Brain uptake and distribution patterns of 2-hydroxypropyl-ß-cyclodextrin after intrathecal and intranasal administration.

OBJECTIVES: Cyclodextrins are increasingly used therapeutically. For example, 2-hydroxypropyl-ß-cyclodextrin (kleptose) is used for the treatment of Niemann-Pick disease. Kleptose crosses the blood-brain barrier poorly, in part because of a central nervous system (CNS)-to-blood (efflux) transporter, and so is administered by the intrathecal (IT) route in the treatment of Niemann-Pick disease. METHODS: Here, we evaluated the uptake and distribution of kleptose by the brain and spinal cord after intranasal (IN) or IT delivery. KEY FINDINGS: Kleptose distributed to all regions of the brain and spinal cord after IN administration, with only 3.27% of the administered dose entering the bloodstream. Intrathecal delivery produced levels in the whole brain about 40 times higher than intranasal administration and about 20 times higher than previously found after intravenous administration. About 70-90% of the IT dose rapidly entered the bloodstream, in part because of the previously described efflux transporter. The uptake by CNS after IN and IT was both non-saturable. The uptake by the olfactory bulb, hypothalamus and pons-medulla was favoured by all routes of administration. CONCLUSIONS: Kleptose was taken up by all regions of the CNS after either IN or IT administration, but IN administration resulted in only a fraction of the uptake of the IT route.

Characterization of systemic immunosuppression by IDH mutant glioma small extracellular vesicles.

BACKGROUND: Gliomas are the most common primary brain tumors and are universally fatal. Mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) define a distinct glioma subtype associated with an immunosuppressive tumor microenvironment. Mechanisms underlying systemic immunosuppression in IDH mutant (mutIDH) gliomas are largely unknown. Here, we define genotype-specific local and systemic tumor immunomodulatory functions of tumor-derived glioma small extracellular vesicles (TEX). METHODS: TEX produced by human and murine wildtype and mutant IDH glioma cells (wtIDH and mutIDH, respectively) were isolated by size exclusion chromatography (SEC). TEX morphology, size, quantity, molecular profiles and biodistribution were characterized. TEX were injected into naive and tumor-bearing mice, and the local and systemic immune microenvironment composition was characterized. RESULTS: Using in vitro and in vivo glioma models, we show that mutIDH TEX are more numerous, possess distinct morphological features and are more immunosuppressive than wtIDH TEX. mutIDH TEX cargo mimics their parental cells, and induces systemic immune suppression in naive and tumor-bearing mice. TEX derived from mutIDH gliomas and injected into wtIDH tumor-bearing mice reduce tumor-infiltrating effector lymphocytes, dendritic cells and macrophages, and increase circulating monocytes. Astonishingly, mutIDH TEX injected into brain tumor-bearing syngeneic mice accelerate tumor growth and increase mortality compared with wtIDH TEX. CONCLUSIONS: Targeting of mutIDH TEX represents a novel therapeutic approach in gliomas.

Transport of Extracellular Vesicles across the Blood-Brain Barrier: Brain Pharmacokinetics and Effects of Inflammation.

Extracellular vesicles can cross the blood-brain barrier (BBB), but little is known about passage. Here, we used multiple-time regression analysis to examine the ability of 10 exosome populations derived from mouse, human, cancerous, and non-cancerous cell lines to cross the BBB. All crossed the BBB, but rates varied over 10-fold. Lipopolysaccharide (LPS), an activator of the innate immune system, enhanced uptake independently of BBB disruption for six exosomes and decreased uptake for one. Wheatgerm agglutinin (WGA) modulated transport of five exosome populations, suggesting passage by adsorptive transcytosis. Mannose 6-phosphate inhibited uptake of J774A.1, demonstrating that its BBB transporter is the mannose 6-phosphate receptor. Uptake rates, patterns, and effects of LPS or WGA were not predicted by exosome source (mouse vs. human) or cancer status of the cell lines. The cell surface proteins CD46, AVß6, AVß3, and ICAM-1 were variably expressed but not predictive of transport rate nor responses to LPS or WGA. A brain-to-blood efflux mechanism variably affected CNS retention and explains how CNS-derived exosomes enter blood. In summary, all exosomes tested here readily crossed the BBB, but at varying rates and by a variety of vesicular-mediated mechanisms involving specific transporters, adsorptive transcytosis, and a brain-to-blood efflux system.

