Thomas P Davis

Interests

Teaching

Pharmacology, Toxicology and Drug Delivery.

Research

Barriers of the CNS in Health and Disease. CNS P-glycoprotein trafficking in disease states. Drug targeting blood brain barrier transporters to treat Stroke. Drug-drug interaction at the BBB. New drug development for stroke.

Courses

Scholarly Contributions

Chapters

• Davis, T. P. (2016). Development and Maintenance of the Blood Brain Barrier. In Caplan's Primer on Cerebrovascular Diseases. 2nd Edition. Academic Press.(pp 50-70). Philadelphia, PA: Elsevier Inc.
• Davis, T. P. (2016). “Glial Support of Blood-Brain Barrier Integrity: Molecular Targets for Novel Therapeutic Strategies in Stroke”. Jun Chen Ph.D. John Zhang Ph.D., and Xiaoming Hu, Ph.D., Editors. pp. 45-80. Springer International Press. Published August 16, 2016.. In Non-Neuronal Mechanisms of Brain Damage and Repair after Stroke.(pp Chapter 3.). Springer International Publishing.
• Ronaldson, P. T., & Davis, T. P. (2016). Mechanisms of Endothelial Injury and Blood-Brain Barrier Dysfunction in Stroke. In Caplan Primer on Cerebrovascular Diseases, 2nd Edition(pp TBD). Philadelphia, PA: Elsevier Inc.
• Witt, K. A., Ronaldson, P. T., Sandoval, K. E., & Davis, T. P. (2010).

  CNS Delivery of Peptides Across the BBB Using the Dual-Artery In Situ Brain Perfusion Model

  . In BBB. Humana Press. doi:10.1007/978-1-60761-529-3_11
• Alberts, D. S., Edwards, L., Peng, Y. M., Serokman, R., Davis, T. P., & Meyskens, F. L. (1986).

  Clinical Toxicology Pharmacokinetics of 13-Cis-Retinoic Acid Administered Chronically at Low Doses Expected for Cancer Chemoprevention Trials

  . In Cancer. Humana Press. doi:10.1007/978-1-4612-5006-7_15
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  This work was supported by research grants CA-27502 and CA-17094 from the National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20205. We would like to thank Dr. Thomas Moon for his excellent scientific advice and Susan Leigh for her outstanding dedication in the coordination of collection of biological samples.

Journals/Publications

• Ronaldson, P. T., Williams, E. I., Betterton, R. D., Stanton, J. A., Nilles, K. L., & Davis, T. P. (2024). CNS Drug Delivery in Stroke: Improving Therapeutic Translation From the Bench to the Bedside. Stroke, 55(1), 190-202.
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  Drug development for ischemic stroke is challenging as evidenced by the paucity of therapeutics that have advanced beyond a phase III trial. There are many reasons for this lack of clinical translation including factors related to the experimental design of preclinical studies. Often overlooked in therapeutic development for ischemic stroke is the requirement of effective drug delivery to the brain, which is critical for neuroprotective efficacy of several small and large molecule drugs. Advancing central nervous system drug delivery technologies implies a need for detailed comprehension of the blood-brain barrier (BBB) and neurovascular unit. Such knowledge will permit the innate biology of the BBB/neurovascular unit to be leveraged for improved bench-to-bedside translation of novel stroke therapeutics. In this review, we will highlight key aspects of BBB/neurovascular unit pathophysiology and describe state-of-the-art approaches for optimization of central nervous system drug delivery (ie, passive diffusion, mechanical opening of the BBB, liposomes/nanoparticles, transcytosis, intranasal drug administration). Additionally, we will discuss how endogenous BBB transporters represent the next frontier of drug delivery strategies for stroke. Overall, this review will provide cutting edge perspective on how central nervous system drug delivery must be considered for the advancement of new stroke drugs toward human trials.
• Betterton, R. D., Williams, E. I., Nilles, K. L., Davis, T. P., & Ronaldson, P. T. (2023). Methods to Study Drug Uptake at the Blood-Brain Barrier Following Experimental Ischemic Stroke: In Vitro and In Vivo Approaches. Methods in molecular biology (Clifton, N.J.), 2616, 403-418.
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  Drug permeability across the blood-brain barrier (BBB) is an important concept in the development of therapeutic strategies to treat neurological diseases such as ischemic stroke. These mechanisms can be evaluated in detail using cultured brain microvascular endothelial cells or intact animals subjected to experimental stroke. Here, we describe state-of-the-art approaches to study BBB transport of therapeutics using our in vitro and in vivo approaches. These methodologies allow for precise determination of transporter kinetic properties for currently marketed therapeutics or for new chemical entities that are under development as stroke drugs.
• Lochhead, J. J., Williams, E. I., Reddell, E. S., Dorn, E., Ronaldson, P. T., & Davis, T. P. (2023). High Resolution Multiplex Confocal Imaging of the Neurovascular Unit in Health and Experimental Ischemic Stroke. Cells, 12(4).
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  The neurovascular unit (NVU) is an anatomical group of cells that establishes the blood-brain barrier (BBB) and coordinates cerebral blood flow in association with neuronal function. In cerebral gray matter, cellular constituents of the NVU include endothelial cells and associated pericytes, astrocytes, neurons, and microglia. Dysfunction of the NVU is a common feature of diseases that affect the CNS, such as ischemic stroke. High-level evaluation of these NVU changes requires the use of imaging modalities that can enable the visualization of various cell types under disease conditions. In this study, we applied our confocal microscopy strategy using commercially available labeling reagents to, for the first time, simultaneously investigate associations between endothelial cells, the vascular basal lamina, pericytes, microglia, astrocytes and/or astrocyte end-feet, and neurites in both healthy and ischemic brain tissue. This allowed us to demonstrate ischemia-induced astrocyte activation, neurite loss, and microglial migration toward blood vessels in a single confocal image. Furthermore, our labeling cocktail enabled a precise quantification of changes in neurites and astrocyte reactivity, thereby showing the relationship between different NVU cellular constituents in healthy and diseased brain tissue. The application of our imaging approach for the simultaneous visualization of multiple NVU cell types provides an enhanced understanding of NVU function and pathology, a state-of-the-art advancement that will facilitate the development of more effective treatment strategies for diseases of the CNS that exhibit neurovascular dysfunction, such as ischemic stroke.
• Schleicher, R. L., Vorasayan, P., McCabe, M. E., Bevers, M. B., Davis, T. P., Griffin, J. H., Hinduja, A., Jadhav, A. P., Lee, J. M., Sawyer, R. N., Zlokovic, B. V., Sheth, K. N., Fedler, J. K., Lyden, P., Kimberly, W. T., & , N. I. (2023). Analysis of brain edema in RHAPSODY. International journal of stroke : official journal of the International Stroke Society, 17474930231187268.
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  Cerebral edema is a secondary complication of acute ischemic stroke, but its time course and imaging markers are not fully understood. Recently, net water uptake (NWU) has been proposed as a novel marker of edema.
• Williams, E. I., Betterton, R. D., Stanton, J. A., Moreno-Rodriguez, V. M., Lochhead, J. J., Davis, T. P., & Ronaldson, P. T. (2023). Oatp (Organic Anion Transporting Polypeptide)-Mediated Transport: A Mechanism for Atorvastatin Neuroprotection in Stroke. Stroke, 54(11), 2875-2885.
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  Drug discovery for stroke is challenging as indicated by poor clinical translatability. In contrast, HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (ie, statins) improve poststroke neurological outcomes. This property requires transport across the blood-brain barrier via an endogenous uptake transporter (ie, Oatp1a4 [organic anion transporting polypeptide 1a4]). Our goal was to study Oatp1a4 as a drug delivery mechanism because the blood-brain barrier cannot be assumed to be completely open for all drugs in ischemic stroke.
• Betterton, R. D., Abdullahi, W., Williams, E. I., Lochhead, J. J., Brzica, H., Stanton, J., Reddell, E., Ogbonnaya, C., Davis, T. P., & Ronaldson, P. T. (2022). Regulation of Blood-Brain Barrier Transporters by Transforming Growth Factor-/Activin Receptor-Like Kinase 1 Signaling: Relevance to the Brain Disposition of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors (i.e., Statins). Drug metabolism and disposition: the biological fate of chemicals, 50(7), 942-956.
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  Our laboratory has shown that activation of transforming growth factor- (TGF- )/activin receptor-like kinase 1 (ALK1) signaling can increase protein expression and transport activity of organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier (BBB). These results are relevant to treatment of ischemic stroke because Oatp transport substrates such as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e., statins) improve functional neurologic outcomes in patients. Advancement of our work requires determination if TGF- /ALK1 signaling alters Oatp1a4 functional expression differently across brain regions and if such disparities affect central nervous system (CNS) statin disposition. Therefore, we studied regulation of Oatp1a4 by the TGF- /ALK1 pathway, in vivo, in rat brain microvessels isolated from cerebral cortex, hippocampus, and cerebellum using the ALK1 agonist bone morphogenetic protein-9 (BMP-9) and the ALK1 inhibitor 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride 193189. We showed that Oatp1a4 protein expression and brain distribution of three currently marketed statin drugs (i.e., atorvastatin, pravastatin, and rosuvastatin) were increased in cortex relative to hippocampus and cerebellum. Additionally, BMP-9 treatment enhanced Oatp-mediated statin transport in cortical tissue but not in hippocampus or cerebellum. Although brain drug delivery is also dependent upon efflux transporters, such as P-glycoprotein and/or Breast Cancer Resistance Protein, our data showed that administration of BMP-9 did not alter the relative contribution of these transporters to CNS disposition of statins. Overall, this study provides evidence for differential regulation of Oatp1a4 by TGF- /ALK1 signaling across brain regions, knowledge that is critical for development of therapeutic strategies to target Oatps at the BBB for CNS drug delivery. SIGNIFICANCE STATEMENT: Organic anion transporting polypeptides (Oatps) represent transporter targets for brain drug delivery. We have shown that Oatp1a4 statin uptake is higher in cortex versus hippocampus and cerebellum. Additionally, we report that the transforming growth factor- /activin receptor-like kinase 1 agonist bone morphogenetic protein-9 increases Oatp1a4 functional expression, but not efflux transporters P-glycoprotein and Breast Cancer Resistance Protein, in cortical brain microvessels. Overall, this study provides critical data that will advance treatment for neurological diseases where drug development has been challenging.
• Betterton, R. D., Davis, T. P., Dorn, E., Lochhead, J. J., Ogbonnaya, C. E., Reddell, E. S., Ronaldson, P. T., Stanton, J. A., Williams, E. I., & Yang, J. (2022).

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

  . Pharmaceutics. doi:10.3390/pharmaceutics14050949
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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 [14C]sucrose and [3H]codeine, an opioid analgesic drug that is co-administered with APAP (i.e., Tylenol #3). Localization and protein expression of tight junction proteins (i.e., claudin-5, occludin, ZO-1) were studied in rat brain microvessels using Western blot analysis and confocal microscopy, respectively. Paracellular [14C]sucrose “leak” and brain [3H]codeine accumulation were significantly enhanced in rats treated with 500 mg/kg APAP only. Additionally, claudin-5 localization and protein expression were altered in brain microvessels isolated from rats administered 500 mg/kg APAP. Our novel and translational data show that BBB integrity is altered following a single high APAP dose, results that are relevant to patients abusing or misusing APAP and/or APAP/opioid combination products.
• Nilles, K. L., Williams, E. I., Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2022). Blood-Brain Barrier Transporters: Opportunities for Therapeutic Development in Ischemic Stroke. International journal of molecular sciences, 23(3).
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  Globally, stroke is a leading cause of death and long-term disability. Over the past decades, several efforts have attempted to discover new drugs or repurpose existing therapeutics to promote post-stroke neurological recovery. Preclinical stroke studies have reported successes in identifying novel neuroprotective agents; however, none of these compounds have advanced beyond a phase III clinical trial. One reason for these failures is the lack of consideration of blood-brain barrier (BBB) transport mechanisms that can enable these drugs to achieve efficacious concentrations in ischemic brain tissue. Despite the knowledge that drugs with neuroprotective properties (i.e., statins, memantine, metformin) are substrates for endogenous BBB transporters, preclinical stroke research has not extensively studied the role of transporters in central nervous system (CNS) drug delivery. Here, we review current knowledge on specific BBB uptake transporters (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents); organic cation transporters (OCTs in humans; Octs in rodents) that can be targeted for improved neuroprotective drug delivery. Additionally, we provide state-of-the-art perspectives on how transporter pharmacology can be integrated into preclinical stroke research. Specifically, we discuss the utility of in vivo stroke models to transporter studies and considerations (i.e., species selection, co-morbid conditions) that will optimize the translational success of stroke pharmacotherapeutic experiments.
• Ronaldson, P. T., & Davis, T. P. (2022). Transport Mechanisms at the Blood-Brain Barrier and in Cellular Compartments of the Neurovascular Unit: Focus on CNS Delivery of Small Molecule Drugs. Pharmaceutics, 14(7).
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  Ischemic stroke is a primary origin of morbidity and mortality in the United States and around the world. Indeed, several research projects have attempted to discover new drugs or repurpose existing therapeutics to advance stroke pharmacotherapy. Many of these preclinical stroke studies have reported positive results for neuroprotective agents; however, only one compound (3K3A-activated protein C (3K3A-APC)) has advanced to Phase III clinical trial evaluation. One reason for these many failures is the lack of consideration of transport mechanisms at the blood-brain barrier (BBB) and neurovascular unit (NVU). These endogenous transport processes function as a "gateway" that is a primary determinant of efficacious brain concentrations for centrally acting drugs. Despite the knowledge that some neuroprotective agents (i.e., statins and memantine) are substrates for these endogenous BBB transporters, preclinical stroke studies have largely ignored the role of transporters in CNS drug disposition. Here, we review the current knowledge on specific BBB transporters that either limit drug uptake into the brain (i.e., ATP-binding cassette (ABC) transporters) or can be targeted for optimized drug delivery (i.e., solute carrier (SLC) transporters). Additionally, we highlight the current knowledge on transporter expression in astrocytes, microglia, pericytes, and neurons with an emphasis on transport mechanisms in these cell types that can influence drug distribution within the brain.
• Stanton, J. A., Williams, E. I., Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2022). Targeting organic cation transporters at the blood-brain barrier to treat ischemic stroke in rats. Experimental neurology, 357, 114181.
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  Drug discovery and development for stroke is challenging as evidenced by few drugs that have advanced beyond a Phase III clinical trial. Memantine is a N-methyl-d-aspartate (NMDA) receptor antagonist that has been shown to be neuroprotective in various preclinical studies. We have identified an endogenous BBB uptake transport system for memantine: organic cation transporters 1 and 2 (Oct1/Oct2). Our goal was to evaluate Oct1/Oct2 as a required BBB mechanism for memantine neuroprotective effects. Male Sprague-Dawley rats (200-250 g) were subjected to middle cerebral artery occlusion (MCAO) for 90 min followed by reperfusion. Memantine (5 mg/kg, i.v.) was administered 2 h following intraluminal suture removal. Specificity of Oct-mediated transport was evaluated using cimetidine (15 mg/kg, i.v.), a competitive Oct1/Oct2 inhibitor. At 2 h post-MCAO, [H]memantine uptake was increased in ischemic brain tissue. Cimetidine inhibited blood-to-brain uptake of [H]memantine, which confirmed involvement of an Oct-mediated transport mechanism. Memantine reduced post-MCAO infarction and brain edema progression as well as improved neurological outcomes during post-stroke recovery. All positive effects of memantine were attenuated by co-administration of cimetidine, which demonstrates that Oct1/Oct2 transport is required for memantine to exert neuroprotective effects in ischemic stroke. Furthermore, Oct1/Oct2-mediated transport was shown to be the dominant mechanism for memantine brain uptake in the MCAO model despite a concurrent increase in paracellular "leak." These novel and translational findings provide mechanistic evidence for the critical role of BBB transporters in CNS delivery of stroke therapeutics, information that can help such drugs advance in clinical trials.
• Yang, J., Betterton, R. D., Williams, E. I., Stanton, J. A., Reddell, E. S., Ogbonnaya, C. E., Dorn, E., Davis, T. P., Lochhead, J. J., & Ronaldson, P. T. (2022). High-Dose Acetaminophen Alters the Integrity of the Blood-Brain Barrier and Leads to Increased CNS Uptake of Codeine in Rats. Pharmaceutics, 14(5).
  More info

  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.
• Abdullahi, W., Brzica, H., Davis, T. P., Reilly, B. G., Ronaldson, P. T., Abdullahi, W., Brzica, H., Davis, T. P., Reilly, B. G., & Ronaldson, P. T. (2021).

  Transport Properties of Statins by Organic Anion Transporting Polypeptide 1A2 and Regulation by Transforming Growth Factor-β Signaling in Human Endothelial Cells.

  . The Journal of pharmacology and experimental therapeutics, 376(2), 148-160. doi:10.1124/jpet.120.000267
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  Our in vivo rodent studies have shown that organic anion transporting polypeptide (Oatp) 1a4 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 ortholog 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 in vitro 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. We showed that statin accumulation in HUVECs requires OATP1A2-mediated uptake but is also affected by efflux transporters (i.e., P-glycoprotein, breast cancer resistance protein). 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 blood-brain barrier. SIGNIFICANCE STATEMENT: Transporter data derived from rodent models requires validation in human models. Using human umbilical vein endothelial cells, this study has shown that statin transport is mediated by OATP1A2. Additionally, we demonstrated that OATP1A2 is regulated by transforming growth factor-β/activin receptor-like kinase 1 signaling. This work emphasizes the need to consider endothelial transporter kinetics and regulation during preclinical drug development. Furthermore, our forward-thinking approach can identify effective therapeutics for diseases for which drug development has been challenging (i.e., neurological diseases).
• Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2021).

  Organic Cation Transporter (OCT/OCTN) Expression at Brain Barrier Sites: Focus on CNS Drug Delivery.

  . Handbook of experimental pharmacology, 266, 301-328. doi:10.1007/164_2021_448
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  Therapeutic delivery to the central nervous system (CNS) continues to be a considerable challenge in the pharmacological treatment and management of neurological disorders. This is primarily due to the physiological and biochemical characteristics of brain barrier sites (i.e., blood-brain barrier (BBB), blood-cerebrospinal fluid barrier (BCSFB)). Drug uptake into brain tissue is highly restricted by expression of tight junction protein complexes and adherens junctions between brain microvascular endothelial cells and choroid plexus epithelial cells. Additionally, efflux transport proteins expressed at the plasma membrane of these same endothelial and epithelial cells act to limit CNS concentrations of centrally acting drugs. In contrast, facilitated diffusion via transporter proteins allows for substrate-specific flux of molecules across the plasma membrane, directing drug uptake into the CNS. Organic Cation Transporters (OCTs) and Novel Organic Cation Transporters (OCTNs) are two subfamilies of the solute carrier 22 (SLC22) family of proteins that have significant potential to mediate delivery of positively charged, zwitterionic, and uncharged therapeutics. While expression of these transporters has been well characterized in peripheral tissues, the functional expression of OCT and OCTN transporters at CNS barrier sites and their role in delivery of therapeutic drugs to molecular targets in the brain require more detailed analysis. In this chapter, we will review current knowledge on localization, function, and regulation of OCT and OCTN isoforms at the BBB and BCSFB with a particular emphasis on how these transporters can be utilized for CNS delivery of therapeutic agents.
• Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2021).

  Organic Cation Transporter (OCT/OCTN) Expression at Brain Barrier Sites: Focus on CNS Drug Delivery.

  . Handbook of experimental pharmacology. doi:10.1007/164_2021_448
  More info

  Therapeutic delivery to the central nervous system (CNS) continues to be a considerable challenge in the pharmacological treatment and management of neurological disorders. This is primarily due to the physiological and biochemical characteristics of brain barrier sites (i.e., blood-brain barrier (BBB), blood-cerebrospinal fluid barrier (BCSFB)). Drug uptake into brain tissue is highly restricted by expression of tight junction protein complexes and adherens junctions between brain microvascular endothelial cells and choroid plexus epithelial cells. Additionally, efflux transport proteins expressed at the plasma membrane of these same endothelial and epithelial cells act to limit CNS concentrations of centrally acting drugs. In contrast, facilitated diffusion via transporter proteins allows for substrate-specific flux of molecules across the plasma membrane, directing drug uptake into the CNS. Organic Cation Transporters (OCTs) and Novel Organic Cation Transporters (OCTNs) are two subfamilies of the solute carrier 22 (SLC22) family of proteins that have significant potential to mediate delivery of positively charged, zwitterionic, and uncharged therapeutics. While expression of these transporters has been well characterized in peripheral tissues, the functional expression of OCT and OCTN transporters at CNS barrier sites and their role in delivery of therapeutic drugs to molecular targets in the brain require more detailed analysis. In this chapter, we will review current knowledge on localization, function, and regulation of OCT and OCTN isoforms at the BBB and BCSFB with a particular emphasis on how these transporters can be utilized for CNS delivery of therapeutic agents.
• Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2021). Organic Cation Transporter (OCT/OCTN) Expression at Brain Barrier Sites: Focus on CNS Drug Delivery. Handbook of experimental pharmacology, 266, 301-328.
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  Therapeutic delivery to the central nervous system (CNS) continues to be a considerable challenge in the pharmacological treatment and management of neurological disorders. This is primarily due to the physiological and biochemical characteristics of brain barrier sites (i.e., blood-brain barrier (BBB), blood-cerebrospinal fluid barrier (BCSFB)). Drug uptake into brain tissue is highly restricted by expression of tight junction protein complexes and adherens junctions between brain microvascular endothelial cells and choroid plexus epithelial cells. Additionally, efflux transport proteins expressed at the plasma membrane of these same endothelial and epithelial cells act to limit CNS concentrations of centrally acting drugs. In contrast, facilitated diffusion via transporter proteins allows for substrate-specific flux of molecules across the plasma membrane, directing drug uptake into the CNS. Organic Cation Transporters (OCTs) and Novel Organic Cation Transporters (OCTNs) are two subfamilies of the solute carrier 22 (SLC22) family of proteins that have significant potential to mediate delivery of positively charged, zwitterionic, and uncharged therapeutics. While expression of these transporters has been well characterized in peripheral tissues, the functional expression of OCT and OCTN transporters at CNS barrier sites and their role in delivery of therapeutic drugs to molecular targets in the brain require more detailed analysis. In this chapter, we will review current knowledge on localization, function, and regulation of OCT and OCTN isoforms at the BBB and BCSFB with a particular emphasis on how these transporters can be utilized for CNS delivery of therapeutic agents.
• Davis, T. P. (2019). A multi-center, Phase 2 study using a continual reassessment method to determine the safety and tolerability of 3K3A-APC, a Recombinant Variant of Human Activated Protein C, in combination with tissue plasminogen activator, mechanical thrombectomy or both in moderate to severe acute ischemic stroke.. Annals of Neurology, 85, 125-136..
• Davis, T. P. (2019). Distribution of insulin in trigeminal nerve and brain after intranasal administration.. Scientific Reports, 9(1), 2621-2622.
• Davis, T. P. (2019). Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake.. PLOS ONE.
• Davis, T. P. (2019). Perivascular and perineural pathways involved in brain delivery and distribution of drugs after intranasal administration.. Pharmaceutics, 12.
• Davis, T. P. (2019). Vascular Dysfunction -The disregarded partner of Alzheimers Disease.. Alzheimers and Dementia, 15(1), 158-167.
• Davis, T. P., & Ronaldson, P. (2020). Regulation of blood brain barrier integrity by microglia in health and disease. Journal of Cerebral Blood Flow and Metabolism.
• Davis, T. P., Lyden, P., Zlokovic, B., & Griffin, J. (2020). Stroke treatment with PAR1 agents that minimize recanalization related hemorrhage.. Frontiers in Neurology.
• Davis, T. P., Ranaldson, P. T., Williams, E. I., & Batterten, R. D. (2019). Transporter-Mediated Delivery of Small Molecule Drugs to the Brain: A Critical Mechanism that can advance Therapeutic Development for Ischemic Stroke.. Pharmaceutics, 12(11), 675-695.
• Davis, T. P., Ronaldson, P., Reilly, B., & Abdullah, W. (2020). Transport properties of statins by OATP1A2 and regulation by transforming growth factor Beta signaling in human endothelial cells.. J. Pharmacology Experimental Therapeutics.
• Lyden, P. D., Pryor, K. E., Minigh, J., Davis, T. P., Griffin, J. H., Levy, H., & Zlokovic, B. V. (2021). Stroke Treatment With PAR-1 Agents to Decrease Hemorrhagic Transformation. Frontiers in neurology, 12, 593582.
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  Ischemic stroke is the most widespread cause of disability and a leading cause of death in developed countries. To date, the most potent approved treatment for acute stroke is recanalization therapy with thrombolytic drugs such as tissue plasminogen activator (rt-PA or tPA) or endovascular mechanical thrombectomy. Although tPA and thrombectomy are widely available in the United States, it is currently estimated that only 10-20% of stroke patients get tPA treatment, in part due to restrictive selection criteria. Recently, however, tPA and thrombectomy selection criteria have loosened, potentially allowing more patients to qualify. The relatively low rate of treatment may also reflect the perceived risk of brain hemorrhage following treatment with tPA. In translational research and a single patient study, protease activated receptor 1 (PAR-1) targeted therapies given along with thrombolysis and thrombectomy appear to reduce hemorrhagic transformation after recanalization. Such adjuncts may likely enhance the availability of recanalization and encourage more physicians to use the recently expanded selection criteria for applying recanalization therapies. This narrative review discusses stroke therapies, the role of hemorrhagic transformation in producing poor outcomes, and presents the data suggesting that PAR-1 acting agents show promise for decreasing hemorrhagic transformation and improving outcomes.
• Ronaldson, P. T., Brzica, H., Abdullahi, W., Reilly, B. G., & Davis, T. P. (2021). Transport Properties of Statins by Organic Anion Transporting Polypeptide 1A2 and Regulation by Transforming Growth Factor- Signaling in Human Endothelial Cells. The Journal of pharmacology and experimental therapeutics, 376(2), 148-160.
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  Our in vivo rodent studies have shown that organic anion transporting polypeptide (Oatp) 1a4 is critical for blood-to-brain transport of statins, drugs that are effective neuroprotectants. Additionally, transforming growth factor- (TGF-) signaling via the activin receptor-like kinase 1 (ALK1) receptor regulates Oatp1a4 functional expression. The human ortholog of Oatp1a4 is OATP1A2. Therefore, the translational significance of our work requires demonstration that OATP1A2 can transport statins and is regulated by TGF-/ALK1 signaling. Cellular uptake and monolayer permeability of atorvastatin, pravastatin, and rosuvastatin were investigated in vitro using human umbilical vein endothelial cells (HUVECs). Regulation of OATP1A2 by the TGF-/ALK1 pathway was evaluated using bone morphogenetic protein 9 (BMP-9), a selective ALK1 agonist, and LDN193189, an ALK1 antagonist. We showed that statin accumulation in HUVECs requires OATP1A2-mediated uptake but is also affected by efflux transporters (i.e., P-glycoprotein, breast cancer resistance protein). 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-/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 blood-brain barrier. SIGNIFICANCE STATEMENT: Transporter data derived from rodent models requires validation in human models. Using human umbilical vein endothelial cells, this study has shown that statin transport is mediated by OATP1A2. Additionally, we demonstrated that OATP1A2 is regulated by transforming growth factor-/activin receptor-like kinase 1 signaling. This work emphasizes the need to consider endothelial transporter kinetics and regulation during preclinical drug development. Furthermore, our forward-thinking approach can identify effective therapeutics for diseases for which drug development has been challenging (i.e., neurological diseases).
• Blawn, K. T., Davis, T. P., Kellohen, K. L., Largent-milnes, T. M., Liktor-busa, E., Palomino, S. M., Vanderah, T. W., Verkhovsky, V., Vivek, A., Wahl, J., & Wiese, B. M. (2020).

  Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake.

  . PloS one, 15(5), e0227463. doi:10.1371/journal.pone.0227463
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  Disruption of blood-brain barrier integrity and dramatic failure of brain ion homeostasis including fluctuations of pH occurs during cortical spreading depression (CSD) events associated with several neurological disorders, including migraine with aura, traumatic brain injury and stroke. NHE1 is the primary regulator of pH in the central nervous system. The goal of the current study was to investigate the role of sodium-hydrogen exchanger type 1 (NHE1) in blood brain barrier (BBB) integrity during CSD events and the contributions of this antiporter on xenobiotic uptake. Using immortalized cell lines, pharmacologic inhibition and genetic knockdown of NHE1 mitigated the paracellular uptake of radiolabeled sucrose implicating functional NHE1 in BBB maintenance. In contrast, loss of functional NHE1 in endothelial cells facilitated uptake of the anti-migraine therapeutic, sumatriptan. In female rats, cortical KCl but not aCSF selectively reduced total expression of NHE1 in cortex and PAG but increased expression in trigeminal ganglia; no changes were seen in trigeminal nucleus caudalis. Thus, in vitro observations may have a significance in vivo to increase brain sumatriptan levels. Pharmacological inhibition of NHE1 prior to cortical manipulations enhanced the efficacy of sumatriptan at early time-points but induced facial sensitivity alone. Overall, our results suggest that dysregulation of NHE1 contributes to breaches in BBB integrity, drug penetrance, and the behavioral sensitivity to the antimigraine agent, sumatriptan.
• Davis, T. P., & Lochhead, J. (2020). Structure, function and regulation of the blood brain barrier tight junction in CNS disorders.. Frontiers in Physiology, 11(914), 1-17.
• Davis, T. P., & Mukherjee, P. (2020). 3K3A - activated protein C variant does not interfere with the plasma clot lysis activity of tenecteplace.. Stroke, 51(1), 2236 - 2239..
• Davis, T. P., Williams, E., & Ronaldson, P. (2020). Transporter - mediated delivery of small molecule drugs to the brain: A critical mechanism that can advance therapeutic development for stroke. Pharmaceutics, 12(2), 154-172.
• Liktor-Busa, E., Blawn, K. T., Kellohen, K. L., Wiese, B. M., Verkhovsky, V., Wahl, J., Vivek, A., Palomino, S. M., Davis, T. P., Vanderah, T. W., & Largent-Milnes, T. M. (2020). Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake. PloS one, 15(5), e0227463.
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  Disruption of blood-brain barrier integrity and dramatic failure of brain ion homeostasis including fluctuations of pH occurs during cortical spreading depression (CSD) events associated with several neurological disorders, including migraine with aura, traumatic brain injury and stroke. NHE1 is the primary regulator of pH in the central nervous system. The goal of the current study was to investigate the role of sodium-hydrogen exchanger type 1 (NHE1) in blood brain barrier (BBB) integrity during CSD events and the contributions of this antiporter on xenobiotic uptake. Using immortalized cell lines, pharmacologic inhibition and genetic knockdown of NHE1 mitigated the paracellular uptake of radiolabeled sucrose implicating functional NHE1 in BBB maintenance. In contrast, loss of functional NHE1 in endothelial cells facilitated uptake of the anti-migraine therapeutic, sumatriptan. In female rats, cortical KCl but not aCSF selectively reduced total expression of NHE1 in cortex and PAG but increased expression in trigeminal ganglia; no changes were seen in trigeminal nucleus caudalis. Thus, in vitro observations may have a significance in vivo to increase brain sumatriptan levels. Pharmacological inhibition of NHE1 prior to cortical manipulations enhanced the efficacy of sumatriptan at early time-points but induced facial sensitivity alone. Overall, our results suggest that dysregulation of NHE1 contributes to breaches in BBB integrity, drug penetrance, and the behavioral sensitivity to the antimigraine agent, sumatriptan.
• 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.
• Mukherjee, P., Lyden, P., Fernández, J. A., Davis, T. P., Pryor, K. E., Zlokovic, B. V., & Griffin, J. H. (2020). 3K3A-Activated Protein C Variant Does Not Interfere With the Plasma Clot Lysis Activity of Tenecteplase. Stroke, 51(7), 2236-2239.
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  A recombinant engineered variant of APC (activated protein C), 3K3A-APC, lacks anticoagulant properties (
• 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 : official journal of the International Society of Cerebral Blood Flow and Metabolism, 40(1_suppl), S6-S24.
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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.
• Williams, E. I., Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2020). Transporter-Mediated Delivery of Small Molecule Drugs to the Brain: A Critical Mechanism That Can Advance Therapeutic Development for Ischemic Stroke. Pharmaceutics, 12(2).
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  Ischemic stroke is the 5th leading cause of death in the United States. Despite significant improvements in reperfusion therapies, stroke patients still suffer from debilitating neurocognitive deficits. This indicates an essential need to develop novel stroke treatment paradigms. Endogenous uptake transporters expressed at the blood-brain barrier (BBB) provide an excellent opportunity to advance stroke therapy via optimization of small molecule neuroprotective drug delivery to the brain. Examples of such uptake transporters include organic anion transporting polypeptides (OATPs in humans; Oatps in rodents) and organic cation transporters (OCTs in humans; Octs in rodents). Of particular note, small molecule drugs that have neuroprotective properties are known substrates for these transporters and include 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins) for OATPs/Oatps and 1-amino-3,5-dimethyladamantane (i.e., memantine) for OCTs/Octs. Here, we review current knowledge on specific BBB transporters that can be targeted for improvement of ischemic stroke treatment and provide state-of-the-art perspectives on the rationale for considering BBB transport properties during discovery/development of stroke therapeutics.
• Davis, T. P., Abdullahi, W., Lochhead, J. J., & Ronaldson, P. T. (2019). Organic anion transporting polypeptide (OATP)-mediated transport at the blood-brain barrier is required for atorvastatin-induced neuroprotection in experimental ischemic stroke. JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM, 39, 59-59.
• 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.
• Cottier, K. E., Galloway, E. A., Calabrese, E. C., Tome, M. E., Liktor-Busa, E., Kim, J., Davis, T. P., Vanderah, T. W., & Largent-Milnes, T. M. (2018). Loss of Blood-Brain Barrier Integrity in a KCl-Induced Model of Episodic Headache Enhances CNS Drug Delivery. eNeuro, 5(4).
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  Cortical spreading depression (CSD) in the CNS is suggested as a common mechanism contributing to headache. Despite strong evidence for CNS involvement in headache disorders, drug development for headache disorders remains focused on peripheral targets. Difficulty in delivering drugs across the blood-brain barrier (BBB) may partially account for this disparity. It is known, however, that BBB permeability is increased during several CNS pathologies. In this study, we investigated BBB changes in response to KCl-induced CSD events and subsequent allodynia in rats. Cortical KCl injection in awake, freely moving rats produced facial allodynia with peak intensity between 1.5 and 3 h and CSD induction within 0.5-2 h postinjection. Brain perfusion of C-sucrose as a marker of BBB paracellular permeability revealed increased leak in the cortex, but not brainstem, beginning 0.5 h post-KCl injection and resolving within 6 h; no changes in tight junction (TJ) proteins occludin or claudin-5 expression were observed. Acute pretreatment with topiramate to inhibit CSD did not prevent the increased BBB paracellular permeability. CNS delivery of the abortive anti-migraine agent sumatriptan was increased in the cortex 1.5 h post-KCl injection. Surprisingly, sumatriptan uptake was also increased in the brainstem following CSD induction, suggesting regulation of active transport mechanisms at the BBB. Together, these results demonstrate the ability of CSD events to produce transient, time-dependent changes in BBB permeability when allodynia is present and to mediate access of clinically relevant therapeutics (i.e., sumatriptan) to the CNS.
• Davis, T. P. (2018). Loss of Blood-Brain Barrier Integrity in a KCl induced model of Episodic Headache enhances CNS drug delivery.. eNEuro, 0116-18.
• Davis, T. P., Time, M., & Schaefer, C. (2018). Acute pain alters P-glycoprotein-containing protein complexes in rat cerebral complexes: Implications for P-glycoprotein trafficking.. J. Cerebral Blood Flow and Metabolism, 38, 2209-2222.
• Lyden, P., Pryor, K. E., Coffey, C. S., Cudkowicz, M., Conwit, R., Jadhav, A., Sawyer, R. N., Claassen, J., Adeoye, O., Song, S., Hannon, P., Rost, N. S., Hinduja, A., Torbey, M., Lee, J. M., Benesch, C., Rippee, M., Rymer, M., Froehler, M. T., , Haley, E. C., et al. (2018). Final Results of the RHAPSODY trial: A multi-center, Phase 2 trial using a continual reassessment method to determine the safety and tolerability of 3K3A-APC, a Recombinant Variant of Human Activated Protein C, in combination with tissue plasminogen activator, mechanical thrombectomy or both in moderate to severe acute ischemic stroke. Annals of neurology.
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  Agonism of the protease activated receptor (PAR) 1 by activated protein C (APC) provides neuroprotection and vasculoprotection in experimental neuro-injury models. The pleiotropic PAR1 agonist, 3K3A-APC, reduces neurologic injury and promotes vascular integrity; 3K3A-APC proved safe in human volunteers. We performed a randomized, controlled, blinded, trial to determine the maximally tolerated dose (MTD) of 3K3A-APC in ischemic stroke patients.
• Schaefer, C. P., Arkwright, N. B., Jacobs, L. M., Jarvis, C. K., Hunn, K. C., Largent-Milnes, T. M., Tome, M. E., & Davis, T. P. (2018). Chronic morphine exposure potentiates p-glycoprotein trafficking from nuclear reservoirs in cortical rat brain microvessels. PloS one, 13(2), e0192340.
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  The rates of opioid prescription and use have continued to increase over the last few decades resulting in a greater number of opioid tolerant patients. Treatment of acute pain from surgery and injury is a clinical challenge for these patients. Several pain management strategies including prescribing increased opioids are used clinically with limited success; all currently available strategies have significant limitations. Many opioids are a substrate for p-glycoprotein (p-gp), an efflux transporter at the blood-brain barrier (BBB). Increased p-gp is associated with a decreased central nervous system uptake and analgesic efficacy of morphine. Our laboratory previously found that acute peripheral inflammatory pain (PIP) induces p-gp trafficking from the nucleus to the luminal surface of endothelial cells making up the BBB concomitant with increased p-gp activity and decreased morphine analgesic efficacy. In the current study, we tested whether PIP-induced p-gp trafficking could contribute to decreased opioid efficacy in morphine tolerant rats. A 6-day continuous dosing of morphine from osmotic minipumps was used to establish morphine tolerance in female rats. PIP induced p-gp trafficking away from nuclear stores showed a 2-fold increase in morphine tolerant rats. This observation suggests that p-gp trafficking contributes to the decreased morphine analgesic effects in morphine tolerant rats experiencing an acute pain stimulus. Attenuating p-gp trafficking during an acute pain stimulus could improve pain management by increasing the amount of opioid that could reach CNS analgesic targets and decrease the need for the dose escalation that is a serious challenge in pain management.
• Tome, M. E., Jarvis, C. K., Schaefer, C. P., Jacobs, L. M., Herndon, J. M., Hunn, K. C., Arkwright, N. B., Kellohen, K. L., Mierau, P. C., & Davis, T. P. (2018). Acute pain alters P-glycoprotein-containing protein complexes in rat cerebral microvessels: Implications for P-glycoprotein trafficking. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 38(12), 2209-2222.
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  P-glycoprotein (PgP) is the major drug efflux pump in human cerebral microvessels. PgP prevents pathogens, toxins and therapeutic drugs from entering the CNS. Understanding the molecular regulation of PgP activity will suggest novel mechanisms to improve CNS drug delivery. Previously, we found that during peripheral inflammatory pain (PIP) (3 h after λ carrageenan injection in the rat paw), PgP traffics to the cortical microvessel endothelial cell plasma membrane concomitant with increased PgP activity. In the current study, we measured the changes in composition of PgP-containing protein complexes after PIP in rat microvessel isolates. We found that a portion of the PgP is contained in a multi-protein complex that also contains the caveolar proteins CAV1, SDPR, PTRF and PRKCDBP. With PIP, total CAV1 bound to PgP was unchanged; however, phosphorylated CAV1 (Y14P-CAV1) in the complex increased. There were few PgP/CAV1 complexes relative to total PgP and CAV1 in the microvessels suggesting CAV1 bound to PgP is unlikely to affect total PgP activity. However, both PgP and CAV1 trafficked away from the nucleus in response to PIP. These data suggest that P-CAV1 bound to PgP potentially regulates PgP trafficking and contributes to the acute PgP activity increase after a PIP stimulus.
• 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).
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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 the functional 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 : official journal of the International Society of Cerebral Blood Flow and Metabolism, 37(7), 2340-2345.
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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 BBB transporter expression and/or activity. 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 ALK1 signaling with BMP-9 modulates BBB Oatp1a4 expression, presenting 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.
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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.
• Cottier, K. E., Davis, T. P., Galoway, E., Kim, J. S., Largent-milnes, T. M., & Vanderah, T. W. (2017).

  (143) Transient Decreases in Blood-Brain Barrier Integrity Correspond with Facial Allodynia Duration in a Model of Migraine with Aura

  . The Journal of Pain, 18(4), S12. doi:10.1016/j.jpain.2017.02.049
• Davis, T. P., Herndon, J. M., & Tome, M. E. (2017).

  Development and Maintenance of the Blood–Brain Barrier

  . Primer on Cerebrovascular Diseases, 51-56. doi:10.1016/b978-0-12-803058-5.00009-6
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  Abstract The blood–brain barrier (BBB) is a specialized vascular structure that serves to restrict the passage of most molecules from the systemic circulation into the CNS. A functional BBB is present shortly after vascularization of the developing embryonic brain and an intact barrier is crucial for proper neuronal function. This barrier is regulated and maintained via inputs from a number of different cell types, including endothelial cells, pericytes, astrocytes, neurons, and microglia, that are collectively known as the neurovascular unit. Disruption or opening of the BBB often proves deleterious and exacerbates CNS disorders such as Alzheimer disease, epilepsy, and stroke. The BBB is critical for CNS homeostasis, but it also presents a significant obstacle for drug delivery to the CNS.
• Davis, T. P., Schaefer, C., & Tome, M. (2017). The opioid epidemic: A central role for the Blood Brain Barrier in opioid analgesia and abuse.. Fluids and Barriers of the CNS, 14(32), 1-11. doi:10.1186/s12987-017-0080-3
• 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.
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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.
• Sandweiss, A. J., Cottier, K. E., McIntosh, M. I., Dussor, G., Davis, T. P., Vanderah, T. W., & Largent-Milnes, T. M. (2017). 17-β-Estradiol induces spreading depression and pain behavior in alert female rats. Oncotarget, 8(69), 114109-114122.
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  Test the putative contribution of 17-β-estradiol in the development of spreading depression (SD) events and head pain in awake, non-restrained rats.
• Schaefer, C. P., Tome, M. E., & Davis, T. P. (2017). The opioid epidemic: a central role for the blood brain barrier in opioid analgesia and abuse. Fluids and barriers of the CNS, 14(1), 32.
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  Opioids are currently the primary treatment method used to manage both acute and chronic pain. In the past two to three decades, there has been a surge in the use, abuse and misuse of opioids. The mechanism by which opioids relieve pain and induce euphoria is dependent on the drug crossing the blood-brain barrier and accessing the central nervous system. This suggests the blood brain barrier plays a central role in both the benefits and risks of opioid use. The complex physiological responses to opioids that provide the benefits and drive the abuse also needs to be considered in the resolution of the opioid epidemic.
• Bosetti, F., Galis, Z. S., Bynoe, M. S., Charette, M., Cipolla, M. J., Del Zoppo, G. J., Gould, D., Hatsukami, T. S., Jones, T. L., Koenig, J. I., Lutty, G. A., Maric-Bilkan, C., Stevens, T., Tolunay, H. E., Koroshetz, W., & , “. B. (2016). "Small Blood Vessels: Big Health Problems?": Scientific Recommendations of the National Institutes of Health Workshop. Journal of the American Heart Association, 5(11).
• Tome, M. E., Herndon, J. M., Schaefer, C. P., Jacobs, L. M., Zhang, Y., Jarvis, C. K., & Davis, T. P. (2016). P-glycoprotein traffics from the nucleus to the plasma membrane in rat brain endothelium during inflammatory pain. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 36(11), 1913-1928.
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  P-glycoprotein (PgP), a drug efflux pump in blood-brain barrier endothelial cells, is a major clinical obstacle for effective central nervous system drug delivery. Identifying PgP regulatory pathways that can be exploited clinically is critical for improving central nervous system drug delivery. We previously found that PgP activity increases in rat brain microvessels concomitant with decreased central nervous system drug delivery in response to acute peripheral inflammatory pain. In the current study, we tested the hypothesis that PgP traffics to the luminal plasma membrane of the microvessel endothelial cells from intracellular stores during peripheral inflammatory pain. Using immunofluorescence microscopy, we detected PgP in endothelial cell nuclei and in the luminal plasma membrane in control animals. Following peripheral inflammatory pain, luminal PgP staining increased while staining in the nucleus decreased. Biochemical analysis of nuclear PgP content confirmed our visual observations. Peripheral inflammatory pain also increased endothelial cell luminal staining of polymerase 1 and transcript release factor/cavin1 and serum deprivation response protein/cavin2, two caveolar scaffold proteins, without changing caveolin1 or protein kinase C delta binding protein/cavin3 location. Our data (a) indicate that PgP traffics from stores in the nucleus to the endothelial cell luminal membrane in response to peripheral inflammatory pain; (b) provide an explanation for our previous observation that peripheral inflammatory pain inhibits central nervous system drug uptake; and (c) suggest a novel regulatory mechanism for PgP activity in rat brain.
• Anandasabapathy, N., Breton, G., Buckley, N., Caskey, M., Davis, T. P., Green, J., Hawthorne, T., Hurley, A., Keler, T., Lyden, D., Marsh, H. C., Matei, I., Pack, M., Pring, J., Sarma, P., Schlesinger, S. J., Trumpfheller, C., & Yellin, M. (2015).

  Efficacy and safety of CDX-301, recombinant human Flt3L, at expanding dendritic cells and hematopoietic stem cells in healthy human volunteers.

  . Bone marrow transplantation, 50(7), 924-30. doi:10.1038/bmt.2015.74
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  Fms-like tyrosine kinase-3 ligand (Flt3L) uniquely binds the Flt3 (CD135) receptor expressed on hematopoietic stem cells (HSCs), early progenitor cells, immature thymocytes and steady-state dendritic cells (DCs) and induces their proliferation, differentiation, development and mobilization in the bone marrow, peripheral blood and lymphoid organs. CDX-301 has an identical amino-acid sequence and comparable biological activity to the previously tested rhuFlt3L, which ceased clinical development over a decade ago. This Phase 1 trial assessed the safety, pharmacokinetic, pharmacodynamic and immunologic profile of CDX-301, explored alternate dosing regimens and examined the impact of rhuFlt3L on key immune cell subsets. Thirty healthy volunteers received CDX-301 (1-75 μg/kg/day) over 5-10 days. One event of Grade 3 community-acquired pneumonia occurred. There were no other infections, dose-limiting toxicities or serious adverse events. CDX-301 resulted in effective peripheral expansion of monocytes, hematopoietic stem and progenitor cells and key subsets of myeloid DCs and plasmacytoid DCs, with no clear effect on regulatory T cells. These data from healthy volunteers support the potential for CDX-301, as monotherapy or in combination with other agents, in various indications including allogeneic HSC transplantation and immunotherapy, but the effects of CDX-301 will need to be investigated in each of these patient populations.
• Davis, T. P., Abbruscato, T. J., & Egleton, R. D. (2015). Peptides at the blood brain barrier: Knowing me knowing you. Peptides, 72, 50-6.
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  When the Davis Lab was first asked to contribute to this special edition of Peptides to celebrate the career and influence of Abba Kastin on peptide research, it felt like a daunting task. It is difficult to really understand and appreciate the influence that Abba has had, not only on a generation of peptide researchers, but also on the field of blood brain barrier (BBB) research, unless you lived it as we did. When we look back at our careers and those of our former students, one can truly see that several of Abba's papers played an influential role in the development of our personal research programs.
• Ronaldson, P. T., & Davis, T. P. (2015). Targeting transporters: promoting blood-brain barrier repair in response to oxidative stress injury. Brain research, 1623, 39-52.
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  The blood-brain barrier (BBB) is a physical and biochemical barrier that precisely regulates the ability of endogenous and exogenous substances to accumulate within brain tissue. It possesses structural and biochemical features (i.e., tight junction and adherens junction protein complexes, influx and efflux transporters) that work in concert to control solute permeation. Oxidative stress, a critical component of several diseases including cerebral hypoxia/ischemia and peripheral inflammatory pain, can cause considerable injury to the BBB and lead to significant CNS pathology. This suggests a critical need for novel therapeutic approaches that can protect the BBB in diseases with an oxidative stress component. Recent studies have identified molecular targets (i.e., putative membrane transporters, intracellular signaling systems) that can be exploited for optimization of endothelial drug delivery or for control of transport of endogenous substrates such as the antioxidant glutathione (GSH). In particular, targeting transporters offers a unique approach to protect BBB integrity by promoting repair of cell-cell interactions at the level of the brain microvascular endothelium. This review summarizes current knowledge in this area and emphasizes those targets that present considerable opportunity for providing BBB protection and/or promoting BBB repair in the setting of oxidative stress. This article is part of a Special Issue entitled SI: Cell Interactions In Stroke.
• Tome, M. E., Schaefer, C. P., Jacobs, L. M., Zhang, Y., Herndon, J. M., Matty, F. O., & Davis, T. P. (2015). Identification of P-glycoprotein co-fractionating proteins and specific binding partners in rat brain microvessels. Journal of neurochemistry, 134(2), 200-10.
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  Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P-glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood-brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP-containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post-translationally regulated at the BBB. The goal of the current study was to identify proteins that co-localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co-localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co-fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post-translational regulation of PgP activity at the BBB.
• Bhardwaj, N., Brody, J., Crowley, E., Davis, T. P., Keler, T., Kim-schulze, S., & Merad, M. (2014).

