(T1130-05-29) Endogenous Blood-Brain Barrier Transporters are Critical Determinants of Neuroprotective Drug Efficacy in Ischemic Stroke

Purpose: Stroke is the fifth leading cause of death in the United States. Available treatment strategies for stroke are focused on rescuing injured brain tissue that surrounds the infarction core (i.e., the ischemic penumbra). There are only two FDA approved therapies for stroke: recombinant tissue plasminogen activator (r-tPA) and endovascular thrombectomy (EVT). These therapies promote reperfusion of ischemic brain tissue; however, reperfusion therapies are associated with exacerbation of neuronal injury and/or death. Additionally, both r-tPA therapy and EVT have a limited therapeutic window of 4 to 6 hours from the onset of stroke due to risk of hemorrhagic transformation. Therefore, there is an unmet clinical need for neuroprotective therapies that can protect neuronal tissue and promote repair in stroke. To date, drug discovery for stroke has been challenging as indicated by poor translatability of compounds from preclinical studies to successful clinical trials. In contrast, some drugs (i.e., 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins); memantine) have utility in improving functional neurological outcomes in stroke patients. This property indicates that both statins and memantine are efficiently delivered across the blood-brain barrier (BBB). In vivo studies conducted in our laboratory have uncovered specific transport mechanisms that enable these drugs to be delivered from the systemic circulation into brain tissue: transport via endogenous BBB uptake transporters organic anion transporting polypeptide 1a4 (Oatp1a4) or organic cation transporters (Octs). The goal of this project was to show that functional expression of Oatp1a4 or Oct1/Oct2 at the BBB are required mechanisms that enable statins and memantine to be effective neuroprotective drugs for ischemic stroke.

Methods: Male Sprague-Dawley rats (200-250 g) were subjected to transient middle cerebral artery occlusion (tMCAO) for 90 minutes followed by reperfusion for 24 h or 72 h. Sham-operated animals (i.e., controls) underwent the same surgical procedure except that the intraluminal suture was not inserted. Atorvastatin (20 mg/kg, i.v.) or memantine (5 mg/kg, i.v.) were injected 2 h following removal of the intraluminal suture (i.e., reperfusion). Oatp1a4, Oct1, and Oct2 protein expression were determined by western blot analysis of isolated brain microvessels. The role of Oatp-mediated transport was determined using the pharmacological Oatp inhibitor fexofenadine (3.2 mg/kg, i.v.) injected at the same time as atorvastatin. Similarly, specificity of Oct1/Oct2-mediated transport was assessed using the competitive Oct inhibitor cimetidine (15 mg/kg, i.v.) injected simultaneously with memantine. Following tMCAO, infarction volume and brain edema ratios were calculated from TTC-stained brain tissue slices. Post-stroke outcomes were assessed after tMCAO via neurological deficit scores (i.e., a scoring system modelled upon clinically relevant stroke assessments such as the modified Rankin Scale) and rotarod analysis (i.e., motor function). BBB transport properties of [³H]atorvastatin (0.3 mCi/ml) and [³H]memantine (0.5 mCi/ml) as well as paracellular “leak” (via [¹⁴C]sucrose (0.3 mCi/ml or 0.5 mCi/ml) were measured using our in situ brain perfusion technique.

Results: Atorvastatin significantly reduced both infarction volume and the brain edema ratio following 24 h reperfusion while memantine decreased both parameters after 72 h reperfusion. Both drugs also improved post-stroke outcomes as determined by neurological deficit scores and rotarod analysis. In the presence of fexofenadine (3.2 mg/kg, i.v.), atorvastatin had no effect on infarction volume, brain edema, or neurocognitive performance. Similarly, cimetidine (15 mg/kg, i.v.) blocked all positive effects of memantine on stroke outcomes. Our in situ perfusion data showed that uptake of [³H]atorvastatin and [³H]memantine in ipsilateral and contralateral cerebral cortices was blocked by pharmacological inhibitors of Oatp1a4 (i.e., fexofenadine) or Oct1/Oct2 (i.e., cimetidine). In these experiments, we observed that the magnitude of neuroprotective drug uptake in ipsilateral cortex was greater than that measured in contralateral cortex under tMCAO conditions. This finding suggests that a component of blood-to-brain atorvastatin and memantine uptake results from non-selective paracellular “leak”, which we confirmed by showing increased uptake of [¹⁴C]sucrose in ipsilateral cortical tissue following tMCAO.

Conclusion: This study has identified BBB transport mediated by Oatp1a4 and Oct1/Oct2 as critical mechanisms that facilitate CNS drug delivery in the setting of ischemic stroke. Specifically, we have shown for the first time that blood-to-brain transport via Oatp1a4 or Oct1/Oct2 is required for atorvastatin or memantine to exert neuroprotective effects in the ischemic brain and to promote post-stroke recovery. This effect occurred despite concurrent paracellular “leak”, which demonstrates that selective BBB transporters dominate over non-selective drug uptake pathways. Our data emphasize the need to assess brain penetration of therapeutic agents during preclinical drug development, a consideration that will likely enable new compounds for ischemic stroke to advance further in clinical trials. Overall, these results strengthen the novel and translational evidence generated by our laboratory that endogenous BBB transport systems can be targeted for CNS drug delivery, thereby providing a platform for development of novel treatment strategies for ischemic stroke.

Acknowledgements: This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01-NS084941) and the American Heart Association (19TPA34910113) to PTR. The authors do not have any conflicts of interest to disclose.