Optimization of the Arg8−Arg9 Cleavage Site in 68Ga-Labeled Neurotensin(6−13) Analogs for NTSR1-Targeted PET Imaging of Cancer

Neurotensin receptor 1 (NTSR1) is overexpressed in many cancers and represents a promising target for PET imaging. Neurotensin (NT)-derived radiopharmaceuticals targeting NTSR1 have been widely investigated; however, their clinical translation is limited in part by rapid proteolytic degradation, particularly at the Arg8−Arg9, Pro10−Tyr11, and Tyr11−Ile12 peptide bonds. In this study, we focused on optimizing the Arg8−Arg9 cleavage site through the incorporation of non-canonical amino acids to improve metabolic stability while maintaining high receptor affinity. A series of 16 DOTA-conjugated NT(6−13) analogs containing modifications at Arg8 and/or Arg9 were synthesized by solid-phase peptide synthesis. The natGa-labeled analogs were evaluated in PC-3 cells using a competition binding assay with [125I-Tyr3]Neurotensin, and compounds with Ki values < 5 nM were selected for 68Ga-radiolabeling, PET imaging, biodistribution, and in vivo stability studies. Ga-SK01033 (Phe(3-guanidine) at Arg8) and Ga-SK02040 (NMe-Orn(Me)3 at Arg8) demonstrated Ki values of 3.35 ± 0.32 and 4.34 ± 1.16 nM, respectively. Both peptides were labeled with 68Ga in 35.0−60.9% decay-corrected radiochemical yields, >108 GBq/μmol molar activity, and >97% radiochemical purity. Despite good binding affinity, [68Ga]Ga-SK01033 exhibited poor in vivo stability and negligible tumor uptake (<1 % ID/g at 1 h p.i., no intact tracer detected at 15 min p.i.). In contrast, [68Ga]Ga-SK02040 displayed improved metabolic stability, with 70.3 ± 5.11% intact tracer at 15 min p.i., resulting in tumor uptake of 11.1 ± 2.64 % ID/g at 1 h p.i. and 9.39 ± 2.64 % ID/g at 3 h p.i. Co-injection of excess natGa-SK02040 reduced tumor uptake by 91.2%, confirming NTSR1-mediated targeting. Overall, these findings demonstrate that while Phe(3-guanidine) substitution improves binding affinity without enhancing stability, incorporation of NMe-Orn(Me)3 at Arg8 simultaneously enhances affinity and metabolic stability, resulting in higher tumor uptake and tumor-to-non-target contrast than previously reported [68Ga]Ga-DOTA-NT-20.3. The present study establishes NMe-Orn(Me)3 as a favorable Arg8 modification that enables the development of NT(6−13)-based PET tracers with improved in vivo performance for targeting NTSR1.