Development of radiolabeled bombesin analogs for the detection of gastrin-releasing peptide receptor expressing prostate cancer

A70

Introduction: Bombesin (BN) is a tetradecapeptide that binds with high affinity to the gastrin-releasing peptide receptor (GRPR), which is expressed on prostate tumors. It has been demonstrated that BN analogs can be radiolabeled with a variety of radiometals for potential diagnosis and treatment of GRPR-positive tumors. We have previously evaluated a BN analog that consists of the eight C-terminal amino acids of BN (BN(7-14)) conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) via an 8 carbon linker (Aoc) to yield DOTA-Aoc-BN(7-14). This was radiolabeled with the positron-emitter, ⁶⁴Cu, and evaluated in mice bearing human prostate cancer xenografts. GRPR-mediated tumor uptake was observed by microPET imaging; however, there was also significant liver and kidney uptake. In the present study, we sought to reduce this liver and kidney uptake by replacing the Aoc linker with amino acid linkers. Experimental Procedures: The peptides were synthesized by solid phase peptide synthesis to contain linkers with either glycine (G), serine (S), or glutamic acid (E). DOTA was conjugated to BN(7-14) using the three amino acid linkers to form DOTA-linker-BN(7-14). The resulting peptides were DOTA-GGG-BN(7-14), DOTA-GSG-BN(7-14), DOTA-GEG-BN(7-14), DOTA-GSS-BN(7-14), and DOTA-GEE-BN(7-14). The unlabeled peptides were evaluated in a competitive binding assay using human PC-3 androgen independent prostate cancer cells to determine IC₅₀ values. These peptides were radiolabeled with ⁶⁴Cu and evaluated for internalization into PC-3 cells and for their biodistribution in mice bearing PC-3 tumor xenografts. Results: The competitive binding assay gave IC₅₀ values of 50, 82, 2156, 32, and 19255 nM for the analogs with the GGG, GSG, GEG, GSS, and GEE linkers respectively. The GGG, GSG, and GSS analogs showed good internalization with 555, 680, and 666 fmol/mg being internalized at 24 h, while GEG and GEE did not internalize. The biodistribution studies showed that the tumor uptake for the GGG, GSG, and GSS analogs at 1 h after injection was 4.6, 2.8, and 3.8 percent of the injected dose per gram of tumor (% ID/g), respectively. This tumor uptake was greater than the uptake in all other tissues except for the liver, kidney and the GRPR-positive pancreas. Only the GGG linker demonstrated better tumor to liver and kidney ratios at 4 h compared to the original Aoc linker. In addition, administration of an excess of unlabeled BN demonstrated that the tumor uptake was GRPR-mediated. Conclusions: The tumor uptake of the GGG, GSG, and GSS analogs indicate that they might be useful for PET imaging of GRPR-expressing prostate cancer. MicroPET imaging studies using these analogs in tumor bearing mice are currently underway.