Human BBB Research Articles

Evaluation of Drug Blood-Brain-Barrier Permeability Using a Microfluidic Chip.

The blood-brain-barrier (BBB) is made up of blood vessels whose permeability enables the passage of some compounds. A predictive model of BBB permeability is important in the early stages of drug development. The predicted BBB permeabilities of drugs have been confirmed using a variety of in vitro methods to reduce the quantities of drug candidates needed in preclinical and clinical trials. Most prior studies have relied on animal or cell-culture models, which do not fully recapitulate the human BBB. The development of microfluidic models of human-derived BBB cells could address this issue. We analyzed a model for predicting BBB permeability using the Emulate BBB-on-a-chip machine. Ten compounds were evaluated, and their permeabilities were estimated. Our study demonstrated that the permeability trends of ten compounds in our microfluidic-based system resembled those observed in previous animal and cell-based experiments. Furthermore, we established a general correlation between the partition coefficient (Kp) and the apparent permeability (Papp). In conclusion, we introduced a new paradigm for predicting BBB permeability using microfluidic-based systems.

Brain-Targeted Liposomes Loaded with Monoclonal Antibodies Reduce Alpha-Synuclein Aggregation and Improve Behavioral Symptoms in Parkinson's Disease.

Monoclonal antibodies (mAbs) hold promise in treating Parkinson's disease (PD), although poor delivery to the brain hinders their therapeutic application. In the current study, it is demonstrated that brain-targeted liposomes (BTL) enhance the delivery of mAbs across the blood-brain-barrier (BBB) and into neurons, thereby allowing the intracellular and extracellular treatment of the PD brain. BTL are decorated with transferrin to improve brain targeting through overexpressed transferrin-receptors on the BBB during PD. BTL are loaded with SynO4, a mAb that inhibits alpha-synuclein (AS) aggregation, a pathological hallmark of PD. It is shown that 100-nm BTL cross human BBB models intact and are taken up by primary neurons. Within neurons, SynO4 is released from the nanoparticles and bound to its target, thereby reducing AS aggregation, and enhancing neuronal viability. In vivo, intravenous BTL administration results in a sevenfold increase in mAbs in brain cells, decreasing AS aggregation and neuroinflammation. Treatment with BTL also improve behavioral motor function and learning ability in mice, with a favorable safety profile. Accordingly, targeted nanotechnologies offer a valuable platform for drug delivery to treat brain neurodegeneration.

Exploring the reactivity of bicyclic α-iminophosphonates to access new imidazoline I2 receptor ligands

Recent studies pointed out the modulation of imidazoline I2 receptors (I2-IR) by selective ligands as a putative strategy to face neurodegenerative diseases. Foregoing the classical 2-imidazoline/imidazole-containing I2-IR ligands, we report a family of bicyclic α-iminophosphonates endowed with high affinity and selectivity upon I2-IR and we advanced a representative compound B06 in preclinical phases. In this paper, we describe the synthetic possibilities of bicyclic α-iminophosphonates by exploring its ambivalent reactivity, leading to unprecedented molecules that showed promising activities as I2-IR ligands in human brain tissues and good BBB permeation capabilities. After in silico ADME prediction studies, we assessed the neuroprotective properties of selected compounds and beneficial effect in an in vitro model of Alzheimeŕs and Parkinson’s disease. Along with their neuroprotective effect, compounds showed a potent anti-inflammatory response when evaluated in a neuroinflammation cellular model. Moreover, this is the first time that the neuroprotective effects of imidazoline I2-IR ligands in a transgenic Alzheimer’s disease Caenorhabditis elegans strain are investigated. Using a thrashing assay, we found a significant cognition improvement in this in vivo model after treatment with the new bicyclic α-phosphoprolines. Therefore, our results confirmed the need of exploring structurally new I2-IR ligands and their potential for therapeutic strategies in neurodegeneration.

