Involvement of a proton‐coupled organic cation antiporter in the blood–brain barrier transport of amantadine

The blood‐to‐brain transport of amantadine, a weak N‐methyl‐d ‐aspartate (NMDA) antagonist, has been shown previously to participate in the cationic drug‐sensitive transport system across the mouse blood–brain barrier (BBB). The purpose of the present study was to characterize the influx transport system by means of both an in situ mouse brain perfusion technique and in vitro studies using rat immortalized brain capillary endothelial cells (GPNT). The observed concentration‐dependent initial uptake rate of [³H]amantadine suggested the involvement of a carrier‐mediated transport mechanism. The normal uptake at physiological pH 7.4 was decreased by 72.9% in acidic perfusate, while it was increased by 35.3% in alkaline perfusate. These results suggest that pH‐dependent transport is regulated by utilizing an oppositely directed proton gradient as a driving force. In addition, the [³H]amantadine uptake was moderately inhibited by the adamantane structural analogs (rimantadine and memantine) and other cationic drugs (pyrilamine, clonidine, nicotine, etc.), but not by substrates or inhibitors of the well‐characterized organic cation transporters (tetraethylammonium, l ‐carnitine and choline). A similar inhibition pattern was observed between the in vivo studies and the in vitro experiments. These results indicate that the influx transport for amantadine across the BBB involves a proton‐coupled organic cation antiporter. Copyright © 2016 John Wiley & Sons, Ltd.     

Enalapril attenuates ischaemic brain oedema and protects the blood–brain barrier in rats via an anti‐oxidant action

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Characterization of endocytosis of transferrin-coated PLGA nanoparticles by the blood–brain barrier

Many studies showed that transferrin increases brain delivery of nanoparticles (NPs) in vivo, however the mechanisms implied in their brain uptake are not yet clearly elucidated. In this study we evaluated the endocytosis of PLGA NPs coated with transferrin on an in vitro model of the blood–brain barrier (BBB) made of a co-culture of brain endothelial cells and astrocytes. PLGA NPs were prepared using DiI as a fluorescent marker and coated with Tween^® 20, BSA and transferrin (Tf). Blank and BSA-NPs served as controls. The cellular toxicity on BBB of the different samples was evaluated following tight junction aperture and due to high toxicity NPs prepared with Tween^® 20 were discarded. The size of the NPs prepared by the solvent diffusion method, varied from 63 to 90 nm depending on DiI incorporation and surface coating. Proteins adsorption on the surface of the NPs was found to be stable for at least 12 days at 37 °C. Contrary to Blank or BSA-NPs, Tf-NPs were found to be highly adsorbed by the cells and endocytosed using an energy-dependent process. Studies in presence of inhibitors suggest that Tf-NPs interact with the cells in a specific manner and enter the cells via the caveolae pathway.     

Cerebral open flow microperfusion: A new in vivo technique for continuous measurement of substance transport across the intact blood–brain barrier

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Involvement of an influx transporter in the blood–brain barrier transport of naloxone

Naloxone, a potent and specific opioid antagonist, has been shown in previous studies to have an influx clearance across the rat blood–brain barrier (BBB) two times greater than the efflux clearance. The purpose of the present study was to characterize the influx transport of naloxone across the rat BBB using the brain uptake index (BUI) method. The initial uptake rate of [³H]naloxone exhibited saturability in a concentration‐dependent manner (concentration range 0.5 µM to 15 mM ) in the presence of unlabeled naloxone. These results indicate that both passive diffusion and a carrier‐mediated transport mechanism are operating. The in vivo kinetic parameters were estimated as follows: the Michaelis constant, K_t, was 2.99±0.71 mM ; the maximum uptake rate, J_max, was 0.477±0.083 µmol/min/g brain; and the nonsaturable first‐order rate constant, K_d, was 0.160±0.044 ml/min/g brain. The uptake of [³H]naloxone by the rat brain increased as the pH of the injected solution was increased from 5.5 to 8.5 and was strongly inhibited by cationic H₁‐antagonists such as pyrilamine and diphenhydramine and cationic drugs such as lidocaine and propranolol. In contrast, the BBB transport of [³H]naloxone was not affected by any typical substrates for organic cation transport systems such as tetraethylammonium, ergothioneine or L ‐carnitine or substrates for organic anion transport systems such as p‐aminohippuric acid, benzylpenicillin or pravastatin. The present results suggest that a pH‐dependent and saturable influx transport system that is a selective transporter for cationic H₁‐antagonists is involved in the BBB transport of naloxone in the rat. Copyright © 2010 John Wiley & Sons, Ltd.     

