Biodistribution of the Bombesin Receptor-Targeted Radiopharmaceutical Precursor BBN/C1-C2 in a Prostate Cancer Model

Gastrin-releasing peptide receptor (GRPR) is a G protein-coupled receptor expressed in the central nervous system, the gastrointestinal tract, the pancreas, and the adrenal cortical tissue regulating their physiological functions. In addition to normal tissues, GRPR is overexpressed in many solid cancers. At present, several radiolabeled ligands targeting GRPR have been introduced into clinical practice for cancer diagnostics and radioligand therapy. However, there were found the high uptake of radiopharmaceuticals in normal organs and low bioavailability of the drugs caused by their stability. Therefore, GRPR radioantagonists with improved proteolytic stability and longer residence time in tumor foci with low uptake in non-target organs are currently being sought to improve the therapeutic efficacy.

The aim of the study was to analyze the biodistribution of the BBN/C1-C2 molecule created on the basis of bombesin and knottin and its accumulation dynamics in the tumor in an in vivo model.

Materials and Methods. The study analyzed the biodistribution of Cy7.5-labeled BBN/C1-C2 peptide based on bombesin and knottin U5-Sth1a and produced using solid-phase peptide synthesis. The investigation was carried out on a mouse model Nu/Nu with a prostate cancer solid tumor (PC-3 culture) transplanted into the right side, expressing GRPR, using real-time surface fluorescent imaging on days 2 and 5 after intravenous administration of the molecules under study.

Results. The biodistribution analysis showed the selective binding of the BBN/C1-C2 molecule to a tumor, as well as its holding power on the tumor surface for up to 5 days without internalization into cells with low accumulation in normal organs and tissues.

Conclusion. We managed to obtain a stable molecule; however, U5-Sth1a toxin tropic to ion channels was used as a scaffold, so the resulting molecule retained the domain responsible for attaching to the target channel and typical for knottin. In the future, when using the molecule as a ligand for radiotherapy, it can have a negative effect on the patient’s heart due to the supplementary radiation load. In this regard, the BBN/C1-C2 molecule requires the extension study.