  Flt3L-primed in situ vaccine for patients with low-grade lymphoma: tumor regression at untreated sites (NCT01976585)

  . F1000Research, 5. doi:10.7490/f1000research.1097381.1
• Davis, T. P., Demarco, K. M., Laracuente, M. L., Ronaldson, P. T., Sanchez-covarrubias, L., Slosky, L. M., Thompson, B. J., & Zhang, Y. (2014).

  P-glycoprotein modulates morphine uptake into the CNS: a role for the non-steroidal anti-inflammatory drug diclofenac.

  . PloS one, 9(2), e88516. doi:10.1371/journal.pone.0088516
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  Our laboratory has previously demonstrated that peripheral inflammatory pain (PIP), induced by subcutaneous plantar injection of λ-carrageenan, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp) that is endogenously expressed at the blood-brain barrier (BBB). The result of increased P-gp functional expression was a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. A major concern in the treatment of acute pain/inflammation is the potential for drug-drug interactions resulting from P-gp induction by therapeutic agents co-administered with opioids. Such effects on P-gp activity can profoundly modulate CNS distribution of opioid analgesics and alter analgesic efficacy. In this study, we examined the ability of diclofenac, a non-steroidal anti-inflammatory drug (NSAID) that is commonly administered in conjunction with the opioids during pain therapy, to alter BBB transport of morphine via P-gp and whether such changes in P-gp morphine transport could alter morphine analgesic efficacy. Administration of diclofenac reduced paw edema and thermal hyperalgesia in rats subjected to PIP, which is consistent with the known mechanism of action of this NSAID. Western blot analysis demonstrated an increase in P-gp expression in rat brain microvessels not only following PIP induction but also after diclofenac treatment alone. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased morphine brain uptake. Critically, morphine analgesia was significantly reduced in animals pretreated with diclofenac (3 h), as compared to animals administered diclofenac and morphine concurrently. These novel findings suggest that administration of diclofenac and P-gp substrate opioids during pain pharmacotherapy may result in a clinically significant drug-drug interaction.
• Davis, T. P., Sanchez-Covarubias, L., & Tome, M. E. (2014). P-glycoprotein trafficking as a therapeutic target to optimize CNS drug delivery. Advances in pharmacology (San Diego, Calif.), 71, 25-44.
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  The primary function of the blood-brain barrier (BBB)/neurovascular unit is to protect the central nervous system (CNS) from potentially harmful xenobiotic substances and maintain CNS homeostasis. Restricted access to the CNS is maintained via a combination of tight junction proteins as well as a variety of efflux and influx transporters that limits the transcellular and paracellular movement of solutes. Of the transporters identified at the BBB, P-glycoprotein (P-gp) has emerged as the transporter that is the greatest obstacle to effective CNS drug delivery. In this chapter, we provide data to support intracellular protein trafficking of P-gp within cerebral capillary microvessels as a potential target for improved drug delivery. We show that pain-induced changes in P-gp trafficking are associated with changes in P-gp's association with caveolin-1, a key scaffolding/trafficking protein that colocalizes with P-gp at the luminal membrane of brain microvessels. Changes in colocalization with the phosphorylated and nonphosphorylated forms of caveolin-1, by pain, are accompanied by dynamic changes in the distribution, relocalization, and activation of P-gp "pools" between microvascular endothelial cell subcellular compartments. Since redox-sensitive processes may be involved in signaling disassembly of higher-order structures of P-gp, we feel that manipulating redox signaling, via specific protein targeting at the BBB, may protect disulfide bond integrity of P-gp reservoirs and control trafficking to the membrane surface, providing improved CNS drug delivery. The advantage of therapeutic drug "relocalization" of a protein is that the physiological impact can be modified, temporarily or long term, despite pathology-induced changes in gene transcription.
• Davis, T. P., Sanchez-covarubias, L., & Tome, M. E. (2014).

  P-glycoprotein trafficking as a therapeutic target to optimize CNS drug delivery.

  . Advances in pharmacology (San Diego, Calif.), 71, 25-44. doi:10.1016/bs.apha.2014.06.009
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  The primary function of the blood-brain barrier (BBB)/neurovascular unit is to protect the central nervous system (CNS) from potentially harmful xenobiotic substances and maintain CNS homeostasis. Restricted access to the CNS is maintained via a combination of tight junction proteins as well as a variety of efflux and influx transporters that limits the transcellular and paracellular movement of solutes. Of the transporters identified at the BBB, P-glycoprotein (P-gp) has emerged as the transporter that is the greatest obstacle to effective CNS drug delivery. In this chapter, we provide data to support intracellular protein trafficking of P-gp within cerebral capillary microvessels as a potential target for improved drug delivery. We show that pain-induced changes in P-gp trafficking are associated with changes in P-gp's association with caveolin-1, a key scaffolding/trafficking protein that colocalizes with P-gp at the luminal membrane of brain microvessels. Changes in colocalization with the phosphorylated and nonphosphorylated forms of caveolin-1, by pain, are accompanied by dynamic changes in the distribution, relocalization, and activation of P-gp "pools" between microvascular endothelial cell subcellular compartments. Since redox-sensitive processes may be involved in signaling disassembly of higher-order structures of P-gp, we feel that manipulating redox signaling, via specific protein targeting at the BBB, may protect disulfide bond integrity of P-gp reservoirs and control trafficking to the membrane surface, providing improved CNS drug delivery. The advantage of therapeutic drug "relocalization" of a protein is that the physiological impact can be modified, temporarily or long term, despite pathology-induced changes in gene transcription.
• Sanchez-Covarrubias, L., Slosky, L. M., Thompson, B. J., Davis, T. P., & Ronaldson, P. T. (2014). Transporters at CNS barrier sites: obstacles or opportunities for drug delivery?. Current pharmaceutical design, 20(10), 1422-49.
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  The blood-brain barrier (BBB) and blood-cerebrospinal fluid (BCSF) barriers are critical determinants of CNS homeostasis. Additionally, the BBB and BCSF barriers are formidable obstacles to effective CNS drug delivery. These brain barrier sites express putative influx and efflux transporters that precisely control permeation of circulating solutes including drugs. The study of transporters has enabled a shift away from "brute force" approaches to delivering drugs by physically circumventing brain barriers towards chemical approaches that can target specific compounds of the BBB and/or BCSF barrier. However, our understanding of transporters at the BBB and BCSF barriers has primarily focused on understanding efflux transporters that efficiently prevent drugs from attaining therapeutic concentrations in the CNS. Recently, through the characterization of multiple endogenously expressed uptake transporters, this paradigm has shifted to the study of brain transporter targets that can facilitate drug delivery (i.e., influx transporters). Additionally, signaling pathways and trafficking mechanisms have been identified for several endogenous BBB/BCSF transporters, thereby offering even more opportunities to understand how transporters can be exploited for optimization of CNS drug delivery. This review presents an overview of the BBB and BCSF barrier as well as the many families of transporters functionally expressed at these barrier sites. Furthermore, we present an overview of various strategies that have been designed and utilized to deliver therapeutic agents to the brain with a particular emphasis on those approaches that directly target endogenous BBB/BCSF barrier transporters.
• Sanchez-Covarrubias, L., Slosky, L. M., Thompson, B. J., Zhang, Y., Laracuente, M. L., DeMarco, K. M., Ronaldson, P. T., & Davis, T. P. (2014). P-glycoprotein modulates morphine uptake into the CNS: a role for the non-steroidal anti-inflammatory drug diclofenac. PloS one, 9(2), e88516.
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  Our laboratory has previously demonstrated that peripheral inflammatory pain (PIP), induced by subcutaneous plantar injection of λ-carrageenan, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp) that is endogenously expressed at the blood-brain barrier (BBB). The result of increased P-gp functional expression was a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. A major concern in the treatment of acute pain/inflammation is the potential for drug-drug interactions resulting from P-gp induction by therapeutic agents co-administered with opioids. Such effects on P-gp activity can profoundly modulate CNS distribution of opioid analgesics and alter analgesic efficacy. In this study, we examined the ability of diclofenac, a non-steroidal anti-inflammatory drug (NSAID) that is commonly administered in conjunction with the opioids during pain therapy, to alter BBB transport of morphine via P-gp and whether such changes in P-gp morphine transport could alter morphine analgesic efficacy. Administration of diclofenac reduced paw edema and thermal hyperalgesia in rats subjected to PIP, which is consistent with the known mechanism of action of this NSAID. Western blot analysis demonstrated an increase in P-gp expression in rat brain microvessels not only following PIP induction but also after diclofenac treatment alone. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased morphine brain uptake. Critically, morphine analgesia was significantly reduced in animals pretreated with diclofenac (3 h), as compared to animals administered diclofenac and morphine concurrently. These novel findings suggest that administration of diclofenac and P-gp substrate opioids during pain pharmacotherapy may result in a clinically significant drug-drug interaction.
• Davis, T. P., & Ronaldson, P. T. (2013).

  Gabapentin and diclofenac reduce opioid consumption in patients undergoing tonsillectomy: a result of altered CNS drug delivery?

  . Archives of trauma research, 2(2), 97-8. doi:10.5812/atr.11011
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  Dear Editor, We have read with great interest the article by Mogadam and colleagues on utilization of gabapentin and diclofenac for management of post-operative pain in patients undergoing tonsillectomy (1). Perhaps the most intriguing aspect of this study was the observation that pre-operative administration of gabapentin or diclofenac resulted in reduced post-operative utilization of meperidine, an opioid analgesic. Optimal therapeutic efficacy of opioids requires that they cross the blood-brain barrier (BBB) and attain effective concentrations in the CNS (2). CNS delivery of opioids is determined by putative membrane transporters localized to the BBB endothelium (3, 4). Both pathophysiological factors (i.e., inflammatory signaling in pain) and pharmacological factors (i.e., use of ancillary pain medications) can modulate mechanisms involved in BBB opioid transport, an effect that can cause profound changes in CNS delivery of traditional opioids (i.e., morphine, meperidine). In fact, our research group has demonstrated that painful and/or inflammatory stimuli in the periphery can significantly change transport mechanisms for opioids at the BBB such as the drug efflux transporter P-glycoprotein (P-gp) (5) and the drug influx transporter organic anion transporting polypeptide 1a4 (6). Of particular note, we have also shown that diclofenac itself can attenuate pain-induced changes in BBB transporter activity (6). Our data suggest that a modulation in post-operative meperidine efficacy may, in part, be the result of altered CNS opioid delivery induced by diclofenac. An additional factor to consider is that some ancillary pain medications may act as chemical inhibitors of the same BBB transporters. In the context of the study by Mogadam and colleagues, this effect may involve the critical BBB efflux transporter P-gp. Specifically, gabapentin is a known P-gp inhibitor (7) while in vitro studies have suggested that meperidine is a P-gp transport substrate (8). Although in vivo studies in mdr1a knockout mice showed no difference in meperidine antinociception (9), a direct analysis of P-gp-mediated transport of meperidine in intact laboratory animals has not been undertaken. Furthermore, this study measured analgesic efficacy using only tail-pinch, a technique that may not have been sensitive enough to detect differences in meperidine analgesia between P-gp-deficient and P-gp-competent mice. Nonetheless, it is highly plausible that gabapentin blocked P-gp-mediated efflux transport at the BBB, an effect that effectively increased CNS meperidine delivery with the clinical manifestation of reduced opioid consumption in the post-operative period. Overall, results obtained from basic science studies of our laboratory and others point towards an explanation of modified post-operative opioid efficacy in terms of altered BBB transport and/or CNS delivery of meperidine induced by ancillary pain medications. Additionally, the paper by Mogadam and colleagues emphasizes the absolute importance of novel translational studies aimed at understanding specific mechanisms involved in CNS opioid delivery, opioid efficacy, and/or drug-opioid interactions.
• Davis, T., Ronaldson, P. T., & Davis, T. P. (2013). Targeted drug delivery to treat pain and cerebral hypoxia. Pharmacological reviews, 65(1).
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  Limited drug penetration is an obstacle that is often encountered in treatment of central nervous system (CNS) diseases including pain and cerebral hypoxia. Over the past several years, biochemical characteristics of the brain (i.e., tight junction protein complexes at brain barrier sites, expression of influx and efflux transporters) have been shown to be directly involved in determining CNS permeation of therapeutic agents; however, the vast majority of these studies have focused on understanding those mechanisms that prevent drugs from entering the CNS. Recently, this paradigm has shifted toward identifying and characterizing brain targets that facilitate CNS drug delivery. Such targets include the organic anion-transporting polypeptides (OATPs in humans; Oatps in rodents), a family of sodium-independent transporters that are endogenously expressed in the brain and are involved in drug uptake. OATP/Oatp substrates include drugs that are efficacious in treatment of pain and/or cerebral hypoxia (i.e., opioid analgesic peptides, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors). This clearly suggests that OATP/Oatp isoforms are viable transporter targets that can be exploited for optimization of drug delivery to the brain and, therefore, improved treatment of CNS diseases. This review summarizes recent knowledge in this area and emphasizes the potential that therapeutic targeting of OATP/Oatp isoforms may have in facilitating CNS drug delivery and distribution. Additionally, information presented in this review will point to novel strategies that can be used for treatment of pain and cerebral hypoxia.
• Davis, T., Slosky, L. M., Thompson, B. J., Sanchez-Covarrubias, L., Zhang, Y., Laracuente, M., Vanderah, T. W., Ronaldson, P. T., & Davis, T. P. (2013). Acetaminophen modulates p-glycoprotein functional expression at the blood-brain barrier by a constitutive androstane receptor-dependent mechanism. Molecular pharmacology, 84(5).
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  Effective pharmacologic treatment of pain with opioids requires that these drugs attain efficacious concentrations in the central nervous system (CNS). A primary determinant of CNS drug permeation is P-glycoprotein (P-gp), an endogenous blood-brain barrier (BBB) efflux transporter that is involved in brain-to-blood transport of opioid analgesics (i.e., morphine). Recently, the nuclear receptor constitutive androstane receptor (CAR) has been identified as a regulator of P-gp functional expression at the BBB. This is critical to pharmacotherapy of pain/inflammation, as patients are often administered acetaminophen (APAP), a CAR-activating ligand, in conjunction with an opioid. Our objective was to investigate, in vivo, the role of CAR in regulation of P-gp at the BBB. Following APAP treatment, P-gp protein expression was increased up to 1.4-1.6-fold in a concentration-dependent manner. Additionally, APAP increased P-gp transport of BODIPY-verapamil in freshly isolated rat brain capillaries. This APAP-induced increase in P-gp expression and activity was attenuated in the presence of CAR pathway inhibitor okadaic acid or transcriptional inhibitor actinomycin D, suggesting P-gp regulation is CAR-dependent. Furthermore, morphine brain accumulation was enhanced by P-gp inhibitors in APAP-treated animals, suggesting P-gp-mediated transport. A warm-water (50°C) tail-flick assay revealed a significant decrease in morphine analgesia in animals treated with morphine 3 or 6 hours after APAP treatment, as compared with animals treated concurrently. Taken together, our data imply that inclusion of APAP in a pain treatment regimen activates CAR at the BBB and increases P-gp functional expression, a clinically significant drug-drug interaction that modulates opioid analgesic efficacy.
• Fernandez, J. A., Mosnier, L. O., Davis, T. P., Zlokovic, B. V., & Griffin, J. H. (2013). Influence of the 3K3A-activated protein C variant on the in vitro fibrinolytic activity of tPA. JOURNAL OF THROMBOSIS AND HAEMOSTASIS, 11, 713-714.
• Guo, H., Zhao, Z., Yang, Q., Wang, M., Bell, R. D., Wang, S., Chow, N., Davis, T. P., Griffin, J. H., Goldman, S. A., & Zlokovic, B. V. (2013). An activated protein C analog stimulates neuronal production by human neural progenitor cells via a PAR1-PAR3-S1PR1-Akt pathway. The Journal of neuroscience : the official journal of the Society for Neuroscience, 33(14), 6181-90.
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  Activated protein C (APC) is a protease with anticoagulant and cell-signaling activities. In the CNS, APC and its analogs with reduced anticoagulant activity but preserved cell signaling activities, such as 3K3A-APC, exert neuroprotective, vasculoprotective, and anti-inflammatory effects. Murine APC promotes subependymal neurogenesis in rodents in vivo after ischemic and traumatic brain injury. Whether human APC can influence neuronal production from resident progenitor cells in humans is unknown. Here we show that 3K3A-APC, but not S360A-APC (an enzymatically inactive analog of APC), stimulates neuronal mitogenesis and differentiation from fetal human neural stem and progenitor cells (NPCs). The effects of 3K3A-APC on proliferation and differentiation were comparable to those obtained with fibroblast growth factor and brain-derived growth factor, respectively. Its promoting effect on neuronal differentiation was accompanied by inhibition of astroglial differentiation. In addition, 3K3A-APC exerted modest anti-apoptotic effects during neuronal production. These effects appeared to be mediated through specific protease activated receptors (PARs) and sphingosine-1-phosphate receptors (S1PRs), in that siRNA-mediated inhibition of PARs 1-4 and S1PRs 1-5 revealed that PAR1, PAR3, and S1PR1 are required for the neurogenic effects of 3K3A-APC. 3K3A-APC activated Akt, a downstream target of S1PR1, which was inhibited by S1PR1, PAR1, and PAR3 silencing. Adenoviral transduction of NPCs with a kinase-defective Akt mutant abolished the effects of 3K3A-APC on NPCs, confirming a key role of Akt activation in 3K3A-APC-mediated neurogenesis. Therefore, APC and its pharmacological analogs, by influencing PAR and S1PR signals in resident neural progenitor cells, may be potent modulators of both development and repair in the human CNS.
• Lyden, P., Levy, H., Weymer, S., Pryor, K., Kramer, W., Griffin, J. H., Davis, T. P., & Zlokovic, B. (2013). Phase 1 safety, tolerability and pharmacokinetics of 3K3A-APC in healthy adult volunteers. Current pharmaceutical design, 19(42), 7479-85.
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  Activated Protein C (APC) stimulates multiple cytoprotective pathways via the protease activated receptor-1 (PAR-1) and promotes anticoagulation. 3K3A-APC was designed for preserved activity at PAR-1 with reduced anticoagulation. This Phase 1 trial characterized pharmacokinetics and anticoagulation effects of 3K3A-APC.
• Mosnier, L. O., Fernandez, J. A., Davis, T. P., Zlokovic, B. V., & Griffin, J. H. (2013). Influence of the 3K3A-activated protein C variant on the plasma clot lysis activity of t-PA and of t-PA on the variant's anticoagulant activity. JOURNAL OF THROMBOSIS AND HAEMOSTASIS, 11(11), 2059-2062.
• Ronaldson, P. T., & Davis, T. P. (2013). Gabapentin and diclofenac reduce opioid consumption in patients undergoing tonsillectomy: a result of altered CNS drug delivery?. Archives of trauma research, 2(2), 97-8.
• Davis, T., McCaffrey, G., & Davis, T. P. (2012). Physiology and pathophysiology of the blood-brain barrier: P-glycoprotein and occludin trafficking as therapeutic targets to optimize central nervous system drug delivery. Journal of investigative medicine : the official publication of the American Federation for Clinical Research, 60(8).
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  The blood-brain barrier (BBB) is a physical and metabolic barrier that separates the central nervous system from the peripheral circulation. Central nervous system drug delivery across the BBB is challenging, primarily because of the physical restriction of paracellular diffusion between the endothelial cells that comprise the microvessels of the BBB and the activity of efflux transporters that quickly expel back into the capillary lumen a wide variety of xenobiotics. Therapeutic manipulation of protein trafficking is emerging as a novel means of modulating protein function, and in this minireview, the targeting of the trafficking of 2 key BBB proteins, P-glycoprotein and occludin, is presented as a novel, reversible means of optimizing central nervous system drug delivery.
• Davis, T., McCaffrey, G., Staatz, W. D., Sanchez-Covarrubias, L., Finch, J. D., Demarco, K., Laracuente, M., Ronaldson, P. T., & Davis, T. P. (2012). P-glycoprotein trafficking at the blood-brain barrier altered by peripheral inflammatory hyperalgesia. Journal of neurochemistry, 122(5).
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  P-glycoprotein (ABCB1/MDR1, EC 3.6.3.44), the major efflux transporter at the blood-brain barrier (BBB), is a formidable obstacle to CNS pharmacotherapy. Understanding the mechanism(s) for increased P-glycoprotein activity at the BBB during peripheral inflammatory pain is critical in the development of novel strategies to overcome the significant decreases in CNS analgesic drug delivery. In this study, we employed the λ-carrageenan pain model (using female Sprague-Dawley rats), combined with confocal microscopy and subcellular fractionation of cerebral microvessels, to determine if increased P-glycoprotein function, following the onset of peripheral inflammatory pain, is associated with a change in P-glycoprotein trafficking which leads to pain-induced effects on analgesic drug delivery. Injection of λ-carrageenan into the rat hind paw induced a localized, inflammatory pain (hyperalgesia) and simultaneously, at the BBB, a rapid change in colocalization of P-glycoprotein with caveolin-1, a key scaffolding/trafficking protein. Subcellular fractionation of isolated cerebral microvessels revealed that the bulk of P-glycoprotein constitutively traffics to membrane domains containing high molecular weight, disulfide-bonded P-glycoprotein-containing structures that cofractionate with membrane domains enriched with monomeric and high molecular weight, disulfide-bonded, caveolin-1-containing structures. Peripheral inflammatory pain promoted a dynamic redistribution between membrane domains of P-glycoprotein and caveolin-1. Disassembly of high molecular weight P-glycoprotein-containing structures within microvascular endothelial luminal membrane domains was accompanied by an increase in ATPase activity, suggesting a potential for functionally active P-glycoprotein. These results are the first observation that peripheral inflammatory pain leads to specific structural changes in P-glycoprotein responsible for controlling analgesic drug delivery to the CNS.
• Davis, T., Ronaldson, P. T., & Davis, T. P. (2012). Blood-brain barrier integrity and glial support: mechanisms that can be targeted for novel therapeutic approaches in stroke. Current pharmaceutical design, 18(25).
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  The blood-brain barrier (BBB) is a critical regulator of brain homeostasis. Additionally, the BBB is the most significant obstacle to effective CNS drug delivery. It possesses specific charcteristics (i.e., tight junction protein complexes, influx and efflux transporters) that control permeation of circulating solutes including therapeutic agents. In order to form this "barrier," brain microvascular endothelial cells require support of adjacent astrocytes and microglia. This intricate relationship also occurs between endothelial cells and other cell types and structures of the CNS (i.e., pericytes, neurons, extracellular matrix), which implies existence of a "neurovascular unit." Ischemic stroke can disrupt the neurovascular unit at both the structural and functional level, which leads to an increase in leak across the BBB. Recent studies have identified several pathophysiological mechanisms (i.e., oxidative stress, activation of cytokine-mediated intracellular signaling systems) that mediate changes in the neurovascular unit during ischemic stroke. This review summarizes current knowledge in this area and emphasizes pathways (i.e., oxidative stress, cytokine-mediated intracellular signaling, glial-expressed receptors/targets) that can be manipulated pharmacologically for i) preservation of BBB and glial integrity during ischemic stroke and ii) control of drug permeation and/or transport across the BBB. Targeting these pathways present a novel opportunity for optimization of CNS delivery of therapeutics in the setting of ischemic stroke.
• Davis, T., Ronaldson, P. T., & Davis, T. P. (2011). Targeting blood-brain barrier changes during inflammatory pain: an opportunity for optimizing CNS drug delivery. Therapeutic delivery, 2(8).
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  The blood-brain barrier (BBB) is the most significant obstacle to effective CNS drug delivery. It possesses structural and biochemical features (i.e., tight-junction protein complexes and, influx and efflux transporters) that restrict xenobiotic permeation. Pathophysiological stressors (i.e., peripheral inflammatory pain) can alter BBB tight junctions and transporters, which leads to drug-permeation changes. This is especially critical for opioids, which require precise CNS concentrations to be safe and effective analgesics. Recent studies have identified molecular targets (i.e., endogenous transporters and intracellular signaling systems) that can be exploited for optimization of CNS drug delivery. This article summarizes current knowledge in this area and emphasizes those targets that present the greatest opportunity for controlling drug permeation and/or drug transport across the BBB in an effort to achieve optimal CNS opioid delivery.
• Davis, T., Ronaldson, P. T., Finch, J. D., Demarco, K. M., Quigley, C. E., & Davis, T. P. (2011). Inflammatory pain signals an increase in functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier. The Journal of pharmacology and experimental therapeutics, 336(3).
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  Pain is a dominant symptom associated with inflammatory conditions. Pharmacotherapy with opioids may be limited by poor blood-brain barrier (BBB) permeability. One approach that may improve central nervous system (CNS) delivery is to target endogenous BBB transporters such as organic anion-transporting polypeptide 1a4 (Oatp1a4). It is critical to identify and characterize biological mechanisms that enable peripheral pain/inflammation to "transmit" upstream signals and alter CNS drug transport processes. Our goal was to investigate, in vivo, BBB functional expression of Oatp1a4 in animals subjected to peripheral inflammatory pain. Inflammatory pain was induced in female Sprague-Dawley rats (200-250 g) by subcutaneous injection of 3% λ-carrageenan into the right hind paw; control animals were injected with 0.9% saline. In rat brain microvessels, Oatp1a4 expression was increased during acute pain/inflammation. Uptake of taurocholate and [d-penicillamine(2,5)]-enkephalin, two established Oatp substrates, was increased in animals subjected to peripheral pain, suggesting increased Oatp1a4-mediated transport. Inhibition of inflammatory pain with the anti-inflammatory drug diclofenac attenuated these changes in Oatp1a4 functional expression, suggesting that inflammation in the periphery can modulate BBB transporters. In addition, diclofenac prevented changes in the peripheral signaling cytokine transforming growth factor-β1 (TGF-β1) levels and brain microvascular TGF-β receptor expression induced by inflammatory pain. Pretreatment with the pharmacological TGF-β receptor inhibitor 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) increased Oatp1a4 functional expression in λ-carrageenan-treated animals and saline controls, suggesting that TGF-β signaling is involved in Oatp1a4 regulation at the BBB. Our findings indicate that BBB transporters (i.e., Oatp1a4) can be targeted during drug development to improve CNS delivery of highly promising therapeutics.
• Willis, C. L., Brooks, T. A., & Davis, T. P. (2011). Chronic Inflammation Pain and the Neurovascular Unit: A Central Role for Glia in Maintaining BBB Integrity (vol 14, pg 1625, 2008). CURRENT PHARMACEUTICAL DESIGN, 17(22), 2367-2367.
• Davis, T., Lochhead, J. J., McCaffrey, G., Quigley, C. E., Finch, J., DeMarco, K. M., Nametz, N., & 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).
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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.
• Davis, T., Willis, C. L., Meske, D. S., & Davis, T. P. (2010). Protein kinase C activation modulates reversible increase in cortical blood-brain barrier permeability and tight junction protein expression during hypoxia and posthypoxic reoxygenation. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 30(11).
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  Hypoxia (Hx) is a component of many disease states including stroke. Ischemic stroke occurs when there is a restriction of cerebral blood flow and oxygen to part of the brain. During the ischemic, and subsequent reperfusion phase of stroke, blood-brain barrier (BBB) integrity is lost with tight junction (TJ) protein disruption. However, the mechanisms of Hx and reoxygenation (HR)-induced loss of BBB integrity are not fully understood. We examined the role of protein kinase C (PKC) isozymes in modifying TJ protein expression in a rat model of global Hx. The Hx (6% O(2)) induced increased hippocampal and cortical vascular permeability to 4 and 10 kDa dextran fluorescein isothiocyanate (FITC) and endogenous rat-IgG. Cortical microvessels revealed morphologic changes in nPKC-θ distribution, increased nPKC-θ and aPKC-ζ protein expression, and activation by phosphorylation of nPKC-θ (Thr538) and aPKC-ζ (Thr410) residues after Hx treatment. Claudin-5, occludin, and ZO-1 showed disrupted organization at endothelial cell margins, whereas Western blot analysis showed increased TJ protein expression after Hx. The PKC inhibition with chelerythrine chloride (5 mg/kg intraperitoneally) attenuated Hx-induced hippocampal vascular permeability and claudin-5, PKC (θ and ζ) expression, and phosphorylation. This study supports the hypothesis that nPKC-θ and aPKC-ζ signaling mediates TJ protein disruption resulting in increased BBB permeability.
• Chow, N., Davis, T. P., Guo, H., Singh, I., Thiyagarajan, M., Wang, Y., & Zlokovic, B. V. (2009).

