Cobiprostone Is a Type-2 Chloride Channel Activator That Protects Against NSAID-Induced Cellular Damage

Abstract

Purpose: Cobiprostone is being clinically tested as a protectant against nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers, and the present study is to determine the cellular and molecular basis of protection by cobiprostone against NSAID-induced cellular damage. Methods: T84 cells were grown to confluence on solid supports. The effects of cobiprostone on indomethacin-induced changes in intracellular Ca2+ levels ([Ca2+]i) using Indo-1/AM (a dye that measures [Ca2+]I), and the effects of cobiprostone on mitochondrial membrane potential using JC-1 (a dye that monitors mitochondrial membrane potential), were tested. The effects of cobiprostone on indomethacin-induced cytochrome c release, cyclic AMP, and cell death and regrowth were also investigated. Results: Five hundred (500) μM indomethacin caused an immediate increase in [Ca2+]i, which became irreversible (calcium deregulation) after approximately 30 minutes. One hundred (100) nM cobiprostone was shown to protect against indomethacin-induced increases in [Ca2+]i, and caused a reversal of indomethacin-induced calcium deregulation. However, cobiprostone did not prevent or reverse the increase in ([Ca2+]i caused by indomethacin in the presence of different Cl− channel blockers, including diphenylamine-2-carboxylate (DPC), niflumic acid or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), suggesting that the protective effects of cobiprostone are due to activation of Cl− channels. Cobiprostone has only a small effect on cAMP levels in T84 cells, precluding a role of cAMP in the protective action of cobiprostone. Indomethacin caused depolarization of the mitochondrial membrane potential. Cobiprostone protected against indomethacin-induced depolarization of mitochondrial membrane potential and reversed the loss of mitochondrial membrane potential. Cobiprostone was also shown to protect against cytochrome c relocation from mitochondria into the cytoplasm, which is associated with apoptosis. Finally, in a novel long-term assay using T84 cell cultures, cobiprostone reduced indomethacin-induced cell death and promoted cell regrowth. Conclusion: Activation of Cl− channels may underlie the protective effects of cobiprostone in protection against NSAID-induced damage. The results further indicate that cobiprostone has the potential to be of benefit against NSAID-induced cell death. This research was funded entirely by Sucampo Pharmaceuticals, Inc., Bethesda, MD.