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Cholesterol 24-hydroxylase (CH24H, CYP46A1) is a cytochrome P450 family enzyme that maintains the homeostasis of brain cholesterol. Soticlestat, a potent and selective CH24H inhibitor, is in development as a therapeutic agent for Dravet syndrome and Lennox-Gastaut syndrome. Herein, we report the discovery of aryl-piperidine derivatives as potent and selective CH24H positron emission tomography (PET) tracers which can be used for dose guidance of a clinical CH24H inhibitor and as a diagnostic tool for CH24H-related pathology. Starting from compound 1 (IC 50  = 16 nM, logD = 1.7), which was reported as a CH24H inhibitor with lower lipophilicity, a 18 F-labeling site (3-fluoroazetidine) was incorporated by structure-based drug design (SBDD) utilizing the co-crystal structure of a compound 1 analog. Subsequent optimization to adjust key parameters for PET tracers, such as potency, lipophilicity, brain penetration, and unbound plasma protein binding, enabled compounds 3f (IC 50  = 8.8 nM) and 3g (IC 50  = 8.7 nM) as PET imaging candidates. Selectivity of these compounds for CH24H was validated by a brain distribution study using CH24H-WT and KO mice. In non-human primate PET imaging, [ 18 F]3f and [ 18 F]3g showed similar regional uptake in the brain, indicating that these tracers were specific to the CH24H-expressed regions and validated the expression of CH24H in the living brain by different tracers., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Purpose: Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that plays a major role in brain cholesterol homeostasis by converting cholesterol into 24S-hydroxycholesterol. The selective CH24H inhibitor soticlestat (TAK-935) is being pursued as a drug for treatment of seizures in developmental and epileptic encephalopathies. Herein, we describe the successful discovery and the preclinical validation of the novel radiolabeled CH24H ligand (3-[ 18 F]fluoroazetidin-1-yl){1-[4-(4-fluorophenyl)pyrimidin-5-yl]piperidin-4-yl}methanone ([ 18 F]T-008) and its tritiated analog, [ 3 H]T-008., Methods: In vitro autoradiography (ARG) studies in the CH24H wild-type (WT) and knockout (KO) mouse brain sections were conducted using [ 3 H]T-008. PET imaging was conducted in two adult rhesus macaques using [ 18 F]T-008. Each macaque received two test-retest baseline scans and a series of two blocking doses of soticlestat administered prior to [ 18 F]T-008 to determine the CH24H enzyme occupancy. PET data were analyzed with Logan graphical analysis using plasma input. A Lassen plot was applied to estimate CH24H enzyme occupancy by soticlestat., Results: In ARG studies, binding of [ 3 H]T-008 was specific to CH24H in the mouse brain sections, which was not observed in CH24H KO or in wild-type mice after pretreatment with soticlestat. In rhesus PET studies, the rank order of [ 18 F]T-008 uptake was striatum > cortical regions > cerebellum, which was consistent with CH24H distribution in the brain. Pre-blocking with soticlestat reduced the maximum uptake and increased the washout in all brain regions in a dose-dependent manner. Calculated global occupancy values for soticlestat at a dose of 0.89 mg/kg were 97-98%, indicating maximum occupancy., Conclusion: The preclinical in vitro and in vivo evaluation of labeled T-008 demonstrates that [ 18 F]T-008 is suitable for imaging CH24H in the brain and warrants further studies in humans., (© 2021. The Author(s).)