Ken A. Witt

Blood-brain barrier tight junction permeability and ischemic stroke

The blood-brain barrier (BBB) is formed by the endothelial cells of cerebral microvessels, providing a dynamic interface between the peripheral circulation and the central nervous system. The tight junctions (TJs) between the endothelial cells serve to restrict blood-borne substances from entering the brain. Under ischemic stroke conditions decreased BBB TJ integrity results in increased paracellular permeability, directly contributing to cerebral vasogenic edema, hemorrhagic transformation, and increased mortality. This loss of TJ integrity occurs in a phasic manner, which is contingent on several interdependent mechanisms (ionic dysregulation, inflammation, oxidative and nitrosative stress, enzymatic activity, and angiogenesis). Understanding the inter-relation of these mechanisms is critical for the development of new therapies. This review focuses on those aspects of ischemic stroke impacting BBB TJ integrity and the principle regulatory pathways, respective to the phases of paracellular permeability.

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

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.

CNS Drug Delivery: Opioid Peptides and the Blood-Brain Barrier

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

Assessment of Stereoselectivity of Trimethylphenylalanine Analogues of δ-Opioid [D-Pen2,D-Pen5]-Enkephalin

Abstract : [D‐Pen2,D‐Pen5]‐Enkephalin (DPDPE) is an enzymatically stable δ‐opioid receptor‐selective peptide, which was modified by the trimethylation of the Phe4 residue to give β‐methyl‐2′,6′‐dimethylphenylalanine (TMP), resulting in four conformations : (2R,3S)‐β‐Phe‐DPDPE, (2R,3R)‐β‐Phe‐DPDPE, (2R,3S)‐β‐Phe‐DPDPE, and (2S,3R)‐β‐Phe‐DPDPE. Synthesis was by solid‐phase techniques using enantiomerically pure amino acids to give the four optically pure diastereoisomer peptides. The potency and selectivity (δ‐ versus μ‐opioid receptor) were evaluated by radioreceptor binding in rat brain, with a μ/δ ratio decrease for all TMP conformations, compared with the parent compound (DPDPE). Octanol/buffer distribution analysis showed enhanced lipophilicity of all TMP forms, with a sixfold enhancement associated with (2S,3S)‐TMP. In situ vascular perfusion in anesthetized rats showed a 1.6‐fold (p < 0.01) increase in the ratio of brain uptake for (2S,3S)‐TMP and a 1.5‐fold (p < 0.01) decrease in uptake for (2R,3R)‐TMP. Saturability of (2S,3S)‐TMP was shown (p < 0.01) against 100 μM unlabeled DPDPE, showing a shared nondiffusionary transport system. P‐glycoprotein affinity was shown in situ for the parent and (2S,3S)‐TMP (p < 0.01). Protein binding capacity of the TMP compounds in rat plasma and in situ mammalian bovine serum albumin‐Ringer showed (2R,3S)‐TMP and (2S,3R)‐TMP with the lowest degree of protein binding (p < 0.01), and (2S,3S)‐TMP and (2R,3R)‐TMP with comparable affinities to DPDPE. Analgesia, via intravenous administration, showed significantly reduced (p < 0.01) end effect and time course for (2R,3R)‐TMP, (2R,3S)‐TMP, and (2S,3R)‐TMP as compared with DPDPE. These results demonstrate that topographical modification in a conformationally restricted peptide can significantly modulate potency and receptor selectivity, binding capacity, enzymatic stability, lipophilicity, P‐glycoprotein affinity, and blood‐brain barrier permeability, resulting in a change of bioavailability, and thereby provides insight for future peptide drug design.

Age and 17β-estradiol effects on blood-brain barrier tight junction and estrogen receptor proteins in ovariectomized rats.

