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Synthesis, Radiolabeling and In vitro GRP Receptor Targeting Studies of 99mTc‐Triaza‐X‐BBN[7‐14]NH2 (X=Serylserylserine, Glycylglycylglycine, Glycylserylglycine, or Beta Alanine)

Gastrin‐releasing peptide (GRP) receptors have been shown to be over‐expressed on several types of human cancer cells including prostate, breast, small cell lung and pancreatic cancers. Bombesin (BBN) is a 14 amino acid peptide that is an analogue of human gastrin‐releasing peptide, binding to GRP receptors (GRPr) with high affinity and specificity. The aim of these studies was to develop new 99mTc‐labeled BBN analogs having high tumor uptake and optimal pharmacokinetics for specific targeting of human prostate cancers. In this study, a new tridentate bifunctional chelating agent, 2‐(4,7‐bis(tert‐butoxycarbonyl)‐1,4,7‐triazonan‐1‐yl)‐acetic acid, was synthesized by first reacting 2 equivalents of BOC‐ON with 1,4,7‐triazacyclanone (Triaza) in CHCl3 at room temperature. The product, N, N′‐bis‐(tert‐butoxycarbonyl)‐1,4,7‐triazacyclanone, was alkylated using BrCH2COOH in acetonitrile. This new ligand framework was characterized by 1H and 13C NMR and electrospray ionization mass spectrometry (ESI‐MS). Solid‐phase peptide synthesis (SPPS) was used to produce Triaza‐X‐BBN[7‐14]NH2 conjugates with the following structure: Triaza‐X‐Q‐W‐A‐V‐G‐H‐L‐M‐(NH2), where the spacer group X=SSS, GGG, GSG and β‐Alanine (SSS=Serylserylserine, GGG=Glycylglycylglycine, and GSG=Glycylserylglycine). These conjugates were purified by reversed phase‐HPLC (RP‐HPLC) and characterized by ESI‐MS. In vitro competitive binding assays, using 125I‐Tyr4‐BBN as the radiolabelling gold standard, demonstrated IC50 values in the nanomolar range for all the new non‐metallated conjugates. For example, IC50s were 1.8±0.4, 3.9.±0.4, 1.9±0.3, and 1.3±0.2 nM for X=SSS, GGG, GSG and β‐Alanine, respectively. The new BBN conjugates were radiolabeled with Tc‐99m in moderate yield via the Isolink® radiolabeling kit available from Tyco Healthcare, St. Louis, MO. In vitro internalization and externalization analyses indicated receptor binding to be receptor specific in human, PC‐3, prostate cancer cells. These studies indicate that future in vivo studies in tumor‐bearing mouse models are justified.