  Differential neuroprotection and risk for bleeding from activated protein C with varying degrees of anticoagulant activity.

  . Stroke, 40(5), 1864-9. doi:10.1161/strokeaha.108.536680
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  Activated protein C (APC), a protease with anticoagulant and cytoprotective activities, protects neurons and endothelium from ischemic injury. Drotrecogin-alfa activated, a hyperanticoagulant form of human recombinant APC, is currently being studied in patients with ischemic stroke. How changes in APC anticoagulant activity influence APC's neuroprotection and risk for bleeding is not clear..We used neuronal and brain endothelial cell injury models and middle cerebral artery occlusion in mice to compare efficacy and safety of drotrecogin-alfa activated and human 3K3A-APC, an APC nonanticoagulant mutant..Drotrecogin-alfa activated and 3K3A-APC exhibited 148% and 10% of plasma-derived APC's anticoagulant activity and differ in the carbohydrate content. 3K3A-APC protected mouse neurons from N-methyl-d-aspartate-induced apoptosis and human brain endothelial cell from oxygen-glucose deprivation with 1.8- and 3.1-fold greater efficacy than drotrecogin-alfa activated. Given 5 minutes before transient middle cerebral artery occlusion, 3K3A-APC and drotrecogin-alfa activated (0.5 and 2 mg/kg intravenously) reduced comparably and dose-dependently the infarction lesion up to 85%. 3K3A-APC, but not drotrecogin-alfa activated, improved neurological score dose-dependently (P
• Davis, T., McCaffrey, G., Willis, C. L., Staatz, W. D., Nametz, N., Quigley, C. A., Hom, S., Lochhead, J. J., & 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).
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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.
• Davis, T., Ronaldson, P. T., Demarco, K. M., Sanchez-Covarrubias, L., Solinsky, C. M., & Davis, T. P. (2009). Transforming growth factor-beta signaling alters substrate permeability and tight junction protein expression at the blood-brain barrier during inflammatory pain. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 29(6).
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  Our laboratory has shown that peripheral inflammatory pain induced by lambda-carrageenan (CIP) can increase blood-brain barrier (BBB) permeability and alter tight junction (TJ) protein expression leading to changes in BBB functional integrity. However, the intracellular signaling mechanisms involved in this pathophysiologic response have not been elucidated. Transforming growth factor (TGF)-beta signaling pathways are known to regulate vascular integrity and permeability. Therefore, we examined the function of TGF-beta signaling at the BBB in rats subjected to CIP. During CIP, serum TGF-beta1 and protein expression of the TGF-beta receptor activin receptor-like kinase-5 (ALK5) were reduced. Brain permeability to (14)C-sucrose was increased and expression of TJ proteins (i.e., claudin-5, occludin, zonula occluden (ZO-1)) were also altered after 3 h CIP. Pharmacological inhibition of ALK5 with the selective inhibitor SB431542 further enhanced brain uptake of (14)C-sucrose, increased TJ protein expression (i.e., claudin-3, claudin-5, occludin, ZO-1), and decreased nuclear expression of TGF-beta/ALK5 signaling molecules (i.e., Smad2, Smad3), which suggests a role for TGF-beta/ALK5 signaling in the regulation of BBB integrity. Interestingly, administration of exogenous TGF-beta1 before CIP activated the TGF-beta/ALK5 pathway and reduced BBB permeability to (14)C-sucrose. Taken together, our data show that TGF-beta/ALK5 signaling is, in part, involved in the regulation of BBB functional integrity.
• Ronaldson, P. T., Demarco, K. M., Sanchez-Covarrubias, L., Solinsky, C. M., & Davis, T. P. (2009). Transforming growth factor-beta (TGF-beta) signaling is involved in the regulation of blood-brain barrier (BBB) functional integrity during peripheral inflammatory pain. JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM, 29, S484-S484.
• Davis, T., Brooks, T. A., Nametz, N., Charles, R., & Davis, T. P. (2008). Diclofenac attenuates the regional effect of lambda-carrageenan on blood-brain barrier function and cytoarchitecture. The Journal of pharmacology and experimental therapeutics, 325(2).
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  The microenvironment of the brain requires tight regulation for proper neuronal function. Protecting the central nervous system (CNS) from the varying concentrations of ions, proteins, and toxins in the periphery is the dynamically regulated blood-brain barrier (BBB). Recent studies have demonstrated significant modulation of the BBB in a number of diseases and physiological states, including pain. This study expands on previous explorations of acute and chronic pain-induced effects on the function and molecular cytoarchitecture of the barrier. It describes the role of cyclooxygenase (COX) up-regulation by blocking with diclofenac (30 mg/kg, i.p.), and it examines the variation in BBB regulation through various brain regions. Edema and hyperalgesia were induced by lambda-carrageenan and attenuated by the additional administration of diclofenac. Examination of unidirectional [14C]sucrose permeability with multitime in situ perfusion studies demonstrated that lambda-carrageenan significantly increased cerebral permeability and decreased brainstem permeability. There were no significant changes in any of the other brain regions examined. These permeability changes correlated with up- and down-regulation of the tight junction (TJ) protein claudin-5 in the cerebrum and brainstem, respectively. Diclofenac administration attenuated the cerebral permeability uptake as well as the claudin-5 up-regulation. In addition, diclofenac reversed the lowered permeability in the brainstem, but it did not attenuate TJ protein expression. These studies demonstrate the complex regulation of the BBB occurring during inflammatory pain and the role of COX in this process. An understanding of BBB regulation during pain states is critically important for pharmacotherapy, and it holds great promise for new therapies to treat central nervous system pathologies.
• Davis, T., Campos, C. R., Ocheltree, S. M., Hom, S., Egleton, R. D., & Davis, T. P. (2008). Nociceptive inhibition prevents inflammatory pain induced changes in the blood-brain barrier. Brain research, 1221.
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  Previous studies by our group have shown that peripheral inflammatory insult, using the lambda-carrageenan inflammatory pain (CIP) model, induced alterations in the molecular and functional properties of the blood-brain barrier (BBB). The question remained whether these changes were mediated via an inflammatory and/or neuronal mechanism. In this study, we investigated the involvement of neuronal input from pain activity on alterations in BBB integrity by peripheral inhibition of nociceptive input. A perineural injection of 0.75% bupivacaine into the right hind leg prior to CIP was used for peripheral nerve block. Upon nerve block, there was a significant decrease in thermal allodynia induced by CIP, but no effect on edema formation 1 h post-CIP. BBB permeability was increased 1 h post-CIP treatment as determined by in situ brain perfusion of [(14)C] sucrose; bupivacaine nerve block of CIP caused an attenuation of [(14)C] sucrose permeability, back to saline control levels. Paralleling the changes in [(14)C] sucrose permeability, we also report increased expression of three tight junction (TJ) proteins, zonula occluden-1 (ZO-1), occludin and claudin-5 with CIP. Upon bupivacaine nerve block, changes in expression were prevented. These data show that the lambda-carrageenan-induced changes in [(14)C] sucrose permeability and protein expression of ZO-1, occludin and claudin-5 are prevented with inhibition of nociceptive input. Therefore, we suggest that nociceptive signaling is in part responsible for the alteration in BBB integrity under CIP.
• Davis, T., McCaffrey, G., Seelbach, M. J., Staatz, W. D., Nametz, N., Quigley, C., Campos, C. R., Brooks, T. A., & Davis, T. P. (2008). Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia. Journal of neurochemistry, 106(6).
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  Tight junctions (TJs) at the blood-brain barrier (BBB) dynamically alter paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS in response to external stressors, such as pain, inflammation, and hypoxia. In this study, we investigated the effect of lambda-carrageenan-induced peripheral inflammatory pain (i.e., hyperalgesia) on the oligomeric assembly of the key TJ transmembrane protein, occludin. Oligomerization of integral membrane proteins is a critical step in TJ complex assembly that enables the generation of tightly packed, large multiprotein complexes capable of physically obliterating the interendothelial space to inhibit paracellular diffusion. Intact microvessels isolated from rat brains were fractionated by detergent-free density gradient centrifugation, and gradient fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/ Western blot. Injection of lambda-carrageenan into the rat hind paw produced after 3 h a marked change in the relative amounts of oligomeric, dimeric, and monomeric occludin isoforms associated with different plasma membrane lipid raft domains and intracellular compartments in endothelial cells at the BBB. Our findings suggest that increased BBB permeability (i.e., leak) associated with lambda-carrageenan-induced peripheral inflammatory pain is promoted by the disruption of disulfide-bonded occludin oligomeric assemblies, which renders them incapable of forming an impermeant physical barrier to paracellular transport.
• Willis, C. L., & Davis, T. P. (2008). Chronic inflammatory pain and the neurovascular unit: A central role for glia in maintaining BBB integrity?. CURRENT PHARMACEUTICAL DESIGN, 14(16), 1625-1643.
• Davis, T., Hom, S., Fleegal, M. A., Egleton, R. D., Campos, C. R., Hawkins, B. T., & Davis, T. P. (2007). Comparative changes in the blood-brain barrier and cerebral infarction of SHR and WKY rats. American journal of physiology. Regulatory, integrative and comparative physiology, 292(5).
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  Hypertension is involved in the exacerbation of stroke. It is unclear how blood-brain barrier (BBB) tight-junction (TJ) and ion transporter proteins critical for maintaining brain homeostasis contribute to cerebral infarction during hypertension development. In the present study, we investigated cerebral infarct volume following permanent 4-h middle cerebral artery occlusion (MCAO) and characterized the expression of BBB TJ and ion transporter proteins in brain microvessels of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats at 5 wk (prehypertension), 10 wk (early-stage hypertension), and 15 wk (later-stage hypertension) of age. Hypertensive SHR show increased infarct volume following MCAO compared with WKY control rats. BBB TJ and ion transporter proteins, known to contribute to edema and fluid volume changes in the brain, show differential protein expression patterns during hypertension development. Western blot analysis of TJ protein zonula occludens-2 (ZO-2) showed decreased expression, while ion transporter, Na(+)/H(+) exchanger 1 (NHE-1), was markedly increased in hypertensive SHR. Expression of TJ proteins ZO-1, occludin, actin, claudin-5, and Na(+)-K(+)-2Cl(-) cotransporter remain unaffected in SHR compared with control. Selective inhibition of NHE-1 using dimethylamiloride significantly attenuated ischemia-induced infarct volume in hypertensive SHR following MCAO, suggesting a novel role for NHE-1 in the brain in the regulation of ischemia-induced infarct volume in SHR.
• Davis, T., McCaffrey, G., Staatz, W. D., Quigley, C. A., Nametz, N., Seelbach, M. J., Campos, C. R., Brooks, T. A., Egleton, R. D., & Davis, T. P. (2007). Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo. Journal of neurochemistry, 103(6).
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  Tight junctions (TJs) are major components of the blood-brain barrier (BBB) that physically obstruct the interendothelial space and restrict paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS. TJs are dynamic structures whose intricate arrangement of oligomeric transmembrane and accessory proteins rapidly alters in response to external stressors to produce changes in BBB permeability. In this study, we investigate the constitutive trafficking of the TJ transmembrane proteins occludin and claudin-5 that are essential for forming the TJ seal between microvascular endothelial cells that inhibits paracellular diffusion. Using a novel, detergent-free OptiPrep density-gradient method to fractionate rat cerebral microvessels, we identify a plasma membrane lipid raft domain that contains oligomeric occludin and claudin-5. Our data suggest that oligomerization of occludin involves disulfide bond formation within transmembrane regions, and that assembly of the TJ oligomeric protein complex is facilitated by an oligomeric caveolin scaffold. This is the first time that distribution of oligomeric TJ transmembrane proteins within plasma membrane lipid rafts at the BBB has been examined in vivo. The findings reported in this study are critical to understand the mechanism of assembly of the TJ multiprotein complex that is essential for maintaining BBB integrity.
• Davis, T., Seelbach, M. J., Brooks, T. A., Egleton, R. D., & Davis, T. P. (2007). Peripheral inflammatory hyperalgesia modulates morphine delivery to the brain: a role for P-glycoprotein. Journal of neurochemistry, 102(5).
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  P-glycoprotein (Pgp, ABCB1) is a critical efflux transporter at the blood-brain barrier (BBB) where its luminal location and substrate promiscuity limit the brain distribution of numerous therapeutics. Moreover, Pgp is known to confer multi-drug resistance in cancer chemotherapy and brain diseases, such as epilepsy, and is highly regulated by inflammatory mediators. The involvement of inflammatory processes in neuropathological states has led us to investigate the effects of peripheral inflammatory hyperalgesia on transport properties at the BBB. In the present study, we examined the effects of lambda-carrageenan-induced inflammatory pain (CIP) on brain endothelium regulation of Pgp. Western blot analysis of enriched brain microvessel fractions showed increased Pgp expression 3 h post-CIP. In situ brain perfusion studies paralleled these findings with decreased brain uptake of the Pgp substrate and opiate analgesic, [(3)H] morphine. Cyclosporin A-mediated inhibition of Pgp enhanced the uptake of morphine in lambda-carrageenan and control animals. This indicates that the CIP induced decrease in morphine transport was the result of an increase in Pgp activity at the BBB. Furthermore, antinociception studies showed decreased morphine analgesia following CIP. The observation that CIP modulates Pgp at the BBB in vivo is critical to understanding BBB regulation during inflammatory disease states.
• Davis, T. P., & Witt, K. A. (2006).

  CNS drug delivery: opioid peptides and the blood-brain barrier.

