MACROPA highly stable chelator of Radium-223 and functionalization attempts for targeted treatment of cancer

Objectives: Radium-223 dichloride was approved as the first alpha particle emitting radionuclide therapy in 2013. It demonstrated treatment efficiency in castrate resistant prostate cancer. Yet, ²²³RaCl₂ does not accumulate directly in cancerous tissues but rather to sites associated with elevated bone turnover, which is not sufficient to trigger an effective therapeutic response for the complete eradication of bone metastases (1). To specifically target cancerous cells, it is essential to re-engineer Radium-223 alpha therapy as a molecularly targeted agent. In the last decade, several attempts to chelate radium into a stable complex have failed. EDTA, DOTA and other macrocyclic structures are unreliable in vivo (2). To the best of our knowledge, we here report for the first time stable radium chelation utilizing an 18-membered bis-picolinate diazacrown ring: MACROPA. Previously, MACROPA has been described with strong chelating features with barium and actinium. Here, we have achieved Radium-223 chelation utilizing MACROPA, converting the radioisotope into an irreversible complex, and challenged its stability in vitro and in vivo. Further, derived biological structures conjugated to MACROPA such as β-Alanine; DUPA (PSMA-targeting molecule) and a dimeric galacto-RGD have been labeled with Radium-223 and investigated.

Methods: Utilizing radioTLCs and size exclusion chromatography, MACROPA chelation of Radium-223 has been evaluated focusing on chelation kinetics as well as chelator concentration required to complete complexation. In vitro stability of the radiocomplex and derived conjugated structures have been assessed utilizing PBS and serum protein challenges. In vivo evaluations of MACROPA complexes have been conducted through radioactive organ distributions counting following intravenous administration of naïve skeletally mature mice (3.7KBq/mice) as compared to ²²³RaCl₂. Results: ²²³Ra-MACROPA was formed with a radiolabeling efficiency of >95% (n>10) (Fig.1A) at room temperature within 5 min post-mixing at physiological pH with a minimum chelator concentration of 10-15 µM and KBqs amount of Radium-223 (Fig.1B). Buffer and human serum protein stability challenges of ²²³Ra-MACROPA showed high stability of >90% over 12 days of incubation. Derived constructs β-Alanine; DUPA presented similar complexing features with a slightly lower stability in vitro >75%. However, the dimeric peptide RGD structure did not label consistently and showed no stability (Fig.1C). In vivo, ²²³Ra-MACROPA displayed a striking contrast in bone uptake with respect to ²²³RaCl₂ administration, revealing only 1.6+/-0.3%IA/g versus 22+/-1%IA/g in femoral uptake after 24h p.i. (Fig.1D). This demonstrates an intact complex structure in circulation; further high renal excretions were observed (276+/-100%IA/g) within 15min p.i. confirming the clearing capability of ²²³Ra-MACROPA complex in contrast to 40% with ²²³RaCl₂. Similar observations were found when evaluating ²²³Ra-β-Alanine; further suggesting adequate chelator stability when functionalized with a short linear chain (Fig.1E). However, DUPA (Fig.1F) and dimeric RGD functionalization resulted in failure to maintain chelation in biological environment. Conclusion: We have shown that ²²³Ra can be chelated with MACROPA, forming a highly stable complex under in vitro and in vivo challenges. Considering the successful conjugation with β-Alanine; further studies are focusing on functionalized ²²³Ra-MACROPA complexes with cancer specific targeting moieties. Acknowledgements: We thank the DOE for ²²⁷Ac/Th supplies as well as NIH/NCI for supporting this research (R01CA229893/R01CA201035-DLJ Thorek) and ERF-Junior faculty award (2015-DS Abou).

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