CPU‐86017 improves the compromised blood–brain barrier permeability mediated by impaired endothelial no system and oxidative stress caused by L‐thyroxine

Impaired endothelial cell (EC) function leads to alterations in the permeability of the blood–brain barrier (BBB). There are two aspects of the transport through the BBB: from the blood to the brain (influx) and from the brain to the blood (efflux). An impaired EC model induced by L ‐thyroxine that compromises the influx and efflux properties of the BBB was used to assess responses to the intervention of CPU‐86017 (an antioxidant and calcium channel blocker) and propranolol. CPU‐86017 (t_1/2=1.5 h) was also used as a target drug, leaving no traces in the brain and blood 24 h after administration. The permeability of the BBB was evaluated by using CPU‐86017 after iv and icv injection and concentrations in the blood and brain being measured by high‐performance liquid chromatography. The bidirectional permeability of CPU‐86017 was impaired and associated with a reduced NO bioavailability assessed functionally by the vasoactivity in the model. Partial relief of NO bioavailability and oxidative stress induced by propranolol was consistent with a recovery of BBB efflux alone. Complete recovery in the efflux and influx of the BBB by CPU‐86017 was a result of the complete restoration of NO bioavailability and reduction in oxidative stress. Normal BBB influx is dependent on an intact endothelial NO system, and efflux could be restored easily by partial improvement of NO bioavailability. CPU‐86017 is thus more effective than propranolol in protecting the endothelium from damage produced by L ‐thyroxine through oxidative stress. Drug Dev. Res. 64:145–156, 2005. © 2005 Wiley‐Liss, Inc.     

Possible involvement of cationic‐drug sensitive transport systems in the blood‐to‐brain influx and brain‐to‐blood efflux of amantadine across the blood–brain barrier

The purpose of this study was to characterize the brain‐to‐blood efflux transport of amantadine across the blood–brain barrier (BBB). The apparent in vivo efflux rate constant for [³H]amantadine from the rat brain (k_eff) was found to be 1.53 × 10^‐2 min^‐1 after intracerebral microinjection using the brain efflux index method. The efflux of [³H]amantadine was inhibited by 1‐methyl‐4‐phenylpyridinium (MPP⁺), a cationic neurotoxin, suggesting that amantadine transport from the brain to the blood across the BBB potentially involves the rat plasma membrane monoamine transporter (rPMAT). On the other hand, other selected substrates for organic cation transporters (OCTs) and organic anion transporters (OATs), as well as inhibitors of P‐glycoprotein (P‐gp), did not affect the efflux transport of [³H]amantadine. In addition, in vitro studies using an immortalized rat brain endothelial cell line (GPNT) showed that the uptake and retention of [³H]amantadine by the cells was not changed by the addition of cyclosporin, which is an inhibitor of P‐gp. However, cyclosporin affected the uptake and retention of rhodamine123. Finally, the initial brain uptake of [³H]amantadine was determined using an in situ mouse brain perfusion technique. Notably, the brain uptake clearance for [³H]amantadine was significantly decreased with the co‐perfusion of quinidine or verapamil, which are cationic P‐gp inhibitors, while MPP⁺ did not have a significant effect. It is thus concluded that while P‐gp is not involved, it is possible that rPMAT and the cationic drug‐sensitive transport system participate in the brain‐to‐blood efflux and the blood‐to‐brain influx of amantadine across the BBB, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Probucol prevents blood–brain barrier dysfunction in wild‐type mice induced by saturated fat or cholesterol feeding

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Transient alterations of the blood–brain barrier tight junction and receptor potential channel gene expression by chlorpyrifos

