ИСТИНА

One of the ways to design radiopharmaceutical is to use biological vector (e.g. peptide), connected with a suitable radionuclide via a stable chelate complex. In this case the key factor of complexes’ applicability is in vivo stability. Radionuclides Zn-62, Zn-63 and Zn-69m has a potential to be used in nuclear medicine. According 3R principles [1] to reduce laboratory animals use we need first to study its stability in vitro, i.e. define complex stability constants, study complex dissociation kinetics in biological media etc. In this work we used long-lived Zn-65 to study its complexes with azacrown-derivative L (fig. 1) as a potential radiopharmaceutical. In particular, labeling conditions and in vitro stability were investigated. Zn-65 was produced by deuteron-induced reaction from copper metal on cyclotrone (beam energy is 14.8 MeV). Zn-65 separation from copper was carried out using Dowex 1x8 anion-exchange resin with two different eluents: 2M HCl (zinc is sorbed on resin and copper passes through the column) and distilled H2O (Zn2+ is desorbed). Thin layer chromatography conditions were defined for Zn-65 complexes with L using cellulose on aluminium plates (Sigma). Labeling efficiency experiments were carried out in 0.15 M sodium acetate buffer solution with final pH 7.0–7.5. Labeling yields for ligands concentrations in a range from 1•10-6 to 1•10-3 M were defined using TLC technique and gamma-spectrometry. It was established that appropriate labeling yield is achieved at L concentrations of 1•10-4 M or higher. In order to analyze it before in vivo experiments, the set of in vitro experiments with fetal bovine serum was performed. Complex for in vitro experiment was prepared in 0.15 M sodium acetate buffer solution with pH 7.0–7.5 and L concentration 1•10-4 M. 100 µl of Zn-65 blank or ZnL was added to 900 µl of fetal bovine serum, mixed and incubated at 37°C. Aliquotes of 100 µl were taken at 1, 5, 15, 30, 60, 120, 180, 240, 360 minutes and 1 day and treated with 300 µl of ethanol for protein precipitation. After centrifugation the supernatant was separated and measured by gamma-spectrometry. As a result, it was shown that ZnL, despite the relatively low stability constant (logK = 12.6 [2] comparing with ZnDOTA complex logK = 18.7 [3]), is highly stable in serum at least for 1 day: no rechelation by serum proteins is observed while Zn-65 in a blank sample quickly binds with serum proteins. Thus, it was shown that ZnL is suitable as a part of radiopharmaceutical and should be further investigated in vivo. The reported study was funded by Russian Foundation for Basic Research according to the research project № 18-33-00824.