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

The accumulation of Tc-99m-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 Tc-99m-labeled probe from a monovalent ligand using D-penicillamine (Pen) as a chelating molecule and c(RGDIK) as a model targeting device. The Pen-conjugated c(RGDfK) via a hexanoate linkage (PenHx-c(RGDfK)) provided a bivalent [Tc-99m]Tc-[(Pen-Hx-c(RGDfK))(2) that possessed much higher integrin alpha(v)beta(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 Tc-99m-Pen(2)-based bivalent probe. The Hx linkage in Pen-Hx-c(RGDfK) was replaced with ace D-serine-D-serineglycine (Ac-ssG) or hexanoyl-D-serine-D-serine-D-serine (Hx-sss) to prepare Pen-Ac-ssG-c(RGDIK) or Pen-Hx-sss-c(RGDfK). PenAc-ssG-c(RGDfK) impaired the complexation ability of Pen-Hx-c(RGDfK), and a monovalent Tc-99m-labeled compound was generated at low ligand concentration. However, Pen-Hx-sss-c(RGDIK) provided the objective bivalent Tc-99m-labeled probe in high radiochemical yields at a concentration similar to that of Pen-Hx-c(RGDfK). [Tc-99m]Tc-[Pen-Hx-sss-c(RGDfK)](2) also possessed stability and integrin alpha(v)beta(3) binding affinity similar to those of [Tc-99m]Tc-[Pen-Hx-c(RGDfIC)](2). As a result, [Tc-99m]Tc-[Pen-Hx-sssc(RGDfK)](2) exhibited tumor and abdominal radioactivity levels similar to and significantly lower than those of [Tc-99m]Tc-[Pen-Hxc(RGDfIC)](2). These findings indicate the incorporation of a tripeptide PK modifier to Pen-Hx-c(RGDIK) preserved the complexation ability and improved the pharmacokinetics of the resulting Tc-99m-labeled bivalent probe without impairing the targeting ability. Thus, the [Pen-Hx-(PK modifier)-(targeting device)] would constitute a basic formulation for preparing the TcPen(2)-based bivalent probes for imaging saturable targets of the body.