Jeffrey James Lochhead

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Chapters

• Lochhead, J. J., Kumar, N. N., Nehra, G., Stenslik, M. J., Bradley, L. H., & Thorne, R. G. (2022). Intranasal Drug Delivery to the Brain. In Drug Delivery to the Brain.
• Lochhead, J. J., & Thorne, R. G. (2014). Intranasal Drug Delivery to the Brain. In Drug Delivery to the Brain. Springer, New York, NY. doi:10.1007/978-1-4614-9105-7_14
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  Drug delivery into the central nervous system (CNS) compartment is often restricted by the blood–brain barrier (BBB) and blood–cerebrospinal fluid barriers (BCSFB) that separate the blood from brain interstitial and cerebrospinal fluids, respectively. New strategies to circumvent the BBB are greatly needed to utilize polar pharmaceuticals and large biotherapeutics for CNS disease treatment because the BBB is typically impermeable to such compounds. Intranasal administration is a noninvasive method of drug delivery that potentially allows even large biotherapeutics access to the CNS along extracellular pathways associated with the olfactory and trigeminal nerves. Rapid effects, ease of self-administration, and the potential for frequent, chronic dosing are among the potential advantages of the intranasal route. This chapter provides an overview of the unique anatomic and physiologic attributes of the nasal mucosa and its associated cranial nerves that allow small but significant fractions of certain intranasally applied drugs to transfer across the nasal epithelia and subsequently be transported directly into the CNS. We also review the preclinical and clinical literature related to intranasal targeting of biotherapeutics to the CNS and speculate on future directions.
• Sparks, D. L., Petanceska, S., Sabbagh, M. N., Connor, D. J., Soares, H., Adler, C. H., Lopez, J., Silverberg, N., Davis, K. A., Stipho-majeed, S., Johnson-traver, S., Volodarsky, P., Ziolkowski, C., Lochhead, J. J., & Browne, P. (2005). Cholesterol, copper and statin therapy in Alzheimer's disease. In Alzheimer's Disease and Related Disorders Annual 5. CRC Press. doi:10.1201/B13935-7
• Sparks, D., Lochhead, J. J., Fisher, A., & Martin, T. A. (2005). Water quality and cholesterol-induced pathology: differential effects of the M-1 muscarinic receptor agonist Af267B on accumulation of Alzheimer-Like amyloid _ In rabbit brain. In Recent Progress in Alzheimer's and Parkinson's Diseases. CRC Press. doi:10.1201/B14441-27

Journals/Publications

• 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., Lochhead, J. J., Williams, E. I., Stanton, J. A., Brzica, H., Reddell, E. S., Davis, T. P., & Ronaldson, P. T. (2021). Involvement of endogenous blood-brain barrier transporters in the regional disposition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in the brain.. Drug Metabolism and Disposition.
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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 (HMG CoA) reductase inhibitors (i.e., statins) improve functional neurological outcomes in patients. Advancement of our work requires determination if TGF-/ALK1 signaling alters Oatp1a4 functional expression differently varies across brain regions and if such disparities affect 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 LDN193189. We showed that Oatp1a4 protein expression and brain distribution of three currently marketed statin drugs (i.e., atorvastatin, pravastatin, 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. While brain drug delivery is also dependent upon efflux transporters such as P-glycoprotein (P-gp) and/or Breast Cancer Resistance Protein (Bcrp), 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.
• 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.
• Kwak, E. A., Pan, C. C., Ramonett, A., Kumar, S., Cruz-Flores, P., Ahmed, T., Ortiz, H. R., Lochhead, J. J., Ellis, N. A., Mouneimne, G., Georgieva, T. G., Lee, Y. S., Vanderah, T. W., Largent-Milnes, T., Mohler, P. J., Hund, T. J., Langlais, P. R., Mythreye, K., & Lee, N. Y. (2022). β-spectrin as a stalk cell-intrinsic regulator of VEGF signaling. Nature communications, 13(1), 1326.
