Insertion of an n-terminal 6-aminohexanoic acid after the 7 amino acid position of glucagon-like peptide-1 produces a long-acting hypoglycemic agent

The use of glucagon-like peptide-1 (GLP-1) as a routine treatment for type 2 diabetes mellitus is undermined by its short biological half-life. A cause of degradation is its cleavage at the N-terminal HAE sequence by the enzyme dipeptidyl peptidase IV (DPP IV). To protect from DPP IV, we have studied the biological activity of a GLP-1 analog in which 6-aminohexanoic acid (Aha) is inserted between histidine and alanine at positions 7 and 8. We have compared the biological activity of this new compound, GLP-1 Aha⁸, with the previously described GLP-1 8-glycine (GLP-1 Gly⁸) analog. GLP-1 Aha⁸ (10 nM) was equipotent with GLP-1 (10 nM) in stimulating insulin secretion in RIN 1046-38 cells. As with GLP-1 Gly⁸, the binding affinity of GLP-1 Aha⁸ for the GLP-1 receptor in intact Chinese hamster ovary (CHO) cells expressing the human GLP-1 receptor (CHO/GLP-1R cells) was reduced (IC₅₀: GLP-1, 3.7 ± 0.2 nM; GLP-1 Gly⁸, 41 ± 9 nM; GLP-1 Aha⁸, 22 ± 7 nM). GLP-1 Aha⁸ was also shown to stimulate intracellular cAMP production 4-fold above basal at concentrations as low as 0.5 nM. However, it exhibited a higher ED₅₀ when compared to GLP-1 and GLP-1 Gly⁸ (ED₅₀: GLP-1, 0.036 ± 0.002 nM, GLP-1 Gly⁸, 0.13 ± 0.02 nM, GLP-1 Aha⁸, 0.58 ± 0.03 nM). A series of D-amino acid-substituted GLP-1 compounds were also examined to assess the importance of putative peptidase-sensitive cleavage sites present in the GLP-1 molecule. They had poor binding affinity for the GLP-1 receptor, and none of these compounds stimulated the production of intracellular cAMP in CHO/GLP-1R cells or insulin secretion in RIN 1046-38 cells. GLP-1 Aha⁸ (24 nmol/kg) administered sc to fasted Zucker (fa/fa) rats (mean blood glucose, 195 ± 32 mg/dl) lowered blood glucose levels to a nadir of 109 ± 3 mg/dl, and it remained significantly lower for 8 h. Matrix-assisted linear desorption ionization-time of flight mass spectrometry of GLP-1 Aha⁸ incubated with DPP IV (37 C, 2 h) did not exhibit an N-terminal degradation product. Taken together, these results show that insertion of Aha after the 7 position in GLP-1 produces an effective, long-acting GLP-1 analog, which may be useful in the treatment of type 2 diabetes mellitus.