Intracellular metabolic fate of radioactivity after injection of technetium-99m-labeled hydrazino nicotinamide derivatized proteins

Hydrazino nicotinate (HYNIC) has been shown to produce technetium-99m (Tc-9 9m)-labeled proteins and peptides of high stability with high specific acti vities. However, persistent localization of radioactivity was observed in n ontarget tissues such as the liver and kidney after administration of [Tc-9 9m]HYNIC-labeled proteins and peptides, which compromises the diagnostic ac curacy of the radiopharmaceuticals. Since lysosomes are the principal sites of intracellular catabolism of proteins and peptides, Tc-99m-HYNIC-labeled galactosyl-neoglycoalbumin (NGA) was prepared using tricine as a co-ligand to investigate the fate of the radiolabel after lysosomal proteolysis in h epatocytes. When injected into mice, over 90% of the injected radioactivity was accumulated in the liver after 10 min injection. At 24 h postinjection , ca. 40% of the injected radioactivity still remained in liver lysosomes. Size-exclusion HPLC analyses of liver homogenates at 24 h postinjection sho wed a broad radioactivity peak ranging from molecular masses of 0.5-50 kDa. RP-HPLC analyses of liver homogenates suggested the presence of multiple r adiolabeled species. However, most of the radioactivity migrated to lower m olecular weight fractions on size-exclusion HPLC after treatment of the liv er homogenates with sodium triphenylphosphine-3-monosulfonate (TPPMS). The TPPMS-treated liver homogenates showed a major peak at a retention time sim ilar to that of [[Tc-99m](HYNIC-lysine)(tricine)(TPPMS)] on RP-HPLC. Simila r results were obtained with urine and fecal samples. These findings sugges ted that the chemical bonding between Tc-99m and HYNIC remains stable in th e lysosomes and following excretion from the body. The persistent localizat ion of radioactivity in the liver could be attributed to the slow eliminati on rate of the final radiometabolite, [[Tc-99m](HYNIC-lysine)(tricine)(2)], from lysosomes, and subsequent dissociation of one of the tricine co-ligan ds in the low pH environment of the lysosomes in the absence of excess co-l igands, followed by binding proteins present in the organelles. The finding s in this study also suggested that the development of appropriate co-ligan ds capable of preserving stable bonding with the Tc center is essential to reduce the residence time of radioactivity in nontarget tissues after admin istration of [Tc-99m]HYNIC-labeled proteins and peptides.