N-terminal His(7)-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity

Abstract

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His 7 -modified analogue of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC 50 values 32·9 and 6·7 nM, respectively) compared with native GLP-1 (IC 50 0·37 nM). Similarly, both analogues stimulated cAMP production with EC 50 values of 16·3 and 27 nM respectively compared with GLP-1 (EC 50 4·7 nM). However, N-acetyl-GLP-1 and Npyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5·6 mM glucose (P,0·05 to P,0·001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes.

Function of a long‐term, GLP‐1‐treated, insulin‐secreting cell line is improved by preventing DPP IV‐mediated degradation of GLP‐1

“…The present investigation has several interesting observations regarding prolonged GLP-1 treatment on an insulin-secreting cell line, particularly when degradation by DPP IV (present in the media) is inhibited/prevented (using either a specific inhibitor, DPA) or a previously characterized DPP IV resistant analogue of GLP-1 with preserved biological activities, N-acetyl-GLP-1 [14,21,28]. First, we have confirmed the previously observed concentration-dependent effects of GLP-1 and N-acetyl-GLP-1 on insulin secretion from an insulin-secreting cell line [14,29]. N-terminal acetylation of GLP-1 did not significantly affect insulinotropic action, contrasting it with the enhanced action observed following acetylation of the sister incretin hormone, GIP [30].…”

“…Synthesized peptides were cleaved from the resin and purified by reversed-phase HPLC and their identity confirmed by electrospray ionization mass spectrometry [12][13][14][15][16].…”

Controlled release of biologics for the treatment of type 2 diabetes

“…These N-terminal His7 modifications include: desamino, N-imidazole, N-α-methyl, N-methyl, D-His7, N-glucitol, N-acetyl, and N-pyroglutamyl[106–109]. All of the analogs listed have partial or complete resistance to DPP-IV with receptor affinities unchanged or reduced, at most, by 15-fold compared to the native peptide[110].…”

Nutrient regulation of pancreatic β-cell function in diabetes: problems and potential solutions

“…Three different types of structural modifications have been investigated to safeguard GLP-1 and GIP against DPPIV degradation. The first involves, extension of the N-terminal His 7 and Tyr 1 positions of GLP-1 and GIP respectively by various chemical modifications [42,43]. The second involves amino acid substitution at Ala 8 [44] and Glu 9 [45,46] of GLP-1, and at Ala 2 [47] and Glu 3 [48,49] of GIP respectively.…”

“…On the whole, the bioactivity of GIP is not as sensitive as GLP-1 to compromise when structural changes are introduced to impart DPPIV stability. Examples of DPPIV-resistant analogues of GLP-1 and GIP which possess enhanced biological activity are (Val 8 )GLP-1 and N-acetyl GIP respectively [42,43]. Administration of (Val 8 )GLP-1 for 3 weeks in ob/ob diabetic mice led to improved glucose tolerance, reduced the glycaemic excursion after feeding, increased the plasma insulin response to glucose and feeding, and improved insulin sensitivity [33].…”