Exploring the GLP-1-GLP-1R axis in porcine pancreas and gastrointestinal tract in vivo by ex vivo autoradiography

Introduction Glucagon-like peptide-1 (GLP-1) increases insulin secretion from pancreatic beta-cells and GLP-1 receptor (GLP-1R) agonists are widely used as treatment for type 2 diabetes mellitus. Studying occupancy of the GLP-1R in various tissues is challenging due to lack of quantitative, repeatable assessments of GLP-1R density. The present study aimed to describe the quantitative distribution of GLP-1Rs and occupancy by endogenous GLP-1 during oral glucose tolerance test (OGTT) in pigs, a species that is used in biomedical research to model humans.Research design and methods GLP-1R distribution and occupancy were measured in pancreas and gastrointestinal tract by ex vivo autoradiography using the GLP-1R-specific radioligand Lu-177-exendin-4 in two groups of pigs, control or bottle-fed an oral glucose load. Positron emission tomography (PET) data from pigs injected with Ga-68-exendin-4 in a previous study were used to retrieve data on biodistribution of GLP-1R in the gastrointestinal tract.Results High homogenous uptake of Lu-177-exendin-4 was found in pancreas, and even higher uptake in areas of duodenum. Low uptake of Lu-177-exendin-4 was found in stomach, jejunum, ileum and colon. During OGTT, there was no increase in plasma GLP-1 concentrations and occupancy of GLP-1Rs was low. The ex vivo autoradiography results were highly consistent with to the biodistribution of Ga-68-exendin-4 in pigs scanned by PET.Conclusion We identified areas with similarities as well as important differences regarding GLP-1R distribution and occupancy in pigs compared with humans. First, there was strong ligand binding in the exocrine pancreas in islets. Second, GLP-1 secretion during OGTT is minimal and GLP-1 might not be an important incretin in pigs under physiological conditions. These findings offer new insights on the relevance of porcine diabetes models

Feasibility of 68Ga-labeled Siglec-9 peptide for the imaging of acute lung inflammation: a pilot study in a porcine model of acute respiratory distress syndrome

There is an unmet need for noninvasive, specific and quantitative imaging of inherent inflammatory activity. Vascular adhesion protein-1 (VAP-1) translocates to the luminal surface of endothelial cells upon inflammatory challenge. We hypothesized that in a porcine model of acute respiratory distress syndrome (ARDS), positron emission tomography (PET) with sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) based imaging agent targeting VAP-1 would allow quantification of regional pulmonary inflammation. ARDS was induced by lung lavages and injurious mechanical ventilation. Hemodynamics, respiratory system compliance (Crs) and blood gases were monitored. Dynamic examination using [15O]water PET-CT (10 min) was followed by dynamic (90 min) and whole-body examination using VAP-1 targeting 68Ga-labeled 1,4,7,10-tetraaza cyclododecane-1,4,7-tris-acetic acid-10-ethylene glycol-conjugated Siglec-9 motif peptide ([68Ga]Ga-DOTA-Siglec-9). The animals received an anti-VAP-1 antibody for post-mortem immunohistochemistry assay of VAP-1 receptors. Tissue samples were collected post-mortem for the radioactivity uptake, histology and immunohistochemistry assessment. Marked reduction of oxygenation and Crs, and higher degree of inflammation were observed in ARDS animals. [68Ga]Ga-DOTA-Siglec-9 PET showed significant uptake in lungs, kidneys and urinary bladder. Normalization of the net uptake rate (Ki) for the tissue perfusion resulted in 4-fold higher uptake rate of [68Ga]Ga-DOTA-Siglec-9 in the ARDS lungs. Immunohistochemistry showed positive VAP-1 signal in the injured lungs. Detection of pulmonary inflammation associated with a porcine model of ARDS was possible with [68Ga]Ga-DOTA-Siglec-9 PET when using kinetic modeling and normalization for tissue perfusion.</p

68Ga-Based Radiopharmaceuticals: Production and Application Relationship

The contribution of 68Ga to the promotion and expansion of clinical research and routine positron emission tomography (PET) for earlier better diagnostics and individualized medicine is considerable. The potential applications of 68Ga-comprising imaging agents include targeted, pre-targeted and non-targeted imaging. This review discusses the key aspects of the production of 68Ga and 68Ga-based radiopharmaceuticals in the light of the impact of regulatory requirements and endpoint pre-clinical and clinical applications

