GLYCOSYLATED INSULINS

Different glycosylated human insulins were synthesized through the covalent attachment of p-succinyl amidophenyl glycopyranoside (SAPG) moiety(ies) to insulin amino groups GlyA1, LysB29 and PheB1. All 7 possible glucosyl derivatives were purified to homogeneity. All but GlyA1, LysB29 disubstituted and trisubstituted products maintained bioactivity similar to native insulin. The mono and diSAPG insulins (especially PheB1 substituted) showed decreased association as determined by GPC and dynamic light scattering. PheB1 substituted and/or di-and trisubstituted insulins demonstrated higher long term stability than native insulin as assessed by the fraction of remaining nonaggregated protein. Because of possible clinical applications, the immunogenicity of different glycosylated insulins was also investigated using two different strains of mice: A/J (H-2(a)) and C57BL/10ScSn (H-2(b)). Generally, monosubstituted derivatives provoked immunological responses in vivo and in vitro comparable to regular insulin whereas disubstituted derivatives displayed elevated responses both in vive and in vitro. Ongoing insulin receptor binding studies revealed that GlyA1 glycosylation negatively affects the binding constant, whereas LysB29 modification in the GlyA1, LysB29 disubstituted derivative has no additional effect. Clinical studies with GlyA1 SAPG insulin demonstrated that important pharmacological parameters were unchanged (blood glucose level, C-peptide concentration) compared to insulin, with the exception of an elevated concentration of GlyA1 SAPG insulin in the blood. This may explain the preserved bioactivity of GlyA1 SAPG insulin despite its attenuated affinity for the insulin receptor.