Search

Glucagon-like peptide-1 (GLP-1) has the ability to lower the blood glucose level, and its regulatory functions make it an attractive therapeutic agent for the treatment of type 2 diabetes. However, its rapid degradation by enzymes like dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11 severely compromises its effective clinical use. Whereas specific DPP-IV inhibitors have been developed, NEP 24.11 targets multiple sites in the GLP-1 sequence, which makes it difficult to block. To address this drawback, we have designed and synthesized conformationally constrained GLP-1 analogues by introducing multiple lactam bridges that stabilized both alpha-helices in the N- and C-terminal regions simultaneously. In addition to improving the receptor activation capability (up to 5-fold) by fixing the alpha-helical conformations required for optimal receptor interaction, the introduced lactam bridges provided outstanding shielding over NEP 24.11 (half-life of96 h). These highly constrained peptides are the first examples of NEP 24.11-resistant GLP-1 analogues.

To elucidate the receptor-bound conformation of glucagon-like peptide-1 (GLP-1), a series of conformationally constrained GLP-1 analogues were synthesized by introducing lactam bridges between Lys(i) and Glu(i)(+4) to form alpha-helices at various positions. The activity and affinity of these analogues to GLP-1 receptors suggested that the receptor-bound conformation comprises two alpha-helical segments between residues 11-21 and 23-34. It is notable that the N-terminal alpha-helix is extended to Thr(11), and that Gly(22) plays a pivotal role in arranging the two alpha-helices. Based on these findings, a highly potent bicyclic GLP-1 analogue was synthesized which is the most conformationally constrained GLP-1 analogue reported to date.

Derived from endocrine pancreatic beta cells, insulinomas express glucagon-like peptide-1 (GLP-1) receptor with high density and incidence. In this study, we labeled a novel GLP-1 analogue, EM3106B, with (18)F and performed PET imaging to visualize insulinoma tumors in an animal model. A GLP-1 analogue that contains multiple lactam bridges, EM3106B, was labeled with (18)F through a maleimide-based prosthetic group, N-2-(4-(18)F-fluorobenzamido)ethylmaleimide ((18)F-FBEM). The newly developed radiotracer was characterized by cell based receptor-binding assay, cell uptake and efflux assay. The stability in serum was evaluated by radio-HPLC analysis. In vivo PET imaging was performed in nude mice bearing subcutaneous INS-1 insulinoma tumors and MDA-MB-435 tumors of melanoma origin. Ex vivo biodistribution study was performed to confirm the PET imaging data. EM3106B showed high binding affinity (IC(50) = 1.38 nM) and high cell uptake (5.25 ± 0.61% after 120 min incubation). (18)F-FBEM conjugation of EM3106B resulted in high labeling yield (24.9 ± 2.4%) and high specific activity (75 GBq/μmol at the end of bombardment). EM3106B specifically bound and was internalized by GLP-1R positive INS-1 cells. After intravenous injection of 3.7 MBq (100 μCi) of (18)F-FBEM-EM3106B, the INS-1 tumors were clearly visible with high contrast in relation to the contralateral background on PET images, and tumor uptake of (18)F-FBEM-EM3106B was determined to be 28.5 ± 4.7 and 25.4 ± 4.1% ID/g at 60 and 120 min, respectively. (18)F-FBEM-EM3106B showed low uptake in MB-MDA-435 tumors with low level of GLP-1R expression. Direct tissue sampling biodistribution experiment confirmed high tracer uptake in INS-1 tumors and receptor specificity in both INS-1 tumor and pancreas. In conclusion, (18)F-FBEM-EM3106B exhibited GLP-1R-receptor-specific targeting properties in insulinomas. The favorable characteristics of (18)F-FBEM-EM3106B, such as high specific activity and high tumor uptake, and high tumor to nontarget uptake, demonstrate that it is a promising tracer for clinical insulinoma imaging.