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

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-Pip(5)-d-Phe(6)-Gln(7)-Trp(8)-Ala(9)-Val(10)-Gly(11)-His(12)-Sta(13)-Leu(14)-NH(2) (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 Gln(7)-Trp(8) bond within the pharmacophore of RM2 can be stabilized via substitution of l-Trp(8) by α-methyl-l-tryptophan (α-Me-l-Trp) and whether the corresponding DOTAGA analog might also be advantageous. A comparative preclinical evaluation of (177)Lu-α-Me-l-Trp(8)-RM2 ((177)Lu-AMTG) and its DOTAGA counterpart ((177)Lu-AMTG2) was performed using (177)Lu-RM2 and (177)Lu-NeoBOMB1 as reference compounds. Methods: Peptides were synthesized by solid-phase peptide synthesis and labeled with (177)Lu. Lipophilicity was determined at pH 7.4 (logD(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 vitro (human plasma, 37°C, 72 ± 2 h) and in 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. Results: Solid-phase peptide synthesis yielded 9%–15% purified labeling precursors. (177)Lu labeling proceeded quantitatively. Compared with (177)Lu-RM2, (177)Lu-AMTG showed slightly improved GRPR affinity, a similar low internalization rate, slightly increased lipophilicity, and considerably improved stability in vitro and in vivo. In vivo, (177)Lu-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, (177)Lu-AMTG also revealed a less rapid clearance from tumor tissue. Compared with (177)Lu-AMTG, (177)Lu-AMTG2 did not show any further benefits. Conclusion: The results of this study, particularly the superior metabolic stability of (177)Lu-AMTG, strongly recommend a clinical evaluation of this novel GRPR-targeted ligand to investigate its potential for radioligand therapy of GRPR-expressing malignancies.