Oral delivery of proteins: Preparation and characterization of an oral dual-controlled release formulation of insulin and ovomucoid

Oral bioavailability of proteins is extremely low due to extensive degradation in the gastrointestinal tract and low epithelial permeability. Further, the structure and conformation of proteins are easily altered when exposed to formulation and process conditions leading to a loss of biological activity. Oral delivery of proteins may be enhanced by the use of absorption modifiers such as enzyme inhibitors and permeation enhancers. In the present study, the role of ovomucoids has been investigated as possible absorption modifiers in the oral delivery of a model protein, insulin. Ovomucoids are enzyme inhibitors isolated from egg white of avian species. They have protease inhibitory activity and also bind to lectins on the cell surfaces through their carbohydrate moeity. However, their role in the oral delivery of proteins has not been investigated. The purpose of this study was to develop a dual controlled release formulation of insulin and ovomucoid. Enzymatic degradation studies revealed that insulin is degraded extensively in the presence of trypsin and a-chymotrypsin. Duck ovomucoid (DkOVM) stabilized insulin against degradation in the presence of trypsin and a-chymotrypsin for an hour. In contrast, chicken ovomucoid (CkOVM) was only effective against trypsin mediated degradation of insulin. Permeability studies of insulin from rat intestinal segments revealed that insulin is absorbed more from the jejunum and ileum than from the duodenum. In the presence of CkOVM and DkOVM, the permeability of insulin decreased, which may be explained in part, by the action of insulin by adipocytes. A coprecipitation technique was successfully applied to prepare microcapsules of insulin with high encapsulation efficiency. Dissolution stability studies of insulin microcapsules in the presence of enzymes revealed considerable improvement in the availability of insulin with ovomucoids even at the end of 6 hours. Characterization of insulin in the microcapsules using DSC, FT-IR, powder x-ray diffraction and size exclusion chromatography revealed that the structure of insulin was conserved after subjecting it to formulation and process conditions. A three-factor, three-level optimization design was used to evaluate the effect of critical process variables including the rate of addition of polymeric solution, compression pressure and volume of water with respect to polymeric solution. Mathematical relationships, contour plots and response surface methods were employed with constrained optimization to predict levels of factors that provide optimum response. The predicted and observed values were in close agreement. The release of DkOVM was delayed from the formulation. The novel formulation incorporates controlled release characteristics of both protein and inhibitor that may enhance the stability and availability of proteins with less potential for inhibitor related toxicity.