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  Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report β-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific β-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, β-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, β-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.
• 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.
• 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.
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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., 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.
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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., & Davis, T. P. (2019). Perivascular and Perineural Pathways Involved in Brain Delivery and Distribution of Drugs after Intranasal Administration. Pharmaceutics, 11(11).
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  One of the most challenging aspects of treating disorders of the central nervous system (CNS) is the efficient delivery of drugs to their targets within the brain. Only a small fraction of drugs is able to cross the blood-brain barrier (BBB) under physiological conditions, and this observation has prompted investigation into the routes of administration that may potentially bypass the BBB and deliver drugs directly to the CNS. One such route is the intranasal (IN) route. Increasing evidence has suggested that intranasally-administered drugs are able to bypass the BBB and access the brain through anatomical pathways connecting the nasal cavity to the CNS. Though the exact mechanisms regulating the delivery of therapeutics following IN administration are not fully understood, current evidence suggests that the perineural and perivascular spaces of the olfactory and trigeminal nerves are involved in brain delivery and cerebral perivascular spaces are involved in widespread brain distribution. Here, we review evidence for these delivery and distribution pathways, and we address questions that should be resolved in order to optimize the IN route of administration as a viable strategy to treat CNS disease states.
• 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.
• Kumar, N. N., Lochhead, J. J., Pizzo, M. E., Nehra, G., Boroumand, S., Greene, G., & Thorne, R. G. (2018). Delivery of immunoglobulin G antibodies to the rat nervous system following intranasal administration: Distribution, dose-response, and mechanisms of delivery. Journal of controlled release : official journal of the Controlled Release Society, 286, 467-484.
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  The intranasal route has been hypothesized to circumvent the blood-brain and blood-cerebrospinal fluid barriers, allowing entry into the brain via extracellular pathways along olfactory and trigeminal nerves and the perivascular spaces (PVS) of cerebral blood vessels. We investigated the potential of the intranasal route to non-invasively deliver antibodies to the brain 30 min following administration by characterizing distribution, dose-response, and mechanisms of antibody transport to and within the brain after administering non-targeted radiolabeled or fluorescently-labeled full length immunoglobulin G (IgG) to normal adult female rats. Intranasal [I]-IgG consistently yielded highest concentrations in the olfactory bulbs, trigeminal nerves, and leptomeningeal blood vessels with their associated PVS. Intranasal delivery also resulted in significantly higher [I]-IgG concentrations in the CNS than systemic (intra-arterial) delivery for doses producing similar endpoint blood concentrations. Importantly, CNS targeting significantly increased with increasing dose only with intranasal administration, yielding brain concentrations that ranged from the low-to-mid picomolar range with tracer dosing (50 μg) up to the low nanomolar range at higher doses (1 mg and 2.5 mg). Finally, intranasal pre-treatment with a previously identified nasal permeation enhancer, matrix metalloproteinase-9, significantly improved intranasal [I]-IgG delivery to multiple brain regions and further allowed us to elucidate IgG transport pathways extending from the nasal epithelia into the brain using fluorescence microscopy. The results show that it may be feasible to achieve therapeutic levels of IgG in the CNS, particularly at higher intranasal doses, and clarify the likely cranial nerve and perivascular distribution pathways taken by antibodies to reach the brain from the nasal mucosae.