Quantification of P-glycoprotein function at the human blood-brain barrier using [18F]MC225 and PET

IntroductionP-glycoprotein (P-gp) is one of the most studied efflux transporters at the blood-brain barrier. It plays an important role in brain homeostasis by protecting the brain from a variety of endogenous and exogeneous substances. Changes in P-gp function are associated both with the onset of neuropsychiatric diseases, including Alzheimer’s disease and Parkinson’s disease, and with drug-resistance, for example in treatment-resistant depression. The most widely used approach to measure P-gp function in vivo is (R)-[11C]verapamil PET. (R)-[11C]verapamil is, however, an avid P-gp substrate, which complicates the use of this tracer to measure an increase in P-gp function as its baseline uptake is already very low. [18F]MC225 was developed to measure both increases and decreases in P-gp function.AimThe aim of this study was (1) to identify the pharmacokinetic model that best describes [18F]MC225 kinetics in the human brain and (2) to determine test-retest variability.MethodsFive (2 male, 3 female) of fourteen healthy subjects (8 male, 6 female, age 67 ± 5 years) were scanned twice (injected dose 201 ± 47 MBq) with a minimum interval of 2 weeks between scans. Each scanning session consisted of a 60-min dynamic [18F]MC225 scan with continuous arterial sampling. Whole brain grey matter data were fitted to a single tissue compartment model, and to reversible and irreversible two tissue-compartment models to obtain various outcome parameters (in particular the volume of distribution (VT), Ki, and the rate constants K1 and k2). In addition, a reversible two-tissue compartment model with fixed k3/k4 was included. The preferred model was selected based on the weighted Akaike Information Criterion (AIC) score. Test-retest variability (TRTV) was determined to assess reproducibility.ResultsSixty minutes post-injection, the parent fraction was 63.8 ± 4.0%. The reversible two tissue compartment model corrected for plasma metabolites with an estimated blood volume (VB) showed the highest AIC weight score of 34.3 ± 17.6%. The TRVT of the VT for [18F]MC225 PET scans was 28.3 ± 20.4% for the whole brain grey matter region using this preferred model.Conclusion[18F]MC225 VT, derived using a reversible two-tissue compartment model, is the preferred parameter to describe P-gp function in the human BBB. This outcome parameter has an average test-retest variability of 28%.Trial registrationEudraCT 2020-001564-28. Registered 25 May 2020.

Toxoplasma Gondii Induces Blood-Brain Barrier Dysfunction in a Human Stem Cell- Derived Model


 
 
 Background:
 Toxoplasma gondii is a single-celled obligate intracellular parasite commonly found in cat feces and animal issues and is known to cause cerebral infections in immune- compromised individuals and neonates. Once ingested, T. gondii reaches the host's blood supply and reaches the brain, where infection ensues. However, it remains unclear how T. gondii crosses the blood-brain barrier to reach the brain. In this study, we utilize a human stem cell-derived BBB model with near in vivo properties to investigate the effects of T. gondii on critical barrier properties that permit entry into the brain parenchyma.
 Methods:
 Human-induced pluripotent stem cells (iPSCs) were used to derive brain microvascular endothelial cells (BMECs). BMECs were exposed to tachyzoites, the infectious rapid replicating stage of T. gondii, at a multiplicity of infection (MOI) of 0.1 and 1.0 for 24 hours. Following exposure, several critical barrier properties were monitored, including trans- endothelial electrical resistance (TEER) and tight junction analysis.
 Results:
 BMECs treated with both MOI of 0.1 and 1.0 showed a significant reduction in all three tight junction proteins (Claudin-5, Occludin, and ZO-1), as indicated by an increase in discontinuous junctions and a decrease in area fraction index. Barrier integrity is directly associated with tight junctional continuity and localization. Furthermore, tachyzoites at an MOI of 0.1 and 1.0 demonstrated a reduction in barrier tightness, as observed by a decrease in TEER.
 Conclusion and Potential Impact:
 These results indicate that T. gondii may, in part, have access to the brain by inducing disruption in tight junction proteins, thus increasing barrier leakiness via a para-cellular route. These data may help guide future investigations on T. gondii's ability to evoke blood-brain barrier dysfunction and reach the vulnerable brain tissue.
 
