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 B-12 (B-12) may be exploited in this area, but an open question is whether B-12-peptide conjugates bound to the B-12 gastric uptake protein intrinsic factor (IF) can provide any stability against proteases. Herein, we describe a new conjugate of B-12 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 beta. Prebinding the B-12 conjugate to IF, however, resulted in up to a 4-fold greater activity of the B-12-Ex-4 conjugate relative to Ex-4, when the IF-B-12-Ex-4 complex was exposed to 22 mu g/mL of trypsin, 2.3-fold greater activity when exposed to 1.25 mu g/mL of chymotrypsin, and there was no decrease in function at up to 5 mu g/mL of meprin beta.