Emmanuel Sevin

A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

Apical-to-Basolateral Transport of Amyloid-β Peptides through Blood-Brain Barrier Cells is Mediated by the Receptor for Advanced Glycation End-Products and is Restricted by P-Glycoprotein

Several studies have highlighted the close relationship between Alzheimers disease (AD) and alterations in the bidirectional transport of amyloid-β (Aβ) peptides across the blood-brain barrier (BBB). The brain capillary endothelial cells (BCECs) that compose the BBB express the receptors and transporters that enable this transport process. There is significant in vivo evidence to suggest that P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) restrict Aβ peptides entry into the brain, whereas the receptor for advanced glycation end-products (RAGE) seems to mediate apical-to-basolateral passage across the BBB. However, deciphering the molecular mechanisms underlying these in vivo processes requires further in vitro characterization. Using an in vitro BBB model and specific competition experiments against RAGE, we have observed a significant decrease in apical-to-basolateral (but not basolateral-to-apical) transport of Aβ1-40 and Aβ1-42 peptides through BCECs. This transport is a caveolae-dependent process and fits with the apical location of RAGE observed in confocal microscopy experiments. Inhibition of P-gp and BCRP using different inhibitors increases transport of Aβ peptides suggesting that these efflux pumps are involved in Aβ peptide transport at the BCECs level. Taken as a whole, these results demonstrate the involvement of the caveolae-dependent transcytosis of Aβ peptides through the BBB in a RAGE-mediated transport process, reinforcing the hypothesis whereby this receptor is a potential drug target in AD.

Transcriptional profiles of receptors and transporters involved in brain cholesterol homeostasis at the blood–brain barrier: Use of an in vitro model

Brain is the most cholesterol rich organ of the whole body and recent studies suggest a role for the blood-brain barrier (BBB) in cerebral cholesterol homeostasis. Low density lipoprotein receptor (LDLR)-related receptors and ATP-binding Cassette (ABC) transporters play an important role in peripheral sterol homeostasis. The purpose of this study was to determine the mRNA expression profiles of ABC transporters (ABCA1, 2, 3, 7 and ABCG1) and low density lipoprotein receptor (LDLR)-related receptors (LDLR, vLDLR, LRP1, LRP2 and LRP8) in BBB endothelium using an in vitro co-culture model of bovine brain capillary endothelial cells (BCECs) and rat glial cells. All transcripts tested are expressed by BCECs and in capillary extract, except vLDLR. Glial cells influence ABCG1, A1, 2, 7 and LRP1 transcription, suggesting a role in cerebral lipid supply/elimination through the modulation of the expression of these transporters and receptors by these cells. Altogether, these results highlight the importance of glial input in the BBB transport phenotype for sterol homeostasis in the central nervous system, and confirm the importance of the BBB in this process.

Catalytic site inhibition of insulin-degrading enzyme by a small molecule induces glucose intolerance in mice

Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-β. Knockout and genetic studies have linked IDE to Alzheimers disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisingly impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.

Actin, gelsolin and filamin‐A are dynamic actors in the cytoskeleton remodelling contributing to the blood brain barrier phenotype

The brain vascular endothelium operates as a dynamic regulatory interface to maintain the cell environment of the nervous system. In the vicinity of astrocytes, brain endothelial cells develop characteristic features conferring a strong cellular impermeability which limits the penetration of various compounds. The aim of our study was to determine by differential proteomic analysis the changes occurring in bovine brain capillary endothelial cells (BBCEC) differentiated in co‐culture with astrocytes compared with endothelial cells cultured alone. In order to obtain reproducible and meaningful protein profiles of in vitro blood–brain barrier models, three sample preparation procedures were carried out to provide the first 2‐D comparative proteomic study of BBCEC. Our study highlights advantages and drawbacks of each procedure. The cellular proteins prepared from mechanical scraping of collagen‐seeded BBCEC were strongly contaminated by serum proteins. Enzymatic dissociation of BBCEC by trypsin or collagenase solved this problem. A comparative 2‐DE profile study of collagenase‐harvested BBCEC revealed that cytoskeleton‐related proteins (actin, gelsolin and filamin‐A) show the most significant quantitative changes in the Triton soluble protein fraction from BBCEC that exhibit characteristics closest to the in vivo situation.

Resveratrol and Grape Extract-loaded Solid Lipid Nanoparticles for the Treatment of Alzheimer’s Disease

The aggregation of amyloid-β peptide (Aβ) has been linked to the formation of neuritic plaques, which are pathological hallmarks of Alzheimer’s disease (AD). Various natural compounds have been suggested as therapeutics for AD. Among these compounds, resveratrol has aroused great interest due to its neuroprotective characteristics. Here, we provide evidence that grape skin and grape seed extracts increase the inhibition effect on Aβ aggregation. However, after intravenous injection, resveratrol is rapidly metabolized into both glucuronic acid and sulfate conjugations of the phenolic groups in the liver and intestinal epithelial cells (within less than 2 h), which are then eliminated. In the present study, we show that solid lipid nanoparticles (SLNs) functionalized with an antibody, the anti-transferrin receptor monoclonal antibody (OX26 mAb), can work as a possible carrier to transport the extract to target the brain. Experiments on human brain-like endothelial cells show that the cellular uptake of the OX26 SLNs is substantially more efficient than that of normal SLNs and SLNs functionalized with an unspecific antibody. As a consequence, the transcytosis ability of these different SLNs is higher when functionalized with OX-26.

