Synthesis and radiopharmacological characterization of radiolabeled EGFR-specific oligopeptides

Autor: Starke, F., Sihver, W., Heldt, J.-M., Sachse, S., Bergmann, R., Pietzsch, H.-J., Steinbach, J.
Jazyk: angličtina
Rok vydání: 2013
Zdroj: Journal of Labelled Compounds and Radiopharmaceuticals (2013)56, S374
20th International Symposium on Radiopharmaceutical Sciences, 12.-17.05.2013, Jeju, Korea
Popis: Objectives: The epidermal growth factor receptor (EGFR) is overexpressed in many types of human epithelial cancers. Because of its important role in the initiation and progression of tumors, it is an interesting molecular target for new radiotracers. [1] Recently, two short peptides for targeting the EGFR have been reported - the D4 peptide (sequence: LARLLT) and the GE11 peptide (sequence: YHWYGYTPQNVI).[2,3] These short oligopeptides should provide a high specific binding to EGFR-positive tumor cells as well as a rapid tumor uptake and fast blood clearance. We investigated the chemical modification and radiolabeling of the two peptides and examined the application of the D4 peptide as a possible agent for diagnosis and therapy of cancer. Methods: The D4 and the GE11 peptide haven been conjugated with several chelators, e.g. NOTA, DOTA and HYNIC, to allow radiolabeling with suitable radionuclides for diagnostic (e.g. 99mTc, 64Cu) or for therapeutic applications (e.g. 90Y). The properties of the conjugates were modified by the previous insertion of the newly developed hydrophilic, heterobifunctional linker 1. Toxicity tests have been made using a cell viability assay MTS on the EGFR-expressing cell lines A431 (epidermoid carcinoma cells) and FaDu (squamous carcinoma cells). Binding assays were performed on membranes of A431-, FaDu- and non-EGFR-expressing MDA-MB435S cells. Kinetics and saturation assays were conducted using the 64Cu-labeled conjugate 2. First biodistribution PET studies in nude mice have been performed. Results: Conjugation of the two peptides with the new linker 1 followed by the reaction with different chelators gave overall yields of 40-65%. The GE11 peptide itself, as well as all of its conjugates which did not contain the hydrophilic linker 1, were not soluble in water and therefore inapplicable for further experiments. The remaining compounds were labeled with 64Cu and 68Ga (NOTA conjugates), 99mTc (HYNIC conjugates) and 90Y (DOTA conjugates) with specific activities of up to 980 GBq/μmol for 64Cu-2. No change of cell viability was observed after treating A431 and FaDu cells with plain D4 and D4-1 for 4 days at concentrations of up to 125 μM. Binding association with 64Cu-2 on A431 membranes reached equilibrium after one hour, on FaDu membranes equilibrium was not complete after two hours. TDiss½ was less than 5 min. In saturation assays, the affinity determination of 64Cu-2 on A431 and FaDu membranes gave Kd values of 28 ± 6 nM and 72 ± 18 nM, respectively. No specific binding of 64Cu-2 to MDA-MB453S cell membranes was observed. First PET studies with healthy nude mice showed a favorable biodistribution. Conclusions: We successfully prepared and radiolabeled several conjugates of the EGFR-affine peptides D4 and GE11 with satisfying yields and high specific activities. The insertion of the new hydrophilic heterobifunctional linker 1 was necessary to achieve high solubility in water. The preservation of cell viability after D4 and D4-1 treatment indicates no toxic effect of the conjugates. The affinity of 64Cu-2 to the high EGFR-expressing A431 cells and the moderate EGFR-expressing FaDu cells was in the expected range. Binding assays with radiolabeled GE11-conjugates as well as PET studies of GE11-conjugates and 64Cu-2 with tumor-bearing nude mice are currently under investigation. References: [1] Cardo-Vila M, et al. (2010) Proc Natl Acad Sci USA, 107, 5118-23. [2] Song S, et al. (2009) FASEB, 23, 1396-404. [3] Li Z, et al. (2005) FASEB, 19, 1978-85.
Databáze: OpenAIRE
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