Design, synthesis, and biological evaluation of the first radio-metalated neurotensin analogue targeting neurotensin receptor 2

Neurotensin receptor 2 (NTS2) is a well-known mediator of central opioid-independent analgesia. Seminal studies have highlighted NTS2 overexpression in a variety of tumors including prostate cancer, pancreas adenocarcinoma, and breast cancer. Herein, we describe the first radiometalated neurotensin analogue targeting NTS2. JMV 7488 (DOTA-(βAla)2-Lys-Lys-Pro-(D)Trp-Ile-TMSAla-OH) was prepared using solid-phase peptide synthesis, then purified, radiolabeled with 68Ga and 111In, and investigated in vitro on HT-29 cells and MCF-7 cells, respectively, and in vivo on HT-29 xenografts. [68Ga]Ga-JMV 7488 and [111In]In-JMV 7488 were quite hydrophilic (logD7.4 = −3.1 ± 0.2 and −2.7 ± 0.2, respectively, p < 0.0001). Saturation binding studies showed good affinity toward NTS2 (KD = 38 ± 17 nM for [68Ga]Ga-JMV 7488 on HT-29 and 36 ± 10 nM on MCF-7 cells; KD = 36 ± 4 nM for [111In]In-JMV 7488 on HT-29 and 46 ± 1 nM on MCF-7 cells) and good selectivity (no NTS1 binding up to 500 nM). On cell-based evaluation, [68Ga]Ga-JMV 7488 and [111In]In-JMV 7488 showed high and fast NTS2-mediated internalization of 24 ± 5 and 25 ± 11% at 1 h for [111In]In-JMV 7488, respectively, along with low NTS2-membrane binding (<8%). Efflux was as high as 66 ± 9% at 45 min for [68Ga]Ga-JMV 7488 on HT-29 and increased for [111In]In-JMV 7488 up to 73 ± 16% on HT-29 and 78 ± 9% on MCF-7 cells at 2 h. Maximum intracellular calcium mobilization of JMV 7488 was 91 ± 11% to that of levocabastine, a known NTS2 agonist on HT-29 cells demonstrating the agonist behavior of JMV 7488. In nude mice bearing HT-29 xenograft, [68Ga]Ga-JMV 7488 showed a moderate but promising significant tumor uptake in biodistribution studies that competes well with other nonmetalated radiotracers targeting NTS2. Significant uptake was also depicted in lungs. Interestingly, mice prostate also demonstrated [68Ga]Ga-JMV 7488 uptake although the mechanism was not NTS2-mediated.