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.

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

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2025年9月1日

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Sebastian Fischer,
Lena Koller,
Sandra Dominelli,
Roswitha Beck,
Hans-Jürgen Wester,
Thomas Günther

摘要

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.