In Vitro and In Vivo Evaluation of a ⁶⁴Cu-Labeled Polyethylene Glycol-Bombesin Conjugate

Abstract

The goal of this study was to synthesize and evaluate a novel bombesin (BN) analogue containing a polyethylene glycol (PEG) linker that can be radiolabeled with 64Cu through the DOTA bifunctional chelate. It is hypothesized that PEG linkers would improve the pharmacokinetics of radiolabeled bombesin analogues to optimize their tumor-to-normal tissue ratios for radiotherapy applications. The formation of this conjugate (DOTA-PEG-BN(7-14)) was confirmed by MALDI-TOF mass spectrometry and was radiolabeled with 64Cu at a specific activity of 2.7 MBq/nmol. DOTA-PEG-BN(7-14) bound specifically to gastrin-releasing peptide receptor (GRPR)-positive PC-3 cells with an IC50 value of 3.9 microM for displacing 125I-Tyr4-BN. Internalization of 64Cu-DOTA-PEG-BN(7-14) into PC-3 cells showed that 5.7%, 13.4%, and 21.0% was internalized at 0.5, 2, and 4 hours, respectively. Biodistribution of 64Cu-DOTA-PEGBN(7-14) was evaluated in normal, athymic nude mice 2, 4, and 24 hours after i.v. injection. This showed that most of the tissues had a similar uptake and clearance of 64Cu-DOTA-PEG-BN(7-14) compared to a control peptide with an alkyl linker (DOTA-Aoc-BN(7-14)) at the given time points. There was uptake of 10.8% ID/g of 64Cu-DOTA-PEG-BN(7-14) 4 hours after i.v. injection in the GRPR-positive pancreas that was inhibited to 2.4% upon injection of an excess of Tyr4-BN. These studies demonstrate that BN analogues can be conjugated with PEG linkers, radiolabeled with 64Cu, and bind to GRPR. Future studies will attempt to increase the affinity of these analogues for GRPR and alter the pharmacokinetics of the 64Cu-labeled conjugates through the use of various sized PEG linkers.

Molecular Assembly of Multifunctional 99mTc Radiopharmaceuticals Using “Clickable” Amino Acid Derivatives

“…Common strategies to extend the serum half-life of peptide-based radiotracers in vivo include PEGylation, the formation of multimers or structural modifications for stabilization against enzymatic degradation. [23,36,37] Alternative approaches make use of albumin-binding molecules. [24,38] By noncovalent interaction with serum albumin, this class of compounds has been shown to prolong the in vivo circulatory half-life of conjugated probes (e.g., fluorophores and MRI contrast agents) as well as antibody fragments.…”

MicroPET Imaging of Breast Cancer Using Radiolabeled Bombesin Analogs Targeting the Gastrin-releasing Peptide Receptor

“…We have previously evaluated a BN analog that consists of the eight C-terminal amino acids of BN (BN (7)(8)(9)(10)(11)(12)(13)(14)) conjugated to the macrocyclic chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) via an 8-carbon linker (Aoc) to yield DOTA-Aoc-BN(7-14) [9]. This was radiolabeled with 64 Cu and evaluated in mice bearing human prostate cancer xenografts.…”

[ 64 Cu-NOTA-8-Aoc-BBN(7-14)NH 2 ] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues

“…Receptor-specific peptide conjugates containing the chelating agents DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) have shown some promise for production of Cu-64-labeled targeting vectors (22)(23)(24)(25)(26). However, 64 Cu 2ϩ complexes of these specific chelating ligands are only moderately stable under in vivo conditions, resulting in demetallation and subsequent accumulation in nontarget tissues such as liver.…”

“…However, ligands of this general type still suffer from difficult synthetic protocols and renal accumulation and retention of 64 Cu radionuclide (27)(28)(29). Thus, there is some impetus to improve the in vivo kinetic stability of 64 Cu-macrocyclic bioconjugates to reduce accumulation in collateral tissues such as liver (22)(23)(24)(25)(26). Furthermore, reduction of uptake in normal kidney would do much to improve the inherent renal toxicities of many peptide-based therapeutic agents (27)(28)(29).…”