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).