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

Abstract

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.

Keywords:

• gastrin‐releasing peptide
• bombesin
• prostate cancer

Acknowledgments

This material is the result of work supported with resources and the use of facilities at the Harry S. Truman Memorial Veterans' Hospital. Columbia, MO, 65201 and the University of Missouri‐Columbia School of Medicine, Columbia, MO 65211, USA. This work was funded in part by grants from the National Institute of Biomedical Imaging and Bioengineering (1R21EB000833‐01A1) and United States Department of Veterans' Affairs VA Merit Award.