  . The AAPS journal, 8(1), E76-88. doi:10.1208/aapsj080109
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  Peptides are key regulators in cellular and intercellular physiological responses and possess enormous promise for the treatment of pathological conditions. Opioid peptide activity within the central nervous system (CNS) is of particular interest for the treatment of pain owing to the elevated potency of peptides and the centrally mediated actions of pain processes. Despite this potential, peptides have seen limited use as clinically viable drugs for the treatment of pain. Reasons for the limited use are primarily based in the physiochemical and biochemical nature of peptides. Numerous approaches have been devised in an attempt to improve peptide drug delivery to the brain, with variable results. This review describes different approaches to peptide design/modification and provides examples of the value of these strategies to CNS delivery of peptide drugs. The various modes of modification of therapeutic peptides may be amalgamated, creating more efficacious "hybrid" peptides, with synergistic delivery to the CNS. The ongoing development of these strategies provides promise that peptide drugs may be useful for the treatment of pain and other neurologically-based disease states in the future.
• Davis, T., Brooks, T. A., Ocheltree, S. M., Seelbach, M. J., Charles, R. A., Nametz, N., Egleton, R. D., & Davis, T. P. (2006). Biphasic cytoarchitecture and functional changes in the BBB induced by chronic inflammatory pain. Brain research, 1120(1).
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  The blood-brain barrier (BBB) is a dynamic system which maintains brain homeostasis and limits CNS penetration via interactions of transmembrane and intracellular proteins. Inflammatory pain (IP) is a condition underlying several diseases with known BBB perturbations, including stroke, Parkinson's, multiple sclerosis and Alzheimer's. Exploring the underlying pathology of chronic IP, we demonstrated alterations in BBB paracellular permeability with correlating changes in tight junction (TJ) proteins: occludin and claudin-5. The present study examines the IP-induced molecular changes leading to a loss in functional BBB integrity. IP was induced by injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the right hindpaw of female Sprague-Dawley rats. Inflammation and hyperalgesia were confirmed, and BBB paracellular permeability was assessed by in situ brain perfusion of [14C]sucrose (paracellular diffusion marker). The permeability of the BBB was significantly increased at 24 and 72 h post-CFA. Analysis of the TJ proteins, which control the paracellular pathway, demonstrated decreased claudin-5 expression at 24 h, and an increase at 48 and 72 h post-injection. Occludin expression was significantly decreased 72 h post-CFA. Expression of junction adhesion molecule-1 (JAM-1) increased 48 h and decreased by 72 h post-CFA. Confocal microscopy demonstrated continuous expression of both occludin and JAM-1, each co-localizing with ZO-1. The increased claudin-5 expression was not limited to the junction. These results provide evidence that chronic IP causes dramatic alterations in specific cytoarchitectural proteins and demonstrate alterations in molecular properties during CFA, resulting in significant changes in BBB paracellular permeability.
• Davis, T., Brooks, T. A., Hawkins, B. T., Huber, J. D., Egleton, R. D., & Davis, T. P. (2005). Chronic inflammatory pain leads to increased blood-brain barrier permeability and tight junction protein alterations. American journal of physiology. Heart and circulatory physiology, 289(2).
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  The blood-brain barrier (BBB) maintains brain homeostasis by limiting entry of substances to the central nervous system through interaction of transmembrane and intracellular proteins that make up endothelial cell tight junctions (TJs). Recently it was shown that the BBB can be modulated by disease pathologies including inflammatory pain. This study examined the effects of chronic inflammatory pain on the functional and molecular integrity of the BBB. Inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into the right plantar hindpaw in female Sprague-Dawley rats under halothane anesthesia; control animals were injected with saline. Edema and hyperalgesia were assessed by plethysmography and infrared paw-withdrawal latency. At 72 h postinjection, significant edema formation and hyperalgesia were noted in the CFA-treated rats. Examination of permeability of the BBB by in situ perfusion of [14C]sucrose while rats were under pentobarbital anesthesia demonstrated that CFA treatment significantly increased brain sucrose uptake. Western blot analysis of BBB TJ proteins showed no change in expression of zonula occludens-1 (an accessory protein) or actin (a cytoskeletal protein) with CFA treatment. Expression of the transmembrane TJ proteins occludin and claudin-3 and -5 significantly changed with CFA treatment with a 60% decrease in occludin, a 450% increase in claudin-3, and a 615% increase in claudin-5 expression. This study demonstrates that during chronic inflammatory pain, alterations in BBB function are associated with changes in specific transmembrane TJ proteins.
• Davis, T., Egleton, R. D., & Davis, T. P. (2005). Development of neuropeptide drugs that cross the blood-brain barrier. NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics, 2(1).
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  In recent years, there have been several important advancements in the development of neuropeptide therapeutics. Nevertheless, the targeting of peptide drugs to the CNS remains a formidable obstacle. Delivery of peptide drugs is limited by their poor bioavailability to the brain due to low metabolic stability, high clearance by the liver, and the presence of the blood brain barrier (BBB). Multiple strategies have been devised in an attempt to improve peptide drug delivery to the brain, with variable results. In this review, we discuss several of the strategies that have been used to improve both bioavailability and BBB transport, with an emphasis on antibody based vector delivery, useful for large peptides/small proteins, and glycosylation, useful for small peptides. Further development of these delivery methods may finally enable peptide drugs to be useful for the treatment of neurological disease states.
• Davis, T., Fleegal, M. A., Hom, S., Borg, L. K., & Davis, T. P. (2005). Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation. American journal of physiology. Heart and circulatory physiology, 289(5).
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  The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-betaII, PKC-gamma, PKC-eta, PKC-mu, and PKC-lambda also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-epsilon and PKC-zeta were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 microM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-gamma and PKC-theta in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.
• Davis, T., Hawkins, B. T., & Davis, T. P. (2005). The blood-brain barrier/neurovascular unit in health and disease. Pharmacological reviews, 57(2).
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  The blood-brain barrier (BBB) is the regulated interface between the peripheral circulation and the central nervous system (CNS). Although originally observed by Paul Ehrlich in 1885, the nature of the BBB was debated well into the 20th century. The anatomical substrate of the BBB is the cerebral microvascular endothelium, which, together with astrocytes, pericytes, neurons, and the extracellular matrix, constitute a "neurovascular unit" that is essential for the health and function of the CNS. Tight junctions (TJ) between endothelial cells of the BBB restrict paracellular diffusion of water-soluble substances from blood to brain. The TJ is an intricate complex of transmembrane (junctional adhesion molecule-1, occludin, and claudins) and cytoplasmic (zonula occludens-1 and -2, cingulin, AF-6, and 7H6) proteins linked to the actin cytoskeleton. The expression and subcellular localization of TJ proteins are modulated by several intrinsic signaling pathways, including those involving calcium, phosphorylation, and G-proteins. Disruption of BBB TJ by disease or drugs can lead to impaired BBB function and thus compromise the CNS. Therefore, understanding how BBB TJ might be affected by various factors holds significant promise for the prevention and treatment of neurological diseases.
• Davis, T., Hawkins, B. T., Egleton, R. D., & Davis, T. P. (2005). Modulation of cerebral microvascular permeability by endothelial nicotinic acetylcholine receptors. American journal of physiology. Heart and circulatory physiology, 289(1).
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  Nicotine increases the permeability of the blood-brain barrier in vivo. This implies a possible role for nicotinic acetylcholine receptors in the regulation of cerebral microvascular permeability. Expression of nicotinic acetylcholine receptor subunits in cerebral microvessels was investigated with immunofluorescence microscopy. Positive immunoreactivity was found for receptor subunits alpha3, alpha5, alpha7, and beta2, but not subunits alpha4, beta3, or beta4. Blood-brain barrier permeability was assessed via in situ brain perfusion with [14C]sucrose. Nicotine increased the rate of sucrose entry into the brain from 0.3 +/- 0.1 to 1.1 +/- 0.2 microl.g(-1).min(-1), as previously described. This nicotine-induced increase in blood-brain barrier permeability was significantly attenuated by both the blood-brain barrier-permeant nicotinic antagonist mecamylamine and the blood-brain barrier-impermeant nicotinic antagonist hexamethonium to 0.5 +/- 0.2 and 0.3 +/- 0.2 microl.g(-1).min(-1), respectively. These data suggest that nicotinic acetylcholine receptors expressed on the cerebral microvascular endothelium mediate nicotine-induced changes in blood-brain barrier permeability.
• Davis, T., Witt, K. A., Mark, K. S., Huber, J., & Davis, T. P. (2005). Hypoxia-inducible factor and nuclear factor kappa-B activation in blood-brain barrier endothelium under hypoxic/reoxygenation stress. Journal of neurochemistry, 92(1).
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  This investigation focuses on transcription factor involvement in blood-brain barrier (BBB) endothelial cell-induced alterations under conditions of hypoxia and post-hypoxia/reoxygenation (H/R), using established in vivo/ex vivo and in vitro BBB models. Protein/DNA array analyses revealed a correlation in key transcription factor activation during hypoxia and H/R, including NFkappaB and hypoxia-inducible factor (HIF)1. Electrophoretic mobility shift assays confirmed NFkappaB and HIF1 binding activity ex vivo and in vitro, under conditions of hypoxia and H/R. Hypoxia- and H/R-treated BBB endothelium showed increased HIF1alpha protein expression in both cytoplasmic and nuclear fractions, in ex vivo and in vitro models. Co-immunoprecipitation of HIF1alpha and HIF1beta was shown in the nuclear fraction under conditions of hypoxia and H/R in both models. Hypoxia- and H/R-treated BBB endothelium showed increased expression of NFkappaB-p65 protein in both cytoplasmic and nuclear fractions. Co-immunoprecipitation of NFkappaB-p65 with NFkappaB-p50 was shown in the nuclear fraction under conditions of hypoxia and H/R in the ex vivo model, and after H/R in the in vitro model. These data offer novel avenues in which to alter and/or investigate BBB activity across model systems and to further our understanding of upstream regulators during hypoxia and H/R.
• Davis, T., Brown, R. C., Egleton, R. D., & Davis, T. P. (2004). Mannitol opening of the blood-brain barrier: regional variation in the permeability of sucrose, but not 86Rb+ or albumin. Brain research, 1014(1-2).
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  Clinically, infusion of hyperosmolar solutions is used to enhance chemotherapeutic drug penetration of the blood-brain barrier (BBB) in patients with malignant brain tumors or metastases. We examined the effect of hyperosmolar BBB disruption on brain permeability of three compounds, 86Rb+, a marker for K+ permeability and transport, [14C]sucrose and Evans blue albumin, using a rat in situ perfusion model. 86Rb+ and [14C]sucrose had increased permeability 20 min after BBB disruption with 1.6 M mannitol. There was no change in Evans blue albumin permeability. Only [14C]sucrose showed regional variation in permeability after mannitol-induced BBB disruption, with the cortex and midbrain having higher sucrose permeability then either the cerebellum or brainstem. These data suggest that the clinical efficacy of hyperosmolar disruption therapy in conjunction with chemotherapeutic agents, of a similar molecular weight to sucrose, may be affected by the location of the tumor within the brain.
• Davis, T., Brown, R. C., Mark, K. S., Egleton, R. D., & Davis, T. P. (2004). Protection against hypoxia-induced blood-brain barrier disruption: changes in intracellular calcium. American journal of physiology. Cell physiology, 286(5).
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  Tissue damage after stroke is partly due to disruption of the blood-brain barrier (BBB). Little is known about the role of calcium in modulating BBB disruption. We investigated the effect of hypoxic and aglycemic stress on BBB function and intracellular calcium levels. Bovine brain microvessel endothelial cells were treated with A-23187 to increase intracellular calcium without hypoxia or treated with a calcium chelator (BAPTA) or calcium channel blockers (nifedipine or SKF-96365) and 6 h of hypoxia. A-23187 alone did not increase paracellular permeability. Hypoxia increased intracellular calcium, and hypoxia or hypoxia-aglycemia increased paracellular permeability. Treatment with nifedipine and SKF-96365 increased intracellular calcium under normoglycemic conditions, instead of blocking calcium influx, and was protective against hypoxia-induced BBB disruption under normoglycemia. Protection by nifedipine and SKF-96365 was not due to antioxidant properties of these compounds. These data indicate that increased intracellular calcium alone is not enough to disrupt the BBB. However, increased intracellular calcium after drug treatment and hypoxia suggests a potential mechanism for these drugs in BBB protection; nifedipine and SKF-96365 plus hypoxic stress may trigger calcium-mediated signaling cascades, altering BBB integrity.
• Davis, T., Hau, V. S., Huber, J. D., Campos, C. R., Davis, R. T., & Davis, T. P. (2004). Effect of lambda-carrageenan-induced inflammatory pain on brain uptake of codeine and antinociception. Brain research, 1018(2).
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  This study investigated the potential clinical implications of lambda-carrageenan-induced inflammatory pain on brain uptake of a commonly used analgesic, codeine, in relation to the fundamental properties of the blood-brain barrier (BBB) correlated to its antinociceptive profile over a 168-h time course. BBB uptake of [14C]sucrose (a membrane impermeant marker) and [3H]codeine were investigated using an in situ brain perfusion model in the rat. Results demonstrated a significantly increased brain uptake of [14C]sucrose at 1, 3, 6 and 48 h (139+/-9%, 166+/-19%, 138+/-13% and 146+/-7% compared with control, respectively) and [3H]codeine at 3 and 48 h (179+/-6% and 179+/-12% compared with control, respectively). Capillary depletion analyses ensured that increased radioisotope associated with the brain was due to increased uptake rather than trapping in the cerebral vasculature. Antinociception studies using a radiant-heat tail flick analgesia method demonstrated that lambda-carrageenan-induced inflammatory pain enhanced the in vivo antinociceptive profile of i.p.-administered codeine (7 mg/kg) at 3 and 48 h (144+/-11% and 155+/-9% compared with control, respectively). This study demonstrated that brain uptake and antinociception of codeine are increased during lambda-carrageenan-induced inflammatory pain, suggesting that the presence of inflammatory pain may be an important consideration in therapeutic drug dosing, potential adverse effects and/or neurotoxicity.
• Davis, T., Hawkins, B. T., Abbruscato, T. J., Egleton, R. D., Brown, R. C., Huber, J. D., Campos, C. R., & Davis, T. P. (2004). Nicotine increases in vivo blood-brain barrier permeability and alters cerebral microvascular tight junction protein distribution. Brain research, 1027(1-2).
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  The blood-brain barrier (BBB) is critical to the health of the central nervous system. The BBB is formed primarily by the presence of tight junctions (TJ) between cerebral microvessel endothelial cells. In light of the known effects of nicotine on endothelial cell biology, the specific effects of nicotine on the in vivo BBB were examined. Using in situ brain perfusion, it was found that continuous administration of nicotine (4.5 mg free base x kg(-1) x day(-1)) for 1 and 7 days led to increased permeability of the BBB to [14C]-sucrose without significant changes in its initial volume of distribution. The expression and distribution of the TJ-associated proteins actin, occludin, claudin-1, -3, and -5, and ZO-1 and -2 were analyzed by Western blot and immunofluorescence microscopy. Though no changes in total protein expression were observed, nicotine treatment was associated with altered cellular distribution of ZO-1 and diminished junctional immunoreactivity of claudin-3. It is proposed that nicotine leads to changes in BBB permeability via the modulation of TJ proteins.
• Davis, T., Mark, K. S., Burroughs, A. R., Brown, R. C., Huber, J. D., & Davis, T. P. (2004). Nitric oxide mediates hypoxia-induced changes in paracellular permeability of cerebral microvasculature. American journal of physiology. Heart and circulatory physiology, 286(1).
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  Ischemic stroke from a reduction in blood flow to the brain microvasculature results in a subsequent decreased delivery of oxygen (i.e., hypoxia) and vital nutrients to endothelial, neuronal, and glial cells. Hypoxia associated with stroke has been shown to increase paracellular permeability of the blood-brain barrier, leading to the release of cellular mediators and brain tissue injury. Whereas reperfusion does not occur in all ischemic strokes, increased permeability has been seen in posthypoxic reoxygenation. Currently, it is unknown whether these deleterious effects result from cellular mechanisms stimulated by decreased oxygen during stroke or posthypoxic reoxygenation stress. This study used primary bovine brain microvessel endothelial cells (BBMECs) to examine the involvement of nitric oxide (NO) as a mediator in hypoxia-induced permeability changes. Hypoxia-induced increased transport of [14C]sucrose across BBMEC monolayers compared with normoxia was attenuated by either posthypoxic reoxygenation or inhibition of NO synthase (NOS). The hypoxia-induced permeability effect was further reduced when NOS inhibition was combined with posthypoxic reoxygenation. Additionally, a significant increase in total NO was seen in BBMECs after hypoxic exposure. This correlation was supported by the increased [14C]sucrose permeability observed when BBMECs were exposed to the NO donor diethylenetriaamine NONOate. Western blot analyses of NOS isoforms showed a significant increase in the inducible isoform after hypoxic exposure with a subsequent reduction in expression on reoxygenation. Results from this study suggest that hypoxia-induced blood-brain barrier breakdown can be diminished by inhibition of NO synthesis, decreased concentration of NO metabolites, and/or reoxygenation.
• Schoenfeldt, M., & Davis, T. P. (2004).

  Clinical trials referral resource. Current clinical trials in multiple myeloma.

  . Oncology (Williston Park, N.Y.), 18(2), 196-8, 203.
• Schoenfeldt, M., & Davis, T. P. (2004).

  Clinical trials referral resource. Current phase III clinical trials for adults with leukemia or lymphoma.

  . Oncology (Williston Park, N.Y.), 18(1), 62, 65, 69.
• Davis, T., Brown, R. C., Mark, K. S., Egleton, R. D., Huber, J. D., Burroughs, A. R., & Davis, T. P. (2003). Protection against hypoxia-induced increase in blood-brain barrier permeability: role of tight junction proteins and NFkappaB. Journal of cell science, 116(Pt 4).
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  Co-culture with glial cells and glia-conditioned media can induce blood-brain barrier properties in microvessel endothelial cells and protect against hypoxia-induced blood-brain barrier breakdown. We examined the effect of two types of glia-conditioned media on brain microvessel endothelial cell permeability and tight junction protein expression, and studied potential mechanisms of action. We found that C6-glioma-conditioned media, but not rat astrocyte-conditioned media, protected against an increase in permeability induced by exposure to 1% oxygen for 24 hours. This hypoxic stress caused an increase in the expression of tight junction proteins claudin-1 and actin, particularly in cells treated with C6-conditioned media. We found that C6-conditioned media has a significantly higher level of both basic fibroblast growth factor and vascular endothelial growth factor. Treatment with C6-conditioned media for 1 or 3 days protects against hypoxia-induced permeability increases, and this protective effect may be mediated by signal transduction pathways terminating at the transcription factor NFkappaB.
• Davis, T., Egleton, R. D., Campos, C. C., Huber, J. D., Brown, R. C., & Davis, T. P. (2003). Differential effects of diabetes on rat choroid plexus ion transporter expression. Diabetes, 52(6).
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  Though diabetes is a disease with vascular complications, little is known about its effects on the blood-brain barrier or the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB is situated at choroid plexuses located in the lateral, third, and fourth ventricles. Choroid plexuses are the primary site of cerebrospinal fluid (CSF) production and express numerous ion transporters. Previous studies have shown a perturbation of ion transport in the periphery and brain during diabetes. In this study, we investigated the effect of diabetes on ion transporters in the choroid plexuses of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Sprague-Dawley rats by intraperitoneal injection of STZ (60 mg/kg in citrate buffer, confirmed by glucose analysis: 601 +/- 22 mg/dl diabetic rats, 181 +/- 46 mg/dl age-matched controls); and at 28 days, rats were killed, choroid plexuses harvested, and protein extracted. Western blot analyses were carried out using antibodies for ion transporters, including Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)-K(+)-ATPase alpha1-subunit. The efflux of the K(+) analog (86)Rb(+) from choroid plexus was also studied. Diabetic rats showed an increase in expression of the Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)-K(+)-ATPase alpha1-subunit, as compared with age-matched controls, a decrease in Na(+)-H(+) exchanger expression, and no change in Na(+)-K(+)-ATPase beta1- or beta2-subunit. The net effect of these changes was a 66% increase in (86)Rb(+) efflux from diabetic choroid plexus compared with controls. These changes in expression may affect choroid plexus ion balance and thus significantly affect CSF production in diabetic rats.
• Davis, T., Huber, J. D., Campos, C. R., Egleton, R. D., Witt, K., Guo, L., Roberts, M. J., Bentley, M. D., & Davis, T. P. (2003). Conjugation of low molecular weight poly(ethylene glycol) to biphalin enhances antinociceptive profile. Journal of pharmaceutical sciences, 92(7).
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  The objectives of this study were to examine the effect of poly(ethylene glycol) (PEG) conjugation on the tyrosine residues of biphalin to determine the proper size PEG for optimal efficacy and investigate the antinociceptive profile of PEG-biphalin against biphalin via three routes of administration. All antinociception evaluations were made using a radiant-heat tail flick analgesia meter. (2 kDa)(2) PEG-biphalin was identified as the optimal size of PEG to enhance the antinociceptive profile following intravenous administration of 685 nmol kg(-1) of biphalin or PEG-biphalin [(1 kDa)(2), (2 kDa)(2), (5 kDa)(2), (12 kDa)(2), (20 kDa)(2)]. (2 kDa)(2) PEG-biphalin displayed an area under the curve (AUC) approximately 2.5 times that of biphalin with enhanced analgesia up to 300 min postinjection. (2 kDa)(2) PEG-biphalin was equipotent to biphalin following intracerebroventricular administration (0.4 nmol kg(-1)). Both biphalin and (2 kDa)(2) PEG-biphalin were effectively antagonized with naloxone (10 mg kg(-1)) and a partial antagonistic effect was seen following pretreatment with naltrindole (20 mg kg(-1)). (2 kDa)(2) PEG-biphalin showed significantly increased potency (A(50)) when administered intravenously and subcutaneously. Additionally, (2 kDa)(2) PEG-biphalin demonstrated a significantly enhanced antinociceptive profile (AUC) via all routes of administration tested. These findings indicate that PEG conjugation to biphalin retains opioid-mediated effects observed with biphalin and is a valuable tool for eliciting potent, sustained analgesia via parenteral routes of administration.
• Davis, T., Witt, K. A., Mark, K. S., Hom, S., & Davis, T. P. (2003). Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression. American journal of physiology. Heart and circulatory physiology, 285(6).
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  Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJs) that are critical for maintaining brain homeostasis. The effects of initial reoxygenation after a hypoxic insult (H/R) on functional and molecular properties of the BBB and TJs remain unclear. In situ brain perfusion and Western blot analyses were performed to assess in vivo BBB integrity on reoxygenation after a hypoxic insult of 6% O2 for 1 h. Model conditions [blood pressure, blood gas chemistries, cerebral blood flow (CBF), and brain ATP concentration] were also assessed to ensure consistent levels and criteria for insult. In situ brain perfusion revealed that initial reoxygenation (10 min) significantly increased the uptake of [14C]sucrose into brain parenchyma. Capillary depletion and CBF analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Hypoxia with reoxygenation (10 min) produced an increase in BBB permeability with associated alterations in tight junctional protein expression. These results suggest that H/R leads to reorganization of TJs and increased paracellular diffusion at the BBB, which is not a result of increased CBF, vascular volume change, or endothelial uptake of marker. Additionally, the tight junctional protein occludin had a shift in bands that correlated with functional changes (i.e., increased permeability) without significant change in expression of claudin-3, zonula occludens-1, or actin. H/R-induced changes in the BBB may result in edema and/or associated pathological outcomes.
• Davis, T., Wolka, A. M., Huber, J. D., & Davis, T. P. (2003). Pain and the blood-brain barrier: obstacles to drug delivery. Advanced drug delivery reviews, 55(8).
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  Delivery of drugs across the blood-brain barrier has been shown to be altered during pathological states involving pain. Pain is a complex phenomenon involving immune and centrally mediated responses, as well as activation of the hypothalamic-pituitary-adrenal axis. Mediators released in response to pain have been shown to affect the structure and function of the blood-brain barrier in vitro and in vivo. These alterations in blood-brain barrier permeability and cytoarchitecture have implications in terms of drug delivery to the central nervous system, since pain and inflammation have the capacity to alter drug uptake and efflux across the blood-brain barrier. An understanding of how blood-brain barrier and central nervous system drug delivery mechanisms are altered during pathological conditions involving pain and/or inflammation is important in designing effective therapeutic regimens to treat disease.
• Davis, T., Brown, R. C., & Davis, T. P. (2002). Calcium modulation of adherens and tight junction function: a potential mechanism for blood-brain barrier disruption after stroke. Stroke; a journal of cerebral circulation, 33(6).
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  This review deals with the role of calcium in endothelial cell junctions of the blood-brain barrier (BBB). Calcium is critical for adherens junction function, but it appears that calcium is also important in regulating tight junction function necessary for the barrier characteristics of cerebral microvessels.
• Davis, T., Hau, V. S., Huber, J. D., Campos, C. R., Lipkowski, A. W., Misicka, A., & Davis, T. P. (2002). Effect of guanidino modification and proline substitution on the in vitro stability and blood-brain barrier permeability of endomorphin II. Journal of pharmaceutical sciences, 91(10).
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  Endomorphin II (ENDII), an endogenous ligand for the mu-opioid receptor, was investigated as a possible analgesic with fewer side effects than morphine. To improve CNS entry of END II, structural modification was also examined to determine whether Pro(4) substitution and cationization affected physico-chemical characteristics, blood-brain barrier (BBB) transport, and analgesic profile. END II and its Pro(4)-substituted analog, Morphiceptin (MOR), were cationized by guanidino (GU)-addition. MOR was seven times less lipophilic than END II, whereas GU-addition decreased lipophilicity of both peptides. MOR did not affect in vitro BBB permeability; however, GU-addition increased permeability of MOR by 31%. MOR decreased protein binding by 23% compared to END II, whereas GU-addition increased protein binding of both peptides by 71 and 113%, respectively. MOR increased brain t(1/2) compared to END II. GU-addition significantly increased t(1/2) of MOR and END II in both brain (sixfold and 10-fold, respectively) and serum (over 10-fold). Pro(4)-substitution and GU-addition enhanced the in vivo analgesia profiles of i.v. administered END II and MOR, but decreased i.c.v. analgesia profiles. This study demonstrates Pro(4)-substitution decreases protein binding and enhances brain stability while cationization enhances both brain and serum stability with variable effects on BBB permeability. The analgesic profiles show that both Pro(4)-substitution and cationization enhance i.v. analgesia and thus, are promising structural modifications for the development of successful opioid drugs.
• Davis, T., Hawkins, B. T., Brown, R. C., & Davis, T. P. (2002). Smoking and ischemic stroke: a role for nicotine?. Trends in pharmacological sciences, 23(2).
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  Cigarette smoking is a preventable risk factor for ischemic stroke. The mechanisms by which smoking contributes to stroke are poorly understood and the role of nicotine in this process is controversial. Although nicotine administered transdermally and orally does not appear to have as many associated health risks as do cigarettes, nicotine does have acute vasoactive and mitogenic effects on vascular tissues. Nicotine might alter the function of the blood-brain barrier and disrupt normal endothelial cell function. Some of the detrimental effects of nicotine are prevented by nicotinic acetylcholine receptor antagonists. However, recent studies indicate that nicotine might also interact with intracellular signaling pathways that are independent of acetylcholine receptors. In light of these recent developments, the impact of nicotine on cerebrovascular pathology should not be dismissed.
• Davis, T., Huber, J. D., Hau, V. S., Mark, K. S., Brown, R. C., Campos, C. R., & Davis, T. P. (2002). Viability of microvascular endothelial cells to direct exposure of formalin, lambda-carrageenan, and complete Freund's adjuvant. European journal of pharmacology, 450(3).
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  We investigated three inflammatory agents to establish if these substances elicit a direct effect on the functional and structural integrity of the blood-brain barrier. Cellular cytotoxicity and paracellular permeability were assessed in vitro using primary bovine brain microvascular endothelial cells exposed to formalin, lambda-carrageenan, or complete Freund's adjuvant for 1, 3, or 72 h, respectively. Results showed that only the highest concentration (0.025%) of formalin produced a decrease in cell viability (approximately 34%) and a significant increase in cell permeability to [(14)C]sucrose at 120 min (approximately 137%). Brain perfusion using female Sprague-Dawley rats showed no difference in paracellular permeability to [(14)C]sucrose for any inflammatory agent. Western blot analyses were performed on isolated rat brain microvessels to assess the structural integrity of blood-brain barrier tight junctions. Results indicate that expression of zonula occludens-1, occludin, claudin-1, and actin remain unchanged following intravenous exposure to inflammatory agents. This study confirms that changes seen at the blood-brain barrier following a peripheral inflammation are due to physiological responses to the given inflammatory agent and not to any direct interaction between the inflammatory agent and the brain microvasculature.
• Davis, T., Mark, K. S., & Davis, T. P. (2002). Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation. American journal of physiology. Heart and circulatory physiology, 282(4).
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  Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are critical for maintaining brain homeostasis and low permeability. Both integral (claudin-1 and occludin) and membrane-associated zonula occluden-1 and -2 (ZO-1 and ZO-2) proteins combine to form these TJ complexes that are anchored to the cytoskeletal architecture (actin). Disruptions of the BBB have been attributed to hypoxic conditions that occur with ischemic stroke, pathologies of decreased perfusion, and high-altitude exposure. The effects of hypoxia and posthypoxic reoxygenation in cerebral microvasculature and corresponding cellular mechanisms involved in disrupting the BBB remain unclear. This study examined hypoxia and posthypoxic reoxygenation effects on paracellular permeability and changes in actin and TJ proteins using primary bovine brain microvessel endothelial cells (BBMEC). Hypoxia induced a 2.6-fold increase in [(14)C]sucrose, a marker of paracellular permeability. This effect was significantly reduced (~58%) with posthypoxic reoxygenation. After hypoxia and posthypoxic reoxygenation, actin expression was increased (1.4- and 2.3-fold, respectively). Whereas little change was observed in TJ protein expression immediately after hypoxia, a twofold increase in expression was seen with posthypoxic reoxygenation. Furthermore, immunofluorescence studies showed alterations in occludin, ZO-1, and ZO-2 protein localization during hypoxia and posthypoxic reoxygenation that correlate with the observed changes in BBMEC permeability. The results of this study show hypoxia-induced changes in paracellular permeability may be due to perturbation of TJ complexes and that posthypoxic reoxygenation reverses these effects.
• Davis, T., Witt, K. A., Huber, J. D., Egleton, R. D., & Davis, T. P. (2002). Pluronic p85 block copolymer enhances opioid peptide analgesia. The Journal of pharmacology and experimental therapeutics, 303(2).
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  Peptide-based drug development is a rapidly growing field within pharmaceutical research. Nevertheless, peptides have found limited clinical use due to several physiological and pathological factors. Pluronic block copolymers represent a growing technology with the potential to enhance efficacy of peptide therapeutics. This investigation assesses Pluronic P85 (P85) and its potential to enhance opioid peptide analgesia. Two opioid peptides, [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE) and biphalin, were examined as to the benefits of P85 coadministration, above (1.0%) and below (0.01%) the critical micelle concentration, with morphine as a nonpeptide control. P85 was examined in vitro to assess blood-brain barrier uptake in association with P-glycoprotein effect, DPDPE and morphine being P-glycoprotein substrates. P85 coadministration with DPDPE and biphalin showed increased (p < 0.01) analgesia with both 0.01 and 1.0% P85. Morphine showed increased (p < 0.01) analgesia with 0.01% P85 only. This increase in analgesia is due to both an increase in peak effect, as well as a prolongation of effect. P85 increased cellular uptake of (125)I-DPDPE and [(3)H]morphine at 0.01% (p < 0.01) and 1.0% (p < 0.01 and p < 0.05, respectively). Cyclosporin-A coadministration with (125)I-DPDPE and [(3)H]morphine increased cellular uptake (p < 0.01 and p < 0.05, respectively). (125)I-DPDPE and [(3)H]morphine coadministered with 0.01% P85 and cyclosporin-A increased cellular uptake compared with control (p < 0.01) and compared with cyclosporin-A coadministration without P85 (p < 0.01 and p < 0.05, respectively). This indicates that, in addition to P-gp inhibition, 0.01% P85 increased (125)I-DPDPE and [(3)H]morphine uptake. In our examination, we determined that P85 enhanced the analgesic profile of biphalin, DPDPE, and morphine, both above and below the critical micelle concentration.
• Davis, T. P., Egleton, R. D., Gillespie, T. J., Huber, J. D., & Witt, K. A. (2001).

