In vitro and in vivo evaluation of the effects of aluminum [¹⁸F]fluoride radiolabeling on an integrin αvβ₆-specific peptide

Introduction: Incorporation of fluorine-18 ((18)F) into radiotracers by capturing ionic [(18)F]-species can greatly accelerate and simplify radiolabeling for this important positron emission tomography (PET) radioisotope. Among the different strategies, the incorporation of aluminum [(18)F]fluoride (Al[(18)F](2+)) into NOTA chelators has recently emerged as a robust approach to peptide radiolabeling. This study presents Al[(18)F](2+)-radiolabeling of an α(v)β₆ integrin-targeted peptide (NOTA-PEG₂₈-A20FMDV2) and its in vitro and in vivo evaluation.

Methods: Aluminum [(18)F]fluoride was prepared at r.t. from [(18)F]fluoride (40 MBq-11 GBq) and introduced into NOTA-PEG₂₈-A20FMDV2 (1) in sodium acetate (pH 4.1; 100°C, 15 min). The radiotracer Al[(18)F] NOTA-PEG₂₈-A20FMDV2 (2) was purified by HPLC, formulated in PBS and evaluated in vitro (stability; binding and internalization in α(v)β₆(+) and α(v)β₆(-) cells) and in vivo (paired α(v)β₆(+) and α(v)β₆(-) xenograft mice: PET/CT, biodistribution, tumor autoradiography and metabolites).

Results: The radiotracer 2 was prepared in 90 ± 6 min (incl. formulation; n=3) in 19.3 ± 5.4% decay corrected radiochemical yield (radiochemical purity: >99%; specific activity: 158 ± 36 GBq/μmol) and was stable in PBS and serum (2 h). During in vitro cell binding studies, 2 showed high, α(v)β₆-targeted binding (α(v)β₆(+): 42.4 ± 1.2% of total radioactivity, ratio (+)/(-)=8.4/1) and internalization (α(v)β₆(+): 28.3 ± 0.5% of total radioactivity, (+)/(-)=11.7/1). In vivo, 2 maintained α(v)β₆-targeted binding (biodistribution; 1 h: α(v)β₆(+): 1.74 ± 0.38% ID/g, (+)/(-)=2.72/1; 4 h: α(v)β₆(+): 1.21 ± 0.56% ID/g, (+)/(-)=4.0/1; 11% intact 2 in tumor at 1 h), with highest uptake around the tumor edge (autoradiography). Most of the radioactivity cleared rapidly in the urine within one hour, but a significant fraction remained trapped in the kidneys (4 h: 229 ± 44% ID/g).

Conclusion: The Al[(18)F]/NOTA-based radiolabeling was rapid and efficient, and the radiotracer 2 showed good α(v)β₆-selectivity in vitro and in vivo. However, in contrast to A20FMDV2 labeled with covalently bound [(18)F]-prosthetic groups (e.g., [(18)F]fluorobenzoic acid), 2 demonstrated significant trapping in kidneys, similar to radiometal-labeled chelator-analogs of 2.