GLYCOSYLATED INSULINS

Different glycosylated human insulins were synthesized through the cov alent attachment of p-succinyl amidophenyl glycopyranoside (SAPG) moie ty(ies) to insulin amino groups GlyA1, LysB29 and PheB1. All 7 possibl e glucosyl derivatives were purified to homogeneity. All but GlyA1, Ly sB29 disubstituted and trisubstituted products maintained bioactivity similar to native insulin. The mono and diSAPG insulins (especially Ph eB1 substituted) showed decreased association as determined by GPC and dynamic light scattering. PheB1 substituted and/or di-and trisubstitu ted insulins demonstrated higher long term stability than native insul in as assessed by the fraction of remaining nonaggregated protein. Bec ause of possible clinical applications, the immunogenicity of differen t glycosylated insulins was also investigated using two different stra ins of mice: A/J (H-2(a)) and C57BL/10ScSn (H-2(b)). Generally, monosu bstituted 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 insuli n receptor binding studies revealed that GlyA1 glycosylation negativel y affects the binding constant, whereas LysB29 modification in the Gly A1, LysB29 disubstituted derivative has no additional effect. Clinical studies with GlyA1 SAPG insulin demonstrated that important pharmacol ogical parameters were unchanged (blood glucose level, C-peptide conce ntration) compared to insulin, with the exception of an elevated conce ntration of GlyA1 SAPG insulin in the blood. This may explain the pres erved bioactivity of GlyA1 SAPG insulin despite its attenuated affinit y for the insulin receptor.