In vitro model for evaluating drug transport across the blood-brain barrier. Adv Drug Deliv Rev

Université d'Artois, Arras, Nord-Pas-de-Calais, France
Advanced drug delivery reviews (Impact Factor: 15.04). 05/1999; 36(2-3):165-178. DOI: 10.1016/S0169-409X(98)00083-0
Source: PubMed


The passage of substances across the blood–brain barrier (BBB) is regulated in the cerebral capillaries, which possess certain distinct different morphological and enzymatic properties compared with the capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies some characteristics of the in vivo BBB are lost. To provide an in vitro system for studying brain capillary functions, we have developed a process of coculture that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In order to assess the drug transport across the blood–brain barrier, we compared the extraction ratios in vivo to the permeability of the in vitro model. The in vivo and the in vitro values showed a strong correlation. The relative ease with which such cocultures can be produced in large quantities facilitates the screening of new centrally active drugs. This model provides an easier, reproducible and mass-production method to study the blood–brain barrier in vitro.

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Available from: Romeo Cecchelli
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    • "Further experiments would be required to definitively assess whether human astrocytes may be used successfully in co-culture with EPDCs. Nevertheless, co-culture models of endothelial cells from human [35] or bovine [17] origin and rat astrocytes have been successfully developed by other groups. These observations suggest that putative species differences are unlikely to impede the establishment of new valuable in vitro models of the BBB. "
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    ABSTRACT: The vascular system is adapted to specific functions in different tissues and organs. Vascular endothelial cells are important elements of this adaptation, leading to the concept of 'specialized endothelial cells'. The phenotype of these cells is highly dependent on their specific microenvironment and when isolated and cultured, they lose their specific features after few passages, making models using such cells poorly predictive and irreproducible. We propose a new source of specialized endothelial cells based on cord blood circulating endothelial progenitors (EPCs). As prototype examples, we evaluated the capacity of EPCs to acquire properties characteristic of cerebral microvascular endothelial cells (blood-brain barrier (BBB)) or of arterial endothelial cells, in specific inducing culture conditions. First, we demonstrated that EPC-derived endothelial cells (EPDCs) co-cultured with astrocytes acquired several BBB phenotypic characteristics, such as restricted paracellular diffusion of hydrophilic solutes and the expression of tight junction proteins. Second, we observed that culture of the same EPDCs in a high concentration of VEGF resulted, through activation of Notch signaling, in an increase of expression of most arterial endothelial markers. We have thus demonstrated that in vitro culture of early passage human cord blood EPDCs under specific conditions can induce phenotypic changes towards BBB or arterial phenotypes, indicating that these EPDCs maintain enough plasticity to acquire characteristics of a variety of specialized phenotypes. We propose that this property of EPDCs might be exploited for producing specialized endothelial cells in culture to be used for drug testing and predictive in vitro assays.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "the beginning and at the end of the experiments . The receiver compartments were sampled at the end of the experiments . The amount of radiotracer in the abluminal compartment was measured in a liquid scintillation analyser ( Packard Instrument Company , Meriden , USA ) . The BBB permeability coefficients to sucrose were calculated as described by Cecchelli et al . 1999 [ 11 ]"
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    ABSTRACT: 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.
    Full-text · Article · Dec 2013 · PLoS ONE
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    • "Cells were treated with LIP when the transendothelial electrical resistance (TEER) value (measured by EVOMX meter, STX2 electrode; World Precision Instruments, Sarasota, FL, USA) was found to be the highest. The functional properties of cell monolayers were assessed by measuring the endothelial permeability (EP) of [14C]-sucrose and [3H]-propranolol (between 0 and 120 minutes) as described previously.25 Radiolabeled LIP (0.5 mL; from 100 to 400 nmols of total lipids/well) were added to the upper chamber and incubated for between 10 and 120 minutes. "
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    ABSTRACT: We investigated the ability of amyloid-β-targeting liposomes, decorated with an anti-transferrin receptor antibody, to cross the blood-brain barrier (BBB), comparing two antibody ligation techniques. Fluorescent or radiolabeled liposomes composed of sphingomyelin/cholesterol and containing phosphatidic acid, known to bind amyloid-β, were further functionalized with the anti-transferrin receptor antibody RI7217. Two different techniques were used to attach RI7217 to the liposomes surface: biotin/streptavidin linkage or thiol-maleimide covalent ligation. Surface plasmon resonance (SPR) and immunoblotting were employed to assess the nanoparticles' binding performances. Confocal microscopy and radiochemical techniques were used for uptake and permeability studies on an in vitro BBB model made of human brain capillary endothelial cells hCMEC/D3. Immunoblotting experiments showed that RI7217-functionalized liposomes bind to transferrin receptor independently of the procedure employed to ligate their surface with the antibody, while SPR experiments showed a slightly higher affinity for covalently functionalized nanoliposomes. The functionalization with RI7217 did not affect the liposomes' affinity for amyloid-β. The functionalization of liposomes with RI7217, independently of the ligation procedure, gave higher values of uptake and permeability across the barrier model in comparison to the nondecorated ones, without cell monolayer alterations. Of note, the best performing particles were those covalently coupled with the antibody. The ratios of the two radiolabeled lipids ((3)H-sphingomyelin and (14)C-phosphatidic acid) present in the liposome bilayer were found to be similar in the apical and in the basolateral compartments of the barrier model, suggesting that liposomes were transported intact across the cell monolayer. Confocal experiments showed no co-localization of RI7217-liposomes with early/late endosomes or early lysosomes. Our results suggest that RI7217 promotes the in vitro barrier crossing of liposomes containing phosphatidic acid, targeting the Alzheimer's disease amyloid-β peptide. Moreover, for the first time, we prove herein the superior efficiency of covalent coupling of RI7217 versus biotin/streptavidin ligation to facilitate liposomes in overcoming the BBB in vitro.
    Full-text · Article · May 2013 · International Journal of Nanomedicine
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