Bis(hydroxamamide)-based bifunctional chelating agent for 99mTc labeling of polypeptides.

To develop chelating molecules that provide 99mTc-labeled polypeptides of high in vivo stability and high specific activities under mild reaction conditions, an asymmetrical bis(benzohydroxamamide) compound with an amine group, 4'-aminomethyl-N,N'-trimethylenedibenzohydroxamamide [NH2-C3(BHam)2], was designed and synthesized. The amine residue of NH2-C3(BHam)2 was converted to a maleimide group by reaction with N-succinimidyl-6-maleimidohexanoate, and the conjugation product was coupled to thiol groups of a monoclonal antibody against osteogenic sarcoma (OST7, IgG1) pretreated with 2-iminothiolane to prepare C3(BHam)2-OST7. 99mTc radiolabeling of C3(BHam)2-OST7 was performed by the exchange reaction with [99mTc]glucoheptonate. [99mTc]C3(BHam)2-OST7 was further characterized using directly radioiodinated OST7 ([125I]OST7) and [111In]labeled OST7 with 1-[4-[(5-maleimidopentyl)amidobenzyl]ethylenediamine-N,N, N'N'-tetraacetic acid (EMCS-Bz-EDTA) as references. [99mTc]C3(BHam)2-OST7 was obtained with radiochemical yields of over 94% at protein concentrations as low as 0.2 mg/mL at room temperature for 1 h. [99mTc]C3(BHam)2-OST7 remained stable after incubation in freshly prepared murine plasma and in the presence of cysteine. Similar binding affinities to tumor cells were observed between [99mTc]C3(BHam)2-OST7 and [125I]OST7. When injected into normal mice, [99mTc]C3(BHam)2-OST7 exhibited radioactivity levels in the blood similar to [111In]-EMCS-Bz-EDTA-OST7 up to 24 h postinjection with significantly faster elimination rate of the radioactivity from the liver. In nude mice bearing osteogenic sarcoma, no significant differences were observed in the radioactivity levels in the blood and the tumor between [99mTc]C3(BHam)2-OST7 and [125I]OST7 at 24 h postinjection. These findings indicated that C3(BHam)2 provided 99mTc chelate of high stability at low concentrations even when conjugated to an intact antibody. Such characteristics render bis(hydroxamamide) compounds useful as chelating molecules for preparation of 99mTc-labeled polypeptides.