Substitution of l-Tryptophan by <b>α</b>-Methyl-l-Tryptophan in ¹⁷⁷Lu-RM2 Results in ¹⁷⁷Lu-AMTG, a High-Affinity Gastrin-Releasing Peptide Receptor Ligand with Improved In Vivo Stability

Abstract

Theranostic applications targeting the gastrin-releasing peptide receptor (GRPR) have shown promising results. When compared with other peptide ligands for radioligand therapy, the most often used GRPR ligand, DOTA-Pip⁵-d-Phe⁶-Gln⁷-Trp⁸-Ala⁹-Val¹⁰-Gly¹¹-His¹²-Sta¹³-Leu¹⁴-NH₂ (RM2), may be clinically impacted by limited metabolic stability. With the aim of improving the metabolic stability of RM2, we investigated whether the metabolically unstable Gln⁷-Trp⁸ bond within the pharmacophore of RM2 can be stabilized via substitution of l-Trp⁸ by α-methyl-l-tryptophan (α-Me-l-Trp) and whether the corresponding DOTAGA analog might also be advantageous. A comparative preclinical evaluation of ¹⁷⁷Lu-α-Me-l-Trp⁸-RM2 (¹⁷⁷Lu-AMTG) and its DOTAGA counterpart (¹⁷⁷Lu-AMTG2) was performed using ¹⁷⁷Lu-RM2 and ¹⁷⁷Lu-NeoBOMB1 as reference compounds. <b>Methods:</b> Peptides were synthesized by solid-phase peptide synthesis and labeled with ¹⁷⁷Lu. Lipophilicity was determined at pH 7.4 (log<i>D</i>_7.4). Receptor-mediated internalization was investigated on PC-3 cells (37°C, 60 min), whereas GRPR affinity (half-maximal inhibitory concentration) was determined on both PC-3 and T-47D cells. Stability toward peptidases was examined in&nbsp;vitro (human plasma, 37°C, 72 ± 2 h) and in&nbsp;vivo (murine plasma, 30 min after injection). Biodistribution studies were performed at 24 h after injection, and small-animal SPECT/CT was performed on PC-3 tumor–bearing mice at 1, 4, 8, 24, and 28 h after injection. <b>Results:</b> Solid-phase peptide synthesis yielded 9%–15% purified labeling precursors. ¹⁷⁷Lu labeling proceeded quantitatively. Compared with ¹⁷⁷Lu-RM2, ¹⁷⁷Lu-AMTG showed slightly improved GRPR affinity, a similar low internalization rate, slightly increased lipophilicity, and considerably improved stability in&nbsp;vitro and in&nbsp;vivo. In vivo, ¹⁷⁷Lu-AMTG exhibited the highest tumor retention (11.45 ± 0.43 percentage injected dose/g) and tumor-to-blood ratio (2,702 ± 321) at 24 h after injection, as well as a favorable biodistribution profile. As demonstrated by small-animal SPECT/CT imaging, ¹⁷⁷Lu-AMTG also revealed a less rapid clearance from tumor tissue. Compared with ¹⁷⁷Lu-AMTG, ¹⁷⁷Lu-AMTG2 did not show any further benefits. <b>Conclusion:</b> The results of this study, particularly the superior metabolic stability of ¹⁷⁷Lu-AMTG, strongly recommend a clinical evaluation of this novel GRPR-targeted ligand to investigate its potential for radioligand therapy of GRPR-expressing malignancies.

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