Studies on the impact of modifications at the Gln-Trp site in RM2-based GRPR ligands.

Background: One of the most studied, and preclinically as well as clinically applied gastrin-releasing peptide receptor (GRPR) ligands represents the antagonist RM2 (DOTA-Pip⁵-D-Phe⁶-Gln⁷-Trp⁸-Ala⁹-Val¹⁰-Gly¹¹-His¹²-Sta¹³-Leu¹⁴-NH₂). As an improved in vivo stability was observed for a RM2 analog comprising the unnatural amino acid α-methyl-L-tryptophan instead of L-Trp, we aimed to elucidate the impact of other unnatural amino acids (homoserine [Hse], β-(3-benzothienyl)alanine [Bta]) at the metabolically less stable Gln-Trp site. Furthermore, we conjugated either DOTA, NOTA or NODAGA to the RM2 peptide and its modified derivatives, and evaluated each analog preclinically using ⁶⁸Ga and ⁶⁴Cu, as well as ¹⁷⁷Lu (only DOTA-comprising compounds).

Results: GRPR affinity and lipophilicity of RM2 derivatives were in a range of 1.2-8.4 nM and - 2.9 to - 1.1 (^nat/68Ga-labeled), 1.7-33.0 nM and - 2.4 to - 1.6 (^nat/64Cu-labeled), as well as 3.0-19.7 nM and - 3.2 to - 1.8 (^nat/177Lu-labeled), respectively. Both, [¹⁷⁷Lu]Lu-[Hse⁷]RM2 and [¹⁷⁷Lu]Lu-[Bta⁸]RM2 revealed distinctly lower in vivo stability (< 20% intact at 15 min post-injection) than [¹⁷⁷Lu]Lu-[α-Me-Trp⁸]RM2 (= [¹⁷⁷Lu]Lu-AMTG) and [¹⁷⁷Lu]Lu-RM2 (> 30% intact at 30 min post-injection). Both [⁶⁸Ga]Ga-RM2 and [⁶⁸Ga]Ga-AMTG exhibited high tumor (~ 15 percentage injected dose per gram, %ID/g) and pancreas uptake (> 25%ID/g), whereas [⁶⁸Ga]Ga-[Hse⁷]RM2 and [⁶⁸Ga]Ga-[Bta⁸]RM2 revealed lower tumor (~ 7.5%ID/g) but also substantially lower pancreas uptake (< 8%ID/g) at 1 h post-injection. At 24 h post-injection (p.i.), [¹⁷⁷Lu]Lu-RM2 and [¹⁷⁷Lu]Lu-AMTG exhibited high (> 8% ID/g) while [¹⁷⁷Lu]Lu-[Hse⁷]RM2 and [¹⁷⁷Lu]Lu-[Bta⁸]RM2 displayed low tumor retention (~ 2%ID/g). All compounds showed low activity levels in the pancreas at 24 h post-injection (< 1%ID/g).

Conclusion: Substitution of the Gln-Trp site in RM2 by artificial amino acids had a distinct impact on overall pharmacokinetics. While Hse (instead of Gln) and Bta (instead of Trp) led to a decreased, α-Me-Trp (instead of Trp) led to an increased in vivo stability, which resulted in improved pharmacokinetics over time in case of the latter. However, at 1 h post-injection both [⁶⁸Ga]Ga-[Hse⁷]RM2 and [⁶⁸Ga]Ga-[Bta⁸]RM2 displayed slightly higher tumor-to-pancreas and tumor-to-intestine ratios, rendering homoserine and β-(3-benzothienyl)alanine potential options for the modification of GRPR ligands with regard to imaging properties.