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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

Diabetes Section (M.E.D., J.A.B., M.B., J.M.E.) and Drug Design and Development Section (H.W.H., N.H.G.), National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224

Address all correspondence and requests for reprints to: Dr. Josephine M. Egan, Diabetes Section 23, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, Maryland 21224. E-mail: eganj{at}vax.grc.nia.nih.gov

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.

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