Background Hitherto, clinically applied 99mTc-based cholecystokinin-2 receptor (CCK-2R) ligands suffer from limited in vivo stability. Therefore, we focused on the development of pharmacokinetically optimized 99mTc-labeled CCK-2R ligands for improved imaging of CCK-2R-overexpressing malignancies via single-photon emission computed tomography (SPECT). The novel CCK-2R ligands were designed based on the recently published compounds CCK-66, rhCCK-18 and rhCCK-84. Results CCK-2R ligands were prepared by solid-phase peptide synthesis (SPPS) and 99mTc-labeled via the tetraamine (N4) chelator (95 °C, 15 min) with final radiochemical purities ≥ 95% (radiochemical yields > 99%, n = 39), as determined by radio-RP-HPLC. Affinity (IC50,inverse) and internalization experiments were performed using CCK-2R+ AR42J cells. Lipophilicity (logD7.4), human serum albumin (HSA) binding and stability in human serum (4 h, 37 °C) was determined as well. Four of the seven novel CCK-2R ligands, i.e. [99mTc]Tc-N4-CCK-66 and [99mTc]Tc-N4-CCK-103 to -105, exhibited similar CCK-2R affinities compared to the reference ligands [177Lu]Lu-DOTA-PP-F11N and [177Lu]Lu-DOTA-CCK-66 (IC50,inverse: 28.2–40.3 nM). [99mTc]Tc-N4-CCK-66 and [99mTc]Tc-N4-CCK-104 demonstrated high CCK-2R-mediated internalization (223.8 ± 0.1% and 145.2 ± 0.1% compared to [177Lu]Lu-DOTA-PP-F11N, respectively), high stability in human serum (> 95%) and similar lipophilicity (logD7.4: − 2.09 to − 2.42) and HSA binding (47.5–53.2%) compared with [177Lu]Lu-DOTA-CCK-66. 4-(Di-tert-butyl(hydroxy)silyl)benzoic acid (SiOH-BA)-bearing compounds [99mTc]Tc-N4-CCK-100 and [99mTc]Tc-N4-CCK-103 showed an elevated lipophilicity (logD7.4: − 1.89 to − 0.95) and HSA binding (78.1–89.1%) as well as a reduced stability in human serum (87.0–93.0%), but higher internalization than [177Lu]Lu-DOTA-PP-F11N (145.0 ± 0.1% and 219.5 ± 0.1%, respectively). Conclusions Several 99mTc-labeled CCK-2R ligands with very favorable in vitro characteristics were successfully developed in this study. In particular, [99mTc]Tc-N4-CCK-66 and [99mTc]Tc-N4-CCK-104 are highly recommended for further evaluation in in vivo studies to examine their potential as 99mTc-based SPECT agents for improved imaging of CCK-2R-expressing malignancies. Furthermore, a possible radiotheranostic use of these N4-conjugated compounds could be explored in future studies using 186/188Re.

Synthesis and in vitro evaluation of pharmacokinetically optimized ^99mTc-labeled cholecystokinin-2 receptor-targeted peptides for improved SPECT imaging

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2026年7月20日

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Veronika Felber,
Nadine Holzleitner,
Thomas Günther

摘要

Background

Hitherto, clinically applied ^99mTc-based cholecystokinin-2 receptor (CCK-2R) ligands suffer from limited in vivo stability. Therefore, we focused on the development of pharmacokinetically optimized ^99mTc-labeled CCK-2R ligands for improved imaging of CCK-2R-overexpressing malignancies via single-photon emission computed tomography (SPECT). The novel CCK-2R ligands were designed based on the recently published compounds CCK-66, rhCCK-18 and rhCCK-84.

Results

CCK-2R ligands were prepared by solid-phase peptide synthesis (SPPS) and ^99mTc-labeled via the tetraamine (N₄) chelator (95 °C, 15 min) with final radiochemical purities ≥ 95% (radiochemical yields > 99%, n = 39), as determined by radio-RP-HPLC. Affinity (IC_50,inverse) and internalization experiments were performed using CCK-2R⁺ AR42J cells. Lipophilicity (logD_7.4), human serum albumin (HSA) binding and stability in human serum (4 h, 37 °C) was determined as well. Four of the seven novel CCK-2R ligands, i.e. [^99mTc]Tc-N₄-CCK-66 and [^99mTc]Tc-N₄-CCK-103 to -105, exhibited similar CCK-2R affinities compared to the reference ligands [¹⁷⁷Lu]Lu-DOTA-PP-F11N and [¹⁷⁷Lu]Lu-DOTA-CCK-66 (IC_50,inverse: 28.2–40.3 nM). [^99mTc]Tc-N₄-CCK-66 and [^99mTc]Tc-N₄-CCK-104 demonstrated high CCK-2R-mediated internalization (223.8 ± 0.1% and 145.2 ± 0.1% compared to [¹⁷⁷Lu]Lu-DOTA-PP-F11N, respectively), high stability in human serum (> 95%) and similar lipophilicity (logD_7.4: − 2.09 to − 2.42) and HSA binding (47.5–53.2%) compared with [¹⁷⁷Lu]Lu-DOTA-CCK-66. 4-(Di-tert-butyl(hydroxy)silyl)benzoic acid (SiOH-BA)-bearing compounds [^99mTc]Tc-N₄-CCK-100 and [^99mTc]Tc-N₄-CCK-103 showed an elevated lipophilicity (logD_7.4: − 1.89 to − 0.95) and HSA binding (78.1–89.1%) as well as a reduced stability in human serum (87.0–93.0%), but higher internalization than [¹⁷⁷Lu]Lu-DOTA-PP-F11N (145.0 ± 0.1% and 219.5 ± 0.1%, respectively).

Conclusions

Several ^99mTc-labeled CCK-2R ligands with very favorable in vitro characteristics were successfully developed in this study. In particular, [^99mTc]Tc-N₄-CCK-66 and [^99mTc]Tc-N₄-CCK-104 are highly recommended for further evaluation in in vivo studies to examine their potential as ^99mTc-based SPECT agents for improved imaging of CCK-2R-expressing malignancies. Furthermore, a possible radiotheranostic use of these N₄-conjugated compounds could be explored in future studies using ^186/188Re.

Synthesis and in vitro evaluation of pharmacokinetically optimized 99mTc-labeled cholecystokinin-2 receptor-targeted peptides for improved SPECT imaging

摘要

Synthesis and in vitro evaluation of pharmacokinetically optimized ^99mTc-labeled cholecystokinin-2 receptor-targeted peptides for improved SPECT imaging