Near-Infrared Fluorescence Bombesin Antagonist Peptides for Molecular Imaging of Prostate Cancer

Objectives: We aimed to develop near-infrared fluorescent (NIRF) bombesin antagonist peptides to target the gastrin releasing peptide receptors (GRPRs) overexpressed in human prostate cancers. Methods: Alexa fluor 750 was conjugated to -DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, with a spacer Gly-4amino_carboxymethyl_piperidine (AF750-G-Pip-RM2-BBN), or -Gly-Ser-Gly- (AF750- GSG-RM2-BBN). The conjugates were purified using HPLC and examined by MS. The binding affinity was evaluated in PC-3 cells using an IC50 assay with 125I-Tyr4-BBN as the competitive agent. The in vitro binding specificity in PC-3 cells was further tested using a NIRF microscope. The in vivo specificity and imaging efficacy were evaluated using an IVIS-Spectrum system in mice bearing PC-3 flank tumors via tail-vein injection of the respective compound. Results were compared to the control groups with a co-injection of the compound and a full length bombesin. Results: AF750-G-Pip-RM2-BBN and AF750-GSG-RM2-BBN were synthesized and purified to be >95%, and molecular weights were verified. AF750-G-Pip-RM2-BBN showed better water solubility than AF750-GSG-RM2-BBN. The IC50 was 9.8±2.9 nM for AF750-G-Pip-RM2-BBN and 16.5±4.6 nM for AF750-GSG-RM2-BBN in PC-3 cells. In vitro NIRF microscopic imaging proved their high binding specificity to the GRPR on the cell membrane of PC-3. In vivo study showed a significant increase of tumor to muscle ratio in the uptake group for AF750-G-Pip-RM2-BBN at 28 hrs (6.82±1.98:1), and for AF750-GSG-RM2-BBN at 20 hrs (7.78:1), as compared to the respective control group. Conclusion: Our in vitro and in vivo data showed a high binding affinity and specificity of AF750-G-Pip-RM2-BBN and AF750- GSG-RM2-BBN in human prostate PC-3 cells.