Patrick T Ronaldson

Interests

Teaching

Pharmacokinetics; Pharmacodynamics; Molecular Pharmacology

Research

Ischemic Stroke; Blood-Brain Barrier; Mechanisms of Drug Transport across Biological Membranes; Regulation of Drug Transport Processes; CNS Drug Delivery; Vascular Protection; Drug-Drug Interactions

Courses

Scholarly Contributions

Chapters

• Sangha, V., Williams, E. I., Ronaldson, P. T., & Bendayan, R. (2022). Drug transport in the brain. In Drug Transporters: Molecular Characterization and Role in Drug Disposition. 3rd Edition..
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  Pharmacotherapy of central nervous system (CNS) diseases remains difficult due to limited drug permeation at the blood–brain barrier (BBB), blood–cerebrospinal fluid barrier (BCSFB), and blood–arachnoid barrier (BAB). Additionally, brain parenchymal cellular compartments can play a critical role in regulating CNS drug distribution. Membrane- associated drug transporters are essential determinants of drug disposition across the BBB, BCSFB, as well as in brain cellular compartments. Tightly regulated influx and efflux transport systems are involved in the movement of solutes and drugs into and out of the brain. Influx transport systems facilitate uptake of nutrients (i.e., amino acids, glucose, nucleosides) and xenobiotics. In contrast, efflux transport systems restrict CNS entry of a broad spectrum of drugs and metabolites and actively extrude various endogenous compounds (i.e., neurotoxins, metabolic products) out of the brain. The objective of this chapter is to review expression, localization, and functional activity of transporters at brain barriers and in brain cellular compartments in normal physiological and pathological conditions.
• Ronaldson, P. T., & Davis, T. P. (2016). Glial support of blood-brain barrier integrity: Molecular targets for novel therapeutic strategies in stroke. In Non-neural mechanisms of brain damage and repair after stroke(pp 45-80). Switzerland: Springer International Publishing. doi:10.1007/978-3-319-32337-4
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  The blood-brain barrier (BBB) regulates CNS homeostasis and is the most significant obstacle to effective brain drug delivery. It possesses characteristics (i.e., tight junction protein complexes, influx and efflux transporters) that control permeation of circulating solutes including therapeutic agents. In order to form this “barrier,” brain microvascular endothelial cells require support of adjacent astrocytes and microglia. This intricate relationship also occurs between endothelial cells and other cell types and structures of the CNS (i.e., pericytes, neurons, extracellular matrix), which implies existence of a “neurovascular unit (NVU).” Ischemic stroke can disrupt the NVU at both the structural and functional level, which leads to considerable BBB dysfunction. Recent studies have identified several pathophysiological mechanisms (i.e., oxidative stress, activation of cytokine-mediated intracellular signaling systems) that mediate NVU changes during ischemic stroke. This chapter summarizes current knowledge in this area and emphasizes pathways (i.e., oxidative stress, cytokine-mediated intracellular signaling, glial-expressed receptors/targets) that can be manipulated pharmacologically for i) preservation of BBB and glial integrity during ischemic stroke and ii) control of drug permeation and/or transport across the BBB in an effort to identify novel molecular targets that will enable improved stroke pharmacotherapy.
• Ronaldson, P. T., & Davis, T. P. (2016). Mechanisms of Endothelial Injury and Blood-Brain Barrier Dysfunction in Stroke. In Caplan Primer on Cerebrovascular Diseases, 2nd Edition(pp TBD). Philadelphia, PA: Elsevier Inc.
• Witt, K. A., Ronaldson, P. T., Sandoval, K. E., & Davis, T. P. (2010). CNS Delivery of Peptides Across the BBB Using the Dual-Artery In Situ Brain Perfusion Model. In Drug Delivery to the Central Nervous System(pp 233-247). Humana Press. doi:10.1007/978-1-60761-529-3_11
• Ashraf, T., Ronaldson, P. T., & Bendayan, R. (2006). Drug Transport in the Brain. In Drug Transporters: Molecular Characterization and Role in Drug Disposition(pp 411-461). John Wiley & Sons, Inc. doi:10.1002/9781118705308.CH14

