Degradation and aggregation of human calcitonin in vitro.
OBJECTIVE
To investigate the degradation of human calcitonin (hCT) by enzymes or mucosa from different gastrointestinal (GI) compartments and evaluate the stabilization effect of a synthetic ionizable copolymer on the stability of hCT in an aqueous solution. These data are a prerequisite for the development of a hydrogel based colon-specific hCT delivery system.


METHODS
Luminal and brush border membrane (BBM) enzymes from the colon and small intestine (SI) of 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 human calcitonin was performed using gradient elution, reverse phase high-pressure liquid chromatography (RP-HPLC). The structure of the hCT fragments was determined by Matrix Assisted Laser Desorption/lonization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) analysis. UV/VIS and fluorescence spectroscopy methods were used to evaluate the influence of a copolymer, possessing the same structure as the primary chains in hydrogels degradable in the colon, on the fibrillation process of hCT.


RESULTS
In vitro results showed that isolated luminal enzymes and BBM enzymes from the SI were more potent in degrading intact hCT, as expected. Moreover, BBM enzymes were far more abundant in the SI than in the colon. Compared with rabbit colonic luminal enzymes, the degradation potency of human fecalase was further abated. Intact mucosal studies revealed extensive degradation by the SI mucosa but not by the colonic mucosa. The primary structures of the peptide fragments were identified by MALDI-TOF MS analysis. Fibrillation studies of hCT indicated that acrylic acid-containing polymeric materials were able to decrease the aggregation of hCT in aqueous solutions.


CONCLUSIONS
Reduced proteolytic activity suggests that the colon is an advantageous site for peptide delivery. The structures of hCT degradation products were identified and the participation of particular enzymes in the degradation process was suggested. In addition, it was determined that an acrylic acid-containing copolymer improved the physical stability of hCT in aqueous solution.
Lu
R H
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Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD, University of Utah, Salt Lake City 84112, USA.
Kopecková
P
P
Kopecek
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002116:Calcitonin / Q000737:chemistry / Q000378:metabolism*; D003106:Colon / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000201:enzymology / Q000378:metabolism*; D007421:Intestine, Small / Q000201:enzymology; D013058:Mass Spectrometry; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D002116:Calcitonin; D010447:Peptide Hydrolases