1. Jensen R.T., Battey J.F., Spindel E.R., Benya R.V. International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states. Pharmacol Rev 2008; 60(1): 1–42, https://doi.org/10.1124/pr.107.07108.
2. Shimoda J. Effects of bombesin and its antibody on growth of human prostatic carcinoma cell lines. Nihon Hinyokika Gakkai Zasshi 1992; 83(9): 1459–1468, https://doi.org/10.5980/jpnjurol1989.83.1459.
3. D’Onofrio A., Engelbrecht S., Läppchen T., Rominger A., Gourni E. GRPR-targeting radiotheranostics for breast cancer management. Front Med (Lausanne) 2023; 10: 1250799, https://doi.org/10.3389/fmed.2023.1250799.
4. Liu P., Tu Y., Tao J., Liu Z., Wang F., Ma Y., Li Z., Han Z., Gu Y. GRPR-targeted SPECT imaging using a novel bombesin-based peptide for colorectal cancer detection. Biomater Sci 2020; 8(23): 6764–6772, https://doi.org/10.1039/d0bm01432j.
5. Kähkönen E., Jambor I., Kemppainen J., Lehtiö K., Grönroos T.J., Kuisma A., Luoto P., Sipilä H.J., Tolvanen T., Alanen K., Silén J., Kallajoki M., Roivainen A., Schäfer N., Schibli R., Dragic M., Johayem A., Valencia R., Borkowski S., Minn H. In vivo imaging of prostate cancer using [68Ga]-labeled bombesin analog BAY86-7548. Clin Cancer Res 2013; 19(19): 5434–5443, https://doi.org/10.1158/1078-0432.CCR-12-3490.
6. Stoykow C., Erbes T., Maecke H.R., Bulla S., Bartholomä M., Mayer S., Drendel V., Bronsert P., Werner M., Gitsch G., Weber W.A., Stickeler E., Meyer P.T. Gastrin-releasing peptide receptor imaging in breast cancer using the receptor antagonist (68)Ga-RM2 and PET. Theranostics 2016; 6(10): 1641–1650, https://doi.org/10.7150/thno.14958.
7. Kurth J., Krause B.J., Schwarzenböck S.M., Bergner C., Hakenberg O.W., Heuschkel M. First-in-human dosimetry of gastrin-releasing peptide receptor antagonist [177Lu]Lu-RM2: a radiopharmaceutical for the treatment of metastatic castration-resistant prostate cancer. Eur J Nucl Med Mol Imaging 2020; 47(1): 123–135, https://doi.org/10.1007/s00259-019-04504-3.
8. Nock B.A., Kaloudi A., Lymperis E., Giarika A., Kulkarni H.R., Klette I., Singh A., Krenning E.P., de Jong M., Maina T., Baum R.P. Theranostic perspectives in prostate cancer with the gastrin-releasing peptide receptor antagonist NeoBOMB1: preclinical and first clinical results. J Nucl Med 2017; 58(1): 75–80, https://doi.org/10.2967/jnumed.116.178889.
9. Linder K.E., Metcalfe E., Arunachalam T., Chen J., Eaton S.M., Feng W., Fan H., Raju N., Cagnolini A., Lantry L.E., Nunn A.D., Swenson R.E. In vitro and in vivo metabolism of Lu-AMBA, a GRP-receptor binding compound, and the synthesis and characterization of its metabolites. Bioconjug Chem 2009; 20(6): 1171–1178, https://doi.org/10.1021/bc9000189.
10. Lymperis E., Kaloudi A., Sallegger W., Bakker I.L., Krenning E.P., de Jong M., Maina T., Nock B.A. Radiometal-dependent biological profile of the radiolabeled gastrin-releasing peptide receptor antagonist SB3 in cancer theranostics: metabolic and biodistribution patterns defined by neprilysin. Bioconjug Chem 2018; 29(5): 1774–1784, https://doi.org/10.1021/acs.bioconjchem.8b00225.
11. Nock B.A., Kaloudi A., Kanellopoulos P., Janota B., Bromińska B., Iżycki D., Mikołajczak R., Czepczynski R., Maina T. [99mTc]Tc-DB15 in GRPR-targeted tumor imaging with SPECT: from preclinical evaluation to the first clinical outcomes. Cancers (Basel) 2021; 13(20): 5093, https://doi.org/10.3390/cancers13205093.
12. Beloborodov E.A., Iurova E.V., Fomin A.N., Saenko Y.V. Development and synthesis of bombesin-based radiopharmaceutical precursors modified with knottin. Sovremennye tehnologii v medicine 2024; 16(2): 5, https://doi.org/10.17691/stm2024.16.2.01.
13. El-Gogary R.I., Rubio N., Wang J.T., Al-Jamal W.T., Bourgognon M., Kafa H., Naeem M., Klippstein R., Abbate V., Leroux F., Bals S., Van Tendeloo G., Kamel A.O., Awad G.A., Mortada N.D., Al-Jamal K.T. Polyethylene glycol conjugated polymeric nanocapsules for targeted delivery of quercetin to folate-expressing cancer cells in vitro and in vivo. ACS Nano 2014; 8(2): 1384–1401, https://doi.org/10.1021/nn405155b.
14. Wang C., Pulli B., Jalali Motlagh N., Li A., Wojtkiewicz G.R., Schmidt S.P., Wu Y., Zeller M.W.G., Chen J.W. A versatile imaging platform with fluorescence and CT imaging capabilities that detects myeloperoxidase activity and inflammation at different scales. Theranostics 2019; 9(25): 7525–7536, https://doi.org/10.7150/thno.36264.
15. Cuttitta F., Carney D.N., Mulshine J., Moody T.W., Fedorko J., Fischler A., Minna J.D. Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer. Nature 1985; 316(6031): 823–826, https://doi.org/10.1038/316823a0.
