A comparison of three 67/68Ga-labelled exendin-4 derivatives for β-cell imaging on the GLP-1 receptor: the influence of the conjugation site of NODAGA as chelator

Abstract

BackgroundVarious diseases derive from pathologically altered β-cells. Their function can be increased, leading to hyperinsulinism, or decreased, resulting in diabetes. Non-invasive imaging of the β-cell-specific glucagon-like peptide receptor-1 (GLP-1R) would allow the assessment of both β-cell mass and derived tumours, potentially improving the diagnosis of various conditions. We tested three new 67/68Ga-labelled derivatives of exendin-4, an agonist of GLP-1R, in vitro and in vivo. We determined the influence of the chelator NODAGA conjugated to resident lysines either at positions 12 and 27 or the C-terminally attached lysine at position 40 on the binding and kinetics of the peptide.MethodsBinding and internalisation of 67Ga-labelled Ex4NOD12, Ex4NOD27 and Ex4NOD40 were tested on Chinese hamster lung (CHL) cells stably transfected to express the GLP-1 receptor (GLP-1R). In vivo biodistribution of 68Ga-labelled peptides was investigated in CD1 nu/nu mice with subcutaneous CHL-GLP-1R positive tumours; the specificity of the binding to GLP-1R was determined by pre-injecting excess peptide.ResultsAll peptides showed good in vitro binding affinities to GLP-1R in the range of 29 to 54 nM. 67/68Ga-Ex4NOD40 and 67/68Ga-Ex4NOD12 show excellent internalisation (>30%) and high specific uptake in GLP-1R positive tissue, but high activity was also found in the kidneys.ConclusionsWe show that of the three peptides, Ga-Ex4NOD40 and Ga-Ex4NOD12 demonstrate the most favourable in vitro properties and in vivo binding to GLP-1R positive tissue. Therefore, we conclude that the lysines at positions 12 and 40 might preferentially be utilised for modifying exendin-4.

Glucagon-Like Peptide-1 Receptor PET/CT with 68Ga-NOTA-Exendin-4 for Detecting Localized Insulinoma: A Prospective Cohort Study

“…Because imaging modalities need to be highly sensitive on account of the small size of insulinoma, PET may offer an advantage over SPECT, with higher spatial resolution, sensitivity, and imaging contrast. So far, exendin-based PET tracers labeled with 68 Ga, 18 F, and 64 Cu have been successfully prepared (14–25). For 64 Cu, its long half-life (12.7 h) and β − emission can lead to an increased radiation burden for the patient.…”

Oxidation of methionine — is it limiting the diagnostic properties of 99mTc-labeled Exendin-4, a Glucagon-Like Peptide-1 receptor agonist?

“…However, so far, there is no published data on the GLP-1 receptor affinity of the oxidized form of Exendin-4. The introduction of chelator in various lysine positions gave compounds with comparable biological activity with the in vitro binding affinities to GLP-1R in the range of 29 to 54 nM; however, only the lysines at positions 12 and 40 were suggested as preferential modification site [18]. In another study, both lysine residues appeared to be important for the affinity to the GLP-1 receptor [25].…”

“…Notably, oxidation of methionine in gastrin analogs dramatically reduces the affinity for CCK-2 receptors [17]. Various approaches were applied to prevent the oxidation of methionine during radiolabeling of peptides, such as addition of antioxidants, including selenomethionine and L-methionine as well as gentisic acid and ascorbic acid [17,18]. The other option is to replace the methionine with another amino acid, which would not be prone to oxidation.…”

GLP-1R is downregulated in beta cells of NOD mice and T1D patients

“…The GLP-1 peptide was purchased from Alta Biosciences (54) For assessment of biological activity 400pmol of unlabelled exendin was injected iv, while up to 4nmol was injected i.v to assess binding of labelled probe as labelled probes have been demonstrated to have a significantly decreased affinity for the receptor (55).…”