Towards the clinical translation of the adenosine A2A receptor (A2AR) radioligand [18F]FLUDA: preclinical perspectives

Introduction
The A2AR has emerged as a potential therapeutic target due to its involvement in basic functions of the neuronal, cardiovascular and immune systems. Blockade of A2AR is regarded as potential tool related to CAR T-cell immunotherapy of cancer. Furthermore, A2AR are believed to regulate myocardial oxygen demand and to increase coronary circulation by vasodilation. With regard to Parkinson’s disease A2AR antagonists, such as the FDA approved Nourianz®, are used as an adjunctive treatment to levopoda®. Therefore noninvasive imaging to monitor changes of receptor density and/or occupancy during the A2aR-tailored therapy is of utmost importance. We recently developed the A2AR PET radiotracer [18F]FLUDA which demonstrated superior pharmacokinetic properties among the radiotracers available. Towards its clinical translation, an automated radiosynthesis was developed and the radiotracer was fully characterized in vitro and in vivo including toxicity and dosimetry1 studies.
Methods
The binding affinity (Ki) of FLUDA towards the human A2AR and A1R subtypes was estimated in vitro by competitive radioligand binding assays. In addition, selectivity studies were performed. An automated two-step one-pot radiosynthesis of [18F]FLUDA was developed. In vitro autoradiography was performed on cryosections of mice brain. Dynamic PET/MR studies under baseline and blocking conditions were assessed. The time-activity curves of the SUV ratio (SUVR) of striatum over cerebellum were used as measure for specific uptake. Metabolism was investigated in CD-1 mice via radio-HPLC analysis of extracted plasma and brain samples. Single-dose acute toxicity of FLUDA was assessed in male and female Wistar rats.
Results
A high A2aR binding affinity and high A1R selectivity were recorded for FLUDA (KiA2aR= 0.60 nM and KiA1R= XX nM). The two-step one-pot automated radiosynthesis of [18F]FLUDA was successfully established (radiochemical yield: 9±1%; radiochemical purity: ≥98%; molar activity= 69 333 GBq/µmol). In vitro autoradiography with [18F]FLUDA revealed 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.1A). In vivo evaluation in mice revealed that only the parent radiotracer was found in plasma and brain samples at 15 min p.i.. PET scans (n=3) showed a selective binding of [18F]FLUDA in striatum (SUVR15 30 min p.i.>8), which was significantly reduced by pre-treatment with 2.5 mg/kg i.p. tozadenant (30%, n=3, p<0.05, Fig.1B/C). The single dose toxicity study of FLUDA did not reveal any adverse effects for ~ 1000-fold of expected human dose.
Conclusion
The fully automated radiosynthesis of [18F]FLUDA allows its translation to clinical radiopharmacy. [18F]FLUDA exhibited a A2AR-specific accumulation in the striatum in vitro and in vivo. No safety concerns are expected upon administration of [18F]FLUDA according to toxicity and dosimetry studies. Altogether, these results encourage the performance of clinical trials to validate the potential of [18F]FLUDA for imaging of neuronal, cardiovascular and cancer-related diseases.
Acknowledgments
The authors thank the European Regional Development Fund and Sächsische Aufbaubank (SAB) for financial support (project no. 100226753).
[1] Kranz, M. et al. (2020) this volume.