Szilvia Veszelka

Comparison of brain capillary endothelial cell-based and epithelial (MDCK-MDR1, Caco-2, and VB-Caco-2) cell-based surrogate blood-brain barrier penetration models.

An accurate means of predicting blood-brain barrier (BBB) penetration and blood-brain partitioning of NCEs (new chemical entities) would fulfill a major need in pharmaceutical research. Currently, an industry-standard BBB drug penetration model is not available. Primary brain capillary endothelial cells, optionally co-cultured with astrocytes and/or pericytes, are the most valued models of BBB. For routine use, establishing and maintaining a co-culture system is too costly and labor intensive. Alternatively, non-cerebral cell lines such as MDCK-MDR1 are used, and most recently, the suitability of native and modified Caco-2 for predicting brain penetration has also come under investigation. This study provides comparative data on the morphology and functionality of the high integrity brain capillary endothelial BBB model (EPA: triple culture of brain capillary endothelial cells with pericytes and astrocytes) and the epithelial cell-based (native Caco-2, high P-glycoprotein expressing vinblastine-treated VB-Caco-2 and MDCK-MDR1) surrogate BBB models. Using a panel of 10 compounds VB-Caco-2 and MDCK-MDR1 cell lines show restrictive paracellular pathway and BBB-like selective passive permeability that makes them comparable to the rat brain BBB model, which gave correlation with the highest r(2) value with in vivo permeability data. In bidirectional assay, the VB-Caco-2 and the MDCK-MDR1 models identified more P-glycoprotein drug substrates than the rat brain BBB model. While the complexity and predictive value of the BBB model is the highest, for the screening of NCEs to determine whether they are efflux substrates or not, the VB-Caco-2 and the MDCK-MDR1 models may provide a simple and inexpensive tool.

The NMDA and AMPA/KA Receptors are Involved in Glutamate-Induced Alterations of Occludin Expression and Phosphorylation in Brain Endothelial Cells

Glutamate levels increase dramatically in cerebral ischemia and stroke. This may lead to opening of the blood–brain barrier (BBB) and induce further brain damage. Because endothelial tight junctions are critical elements of the BBB integrity, the aim of this study was to investigate the mechanisms of glutamate-induced alterations of the tight-junction protein occludin in cultured brain microvascular endothelial cells (BMECs). Transient exposure to glutamate resulted in cellular redistribution of occludin, followed by a decrease in the total level of this protein and diminished barrier function of BMECs. Inhibition of the N-methyl-d-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate (AMPA/KA) receptors attenuated glutamate-induced changes in occludin redistribution but not in the total protein levels. Treatment with glutamate also increased tyrosine phosphorylation and decreased threonine phosphorylation of occludin. Inhibition of the NMDA receptors by MK-801 partially protected against glutamate-induced elevation of occludin tyrosine phosphorylation. In addition, pretreatment with MK-801-attenuated glutamate-mediated disruption of endothelial barrier function. Blocking of the AMPA/KA receptors by 6,7-dinitroquinoxaline-2. 3-dione (DNQX) protected against hypophosphorylation of threonine residues of occludin; however, it did not affect disruption of endothelial integrity. These findings indicate the opposite effects of the NMDA and AMPA/KA receptors on occludin phosphorylation and disruption of the BBB functions.

Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages

Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.

Evaluation of cytotoxicity of surfactants used in self-micro emulsifying drug delivery systems and their effects on paracellular transport in Caco-2 cell monolayer

