Cu-64-labelled compounds with dithiocarbamate ligands for theragnostic applications: preliminary in vitro studies

Aim. Recently DEDC (diethyl-dithiocarbamate) and PDTC (pyrrolidinedithiocarbamate) have been recognized to act as copper chelators able to spontaneously form stable Cu(II)-complexes. It was found that Cu-DEDC and Cu- PDTC complexes display potent proteasome-inhibitory and apoptosis-inducing activities in cultured human cancer but not in normal cells [1, 2]. Despite the invitro investigation of the mechanism responsible of the cytotoxicity of these compounds is well documented, little is known of the mechanism involved in the cell uptake, accumulation patterns and efflux of this family of compounds as well as their in- vivo pharmacokinetic profile.Using the corresponding 64Cu-complexes, as chemical probes, we investigate here the accumulation of 64Cu-DEDC and 64Cu- PDTC in human normal and cancer cells under a range of conditions. Method. The cellular accumulation of both Cu-complexes was assessed, working both in carried added (64Cu/Cu) and carrier free condition (64Cu), in Human Embryonic Kidney (HEK293) and human breast cancer cell lines (MCF-7), at 4 and 37 °C, in presence or not of copper-transporter 1 (Ctr1) inhibitor. The chemical integrity of the 64Cucomplexes after cellular incubation and in cytosolic fraction was determined. Results. A selective accumulation of the 64Cu-complexes was found in human cancer cell (% cell uptake in MCF7 = 40 ± 5; % cell uptake HEK= 7 ± 2.02). For both 64Cu-complexes a linear increase of the cellular accumulation was observed over the time and did not reach a plateau even in carried added conditions where a relatively high concentration of compounds (50 ?M) was used. After 15 min of incubation the % of cell uptake was quantified to be 20%, at 60 min this amount was ranging 30-40%. No variation of the cellular accumulation was observed in temperature-dependent experiments suggesting for these compounds a passive mechanism of uptake. The complexes were effluxed from the cells in the form of ionic Cu. Conclusion. These results support a model in which the ligand acts as an ionophore for the metal ion. Intracellular metal accumulation is maximized when the cells were treated with the Cu-complexes; copper and ligand follow different uptake kinetics and reach different intracellular concentrations. The high lipid solubility of these complexes allows them to rapidly cross the membrane reaching a high intracellular concentration. Copper overload, originated by the complex dissociation could be responsible for the cytotoxic effects. 1. Giovagnini L et. al. Inorg Chem, 2008, 47, 6336-6343. 2. Yu Z., et al. Int. J Mol Med, 2007, 20: 919-925.