Evaluation of Bovine Insulin Compatibility with Protease Inhibitor and Absorption Enhancer Compounds for an Oral Colon Delivery System

Purpose. In view of developing an insulin-containing oral colon delivery system, the compatibility of the protein with selected protease inhibitor and absorption enhancer compounds, i.e. camostat mesilate (CM) and sodium glycocholate (NaGly), was assessed. An HPLC evaluation of insulin and its main degradation products, namely A21-desamido insulin (A21), Other Insulin Related Compounds (OIRC) and High Molecular Weight Proteins (HMWP), was carried out. Thermal analyses were also performed to support the chromatographic investigation. Methods. Insulin-CM, insulin-NaGly (both 1:10 by weight) and insulin-CM-NaGly (1:5:10 by weight) physical mixtures were prepared and stored for a one-year period in glass vials at 4°C. HPLC analyses were performed at 0, 1, 2, 3, 6 and 12 months according to Eur. Ph. 6th Ed. For the DSC study, insulin and CM or NaGly mixtures, in which the protein amount ranged between 0 and 100%, were weighed in aluminium pans that were subsequently sealed and perforated. Analyses were immediately performed under 70 mL/min nitrogen purge at the heating rate of 10°C/min. Results. Insulin content did not vary in any sample type throughout the study period, whereas all assayed degradation products tended to increase slightly. However, none exceeded the limits set by Eur. Ph. 6th Ed. for raw bovine insulin (3% for A21 and OIRC, 1% for HMWP). As far as calorimetric analyses are concerned, the melting temperature and heat of fusion of CM were not affected by increasing amounts of protein in insulin-CM mixtures. Indeed, a linear relationship was found between the melting enthalpy of CM and its percentage in the mixtures. With respect to NaGly, which is in the form of a hydrate, the crystallization event following water loss was considered. No shift in the crystallization temperature was noticed, and the relevant enthalpy was shown to increase as a linear function of its percentage in the mixtures. Conclusion. The overall results indicated that the selected adjuvants could be included in an insulin solid formulation intended for oral colon delivery without having a significant impact on the protein stability.