 

Ethanol Induces Blood-Brain Barrier Dysfunction in a Familial Alzheimer’s Human Stem Cell-Derived Model


 
 
 Background:
 Alcohol consumption has been directly linked to neurodegenerative diseases, including Alzheimer's disease (AD). Additionally, heavy consumption of alcohol has been shown to cause a faster cognitive decline in AD patients. A subset of studies demonstrated that alcohol can diminish BBB integrity and independently AD patients have suppressed barrier properties, but the direct effect of alcohol on barrier integrity in AD patients remains unclear. In this study, we utilize a human stem cell-derived AD BBB model with near in vivo properties to investigate the effects of alcohol on critical barrier properties.
 Methods:
 Brain microvascular endothelial cells (BMECs) were derived from healthy (IMR90) and AD (PSEN 1, PSEN 2, and APP) human induced pluripotent stem cells (iPSCs). Healthy and AD cell lines were treated with physiologically relevant concentrations of alcohol (5, 25, and 50 mM). Following exposure, several critical barrier properties were monitored for up to 5 days post-exposure, including trans-endothelial electrical resistance (TEER), sodium fluorescein permeability, and tight junction localization.
 Results:
 Moderate to severe alcohol exposure (25mM and 50mM) decreased barrier integrity in both healthy and AD-derived BMECs, as observed by an increase in sodium fluorescein permeability and a reduction in TEER. Furthermore, alcohol increased the number of discontinuous tight junctions directly contributing to the diminished barrier integrity. Interestingly, our preliminary results demonstrate that AD-derived BMECs are more susceptible to ethanol- induced barrier injury at lower concentrations of ethanol (5mM) compared to healthy-derived BMECs.
 Conclusion and Potential Impact:
 Our results indicate that alcohol can diminish critical barrier properties in healthy-derived BMECs similarly to other non-human established BBB models. For the first time, we observed an increase sensitivity to alcohol-induced BBB dysfunction in a familial AD-derived BBB model. These data suggest that mild alcohol consumption could significantly alter the BBB and contribute to the development or exacerbation of AD-induced barrier dysfunction.
 
 

Role of pericytes in blood–brain barrier preservation during ischemia through tunneling nanotubes

Crosstalk mechanisms between pericytes, endothelial cells, and astrocytes preserve integrity and function of the blood-brain-barrier (BBB) under physiological conditions. Long intercellular channels allowing the transfer of small molecules and organelles between distant cells called tunneling nanotubes (TNT) represent a potential substrate for energy and matter exchanges between the tripartite cellular compartments of the BBB. However, the role of TNT across BBB cells under physiological conditions and in the course of BBB dysfunction is unknown. In this work, we analyzed the TNT’s role in the functional dialog between human brain endothelial cells, and brain pericytes co-cultured with human astrocytes under normal conditions or after exposure to ischemia/reperfusion, a condition in which BBB breakdown occurs, and pericytes participate in the BBB repair. Using live time-lapse fluorescence microscopy and laser-scanning confocal microscopy, we found that astrocytes form long TNT with pericytes and endothelial cells and receive functional mitochondria from both cell types through this mechanism. The mitochondrial transfer also occurred in multicellular assembloids of human BBB that reproduce the three-dimensional architecture of the BBB. Under conditions of ischemia/reperfusion, TNT formation is upregulated, and astrocytes exposed to oxygen-glucose deprivation were rescued from apoptosis by healthy pericytes through TNT-mediated transfer of functional mitochondria, an effect that was virtually abolished in the presence of TNT-destroying drugs. The results establish a functional role of TNT in the crosstalk between BBB cells and demonstrate that TNT-mediated mitochondrial transfer from pericytes rescues astrocytes from ischemia/reperfusion-induced apoptosis. Our data confirm that the pericytes might play a pivotal role in preserving the structural and functional integrity of BBB under physiological conditions and participate in BBB repair in brain diseases.