• Lochhead, J. J. (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
• Wolak, D. J., Thorne, R. G., Singh, V., Lochhead, J. J., Kumar, N. N., Ithapu, V. K., & Gautam, M. (2016). Relative vascular permeability and vascularity across different regions of the rat nasal mucosa: implications for nasal physiology and drug delivery.. Scientific reports, 6(1), 31732. doi:10.1038/srep31732
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  Intranasal administration provides a non-invasive drug delivery route that has been proposed to target macromolecules either to the brain via direct extracellular cranial nerve-associated pathways or to the periphery via absorption into the systemic circulation. Delivering drugs to nasal regions that have lower vascular density and/or permeability may allow more drug to access the extracellular cranial nerve-associated pathways and therefore favor delivery to the brain. However, relative vascular permeabilities of the different nasal mucosal sites have not yet been reported. Here, we determined that the relative capillary permeability to hydrophilic macromolecule tracers is significantly greater in nasal respiratory regions than in olfactory regions. Mean capillary density in the nasal mucosa was also approximately 5-fold higher in nasal respiratory regions than in olfactory regions. Applying capillary pore theory and normalization to our permeability data yielded mean pore diameter estimates ranging from 13-17 nm for the nasal respiratory vasculature compared to
• Wolak, D. J., Thorne, R. G., Pizzo, M. E., & Lochhead, J. J. (2015). Rapid transport within cerebral perivascular spaces underlies widespread tracer distribution in the brain after intranasal administration.. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 35(3), 371-81. doi:10.1038/jcbfm.2014.215
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  The intranasal administration route is increasingly being used as a noninvasive method to bypass the blood-brain barrier because evidence suggests small fractions of nasally applied macromolecules may reach the brain directly via olfactory and trigeminal nerve components present in the nasal mucosa. Upon reaching the olfactory bulb (olfactory pathway) or brainstem (trigeminal pathway), intranasally delivered macromolecules appear to rapidly distribute within the brains of rodents and primates. The mechanisms responsible for this distribution have yet to be fully characterized. Here, we have used ex vivo fluorescence imaging to show that bulk flow within the perivascular space (PVS) of cerebral blood vessels contributes to the rapid central distribution of fluorescently labeled 3 and 10 kDa dextran tracers after intranasal administration in anesthetized adult rats. Comparison of tracer plasma levels and fluorescent signal distribution associated with the PVS of surface arteries and internal cerebral vessels showed that the intranasal route results in unique central access to the PVS not observed after matched intravascular dosing in separate animals. Intranasal targeting to the PVS was tracer size dependent and could be regulated by modifying nasal epithelial permeability. These results suggest cerebral perivascular convection likely has a key role in intranasal drug delivery to the brain.
• Sanchez-covarrubias, L., Ronaldson, P. T., Quigley, C. E., Mccaffrey, G., Lochhead, J. J., Finch, J. D., Demarco, K. M., & Davis, T. P. (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), H582-93. doi:10.1152/ajpheart.00889.2011
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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.
• Thorne, R. G., & Lochhead, J. J. (2012). Intranasal delivery of biologics to the central nervous system.. Advanced drug delivery reviews, 64(7), 614-28. doi:10.1016/j.addr.2011.11.002
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  Treatment of central nervous system (CNS) diseases is very difficult due to the blood-brain barrier's (BBB) ability to severely restrict entry of all but small, non-polar compounds. Intranasal administration is a non-invasive method of drug delivery which may bypass the BBB to allow therapeutic substances direct access to the CNS. Intranasal delivery of large molecular weight biologics such as proteins, gene vectors, and stem cells is a potentially useful strategy to treat a variety of diseases/disorders of the CNS including stroke, Parkinson's disease, multiple sclerosis, Alzheimer's disease, epilepsy, and psychiatric disorders. Here we give an overview of relevant nasal anatomy and physiology and discuss the pathways and mechanisms likely involved in drug transport from the nasal epithelium to the CNS. Finally we review both pre-clinical and clinical studies involving intranasal delivery of biologics to the CNS.
