antagonist radioligands ͉ tumor targeting ͉ peptide hormones ͉ neuropeptides ͉ receptor internalization P eptide receptor targeting in vivo is a successful method to image and treat various types of cancers (1). The best example is somatostatin receptor targeting with 111 In-, 90 Y-, or 177 Lu-labeled somatostatin radioligands that are injected into the patients intravenously and accumulate in their somatostatin receptor-expressing tumors. For this purpose, agonists have been selected. The rationale is that agonists, after high-affinity binding to the receptor, usually trigger internalization of the ligandreceptor complex (2). This process of internalization is the basis for an efficient accumulation of the radioligand in a cell over time (1,(3)(4)(5), and it has been considered a crucial step in the process of in vivo receptor targeting with radiolabeled peptides (4-6). Recently, a highly significant correlation between the rate of ligand internalization in vitro into AR42J cells expressing somatostatin receptor subtype 2 (sst 2 ) and the in vivo uptake in the sst 2 -expressing rat tumor model has been reported

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.

RM2 was shown to be a potent GRPr antagonist. Pharmacokinetics and imaging studies indicate that (111)In-RM2 and (68)Ga-RM2 are ideal candidates for clinical SPECT and PET studies.

Somatostatin-based radiolabeled peptides have been successfully introduced into the clinic for targeted imaging and radionuclide therapy of somatostatin receptor (sst)-positive tumors, especially of subtype 2 (sst2). The clinically used peptides are exclusively agonists. Recently, we showed that radiolabeled antagonists may be preferable to agonists because they showed better pharmacokinetics, including higher tumor uptake. Factors determining the performance of radioantagonists have only scarcely been studied. Here, we report on the development and evaluation of four 64 Cu or 68 Ga radioantagonists for PET of sst2-positive tumors. Methods: The novel antagonist p-Cl-Phecyclo(D-Cys-Tyr-D-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)D-Tyr-NH 2 (LM3) was coupled to 3 macrocyclic chelators, namely 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A), 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), and DOTA. 64/nat Cu-and 68/nat Ga-NODAGA-LM3 were prepared at room temperature, and 64/nat Cu-CB-TE2A-LM3 and 68/nat Ga-DOTA-LM3 were prepared at 95°C. Binding affinity and antagonistic properties were determined with receptor autoradiography and immunofluorescence microscopy using human embryonic kidney (HEK)-sst2 cells. In vitro internalization and dissociation was evaluated using the same cell line. Biodistribution and small-animal PET studies were performed with HEK-sst2 xenografts. Results: All metallopeptides demonstrated antagonistic properties. The affinities depend on chelator and radiometal and vary about 10-fold; 68/nat Ga-NODAGA-LM3 has the lowest half maximal inhibitory concentration (1.3 6 0.3 nmol/L). The biodistribution studies show impressive tumor uptake at 1 h after injection, particularly of 64 Cu-and 68 Ga-NODAGA-LM3 (;40 percentage injected dose per gram of tissue [%ID/g]), which were proven to be specific. Background clearance was fast and the tumor washout relatively slow for 64 Cu-NODAGA-LM3 (;15 %ID/g, 24 h after injection) and almost negligible for 64 Cu-CB-TE2A-LM3 (26.9 6 3.3 %ID/g and 21.6 6 2.1 %ID/g, 4 and 24 h after injection, respectively). Tumor-to-normal-tissue ratios were significantly higher for 64 Cu-NODAGA-LM3 than for 64 Cu-CB-TE2A-LM3 (tumor-to-kidney, 12.8 6 3.6 and 1.7 6 0.3, respectively; tumor-to-muscle, 1,342 6 115 and 75.2 6 8.5, respectively, at 24 h, P , 0.001). Small-animal PET shows clear tumor localization and high image contrast, especially for 64 Cu-and 68 Ga-NODAGA-LM3. Conclusion: This article demonstrates the strong dependence of the affinity and pharmacokinetics of the somatostatin-based radioantagonists on the chelator and radiometal. 64 Cu-and 68 Ga-NODAGA-LM3 and 64 Cu-CB-TE2A-LM3 are promising candidates for clinical translation because of their favorable pharmacokinetics and the high image contrast on PET scans.

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