Journals/Publications

• Hammer, M. F., Bahramnejad, E., Watkins, J. C., & Ronaldson, P. T. (2024). Candesartan restores blood-brain barrier dysfunction, mitigates aberrant gene expression, and extends lifespan in a knockin mouse model of epileptogenesis. Clinical science (London, England : 1979), 138(17), 1089-1110.
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  Blockade of Angiotensin type 1 receptor (AT1R) has potential therapeutic utility in the treatment of numerous detrimental consequences of epileptogenesis, including oxidative stress, neuroinflammation, and blood-brain barrier (BBB) dysfunction. We have recently shown that many of these pathological processes play a critical role in seizure onset and propagation in the Scn8a-N1768D mouse model. Here we investigate the efficacy and potential mechanism(s) of action of candesartan (CND), an FDA-approved angiotensin receptor blocker (ARB) indicated for hypertension, in improving outcomes in this model of pediatric epilepsy. We compared length of lifespan, seizure frequency, and BBB permeability in juvenile (D/D) and adult (D/+) mice treated with CND at times after seizure onset. We performed RNAseq on hippocampal tissue to quantify differences in genome-wide patterns of transcript abundance and inferred beneficial and detrimental effects of canonical pathways identified by enrichment methods in untreated and treated mice. Our results demonstrate that treatment with CND gives rise to increased survival, longer periods of seizure freedom, and diminished BBB permeability. CND treatment also partially reversed or 'normalized' disease-induced genome-wide gene expression profiles associated with inhibition of NF-κB, TNFα, IL-6, and TGF-β signaling in juvenile and adult mice. Pathway analyses reveal that efficacy of CND is due to its known dual mechanism of action as both an AT1R antagonist and a PPARγ agonist. The robust effectiveness of CND across ages, sexes and mouse strains is a positive indication for its translation to humans and its suitability of use for clinical trials in children with SCN8A epilepsy.
• Hammer, M. F., Krzyzaniak, C. T., Bahramnejad, E., Smelser, K. J., Hack, J. B., Watkins, J. C., & Ronaldson, P. T. (2024). Sex differences in physiological response to increased neuronal excitability in a knockin mouse model of pediatric epilepsy. Clinical science (London, England : 1979), 138(4), 205-223.
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  Epilepsy is a common neurological disease; however, few if any of the currently marketed antiseizure medications prevent or cure epilepsy. Discovery of pathological processes in the early stages of epileptogenesis has been challenging given the common use of preclinical models that induce seizures in physiologically normal animals. Moreover, despite known sex dimorphism in neurological diseases, females are rarely included in preclinical epilepsy models.
• Lochhead, J. J., Ronaldson, P. T., & Davis, T. P. (2024). The role of oxidative stress in blood-brain barrier disruption during ischemic stroke: Antioxidants in clinical trials. Biochemical pharmacology, 228, 116186.
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  Ischemic stroke is one of the leading causes of death and disability. Occlusion and reperfusion of cerebral blood vessels (i.e., ischemia/reperfusion (I/R) injury) generates reactive oxygen species (ROS) that contribute to brain cell death and dysfunction of the blood-brain barrier (BBB) via oxidative stress. BBB disruption influences the pathogenesis of ischemic stroke by contributing to cerebral edema, hemorrhagic transformation, and extravasation of circulating neurotoxic proteins. An improved understanding of mechanisms for ROS-associated alterations in BBB function during ischemia/reperfusion (I/R) injury can lead to improved treatment paradigms for ischemic stroke. Unfortunately, progress in developing ROS targeted therapeutics that are effective for stroke treatment has been slow. Here, we review how ROS are produced in response to I/R injury, their effects on BBB integrity (i.e., tight junction protein complexes, transporters), and the utilization of antioxidant treatments in ischemic stroke clinical trials. Overall, knowledge in this area provides a strong translational framework for discovery of novel drugs for stroke and/or improved strategies to mitigate I/R injury in stroke patients.
• Ronaldson, P. T., & Davis, T. P. (2024). Blood-brain barrier transporters: a translational consideration for CNS delivery of neurotherapeutics. Expert opinion on drug delivery, 1-19.
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  Successful neuropharmacology requires optimization of CNS drug delivery and, by extension, free drug concentrations at brain molecular targets. Detailed assessment of blood-brain barrier (BBB) physiological characteristics is necessary to achieve this goal. The 'next frontier' in CNS drug delivery is targeting BBB uptake transporters, an approach that requires evaluation of brain endothelial cell transport processes so that effective drug accumulation and improved therapeutic efficacy can occur.
• Ronaldson, P. T., Williams, E. I., Betterton, R. D., Stanton, J. A., Nilles, K. L., & Davis, T. P. (2024). CNS Drug Delivery in Stroke: Improving Therapeutic Translation From the Bench to the Bedside. Stroke, 55(1), 190-202.
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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.
• Yarbro, J. M., Han, X., Dasgupta, A., Yang, K., Liu, D., Shrestha, H. K., Zaman, M., Wang, Z., Yu, K., Lee, D. G., Vanderwall, D., Niu, M., Sun, H., Xie, B., Chen, P. C., Jiao, Y., Zhang, X., Wu, Z., Fu, Y., , Li, Y., et al. (2024). Human-mouse proteomics reveals the shared pathways in Alzheimer's disease and delayed protein turnover in the amyloidome. bioRxiv : the preprint server for biology.
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  Murine models of Alzheimer's disease (AD) are crucial for elucidating disease mechanisms but have limitations in fully representing AD molecular complexities. We comprehensively profiled age-dependent brain proteome and phosphoproteome ( > 10,000 for both) across multiple mouse models of amyloidosis. We identified shared pathways by integrating with human metadata, and prioritized novel components by multi-omics analysis. Collectively, two commonly used models (5xFAD and APP-KI) replicate 30% of the human protein alterations; additional genetic incorporation of tau and splicing pathologies increases this similarity to 42%. We dissected the proteome-transcriptome inconsistency in AD and 5xFAD mouse brains, revealing that inconsistent proteins are enriched within amyloid plaque microenvironment (amyloidome). Determining the 5xFAD proteome turnover demonstrates that amyloid formation delays the degradation of amyloidome components, including Aβ-binding proteins and autophagy/lysosomal proteins. Our proteomic strategy defines shared AD pathways, identify potential new targets, and underscores that protein turnover contributes to proteome-transcriptome discrepancies during AD progression.
• Betterton, R. D., Williams, E. I., Nilles, K. L., Davis, T. P., & Ronaldson, P. T. (2023). Methods to Study Drug Uptake at the Blood-Brain Barrier Following Experimental Ischemic Stroke: In Vitro and In Vivo Approaches. Methods in molecular biology (Clifton, N.J.), 2616, 403-418.
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  Drug permeability across the blood-brain barrier (BBB) is an important concept in the development of therapeutic strategies to treat neurological diseases such as ischemic stroke. These mechanisms can be evaluated in detail using cultured brain microvascular endothelial cells or intact animals subjected to experimental stroke. Here, we describe state-of-the-art approaches to study BBB transport of therapeutics using our in vitro and in vivo approaches. These methodologies allow for precise determination of transporter kinetic properties for currently marketed therapeutics or for new chemical entities that are under development as stroke drugs.
• Lochhead, J. J., Williams, E. I., Reddell, E. S., Dorn, E., Ronaldson, P. T., & Davis, T. P. (2023). High Resolution Multiplex Confocal Imaging of the Neurovascular Unit in Health and Experimental Ischemic Stroke. Cells, 12(4).
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  The neurovascular unit (NVU) is an anatomical group of cells that establishes the blood-brain barrier (BBB) and coordinates cerebral blood flow in association with neuronal function. In cerebral gray matter, cellular constituents of the NVU include endothelial cells and associated pericytes, astrocytes, neurons, and microglia. Dysfunction of the NVU is a common feature of diseases that affect the CNS, such as ischemic stroke. High-level evaluation of these NVU changes requires the use of imaging modalities that can enable the visualization of various cell types under disease conditions. In this study, we applied our confocal microscopy strategy using commercially available labeling reagents to, for the first time, simultaneously investigate associations between endothelial cells, the vascular basal lamina, pericytes, microglia, astrocytes and/or astrocyte end-feet, and neurites in both healthy and ischemic brain tissue. This allowed us to demonstrate ischemia-induced astrocyte activation, neurite loss, and microglial migration toward blood vessels in a single confocal image. Furthermore, our labeling cocktail enabled a precise quantification of changes in neurites and astrocyte reactivity, thereby showing the relationship between different NVU cellular constituents in healthy and diseased brain tissue. The application of our imaging approach for the simultaneous visualization of multiple NVU cell types provides an enhanced understanding of NVU function and pathology, a state-of-the-art advancement that will facilitate the development of more effective treatment strategies for diseases of the CNS that exhibit neurovascular dysfunction, such as ischemic stroke.
• Williams, E. I., Betterton, R. D., Stanton, J. A., Moreno-Rodriguez, V. M., Lochhead, J. J., Davis, T. P., & Ronaldson, P. T. (2023). Oatp (Organic Anion Transporting Polypeptide)-Mediated Transport: A Mechanism for Atorvastatin Neuroprotection in Stroke. Stroke, 54(11), 2875-2885.
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  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.
• Betterton, R. D., Abdullahi, W., Williams, E. I., Lochhead, J. J., Brzica, H., Stanton, J., Reddell, E., Ogbonnaya, C., Davis, T. P., & Ronaldson, P. T. (2022). Regulation of Blood-Brain Barrier Transporters by Transforming Growth Factor-/Activin Receptor-Like Kinase 1 Signaling: Relevance to the Brain Disposition of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors (i.e., Statins). Drug metabolism and disposition: the biological fate of chemicals, 50(7), 942-956.
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  Our laboratory has shown that activation of transforming growth factor- (TGF- )/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- /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- /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- /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- /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.
• Nilles, K. L., Williams, E. I., Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2022). Blood-Brain Barrier Transporters: Opportunities for Therapeutic Development in Ischemic Stroke. International journal of molecular sciences, 23(3).
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  Globally, stroke is a leading cause of death and long-term disability. Over the past decades, several efforts have attempted to discover new drugs or repurpose existing therapeutics to promote post-stroke neurological recovery. Preclinical stroke studies have reported successes in identifying novel neuroprotective agents; however, none of these compounds have advanced beyond a phase III clinical trial. One reason for these failures is the lack of consideration of blood-brain barrier (BBB) transport mechanisms that can enable these drugs to achieve efficacious concentrations in ischemic brain tissue. Despite the knowledge that drugs with neuroprotective properties (i.e., statins, memantine, metformin) are substrates for endogenous BBB transporters, preclinical stroke research has not extensively studied the role of transporters in central nervous system (CNS) drug delivery. Here, we review current knowledge on specific BBB uptake transporters (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents); organic cation transporters (OCTs in humans; Octs in rodents) that can be targeted for improved neuroprotective drug delivery. Additionally, we provide state-of-the-art perspectives on how transporter pharmacology can be integrated into preclinical stroke research. Specifically, we discuss the utility of in vivo stroke models to transporter studies and considerations (i.e., species selection, co-morbid conditions) that will optimize the translational success of stroke pharmacotherapeutic experiments.
• Ronaldson, P. T., & Davis, T. P. (2022). Transport Mechanisms at the Blood-Brain Barrier and in Cellular Compartments of the Neurovascular Unit: Focus on CNS Delivery of Small Molecule Drugs. Pharmaceutics, 14(7).
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  Ischemic stroke is a primary origin of morbidity and mortality in the United States and around the world. Indeed, several research projects have attempted to discover new drugs or repurpose existing therapeutics to advance stroke pharmacotherapy. Many of these preclinical stroke studies have reported positive results for neuroprotective agents; however, only one compound (3K3A-activated protein C (3K3A-APC)) has advanced to Phase III clinical trial evaluation. One reason for these many failures is the lack of consideration of transport mechanisms at the blood-brain barrier (BBB) and neurovascular unit (NVU). These endogenous transport processes function as a "gateway" that is a primary determinant of efficacious brain concentrations for centrally acting drugs. Despite the knowledge that some neuroprotective agents (i.e., statins and memantine) are substrates for these endogenous BBB transporters, preclinical stroke studies have largely ignored the role of transporters in CNS drug disposition. Here, we review the current knowledge on specific BBB transporters that either limit drug uptake into the brain (i.e., ATP-binding cassette (ABC) transporters) or can be targeted for optimized drug delivery (i.e., solute carrier (SLC) transporters). Additionally, we highlight the current knowledge on transporter expression in astrocytes, microglia, pericytes, and neurons with an emphasis on transport mechanisms in these cell types that can influence drug distribution within the brain.
