1,4,7-triazacyclononane ligands as versatile platform for radiocopper-labeled agents

Objectives: Pyridine containing bifunctional chelating agents (BFCA) based on 1, 4, 7 – triazacyclononane backbones (DMPTACN) are suitable platforms for copper (II) complexes. These ligands rapidly form highly stable, inert square pyramidal complexes that are resistant to metal leaching.¹ The ligand structure enables convenient introduction of additional conjugatable functional groups like -COOH end groups to allow a regioselective coupling to various biomolecules for e.g. bombesin.² Since Cu-64 is a highly useful PET (positron emission tomography) radioisotope because of its intermediate half-life (⁶⁴Cu, t_1/2 -12.7 h) and a high resolution image, these conjugatable DMPTACN chelators can be coupled with various vectors for e.g., proteins, dendritic polymers in order to understand their detailed in vivo pharmacokinetic properties with respect to their ADME behavior. The current objective of the work was to synthesize new maleimide and -SCN derivatives of DMPTACN ligands which could be employed to be conjugated to anti-inflammatory dendritic polyglcerol sulfate (dPGS) & neutral dPG derivatives under mild conditions in order to study the metabolic fate of these potentially therapeutic polymers using positron emission tomography.
Methods: DMPTACN was synthesized by a 10-step process using an established protocol.¹ Two coupling groups such as maleimide (1) or isothiocyanate (2) have been attached for further conjugation. These ligands were then conjugated to the thiolated dPGS via Michael addition of 1 and also via direct labeling of 2 to dPGS amine to yield highly stable conjugates. ⁶⁴Cu was produced following an already established protocol with high specific activities of 150-250 GBq/µmol.^{3 64}Cu-labeling of the conjugates were performed using [⁶⁴Cu]CuCl₂ at ambient temperature in aqueous buffer solution (0.1 M MES/NaOH) and resulted in a radiochemical purity of ≥99% within a few minutes. Challenge experiments were conducted in presence of EDTA or copper seeking superoxide dismutase (SOD) for evaluation of in vitro stability.⁴ Biodistribution and PET studies were conducted in male Wistar rats.
Results: DMPTACN-dPG/S conjugates form highly stable metal complexes with ⁶⁴Cu under mild conditions (ambient temperature, aqueous solution) showing resistance to demetalation in vitro and high in vivo stability. Biodistribution data and PET experiments show a charge dependant excretion of the dPGS and dPG macromolecules.
Conclusions: DMPTACN ligands are a versatile platform which can be applied for labeling small molecules, proteins as well as polymers for detailed insight on their biodistribution profiles using ⁶⁴Cu that allows studying relatively long biochemical processes and thus, prove to be an attractive candidate for PET imaging.
Acknowledgements: This study is part of a research initiative “Technologie und Medizin – Multimodale Bildgebung zur Aufklärung des in-vivo Verhaltens von polymeren Biomaterialien” of the Helmholtz-Portfoliothema.
References:
[1] Gasser G., et al. (2008) Bioconjugate Chem. 19, 719-730.
[2] Bergmann R., et al. (2013) Eur. J. Med. Chem. 70, 434-446.
[3] Thieme S., et al. (2012) Appl. Radiat. Isot. 70, 602-608.
[4] Zarschler K., et al. (2013) RSC Adv. 4, 10157-10164.