Oral Delivery of Proteins: Effect of Chicken and Duck Ovomucoid on the Stability of Insulin in the Presence ofα-Chymotrypsin and Trypsin

Abstract

The in-vitro stability of insulin in the presence of α-chymotrypsin and trypsin has been evaluated in the presence of different concentrations of chicken and duck ovomucoid (CkOVM and DkOVM), a new class of enzyme inhibitor derived from the egg white of avian species. The inhibitory effect was compared with that of aprotinin. The effectiveness of DkOVM was also determined in the presence of agents that accelerate α-chymotrypsin-mediated degradation of insulin in solution by deaggregation. Insulin solutions (18μM) were incubated at 37°C with 0.1 μM chymotrypsin and 0.5 μM trypsin in lOOmM Tris buffer containing 1 mM calcium chloride and different concentrations of CkOVM and DkOVM. Samples were treated with cold Tris containing 1% (v/v) trifluoroacetic acid to stop the enzyme action and analysed by reversed-phase high-performance liquid chromatography. Similar studies were performed with aprotinin, EDTA (0.05 mM) and sodium glycocholate (30mM) in the presence of α-chymotrypsin and DkOVM. DkOVM was effective against α-chymotrypsin-mediated degradation of insulin at enzyme-to-inhibitor ratios of 1:0–5, 1:1 and 1:2. CkOVM was ineffective against α-chymotrypsin even at an enzyme-to-inhibitor ratio of 1:4. In contrast, both DkOVM and CkOVM were completely effective against trypsin-mediated degradation of insulin at an enzyme-to-inhibitor ratio of 1:1. This effect was comparable with that of aprotinin at an enzyme-to-inhibitor ratio of 1:1. Inhibition of the enzyme was reduced in the presence of sodium glycocholate and EDTA. DkOVM effectively stabilized insulin against degradation for a study period of 1 h in the presence of α-chymotrypsin and trypsin. Because insulin is extensively degraded by α-chymotrypsin, DkOVM might be used to enhance the oral delivery of insulin.