The objective of this study was to examine the cellular effects of the members of two non-ionic amphiphilic tenside groups and their mixtures on human Caco-2 cell monolayers as dependent upon their chemical structures and physicochemical properties. The first group of polyethylene glycol esters is represented by Polysorbates and Labrasol alone and in blends, while the members of the second group. Capryol 90, Capryol PGMC, Lauroglycol 90 and Lauroglycol FCC were used as propylene glycol esters. They are increasingly used in SMEDDS as recent tensides or co-tensides to increase hydrophobic bioavailability of a drug. Critical micelle concentration was measured by determination of surface tension. CMC refers to the ability of solubilization of surfactants. Cytotoxicity tests were performed on Caco-2 cell monolayers by MTT and LDH methods. Paracellular permeability as a marker of the integrity of cell monolayers, was examined with Lucifer yellow assays combined with TransEpithelial Electrical Resistance (TEER) measurements. The effect of these surfactants on tight junctions as evidence for paracellular pathway was also characterized. The results of cytotoxicity assays were in agreement, and showed significant differences among the cytotoxic properties of surfactants in a concentration-dependent manner. Polysorbates 20, 60, 80 are the most toxic compounds. In the case of Labrasol, the degree of esterification and lack of sorbit component decreased cytotoxicity. If the hydrophyl head was changed from polyethylene glycol to propylene glycol the main determined factor of cytotoxicity was the monoester content and the length of carbon chain. In our CMC experiments, we found that only Labrasol showed expressed cytotoxicity above the CMC. It refers to good ability of micelle solubilization of Labrasol. In our paracellular transport experiments each of polyethylene glycol surfactants (Polysorbates and Labrasol) altered TEER values, but propylene glycol esters did not modify the monolayer integrity. Polyethylene glycol esters alone and in blends (0.05% Labrasol--0.001% Polysorbates 20, 60, 80) were able to increase Lucifer yellow permeability significantly below the IC₅₀ concentration. On the other hand Labrasol and Polysorbates 20 have expressed effect on tight junctions of Caco-2 monolayer. It could be concluded that polyethylene glycol ester-type tensides were able to enhance the paracellular permeability by the redistribution of junctional proteins. Our results might ensure useful data for selection of suitable tensides, co-tensides and tenside mixtures for SMEDDS formulations.

Sodium hyaluronate as a mucoadhesive component in nasal formulation enhances delivery of molecules to brain tissue

Intranasal administration of molecules has been investigated as a non-invasive way for delivery of drugs to the brain in the last decade. Circumvention of both the blood-brain barrier and the first-pass elimination by the liver and gastrointestinal tract is considered as the main advantages of this method. Because of the rapid mucociliary clearance in the nasal cavity, bioadhesive formulations are needed for effective targeting. Our goal was to develop a formulation containing sodium hyaluronate, a well-known mucoadhesive molecule, in combination with a non-ionic surfactant to enhance the delivery of hydrophilic compounds to the brain via the olfactory route. Fluorescein isothiocyanate-labeled 4 kDa dextran (FD-4), used as a test molecule, was administered nasally in different formulations to Wistar rats, and detected in brain areas by fluorescent spectrophotometry. Hyaluronan increased the viscosity of the vehicles and slowed down the in vitro release of FD-4. Significantly higher FD-4 transport could be measured in the majority of brain areas examined, including olfactory bulb, frontal and parietal cortex, hippocampus, cerebellum, midbrain and pons, when the vehicle contained hyaluronan in combination with absorption enhancer. The highest concentrations of FD-4 could be detected in the olfactory bulbs, frontal and parietal cortex 4h after nasal administration in the mucoadhesive formulation. Intravenous administration of a hundred times higher dose of FD-4 resulted in a lower brain penetration as compared to nasal formulations. Morphological examination of the olfactory system revealed no toxicity of the vehicles. Hyaluronan, a non-toxic biomolecule used as a mucoadhesive in a nasal formulation, increased the brain penetration of a hydrophilic compound, the size of a peptide, via the nasal route.

Exposure to lipopolysaccharide and/or unconjugated bilirubin impair the integrity and function of brain microvascular endothelial cells.

Background Sepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS) is known to alter the integrity of the blood-brain barrier (BBB), little is known on the effects of unconjugated bilirubin (UCB) and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC). Methodology/Principal Findings Monolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB. Conclusions LPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period.

Human serum amyloid P component attenuates the bacterial lipopolysaccharide-induced increase in blood-brain barrier permeability in mice.

Endotoxin challenge leads to septic shock, multi-organ failure and death in mice. Permeability of the blood-brain barrier (BBB) is increased by endotoxemia. Serum amyloid P component (SAP) is a lipopolysaccharide (LPS)-binding protein that can modulate the host reactions during infections. It is controversial whether SAP can protect from LPS toxicity in vivo or not. We have tested the effect of human SAP on BBB permeability of Salmonella typhimurium LPS-injected mice. The animals showed signs of sickness behaviour including immobility, anorexia, and diarrhoea. Intraperitoneally administered LPS increased the BBB permeability for sodium fluorescein for about 4-fold, and for albumin for more than 2-fold in brain cortex. SAP, given intravenously, had no effect on basal BBB permeability for albumin, although it decreased sodium fluorescein extravasation to brain tissue. In LPS-treated mice, SAP administration alleviated the symptoms of septic shock, and significantly inhibited the enhanced BBB permeability for both tracers. Our data indicate that human SAP may counteract the toxic effects of LPS during septic shock.

