Synthesis and Evaluation of Novel ⁶⁸ Ga-Labeled GRPR-Targeted PET Tracers Derived from [ <scp>d</scp> -Phe ⁶ ,Pro ¹⁴ ]Bombesin(6–14) and [ <scp>d</scp> -Phe ⁶ ,des-Met ¹⁴ ]Bombesin(6–14) Sequences

Gastrin-releasing peptide receptor (GRPR) is a G-protein-coupled receptor that is overexpressed in several malignancies, rendering it an ideal target for cancer imaging and therapy. In this report, we conducted structure–activity relationship studies by optimizing the substitution of Pro 14 based on an agonist sequence, [ d -Phe 6,Pro 14 ]Bombesin(6–14), and optimizing the substitution of the C -terminal Leu 13 -NH 2 based on an antagonist sequence, [ d -Phe 6,des-Met 14 ]Bombesin(6–14). PEP-1–PEP-5 derived from the agonist sequence and PEP-6–PEP-14 derived from the antagonist sequence were synthesized by a solid-phase approach and were obtained in 10–60% yield. In vitro competition binding assays showed that the K i values of PEP-1–PEP-14 were in the range of 1.16 nM to 266 nM. PEP-3, PEP-4, PEP-8, PEP-9, and PEP-10 with K i < 6 nM were selected for elongation with Ga-DOTA complex and Pip linker at their N -terminus. Calcium release assays confirmed the agonist-antagonist nature of Ga-DOTA-Pip-conjugated peptides. Ga-DOTA-Pip-PEP-4, Ga-DOTA-Pip-PEP-9, and Ga-DOTA-Pip-PEP-10 retaining high GRPR binding affinity (K i < 10 nM) were selected for 68 Ga labeling for further evaluation. [ 68 Ga]Ga-DOTA-Pip-PEP-4, [ 68 Ga]Ga-DOTA-Pip-PEP-9, and [ 68 Ga]Ga-DOTA-Pip-PEP-10 were obtained in 16–44% decay-corrected radiochemical yields with ≥64 GBq/μmol molar activity and ≥ 97% radiochemical purity. PET imaging and ex vivo biodistribution studies were conducted in PC-3 tumor-bearing mice at 1 h postinjection. All [ 68 Ga]Ga-DOTA-Pip-PEP-4, [ 68 Ga]Ga-DOTA-Pip-PEP-9, and [ 68 Ga]Ga-DOTA-Pip-PEP-10 were excreted mainly via the renal pathway and enabled clear visualization of tumor xenografts in PET images with good tumor-to-background contrast. The pancreas uptake of the agonist tracer, [ 68 Ga]Ga-DOTA-Pip-PEP-4, was much lower than that of the clinically evaluated agonist tracer, [ 68 Ga]Ga-AMBA. Similarly, the pancreas uptake of the antagonist tracers, [ 68 Ga]Ga-DOTA-Pip-PEP-9, and [ 68 Ga]Ga-DOTA-Pip-PEP-10, was also much lower than that of the clinically evaluated antagonist tracers, [ 68 Ga]Ga-RM2, [ 68 Ga]Ga-SB3, and [ 68 Ga]Ga-NeoB. Our data demonstrate that the sequences of Ga-DOTA-Pip-PEP-4, Ga-DOTA-Pip-PEP-9, and Ga-DOTA-Pip-PEP-10 are promising templates for the development of radiopharmaceuticals targeting GRPR-expressing cancer.