Intracellular Metabolic Fate of Radioactivity after Injection of Technetium-99m-Labeled Hydrazino Nicotinamide Derivatized Proteins

Abstract

Hydrazino nicotinate (HYNIC) has been shown to produce technetium-99m (99mTc)-labeled proteins and peptides of high stability with high specific activities. However, persistent localization of radioactivity was observed in nontarget tissues such as the liver and kidney after administration of [99mTc]HYNIC-labeled proteins and peptides, which compromises the diagnostic accuracy of the radiopharmaceuticals. Since lysosomes are the principal sites of intracellular catabolism of proteins and peptides, 99mTc-HYNIC-labeled galactosyl-neoglycoalbumin (NGA) was prepared using tricine as a co-ligand to investigate the fate of the radiolabel after lysosomal proteolysis in hepatocytes. 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 showed a broad radioactivity peak ranging from molecular masses of 0.5-50 kDa. RP-HPLC analyses of liver homogenates suggested the presence of multiple radiolabeled species. However, most of the radioactivity migrated to lower molecular weight fractions on size-exclusion HPLC after treatment of the liver homogenates with sodium triphenylphosphine-3-monosulfonate (TPPMS). The TPPMS-treated liver homogenates showed a major peak at a retention time similar to that of [[99mTc](HYNIC-lysine)(tricine)(TPPMS)] on RP-HPLC. Similar results were obtained with urine and fecal samples. These findings suggested that the chemical bonding between 99mTc and HYNIC remains stable in the lysosomes and following excretion from the body. The persistent localization of radioactivity in the liver could be attributed to the slow elimination rate of the final radiometabolite, [[99mTc](HYNIC-lysine)(tricine)2], from lysosomes, and subsequent dissociation of one of the tricine co-ligands in the low pH environment of the lysosomes in the absence of excess co-ligands, followed by binding proteins present in the organelles. The findings in this study also suggested that the development of appropriate co-ligands capable of preserving stable bonding with the Tc center is essential to reduce the residence time of radioactivity in nontarget tissues after administration of [99mTc]HYNIC-labeled proteins and peptides.

Biodistribution of Radioiodinated Adenovirus Fiber Protein Knob Domain after Intravenous Injection in Mice

“…Ad5 knob was labeled with 99m Tc by an indirect method using the chelator succinimidyl 6-hydrazinonicotinate (HYNIC), which was incorporated into knob protein by chemical modification (51). Catabolism of 99m Tc-HYNIC-labeled proteins in hepatocyte microsomes has been reported in other studies (34) to generate a stable radiometabolite, 99m Tc-(HYNIC-lysine)(tricine)(tricine), which is eliminated from the liver only very slowly. Because these stable radiometabolites are retained in the liver, it is not possible to determine from tissue counting or in vivo imaging whether radioactivity corresponds to intact radiolabeled knob protein or to products of knob degradation.…”

6-Hydrazinonicotinamide Derivatives as Bifunctional Coupling Agents for 99mTc-Labeling of Small Biomolecules

“…Since then, the HYNIC technology has successfully been used for the 99m Tc-labeling of antibodies [3][4][5][6][7][8] and small biomolecules (BMs), including chemotactic peptides [9][10][11][12][13][14][15][16][17][18], somatostatin analogs [19][20][21][22][23][24][25],"stealth" liposomes [26,27], antisense oligonucleotides [28][29][30][31], a folate receptor ligand [32], and polypeptides [32][33][34]. A ternary ligand system (HYNIC, tricine, and trisodium triphenylphosphine-3,3¢,3≤-trisulfonate, TPPTS) has been used for the 99m Tc-labeling of chemotactic peptides [35] and leukotriene B 4 (LTB 4 ) receptor antagonists [36][37][38][39] for imaging infection and inflammation, integrin a v b 3 receptor antagonists for tumor imaging [40], and a glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonist for imaging thrombosis [41][42][43][44][45][46]…”

An unexpected ‘4+2’ [N3S]/[NS] rhenium(IV) complex formed upon cleavage of a Re(V) imido bond

“…The most successful strategy to the development of diagnostic imaging agents of Tc and potential therapeutic reagents based on Re has exploited ligands to stabilize the oxometal [MO] 3+ core (where M = Tc and Re), utilizing tetradentate N x S( 4−x ) (x = 0 to 3) chelates [9], '3+1' [10] and '3+2' [11] mixed thiolate ligands. However, the recent introduction of technetium and rhenium complexes with alternative functional cores, such as diazenido-, hydrazido-, imido-and nitrido- [12], at the 'carrier free' level has made this class of compounds featuring M-N multiple bonds more attractive for the development of 99m Tc and 186/188 Re radiopharmaceuticals, whose production had been focused in the past primarily on oxometal complexes(Scheme 1).…”