Quantifying the Lymphatic Transport of Model Therapeutics from the Brain in Rats.

In recent years, the drainage of fluids, immune cells, antigens, fluorescent tracers, and other solutes from the brain has been demonstrated to occur along lymphatic outflow pathways to the deep cervical lymph nodes in the neck. To the best of our knowledge, no studies have evaluated the lymphatic transport of therapeutics from the brain. The objective of this study was to determine the lymphatic transport of model therapeutics of different molecular weights and lipophilicity from the brain using cervical lymph cannulation and ligation models in rats. To do this, anesthetized Sprague-Dawley rats were cannulated at the carotid artery and cannulated, ligated, or left intact at the cervical lymph duct. Rats were administered 14C-ibuprofen (206.29 g/mol, logP 3.84), 3H-halofantrine HCl (536.89 g/mol, logP 8.06), or 3H-albumin (∼65,000 g/mol) via direct injection into the brain striatum at a rate of 0.5 µL/min over 16 min. Plasma or cervical lymph samples were collected for up to 6-8 h following dosing, and brain and lymph nodes were collected at 6 or 8 h. Samples were subsequently analyzed for radioactivity levels via scintillation counting. For 14C-ibuprofen, plasma concentrations over time (plasma AUC0-6h) were >2 fold higher in lymph-ligated rats than in lymph-intact rats, suggesting that ibuprofen is cleared from the brain primarily via nonlymphatic routes (e.g., across the blood-brain barrier) but that this clearance is influenced by changes in lymphatic flow. For 3H-halofantrine, >73% of the dose was retained at the brain dosing site in lymph-intact and lymph-ligated groups, and plasma AUC0-8h values were low in both groups (<0.3% dose.h/mL), consistent with the high retention in the brain. It was therefore not possible to determine whether halofantrine undergoes lymphatic transport from the brain within the duration of the study. For 3H-albumin, plasma AUC0-8h values were not significantly different between lymph-intact, lymph-ligated, and lymph-cannulated rats. However, >4% of the dose was recovered in cervical lymph over 8 h. Lymph/plasma concentration ratios of 3H-albumin were also very high (up to 53:1). Together, these results indicate that 3H-albumin is transported from the brain not only via lymphatic routes but also via the blood. Similar to other tissues, the lymphatics may thus play a significant role in the transport of macromolecules, including therapeutic proteins, from the brain but are unlikely to be a major transport pathway from the brain for small molecule drugs that are not lipophilic. Our rat cervical lymph cannulation model can be used to quantify the lymphatic drainage of different molecules and factors from the brain.

Sex-Dependent Changes to the Intestinal and Hepatic Abundance of Drug Transporters and Metabolizing Enzymes in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by death and dysfunction of motor neurons that result in a rapidly progressing loss of motor function. While there are some data on alterations at the blood-brain barrier (BBB) in ALS and their potential impact on CNS trafficking of drugs, little is reported on the impact of this disease on the expression of drug-handling proteins in the small intestine and liver. This may impact the dosing of the many medicines that individuals with ALS are prescribed. In the present study, a proteomic evaluation was performed on small intestine and liver samples from postnatal day 120 SOD1G93A mice (a model of familial ALS that harbors a human mutant form of superoxide dismutase 1) and wild-type (WT) littermates (n = 7/genotype/sex). Untargeted, quantitative proteomics was undertaken using either label-based [tandem mass tag (TMT)] or label-free [data-independent acquisition (DIA)] acquisition strategies on high-resolution mass spectrometric instrumentation. Copper chaperone for superoxide dismutase (CCS) was significantly higher in SOD1G93A samples compared to the WT samples for both sexes and tissues, therefore representing a potential biomarker for ALS in this mouse model. Relative to WT mice, male SOD1G93A mice had significantly different proteins (Padj < 0.05, |fold-change|>1.2) in the small intestine (male 22, female 1) and liver (male 140, female 3). This included an up-regulation of intestinal transporters for dietary glucose [solute carrier (SLC) SLC5A1] and cholesterol (Niemann-Pick c1-like 1), as well as for several drugs (e.g., SLC15A1), in the male SOD1G93A mice. There was both an up-regulation (e.g., SLCO2A1) and down-regulation (ammonium transporter rh type b) of transporters in the male SOD1G93A liver. In addition, there was both an up-regulation (e.g., phosphoenolpyruvate carboxykinase) and down-regulation (e.g., carboxylesterase 1) of metabolizing enzymes in the male SOD1G93A liver. This proteomic data set identified male-specific changes to key small intestinal and hepatic transporters and metabolizing enzymes that may have important implications for the bioavailability of nutrients and drugs in individuals with ALS.

