Biodistribution and Efficacy of an NF‐κB‐Targeted Biologic for Therapy of Ischemic Stroke

Strokes are the fifth leading cause of death in the United States, and ~87% of strokes are ischemic, meaning that they are caused by occlusion of a cerebral artery or its branches. This occlusion results in a lack of oxygen reaching the tissue, increases in intracellular calcium and glutamate release, and the activation of inflammatory processes, which can cause additional tissue loss and worsen neurological condition. The nuclear factor kappa B (NF-κB) cascade is an inflammatory pathway activated by many cytokines and inflammatory signals that are elevated following ischemic stroke, making it a central hub for post-stroke inflammation. While there are current therapies to treat the occlusion causing an ischemic stroke, they do not treat the inflammation induced injury that occurs. We have designed a novel inhibitor of the NF-κB cascade attached to the carrier protein elastin-like polypeptide (ELP) as an adjuvant to current revascularization therapies. ELP is based on the sequence of human elastin, has a long plasma half-life, is thermally responsive, and has a tunable size. We hypothesize that ELPs can be used to deliver our inhibitory peptide SynB1-ELP-p50i to the infarcted region after ischemic stroke, reduce inflammation, and spare brain tissue. To begin, BV2 microglia, C8-D1A astrocytes, and SH-SY5Y neurons were treated with rhodamine labeled SynB1-ELP-p50i for 24 hours and imaged with confocal microscopy to determine if the protein was able to enter the cells. SynB1-ELP-p50i entered the cells and was primarily localized to the cytoplasm. Next, using the middle cerebral artery occlusion (MCAO) model of ischemic stroke in spontaneously hypertensive rats (SHRs), biodistribution and pharmacokinetics were assessed following a bolus administration of 50 mg/kg rhodamine labeled SynB1-ELP-p50i, using two routes of administration (intravenous (IV, femoral vein) and intraarterial (IA, carotid catheter)). SynB1-ELP-p50i accumulated in the ischemic hemisphere at levels nearly 8 fold higher than in the contralateral hemisphere following MCAO (two-way ANOVA F(1,12) = 63.08; Sidak's p < 0.0001). There was no difference in intrabrain deposition between IV and IA routes. To assess the efficacy of SynB1-ELP-p50i (50 mg/kg, IV) to reduce infarct size and inflammation following MCAO, the brains were stained, and infarct size was measured using Image J. There was no effect of SynB1-ELP-p50i treatment on infarct size compared to saline controls, and the baseline mean arterial pressures were the same between treatment groups. qPCR analysis of downstream targets of the NF-κB cascade are ongoing to determine if SynB1-ELP-p50i treatment affected the production of inflammatory cytokines following MCAO. Future studies will assess effects of SynB1-ELP-p50i treatment on behavior and neurological deficits following MCAO in SHRs.