In Vitro and In Vivo Evaluation of a 64 Cu-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 μM 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.