Laurence Fenart

Modelling of the blood–brain barrier in drug discovery and development

The market for neuropharmaceuticals is potentially one of the largest sectors of the global pharmaceutical market owing to the increase in average life expectancy and the fact that many neurological disorders have been largely refractory to pharmacotherapy. The brain is a delicate organ that can efficiently protect itself from harmful compounds and precisely regulate its microenvironment. Unfortunately, the same mechanisms can also prove to be formidable hurdles in drug development. An improved understanding of the regulatory interfaces that exist between blood and brain may provide novel and more effective strategies to treat neurological disorders.

Indirect evidence that drug brain targeting using polysorbate 80-coated polybutylcyanoacrylate nanoparticles is related to toxicity.

AbstractPurpose. To investigate the mechanism underlying the entry of the analgesic peptide dalargin into brain using biodegradable polybutylcyanoacrylate (PBCA) nanoparticles (NP) overcoated with polysorbate 80. Methods. The investigations were carried out with PBCA NP and with non biodegradable polystyrene (PS) NP (200 nm diameter). Dalargin adsorption was assessed by HPLC. Its entry into the CNS in mice was evaluated using the tail-flick procedure. Locomotor activity measurements were performed to compare NP toxicities. BBB permeabilization by PBCA NP was studied in vitro using a coculture of bovine brain capillary endothelial cells and rat astrocytes. Results. Dalargin loading was 11.7 µg/mg on PBCA NP and 16.5µg/ mg on PS NP. Adding polysorbate 80 to NP led to a complete desorption. Nevertheless, dalargin associated with PBCA NP and polysorbate 80 induced a potent and prolonged analgesia, which could not be obtained using PS NP in place of PBCA NP. Locomotor activity dramatically decreased in mice dosed with PBCA NP, but not with PS NP. PBCA NP also caused occasional mortality. In vitro, PBCA NP (10 µg/ml) induced a permeabilization of the BBB model. Conclusions. A non specific permeabilization of the BBB, probably related to the toxicity of the carrier, may account for the CNS penetration of dalargin associated with PBCA NP and polysorbate 80.

High transcytosis of melanotransferrin (P97) across the blood–brain barrier

The blood–brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14‐fold higher than that of holo‐transferrin, with no apparent intra‐endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration‐ and conformation‐dependent, suggesting that the transport of P97 is due to receptor‐mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low‐density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.

Prediction of Drug Transport Through the Blood-Brain Barrier in Vivo: A Comparison Between Two in Vitro Cell Models

AbstractPurpose. Studies were conducted to evaluate whether the use of an in vitro model of the blood-brain barrier (BBB) resulted in more accurate predictions of the in vivo transport of compounds compared to the use of a human intestinal cell line (Caco-2). Methods. The in vitro BBB model employs bovine brain capillary endothelial cells co-cultured with primary rat astrocytes. The Caco-2 cells originate from a human colorectal carcinoma. The rat was used as experimental animal for the in vivo studies. Results. Strong correlations (r = 0.93-0.95) were found between the results generated by the in vitro model of the BBB and two different methodologies to measure the permeability across the BBB in vivo. In contrast, a poor correlation (r = 0.68) was obtained between Caco-2 cell data and in vivo BBB transport. A relatively poor correlation (r = 0.74) was also found between the two in vitro models. Conclusion. The present study illustrates the limitations of the Caco-2 model to predict BBB permeability of compounds in vivo. The results emphasize the fact that the BBB and the intestinal mucosa are two fundamentally different biologic barriers, and to be able to make accurate predictions about the in vivo CNS penetration of potential drug candidates, it is important that the in vitro model possesses the main characteristics of the in vivo BBB.

Contribution of glial cells and pericytes to the mRNA profiles of P-glycoprotein and multidrug resistance-associated proteins in an in vitro model of the blood-brain barrier.

P-glycoprotein (P-gp) and the multidrug resistance-associated proteins (MRP), whose expression is associated with multidrug resistance, have been recently located in the brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB), without taking into account a possible influence or contribution of glial cells and pericytes. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), the present study analysed the transcriptional expression of P-gp and the seven homologues of MRP transporters in BCECs in solo culture or in an in vitro model of the BBB consisting of a co-culture of BCECs and glial cells. Pericytes, glial cells, isolated brain capillaries and bovine grey matter extracts were also tested. P-gp mRNA, absent in glial cells, was found in brain capillaries and in co-cultured BCECs with an increased signal compared to the in solo culture. No amplification was observed in pericytes or grey matter. While MRP2, MRP3 and MRP7 remained undetected, MRP1, absent in capillaries or grey matter, was amplified in BCECs, glial cells and pericytes. MRP4 gave a low signal in most cultures. MRP5 was ubiquitously expressed, displaying a potent signal in all conditions. In spite of its presence in cultured glial cells, MRP6 mRNA expression appeared to be restricted to BCECs, with the same upregulation in the co-cultured condition as observed with P-gp. Moreover, MRP6 was the only transporter whose endothelial mRNA expression was influenced by the presence of pericytes. The tissue distribution of the expression of these transporters and the contribution of the different cell populations are discussed.

