Radiosynthesis and characterization of a carbon-11 PET tracer for receptor-interacting protein kinase
Radiosynthesis and characterization of a carbon-11 PET tracer for receptor-interacting protein kinase
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DOI：
10.1016/j.nucmedbio.2022.04.006
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发表时间：
2022-04-25
影响因子：
3.1
通讯作者：
Tu, Zhude
中科院分区：
文献类型：
--
作者：
Huang, Tianyu;Gu, Jiwei;Tu, Zhude
受体相互作用蛋白激酶1 （RIPK1）通过激活一组下游免疫受体而成为坏死性下垂和炎症反应的重要调节因子。它已被认为是在各种生理和病理过程中细胞死亡和炎症的关键因素。人类RIPK1缺乏或失调可导致严重的免疫缺陷和神经退行性疾病，如多发性硬化症和肌萎缩侧索硬化症。最近，不同结构的RIPK1抑制剂已被开发为神经退行性疾病和其他病理性炎症过程的潜在治疗药物。7-氧-2,4,5,7-四氢- 6h -pyrazolo[3,4-c]吡啶（Compound 5或TZ7774）是一种新型RIPK1抑制剂，Ki值为0.91 nM，可抑制小鼠和人细胞的坏死。为了开发一种用于体内研究RIPK1的放射性示踪剂，我们对[11C]TZ7774进行了放射性合成，并在啮齿动物和猕猴中进行了初步的体外和体内评价。方法：对去甲基前体TZ7790的合成工艺进行优化。[11C] TZ7774的放射性合成是通过TZ7790与[11C]碘化甲基n -甲基化反应实现的。[11C]TZ7774在正常Sprague-Dawley大鼠体内进行体外生物分布。通过正常和LPS处理（10 mg/ kg） C57BL/6小鼠的体外生物分布研究，以及脾脏的体外放射自显影和免疫组织化学，研究[11C]TZ7774对炎症反应的反应。对猕猴[11C]TZ7774进行了微pet脑研究。结果与结论：[11C]TZ7774的放射合成具有良好的放射化学产率（30-40%，衰变校正到轰击结束（EOB）），高化学纯度（>90%），高放射化学纯度（>99%）和高摩尔活性（>207 GBq/mu mol，衰变校正到EOB）。Sprague-Dawley大鼠的生物分布研究显示[11C]TZ7774在注射后5分钟的脑摄取率为0.53 (%ID/g)；胰腺、脾脏、肾脏和肝脏也显示出相对较高的初始摄食量，5分钟时分别为0.49、0.41、0.62和0.95。lps处理的C57BL/6小鼠肝脏、脾脏和肾脏对[11C]TZ7774的摄取分别增加了40.9%、90.4%和54.9%。体外放射自显影研究也显示lps处理小鼠脾脏对[11C] TZ7774的摄取增加。免疫组织化学进一步表征证实，LPS预处理小鼠脾脏红髓和白髓中RIPK1表达增加。MicroPET显示[11C]TZ7774在6-10 min时具有良好的初始脑摄取，（SUV）接近3.7，并迅速从大脑中冲洗出来。这些数据证实了RIPK1特异性放射性示踪剂[11C]TZ7774的成功放射性合成。我们的初步研究表明，啮齿动物对脂多糖诱导的炎症反应良好，猕猴大脑对脂多糖的吸收良好。[11C]TZ7774具有成像RIPK1相关的坏死下垂和炎症过程的潜力。
Introduction: Receptor-interacting protein kinase 1 (RIPK1) has emerged as a crucial regulator of necroptosis and the inflammatory response by activating a group of downstream immune receptors. It has been recognized as a pivotal contributor to cell death and inflammation in various physiological and pathological processes. RIPK1 deficiency or dysregulation in humans can cause severe immunodeficiency and neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis. Recently, diverse structures of RIPK1 inhibitors have been developed as potential therapeutics for neurodegenerative diseases and other pathological inflammatory processes. 7-oxo-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine (Compound 5 or TZ7774) was reported as a novel RIPK1 inhibitor with a Ki of 0.91 nM that can suppress necroptosis in mouse and human cells. To develop a radiotracer for investigating the RIPK1 in vivo, we radiosynthesized [11C]TZ7774 and performed preliminary in vitro and in vivo evaluations in rodents and macaque. Methods: Synthesis of the desmethyl precursor TZ7790 was performed and optimized. The radiosynthesis of [11C] TZ7774 was achieved through TZ7790 reacting with [11C]methyl iodide via N-methylation. Ex vivo biodistribution of [11C]TZ7774 was performed in normal Sprague-Dawley rats. Characterization of [11C]TZ7774 in response to inflammation was performed using ex vivo biodistribution study in normal and LPS treated (10 mg/ kg) C57BL/6 mice, and in vitro autoradiography and immunohistochemistry of the spleen. MicroPET brain study of [11C]TZ7774 in the macaque was also performed. Results and conclusions: The radiosynthesis of [11C]TZ7774 was achieved with good radiochemical yield (30-40%, decay corrected to the end of bombardment (EOB)), high chemical purity (>90%), high radiochemical purity (>99%), and high molar activity (>207 GBq/mu mol, decay corrected to EOB). Biodistribution studies in Sprague-Dawley rats showed [11C]TZ7774 has a high brain uptake of 0.53 (%ID/g) at 5 min post injection; pancreas, spleen, kidney, and liver also showed a relatively high initial uptake of 0.49, 0.41, 0.62, and 0.95 at 5 min respectively. Uptake of [11C]TZ7774 increased in LPS-treated C57BL/6 mice by 40.9%, 90.4%, and 54.9% in liver, spleen, and kidney respectively. In vitro autoradiography study also revealed increased uptake of [11C] TZ7774 in the spleen of LPS-treated mice. Further characterization with immunohistochemistry confirmed increased expression of RIPK1 in red and white pulp of the spleen for mice pre-treated with LPS. MicroPET demonstrated that [11C]TZ7774 had good initial brain uptake in macaque with an (SUV) of similar to 3.7 at 6-10 min, and quickly washed out from brain. These data confirm successful radiosynthesis of a RIPK1 specific radiotracer [11C]TZ7774. Our preliminary studies showed good response to LPS-induced inflammation in rodents and good uptake in macaque brain. [11C]TZ7774 has a potential to image RIPK1 related necroptosis and inflammatory processes.