Bovine Brain Microvessel Endothelial Cells Research Articles

Methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxypyrovalerone (MDPV) induce differential cytotoxic effects in bovine brain microvessel endothelial cells

Designer drugs such as synthetic psychostimulants are indicative of a worldwide problem of drug abuse and addiction. In addition to methamphetamine (METH), these drugs include 3,4-methylenedioxy-methamphetamine (MDMA) and commercial preparations of synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), typically referred to as “bath salts.” These psychostimulants exert neurotoxic effects by altering monoamine systems in the brain. Additionally, METH and MDMA adversely affect the integrity of the blood-brain barrier (BBB): there are no current reports on the effects of MDPV on the BBB. The aim of this study was to compare the effects of METH, MDMA and MDPV on bovine brain microvessel endothelial cells (bBMVECs), an accepted in vitro model of the BBB. Confluent bBMVEC monolayers were treated with METH, MDMA and MDPV (0.5mM–2.5mM) for 24h. METH and MDMA increased lactate dehydrogenase release only at the highest concentration (2.5mM), whereas MDPV induced cytotoxicity at all concentrations. MDMA and METH decreased cellular proliferation only at 2.5mM, with similar effects observed after MDPV exposures starting at 1mM. Only MDPV increased reactive oxygen species production at all concentrations tested whereas all 3 drugs increased nitric oxide production. Morphological analysis revealed different patterns of compound-induced cell damage. METH induced vacuole formation at 1mM and disruption of the monolayer at 2.5mM. MDMA induced disruption of the endothelial monolayer from 1mM without vacuolization. On the other hand, MDPV induced monolayer disruption at doses ≥0.5mM without vacuole formation; at 2.5mM, the few remaining cells lacked endothelial morphology. These data suggest that even though these synthetic psychostimulants alter monoaminergic systems, they each induce BBB toxicity by different mechanisms with MDPV being the most toxic.

Fucoidan Extracted from Hijiki Protects Brain Microvessel Endothelial Cells Against Diesel Exhaust Particle Exposure-Induced Disruption.

This study was performed to evaluate the protective effects of fucoidan against the decreased function of primary cultured bovine brain microvessel endothelial cells (BBMECs) after exposure to diesel exhaust particles (DEPs). BBMECs were extracted from bovine brains and cultured until confluent. To evaluate the function of BBMECs, we performed a permeability test using cell-by-cell equipment and by Western blot analysis for zonular occludens-1 (ZO-1), which is a tight junction protein of BMECs, and evaluated oxidative stress in BBMECs using the DCFH-DA assay and the CUPRAC-BCS assay. The increased oxidative stress in BBMECs following DEP exposure was suppressed by fucoidan. In addition, permeability of BBMECs induced by DEP exposure was decreased by fucoidan treatment. Our results showed that fucoidan protects against BBMEC disruption induced by DEP exposure. This study provides evidence that fucoidan might protect the central nervous system (CNS) against DEP exposure.

Toxicity of low doses of ultrafine diesel exhaust particles on bovine brain microvessel endothelial cells

Diesel exhaust particles (DEPs) are an important risk factor for various health conditions including neurodegenerative diseases. However, information regarding the toxicity of low doses of ultrafine DEPs in relation to the brain is limited. This study was performed to evaluate the neurotoxicity of low doses of ultrafine DEPs (ng/mL level) using bovine brain microvessel endothelial cells (BMECs) as a model of the blood-brain barrier (BBB). The cells were exposed to various concentrations of DEPs (from 1.28 ng/mL to 20 μg/mL) for 24 h, and cytotoxicity was evaluated by assaying lactate dehydrogenase activity, the generation of reactive oxygen species (ROS) by the dichlorofluorescein diacetate method, total antioxidant capacity (TAC) using bathocuproinedisulfonic acid disodium salt, and cell permeability by measuring the flux of fluorescein. The results showed that low doses of DEPs decreased cell viability and induced oxidative stress by increasing ROS generation and decreasing TAC in a dose-dependent manner. Furthermore, DEPs exposure increased BMECs permeability. This study shows that exposure to low doses of ultrafine DEPs might disrupt the function of BMECs and generate oxidative stress, suggesting that exposure to DEPs even at extremely low doses may compromise the integrity of the BBB and induce adverse effects in the central nervous system.

Non-aggregated protamine-coated poly(lactide-co-glycolide) nanoparticles of cisplatin crossed blood–brain barrier, enhanced drug delivery and improved therapeutic index in glioblastoma cells: in vitro studies

Background and objectives: Non-aggregated protamine impregnated poly(lactide-co-glycolide) nanoparticles of cisplatin (Pt-PLGA NPs) were synthesized to augment brain delivery. Methods and results: The mean particle size of Pt-PLGA NPs and PLGA NPs were observed to be 173.2 ± 7.9 nm and 140 ± 10.2 nm, respectively. The Pt-PLGA NPs significantly (p < 0.05, one-way analysis of variance; ANOVA) delivered higher amount (172.41 ± 15.04 μg) of cisplatin in comparison to 110.48 ± 4.71 μg by PLGA NPs and 20.83 ± 1.65 μg by cisplatin solution across in vitro bovine brain microvessel endothelial cells. Cisplatin bearing Pt-PLGA NPs was found to be highly cytotoxic to U87 glioblastoma cells with an IC50 of 2.1 μM as compared (one-way ANOVA, p < 0.05) to PLGA NPs (3.9 μM) and cisplatin alone (13.33 μM). Impregnation with Pt enhanced the uptake of PLGA NPs in U87 glioblastoma cells as compared to PLGA NPs by following endocytosis mechanism. Conclusion: Cisplatin-loaded Pt-PLGA NPs compel preclinical tumour regression study to further improve its utility against glioblastoma.