• Stanton, J. A., Williams, E. I., Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2022). Targeting organic cation transporters at the blood-brain barrier to treat ischemic stroke in rats. Experimental neurology, 357, 114181.
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  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, [H]memantine uptake was increased in ischemic brain tissue. Cimetidine inhibited blood-to-brain uptake of [H]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.
• Yang, J., Betterton, R. D., Williams, E. I., Stanton, J. A., Reddell, E. S., Ogbonnaya, C. E., Dorn, E., Davis, T. P., Lochhead, J. J., & Ronaldson, P. T. (2022). High-Dose Acetaminophen Alters the Integrity of the Blood-Brain Barrier and Leads to Increased CNS Uptake of Codeine in Rats. Pharmaceutics, 14(5).
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  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 [C]sucrose and [H]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 [C]sucrose "leak" and brain [H]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.
• Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2021). Organic Cation Transporter (OCT/OCTN) Expression at Brain Barrier Sites: Focus on CNS Drug Delivery. Handbook of experimental pharmacology, 266, 301-328.
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  Therapeutic delivery to the central nervous system (CNS) continues to be a considerable challenge in the pharmacological treatment and management of neurological disorders. This is primarily due to the physiological and biochemical characteristics of brain barrier sites (i.e., blood-brain barrier (BBB), blood-cerebrospinal fluid barrier (BCSFB)). Drug uptake into brain tissue is highly restricted by expression of tight junction protein complexes and adherens junctions between brain microvascular endothelial cells and choroid plexus epithelial cells. Additionally, efflux transport proteins expressed at the plasma membrane of these same endothelial and epithelial cells act to limit CNS concentrations of centrally acting drugs. In contrast, facilitated diffusion via transporter proteins allows for substrate-specific flux of molecules across the plasma membrane, directing drug uptake into the CNS. Organic Cation Transporters (OCTs) and Novel Organic Cation Transporters (OCTNs) are two subfamilies of the solute carrier 22 (SLC22) family of proteins that have significant potential to mediate delivery of positively charged, zwitterionic, and uncharged therapeutics. While expression of these transporters has been well characterized in peripheral tissues, the functional expression of OCT and OCTN transporters at CNS barrier sites and their role in delivery of therapeutic drugs to molecular targets in the brain require more detailed analysis. In this chapter, we will review current knowledge on localization, function, and regulation of OCT and OCTN isoforms at the BBB and BCSFB with a particular emphasis on how these transporters can be utilized for CNS delivery of therapeutic agents.
• Lochhead, J. J., Yang, J., Ronaldson, P. T., & Davis, T. P. (2020). Structure, Function, and Regulation of the Blood-Brain Barrier Tight Junction in Central Nervous System Disorders. Frontiers in Physiology, 11, 914. doi:0.3389/fphys.2020.00914
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  The blood-brain barrier (BBB) allows the brain to selectively import nutrients and energy critical to neuronal function while simultaneously excluding neurotoxic substances from the peripheral circulation. In contrast to the highly permeable vasculature present in most organs that reside outside of the central nervous system (CNS), the BBB exhibits a high transendothelial electrical resistance (TEER) along with a low rate of transcytosis and greatly restricted paracellular permeability. The property of low paracellular permeability is controlled by tight junction (TJ) protein complexes that seal the paracellular route between apposing brain microvascular endothelial cells. Although tight junction protein complexes are principal contributors to physical barrier properties, they are not static in nature. Rather, tight junction protein complexes are highly dynamic structures, where expression and/or localization of individual constituent proteins can be modified in response to pathophysiological stressors. These stressors induce modifications to tight junction protein complexes that involve synthesis of new protein or discrete trafficking mechanisms. Such responsiveness of BBB tight junctions to diseases indicates that these protein complexes are critical for maintenance of CNS homeostasis. In fulfillment of this vital role, BBB tight junctions are also a major obstacle to therapeutic drug delivery to the brain. There is an opportunity to overcome this substantial obstacle and optimize neuropharmacology acquisition of a detailed understanding of BBB tight junction structure, function, and regulation. In this review, we discuss physiological characteristics of tight junction protein complexes and how these properties regulate delivery of therapeutics to the CNS for treatment of neurological diseases. Specifically, we will discuss modulation of tight junction structure, function, and regulation both in the context of disease states and in the setting of pharmacotherapy. In particular, we will highlight how these properties can be potentially manipulated at the molecular level to increase CNS drug levels paracellular transport to the brain.
• Ronaldson, P. T., & Davis, T. P. (2020). Regulation of blood-brain barrier integrity by microglia in health and disease: A therapeutic opportunity. Journal of Cerebral Blood Flow and Metabolism, 40(1(Suppl)), S6-S24. doi:10.1177/0271678X20951995
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  The blood-brain barrier (BBB) is a critical regulator of CNS homeostasis. It possesses physical and biochemical characteristics (i.e. tight junction protein complexes, transporters) that are necessary for the BBB to perform this physiological role. Microvascular endothelial cells require support from astrocytes, pericytes, microglia, neurons, and constituents of the extracellular matrix. This intricate relationship implies the existence of a neurovascular unit (NVU). NVU cellular components can be activated in disease and contribute to dynamic remodeling of the BBB. This is especially true of microglia, the resident immune cells of the brain, which polarize into distinct proinflammatory (M1) or anti-inflammatory (M2) phenotypes. Current data indicate that M1 pro-inflammatory microglia contribute to BBB dysfunction and vascular "leak", while M2 anti-inflammatory microglia play a protective role at the BBB. Understanding biological mechanisms involved in microglia activation provides a unique opportunity to develop novel treatment approaches for neurological diseases. In this review, we highlight characteristics of M1 proinflammatory and M2 anti-inflammatory microglia and describe how these distinct phenotypes modulate BBB physiology. Additionally, we outline the role of other NVU cell types in regulating microglial activation and highlight how microglia can be targeted for treatment of disease with a focus on ischemic stroke and Alzheimer's disease.
• Ronaldson, P. T., Brzica, H., Abdullahi, W., Reilly, B. G., & Davis, T. P. (2020). Transport Properties of Statins by OATP1A2 and Regulation by Transforming Growth Factor-beta (TGF-beta) Signaling in Human Endothelial Cells. The Journal of Pharmacology and Experimental Therapeutics. doi:10.1124/jpet.120.000267
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  Our rodent studies have shown that Organic Anion Transporting Polypeptide 1a4 (Oatp1a4) is critical for blood-to-brain transport of statins, drugs that are effective neuroprotectants. Additionally, Transforming Growth Factor-β (TGF-β) signaling via the activin receptor-like kinase 1 (ALK1) receptor regulates Oatp1a4 functional expression. The human orthologue of Oatp1a4 is OATP1A2. Therefore, the translational significance of our work requires demonstration that OATP1A2 can transport statins and is regulated by TGF-β/ALK1 signaling. Cellular uptake and monolayer permeability of atorvastatin, pravastatin, and rosuvastatin were investigated, using human umbilical vein endothelial cells (HUVECs). Regulation of OATP1A2 by the TGF-β/ALK1 pathway was evaluated using bone morphogenetic protein 9 (BMP-9), a selective ALK1 agonist, and LDN193189, an ALK1 antagonist. Statin accumulation in HUVECs requires OATP1A2-mediated uptake but is also affected by efflux transporters (i.e., P-glycoprotein (P-gp), Breast Cancer Resistance Protein (BCRP)). Absorptive flux (i.e., apical-to-basolateral) for all statins was higher than secretory flux (i.e., basolateral-to-apical) and was decreased by an OATP inhibitor (i.e., estrone-3-sulfate). OATP1A2 protein expression, statin uptake, and cellular monolayer permeability were increased by BMP-9 treatment. This effect was attenuated in the presence of LDN193189. Apical-to-basolateral statin transport across human endothelial cellular monolayers requires functional expression of OATP1A2, which can be controlled by therapeutically targeting TGF-β/ALK1 signaling. Taken together with our previous work, the present data show that OATP-mediated drug transport is a critical mechanism in facilitating neuroprotective drug disposition across endothelial barriers of the BBB. Transporter data derived from rodent models requires validation in human models. Using human umbilical vein endothelial cells (HUVECs), we have shown that statin uptake transport is mediated by OATP1A2. Additionally, we demonstrated that OATP1A2 is regulated by TGF-β/ALK1 signaling. This work emphasizes the need to consider endothelial transporter kinetics and regulation during preclinical drug development. Furthermore, our forward-thinking approach can identify drugs that are more likely to be effective in diseases where drug development has been challenging (i.e., neurological diseases).
• Ronaldson, P. T., Lochhead, J. J., & Davis, T. P. (2020). Abstract TP121: Organic Anion Transporting Polypeptide (Oatp)-Mediated Transport is Required for Statin-Induced Neuroprotection: A Role for Blood-Brain Barrier Transporters in Stroke Treatment. Stroke, 51(Suppl_1). doi:10.1161/str.51.suppl_1.tp121
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  Objectives: Treatment approaches for stroke include reperfusion therapies (i.e., recombinant tissue plasminogen activator, endovascular thrombectomy); however, many stroke patients still experience...
• Lochhead, J. J., Kellohen, K. L., Ronaldson, P. T., & Davis, T. P. (2019). Distribution of insulin in trigeminal nerve and brain after intranasal administration. Scientific reports, 9(1), 2621.
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  In the brain, insulin acts as a growth factor, regulates energy homeostasis, and is involved in learning and memory acquisition. Many central nervous system (CNS) diseases are characterized by deficits in insulin signaling. Pre-clinical studies have shown that intranasal insulin is neuroprotective in models of Alzheimer's disease, Parkinson's disease, and traumatic brain injury. Clinical trials have also shown that intranasal insulin elicits beneficial cognitive effects in patients with Alzheimer's disease. It is known that insulin can be detected in the CNS within minutes following intranasal administration. Despite these advances, the anatomical pathways that insulin utilizes to reach the CNS and the cellular CNS targets after intranasal administration are not fully understood. Here, we intranasally administered fluorescently labeled insulin and imaged its localization within the brain and trigeminal nerves. Our data indicates that intranasal insulin can reach cellular CNS targets along extracellular components of the trigeminal nerve. Upon CNS entry, we found insulin significantly increased levels of an activated form of the insulin receptor. These findings suggest that the intranasal route of administration is able to effectively deliver insulin to CNS targets in a biologically active form.
• Abdullahi, W., Brzica, H., Hirsch, N. A., Reilly, B. G., & Ronaldson, P. T. (2018). Functional Expression of Organic Anion Transporting Polypeptide 1a4 Is Regulated by Transforming Growth Factor-beta/Activin Receptor-like Kinase 1 Signaling at the Blood-Brain Barrier. Molecular Pharmacology, 94(6), 1321-1333. doi:10.1124/mol.118.112912
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  Central nervous system (CNS) drug delivery can be achieved by targeting drug uptake transporters such as Oatp1a4. In fact, many drugs that can improve neurologic outcomes in CNS diseases [3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (i.e., statins)] are organic anion transporting polypeptide (OATP) transport substrates. To date, transport properties and regulatory mechanisms of Oatp1a4 at the blood-brain barrier (BBB) have not been rigorously studied. Such knowledge is critical to develop Oatp1a4 for optimization of CNS drug delivery and for improved treatment of neurological diseases. Our laboratory has demonstrated that the transforming growth factor-beta (TGF-beta)/activin receptor-like kinase 1 (ALK1) signaling agonist bone morphogenetic protein 9 (BMP-9) increases functional expression of Oatp1a4 in rat brain microvessels. Here, we expand on this work and show that BMP-9 treatment increases blood-to-brain transport and brain exposure of established OATP transport substrates (i.e., taurocholate, atorvastatin, and pravastatin). We also demonstrate that BMP-9 activates the TGF-beta/ALK1 pathway in brain microvessels as indicated by increased nuclear translocation of specific Smad proteins associated with signaling mediated by the ALK1 receptor (i.e., pSmad1/5/8). Furthermore, we report that an activated Smad protein complex comprised of phosphorylated Smad1/5/8 and Smad4 is formed following BMP-9 treatment and binds to the promoter of the gene (i.e., the gene that encodes Oatp1a4). This signaling mechanism causes increased expression of mRNA. Overall, this study provides evidence that Oatp1a4 transport activity at the BBB is directly regulated by TGF-beta/ALK1 signaling and indicates that this pathway can be targeted for control of CNS delivery of OATP substrate drugs.
