RESUMO
Aptamers are single-stranded oligonucleotides that recognize molecular targets with high affinity and specificity. Aptamer that selectively bind to the protein tyrosine kinase-7 (PTK7) receptor, overexpressed on many cancers, has been labelled as probes for molecular imaging of cancer. Two new PTK7-targeting aptamer probes were developed by coupling frameworks from the fluorescent dye AlexaFluor647 or the 6-hydrazinonicotinamide (HYNIC) chelator-labelled to 99mTc. The derivatizations via a 5'-aminohexyl terminal linker were done at room temperature and under mild buffer conditions. Physicochemical and biological controls for both imaging agents were performed verifying the integrity of the aptamer-conjugates by HPLC. Recognition of melanoma (B16F1) and lymphoma (A20) mouse cell lines by the aptamer was studied using cell binding, flow cytometry and confocal microscopy. Finally, in vivo imaging studies in tumour-bearing mice were performed. The new probes were able to bind to melanoma and lymphoma cell lines in vitro, the in vivo imaging in tumour-bearing mice showed different uptake behaviours showing for the fluorescent conjugate good uptake by B cell lymphoma while the radiolabelled conjugate did not display tumour uptake due to its high extravascular distribution, and both showed rapid clearance properties in tumour-bearing mice.
Assuntos
Aptâmeros de Nucleotídeos/farmacocinética , Moléculas de Adesão Celular/antagonistas & inibidores , Corantes Fluorescentes/farmacocinética , Linfoma/diagnóstico , Melanoma/diagnóstico , Impressão Molecular , Inibidores de Proteínas Quinases/farmacocinética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/química , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Neoplasias Experimentais/diagnóstico , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
In this work Nimotuzumab (monoclonal antibody, recognizes the EGF-R) was radiolabeled with (177)Lu as a potential cancer therapy radiopharmaceutical. In-vitro cell binding studies and in-vivo biodistribution and imaging studies were performed to determine the radiochemical stability, targeting specificity and pharmacokinetics of the (177)Lu-labeled antibody. Nimotuzumab was derivatized with DOTA-NHS at room temperature for 2 hours. DOTA-Nimotuzumab was radiolabeled with (177)LuCl3 (15 MBq/mg) at 37°C for 1 h. The radiochemical purity was assessed by ITLC, silica gel and by RP-HPLC. Binding specificity studies were performed with EGF-R positive A431 human epithelial carcinoma and EGF-R negative MDA-MB-435 breast carcinoma cells. Biodistribution studies were performed in healthy female CD-1 mice at 1 h, 4 h, 24 h, and A431 xenografted nude mice at 10 min, 1 h, 4 h, 24 h, 48 h, and 96 h. SPECT-CT imaging studies were performed in A431 xenografted mice at 24 h post injection. DOTA-Nimotuzumab was efficiently labeled with (177) LuCl(3) at 37°C. The in vitro stability of labeled product was optimal over 24 h in buffered saline and mouse serum. Specific recognition of EGF-R by (177)Lu-DOTA-Nimotuzumab was observed in A431 cell binding studies. Biodistribution studies demonstrated increasing tumor uptake of (177)Lu-DOTA-Nimotuzumab over time, with tumor to muscle ratios of 6.26, 10.68, and 18.82 at 4 h, 24 h, and 96 h post injection. Imaging of A431 xenografted mice showed high uptake in the tumor. (177)Lu-DOTA-Nimotuzumab has the potential to be a promising therapy agent, which may be useful in the treatment of patients with EGF-R positive cancer.