Zirconium-89 tetraazamacrocycle complexes: the influence of macrocycle structure on stability

Introduction: The development of bifunctional chelators (BFCs) that can stably chelate zirconium-89 (⁸⁹Zr) while being conjugated to targeting molecules is an area of active research (1). A recent publication described the extraordinary stability of the radiolabeled tetraazamacrocycle, ⁸⁹Zr-DOTA in vivo (2). This work extends this research and examines the influence of macrocycle structure on the stability of these radiometal complexes.

Methods: The non-radioactive Zr-tetraazamacrocycle complexes, Zr-NOTA, Zr-PCTA and Zr-TRITA were prepared using standard procedures, and the molecular structure of each complex was elucidated using single crystal x-ray diffraction analysis. The radioactive analogs were prepared using ⁸⁹ZrCl₄. In vitro, the radiometal complexes were challenged with exogenous chelating ligands, biologically relevant metal ions and human serum. Biodistribution and small animal PET studies were conducted in normal mice to examine the in vivo behavior of each radiometal complex.

Results: Single crystal analysis revealed each Zr-complex to be octa-coordinate. Radiochemical studies revealed ⁸⁹Zr-PCTA and ⁸⁹Zr-NOTA to be extraordinarily inert to exogenous ligand, metal and serum challenge in vitro. Furthermore, biodistribution studies revealed rapid systemic clearance and low tissue retention of radioactivity. For example, mice injected with ⁸⁹Zr-PCTA or ⁸⁹Zr-NOTA retained less radioactivity in their kidney tissue than did mice receiving ⁸⁹Zr-DFO (⁸⁹Zr-PCTA vs. ⁸⁹Zr-NOTA vs. ⁸⁹Zr-DFO; mean %ID/g ± SD; [one-way ANOVA value, p value]: kidney (72 h): 0.15 ± 0.01 vs. 0.30 ± 0.023 vs. 0.69 ± 0.098; [F(3, 136), p <0.0001]). Conclusion: The structural characterization of Zr-NOTA, Zr-PCTA and Zr-TRITA using single crystal x-ray diffraction and the use of these tetraazamacrocycles as ⁸⁹Zr chelators is described. In all studies, ⁸⁹Zr-PCTA or ⁸⁹Zr-NOTA demonstrated excellent in vitro and in vivo stability. These results extend the number of tetraazamacrocycles that may be considered useful in ⁸⁹Zr-radiopharmaceutical development.