• Abdullahi, W., Tripathi, D., & Ronaldson, P. T. (2018). Blood-brain barrier dysfunction in ischemic stroke: Targeting tight junctions and transporters for vascular protection. American Journal of Physiology. Cell Physiology, 315(3), C343-C356. doi:10.1152/ajpcell.00095.2018
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  The blood-brain barrier (BBB) is a physical and biochemical barrier that precisely controls cerebral homeostasis. It also plays a central role in the regulation of blood-to-brain flux of endogenous and exogenous xenobiotics and associated metabolites. This is accomplished by molecular characteristics of brain microvessel endothelial cells such as tight junction protein complexes and functional expression of influx and efflux transporters. One of the pathophysiological features of ischemic stroke is disruption of the BBB, which significantly contributes to development of brain injury and subsequent neurological impairment. Biochemical characteristics of BBB damage include decreased expression and altered organization of tight junction constituent proteins as well as modulation of functional expression of endogenous BBB transporters. Therefore, there is a critical need for development of novel therapeutic strategies that can protect against BBB dysfunction (i.e., vascular protection) in the setting of ischemic stroke. Such strategies include targeting tight junctions to ensure that they maintain their correct structure or targeting transporters to control flux of physiological substrates for protection of endothelial homeostasis. In this review, we will describe the pathophysiological mechanisms in cerebral microvascular endothelial cells that lead to BBB dysfunction following onset of stroke. Additionally, we will utilize this state-of-the-art knowledge to provide insights on novel pharmacological strategies that can be developed to confer BBB protection in the setting of ischemic stroke.
• Brzica, H., Abdullahi, W., Reilly, B. G., & Ronaldson, P. T. (2018). A simple and reproducible method to prepare membrane samples from freshly isolated rat brain micro vessels. Journal of Visualized Experiments. doi:10.3791/57698
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  The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of CNS diseases. Many BBB changes that affect pharmacotherapy involve membrane proteins that are localized and expressed at the level of the endothelial cell. Indeed, such knowledge on physiology of the BBB in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for simple but robust and reproducible methods for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from these freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to generate samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this article, we provide details of the method routinely utilized in our laboratory for isolation of rat brain microvessels and preparation of membrane samples. This approach is used for our molecular pharmacology studies involving analysis of expression of drug transport proteins at the BBB. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from western blot experiments that can greatly aid our understanding of BBB responses to pathophysiological and pharmacological stimuli.
• Brzica, H., Abdullahi, W., Reilly, B. G., & Ronaldson, P. T. (2018). Sex-specific differences in organic anion transporting polypeptide 1a4 (Oatp1a4) functional expression at the blood-brain barrier in Sprague-Dawley rats. Fluids and barriers of the CNS, 15(1), 25. doi:10.1186/s12987-018-0110-9
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  BACKGROUND: Targeting endogenous blood-brain barrier (BBB) transporters such as organic anion transporting polypeptide 1a4 (Oatp1a4) can facilitate drug delivery for treatment of neurological diseases. Advancement of Oatp targeting for optimization of CNS drug delivery requires characterization of sex-specific differences in BBB expression and/or activity of this transporter.METHODS: In this study, we investigated sex differences in Oatp1a4 functional expression at the BBB in adult and prepubertal (i.e., 6-week-old) Sprague-Dawley rats. We also performed castration or ovariectomy surgeries to assess the role of gonadal hormones on Oatp1a4 protein expression and transport activity at the BBB. Slco1a4 (i.e., the gene encoding Oatp1a4) mRNA expression and Oatp1a4 protein expression in brain microvessels was determined using quantitative real-time PCR and western blot analysis, respectively. Oatp transport function at the BBB was determined via in situ brain perfusion using [3H]taurocholate and [3H]atorvastatin as probe substrates. Data were expressed as mean ± SD and analyzed via one-way ANOVA followed by the post hoc Bonferroni t-test.RESULTS: Our results showed increased brain microvascular Slco1a4 mRNA and Oatp1a4 protein expression as well as increased brain uptake of [3H]taurocholate and [3H]atorvastatin in female rats as compared to males. Oatp1a4 expression at the BBB was enhanced in castrated male animals but was not affected by ovariectomy in female animals. In prepubertal rats, no sex-specific differences in brain microvascular Oatp1a4 expression were observed. Brain accumulation of [3H]taurocholate in male rats was increased following castration as compared to controls. In contrast, there was no difference in [3H]taurocholate brain uptake between ovariectomized and control female rats.CONCLUSIONS: These novel data confirm sex-specific differences in BBB Oatp1a4 functional expression, findings that have profound implications for treatment of CNS diseases. Studies are ongoing to fully characterize molecular pathways that regulate sex differences in Oatp1a4 expression and activity.
• Yang, J., Reilly, B. G., Davis, T. P., & Ronaldson, P. T. (2018). Modulation of Opioid Transport at the Blood-Brain Barrier by Altered ATP-Binding Cassette (ABC) Transporter Expression and Activity. Pharmaceutics, 10(4), E192. doi:10.3390/pharmaceutics10040192
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  Opioids are highly effective analgesics that have a serious potential for adverse drug reactions and for development of addiction and tolerance. Since the use of opioids has escalated in recent years, it is increasingly important to understand biological mechanisms that can increase the probability of opioid-associated adverse events occurring in patient populations. This is emphasized by the current opioid epidemic in the United States where opioid analgesics are frequently abused and misused. It has been established that the effectiveness of opioids is maximized when these drugs readily access opioid receptors in the central nervous system (CNS). Indeed, opioid delivery to the brain is significantly influenced by the blood-brain barrier (BBB). In particular, ATP-binding cassette (ABC) transporters that are endogenously expressed at the BBB are critical determinants of CNS opioid penetration. In this review, we will discuss current knowledge on the transport of opioid analgesic drugs by ABC transporters at the BBB. We will also examine how expression and trafficking of ABC transporters can be modified by pain and/or opioid pharmacotherapy, a novel mechanism that can promote opioid-associated adverse drug events and development of addiction and tolerance.
• Abdullahi, W., Brzica, H., Ibbotson, K., Davis, T. P., & Ronaldson, P. T. (2017). Bone Morphogenetic Protein-9 Increases Expression of Organic Anion Transporting Polypeptide 1a4 at the Blood-Brain Barrier via the Activin Receptor-Like Kinase-1 Receptor. Journal of Cerebral Blood Flow and Metabolism, 37(7), 2340-2345. doi:10.1177/0271678X17702916
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  Targeting uptake transporters such as organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier (BBB) can facilitate central nervous system (CNS) drug delivery. Effective blood-to-brain drug transport via this strategy requires characterization of mechanisms that modulate transporter expression and/or activity at the BBB. Here, we show that activation of activin receptor-like kinase (ALK)-1 using Bone Morphogenetic Protein (BMP)-9 increases Oatp1a4 protein expression in rat brain microvessels in vivo. These data indicate that targeting BMP-9/ALK1 signaling modulates BBB Oatp1a4 expression, a unique opportunity to optimize drug delivery and improve pharmacotherapy for CNS diseases.
• Abdullahi, W., Davis, T. P., & Ronaldson, P. T. (2017). Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?. The AAPS journal, 19(4), 931-939. doi:10.1208/s12248-017-0081-9
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  Drug delivery to the central nervous system (CNS) is greatly limited by the blood-brain barrier (BBB). Physical and biochemical properties of the BBB have rendered treatment of CNS diseases, including those with a hypoxia/reoxygenation (H/R) component, extremely difficult. Targeting endogenous BBB transporters from the ATP-binding cassette (ABC) superfamily (i.e., P-glycoprotein (P-gp)) or from the solute carrier (SLC) family (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents)) has been suggested as a strategy that can improve delivery of drugs to the brain. With respect to P-gp, direct pharmacological inhibition using small molecules or selective regulation by targeting intracellular signaling pathways has been explored. These approaches have been largely unsuccessful due to toxicity issues and unpredictable pharmacokinetics. Therefore, our laboratory has proposed that optimization of CNS drug delivery, particularly for treatment of diseases with an H/R component, can be achieved by targeting Oatp isoforms at the BBB. As the major drug transporting Oatp isoform, Oatp1a4 has demonstrated blood-to-brain transport of substrate drugs with neuroprotective properties. Furthermore, our laboratory has shown that targeting Oatp1a4 regulation (i.e., TGF-β signaling mediated via the ALK-1 and ALK-5 transmembrane receptors) represents an opportunity to control Oatp1a4 functional expression for the purpose of delivering therapeutics to the CNS. In this review, we will discuss limitations of targeting P-gp-mediated transport activity and the advantages of targeting Oatp-mediated transport. Through this discussion, we will also provide critical information on novel approaches to improve CNS drug delivery by targeting endogenous uptake transporters expressed at the BBB.
• Abdullahi, W., Ronaldson, P. T., & Brzica, H. (2017). Abstract WP433: Targeting Organic Anion Transporting Polypeptide 1a4 (Oatp1a4) Expression at the Blood-brain Barrier: Implications for Treatment of Ischemic Stroke. Stroke, 48(suppl_1). doi:10.1161/str.48.suppl_1.wp433
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  Introduction: Stroke is a leading cause of mortality and morbidity. Several drugs with neuroprotective properties have been proposed for stroke treatment but many have failed in clinical trials. Th...
• Brzica, H., Abdullahi, W., Ibbotson, K., & Ronaldson, P. T. (2017). Role of Transporters in CNS Drug Delivery and Blood-Brain Barrier Protection: Relevance to Treatment of Stroke. Journal of Central Nervous System Diseases, 9, 1-12. doi:10.1177/1179573517693802
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  Ischemic stroke is a leading cause of mortality and morbidity in the United States. The blood brain barrier (BBB) is a critical modifier of stroke therapy that limits CNS drug delivery. The only approved pharmacological treatment for ischemic stroke is recombinant tissue plasminogen activator (r-tPA), a thrombolytic drug. However, a short therapeutic window and adverse events including hemorrhage and potential enhancement of excitotoxicity limit r-tPA therapy. Transporters expressed at the BBB can be used to deliver neuroprotective drugs and thereby expand treatment options. Organic anion transporting polypeptides (Oatps) and organic cation transporters (Octs) facilitate delivery of drugs with neuroprotective properties such as statins and/or mementine. Additionally, multidrug resistance proteins (Mrps) can be targeted to reduce endogenous antioxidant loss from brain microvascular endothelial cells and preserve vascular integrity. Here, we review current knowledge on BBB transporters and demonstrate how transporter targeting can be exploited for novel approaches to treat ischemic stroke.
• Ibbotson, K., Yell, J. A., & Ronaldson, P. T. (2017). Nrf2 Signaling Increases Expression of ATP-Binding Cassette Subfamily C mRNA Transcripts at the Blood-Brain Barrier following Hypoxia-Reoxygenation Stress. Fluids and Barriers of the CNS, 14(1), 6. doi:10.1186/s12987-017-0055-4
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  Background: Strategies to maintain BBB integrity in diseases with a hypoxia/reoxygenation (H/R) component involve preventing glutathione (GSH) loss from endothelial cells. GSH efflux transporters include multidrug resistance proteins (Mrps). Therefore, characterization of Mrp regulation at the BBB during H/R is required to advance these transporters as therapeutic targets. Our goal was to investigate, in vivo, regulation of Abcc1, Abcc2, and Abcc4 mRNA expression (i.e., genes encoding Mrp isoforms that transport GSH) by nuclear factor E2-related factor (Nrf2) using a well-established H/R model. Methods: Female Sprague-Dawley rats (200-250 g) were subjected to normoxia (Nx, 21% O2, 60 min), hypoxia (Hx, 6% O2, 60 min) or H/R (6% O2, 60 min followed by 21% O2, 10 min, 30 min, or 1 h) or were treated with the Nrf2 activator sulforaphane (25 mg/kg, i.p.) for 3 h. Abcc mRNA expression in brain microvessels was determined using quantitative real-time PCR. Nrf2 signaling activation was examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) respectively. Data were expressed as mean ± S.D. and analyzed via ANOVA followed by the post-hoc Bonferroni t-test.Results: We observed increased microvascular expression of Abcc1, Abcc2, and Abcc4 mRNA following H/R treatment with reoxygenation times of 10 min, 30 min, and 1 h and in animals treated with sulforaphane. Using a biotinylated Nrf2 probe, we observed an upward band shift in microvessels isolated from H/R animals or animals administered sulforaphane. ChIP studies showed increased Nrf2 binding to antioxidant response elements on Abcc1, Abcc2, and Abcc4 promoters following H/R or sulforaphane treatment, suggesting a role for Nrf2 signaling in Abcc gene regulation. Conclusions: Our data show increased Abcc1, Abcc2, and Abcc4 mRNA expression at the BBB in response to H/R stress and that Abcc gene expression is regulated by Nrf2 signaling. Since these Mrp isoforms transport GSH, these results may point to endogenous transporters that can be targeted for BBB protection during H/R stress. Future studies are ongoing to examine functional implications of Nrf2-mediated increases in Abcc transcript expression in order to determine the utility of targeting Mrp isoforms for BBB protection in diseases with an H/R component.
