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-[18F]fluoroazetidin-1-yl){1-[4-(4-fluorophenyl)pyrimidin-5-yl]piperidin-4-yl}methanone ([18F]T-008) and its tritiated analog, [3H]T-008. METHODS In vitro autoradiography (ARG) studies in the CH24H wild-type (WT) and knockout (KO) mouse brain sections were conducted using [3H]T-008. PET imaging was conducted in two adult rhesus macaques using [18F]T-008. Each macaque received two test-retest baseline scans and a series of two blocking doses of soticlestat administered prior to [18F]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 [3H]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 [18F]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 [18F]T-008 is suitable for imaging CH24H in the brain and warrants further studies in humans.

中文翻译：

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[18F]T-008 的临床前表征，一种用于胆固醇 24-羟化酶的新型 PET 成像放射性配体。

用途 胆固醇 24-羟化酶 (CH24H) 是一种大脑特异性酶，通过将胆固醇转化为 24S-羟基胆固醇，在大脑胆固醇稳态中起主要作用。选择性 CH24H 抑制剂 soticlestat (TAK-935) 正在被用作治疗发育性和癫痫性脑病癫痫发作的药物。在此，我们描述了新型放射性标记 CH24H 配体 (3-[18F]fluoroazetidin-1-yl){1-[4-(4-fluorophenyl)pyrimidin-5-yl]piperidin-4- 的成功发现和临床前验证基}甲酮（[18F]T-008）及其氚代类似物，[3H]T-008。方法 使用 [3H]T-008 对 CH24H 野生型 (WT) 和敲除 (KO) 小鼠脑切片进行体外放射自显影 (ARG) 研究。使用 [18F]T-008 在两只成年恒河猴中进行 PET 成像。每只猕猴在 [18F]T-008 之前接受了两次重测基线扫描和一系列两次阻断剂量的 soticlestat，以确定 CH24H 酶的占有率。PET 数据通过使用血浆输入的 Logan 图形分析进行分析。应用拉森图来估计 soticlestat 的 CH24H 酶占有率。结果 在 ARG 研究中，[3H]T-008 的结合对小鼠脑切片中的 CH24H 具有特异性，而在用 Soticlestat 预处理后，在 CH24H KO 或野生型小鼠中未观察到这种结合。在恒河猴PET研究中，[18F]T-008摄取的排列顺序为纹状体>皮质区>小脑，与脑内CH24H分布一致。使用 Soticlestat 进行预阻断降低了最大摄取并以剂量依赖性方式增加了所有大脑区域的冲洗。以 0.89 mg/kg 的剂量计算的 soticlestat 的全局占用率为 97-98%，表明最大占用率。结论 标记的 T-008 的临床前体外和体内评估表明，[18F]T-008 适用于大脑中的 CH24H 成像，值得在人体中进行进一步研究。