Imaging of neurotensin receptors in tumors by a novel stabilized ⁶⁴Cu-DOTA-neurotensin analog

Background:

Neurotensin (NT) and its receptors (NTR) are overexpressed in various tumors (breast, prostate, lung, ductal pancreas, pituitary) and play a crucial role in tumor progression and malignancy. For tumor diagnosis and optimized targeted, individualized therapy it is important to image and quantify functional expression of these receptors. The development and radiopharmacological characterization of a novel stable neurotensin analog radiolabeled with ⁶⁴Cu is described.

Material and methods:

The peptide (ArgΨ(CH₂NH)ArgProdmTyrtLeuLeu-OH) was synthesized by manual solid phase synthesis on a Merrifield-resin and conjugated with DOTA (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid). Radiolabeling of the peptide (3 nmol) with ⁶⁴CuCl₂ was carried out in 0.1 M ammonium acetate at pH 5.5, 37°C and 15 min. The IC₅₀ was determined on HT-29 cell membranes. Cell uptake and internalization was studied in HT-29 and PC3 cells. The biodistribution of the radiotracer was investigated in HT-29 tumor bearing NMRI nu/nu mice (5 min, 60 min p.i.; 4 animals per time point) and imaged by small animal PET (8 animals). The metabolic stability was analyzed in Wistar rats.

Results:

The binding affinity of the radiotracer towards NTR1 was 7 nM (4-12 nM, 95% confidence interval). The radiochemical purity after one step radiolabeling was greater than 92%. After single intravenous administration the activity concentration increased fast in the tumor (0.8±0.1 SUV, 5 min p.i.) and decreased to 0.3±0.1 SUV (60 min). At 60 min p.i. the tumor to organ ratios were 2.8±0.7 (blood), 5.2±0.9 (muscle), 4.2±0.6 (pancreas), 0.6±0.5 (liver), and 0.4±0.4 (kidneys). The radiotracer was fast accumulated in the kidneys (3.7±0.6 SUV, 5 min p.i.; 0.8±0.1 SUV, 60 min p.i.) and eliminated in the urine (60±6% injected dose, 60 min p.i.). The tumors were clearly delineated in the PET images. The tumor uptake of the radiotracer was competitively inhibited by 73% by simultaneous injection of the neurotensin derivative 8-13. In rat plasma 33% of the radioactivity accounted for the original compound at 60 min p.i.

Conclusions:

The novel ⁶⁴Cu-neurotensin analog with good stability and high receptor affinity allows for the in vivo imaging and functional characterization of NTR1 receptor overexpressing tumors. These findings are a prerequisite for other imaging applications, e.g., using SPECT radionuclides (¹¹¹In), and potentially also for targeted radionuclide therapy (⁶⁷Cu, ⁹⁰Y or ¹⁷⁷Lu).
Acknowledgement: This project was supported in part by the EC (Grant agreement no. 223057, GIPIO).