• Lochhead, J. J., Ronaldson, P. T., & Davis, T. P. (2017). Hypoxic Stress and Inflammatory Pain Disrupt Blood-Brain Barrier Tight Junctions: Implications for Drug Delivery to the Central Nervous System. The AAPS journal, 19(4), 910-920. doi:10.1208/s12248-017-0076-6
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  A functional blood-brain barrier (BBB) is necessary to maintain central nervous system (CNS) homeostasis. Many diseases affecting the CNS, however, alter the functional integrity of the BBB. It has been shown that various diseases and physiological stressors can impact the BBB's ability to selectively restrict passage of substances from the blood to the brain. Modifications of the BBB's permeability properties can potentially contribute to the pathophysiology of CNS diseases and result in altered brain delivery of therapeutic agents. Hypoxia and/or inflammation are central components of a number of diseases affecting the CNS. A number of studies indicate hypoxia or inflammatory pain increase BBB paracellular permeability, induce changes in the expression and/or localization of tight junction proteins, and affect CNS drug uptake. In this review, we look at what is currently known with regard to BBB disruption following a hypoxic or inflammatory insult in vivo. Potential mechanisms involved in altering tight junction components at the BBB are also discussed. A more detailed understanding of the mediators involved in changing BBB functional integrity in response to hypoxia or inflammatory pain could potentially lead to new treatments for CNS diseases with hypoxic or inflammatory components. Additionally, greater insight into the mechanisms involved in TJ rearrangement at the BBB may lead to novel strategies to pharmacologically increase delivery of drugs to the CNS.
• Ronaldson, P. T., Bauer, B., El-Kattan, A. F., Shen, H., Salphati, L., & Louie, S. W. (2016). Highlights From the American Association of Pharmaceutical Scientists/ International Transporter Consortium Joint Workshop on Drug Transporters in Absorption, Distribution, Metabolism, and Excretion: From the Bench to the Bedside - Clinical Pharmacology Considerations. Clinical pharmacology and therapeutics, 100(5), 419-422. doi:10.1002/cpt.439
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  The American Association of Pharmaceutical Scientists/International Transporter Consortium Joint Workshop on Drug Transporters in absorption, distribution, metabolism, and excretion was held with the objective of discussing innovative advances in transporter pharmacology. Specific topics included (i) transporters at the blood-brain barrier (BBB); (ii) emerging transport proteins; (iii) recent advances in achieving hepatoselectivity and optimizing clearance for organic anion-transporting polypeptide (OATP) substrates; (iv) utility of animal models for transporter studies; and (v) clinical correlation of transporter polymorphisms. Here, we present state-of-the-art highlights from this workshop in these key areas of focus.
• Davis, T. P. (2015). Targeting transporters: Promoting blood-brain barrier repair in response to oxidative stress injury. Brain Research, 1623, 39-52. doi:10.1016/j.brainres.2015.03.018
• Thompson, B. J., & Ronaldson, P. T. (2014). Drug delivery to the ischemic brain.. Advances in pharmacology (San Diego, Calif.), 71, 165-202. doi:10.1016/bs.apha.2014.06.013
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  Cerebral ischemia occurs when blood flow to the brain is insufficient to meet metabolic demand. This can result from cerebral artery occlusion that interrupts blood flow, limits CNS supply of oxygen and glucose, and causes an infarction/ischemic stroke. Ischemia initiates a cascade of molecular events in neurons and cerebrovascular endothelial cells including energy depletion, dissipation of ion gradients, calcium overload, excitotoxicity, oxidative stress, and accumulation of ions and fluid. Blood-brain barrier (BBB) disruption is associated with cerebral ischemia and leads to vasogenic edema, a primary cause of stroke-associated mortality. To date, only a single drug has received US Food and Drug Administration (FDA) approval for acute ischemic stroke treatment, recombinant tissue plasminogen activator (rt-PA). While rt-PA therapy restores perfusion to ischemic brain, considerable tissue damage occurs when cerebral blood flow is reestablished. Therefore, there is a critical need for novel therapeutic approaches that can "rescue" salvageable brain tissue and/or protect BBB integrity during ischemic stroke. One class of drugs that may enable neural cell rescue following cerebral ischemia/reperfusion injury is the HMG-CoA reductase inhibitors (i.e., statins). Understanding potential CNS drug delivery pathways for statins is critical to their utility in ischemic stroke. Here, we review molecular pathways associated with cerebral ischemia and novel approaches for delivering drugs to treat ischemic disease. Specifically, we discuss utility of endogenous BBB drug uptake transporters such as organic anion transporting polypeptides and nanotechnology-based carriers for optimization of CNS drug delivery. Overall, this chapter highlights state-of-the-art technologies that may improve pharmacotherapy of cerebral ischemia.
• Zhang, Y., Vanderah, T. W., Thompson, B. J., Slosky, L. M., Sanchez-covarrubias, L., Ronaldson, P. T., Laracuente, M. L., & Davis, T. P. (2013). Acetaminophen modulates P-glycoprotein functional expression at the blood-brain barrier by a constitutive androstane receptor-dependent mechanism.. Molecular pharmacology, 84(5), 774-86. doi:10.1124/mol.113.086298
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  Effective pharmacologic treatment of pain with opioids requires that these drugs attain efficacious concentrations in the central nervous system (CNS). A primary determinant of CNS drug permeation is P-glycoprotein (P-gp), an endogenous blood-brain barrier (BBB) efflux transporter that is involved in brain-to-blood transport of opioid analgesics (i.e., morphine). Recently, the nuclear receptor constitutive androstane receptor (CAR) has been identified as a regulator of P-gp functional expression at the BBB. This is critical to pharmacotherapy of pain/inflammation, as patients are often administered acetaminophen (APAP), a CAR-activating ligand, in conjunction with an opioid. Our objective was to investigate, in vivo, the role of CAR in regulation of P-gp at the BBB. Following APAP treatment, P-gp protein expression was increased up to 1.4-1.6-fold in a concentration-dependent manner. Additionally, APAP increased P-gp transport of BODIPY-verapamil in freshly isolated rat brain capillaries. This APAP-induced increase in P-gp expression and activity was attenuated in the presence of CAR pathway inhibitor okadaic acid or transcriptional inhibitor actinomycin D, suggesting P-gp regulation is CAR-dependent. Furthermore, morphine brain accumulation was enhanced by P-gp inhibitors in APAP-treated animals, suggesting P-gp-mediated transport. A warm-water (50°C) tail-flick assay revealed a significant decrease in morphine analgesia in animals treated with morphine 3 or 6 hours after APAP treatment, as compared with animals treated concurrently. Taken together, our data imply that inclusion of APAP in a pain treatment regimen activates CAR at the BBB and increases P-gp functional expression, a clinically significant drug-drug interaction that modulates opioid analgesic efficacy.
• Ronaldson, P. T., & Davis, T. P. (2012). Blood-brain barrier integrity and glial support: mechanisms that can be targeted for novel therapeutic approaches in stroke.. Current pharmaceutical design, 18(25), 3624-44. doi:10.2174/138161212802002625
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  The blood-brain barrier (BBB) is a critical regulator of brain homeostasis. Additionally, the BBB is the most significant obstacle to effective CNS drug delivery. It possesses specific charcteristics (i.e., tight junction protein complexes, influx and efflux transporters) that control permeation of circulating solutes including therapeutic agents. In order to form this "barrier," brain microvascular endothelial cells require support of adjacent astrocytes and microglia. This intricate relationship also occurs between endothelial cells and other cell types and structures of the CNS (i.e., pericytes, neurons, extracellular matrix), which implies existence of a "neurovascular unit." Ischemic stroke can disrupt the neurovascular unit at both the structural and functional level, which leads to an increase in leak across the BBB. Recent studies have identified several pathophysiological mechanisms (i.e., oxidative stress, activation of cytokine-mediated intracellular signaling systems) that mediate changes in the neurovascular unit during ischemic stroke. This review summarizes current knowledge in this area and emphasizes pathways (i.e., oxidative stress, cytokine-mediated intracellular signaling, glial-expressed receptors/targets) that can be manipulated pharmacologically for i) preservation of BBB and glial integrity during ischemic stroke and ii) control of drug permeation and/or transport across the BBB. Targeting these pathways present a novel opportunity for optimization of CNS delivery of therapeutics in the setting of ischemic stroke.
• Ronaldson, P., Lochhead, J. J., McCaffrey, G., Sanchez-Covarrubias, L., Finch, J. D., Demarco, K. M., Quigley, C. E., Davis, T. P., & Ronaldson, P. T. (2012). Tempol modulates changes in xenobiotic permeability and occludin oligomeric assemblies at the blood-brain barrier during inflammatory pain. American journal of physiology. Heart and circulatory physiology, 302(3).
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  Our laboratory has shown that λ-carrageenan-induced peripheral inflammatory pain (CIP) can alter tight junction (TJ) protein expression and/or assembly leading to changes in blood-brain barrier xenobiotic permeability. However, the role of reactive oxygen species (ROS) and subsequent oxidative stress during CIP is unknown. ROS (i.e., superoxide) are known to cause cellular damage in response to pain/inflammation. Therefore, we examined oxidative stress-associated effects at the blood-brain barrier (BBB) in CIP rats. During CIP, increased staining of nitrosylated proteins was detected in hind paw tissue and enhanced presence of protein adducts containing 3-nitrotyrosine occurred at two molecular weights (i.e., 85 and 44 kDa) in brain microvessels. Tempol, a pharmacological ROS scavenger, attenuated formation of 3-nitrotyrosine-containing proteins in both the hind paw and in brain microvessels when administered 10 min before footpad injection of λ-carrageenan. Similarly, CIP increased 4-hydroxynoneal staining in brain microvessels and this effect was reduced by tempol. Brain permeability to [(14)C]sucrose and [(3)H]codeine was increased, and oligomeric assemblies of occludin, a critical TJ protein, were altered after 3 h CIP. Tempol attenuated both [(14)C]sucrose and [(3)H]codeine brain uptake as well as protected occludin oligomers from disruption in CIP animals, suggesting that ROS production/oxidative stress is involved in modulating BBB functional integrity during pain/inflammation. Interestingly, tempol administration reduced codeine analgesia in CIP animals, indicating that oxidative stress during pain/inflammation may affect opioid delivery to the brain and subsequent efficacy. Taken together, our data show for the first time that ROS pharmacological scavenging is a viable approach for maintaining BBB integrity and controlling central nervous system drug delivery during acute inflammatory pain.
• Ronaldson, P. T., Piquette-miller, M., Gingras, D., Bendayan, R., & Bendayan, M. (2004). Cellular localization and functional expression of P-glycoprotein in rat astrocyte cultures.. Journal of neurochemistry, 89(3), 788-800. doi:10.1111/j.1471-4159.2004.02417.x
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  We investigated the cellular/subcellular localization and functional expression of P-glycoprotein, an ATP-dependent membrane-associated efflux transporter, in astrocytes, a brain parenchyma compartment that is poorly characterized for the expression of membrane drug transporters. Analyses were carried out on primary cultures of astrocytes isolated from the cerebral cortex of neonatal Wistar rats and CTX TNA2, an immortalized rat astrocyte cell line. Both cell cultures display morphological features typical of type I astrocytes. RT-PCR analysis revealed mdr1a and mdr1b mRNA in primary cultures of astrocytes and in CTX TNA2 cells. Western blot analysis using the P-glycoprotein monoclonal C219 antibody detected a single band of appropriate size in both cell systems. Immunocytochemical analysis using the monoclonal antibodies C219 and MRK16 labeled P-glycoprotein along the plasma membrane, caveolae, coated vesicles and nuclear envelope. Immunoprecipitation studies using the caveolin-1 polyclonal H-97 antibody demonstrated that P-glycoprotein is physically associated with caveolin-1 in both cell culture systems. The accumulation of [(3)H]digoxin (an established P-glycoprotein substrate) by the astrocyte cultures was significantly enhanced in the presence of standard P-glycoprotein inhibitors and an ATP depleting agent. These results demonstrate the cellular/subcellular location and functional expression of P-glycoprotein in rat astrocytes and suggest that this glial compartment may play an important role in the regulation of drug transport in the CNS.
• Ronaldson, P. T., Pallapothu, M., Dallas, S., & Bendayan, R. (2002). Functional expression of P-glycoprotein in primary rat astrocyte cultures and an immortalized rat astrocyte cell line. Clinical Pharmacology & Therapeutics, 71(2).