• Davis, T. P., Quigley, C. E., Nametz, N., Mccaffrey, G., Lochhead, J. J., Finch, J. D., Demarco, K. M., & Davis, T. P. (2011). Erratum: Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation (Journal of Cerebral Blood Flow and Metabolism (2010) 30 (1625-1636) DOI: 10.1038/jcbfm.2010.29). Journal of Cerebral Blood Flow and Metabolism, 31(2), 790-791. doi:10.1038/jcbfm.2010.177
• Quigley, C. E., Nametz, N., Mccaffrey, G., Lochhead, J. J., Finch, J. D., Demarco, K. M., & Davis, T. P. (2010). Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation.. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 30(9), 1625-36. doi:10.1038/jcbfm.2010.29
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  The blood-brain barrier (BBB) has a critical role in central nervous system homeostasis. Intercellular tight junction (TJ) protein complexes of the brain microvasculature limit paracellular diffusion of substances from the blood into the brain. Hypoxia and reoxygenation (HR) is a central component to numerous disease states and pathologic conditions. We have previously shown that HR can influence the permeability of the BBB as well as the critical TJ protein occludin. During HR, free radicals are produced, which may lead to oxidative stress. Using the free radical scavenger tempol (200 mg/kg, intraperitoneal), we show that oxidative stress produced during HR (6% O(2) for 1 h, followed by room air for 20 min) mediates an increase in BBB permeability in vivo using in situ brain perfusion. We also show that these changes are associated with alterations in the structure and localization of occludin. Our data indicate that oxidative stress is associated with movement of occludin away from the TJ. Furthermore, subcellular fractionation of cerebral microvessels reveals alterations in occludin oligomeric assemblies in TJ associated with plasma membrane lipid rafts. Our data suggest that pharmacological inhibition of disease states with an HR component may help preserve BBB functional integrity.
• Hom, S., Willis, C. L., Staatz, W. D., Quigley, C. A., Nametz, N., Mccaffrey, G., Lochhead, J. J., Hom, S., & Davis, T. P. (2009). Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress.. Journal of neurochemistry, 110(1), 58-71. doi:10.1111/j.1471-4159.2009.06113.x
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  Hypoxic (low oxygen) and reperfusion (post-hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood-brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O(2), 60 min), Hx (6% O(2), 60 min), or hypoxia/reoxygenation (H/R, 6% O(2), 60 min followed by 21% O(2), 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent-free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro-octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non-reducing and reducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis/western blot of PFO-solubilized occludin revealed that occludin oligomeric assemblies co-localizing with 'TJ-associated' raft domains contained a high molecular weight 'structural core' that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO-solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non-covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide-bonded inner core, and dispersal of these non-covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.
• Ziolkowski, C., Sparks, D. L., Soares, H., Sabbagh, M. N., Petanceska, S., Lopez, J., Lochhead, J. J., Connor, D. J., Browne, P., & Adler, C. H. (2005). Cholesterol, copper and Abeta in controls, MCI, AD and the AD cholesterol-lowering treatment trial (ADCLT).. Current Alzheimer research, 2(5), 527-39. doi:10.2174/156720505774932296
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  Cholesterol clearly plays an influential role in promoting the production of amyloid beta (Abeta) and possibly the progression of Alzheimer's Disease (AD). The AD Cholesterol-Lowering Treatment trial (ADCLT; 1 year duration) tested atorvastatin and found significant benefit on measures of cognition and depressive symptoms in treated patients (N = 32) compared to placebo (N = 31). We assessed the circulating levels of Abeta(1-40), Abeta(1-42), ceruloplasmin (copper chaperone), apolipoprotein E and HDL-cholesterol in blood collected at each clinical visit during the ADCLT. We also determined the circulating cholesterol, ceruloplasmin, and Abeta levels in AD and MCI (mild cognitive impairment) patients, and controls (two groups stratified by function; high and low) participating in our Brain Bank Program. Each Brain Bank individual was clinically assessed for performance on the Mini-Mental Status Exam (MMSE), Rey auditory verbal learning test (AVLT), Clock draw, and UPSIT (smell identification test). Among individuals of equal age and education, scores on the MMSE were significantly reduced in AD compared to both MCI and controls, as were scores on the UPSIT. Ability on delayed verbal recall was significantly reduced in AD compared to MCI, and in MCI compared to both control groups. Performance on the Clock draw was similar for AD and MCI patients, but was significantly