Translational Preclinical\nPET Imaging and Metabolic\nEvaluation of a New Cannabinoid 2 Receptor (CB<sub>2</sub>R) Radioligand,\n(<i>Z</i>)‑<i>N</i>‑(3-(2-(2‑[<sup>18</sup>F]Fluoroethoxy)ethyl)-4,5-dime

We have previously developed seven fluorinated analogues\nof A-836339\nas new PET tracers for cannabinoid type 2 receptor (CB<sub>2</sub>R) imaging, among which (<i>Z</i>)-<i>N</i>-(3-(2-(2-[<sup>18</sup>F]fluoroethoxy)ethyl)-4,5-dimethylthiazol-2(3<i>H</i>)-ylidene)-2,2,3,3-tetramethylcyclopropane-1-carboxamide ([<sup>18</sup>F]FC0324) displayed high affinity and selectivity for CB<sub>2</sub>R in healthy rats. In the present study, we have further evaluated\nthe imaging and metabolic properties of [<sup>18</sup>F]FC0324 in\na rat model of human CB<sub>2</sub>R overexpression in the brain (AAV-<i>h</i>CB<sub>2</sub>) and in non-human primates (NHPs). Autoradiography\nwith AAV-<i>h</i>CB<sub>2</sub> rat brain sections exhibited\na signal of [<sup>18</sup>F]FC0324 8-fold higher in the ipsilateral\nregion than in the contralateral region. Blocking with NE40, a CB<sub>2</sub>R-specific agonist, resulted in a 91% decrease in the radioactivity.\nPET experiments showed a signal 7-fold higher in the ipsilateral region,\nand the specificity of [<sup>18</sup>F]FC0324 for <i>h</i>CB<sub>2</sub>R in vivo was confirmed by the 80% decrease after blocking\nwith NE40. In NHPs, brain time-activity curves displayed a fast and\nhomogeneous distribution followed by a rapid washout, in accordance\nwith the low amount of CB<sub>2</sub>Rs in healthy brain. Whole-body\nPET-CT suggested a high and specific uptake of the radiotracer in\nthe spleen, a CB<sub>2</sub>R-rich organ, and in the organs involved\nin metabolism and excretion, with a low bone uptake. In vitro metabolism\nwith monkey liver microsomes (MLMs) led to the formation of six main\nhydroxylated metabolites of FC0324. Five of them were produced by\nhuman liver microsomes, being much less active than MLMs. In vivo,\nin NHPs, the main radiometabolite was likely to result from further\noxidation of hydroxylated compounds, and parent [<sup>18</sup>F]FC0324\naccounted for 8 ± 3% of plasma radioactivity (at 120 min p.i.)\nwith a low level of potential interfering radiometabolites. Furthermore,\nthis metabolism should be significantly reduced in humans due to species\ndifferences. In conclusion, [<sup>18</sup>F]FC0324 appears to be a\npromising candidate for further human studies with suitable kinetics,\nselectivity, and metabolic profile for CB<sub>2</sub>R PET imaging.