3p-C-NETA-TATE: a versatile somatostatin analogue for Al18F-labeled and therapeutic SSTR2 targeting radiopharmaceuticals

Background: Somatostatin-based radiopharmaceuticals (e.g. [68Ga]Ga-DOTATATE and [177Lu]Lu-DOTATATE) have been used to diagnose, monitor, and treat neuroendocrine tumour patients with great success. [18F]AlF-NOTA-octreotide, a promising 18F-labeled somatostatin analogue and potential alternative for 68Ga-DOTA-peptides, is under clinical evaluation [1]. However, ideally, the same precursor (combination of chelator-linker-vector) can be used for production of both diagnostic and therapeutic radioprobes with very similar (e.g. Al18F/213Bi/177Lu) or identical (e.g. complementary Tb-radionuclides) pharmacokinetic properties, allowing for accurate personalised dosimetry estimation, and radionuclide therapy of NET patients (Figure 1) [2]. In this study we evaluated a potentially versatile and effective chelator 3p-C-NETA [3] and present first results of radiosynthesis and preclinical evaluation of [18F]AlF-3p-C-NETA-TATE.
Methods: 3p-C-NETA was radiolabeled with diagnostic (68Ga, Al18F) or therapeutic (177Lu, 161Tb, 213Bi, and 67Cu) radionuclides at different temperatures. The in vitro stability of the corresponding radiocomplexes was determined in PBS and human serum at 37 °C. 3p-C-NETA-TATE was synthesized using standard solid/liquid-phase peptide synthesis and purified using HPLC. [18F]AlF-3p-C-NETA-TATE was synthesized in an automated AllinOne® synthesis module (Trasis, Belgium), analyzed using radio-HPLC and the in vitro stability of [18F]AlF -3p-C-NETA-TATE was evaluated in formulation buffer, PBS and human serum at 37 °C. In vitro cell binding and internalization was performed with [18F]AlF-3p-C-NETA-TATE using SSTR2 expressing cells (BON-1.SSTR2) and the pharmacokinetics were evaluated using µPET/MRI and µPET/CT in healthy rats and tumor mice (BON1.SSTR2) respectively, with [18F]AlF-NOTA-Octreotide as benchmark [1].
Results: 3p-C-NETA was efficiently labeled with 177Lu and 213Bi (RCY>95%) at 25°C and with 161Tb (>95%) and 68Ga (>90%) at 55 °C. 67Cu-labeling resulted in RCYs of 90 ± 1.9 % and 96 ± 0.9% at 25°C and 95°C respectively. Al18F-labeling required 95°C to achieve good yields (>85%). The 177Lu- and 161Tb-3p-C-NETA-complex showed excellent in vitro stability in both PBS and human serum over a period of eight days (97% intact). We also observed high in vitro stability up to 2 h for [18F]AlF-3p-C-NETA (>93% intact in PBS and human serum). In contrast, 67Cu- and 68Ga-3p-C-NETA were stable in PBS (>90% intact), but not in human serum (< 80% intact after 24h and 2h for 67Cu and 68Ga, respectively). [18F]AlF-3p-C-NETA-TATE was obtained in good RCY (56.4 ± 10.3 %) and radiochemical purity (>98%). [18F]AlF-3p-C-NETA-TATE displayed excellent in vitro stability with >95% intact tracer after 4 hours in all tested conditions. High SSTR2 specific cell binding and internalization (16.3 ± 1.9 % of which 80.1 ± 1.9 % is internalized) was observed after 60 min incubation for [18F]AlF-3p-C-NETA-TATE. Finally, [18F]AlF-3p-C-NETA-TATE showed excellent pharmacokinetic properties and tumor accumulation, comparable with the results obtained for [18F]AlF -NOTA-Octreotide.

Conclusions: 3p-C-NETA is an excellent versatile chelator that can be used for both targeted radionuclide therapy (177Lu, 213Bi, 161Tb) and diagnostic applications (Al18F) and has the potential to replace DOTA analogues in current clinical use. [18F]AlF-3p-C-NETA-TATE and [213Bi]Bi-3p-C-NETA-TATE will be further evaluated as potential theranostic pair in SSTR2 expressing tumor mice.

Reference
1. Eur J Nucl Med Mol Imaging 2020, 47, 3033–46.
2. Pharmaceutics 2021, 13, 599.
3. Bioorg Med Chem Lett. 2008, 18, 3436–9.