Purpose

The monoacylglycerol lipase (MAGL) plays a pivotal role in modulating the endocannabinoid system and is considered an attractive therapeutic target for diseases in both the central nervous system and periphery. The current study aimed to develop and evaluate a suitable carbon-11 labeled tracer for imaging MAGL in preclinical studies.

Methods

(R)-YH168 was synthesized via a multi-step pathway and its half-maximal inhibitory concentration (IC₅₀) values were measured using an enzymatic assay. Radiosynthesis of (R)-[¹¹C]YH168 was accomplished by ¹¹C-methylation via Suzuki cross-coupling of a pinacol boron precursor. In vitro autoradiography was performed using brain tissues from MAGL knockout and the corresponding wild-type mice. The metabolic stability of (R)-[¹¹C]YH168 in mouse brain and plasma was assessed 5 min after injection. Dynamic PET scans were conducted on anesthetized mice and rhesus monkey. For studies in non-human primates, arterial blood samples were analyzed to obtain the input function for kinetic modeling. Blocking studies with the irreversible MAGL inhibitor PF-06795071 were performed to assess the binding specificity of (R)-[¹¹C]YH168.

Results

(R)-[¹¹C]YH168 was synthesized via Suzuki coupling of the phenyl boronic ester with [¹¹C]CH₃I in the presence of palladium catalyst. In vitro autoradiography revealed a heterogeneous distribution pattern of (R)-[¹¹C]YH168 with higher binding to MAGL-rich brain regions in wild-type mouse brain slices compared to that of MAGL knockout mice. Dynamic PET imaging in wild-type and MAGL knockout mice confirmed its high specificity and selectivity in mouse brains. In the rhesus monkey, (R)-[¹¹C]YH168 displayed good brain permeability. High levels of radioactivity uptake were seen in the cingulate cortex, frontal cortex, cerebellum, occipital cortex, and hippocampus, consistent with MAGL expression. The one-tissue compartment model was appropriate for fitting the regional time-activity curves and provided reliable volume of distribution values across all brain regions. Pretreatment with PF-06795071 (0.1 mg/kg) resulted in almost complete blockade (> 95%) of radioactivity uptake, demonstrating binding specificity of (R)-[¹¹C]YH168 to MAGL in the non-human primate brain. The regional non-displaceable binding potential follows the rank order of cingulate cortex ~ frontal cortex ~ insula > putamen > temporal cortex > caudate ~ occipital cortex ~ thalamus > nucleus accumbens ~ hippocampus ~ cerebellum ~ globus pallidus > substantia nigra > amygdala.

Conclusion

(R)-[¹¹C]YH168 is a promising PET probe for imaging and quantifying MAGL in the brains of mice and non-human primates. This ¹¹C-labeled tracer holds great potential for translation into human subjects and offers the possibility of performing multiple PET scans on the same subject within a single day.

中文翻译：

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探索 （R）-[11C]YH168 作为 PET 示踪剂对大脑中单酰基甘油脂肪酶进行成像：从小鼠到非人灵长类动物

 目的

单酰基甘油脂肪酶 （MAGL） 在调节内源性大麻素系统中起着关键作用，被认为是中枢神经系统和外周疾病的有吸引力的治疗靶点。目前的研究旨在开发和评估一种合适的碳 11 标记示踪剂，用于临床前研究中对 MAGL 进行成像。

 方法

（R）-YH168 通过多步途径合成，并使用酶法测定其半数最大抑制浓度 （IC₅₀） 值。（R）-[¹¹C]YH168 的放射合成是通过 Pinacol 硼前驱体的 Suzuki 交叉偶联的 ¹¹C-甲基化完成的。使用来自 MAGL 敲除的脑组织和相应的野生型小鼠进行体外放射自显影。注射后 5 min 评估 （R）-[¹¹C]YH168 在小鼠脑和血浆中的代谢稳定性。对麻醉小鼠和恒河猴进行动态 PET 扫描。对于非人类灵长类动物的研究，分析了动脉血样本以获得动力学建模的输入函数。使用不可逆的 MAGL 抑制剂 PF-06795071 进行阻断研究，以评估 （R）-[¹¹C]YH168 的结合特异性。

 结果

在钯催化剂存在下，通过苯基硼酯与 [¹¹C]CH₃I 的 Suzuki 偶联合成 （R）-[¹¹C]YH168。体外放射自显影显示 （R）-[¹¹C]YH168 的异质性分布模式，与 MAGL 敲除小鼠相比，与野生型小鼠脑切片中富含 MAGL 的脑区的结合更高。野生型和 MAGL 敲除小鼠的动态 PET 成像证实了其在小鼠大脑中的高特异性和选择性。在恒河猴中，（R）-[¹¹C]YH168 表现出良好的脑通透性。在扣带皮层、额叶皮层、小脑、枕叶皮层和海马体中观察到高水平的放射性吸收，与 MAGL 表达一致。单组织隔室模型适用于拟合区域时间 - 活动曲线，并在所有大脑区域提供可靠的分布体积值。用 PF-06795071 （0.1 mg/kg） 预处理导致放射性吸收几乎完全阻断 （> 95%），证明 （R）-[¹¹C]YH168 与非人灵长类动物大脑中 MAGL 的结合特异性。区域不可移位的结合电位遵循扣带皮层 ~ 额叶皮层 ~ 岛叶 > 壳核 > 颞叶皮层 > 尾状核 ~ 枕叶皮层 ~ 丘脑 > 伏隔核 ~ 海马体 ~ 小脑 ~ 苍白球 > 黑质 > 杏仁核。

 结论

（R）-[¹¹C]YH168 是一种很有前途的 PET 探针，用于对小鼠和非人灵长类动物大脑中的 MAGL 进行成像和定量。这种 ¹¹C 标记的示踪剂具有转化为人类受试者的巨大潜力，并提供了在一天内对同一受试者进行多次 PET 扫描的可能性。