Coordination-Mediated Synthesis of Purification-Free Bivalent ^99mTc-Labeled Probes for in Vivo Imaging of Saturable...

In the synthesis of technetium-99m (^99mTc) labeled target-specific ligands, the presence of a large excess of unlabeled ligands over ^99mTc in the injectate hinders target accumulation of ^99mTc-labeled ligands by competing for target molecules. To circumvent the problem, we recently developed a concept of the metal coordination-mediated multivalency, and proved the concept with a ^99mTc-labeled trivalent compound [^99mTc(CO)₃(CN-RGD)₃]⁺. In this study, D-penicillamine (Pen) was selected as a chelating molecule and a cyclic RGDfK peptide was conjugated to Pen via a hexanoic linkage (Pen-Ahx-c(RGDfK)). ^99mTc complexation reaction, and the stability, integrin α_vβ₃ binding affinity, and biodistribution of the ^99mTc-labeled probe were investigated to evaluate the applicability of the concept to bivalent probes. ^99mTc-[Pen-Ahx-c(RGDfK)]₂ was obtained over 95% radiochemical yields under low Pen-Ahx-c(RGDfK) concentration (50 μM). ^99mTc-[Pen-Ahx-c(RGDfK)]₂ showed approximately 10-times higher integrin α_vβ₃ binding affinity than the monovalent compounds, Pen-Ahx-c(RGDfK) and c(RGDyV). In biodistribution studies, the tumor accumulation of ^99mTc-[Pen-Ahx-c(RGDfK)]₂ was decreased to 77% and 43% of HPLC-purified (Pen-Ahx-c(RGDfK)-free) ^99mTc-[Pen-Ahx-c(RGDfK)]₂ by the presence of 5 nmol of unlabeled Pen-Ahx-c(RGDfK) and Re-[Pen-Ahx-c(RGDfK)]₂, respectively. ^99mTc-[Pen-Ahx-c(RGDfK)]₂ provided tumor image without removing unlabeled ligand, while a ^99mTc-labeled monovalent probe prepared from a monovalent ligand could not. These findings indicate the availability of the design concept to prepare ^99mTc-labeled bivalent probes with a variety of ^99mTc core and other metallic radionuclides of clinical relevance.