CNS Drug Delivery in Stroke: Improving Therapeutic Translation From the Bench to the Bedside.

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.

Virologs, viral mimicry, and virocell metabolism: the expanding scale of cellular functions encoded in the complex genomes of giant viruses.

The phylum Nucleocytoviricota includes the largest and most complex viruses known. These "giant viruses" have a long evolutionary history that dates back to the early diversification of eukaryotes, and over time they have evolved elaborate strategies for manipulating the physiology of their hosts during infection. One of the most captivating of these mechanisms involves the use of genes acquired from the host-referred to here as viral homologs or "virologs"-as a means of promoting viral propagation. The best-known examples of these are involved in mimicry, in which viral machinery "imitates" immunomodulatory elements in the vertebrate defense system. But recent findings have highlighted a vast and rapidly expanding array of other virologs that include many genes not typically found in viruses, such as those involved in translation, central carbon metabolism, cytoskeletal structure, nutrient transport, vesicular trafficking, and light harvesting. Unraveling the roles of virologs during infection as well as the evolutionary pathways through which complex functional repertoires are acquired by viruses are important frontiers at the forefront of giant virus research.

Oatp (Organic Anion Transporting Polypeptide)-Mediated Transport: A Mechanism for Atorvastatin Neuroprotection in Stroke.

BACKGROUND: Drug discovery for stroke is challenging as indicated by poor clinical translatability. In contrast, HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (ie, statins) improve poststroke neurological outcomes. This property requires transport across the blood-brain barrier via an endogenous uptake transporter (ie, Oatp1a4 [organic anion transporting polypeptide 1a4]). Our goal was to study Oatp1a4 as a drug delivery mechanism because the blood-brain barrier cannot be assumed to be completely open for all drugs in ischemic stroke. METHODS: Male Sprague-Dawley rats (200-250 g) were subjected to middle cerebral artery occlusion (90 minutes) followed by reperfusion for up to 7 days. Atorvastatin (20 mg/kg, IV) was administered 2 hours following intraluminal suture removal. Involvement of Oatp-mediated transport was determined using fexofenadine (3.2 mg/kg, IV), a competitive Oatp inhibitor. Oatp1a4 transport activity was measured by in situ brain perfusion. Infarction volumes/brain edema ratios and neuronal nuclei expression were determined using 2,3,5-triphenyltetrazolium chloride-stained brain tissue slices and confocal microscopy, respectively. Poststroke functional outcomes were assessed via neurological deficit scores and rotarod analysis. RESULTS: At 2-hour post-middle cerebral artery occlusion, [3H]atorvastatin uptake was increased in ischemic brain tissue. A single dose of atorvastatin significantly reduced post-middle cerebral artery occlusion infarction volume, decreased brain edema ratio, increased caudoputamen neuronal nuclei expression, and improved functional neurological outcomes. All middle cerebral artery occlusion positive effects of atorvastatin were attenuated by fexofenadine coadministration (ie, an Oatp transport inhibitor). CONCLUSIONS: Our data demonstrate that neuroprotective effects of atorvastatin may require central nervous system delivery by Oatp-mediated transport at the blood-brain barrier, a mechanism that persists despite increased cerebrovascular permeability in ischemic stroke. These novel and translational findings support utility of blood-brain barrier transporters in drug delivery for neuroprotective agents.

Targeting organic cation transporters at the blood-brain barrier to treat ischemic stroke in rats.

Drug discovery and development for stroke is challenging as evidenced by few drugs that have advanced beyond a Phase III clinical trial. Memantine is a N-methyl-d-aspartate (NMDA) receptor antagonist that has been shown to be neuroprotective in various preclinical studies. We have identified an endogenous BBB uptake transport system for memantine: organic cation transporters 1 and 2 (Oct1/Oct2). Our goal was to evaluate Oct1/Oct2 as a required BBB mechanism for memantine neuroprotective effects. Male Sprague-Dawley rats (200-250 g) were subjected to middle cerebral artery occlusion (MCAO) for 90 min followed by reperfusion. Memantine (5 mg/kg, i.v.) was administered 2 h following intraluminal suture removal. Specificity of Oct-mediated transport was evaluated using cimetidine (15 mg/kg, i.v.), a competitive Oct1/Oct2 inhibitor. At 2 h post-MCAO, [3H]memantine uptake was increased in ischemic brain tissue. Cimetidine inhibited blood-to-brain uptake of [3H]memantine, which confirmed involvement of an Oct-mediated transport mechanism. Memantine reduced post-MCAO infarction and brain edema progression as well as improved neurological outcomes during post-stroke recovery. All positive effects of memantine were attenuated by co-administration of cimetidine, which demonstrates that Oct1/Oct2 transport is required for memantine to exert neuroprotective effects in ischemic stroke. Furthermore, Oct1/Oct2-mediated transport was shown to be the dominant mechanism for memantine brain uptake in the MCAO model despite a concurrent increase in paracellular "leak." These novel and translational findings provide mechanistic evidence for the critical role of BBB transporters in CNS delivery of stroke therapeutics, information that can help such drugs advance in clinical trials.