Effect of Apolipoprotein E isoforms on the Abundance and Function of P-glycoprotein in Human Brain Microvascular Endothelial Cells.

BACKGROUND: Individuals with Alzheimer's disease (AD) often require many medications; however, these medications are dosed using regimens recommended for individuals without AD. This is despite reduced abundance and function of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) in AD, which can impact brain exposure of drugs. The fundamental mechanisms leading to reduced P-gp abundance in sporadic AD remain unknown; however, it is known that the apolipoprotein E (apoE) gene has the strongest genetic link to sporadic AD development, and apoE isoforms can differentially alter BBB function. The aim of this study was to assess if apoE affects P-gp abundance and function in an isoform-dependent manner using a human cerebral microvascular endothelial cell (hCMEC/D3) model. METHODS: This study assessed the impact of apoE isoforms on P-gp abundance (by western blot) and function (by rhodamine 123 (R123) uptake) in hCMEC/D3 cells. Cells were exposed to recombinant apoE3 and apoE4 at 2 - 10 µg/mL over 24 - 72 hours. hCMEC/D3 cells were also exposed for 72 hours to astrocyte-conditioned media (ACM) from astrocytes expressing humanised apoE isoforms. RESULTS: P-gp abundance in hCMEC/D3 cells was not altered by recombinant apoE4 relative to recombinant apoE3, nor did ACM containing human apoE isoforms alter P-gp abundance. R123 accumulation in hCMEC/D3 cells was also unchanged with recombinant apoE isoform treatments, suggesting no change to P-gp function, despite both abundance and function being altered by positive controls SR12813 (5 µM) and PSC 833 (5 µM), respectively. CONCLUSIONS: Different apoE isoforms have no direct influence on P-gp abundance or function within this model, and further in vivo studies would be required to address whether P-gp abundance or function are reduced in sporadic AD in an apoE isoform-specific manner.

Comparison of the Anti-inflammatory Activity and Cellular Interaction of Brush Polymer-N-Acetyl Cysteine Conjugates in Human and Murine Microglial Cell Lines.

Microglia-mediated neuroinflammation is commonly associated with neurodegeneration and has been implicated in several neurological disorders, such as Alzheimer's disease and Parkinson's disease. Therefore, it is crucial to develop a detailed understanding of the interaction of potential nanocarriers with microglial cells to efficiently deliver anti-inflammatory molecules. In this study, we applied brush polymers as a modular platform to systematically investigate their association with murine (BV-2) and human (HMC3) microglial cell lines in the presence and absence of the pro-inflammatory inducer lipopolysaccharide (LPS) using flow cytometry. Brush polymers of different sizes and shapes, ranging from ellipsoid to worm-like cylinders, were prepared through a combination of the two building blocks carboxylated N-acylated poly(aminoester)s (NPAEs)-based polymers and poly(2-ethyl-2-oxazoline)-NH2 (PEtOx-NH2) and characterized by 1H NMR spectroscopy, size exclusion chromatography, and small-angle neutron scattering. Generally, ellipsoidal particles showed the highest cellular association. Moreover, while no significant differences in murine cell association were observed, the brush polymers revealed a significant accumulation in LPS-activated human microglia compared to resting cells, emphasizing their higher affinity to activated HMC3 cells. Brush polymers with the highest cell association were further modified with the anti-inflammatory agent N-acetyl cysteine (NAC) in a reversible manner. The brush polymer-NAC conjugates were found to significantly attenuate the production of interleukin 6 (p < 0.001) in LPS-activated HMC3 cells compared to LPS-activated BV-2 cells. Thus, the presented brush polymer-NAC conjugates showed a high anti-inflammatory activity in human microglia, suggesting their potential for disease-targeted therapy of microglial-mediated neuroinflammation in the future.

