Improved in vivo PET imaging of the adenosine A2A receptor in the brain using [18F]FLUDA, a deuterated radiotracer with high metabolic stability

Abstract

Purpose: The adenosine A_2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A_2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A_2A receptor radiotracer and report herein the preclinical evaluation of [¹⁸F]FLUDA, a deuterated isotopologue of [¹⁸F]FESCH.

Methods: [¹⁸F]FLUDA was synthesized by a two-step one-pot approach and evaluated in vitro by autoradiographic studies as well as in vivo by metabolism and dynamic PET/MRI studies in mice and piglets under baseline and blocking conditions. A single-dose toxicity study was performed in rats.

Results: [¹⁸F]FLUDA was obtained with a radiochemical yield of 19% and molar activities of 72-180 GBq/μmol. Autoradiography proved A_2A receptor-specific accumulation of [¹⁸F]FLUDA in the striatum of a mouse and pig brain. In vivo evaluation in mice revealed improved stability of [¹⁸F]FLUDA compared to that of [¹⁸F]FESCH, resulting in the absence of brain-penetrant radiometabolites. Furthermore, the radiometabolites detected in piglets are expected to have a low tendency for brain penetration. PET/MRI studies confirmed high specific binding of [¹⁸F]FLUDA towards striatal A_2A receptor with a maximum specific-to-non-specific binding ratio in mice of 8.3. The toxicity study revealed no adverse effects of FLUDA up to 30 μg/kg, ~ 4000-fold the dose applied in human PET studies using [¹⁸F]FLUDA.

Conclusions: The new radiotracer [¹⁸F]FLUDA is suitable to detect the availability of the A_2A receptor in the brain with high target specificity. It is regarded ready for human application.

Keywords: A2A receptor; Adenosine receptors; FESCH; Fluorine-18; Neurodegeneration; Positron-emission tomography.

Fig. 1

Representative radiotracers for PET imaging of the A_2A receptor and the herein reported [¹⁸F]FLUDA

Scheme 1

Synthesis of FLUDA, reagents and conditions. a p-TsCl, NEt₃, CH₂Cl₂, 0 °C, 3 h, 68% yield. b TBAF, MeCN/THF, 90 °C, 15 min, 36% yield. c Desmethyl SCH442416, Cs₂CO₃, MeOH, microwave heating (1 h, 100 °C, 100 W), 37% yield

Fig. 2

a Scheme of the two-step one-pot procedure for the radiosynthesis of [¹⁸F]FLUDA. b Representative chromatograms of isolation of [¹⁸F]FLUDA by semi-preparative HPLC. c Identification of [¹⁸F]FLUDA. HPLC chromatograms obtained by co-injection with the reference compound FLUDA

Fig. 3

Representative RP-HPLC radiochromatograms of plasma samples after administration of [¹⁸F]FLUDA to a mouse and piglet

Fig. 4

Representative in vitro autoradiographic images of the binding pattern of [¹⁸F]FLUDA (0.93 nM) in horizontal mouse brain slices. The highest accumulation of activity in the striatum (a, red). The binding is completely blocked by co-administration of 10 μM of the A_2A receptor antagonist ZM241385 (b). For annotation of the brain regions, the slices were Nissl-stained after autoradiography (c). St striatum, Cb cerebellum, and Cx cortex, Th thalamus. Representative competition curve (d). K_D and B_max calculated from the homologous completion of [¹⁸F]FLUDA with FLUDA (Cheng-Prusoff equation K_D = IC₅₀− [[¹⁸F]FLUDA] and B_max = top – bottom ∙ (K_D + [[¹⁸F]FLUDA])/[[¹⁸F]FLUDA] [20].

Fig. 5

In vitro autoradiographic images of the binding pattern of [¹⁸F]FLUDA (0.64 nM) in sagittal pig brain slices. The highest accumulation of activity is in the striatum (a, red, St striatum, Cb cerebellum, Cx cortex, and Th thalamus). The binding is completely blocked by co-administration of 10 μM of the A_2A receptor antagonist ZM241385 (b). Homologous competition curve (c)

Fig. 6

a Representative horizontal PET images (0–60 min) of [¹⁸F]FLUDA in the brain of CD-1 mice (striatum: red, cerebellum: yellow). b TACs at baseline for CD-1 mice (n = 4) in different brain regions after injection of [¹⁸F]FLUDA; TACs of c SUVr_St/Cb, d SUV striatum, and e SUV cerebellum: after pre-treatment with vehicle (red square, n = 8), tozadenant (2.5 mg/kg bw, blue circle, n = 4) and istradefylline (1.0 mg/kg bw, green triangle, n = 4)

Fig. 7

a Representative horizontal PET images (0–60 min) of [¹⁸F]FLUDA in the brain of piglets (striatum: red, cerebellum: yellow). b TACs of SUVr_St/Cb of [¹⁸F]FLUDA in the pig brain after administration of vehicle (red square, n = 1) or blocking with tozadenant (blue circle, n = 1)