Novel Somatostatin Receptor-4 Agonist SM-I-26 Mitigates Lipopolysaccharide-Induced Inflammatory Gene Expression in Microglia.

Somatostatin receptor subtype 4 (SSTR4) is expressed in BV2 microglia, suggesting that SSTR4 agonists may impact microglia function. This study assessed the high-affinity SSTR4 agonist SM-I-26 (SMI) (0 nM, 10 nM, 1000 nM) against lipopolysaccharide (LPS)-induced inflammation (0, 10 or 100 ng/ml) over 6 or 24 h in BV2 microglia. Cell viability, nitrite output and mRNA expression changes of genes associated with our target (Sstr4), inflammation (Tnf-α, Il-6, Il-1ß, inos), anti-inflammatory and anti-oxidant actions (Il-10, Catalase), and mediators of Aß binding/phagocytosis (Msr1, Cd33, Trem1, Trem2) were measured. At 6 h SMI showed no effect across all conditions. At 24 h SMI (10 and 1000 nM) upregulated Sstr4 expression under inflammatory and non-inflammatory conditions. At 24 h SMI downregulated expression of the inflammatory cytokines Tnf-α (1000 nM within all LPS concentrations) and Il-6 (10 nM within 0 and 10 ng/ml LPS). At 24 h 10 nM SMI upregulated Il-10, while 1000 nM upregulated Catalase under inflammatory and non-inflammatory conditions. At 24 h Msr1 and Cd33 were upregulated by 1000 nM SMI under non-inflammatory conditions, while Trem1 was downregulated by 10 and 1000 nM SMI under mildly inflammatory and non-inflammatory conditions. These results show that SMI had concentration and time-dependent effects on mRNA expression of genes associated with different states of microglial activation. The SMI reduced Tnf-α and Il-6 inflammatory gene expression, and increased Il-10 anti-inflammatory gene expression, identifies anti-inflammatory actions of SSTR4 agonists extend to microglia.

Age and 17ß-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 17ß-estradiol or placebo treatment. Middle-aged rats showed decreased protein expression of occludin with 17ß-estradiol (55 kDa band) or placebo (45, 55, 60 kDa bands) treatment compared to respective young. In young animals, 17ß-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 17ß-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 17ß-estradiol treatment alters the expression of ERα and distinct BBB TJ protein isoforms without altering functional paracellular permeability.

Synthesis and structure-activity relationships of 3,4,5-trisubstituted-1,2,4-triazoles: high affinity and selective somatostatin receptor-4 agonists for Alzheimer's disease treatment.

Somatostatin receptor-4 (SST4) is highly expressed in brain regions affiliated with learning and memory. SST4 agonist treatment may act to mitigate Alzheimer's disease (AD) pathology. An integrated approach to SST4 agonist lead optimization is presented herein. High affinity and selective agonists with biological efficacy were identified through iterative cycles of a structure-based design strategy encompassing computational methods, chemistry, and preclinical pharmacology. 1,2,4-Triazole derivatives of our previously reported hit (4) showed enhanced SST4 binding affinity, activity, and selectivity. Thirty-five compounds showed low nanomolar range SST4 binding affinity, 12 having a K i < 1 nM. These compounds showed >500-fold affinity for SST4 as compared to SST2A. SST4 activities were consistent with the respective SST4 binding affinities (EC50 < 10 nM for 34 compounds). Compound 208 (SST4 K i = 0.7 nM; EC50 = 2.5 nM; >600-fold selectivity over SST2A) display a favorable physiochemical profile, and was advanced to learning and memory behavior evaluations in the senescence accelerated mouse-prone 8 model of AD-related cognitive decline. Chronic administration enhanced learning with i.p. dosing (1 mg kg-1) compared to vehicle. Chronic administration enhanced memory with both i.p. (0.01, 0.1, 1 mg kg-1) and oral (0.01, 10 mg kg-1) dosing compared to vehicle. This study identified a novel series of SST4 agonists with high affinity, selectivity, and biological activity that may be useful in the treatment of AD.

