Improvement of the physicochemical and biopharmaceutical properties of insulin by poly(ethylene glycol) conjugation

Two bovine insulin derivatives were obtained by covalent attachment of one or two monomethoxy poly(ethylene glycol) molecules with a molecular weight of 5000 Da. Circular dichroism studies indicated that the binding of one polymer chain induced only negligible changes in the peptide's secondary structure, while an increased alpha helix conformation was observed in the other conjugate. Proteolytic digestion studies carried out with trypsin, chymotrypsin and pepsin showed a significantly higher stability of the the conjugates with respect to the native peptide; furthermore, the stability towards pretenses was found to depend on the number of polymer chains bound to the peptide. The native peptide completely degraded after 3 h of incubation with the proteolytic enzymes; at the same time, adegradation of 67, 65 and 100% was observed in the case of the one poly(ethylene glycol) derivative, and 0, 28 and 0% in the case of the two poly(ethylene glycol) derivatives after incubation with trypsin, chymotrypsin and pepsin, respectively. In vivo studies carried out by subcutaneous and intravenous administration to diabetic rats of the native and modified peptide demonstrated that the polymer conjugation could affect the hypoglycaemic profile of the homone. By intravenous administration, the derivative with one polymer molecule exhibited about 10% less of the maximal activity of the native hormone, whereas 20% of maximal activity reduction was observed for the conjugate with two polymer,molecules. Interestingly, after subcutaneous administration, superimposed hypoglycemic profiles, were found with the conjugate bearing one polymer molecule and the native insulin, while the other conjugate showed a significantly lower efficacy, although the pharmacological effect could be maintained for a longer time.