The blood–brain barrier (BBB) is formed by specialized endothelial cells lining capillaries in the central nervous system (CNS). We previously demonstrated that exposure to very low concentrations of the organophosphorus insecticide chlorpyrifos (CPF) decreased electrical resistance across the BBB in vitro, indicating a loss of BBB integrity. The present study examined the transient effects of CPF on expression of genes contributing to tight junctions of the BBB. Rat brain endothelial cells (RBE4) were co‐cultured with rat astrocytes on membrane inserts to form an in vitro BBB. The RBE4 cells in the BBB were then exposed to CPF for 2, 4 and 12 h. Total RNA was extracted from RBE4 cells and quantitative real‐time PCR (qRT‐PCR) was used to quantify levels of gene expression of tight junction proteins claudin5, scaffold proteins zona occludens (ZO1) and transient receptor potential (canonical) channels (TRPC4). Gene expression decreased 2 h after exposure to CPF, especially TRPC4, but the effects were reversed 12 h later. CPF exposure for only 15 min caused less effect than longer exposures, with TRPC4 gene expression above the control at 4 h. These results suggest that altering gene expression for claudin5, TRPC4 and ZO1 by CPF may directly contribute to BBB disruption, and that the alteration is reversible upon removal of CPF. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of an in vitro blood-brain barrier model based on immortalized porcine brain microvascular endothelial cells

Immortalized porcine brain microvessel endothelial cells (PBMEC/C1-2) were used to develop a model for measurement of blood-brain barrier permeation of central nervous system active drugs. Previous studies showed that a system using C6 astrocyte glioma conditioned medium leads to cell layers with transendothelial electrical resistance values up to 300 Omega cm(2) and a permeability coefficient P(e) of 3.24 +/- 0.14 x 10(-4) cm/min for U-[(14)C]sucrose, which is in good agreement to published values and thus indicates the formation of tight junctions in vitro. However, commercially available inserts for the Transwell system were not permeable for highly lipophilic compounds, such as diazepam. Systematic studies with different insert showed, that inserts with a pore width of 1 microm proved to be optimal for permeation studies of lipophilic compounds. Permeability studies with a set of three benzodiazepines further supported this finding.     

Temporal expression of transporters and receptors in a rat primary co-culture blood–brain barrier model

Abstract

1.?The more relevant primary co-cultures of brain microvessel endothelial cells and astrocytes (BMEC) are less utilized for screening of potential CNS uptake when compared to intestinal and renal cell lines.

2.?In this study, we characterized the temporal mRNA expression of major CNS transporters and receptors, including the transporter regulators Pxr, Ahr and Car in a rat BMEC co-cultured model. Permeability was compared with the Madin–Darby canine kidney (MDCKII)-MDR1 cell line and rat brain in situ perfusion model.

3.?Our data demonstrated differential changes in expression of individual transporters and receptors over the culture period. Expression of ATP-binding cassette transporters was better retained than that of solute carrier transporters. The insulin receptor (IR) was best maintained among investigated receptors. AhR demonstrated high mRNA expression in rat brain capillaries and expression was better retained than Pxr or Car in culture. Mdr1b expression was up-regulated during primary culture, albeit Mdr1a mRNA levels were much higher. P-gp and Bcrp-1 were highly expressed and functional in this in vitro system.

4.?Permeability measurements with 18 CNS marketed drugs demonstrated weak correlation between rBMEC model and rat in situ permeability and moderate correlation with MDCKII-MDR1 cells.

5.?We have provided appropriate methodologies, as well as detailed and quantitative characterization data to facilitate improved understanding and rational use of this in vitro rat BBB model.     

The Application of Bovine Brain Microvessel Endothelial-Cell Monolayers Grown onto Polycarbonate Membranes in Vitro to Estimate the Potential Permeability of Solutes Through the Blood–Brain Barrier

