Evaluation of DOTA-chelated neurotensin analogs with spacer-enhanced biological performance for neurotensin-receptor-1-positive tumor targeting

Abstract

Introduction Neurotensin receptor 1 (NTR1) is overexpressed in many cancers types. Neurotensin (NT), a 13 amino acid peptide, is the native ligand for NTR1 and exhibits high (nM) affinity to the receptor. Many laboratories have been investigating the development of diagnostic and therapeutic radiopharmaceuticals for NTR1-positive cancers based on the NT peptide. To improve the biological performance for targeting NTR1, we proposed NT analogs with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelation system and different lengths of spacers. Methods We synthesized four NTR1-targeted conjugates with spacer lengths from 0 to 9 atoms (null (N0), β-Ala-OH (N1), 5-Ava-OH (N2), and 8-Aoc-OH (N3)) between the DOTA and the pharmacophore. In vitro competitive binding, internalization and efflux studies were performed on all four NT analogs. Based on these findings, metabolism studies were carried out on our best performing conjugate, 177Lu-N1. Lastly, in vivo biodistribution and SPECT/CT imaging studies were performed using 177Lu-N1 in an HT-29 xenograft mouse model. Results As shown in competitive binding assay, the NT analogs with different spacers (N1, N2 and N3) exhibited lower IC50 values than the NT analog without a spacer (N0). Furthermore, N1 revealed higher retention in HT-29 cells with more rapid internalization and slower efflux than the other NT analogs. In vivo biodistribution and SPECT/CT imaging studies of 177Lu-N1 demonstrated excellent accumulation (3.1 ± 0.4 %ID/g) in the NTR1-positive tumors at 4 h post-administration. Conclusions The DOTA chelation system demonstrated some modest steric inhibition of the pharmacophore. However, the insertion of a 4-atom hydrocarbon spacer group restored optimal binding affinity of the analog. The in vivo assays indicated that 177Lu-N1 could be used for imaging and radiotherapy of NTR1-positive tumors.

Investigation of the Biological Impact of Charge Distribution on a NTR1-Targeted Peptide

“…The in vitro internalization and efflux studies were performed as stated previously 38, 39 . HT-29 cells (~1×10 6 ) were suspended in 100 μL of McCoy’s 5A medium (pH 7.4, 4.8 mg/mL HEPES, and 2 mg/mL BSA).…”

Radiopharmaceuticals for imaging and endoradiotherapy of neurotensin receptor‐positive tumors

“…Most importantly, the peptide with the PEG 4 ‐spacer linked to the DOTA chelator was found to have a more favorable receptor affinity by a factor of 4 compared with NT(8–13) without the spacer, and the presence of the spacer did not significantly affect the lipophilicity of the peptide. Similarly, the “spacer‐enhanced” in vivo performance of DOTA‐linked NT analogs has also been described by Jia et al, who found that a 4‐atom hydrocarbon spacer length was optimal for obtaining 177 Lu‐labeled DOTA NT peptides with adequate receptor binding affinity and tumor accumulation in vivo.…”

Analysis of argentinated peptide complexes using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: Peptide = oxytocin, arg8‐vasopressin, bradykinin, bombesin, somatostatin, neurotensin

“…These NT receptors are over‐expressed in various tumors and cancerous cells/tissues . Therefore, various NT‐based radio‐labeled pharmaceutics using 188 Re, 177 Lu, 123/ 125 I, 111 In, 99m Tc, 64 Cu, and 18 F are being developed to target these cancer sites/tumors for diagnosis and therapy applications . The C‐terminal segment (NT8‐13) is more important for the receptor binding as compared to the N‐terminal.…”

“…[7,9,10] Therefore, various NT-based radio-labeled pharmaceutics using 188 Re, 177 Lu, 123/125 I, 111 In, 99m Tc, 64 Cu, and 18 F are being developed to target these cancer sites/tumors for diagnosis and therapy applications. [7,[78][79][80][81] The C-terminal segment (NT8-13) is more important for the receptor binding as compared to the N-terminal. Among them, Arg 8 , Tyr 11 , Leu 13 -COOH play a key role for its activity.…”