16. Begum A.A., Moyle P.M., Toth I. Investigation of bombesin peptide as a targeting ligand for the gastrin releasing peptide (GRP) receptor. Bioorg Med Chem 2016; 24(22): 5834–5841, https://doi.org/10.1016/j.bmc.2016.09.039.
17. Okarvi S.M., Jammaz I.A. Preparation and evaluation of bombesin peptide derivatives as potential tumor imaging agents: effects of structure and composition of amino acid sequence on in vitro and in vivo characteristics. Nucl Med Biol 2012; 39(6): 795–804, https://doi.org/10.1016/j.nucmedbio.2012.01.002.
18. Maina T., Nock B.A. From bench to bed: new gastrin-releasing peptide receptor-directed radioligands and their use in prostate cancer. PET Clin 2017; 12(2): 205–217, https://doi.org/10.1016/j.cpet.2016.12.002.
19. Baratto L., Jadvar H., Iagaru A. Prostate cancer theranostics targeting gastrin-releasing peptide receptors. Mol Imaging Biol 2018; 20(4): 501–509, https://doi.org/10.1007/s11307-017-1151-1.
20. Maddalena M.E., Fox J., Chen J., Feng W., Cagnolini A., Linder K.E., Tweedle M.F., Nunn A.D., Lantry L.E. 177Lu-AMBA biodistribution, radiotherapeutic efficacy, imaging, and autoradiography in prostate cancer models with low GRP-R expression. J Nucl Med 2009; 50(12): 2017–2024, https://doi.org/10.2967/jnumed.109.064444.
21. Wild D., Frischknecht M., Zhang H., Morgenstern A., Bruchertseifer F., Boisclair J., Provencher-Bolliger A., Reubi J.C., Maecke H.R. Alpha- versus beta-particle radiopeptide therapy in a human prostate cancer model (213Bi-DOTA-PESIN and 213Bi-AMBA versus 177Lu-DOTA-PESIN). Cancer Res 2011; 71(3): 1009–1018, https://doi.org/10.1158/0008-5472.CAN-10-1186.
22. Baum R., Prasad V., Mutloka N., Frischknecht M., Maecke H., Reubi J.C. Molecular imaging of bombesin receptors in various tumors by Ga-68 AMBA PET/CT: first results. J Nucl Med 2007; 48(Suppl 2): 79.
23. Bodei L., Ferrari M., Nunn A., Llull J., Cremonesi M., Martano L., Laurora G., Scardino E., Tiberini S., Bufi G., Eaton S., de Cobelli O., Paganelli G. Lu-177-AMBA bombesin analogue in hormone refractory prostate cancer patients: a phase I escalation study with single-cycle administrations. Eur J Nucl Med Mol 2007; 34 (Suppl S2): S221.
24. Cornelio D.B., Roesler R., Schwartsmann G. Gastrin-releasing peptide receptor as a molecular target in experimental anticancer therapy. Ann Oncol 2007; 18(9): 1457–1466, https://doi.org/10.1093/annonc/mdm058.
25. Kenakin T. The potential for selective pharmacological therapies through biased receptor signaling. BMC Pharmacol Toxicol 2012; 13: 3, https://doi.org/10.1186/2050-6511-13-3.
26. Thundimadathil J. Cancer treatment using peptides: current therapies and future prospects. J Amino Acids 2012; 2012: 967347, https://doi.org/10.1155/2012/967347.
27. Hoppenz P., Els-Heindl S., Beck-Sickinger A.G. Identification and stabilization of a highly selective gastrin-releasing peptide receptor agonist. J Pept Sci 2019; 25(12): e3224, https://doi.org/10.1002/psc.3224.
28. Valverde I.E., Bauman A., Kluba C.A., Vomstein S., Walter M.A., Mindt T.L. 1,2,3-triazoles as amide bond mimics: triazole scan yields protease-resistant peptidomimetics for tumor targeting. Angew Chem Int Ed Engl 2013; 52(34): 8957–8960, https://doi.org/10.1002/anie.201303108.
29. De K., Banerjee I., Sinha S., Ganguly S. Synthesis and exploration of novel radiolabeled bombesin peptides for targeting receptor positive tumor. Peptides 2017; 89: 17–34, https://doi.org/10.1016/j.peptides.2017.01.002.
30. Tu Y., Tao J., Wang F., Liu P., Han Z., Li Z., Ma Y., Gu Y. A novel peptide targeting gastrin releasing peptide receptor for pancreatic neoplasm detection. Biomater Sci 2020; 8(9): 2682–2693, https://doi.org/10.1039/d0bm00162g.
31. Iurova E., Beloborodov E., Tazintseva E., Fomin A., Shutov A., Slesarev S., Saenko Y., Saenko Y. Arthropod toxins inhibiting Ca2+ and Na+ channels prevent AC-1001 H3 peptide-induced apoptosis. J Pept Sci 2021; 27(1): e3288, https://doi.org/10.1002/psc.3288.
32. Aronov D.M., Lupanov V.P. Calcium antagonists in the treatment of patients with cardiovascular diseases. Focus on amlodipine. Russkij medicinskij zurnal 2007; 15(4): 275–281.

Beloborodov E.A., Iurova E.V., Dolgova D.R., Ermakova A.A., Sugak D.E., Fomin A.N., Saenko Y.V. Biodistribution of the Bombesin Receptor-Targeted Radiopharmaceutical Precursor BBN/C1-C2 in a Prostate Cancer Model. Sovremennye tehnologii v medicine 2025; 17(6): 25, https://doi.org/10.17691/stm2025.17.6.03