Age and estrogen levels alter blood-brain barrier (BBB) tight junction (TJ) regulation, impacting brain homeostasis and pathological outcomes. This examination evaluated BBB TJ and estrogen receptor (ER) protein expression changes in young (8-10 week) and middle-aged (10-12 month) ovariectomized female Fisher-344 rats with chronic 17β-estradiol or placebo treatment. Middle-aged rats showed decreased protein expression of occludin with 17β-estradiol (55 kDa band) or placebo (45, 55, 60 kDa bands) treatment compared to respective young. In young animals, 17β-estradiol treatment increased expression of the occludin 55 kDa band over placebo; however, this effect was lost in the middle-aged animals. In both young and middle-aged animals, expression of claudin-5 (23, 32 kDa bands) and ERα (66 kDa) increased with 17β-estradiol treatment, while junctional adhesion molecule-A showed no change across all groups. However, ERα expression (66 kDa) was significantly reduced in the middle-aged animals compared to young placebo treated animals. Measurement of BBB TJ permeability via in situ perfusion of (14)C-sucrose showed no change with age or treatment. Our results show that increasing age and 17β-estradiol treatment alters the expression of ERα and distinct BBB TJ protein isoforms without altering functional paracellular permeability.

Chapter Twelve - Steroids and the Blood–Brain Barrier: Therapeutic Implications

Steroids have a wide spectrum of impact, serving as fundamental regulators of nearly every physiological process within the human body. Therapeutic applications of steroids are equally broad, with a diverse range of medications and targets. Within the central nervous system (CNS), steroids influence development, memory, behavior, and disease outcomes. Moreover, steroids are well recognized as to their impact on the vascular endothelium. The blood-brain barrier (BBB) at the level of the brain microvascular endothelium serves as the principle interface between the peripheral circulation and the brain. Steroids have been identified to impact several critical properties of the BBB, including cellular efflux mechanisms, nutrient uptake, and tight junction integrity. Such actions not only influence brain homeostasis but also the delivery of CNS-targeted therapeutics. A greater understanding of the respective steroid-BBB interactions may shed further light on the differential treatment outcomes observed across CNS pathologies. In this chapter, we examine the current therapeutic implications of steroids respective to BBB structure and function, with emphasis on glucocorticoids and estrogens.

Chronic peripheral administration of somatostatin receptor subtype-4 agonist NNC 26-9100 enhances learning and memory in SAMP8 mice

Selective somatostatin receptor subtype agonists have been proposed as a means to mitigate learning and memory loss associated with Alzheimers disease. The first aim of this study evaluated blood-to-brain transport and regional brain distribution of NNC 26-9100, a selective somatostatin subtype-4 (sst4) receptor agonist. The entry rate of (131)I-NNC 26-9100 was K(i)=0.25 μl/g min, with an ~93% association with the parenchymal component. The second goal of this study was to evaluate the effect of chronic NNC 26-9100 administration (i.p.) on learning and memory, brain Aβ(x-42) levels, and protein expression of sst4 receptor and amyloid precursor protein (APP) in the senescence-accelerated mouse p8 (SAMP8) model of Alzheimers disease. Mice chronically treated with NNC 26-9100 showed improved learning (day 21) and memory (day 28) using the T-maze paradigm (20 and 200 μg). Ex vivo tissue analyses showed a decline in Aβ(x-42) levels at the 20 μg dose, while no alterations were observed in sst4 receptor or APP protein expression compared to vehicle controls. These findings indicate NNC 26-9100 is taken up into key brain regions associated with learning and memory. Furthermore, chronic administration of NNC 26-9100 improved learning and memory and decreased Aβ(x-42) brain levels. These results suggest sst4 receptor agonists may provide a viable therapy in the treatment of Alzheimers disease and other forms of cognitive impairment.

Steroids and the Blood–Brain Barrier: Therapeutic Implications

Steroids have a wide spectrum of impact, serving as fundamental regulators of nearly every physiological process within the human body. Therapeutic applications of steroids are equally broad, with a diverse range of medications and targets. Within the central nervous system (CNS), steroids influence development, memory, behavior, and disease outcomes. Moreover, steroids are well recognized as to their impact on the vascular endothelium. The blood-brain barrier (BBB) at the level of the brain microvascular endothelium serves as the principle interface between the peripheral circulation and the brain. Steroids have been identified to impact several critical properties of the BBB, including cellular efflux mechanisms, nutrient uptake, and tight junction integrity. Such actions not only influence brain homeostasis but also the delivery of CNS-targeted therapeutics. A greater understanding of the respective steroid-BBB interactions may shed further light on the differential treatment outcomes observed across CNS pathologies. In this chapter, we examine the current therapeutic implications of steroids respective to BBB structure and function, with emphasis on glucocorticoids and estrogens.

