The effect of macrocyclic chelators on the targeting properties of the ⁶⁸Ga-labeled gastrin releasing peptide receptor antagonist PEG₂-RM26

Introduction: Overexpression of gastrin-releasing peptide receptors (GRPR) has been reported in several cancers. Bombesin (BN) analogs are short peptides with a high affinity for GRPR. Different BN analogs were evaluated for radionuclide imaging and therapy of GRPR-expressing tumors. We have previously investigated an antagonistic analog of BN (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂, RM26) conjugated to NOTA via a PEG₂ spacer (NOTA-PEG₂-RM26) labeled with ⁶⁸Ga, ¹¹¹In and Al¹⁸F. ⁶⁸Ga-labeled NOTA-PEG₂-RM26 showed high tumor-to-organ ratios. Methods: The influence of different macrocyclic chelators (NOTA, NODAGA, DOTA and DOTAGA) on the targeting properties of ⁶⁸Ga-labeled PEG₂-RM26 was studied in vitro and in vivo. Results: All conjugates were labeled with generator-produced ⁶⁸Ga with high yields and demonstrated high stability and specific binding to GRPR. The IC₅₀ values of ^natGa-X-PEG₂-RM26 (X = NOTA, DOTA, NODAGA, DOTAGA) were 2.3 ± 0.2, 3.0 ± 0.3, 2.9 ± 0.3 and 10.0 ± 0.6 nM, respectively. The internalization of the conjugates by PC-3 cells was low. However, the DOTA-conjugated analog demonstrated a higher internalization rate compared to other analogs. GRPR-specific uptake was found in receptor-positive normal tissues and PC-3 xenografts for all conjugates. The biodistribution of the conjugates was influenced by the choice of the chelator moiety. Although all radiotracers cleared rapidly from the blood, [⁶⁸Ga]Ga-NOTA-PEG₂-RM26 showed significantly lower uptake in lung, muscle and bone compared to the other analogs. The uptake in tumors (5.40 ± 1.04 %ID/g at 2 h p.i.) and the tumor-to-organ ratios (25 ± 3, 157 ± 23 and 39 ± 4 for blood, muscle and bone, respectively) were significantly higher for the NOTA-conjugate than the other analogs. Conclusions: Chelators had a clear influence on the biodistribution and targeting properties of ⁶⁸Ga-labeled antagonistic BN analogs. Positively charged [⁶⁸Ga]Ga-NOTA-PEG₂-RM26 provided a low kidney radioactivity uptake, high affinity, high tumor uptake and high image contrast