reduced when comparing MCI to control. Both cholesterol and ceruloplasmin levels were significantly increased in low-function controls compared to the high-function control group, but were not different from levels identified in the MCI and AD patients. Significantly increased levels of Abeta(1-40) occurred in low- compared to high-function controls, with a further significant increase in MCI compared to low-function controls. Circulating Abeta(1-40) levels were decreased in AD compared to MCI. Levels of Abeta(1-42) were not significantly different between the groups. The slight gradual increase in circulating Abeta(1-40) and Abeta(1-42) levels produced by atorvastatin treatment in the ADCLT were not significant compared placebo. There was a trend for significant reduction in circulating ceruloplasmin levels after a year of atorvastatin therapy compared to levels observed at screen. The levels of HDL-cholesterol remained stable in the atorvastatin treated AD patients for 9 months and then decreased significantly compared to the placebo group at the 1-year time-point. The combined data support a role for cholesterol in AD and a possible influence of increasing circulating copper levels. The deterioration of function in controls and transition to MCI may be associated with concomitant incremental increases in circulating Abeta(1-40) levels. Increased cholesterol and ceruloplasmin levels may be associated with slight deterioration in function among controls as a precursor to impairment considered MCI. The clinical benefit of atorvastatin therapy is clearly not associated with decreased circulating Abeta or increased HDL-cholesterol, but a positive influence of reduced copper (ceruloplasmin) levels may be a consideration.
• Ziolkowski, C., Wassar, D., Sparks, D. L., Sabbagh, M. N., Petanceska, S., Lopez, J., Lochhead, J. J., Launer, L. J., Johnson-traver, S., Connor, D. J., & Browne, P. (2005). Atorvastatin therapy lowers circulating cholesterol but not free radical activity in advance of identifiable clinical benefit in the treatment of mild-to-moderate AD.. Current Alzheimer research, 2(3), 343-53. doi:10.2174/1567205054367900
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  Cholesterol-induced production of amyloid beta (Abeta) as a putative neurotoxin in Alzheimer's disease (AD), along with epidemiological evidence, suggests that statin drugs may provide benefit in treatment of the disorder. We tested the effect of once daily atorvastatin calcium (80 mg; two 40 mg tablets) on cognitive and/or behavioral decline in patients with mild-to-moderate AD. The study was designed as a pilot intention-to-treat, proof-of-concept, double-blind, placebo-controlled, randomized (1:1) trial with a 1-year exposure to study medication employing last-observation-carried-forward (LOCF) ANCOVA as the primary statistical method of assessment. Alternate statistical methods were employed to further explore the effect of atorvastatin treatment on progression of deterioration. Of the 98 individuals with mild-to-moderate AD (Mini-Mental State Examination score of 12-28) providing Informed Consent, 71 were eligible for randomization, 67 were randomized and 63 completed the 3-month visit and were statistically evaluable. The primary outcome measures were change in the Alzheimer Disease Assessment Scale-Cognitive (ADAS-cog) performance and the Clinical Global Impression of Change (CGIC). Secondary outcome measures included the MMSE, Geriatric Depression Scale (GDS), the Neuropsychiatric Inventory (NPI) and the ADCS Activities of Daily Living inventory (ADCS-ADL). Tertiary outcome measures included levels of total circulating cholesterol, LDL and VLDL, and circulating activity of the free radical scavenger enzymes superoxide dismutase (SOD) and glutathione peroxidase (GpX). Atorvastatin reduced circulating cholesterol levels and produced a positive signal on each of the clinical outcome measures compared to placebo, but did not elicit a difference in circulating SOD or GpX activities. The observed beneficial clinical effect reached significance for the GDS (p = 0.040) and the ADAS-cog at 6 months (p = 0.003), was all but significant for the ADAS-cog (p = 0.055) at 12 months, and was of marginal significance for the CGIC (p = 0.073) and NPI (p = 0.071) at 12 months when employing the primary statistical approach (ANCOVA with LOCF). Application of repeated measures ANCOVA statistics revealed the difference was significant for the CGIC and marginally significant for the ADAS-cog, but not significant for the other clinical indices. This evaluation indicated significant time-by-treatment interactions (altered progression) for the ADAS-cog and MMSE, but not the CGIC. Application of random intercept regression analysis revealed a significant difference for the CGIC, ADAS-cog and MMSE. Regression analysis also indicated that atorvastatin produced change in the slope of deterioration on the MMSE. Accordingly, atorvastatin therapy may be an effective treatment and may slow the progression of AD among mild-to-moderately affected patients.
