Enhanced Peptide Stability Against Protease Digestion Induced by Intrinsic Factor Binding of a Vitamin B₁₂ Conjugate of Exendin-4

Peptide digestion from proteases is a significant limitation in peptide therapeutic development. It has been hypothesized that the dietary pathway of vitamin B₁₂ (B₁₂) may be exploited in this area, but an open question is whether B₁₂-peptide conjugates bound to the B₁₂ gastric uptake protein intrinsic factor (IF) can provide any stability against proteases. Herein, we describe a new conjugate of B₁₂ with the incretin peptide exendin 4 that demonstrates picomolar agonism of the glugacon-like peptide-1 receptor (GLP1-R). Stability studies reveal that Ex-4 is digested by pancreatic proteases trypsin and chymotrypsin and by the kidney endopeptidase meprin β. Prebinding the B₁₂ conjugate to IF, however, resulted in up to a 4-fold greater activity of the B₁₂-Ex-4 conjugate relative to Ex-4, when the IF-B₁₂-Ex-4 complex was exposed to 22 μg/mL of trypsin, 2.3-fold greater activity when exposed to 1.25 μg/mL of chymotrypsin, and there was no decrease in function at up to 5 μg/mL of meprin β.