Oral delivery and formulation of salmon calcitonin: Protection from serine proteases with ovomucoids



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Texas Tech University


Oral bioavailability of peptides and proteins is extremely low due to extensive degradation in the gastrointestinal tract and low epithelial permeability. Further, the structure and conformation of proteins are prone to alteration when exposed to formulation and process conditions. The conformation changes might lead to a loss of biological activity. Oral delivery of proteins may be enhanced by the use of enzyme inhibitors and absorption enhancers. In the present study, ovomucoids has been investigated as enzyme inhibitor in the oral delivery of salmon calcitonin. Glycyrrhetinic acid was employed as the absorption enhancer. Enzymatic degradation studies revealed that salmon calcitonin is degraded extensively by intestinal serine proteases such as trypsin, a-chymotrypsin, and elastase. Various ovomucoid species such as chicken, duck and turkey ovomucoids were investigated for their inhibitory action against the serine proteases. Duck and turkey ovomucoids stabilized salmon calcitonin against degradation in the presence of the serine proteases for an hour. In contrast, chicken ovomucoid was ineffective against a-chymotrypsin mediated degradation. The cytotoxicity studies on various enzyme inhibitors and absorption enhancers revealed that ovomucoids were safer as compared to aprotinin and protease inhibitor cocktail for long-term use up to 25 pM concentrations. Among the absorption enhancers, octylglucoside and glycyrrhetinic acid did not show any cytotoxicity on Caco-2 cell monolayer for a period of 24 hrs. Permeability studies of salmon calcitonin in Caco-2 cell monolayer suggested that salmon calcitonin is transported via passive diffusion mechanism. The permeability was enhanced in the presence of glycyrrhetinic acid. Regional permeability in rat gastrointestinal tract segments revealed that salmon calcitonin is permeated more from ileum followed by colon, jejunum, duodenum, and stomach. Therefore, the formulation of salmon calcitonin was made to bypass duodenum region and it was targeted to jejunum region. The preclinical in vivo absorption studies in rats revealed that tOVM and glycyrrhetinic acid increased the oral absorption of sCT as compared to the control without these additives. An osmotically controlled bilayered tablet coated with enteric polymers was successfully applied to prepare salmon calcitonin formulation with turkey ovomucoid and glycyrrhetinic acid. Dissolution studies were performed for a period of 4 hrs which showed dual controlled release of the drug and the inhibitor. Characterization of salmon calcitonin in the formulation using DSC, FT-IR, powder X-ray diffraction and gel electrophoresis studies revealed that the structure of salmon calcitonin was conserved after subjecting to formulation and process conditions. A seven-factor, two-level Plackett-Burman screening design and a three-factor, three-level Box-Behnken optimization design was used to evaluate the effect of critical process variables including the orifice size, coating level, amounts of sodium chloride, Polyox® NIO, Poly ox N80, Carbopol® 934P, Carbopol® 974P. Mathematical relationships, contour plots and response surface methods were employed with constrained optimization to predict levels of factors that provided optimum response. The novel formulation incorporates sCT, tOVM, and glycyrrhetinic acid with a dual-controlled release characteristics for the drug and the enzyme inhibitor.