Evaluation of ⁶⁸Ga- and ⁶⁴Cu-labeled PSMA-targeting conjugates comprising a novel chelating system

Objectives We recently developed a promising chelating system based on 1,4,7-triazacyclononane with pendant five-membered aromatic azaheterocycles for stable ⁶⁴Cu chelation.^[1] The aims of the present study were (1) to determine the labeling properties of the chelating system with ⁶⁸Ga and (2) to evaluate the biological properties of the chelate in vivo in combination with a prostate specific membrane antigen (PSMA) targeting vector as a model compound.

Methods NODIA-Me-2NaI-Ahx-Lys-urea-Glu ((4S,18S,22S)-1-(4,7-bis((1-methyl-1H-imidazol-2-yl)methyl)-1,4,7-triazonan-1-yl)-4-(naphthalen-2-ylmethyl)-2,5,12,20-tetraoxo-3,6,13,19,21-pentaazatetracosane-18,22,24-tricarboxylic acid) was obtained via standard peptide chemistry in 45% overall yield. Radiolabeling with ⁶⁴Cu was performed manually at r.t. in ammonium acetate buffer (pH 8.0). ⁶⁸Ga labeling was accomplished in a fully automated process using an Eckert & Ziegler Pharmtracer module at 95 °C for 10 min (sodium acetate buffer pH 4.5). The stability of ⁶⁸Ga and ⁶⁴Cu labeled NODIA-Me-2NaI-Ahx-Lys-urea-Glu was assessed in human serum at 37 °C. In vivo properties of ⁶⁸Ga- and ⁶⁴Cu-labeled NODIA-Me-2NaI-Ahx-Lys-urea-Glu were assessed by ex vivo biodistribution and PET imaging studies in LNCaP and PC-3 xenograft bearing mice.

Results NODIA-Me-2NaI-Ahx-Lys-urea-Glu radiolabeled readily with ⁶⁴Cu in quantitative yield in less than 5 min at r.t. with no need for further purification. Quantitative labeling yields were also obtained for ⁶⁸Ga. Without optimization, specific activities of >30 MBq/nmol of peptide could be achieved for both radiometals tested. No significant decomposition was observed for the ⁶⁸Ga- (4 h) and ⁶⁴Cu-conjugates (24 h) after incubation in human serum. Ex vivo biodistribution revealed low uptake (<1% IA/g) in PSMA negative tissues/organs at 1 h p.i. for both radiolabeled compounds. Kidney uptake at 1 h p.i. was 6.28 ± 0.92% IA/g and 4.96 ± 0.79% IA/g and tumor uptake was 1.33 ± 0.46% IA/g and 2.15 ± 0.38% IA/g for the ⁶⁸Ga- and ⁶⁴Cu-conjugates, respectively. Consecutive PET imaging showed rapid clearance from normal organs and prolonged retention in the tumor. Specific uptake was confirmed by blocking studies (PMPA) and PSMA negative xenografts (PC-3).

Conclusions Our novel azaheterocycle chelating system can be labeled with ⁶⁸Ga. In vitro and in vivo studies demonstrate promise for developing a range of radiotracers by combining the new chelator with appropriate biological targeting vectors such as PSMA-targeting peptides. ACKNOWLEDGEMENTS: MDB thanks the Research Commission of the University Freiburg as well as the Fonds der chemischen Industrie for funding. JPH thanks the DKFZ for funding. We thank Prof. Helmut Maecke for helpful discussions. REFERENCES: [1] Gotzmann, C.; Braun, F.; Bartholomä M.D. (2016), RSC Advances, 6, 119-131.

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