Stable Chelation of Zr-89 and Ac-225 with Dual Size-Selective Macrocyclic py₂-macrodipa for ²²⁵Ac/⁸⁹Zr Paired...

Radiotheranostic agents combine targeted therapeutics and diagnostic imaging, offering more efficient and personalized treatment strategies. Positron emission tomography provides high sensitivity and quantitative accuracy; however, its integration with α-particle radiotherapy remains limited by the lack of chelators capable of stabilizing radionuclides with widely different ionic radii. ⁸⁹Zr and ²²⁵Ac are promising radionuclides for PET imaging and targeted α-therapy, respectively, but require robust and versatile chelation strategies. Herein, we report stable coordination using the 18-membered macrocyclic dual size-selective chelator py₂-macrodipa with [⁸⁹Zr]Zr⁴⁺and [²²⁵Ac]Ac³⁺ ions. The chelator demonstrated efficient radiolabeling with both ⁸⁹Zr and ²²⁵Ac, exhibiting ⁸⁹Zr labeling efficiency comparable to the state-of-the-art chelator DFO at micromolar concentrations. The [⁸⁹Zr][Zr(py₂-macrodipa)]²⁺ complex showed high stability in human serum and in vivo, with minimal uptake in nonspecific tissues such as bone and other organs. Similarly, the [²²⁵Ac][Ac(py₂-macrodipa)]⁺ complex displayed robust in vitro and in vivo stability along with a biodistribution clearance profile similar to its ⁸⁹Zr analogue. In addition, a squaramide ethyl ester-conjugated bifunctional chelator (py₂-macrodipa-sq) was synthesized for antibody conjugation and assessed using the PD-1 targeting antibody Opdivo. Both ⁸⁹Zr- and ²²⁵Ac-labeled opdivo-py₂-macrodipa-sq conjugates were obtained with >98% radiochemical purity and demonstrated high stability in vitro and in vivo. Although the therapeutic construct exhibited higher activity levels in blood and other organs than the diagnostic analogue, both cleared over time with similar distribution patterns and low bone uptake. These findings establish py₂-macrodipa and its bifunctional derivative, py₂-macrodipa-sq, as potential chelation platforms for the development of matched ⁸⁹Zr/²²⁵Ac radiotheranostic agents, demonstrating stable and comparable in vivo behavior.