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Laverman P.,Radboud University Nijmegen | Joosten L.,Radboud University Nijmegen | Eek A.,Radboud University Nijmegen | Roosenburg S.,Radboud University Nijmegen | And 10 more authors.
European Journal of Nuclear Medicine and Molecular Imaging | Year: 2011

Purpose Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in various tumours such as medullary thyroid carcinomas and small-cell lung cancers. Due to this high expression, CCK-2 receptors might be suitable targets for radionuclide imaging and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed and some have been tested in patients. Here we aimed to compare the in vivo tumour targeting properties of 12 111Inlabelled 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (DOTA)-conjugated gastrin/CCK2 receptor-binding peptides. Methods Two CCK8-based peptides and ten gastrin-based peptide analogues were tested. All peptides were conjugated with DOTA and labelled with 111In. Biodistribution studies were performed in mice with subcutaneous CCK2/ gastrin receptor-expressing tumours and with receptornegative tumours contralaterally. Biodistribution was studied by counting dissected tissues at 1 and 4 h after injection. Results Both the CCK analogues displayed relatively low tumour uptake (approximately 2.5%ID/g) as compared to minigastrin analogues. Two linear minigastrin peptides (MG0 and sargastrin) displayed moderate tumour uptake at both 1 and 4 h after injection, but also very high kidney uptake (both higher than 48%ID/g). The linear MG11, lacking the penta-Glu sequence, showed lower tumour uptake and also low kidney uptake. Varying the N-terminal Glu residues in the minigastrin analogues led to improved tumour targeting properties, with PP-F11 displaying the optimal biodistribution. Besides the monomeric linear peptides, a cyclized peptide and a divalent peptide were tested. Conclusion Based on these studies, optimal peptides for peptide receptor radionuclide targeting of CCK2/gastrin receptor-expressing tumours were the linear minigastrin analogue with six D-Glu residues (PP-F11), the divalent analogue MGD5 and the cyclic peptide cyclo-MG1. These peptides combined high tumour uptake with low kidney retention, and may therefore be good candidates for future clinical studies. © The Author(s) 2011. Source

Ocak M.,Innsbruck Medical University | Ocak M.,Istanbul University | Helbok A.,Innsbruck Medical University | Rangger C.,Innsbruck Medical University | And 8 more authors.
European Journal of Nuclear Medicine and Molecular Imaging | Year: 2011

Purpose Stability of radiolabelled cholecystokinin 2 (CCK2) receptor targeting peptides has been a major limitation in the use of such radiopharmaceuticals especially for targeted radionuclide therapy applications, e.g. for treatment of medullary thyroid carcinoma (MTC). The purpose of this study was to compare the in vitro stability of a series of peptides binding to the CCK2 receptor [selected as part of the COST Action on Targeted Radionuclide Therapy (BM0607)] and to identify major cleavage sites. Methods Twelve different 1,4,7,10-tetraazacyclododecane- N,N',N'',N'''-tetraacetic acid (DOTA)-minigastrin/CCK conjugates were provided within an European COST Action (BM0607) by different laboratories and radiolabelled with 177Lu. Their in vitro stabilities were tested in fresh human serum. Radiochemical yields (RCY) and intact radioligands for half-life calculations were determined by radio-HPLC. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis of metabolites was performed to identify cleavage products using conjugates labelled with excess stable natLu, incubated in serum at 37°C. Urine metabolite analysis after injection in normal mice was performed by radio-HPLC analysis.Results Variable stability in human serum was found for the different peptides with calculated half-lives between 4.5± 0.1 h and 198±0.1 h (n=2). In urine of normal mice only metabolised peptide fragments were detected even at short times after injection for all peptides. MALDI-TOF MS revealed a major cleavage site of all minigastrin derivatives between Asp and Phe-NH2 at the C-terminal end. Conclusion Development of CCK2 receptor ligands especially for therapeutic purposes in patients with MTC or small cell lung cancer (SCLC) is still ongoing in different laboratories. This comparative study provided valuable insight into the importance of biological stability especially in the context of other results of this comparative trial within the COST Action BM0607. © Springer-Verlag 2011. Source

Aloj L.,AF Medicina Nucleare | Aurilio M.,AF Medicina Nucleare | Rinaldi V.,AF Medicina Nucleare | D'ambrosio L.,AF Medicina Nucleare | And 15 more authors.
European Journal of Nuclear Medicine and Molecular Imaging | Year: 2011