High-Dose Acetaminophen Alters the Integrity of the Blood-Brain Barrier and Leads to Increased CNS Uptake of Codeine in Rats.

The consumption of acetaminophen (APAP) can induce neurological changes in human subjects; however, effects of APAP on blood-brain barrier (BBB) integrity are unknown. BBB changes by APAP can have profound consequences for brain delivery of co-administered drugs. To study APAP effects, female Sprague-Dawley rats (12-16 weeks old) were administered vehicle (i.e., 100% dimethyl sulfoxide (DMSO), intraperitoneally (i.p.)) or APAP (80 mg/kg or 500 mg/kg in DMSO, i.p.; equivalent to a 900 mg or 5600 mg daily dose for a 70 kg human subject). BBB permeability was measured via in situ brain perfusion using [14C]sucrose and [3H]codeine, an opioid analgesic drug that is co-administered with APAP (i.e., Tylenol #3). Localization and protein expression of tight junction proteins (i.e., claudin-5, occludin, ZO-1) were studied in rat brain microvessels using Western blot analysis and confocal microscopy, respectively. Paracellular [14C]sucrose "leak" and brain [3H]codeine accumulation were significantly enhanced in rats treated with 500 mg/kg APAP only. Additionally, claudin-5 localization and protein expression were altered in brain microvessels isolated from rats administered 500 mg/kg APAP. Our novel and translational data show that BBB integrity is altered following a single high APAP dose, results that are relevant to patients abusing or misusing APAP and/or APAP/opioid combination products.

Regulation of Blood-Brain Barrier Transporters by Transforming Growth Factor-ß/Activin Receptor-Like Kinase 1 Signaling: Relevance to the Brain Disposition of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors (i.e., Statins).

Our laboratory has shown that activation of transforming growth factor- ß (TGF- ß )/activin receptor-like kinase 1 (ALK1) signaling can increase protein expression and transport activity of organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier (BBB). These results are relevant to treatment of ischemic stroke because Oatp transport substrates such as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e., statins) improve functional neurologic outcomes in patients. Advancement of our work requires determination if TGF- ß /ALK1 signaling alters Oatp1a4 functional expression differently across brain regions and if such disparities affect central nervous system (CNS) statin disposition. Therefore, we studied regulation of Oatp1a4 by the TGF- ß /ALK1 pathway, in vivo, in rat brain microvessels isolated from cerebral cortex, hippocampus, and cerebellum using the ALK1 agonist bone morphogenetic protein-9 (BMP-9) and the ALK1 inhibitor 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride 193189. We showed that Oatp1a4 protein expression and brain distribution of three currently marketed statin drugs (i.e., atorvastatin, pravastatin, and rosuvastatin) were increased in cortex relative to hippocampus and cerebellum. Additionally, BMP-9 treatment enhanced Oatp-mediated statin transport in cortical tissue but not in hippocampus or cerebellum. Although brain drug delivery is also dependent upon efflux transporters, such as P-glycoprotein and/or Breast Cancer Resistance Protein, our data showed that administration of BMP-9 did not alter the relative contribution of these transporters to CNS disposition of statins. Overall, this study provides evidence for differential regulation of Oatp1a4 by TGF- ß /ALK1 signaling across brain regions, knowledge that is critical for development of therapeutic strategies to target Oatps at the BBB for CNS drug delivery. SIGNIFICANCE STATEMENT: Organic anion transporting polypeptides (Oatps) represent transporter targets for brain drug delivery. We have shown that Oatp1a4 statin uptake is higher in cortex versus hippocampus and cerebellum. Additionally, we report that the transforming growth factor- ß /activin receptor-like kinase 1 agonist bone morphogenetic protein-9 increases Oatp1a4 functional expression, but not efflux transporters P-glycoprotein and Breast Cancer Resistance Protein, in cortical brain microvessels. Overall, this study provides critical data that will advance treatment for neurological diseases where drug development has been challenging.

Wisdom of generations: a pilot study of the values transmitted in ethical wills of nursing home residents and student volunteers.

PURPOSE: This is a pilot study that provides a description of the values older persons report in ethical wills and their reasoning for the values they chose, and compares the values in ethical wills of seniors and students. Nursing home residents rarely get the opportunity or venue to discuss these topics and the ethical will enables them to have conversations about issues they feel are important. DESIGN AND METHODS: The 22 Questions for Ethical Wills(c) assessment was administered to 15 residents of a large nursing home in suburban Maryland and to 11 student volunteers. Raters identified recurring themes and independently analyzed the text from the ethical wills based on the themes. Questions that prompted similar responses were combined in the analyses. RESULTS: The most prevalent theme among both nursing home residents and students was "interpersonal relations." Differences between groups of respondents were especially evident for the theme of "education," which was given by over a half of the older persons and none of the students. Greater variability was found in the responses of the older participants, as was a greater likelihood of providing detailed explanations for their relayed values. IMPLICATIONS: The 22 Questions for Ethical Wills(c) is a useful methodology to elicit meaningful discussions of values and life lessons in persons both young and old. This process offers an intriguing comparison between the similarities and differences of life views of persons at opposite ends of the age spectrum.