In view of the excellent copper(ii) and 64-copper(ii) complexation of a TE1PA ligand, a monopicolinate cyclam, in both aqueous medium and in vivo, we looked for a way to make it bifunctional, while maintaining its chelating properties. Overcoming the already known drawback of grafting via its carboxyl group, which is essential to the overall properties of the ligand, a TE1PA bifunctional derivative bearing an additional isothiocyanate coupling function on a carbon atom of the macrocyclic ring was synthesized. This led to an architecture that is comparable to that of other commercially available bifunctional copper(ii) chelators such as p-SCN-Bn-DOTA already used in clinical trials for 64Cu-immuno-PET imaging. The C-functionalization of TE1PA on one carbon atom in the β-N position of the cyclam backbone was successfully achieved by adapting our patented methodology to the huge challenge, allowing the regiospecific mono-N-functionalization of the unsymmetrical ligand. The obtained ligand p-SCN-Bn-TE1PA was coupled to a 9E7.4 murine antibody (mAb), an IgG2a anti CD-138 for multiple myeloma (MM) targeting. The conjugation efficiency was assessed by looking at the 64Cu radiolabeling and the radiopharmaceutical 64Cu-9E7.4-p-SCN-Bn-TE1PA immunoreactivity, and in particular by comparing with 9E7.4-p-SCN-Bn-NOTA and 9E7.4-p-SCN-Bn-DOTA obtained from commercial and presumably highly efficient chelators NOTA and DOTA, respectively. The results are quite clear, showing that p-SCN-Bn-TE1PA has a coupling rate 5 times higher and an immunoreactivity 1.5 to 2 times greater than those of its two competitors. p-SCN-Bn-TE1PA also outperforms TE1PA conjugated via its carboxylic function on the same antibody. The first 64Cu-immuno-PET preclinical study in a syngeneic model of MM was performed, confirming the good in vivo properties of 64Cu-9E7.4-p-SCN-Bn-TE1PA for PET imaging, considering the high clearance even after 24 h and the particularly important tumor-to-liver ratio that was increasing at 48 h.

Following the successful synthesis of a C-functionalized version of the TE1PA ligand, a monopicolinate cyclam, we looked to demonstrate its in vivo properties versus DOTA and NOTA, after conjugation on the 9E7.4 rat antibody, an IgG 2a against CD138 murine, which has relevant properties for multiple myeloma targeting. For each ligand, different conjugation approaches had been considered to select the most appropriate for the comparative study. The p-SCN-Bn-TE1PA, NHS-DOTA, and p-SCN-Bn-NOTA were finally chosen for conjugation and radiolabeling tests. For in vivo comparison, we used a model of subcutaneous grafted mice with 5T33 tumor cells. In vitro tests and immuno-PET study highlighted 64 Cu-9E7.4-p-SCN-Bn-NOTA as the least attractive. Further competitive biodistribution and hepatic metabolic studies at 2, 24, and 48 h post-injection (100 μg radiolabeled with 10 MBq of 64 Cu) were then performed with the 64 Cu-9E7.4-p-SCN-Bn-TE1PA and 64 Cu-9E7.4-NHS-DOTA. Results show a better in vivo resistance of 64 Cu-9E7.4-p-SCN-Bn-TE1PA to transchelation compared to 64 Cu-9E7.4-NHS-DOTA, especially at later times. This was confirmed with 64 Cu-9E7.4p-SCN-Bn-NOTA at 48 h PI. 64 Cu-9E7.4-p-SCN-Bn-TE1PA also demonstrated an excellent hepatic clearance. 64 Cu-9E7.4-p-SCN-Bn-TE1PA displayed an overall superiority compared to 64 Cu-9E7.4-NHS-DOTA and 64 Cu-9E7.4-p-SCN-Bn-NOTA in terms of in vivo stability, reinforcing the usefulness of the p-SCN-Bn-TE1PA ligand for 64 Cu immuno-PET imaging.

Herein we present the preparation of two novel cyclam-based macrocycles (te1pyp and cb-te1pyp), bearing phosphonate-appended pyridine side arms for the coordination of copper­(II) ions in the context of 64Cu PET imaging. The two ligands have been prepared through conventional protection–alkylation sequences on cyclam, and their coordination properties have been thoroughly investigated. The corresponding copper complexes have been fully characterized in the solid state (X-ray diffraction analysis) and in solution (EPR and UV–vis spectroscopies). Potentiometric studies combined with spectrometry have also allowed us to determine their thermodynamic stability constants, confirming their high affinity for copper­(II) cations. The kinetic inertness of the complexes has been verified by acid-assisted dissociation experiments, enabling their use in 64Cu-PET imaging in mice for the first time. Indeed, the two ligands could be quantitatively radiolabeled under mild conditions, and the resulting 64Cu complexes have demonstrated excellent stability in serum. PET imaging demonstrated a set of features emerging from the combination of picolinates and phosphonate units: high stability in vivo, fast clearance from the body via renal elimination, and most interestingly, very low fixation in the liver. This is in contrast with what was observed for monopicolinate cyclam (te1pa), which had a non-negligible accumulation in the liver, owing probably to its different charge and lipophilicity. These results thus pave the way for the use of such phosphonated pyridine chelators for in vivo 64Cu-PET imaging.