Protein kinase C-β distinctly regulates blood-brain barrier-forming capacity of Brain Microvascular endothelial cells and outgrowth endothelial cells

Outgrowth endothelial cells (OECs) provide an endogenous repair mechanism and thus maintain endothelial barrier integrity. As inhibition of protein kinase C-β (PKC-β) activity has been shown to attenuate endothelial damage in various pathological conditions including hyperglycaemia and ischaemic injury, the present study comparatively assessed the effect of LY333531, a PKC-β inhibitor, on the cerebral barrier integrity formed by OECs or human brain microvascular endothelial cells (HBMECs). To this end, an in vitro model of human BBB established by co-culture of astrocytes and pericytes with either OECs or HBMECs was exposed to 4 h of oxygen-glucose deprivation with/out LY333531 (0.05 µM). The inhibition of PKC-β protected the integrity and function of the BBB formed by HBMECs, as evidenced by increases in transendothelial electrical resistance and decreases in sodium fluorescein flux. It also attenuated ischaemia-evoked actin cytoskeleton remodelling, oxidative stress, and apoptosis in HBMECs. In contrast, treatments with LY333531 exacerbated the deleterious effect of ischaemia on the integrity and function of BBB formed by OECs while augmenting the levels of oxidative stress, apoptosis, and cytoskeletal reorganisation in OECs. Interestingly, the magnitude of damage in all aforementioned parameters, notably oxidative stress, was lower with low dose of LY333531 (0.01 µM). It is therefore possible that the therapeutic concentration of LY333531 (0.05 µM) may neutralise the activity of NADPH oxidase and thus trigger a negative feedback mechanism which in turn exacerbate the detrimental effects of ischaemic injury. In conclusion, targeting PKC-β signalling pathway in ischaemic settings requires close attention while using OECs as cellular therapeutic.

Construction and Functional Evaluation of a Three-Dimensional Blood–Brain Barrier Model Equipped With Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells

PurposeThe purpose of this study was to construct and validate an in vitro three-dimensional blood–brain barrier (3DBBB) model system equipped with brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs).MethodsThe 3D-BBB system was constructed by seeding hiPS-BMECs onto the capillary lane of a MIMETAS OrganoPlate® 3-lane coated with fibronectin/collagen IV. hiPS-BMECs were incubated under continuous switchback flow with an OrganoFlow® for 2 days. The 3D capillary structure and expression of tight-junction proteins and transporters were confirmed by immunocytochemistry. The mRNA expression of transporters in the 3D environment was determined using qRT-PCR, and the permeability of endogenous substances and drugs was evaluated under various conditions.Results and DiscussionThe expression of tight-junction proteins, including claudin-5 and ZO-1, was confirmed by immunohistochemistry. The permeability rate constant of lucifer yellow through hiPS-BMECs was undetectably low, indicating that paracellular transport is highly restricted by tight junctions in the 3D-BBB system. The mRNA expression levels of transporters and receptors in the 3D-BBB system differed from those in the 2D-culture system by 0.2- to 5.8-fold. The 3D-cultured hiPS-BMECs showed asymmetric transport of substrates of BCRP, CAT1 and LAT1 between the luminal (blood) and abluminal (brain) sides. Proton-coupled symport function of MCT1 was also confirmed.ConclusionThe 3D-BBB system constructed in this study mimics several important characteristics of the human BBB, and is expected to be a useful high-throughput evaluation tool in the development of CNS drugs.

DDRE-25. WSD0628: A BRAIN PENETRABLE ATM INHIBITOR AS A RADIOSENSITIZER FOR THE TREATMENT OF GBM AND METASTATIC CNS TUMOR

Abstract ATM (ataxia telangiectasia mutated) kinase, activated by DNA double-strand breaks, promotes DNA repair as well as activates DNA damage checkpoint and plays a key role for resistance to radiotherapy and chemotherapy. ATM function loss confers hypersensitivity to ionizing radiation evidenced by ataxia-telangiectasia (A-T) cells. Thus, pharmacological inhibition of ATM kinase is expected to suppress DSB DNA repair, block checkpoint controls and enhance the therapeutic effect of radiotherapy and other DNA double-strand breaks-inducing chemotherapy. Herein, we report a discovery of a potent, selective, orally bioavailable, and brain penetrable ATM inhibitor WSD0628, as a radiosensitizer for GBM and metastatic CNS tumors with IC50 against ATM < 1nM with high selectivity ( >400 folds) for ATR and DNA-PK. WSD0628 is highly selective over other kinases. In-vitro MDCKII transfected cells and Caco-2 assays have shown that WSD0628 is highly permeable and not a substrate of P-gp or BCRP, two main efflux transporters expressed on human BBB. Preclinical CNS PK studies in rat and mouse confirmed good brain penetration of WSD0628 with Kp,uu,brain and Kp,uu,csf > 0.3. Significant prolongation of overall survival for mice bearing GBM PDX intracranial model was achieved by treatment with WSD0628 (5mpk, QD) combo with radiation. Moreover, WSD0628 shows low PK variation liability without aldehyde oxidase (AO) metabolism, low hERG liability ( >30 uM), and good safety window based on DRF studies. Taken together, our data provide a good rationale for WSD0628 to be developed toward clinic combo with radiation for the treatment of patients with GBM and cancers with CNS metastasis.

