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以及失败的、废弃的、 或尚未寻求临床开发候选者，以获得新的临床适应症。这一想法越来越受欢迎。 近年来的牵引，包括中风等中枢神经系统疾病的治疗。小胶质细胞(MG)，是小胶质细胞的常驻免疫细胞 中枢神经系统和浸润性巨噬细胞在缺血性脑损伤中起关键作用.调制MG/MP 表型是开发中风新疗法的一条很有前途的途径。KCa3.1是一种钙激活的 在反应性MG/MP中高表达的钾通道。先前的研究使用基因缺失或 KCa3.1的药物消融已经证明它会导致神经炎症和加重 中风后受伤。Senicapoc是一种KCa3.1特异性抑制剂，已用于非人类临床试验 神经学的指征。该药物已证明有安全记录，并有实质性的药代动力学(PK)， 药效学(PD)和细胞类型特定表达数据已完成，并可在该试剂上获得。目标 该建议的R61阶段包括：(1)量化senicapoc的PK轮廓，特别是在卒中的设置中 (小鼠短暂性大脑中动脉阻塞/再灌注模型)，并确定最佳给药剂量和 缺血性脑损伤的暂时性给药范式，(2)确定Senicapoc在减轻脑损伤中的疗效 应用尖端神经成像技术和神经成像技术对青年成年野生型小鼠卒中致脑损伤的研究 感觉-运动、神经行为和认知的纵向评估和(3)确定老年人的帕金森病 使用最先进的神经免疫学生物标记物描述和量化药物的CNS靶点参与。pk 评估将包括使用高效液相色谱/质谱仪来量化脑和血浆中老年载脂蛋白的总水平和游离水平。 光谱学。此外，我们将量化促炎细胞因子和趋化因子的水平 免疫分析。疗效评估将包括多参数14T磁共振成像以量化梗死体积(DWI/T2)， 脑水肿(T2/FLAIR)和脑白质完整性(DTI/气管造影术)。感觉-运动、神经行为和 认知测量将包括观察性评估和动态评估。对于PD研究，我们将检查 以MG/MP为靶点的老年载脂蛋白对卒中后神经免疫反应的影响 TSPO-放射性配基和正电子发射光谱(PET)以及用流式细胞术进行的体外研究 和免疫荧光显微镜。对于项目的R33阶段，我们将确定senicapoc的有效性 卒中患者的PD谱发生改变：(I)在不同年龄和/或性别的小鼠中，(Ii)在自发性高血压患者中， 病态的老鼠。总体而言，这些研究将提供对老年人口委员会能力的全面评估 影响卒中后中枢神经系统的变化，为今后的临床试验评估提供科学依据 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).