Design and feasibility study of calcitonin and cathepsin K inhibitor for colon delivery

The degradation of human calcitonin (hCT) by enzymes and mucosa from different gastrointestinal (GI) compartments was studied. The data from these studies are essential for the development of a hydrogel based colon-specific hCT delivery system. Lulminal and brush border membrane (BBM) enzymes from the colon and small intestine (SI) or the rabbit were isolated, and their enzymatic activity toward hCT in vitro was evaluated. Human fecalase was used to mimic the luminal enzymatic activity in the human colon, and its degradation ability was assessed. Excised intact rabbit intestinal tissues from both the colon and the SI were used to study the degradation patterns of hCT by intact mucosa. Detection of intact hCT was performed using reverse phase high performance liquid chromatography (HPLC). In vitro study showed that isolated luminal enzymes and BBM enzymes from the SI were more potent id degrading intact hCT. Moreover, BBM enzymes were for more abundant in the SI than in the colon. Compared with rabbit colonic enzymes, the degradation potency of human fecalase was further abated. Reduced proteolytic activity suggests that the colon is an advantageous site of peptide delivery. The primary structures of the peptide fragments were identified by Matrix Assisted desorption/Ionization Time-of-Flight (MALDI) Mass Spectrometry analysis. A synthetic copolymer, which has the same structure as the primary chain in hydrogels degradation in the colon, was synthesized by free radical precipitation copolymerization of N, N-dimethylacrylamide, acrylic acid, N-tert-butylacrylamide, and N-methacryloylglycylglycine p-nitrohenyl ester, followed by aminolysis to convert the nitrophenyl ester group into hydroxypropionamide. Fibrillation study using UV/Vis and fluorescence spectroscopy methods indicates incorporation of polymer into the buffer solution can significantly decrease the aggregation of hCT thus improving the physical stability of hCT in aqueous solution. These results suggest the hydrogel system will have the synergetic effect on physical stabilization of hCT as a delivery system. A polymetric cathepsin K inhibitor was designed and synthesized to inhibit the cathepsin K activity in osteoclasts thus deterring the collagen degradation. The inhibitor was synthesized by conjugating a special designed short peptide to a functionalized semitelechelic poly[N-(2-hydroxypropyl)methacrylamide. A competitive inhibition of the surrogate enzyme papain indicates this polymeric inhibitor has the potential to be a potent antiosteoclastic drug in the treatment of osteoclast by co administrating with hCT. Furthermore, the colon specific biodegradable hydrogel will protect the cathepsin K inhibitor in the same manner as hCT through the GI tract, and consequently enhances its clinical efficacy.