EXTH-16. LP-184, A NOVEL ALKYLATING AGENT, IS EFFECTIVE IN GLIOBLASTOMA

Abstract Temozolomide (TMZ) is currently the most effective standard-of-care chemotherapy based on its ability to prolong survival of patients with newly diagnosed MGMT-methylated GBM. Blood-brain barrier permeable agents effective against TMZ-resistant GBMs (i.e. recurrent GBM, MGMT unmethylated GBM) are desperately needed. Lantern Pharma is currently developing LP-184, a hydroxyurea methyl acylfulvene derivative of irofulven guided by its AI platform RADRⓇ analysis showing that tumor cell sensitivity to LP-184 correlates positively with genes commonly upregulated in GBM and either associated with TMZ resistance (e.g. MGMT, UGDH) or tumor promotion (e.g. EGFR, ANXA2). In an in vitro 3D model that mimics the human BBB, the apparent permeability for LP-184 was 1.53E-04 cm/s at 30 minutes, comparable to 1.72E-04 cm/s for TMZ under identical conditions. LP-184 predominantly alkylates DNA at 3’-adenine predicting insensitivity to MGMT expression. In vitro cell toxicity assays in a panel of GBM cell lines and neurospheres reveal an IC50 range of 30 - 400 nM with the MGMT unmethylated cell line LN-18 being among the most sensitive. A single cycle of LP-184 (4 mg/kg, i.v, every other day X 4 doses) induced rapid and near complete regression of subcutaneous U87 xenografts pre-established in immune-deficient mice and statistically significantly prolonged survival of mice bearing pre-established orthotopic U87 xenografts (p < 0.0001). LP-184-induced DNA damage is associated with synthetic lethality in tumor cell lines with decreased expression of Nucleotide Excision Repair (NER) genes. Furthermore, interrogation of clinical databases for expression of 112 NER genes revealed that 25 - 37% of clinical GBM clusters in a low NER expression subgroup, predicting high sensitivity of low NER GBM subsets to LP-184. These findings identify LP-184 as a promising new alkylating agent and support its further development for GBM therapy.

Multifunctional silica-coated mixed polymeric micelles for integrin-targeted therapy of pediatric patient-derived glioblastoma

Glioblastoma multiforme (GBM) remains a major cause of mortality because treatments are precluded by to the limited transport and penetration of chemotherapeutics across the blood–brain barrier. Pitavastatin (PTV) is a hydrophobic Food and Drug Administration (FDA)-approved anticholesterolemic agent with reported anti-GBM activity. In the present study, we encapsulate PTV in silica-coated polymeric micelles (SiO2 PMs) surface-modified with the cyclic peptide Arg-Gly-Asp-Phe-Val (cRGDfV) that actively targets the αvβ3 integrin overexpressed in the BBB endothelium and GBM. A central composite design is utilized to optimize the preparation process and improve the drug encapsulation ratio from 131 to 780 μg/mL. The silica shell provides full colloidal stability upon extreme dilution and enables a better control of the release kinetics in vitro with 28% of the cargo released after 12 h. Furthermore, SiO2 PMs show excellent compatibility and are internalized by human BBB endothelial cells, astrocytes and pericytes, as shown by confocal laser scanning fluorescence microscopy and flow cytometry. Finally, the anticancer efficacy is assessed in a pediatric patient-derived glioma cell line expressing high levels of the integrin subunits αv, β3 and β5. This PTV-loaded nanocarrier triggers apoptosis by reducing the mRNA level of anti-apoptotic genes NF-kβ, IL-6, BIRC1 and BIRC5 by 89%, 33%, 81% and 63%, respectively, and the cell viability by >60%. Overall, our results suggest the potential of these hybrid nanocarriers for the targeted therapy of GBM and other tumors overexpressing integrin receptors.

