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Background: The beneficial role of glucose-dependent insulinotropic polypeptide receptor (GIPR) in weight control and maintaining glucose levels has led to the development of several multi-agonistic peptide drug candidates, targeting GIPR and glucagon like peptide 1 receptor (GLP1R) and/or the glucagon receptor (GCGR). The in vivo quantification of target occupancy by these drugs would accelerate the development of new drug candidates. The aim of this study was to evaluate a novel peptide (GIP1234), based on previously reported ligand DOTA-GIP-C803, modified with a fatty acid moiety to prolong its blood circulation. It would allow higher target tissue exposure and consequently improved peptide uptake as well as in vivo PET imaging and quantification of GIPR occupancy by novel drugs of interest., Method: A 40 amino acid residue peptide (GIP1234) was synthesized based on DOTA-GIP-C803, in turn based on the sequences of endogenous GIP and Exendin-4 with specific amino acid modifications to obtain GIPR selectivity. A palmitoyl fatty acid chain was furthermore added at Lys14 via a glutamic acid linker to prolong its blood circulation time by the interaction with albumin. GIP1234 was conjugated with a DOTA chelator at the C-terminal cysteine residue to achieve 68 Ga radiolabeling. The resulting PET probe, [ 68 Ga]Ga-DOTA-GIP1234 was evaluated for receptor binding specificity and selectivity using HEK293 cells transfected with human GIPR, GLP1R, or GCGR. Blocking experiments with tirzepatide (2 μM) were conducted using huGIPR HEK293 cells to investigate binding specificity. Ex vivo and in vivo organ distribution of [ 68 Ga]Ga-DOTA-GIP1234 was studied in rats and a pig in comparison to [ 68 Ga]Ga-DOTA-C803-GIP. Binding of [ 68 Ga]Ga-DOTA-GIP1234 to albumin was assessed in situ using polyacrylamide gel electrophoresis (PAGE). The stability was tested in formulation buffer and rat blood plasma., Results: [ 68 Ga]Ga-DOTA-GIP1234 was synthesized with non-decay corrected radiochemical yield of 88 ± 3.7 % and radiochemical purity of 97.8 ± 0.8 %. The molar activity for the radiotracer was 8.1 ± 1.1 MBq/nmol. [ 68 Ga]Ga-DOTA-GIP1234 was stable and maintained affinity to huGIPR HEK293 cells (dissociation constant (K d ) = 40 ± 12.5 nM). The binding of [ 68 Ga]Ga-DOTA-GIP1234 to huGCGR and huGLP1R cells was insignificant. Pre-incubation of huGIPR HEK293 cell sections with tirzepatide resulted in the decrease of [ 68 Ga]Ga-DOTA-GIP1234 binding by close to 90 %. [ 68 Ga]Ga-DOTA-GIP1234 displayed slow blood clearance in pigs with SUV = 3.5 after 60 min. Blood retention of the tracer in rat was 2-fold higher than that of [ 68 Ga]Ga-DOTA-C803-GIP. [ 68 Ga]Ga-DOTA-GIP1234 also demonstrated strong liver uptake in both pig and rat combined with decreased renal excretion. The concentration dependent binding of [ 68 Ga]Ga-DOTA-GIP1234 to albumin was confirmed in situ by PAGE., Conclusion: [ 68 Ga]Ga-DOTA-GIP1234 demonstrated nanomolar affinity and selectivity for huGIPR in vitro. Addition of a fatty acid moiety prolonged blood circulation time and tissue exposure in both rat and pig in vivo. However, the liver uptake was also increased which may make PET imaging of abdominal tissues such as pancreas challenging. The investigation of the influence of fatty acid moiety on the biological performance of the peptide ligand paved the way for further rational design of GIPR ligand analogues with improved characteristics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Martin Bossart is an employee of Sanofi Germany. Michael Wagner is an employee of Dewpoint Therapeutics. Olof Eriksson is an employee of Antaros Medical AB and a shareholder of Antaros Tracer AB. No other potential conflicts of interest relevant to this article exist., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Imaging and radiotherapy targeting the glucose-dependent insulinotropic polypeptide receptor (GIPR) could potentially benefit the management of neuroendocrine neoplasms (NENs), complementing clinically established radiopharmaceuticals. The aim of this study was to evaluate a GIPR-targeting positron emission tomography (PET) radioligand with receptor-specific binding, fast blood clearance, and low liver background uptake. The peptide DOTA-bioconjugate, C803-GIP, was developed based on the sequence of the endogenous GIP(1-30) and synthetic exendin-4 peptides with selective amino acid mutations to combine their specificity for the GIPR and in vivo stability, respectively. The 68Ga-labeled bioconjugate was evaluated in vitro in terms of binding affinity, specificity, and internalization in HEK293 cells transfected with the human GIPR, GLP1, or GCG receptors and in sections of human insulinoma and NENs. In vivo binding specificity, biodistribution, and tissue background were investigated in mice bearing huGIPR-HEK293 xenografts and in a pig. Ex vivo organ distribution, pharmacokinetics, and dosimetry were studied in normal rats. [68Ga]Ga-C803-GIP was stable and demonstrated a high affinity to the huGIPR-HEK293 cells. Binding specificity was demonstrated in vitro in frozen sections of NENs and huGIPR-HEK293 cells. No specific uptake was observed in the negative controls of huGLP1R and huGCGR cells. A novel rationally designed PET radioligand, [68Ga]Ga-C803-GIP, demonstrated promising binding characteristics and specificity towards the GIPR. [ABSTRACT FROM AUTHOR]