A synthetic glucagon-like peptide-1 analog with improved plasma stability

Abstract

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser 8 ]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser 8 ]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser 8 ]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser 8 ]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone.[Ser 8 ]GLP-1(7-36)amide induced a 1·5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser 8 ]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

“…This suggests that increasing the size of the amino acid side chain at Ala 8 , achieved by substituting for either valine or 2-aminobutyric acid, drastically reduces the specificity of DPP IV for GLP-1. This finding is in accordance with previous studies, where Ala 8 of GLP-1 was substituted with glycine (Deacon et al 1998, Burcelin et al 1999, Siegel et al 1999, Doyle et al 2001, serine (Deacon et al 1998, Ritzel et al 1998, Siegel et al 1999, -alanine (Siegel et al 1999), threonine or -aminoisobutyric acid (Deacon et al 1998).…”

“…(Gly 8 )GLP-1 lowered blood glucose (Burcelin et al 1999) and increased insulin secretion (Doyle et al 2001) in diabetic mice and rats; however, this was less effective than native peptide. Also notable was (Ser 8 )GLP-1 which possessed enhanced insulinotropic and glucose-lowering activity in normal animals (Ritzel et al 1998 …”

Improved biological activity of Gly2- and Ser2-substituted analogues of glucose-dependent insulinotrophic polypeptide

“…The reason why (Ser 2 )GIP should elicit such a marked insulin response is unclear, especially when weighed against its cAMP-stimulating ability, but one explanation might be that its additional hydrophilicity may be linked to activation of phospholipase A 2 or other signal transduction pathways (Lu et al 1993, Ehses et al 2001. Unlike their DPP IV-resistant GLP-1 counterparts (Deacon et al 1998, Ritzel et al 1998, Burcelin et al 1999, both GIP analogues displayed increased biological activity in vitro. Since no significant degradation of GIP occurred during these incubations, these data suggest that the combination of increased DPP IV resistance and improved biological potency at the cellular level should provide analogues with greatly enhanced activity in vivo.…”

“…Substituting the alkyl side-chain methyl-group (CH 3 -) of the native Ala 2 with a hydrogen group (H-), as is the case for (Gly 2 )GIP, makes this analogue less susceptible to degradation by DPP IV. Similarly, substitution of the side-chain CH 3 -group with a hydroxy (OH-) group, as in (Ser 2 )GIP, renders this analogue more hydrophilic and a poor substrate for DPP IV (Ritzel et al 1998). The resulting plasma stability, therefore, prevents removal of the NH 2 -terminal dipeptide, preventing the formation of the GIP(3-42) antagonist (Gault et al 2002).…”

International Union of Pharmacology. XXXV. The Glucagon Receptor Family

“…Since the half-life of the naturally occurring peptide in plasma is too short for optimal clinical use, long-acting degradation resistant GLP-1 analogs have now been developed (Deacon et al, 1998;Ritzel et al, 1998;Burcelin et al, 1999a;Siegel et al, 1999;Doyle et al, 2001;Xiao et al, 2001), and these analogs, together with the lizard peptide exendin-4, are being assessed in studies of patients with type 2 diabetes Egan et al, 2002;Juhl et al, 2002).…”