Optimization of dose and route of administration of the P‐glycoprotein inhibitor, valspodar (PSC‐833) and the P‐glycoprotein and breast cancer resistance protein dual‐inhibitor, elacridar (GF120918) as dual infusion in rats

Transporters can play a key role in the absorption, distribution, metabolism, and excretion of drugs. Understanding these contributions early in drug discovery allows for more accurate projection of the clinical pharmacokinetics. One method to assess the impact of transporters in vivo involves co‐dosing specific inhibitors. The objective of the present study was to optimize the dose and route of administration of a P‐glycoprotein (P‐gp) inhibitor, valspodar (PSC833), and a dual P‐gp/breast cancer resistance protein (BCRP) inhibitor, elacridar (GF120918), by assessing the transporters’ impact on brain penetration and absorption. A dual‐infusion strategy was implemented to allow for flexibility with dose formulation. The chemical inhibitor was dosed intravenously via the femoral artery, and a cassette of known substrates was infused via the jugular vein. Valspodar or elacridar was administered as 4.5‐hour constant infusions over a range of doses. To assess the degree of inhibition, the resulting ratios of brain and plasma concentrations, Kp's, of the known substrates were compared to the vehicle control. These data demonstrated that doses greater than 0.9 mg/hr/kg valspodar and 8.9 mg/hr/kg elacridar were sufficient to inhibit P‐gp‐ and BCRP‐mediated efflux at the blood‐brain barrier in rats without any tolerability issues. Confirmation of BBB restriction by efflux transporters in preclinical species allows for subsequent prediction in humans based upon the proteomic expression at rodent and human BBB. Overall, the approach can also be applied to inhibition of efflux at other tissues (gut absorption, liver clearance) or can be extended to other transporters of interest using alternate inhibitors.

Transient Receptor Potential Vanilloid in the Brain Gliovascular Unit: Prospective Targets in Therapy.

The gliovascular unit (GVU) is composed of the brain microvascular endothelial cells forming blood–brain barrier and the neighboring surrounding “mural” cells (e.g., pericytes) and astrocytes. Modulation of the GVU/BBB features could be observed in a variety of vascular, immunologic, neuro-psychiatric diseases, and cancers, which can disrupt the brain homeostasis. Ca2+ dynamics have been regarded as a major factor in determining BBB/GVU properties, and previous studies have demonstrated the role of transient receptor potential vanilloid (TRPV) channels in modulating Ca2+ and BBB/GVU properties. The physiological role of thermosensitive TRPV channels in the BBB/GVU, as well as their possible therapeutic potential as targets in treating brain diseases via preserving the BBB are reviewed. TRPV2 and TRPV4 are the most abundant isoforms in the human BBB, and TRPV2 was evidenced to play a main role in regulating human BBB integrity. Interspecies differences in TRPV2 and TRPV4 BBB expression complicate further preclinical validation. More studies are still needed to better establish the physiopathological TRPV roles such as in astrocytes, vascular smooth muscle cells, and pericytes. The effect of the chronic TRPV modulation should also deserve further studies to evaluate their benefit and innocuity in vivo.

Modeling Nanocarrier Transport across a 3D In Vitro Human Blood-Brain-Barrier Microvasculature.

