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 ami no groups (seven possible derivatives), and resulting insulin conjugates pu rified to homogeneity. In vivo bioactivity in rats of most derivatives was preserved while disubstituted PEG-insulins showed decreased potency. Only s ite-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 inv olved in the fibrillation process. Immunogenicity in vivo and in vitro of m onoglycosylated insulins was comparable to that of insulin, diglycosylated insulins showed immunogenicity enhancement. Immunogenicity of PEG-insulins was significantly suppressed. PheB1-glycosylated insulins administered subc utaneously in dogs displayed extended action profiles, the most effective b eing 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 incre ased, providing some explanation for prolonged action profile. Lectin-speci fic binding as a retardation basis is not likely involved. In support of th is, subcutaneously administered PheB1-PEG(600)insulin showed an even more p rotracted action profile, suggesting that the basis of retardation is physi cal and nonspecific. This implies that by increasing PEG chain MW further d elay/prolongation of action can be achieved to yield new soluble basal insu lin substitutes with potential clinical applications. (C) 1999 Elsevier Sci ence B.V. All rights reserved.