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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.

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.

Aloj L.,Instituto Nazionale Tumori | Aurilio M.,Instituto Nazionale Tumori | Rinaldi V.,Instituto Nazionale Tumori | D'ambrosio L.,Instituto Nazionale Tumori | 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.

Zoppellaro G.,Palacky University | Kolokithas-Ntoukas A.,University of Patras | Polakova K.,Palacky University | Tucek J.,Palacky University | And 9 more authors.
Chemistry of Materials | Year: 2014

Clustering of biocompatible magnetic iron oxide nanocrystallites (MIONs) is a synthetic strategy which improves magnetic manipulation, imaging, and sensing for biomedical applications. In this work we describe the synthesis of condensed clustered MIONs obtained through biomineralization and epitaxial aggregation in the presence of alginate at ambient conditions, mimicking the process that so far has been achieved only by nature, in iron-oxidizing bacteria. These condensed-type magnetic nanostructures exhibit higher magnetophoretic responses compared to other types of magnetic colloids and clustered systems. The soft environmental conditions used for the synthesis of the magnetic nanosystems enables the alginate coating material to retain high drug loading ability for the doxorubicin molecule as well as strong binding proclivity for radionuclides. The strong binding of doxorubicin forms the physical basis to obtain magnetic nanocarriers, where the selective release of the drug occurs only under the action of external stimuli, such as remote magnetic hyperthermia or increased temperature (i.e., inflamed tissue). Furthermore, the strong binding proclivity of radionuclides facilitates in vivo SPECT imaging. The witnessed properties are obtained by using only ∼17 wt % alginate content in the magnetic superstructures; thus, very high saturation magnetization value is imparted to the condensed system, expressed in terms of the hybrid's mass. In spite of the fact that the magnetic nanoassemblies are characterized by low hydrodynamic diameter, ∼45 nm, the transverse relaxivity reaches the remarkable value of 250 s-1 mM -1Fe (for negative MION contrast agents of this size), a property that validates the use of these nanostructures as effective MRI contrast agents. © 2014 American Chemical Society.

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.

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.

Tsiapa I.,University of Patras | Loudos G.,Technological Educational Institute of Athens | Varvarigou A.,Institute of Radioisotopes Radiodiagnostic Products | Fragogeorgi E.,Technological Educational Institute of Athens | And 10 more authors.
Nuclear Medicine and Biology | Year: 2013

Introduction: Radiolabeled RGD peptides that specifically target integrin ανβ3 have great potential in early tumor detection through noninvasive monitoring of tumor angiogenesis. Based on previous findings of our group on radiopeptides containing positively charged aminoacids, we developed a new cyclic cRGDfK derivative, c(RGDfK)-(Orn)3-CGG. This new peptide availing the polar linker (Orn)3 and the 99mTc-chelating moiety CGG (Cys-Gly-Gly) is appropriately designed for 99mTc-labeling, as well as consequent conjugation onto nanoparticles. Methods: A tumor imaging agent, c(RGDfK)-(Orn)3-[CGG-99mTc], is evaluated with regard to its radiochemical, radiobiological and imaging characteristics. Results: The complex c(RGDfK)-(Orn)3-[CGG-99mTc] was obtained in high radiochemical yield (>98%) and was stable in vitro and ex vivo. It presented identical to the respective, fully analytically characterized 185/187Re complex retention time in RP-HPLC. In contrary to other RGD derivatives, we showed that the new radiopeptide exhibits kidney uptake and urine excretion due to the ornithine linker. High tumor uptake (3.87±0.48% ID/g at 60min p.i.) was observed and was maintained relatively high even at 24h p.i. (1.83±0.05 % ID/g), thus providing well-defined scintigraphic imaging. Accumulation in other organs was negligible. Blocking experiments indicated target specificity for integrin receptors in U87MG glioblastoma cells. Conclusion: Due to its relatively high tumor uptake, renal elimination and negligible abdominal localization, the new 99mTc-RGD peptide is considered promising in the field of imaging ανβ3-positive tumors. However, the preparation of multifunctional SPECT/MRI contrast agents (RGD-conjugated nanoparticles) for dual modality imaging of integrin expressing tumors should be further investigated. © 2013 Elsevier Inc.