Polymer nanoparticles (NPs), due to their small size and surface functionalization potential have demonstrated effective drug transport across the blood-brain-barrier (BBB). Currently, the lack of in vitro BBB models that closely recapitulate complex human brain microenvironments contributes to high failure rates of neuropharmaceutical clinical trials. In this work, a previously established microfluidic 3D in vitro human BBB model, formed by the self-assembly of human-induced pluripotent stem cell-derived endothelial cells, primary brain pericytes, and astrocytes in triculture within a 3D fibrin hydrogel is exploited to quantify polymer NP permeability, as a function of size and surface chemistry. Microvasculature are perfused with commercially available 100-400 nm fluorescent polystyrene (PS) NPs, and newly synthesized 100 nm rhodamine-labeled polyurethane (PU) NPs. Confocal images are taken at different timepoints and computationally analyzed to quantify fluorescence intensity inside/outside the microvasculature, to determine NP spatial distribution and permeability in 3D. Results show similar permeability of PS and PU NPs, which increases after surface-functionalization with brain-associated ligand holo-transferrin. Compared to conventional transwell models, the method enables rapid analysis of NP permeability in a physiologically relevant human BBB set-up. Therefore, this work demonstrates a new methodology to preclinically assess NP ability to cross the human BBB.

Outgrowth endothelial cells form a functional cerebral barrier and restore its integrity after damage.

Breakdown of blood-brain barrier, formed mainly by brain microvascular endothelial cells (BMECs), represents the major cause of mortality during early phases of ischemic strokes. Hence, discovery of novel agents that can effectively replace dead or dying endothelial cells to restore blood-brain barrier integrity is of paramount importance in stroke medicine. Although endothelial progenitor cells (EPCs) represent one such agents, their rarity in peripheral blood severely limits their adequate isolation and therapeutic use for acute ischemic stroke which necessitate their ex vivo expansion and generate early EPCs and outgrowth endothelial cells (OECs) as a result. Functional analyses of these cells, in the present study, demonstrated that only OECs endocytosed DiI-labelled acetylated low-density lipoprotein and formed tubules on matrigel, prominent endothelial cell and angiogenesis markers, respectively. Further analyses by flow cytometry demonstrated that OECs expressed specific markers for stemness (CD34), immaturity (CD133) and endothelial cells (CD31) but not for hematopoietic cells (CD45). Like BMECs, OECs established an equally tight in vitro model of human BBB with astrocytes and pericytes, suggesting their capacity to form tight junctions. Ischemic injury mimicked by concurrent deprivation of oxygen and glucose (4 hours) or deprivation of oxygen and glucose followed by reperfusion (20 hours) affected both barrier integrity and function in a similar fashion as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux, respectively. Wound scratch assays comparing the vasculoreparative capacity of cells revealed that, compared to BMECs, OECs possessed a greater proliferative and directional migratory capacity. In a triple culture model of BBB established with astrocytes, pericytes and BMEC, exogenous addition of OECs effectively repaired the damage induced on endothelial layer in serum-free conditions. Taken together, these data demonstrate that OECs may effectively home to the site of vascular injury and repair the damage to maintain (neuro)vascular homeostasis during or after a cerebral ischemic injury.

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Excess glutamate secreted from astrocytes drives upregulation of P-glycoprotein in endothelial cells in amyotrophic lateral sclerosis

In amyotrophic lateral sclerosis (ALS), upregulation in expression and activity of the ABC transporter P-glycoprotein (P-gp) driven by disease advancement progressively reduces CNS penetration and efficacy of the ALS drug, riluzole. Post-mortem spinal cord tissues from ALS patients revealed elevated P-gp expression levels in endothelial cells of the blood-spinal cord barrier compared to levels measured in control, non-diseased individuals. We recently found that astrocytes expressing familial ALS-linked SOD1 mutations regulate expression levels of P-gp in endothelial cells, which also exhibit a concomitant, significant increase in reactive oxygen species production and NFκB nuclear translocation when exposed to mutant SOD1 astrocyte conditioned media. In this study, we found that glutamate, which is abnormally secreted by mutant SOD1 and sporadic ALS astrocytes, drives upregulation of P-gp expression and activity levels in endothelial cells via activation of N-Methyl-D-Aspartic acid (NMDA) receptors. Surprisingly, astrocyte-secreted glutamate regulation of endothelial P-gp levels is not a mechanism shared by all forms of ALS. C9orf72-ALS astrocytes had no effect on endothelial cell P-gp expression and did not display increased glutamate secretion. Utilizing an optimized in vitro human BBB model consisting of patient-derived induced pluripotent stem cells, we showed that co-culture of endothelial cells with patient-derived astrocytes increased P-gp expression levels and transport activity, which was significantly reduced when endothelial cells were incubated with the NMDAR antagonist, MK801. Overall, our findings unraveled a complex molecular interplay between astrocytes of different ALS genotypes and endothelial cells potentially occurring in disease that could differentially impact ALS prognosis and efficacy of pharmacotherapies.

