Value of the radiolabelled GLP-1 receptor antagonist exendin(9-39) for targeting of GLP-1 receptor-expressing pancreatic tissues in mice and humans

Purpose: Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. Moreover, it was recently reported that antagonist tracers were superior to agonist tracers for somatostatin and gastrin-releasing peptide receptor targeting of tumours. The present preclinical study determines therefore the value of an established GLP-1 receptor antagonist for the in vitro visualization of GLP-1 receptor-expressing tissues in mice and humans. Methods: Receptor autoradiography studies with 125I-GLP-1(7-36)amide agonist or 125I-Bolton-Hunter-exendin(9-39) antagonist radioligands were performed in mice pancreas and insulinomas as well as in human insulinomas; competition experiments were performed in the presence of increasing concentration of GLP-1(7-36)amide or exendin(9-39). Results: The antagonist 125I-Bolton-Hunter-exendin(9-39) labels mouse pancreatic β-cells and mouse insulinomas, but it does not label human pancreatic β-cells and insulinomas. High affinity displacement (IC50 approximately 2nM) is observed in mouse β-cells and insulinomas with either the exendin(9-39) antagonist or GLP-1(7-36)amide agonist. For comparison, the agonist 125I-GLP-1(7-36)amide intensively labels mouse pancreatic β-cells, mouse insulinoma and human insulinomas; high affinity displacement is observed for the GLP-1(7-36)amide in all tissues; however, a 5 and 20 times lower affinity is found for exendin(9-39) in the mouse and human tissues, respectively. Conclusion: This study reports a species-dependent behaviour of the GLP-1 receptor antagonist exendin(9-39) that can optimally target GLP-1 receptors in mice but not in human tissue. Due to its overly low binding affinity, this antagonist is an inadequate targeting agent for human GLP-1 receptor-expressing tissues, as opposed to the GLP-1 receptor agonist, GLP-1(7-36)amid

Co-expressed peptide receptors in breast cancer as a molecular basis for in vivo multireceptor tumour targeting

Breast cancers can express different types of peptide receptors such as somatostatin, vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP) and NPY(Y1) receptors. The aim of this in vitro study was to evaluate which is the most appropriate peptide receptor or peptide receptor combination for in vivo diagnostic and therapeutic targeting of breast cancers. Seventy-seven primary breast cancers and 15 breast cancer lymph node metastases were investigated in vitro for their expression of somatostatin, VPAC1, GRP and NPY(Y1) receptors using in vitro receptor autoradiography on successive tissue sections with 125I-[Tyr3]-octreotide, 125I-VIP, 125I-[Tyr4]-bombesin and 125I-[Leu31,Pro34]-PYY respectively. This study identified two groups of tumours: a group of 68 tumours (88%) with at least one receptor expressed at high density (>2,000dpm/mg tissue) that may provide a strong predictive value for successful in vivo targeting, and a group of nine tumours (12%) with no receptors or only a low density of them (<2,000dpm/mg tissue). In the group with high receptor density, 50 of the 68 tumours (74%) expressed GRP receptors, 45 (66%) expressed NPY(Y1) receptors, 25 (37%) expressed VPAC1 receptors and 14 (21%) expressed somatostatin receptors. Mean density was 9,819±530dpm/mg tissue for GRP receptors, 9,135±579dpm/mg for NPY(Y1) receptors, 4,337±528dpm/mg for somatostatin receptors and 3,437±306dpm/mg for VPAC1 receptors. It is of note that tumours expressing NPY(Y1) or GRP receptors, or both, were found in 63/68 (93%) cases. Lymph node metastases showed a similar receptor profile to the corresponding primary tumour. This in vitro study strongly suggests that the combination of radiolabelled GRP and Y1 analogues should allow targeting of breast carcinomas and their lymph node metastases for in vivo peptide receptor scintigraphy and radiotherap

Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

Purpose: Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the 125iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer 125I-GLP-1(7-36)amide. Methods: Receptor autoradiography studies with 125I-GLP-1(7-36)amide agonist or 125I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. Results: The antagonist 125I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer 125I-GLP-1(7-36)amide. For comparison, 125I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. Conclusion: The GLP-1 receptor antagonist exendin(9-39) labelled with 125I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients

Selective in vitro targeting of GRP and NMB receptors in human tumours with the new bombesin tracer 177Lu-AMBA

Purpose: To investigate the in vitro binding properties of a novel radiolabelled bombesin analogue, 177Lu-AMBA, in human neoplastic and non-neoplastic tissues selected for their expression of the bombesin receptor subtypes GRP-R, NMB-R and BRS-3. Methods: In vitro receptor autoradiography was performed in cancers expressing the various bombesin receptor subtypes. The novel radioligand 177Lu-AMBA was used and compared with established bombesin radioligands such as 125I-Tyr4-bombesin and 125I-[D-Tyr6,β-Ala11,Phe13,Nle14]-bombesin(6-14). In vitro incidence of detection of each of the three bombesin receptor subtypes was evaluated in each tumour. Results: 177Lu-AMBA identified all GRP-R-expressing tumours, such as prostatic, mammary and renal cell carcinomas as well as gastrointestinal stromal tumours. 177Lu-AMBA also identified all NMB-expressing tumours, but did not detect BRS-3-expressing tumours or BRS-3-expressing pancreatic islets. GRP-R-expressing peritumoural vessels were heavily labelled with 177Lu-AMBA. In contrast to the strongly GRP-R-positive mouse pancreas, the human pancreas was not labelled with 177Lu-AMBA unless chronic pancreatitis was diagnosed. In general, the sensitivity was slightly better with 177Lu-AMBA than with the conventional bombesin radioligands. Conclusion: The present in vitro study suggests that 177Lu-AMBA may be a very useful in vivo targeting agent for GRP-R-expressing tumours, NMB-R-expressing tumours and GRP-R-expressing neoangiogenic vessel

