Preclinical results of novel GRPR-targeted antagonists with modified binding sequences

Objectives: In addition to previous approaches aiming to improve the metabolic stability and pharmacokinetics of GRPR antagonists by C- or N-terminal modifications, we investigated in this study the introduction of unnatural amino acids (homoserine [Hse], citrulline [Cit], β-(3-benzo­thienyl)alanine [Bta] and α-methyl tryptophan [α-Me-Trp]) at the metabolically unstable Gln⁷-Trp⁸ sequence within the typically used Gastrin-releasing peptide receptor (GRPR) antagonist RM2 (DOTA-Pip⁵-D-Phe⁶-Gln⁷-Trp⁸-Ala⁹-Val¹⁰-Gly¹¹-His¹²-Sta¹³-Leu¹⁴-NH₂). The results of the development and evaluation of the five novel candidates for diagnosis and therapy of GRPR-based malignancies are given. Methods: All RM2-based compounds were synthesized by standard Fmoc-based solid-phase peptide synthesis (SPPS). Labeling with ¹⁷⁷Lu was carried out at 95 °C within 10 min (1.0 M sodium acetate buffer, pH = 5.5, 0.1 M sodium ascorbate). GRPR affinity (IC₅₀) and internalization (37 °C, 60 min) were determined using PC-3 cells. In addition, the lipophilicities (expressed as n-octanol/PBS partition coefficient; logD_7.4) were determined and metabolic stabilities in vitro in human as well as murine plasma (3 d, 37 °C) were examined. Biodistribution studies were carried out at 1 h and 24 h p.i. on PC-3 tumor-bearing CB17-SCID mice. Results: Similar to the production of parent compound, the new RM2 derivatives were easily accessible by SPPS, resulting in 6-11% HPLC purified labeling precursor. Each of ¹⁷⁷Lu-labeled compounds revealed suitable hydrophilicity (logD_7.4) (¹⁷⁷Lu-RM2: - 2.51 ± 0.02, ¹⁷⁷Lu-Hse⁷-RM2: - 2.25 ± 0.06, ¹⁷⁷Lu-Cit⁷-RM2: - 3.22 ± 0.15, ¹⁷⁷Lu-Bta⁸-RM2: - 1.81 ± 0.02, ¹⁷⁷Lu-α-Me-Trp⁸-RM2 = AMTG: - 2.28 ± 0.06 and ¹⁷⁷Lu-DOTAGA-α-Me-Trp⁸-RM2 = AMTG2: - 2.51 ± 0.11. The GRPR affinity (IC₅₀ [nM]) of the new compounds was high to moderate (Lu-RM2: 3.45 ± 0.18, Lu-Hse⁷-RM2: 19.7 ± 1.6; Lu-Cit⁷-RM2: 11.6 ± 2.1; Lu-Bta⁸-RM2: 4.63 ± 0.23; Lu-AMTG: 3.04 ± 0.08; Lu-AMTG2: 4.74 ± 0.23). Furthermore, internalization studies at 1 h confirmed antagonistic behavior for all evaluated compounds (specific internalization of ¹⁷⁷Lu-RM2: 2.92 ± 0.20%; ¹⁷⁷Lu-Hse⁷-RM2: 1.40 ± 0.16%; ¹⁷⁷Lu-Bta⁸-RM2: 2.26 ± 0.18% and ¹⁷⁷Lu-AMTG: 3.03 ± 0.18%). Compared to ¹⁷⁷Lu-RM2, improved tumor-to-background (T/B) ratios at 1 h p.i. in mice was found in all organs for ¹⁷⁷Lu-Hse⁷-RM2 and ¹⁷⁷Lu-Bta⁸-RM2, whereas ¹⁷⁷Lu-AMTG and ¹⁷⁷Lu-AMTG2 showed enhanced T/B ratios in most organs (especially in blood and muscle) at 24 h p.i. Interestingly, studies on the metabolic stability of ¹⁷⁷Lu-RM2 and ¹⁷⁷Lu-AMTG in vitro in human and murine plasma revealed similar stability of both derivatives after 3 d (67% and 59%, respectively) in murine plasma, whereas stability in human plasma was significantly higher for ¹⁷⁷Lu-AMTG after 3 d of incubation at 37 °C (36% and 92%, for ¹⁷⁷Lu-RM2 and ¹⁷⁷Lu-AMTG, respectively). Conclusion: Based on its high affinity, excellent biodistribution and improved metabolic stability, the novel GRPR-targeted antagonist AMTG as well as AMTG2 could be promising candidates for diagnosis and therapy of GRPR-expressing malignancies. In addition, Hse⁷-RM2 and Bta⁸-RM2 could be appropriate ligands for diagnostic purposes.