Novel mono-lipidated dimeric glucagon-like peptide-1 receptor agonist with improved long-acting and hypoglycemic activity

Abstract

Dimerization is a useful tool to boost ligand-receptor interaction. Both lipidation and dimerization effectively prolong the short half-life (t _1/2) of peptides by facilitating binding with serum albumin and increasing hydrodynamic size. Here, we described two novel GLP-1 conjugates with high glucagon-like peptide-1 (GLP-1) receptor activation potencies, dimerized GLP-1 (Di-GLP-1) and lipidated Di-GLP-1 (Lip-Di-GLP-1). Di-GLP-1 and Lip-Di-GLP-1 were prepared through cysteine-maleimide specific coupling reactions using Gly₈-Cys₃₁-GLP-1, bis-maleimide amine, and activated palmitic acid. The receptor activation potencies of Di-GLP-1 and Lip-Di-GLP-1 were 13.6-fold and 9.5-fold higher than GLP-1, respectively. The in vivo hypoglycemic and insulinotropic activities of Di-GLP-1 and Lip-Di-GLP-1 were also better than GLP-1 in db/db mice. Furthermore, Lip-Di-GLP-1 was found to have greater circulating t _1/2 than synthesized liraglutide by 1.8-fold. Accordingly, the improved pharmacokinetic profiles of Lip-Di-GLP-1 resulted in protracted antidiabetic effects as confirmed by hypoglycemic duration test. Moreover, Lip-Di-GLP-1 administered in mice potently inhibits gastric emptying and reduce food intake. Chronic Lip-Di-GLP-1 treatment in db/db mice resulted in significant improvements in food intake, body weight, pancreatic function and corrected hyperglycemia, which was more effective than synthesized liraglutide. Our research indicated that combined dimerization and lipidation were effectively applied to GLP-1, and the preclinical results suggested the potential usage of Lip-Di-GLP-1 as a long-acting antidiabetic agent.

Fig. 1. Structures (A) and biological activity tests of Di-GLP-1 and Lip-Di-GLP-1. (B) Dose–response relationship of GLP-1, Di-GLP-1 and Lip-Di-GLP-1 on GLP-1 receptor. Experiments and performed in triplicate and repeated three times. (C) Acute glucose-lowering effects of GLP-1, Di-GLP-1 and Lip-Di-GLP-1 in Kunming mice and calculated AUC_glucose values during 0–120 min. Hypoglycemic and insulinotropic activities of GLP-1, Di-GLP-1 and Lip-Di-GLP-1 were measured by IPGTT in db/db mice. (D) Time–blood glucose and (E) time–plasma insulin in db/db mice and the corresponding AUC values of glucose and insulin. Means ± SD, n = 6. (F) PK profiles of Lip-Di-GLP-1 in Kunming mice. Means ± SD, n = 3. ***P < 0.001 vs. saline, ^cP < 0.001 vs. GLP-1, liraglutide (chemical) and liraglutide (biotechnological).

Fig. 2. In vitro toxicity effect of Di-GLP-1 (A) and Lip-Di-GLP-1 (B). Dose–response relationship of Di-GLP-1 and Lip-Di-GLP-1 on INS-1 cells were observed under concentrations of 10, 100 and 1000 nM. Experiments and performed in triplicate and repeated three times. Preliminary in vivo toxicity study of Di-GLP-1 and Lip-Di-GLP-1 was evaluated in db/db mice. ALT and AST (C) were key parameters indicative of liver toxicity, BUN (D) and SCr (E) were key parameters indicative of renal toxicity. These data were determined using automatic biochemical analyzer. Means ± SD, n = 6.

Fig. 3. Dose–response studies for glucose lowering and anorectic effects of Lip-Di-GLP-1 and synthesized liraglutide in db/db mice at doses of 10 nmol kg⁻¹ (A and E), 25 nmol kg⁻¹ (B and F), and 100 nmol kg⁻¹ (C and G). (D) Paracetamol absorption test of Lip-Di-GLP-1 and synthesized liraglutide in Kunming mice. Means ± SD, n = 6.

Fig. 4. Chronic treatment effects of Lip-Di-GLP-1 on HbA1c (A and B), food intake (C), body weight (D), blood glucose (E) and fasted insulin (F) in db/db mice. (G) IPGTT was performed at the end of the study. Means ± SD, n = 6. ***P < 0.001 vs. saline, ^bP < 0.01 vs. GLP-1.

Fig. 5. Treatment effects of Lip-Di-GLP-1 and synthesized liraglutide on pancreatic islets in db/db mice. Representative images of insulin staining in saline (A), liraglutide (B) and Lip-Di-GLP-1 (C) groups. (D) IOD values of insulin in histological analysis. Means ± SD, n = 6. ***P < 0.001 vs. saline.

Fig. 6. Biochemical analysis results of synthesized liraglutide and Lip-Di-GLP-1 on serum biomarkers of db/db mice after 6 weeks treatment. (A) ALT and AST. (B) SCr. (C) BUN. Means ± SD, n = 6.