Gastrin-releasing peptide receptors (GRPRs) expressed on human tumors can serve as molecular targets for radiolabeled peptide analogs based on the frog tetradecapeptide bombesin (BBN). We have recently expanded this approach toward human GRP(18-27) sequences and introduced 99mTc-demomedin C, our first radiotracer based on GRP(18-27), showing favorable biologic characteristics during preclinical evaluation in rodents. We now present a series of 99mTc-demomedin C analogs, generated by single-Gly 24 or double-Gly24/Met27 substitutions in the peptide chain, and compare their performance in GRPR-positive in vitro and in vivo models. Methods: The SARNC ([(N4)Gly18]GRP(18-27)) analogs (SARNC2 dAla24, SARNC3 dAla24/Nle27, SARNC4 dAla24/Leu27, SARNC5 βAla24, and SARNC6 Sar24) were synthesized on the solid support and purified by high-performance liquid chromatography (HPLC). Competition binding experiments against [125I-Tyr4]BBN were conducted in GRPR-positive PC-3 cell membranes. Internalization of 99mTc radioligands was compared in PC-3 cells at 37°C. Metabolic stability was studied by HPLC analysis of blood samples collected 5 min after injection of radiopeptides in mice. Biodistribution was performed by injecting a 99mTc-SARNC bolus (185 kBq [5 μCi], 100 μL, 10 pmol of peptide ± 40 nmol of Tyr 4-BBN: in vivo GRPR blockade) in severe combined immune deficient mice bearing PC-3 xenografts. Results: SARNCs bound to GRPR with high affinity (range of 50% inhibitory concentration [IC50] values, 0.3 nM [SARNC5] to 9.3 nM [SARNC4]). 99mTc-SARNCs specifically internalized in PC-3 cells, with 99mTc-SARNC5 displaying the fastest internalization rate. 99mTc-SARNCs showed distinct degradation rates (17% [ 99mTc-SARNC3] to >50% [99mTc-SARNC4] remaining intact). All 99mTc-SARNCs efficiently and specifically localized in GRPR-positive PC-3 xenografts in mice (4.4 percentage injected dose per gram [%ID/g] [99mTc-SARNC4] to 12.0 %ID/g [99mTc- SARNC2] at 4 h after injection). 99mTc-SARNC6 displayed the highest tumor-to-nontumor ratios followed by 99mTc-SARNC2. Conclusion: This structure-activity relationship study has shown the impact of single-Gly 24 or double-Gly24/Met27 substitutions in the 99mTc-SARNC1 motif on key biologic parameters, including GRPR affinity, internalization efficiency, and in vivo stability, which eventually determine the pharmacokinetic profile of resulting radiopeptides. By revealing improved analogs, this study has strengthened the applicability perspectives of radioligands based on human GRP sequences in the detection and therapy of GRPRexpressing tumors in humans. COPYRIGHT

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The Journal of Nuclear Medicine
Department of Nuclear Medicine

Marsouvanidis, I-P, Maina, T, Sallegger, W, Krenning, E.P, de Jong, M, & Nock, B.A. (2013). 99mTc radiotracers based on human GRP(18-27): Synthesis and comparative evaluation. The Journal of Nuclear Medicine, 54(10), 1797–1803. doi:10.2967/jnumed.112.118695