Substitution of l-Tryptophan by α-Methyl-l-Tryptophan in 177Lu-RM2 Results in 177Lu-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-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.

Methods

Peptides were synthesized by solid-phase peptide synthesis and labeled with 177Lu. 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. 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 vitro and in 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.

Conclusion

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.