• Ziolwolski, C., Wasser, D., Sparks, D. L., Sabbagh, M. N., Lopez, J., Lochhead, J. J., Launer, L. J., Johnson-traver, S., Connor, D. J., & Browne, P. (2005). Atorvastatin for the treatment of mild to moderate Alzheimer disease: preliminary results.. Archives of neurology, 62(5), 753-7. doi:10.1001/archneur.62.5.753
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  Laboratory evidence of cholesterol-induced production of amyloid beta as a putative neurotoxin precipitating Alzheimer disease, along with epidemiological evidence, suggests that cholesterol-lowering statin drugs may favorably influence the progression of the disorder..To determine if treatment with atorvastatin calcium affects the cognitive and/or behavioral decline in patients with mild to moderate Alzheimer disease..Pilot intention-to-treat, proof-of-concept, double-blind, placebo-controlled, randomized (1:1) trial with a 1-year exposure to once-daily atorvastatin calcium (80 mg; two 40-mg tablets) or placebo using last observation carried forward analysis of covariance as the primary method of statistical assessment..Individuals with mild to moderate Alzheimer disease (Mini-Mental State Examination score of 12-28) were recruited. Of the 98 participants providing informed consent, 71 were eligible for randomization, 67 were randomized, and 63 subjects completed the 3-month visit and were considered evaluable..The primary outcome measures were change in Alzheimer's Disease Assessment Scale-cognitive subscale and the Clinical Global Impression of Change Scale scores. The secondary outcome measures included scores on the Mini-Mental State Examination, Geriatric Depression Scale, the Neuropsychiatric Inventory Scale, and the Alzheimer's Disease Cooperative Study-Activities of Daily Living Inventory. The tertiary outcome measures included total cholesterol, low-density lipoprotein cholesterol, and very low-density lipoprotein cholesterol levels..Atorvastatin reduced circulating cholesterol levels and produced a positive signal on each of the clinical outcome measures compared with placebo. This beneficial effect reached significance for the Geriatric Depression Scale and the Alzheimer's Disease Assessment Scale-cognitive subscale at 6 months and was significant at the level of a trend for the Alzheimer's Disease Assessment Scale-cognitive subscale, Clinical Global Impression of Change Scale, and Neuropsychiatric Inventory Scale at 12 months assessed by analysis of covariance with last observation carried forward..Atorvastatin treatment may be of some clinical benefit and could be established as an effective therapy for Alzheimer disease if the current findings are substantiated by a much larger multicenter trial.
