Preparation and characterization of monomethoxypoly(ethylene glycol)-insulin conjugates.

Insulin was modified with monomethoxypoly(ethylene glycol) 5000 (MPEG5000) by reaction of insulin amino groups with activated MPEG5000, and mono-, di- and tri-PEGylated insulin were obtained. Circular dichroism demonstrated that the attachment of MPEG5000 to insulin did not alter the tertiary structure of insulin. PEGylation of insulin could inhibit the self-association tendency of insulin, which decreased with the increase of substitution degree. The physical stability and proteolytic stability of insulin increased after MPEG conjugation, and the stabilities of the conjugates depended on the PEGylation degree. In vivo biological activity of mono-PEGylated insulin following parenteral administration both in mice and rats was preserved while di- and tri-PEGylated insulin showed a great loss in biological activity. In addition, mono-PEGylated insulin administered subcutaneously in mice as well as intravenously in rats displayed extended action profiles. These results indicated that mono-substituted MPEG5000-insulin conjugate was a good candidate for insulin replacement therapy.