Bispidines as versatile bifunctional chelators for ⁶⁴Cu PET imaging

Objectives: Ligands based on 3,7-diazabicyclo[3.3.1]nonane (bispidine) form very stable coordination compounds, particularly with Cu^II. Due to the formation of thermodynamically and kinetically very stable ⁶⁴Cu^II complexes, pentadentate I, hexadentate II and macrocyclic bispidines III are well suited for in vivo application. The bispidine scaffold has a number of options for derivatization that permit the introduction of additional functions such as biological vectors and fluorescent molecules. This allows the adjustment of defined features of solubility and of selective binding properties for in vivo applications with controllable targeting.

Methods: Bispidine ligands I and II were synthesized by two consecutive Mannich reactions. The bispidine dioxotetraaza macrocycle III was synthesized by cyclization of 5,7-dimethyl-1,3-diazabicyclononane with the bis(α-chloroacetamide) of o-phenylendiamine. ⁶⁴Cu was produced following an established protocol with high specific activities of 150-250 GBq/µmol. ⁶⁴Cu-labeling of bispidine ligands and peptide conjugates were performed using [64Cu]CuCl2 dissolved in 0.05 M 2-[N-morpholino]ethansulfonic acid (MES)-NaOH buffer (pH 5.5, 6.0, and 6.5). The stability of the ⁶⁴Cu^II-labeled bispidine ligands I – III was studied in the presence of an excess of human superoxide dismutase (SOD) and human serum using standard gel electrophoresis techniques. Biodistribution and PET studies were conducted in male Wistar rats. Small animal PET studies were performed in tumor (PC3, MPC^#) bearing mice.

Results: Bispidine ligands I and II form highly stable metal complexes with ⁶⁴Cu^II under mild conditions (ambient temperature, aqueous solution). ⁶⁴Cu-labeling of bispidine dioxotetraaza macrocycles III shows relatively rapid complex formation at 50°C. Challenge experiments with SOD and human serum indicate a high in vitro stability of ⁶⁴Cu^II complexes with bispidine ligands I – III. Biodistribution studies, showing rapid blood and tissue clearance, support the high complex stability in vivo. ⁶⁴Cu-labeled bispidine bioconjugates, incorporating specific vector molecules, such as bombesin and TATE peptides, permit clear tumor visualization with high target-to-background ratio.
Conclusions: Bifunctional bispidine ligands I - III represent a versatile platform for the development of new copper radiopharmaceuticals. The bispidine scaffold holds promising potential to tune the charge and lipophilicity of the radiocopper complexes and consequently to influence the biodistribution and pharmacokinetic properties. Moreover, bispidine ligands can be readily modified with appropriate vector molecules and fluorescence tags.
Acknowledgements: Financial support by the Helmholtz Virtual Institute NanoTracking (Agreement Number VH-VI-421) is gratefully acknowledged.
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