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

• 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.

Journals/Publications

• 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).
• 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.
• 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
• 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.

Presentations

• 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. (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

• 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.
• 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.