Abstract 4786: WSD0922: A novel, oral bioavailable, brain penetrable and reversible EGFRm+ inhibitor for the treatment of primary and metastatic brain tumors

Abstract Epidermal growth factor receptor (EGFR) is a cell surface protein that regulates normal cellular growth in tissues of epithelial origin. Des-regulated EGFR signaling (caused by mechanisms of overexpression, mutation, autocrine activation) contributes for oncogenesis by inducing cells proliferation and resisting apoptosis. In patients with NSCLC, activating mutants (EGFRm+) occur within exon 18-21 with increased EGFR activity. About 90% of these mutations (G719x, exon 19 deletions and exon 21 mutation) are sensitizing EGFRm+. EGFR TKIs have shown good performance in clinic for systemic NSCLC anchoring EGFRm+. However, up to 65% of NSCLC patients will develop brain metastasis (BM) ultimately. Current approved EGFR TKIs are not so effective for BM due to limited brain penetration as they are substrates of P-gp or BCRP, two main efflux transporters expressed on human BBB. In patients with GBM, the most common mutation is EGFRvIII (exon2-7 deletion) with an overall frequency of 25–64%. Current EGFR TKIs are ineffective in GBM treatment probably due to resistance caused by EGFRvIII as well as limited brain penetration. One-year survival of GBM patients is approximate 43%. Herein, we report a discovery of brain penetrable EGFR inhibitor WSD0922 for NSCLC BM and GBM with IC50 against EGFRm is <10nM. The ex-vivo antiproliferative GI50 in GBM tissues with EGFRvIII amplification is <100nM. WSD0922 is highly selective over other kinases. In-vitro MDCKII transfected cells and Caco-2 assays showed that WSD0922 is highly permeable and not a substrate of P-gp or BCRP. Preclinical CNS PK studies confirmed good brain penetration of WSD0922 with Kp,uu,brain close to unity. Treatment of mice bearing PC-9 (exon 19 del of NSCLC) in both subcutaneous (SC) and intracranial models with WSD0922 at 1-10mpk BID via PO resulted in dose dependent tumor growth inhibition (TGI) or regression with statistically significant survival benefit. Moreover, significant TGI for mice bearing GBM PDX model was achieved by treatment with WSD0922. No toxicity was observed in the animals during the treatment. Predicted human PK properties are very promising to offer sufficient target engagement in clinic. Taken together, our data provide a good rationale for WSD0922 to be developed toward clinic for the treatment of patients with BM of NSCLC or GBM harboring EGFRm+. Citation Format: Wei Zhong, Jinqiang Zhang, Zhihua Mu, Claire Sun. WSD0922: A novel, oral bioavailable, brain penetrable and reversible EGFRm+ inhibitor for the treatment of primary and metastatic brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4786.

A novel human induced pluripotent stem cell blood-brain barrier model: Applicability to study antibody-triggered receptor-mediated transcytosis

We have developed a renewable, scalable and transgene free human blood-brain barrier model, composed of brain endothelial cells (BECs), generated from human amniotic fluid derived induced pluripotent stem cells (AF-iPSC), which can also give rise to syngeneic neural cells of the neurovascular unit. These AF-iPSC-derived BECs (i-BEC) exhibited high transendothelial electrical resistance (up to 1500 Ω cm2) inducible by astrocyte-derived molecular cues and retinoic acid treatment, polarized expression of functional efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. In vitro human BBB models enable pre-clinical screening of central nervous system (CNS)-targeting drugs and are of particular importance for assessing species-specific/selective transport mechanisms. This i-BEC human BBB model discriminates species-selective antibody- mediated transcytosis mechanisms, is predictive of in vivo CNS exposure of rodent cross-reactive antibodies and can be implemented into pre-clinical CNS drug discovery and development processes.