Development and biological evaluation of [18F]FLUDA for clinical translation to image the adenosine A2A receptor with PET

Introduction
The adenosine A2A receptor (A2AR) is related to the pathogenesis of several brain diseases and is assumed to mediate immunsuppressive processes related to cancer pathology. Thus, A2AR radiotracers for PET imaging are promising candidates for the differential diagnosis of neurodegenerative diseases, in particular Parkinson’s disease, and to study the A2AR within the tumor environment. We developed [18F]FLUDA based on the deuteration of the [18F]fluoroethoxy chain of [18F]FESCH [1,2] to improve imaging properties in mice. [18F]FLUDA was evaluated pre-clinically prior to a first-in-human trial.

Methods
Binding affinities of FLUDA towards the human A2AR and A1R subtypes were estimated in vitro by competitive radioligand binding assays. [18F]FLUDA was synthesized by a two-step one-pot approach. In vitro autoradiography of [18F]FLUDA was performed on mice brain cryosections. In vivo evaluation of [18F]FLUDA was carried out in a CD-1 mouse by radio-HPLC analysis of plasma and brain samples (15 min p.i.) and dynamic PET/MR studies under baseline (n = 4) and blocking conditions (2.5 mg tozadenant per kg, 15 min before tracer, n = 4). The cerebellum was used as a reference tissue. The time-activity curves were obtained and the SUV ratio (SUVR) of striatum over cerebellum were used as measure for specific uptake.

Results/discussion
In vitro binding studies revealed no influence of deuteration on the estimated binding affinities with Ki values of FLUDA and FESCH towards human A2AR of 0.60 nM and 0.61 nM, respectively. The radiosynthesis of [18F]FLUDA was successfully established (Fig.1). A single experiment indicates that [18F]FLUDA is metabolically more stable in mouse than [18F]FESCH. While no radiometabolites were found for [18F]FLUDA in plasma and brain samples at 15 min p.i., for [18F]FESCH the percentages of intact radiotracer were 71% and 41% in brain and plasma samples, respectively. By in vitro autoradiography [18F]FLUDA demonstrated a specific accumulation in the striatum, which is characterized by the binding parameters KD = 4.3 ± 0.7 nM and Bmax = 556 ± 143 fmol/mg wet weight (Fig.2A). PET scans revealed a selective binding of [18F]FLUDA in striatum (SUVR15 30 min p.i. >8), which was significantly reduced by tozadenant pre-treatment by 30% (p<0.05, Fig.2B/C).

Conclusion
The two-step one-pot radiosynthesis of [18F]FLUDA was successfully implemented for preclinical studies. Due to the promising preclinical results in mice, we focus on the clinical translation of [18F]FLUDA. Based on the performed single dose toxicity study of FLUDA we don’t expect any adverse effects. Currently, we are working on the radiation dosimetry of [18F]FLUDA in piglets and its implementation for clinical application.

Acknowledgments
The authors thank the European Regional Development Fund and Sächsische Aufbaubank (SAB) for financial support (project no. 100226753).

References
(1) Bhattacharjee et al., Nucl Med Biol 2011, 38, 897-906; (2) Khanapur et al., J Med Chem 2014, 57, 6765-80