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) Sequence

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 Pro¹⁴ based on an agonist sequence, [d-Phe⁶,Pro¹⁴]Bombesin(6-14), and optimizing the substitution of the <i>C</i>-terminal Leu¹³-NH² based on an antagonist sequence, [d-Phe⁶,des-Met¹⁴]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. <i>In vitro</i> 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 <i>N</i>-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 ⁶⁸Ga labeling for further evaluation. [⁶⁸Ga]Ga-DOTA-Pip-PEP-4, [⁶⁸Ga]Ga-DOTA-Pip-PEP-9, and [⁶⁸Ga]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 <i>ex vivo</i> biodistribution studies were conducted in PC-3 tumor-bearing mice at 1 h postinjection. All [⁶⁸Ga]Ga-DOTA-Pip-PEP-4, [⁶⁸Ga]Ga-DOTA-Pip-PEP-9, and [⁶⁸Ga]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, [⁶⁸Ga]Ga-DOTA-Pip-PEP-4, was much lower than that of the clinically evaluated agonist tracer, [⁶⁸Ga]Ga-AMBA. Similarly, the pancreas uptake of the antagonist tracers, [⁶⁸Ga]Ga-DOTA-Pip-PEP-9, and [⁶⁸Ga]Ga-DOTA-Pip-PEP-10, was also much lower than that of the clinically evaluated antagonist tracers, [⁶⁸Ga]Ga-RM2, [⁶⁸Ga]Ga-SB3, and [⁶⁸Ga]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.