The effect of sucrose esters on a culture model of the nasal barrier

Sucrose esters are effective solubilizers and there is an interest to use them as pharmaceutical excipients for nasal drug delivery. We have determined for the first time the non-toxic doses of laurate and myristate sucrose esters by four independent methods, and their effects on epithelial permeability using RPMI 2650 human nasal epithelial cell line. Based on real-time cell electronic sensing, MTT dye conversion and lactate dehydrogenase release methods reference surfactant Cremophor RH40 proved to be the least toxic excipient, and could be used at 5mg/mL concentration for 1h in epithelial cells without cellular damage. The non-toxic dose of Tween 80 was 1 mg/mL, while the dose of laurate and myristate sucrose esters that could be safely used on cells for 1 h was 0.1 mg/mL. Both the reference surfactants and the sucrose esters significantly enhanced the permeability of epithelial cell layers for the paracellular marker FITC-labelled 4.4 kDa dextran at 0.1 mg/mL concentration. The effects of sucrose esters on epithelial permeability were dose-dependent. These data indicate that laurate and myristate sucrose esters can be potentially used as permeability enhancers in nasal formulations to augment drug delivery to the systemic circulation.

Effect of nicotine and polyaromtic hydrocarbons on cerebral endothelial cells

The present study was designed to investigate the effect of nicotine and polyaromatic hydrocarbon compounds on cerebral endothelial cells (CECs). Nicotine treatments from 15 min to 5 h did not cause any changes in the expression and localization of principal junctional proteins. One day of treatment with a relatively high concentration of nicotine induced a decrease in the expression of the tight junction protein ZO‐1, occludin, and the adherens junction protein, cadherin. Treatment with 3 × 10−5 M phenanthrene for 24 h caused a redistribution of occludin from the Triton X‐100 insoluble to the Triton X‐100 soluble fraction. Transendothelial electrical resistance was not significantly affected by 24 h treatments with nicotine, methylanthracene or phenanthrene. However, 24 h nicotine treatment increased transendothelial permeability in CECs exposed to oxidative stress. Both nicotine and phenanthrene were able to regulate the expression of a large number of proteins as revealed by 2D electrophoresis. Our experiments suggest that tobacco smoking may affect the junctional complex of CECs, and that this effect is enhanced by oxidative stress.

Docosahexaenoic Acid Reduces Amyloid-β Induced Toxicity in Cells of the Neurovascular Unit

Alzheimers disease (AD) is characterized by the accumulation of amyloid-β peptides (Aβ) as perivascular deposits and senile plaques in the brain. The intake of the polyunsaturated fatty acid docosahexaenoic acid (DHA) has been associated with decreased amyloid deposition and reduced risk in AD in several epidemiological trials; however the exact underlying molecular mechanism remains to be elucidated. The aim of the study was to test whether DHA can exert a direct protective effect on the elements of the neurovascular unit, such as neurons, glial cells, brain endothelial cells, and pericytes, treated with Aβ42 (15 μM). A dose-dependent high cellular toxicity was found in viability assays in all cell types and on acute hippocampal slices after treatment with Aβ42 small oligomers prepared in situ from an isopeptide precursor. The cell morphology also changed dramatically in all cell types. In brain endothelial cells, damaged barrier function and increased para- and transcellular permeability were observed after peptide treatment. The production of reactive oxygen species was elevated in pericytes and endothelial and glial cells. DHA (30 μM) significantly decreased the Aβ42-induced toxic effects in all cell types measured by viability assays, and protected the barrier integrity and functions of brain endothelial cells. DHA also decreased the elevated rhodamine 123 accumulation in brain endothelial cells pre-treated with Aβ42 indicating an effect on efflux pump activity. These results indicate for the first time that DHA can protect not only neurons but also the other elements of the neurovascular unit from the toxic effects of Aβ42 and this effect may be beneficial in AD.