Previously our laboratory (Rim et aL, Int. J. Pharm. 32:79–84, 1986) described an in vitro blood-brain barrier (BBB) model consisting of cultured bovine brain microvessel endothelial cells (BMECs) grown onto regenerated cellulose acetate membranes. However, the utility of this in vitro BBB model system was limited because the regenerated cellulose acetate membrane and not the monolayer of bovine BMECs was rate limiting for the permeability of very lipophilic compounds. Therefore, in this study we have evaluated polycarbonate membranes as supports for growing bovine BMECs and for conducting in vitro drug permeability studies. Bovine BMECs were cultured on collagen-coated polycarbonate membranes (13-mm diameter, 12-µm pore size) which were then mounted into side-by-side diffusion cells for transport studies. The permeabilities of a series of solutes of varying lipophilicity (progesterone, estrone, testosterone, haloperidol, propranolol, antipyrine, caffeine, urea, acyclovir, ganciclovir, ribavirin, and glycerol) were determined and an excellent correlation (r = 0.97) was established between the permeability coefficients of the solutes and their log partition coefficients (PC)/(MW)^1/2. These results suggest that bovine BMECs cultured onto polycarbonate membranes can be used as an in vitro model system for estimating the potential permeability of a solute through the BBB in vivo.     

Uptake of apolipoprotein E fragment coupled liposomes by cultured brain microvessel endothelial cells and intact brain capillaries

The suitability of surface modified liposomes as drug carriers for brain-specific targeting was investigated using apolipoprotein E fragments as brain-directed vectors. Liposomes coated with polyethylene glycol-2000 (sterically stabilized, PEGylated liposomes) were prepared from hydrogenated egg phosphatidylcholine, cholesterol, and a PEG-derivatized phospholipid. Liposomes were covalently coupled to a peptide of 26 amino acids length, derived from the binding site of human apolipoprotein E4 (ApoE4) and a peptide of random amino acid sequence, respectively. Rhodamine-labeled dipalmitoylphosphatidylethanolamine was incorporated into the lipid bilayer in order to visualize the liposomal interaction with brain capillary endothelial cell monolayers. The interaction of the liposomes with monolayers of porcine brain capillary endothelial cells (BCEC), the rodent cell line RBE4, and freshly isolated porcine brain capillaries was studied by means of confocal laser scanning fluorescence microscopy. In contrast to random peptide coupled liposomes, the ApoE4-fragment coupled liposomes were rapidly taken up by cultured BCECs and RBE4 cells. Uptake could be inhibited by ApoE4, free peptide, and antibodies against the LDL receptor in a concentration-dependent manner. The results indicate that the liposomes are internalized via the LDL receptor, which is expressed at the blood?brain barrier. In conclusion, liposomes coupled to ApoE4 fragments are taken up into brain endothelium via an endocytotic pathway and may therefore be a suitable carrier for drug delivery to the brain.     

Characterisation of a mouse cerebral microvascular endothelial cell line (bEnd.3) after oxygen glucose deprivation and reoxygenation

Studies have utilised immortalised mouse cerebral endothelial cells (bEnd.3) exposed to oxygen glucose deprivation (OGD) to study blood–brain barrier (BBB) disruption after ischaemia. However, there is a paucity of literature describing the duration of OGD (and reoxygenation [RO]) required to best simulate BBB disruption in vivo. In this study we assessed BBB disruption in bEnd.3 cells after exposure to a range of OGD periods, and also after OGD + RO. Exposure of bEnd.3 monolayers to 4, 6, 16, or 24 hours of OGD resulted in a significant increase in permeability. The hyperpermeability after 16 or 24 hours was associated with decreased expression of tight junction proteins (occludin and claudin‐5). Furthermore, there was a decrease in cell viability and increased expression of the pro‐apoptotic protein, cleaved caspase‐3. Exposure of bEnd.3 monolayers to 1 hour OGD+ 23 hours RO exacerbated hyperpermeability relative to 1 hour OGD, which was associated with decreased expression levels of occludin and ZO‐1, but no change in cell viability or caspase‐3. 4 hours OGD + 23 hours RO exacerbated hyperpermeability, decreased expression levels of tight junction proteins, decreased cell viability, and increased caspase‐3 expression. Thus, bEnd.3 cells exhibit hyperpermeability, a loss of tight junction proteins, and undergo cell death, after exposure to prolonged periods of OGD. Moreover, they exhibit exacerbated hyperpermeability, a loss of tight junction proteins, and increased expression of caspase‐3 after OGD + RO. These findings will facilitate the use of this cell line in studies of BBB disruption and for the testing of therapeutics.