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Drug development for ischemic stroke is challenging as evidenced by the paucity of therapeutics that have advanced beyond a phase III trial. There are many reasons for this lack of clinical translation including factors related to the experimental design of preclinical studies. Often overlooked in therapeutic development for ischemic stroke is the requirement of effective drug delivery to the brain, which is critical for neuroprotective efficacy of several small and large molecule drugs. Advancing central nervous system drug delivery technologies implies a need for detailed comprehension of the blood-brain barrier (BBB) and neurovascular unit. Such knowledge will permit the innate biology of the BBB/neurovascular unit to be leveraged for improved bench-to-bedside translation of novel stroke therapeutics. In this review, we will highlight key aspects of BBB/neurovascular unit pathophysiology and describe state-of-the-art approaches for optimization of central nervous system drug delivery (ie, passive diffusion, mechanical opening of the BBB, liposomes/nanoparticles, transcytosis, intranasal drug administration). Additionally, we will discuss how endogenous BBB transporters represent the next frontier of drug delivery strategies for stroke. Overall, this review will provide cutting edge perspective on how central nervous system drug delivery must be considered for the advancement of new stroke drugs toward human trials.

Objectives:We have shown that transforming growth factor-β (TGF-β)/activin-like kinase 1 (ALK1) signaling at the blood-brain barrier (BBB) can regulate organic anion transporting polypeptide 1a4 (Oatp1a4), a transporter involved in brain uptake of drugs relevant to stroke pharmacotherapy (i.e., 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins)). To date, the involvement of endoglin (ENG), a co-receptor that can modulate the TGF-β/ALK1 pathway, in regulation of Oatp1a4 expression/activity has not been elucidated. The objective of this study is to evaluate whether ENG plays a role in the control of BBB transport mechanisms for stroke therapeutics.Methods:Our studies were conducted using the immortalized mouse brain endothelial cell line bEnd.3. Endoglin knockdown mutants were generated through lentiviral transfection of ENG-shRNA and validated using confocal microscopy and western blot analysis. Cells were then treated with bone morphogenetic protein-9 (BMP-9; 1 nM), a known TGF-β/ALK1 agonist. Oatp1a4 protein expression and transport activity were measured using western blot analysis and in vitrouptake assays using the known Oatp substrate drug [3H]atorvastatin. Additionally, ENG expression in bEnd.3 cells was assessed under normoxic and oxygen/glucose deprivation (OGD) conditions (i.e., 1% O2in glucose-free medium).Results:BMP-9 treatment in non-transfected bEnd.3 cells resulted in increased Oatp1a4 protein expression and increased cellular uptake of [3H]atorvastatin. These enhancements in Oatp1a4 functional expression were attenuated in ENG-shRNA knockdown bEnd.3 cells treated with BMP-9. Confocal microscopy and western blot analysis confirmed decreased ENG protein expression following shRNA transfection. Under OGD conditions, expression of ENG in bEnd.3 cells was significantly increased.Conclusions:Our data provide the first evidence for involvement of ENG in regulation of Oatp1a4 by TGF-β/ALK1 signaling in brain microvessel endothelial cells. These observations have clear implications for CNS delivery of drugs relevant to treatment of stroke. Studies are ongoing in our laboratory to fully evaluate ENG regulation of BBB transport mechanisms in the setting of ischemic stroke.

Purpose:Cationic drugs have often been considered for stroke treatment but their utility is dependent upondelivery across the blood-brain barrier (BBB). This process can involve transporters for cationic substratessuch as organic cation transporters 1 and 2 (Oct1 and Oct2) as well as the multidrug and toxin extruder 1(Mate1). The goal of this study was to show that the Oct/Mate system at the BBB represents an endogenoustransporter mechanism that can be exploited for efficient brain delivery of cationic drugs relevant to stroketherapy.Methods:Male Sprague-Dawley rats (200-250 g) were subjected to middle cerebral artery occlusion (MCAO;90 minutes) followed by reperfusion. Sham-operated rats were used as controls. Memantine (5 mg/kg, i.v.), arepresentative cationic neuroprotective drug, was injected 2 h following intraluminal suture removal in thepresence and absence of cimetidine (15 mg/kg, i.v.), an inhibitor of cation transporters. Localization andprotein expression of Oct1, Oct2, and Mate1 were evaluated by confocal microscopy and western blottingrespectively. Progression of cerebral infarction volume was determined via TTC-stained brain slices.Neurological and motor outcomes during the acute (1 day) and subacute (3-7 days) recovery period post-stroke were assessed using functional neuroscores (i.e., based upon the modified Rankin Scale) and rotorodanalysis.Results:Localization and protein expression of Oct1, Oct2, and Mate1 were confirmed in cerebralmicrovessels isolated from Sprague-Dawley rats subjected to MCAO and in Sham controls. Memantinereduced progression of cerebral infarction, reduced neurological scores, and improved rotorod performanceduring the acute and subacute recovery periods following MCAO. Pharmacological effectiveness of memantinewas decreased in the presence of cimetidine.Conclusions:Our data show that memantine cannot exert beneficial effects during acute and subacute post-stroke recovery without functional expression of endogenous transporters for organic cations (i.e., Oct1, Oct2,and Mate1) at the BBB. This work is highly translational and emphasizes the critical need to evaluate BBBtransport mechanisms during preclinical development of drugs for ischemic stroke treatment.

Clinical development of new drugs for ischemic stroke has been challenging. In contrast, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e., statins) are routinely administered to stroke patients because they improve neurological outcomes. This property of statins requires delivery across the blood-brain barrier (BBB). We have uncovered a BBB uptake transporter for statins: organic anion transporting polypeptide 1a4 (Oatp1a4). Our goal was to determine whether Oatp-mediated transport at the BBB is a required mechanism for a commonly prescribed statin (i.e., atorvastatin; ATV) to exert neuroprotective effects following ischemic stroke.Methods:Male Sprague-Dawley rats (200-250 g) were subjected to middle cerebral artery occlusion (MCAO; 90 min) followed by reperfusion. ATV (20 mg/kg, i.v.) was administered 2 h following intraluminal suture removal. Involvement of Oatp-mediated transport was determined using fexofenadine (FEX; 3.2 mg/kg, i.v.), a competitive Oatp inhibitor. Oatp1a4 transport activity was measured by in situbrain perfusion. Following MCAO/reperfusion, infarction volumes and brain edema ratios were calculated from TTC-stained brain tissue slices. Hippocampal neuronal cell numbers were assessed by immunofluorescence imaging of neuronal nuclei (NeuN) staining. Post-stroke functional outcomes were assessed via neurological deficit scores and rotarod analysis. At 2 h post-MCAO, ATV uptake was increased in ischemic brain tissue. FEX blocked blood-to-brain uptake of ATV, which confirmed involvement of an Oatp-mediated transport mechanism. ATV reduced post-MCAO infarction volume and brain edema ratio, increased hippocampal NeuN staining, and improved neurological outcomes. All positive effects of ATV were attenuated by co-administration of FEX.Conclusions:Our data demonstrate ATV requires functional expression of an endogenous BBB transporter (i.e., Oatp1a4) to exert neuroprotective effects and promote post-stroke recovery. These novel and translational findings provide mechanistic evidence on the critical role of BBB transporters in delivery of stroke drugs to the ischemic brain, information that can facilitate therapeutic advancement in clinical trials.