A Biodistribution Study of the Radiolabeled Kv1.3-Blocking Peptide DOTA-HsTX1[R14A] Demonstrates Brain Uptake in a Mouse Model of Neuroinflammation.

The voltage-gated potassium channel Kv1.3 regulates the pro-inflammatory function of microglia and is highly expressed in the post-mortem brains of individuals with Alzheimer's and Parkinson's diseases. HsTX1[R14A] is a selective and potent peptide inhibitor of the Kv1.3 channel (IC50 ∼ 45 pM) that has been shown to decrease cytokine levels in a lipopolysaccharide (LPS)-induced mouse model of inflammation. Central nervous system exposure to HsTX1[R14A] was previously detected in this mouse model using liquid chromatography with tandem mass spectrometry, but this technique does not report on the spatial distribution of the peptide in the different brain regions or peripheral organs. Herein, the in vivo distribution of a [64Cu]Cu-labeled DOTA conjugate of HsTX1[R14A] was observed for up to 48 h by positron emission tomography (PET) in mice. After subcutaneous administration to untreated C57BL/6J mice, considerable uptake of the radiolabeled peptide was observed in the kidney, but it was undetectable in the brain. Biodistribution of a [68Ga]Ga-DOTA conjugate of HsTX1[R14A] was then investigated in the LPS-induced mouse model of neuroinflammation to assess the effects of inflammation on uptake of the peptide in the brain. A control peptide with very weak Kv1.3 binding, [68Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC50 ∼ 6 µM), was also tested. Significantly increased uptake of [68Ga]Ga-DOTA-HsTX1[R14A] was observed in the brains of LPS-treated mice compared to mice treated with control peptide, implying that the enhanced uptake was due to increased Kv1.3 expression rather than simply increased blood-brain barrier disruption. PET imaging also showed accumulation of [68Ga]Ga-DOTA-HsTX1[R14A] in inflamed joints and decreased clearance from the kidneys in LPS-treated mice. These biodistribution data highlight the potential of HsTX1[R14A] as a therapeutic for the treatment of neuroinflammatory diseases mediated by overexpression of Kv1.3.

Reduction in ABCG2 mRNA Expression in Human Immortalised Brain Microvascular Endothelial Cells by Ferric Ammonium Citrate is Mediated by Reactive Oxygen Species and Activation of ERK1/2 Signalling.

PURPOSE: The ATP-binding cassette (ABC) transport protein ABCG2 (also known as breast cancer resistance protein (BCRP)) is expressed at the luminal face of the blood-brain barrier (BBB), where it limits the brain uptake of a number of therapeutic drugs. We recently reported that the ABC efflux transporter P-glycoprotein (P-gp) was downregulated in human immortalised brain endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC). The aim of the present study, therefore, was to assess whether BCRP expression is also affected by FAC and identify any signalling mechanisms involved. METHODS: ABCG2 mRNA was assessed by RT-qPCR. Protein levels of BCRP, phosphorylated extracellular-regulated kinases 1 and 2 (p-ERK1/2) and total ERK 1/2 were assessed by Western blot. Reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate. RESULTS: Treatment of hCMEC/D3 cells with FAC (250 µM, 72 h) significantly reduced ABCG2 mRNA levels (32.2 ± 3.7%) without a concomitant reduction in BCRP protein expression. ABCG2 mRNA levels were restored to control levels when co-treated with the antioxidant N-acetylcysteine (NAC), suggesting the effect of FAC was mediated by a ROS-sensitive pathway. We also found that FAC-treatment was associated with increased levels of p-ERK1/2, suggesting involvement of the ERK1/2 signalling pathway in the observed ABCG2 mRNA downregulation. The ERK1/2 signalling pathway inhibitor U0126 restored p-ERK1/2 levels and partially attenuated the FAC-induced reduction in ABCG2 mRNA. CONCLUSIONS: This study suggests that FAC-induced downregulation of ABCG2 mRNA is driven by ROS and ERK1/2 signalling, mechanisms which may be exploited to modulate BCRP expression at the BBB.

Copper bis(thiosemicarbazone) complexes modulate P-glycoprotein expression and function in human brain microvascular endothelial cells.