Modest Blood-Brain Barrier Permeability of the Cyclodextrin Kleptose: Modification by Efflux and Luminal Surface Binding.

Cyclodextrins (CDs) have a variety of uses from acting as excipients to aiding the ability of lipid soluble drugs to cross the blood-brain barrier (BBB). They are being investigated as an active pharmaceutical ingredient, most recently for the treatment of Niemann-Pick disease, a lysosomal storage disease. Cyclodextrins are helpful in animal models and human subjects/patients afflicted with Neimann-Pick disease, including improving the neurologic component of the disease. The improvement in brain disease by intravenous administration implies that CDs can cross the BBB; however, there are only a few studies that have directly addressed this. In the current studies, multiple-time regression analysis indicated that 2-hydroxypropyl-ß-cyclodextrin [Kleptose (Klep)] radioactively labeled with 14C (C-Klep) crossed the BBB at a slow rate by a nonsaturable mechanism consistent with transcellular diffusion. However, the rate of transport varied greatly by the brain region with no detectable uptake by the spinal cord; additionally, many regions rapidly reached equilibrium between the brain and blood. The presence of a brain-to-blood efflux system was also detected and much of the C-Klep did not completely cross the BBB, but loosely adhered to the luminal surface of brain endothelial cells. Peripheral tissues also took up C-Klep, with the kidney taking up the most, which is consistent with renal clearance. In conclusion, we demonstrated minimal uptake of the ß-cyclodextrin Kleptose by the brain with accumulation being affected by efflux and reversible luminal binding. SIGNIFICANCE STATEMENT: This cyclodextrin, which produces therapeutic effects on the central nervous system after peripheral administration, penetrates the BBB poorly. Uptake by the brain to a therapeutic level will likely be difficult to achieve without giving high peripheral doses, bypassing the BBB, or otherwise altering penetration into the brain.

Resistance to the sympathoexcitatory effects of insulin and leptin in late pregnant rats.

KEY POINTS: Pregnancy increases sympathetic nerve activity (SNA), although the mechanisms responsible for this remain unknown. We tested whether insulin or leptin, two sympathoexcitatory hormones increased during pregnancy, contribute to this. Transport of insulin across the blood-brain barrier in some brain regions, and into the cerebrospinal fluid (CSF), was increased, although brain insulin degradation was also increased. As a result, brain and CSF insulin levels were not different between pregnant and non-pregnant rats. The sympathoexcitatory responses to insulin and leptin were abolished in pregnant rats. Blockade of arcuate nucleus insulin receptors did not lower SNA in pregnant or non-pregnant rats. Collectively, these data suggest that pregnancy renders the brain resistant to the sympathoexcitatory effects of insulin and leptin, and that these hormones do not mediate pregnancy-induced sympathoexcitation. Increased muscle SNA stimulates glucose uptake. Therefore, during pregnancy, peripheral insulin resistance coupled with blunted insulin- and leptin-induced sympathoexcitation ensures adequate delivery of glucose to the fetus. ABSTRACT: Pregnancy increases basal sympathetic nerve activity (SNA), although the mechanism responsible for this remains unknown. Insulin and leptin are two sympathoexcitatory hormones that increase during pregnancy, yet, pregnancy impairs central insulin- and leptin-induced signalling. Therefore, to test whether insulin or leptin contribute to basal sympathoexcitation or, instead, whether pregnancy induces resistance to the sympathoexcitatory effects of insulin and leptin, we investigated α-chloralose anaesthetized late pregnant rats, which exhibited increases in lumbar SNA (LSNA), splanchnic SNA and heart rate (HR) compared to non-pregnant animals. In pregnant rats, transport of insulin into cerebrospinal fluid and across the blood-brain barrier in some brain regions increased, although brain insulin degradation was also increased; brain and cerebrospinal fluid insulin levels were not different between pregnant and non-pregnant rats. Although i.c.v. insulin increased LSNA and HR and baroreflex control of LSNA and HR in non-pregnant rats, these effects were abolished in pregnant rats. In parallel, pregnancy completely prevented the actions of leptin with respect to increasing lumbar, splanchnic and renal SNA, as well as baroreflex control of SNA. Blockade of insulin receptors (with S961) in the arcuate nucleus, the site of action of insulin, did not decrease LSNA in pregnant rats, despite blocking the effects of exogenous insulin. Thus, pregnancy is associated with central resistance to insulin and leptin, and these hormones are not responsible for the increased basal SNA of pregnancy. Because increases in LSNA to skeletal muscle stimulates glucose uptake, blunted insulin- and leptin-induced sympathoexcitation reinforces systemic insulin resistance, thereby increasing the delivery of glucose to the fetus.

