Two bombesin analogs, Demobesin 4 and Demobesin 1, were characterized in vitro as gastrin-releasing peptide (GRP) receptor agonist and antagonist, respectively, and were compared as 99m Tc-labeled ligands for their in vitro and in vivo tumortargeting properties. Methods: N 4 -[Pro 1 ,Tyr 4 ,Nle 14 ]Bombesin (Demobesin 4) and N 4 -[D-Phe 6 ,Leu-NHEt 13 ,des-Met 14 ]bombesin(6-14) (Demobesin 1) were characterized in vitro for their binding properties with GRP receptor autoradiography using GRP receptor-transfected HEK293 cells, PC3 cells, and human prostate cancer specimens. Their ability to modulate calcium mobilization in PC3 and transfected HEK293 cells was analyzed as well as their ability to trigger internalization of the GRP receptor in transfected HEK293 cells, as determined qualitatively by immunofluorescence microscopy and quantitatively by enzyme-linked immunosorbent assay (ELISA). Further, their internalization properties as 99m Tc-labeled radioligands were tested in vitro in both cell lines. Finally, their biodistribution was analyzed in PC3 tumor-bearing mice. Results: A comparable binding affinity with the 50% inhibitory concentration (IC 50 ) in the nanomolar range was measured for Demobesin 4 and Demobesin 1 in all tested tissues. Demobesin 4 behaved as an agonist by strongly stimulating calcium mobilization and by triggering GRP receptor internalization. Demobesin 1 was ineffective in stimulating calcium mobilization and in triggering GRP receptor internalization. However, in these assays, it behaved as a competitive antagonist as it reversed completely the agonist-induced effects in both systems. 99m Tc-Labeled Demobesin 1 was only weakly taken up by PC3 cells or GRP receptor-transfected HEK293 cells (10% and 5%, respectively, of total added radioactivity) compared with 99m Tc-labeled Demobesin 4 (45% of total added radioactivity in both cell lines). Remarkably, the biodistribution study revealed a much more pronounced uptake at 1, 4, and 24 h after injection of 99m Tc-labeled Demobesin 1 in vivo into PC3 tumors than 99m Tc-labeled Demobesin 4. In vivo competition experiments demonstrated a specific uptake in PC3 tumors and in physiologic GRP receptor-expressing tissues. The tumor-tokidney ratios were 0.7 for Demobesin 4 and 5.2 for Demobesin 1 at 4 h. Conclusion: This comparative in vitro/in vivo study with Demobesin 1 and Demobesin 4 indicates that GRP receptor antagonists may be superior targeting agents to GRP receptor agonists, suggesting a change of paradigm in the field of bombesin radiopharmaceuticals.

Four open chain tetraamine-functionalized bombesin (BB) analogues were synthesized [parent tetradecapeptide-based Demobesin 3 and 4 and BB(7-14)-based Demobesin 5 and 6]. Labeling with (99m)Tc afforded high-purity and high specific activity radiotracers. Peptides showed high affinity for the human GRP-R (GRP-R = gastrin releasing peptide receptor) expressed in PC-3 cells. In human tumors preferentially expressing single bombesin receptor subtypes, they showed high affinity for the GRP-R, less affinity for the NMB-R (NMB-R = neuromedin B receptor) and no affinity for the orphan BB(3)-R (bombesin subtype 3 receptor). [(99m)Tc]Demobesin 3-6 efficiently internalized in a time- and dose-dependent manner in PC-3 cells and showed a high and specific uptake in human PC-3 xenografts and the pancreas of nude mice. [(99m)Tc]Demobesin 3 and 4 were rapidly excreted via the kidneys while the truncated analogues were predominantly processed by the hepatobiliary system. Patient studies are scheduled for validating the suitability of [(99m)Tc]Demobesin 3 and 4 in the GRP-R-targeted imaging of tumors.

Demobesin 1 is a potent new GRP-R-selective bombesin (BN) analogue containing an open chain tetraamine chelator for stable technetium-99m binding. Following a convenient labelling protocol, the radiopeptide, [(99m)Tc]Demobesin 1, formed in nearly quantitative yields and with high specific activities. Both unlabelled and labelled peptide demonstrated high-affinity binding in membrane preparations of the human androgen-independent prostate adenocarcinoma PC-3 cell line. The IC(50) values determined for Demobesin 1 and [Tyr(4)]BN were 0.70+/-0.08 n M and 1.5+/-0.20 n M, respectively, while the K(d) defined for [(99m)Tc/(99g)Tc]Demobesin 1 was 0.67+/-0.10 n M. [(99m)Tc]Demobesin 1 was rather stable in murine plasma, whereas it degraded rapidly in kidney and liver homogenates. After injection in healthy Swiss albino mice, [(99m)Tc]Demobesin 1 accumulated very efficiently in the target organs (pancreas, intestinal tract) via a GRP-R-mediated process, as shown by in vivo receptor blocking experiments. An equally high and GRP-R-mediated uptake was exhibited by [(99m)Tc]Demobesin 1 after injection in PC-3 tumour-bearing athymic mice. The initial high radioligand uptake of 16.2+/-3.1%ID/g in the PC-3 xenografts at 1 h p.i. remained at a similar level (15.61+/-1.19%ID/g) at 4 h p.i. Even after 24 h p.i., when the radioactivity had cleared from all other tissues, a value of 5.24+/-0.67%ID/g was still observed in the tumour. The high and prolonged localization of [(99m)Tc]Demobesin 1 at the tumour site and its rapid background clearance are very promising qualities for GRP-R-targeted tumour imaging in man.