Purpose Specific overexpression of cholecystokinin 2 (CCK2)/gastrin receptors has been demonstrated in several tumours of neuroendocrine origin. In some of these cancer types, such as medullary thyroid cancer (MTC), a sensitive diagnostic modality is still unavailable and therapeutic options for inoperable lesions are needed. Peptide receptor radionuclide therapy (PRRT) may be a viable therapeutic strategy in the management of these patients. Several CCK2R-targeted radiopharmaceuticals have been described in recent years. As part of the European Union COSTAction BM0607 we studied the in vitro and in vivo characteristics of 12 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R binding peptides. In the presentstudy, we analysed binding and internalization characteristics. Stability, biodistribution and imaging studies have been performed in parallel by other centres involved in the project. Methods Determination of IC50 values was performed using autoradiography, with DOTA-peptides displacing 125I-CCK from receptors on tissue sections from human tumours. Saturation binding and internalization experiments were performed using 111In-labelled peptides. The rat AR42J cell line and the human A431-CCK2R transfected cell line were utilized for in vitro experiments; dissociation constants (Kd) and apparent number of binding sites (Bmax) were determined. Internalization was determined in receptor-expressing cells by incubating with tracer amounts of peptide at 37 and 4°C for different times up to 120 min. Surface-bound peptide was then stripped either by acid wash or subsequent incubation with 1 μM unlabelled peptide at 4°C. Results All peptides showed high receptor affinity with IC50 values ranging from 0.2 to 3.4 nM. Saturation experiments also showed high affinity with Kd values in the 10-9-10-8 M range. Bmax values estimated in A431- CCK2R cells ranged from 0.6 to 2.2×106 per cell. All peptides showed high levels of internalization when incubated at 37°C. Conclusion All DOTA-conjugated peptides showed high receptor binding and internalization properties and appear suitable for further characterization, as described in other articles of this issue. © Springer-Verlag 2011. Source

Psimadas D.,University Hospital of Larissa | Psimadas D.,Institute of Radioisotopes Radiodiagnostic Products | Psimadas D.,Technological Educational Institute of Athens | Baldi G.,CERICOL | And 6 more authors.
Journal of Biomedical Nanotechnology | Year: 2012

Magnetic nanoparticles have become important tools for imaging a wide range of diseases, improving drug delivery and applying hyperthermic treatment. Iron oxide based nanoparticles have been widely examined, unlike cobalt ferrite based ones. Herein, monodisperse and stable CoFe 2O 4nanoparticles have been produced, coated and further stabilized using ethyl 12-(hydroxyamino)-12-oxododecanoate, poly(lactic-co-glycolic acid) and bovine serum albumin. The final product, NBRh1, was fully characterized and has been directly radiolabeled with 99mTc using SnCl 2 as the reducing agent in high yields. In vitro stability and hyperthermic properties of 99mTc-NBRh1 were encouraging for further application in low frequencies hyperthermia and biomagnetic applications. In vivo evaluation followed after injection in healthy mice. The planar and SPECT imaging data as well as the biodistribution results were in accordance, showing high liver and spleen uptake as expected starting almost immediately after administration. In conclusion the preliminary results for nanoparticles bearing a cobalt ferrite core justify further investigations towards potential hyperthermic applications, drug transportation and liver or spleen imaging. Copyright © 2012 American Scientific Publishers All rights reserved. Source

Psimadas D.,Institute of Radioisotopes Radiodiagnostic Products | Psimadas D.,University Hospital of Larissa | Fani M.,Institute of Radioisotopes Radiodiagnostic Products | Fani M.,University Hospital Freiburg | And 7 more authors.
Bioorganic and Medicinal Chemistry | Year: 2012

During the past decade radiolabeled RGD-peptides have been extensively studied to develop site-directed targeting vectors for integrins. Integrins are heterodimeric cell-surface adhesion receptors, which are upregulated in cancer cells and neovasculature during tumor angiogenesis and recognize the RGD aminoacid sequence. In the present study, we report the synthesis and development of two derivatives of the N-Lys derivatized cyclic Arg-Gly-Asp-d-Phe-Lys peptide, namely of cRGDfKHis and cRGDfK-CPA (CPA: 3-l-Cysteine Propionic Acid), radiolabeled via the [ 99mTc(H 2O) 3(CO) 3] + metal aquaion at a high yield even at low concentrations of 10-5 M (>87%) for cRGDfK-10-5 M (>93%) for cRGDfK-CPA. Radiolabeled peptides were characterized with regard to their stability in saline, in His/Cys solutions, as well as in plasma, serum and tissue homogenates and were found to be practically stable. Internalization and efflux assays using αvβ3-receptor-positive MDA-MB 435 breast cancer cells showed a good percentage of quick internalization (29.1 ± 9.8% for 99mTc-HiscRGDfK and 37.0 ± 0.7% for 99mTc-CPA- cRGDfK at 15 min) and no retention of radioactivity for both derivatives. Their in vivo behavior was assessed in normal mice and pathological SCID mice bearing MDA-MB 435 ανβ3 positive breast tumors. Both presented fast blood clearance and elimination via both the urinary and hepatobiliary systems, with 99mTc-His-cRGDfK remaining for a longer time than 99mTc-CPA-cRGDfK in all organs examined. Tumor uptake 30 min pi was higher for 99mTc-CPAcRGDfK (4.2 ± 1.5% ID/g) than for 99mTc-His-cRGDfK (2.8 ± 1.5% ID/g). Dynamic scintigraphic studies showed that the tumor could be visualized better between 15 and 45 min pi for both radiolabeled compounds but low delineation occurred due to high abdominal background. It was finally noticed that the accumulated activity on the tumor site was depended on the size of the experimental tumor; the smaller the size, the higher was the radioactivity concentration. © 2012 Published by Elsevier Ltd. All rights reserved. Source

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