  Peptide drug modifications to enhance bioavailability and blood-brain barrier permeability.

  . Peptides, 22(12), 2329-43. doi:10.1016/s0196-9781(01)00537-x
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  Peptides have the potential to be potent pharmaceutical agents for the treatment of many central nervous system derived maladies. Unfortunately peptides are generally water-soluble compounds that will not enter the central nervous system, via passive diffusion, due to the existence of the blood-brain barrier. Peptides can also undergo metabolic deactivation by peptidases, thus further reducing their therapeutic benefits. In targeting peptides to the central nervous system consideration must be focused both on increasing bioavailability and enhancing brain uptake. To date multiple strategies have been examined with this focus. However, each strategy comes with its own complications and considerations. In this review we assess the strengths and weaknesses of many of the methods currently being examined to enhance peptide entry into the central nervous system.
• Hom, S., Davis, T. P., Egleton, R. D., & Huber, J. D. (2001).

  Effect of reduced flow on blood-brain barrier transport systems.

  . Brain research, 890(1), 38-48. doi:10.1016/s0006-8993(00)03027-4
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  Pathological states (i.e. stroke, cardiac arrest) can lead to reduced blood flow to the brain potentially altering blood-brain barrier (BBB) permeability and regulatory transport functions. BBB disruption leads to increased cerebrovascular permeability, an important factor in the development of ischemic brain injury and edema formation. In this study, reduced flow was investigated to determine the effects on cerebral blood flow (CBF), pressure, basal BBB permeability, and transport of insulin and K+ across the BBB. Anesthetized adult female Sprague-Dawley rats were measured at normal flow (3.1 ml min(-1)), half flow (1.5 ml min(-1)), and quarter flow (0.75 ml min(-1)), using bilateral in situ brain perfusion for 20 min followed by capillary depletion analysis. Reduction in perfusion flow rates demonstrated a modest reduction in CBF (1.27-1.56 ml min(-1) g(-1)), a decrease in pressure, and no significant effect on basal BBB permeability indicating that autoregulation remained functional. In contrast, there was a concomittant decrease in BBB transport of both insulin and K+ with reduced flow. At half and quarter flow, insulin transport was significantly reduced (R(Br)%=17.2 and R(Br)%=16.2, respectively) from control (R(Br)%=30.4). Additionally, a significant reduction in [86Rb+] was observed at quarter flow (R(Br)%=2.5) as compared to control (R(Br)%=4.8) suggesting an alteration in ion homeostasis as a result of low flow. This investigation suggests that although autoregulation maintains CBF, BBB transport mechanisms were significantly compromised in states of reduced flow. These flow alterations may have a significant impact on brain homeostasis in pathological states.
• Davis, T. P., & Egleton, R. D. (1999).

  Transport of the delta-opioid receptor agonist [D-penicillamine2,5] enkephalin across the blood-brain barrier involves transcytosis1.

  . Journal of pharmaceutical sciences, 88(4), 392-7. doi:10.1021/js980410+
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  The delta opioid receptor antagonist [D-penicillamine2,5]enkephalin (DPDPE) is an enzymatically stable peptide analogue of Met-enkephalin. DPDPE uses a saturable transport mechanism to cross the blood-brain barrier (BBB), though the exact mechanism is not fully understood. The aim of the present study was to identify the mechanism by which DPDPE enters the brain. The effect of phenylarsine oxide (PAO), an endocytosis inhibitor, on the transport of [3H]DPDPE was investigated using both in vitro and in situ transport studies. Two in vitro models of the BBB utilizing primary bovine brain microvascular endothelial cells (BBMEC) were studied. [3H]DPDPE permeability across monolayers of BBMEC grown on polycarbonate filters was studied. PAO significantly reduced the permeability of [3H]DPDPE across the monolayer. PAO also reduced the uptake of [3H]DPDPE into BBMEC cells, without affecting binding to the cells. The in situ perfusion model of the BBB was also studied, PAO reduced DPDPE uptake by the brain in a dose-dependent manner. These studies indicate that DPDPE enters the brain via an energy-dependent transcytotic mechanism.
• Davis, T. P., Koldovsky, O., Rao, R. K., & Williams, C. S. (1999).

  Effect of milk on somatostatin degradation in suckling rat jejunum in vivo.

  . Journal of pediatric gastroenterology and nutrition, 28(1), 84-94. doi:10.1097/00005176-199901000-00019
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  Somatostatin-14 is present in breast milk, and intact somatostatin-14 has been recovered from gastric lumen of infants. Studies have shown that somatostatin-14 is metabolized in the intestinal luminal contents in vitro, which could be prevented by the presence of breast milk. In this study, the effect of milk on stability of somatostatin-14 in suckling rat jejunum in vivo was examined..125I-Somatostatin-14[Tyr 11] was administered to the isolated jejunal loops in anesthetized suckling rats in the absence or presence of milk, fractions of milk, or known protease-peptidase inhibitors. Structural integrity of 125I-somatostatin-14[Tyr 11] recovered from tissues at different intervals was analyzed by gel filtration and high-performance liquid chromatography..Radioactivity rapidly disappeared from the jejunal lumen with a 50% clearance achieved by 1.2 minutes. Gel filtration and high-performance liquid chromatography analyses showed that 125I-somatostatin- 14[Tyr 11] was rapidly degraded into smaller fragments. At 1 minute, jejunal luminal radioactivity was eluted in a major peak with retention time of 42.4 minutes, along with other minor peaks (retention time, 5.6, 8.0, 10.4, and 14.4 minutes); only a trace amount of intact 125I-somatostatin-14[Tyr 11] (retention time, 44.8 minutes) was present. Coadministration of rat's milk or its soluble fraction increased the level of intact 125I-somatostatin-14[Tyr 11] in the jejunal lumen and jejunal tissue. Presence of rat's milk-casein or peptidase inhibitors (bestatin, phosphoramidon, or Bowman-Birk inhibitor), however, failed to increase the level of intact 125I-somatostatin-14[Tyr 11]..These results suggest that somatostatin-14 is rapidly degraded in the jejunal lumen of suckling rats, and that milk-borne peptidase inhibitors prevent this somatostatin-14 degradation.
• Hruby, V. J., Abbruscato, T. J., Bechowski, H. B., Davis, T. P., Egleton, R. D., Gentry, C. L., & Gillespie, T. J. (1999).

  The effect of halogenation on blood-brain barrier permeability of a novel peptide drug.

  . Peptides, 20(10), 1229-38. doi:10.1016/s0196-9781(99)00127-8
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  The utility of a drug depends on its ability to reach appropriate receptors at the target tissue and remain metabolically stable to produce the desired effect. To improve central nervous system entry of the opioid analgesic [D-Pen2, L-Pen5, Phe6] Enkephalin (DPLPE-Phe), our research group synthesized analogs that had chloro, bromo, fluoro, and iodo halogens on the para positions of the phenylalanine-4 residue. This study reports on investigation of the effect of halogenation on stability, lipophilicity, and in vitro blood-brain barrier permeability of a novel enkephalin analog DPLPE-Phe. The stability of each halogenated DPLPE-Phe analog as well as the amidated and nonamidated parent peptide was tested in plasma and brain. All peptides tested had a half-time disappearance >300 min except for DPLPE-Phe-NH2, which was found to have a half-life of 30 min in plasma. Octanol/saline distribution studies indicated addition of halogens to DPLPE-Phe-OH significantly increased lipophilicity except for p-[F-Phe4]DPLPE-Phe-OH. p-[Cl-Phe4]DPLPE-Phe-OH exhibited the most pronounced increase in lipophilicity. Para-bromo and para-chloro halogen additions significantly enhanced in vitro blood-brain barrier permeability, providing evidence for improved delivery to the central nervous system.
• Abbruscato, T. J., Davis, T. P., Egleton, R. D., & Thomas, S. A. (1998).

  Transport of opioid peptides into the central nervous system.

  . Journal of pharmaceutical sciences, 87(11), 1433-9. doi:10.1021/js980062b
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  Peptide hormones and neurotransmitters play crucial roles in the maintenance of physiological function at both the cellular and organ level. Although peptide neuropharmaceuticals have enormous potential in the treatment of disease states, the blood-brain barrier (BBB) generally prevents the entry of peptides into the brain either by enzyme degradation or by specific properties of the BBB. Peptides that act at opioid receptors are currently being designed for analgesia and to reduce the unwanted side effects associated with morphine, such as addiction and inhibition of gastric motility. It has been the focus of our group to produce stabile peptide analogues of Met-enkephalin, that lead to analgesia without side effects. In this paper we present the methodologies that have been used to elucidate the transport mechanisms of three peptides across the BBB. Using a primary endothelial cell culture model of the BBB, in situ perfusion, and kinetic analysis we show that D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) crosses the BBB via diffusion, [D-penicillamine2,5]enkephalin uses a combination of diffusion and a saturable transport mechanism, and biphalin ([Tyr-D-Ala-Gly-Phe-NH]2) uses diffusion and the large neutral amino acid carrier. Understanding BBB transport mechanisms for peptides will aid in the rational design of peptides targeted to the brain.
• Hruby, V. J., Yamamura, H. I., Weber, S. J., Slaninova, J., Misicka, A., Lipkowski, A. W., Knapp, R. J., Hruby, V. J., Davis, T. P., & Appleyard, S. M. (1998). [125I-Tyr1]biphalin binding to opioid receptors of rat brain and NG108-15 cell membranes.. Life sciences, 62(14), PL199-204. doi:10.1016/s0024-3205(98)00058-7
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  Mono iodinated analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2], both nonradioactive [I-Tyr1]biphalin and radioactive [125I-Tyr1]biphalin have been synthesized. The radioligand binding profiles of these compounds for two types of tissues, rat brain membranes, and NG108-15 cell membranes were identical to the parent biphalin. This is additional evidence for the hypothesis that biphalin behaves like a monomeric ligand and that only one intact tyrosine is necessary for high biological activity. The second tyrosine could be used for successful radioiodination which may greatly simplify biochemical and pharmacological studies of biphalin. The results of receptor binding studies show that the binding of both biphalin and [I-Tyr1]biphalin to the delta and mu opioid receptors are not independent. [125I-Tyr1]Biphalin binds to delta receptors as shown in NG108-15 cell membranes. Nevertheless, [125I]biphalin binding to delta receptors in rat brain membranes was hardly evident and mu receptor binding predominated or at least was much more readily detectable in this preparation.
• Bell, I. R., Bootzin, R. R., Davis, T. P., Hau, V. S., & Schwartz, G. E. (1997).

  Elevation of plasma beta-endorphin levels of shy elderly in response to novel laboratory experiences.

  . Behavioral medicine (Washington, D.C.), 22(4), 168-73. doi:10.1080/08964289.1997.10543550
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  Heightened psychophysiological reactivity to the novel or unfamiliar is a leading characteristic of sky or behaviorally inhibited individuals. To assess one aspect of the physiological stress response in shyness, the authors compared the morning plasma beta-endorphin levels of 15 extremely sky, healthy elderly individuals with beta-endorphin levels of 15 extremely outgoing persons on three pairs of 2 successive days. The primary finding was that sky participants exhibited significantly higher levels of beta-endorphin on the 1st days of each pair of days, compared with the 2nd days in the laboratory. No main effect for shyness or interaction between shyness and diet on endorphin levels was found. The findings are consistent with a peripheral opioid hyperreactivity to novelty in shy elderly persons. Shyness may constitute a risk factor for panic disorder in younger adults and for nasal allergies and certain cancers in older adults. Experimental design and interpretation of future studies of shy individuals' stress responses may need to consider novelty versus familiarity of the procedures and setting.
• Davis, T. P., Hruby, V. J., McKinley, B. D., Patel, D., Porreca, F., & Yamamura, H. I. (1997).

  Peptide Targeting and Delivery across the Blood−Brain Barrier Utilizing Synthetic Triglyceride Esters:  Design, Synthesis, and Bioactivity

  . Bioconjugate Chemistry, 8(3), 434-441. doi:10.1021/bc970027g
• Davis, T. P., Rounseville, M. P., & Waters, S. M. (1997).

  Effect of dopaminergic drugs on processing and degradative neuropeptidase mRNA in rat frontal cortex and caudate-putamen.

  . Brain research, 754(1-2), 28-34. doi:10.1016/s0006-8993(97)00038-3
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  Drugs which act upon central dopamine receptors alter the level, mRNA expression and in vitro degradation of neuropeptides associated with dopamine neuron regulation. Changes in the degradation of certain neuropeptides are correlated with significant alterations in the activity of specific neuropeptidases, namely aminopeptidase N (APN) and neutral endopeptidase 24.11 (NEP 24.11). In the present study, we sought to examine the molecular mechanism of neuropeptidase activity changes in response to dopaminergic drug treatment. The effects of dopaminergic drugs on the mRNA level of APN and NEP 24.11 were determined by RNase protection assays of RNA extracted from rat frontal cortex and caudate-putamen. Additionally, the effects of dopaminergic drugs on the mRNA expression for the neuropeptide processing enzymes, prohormone convertase 1 (PC1) and PC2, were determined. After 7-day administration of the dopamine receptor antagonist, haloperidol (1 mg/kg), no effect on the mRNA expression of APN, NEP 24.11, PC1 or PC2 was observed in either of the rat brain regions studied. Administration of the dopamine receptor agonist, apomorphine (5 mg/kg, bid), altered only the expression of APN mRNA in rat caudate-putamen, where the greatest effect on APN activity has been previously observed. These results suggest that alterations in other post-transcriptional events, such as mRNA translation or insertion of neuropeptidase protein into the membrane, likely play a larger role than changes in mRNA expression in the modulation of neuropeptidase activity.
• Hruby, V. J., Davis, T. P., O'brien, D. F., Porreca, F., & Yamamura, H. I. (1997).

  Design of peptides and peptidomimetics that are selective, stable and can cross membrane barriers

  . Protein Engineering, 10(3333).
• Botros, I. W., Davis, T. P., Day, R. W., Day, R., & Mania-farnell, B. L. (1996).

  Differential modulation of prohormone convertase mRNA by second messenger activators in two cholecystokinin-producing cell lines.

  . Peptides, 17(1), 47-54. doi:10.1016/0196-9781(95)02044-6
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  Regulation of prohormone convertase expression was studied in two neuropeptide-producing cell lines, the human neuroepithelioma cell line SK-N-MCIXC and the rat medullary thyroid carcinoma cell line WE 4/2. The cells were treated with the phosphodiesterase inhibitor isobutyl-methylxanthine and the tumor-promoting phorbol ester, phorbol-12-myristate-13 acetate, activators of the cyclic AMP (cAMP) and protein kinase C (PKC) second messenger pathways, respectively. mRNA levels of prohormone convertase 1 (PC1), prohormone convertase 2 (PC2), and furin were determined after 3- and 6-h treatments, using Northern analysis. Activation of both cAMP and PKC pathways increased PC1, but not PC2 or furin mRNA levels in SK-N-MCIXC cells. Activation of the cAMP pathway increased PC1, PC2, and furin mRNA levels in WE 4/2 cells, whereas activation of the PKC pathway did not change prohormone convertase mRNA levels in this cell line. These results indicate that prohormone convertases may be differentially regulated by cAMP and PKC mechanisms and regulation may be tissue specific.
• Davis, T. P., & Konkoy, C. S. (1996).

  Ectoenzymes as sites of peptide regulation.

  . Trends in pharmacological sciences, 17(8), 288-94. doi:10.1016/0165-6147(96)10036-5
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  The ectoenzyme-mediated metabolism of neuropeptides may be an important regulatory site of peptide-mediated activity. These membrane-bound, extracellularly oriented peptidases are not only responsible for inactivating peptide substrates, but also lead to the formation of metabolic fragments. Peptide fragments formed after enzymatic proteolysis have been shown to display novel bioactivity as a consequence of a shift in receptor selectivity. This example of nervous system plasticity through peptide biotransformation can have multiple consequences. Centrally acting drugs have been shown to have profound effects on peptide-mediated systems throughout the brain and spinal cord including a differential alteration in ectoenzyme activity and ectoenzyme-mediated metabolism of neuropeptides. In this review, Tom Davis and Chris Konkoy suggest that the modulation of ectoenzyme-mediated peptide metabolism represents an additional level at which the concentration of extracellular neuropeptides, and thus peptide-mediated transmission, can be regulated.
• Davis, T. P., Konkoy, C. S., & Waters, S. M. (1996).

  Haloperidol and apomorphine differentially affect neuropeptidase activity.

  . The Journal of pharmacology and experimental therapeutics, 277(1), 113-20.
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  In addition to their well characterized effects at dopamine receptors, neuroleptic drugs have been shown to affect the level and in vitro metabolism of neuropeptides. In the present study, the effect of acute and subchronic administration of the neuroleptic haloperidol and the nonselective, dopamine agonist apomorphine on neuropeptidase activity was determined in regional, rat brain P2 membranes. Subchronic administration of haloperidol decreased the activity of aminopeptidase N in the frontal cortex and caudate-putamen. In contrast, subchronic administration of apomorphine increased aminopeptidase N activity in the frontal cortex and caudate-putamen. Neutral endopeptidase 24.11 also was affected differentially in the caudate-putamen, but both subchronic haloperidol and apomorphine decreased neutral endopeptidase 24.11 activity in the frontal cortex. Metalloendopeptidase 24.15 activity was decreased in the caudate-putamen after acute haloperidol and increased in the frontal cortex after acute apomorphine administration; however, no effect was noted after subchronic administration of either drug. Angiotensin converting enzyme was not affected by any treatment. Therefore, neuroleptic-induced alterations in aminopeptidase N, neutral endopeptidase 24.11 and metalloendopeptidase 24.15 activity may account for previously reported alterations in neuropeptide degradation. In view of the interaction between mesocorticolimbic dopamine neurons and neuropeptides, e.g., substance P, neurotensin and enkephalins, neuroleptic-induced alterations in the activities of neuropeptidases, and thus neuropeptide metabolism can, in turn, play a role in modulating midbrain dopaminergic activity.
• Hom, S., Hruby, V. J., Abbruscato, T. J., Bartosz, H., Davis, T. P., Gillespie, T. J., Greene, D. L., Hau, V. S., Lipkowski, A. W., & Misicka, A. (1996).

  Enkephalin analog prodrugs: assessment of in vitro conversion, enzyme cleavage characterization and blood-brain barrier permeability.

  . The Journal of pharmacology and experimental therapeutics, 277(3), 1366-75.
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  To improve the blood-brain barrier penetration of the delta-opioid receptor peptides [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Pen2, L-Cys5]enkephalin (DPLCE), various prodrug forms were synthesized to increase lipophilicity and drug delivery to the brain. The aims of this study were 3-fold, 1) to assess the metabolic conversion of various DPDPE and DPLCE prodrugs in vitro using mouse brain homogenate and mouse serum, 2)to characterize the proteolytic enzymes responsible for cleaving prodrugs to the parent compounds using select peptidase inhibitors and 3)to assess the blood-brain barrier permeability of prodrugs, compared with their parent compounds, using the in vitro bovine brain microvessel endothelial cell culture model. The prodrugs with carboxyl-terminal phenylalanine residues (DPDPE-Phe and DPLCE-Phe) had significantly longer metabolic conversion times in both mouse serum and brain homogenates than did the prodrugs with amino-terminal phenylalanine residues. Inhibition of leucine aminopeptidase with bestatin in the serum increased the conversion time of Phe0-DPDPE from 6.8 min to 92.2 min. Inhibition of aminopeptidase M with amastatin in the brain homogenate increased the conversion time of Phe0-DPDPE from 3.9 min to > 450 min. The long half-life of DPLCE-Arg-Pro-Ala in serum (317 min) vs. brain (9.2 min) can be explained by the high levels of the degradative endopeptidase 24.15 (EC 3.4.24.15) in the central nervous system but not in plasma. The data also showed that, for specific prodrugs of DPDPE such as Phe0-DPDPE and DPDPE-Arg-Gly, the prodrug shows a significant improvement in permeability, compared with the parent compound. Therefore, these data provide evidence that prodrugs or prodrug-enzyme inhibitor combinations may optimize the delivery of peptide and/or protein drugs to the central nervous system.
• Ritenbaugh, C., Bell, I. R., Bootzin, R. R., Davis, T. P., Hau, V. S., Johnson, K. A., & Schwartz, G. E. (1996).

  Time-dependent sensitization of plasma beta-endorphin in community elderly with self-reported environmental chemical odor intolerance.

  . Biological psychiatry, 40(2), 134-43. doi:10.1016/0006-3223(95)00331-2
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  This study examined plasma beta-endorphin as a marker of the physiological stress response in community elderly who were either high (n = 15) or low (n = 15) in self-rated frequency of illness from environmental chemical odors. Individuals who report nonatopic multiple sensitivities to or intolerances for low levels of environmental chemicals also claim high rates of comorbid food sensitivities or intolerances. Subjects gave 9 AM blood samples for plasma beta-endorphin 90 min after ingesting either 1% fat cow's milk or a soy-based nondairy drink, on six different mornings in the laboratory after all-night sleep recordings. The six sessions-were divided into three sets of two successive days each, with each set [involving baseline (ad lib milk), nondairy (soy-based), and dairy diets] separated from the next by 3 weeks. In the chemically tolerant subjects, stably lower beta-endorphin levels suggested that milk may have been a physiologically less stressful beverage than was the soy drink. In contrast, the chemical odor intolerant group exhibited a) increased levels of plasma beta-endorphin averaged over the 6 days (p = .02); and b) marked fluctuations in endorphin from one laboratory day to the next (Group x Diet x Day interaction, p = .005). The findings were consistent with time-dependent, context-dependent sensitization of beta-endorphin in the chemical odor intolerant individuals.
• Chorev, M., Davis, T. P., Mayr, C. A., Rosenblatt, M., Schetz, J. A., & Taylor, J. E. (1995).

  Distribution and pharmacokinetics of a potent peptide antagonist of parathyroid hormone and parathyroid hormone-related protein in the rat.

  . The Journal of pharmacology and experimental therapeutics, 274(3), 1456-62.
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  Humoral hypercalcemia of malignancy results from the production by cancer cells of parathyroid hormone related protein that activates receptors in bone. Peptide antagonists that block parathyroid hormone receptors in vivo would be instrumental in the clinical treatment of humoral hypercalcemia of malignancy. We report the in vivo whole body distribution and blood plasma pharmacokinetics of the parathyroid hormone receptor antagonist [Nle8,18,D-Trp12,monoiodinated Tyr34]bPTH(7-34)amide to determine parameters that are likely to affect its administration regimen. A single intravenously injected dose of [Nle8,18,D-Trp12,monoiodinated Tyr34]bPTH(7-34)amide was rapidly cleared from blood plasma. The plasma concentration reaches a maximum at 10 min (Cmax = 1.93 +/- 0.27% of total injected CPM/ml), and the intact PTH derivative was detectable in plasma by HPLC analysis at this time. In vivo binding to plasma proteins was noncovalent. The peptide was rapidly cleared from blood by the liver, and more slowly by the kidney. Radiolabel was detected in excreted feces at 8 hr, but the preferred route of excretion was renal as judged by significant counts in excreted urine. Absorption of labeled peptide by skin and bone was sustained. Strong and sustained absorption also occurred in the vas deferens, seminal vesicle and hypothalamus. Given the rapid clearance of antagonist, multiple or sustained dosing schemes might be necessary to achieve the desired pharmacological effect. The high counts in liver at early time points after i.v. injection suggest that other routes of administration that do not bypass the hepatic first-pass effect would result in very low blood levels of drug.
• Hruby, V. J., Abbruscato, T. J., Brownson, E. A., & Davis, T. P. (1995).

  Conformationally constrained peptide drugs targeted at the blood-brain barrier.

  . NIDA research monograph, 154(154), 47-60.
• Hruby, V. J., Yamamura, H. I., Weber, S. J., Slaninova, J., Knapp, R. J., Hruby, V. J., Fang, S. N., & Davis, T. P. (1995). [125I]SNF 8702: a selective radioligand for CCKB receptors.. Peptides, 16(2), 221-4. doi:10.1016/0196-9781(94)00168-5
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  The CCK-8 analogue, SNF 8702, was radioiodinated. [125I]SNF 8702 showed high-affinity specific binding for both guinea pig and mouse brain tissues, whereas no specific binding was seen for guinea pig pancreatic tissue. The properties of the site labeled by [125I]SNF 8702 were characterized by binding inhibition studies for a series of CCKA and CCKB receptor ligands. The binding selectivity profile corresponded to that for the CCKB receptor. The labeled compound is stable for more than 6 weeks during storage at -20 degrees C.
• Davis, T. P., & Oakes, M. G. (1994).

  The ontogeny of enzymes involved in post-translational processing and metabolism of neuropeptides.

