Site-specific insulin conjugates with enhanced stability and extended action profile.

Two different hydrophilic moieties, carboxyl derivatives of monosaccharidic (Glc, Gal, Man, Fuc) glycosides and methoxypolyethylene glycols of varying MW, were covalently attached to the insulin GlyA1, PheB1 and/or LysB29 amino groups (seven possible derivatives), and resulting insulin conjugates purified to homogeneity. In vivo bioactivity in rats of most derivatives was preserved while disubstituted PEG-insulins showed decreased potency. Only site-specific modification of PheB1 amino group with either moiety resulted in pronouncedly increased resistance of insulin to fibrillation, indicating that the B-chain N-terminus of the insulin molecule is mechanistically involved in the fibrillation process. Immunogenicity in vivo and in vitro of monoglycosylated insulins was comparable to that of insulin, diglycosylated insulins showed immunogenicity enhancement. Immunogenicity of PEG-insulins was significantly suppressed. PheB1-glycosylated insulins administered subcutaneously in dogs displayed extended action profiles, the most effective being PheB1-galactosylated insulin, resembling the pharmacodynamic response of intermediate-acting insulin preparations. The pharmacokinetic parameters of these insulin derivatives were not significantly different from that of insulin even though absorption and residence time and clearance were increased, providing some explanation for prolonged action profile. Lectin-specific binding as a retardation basis is not likely involved. In support of this, subcutaneously administered PheB1-PEG(600)-insulin showed an even more protracted action profile, suggesting that the basis of retardation is physical and nonspecific. This implies that by increasing PEG chain MW, further delay/prolongation of action can be achieved to yield new soluble basal insulin substitutes with potential clinical applications.