Genetics and sex influence peripheral and central innate immune responses and blood-brain barrier integrity.

Lipopolysaccharide (LPS) is a stimulator of the innate immune system and is routinely used in animal models to study blood-brain barrier (BBB) dysfunction under inflammatory conditions. It is appreciated that both humans and mice have sexually dimorphic immune responses, which could influence the brain's response to a systemic inflammatory insult. Mouse strain is also an important factor that can contribute to pathophysiological responses to inflammatory stimuli. Therefore, we aimed to test whether BBB disruption and the associated cytokine profiles in response to LPS differed in male and female mice from two mouse strains most commonly used in blood-brain barrier studies: CD-1 and C57BL6/J (C57). Mice were treated with saline, a single injection of 0.3, or 3mg/kg LPS, or three injections of 3mg/kg LPS, and studied 28 hours after the first LPS injection. To assay BBB disruption, we utilized the tracer 99mTc-DTPA. A 23-plex panel of cytokines was assayed in brain and blood of the same cohort of mice, which allowed us to compare differences in the levels of individual cytokines as well as correlations among cytokines and 99mTc-DTPA uptake. We found that only the three-injection dose of LPS induced significant BBB disruption in all sexes and strains. The treatment, strain, and sex, as well as treatment-by- strain and treatment-by-sex interactions significantly contributed to the variance. The mean brain/serum ratios of 99mTc-DTPA in the three-injection LPS group were ranked CD-1 male < CD-1 female < C57 male < C57 female. There were significant sex and strain differences in cytokine profiles in brain and blood, and pro-inflammatory cytokines and chemokines in brain were most strongly correlated with 99mTc-DTPA brain/serum ratios.

Blast exposure elicits blood-brain barrier disruption and repair mediated by tight junction integrity and nitric oxide dependent processes.

Mild blast-induced traumatic brain injury (TBI) is associated with blood-brain barrier (BBB) disruption. However, the mechanisms whereby blast disrupts BBB integrity are not well understood. To address this issue BBB permeability to peripherally injected 14C-sucrose and 99mTc-albumin was quantified in ten brain regions at time points ranging from 0.25 to 72 hours. In mice, repetitive (2X) blast provoked BBB permeability to 14C-sucrose that persisted in specific brain regions from 0.25 to 72 hours. However, 99mTc-albumin revealed biphasic BBB disruption (open-closed-open) over the same interval, which was most pronounced in frontal cortex and hippocampus. This indicates that blast initiates interacting BBB disruption and reparative processes in specific brain regions. Further investigation of delayed (72 hour) BBB disruption revealed that claudin-5 (CLD5) expression was disrupted specifically in the hippocampus, but not in dorsal striatum, a brain region that showed no blast-induced BBB permeability to sucrose or albumin. In addition, we found that delayed BBB permeability and disrupted CLD5 expression were blocked by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). These data argue that latent nitric oxide-dependent signaling pathways initiate processes that result in delayed BBB disruption, which are manifested in a brain-region specific manner.

Effect of controlled cortical impact on the passage of pituitary adenylate cyclase activating polypeptide (PACAP) across the blood-brain barrier.

Injuries to the central nervous system can affect the blood-brain barrier (BBB), including disruption and influencing peptide transport across the BBB. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) is a potent neurotrophic and neuroprotective peptide currently being investigated for its therapeutic role following injury to the central nervous system and can cross the BBB in a saturable manner. The goal of the current study was to investigate for the first time PACAP38 uptake by the brain following traumatic brain injury (TBI). Using radioactively labeled PACAP38, we measured the levels of PACAP38 present in the injured, ipsilateral cortex in Sham-treated mice compared to mice receiving a controlled cortical impact (CCI), a model of TBI. Experiments were conducted at 6 different time points (from 2h up to 4 weeks) following CCI to determine temporal changes in PACAP38 transport. PACAP38 uptake was increased at 2 and 72h post-CCI compared to Sham. We did not detect changes in PACAP38 uptake in the contralateral cortex and cerebellum between Sham and CCI-treatment. The rate of PACAP38 transport into the ipsilateral cortex following CCI was increased 3.6-fold 72h after compared to 2h post-CCI. In addition, the rate of transport into the cerebellum was greater than that of the cortices. The data presented here shows PACAP38 transport is temporally altered following CCI-treatment and PACAP38 uptake is greater in the cerebellum compared to the cortices.