Synthesis, Characterisation and Application of 68Ga-labelled Macromolecules

The positron emitting radionuclide 68Ga (T1/2 = 68 min) might become of practical interest for clinical positron emission tomography (PET). The metallic cation, 68Ga(III), is suitable for complexation with chelators, either naked or conjugated with biological macromolecules. Such labelling procedures require pure and concentrated preparations of 68Ga(III), which cannot be sufficiently fulfilled by the presently available 68Ge/68Ga generator eluate. This thesis presents methods to increase the concentration and purity of 68Ga obtained from a commercial 68Ge/68Ga generator. The use of the preconcentrated and purified 68Ga eluate along with microwave heating allowed quantitative 68Ga-labelling of peptide conjugates within 15 min. The specific radioactivity of the radiolabelled peptides was improved considerably compared to previously applied techniques using non-treated generator eluate and conventional heating. A commercial 68Ge/68Ga generator in combination with the method for preconcentration/purification and microwave heated labelling might result in an automated device for 68Ga-based radiopharmaceutical kit production with quantitative incorporation of 68Ga(III). Macromolecules were labelled with 68Ga(III) either directly or via a chelator. The bifunctional chelator, DOTA, was conjugated in solution to peptides, an antibody and oligonucleotides. The peptides had varied pI values, constitution, and length ranging from 8 to 53 amino acid residues. The oligonucleotides were of various sequences and length with modifications in backbone, sugar moiety and both 3' and 5' ends with a molecular weight up to 9.8 kDa. The bioconjugates were labeled with 68Ga(III), and the resulting tracers were characterised chemically and biologically. The identity of the 68Ga-labelled bioconjugates was verified. The tracers were found to be stable and their biological activity maintained. Specific radioactivity was shown to be an important parameter influencing the feasibility of accurate imaging data quantification. Furthermore, 68Ga-labelled peptide imaging was shown to be a useful tool to study peptide adsorption to microstructures in a chemical analysis device

Synthesis, Characterization and Application of 68Ga-labelled Peptides and Oligonucleotides

The positron emitting 68Ga radionuclide (T1/2 = 68 min) has the potential of practical interest for clinical PET. The metallic cation, 68Ga3+, is suitable for complexation reactions with chelators either naked or conjugated with macromolecules such as peptides and oligonucleotides. Such labeling procedures require pure and concentrated radiometal preparations, which cannot be sufficiently fulfilled by the presently available 68Ge/68Ga generator eluate. This thesis presents a method to increase the concentration and purity of 68Ga obtained from a commercial 68Ge/68Ga generator. DOTATOC (DOTA = 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid, TOC = D-Phe1-Tyr3–Octreotide) was used as a test molecule for comparing the labeling properties of different 68Ga preparations. In addition, DOTA-RDG (RGD = Cys2-6; c[CH2CO-Lys(DOTA)-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-CCX6-NH2) and NODAGATATE (NODAGA = 1,4,7-triazacyclononane-1,4,7-triacetic, TATE = Tyr3 - Octreotate) were used to prove the concept. The use of the concentrated and purified 68Ga eluate along with microwave activation allowed quantitative 68Ga-labelling of peptide conjugates of ≤1 nanomolar quantities within 10 min. The specific radioactivity of the radiolabelled peptides was improved by a factor of &gt;100 compared to previously applied techniques using non-treated generator eluate and conventional heating. A commercial 68Ge/68Ga generator in combination with this method for purification, concentration and microwave activated labeling resulted in a kit technology for 68Ga-tracer production.Four 17-mer oligonucleotides modified and functionalised with an hexylamine group in the 3'- or 5'- position were conjugated with DOTA and labelled with 68Ga using microwave activation. Chemical modification of the oligonucleotide backbone or sugar moiety did not influence the labelling nor the hybridisation ability of the oligonucleotides. However, the radioactivity organ biodistribution in rats differed dependent on the oligonucleotide structure. This indicated that metabolism and non-specific binding were affected by the backbone and sugar moiety structure

Prospective of 68

During the last decade, the utilization of 68Ga for the development of imaging agents has increased considerably with the leading position in the oncology. The imaging of infection and inflammation is lagging despite strong unmet medical needs. This review presents the potential routes for the development of 68Ga-based agents for the imaging and quantification of infection and inflammation in various diseases and connection of the diagnosis to the treatment for the individualized patient management