New complexing agent for one-step Al18F-labeling of heat-sensitive biomolecules: RESCA1 (2016)

330 Objectives The Al18F-labeling strategy involves chelation in aqueous medium of aluminium mono[18F]fluoride ({Al18F}2+) by a suitable chelator conjugated to a biomolecule.1 McBride et al. pioneered this method using the hexadentate ligand 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and the pentadentate ligand 1,4,7-triazacyclononane-1,4-diacetic acid (NODA). However, the need for elevated temperatures (100-120 °C) limits its widespread use. Therefore, we designed new restrained complexing agents (RESCAs) for application of the AlF strategy at room temperature. RESCA1 is an acyclic pentadentate ligand with an N2O3 coordinative set. Here we describe the evaluation of RESCA1 as an Al18F-chelator and as a proof of principle, RESCA1 was conjugated to human serum albumin (HSA) and the conjugate was labeled with {Al18F}2+. Methods The effectiveness of RESCA1 to complex {Al18F}2+ was tested in buffers with pH ranging from 3-8. HSA was reacted with RESCA1-TFP to obtain RESCA1-HSA. RESCA1 (0.057 mg, 150 nmol) or RESCA1-HSA (7.5 mg, 110 nmol) in 750 μl sodium acetate buffer (0.1 M, pH 4.5) was added to a freshly prepared {Al18F}2+ solution (1.4 GBq, 50 nmol AlCl3, 12 min, RT). Al18F-RESCA1-HSA was purified using a PD-10 column and RCP was determined with SEC-HPLC. To test stability in serum, Al18F-RESCA1 or Al18F-RESCA1-HSA in 100 µl PBS was added to rat serum (900 µl), kept at 37 °C and monitored up to 4 h. Ex vivo biodistribution was studied at 10 min and 60 min p.i. of Al18F-RESCA1 (2.5 MBq) in healthy mice or at 1 h, 3 h and 6 h p.i. of Al18F-RESCA1-HSA (2-7.5 MBq) in healthy female rats. Small-animal whole-body PET imaging was performed on rats using a FOCUS 220 tomograph.
Results: RESCA1 was synthesized in a three-step reaction starting from commercially available chemicals with an overall yield of 65%. The new ligand RESCA1 was able to complex {Al18F}2+ with excellent yields (>90%) at room temperature. The best RCYs were obtained in sodium acetate buffer in the range of pH 4.5- 5.5. Al18F-RESCA1 displayed excellent stability for at least four hours in PBS (>95%) and rat serum (>90%). The distribution of Al18F-RESCA1 in mice showed high in vivo stability, since bone uptake at 10 min and 60 min p.i., which would be an indication of release of fluorine-18, was negligible (0.41 ± 0.09% ID/g and 0.49 ± 0.05% ID/g, respectively).RESCA1-TFP ester was conjugated to HSA providing RESCA1-HSA with a chelator-to-protein ratio of 3, estimated by ESI-TOF-HRMS analysis. Al18F-RESCA-HSA was prepared in high RCY (>70%) and purity (>95%) in < 30 min. After 4 h incubation in rat serum 91% of Al18F-RESCA-HSA was still intact. Biodistribution in rats showed high retention in blood with 5.42 ± 0.23% ID/g, 4.93 ± 0.22% ID/g and 3.66 ± 0.06% ID/g at 1h, 3h and 6 h, respectively, from which the blood biological half-life was calculated to be 8.6 h. No significant increase in bone uptake was observed, indicating excellent in vivo stability of the Al18F-labeled construct.
Conclusions: The chelator RESCA1 proves to be a good lead candidate for efficient labeling of heat-sensitive biomolecules via the Al18F-method. We successfully labeled for the first time a heat-sensitive biomolecule via the Al18F-method in one radiolabeling step. Moreover Al18F- RESCA1-HSA showed excellent stability and favorable properties for PET blood pool imaging applications.
References:
1.McBride, W. J., Sharkey, R. M., and Goldenberg, D. M. [2013], EJNMMI Res.3, 36.
Figure 1: Chemical structure of RESCA1 and Al18F-RESCA1-HSA, in vivo biodistribution (%ID/g) of Al18F-RESCA1-HSA in healthy rats and whole-body PET image of a healthy rat 180 min after intravenous injection of Al18F-RESCA1-HSA