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Angiogenesis, the process whereby new capillaries are formed by outgrowth from existing microvessels, is required for tumor growth and metastasis. The transmembrane cell-surface receptor ?V?3 has recently received increasingly attention, because of the critical role in tumor associated angiogenesis and metastasis formation . The restricted expression of integrin ?V?3 during tumor growth, invasion, and metastasis presents an interesting target for both detection and treatment of solid tumors. Targeting ?V?3 with radiolabelled ligands may provide information about the receptor status and enable the planning and the monitoring of therapeutic approaches. Recently we developed a novel ?V?3 antagonist that showed a high selectivity for the receptor. Adhesion assays, competitive binding assays and cross-linking experiments performed in human erythroleukemia K562 cells, stably cotransfected with cDNA of ?v or ?IIb and ?3, demonstrated the high selectivity for ?V?3 integrin. Starting from these evidences, RGDechi was covalently bound to the chelating agent DTPA able to give stable complexes of radionuclides, such as 111In and 90Y . The final goal has been to obtain a radiolabelled compound to be used in nuclear medicine as a diagnostic and therapeutic agent. In particular DTPA-RGDechi has been labeled with 111In and used in SPECT for diagnostic purpose. Moreover, the ligand RGDechi has been labeled with 18F for micro-PET in order to evaluate the use of this peptide for in vivo imaging.

Bressanone 4-7/ 09/2002

Bressanone 4-7/09/2002

Aim. The alpha(v)beta(3) integrin is a cell adhesion receptor involved in angiogenesis, tumor cell migration and metastatic dissemination. The tripeptide sequence RGD binds to alpha(v)beta(3) but also interacts with other integrins. We developed a novel cyclized RGD pentapeptide covalently linked by a spacer to an echistatin domain that showed a high selectivity for alpha(v)beta(3) integrin ( J Med Chem 2006, 49:3416-3420). Materials and Methods. In the present study we evaluated this chimeric RGD peptide (RGDechi) in human erythroleukemia K562 cells, stably cotransfected with cDNA of alpha(v) or alpha(IIb) and beta(3) or beta(5) subunits by adhesion assays, competitive binding assays and cross-linking experiments. RGDechi was then conjugated with DTPA and labeled with 111In for SPECT imaging whereas a one-step procedure was used for labeling the chimeric peptide with 18F for PET imaging. K562 cells overexpressing alpha(v)beta(3) or alpha(v)beta(5) were subcutaneously injected into opposite flanks of individual nude mice and allowed to grow up to 0.5 cm in size. Alternatively, U87MG human glioblastoma cells and A431 human epidermoid cells endogeneously expressing high levels of alpha(v)beta(3) and alpha(v)beta(5), respectively, were used to develop xenografts in nude mice. Imaging studies were then performed using both SPECT and microPET. Results. Adhesion assays showed that the chimeric RGDechi was able to inhibit adhesion of alpha(v)beta(3) overexpressing cells but not alpha(IIb)beta(3) and alpha(v)beta(5) overexpressing clones to native ligands. Nude mice bearing K562 and U87MG tumor xenografts, both overexpressing alpha(v)beta(3), showed a high tumor uptake of 111In-labeled and 18F-labeled RGDechi assessed by SPECT and microPET, respectively. No tumor uptake of radiolabeled RGDechi could be observed in K562 and A431 tumor xenografts overexpressing alpha(v)beta(5). Conclusion. Our findings indicate that chimeric RGDechi is suitable for in vivo selective alpha(v)beta(3) receptor imaging.

Regulatory peptides and their analogs are being extensively investigated as radiopharmaceuticals for cancer imaging. In particular, cholecystokinin (CCK) receptors of the subtype B (CCK-BR) have been shown to be overexpressed in certain neuroendocrine tumors including medullary thyroid cancer. Our recent work has focused on new methods to radiolabel the CCK8 peptide with (111)In or (99m)Tc for the purpose of developing radiopharmaceuticals for in vivo CCK-B receptor imaging. Labeling of CCK8 with (111)In was achieved at the N-terminus of the peptide by adding, in solid phase, a glutamate coupled diethylenetriaminepentaacetic acid (DTPA) moiety through a glycine linker, yielding DTPA-Glu-G-CCK8. For labeling with (99m)Tc, the CCK8 peptide was modified at its N-terminus by introducing, in the following order--cysteine, glycine, and a diphenylphosphinopropionyl moiety--giving a 10-residue peptide derivative, Phos-GC-CCK8. A cell culture model was developed for the purpose of evaluating the binding properties of these two ligands. The human epidermoid carcinoma cell line, A431, was transfected with a plasmid containing the full coding sequence of the human CCK-BR under a strong viral promoter, obtaining a number of receptors in the range of 2-5 x 10(6) per cell. Control cells were transfected with vector alone. An animal tumor model utilizing these two cell lines was developed to evaluate the specificity of interaction with the CCK-BR and biodistribution properties of the compounds. CCK-BR positive and control cells were subcutaneously injected in opposite flanks of CD1 female nude mice in order to obtain xenografts differing only in their ability to express CCK-B receptors. High performance liquid chromatography (HPLC) and other chromatographic methods were utilized to assess stability of the radiolabeled compounds after injection. Both (111)In-DTPA-Glu-G-CCK8 and (99m)Tc-Phos-GC-CCK8 showed similar binding affinities for cultured CCK-BR expressing cells, with dissociation constants in the range of 20-40 nM. With the two xenograft approach, we were able to demonstrate specific interaction with the receptor of both CCK analogs in our animal model. The data obtained shows rapid specific localization of both compounds on the CCK-BR overexpressing xenografts. Both tracers show rapid plasma clearance of unbound peptide. Clearance of (111)In-DTPA-Glu-G-CCK8 appears to be preferentially through the kidneys, whereas (99m)Tc-Phos-GC-CCK8 clearance occurs both through kidneys and the hepatobiliary system. Both our labeling approaches appear adequate for clinical use of peptide based radiopharmaceuticals, although (99m)Tc-Phos-GC-CCK8 shows elevated accumulation in the gastrointestinal tract, which causes high background activity.