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.