  . Brain research. Developmental brain research, 80(1-2), 127-36. doi:10.1016/0165-3806(94)90096-5
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  We quantitated the level of activity of several peptidases to determine if enzymes involved in the post-translational processing and metabolism of peptides are detectable and are altered developmentally in specific regions of the rat brain. Carboxypeptidase H (EC 3.4.17.10), a processing enzyme, located in chromaffin secretory granules was expressed at high levels on postnatal day 0 (P 0, birth) in hypothalamus, cortex and cerebellum (3.48, 4.98, 4.29 pmol/mg/min, respectively). An increase of activity occurred from P 0 to P 7 in both hypothalamus and cortex (7.68, 6.94) with a decrease shown in cerebellum (3.89). After P 7, activity increased by P 90 (adult) in the hypothalamus (7.65), decreased to birth levels in the cortex (4.79) and decreased below birth levels in the cerebellum (2.76). This regional pattern of carboxypeptidase H activity may signify its involvement throughout the life of the rat in the synthesis of specific regional neuropeptides important in development. Enzyme activity for the degradative enzymes, neutral endopeptidase (EC 3.4.24.11) and metallo endopeptidase (EC 3.4.24.15) did not present the same developmental pattern as seen with the processing enzyme. Neutral endopeptidase activity doubled in the hypothalamus from P 0 to P 7 (3080 pmol/mg/min) and remained constant throughout the maturation of the animal. In the cortex, activity increased significantly from P 0 to P 30 (1171) and remained at that level to P 90. In the cerebellum, activity decreased from P 0 to P 30 (320) and remained at that level to P 90 (304). At birth, metallo endopeptidase activity was highest in cortex (2702 pmol/mg/min), intermediate in hypothalamus (1658) and lowest in cerebellum (1410).(ABSTRACT TRUNCATED AT 250 WORDS)
• Hruby, V. J., Abbruscato, T. J., Bilsky, E. J., Davis, T. P., Davis, P., Harvath, R., Polt, R., Porreca, F., Szabo, L. Z., & Yamamura, H. I. (1994).

  Glycopeptide enkephalin analogues produce analgesia in mice: evidence for penetration of the blood-brain barrier.

  . Proceedings of the National Academy of Sciences of the United States of America, 91(15), 7114-8. doi:10.1073/pnas.91.15.7114
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  Most peptides have not proved useful as neuroactive drugs because they are blocked by the blood-brain barrier and do not reach their receptors within the brain. Intraperitoneally administered L-serinyl beta-D-glucoside analogues of [Met5]enkephalin (glycopeptides) have been shown to be transported across the blood-brain barrier to bind with targeted mu- and delta-opioid receptors in the mouse brain. The opioid nature of the binding has been demonstrated with intracerebroventricularly administered naloxone. Paradoxically, glucosylation decreases the lipophilicity of the peptides while promoting transport across the lipophilic endothelial layer. It is suggested that glucose transporter GLUT-1 is responsible for the transport of the peptide message. Profound and long-lasting analgesia has been observed in mice (tail-flick and hot-plate assays) with two of the glycopeptide analogues when administered intraperitoneally.
• Hruby, V. J., Porreca, F., Collins, N., Davis, T. P., Lung, F. D., Meyer, J. P., Porreca, F., & Yamamura, H. I. (1994).

  Design of peptides and peptidomimetics for delta and kappa opioid receptor subtypes

  . Regulatory Peptides, 54(1), 123-124. doi:10.1016/0167-0115(94)90420-0
• Davis, T. P., & Konings, P. N. (1993).

  Peptidases in the CNS: formation of biologically active, receptor-specific peptide fragments.

  . Critical reviews in neurobiology, 7(3-4), 163-74.
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  Peptides function as chemical signals between cells of multicellular organisms, or different organisms, via specific receptors on target cells. Many hormones, neuromodulators, and growth factors are peptides. Because there is no known reuptake system for peptides at the nerve terminal, the biological activity of peptides in the extracellular space is regulated by enzymatic degradation and extracellular metabolism. For example, angiotensin I is processed extracellularly in the lung by angiotensin-converting enzyme (ACE; E.C. 3.4.15.1), a peptidyl dipeptidase, to form the potent vasoconstrictor hormone angiotensin II. When neuropeptides are released from neurons into the extracellular space, specific peptidases also can modulate the peptidergic signal by generating smaller, biologically active fragments via products with similar or dissimilar characteristics of the parent peptide. Therefore, receptor-binding selectivity of a released peptide hormone can be regulated by peptidases. Because peptidases may play a key role in the extracellular regulation of peptidergic signaling, alterations in peptidase activities by drugs or disease states may lead to disruptions in biological homeostasis. The subject of this article is the role of peptidases in the central nervous system in the formation of biologically active, receptor-specific peptides from peptide E, beta-endorphin, neurotensin, and cholecystokinin.
• Davis, T. P., Koldovsky, O., & Rao, R. K. (1993).

  Fate of intraduodenally administered somatostatin in rats in vivo.

  . Peptides, 14(6), 1199-203. doi:10.1016/0196-9781(93)90176-h
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  Intraduodenally administered somatostatin-14 (a milk-borne peptide) has been shown to influence the pancreatic secretions in rats and dogs. To delineate the mechanism involved in the intraduodenal somatostatin-14, the fate of intraduodenal somatostatin-14 was investigated by administering [125I][Tyr11]somatostatin-14 ([125I][Tyr11]SS14) into the lumen of isolated duodenum in vivo of suckling and adult rats. At 2, 5, 10, and 30 min after administration, the radioactivity in the duodenal contents, duodenal wall, blood, liver, and kidney was measured, and the extracted radioactivity was analyzed for intact [125I][Tyr11]SS14 by reversed-phase high performance liquid chromatography. Radioactivity disappeared rapidly from the duodenal lumen with 50% of loss occurring at 2 min. No trace of intact [125I][Tyr11]SS14 was detected in any of the samples, except duodenal contents of suckling rats at 2 min, suggesting a rapid metabolism of SS14 in the duodenal lumen and a lack of duodenal absorption of intact SS14 in the rat.
• Davis, T. P., Konkoy, C. S., & Oakes, M. G. (1993).

  Chronic treatment with neuroleptics alters neutral endopeptidase 24.11 activity in rat brain regions.

  . Peptides, 14(5), 1017-20. doi:10.1016/0196-9781(93)90080-z
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  Chronic administration of neuroleptics has been shown to affect the endogenous levels, mRNA, posttranslational processing, and metabolism of neuropeptides in specific regions of rat brain. Neutral endopeptidase 24.11 (NEP) is known to metabolize a variety of neuropeptide substrates, including the enkephalins and neurotensin, thus modifying or terminating the bioactivity of such peptides. In the present study, chronic treatment with haloperidol (1 mg/kg/day, 12 days) increased NEP activity in nucleus accumbens, and chronic treatment with chlorpromazine (4 mg/kg/day, 12 days) increased NEP activity in caudate putamen. Higher dosages with either compound did not significantly alter NEP activity, and none of the treatments altered NEP activity in the hypothalamus. Chronic treatment with apomorphine (5 mg/kg/day, 12 days) decreased NEP activity in both nucleus accumbens and caudate putamen. These data suggest that chronic treatment with neuroleptic drugs may lead to regionally specific alterations in the metabolism of neuropeptides.
• Audus, K. L., Banks, W. A., & Davis, T. P. (1992).

  Permeability of the blood-brain barrier to peptides: an approach to the development of therapeutically useful analogs.

  . Peptides, 13(6), 1289-94. doi:10.1016/0196-9781(92)90037-4
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  Peptides have been shown in both in vivo and in vitro systems to cross the blood-brain barrier (BBB) and so affect function on the side contralateral to their origin. Some peptides cross primarily by transmembrane diffusion, a nonsaturable mechanism largely dependent on the lipid solubility of the peptide. Other peptides are transported by saturable systems across the BBB. These transport systems can be in the CNS to blood direction, as in the cases of Tyr-MIF-1 and methionine enkephalin, in the blood to CNS direction, as in the case of peptide T, or bidirectional, as in the case of LHRH. Other factors that also affect the amount of peptide crossing the BBB include binding in blood, volume of distribution, enzymatic resistance, and half-time disappearance from the blood. An in vitro model of the BBB has been characterized and used to confirm that peptides can cross the BBB. Results with the model agree with those obtained in vivo and have been used to study the permeability of the BBB to peptides, the effect of peptides on BBB integrity, the cellular pathway peptides and proteins use to cross the BBB, and the ability of the BBB to degrade peptides. The in vivo and in vitro methods have been used together to develop halogenated enkephalin analogs that are enzymatically resistant, cross the BBB readily to accumulate in areas of the brain rich in opiate receptors, and are powerful analgesics.(ABSTRACT TRUNCATED AT 250 WORDS)
• Culling-berglund, A. J., Davis, T. P., & Newcomb, S. A. (1990).

  Endogenous levels of beta-carotene in human buccal mucosa cells by reversed-phase high-performance liquid chromatography.

  . Journal of chromatography, 526(1), 47-58. doi:10.1016/s0378-4347(00)82482-2
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  We have developed a reversed-phase high-performance liquid chromatographic assay for the measurement of low nanogram levels of beta-carotene in a single sample of human buccal mucosa cells. The method includes a simple sonification step for cell disruption and release of the compounds into the supernatant. The limits of detection were 0.02, 0.02 and 0.07 ng/mg of protein for beta-carotene, retinol and retinol palmitate, respectively. Two patient populations were analysed. Average endogenous levels for beta-carotene normalized to protein were 0.25 ng/mg of protein (range 0.04-1.9 ng/mg, twelve patients). No evidence of endogenous retinol or retinol palmitate could be detected in the human samples. An oral dosing study of four normal individuals showed a wide variation of beta-carotene uptake. This rapid and sensitive method will enable investigators to use the non-invasive technique of buccal mucosa cell harvesting to determine cellular depot levels of beta-carotene in various patient populations.
• Culling-berglund, A., Davis, T. P., & Konings, P. N. (1990).

  Chronic haloperidol and chlorpromazine treatment alters in vitro beta-endorphin metabolism in rat brain.

  . European journal of pharmacology, 191(2), 115-28. doi:10.1016/0014-2999(90)94139-o
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  To determine if chronic haloperidol (3.0 mg/kg per day) or chlorpromazine (4.2 mg/kg per day) treatment alters central beta-endorphin metabolism, haloperidol and chlorpromazine were perfused via Alzet minipumps into male Sprague-Dawley rats for 8 days. Crude twice-washed membranes, purified synaptic plasma membranes and Golgi-enriched membranes, respectively, were isolated from rat brains and time course incubated with beta-endorphin. All samples were analyzed by high resolution, reversed-phase high performance liquid chromatography. The half-lives of beta-endorphin for animals treated with haloperidol or chlorpromazine were not statistically different from control animals at the crude washed membranes. At the purified synaptic plasma membranes, however, the half-lives of beta-endorphin from haloperidol (t 1/2 = 45.1 min)- and chlorpromazine (t1/2 = 47.0 min)-treated animals were significantly decreased as compared to the control animals (t1/2 = 78.0 min). The half-life of beta-endorphin at the Golgi-enriched membranes was increased for haloperidol (t1/2 = 112.3 min) and chlorpromazine (t1/2 = 103.0 min)-treated animals when compared to control animals (t1/2 = 80.2 min). The findings indicate a differential effect of the dopamine receptor antagonists haloperidol and chlorpromazine on the extracellular fate at the synaptic plasma membranes of beta-endorphin and the intracellular processing at the Golgi-enriched membranes in vitro.
• Davis, T. P., Louis, R. B., & Morril, M. (1990).

  Haloperidol, chlorpromazine and apomorphine alter central regional neuropeptidase activity

  . European Journal of Pharmacology, 183(6), 2318-2319. doi:10.1016/0014-2999(90)93875-q
• Alberts, D. S., Davis, T. P., Meyskens, F. L., Peng, Y. M., Plezia, P. M., Sayers, S., Surwit, E. A., & Xu, M. J. (1989).

  The role of serum and tissue pharmacology studies in the design and interpretation of chemoprevention trials.

  . Preventive medicine, 18(5), 680-7. doi:10.1016/0091-7435(89)90039-x
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  The design and interpretation of chemoprevention trials are challenging tasks. Innovative methodological approaches to these investigations are in initial stages of development. Important pharmacologic issues should be addressed as early as possible in these trials to facilitate the optimal design of large, Phase III, randomized trials. These include determining the optimal dose of the compound and the toxicity profile. Other key areas involve the use of serum concentrations to monitor subject compliance, the evaluation of concentration of the chemopreventive agent in the target tissue, adequate assessment of the drug delivery systems, and the evaluation of the relationship between the dose administered and the serum or tissue concentrations achieved. Whenever possible the investigation of the relationship between serum or tissue concentrations of a chemopreventive agent vs its biologic activity should be determined. Specific examples involving the retinoids and carotenoids are presented.
• Burgess, H. S., Crowell, S. L., & Davis, T. P. (1989).

  The effect of mycoplasma on the autocrine stimulation of human small cell lung cancer in vitro by bombesin and beta-endorphin.

  . Life sciences, 45(25), 2471-6. doi:10.1016/0024-3205(89)90013-1
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  The tumor stem cell clonogenic assay was utilized to investigate the autocrine growth response of small cell lung cancer (SCLC) to bombesin (BN) and beta-endorphin (beta-E). Mycoplasma contamination was detected in the human SCLC cell line NCl-H345 by a nucleic acid hybridization assay which detects mycoplasma ribosomal RNA. Clonogenic assays of mycoplasma (+) cells were compared to assays of the same cell line following treatment for mycoplasma. Concentrations of beta-E ranging from 0.1nM to 25nM or BN (0.1nM-100nM) were added to cells, media and agarose and applied to prepared base layers. Following incubation for 12-14 days at 37 degrees C, the degree of clonal growth stimulation was determined by colony counts greater than or equal to 42 mu. The non-infected cell population grew in the presence of 25nM BN up to 69% over control growth. The infected cells, however, did not grow more than 27% above control. In the presence of 10nM beta-E, colony counts of non-infected cells exceeded the control values by up to 187% whereas the mycoplasma (+) colonies did not grow more than 20% over the control values. These results indicate a marked reduction in the response of SCLC cell lines to the peptides BN and beta-E when infected with mycoplasma. Since infecting mycoplasma typically adhere to cellular membranes, these adherent mycoplasma may interfere with membrane receptors or alter signal transduction, thus, inhibiting the development of the autocrine response.
• Davis, T. P. (1989).

  Peptide hormones as prohormones: Processing, biological activity, pharmacology: Edited by Jean Martinez, Ellis Horwood, 1989. £45.00 (x + 354 pages) ISBN 0 7458 0222 2

  . Trends in Pharmacological Sciences, 10(10), 424. doi:10.1016/0165-6147(89)90193-4
• Davis, T. P., Gillespie, T. J., & Porreca, F. (1989).

  Peptide fragments derived from the beta-chain of hemoglobin (hemorphins) are centrally active in vivo.

  . Peptides, 10(4), 747-51. doi:10.1016/0196-9781(89)90107-1
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  A novel tetrapeptide (hemorphin-4) and pentapeptide (hemorphin-5), derived from the beta-chain of hemoglobin, were synthesized by solid-phase methodology, purified and the amino acid sequences confirmed. The central (ICV) effects of hemorphin-4 and -5 were studied in two models of phasic and tonic nociception, the mouse warm water tail-flick assay and hindpaw formalin assay, respectively. Additionally, two physiological endpoints, central modulation of bladder motility and central effects on intestinal propulsion, were studied in rats and mice, respectively. In the tail-flick assay, both peptides (40-100 nmoles) produced a dose-related naloxone-reversible antinociceptive effect when tested 10 min after peptide administration, with the tetrapeptide being slightly more potent than the pentapeptide. No effect was noted for either peptide using the tonic nociception assay, except at a dose of 150 nmoles for hemorphin-5. Inhibition of gastrointestinal propulsion was also not affected by either peptide. However, both peptides (10-40 nmoles) inhibited micturition contractions in a dose-related and naloxone-reversible fashion, with the tetrapeptide being twice as potent as the pentapeptide. These findings provide evidence that hemorphin-4 and -5 exert naloxone-reversible opioid actions in vivo and, therefore, may be physiologically important blood-borne peptides.
• Carrier, M., Copeland, J. G., Davis, T. P., Emery, R. W., & Russell, D. H. (1988).

  Urinary polyamines as markers of cardiac allograft rejection. A clinical evaluation.

  . The Journal of Thoracic and Cardiovascular Surgery, 96(5), 806-810. doi:10.1016/s0022-5223(19)35192-x
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  Histologic evaluation of endomyocardial biopsy specimens is the current method of monitoring rejection after cardiac transplantation. Unfortunately, this technique gives a discontinuous evaluation of the recipient immunologic status. A noninvasive marker of immunologic activation and of allograft rejection that would permit a more continuous monitoring than the biopsy technique would be clinically useful. Urinary polyamine excretion reflects cellular proliferation or degeneration and, as a marker of cellular metabolic activity, may also reflect lymphocyte proliferation and organ rejection. From July 1985 to December 1986, urinary polyamines were studied in 18 patients during hospitalization for heart and heart-lung transplantation. Endomyocardial biopsy was performed twice a week and histologic rejection was characterized by standard criteria. Urinary specimens were collected daily and analyzed for polyamines by high-pressure liquid chromatography. Concentrations of acetylputrescine and total urinary polyamines were significantly higher before the 20 rejection episodes than before the 80 biopsies yielding negative results. So that their clinical usefulness could be evaluated, an elevation of polyamines and a daily level variability of 28% or more was chosen to indicate increased metabolic cellular activity and to predict rejection in the next 8 days. On the basis of these definitions, the sensitivity of polyamine assays to predict rejection was 85%, the specificity 88%, and the positive predictive value 79%. Therefore, serial measurements of urinary polyamines may provide daily information on the recipient’s immunologic status after cardiac transplantation.
• Carrier, M., Copeland, J. G., Davis, T. P., Emery, R. W., & Russell, D. H. (1988).

  Value of urinary polyamines as noninvasive markers of cardiac allograft rejection in the dog.

  . The Annals of thoracic surgery, 45(2), 158-63. doi:10.1016/s0003-4975(10)62428-9
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  A noninvasive marker of cardiac allograft rejection would be useful clinically. Lymphocyte proliferation and organ rejection may cause changes in urinary polyamine excretion. To test this hypothesis, cervical heterotopic heart transplantations were performed in a group of 6 nonimmunosuppressed dogs and in a group of 9 dogs treated with cyclosporine (N = 3) or cyclosporine and steroids (N = 6). A group (N = 3) having a sham operation was also studied. Serial biopsies of the transplanted hearts were performed. Urinary polyamine levels were measured daily by high-pressure liquid chromatography of urine specimens. Between 2 and 4 days after transplantation, the transplanted hearts of all animals without immunosuppression demonstrated histological rejection. An early increase in putrescine levels and in total urinary polyamine levels was observed in this group. In the treated groups, histological rejection appeared from the second to the eighth day after transplantation. Each episode of rejection occurred from 1 day to 4 days after a significant increase in urinary polyamine levels compared with the preoperative baseline level (p less than 0.01). In contrast, polyamine excretion in 3 dogs after sham operations remained unchanged. Thus, urinary excretion of polyamines increases before the appearance of histological rejection; this suggests that changes in urinary polyamine levels may be a useful marker of cardiac allograft rejection.
• Cork, R. C., Crago, B. R., Davis, T. P., Hameroff, S. R., Weiss, J. L., & Weiss, J. L. (1987).

  Doxepin Effects on Chronic Pain and Depression: A Controlled Study

  . Survey of Anesthesiology, 31(1), 54. doi:10.1097/00132586-198702000-00051
• Culling-berglund, A. J., Davis, T. P., Gillespie, T. J., & Smith, T. L. (1987).

  Ethanol treatment alters beta-endorphin metabolism by purified synaptosomal plasma membranes.

  . Peptides, 8(3), 467-72. doi:10.1016/0196-9781(87)90011-8
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  Ethanol administration has been shown to affect beta-endorphin (beta-E) levels in most brain areas. Chronic ethanol treatment has also lead to changes in the levels of Met- and Leu-enkephalin which may be due to recent finding that enkephalin A activity is significantly altered. To determine if proteolytic enzymes responsible for beta-E metabolism at the pSPM are also altered, we studied the effect of chronic ethanol (7% v/v; 8 days) administration on in vitro central beta-E metabolism in male C57/BL mice. Purified SPM was time-course incubated with beta-E (20 microM) for 30-120 min and subjected to HPLC analyses for determination of beta-endorphin and related fragments. Chronic ethanol significantly reduced the half-life for beta-E at the pSPM (T1/2 = 50/min) versus controls (T1/2 = 100.4 min). Chronic ethanol also caused significant accumulation of the behaviorally active alpha- and gamma-type endorphins formed at the pSPM. These results suggest that chronic ethanol treatment leads to an increase in the activity of peptidases responsible for beta-E metabolism at pSPM leading to an increased formation of both alpha- and gamma-type endorphins which may affect alcohol related behaviors.
• Burks, T. F., Davis, T. P., Dray, A., & Porreca, F. (1985).

  The proenkephalin A fragment, peptide E: central processing and CNS activity in vivo.

  . European journal of pharmacology, 111(2), 177-83. doi:10.1016/0014-2999(85)90754-x
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  The proenkephalin A derivative, peptide E, delayed gastrointestinal transit in mice and inhibited the micturition reflex in anesthetized rats after intracerebroventricular (i.c.v.) administration. BAM22P, BAM12P and [Met5]enkephalin, possible processing fragments of peptide E, were also compared in the two test systems. Of these peptides, peptide E and BAM 22P were found to have the greatest potency and activity. Studies in vitro of peptide E metabolism by enzyme homogenates of mouse brain using HPLC techniques revealed that peptide E is bound to the membrane homogenate avidly for an extended period of time. The total formation of BAM22P, BAM12P, [Met5]enkephalin and all other peptide fragments during a 40 min incubation period accounted for only 8% of the total peptide E added to the homogenates. Thus, peptide E, rather than one of its known metabolites, appears to be of primary importance in the initiation of CNS-mediated effects. Further, these effects are probably the result of mu-opioid receptor activation.
• Culling-berglund, A., & Davis, T. P. (1985).

  High-performance liquid chromatographic analysis of in vitro central neuropeptide processing.

  . Journal of chromatography, 327, 279-92. doi:10.1016/s0021-9673(01)81657-5
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  Reversed-phase high-performance liquid chromatography (HPLC) was used to study and characterize the in vitro proteolytic processing of beta-endorphin by twice-washed membrane homogenates. A high-resolution method, capable of separating over 30 different human beta-endorphin-related fragments in a single analysis, was used to study the time course of production of specific, biologically active endorphin fragments by membrane-associated proteases. The results demonstrate that frozen (-37 degrees C), postmortem human and rat brains are viable for processing studies and that metabolism proceeds similarly to that in fresh brain homogenates or slices. Significant differences were noted in the formation rates of putative neuroleptic peptides between sex- and age-matched postmortem brain tissues from controls versus postmortem brain tissues from neuropsychiatric patients or drug-treated animals. These data suggest that using HPLC to characterize neuropeptide processing in human or rat membrane-associated enzyme homogenates is both descriptive and quantitative and offers insight into the central regulation of neuropeptide metabolism.
• Davis, T. P., & Dray, A. (1985).

  The proenkephalin A fragment metorphamide shows supraspinal and spinal opioid activity in vivo.

  . Peptides, 6(2), 217-21. doi:10.1016/0196-9781(85)90043-9
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  Metorphamide (Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH2) a novel amidated octapeptide fragment of proenkephalin A was synthesized, purified and subsequently shown to inhibit the reflex contractions of the rat urinary bladder following intracerebroventricular and spinal intrathecal microinjections. The effects of metorphamide were consistently antagonized by naloxone but not by the delta-opioid receptor antagonist ICI 174,864. Comparison of metorphamide with other proenkephalin A fragments suggested that the activity of this peptide was not due to in vivo processing to other active fragments. These data suggest that metorphamide has potent in vivo mu-opioid activity but little delta-opioid receptor activity.
• Hameroff, S. R., Cork, R. C., Crago, B. R., Davis, T. P., & Weiss, J. L. (1985).

  Doxepin effects on chronic pain and depression: A controlled study

  . The Clinical Journal of Pain, 1(3), 171-176. doi:10.1097/00002508-198501030-00008
• Davis, T. P. (1984).

  Heat Shock Proteins

  . BioScience, 34(9), 590-590. doi:10.2307/1309611
• Davis, T. P., & Schoemaker, H. (1984).

  Differential in vitro metabolism of beta-endorphin in schizophrenia.