P-glycoprotein (P-gp) is an efflux transporter at the blood-brain barrier (BBB) that hinders brain access of substrate drugs and clears endogenous molecules such as amyloid beta (Aß) from the brain. As biometals such as copper (Cu) modulate many neuronal signalling pathways linked to P-gp regulation, it was hypothesised that the bis(thiosemicarbazone) (BTSC) Cu-releasing complex, copper II glyoxal bis(4-methyl-3-thiosemicarbazone) (CuII [GTSM]), would enhance P-gp expression and function at the BBB, while copper II diacetyl bis(4-methyl-3-thiosemicarbazone) (CuII [ATSM]), which only releases Cu under hypoxic conditions, would not modulate P-gp expression. Following treatment with 25-250 nM CuII (BTSC)s for 8-48 h, expression of P-gp mRNA and protein in human brain endothelial (hCMEC/D3) cells was assessed by RT-qPCR and Western blot, respectively. P-gp function was assessed by measuring accumulation of the fluorescent P-gp substrate, rhodamine 123 and intracellular Cu levels were quantified by inductively coupled plasma mass spectrometry. Interestingly, CuII (ATSM) significantly enhanced P-gp expression and function 2-fold and 1.3-fold, respectively, whereas CuII (GTSM) reduced P-gp expression 0.5-fold and function by 200%. As both compounds increased intracellular Cu levels, the effect of different BTSC backbones, independent of Cu, on P-gp expression was assessed. However, only the Cu-ATSM complex enhanced P-gp expression and this was mediated partly through activation (1.4-fold) of the extracellular signal-regulated kinase 1 and 2, an outcome that was significantly attenuated in the presence of an inhibitor of the mitogen-activated protein kinase regulatory pathway. Our findings suggest that CuII (ATSM) and CuII (GTSM) have the potential to modulate the expression and function of P-gp at the BBB to impact brain drug delivery and clearance of Aß.

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3.

Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against KV1.3 (IC50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.

Nitrile-Functionalized Poly(2-oxazoline)s as a Versatile Platform for the Development of Polymer Therapeutics.

In recent years, polymers bearing reactive groups have received significant interest for biomedical applications. Numerous functional polymer platforms have been introduced, which allow for the preparation of materials with tailored properties via post-polymerization modifications. However, because of their reactivity, many functional groups are not compatible with the initial polymerization. The nitrile group is a highly interesting and relatively inert functionality that has mainly received attention in radical polymerizations. In this Article, a nitrile-functionalized 2-oxazoline monomer (2-(4-nitrile-butyl)-2-oxazoline, BuNiOx) is introduced, and its compatibility with the cationic ring-opening polymerization is demonstrated. Subsequently, the versatility of nitrile-functionalized poly(2-oxazoline)s (POx) is presented. To this end, diverse (co)polymers are synthesized and characterized by nuclear resonance spectroscopy, size-exclusion chromatography, and mass spectrometry. Amphiphilic block copolymers are shown to efficiently encapsulate the hydrophobic drug curcumin (CUR) in aqueous solution, and the anti-inflammatory effect of the CUR-containing nanostructures is presented in BV-2 microglia. Furthermore, the availability of the BuNiOx repeating units for post-polymerization modifications with hydroxylamine to yield amidoxime (AO)-functionalized POx is demonstrated. These AO-containing POx were successfully applied for the complexation of Fe(III) in a quantitative manner. In addition, AO-functionalized POx were shown to release nitric oxide intracellularly in BV-2 microglia. Thus nitrile-functionalized POx represent a promising and robust platform for the design of polymer therapeutics for a wide range of applications.

Increasing Intracellular Levels of Iron with Ferric Ammonium Citrate Leads to Reduced P-glycoprotein Expression in Human Immortalised Brain Microvascular Endothelial Cells.