Presentations

• Ronaldson, P. T. (2022, May). Blood-Brain Barrier Transporters are Required for Therapeutic Effectiveness of Statins in Ischemic Stroke. University of Arizona College of Medicine Research Day. Tucson, AZ: University of Arizona College of Medicine.
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  This presentation discussed current research in the Ronaldson laboratory that is focused on understanding the role of blood-brain barrier (BBB) transporters in drug delivery to the brain.
• Ronaldson, P. T. (2022, October). Endogenous Blood-Brain Barrier Transporters are Critical Determinants of Neuroprotective Drug Efficacy in Ischemic Stroke. PharmSci360. Boston, MA: American Association of Pharmaceutical Scientists (AAPS).
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  This presentation highlighted current research in Dr. Ronaldson's laboratory demonstrating the utility of targeting SLC transporters for CNS delivery of neuroprotective drugs. Of note, information presented in this talk emphasized that transporters remain the key driver of drug uptake into the brain even in diseases characterized by blood-brain barrier (BBB) dysfunction such as ischemic stroke.
• Ronaldson, P. T. (2022, September). Targeting Blood-Brain Barrier Transporters: A Therapeutic Opportunity for CNS Delivery of Small Molecule Drugs. Research Seminar. San Francisco, CA: ORIC Pharmaceuticals, Inc..
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  This invited seminar highlighted research progress in Dr. Ronaldson's laboratory. Specifically, the concept of targeting SLC transporters for improved drug delivery to the brain was discussed. Emphasis was placed on how these transporters present an opportunity to optimize treatment of neurological diseases via more efficient and effective drug uptake into brain tissue. The presentation was conducted virtually via Zoom.
• Ronaldson, P. T. (2020, December). Transporter-Mediated Delivery of Small Molecule Across the Blood-Brain Barrier: Relevance to the Treatment of Stroke. Chongqing University and BayRay Innovation Center. Chongqing, China (Virtual Presentation): BayRay Innovation Center.
• Ronaldson, P. T. (2020, February). Blood-Brain Barrier Transporters Determine Effects of Statins in Ischemic Stroke. University of Mississippi Medical Center. Jackson, MS: University of Mississippi Medical Center.
• Ronaldson, P. T. (2020, June). Targeting Blood-Brain Barrier Transporters: A Therapeutic Opportunity for Stroke. Leslie Dan Faculty of Pharmacy, University of Toronto. Toronto, Canada: Leslie Dan Faculty of Pharmacy at the University of Toronto.
• Ronaldson, P. T. (2020, November). Transporter-Mediated Delivery of Small Molecules Across the Blood-Brain Barrier. AAPS 360. New Orleans, LA (Virtual Presentation): American Association of Pharmaceutical Scientists (AAPS).
• Ronaldson, P. T. (2019, April). Endogenous blood-brain barrier transporters are critical determinants of statin neuroprotective effects in stroke. Center for Natural Products, Drug Discovery, and Development Seminar Series. Gainesville, FL: University of Florida College of Pharmacy.
• Ronaldson, P. T. (2019, April). Targeting the Blood-Brain Barrier for Delivery of Neuroprotective Drugs in Ischemic Stroke. Experimental Biology 2019. Orlando, FL: American Society of Pharmacology and Experimental Therapeutics.
• Ronaldson, P. T. (2019, April). Transporter-mediated uptake of small molecules at the blood-brain barrier: Focus on organic anion transporting polypeptides. AAPS/IBBS Joint Workshop on Novel Approaches Targeting Brain Barriers for Effective Delivery of Therapeutics. Herndon, VA: American Association of Pharmaceutical Scientists and International Brain Barriers Society.
• Ronaldson, P. T. (2019, February). Pericytes and Transporters: Focus on Drug Delivery to the Ischemic Brain. International Stroke Conference. Honolulu, HI: American Heart Association.
• Ronaldson, P. T. (2019, September). Blood-brain barrier transporters in ischemic stroke: Focus on organic anion transporting polypeptides (Oatps). Solvo Biotechnology Meet the Experts Transporter Conference. Boston, MA: Solvo Biotechnology.
• Ronaldson, P. T. (2018, April). Regulation of Oatp1a4 Functional Expression by Transforming Growth Factor-Beta Signaling at the Blood-Brain Barrier. AAPS Workshop on Drug Transporters in ADME: From the Bench to the Bedside. Dulles, VA: American Association of Pharmaceutical Scientists (AAPS).
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  This invited presentation outlined key findings from the Ronaldson laboratory on the role of transforming growth factor-beta signaling on the regulation of endogenous transport mechanisms at the blood-brain barrier. In particular, data discussed during this presentation emphasized how transporters can be targeted for improved CNS drug delivery in diseases such as ischemic stroke.
• Ronaldson, P. T. (2018, April). Targeting Blood-Brain Barrier Transporters for CNS Drug Delivery: Role of Transforming Growth Factor-β Signaling. Experimental Biology 2018. San Diego, CA: American Society of Pharmacology and Experimental Therapeutics.
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  The presentation highlighted key research in Dr. Ronaldson's laboratory. Specifically, the focus was on regulation of blood-brain barrier transport at the molecular level by transforming growth factor-beta signaling and how this pathway can be targeted to optimize CNS drug delivery.
• Ronaldson, P. T. (2018, October). Targeting Blood-Brain Barrier Transporters to Treat Ischemic Stroke. Stroke Translational Research Advancement Workshop. Lexington, KY: University of Kentucky.
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  The presentation discussed novel approaches to deliver neuroprotective drugs to the brain for treatment of ischemic stroke. The focus was on endogenous transport systems expressed at the blood-brain barrier and how such biology can be exploited to optimize pharmacology.
• Ronaldson, P. T. (2017, February). Targeting Blood-Brain Barrier Transporters: Implications for Treatment of Diseases with a Hypoxia/Reoxygenation Component. Invited Seminar. Amarillo, TX: Texas Tech University Health Sciences Center.
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  This was an invited seminar that was presented to doctoral students at the Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center (Amarillo, TX). The presentation was also podcast to students at Texas Tech University sites in Lubbock, Abilene, and Dallas.
• Ronaldson, P. T. (2017, November). Targeting Blood-Brain Barrier Transporters for CNS Drug Delivery. 12th International Conference on Cerebral Vascular Biology. Melbourne, Australia: International Brain Barriers Society.
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  This was an invited talk at the 12th International Conference on Cerebral Vascular Biology, which was held November 27-December 1, 2017 in Melbourne, Australia.
• Ronaldson, P. T. (2016, February). Blood-Brain Barrier Drug Transporters in Cerebral Hypoxia: Implications for Ischemic Stroke Treatment. 2016 International Stroke Conference. Los Angeles, CA: American Heart Association.
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  Cerebral ischemia occurs when blood flow to the brain is insufficient to meet metabolic demand. This can result from cerebral artery occlusion that interrupts blood flow, limits CNS supply of oxygen and glucose, and causes an infarction/ischemic stroke. Ischemia initiates a cascade of molecular events in neurons and cerebrovascular endothelial cells including energy depletion, dissipation of ion gradients, calcium overload, excitotoxicity, oxidative stress, and accumulation of ions and fluid. Blood-brain barrier (BBB) dysfunction is associated with cerebral ischemia and leads to vasogenic edema, a leading cause of stroke-associated mortality. To date, only a single drug has recieved US Food and Drug Administration (FDA) approval for acute ischemic stroke treatment, recombinant tissue plasminogen activator (rt-PA). While rt-PA therapy restores perfusion to ischemic brain, considerable tissue damage occurs when cerebral blood flow is re-established. Therefore, there is a critical need for novel therapeutic approaches that can “rescue” salvageable brain tissue and/or protect BBB integrity during ischemic stroke. One class of drugs that may enable neural cell rescue following cerebral ischemia/reperfusion are the HMG-CoA reductase inhibitors (i.e., statins). Understanding potential CNS drug delivery pathways for statins is critical to their utility in ischemic stroke. In this invited presentation at the 2016 International Stroke Conference, molecular pathways associated with cerebral ischemia/hypoxia and novel approaches for delivering drugs to treat ischemic disease were discussed. Specifically, utility of endogenous BBB drug uptake transporters such as organic anion transporting polypeptides (OATPs/Oatps) for optimization of CNS drug delivery were examined in detail. Overall, this presentation highlights state-of-the-art technologies that can be extended to improve treatment of ischemic stroke.