  . Peptides, 5(6), 1049-54. doi:10.1016/0196-9781(84)90169-4
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  Biologically active peptide fragments derived from the proteolytic cleavage of beta-endorphin (beta E) have been shown to be present in the brain. Based on clinical results using some of these fragments in neuropsychiatric disease studies we investigated the in vitro metabolism of beta E by twice-washed membrane homogenates of postmortem putamen from sex and age matched controls versus subjects with a diagnosis of schizophrenia. The present study demonstrates that frozen (-80 degrees C) postmortem human tissues are viable for these studies and that metabolism in control tissue proceeds similarly to fresh tissues. Furthermore, a significant increase in the formation of the putative neuroleptic-like peptide fragment des-enkephalin-gamma-endorphin in postmortem schizophrenic putamen versus controls was shown. A significant decrease in the formation of beta E was also reported. These data suggest that an approach using postmortem human brain is possible in studying beta-endorphin catabolism and is therefore applicable to other neuropeptide systems.
• Davis, T. P., Dray, A., & Metsch, R. (1984).

  Endorphins and the central inhibition of urinary bladder motility.

  . Peptides, 5(3), 645-7. doi:10.1016/0196-9781(84)90097-4
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  The involvement of endogenous opioid mechanisms in the central neurogenic control of urinary bladder function has been examined in anesthetized rats. Intracerebroventricular (ICV) microinjections of beta-endorphin (0.5-2.0 micrograms) produced powerful inhibition of rhythmic bladder contractions initiated by central reflex activity. The peptide fragments gamma-endorphin and alpha-endorphin (4-16 micrograms), formed by the processing of beta-endorphin by membrane homogenates of brain, were less active than the parent compound. The inhibitory effects of beta-endorphin was reversed by ICV naloxone (1-2 micrograms) but higher doses were required to reverse gamma- or alpha-endorphin effects. ICV naloxone administered alone increased intravesicular pressure and bladder contraction frequency. These observations support the hypothesis that the endorphins have a physiological role in the central regulation of urinary bladder activity.
• Davis, T. P., Gehrke, C. W., & Johnson, H. D. (1984).

  Effect of temperature stress on circulating biogenic amines in bovine.

  . Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology, 79(2), 369-73. doi:10.1016/0742-8413(84)90216-0
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  A sensitive, simple and selective chromatographic method using high performance liquid chromatography was developed to measure circulating levels of histamine (HI), norepinephrine (NE), dopamine (DA) and serotonin (5-HT) as indicators of response to thermal stress in two breeds of cattle. Duplicate exposures for 8 hr to 13 degrees C resulted in significantly elevated plasma NE and DA in longhorns but not in Herefords and suggest a relatively greater sensitivity to the cold (13 degrees C) in the longhorn. Environmental temperatures of 32 and 42 degrees C significantly increased rectal temperatures of both breeds with much higher rectal temperatures in Herefords. The 32 and 42 degrees C effects on circulating NE and DA of Herefords were highly significant but not in the longhorn. However, heat stress significantly elevated HI and 5-HT in the longhorn which may account for their relatively greater heat tolerance based on their lower rectal temperatures.
• Hameroff, S. R., Cork, R. C., Crago, B. R., Davis, T. P., Lerman, J. C., Neuman, C. P., Watts, K. S., Weiss, J. L., & Womble, J. R. (1984).

  Doxepin??s effects on chronic pain and depression: a controlled study

  . Journal of Clinical Psychopharmacology, 4(4), 234. doi:10.1097/00004714-198408000-00024
• Alberts, D. S., Beaudry, J. N., Davis, T. P., & Peng, Y. M. (1983).

  High-performance liquid chromatography of the provitamin A beta-carotene in plasma.

  . Journal of chromatography, 273(2), 410-4. doi:10.1016/s0378-4347(00)80962-7
• Alberts, D. S., Davis, T. P., Leigh, S., Peng, Y. M., & Woodward, D. L. (1983).

  Disposition of mitoxantrone in patients.

  . Cancer treatment reviews, 10 Suppl B, 23-7. doi:10.1016/0305-7372(83)90018-x
• Davis, T. P., Dill, D. B., Goldman, A., Hillyard, S. D., & Yousef, M. K. (1983).

  Volume and composition of hand sweat of White and Black men and women in desert walks.

  . American journal of physical anthropology, 61(1), 67-73. doi:10.1002/ajpa.1330610107
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  Many investigators have sought, but failed to find, ethnic differences in the number and regional distribution of active sweat glands. In this study measurements have been made of sweat secreted on one hand and also on the whole body of Whites and Blacks walking in desert heat. Whites numbered 31 men and 27 women, ages 30 to 88 years; there were 21 Black men and 31 Black women, ages 16 to 61 years. Each walked on three occasions for 1 hour at a rate that required an oxygen consumption of about 40% of aerobic capacity. Ambient temperature ranged from 32 to 44 degrees C in 1979 and 1980; means were 38.4 degrees C in 1979 and 36.7 degrees C in 1980. There was no sweat in the gloves of many Blacks; this was true of only a few Whites. Volume of body sweat increased in both races with rate of walking; volume of hand sweat increased more in Whites than in Blacks. The Mann-Whitney test revealed that volumes of hand sweat were significantly greater for Whites than for Blacks. It was concluded that in desert walks most Whites and few Blacks sweat freely on their hands. In samples of hand sweat, Na+, K+, and Cl- were determined. Concentrations of each ion varied widely in both races, and were unrelated to race. Concentrations of Na+ and Cl- generally are somewhat higher in hand sweat than in body sweat; concentrations of K+ are much higher. It follows that the values for concentration of Na+ and Cl- reported in Table 3 probably are somewhat higher than would have been found in body sweat, and concentrations of K+ are probably much higher.
• Brothman, A. R., Davis, T. P., Duffy, J. J., & Lindell, T. J. (1982).

  Development of an antibody to actinomycin D and its application for the detection of serum levels by radioimmunoassay.

  . Cancer research, 42(3), 1184-7.
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  An antibody specific for actinomycin D (Act D) has been developed and used in a rapid, sensitive radioimmunoassay for detection of this anticancer drug in serum. The 2-amino group of the heterocyclic chromophore of Act D was covalently coupled to available free carboxyl groups of bovine serum albumin with carbodiimide. The resulting complex was then used for the production of a specific antibody to Act D in two male New Zealand rabbits. Antibody production was of sufficient titer in both rabbits to allow the development of a radioimmunoassay for the free drug which is rapid and sensitive enough to accurately measure 0.1 pmol of Act D. The antibody produced was characterized to be immunoglobulin G by virtue of its ability to bind to Protein A:Sepharose columns. With the use of Act-D analog, actinomine, the antibody was characterized to be specific for the pentapeptide portion of the molecule. Pharmacokinetic analysis of serial serum samples obtained from a patient who received the drug i.v. revealed a biphasic response with an alpha-serum half-life of 1.78 and a beta serum half-life of 34 min. An i.v. injection of Act D into a dog and assay of serum concentration revealed a similar biphasic response with an alpha serum half-life of 0.78 min and a beta-serum half-life of 208 min.
• Chen, A., Davis, T. P., Schoemaker, H., & Yamamura, H. I. (1982).

  High performance liquid chromatography of pharmacologically active amines and peptides in biological materials.

  . Life sciences, 30(12), 971-87. doi:10.1016/0024-3205(82)90515-x
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  Precise and quantitative reversed-phase high performance liquid chromatographic (HPLC) procedures are described which can be used in biogenic amine and neuropeptide research. The amine procedure was applied to various pharmacological matrices including plasma, heart tissue and brain. The use of peptide HPLC as an analytical tool for various neuropeptides is illustrated by studies on des-tyrosine-gamma-endorphin (DT gamma E) metabolism in the brain and the stability of an ACTH (ORG-2766) analogue during a chronic infusion in rats. The power of HPLC as a research tool in peptide pharmacology is described, discussed and demonstrated as an aid in the understanding of the pharmacological effects of exogenous peptides and the function of the brain.
• Hameroff, S. R., Scherer, K., Cork, R. C., Crago, B. R., Davis, T. P., Neuman, C. P., Scherer, K., & Womble, J. R. (1982).

  Doxepin effects on chronic pain, depression and plasma opioids.

  . The Journal of clinical psychiatry, 43(8 Pt 2), 22-7.
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  Thirty patients with chronic low back or cervical pain combined with clinical depression were studied in a six-week, randomized, double-blind comparison of doxepin and placebo. Dependent variables included Hamilton Depression Scores, the Clinical Global Assessment Scale, and Profile of Mood States (POMS), and subjective ratings (visual analogue scales) of pain severity, percent of time pain felt, and effect of pain on activity, muscle tension, sleep, mood, and analgesic drug consumption. Plasma levels of doxepin, desmethyldoxepin, beta-endorphin, and enkephalin-like activity were also measured. Significant improvements in the doxepin-treated group compared to the placebo group were seen in Hamilton scores, Global Assessment Scale, Profile of Mood States, percent of time pain felt, and effect of pain on sleep, muscle tension, and mood. Some improvement was observed after 1 week, although most improvement occurred at 6 weeks, when the mean doxepin dose was 2.5 mg/kg and plasma doxepin and desmethyldoxepin averaged 70 ng/ml. Nonspecific enkephalin-like activity (but not beta-endorphins) increased for the treatment group and decreased for the placebo group. The efficacy of doxepin compared with that of placebo was thus documented in several depressive and pain parameters, indicating that doxepin is a valuable treatment for patients with chronic pain and depression.
• Alberts, D. S., Davis, T. P., & Peng, Y. M. (1981).

  High performance liquid chromatography of a new anticancer drug, ADCA--physicochemical properties and pharmacokinetics.

  . Life sciences, 29(4), 361-9. doi:10.1016/0024-3205(81)90329-5
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  Abstract We have developed a simple, precise and sensitive high performance liquid chromatographic method for a new anticancer drug, ADCA, in biological samples. The assay has an average recovery of 80 ± 1.6% at physiological concentrations, a precision of
• Davis, T. P., Davis, J. O., Forrest, J. M., Freeman, R. H., Rowe, B. P., Seymour, A. A., & Williams, G. M. (1980).

  Effects of indomethacin and meclofenamate on renin release and renal hemodynamic function during chronic sodium depletion in conscious dogs.

  . Circulation research, 47(1), 99-107. doi:10.1161/01.res.47.1.99
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  We studied the control of renin release and renal hemodynamic function by administering prostaglandin synthetase inhibitors to conscious sodium-depleted dogs with blockade of the adrenergic nervous system induced by bilateral renal denervation and propranolol administration. Indomethacin (10 mg/kg) reduced plasma renin activity (PRA) by 59% from a high sodium-depleted value, but PRA was still 3 times the normal sodium-repleted level. Arterial pressure, CCr, CPAH, urine flow, and potassium excretion fell strikingly. Similar results were obtained with meclofenamate. When SQ 14,225 was given to another group of conscious, sodium-depleted dogs with adrenergic nervous system blockade, PRA increased from the high sodium-depleted level of 5.7 to 29.3 ng of Angiotensin I (AI)/ml per hour; indomethacin (10 mg/kg) appeared to reduce PRA (0.05 less than P less than 0.1) but to only 12.1 ng of AI/ ml per hour, which is 17 times the normal level. This high level of PRA after blockade of the adrenergic nervous system and injection of indomethacin suggests that important mechanisms other than norepinephrine and renal prostaglandins control renin release; it is proposed that both the renal vascular receptor and the macula densa are involved. The marked decreases in CCr and CPAH in response to indomethacin emphasize the important role of renal prostaglandins in the control of renal hemodynamic function during sodium depletion.
• Cunningham, T. D., Davis, T. P., Gehrke, C. W., Gehrke, C. W., Gerhardt, K. O., Johnson, H. D., Kuo, K. C., & Williams, C. H. (1979).

  High-performance liquid chromatographic analysis of biogenic amines in biological materials as o-phthalaldehyde derivatives.

  . Journal of chromatography, 162(3), 293-310. doi:10.1016/s0378-4347(00)81516-9
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  A remarkably sensitive, simple and selective reversed-phase high-performance liquid chromatographic (HPLC) method has been developed, allowing, for the first time, the direct measurement of histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin and tyramine in a single sample of plasma (2 ml), tissue (0.2 g), or urine. The biogenic amines were modified by pre-column derivatization with o-phthalaldehyde which stabilizes the molecules, aids in extraction, and improves HPLC detection at the nanogram level. To minimize losses during the sampling procedure a careful collection procedure was designed. We developed a simple sample cleanup in which the samples were thawed, neutralized with KOH, immediately derivatized, extracted into ethyl acetate (EtOAc) and then chromatographed by HPLC. The derivatives were stable in EtOAc for more then 24 h. Interfering amino acids were removed from the EtOAc by partitioning twice with Na2HPO4 buffer (pH 10.0). Complete separation was achieved in ca. 60--90 min on a muBondapak phenyl column using a stepwise gradient of acetonitrile and/or methanol-phosphate buffer (pH 5.1). A variable wavelength fluorometer with a 5-microliter flow-cell was used (excitation 340 nm; emission 480 nm). Linearity ranged from 200 pg to 50 ng onto the column. Precision (R.S.D.) for retention times was 1% and for derivatization and injection 2.5%. Recoveries of the seven biogenic amines from plasma spiked with 25 ng/ml averaged 70%, with a relative standard deviation of 6%. Separation studies were also done using a muBondapak C18 column. The effects of various eluents are presented. Gas-liquid chromatography was also investigated but lacked the sensitivity achieved by HPLC. The HPLC method is used routinely for the determination of biogenic amines in plasma from pigs with malignant hyperthemia and thermally stressed bovine. Significant differences in levels of biogenic amines were noted between stressed and non-stressed animals. Data on rat brain tissue samples were compared with the trihydroxyindole method and canine heart tissue was analyzed for ventricular norepinephrine and dopamine. Application of the method to urine from normal persons and a patient with a brain tumor has been demonstrated.
• Davis, T. P., Johnson, H. D., & Yousef, M. K. (1978).

  Partition of Body Fluids in the Burro

  . Journal of Wildlife Management, 42(4), 923. doi:10.2307/3800788
• Gerhardt, K. O., Williams, C. H., Cunningham, T. D., Davis, T. P., Gehrke, C. W., Gehrke, C. W., Gerhardt, K. O., Johnson, H. D., Kuo, K. C., & Williams, C. H. (1978).

  High-performance liquid-chromatographic separation and fluorescence measurement of biogenic amines in plasma, urine, and tissue.

  . Clinical Chemistry, 24(8), 1317-1324. doi:10.1093/clinchem/24.8.1317
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  We describe a high-performance liquid-chromatographic method for measuring histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin, and tyramine in plasma (2 ml), brain (0.2 g), or urine. These amines are modifed by pre-column derivatization with o-phthalaldehyde, which stabilizes the molecules, facilitates extraction, and improves detection of nanogram amounts. Before separation, samples were neutralized with KOH and immediately derivatized and extracted into ethyl acetate, in which derivatives were stable for longer than 24 h. Interfering amino acids were removed from ethyl acetate by partitioning twice with Na2HPO4 buffer (pH 10.0). Separation was complete in about 90 min on a "mu Bondapak/phenyl" column, with which a stepwise gradient of methanol/phosphate buffer (pH 5.1) was used. A variable-wavelength fluorometer was used (exciting wavelength, 340 nm; emission wavelength, 480 nm). Amount and response were linearly related from 1 to 200 pmol. Precision (CV) for retention times was 1%, for derivatization and injection 2.5%. Analytical recoveries of the seven amines from 2 ml of plasma fortified with 200 pmol averaged 65% (CV approximately 8%). Data on rat-brain tissue samples are compared with results by the trihydroxyindole method. Application of the method to urine from normal persons and a patient with a brain tumor is demonstrated.

Poster Presentations

• Ronaldson, P. T., Williams, E. I., Stanton, J. A., Betterton, R. D., & Davis, T. P. (2022, October). Endogenous Blood-Brain Barrier Transporters are Critical Determinants of Neuroprotective Drug Efficacy in Ischemic Stroke.. PharmSci360. Boston, MA: American Association of Pharmaceutical Scientists' Annual Meeting.
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  Purpose: Stroke is the fifth leading cause of death in the United States. Available treatment strategies for stroke are focused on rescuing injured brain tissue that surrounds the infarction core (i.e., the ischemic penumbra). There are only two FDA approved therapies for stroke: recombinant tissue plasminogen activator (r-tPA) and endovascular thrombectomy (EVT). These therapies promote reperfusion of ischemic brain tissue; however, reperfusion therapies are associated with exacerbation of neuronal injury and/or death. Additionally, both r-tPA therapy and EVT have a limited therapeutic window of 4 to 6 hours from the onset of stroke due to risk of hemorrhagic transformation. Therefore, there is an unmet clinical need for neuroprotective therapies that can protect neuronal tissue and promote repair in stroke. To date, drug discovery for stroke has been challenging as indicated by poor translatability of compounds from preclinical studies to successful clinical trials. In contrast, some drugs (i.e., 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins); memantine) have utility in improving functional neurological outcomes in stroke patients. This property indicates that both statins and memantine are efficiently delivered across the blood-brain barrier (BBB).  In vivo studies conducted in our laboratory have uncovered specific transport mechanisms that enable these drugs to be delivered from the systemic circulation into brain tissue: transport via endogenous BBB uptake transporters organic anion transporting polypeptide 1a4 (Oatp1a4) or organic cation transporters (Octs). The goal of this project was to show that functional expression of Oatp1a4 or Oct1/Oct2 at the BBB are required mechanisms that enable statins and memantine to be effective neuroprotective drugs for ischemic stroke. Methods: Male Sprague-Dawley rats (200-250 g) were subjected to transient middle cerebral artery occlusion (tMCAO) for 90 minutes followed by reperfusion for 24 h or 72 h. Sham-operated animals (i.e., controls) underwent the same surgical procedure except that the intraluminal suture was not inserted. Atorvastatin (20 mg/kg, i.v.) or memantine (5 mg/kg, i.v.) were injected 2 h following removal of the intraluminal suture (i.e., reperfusion). Oatp1a4, Oct1, and Oct2 protein expression were determined by western blot analysis of isolated brain microvessels. The role of Oatp-mediated transport was determined using the pharmacological Oatp inhibitor fexofenadine (3.2 mg/kg, i.v.) injected at the same time as atorvastatin. Similarly, specificity of Oct1/Oct2-mediated transport was assessed using the competitive Oct inhibitor cimetidine (15 mg/kg, i.v.) injected simultaneously with memantine. Following tMCAO, infarction volume and brain edema ratios were calculated from TTC-stained brain tissue slices. Post-stroke outcomes were assessed after tMCAO via neurological deficit scores (i.e., a scoring system modelled upon clinically relevant stroke assessments such as the modified Rankin Scale) and rotarod analysis (i.e., motor function). BBB transport properties of [3H]atorvastatin (0.3 mCi/ml) and [3H]memantine (0.5 mCi/ml) as well as paracellular “leak” (via [14C]sucrose (0.3 mCi/ml or 0.5 mCi/ml) were measured using our in situ brain perfusion technique. Results: Atorvastatin significantly reduced both infarction volume and the brain edema ratio following 24 h reperfusion while memantine decreased both parameters after 72 h reperfusion. Both drugs also improved post-stroke outcomes as determined by neurological deficit scores and rotarod analysis. In the presence of fexofenadine (3.2 mg/kg, i.v.), atorvastatin had no effect on infarction volume, brain edema, or neurocognitive performance. Similarly, cimetidine (15 mg/kg, i.v.) blocked all positive effects of memantine on stroke outcomes. Our in situ perfusion data showed that uptake of [3H]atorvastatin and [3H]memantine in ipsilateral and contralateral cerebral cortices was blocked by pharmacological inhibitors of Oatp1a4 (i.e., fexofenadine) or Oct1/Oct2 (i.e., cimetidine). In these experiments, we observed that the magnitude of neuroprotective drug uptake in ipsilateral cortex was greater than that measured in contralateral cortex under tMCAO conditions. This finding suggests that a component of blood-to-brain atorvastatin and memantine uptake results from non-selective paracellular “leak”, which we confirmed by showing increased uptake of [14C]sucrose in ipsilateral cortical tissue following tMCAO. Conclusions: This study has identified BBB transport mediated by Oatp1a4 and Oct1/Oct2 as critical mechanisms that facilitate CNS drug delivery in the setting of ischemic stroke. Specifically, we have shown for the first time that blood-to-brain transport via Oatp1a4 or Oct1/Oct2 is required for atorvastatin or memantine to exert neuroprotective effects in the ischemic brain and to promote post-stroke recovery. This effect occurred despite concurrent paracellular “leak”, which demonstrates that selective BBB transporters dominate over non-selective drug uptake pathways. Our data emphasize the need to assess brain penetration of therapeutic agents during preclinical drug development, a consideration that will likely enable new compounds for ischemic stroke to advance further in clinical trials. Overall, these results strengthen the novel and translational evidence generated by our laboratory that endogenous BBB transport systems can be targeted for CNS drug delivery, thereby providing a platform for development of novel treatment strategies for ischemic stroke. This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01-NS084941) and the American Heart Association (19TPA34910113) to PTR.
• Ronaldson, P. T., Davis, T. P., Abdullahi, W., Yang, J., Williams, E. I., Lochhead, J. J., & Betterton, R. D. (2021, October 2021). Targeting transforming growth factor- signaling to modulate organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier: Relevance to the Treatment of Ischemic Stroke.. PharmSci 360. Virtual Meeting: American Association of Pharmaceutical Scientists (AAPS).
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  Objectives: Our laboratory has shown that activation of the transforming growth factor-β (TGF-β )/Activin-like Kinase 1 (ALK1) pathway can increase functional expression of organic anion transporting peptide 1a4 (Oatp1a4) at the blood-brain barrier (BBB). This finding is relevant to treatment of ischemic stroke because 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins), drugs that have been demonstrated to be transport substrates for Oatp1a4 at the BBB, are well known to improve post-stroke functional neurological outcomes in patients. We hypothesize that TGF-β/ALK1 signaling can be targeted to control Oatp-mediated delivery of statins for stroke treatment; however, a detailed understanding of the molecular characteristics of this pathway, including the role of the TGF-β/ALK1 co-receptor endoglin, has not been elucidated. Furthermore, it is unknown if signaling can differentially regulate Oatp1a4 in different brain regions. The objective of this study is to evaluate effects of TGF-β/ALK1 signaling on Oatp1a4 functional expression in cerebral cortex, hippocampus, and cerebellum and to study the role of endoglin in mediating the TGF-β/ALK1 pathway under ischemic conditions.Methods: In vivo experiments were conducted using female Sprague-Dawley rats (200-250 g) administered with bone morphogenetic protein-9 (BMP-9; 0-5 μg/kg, i.p.), an established ALK1 agonist and/or LDN193189 (10 mg/kg, i.p.), an established ALK1 antagonist. Localization and protein expression of Oatp1a4 were examined using confocal microscopy and western blot analysis, respectively. Oatp-mediated uptake of [3H]atorvastatin was determined using our established in situ perfusion technique. In vitro experiments were conducted using an immortalized mouse brain endothelial cell line (bEnd.3) that were subjected to oxygen-glucose deprivation (OGD) conditions to study regulation of endoglin under ischemic conditions. Results: Immunostaining corresponding to Oatp1a4 was increased in rat brain microvessels isolated from animals treated with BMP-9 (1 μg/kg, i.p.), results that corroborate our previously published data. BMP-9 treatment increased Oatp1a4 protein expression in brain microvessels isolated from cerebral cortex but had no effect on Oatp1a4 expression in microvessels derived from hippocampal or cerebellar tissue. In cortical microvessels, LDN193189 (10 mg/kg, i.p.) attenuated the increase in Oatp1a4 protein expression, which confirms that TGF-β/ALK1 signaling is involved in regulation of Oatp1a4. The BMP-9-mediated increase in cortical Oatp1a4 protein corresponded with a significant enhancement in [3H]atorvastatin uptake, suggesting that TGF-β/ALK1 signaling can control delivery of statin drugs primarily to the cerebral cortex. Since we propose that targeting the TGF-/ALK1 pathway can control statin drug delivery in stroke, we performed in vitro OGD experiments in bEnd.3 cells to evaluate endoglin expression under ischemic conditions. Following OGD treatment, we observed increased expression of the TGF- signaling co-receptor endoglin (CD105).Conclusions: Our data show, for the first time, evidence for regional differences in Oatp1a4 regulation by TGF-β/ALK1 signaling in brain microvessels. These results have direct implications for CNS drug delivery as they imply that targeting the TGF-β/ALK1 pathway for improved brain delivery of statins will be effective in the cerebral cortex but not in the hippocampus or cerebellum. Increased expression of endoglin under OGD conditions provides an impetus to evaluate the molecular pharmacology of the TGF-β/ALK1 pathway in the setting of ischemia. Studies are ongoing in the laboratory to rigorously study the role of endoglin in TGF-β/ALK1-mediated regulation of Oatp1a4 at the BBB. This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01-NS084941) to PTR and the National Institute on Drug Abuse (R01-DA05181) to TPD and PTR.
• Williams, E. I., Ronaldson, P. T., Lochhead, J. J., Davis, T. P., Davis, T. P., Lochhead, J. J., Williams, E. I., & 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.
• 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., Davis, T. P., Reilly, B. G., Betterton, R. D., & Yang, J. (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.