PURPOSE: P-glycoprotein (P-gp) at the blood-brain barrier (BBB) precludes the brain penetration of many xenobiotics and mediates brain-to-blood clearance of ß-amyloid, which accumulates in the Alzheimer's disease (AD) brain. Zinc and copper are reported to modulate BBB expression and function of P-gp; however, the impact of exogenous iron, which accumulates in AD, on P-gp dynamics remains unknown. METHODS: P-gp protein and MDR1 transcript levels were assessed in immortalised human cerebral microvascular endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC; 250 µM, 72 h), by Western blotting and RT-qPCR, respectively. P-gp function was assessed using rhodamine-123 and [3H]-digoxin accumulation. Intracellular reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate and intracellular iron levels quantified using a ferrozine assay. RESULTS: FAC treatment significantly reduced P-gp protein (36%) and MDR1 mRNA (16%) levels, with no significant change in rhodamine-123 or [3H]-digoxin accumulation. While P-gp/MDR1 downregulation was associated with elevated ROS and intracellular iron, MDR1 downregulation was not attenuated with the antioxidant N-acetylcysteine nor the iron chelators desferrioxamine and deferiprone, suggesting the involvement of a ROS-independent mechanism or incomplete iron chelation. CONCLUSIONS: These studies demonstrate that iron negatively regulates P-gp expression at the BBB, potentially impacting CNS drug delivery and brain ß-amyloid clearance.

Pioglitazone Increases Blood-Brain Barrier Expression of Fatty Acid-Binding Protein 5 and Docosahexaenoic Acid Trafficking into the Brain.

Brain levels of docosahexaenoic acid (DHA), an essential cognitively beneficial fatty acid, are reduced in Alzheimer's disease (AD). We have demonstrated in an AD mouse model that this is associated with reduced blood-brain barrier (BBB) transport of DHA and lower expression of the key DHA-trafficking protein, fatty acid-binding protein 5 (FABP5). This study focused on assessing the impact of activating peroxisome proliferator-activated receptor (PPAR) isoforms on FABP5 expression and function at the BBB. Using immortalized human brain endothelial (hCMEC/D3) cells, a 72 h treatment with the PPARα agonist clofibrate (100 µM), and PPARß/δ agonists GW0742 (1 µM) and GW501506 (0.5 µM), did not affect FABP5 protein expression. In contrast, the PPARγ agonists rosiglitazone (5 µM), pioglitazone (25 µM), and troglitazone (1 µM) increased FABP5 protein expression by 1.15-, 1.18-, and 1.24-fold in hCMEC/D3 cells, respectively, with rosiglitazone and pioglitazone also increasing mRNA expression of FABP5. In line with an increase in FABP5 expression, pioglitazone increased 14C-DHA uptake into hCMEC/D3 cells 1.20- to 1.33-fold over a 2 min period, and this was not associated with increased expression of membrane transporters involved in DHA uptake. Furthermore, treating male C57BL/6J mice with pioglitazone (40 mg/kg/day for 7 days) led to a 1.79-fold increase in BBB transport of 14C-DHA over 1 min, using an in situ transcardiac perfusion technique, which was associated with a 1.82-fold increase in brain microvascular FABP5 protein expression. Overall, this study demonstrated that PPARγ can regulate FABP5 at the BBB and facilitate DHA transport across the BBB, important in restoring brain levels of DHA in AD.

Intestinal Permeability and Oral Absorption of Selected Drugs Are Reduced in a Mouse Model of Familial Alzheimer's Disease.

Compared with the significant number of studies reporting altered abundance and function of drug transporters at the blood-brain barrier (BBB) in Alzheimer's disease (AD), the impact of AD on the abundance of intestinal drug transporters and the subsequent effects on oral drug absorption have received little attention. We have reported the altered abundance of some small intestinal drug transporters in a familial mouse model of AD; however, whether this leads to altered oral drug absorption is unknown. The current study examined plasma concentrations of caffeine and diazepam (markers for transcellular passive transport), digoxin (P-glycoprotein substrate), and valsartan (multidrug resistance-associated protein 2 substrate) following oral administration to 8-10 month old female wild-type (WT) and APPswe/PSEN1dE9 (APP/PS1) transgenic mice, a commonly used mouse model of familial AD. The plasma exposure of valsartan and digoxin was significantly (p < 0.05) lower in APP/PS1 animals compared with WT mice, whereas the plasma concentrations of the passive transcellular markers caffeine and diazepam did not significantly differ between the two genotypes. To assess whether the reduced oral absorption of valsartan and digoxin was due to decreased intestinal transport, the ex vivo transport of the previously mentioned drugs and mannitol (a marker of paracellular transport) across the jejunum of WT and APP/PS1 mice was assessed over 120 min. In line with the in vivo absorption studies, the permeability of caffeine and diazepam did not significantly differ between WT and APP/PS1 mice. The permeability of 3H-digoxin through the APP/PS1 mouse jejunum was lower than that measured through the WT jejunum; the average amount (relative to dose applied) permeating the tissue over 120 min was 0.22 ± 0.11% (mean ± SD) for the APP/PS1 jejunum and 0.85 ± 0.3% for the WT jejunum. A 1.9-fold reduction in the average amount of valsartan permeating the jejunum of APP/PS1 mice relative to that of WT mice was also detected. Although no apparent morphological alterations were observed in the jejunal tissue of APP/PS1 mice, the permeability of 14C-mannitol across the jejunum from APP/PS1 mice was lower than that across the WT jejunum (Papp= 10.7 ± 3.7 × 10-6 and 6.0 ± 3.4 × 10-6 cm/s, respectively), suggesting tightened paracellular junctions in APP/PS1 mice. These studies are the first to demonstrate, in APP/PS1 mice, reduced intestinal permeability and the absorption of drugs commonly prescribed to people with AD for their comorbidities. If these findings translate to people with AD, then modified dosing regimens may be necessary for selected drugs to ensure that their plasma concentrations remain in the effective range.

Impact of reduced P-glycoprotein function on digoxin concentrations in patients with dementia.

AIMS: Patients with Alzheimer's disease (AD), the most common form of dementia, have reduced P-glycoprotein (P-gp) function at the blood-brain barrier. However, the effect of AD on P-gp function in peripheral organs, and the impact on medication efficacy and toxicity is unknown. In this study, clinical chart review and physiologically based pharmacokinetic (PBPK) modelling were employed to determine whether disease-associated changes in P-gp could be assessed from clinically measured digoxin concentrations in patients without and with dementia. METHODS: A retrospective chart review was conducted to compare digoxin dose and concentrations between cohorts. A PBPK model was developed to simulate changes in digoxin concentrations at single and multiple 62.5 and 125 µg/d doses due to reduced P-gp function in peripheral organs. RESULTS: Digoxin concentrations were similar between the nondementia (n = 75) and dementia (n = 72) cohorts (mean ± standard deviation; 0.64 ± 0.31 and 0.60 ± 0.34 ng/mL, respectively; -0.06 to 0.15, 95% confidence interval of difference). PBPK simulations showed that reduced P-gp function resulted in a significant increase in digoxin exposure (AUC), but not in Cmax . For example, when a 2-fold reduction in P-gp function was simulated in older people following multiple 125 µg/d digoxin doses, the AUC over the last dosing interval was increased compared to baseline (24.29 ± 3.94 vs 17.04 ± 3.46 ng/mL*h; 4.52 to 9.98); however, Cmax was similar (1.38 ± 0.20 vs 0.99 ± 0.18 ng/mL; -2.33 to 3.13). CONCLUSION: Clinically measured digoxin concentrations were not statistically different in patients with dementia. Based on PBPK simulations, digoxin AUC may need to be evaluated to adequately assess the impact of reduced P-gp function in peripheral organs.

Dietary docosahexaenoic acid supplementation enhances expression of fatty acid-binding protein 5 at the blood-brain barrier and brain docosahexaenoic acid levels.

The cytoplasmic trafficking of docosahexaenoic acid (DHA), a cognitively beneficial fatty acid, across the blood-brain barrier (BBB) is governed by fatty acid-binding protein 5 (FABP5). Lower levels of brain DHA have been observed in Alzheimer's disease (AD), which is associated with diminished BBB expression of FABP5. Therefore, up-regulating FABP5 expression at the BBB may be a novel approach for enhancing BBB transport of DHA in AD. DHA supplementation has been shown to be beneficial in various mouse models of AD, and therefore, the aim of this study was to determine whether DHA has the potential to up-regulate the BBB expression of FABP5, thereby enhancing its own uptake into the brain. Treating human brain microvascular brain endothelial (hCMEC/D3) cells with the maximum tolerable concentration of DHA (12.5 µM) for 72 h resulted in a 1.4-fold increase in FABP5 protein expression. Associated with this was increased expression of fatty acid transport proteins 1 and 4. To study the impact of dietary DHA supplementation, 6- to 8-week-old C57BL/6 mice were fed with a control diet or a DHA-enriched diet for 21 days. Brain microvascular FABP5 protein expression was up-regulated 1.7-fold in mice fed the DHA-enriched diet, and this was associated with increased brain DHA levels (1.3-fold). Despite an increase in brain DHA levels, reduced BBB transport of 14 C-DHA was observed over a 1 min perfusion, possibly as a result of competitive binding to FABP5 between dietary DHA and 14 C-DHA. This study has demonstrated that DHA can increase BBB expression of FABP5, as well as fatty acid transporters, overall increasing brain DHA levels.

Reduced blood-brain barrier expression of fatty acid-binding protein 5 is associated with increased vulnerability of APP/PS1 mice to cognitive deficits from low omega-3 fatty acid diets.

Lower levels of the cognitively beneficial docosahexaenoic acid (DHA) are often observed in Alzheimer's disease (AD) brains. Brain DHA levels are regulated by the blood-brain barrier (BBB) transport of plasma-derived DHA, a process facilitated by fatty acid-binding protein 5 (FABP5). This study reports a 42.1 ± 12.6% decrease in the BBB transport of 14 C-DHA in 8-month-old AD transgenic mice (APPswe,PSEN1∆E9) relative to wild-type mice, associated with a 34.5 ± 6.7% reduction in FABP5 expression in isolated brain capillaries of AD mice. Furthermore, short-term spatial and recognition memory deficits were observed in AD mice on a 6-month n-3 fatty acid-depleted diet, but not in AD mice on control diet. This intervention led to a dramatic reduction (41.5 ± 11.9%) of brain DHA levels in AD mice. This study demonstrates FABP5 deficiency and impaired DHA transport at the BBB are associated with increased vulnerability to cognitive deficits in mice fed an n-3 fatty acid-depleted diet, in line with our previous studies demonstrating a crucial role of FABP5 in BBB transport of DHA and cognitive function.

Commentary on the 2018 Named Series on blood-brain interfaces: Roles of neuroimmunomodulation in health and disease.

This year's 2018 Named Series on blood-brain interfaces highlights the importance of brain barriers as mediators of neuroimmune communication and regulators of neurological function. The term "brain interfaces" reflects our growing understanding that brain barriers such as the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) are not only gatekeepers, but facilitators of bidirectional communication between the brain and periphery. There is also an emerging appreciation that CNS sites that are exposed to blood-borne immune molecules and cells, such as the leptomeninges and circumventricular organs, may also be considered brain interfaces with important homeostatic and pathological functions. The work featured in this Series covers novel aspects of brain interface functions that focus on mechanisms regulating barrier integrity and transporter activities, downstream consequences of neurovascular injury, peripheral organ infection/injury, and clearance of pathogenic proteins. Results of these studies have emphasized new mechanisms by which brain interface dysfunction could contribute to neuroinflammation and CNS damage in eclampsia, fetal and adult hypoxic/ischemic injury, traumatic brain injury, Helicobacter infections, acute lung injury, multiple sclerosis, and Alzheimer's disease. This body of work emphasizes that brain interfaces may themselves be important therapeutic targets for a variety of CNS diseases that are associated with immune dyshomeostasis. Future works are warranted to further investigate brain interface functions in health and disease.

Cognitive benefits of lithium chloride in APP/PS1 mice are associated with enhanced brain clearance of ß-amyloid.

Epidemiological evidence suggests that people with bipolar disorder prescribed lithium exhibit a lower risk of Alzheimer's disease (AD) relative to those prescribed other mood-stabilizing medicines. Lithium chloride (LiCl) reduces brain ß-amyloid (Aß) levels, and the brain clearance of Aß is reduced in AD. Therefore, the purpose of this study was to assess whether the cognitive benefits of LiCl are associated with enhanced brain clearance of exogenously-administered Aß. The brain clearance of intracerebroventricularly (icv) administered 125I-Aß42 was assessed in male Swiss outbred mice administered daily oral NaCl or LiCl (300 mg/kg for 21 days). LiCl exhibited a 31% increase in the brain clearance of 125I-Aß42 over 10 min, which was associated with a 1.6-fold increase in brain microvascular expression of the blood-brain barrier efflux transporter low density lipoprotein receptor-related protein 1 (LRP1) and increased cerebrospinal fluid (CSF) bulk-flow. 8-month-old female wild type (WT) and APP/PS1 mice were also administered daily NaCl or LiCl for 21 days, which was followed by cognitive assessment by novel object recognition and water maze, and measurement of soluble Aß42, plaque-associated Aß42, and brain efflux of 125I-Aß42. LiCl treatment restored the long-term spatial memory deficit observed in APP/PS1 mice as assessed by the water maze (back to similar levels of escape latency as WT mice), but the short-term memory deficit remained unaffected by LiCl treatment. While LiCl did not affect plaque-associated Aß42, soluble Aß42 levels were reduced by 49.9% in APP/PS1 mice receiving LiCl. The brain clearance of 125I-Aß42 decreased by 27.8% in APP/PS1 mice, relative to WT mice, however, LiCl treatment restored brain 125I-Aß42 clearance in APP/PS1 mice to a rate similar to that observed in WT mice. These findings suggest that the cognitive benefits and brain Aß42 lowering effects of LiCl are associated with enhanced brain clearance of Aß42, possibly via brain microvascular LRP1 upregulation and increased CSF bulk-flow, identifying a novel mechanism of protection by LiCl for the treatment of AD.

Altered Expression of Small Intestinal Drug Transporters and Hepatic Metabolic Enzymes in a Mouse Model of Familial Alzheimer's Disease.

Drug transporter expression and function at the blood-brain barrier is altered in Alzheimer's disease (AD). However, the impact of AD on the expression of transporters and metabolizing enzymes in peripheral tissues has received little attention. The current study evaluated the expression of drug transporters and metabolizing enzymes in the small intestine and liver from 8- to 9-month-old female wild-type (WT) and APPswe/PSEN 1dE9 (APP/PS1) transgenic mice, a widely used AD model, using a quantitative targeted absolute proteomics (QTAP) approach. Furthermore, the general morphological appearance of the liver was assessed by immunohistochemistry, and lipid content was visualized using Oil Red O staining. The small intestines of APP/PS1 mice exhibited a significant 2.3-fold increase in multidrug resistance-associated protein 2 (Mrp2), a 1.9-fold decrease in monocarboxylate transporter 1 (Mct1), and a 3.6-fold increase in UDP-glucuronosyltransferase (Ugt) 2b5 relative to those from WT mice based on QTAP analysis. While the liver from APP/PS1 mice exhibited no changes in drug transporter expression, there was a 1.3-fold elevation in cytochrome P450 (Cyp) 51a1 and a 1.2-fold reduction in Cyp2c29 protein expression, and this was associated with morphological alterations including accumulation of hepatocyte lipids. These studies are the first to demonstrate that the protein expression of transporters and metabolizing enzymes important in oral drug absorption are modified in a mouse model of familial AD, which may lead to altered disposition of some orally administered drugs in AD.

Effect of Permeation Enhancers on the Buccal Permeability of Nicotine: Ex vivo Transport Studies Complemented by MALDI MS Imaging.

PURPOSE: The purpose of this study was to assess the effect of several chemical permeation enhancers on the buccal permeability of nicotine and to image the spatial distribution of nicotine in buccal mucosa with and without buccal permeation enhancers. METHODS: The impact of sodium taurodeoxycholate (STDC), sodium dodecyl sulphate (SDS), dimethyl sulfoxide (DMSO) and Azone® on the permeability of [3H]-nicotine and [14C]-mannitol (a paracellular marker) across porcine buccal mucosa was studied ex vivo in modified Ussing chambers. The distribution of nicotine, mannitol and permeation enhancers was imaged using using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI). RESULTS: Despite STDC significantly increasing permeability of [14C]-mannitol, no enhancing effect was seen on [3H]-nicotine permeability with any of the permeation enhancers. Rather, SDS and DMSO retarded nicotine permeability, likely due to nicotine being retained in the donor compartment. The permeability results were complemented by the spatial distribution of nicotine and mannitol determined with MALDI MSI. CONCLUSIONS: The buccal permeability of nicotine was affected in an enhancer specific manner, suggesting that nicotine primarily diffuses via the transcellular pathway. MALDI MSI was shown to complement ex vivo permeability studies and to be a useful qualitative tool for visualizing drug and penetration enhancer distribution in buccal mucosa.

Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells.

PURPOSE: Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown. METHODS: Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking. RESULTS: A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 µM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold). CONCLUSIONS: Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.