[CANCER RESEARCH 64, 6707– 6715, September 15, 2004] Synthesis and Evaluation of Bombesin Derivatives on the Basis of Pan-Bombesin Peptides Labeled with Indium-111, Lutetium-177, and Yttrium-90 for Targeting Bombesin Receptor-Expressing Tumors Hanwen Zhang, 1 Jianhua Chen, 1 Christian Waldherr, 1 Karin Hinni, 1 Beatrice Waser, 2 Jean Claude Reubi, 2 and Helmut R. Maecke 1 1 Division of Radiological Chemistry, Institute of Nuclear Medicine, Department of Radiology, University Hospital, Basel; and 2 Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Berne, Switzerland ABSTRACT Bombesin receptors are overexpressed on a variety of human tumors like prostate, breast, and lung cancer. The aim of this study was to develop radiolabeled (Indium-111, Lutetium-177, and Yttrium-90) bombesin an- alogues with affinity to the three bombesin receptor subtypes for targeted radiotherapy. The following structures were synthesized: diethylenetri- aminepentaacetic acid--aminobutyric acid-[D-Tyr 6 , -Ala 11 , Thi 13 , Nle 14 ] bombesin (6 –14) (BZH1) and 1,4,7,10-tetraazacyclododecane-N,N,N,N -tetraacetic acid--aminobutyric acid-[D-Tyr 6 , -Ala 11 , Thi 13 , Nle 14 ] bombesin (6 –14) (BZH2). [ 111 In]-BZH1 and in particular [ 90 Y]-BZH2 were shown to have high affinity to all three human bombesin receptor subtypes with binding affinities in the nanomolar range. In human serum metabolic cleavage was found between -Ala 11 and His 12 with an approx- imate half-life of 2 hours. The metabolic breakdown was inhibited by EDTA and -Ala 11 -His 12 (carnosine) indicating that carnosinase is the active enzyme. Both 111 In-labeled peptides were shown to internalize into gastrin- releasing peptide-receptor–positive AR4 –2J and PC-3 cells with similar high rates, which were independent of the radiometal. The biodistribution studies of [ 111 In]-BZH1 and [ 111 In]-BZH2 ([ 177 Lu]-BZH2) in AR4 –2J tumor-bearing rats showed specific and high uptake in gastrin-releasing peptide-receptor–positive organs and in the AR4 –2J tumor. A fast clear- ance from blood and all of the nontarget organs except the kidneys was found. These radiopeptides were composed of the first pan-bombesin radioligands, which show great promise for the early diagnosis of tumors bearing not only gastrin-releasing peptide-receptors but also the other two bombesin receptor subtypes and may be of use in targeted radiotherapy of these tumors. INTRODUCTION The development of ligand-targeted therapeutics in anticancer ther- apy including drug-ligand conjugates has gained momentum in recent years (1). Systemic cytotoxic chemotherapy shows little selectivity and is limited by potentially serious side effects. One strategy to improve the lack of selectivity is to couple therapeutics to vectors like monoclonal antibodies, their fragments, or even smaller molecules (2). The cytotoxic drug part of conjugates used in ligand-targeted therapeutics is often composed of a therapeutic radiometal encapsu- lated by its bifunctional chelator. A very promising group of small targeting ligands is composed of regulatory peptides (3). A high number of peptide receptors were shown to be overexpressed in various human tumors (4). They are promising targets for molecular imaging and targeted therapy of cancer, because they are located on the plasma membrane and, upon binding of a ligand, the receptor-ligand complex is internalized. These findings were the basis for the development of diagnostic and thera- peutic radiopeptides useful in peptide receptor scintigraphy and tar- geted radiotherapy (5–10). Among the most relevant peptide recep- tors, the bombesin receptors are of major interest, because they were found to be overexpressed in various important cancers like prostate (11, 12), breast (13, 14), and small cell lung cancer (15). The human counterparts of bombesin, namely gastrin-releasing peptide (16) and neuromedin B (17), have been found in mammalian tissue. They bind to different bombesin receptor subtypes, such as the neuromedin B preferring receptor (BB1 receptor; ref. 18), the gastrin-releasing pep- tide preferring receptor (BB2; ref. 19), as well as the orphan bombesin receptor subtype-3 (BB3 receptor; ref. 20) and the BB4 receptor (21). The BB1, BB2, and BB3 receptors have been shown recently to be overexpressed on different human tumors (22). Gastrin-releasing pep- tide receptors were predominantly expressed in human prostate cancer (100%), gastrinoma (100%), and breast cancer (70%), whereas con- comitant expression of gastrin-releasing peptide receptor (33%) and BB3 receptor (40%) were found in small cell lung cancer. Also gastrin-releasing peptide receptor (40%) and BB3 (25%) were found concomitantly in renal cell carcinoma. Preferential expression of BB1 was found in intestinal carcinoids (11 of 24), and bronchial carcinoids had preferential BB3 receptor expression (9 of 26). These findings provide a possibility to apply bombesin-like pep- tides as a vehicle for delivering cytotoxic drugs (23–25) into tumor cells. In addition, radiolabeling may allow us to diagnose and treat these tumors (10, 26 –37). The sequence bombesin(7–14) was re- garded to be sufficient for the specific binding interaction with the gastrin-releasing peptide receptor (38, 39). Therefore, most radiola- beled bombesin-like peptides are based on the sequence bombesin (7–14) (10, 28 –31, 33–35). For example, different conjugates were developed using bifunctional chelators for labeling with 99m Tc, like N 2 S 2 (29), N 3 S (31), N  -histidinyl acetate (35), and diaminopropionic acid (36), using the carbonyl approach. Also, diethylenetriaminepen- taacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-N,N',N,N- tetraacetic acid (DOTA) were coupled to this sequence for labeling with hard Lewis acid radiometals like 111 In, 67, 68 Ga, 90 Y, and the lanthanides. Some 99m Tc-labeled peptides have been or are currently being investigated in gastrin-releasing peptide receptor-positive tu- mors in patients (30, 31, 33). Recently, a universal ligand, (D-Tyr 6 , -Ala 11 , Phe 13 , Nle 14 ) bomb- esin (6 –14), has been developed by Mantey et al. (40) and Pradhan et al. (41), which has high affinity to all of the bombesin receptor subtypes. The finding that not only the gastrin-releasing peptide receptor is overexpressed on human tumors but in some cases also neuromedin B and BB3 receptor subtypes prompted us to develop conjugates based on the slightly modified (Thi 13 versus Phe 13 ) universal bombesin ligand [D-Tyr 6 , -Ala 11 , Phe 13 , Nle 14 ] bombe- sin (6 –14) that can be labeled with hard Lewis acid-type metallic Received 12/09/03; revised 4/22/04; accepted 7/8/04. Grant support: Supported analytically by Novartis and financially by the Swiss National Science Foundation (Grant Nr. 3100A0 –100390), by the Amt fu ¨r Ausbildungs- beitra ¨ge (H. Zhang), the Commission for Technology and Innovation (KTI-project 4668 –1 EUS), and by Mallinckrodt Med. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Helmut Maecke, Division of Radiological Chemistry, Institute of Nuclear Medicine, Department of Radiology, University Hospital, Petersgraben 4, CH-4031 Basel, Switzerland. Phone: 41-61-265-46-99; Fax: 41-61-265-55-59; E-mail: hmaecke@uhbs.ch. ©2004 American Association for Cancer Research. 6707 Research. on April 22, 2016. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from