Poster Presentations

• Ronaldson, P. T., Williams, E. I., Stanton, J. A., Betterton, R. D., & Davis, T. P. (2022, October). Endogenous Blood-Brain Barrier Transporters are Critical Determinants of Neuroprotective Drug Efficacy in Ischemic Stroke.. PharmSci360. Boston, MA: American Association of Pharmaceutical Scientists' Annual Meeting.
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  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. Conclusions: 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. 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.
• Betterton, R. D., Lochhead, J. J., Williams, E. I., Yang, J., Abdullahi, W., Davis, T. P., & Ronaldson, P. T. (2021, October 2021). Targeting transforming growth factor- signaling to modulate organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier: Relevance to the Treatment of Ischemic Stroke.. PharmSci 360. Virtual Meeting: American Association of Pharmaceutical Scientists (AAPS).
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  Objectives: Our laboratory has shown that activation of the transforming growth factor-β (TGF-β )/Activin-like Kinase 1 (ALK1) pathway can increase functional expression of organic anion transporting peptide 1a4 (Oatp1a4) at the blood-brain barrier (BBB). This finding is relevant to treatment of ischemic stroke because 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins), drugs that have been demonstrated to be transport substrates for Oatp1a4 at the BBB, are well known to improve post-stroke functional neurological outcomes in patients. We hypothesize that TGF-β/ALK1 signaling can be targeted to control Oatp-mediated delivery of statins for stroke treatment; however, a detailed understanding of the molecular characteristics of this pathway, including the role of the TGF-β/ALK1 co-receptor endoglin, has not been elucidated. Furthermore, it is unknown if signaling can differentially regulate Oatp1a4 in different brain regions. The objective of this study is to evaluate effects of TGF-β/ALK1 signaling on Oatp1a4 functional expression in cerebral cortex, hippocampus, and cerebellum and to study the role of endoglin in mediating the TGF-β/ALK1 pathway under ischemic conditions.Methods: In vivo experiments were conducted using female Sprague-Dawley rats (200-250 g) administered with bone morphogenetic protein-9 (BMP-9; 0-5 μg/kg, i.p.), an established ALK1 agonist and/or LDN193189 (10 mg/kg, i.p.), an established ALK1 antagonist. Localization and protein expression of Oatp1a4 were examined using confocal microscopy and western blot analysis, respectively. Oatp-mediated uptake of [3H]atorvastatin was determined using our established in situ perfusion technique. In vitro experiments were conducted using an immortalized mouse brain endothelial cell line (bEnd.3) that were subjected to oxygen-glucose deprivation (OGD) conditions to study regulation of endoglin under ischemic conditions. Results: Immunostaining corresponding to Oatp1a4 was increased in rat brain microvessels isolated from animals treated with BMP-9 (1 μg/kg, i.p.), results that corroborate our previously published data. BMP-9 treatment increased Oatp1a4 protein expression in brain microvessels isolated from cerebral cortex but had no effect on Oatp1a4 expression in microvessels derived from hippocampal or cerebellar tissue. In cortical microvessels, LDN193189 (10 mg/kg, i.p.) attenuated the increase in Oatp1a4 protein expression, which confirms that TGF-β/ALK1 signaling is involved in regulation of Oatp1a4. The BMP-9-mediated increase in cortical Oatp1a4 protein corresponded with a significant enhancement in [3H]atorvastatin uptake, suggesting that TGF-β/ALK1 signaling can control delivery of statin drugs primarily to the cerebral cortex. Since we propose that targeting the TGF-/ALK1 pathway can control statin drug delivery in stroke, we performed in vitro OGD experiments in bEnd.3 cells to evaluate endoglin expression under ischemic conditions. Following OGD treatment, we observed increased expression of the TGF- signaling co-receptor endoglin (CD105).Conclusions: Our data show, for the first time, evidence for regional differences in Oatp1a4 regulation by TGF-β/ALK1 signaling in brain microvessels. These results have direct implications for CNS drug delivery as they imply that targeting the TGF-β/ALK1 pathway for improved brain delivery of statins will be effective in the cerebral cortex but not in the hippocampus or cerebellum. Increased expression of endoglin under OGD conditions provides an impetus to evaluate the molecular pharmacology of the TGF-β/ALK1 pathway in the setting of ischemia. Studies are ongoing in the laboratory to rigorously study the role of endoglin in TGF-β/ALK1-mediated regulation of Oatp1a4 at the BBB. This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01-NS084941) to PTR and the National Institute on Drug Abuse (R01-DA05181) to TPD and PTR.
• Williams, E. I., Ronaldson, P. T., Davis, T. P., Lochhead, J. J., Lochhead, J. J., Davis, T. P., Williams, E. I., Ronaldson, P. T., Ronaldson, P. T., Davis, T. P., Lochhead, J. J., & Williams, E. I. (2021, October 2021). Acute neuroprotective effects of statins in ischemic stroke is dependent upon an Oatp-mediated transport mechanism at the BBB.. PharmSci 360. Virtual Meeting: American Association of Pharmaceutical Scientists (AAPS).
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  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 are designed to 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, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins) are commonly administered following ischemic stroke due to their utility in improving functional neurological outcomes in stroke patients. This property indicates that neuroprotective effectiveness of statins requires efficient delivery across the blood-brain barrier (BBB). In vivo studies in our laboratory have uncovered a specific biological mechanism that enables statins to be delivered from the systemic circulation into brain tissue: transport via the endogenous BBB uptake transporter organic anion transporting polypeptide 1a4 (Oatp1a4). The goal of this project was to show that functional expression of Oatp1a4 at the BBB is a required mechanism that enables statins to be effective neuroprotective drugs for stroke. Methods: Male and female Sprague-Dawley rats (200-250 g) were subjected to transient middle cerebral artery occlusion (tMCAO) for 90 minutes followed by 22.5 h reperfusion. 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.) was injected 2 h following removal of the intraluminal suture (i.e., reperfusion). Oatp1a4 protein expression was 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. 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), the adhesive removal test (i.e., sensorimotor function), and rotorod analysis (i.e., motor function).Results: Atorvastatin (20 mg/kg, i.v.) significantly reduced both infarction volume and the brain edema ratio. Atorvastatin also improved post-stroke outcomes as determined by neurological deficit scores, the adhesive removal test, and rotorod analysis. In the presence of fexofenadine (3.2 mg/kg, i.v.), atorvastatin had no effect on infarction volume or the brain edema ratio. Similarly, positive effects of atorvastatin on post-stroke outcomes were attenuated by fexofenadine.Conclusions: Our data provide evidence that atorvastatin requires functional expression of Oatp1a4 at the blood brain barrier to exert neuroprotective effects in the setting of ischemic stroke. Furthermore, neurological and sensorimotor performance 24 hours post-tMCAO can be improved following administration of a single intravenous dose of atorvastatin in both male and female rats. Since many patients are unable to swallow immediately following onset of stroke symptoms, intravenous statin delivery will enable early administration of these effective therapeutics to all stroke patients. Overall, our studies on Oatp1a4 imply that this critical endogenous BBB transporter can be exploited to optimize drug delivery to the ischemic brain. These findings are novel and have unique implications for clinical stroke treatment. This work was supported by grants from the Arizona Biomedical Research Commission (ABRC Grant #ADHS16-162406), the American Heart Association (19TPA34910113), and the National Institute of Neurological Diseases and Stroke (R01-NS084941) and) to PTR.
• Apostol, C., Liu, C., Bartlett, M. J., Bernard, K., Molnar, G., Szabo, L., Rowe, R., Ronaldson, P. T., Streicher, J. M., Falk, T., Heien, M. L., & Polt, R. L. (2020, Summer). Glycosylated PACAP Hormones as Potential Therapy for Parkinsonism, Stroke and Traumatic Brain Injury.. American Chemical Society National Meeting.
• Bernard, K., Apostol, C., Liu, C., Bartlett, M. J., Molnar, G., Szabo, L., Ronaldson, P. T., Streicher, J. M., Heien, M. L., Polt, R. L., & Falk, T. (2020, Summer). Preclinical evaluation of glycosylated PACAP Hormones to treat Parkinson’s disease and Stroke.. Arizona Alzheimer’s Consortium Annual Abstracts, 2020.
• Lochhead, J. J., Williams, E. I., Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2020, February). Organic Anion Transporting Polypeptide 1a4: A Critical Determinant of Neuroprotective Drug Efficacy in Stroke. 5th Annual Arizona Biomedical Research Commission (ABRC)-Flinn Research Conference. Phoenix, Arizona: ABRC.
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  Background and Knowledge Gap: Stroke is the 5th leading cause of death in the United States. Despite significant advances in reperfusion therapies (i.e., thrombolytic drug therapy, mechanical endovascular thrombectomy), stroke patients still experience considerable neurological deficits despite these interventions. To date, drug discovery for stroke treatment has been challenging as indicated by poor translatability of compounds from preclinical studies to successful Phase III clinical trials. In contrast, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins) are routinely given to stroke patients because they are known to improve post-stroke outcomes; however, statin use in stroke patients is limited to patients that can swallow since statins are formulated for oral administration only. Indeed, neuroprotective effectiveness of statins requires efficient delivery across the blood-brain barrier (BBB). Our laboratory has shown, in vivo, that the endogenous BBB uptake transporter Oatp1a4 facilitates blood-to-brain transport of currently marketed statins (i.e., atorvastatin, pravastatin); however, little is known regarding the effects of this endogenous BBB transporter on CNS drug disposition in the setting of ischemic stroke, a significant knowledge gap.Hypothesis: We hypothesize that functional expression of Oatp1a4 at the BBB is a required mechanism that enables efficient statin delivery to the brain, thereby enabling these drugs to be effective neuroprotective agents. Methods: Male and female Sprague-Dawley rats (200-250 g) were subjected to transient middle cerebral artery occlusion (tMCAO) for 90 minutes followed by 22.5 h reperfusion. 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.) was injected 2 h following removal of the intraluminal suture (i.e., reperfusion). Oatp1a4 protein expression was 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. 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, the adhesive removal test (i.e., sensorimotor function), and rotorod analysis (i.e., motor function). Results: In tMCAO animals, Oatp1a4 protein expression was increased in microvessels from ischemic cortex (i.e., ipsilateral cortex) but not in contralateral cortex. Atorvastatin significantly reduced both infarction volume and the brain edema ratio. Atorvastatin also improved post-stroke outcomes as determined by neurological deficit scores, the adhesive removal test, and rotorod analysis. In the presence of fexofenadine, atorvastatin had no effect on infarction volume or the brain edema ratio. Similarly, positive effects of atorvastatin on post-stroke outcomes were attenuated by fexofenadine. Conclusions: Our data indicate that neuroprotective effects of atorvastatin in experimental stroke require functional expression of Oatp1a4 at the BBB. Of particular significance, our results suggest that intravenous atorvastatin administered at an early time point following reperfusion (i.e., 2 h) can provide effective neuroprotection in male and female Sprague-Dawley rats subjected to tMCAO. Studies are ongoing in the laboratory to rigorously study regulation and functional expression of Oatp isoforms at the BBB in the tMCAO model.
