Manipulating Pharmacokinetics of Purification-Free 99m Tc-Labeled Bivalent Probes for In Vivo Imaging of Saturable Targets.

The accumulation of ^99m Tc-labeled probes targeting saturable systems of the body is hindered by the presence of a large excess of unlabeled ligands needed to ensure high radiochemical yields in a short reaction time. To address the issue, we recently reported a novel concept of a metal-coordination-mediated synthesis of a bivalent ^99m Tc-labeled probe from a monovalent ligand using d-penicillamine (Pen) as a chelating molecule and c(RGDfK) as a model targeting device. The Pen-conjugated c(RGDfK) via a hexanoate linkage (Pen-Hx-c(RGDfK)) provided a bivalent [ ^99m Tc]Tc-[(Pen-Hx-c(RGDfK)) ₂ that possessed much higher integrin α _v β ₃ binding affinity than Pen-Hx-c(RGDfK) and visualized a murine tumor without purification. However, high radioactivity levels were observed in the abdominal regions, which necessitated improved pharmacokinetics of the probes for practical applications. In this study, a pharmacokinetic (PK) modifier was introduced to manipulate the pharmacokinetics of the ^99m Tc-Pen ₂ -based bivalent probe. The Hx linkage in Pen-Hx-c(RGDfK) was replaced with acetyl-d-serine-d-serine-glycine (Ac-ssG) or hexanoyl-d-serine-d-serine-d-serine (Hx-sss) to prepare Pen-Ac-ssG-c(RGDfK) or Pen-Hx-sss-c(RGDfK). Pen-Ac-ssG-c(RGDfK) impaired the complexation ability of Pen-Hx-c(RGDfK), and a monovalent ^99m Tc-labeled compound was generated at low ligand concentration. However, Pen-Hx-sss-c(RGDfK) provided the objective bivalent ^99m Tc-labeled probe in high radiochemical yields at a concentration similar to that of Pen-Hx-c(RGDfK). [ ^99m Tc]Tc-[Pen-Hx-sss-c(RGDfK)] ₂ also possessed stability and integrin α _v β ₃ binding affinity similar to those of [ ^99m Tc]Tc-[Pen-Hx-c(RGDfK)] ₂ . As a result, [ ^99m Tc]Tc-[Pen-Hx-sss-c(RGDfK)] ₂ exhibited tumor and abdominal radioactivity levels similar to and significantly lower than those of [ ^99m Tc]Tc-[Pen-Hx-c(RGDfK)] ₂ . These findings indicate the incorporation of a tripeptide PK modifier to Pen-Hx-c(RGDfK) preserved the complexation ability and improved the pharmacokinetics of the resulting ^99m Tc-labeled bivalent probe without impairing the targeting ability. Thus, the [Pen-Hx-(PK modifier)-(targeting device)] would constitute a basic formulation for preparing the ^99m Tc-Pen ₂ -based bivalent probes for imaging saturable targets of the body.