Novel Therapeutics for Treating Neuroinflammation Following Ischemic Stroke

Abstract

Ischemic strokes are the fifth leading cause of death in the United States and are caused by the blockage of a major cerebral artery or one of 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 (NFkB) 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. We have designed a novel inhibitor of the NFkB cascade attached to drug carrier elastin-like polypeptide (ELP). ELP is based on the sequence of human elastin, has a long plasma half-life, is thermally responsive, and has a tunable size. Our hypothesis is that ELPs can be used to deliver NFkB inhibitory peptide SynB1-ELP-p50i to the infarct region after ischemic stroke, blocking inflammation and ultimately sparing brain tissue. To determine if SynB1-ELP-p50i is an effective inhibitor of the NFkB cascade, we assessed its efficacy in in vitro experiments. RAW 264.7 cells were treated with 1-50 mM SynB1-ELP-p50i then stimulated with lipopolysaccharide (LPS). TNF-a produced in response to LPS stimulation was measured using a TNF-a ELISA with the cell media. SynB1-ELP-p50i treatment completely blocked LPS-stimulated TNF-a release at a concentration of 10 mM. SynB1-ELP-p50i was determined to be an effective inhibitor for translation to in vivo efficacy studies using the middle cerebral artery occlusion (MCAO) model of ischemic stroke. Biodistribution and pharmacokinetic studies were conducted comparing two routes of delivery (intravenous, via the femoral vein, or intraarterial, via a catheter in the external carotid artery) of 50 mg/kg fluorescently labeled SynB1-ELP-p50i after two hours of MCAO. Our results show significant accumulation of SynB1-ELP-p50i in the ischemic hemisphere following MCAO (two-way ANOVA F(1,12) = 10.34; Sidak's p = 0.045). However, there was no difference in intrabrain deposition between intravenous and intraarterial routes of delivery. Future studies will assess the effects of SynB1-ELP-p50i treatment on infarct size, inflammatory cytokine production, and neurological deficits following MCAO in Wistar rats.