Mouse syngenic in vitro blood-brain barrier model: a new tool to examine inflammatory events in cerebral endothelium

Although cerebral endothelium disturbance is commonly observed in central nervous system (CNS) inflammatory pathologies, neither the cause of this phenomenon nor the effective participation of blood–brain barrier (BBB) in such diseases are well established. Observations were mostly made in vivo using mouse models of chronic inflammation. This paper presents a new mouse in vitro model suitable for the study of underlying mechanistic events touching BBB functions during CNS inflammatory disturbances. This model consists of a coculture with both primary cell types isolated from mice. Mouse brain capillary endothelial cell (MBCEC)s coming from brain capillaries are in culture with their in vivo partners and form differentiated monolayers that retain endothelial markers and numerous phenotypic properties of in vivo cerebral endothelium, such as: (1) peripheral distribution of tight junction proteins (occludin, claudin-5, claudin-3 and JAM-1); (2) high trans-endothelium electrical resistance value; (3) attenuated paracellular flux of sucrose and inulin; (4) P-gp expression; (5) no MECA-32 expression. Furthermore, this endothelium expresses cell adhesion molecules described in vivo and shows intracellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 upregulation under lipopolysaccharide-treatment. Therefore, this well-differentiated model using autologous cells appears as a suitable support to reconstitute pathological in vitro BBB model.

An in vitro blood-brain barrier model for high throughput (HTS) toxicological screening

There is a growing interest to use in vitro BBB cell assays in early safety assessment of compounds. By modifying a well-validated co-culture model of brain capillary endothelial and glial cells, developed by Dehouck et al. [Dehouck, M.P., Meresse, S., Delorme, P., Fruchart, J.C., Cecchelli, R., 1990. An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro. Journal of Neurochemistry 54 (5), 1798-1801], it has been possible to develop a new in vitro BBB system suitable for high throughput screening (HTS). In addition, this new procedure substantially reduces the use of experimental animals and considerably facilitates the process of obtaining a functional in vitro BBB model. The model is ready to use after only 4 days of culture and then shows the typical expression and localization of tight junction proteins. The function of the P-glycoprotein and the transcriptional expression of other efflux transporters such as MRP 1, 4 and 5 have been demonstrated. In addition, the model produces a good in vitro/in vivo correlation for 10 compounds (R2=0.81). Furthermore, studies were undertaken within the European ACuteTox consortium with the objective to assess BBB toxicity and make risk assessments of potentially toxic compounds according to their predicted ability to reach the CNS compartment. These investigations demonstrated that the results produced in the HTS BBB model were similar to the standard co-culture model.

P-glycoprotein in blood-brain barrier endothelial cells: Interaction and oligomerization with caveolins

P‐glycoprotein (P‐gp), an adenosine triphosphate (ATP)‐binding cassette transporter which acts as a drug efflux pump, is highly expressed at the blood–brain barrier (BBB) where it plays an important role in brain protection. Recently, P‐gp has been reported to be located in the caveolae of multidrug‐resistant cells. In this study, we investigated the localization and the activity of P‐gp in the caveolae of endothelial cells of the BBB. We used an in vitro model of the BBB which is formed by co‐culture of bovine brain capillary endothelial cells (BBCEC) with astrocytes. Caveolar microdomains isolated from BBCEC are enriched in P‐gp, cholesterol, caveolin‐1, and caveolin‐2. Moreover, P‐gp interacts with caveolin‐1 and caveolin‐2; together, they form a high molecular mass complex. P‐gp in isolated caveolae is able to bind its substrates, and the caveolae‐disrupting agents filipin III and nystatin decrease P‐gp transport activity. In addition, mutations in the caveolin‐binding motif present in P‐gp reduced the interaction of P‐gp with caveolin‐1 and increased the transport activity of P‐gp. Thus, P‐gp expressed at the BBB is mainly localized in caveolae and its activity may be modulated by interaction with caveolin‐1.

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.

Inhibition of P-glycoprotein : Rapid assessment of its implication in Blood-Brain Barrier integrity and drug transport to the brain by an in vitro model of the Blood-Brain Barrier

AbstractPurpose. The objective of this work was to assess, in vitro, the passage of P-glycoprotein dependent drugs across brain capillary endothelial cells, when these drugs are associated with a reversing agent. Methods. An in vitro model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes was used. Results. We demonstrate that P-glycoprotein expression is upregulated by the presence of astrocytes. Uptake in the cells and transport across endothelial cell monolayers of vincristine, cyclosporin A and doxorubicin were studied. Using S9788 or verapamil as reversing agents, we found an increase in vincristine transport across the endothelial cell monolayers. On the other hand, the association of S9788 or verapamil with cyclosporin A failed to increase the transport of this drug. An increase in the transport of doxorubicin from luminal to abluminal compartment was also observed, due to endothelial cell monolayer breakdown. Conclusions. Using this model, it is possible to predict the passage of a P-glycoprotein dependent drug to the brain or its sequestration in brain capillary endothelial cells when this drug is associated with a reversing agent, or its toxicity on the blood-brain barrier integrity.