Tatsi A.,Institute of Radioisotopes Radiodiagnostic Products | Tatsi A.,University of Patras | Maina T.,Institute of Radioisotopes Radiodiagnostic Products | Cescato R.,University of Bern | And 6 more authors.
EJNMMI Research | Year: 2012

Background In this study, we report on the synthesis, radiolabeling, and biological evaluation of two new somatostatin-14 (SS14) analogs, modified with the universal chelator DOTA. We were interested to investigate if and to what extent such radiotracer prototypes may be useful for targeting sst1-5-expressing tumors in man but, most importantly, to outline potential drawbacks and benefits associated with their use. Methods AT1S and AT2S (DOTA-Ala1-Gly2-c[Cys3-Lys4-Asn5-Phe6-Phe7-Trp8/DTRP 8-Lys9-Thr10- Phe11-Thr12-Ser13-Cys14-OH], respectively) were synthesized on the solid support and labeled with 111In. The sst1-5 affinity profile of AT1S/AT2S was determined by receptor autoradiography using [Leu8,DTrp22,125I-Tyr25]SS28 as radioligand. The ability of AT2S to stimulate sst2 or sst3 internalization was qualitatively analyzed by an immunofluorescencebased internalization assay using hsst2- or hsst3-expressing HEK293 cells. Furthermore, the internalization of the radioligands [111In]AT1S and [111In]AT2S was studied at 37°C in AR4- 2J cells endogenously expressing sst2. The in vivo stability of [111In]AT1S and [111In]AT2S was tested by high-performance liquid chromatography analysis of mouse blood collected 5 min after radioligand injection, and biodistribution was studied in normal mice. Selectively for [111In]AT2S, biodistribution was further studied in SCID mice bearing AR4-2J, HEK293- hsst2A +, -hsst3 + or -hsst5 + tumors. Results The new SS14-derived analogs were obtained by solid phase peptide synthesis and were easily labeled with 111In. Both SS14 conjugates, AT1S, and its DTrp8 counterpart, AT2S, showed a pansomatostatin affinity profile with the respective hsst1-5 IC50 values in the lower nanomolar range. In addition, AT2S behaved as an agonist for sst2 and sst3 since it stimulated receptor internalization. The 111In radioligands effectively and specifically internalized into rsst2A-expressing AR4-2J cells with [111In]AT2S internalizing faster than [111In]AT1S. Ex vivo mouse blood analysis revealed a rapid degradation of both radiopeptides in the bloodstream with the DTrp8 analog showing higher stability. Biodistribution results in healthy mice were consistent with these findings with only [111In]AT2S showing specific uptake in the sst2-rich pancreas. Biodistribution of [111In]AT2S in tumor-bearing mice revealed receptor-mediated uptake in the AR4-2J (1.82 ± 0.36 - block 0.21 ± 0.17 at 4 h post injection (pi)), the HEK293-hsst2A + (1.49 ± 0.2 - block 0.27 ± 0.20 at 4 h pi), the HEK293-hsst3 + (1.24 ± 0.27 - block 0.32 ± 0.06 at 4 h pi), and the HEK293-hsst5 + tumors (0.41 ± 0.12 - block 0.22 ± 0.006 at 4 h pi). Radioactivity washed out from blood and background tissues via the kidneys. Conclusions This study has revealed that the native SS14 structure can indeed serve as a motif for the development of promising pansomatostatin-like radiotracers. Further peptide stabilization is required to increase in vivo stability and, consequently, to enhance in vivo delivery and tumor targeting. © 2012 Tatsi et al.

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