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

IntroductionNeurotensin receptor 1 (NTR1) is overexpressed in many cancer 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 a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelation system and different lengths of spacers.MethodsWe 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.ResultsAs shown in the competitive binding assays, 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.ConclusionsThe 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.