High expression of peptide receptors as a novel target in gastrointestinal stromal tumours

Recent significant advances in understanding the biology of gastrointestinal stromal tumours (GIST) have led to the introduction of a new targeted therapy (imatinib mesylate, Glivec). Hopes of a new era of a specific cancer therapy, however, have been tempered by the recognition that a significant proportion of patients who initially respond to the drug eventually become resistant to it. Given the successful development of peptide receptor scintigraphy and radiotherapy for neuroendocrine tumours, we postulated that a similar approach could offer a valid alternative in the diagnosis and therapy of GIST. Using in vitro receptor autoradiography to measure peptide receptors, we found that 16/19 GIST expressed bombesin subtype 2 receptors, 16/19 expressed vasoactive intestinal peptide subtype 2 receptors (VPAC2) and 12/19 expressed cholecystokinin subtype 2 receptors, in most cases in extremely high densities. All GIST metastases were shown to express two or more of these peptide receptors in very high density. Receptors were also expressed in non-responders to Glivec or after chemo-embolisation. Conversely, somatostatin subtype 2, cholecystokinin subtype 1, bombesin subtype 1 and 3, and neuropeptide Y subtype Y1 and Y2 receptors were not or only rarely expressed. These data represent a strong molecular basis for the use of radiolabelled bombesin, vasoactive intestinal peptide and/or cholecystokinin analogues as targeting agents to localise GIST tumours in patients by in vivo scintigraphy and/or to perform targeted radiotherapy to destroy GIST primaries, metastases and recurrences, including those resistant to Glive

Switch from antagonist to agonist after addition of a DOTA chelator to a somatostatin analog

Purpose: Peptide receptor targeting has become an increasingly attractive method to target tumors diagnostically and radiotherapeutically. Peptides linked to a variety of chelators have been developed for this purpose. They have, however, rarely been tested for their agonistic or antagonistic properties. We report here on a somatostatin antagonist that switched to an agonist upon coupling to a DOTA chelator. Methods: Two novel somatostatin analogs, 406-040-15 and its DOTA-coupled counterpart 406-051-20, with and without cold Indium labeling, were tested for their somatostatin receptor subtypes 1-5 (sst1-sst5) binding affinity using receptor autoradiography. Moreover, they were tested functionally for their ability to affect sst2 and sst3 internalization in vitro in HEK293 cells stably expressing the human sst2 or sst3 receptor, using an immunofluorescence microscopy-based internalization assay. Results: All three compounds were characterized as pan-somatostatin analogs having a high affinity for all five sst. In the sst2 internalization assay, all three compounds showed an identical behavior, namely, a weak agonistic effect complemented by a weak antagonistic effect, compatible with the behavior of a partial agonist. Conversely, in the sst3 internalization assay, 406-040-15 was a full antagonist whereas its DOTA-coupled counterpart, 406-051-20, with and without Indium labeling, switched to a full agonist. Conclusion: Adding the DOTA chelator to the somatostatin analog 406-040-15 triggers a switch at sst3 receptor from an antagonist to an agonist. This indicates that potential radioligands for tumor targeting should always be tested functionally before further development, in particular if a chelator is adde

DOTA-PESIN, a DOTA-conjugated bombesin derivative designed for the imaging and targeted radionuclide treatment of bombesin receptor-positive tumours

Purpose: We aimed at designing and developing a novel bombesin analogue, DOTA-PEG4-BN(7-14) (DOTA-PESIN), with the goal of labelling it with 67/68Ga and 177Lu for diagnosis and radionuclide therapy of prostate and other human cancers overexpressing bombesin receptors. Methods: The 8-amino acid peptide bombesin (7-14) was coupled to the macrocyclic chelator DOTA via the spacer 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG4). The conjugate was complexed with Ga(III) and Lu(III) salts. The GRP receptor affinity and the bombesin receptor subtype profile were determined in human tumour specimens expressing the three bombesin receptor subtypes. Internalisation and efflux studies were performed with the human GRP receptor cell line PC-3. Xenografted nude mice were used for biodistribution. Results: [GaIII/LuIII]-DOTA-PESIN showed good affinity to GRP and neuromedin B receptors but no affinity to BB3. [67Ga/177Lu]-DOTA-PESIN internalised rapidly into PC-3 cells whereas the efflux from PC-3 cells was relatively slow. In vivo experiments showed a high and specific tumour uptake and good retention of [67Ga/177Lu]-DOTA-PESIN. [67Ga/177Lu]-DOTA-PESIN highly accumulated in GRP receptor-expressing mouse pancreas. The uptake specificity was demonstrated by blocking tumour uptake and pancreas uptake. Fast clearance was found from blood and all non-target organs except the kidneys. High tumour-to-normal tissue ratios were achieved, which increased with time. PET imaging with [68Ga]-DOTA-PESIN was successful in visualising the tumour at 1h post injection. Planar scintigraphic imaging showed that the 177Lu-labelled peptide remained in the tumour even 3days post injection. Conclusion: The newly designed ligands have high potential with regard to PET and SPECT imaging with 68/67Ga and targeted radionuclide therapy with 177L

Novel dimeric DOTA-coupled peptidic Y1-receptor antagonists for targeting of neuropeptide Y receptor-expressing cancers

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