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.

Mfsd2a and Glut1 Brain Nutrient Transporters Expression Increase with 32-Week Low and High Lard Compared with Fish-Oil Dietary Treatment in C57Bl/6 Mice.

BACKGROUND: Diet-mediated alterations of critical brain nutrient transporters, major facilitator super family domain-containing 2a (Mfsd2a) and glucose transporter 1 (Glut1), have wide reaching implications in brain health and disease. OBJECTIVE: The aim of the study was to examine the impact of long-term low- and high-fat diets with lard or fish oil on critical brain nutrient transporters, Mfsd2a and Glut1. METHODS: Eight-week-old male C57BL/6 mice were fed 1 of the following 4 diets for 32 wk: 10% of kcal from lard, 10% of kcal from fish oil, 41% of kcal from lard, or 41% of kcal from fish oil. Body weight and blood chemistries delineated dietary effects. Cortical and subcortical Mfsd2a and Glut1 mRNA and protein expression were evaluated, with other supportive nutrient-sensitive targets also assessed for mRNA expression changes. RESULTS: Fish-oil diets increased cortical Mfsd2a mRNA expression compared with lard diets. Subcortical Mfsd2a mRNA expression decreased as the percentage of fat in the diet increased. There was an interaction between the type and percentage of fat with cortical and subcortical Mfsd2a and cortical Glut1 protein expression. In the lard diet groups, protein expression of cortical and subcortical Mfsd2a and cortical Glut1 significantly increased as fat percentage increased. As the fat percentage increased in the fish-oil diet groups, protein expression of cortical and subcortical Mfsd2a and cortical Glut1 did not change. When comparing the fish-oil groups with 10% lard, cortical Mfsd2a protein expression was significantly higher in the 10% and 41% fish-oil groups, whereas cortical Glut1 protein expression was significantly higher in only the 10% fish-oil group. A positive correlation between cortical peroxisome proliferator-activated receptor γ mRNA expression and Mfsd2a protein expression was shown. CONCLUSION: Corresponding to chronic dietary treatment, an interaction between the type of fat and the percentage of fat exists respective to changes in brain expression of the key nutrient transporters Mfsd2a and Glut1.

Somatostatin sst4 ligands: chemistry and pharmacology.

Several classes of compounds (thioureas, ureas, beta-glucosides, sulfonamides, and cyclic peptides) show enhanced binding affinity and selectivity at somatostatin subtype 4 receptors (sst4). Pharmacophore models have recently been proposed to explain receptor subtype selectivity. The chemistry and therapeutic potential of sst4 ligands will be the subject of this review.

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

Glycogen synthase kinase (GSK)-3ß is a multifunctional protein that has been implicated in the pathological characteristics of Alzheimer's disease (AD), including the heightened levels of neurofibrillary tangles, amyloid-beta (Aß), and neurodegeneration. We have previously shown that an antisense oligonucleotide directed at the Tyr 216 site on GSK-3ß (GAO) when injected centrally can decrease GSK-3ß 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-3ß activation, and the phosphorylated-Ser9 residue, which reduces GSK-3ß 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-3ß expression. Results demonstrate that peripheral administration of GAO improves learning and memory, corresponding with alterations in GSK-3ß phosphorylation state. This study supports peripherally administered GAO as a viable means to mediate GSK-3ß activity within the brain and a possible treatment for AD.

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 mitigates the effect of soluble Aß(42) oligomers via a metalloproteinase-dependent mechanism.

