Plasma protein binding of 99mTc-labeled hydrazino nicotinamide derivatized polypeptides and peptides.


6-Hydrazinopyridine-3-carboxylic acid (HYNIC) constitutes one of the most attractive reagents to prepare 99mTc-labeled polypeptides and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed, 99mTc-HYNIC-conjugated IgG showed biodistribution of radioactivity similar to that of 111In-DTPA-conjugated IgG. However, recent studies indicated significant plasma protein binding when the 99mTc labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of 99mTc-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this 99mTc-labeling method. In mice, [99mTc](HYNIC-IgG)(tricine)2 and [99mTc](HYNIC-Fab)(tricine)2 showed persistent localization of radioactivity in tissues when compared with their 125I-labeled counterparts. [99mTc](HYNIC-IgG)(tricine)2 eliminated from the blood at a rate similar to that of 125I-labeled IgG, while [99mTc](HYNIC-Fab)(tricine)2 showed significantly slower clearance of the radioactivity than 125I-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of [99mTc](HYNIC-IgG)(tricine)2 in murine plasma. However, [99mTc](HYNIC-Fab)(tricine)2 and [99mTc](HYNIC-RC160)(tricine)2 demonstrated significant increases in the radioactivity in higher molecular weight fractions in plasma. Formation of higher molecular weight species was reduced when [99mTc](HYNIC-RC160)(tricine)2 was stabilized with nicotinic acid (NIC) to generate [99mTc](HYNICRC160)(tricine)(NIC). [99mTc](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the blood than [99mTc](HYNIC-RC160)(tricine)2. These findings suggested that one of the tricine coligands in 99mTc-HYNIC-labeled (poly)peptides would be replaced with plasma proteins to generate higher molecular weight species that exhibit slow blood clearance. In addition, the molecular sizes of parental peptides played an important role in the progression of the exchange reaction of one of the tricine coligands with plasma proteins.