Case study: adapting in vitro blood–brain barrier models for use in early-stage drug discovery

Several parameters influencing the brain distribution of compounds must be considered when designing potential neuropharmaceuticals in early-stage drug discovery. The blood-brain barrier (BBB) represents an obstacle for drug penetration into the brain. Many in vitro BBB models have proven useful for predicting the BBB permeation rate, but do not meet all criteria for use in early-stage drug discovery: feasibility, rapidity, reliability and a low requirement for human resources. To meet this demand, we have developed a robust, higher-throughput, cell-based model exhibiting BBB features (low paracellular permeability, functional efflux pumps and the correct endothelial phenotype). This system comes in a ready-to-use, frozen format, appropriate for in-house use by large pharmaceutical firms and small biotech companies during early-stage drug discovery.

Accelerated Caco-2 cell permeability model for drug discovery.

INTRODUCTION By culturing Caco-2 cells according to a new and optimized protocol, it has been possible to accelerate the cell culture process in such a way that the cells can be used for experiments after only 6 days. The accelerated Caco-2 model has been compared to the traditional model (requiring 21-25 days of culture) in terms of tightness of the junctions, ability to rank chemical compounds for apparent permeability, active efflux and to discriminate P-gp substrates. METHODS AND RESULTS In the new protocol, Caco-2 cells were cultured with the classical Caco-2 medium supplemented with puromycin. The initial cell seeding density was increased two times compared to the traditional procedure and the presence of a low concentration of puromycin in the culture medium reduced the Caco-2 permeability of mannitol. Bi-directional studies were performed with known P-gp substrates (rhodamine 123, digoxin and saquinavir) and with a total of 20 marketed drugs covering a wide range of physicochemical characteristics and therapeutic indications. Strong correlations were obtained between the apparent permeability in absorptive (Papp A→B) or secretory (Papp B→A) of the drugs in the accelerated model and in the traditional models and comparable efflux ratios were observed in the two studied models. DISCUSSION The new protocol reduces costs for screening and leads to higher throughput compared to traditional Caco-2 cell models. This accelerated model provides short time-feedback to the drug design during the early stage of drug discovery.

Bexarotene Promotes Cholesterol Efflux and Restricts Apical-to-Basolateral Transport of Amyloid-β Peptides in an In Vitro Model of the Human Blood-Brain Barrier

One of the prime features of Alzheimers disease (AD) is the excessive accumulation of amyloid-β (Aβ) peptides in the brain. Several recent studies suggest that this phenomenon results from the dysregulation of cholesterol homeostasis in the brain and impaired bidirectional Aβ exchange between blood and brain. These mechanisms appear to be closely related and are controlled by the blood-brain barrier (BBB) at the brain microvessel level. In animal models of AD, the anticancer drug bexarotene (a retinoid X receptor agonist) has been found to restore cognitive functions and decrease the brain amyloid burden by regulating cholesterol homeostasis. However, the drugs therapeutic effect is subject to debate and the exact mechanism of action has not been characterized. Therefore, the objective of this present study was to determine bexarotenes effects on the BBB. Using an in vitro model of the human BBB, we investigated the drugs effects on cholesterol exchange between abluminal and luminal compartments and the apical-to-basolateral transport of Aβ peptides across the BBB. Our results demonstrated that bexarotene induces the expression of ABCA1 but not ApoE. This upregulation correlates with an increase in ApoE2-, ApoE4-, ApoA-I-, and HDL-mediated cholesterol efflux. Regarding the transport of Aβ peptides, bexarotene increases the expression of ABCB1, which in turn decreases Aβ apical-to-basolateral transport. Our results showed that bexarotene not only promotes the cholesterol exchange between the brain and the blood but also decreases the influx of Aβ peptides across BBB, suggesting that bexarotene is a promising drug candidate for the treatment of AD.

A Simple Method for Assessing Free Brain/Free Plasma Ratios Using an In Vitro Model of the Blood Brain Barrier

Historically, the focus has been to use in vitro BBB models to optimize rate of drug delivery to the CNS, whereas total in vivo brain/plasma ratios have been used for optimizing extent. However, these two parameters do not necessarily show good correlations with receptor occupancy data or other pharmacological readouts. In line with the free drug hypothesis, the use of unbound brain concentrations (Cu,br) has been shown to provide the best correlations with pharmacological data. However, typically the determination of this parameter requires microdialysis, a technique not ideally suited for screening in early drug development. Alternative, and less resource-demanding methodologies to determine Cu,br employ either equilibrium dialysis of brain homogenates or incubations of brain slices in buffer to determine fraction unbound brain (fu,br), which is subsequently multiplied by the total brain concentration to yield Cu,br. To determine Cu,br/Cu,pl ratios this way, still requires both in vitro and in vivo experiments that are quite time consuming. The main objective of this study was to explore the possibility to directly generate Cu,br/Cu,pl ratios in a single in vitro model of the BBB, using a co-culture of brain capillary endothelial and glial cells in an attempt to mimick the in vivo situation, thereby greatly simplifying existing experimental procedures. Comparison to microdialysis brain concentration profiles demonstrates the possibility to estimate brain exposure over time in the BBB model. A stronger correlation was found between in vitro Cu,br/Cu,pl ratios and in vivo Cu,br/Cu,pl obtained using fu,br from brain slice than with fu,br from brain homogenate for a set of 30 drugs. Overall, Cu,br/Cu,pl ratios were successfully predicted in vitro for 88% of the 92 studied compounds. This result supports the possibility to use this methodology for identifying compounds with a desirable in vivo response in the CNS early on in the drug discovery process.