Macrophage exosomes as natural nanocarriers for protein delivery to inflamed brain.

Recent work has stimulated interest in the use of exosomes as nanocarriers for delivery of small drugs, RNAs, and proteins to the central nervous system (CNS). To overcome the blood-brain barrier (BBB), exosomes were modified with brain homing peptides that target brain endothelium but likely to increase immune response. Here for the first time we demonstrate that there is no need for such modification to penetrate the BBB in mammals. The naïve macrophage (Mϕ) exosomes can utilize, 1) on the one hand, the integrin lymphocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), and, 2) on the other hand, the carbohydrate-binding C-type lectin receptors, to interact with brain microvessel endothelial cells comprising the BBB. Notably, upregulation of ICAM-1, a common process in inflammation, promotes Mϕ exosomes uptake in the BBB cells. We further demonstrate in vivo that naïve Mϕ exosomes, after intravenous (IV) administration, cross the BBB and deliver a cargo protein, the brain derived neurotrophic factor (BDNF), to the brain. This delivery is enhanced in the presence of brain inflammation, a condition often present in CNS diseases. Taken together, the findings are of interest to basic science and possible use of Mϕ-derived exosomes as nanocarriers for brain delivery of therapeutic proteins to treat CNS diseases.

Intranasal delivery of N-terminal modified leptin-pluronic conjugate for treatment of obesity.

Leptin is an adipocyte-secreted hormone that is delivered via a specific transport system across the blood-brain barrier (BBB) to the brain where it acts on the hypothalamus receptors to control appetite and thermogenesis. Peripheral resistance to leptin due to its impaired brain delivery prevents therapeutic use of leptin in overweight and moderately obese patients. To address this problem, we modified the N-terminal amine of leptin with Pluronic P85 (LepNP85) and administered this conjugate intranasally using the nose-to-brain (INB) route to bypass the BBB. We compared this conjugate with the native leptin, the N-terminal leptin conjugate with poly(ethylene glycol) (LepNPEG5K), and two conjugates of leptin with Pluronic P85 attached randomly to the lysine amino groups of the hormone. Compared to the random conjugates of leptin with P85, LepNP85 has shown higher affinity upon binding with the leptin receptor, and similarly to native hormone activated hypothalamus receptors after direct injection into brain. After INB delivery, LepNP85 conjugate was transported to the brain and accumulated in the hypothalamus and hippocampus to a greater extent than the native leptin and LepNPEG5K and activated leptin receptors in hypothalamus at lower dose than native leptin. Our work suggests that LepNP85 can access the brain directly after INB delivery and confirms our hypothesis that the improvement in brain accumulation of this conjugate is due to its enhanced brain absorption. In conclusion, the LepNP85 with optimized conjugation chemistry is a promising candidate for treatment of obesity.

Tau Proteins Cross the Blood-Brain Barrier.

Tauopathies are a hallmark of many neurodegenerative diseases, including Alzheimer's disease and traumatic brain injuries. It has been demonstrated that amyloid-beta peptides, alpha-synuclein, and prion proteins cross the blood-brain barrier (BBB), contributing to their abilities to induce disease. Very little is known about whether tau proteins can cross the BBB. Here we systematically characterized several key forms of tau proteins to cross the BBB, including Tau-441 (2N4R), Tau-410 (2N3R), truncated tau 151-391 (0N4R), and truncated tau 121-227. All of these tau proteins crossed the BBB readily and bidirectonally; however, only Tau-410 had a saturable component to its influx. The tau proteins also entered the blood after their injection into the brain, with Tau 121-227 having the slowest exit from brain. The tau proteins varied in regards to their enzymatic stability in brain and blood and in their peripheral pharmacokinetics. These results show that blood-borne tau proteins could contribute to brain tauopathies. The result also suggest that the CNS can contribute to blood levels of tau, raising the possibility that, as suggested for other misfolded proteins, blood levels of tau proteins could be used as a biomarker of CNS disease.