Somatostatin receptor subtype-4 agonist NNC 26–9100 decreases extracellular and intracellular Aβ1–42 trimers

Soluble amyloid β-protein (Aβ) oligomers are primary mediators of synaptic dysfunction associated with the progression of Alzheimers disease. Such Aβ oligomers exist dependent on their rates of aggregation and metabolism. Use of selective somatostatin receptor-subtype agonists have been identified as a potential means to mitigate Aβ accumulation in the brain, via regulation of the enzyme neprilysin. Herein, we first evaluated the impact of the somatostatin receptor subtype-4 agonist 1-[3-[N-(5-Bromopyridin-2-yl)-N-(3,4-dichlorobenzyl)amino]propyl]-3-[3-(1H-imidazol-4-yl)propyl]thiourea (NNC 26-9100) on learning and memory in 12-month SAMP8 mice (i.c.v. injection). NNC 26-9100 (0.2 μg-dose) was shown to enhance both learning (T-maze) and memory (object recognition) compared to vehicle controls. Cortical and hippocampal tissues were evaluated subsequent to NNC 26-9100 (0.2 μg) or vehicle administration for changes in neprilysin activity, along with protein expression of amyloid-precursor protein (APP), neprilysin, and Aβ₁₋₄₂ oligomers within respective cellular fractions (extracellular, intracellular and membrane). NNC 26-9100 increased neprilysin activity in cortical tissue, with an associated protein expression increase in the extracellular fraction and decreased in the intracellular fraction. A decrease in intracellular APP expression was found with treatment in both cortical and hippocampal tissues. NNC 26-9100 also significantly decreased expression of Aβ₁₋₄₂ trimers within both the extracellular and intracellular cortical fractions. No expression changes were found in membrane fractions for any protein. These finding suggest the potential use of selective SSTR4 agonists to mitigate toxic oligomeric forms of Aβ₁₋₄₂ in critical regions of the brain identified with learning and memory decline.

Peripheral Administration of GSK-3β Antisense Oligonucleotide Improves Learning and Memory in SAMP8 and Tg2576 Mouse Models of Alzheimer’s Disease

Glycogen synthase kinase (GSK)-3β is a multifunctional protein that has been implicated in the pathological characteristics of Alzheimers disease (AD), including the heightened levels of neurofibrillary tangles, amyloid-beta (Aβ), and neurodegeneration. We have previously shown that an antisense oligonucleotide directed at the Tyr 216 site on GSK-3β (GAO) when injected centrally can decrease GSK-3β levels, improve learning and memory, and decrease oxidative stress. In addition, we showed that GAO can cross the blood-brain barrier. Herein the impact of peripherally administered GAO in both the non-transgenic SAMP8 and transgenic Tg2576 (APPswe) models of AD were examined respective to learning and memory. Brain tissues were then evaluated for expression changes in the phosphorylated-Tyr 216 residue, which leads to GSK-3β activation, and the phosphorylated-Ser9 residue, which reduces GSK-3β activity. SAMP8 GAO-treated mice showed improved acquisition and retention using aversive T-maze, and improved declarative memory as measured by the novel object recognition (NOR) test. Expression of the phosphorylated-Tyr 216 was decreased and the phosphorylated-Ser9 was increased in GAO-treated SAMP8 mice. Tg2576 GAO-treated mice improved acquisition and retention in both the T-maze and NOR tests, with an increased phosphorylated-Ser9 GSK-3β expression. Results demonstrate that peripheral administration of GAO improves learning and memory, corresponding with alterations in GSK-3β phosphorylation state. This study supports peripherally administered GAO as a viable means to mediate GSK-3β activity within the brain and a possible treatment for AD.