RESUMEN
PURPOSE: Melanocortin receptor 1 (MC1R) is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68Gallium (68Ga) labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography (PET) imaging. Aim of this study was to compare a standard labeling protocol against the 68Ga-DOTA peptide purified from the excess of unlabeled peptide. PROCEDURES: The MC1R ligand DOTA-NAPamide was labeled with 68Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and 68Ga (95°C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors. RESULTS: In biodistribution studies, non-purified 68Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracer preparations. Purified 68Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake. CONCLUSIONS: We demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as 68Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Melanoma Experimental/metabolismo , Compuestos Organometálicos/química , Fragmentos de Péptidos/química , Ensayos Antitumor por Modelo de Xenoinjerto , alfa-MSH/análogos & derivados , Animales , Cromatografía de Fase Inversa , Cinética , Ratones , Compuestos Organometálicos/farmacocinética , Receptor de Melanocortina Tipo 1/metabolismo , Distribución Tisular , alfa-MSH/química , alfa-MSH/farmacocinéticaRESUMEN
Background: Molecular targeting remains to be a promising approach in oncology. Overexpression of G protein-coupled receptors (GPCRs) in human cancer is offering a powerful opportunity for tumor-selective imaging and treatment employing nuclear medicine. We utilized novel chemerin-based peptide conjugates for chemokine-like receptor 1 (CMKLR1) targeting in a breast cancer xenograft model. Methods: By conjugation with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), we obtained a family of five highly specific, high-affinity tracers for hybrid positron emission tomography/magnetic resonance (PET/MR) imaging. A xenograft model with target-positive DU4475 and negative A549 tumors in immunodeficient nude mice enabled CMKLR1-specific imaging in vivo. We acquired small animal PET/MR images, assessed biodistribution by ex vivo measurements and investigated the tracer specificity by blocking experiments. Results: Five CMKLR1-targeting peptide tracers demonstrated high biological activity and affinity in vitro with EC50 and IC50 values below 2 nM. Our target-positive (DU4475) and target-negative (A549) xenograft model could be validated by ex vivo analysis of CMKLR1 expression and binding. After preliminary PET imaging, the three most promising tracers [68Ga]Ga-DOTA-AHX-CG34, [68Ga]Ga-DOTA-KCap-CG34 and [68Ga]Ga-DOTA-ADX-CG34 with best tumor uptake were further analyzed. Hybrid PET/MR imaging along with concomitant biodistribution studies revealed distinct CMKLR1-specific uptake (5.1% IA/g, 3.3% IA/g and 6.2% IA/g 1 h post-injection) of our targeted tracers in DU4475 tumor tissue. In addition, tumor uptake was blocked by excess of unlabeled peptide (6.4-fold, 5.5-fold and 3.4-fold 1 h post-injection), further confirming CMKLR1 specificity. Out of five tracers, we identified these three tracers with moderate, balanced hydrophilicity to be the most potent in receptor-mediated tumor targeting. Conclusion: We demonstrated the applicability of 68Ga-labeled peptide tracers by visualizing CMKLR1-positive breast cancer xenografts in PET/MR imaging, paving the way for developing them into theranostics for tumor treatment.
Asunto(s)
Neoplasias de la Mama/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Tomografía de Emisión de Positrones/métodos , Radiofármacos/química , Receptores de Quimiocina/metabolismo , Animales , Línea Celular , Femenino , Radioisótopos de Galio , Humanos , Ratones Desnudos , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This study investigated the role of individual U-II amino acid positions and side chain characteristics important for U-IIR activation. A complete permutation library of 209 U-II variants was studied in an activity screen that contained single substitution variants of each position with one of the other 19 proteinogenic amino acids. Receptor activation was measured using a cell-based high-throughput fluorescence calcium mobilization assay. We generated the first complete U-II substitution map for U-II receptor activation, resulting in a detailed view into the structural features required for receptor activation, accompanied by complementary information from receptor modeling and ligand docking studies. On the basis of the systematic SAR study of U-II, we created 33 further short and linear U-II variants from eight to three amino acids in length, including d- and other non-natural amino acids. We identified the first high-potency linear U-II analogues. Urolinin, a linear U-II agonist (nWWK-Tyr(3-NO2)-Abu), shows low nanomolar potency as well as improved metabolic stability.