Towards the stable chelation of radium for biomedical applications with an 18-membered macrocyclic ligand

Abstract

Targeted alpha therapy is an emerging strategy for the treatment of disseminated cancer. [²²³Ra]RaCl₂ is the only clinically approved alpha particle-emitting drug, and it is used to treat castrate-resistant prostate cancer bone metastases, to which [²²³Ra]Ra²⁺ localizes. To specifically direct [²²³Ra]Ra²⁺ to non-osseous disease sites, chelation and conjugation to a cancer-targeting moiety is necessary. Although previous efforts to stably chelate [²²³Ra]Ra²⁺ for this purpose have had limited success, here we report a biologically stable radiocomplex with the 18-membered macrocyclic chelator macropa. Quantitative labeling of macropa with [²²³Ra]Ra²⁺ was accomplished within 5 min at room temperature with a radiolabeling efficiency of >95%, representing a significant advancement over conventional chelators such as DOTA and EDTA, which were unable to completely complex [²²³Ra]Ra²⁺ under these conditions. [²²³Ra][Ra(macropa)] was highly stable in human serum and exhibited dramatically reduced bone and spleen uptake in mice in comparison to bone-targeted [²²³Ra]RaCl₂, signifying that [²²³Ra][Ra(macropa)] remains intact in vivo. Upon conjugation of macropa to a single amino acid β-alanine as well as to the prostate-specific membrane antigen-targeting peptide DUPA, both constructs retained high affinity for ²²³Ra, complexing >95% of Ra²⁺ in solution. Furthermore, [²²³Ra][Ra(macropa-β-alanine)] was rapidly cleared from mice and showed low ²²³Ra bone absorption, indicating that this conjugate is stable under biological conditions. Unexpectedly, this stability was lost upon conjugation of macropa to DUPA, which suggests a role of targeting vectors in complex stability in vivo for this system. Nonetheless, our successful demonstration of efficient radiolabeling of the β-alanine conjugate with ²²³Ra and its subsequent stability in vivo establishes for the first time the possibility of delivering [²²³Ra]Ra²⁺ to metastases outside of the bone using functionalized chelators, marking a significant expansion of the therapeutic utility of this radiometal in the clinic.

Fig. 1. (a) Structure of macropa and its coordination to radium. (b) Structures of DOTA and bifunctional constructs of macropa explored in this work for ²²³Ra chelation.

Fig. 2. Formation of [²²³Ra][Ra(macropa)] under different conditions. (a) Migration of [²²³Ra]RaCl₂ (top) and [²²³Ra][Ra(macropa)] (bottom) on DGA-coated chromatographic strips, which were developed using a mobile phase of 0.1 M NaOH and visualized using autoradiographic quantification profiles. (b) RL% measured as a function of time at room temperature, pH 6, with varying concentration of macropa. (c) RL% as a function of macropa concentration 5 min after co-mixing; the ligand concentration required for 50% RL% (RL_50%) was found to be 13 μM, and >80% RL% was achieved at a concentration of 18 μM.

Fig. 3. In vitro stability of [²²³Ra][Ra(macropa)]. Size exclusion chromatography (SEC) of (a) [²²³Ra]RaCl₂ mixed in serum, detected at 280 nm (dashed line) and by gamma counting (solid line); (b) [²²³Ra][Ra(macropa)] after 2 h in human serum; (c) percent intact complex following human serum challenge over 12 days.

Fig. 4. In vivo evaluation of [²²³Ra][Ra(macropa)]. (a) [²²³Ra]RaCl₂ and (b) [²²³Ra][Ra(macropa)] radioactive organ distribution (% injected activity normalized to weight; % IA per g) was assessed utilizing healthy, skeletally mature mice sacrificed at 15 min and 24 h p.i. Differences in splenic, renal, and bone uptake were observed between the two groups. (c) Mice administered [²²³Ra][Ra(macropa)] exhibited 10 to 25-fold lower osseous uptake than that of free ²²³Ra at 15 min and 24 h p.i. (**p = 0.0096; ****p < 0.0001).

Fig. 5. Alkaline earth metal complexes of macropa-β-alanine. (a) Species distribution diagrams of macropa (left) and macropa-β-alanine (right) in the presence of Ba²⁺ at [Ba²⁺]_tot = [L]_tot = 1.0 mM, I = 0.1 M KCl, and 25 °C. (b) X-ray crystal structure of [Ba(macropa-β-alanine)(DMSO)]. Ellipsoids are drawn at the 50% probability level. Nonacidic hydrogen atoms are omitted for clarity. A full discussion of the crystallographic disorder of the structure is provided in the ESI. (c) Comparison of the immediate coordination sphere of the Ba²⁺ center in the crystal structures of [Ba(Hmacropa)(DMF)]⁺ and [Ba(macropa-β-alanine)(DMSO)].

Fig. 6. In vitro and in vivo evaluation of [²²³Ra][Ra(macropa-β-alanine)]. (a) Stability of [²²³Ra][Ra(macropa-β-alanine)] over the course of 12 days in human serum at 37 °C. (b) Organ distribution of [²²³Ra][Ra(macropa-β-alanine)] at 24 h p.i. Significant differences in osseous (*p < 0.005), splenic, and renal uptake were observed in comparison to the control [²²³Ra]RaCl₂.