School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland.
Pharm Res. 2012 Jan;29(1):332-41. doi: 10.1007/s11095-011-0553-z. Epub 2011 Aug 5.
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 37°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.
研究吸入鲑鱼降钙素（sCT）在呼吸上皮屏障中的命运，特别强调其被胰蛋白酶、糜蛋白酶和中性粒细胞弹性蛋白酶降解的情况。
在 37°C 下孵育 2 小时，通过 HPLC 在人呼吸道上皮细胞（hBEpC、Calu-3、16HBE14o-、A549）和 Caco-2 的细胞匀浆、上清液和完整单层中评估 sCT 的降解情况，并进行比较。研究了 sCT 被胰蛋白酶、糜蛋白酶和中性粒细胞弹性蛋白酶分解的情况。通过免疫印迹研究细胞上清液和匀浆中酶的存在情况，通过模型底物测定法研究酶活性。进行 Calu-3 单层的转运研究。
sCT 在孵育上清液或与细胞单层时，在 2 小时内浓度保持不变，与所研究的细胞类型无关。当使用细胞匀浆时，sCT 的浓度会被不同程度地降低。sCT 单独孵育时会被降解。Western blot 显示所有蛋白酶在细胞匀浆中的丰度较高，而在上清液中的表达较弱。转运研究表明 sCT 具有净吸收的转运；存在杆菌肽会导致接收室中 sCT 的量增加。
上皮蛋白酶在 sCT 经肺部给药后的处置中发挥作用。
