Development of a hydroxamamide-based bifunctional chelating agent to prepare technetium-99m-labeled bivalent ligand...

Hydroxamamide (Ham) is a thiol-free chelating agent that forms technetium-99m (^99mTc)-complexes with a metal-to-ligand ratio of 1:2 under moderate reaction conditions. Therefore, Ham-based chelating agents will produce ^99mTc-labeled compounds with a bivalent targeting scaffold. For their universal usage, we developed a novel Ham-based bifunctional chelating agent, "Ham-Mal", with a maleimide group that can easily conjugate with a thiol group, for to preparing ^99mTc-labeled bivalent ligand probes. Ham-Mal was synthesized by a four-step reaction, and then reacted with cysteine or c(RGDfC) to produce Ham-Cys or Ham-RGD. These precursors were reacted with ^99mTcO₄^- for 10 min under room temperature to obtain ^99mTc-(Ham-Cys)₂ and ^99mTc -(Ham-RGD)₂. The cellular uptake level of ^99mTc-(Ham-RGD)₂ by U87MG (high Integrin ɑ_vβ₃ expression) cells was significantly higher than that by PC3 (low Integrin ɑ_vβ₃ expression) cells at 60 min after the incubation, and the uptake was significantly suppressed by pre-treatment for 15 min with excess c(RGDfK) peptide. In the in vivo study with U87MG/PC3 dual xenografted BALB/c-nu mice, the radioactivity of U87MG tumor tissue was significantly higher than that of PC3 tumor tissue at 360 min after the administration of ^99mTc-(Ham-RGD)₂. These results suggest Ham-Mal may have potential as a bifunctional chelating agent for ^99mTc-labeled bivalent ligand probes.