title: 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.
abstract: 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 Aha(8), with the previously described GLP-1 8-glycine (GLP-1 Gly(8)) analog. GLP-1 Aha(8) (10 nM) was equipotent with GLP-1 (10 nM) in stimulating insulin secretion in RIN 1046-38 cells. As with GLP-1 Gly(8), the binding affinity of GLP-1 Aha(8) for the GLP-1 receptor in intact Chinese hamster ovary (CHO) cells expressing the human GLP-1 receptor (CHO/GLP-1R cells) was reduced (IC(50): GLP-1, 3.7 +/- 0.2 nM; GLP-1 Gly(8), 41 +/- 9 nM; GLP-1 Aha(8), 22 +/- 7 nM). GLP-1 Aha(8) 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 ED(50) when compared to GLP-1 and GLP-1 Gly(8) (ED(50): GLP-1, 0.036 +/- 0.002 nM, GLP-1 Gly(8), 0.13 +/- 0.02 nM, GLP-1 Aha(8), 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 Aha(8) (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 Aha(8) 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.
authors.affiliation: Diabetes Section, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: M E
authors.initials: ME
authors.lastname: Doyle
authors.forename: N H
authors.initials: NH
authors.lastname: Greig
authors.forename: H W
authors.initials: HW
authors.lastname: Holloway
authors.forename: J A
authors.initials: JA
authors.lastname: Betkey
authors.forename: M
authors.initials: M
authors.lastname: Bernier
authors.forename: J M
authors.initials: JM
authors.lastname: Egan
mesh_terms: D000595:Amino Acid Sequence; D015119:Aminocaproic Acid; D000818:Animals; D002460:Cell Line; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy / Q000378:metabolism; D005934:Glucagon / Q000235:genetics / Q000494:pharmacology* / Q000627:therapeutic use; D052216:Glucagon-Like Peptide 1; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology* / Q000627:therapeutic use; D011498:Protein Precursors / Q000235:genetics / Q000494:pharmacology* / Q000627:therapeutic use; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D007004:Hypoglycemic Agents; D010446:Peptide Fragments; D011498:Protein Precursors; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D015119:Aminocaproic Acid