Repurposing of KCa3.1 Inhibitor Senicapoc for Stroke: A Pre-clinical Study

药物再利用的概念是指对现有药物的评估和使用，以及失败的，废弃的， 或尚未进行临床开发的候选药物。这一想法越来越受到 近年来，包括用于中枢神经系统疾病如中风的牵引。小胶质细胞（MG）， CNS和浸润性巨噬细胞（MP）在缺血性脑损伤中是关键的。调节MG/MP 表型是开发中风新疗法的有希望的途径。KCa3.1是一种钙激活剂， 钾离子通道在反应性MG/MP中高度表达。先前的研究使用基因缺失或 KCa3.1的药物消融已经证明它有助于神经炎症和恶化 中风后的损伤Senicapoc是一种KCa3.1特异性抑制剂，已用于人类临床试验，用于非肿瘤治疗。 神经系统症状该药物具有经证实的安全性记录，并且具有显著的药代动力学（PK）， 已完成并可获得该药物的药效学（PD）和细胞类型特异性表达数据。的目标 该建议的R61阶段的主要目标是：（1）量化senicapoc的PK特征，特别是在中风的情况下 （小鼠短暂性大脑中动脉闭塞/再灌注模型），并确定最佳剂量和 缺血性脑损伤的时间给药模式，（2）确定senicapoc在减少 使用尖端神经成像技术在年轻成年野生型小鼠中进行中风诱导的脑损伤， 感觉-运动、神经行为和认知纵向评估和（3）确定senicapoc的PD 使用最先进的神经免疫学生物标志物分析和量化药物的CNS靶点参与。PK 评估将包括使用HPLC/质谱法定量脑和血浆中senicapoc的总水平和游离水平。 谱此外，我们还将使用免疫组织化学方法定量促炎细胞因子和趋化因子的水平。 免疫测定。疗效评估将包括多参数14 T MRI，以量化梗死体积（DWI/T2）， 脑水肿（T2/FLAIR）和白色完整性（DTI/纤维束成像）。感觉运动、神经行为和 认知测量将包括观察和动态评估。对于PD研究，我们将检查 使用MG/MP靶向的体内神经免疫应答中senicapoc对中风诱导的变化的影响 TSPO-放射性配体和正电子发射光谱（PET）以及使用流式细胞术的离体研究 和免疫荧光显微镜。对于该项目的R33阶段，我们将确定塞尼卡泊克的疗效是否 中风中的PD和PD特征改变：（i）在小鼠中，年龄和/或性别改变;（ii）在自发性高血压中， 病态的老鼠总的来说，这些研究将提供一个全面的评估能力的senicapoc， 影响中枢神经系统中风引起的变化，并为未来的临床试验提供科学基础， 药物（senicapoc）和靶点（KCa3.1）。

The concept of drug repurposing refers to the evaluation and use of existing drugs, as well as failed, abandoned, or not yet pursued clinical development candidates, for new clinical indications. This idea has gained increasing traction in recent years including for CNS disorders such as stroke. Microglia (MG), the resident immune cells of the CNS, and infiltrating macrophages (MP) are critical in ischemic brain injury. Modulating the MG/MP phenotype is a promising avenue for developing novel therapeutics for stroke. KCa3.1 is a calcium activated potassium channel that is highly expressed in reactive MG/MP. Prior studies using either genetic deletion or pharmacologic ablation of KCa3.1 have demonstrated that it contributes to neuroinflammation and exacerbation of post-stroke injury. Senicapoc is a KCa3.1-specific inhibitor that has been used in human clinical trials for non- neurological indications. The drug has a proven safety record and there is substantial pharmacokinetic (PK), pharmacodynamic (PD) and cell type specific expression data completed and available on this agent. The goals of the R61 phase of this proposal are: (1) Quantify senicapoc's PK profile specifically in the setting of stroke (mouse transient middle cerebral artery occlusion/reperfusion model) and identify an optimal dosing and temporal administration paradigm for ischemic brain injury, (2) Determine the efficacy of senicapoc in reducing stroke-induced brain injury in young adult wild-type mice using cutting-edge neuroimaging technology as well as sensory-motor, neurobehavioral and cognitive longitudinal assessments and (3) Determine senicapoc's PD profile and quantify the drug's CNS target engagement using state-of-the-art neuroimmunology biomarkers. PK assessments will include quantifying total and free levels of senicapoc in brain and plasma using HPLC/mass spectroscopy. In addition we will quantify levels of pro-inflammatory cytokines and chemokines using immunoassays. Efficacy assessments will include multiparametric 14 T MRI to quantify infarct volume (DWI/T2), cerebral edema (T2/FLAIR) and white matter integrity (DTI/tractography). Sensory-motor, neurobehavioral and cognitive measures will include both observational and dynamic assessments. For PD studies, we will examine the effect of senicapoc on stroke-induced changes in the neuroimmune response in vivo using MG/MP-targeted TSPO-radioligand and positron emission spectroscopy (PET) as well as ex vivo studies using flow cytometry and immunofluorescent microscopy. For the R33 phase of the project, we will determine if senicapoc's efficacy and PD profiles in stroke are altered: (i) in mice by age and/or sex and (ii) in spontaneously hypertensive, co- morbid rats. Overall, these studies will provide a comprehensive assessment of the ability of senicapoc to influence stroke-induced changes in the CNS and provide a scientific foundation for future clinical trials assessing both drug (senicapoc) and target (KCa3.1).