Soluble amyloid-ß peptide (Aß) oligomers have been hypothesized to be primary mediators of Alzheimer's disease progression. In this regard, reduction of soluble Aß-oligomers levels within the brain may provide a viable means in which to treat the disease. Somatostatin receptor subtype-4 (SSTR4) agonists have been proposed to reduce Aß levels in the brain via enhancement of enzymatic degradation. Herein we evaluated the effect of selective SSTR4 agonist NNC 26-9100 on the changes in learning and soluble Aß42 oligomer brain content with and without co-administration of the M13-metalloproteinase family enzyme-inhibitor phosphoramidon, using the senescence-accelerated mouse prone-8 (SAMP8) model. NNC 26-9100 treatment (0.2 µg i.c.v. in 2 µL) improved learning, which was blocked by phosphoramidon (1 and 10mM, respectively). NNC 26-9100 decreased total soluble Aß42, an effect which was blocked by phosphoramidon (10mM). Extracellular, intracellular, and membrane fractions were then isolated from cortical tissue and assessed for soluble oligomer alterations. NNC 26-9100 decreased the Aß42 trimeric (12 kDa) form within the extracellular and intracellular fractions, and produced a band-split effect of the Aß42 hexameric (25 kDa) form within the extracellular fraction. These effects were also blocked by phosphoramdon (1 and 10mM, respectively). Subsequent evaluation of NNC 26-9100 in APPswe Tg2576 transgenic mice showed a similar learning improvement and corresponding reduction in soluble Aß42 oligomers within extracellular, intracellular, and membrane fractions. These data support the hypothesis that NNC 26-9100 reduces soluble Aß42 oligomers and enhances learning through a phosphoramidon-sensitive metalloproteinase-dependent mechanism.

Somatostatin receptor subtype-4 agonist NNC 26-9100 decreases extracellular and intracellular Aß1₋42 trimers.

Soluble amyloid ß-protein (Aß) oligomers are primary mediators of synaptic dysfunction associated with the progression of Alzheimer's disease. Such Aß 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 Aß 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 Aß1₋42 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 Aß1₋42 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 Aß1₋42 in critical regions of the brain identified with learning and memory decline.

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 Alzheimer's 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 Aß(x-42) levels, and protein expression of sst4 receptor and amyloid precursor protein (APP) in the senescence-accelerated mouse p8 (SAMP8) model of Alzheimer's 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 Aß(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 Aß(x-42) brain levels. These results suggest sst4 receptor agonists may provide a viable therapy in the treatment of Alzheimer's disease and other forms of cognitive impairment.

Reoxygenation stress on blood-brain barrier paracellular permeability and edema in the rat.

The blood-brain barrier (BBB) serves as a critical regulator of brain homeostasis. Following hypoxia (i.e. 6% oxygen/1 h) and reoxygenation (H/R), the BBB tight junctional complex is disrupted, resulting in increased BBB permeability and the development of vasogenic brain edema. In this study, we examined the effect of H/R on the in vivo rat BBB over a 36 h time course in conjunction with paracellular permeability, gray matter edema, and systemic inflammatory activity. A biphasic increase was observed in the brain uptake of (14)C-sucrose, a paracellular permeability marker; with the first increase at the 10 min reoxygenation time point, and the second increase at the 6-18 h time points. Increased brain water weight gain (edema) also showed a biphasic response with the first increase at the 10 min-1 h reoxygenation time points; and the second increase at only the 24 h time point. Analysis of serum derived cytokines (IL-1beta, TNFalpha, IL-6, IL-10, and IFNgamma) demonstrated that only IL-1beta and IL-6 were at detectable levels, but these levels were similar to controls. White blood cell counts showed significant decreases in lymphocytes (10 min-3 h), increases in monocytes (10 min-3 h and 12 h), and increases in polymorphonuclear cells (1 h and 3 h). We have shown that H/R elicits a biphasic increase in paracellular permeability and edema, which parallel to post-stroke sequelae, despite the lack of occlusion or complete depletion of oxygen.

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.

Hypoxia-inducible factor and nuclear factor kappa-B activation in blood-brain barrier endothelium under hypoxic/reoxygenation stress.

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.

Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression.

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.

Pluronic p85 block copolymer enhances opioid peptide analgesia.

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.