CNS tau efflux via exosomes is likely increased in Parkinson's disease but not in Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) and Parkinson's disease (PD) involve tau pathology. Tau is detectable in blood, but its clearance from neuronal cells and the brain is poorly understood. METHODS: Tau efflux from the brain to the blood was evaluated by administering radioactively labeled and unlabeled tau intracerebroventricularly in wild-type and tau knock-out mice, respectively. Central nervous system (CNS)-derived tau in L1CAM-containing exosomes was further characterized extensively in human plasma, including by single molecule array technology with 303 subjects. RESULTS: The efflux of Tau, including a fraction via CNS-derived L1CAM exosomes, was observed in mice. In human plasma, tau was explicitly identified within L1CAM exosomes. In contrast to AD patients, L1CAM exosomal tau was significantly higher in PD patients than controls and correlated with cerebrospinal fluid tau. CONCLUSIONS: Tau is readily transported from the brain to the blood. The mechanisms of CNS tau efflux are likely different between AD and PD.

Central Nervous System Delivery of Intranasal Insulin: Mechanisms of Uptake and Effects on Cognition.

Intranasal insulin has shown efficacy in patients with Alzheimer's disease (AD), but there are no preclinical studies determining whether or how it reaches the brain. Here, we showed that insulin applied at the level of the cribriform plate via the nasal route quickly distributed throughout the brain and reversed learning and memory deficits in an AD mouse model. Intranasal insulin entered the blood stream poorly and had no peripheral metabolic effects. Uptake into the brain from the cribriform plate was saturable, stimulated by PKC inhibition, and responded differently to cellular pathway inhibitors than did insulin transport at the blood-brain barrier. In summary, these results show intranasal delivery to be an effective way to deliver insulin to the brain.

Sleep fragmentation and sepsis differentially impact blood-brain barrier integrity and transport of tumor necrosis factor-α in aging.

The factors by which aging predisposes to critical illness are varied, complex, and not well understood. Sepsis is considered a quintessential disease of old age because the incidence and mortality of severe sepsis increases in old and the oldest old individuals. Aging is associated with dramatic changes in sleep quality and quantity and sleep increasingly becomes fragmented with age. In healthy adults, sleep disruption induces inflammation. Multiple aspects of aging and of sleep dysregulation interact via neuroimmune mechanisms. Tumor necrosis factor-α (TNF), a cytokine involved in sleep regulation and neuroimmune processes, exerts some of its effects on the CNS by crossing the blood-brain barrier (BBB). In this study we examined the impact of sepsis, sleep fragmentation, and aging on BBB disruption and TNF transport into brain. We used the cecal ligation and puncture (CLP) model of sepsis in young and aged mice that were either undisturbed or had their sleep disrupted. There was a dichotomous effect of sepsis and sleep disruption with age: sepsis disrupted the BBB and increased TNF transport in young mice but not in aged mice, whereas sleep fragmentation disrupted the BBB and increased TNF transport in aged mice, but not in young mice. Combining sleep fragmentation and CLP did not produce a greater effect on either of these BBB parameters than did either of these manipulations alone. These results suggest that the mechanisms by which sleep fragmentation and sepsis alter BBB functions are fundamentally different from one another and that a major change in the organism's responses to those insults occurs with aging.

Alpha synuclein is transported into and out of the brain by the blood-brain barrier.

Alpha-synuclein (α-Syn), a small protein with multiple physiological and pathological functions, is one of the dominant proteins found in Lewy Bodies, a pathological hallmark of Lewy body disorders, including Parkinson's disease (PD). More recently, α-Syn has been found in body fluids, including blood and cerebrospinal fluid, and is likely produced by both peripheral tissues and the central nervous system. Exchange of α-Syn between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications. However, little is known about the ability of α-Syn to cross the blood-brain barrier (BBB). Here, we found that radioactively labeled α-Syn crossed the BBB in both the brain-to-blood and the blood-to-brain directions at rates consistent with saturable mechanisms. Low-density lipoprotein receptor-related protein-1 (LRP-1), but not p-glycoprotein, may be involved in α-Syn efflux and lipopolysaccharide (LPS)-induced inflammation could increase α-Syn uptake by the brain by disrupting the BBB.

Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson's disease.

Extracellular α-synuclein is important in the pathogenesis of Parkinson's disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum α-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived α-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled α-synuclein into mouse brain, that CSF α-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of α-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF α-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal α-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF α-synuclein, a significant correlation between plasma exosomal α-synuclein and disease severity (r = 0.176, p = 0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal α-synuclein were comparable to those determined by CSF α-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS α-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically.