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Background: Iron overload is implicated in many neurodegenerative diseases, where there is also blood-brain barrier (BBB) dysfunction. As there is a growing interest in the role of iron in modulating key BBB proteins, this study assessed the effect of iron on the expression and function of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and claudin-5 in primary mouse brain endothelial cells (MBECs) and their abundance in mouse brain microvessel-enriched membrane fractions (MVEFs)., Methods: Following a 48 h treatment with ferric ammonium citrate (FAC, 250 µM), MBEC protein abundance (P-gp, BCRP and claudin-5) and mRNA (abcb1a, abcg2, and cldn5) were assessed by western blotting and RT-qPCR, respectively. Protein function was evaluated by assessing transport of substrates 3 H-digoxin (P-gp), 3 H-prazosin (BCRP) and 14 C-sucrose (paracellular permeability). C57BL/6 mice received iron dextran (100 mg/kg, intraperitoneally) over 4 weeks, and MVEF protein abundance and iron levels (in MVEFs and plasma) were quantified via western blotting and inductively coupled plasma-mass spectrometry (ICP-MS), respectively., Results: FAC treatment reduced P-gp protein by 50% and abcb1a mRNA by 43%, without affecting 3 H-digoxin transport. FAC did not alter BCRP protein or function, but decreased abcg2 mRNA by 59%. FAC reduced claudin-5 protein and cldn5 mRNA by 65% and 70%, respectively, resulting in a 200% increase in 14 C-sucrose permeability. In vivo, iron dextran treatment significantly elevated plasma iron levels (2.2-fold) but did not affect brain MVEF iron content or alter P-gp, BCRP or claudin-5 protein abundance., Conclusions: Iron overload modulates BBB transporters and junction proteins in vitro, highlighting potential implications for CNS drug delivery in neurodegenerative diseases., Competing Interests: Declarations. Professor Ashley I. Bush has a profit share interest in, Collaborative Medicinal Development LLC and is a shareholder in Alterity Ltd., Cogstate Ltd. and Mesoblast Ltd., (© 2025. The Author(s).)

Exercise improves cognitive function in Alzheimer's disease (AD) via mechanism that are not fully clear. Here, we first examined the effect of voluntary exercise training (VET) on energy metabolism and cognitive function in the APP/PS1 transgenic mouse (Tg) model of familial AD. Next, we profiled extracellular vesicles (EVs) and examined whether they may play a role in the protective effects of VET via intranasal administration of EVs, purified from the blood of sedentary (sEV) and/or acutely exercised (eEV) donor wild-type mice into APP/PS1Tg mice. We show that VET reduced resting energy expenditure (REE) and improved cognition in APP/PS1 Tg mice. Administration of eEV, but not sEV, also reduced REE, but had no effect on cognition. Taken together, these data show that exercise is effective intervention to improve symptoms of AD in APP/PS1Tg mice. In addition, eEVs mediate some of these effects, implicating EVs in the treatment of age-related neurodegenerative diseases., Competing Interests: M.A.F. is a shareholder and scientific advisor for N-Gene Pharmaceuticals. M.A.F. is the founder and shareholder of Celesta Therapeutics., (© 2025 The Authors.)

Microfluidic BBB-on-a-chip models (μBBB) aim to recapitulate the organotypic features of the human BBB with great potential to model CNS diseases and advance CNS therapeutics. Nevertheless, their predictive capacity for drug uptake into the brain remains uncertain due to limited evaluation with only a small number of model drugs. Here, the in vivo brain uptake of a panel of nine radiolabeled compounds is evaluated in Swiss-outbred mice following a single intravenously administered dose and compared against results from the microfluidic μBBB platform and the conventional Transwell BBB model. Radioisotopic measurements are employed to calculate brain-to-plasma concentration ratios (B/P) of the compounds both in vivo and in vitro. The in vitro-in vivo correlation plots of the B/P ratios revealed a strong positive correlation (r = 0.8081, R 2 = 0.6530) for the μBBB, suggesting a high degree of predictive ability for drug permeability into the brain. In contrast, the Transwell assay showed a weaker in vitro-in vivo correlation (r = 0.6467, R 2 = 0.4182). Finally, brain uptake of radiolabeled, brain-targeted, angiopep2-conjugated nanoparticles (ANG2-NP) is assessed in the μBBB and results mirrored the in vivo uptake, while the Transwell model failed to resolve the differences between the targeted and non-targeted NPs., (© 2024 Wiley‐VCH GmbH.)

The drainage of fluid and solutes along lymphatic pathways from the brain has been found to be impaired in mouse models of multiple sclerosis, Alzheimer's disease, and Parkinson's disease where neuroinflammation is present. We recently demonstrated that 3 H-albumin, a model therapeutic protein (∼65 kDa), undergoes preferential lymphatic transport from the brain using a cervical lymph cannulation model in healthy rats. We thus hypothesized that neuroinflammation would impede the lymphatic transport of 3 H-albumin from the brain. Our aim was to quantify the impact of acute neuroinflammation on drainage of the model therapeutic protein ( 3 H-albumin) from the rat brain into blood and deep cervical lymph. To establish the required neuroinflammation model, male Sprague-Dawley rats were administered an intraperitoneal (IP) dose of 0.5-2 mg/kg lipopolysaccharide (LPS, Escherichia coli ) or a saline control. After 12 or 24 h, brain samples were collected and analyzed for concentrations of interferon gamma (IFN-γ) using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The impact of neuroinflammation on the drainage of 3 H-albumin from the brain was determined via IP administration of 2 mg/kg LPS or saline followed by cannulation of the carotid artery for blood collection 24 h later with/without cannulation or ligation at the efferent deep cervical lymph trunk. Rats were then administered 3 H-albumin via direct injection into the brain striatum or via intravenous (IV) injection (lymph-intact group only). Blood ± lymph samples were collected for up to 8 h following dosing. At the end of the study, brain and lymph node samples were harvested for biodistribution analysis, with samples analyzed for radioactivity levels via scintillation counting. Brain concentrations of the pro-inflammatory cytokine IFN-γ were only significantly elevated 24 h after IP administration of 2 mg/kg LPS compared to saline control. Therefore, this induction regimen was utilized for subsequent studies. The plasma concentrations of 3 H-albumin over time were elevated in LPS-induced rats compared to saline-injected rats in the lymph-intact and lymph-ligated groups but not in the lymph-cannulated group. In the deep cervical lymph-cannulated animals, the lymph transport of 3 H-albumin was not increased and appeared to be slower in the LPS-administered rats. Acute LPS-induced neuroinflammation therefore led to an enhanced overall transport of 3 H-albumin from the brain into the systemic circulation. This appeared to be primarily due to increased transport of 3 H-albumin from the brain directly into the blood circulation as 3 H-albumin transport from the brain via the lymphatics was not increased in the LPS-induced neuroinflammation model. Such changes in the clearance of therapeutic proteins from the brain in the setting of neuroinflammation may impact the therapeutic efficacy and safety.

Modifications to the small intestine and liver are known to occur during the symptomatic disease period of amyotrophic lateral sclerosis (ALS), a member of the motor neuron disease (MND) family of neurodegenerative disorders. How these modifications impact on oral absorption and pharmacokinetics of drugs remains unknown. In this study, model drugs representing different mechanisms of intestinal transport (caffeine for passive diffusion, digoxin for P-glycoprotein efflux, and sulfasalazine for breast cancer resistance protein efflux) were administered via oral gavage to postnatal day 114-120 male and female SOD1 G93A mice (model of familial ALS) and wild-type (WT) littermates. Samples of blood, brain and spinal cord were taken at either 15, 30, 60 or 180 min after administration. In addition, the in vivo gastric emptying of 70 kDa fluorescein isothiocyanate-dextran (FITC-dextran) and the ex vivo intestinal permeability of caffeine were assessed. The area under the plasma concentration-time curves (AUC plasma ) of digoxin and sulfasalazine were not significantly different between SOD1 G93A and WT mice for both sexes. However, the AUC plasma of caffeine was significantly lower (female: 0.79-fold, male: 0.76-fold) in SOD1 G93A compared to WT mice, which was associated with lower AUC brain (female: 0.76-fold, male: 0.80-fold) and AUC spinal cord (female: 0.81-fold, male: 0.82-fold). The AUC stomach of caffeine was significantly higher (female: 1.5-fold, male: 1.9-fold) in SOD1 G93A compared to WT mice, suggesting reduced gastric emptying in SOD1 G93A mice. In addition, there was a significant reduction in gastric emptying of FITC-dextran (0.66-fold) and ex vivo intestinal permeability of caffeine (0.52-fold) in male SOD1 G93A compared to WT mice. Reduced systemic and brain/spinal cord exposure of caffeine in SOD1 G93A mice may therefore result from alterations to gastric emptying and small intestinal permeability. Specific dosing requirements may therefore be required for certain medicines in ALS to ensure that they remain in a safe and effective concentration range., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Alzheimer's disease (AD) is associated with brain accumulation of amyloid-beta (Aβ) and neurofibrillary tangle formation, in addition to reduced brain docosahexaenoic acid (DHA) and increased brain iron levels. DHA requires access across the blood-brain barrier (BBB) to enter the brain, and iron has been shown to affect the expression and function of a number of BBB transporters. Therefore, this study aimed to assess the effect of iron on the expression and function of fatty acid binding protein 5 (FABP5) and fatty acid transport protein 1 (FATP1), both which mediate brain endothelial cell trafficking of DHA., Methods: The mRNA and protein levels of FABP5 and FATP1 in human cerebral microvascular endothelial (hCMEC/D3) cells was assessed by RT-qPCR and Western blot, respectively following ferric ammonium citrate (FAC) treatment (up to 750 µM, 72 h). The function of FABP5 and FATP1 was assessed via uptake and efflux of radiolabelled 3 H-oleic acid and 14 C-DHA., Results: FAC (500 µM, 72 h) had no impact on the expression of FABP5 at the protein and mRNA level in hCMEC/D3 cells, which was associated with a lack of effect on the uptake of 14 C-DHA. FAC led to a 19.7% reduction in FATP1 protein abundance in hCMEC/D3 cells with no impact on mRNA levels, and this was associated with up to a 32.6% reduction in efflux of 14 C-DHA., Conclusions: These studies demonstrate a role of iron in down-regulating FATP1 protein abundance and function at the BBB, which may have implications on fatty acid access to the brain., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Over the past decades, evidence has consistently shown that treatment of central nervous system (CNS)-related disorders, including Alzheimer's disease, Parkinson's disease, stroke, multiple sclerosis, and brain cancer, is limited due to the presence of the blood-brain barrier (BBB). To assist with the development of new therapeutics, it is crucial to engineer a drug delivery system that can cross the BBB efficiently and reach target cells within the brain. In this study, we present a potentially efficient strategy for targeted brain delivery through utilization of folic acid (FA)-conjugated brush polymers, that specifically target the reduced folate carrier (RFC, SLC19A1) expressed on brain endothelial cells. Here, azide (N 3 )-decorated brush polymers were prepared in a straightforward manner coupling a heterotelechelic α-NH 2 , ω-N 3 -poly(2-ethyl-2-oxazoline) (NH 2 -PEtOx-N 3 ) to N-acylated poly(amino ester) (NPAE)-based brushes. Strain-promoted azide-alkyne cycloaddition (SPAAC) 'click chemistry' with DBCO-folic acid (FA) yielded FA-brush polymers. Interestingly, while azide functionalization of the brush polymers dramatically reduced their association to brain microvascular endothelial cells (hCMEC/D3), the introduction of FA to azide led to a substantial accumulation of the brush polymers in hCMEC/D3 cells. The ability of the polymeric brush polymers to traverse the BBB was quantitatively assessed using different in vitro BBB models including static Transwell and microfluidic platforms. FA-brush polymers showed efficient transport across hCMEC/D3 cells in a manner dependent on FA composition, whereas nonfunctionalized brush polymers exhibited limited trafficking under the same conditions. Further, cellular uptake inhibition studies suggested that the interaction and transport pathway of FA-brush polymers across BBB relies on the RFC-mediated pathways. The potential application of the developed FA-brush polymers in brain cancer delivery was also investigated in a microfluidic model of BBB-glioblastoma. Brush polymers with more FA units successfully presented an enhanced accumulation into U-87 MG glioma cells following its BBB crossing, compared to controls. These results demonstrate that FA-modified brush polymers hold a great potential for more efficient delivery of future brain therapeutics.

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 14 C-ibuprofen (206.29 g/mol, logP 3.84), 3 H-halofantrine HCl (536.89 g/mol, logP 8.06), or 3 H-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 14 C-ibuprofen, plasma concentrations over time (plasma AUC 0-6h ) 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 3 H-halofantrine, >73% of the dose was retained at the brain dosing site in lymph-intact and lymph-ligated groups, and plasma AUC 0-8h 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 3 H-albumin, plasma AUC 0-8h 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 3 H-albumin were also very high (up to 53:1). Together, these results indicate that 3 H-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.

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 SOD1 G93A 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 SOD1 G93A 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 SOD1 G93A mice had significantly different proteins ( P adj < 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 SOD1 G93A mice. There was both an up-regulation (e.g., SLCO2A1) and down-regulation (ammonium transporter rh type b) of transporters in the male SOD1 G93A 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 SOD1 G93A 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.

Prolonged activation of microglia leads to excessive release of proinflammatory mediators, which are detrimental to brain health. Therefore, there are significant efforts to identify pathways mediating microglial activation. Recent studies have demonstrated that fatty acid-binding protein 4 (FABP4), a lipid binding protein, is a critical player in macrophage-mediated inflammation. Given that we have previously identified FABP4 in microglia, the aim of this study was to assess whether FABP4 activity contributed to inflammation, metabolism and immune function (i.e. immunometabolism) in immortalised mouse microglia (BV-2 cells) using the proinflammatory stimulus lipopolysaccharide (LPS) to induce general microglial activation. Microglial FABP4 expression was significantly increased following exposure to LPS, an outcome associated with a significant increase in microglial proliferation rate. LPS-stimulated BV-2 microglia demonstrated a significant increase in the production of reactive oxygen species (ROS) and tumour necrosis factor-alpha (TNF-α), phosphorylation of c-Jun N-terminal kinase (JNK), increased expression of Toll-like receptor 4 (TLR4), and reduced expression of uncoupling protein 2 (UCP2), all of which were reversed following FABP4 genetic silencing and chemical inhibition with BMS309403. The oxidation rate of 3 H-oleic acid and microglial uptake of 3 H-2-deoxy-D-glucose were modulated with LPS activation, processes which were restored with genetic and chemical inhibition of FABP4. This is the first study to report on the critical role of FABP4 in mediating the deleterious effects of LPS on microglial immunometabolism, suggesting that FABP4 may present as a novel therapeutic target to alleviate microglia-mediated neuroinflammation, a commonly reported factor in multiple neurodegenerative diseases., (© 2023. The Author(s).)

P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), is critical in preventing brain access to substrate drugs and effluxing amyloid beta (Aβ), a contributor to Alzheimer's disease (AD). Strategies to regulate P-gp expression therefore may impact central nervous system (CNS) drug delivery and brain Aβ levels. As we have demonstrated that the copper complex copper diacetyl bis(4-methyl-3-thiosemicarbazone) (Cu(ATSM)) increases P-gp expression and function in human brain endothelial cells, the present study assessed the impact of Cu(ATSM) on expression and function of P-gp in mouse brain endothelial cells (mBECs) and capillaries in vivo, as well as in peripheral organs. Isolated mBECs treated with Cu(ATSM) (100 nM for 24 h) exhibited a 1.6-fold increase in P-gp expression and a 20% reduction in accumulation of the P-gp substrate rhodamine 123. Oral administration of Cu(ATSM) (30 mg/kg/day) for 28 days led to a 1.5 & 1.3-fold increase in brain microvascular and hepatic expression of P-gp, respectively, and a 20% reduction in BBB transport of [3H]-digoxin. A metallomic analysis showed a 3.5 and 19.9-fold increase in Cu levels in brain microvessels and livers of Cu(ATSM)-treated mice. Our findings demonstrate that Cu(ATSM) increases P-gp expression and function at the BBB in vivo, with implications for CNS drug delivery and clearance of Aβ in AD.

Increased expression of the voltage-gated potassium channel Kv1.3 in activated microglia, and the subsequent release of pro-inflammatory mediators, are closely associated with the progression of Alzheimer's disease (AD). Studies have shown that reducing neuroinflammation through the non-selective blockade of microglial Kv1.3 has the potential to improve cognitive function in mouse models of familial AD. We have previously demonstrated that a potent and highly-selective peptide blocker of Kv1.3, HsTX1[R14A], not only entered the brain parenchyma after peripheral administration in a lipopolysaccharide (LPS)-induced mouse model of inflammation, but also significantly reduced pro-inflammatory mediator release from activated microglia. In this study, we show that microglial expression of Kv1.3 is increased in senescence accelerated mice (SAMP8), an animal model of sporadic AD, and that subcutaneous dosing of HsTX1[R14A] (1 mg/kg) every other day for 8 weeks provided a robust improvement in cognitive deficits in SAMP8 mice. The effect of HsTX1[R14A] on the whole brain was assessed using transcriptomics, which revealed that the expression of genes associated with inflammation, neuron differentiation, synapse function, learning and memory were altered by HsTX1[R14A] treatment. Further study is required to investigate whether these changes are downstream effects of microglial Kv1.3 blockade or a result of alternative mechanisms, including any potential effect of Kv1.3 blockade on other brain cell types. Nonetheless, these results collectively demonstrate the cognitive benefits of Kv1.3 blockade with HsTX1[R14A] in a mouse model of sporadic AD, demonstrating its potential as a therapeutic candidate for this neurodegenerative disease., (© 2023. The Author(s).)

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)-NH 2 (PEtOx-NH 2 ) and characterized by 1 H 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.

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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Fluids, solutes and immune cells have been demonstrated to drain from the brain and surrounding structures to the cervical lymph vessels and nodes in the neck via meningeal lymphatics, nasal lymphatics and/or lymphatic vessels associated with cranial nerves. A method to cannulate the efferent cervical lymph duct for continuous cervical lymph fluid collection in rodents has not been described previously and would assist in evaluating the transport of molecules and immune cells from the head and brain via the lymphatics, as well as changes in lymphatic transport and lymph composition with different physiological challenges or diseases. Aim: To develop a novel method to cannulate and continuously collect lymph fluid from the cervical lymph duct in rats and to analyze the protein, lipid and immune cell composition of the collected cervical lymph fluid. Methods: Male Sprague-Dawley rats were cannulated at the carotid artery with or without cannulation or ligation at the cervical lymph duct. Samples of blood, whole lymph and isolated lipoprotein fractions of lymph were collected and analyzed for lipid and protein composition using commercial kits. Whole lymph samples were centrifuged and isolated pellets were stained and processed for flow cytometry analysis of CD3 + , CD4 + , CD8a + , CD45R + (B220) and viable cell populations. Results: Flow rate, phospholipid, triglyceride, cholesterol ester, free cholesterol and protein concentrations in cervical lymph were 0.094 ± 0.014 mL/h, 0.34 ± 0.10, 0.30 ± 0.04, 0.07 ± 0.02, 0.02 ± 0.01 and 16.78 ± 2.06 mg/mL, respectively. Protein was mostly contained within the non-lipoprotein fraction but all lipoprotein types were also present. Flow cytometry analysis of cervical lymph showed that 67.1 ± 7.4% of cells were CD3 + /CD4 + T lymphocytes, 5.8 ± 1.6% of cells were CD3 + /CD8 + T lymphocytes, and 10.8 ± 4.6% of cells were CD3 - /CD45R + B lymphocytes. The remaining 16.3 ± 4.6% cells were CD3 - /CD45 - and identified as non-lymphocytes. Conclusion: Our novel cervical lymph cannulation method enables quantitative analysis of the lymphatic transport of immune cells and molecules in the cervical lymph of rats for the first time. This valuable tool will enable more detailed quantitative analysis of changes to cervical lymph composition and transport in health and disease, and could be a valuable resource for discovery of biomarkers or therapeutic targets in future studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hoang, Cao, Gracia, Nicolazzo and Trevaskis.)

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 (IC 50 ∼ 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 [ 64 Cu]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 [ 68 Ga]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, [ 68 Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC 50 ∼ 6 μM), was also tested. Significantly increased uptake of [ 68 Ga]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 [ 68 Ga]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.

For peripherally administered drugs to reach the central nervous system (CNS) and treat amyotrophic lateral sclerosis (ALS), they must cross the blood-brain barrier (BBB). As mounting evidence suggests that the ultrastructure of the BBB is altered in individuals with ALS and in animal models of ALS (e.g., SOD1 G93A mice), we characterized BBB transporter expression and function in transgenic C9orf72 BAC (C9-BAC) mice expressing a hexanucleotide repeat expansion, the most common genetic cause of ALS. Using an in situ transcardiac brain perfusion technique, we identified a 1.4-fold increase in 3 H-2-deoxy-D-glucose transport across the BBB in C9-BAC transgenic (C9) mice, relative to wild-type (WT) mice, which was associated with a 1.3-fold increase in brain microvascular glucose transporter 1 expression, while other general BBB permeability processes (passive diffusion, efflux transporter function) remained unaffected. We also performed proteomic analysis on isolated brain microvascular endothelial cells, in which we noted a mild (14.3%) reduction in zonula occludens-1 abundance in C9 relative to WT mice. Functional enrichment analysis highlighted trends in changes to various BBB transporters and cellular metabolism. To our knowledge, this is the first study to demonstrate altered BBB function in a C9orf72 repeat expansion model of ALS, which has implications on how therapeutics may access the brain in this mouse model.

Information on master's programs in the pharmaceutical sciences is lacking; this manuscript addresses this gap in the literature, by reporting on the results of an international survey performed in 2021 of master's programs in the pharmaceutical sciences offered at Schools/Colleges of Pharmacy. Ninety-six responses were received from universities from 23 countries, with the greatest number of responses obtained from India, followed by the United States and Japan. Master's programs in the pharmaceutical sciences are generally full time and 2 years in duration. Only 3% of programs were reported to be examination-based, while the remaining 97% had a research component with 70% of programs having a thesis defense with external and/or internal examiners. Master's programs tended to be larger in Asia and Europe than in North America; as well, programs in North America tended to have more international students. Didactic coursework was included in 96% of master's programs in North America, but only in 38% of Asian and 58% of European programs. The predominant placement of graduates from master's programs in Asia was in the pharmaceutical industry (70%); this contrasted with programs in Europe, Africa and North America where 28-36% enter careers in the pharmaceutical industry and higher percentages enter Ph.D. programs. The major challenge identified by programs was funding of faculty and of graduate students, although decreasing career opportunities was identified as a challenge in Asia and Africa., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare no conflict of interest., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

This publication represents the first to report global information on characteristics and requirements of doctoral programs in the pharmaceutical sciences in schools/colleges of Pharmacy. Survey responses (140 responses) were received from doctoral programs in 23 countries, with the greatest number of responses obtained from Japan, followed by India and the United States. Program characteristics and requirements, and student and faculty information, including graduate placement, in programs in Asia, North America, Europe, Africa and Australia were compared. Survey responses indicated differences in entrance requirements for doctoral programs with minimum requirements being a bachelor's degree, pharmacy degree or master's degree, including a M.Phil. degree. Programs differed widely in size in all geographical areas, but there was a similar emphasis on core educational learning outcomes (core competencies) and Ph.D. graduation requirements including qualifying examinations, thesis defense with internal and external reviewers and requirements for peer-reviewed publications. Additionally, three-quarters of programs indicated that there was external review of their programs every 2-4 or 5-7 years. Female students and female faculty mentors represented about 50% of students/faculty in programs in most geographical areas. Placement of students after graduation indicated that the highest percentage went into the pharmaceutical industry in Asia (predominantly India) and North America, with a lower percentage in Europe, Africa and Australia., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Upregulation of the voltage-gated potassium channel K V 1.3 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 K V 1.3 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 K V 1.3 with high affinity (IC 50 of 45 pM) and selectivity (2000-fold for K V 1.3 over K V 1.1). 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 K V 1.3 (IC 50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (K V 1.2, K V 1.4, K V 1.5, K V 1.6, K Ca 1.1 and K Ca 3.1), as well as selectivity against heteromeric channels assembled from K V 1.3/K V 1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged K V 1.3 shows co-localization of Cy5-HsTX1[R14A] and K V 1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect K V 1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of K V 1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify K V 1.3 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 K V 1.3, with broad applicability in fundamental investigations of K V 1.3 biology, and the validation of novel disease indications where K V 1.3 inhibition may be of therapeutic value.

A core competency of Australian Pharmacy graduates is to prepare and compound extemporaneous formulations. Students in our pharmacy course would traditionally formulate extemporaneous products in laboratory classes while simultaneously preparing a handwritten label, with students divorcing this laboratory activity from the entire dispensing process. As a way to incorporate the dispensing process into the preparation of extemporaneous products without adding excessive time to the laboratory, we integrated MyDispense, a virtual pharmacy simulation, in pre-laboratory activities. This meant that students could complete all the dispensing activities for prescribed extemporaneous formulations prior to attending the laboratory. Prescriptions for solutions, suspensions, creams and ointments were developed in MyDispense, including essential components for dispensing an extemporaneous formulation (e.g., formulation name, dosing instructions). These prescriptions were provided to students at least 1 week prior to their laboratory classes, whereas for the laboratory assessments, the prescription was provided at the commencement of the extemporaneous exam. Due to the implementation of dispensing via MyDispense, we found that students demonstrated pre-laboratory engagement as all students presented their printed labels upon entering the laboratory. We also observed an increase in interaction between students and laboratory facilitators, mainly focused on the principles of formulation integrated around patient outcomes. Virtual simulations such as MyDispense can therefore provide a guided realistic learning experience, whilst overcoming time pressures associated with laboratory timetabling. This approach also encourages students to engage in the dispensing process prior to extemporaneous laboratories providing more opportunity to discuss higher-level formulation principles and patient-centred outcomes during laboratory classes.

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 (Aβ) 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) (Cu II [GTSM]), would enhance P-gp expression and function at the BBB, while copper II diacetyl bis(4-methyl-3-thiosemicarbazone) (Cu II [ATSM]), which only releases Cu under hypoxic conditions, would not modulate P-gp expression. Following treatment with 25-250 nM Cu II (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, Cu II (ATSM) significantly enhanced P-gp expression and function 2-fold and 1.3-fold, respectively, whereas Cu II (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 Cu II (ATSM) and Cu II (GTSM) have the potential to modulate the expression and function of P-gp at the BBB to impact brain drug delivery and clearance of Aβ., (© 2022 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

The access of drugs into the central nervous system (CNS) is regulated by the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB). A large body of evidence supports perturbation of these barriers in neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Modifications to the BBB and BSCB are also reported in amyotrophic lateral sclerosis (ALS), albeit these modifications have received less attention relative to those in other neurodegenerative diseases. Such alterations to the BBB and BSCB have the potential to impact on CNS exposure of drugs in ALS, modulating the effectiveness of drugs intended to reach the brain and the toxicity of drugs that are not intended to reach the brain. Given the clinical importance of these phenomena, this review will summarise reported modifications to the BBB and BSCB in ALS, discuss their impact on CNS drug exposure, and suggest further research directions so as to optimise medicine use in people with ALS., (© 2022 The British Pharmacological Society.)

Alzheimer's disease (AD) has traditionally been considered solely a neurological condition. Therefore, numerous studies have been conducted to identify the existence of pathophysiological changes affecting the brain and the blood-brain barrier in individuals with AD. Such studies have provided invaluable insight into possible changes to the central nervous system exposure of drugs prescribed to individuals with AD. However, there is now increasing recognition that extra-neurological systems may also be affected in AD, such as the small intestine, liver, and kidneys. Examination of these peripheral pathophysiological changes is now a burgeoning area of scientific research, owing to the potential impact of these changes on the absorption, distribution, metabolism, and excretion (ADME) of drugs used for both AD and other concomitant conditions in this population. The purpose of this review is to identify and summarise available literature reporting alterations to key organs influencing the pharmacokinetics of drugs, with any changes to the small intestine, liver, kidney, and circulatory system on the ADME of drugs described. By assessing studies in both rodent models of AD and samples from humans with AD, this review highlights possible dosage adjustment requirements for both AD and non-AD drugs so as to ensure the achievement of optimum pharmacotherapy in individuals with AD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The expression of voltage-gated potassium Kv1.3 channels is increased in activated microglia, with non-selective blockade reported to attenuate microglial-mediated neuroinflammation. In this study, we evaluated the impact of a potent and selective peptidic blocker of Kv1.3 channels, HsTX1[R14A], on microglial-mediated neuroinflammation in vitro and in vivo. Treatment with both 0.1 and 1 µg/mL lipopolysaccharide (LPS) significantly (p < 0.05) increased Kv1.3 abundance on the surface of BV-2 microglia in association with increased levels of mRNA for tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The increased transcription of TNF-α and IL-6 was significantly attenuated (by 24.9 and 20.2%, respectively) by HsTX1[R14A] (100 nM). The concomitant increase in TNF-α and IL-6 release from BV-2 microglia was significantly attenuated by HsTX1[R14A] by 10.7 and 12.6%, respectively. In LPS-treated primary mouse microglia, the levels of TNF-α and nitric oxide were also attenuated by HsTX1[R14A] (26.1 and 20.4%, respectively). In an LPS-induced mouse model of neuroinflammation, both an immediate and delayed subcutaneous dose of HsTX1[R14A] (2 mg/kg) significantly reduced plasma and brain levels of the pro-inflammatory mediators TNF-α, IL-1β and IL-6, with no impact on the anti-inflammatory IL-10. These results demonstrate that HsTX1[R14A] is a promising therapeutic candidate for the treatment of diseases with a neuroinflammatory component., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Pharmacy schools are generating significant amounts of data across the training continuum, including data about student selection, performance, and job placement. However, current data practices limit the Academy's ability to effectively leverage the vast amounts of data available within and across pharmacy institutions. To improve data practices and promote the quality and reusability of data, a set of guiding principles for data management and stewardship were developed and published in 2016. The FAIR principles state that digital objects should be findable (ie, data have a unique identifier and are registered in a searchable resource), accessible (ie, data are retrievable by their identifier using an open, free, standardized protocol), interoperable (ie, data use a formal, accessible, shared, and broadly applicable language, and include qualified references to other data), and reusable (ie, data are described with accurate and relevant attributes, released with a data usage license, and meet domain-relevant community standards). This commentary advocates for improved data practices and provides recommendations for advancing FAIR data principles in pharmacy education., (© 2022 American Association of Colleges of Pharmacy.)

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.

Whilst curriculum revision is commonplace, whole degree transformation is less so. In this paper we discuss the rationale, design and implementation of a unique pharmacy program by a research-intensive faculty. The new Monash pharmacy curriculum, which had its first intake in 2017, was built using a range of key innovations that aimed to produce graduates that demonstrate key conceptual understanding and all the skills required to deliver world-best patient outcomes. The key elements of the re-design are outlined and include the process and principles developed, as well as key features such as a student-centred individualised program of development arranged around specific, authentic tasks for each skill and earlier enhanced experiential placements where students become proficient in entrustable professional activities. It is hoped the dissemination of this process, as well as the lessons learnt in the process, will be useful to others looking to transform a health curriculum.

While the processes governing docosahexaenoic acid (DHA) trafficking across the blood-brain barrier have been elucidated, factors governing DHA uptake into microglia, an essential step for this fatty acid to exert its anti-inflammatory effects, are unknown. This study assessed the mRNA and protein expression of fatty acid-binding proteins (FABPs) and fatty acid transport proteins (FATPs) in mouse BV-2 cells and their mRNA expression in primary mouse microglia. The microglial uptake of DHA-d5, a surrogate of DHA, was assessed by LC-MS/MS following interventions including temperature reduction, silencing of various FABP isoforms, competition with DHA, and metabolic inhibition. It was found that DHA-d5 uptake at 4°C was 39.6% lower than at 37°C, suggesting that microglial uptake of DHA-d5 likely involves passive and/or active uptake mechanisms. Of all FABP and FATP isoforms probed, only FABP3, FABP4, FABP5, FATP1, and FATP4 were expressed at both the mRNA and protein level. Silencing of FABP3, FABP4, and FABP5 resulted in no change in cellular DHA-d5 uptake, nor did concomitant DHA administration or the presence of 0.1% sodium azide/50 mM 2-deoxy-D-glucose. This study is the first to identify the presence of FABPs and FATPs in mouse microglia, albeit these proteins are not involved in the microglial uptake of DHA-d5., (Copyright © 2021. Published by Elsevier Ltd.)

K V 1.3 is a voltage-gated potassium channel that is upregulated in neuroinflammatory conditions, such as Alzheimer's disease and Parkinson's disease. HsTX1[R14A] is a potent and selective peptide blocker of K V 1.3 with the potential to block microglial K V 1.3, but its brain uptake is expected to be limited owing to the restrictive nature of the blood-brain barrier. To assess its peripheral and brain exposure, a LC-MS/MS assay was developed to quantify HsTX1[R14A] concentrations in mouse plasma and brain homogenate that was reliable and reproducible in the range of 6.7-66.7 nM (r 2  = 0.9765) and 15-150 pmol/g (r 2  = 0.9984), respectively. To assess if neuroinflammation affected HsTX1[R14A] disposition, C57BL/6 mice were administered HsTX1[R14A] subcutaneously (2 mg/kg) 24 h after an intraperitoneal dose of Escherichia coli lipopolysaccharide (LPS), which is commonly used to induce neuroinflammation; brain and plasma concentrations of HsTX1[R14A] were then quantified over 120 min. LPS treatment significantly retarded the decline in HsTX1[R14A] plasma concentrations, presumably as a result of reducing renal clearance, and led to substantial brain uptake of HsTX1[R14A], presumably through disruption of brain inter-endothelial tight junctions. This study suggests that HsTX1[R14A] may reach microglia in sufficient concentrations to block K V 1.3 in neuroinflammatory conditions, and therefore has the potential to reduce neurodegenerative diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Antisense oligonucleotides (ASOs) are an emerging class of gene-specific therapeutics for diseases associated with the central nervous system (CNS). However, ASO delivery across the blood-brain barrier (BBB) to their CNS target cells remains a major challenge. Since ASOs are mainly taken up into the brain capillary endothelial cells interface through endosomal routes, entrapment in the endosomal compartment is a major obstacle for efficient CNS delivery of ASOs. Therefore, we evaluated the effectiveness of a panel of cell-penetrating peptides (CPPs) bearing several endosomal escape domains for the intracellular delivery, endosomal release and antisense activity of FDA-approved Spinraza (Nusinersen), an ASO used to treat spinal muscular atrophy (SMA). We identified a CPP, HA2-ApoE(131-150), which, when conjugated to Nusinersen, showed efficient endosomal escape capability and significantly increased the level of full-length functional mRNA of the survival motor neuron 2 (SMN2) gene in SMA patient-derived fibroblasts. Treatment of SMN2 transgenic adult mice with this CPP-PMO conjugate resulted in a significant increase in the level of full-length SMN2 in the brain and spinal cord. This work provides proof-of-principle that integration of endosomal escape domains with CPPs enables higher cytosolic delivery of ASOs, and more importantly enhances the efficiency of BBB-permeability and CNS activity of systemically administered ASOs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study evaluated the impact of poly(lactic-co-glycolic acid) (PLGA) microsphere formulations on in vitro release and in vivo plasma exposure of HsTX1[R14A], a potent inhibitor of the voltage-gated potassium channel Kv1.3, with potential to treat autoimmune conditions. Microspheres containing HsTX1[R14A] were prepared using different PLGA materials, including Resomer® RG502H, RG503H and PURASORB® PDLG 5004 (Purac). After assessing encapsulation efficiency and in vitro release, plasma concentrations of HsTX1[R14A] were quantified by LCMS/MS following subcutaneous administration of HsTX1[R14A]-loaded RG503H microspheres (15 mg/kg) or HsTX1[R14A] solution (4 mg/kg) to Sprague-Dawley rats. Microspheres prepared with Purac exhibited the greatest encapsulation efficiency (45.5 ± 2.4% (mean ± SD)) and RG502H the lowest (22.0 ± 6.4%). Release of HsTX1[R14A] was fastest in vitro for RG502H microspheres (maximum release at 31 days) and slowest for Purac (82 days). With a relatively rapid burst release of 20.0 ± 0.4% and a controlled release profile of up to 41 days, HsTX1[R14A]-loaded RG503H microspheres were selected for subcutaneous administration, resulting in detectable plasma concentrations for 11 days relative to 8 h following subcutaneous administration of HsTX1[R14A] solution. Therefore, subcutaneous administration of RG503H PLGA microspheres is a promising approach to be exploited for delivery of this immune modulator., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

In Alzheimer's disease (AD), excessive amounts of quinolinic acid (QUIN) accumulate within the brain parenchyma and dystrophic neurons. QUIN also regulates glutamate uptake into neurons, which may be due to modulation of Na + -dependent excitatory amino acid transporters (EAATs). To determine the biological relationships between QUIN and glutamate dysfunction, we first quantified the functionality and kinetics of [ 3 H]QUIN uptake in primary human neurons using liquid scintillation. We then measured changes in the protein expression of the glutamate transporter EAAT3 and EAAT1b in primary neurons treated with QUIN and the EAAT inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (2,4-PDC) using western blotting and immunohistochemistry. Immunohistochemistry was further used to elucidate intracellular transport of exogenous QUIN and the lysosomal-associated membrane protein 2 (LAMP2). Structural insights into the binding between QUIN and EAAT3 were further investigated using molecular docking techniques. We report significant temperature-dependent high-affinity transport leading to neuronal uptake of [ 3 H]QUIN with a Km of 42.2 μM, and a V max of 9.492 pmol/2 min/mg protein, comparable with the uptake of glutamate. We also found that QUIN increases expression of the EAAT3 monomer while decreasing the functional trimer. QUIN uptake into primary neurons was shown to involve EAAT3 as uptake was significantly attenuated following EAAT inhibition. We also demonstrated that QUIN increases the expression of aberrant EAAT1b protein in neurons further implicating QUIN-induced glutamate dysfunction. Furthermore, we demonstrated that QUIN is metabolised exclusively in lysosomes. The involvement of EAAT3 as a modulator for QUIN uptake was further confirmed using molecular docking. This study is the first to characterise a mechanism for QUIN uptake into primary human neurons involving EAAT3, opening potential targets to attenuate QUIN-induced excitotoxicity in neuroinflammatory diseases.

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 β-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 [ 3 H]-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 [ 3 H]-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 β-amyloid clearance.

Modulating the abundance of the blood-brain barrier (BBB) efflux transporter breast cancer resistance protein (BCRP) has the potential to impact brain levels of drugs and endogenous substrates. Studies have demonstrated that the metal ionophore clioquinol (CQ) increases BBB abundance of P-glycoprotein (P-gp), an effect associated with increased endothelial cell levels of Cu 2+ . This study therefore assessed whether human brain endothelial (hCMEC/D3) cell abundance and function of BCRP is modulated by CQ. hCMEC/D3 cells were treated with CQ, Zn 2+ and Cu 2+ (CZC) (0.5 μM, 0.5 μM, 0.1 μM, respectively) for 24 h and BCRP mRNA and protein abundance was determined by Western blot and qPCR, respectively. After a series of optimisation studies assessing specificity of bodipy prazosin (BP) and Ko143 as a substrate and inhibitor of BCRP, respectively, the impact of CZC on BP uptake was assessed. While CZC did not increase mRNA expression of BCRP, BCRP abundance was increased 1.8 ± 0.1-fold; this was associated with a 68.1 ± 3.3% reduction in accumulation of BP in hCMEC/D3 cells. This is the first study to demonstrate that augmenting metal ion availability enhances protein abundance and function of BCRP at the BBB, which may be exploited to modulate CNS access of therapeutics and endogenous substrates., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid with the capacity to reduce the proinflammatory response of activated microglia that occurs during neuroinflammation. The uptake of DHA into microglia is essential for it to exert its neuroprotective effects. However, quantifying the uptake of DHA into microglia is complicated by the presence of endogenous DHA interfering with any quantification technique. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was therefore developed and validated in order to assess the microglial uptake of docosahexaenoic acid-d5 (DHA-d5) as a surrogate for DHA. Using a mobile phase consisting of 90 % (v/v) acetonitrile and 10 % (v/v) water containing 2 mM ammonium acetate, a flow rate of 0.3 mL/min, and MS/MS detection in the negative ionization mode, DHA-d5 was detected at m/z transitions of 332.1/228.3/234.2, with good linearity between chromatographic area under the curve (AUC) and DHA-d5 mass (R 2 = 0.999) over the range of 0.0063-0.1 ng. The precision and accuracy values for the quality control samples (0.0063, 0.025, and 0.1 ng) were less than 9.3 % and 96.6-109.8 %, respectively, and a comparison of DHA-d5 AUC when prepared in PBS or in microglial cell lysate demonstrated no significant difference between quantification of these quality control samples. Utilizing this quantification approach (with preparation of DHA-d5 calibration standards in PBS), the uptake of DHA-d5 into BV-2 microglial cells over a 15 min period was assessed, following the spiking of DHA-d5 at 50 ng/mL. Following protein normalization using a BCA protein assay, a rapid and linear uptake of DHA-d5 into BV-2 cells was observed in the first 2 min, after which a plateau in uptake was observed, in line with that reported for DHA uptake in other cell types. This novel LC-MS/MS technique can now be exploited to unravel the processes involved in microglial uptake of DHA, insights that may be used to maximize the anti-inflammatory effects of DHA in neuroinflammation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background and Purpose: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by four genes (HCN1-4) with distinct biophysical properties and functions within the brain. HCN4 channels activate slowly at robust hyperpolarizing potentials, making them more likely to be engaged during hyperexcitable neuronal network activity seen during seizures. HCN4 channels are also highly expressed in thalamic nuclei, a brain region implicated in seizure generalization. Here, we assessed the utility of targeting the HCN4 channel as an anti-seizure strategy using pharmacological and genetic approaches., Experimental Approach: The impact of reducing HCN4 channel function on seizure susceptibility and neuronal network excitability was studied using an HCN4 channel preferring blocker (EC18) and a conditional brain specific HCN4 knockout mouse model., Key Results: EC18 (10 mg·kg -1 ) and brain-specific HCN4 channel knockout reduced seizure susceptibility and proconvulsant-mediated cortical spiking recorded using electrocorticography, with minimal effects on other mouse behaviours. EC18 (10 μM) decreased neuronal network bursting in mouse cortical cultures. Importantly, EC18 was not protective against proconvulsant-mediated seizures in the conditional HCN4 channel knockout mouse and did not reduce bursting behaviour in AAV-HCN4 shRNA infected mouse cortical cultures., Conclusions and Implications: These data suggest the HCN4 channel as a potential pharmacologically relevant target for anti-seizure drugs that is likely to have a low side-effect liability in the CNS., (© 2020 The British Pharmacological Society.)

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 3 H-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 14 C-mannitol across the jejunum from APP/PS1 mice was lower than that across the WT jejunum ( P app = 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.

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 PPARβ/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 14 C-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 14 C-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.

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 C max . 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, C max 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., (© 2019 The British Pharmacological Society.)

Previous studies have demonstrated that the ionophore clioquinol (CQ), in conjunction with the biometals copper and zinc, increases the expression of P-glycoprotein (P-gp) in human cerebral microvascular endothelial (hCMEC/D3) cells. As P-gp expression and function at the blood-brain barrier (BBB) is of great interest regarding CNS drug access and endogenous toxin trafficking (e.g., amyloid beta), the present study assessed the in vivo translation of these previous in vitro findings. Swiss outbred mice received an 11-day treatment of CQ (30 mg/kg) by oral gavage, after which brain microvessel-enriched fractions (MEFs) and surrounding interfaces (subcortical brain tissue and plasma) were extracted. P-gp expression was quantified in the MEF, and biometal concentrations in all 3 compartments were assessed via inductively coupled plasma mass spectrometry. CQ treatment did not modify the expression of P-gp, nor copper or zinc concentrations in the brain MEF under this treatment regime. Metallomic analysis revealed, however, that CQ reduced potassium and magnesium levels in the brain MEF and also lowered brain iron levels. This study has shown that under this dosing regimen, CQ does not increase BBB P-gp expression in Swiss outbred mice, but that CQ facilitates redistribution of certain metal ions within the brain MEF, plasma, and brain parenchyma., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Tonic inhibitory currents, mediated by extrasynaptic GABA A receptors, are elevated at a delay following stroke. Flavonoids minimise the extent of cellular damage following stroke, but little is known about their mode of action. We demonstrate that the flavonoid, 2'-methoxy-6-methylflavone (0.1-10 µM; 2'MeO6MF), increases GABA A receptor tonic currents presumably via δ-containing GABA A receptors. Treatment with 2'MeO6MF 1-6 h post focal ischaemia dose dependently decreases infarct volume and improves functional recovery. The effect of 2'MeO6MF was attenuated in δ -/- mice, indicating that the effects of the flavonoid were mediated via δ-containing GABA A receptors. Further, as flavonoids have been shown to have multiple modes of action, we investigated the anti-inflammatory effects of 2'MeO6MF. Using a macrophage cell line, we show that 2'MeO6MF can dampen an LPS-induced elevation in NFkB activity. Assessment of vehicle-treated stroke animals revealed a significant increase in circulating IL1β, TNFα and IFγ levels. Treatment with 2'MeO6MF dampened the stroke-induced increase in circulating cytokines, which was blocked in the presence of the pan-AKT inhibitor, GSK690693. These studies support the hypothesis that compounds that potentiate tonic inhibition via δ-containing GABA A receptors soon after stroke can afford neuroprotection.

It is well established that the expression and function of drug transporters at the blood-brain barrier are altered in Alzheimer's disease (AD). However, we recently demonstrated in a mouse model of AD that the expression of key drug transporters and metabolizing enzymes was modified in peripheral organs, such as the small intestine and liver, suggesting that systemic drug absorption may be altered in AD. The purpose of this study was to determine whether the expression of drug transporters in the kidneys differed between 8- to 9-month-old wild-type mice and APPswe/PSEN1dE9 (APP/PS1) transgenic mice, a mouse model of familial AD, using a quantitative targeted absolute proteomics approach. The protein expression of the drug transporters-multidrug resistance-associated protein 2, organic anion transporter 3, and organic cation transporter 2-was upregulated 1.6-, 1.3-, and 1.4-fold, respectively, in kidneys from APP/PS1 mice relative to wild-type mice. These results suggest that in addition to modified oral absorption of certain drugs, it is possible that the renal excretion of drugs that are multidrug resistance-associated protein 2, organic anion transporter 3, and organic cation transporter 2 substrates could be altered in AD. These changes could affect the interpretation of studies conducted during drug development using this mouse model of AD and potentially impact dosage regimens of such drugs prescribed in this patient population., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

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., (Published by Elsevier Inc.)

Older people are at a greater risk of medicine-induced toxicity resulting from either increased drug sensitivity or age-related pharmacokinetic changes. The scenario is further complicated with the two most prevalent age-related neurodegenerative diseases, Alzheimer's disease (AD) and Parkinson's disease (PD). With aging, AD and PD, there is growing evidence of altered structure and function of the blood-brain barrier (BBB), including modifications to tight junctions and efflux transporters, such as P-glycoprotein. The subsequent impact on CNS drug exposure and risk of neurotoxicity from systemically-acting medicines is less well characterized. The purpose of this review, therefore, is to provide an overview of the multiple changes that occur to the BBB as a result of aging, AD and PD, and the impact that such changes have on CNS exposure of drugs, based on studies conducted in aged rodents or rodent models of disease, and in elderly people with and without AD or PD., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)