• Sparks, D. L., Smith-bell, C. A., Schreurs, B. G., & Lochhead, J. J. (2003). Cholesterol modifies classical conditioning of the rabbit (Oryctolagus cuniculus) nictitating membrane response.. Behavioral neuroscience, 117(6), 1220-32. doi:10.1037/0735-7044.117.6.1220
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  Cholesterol plays an important role in synapse formation, receptor function, and synaptic plasticity, and animal studies show that modifying cholesterol may improve learning and memory. Other data show that feeding animals cholesterol can induce beta amyloid accumulation. Rabbits (Oryctolagus cuniculus) fed 2% cholesterol for 8 weeks were given trace conditioning of the nictitating membrane response using a 100-ms tone, a 700-ms trace, and periorbital electrical stimulation or airpuff. Rabbits fed cholesterol showed significant facilitation of trace conditioning to airpuff and conditioning-specific reflex modification to periorbital electrical stimulation and airpuff. The cholesterol-fed rabbits had beta amyloid accumulation in the cortex, but little in the hippocampus. The data suggest cholesterol had facilitative effects that outweighed potential amnesic effects of cortical beta amyloid.
• Martin, T., Wagoner, T., Sparks, D. L., Martin, T. A., Lochhead, J. J., & Horstman, D. (2002). Water quality has a pronounced effect on cholesterol-induced accumulation of Alzheimer amyloid beta (Abeta) in rabbit brain.. Journal of Alzheimer's disease : JAD, 4(6), 523-9. doi:10.3233/jad-2002-4609
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  Increased circulating cholesterol is known to promote risk of coronary artery disease. It is now emerging that cholesterol promotes production and accumulation of amyloid beta (Abeta) deposited in the hallmark pathologic lesion of Alzheimer's disease (AD), the senile plaque, perhaps by shifting away from normal metabolism of amyloid beta protein precursor (AbetaPP) to beta. Previous studies employing the cholesterol-fed rabbit model of AD demonstrated that induction of AD-like Abeta accumulation in brain could be reversed by co-administration of cholesterol lowering drugs or removing cholesterol, prompted initiation of an AD Cholesterol-Lowering (Statin) Treatment Trial. We now present data that identify a previously unrecognized role for dietary water quality on the severity of neuropathology induced by elevated cholesterol. Neuronal accumulation of Abeta induced by increased circulating concentrations of cholesterol in the New Zealand white rabbit is attenuated when distilled drinking water is administered compared to use of tap water. The numbers of neurons in cholesterol-fed rabbits that exhibited Abeta immunoreactivity, relative to normal chow-fed controls, increased approximately 2.5 fold among animals on tap water but only approximately 1.9 fold among animals on distilled water. This yielded a statistically significant approximately 28% reduction due to the use of distilled water. In addition, the subjectively assessed intensity of neuronal Abeta immunoreactivity was consistently reduced among cholesterol-fed rabbits allowed distilled drinking water compared to cholesterol-fed rabbits on tap water. As intensity of antibody immunoreactivity is likely related to concentration of antigen, the identified difference among cholesterol-fed rabbits allowed distilled drinking water may hold greater importance than a significant reduction in numbers of affected neurons. The effect on neuronal Abeta immunoreactivity intensity was observable among cholesterol-fed rabbits reared and allowed tap water when performing studies in three distinct locales. Pilot data suggest the possibility of increased clearance of Abeta from the brain, identified as increased blood levels, among cholesterol-fed rabbits administered distilled water compared to animals on tap water. The agent(s) occurring in tap water, excluded by distillation, promoting accumulation of neuronal Abeta immunoreactivity is(are) yet undisclosed, but arsenic, manganese, aluminum, zinc, mercury, iron and nitrate have tentatively been excluded because they were not identifiable (below detection limits) in the tap water of the three locales where the cholesterol-induced neuropathologic difference was observable. These findings suggest that water quality may impact on human health in the setting of increased circulating cholesterol levels, and could illustrate a truly simple life-style change that could be of benefit in AD.

Poster Presentations

• 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., Williams, E. I., Lochhead, J. J., Lochhead, J. J., Davis, T. P., Davis, T. P., Ronaldson, P. T., & Ronaldson, P. T. (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.
• Davis, T. P., Lochhead, J. J., & Ronaldson, P. T. (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.
• Ronaldson, P. T., Davis, T. P., Betterton, R. D., Williams, E. I., & Lochhead, J. J. (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.