Novel bifunctional tetrahydroximate chelators for labeling antibodies with Zr-89 for imaging with positron emission tomography

Aim: Due to its suitable half-life (78.4 h) and high positron yield (99% per decay) ⁸⁹Zr is the most attractive positron emitter for PET imaging using radiolabeled antibodies. In previous studies we reported synthesis and evaluation of tetrahydroxamate chelators for ⁸⁹Zr labeling. Chelators based on an iminodiacetamide backbone (1 and 2) were synthesized with an extended dipropylenetriamine and iminodipropionamide backbone: 3 and 4, respectively. The extension of the chelators greatly improved stability of ⁸⁹Zr Trastuzumab complexes but their demetalation remained higher and faster than ⁸⁹Zr-deferoxamine(DFO)-Trastuzumab in vivo. Molecular modeling showed that further elongation of arm length might be needed to reach a better Zr-coordination configuration and improve in vivo stability. We therefore synthesized two new bifunctional tetrahydroxamate chelators 5 and 6 with an extended linker length between hydroxamate motifs and evaluated their potential for ⁸⁹Zr immuno-PET imaging. Materials and Methods: 5 and 6 were synthesized via multi-step organic synthesis. Conjugation of 5, 6 and DFO (deferoxamine) to Trastuzumab was performed at 37°C in PBS pH 9 with 5:1 chelator-to-Trastuzumab ratio. Immunoconjugates were radiolabeled with ⁸⁹Zr at pH 7 for 1 h at room temperature and purified using PD-10 desalting column and concentrated using a 50 kDa molecular weight cut off filter. Radiochemical purity was determined by iTLC-SG chromatography and specific activity by size exclusion chromatography using HPLC. The stability of the complexes was assessed in mouse plasma at 37°C and was monitored by iTLC-SG. PET imaging and biodistribution studies were carried out in a SKOV-3 ovarian cancer xenografts model in mice (35±2 μg, 4.9±1.0 MBq, n=4/group). Density functional theory (DFT) calculations were used to simulate the coordination geometry of Zr complexes.

Results: Compounds 5 and 6 were successfully synthesized, and their identities were confirmed by both NMR and MS analysis. 5 and 6 immunoconjugates were effectively radiolabeled with ⁸⁹Zr: radiochemical yields >96% (versus 70% for DFO), radiochemical purity >99% after purification, specific activity 3.7-4.8 MBq/μg. Progressive demetalation of their ⁸⁹Zr complexes was observed in mouse plasma over time. At Day 6, the remaining intact fraction values were 90.3±1.1 % for ⁸⁹Zr-DFO-Trastuzumab, 71.1±1.7 % for ⁸⁹Zr-5-Trastuzumab and 52.2±4.6 % for ⁸⁹Zr-6-Trastuzumab. At Day 3, the SKOV-3 tumor uptakes were 33.7±7.5, 42.0±8.7 and 24.0±7.1 %ID/g for ⁸⁹Zr-DFO-Trastuzumab, ⁸⁹Zr-5-Trastuzumab and ⁸⁹Zr-6-Trastuzumab respectively (as compare to DFO, p=0.0940 for 5 and p=0.0430 for 6). The bone uptake were 15.3±1.6 %ID/g for ⁸⁹Zr-DFO-Trastuzumab, 26.4±5.0 %ID/g for ⁸⁹Zr-5-Trastuzumab and 22.0±2.0 %ID/g for ⁸⁹Zr-6-Trastuzumab (p=0.0185 and p=0.2045 for 5 and 6 as compare to DFO). At Day 5, significant higher uptake in bone was observed for both chelators: 29.5±6.4 and 31.1±4.8 %ID/g for ⁸⁹Zr-5-Trastuzumab and ⁸⁹Zr-6-Trastuzumab respectively as compare to 15.3±1.2 for ⁸⁹Zr-DFO-Trastuzumab (p=0.004 for 5 and p=0.0013 for 6). DFT calculations showed that unlike 3 and 4 which were not coordinated in an optimal configuration, the longer length between the hydroxamate motifs of 5 (7-atom long) and 6 (9-atom long) was more optimal and closer to the configuration of DFO* (9-atom long).

Conclusions: ⁸⁹Zr-complexes of 5 and 6 showed greatly improved stability in mouse plasma compared to previously reported 1 - 4. Nevertheless, the stability of ⁸⁹Zr-complexes of chelators 5 and 6 were still not as good as that of the DFO both in vitro and in vivo. This suggests that thorough structure-activity-relationship study is needed for the tetrahydroxamate chelators based on the iminodipropionamide backbone.

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