• Ronaldson, P. T., Lochhead, J. J., & Davis, T. P. (2020, February). Organic Anion Transporting Polypeptide (Oatp)-Mediated Transport is required for Statin-Induced Neuroprotection: A Role for Blood-Brain Barrier Transporters in Stroke Treatment. International Stroke Conference 2020. Los Angeles, California: American Heart Association.
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  Objectives: Treatment approaches for stroke include reperfusion therapies (i.e., recombinant tissue plasminogen activator, endovascular thrombectomy); however, many stroke patients still experience disability. This indicates a need to develop neuroprotective treatments that are effective in the setting of successful recanalization. Post-stroke outcomes are improved by treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins). We have shown that the endogenous blood-brain barrier (BBB) uptake transporter Oatp1a4 facilitates blood-to-brain transport of atorvastatin (ATV). The objective of this study was to show that Oatp-mediated transport at the BBB is an absolute requirement for ATV neuroprotective effectiveness in stroke.Methods: Male and female Sprague-Dawley rats (200-250 g) were subjected to transient middle cerebral artery occlusion (tMCAO) for 90 minutes followed by 22.5 h reperfusion. Sham-operated animals were used as controls. ATV (20 mg/kg, i.v.) was injected 2 h following reperfusion. The role of Oatp-mediated transport was determined using the Oatp transport inhibitor fexofenadine (FEX; 3.2 mg/kg, i.v.) injected at the same time as ATV. Following tMCAO, infarction volume and brain edema ratios were calculated from TTC-stained brain slices. Post-stroke outcomes were assessed via measurement of neurological deficit scores, by the adhesive removal test (i.e., sensorimotor function), and by the rotarod performance test (i.e., motor function). Results: In tMCAO animals, ATV reduced (p < 0.01) both infarction volume and brain edema ratio in both sexes. ATV improved neurological deficit scores and well as sensorimotor function and motor performance. In the presence of FEX, ATV had no effect on infarction volume or brain edema ratio. Similarly, positive effects of ATV on post-stroke outcomes were attenuated by FEX. Conclusions: Our data indicate that pharmacological inhibition of Oatp-mediated transport at the BBB prevents ATV from exerting neuroprotective effects in rats following tMCAO. Our results also suggest that i.v. ATV administered at an early time point following reperfusion (i.e., 2 h) can provide effective neuroprotection in male and female rats subjected to tMCAO.
• Betterton, R. D., Abdullahi, W., Yang, J., Reilly, B. G., Serna, S., & Ronaldson, P. T. (2019, June). Modulation of the transforming growth factor-beta (TGF-beta) co-receptor endoglin (CD105) by oxygen/glucose deprivation in cultured rat brain endothelial cells: Relevance to ischemic stroke treatment. 13th International Conference on Cerebral Vascular Biology. Miami, FL: International Brain Barriers Society.
• Davis, T. P., Abdullahi, W., Lochhead, J. J., & Ronaldson, P. T. (2019, July). Organic anion transporting polypeptide (Oatp)-mediated transport at the blood-brain barrier is required for atorvastatin-induced neuroprotection in experimental ischemic stroke. BRAIN and BRAIN PET 2019. Yokohama, Japan: International Society for Cerebral Blood Flow and Metabolism.
• Reilly, B. G., Betterton, R. D., Brzica, H., & Ronaldson, P. T. (2019, June). Expression of glucose transporters in an immortalized rat brain endothelial cell line (RBE4) subjected to in vitro stroke conditions. 13th International Conference on Cerebral Vascular Biology. Miami, FL: International Brain Barriers Society.
• Ronaldson, P. T., Abdullahi, W., Lochhead, J. J., & Davis, T. P. (2019, June). Neuroprotective effects of atorvastatin in experimental stroke requires organic anion transporting polypeptide (Oatp)-mediated transport at the blood-brain barrier.. 13th International Conference on Cerebral Vascular Biology. Miami, FL: International Brain Barriers Society.
• Serna, S., Brzica, H., Becktel, D., Reilly, B. G., Yang, J., Betterton, R. D., & Ronaldson, P. T. (2019, June). Sex differences in multidrug resistance protein 4 (Mrp4) expression at the blood-brain barrier in Sprague-Dawley rats. 13th International Conference on Cerebral Vascular Biology. Miami, FL: International Brain Barriers Society.
• Yang, J., Betterton, R. D., Reilly, B. G., Lochhead, J. J., Davis, T. P., & Ronaldson, P. T. (2019, June). Acetaminophen modulates blood-brain barrier permeability by altering tight junction protein expression in brain vasculature. 13th International Conference on Cerebral Vascular Biology. Miami, FL: International Brain Barriers Society.
• Brzica, H., Abdullahi, W., Reilly, B. G., & Ronaldson, P. T. (2018, June). Sex Differences in Functional Expression of Organic Anion Transporting Polypeptide 1a4 (Oatp1a4) at the Blood-Brain Barrier: Relevance to Treatment of Ischemic Stroke. Gordon Research Conference on Barriers of the CNS 2018. New London, NH: Gordon Research Conferences.
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  Ischemic stroke is considerably more prevalent in individuals over the age of 65, a rapidly growing component of the State of Arizona population. This health impact indicates a critical need to identify and characterize novel methodologies for ischemic stroke treatment. Many such methodologies involve CNS delivery of neuroprotective drugs. Endogenous BBB transporters such as organic anion transporting polypeptide 1a4 (Oatp1a4) can facilitate blood-to-brain transport of therapeutics with neuroprotective properties such as 3-hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins). In order to advance targeting of Oatp1a4 for CNS drug delivery, it is critical to determine sex differences in expression and/or function of this transporter at the BBB. Evidence in the scientific literature indicates that differences in Oatp1a4 mRNA and protein between males and females exist in liver; however, a significant knowledge gap exists because similar studies have not been conducted in brain microvasculature.
• Brzica, H., Abdullahi, W., Reilly, B. G., & Ronaldson, P. T. (2018, March). Sex Differences in Functional Expression of Organic Anion Transporting Polypeptide 1a4 (Oatp1a4) at the Blood-Brain Barrier: Relevance to Treatment of Ischemic Stroke. 3rd Annual ABRC Research Conference. Phoenix, AZ: Arizona Biomedical Research Commission.
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  Ischemic stroke is considerably more prevalent in individuals over the age of 65, a rapidly growing component of the State of Arizona population. This health impact indicates a critical need to identify and characterize novel methodologies for ischemic stroke treatment. Many such methodologies involve CNS delivery of neuroprotective drugs. Endogenous BBB transporters such as organic anion transporting polypeptide 1a4 (Oatp1a4) can facilitate blood-to-brain transport of therapeutics with neuroprotective properties such as 3-hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins). In order to advance targeting of Oatp1a4 for CNS drug delivery, it is critical to determine sex differences in expression and/or function of this transporter at the BBB. Evidence in the scientific literature indicates that differences in Oatp1a4 mRNA and protein between males and females exist in liver; however, a significant knowledge gap exists because similar studies have not been conducted in brain microvasculature.
• Yang, J., Betterton, R. D., Reilly, B. G., Davis, T. P., & Ronaldson, P. T. (2018, September). Acetaminophen Modulates Transmembrane Tight Junction Proteins Claudin-5 and Occludin at the Blood-Brain Barrier. Mountain West Society for Toxicology Meeting. Phoenix, AZ: Society for Toxicology.
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  Opioids are effective as analgesics for treatment of chronic non-cancer pain; however, they cause clinically significant adverse events such as respiratory depression and development of tolerance. Acetaminophen (APAP) has been incorporated into many therapeutic products with opioids, or used in conjunction with opioids, in an effort to provide effective analgesia while reducing opioid dosages (i.e., opioid sparing effect). In 2011, the Food and Drug Administration (FDA) limited the dose of APAP that can be included in combination productions to 325 mg per tablet due to concerns related to liver injury; however, many patients who are prescribed combination products for management of moderate to severe non-cancer pain also consume APAP in excess of the maximum daily limit of 4000 mg/day. Overall use of opioids for chronic non-cancer pain has increased in the United States over the past two decades (Kaye et al. Pain Physician. 20: S93-S109, 2017). Additionally, prescription pain relievers are often used for non-medical purposes (i.e., opioid misuse), an established characteristic of the prescription drug abuse problem in the United States (Vowles et al. Pain. 156: 569-576, 2015). Of particular significance, there is a disproportionate increase in misuse of APAP-containing combination opioid products (Bond et al. Drug Saf. 35: 149-157, 2012). Therefore, it is essential to understand how high doses of APAP and/or high frequency of consumption of combination products containing APAP and opioids can cause injury to body systems other than the liver. Such knowledge is critical to inform development of dosing strategies to counteract misuse of analgesics and to produce safer medications that can be used for treatment of acute and chronic non-cancer pain.