Development of lutetium-labeled bombesin derivates: relationship between structure and diagnostic-therapeutic activity for prostate tumor

Bombesin (BBN) receptors - in particular, the gastrin-releasing peptide (GRP) receptor peptide - have been shown to be massively over expressed in several human tumors types, including prostate cancer, and could be an alternative as target for its treatment by radionuclide therapy (RNT). A large number of BBN analogs had already been synthesized for this purpose and have shown to reduce tumor growth in mice. Nevertheless, most of the studied analogs exhibit high abdominal accumulation, especially in pancreas. This abdominal accumulation may represent a problem in clinical use of radiolabeled bombesin analogs probably due to serious side effects to patients. The goal of the present work was to radiolabel a novel series of bombesin derivatives with lutetium-177 and to evaluate the relationship between their structure and diagnostic-therapeutic activity for prostate tumor. The generic structure of studied peptides is DOTA-Phe-(Gly)n-BBN(6-14), where DOTA is the chelator, n is the number of glycine amino acids of Phe-(Gly)n spacer and BBN(6-14) is the bombesin sequence from the amino acid 6 to the amino acid 14. Preliminary studies were done to establish the ideal labeling conditions for obtaining the highest yield of labeled bombesin derivatives, determined by instant thin layer chromatography (ITLC-SG) and high performance liquid chromatography (HPLC). The stability of the preparations was evaluated either after storing at 2-8 degree C or incubation in human serum at 37 degree C and the partition coefficient was determined in n:octanol:water. In vivo studies were performed in both healthy Balb-c and Nude mice bearing PC-3 xenografts, in order to characterize the biological properties of labeled peptides. In vitro studies involved the evaluation of cold bombesin derivatives effect in PC-3 cells proliferation. Bombesin derivatives were successfully labeled with high yield at optimized conditions and exhibited high stability at 4 degree C. The analysis of the stability in human serum suggested a time-course metabolic degradation of labeled peptides by serum enzymes. The addition of four glycine amino acids in the spacer of bombesin derivatives resulted in slower degradation by human serum enzymes after 4 hours of incubation. HPLC and partition coefficient studies showed that bombesin derivatives present low lipophilicity and the increase of glycine amino acids number in peptides spacers slightly reduced their lipophilicity. The radiolabeled bombesin derivatives presented fast blood clearance, rapid excretion, performed mainly by renal pathway, and low abdominal accumulation in in vivo studies in Balb-c mice. Investigations in Nude mice bearing PC-3 tumor showed that the radio peptides can target tumor cells. Higher tumor uptake was observed with the derivatives of larger spacers at 1 hour post injection, but higher tumor retention after 4 and 24 hours was observed with the derivative of smaller spacer. In addition, tumor uptake showed to be specific and allowed tumor detection by scintigraphy imaging, especially 30 minutes post injection. In addition, the studied bombesin derivatives did not present proliferative or cytotoxic effect to PC-3 cells in vitro and can be characterized as weak agonists or antagonists of bombesin receptors. The results of this work showed that phenyl-glycine extended bombesin derivatives are promising for in vivo applications. The higher tumor uptake of derivatives with larger spacer suggests that these molecules are more applicable in diagnostic procedures. The derivative with smaller spacer is more useful to be applied in radionuclide therapy, because of its higher tumor retention. (author)