delivery.