Prolactin and Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) consists in part of a highly specialized set of cells which separates the brain from the vascular system. The BBB controls the entry and exit of substances from the brain tissue through tight junctions (TJs) between endothelial cells. It is known that the hormone prolactin (PRL) is able to regulate endothelial-dependent processes, like the balance between proliferation and apoptosis and the mammary epithelial permeability. However, the effects of PRL and the role it plays in the BBB permeability are still not well understood. A primary culture of bovine brain microvessel endothelial cells was used as in vitro model of BBB. Cells were treated with PRL (0.1, 1, 10 and 100 nM) for 24 hours. PRL significantly increased cellular proliferation at 10 and 100 nM, but did not modify basal apoptosis. These effects were dependent on the production of the mitogenic factor nitric oxide (NO). PRL significantly decreased the permeability and promoted an increase in trans-endothelial electrical resistance in a NO-independent way. PRL also increased the expression of the TJs proteins claudin-5 and occludin. The short form of the PRL receptor was detected in these cells but its expression was not modified by PRL. Together, these results suggest that PRL has the ability to increase cellular proliferation associated with a decrease on BBB permeability by increasing the expression of TJs proteins.

Prolactin Protects Against the Methamphetamine-Induced Cerebral Vascular Toxicity

Methamphetamine (Meth) is a highly addictive drug of abuse which alters the dopaminergic system and damages the blood-brain barrier (BBB), structure that protects the brain tissue from the circulating substances in the blood, keeping a low permeability through the presence of tight junctions (TJs) between endothelial cells. Meth increases BBB permeability by decreasing the TJs proteins claudin-5 and occludin and by decreasing the viability of endothelial cells. Individuals abused of Meth have increased blood concentrations of prolactin (PRL); hormone related with milk production, but able to increase the expression of TJs proteins and to decrease permeability on the mammary epithelium and brain endothelial cells. However, the effects of PRL on the permeability of the BBB in the presence of Meth have not been studied. Here, we report Meth-induced apoptosis and decreased cellular proliferation as well as the trans-endothelial electrical resistance (TEER), related to a decrease of claudin-5 and occludin in primary cultured bovine brain microvessel endothelial cells. The expression of the PRL receptor was not altered. Administration of PRL prevented a decrease in cellular proliferation, an increase in apoptosis and restored the TEER and TJs proteins to basal levels. This protection was absent at high Meth concentrations. These data suggest that PRL protects brain endothelial cells against the Meth-induced toxicity. Further investigation is required to study the mechanisms involved and to confirm these effects in vivo.

Re-evaluation of the role of P-glycoprotein in in vitro drug permeability studies with the bovine brain microvessel endothelial cells

1. Currently available in vitro blood-brain barrier models all have recognized restrictions. In addition to leakiness, inconsistent data about P-glycoprotein mediated efflux limit the attractiveness of the primary bovine brain microvessel endothelial cells (BBMECs). Therefore, we re-evaluated the role of P-glycoprotein mediated efflux with two culture conditions in BBMECs for prediction of drug permeability of potential P-glycoprotein substrates.2. BBMECs were monocultured on filters on petri dishes and on filter inserts, and expression and localization of P-glycoprotein were compared by using western blot and confocal microscopy, respectively. The functionality of P-glycoprotein was assessed by using cellular uptake, calcein-AM and bidirectional transport assays.3. P-glycoprotein expression was higher in BBMECs cultured on filter inserts decreasing the permeability of digoxin and paclitaxel, but not the permeability of vinblastine. However, the monocultured BBMECs were not able to demonstrate efflux in the bidirectional transport assays. Under certain culture conditions, occludin may not be correctly located, perhaps explaining in part the leakiness of BBMECs.4. In conclusion, BBMECs, despite possessing a functional P-glycoprotein, under certain culture conditions may not be a suitable in vitro model for the bidirectional transport assays and for predicting the permeability of drugs and xenobiotics that are potential P-glycoprotein substrates.

The permeation of dynorphin A 1–6 across the blood brain barrier and its effect on bovine brain microvessel endothelial cell monolayer permeability

Dynorphin A 1–17 (Dyn A 1–17) is an endogenous neuropeptide known to act at the kappa opioid receptor; it has been implicated in a number of neurological disorders, including neuropathic pain, stress, depression, and Alzheimer's and Parkinson's diseases. The investigation of Dyn A 1–17 metabolism at the blood–brain barrier (BBB) is important since the metabolites exhibit unique biological functions compared to the parent compound. In this work, Dyn A 1–6 is identified as a metabolite of Dyn A 1–17 in the presence of bovine brain microvessel endhothelial cells (BBMECs), using LC–MS/MS. The transport of Dyn A 1–6 at the BBB was examined using this in vitro cell culture model of the BBB. Furthermore, the permeation of the BBB by the low molecular weight permeability marker fluorescein was characterized in the presence and absences of Dyn A 1–6.

Similar molecular descriptors determine the in vitro drug permeability in endothelial and epithelial cells

Endothelial and epithelial cells are commonly used for assessing blood-brain barrier (BBB) permeability of the drug candidates. However, the additional value provided by the endothelial cells as an in vitro BBB drug permeability model is not clear. The aim of the study was to identify the molecular descriptors that impact on drug permeability through the primary bovine brain microvessel endothelial cell model (BBMEC) and compare descriptors with those determined for epithelial cell models. In addition, we intended to clarify the reasons for previously reported similar in vitro-in vivo correlations between endothelial and epithelial cell models and to evaluate whether BBMEC can provide additional value. The permeability of model drugs through the BBMEC was determined. Principal component analysis (PCA) model was created with twenty-two model drugs and this model was used to interpret the molecular descriptors. The present study demonstrates that hydrophobic interactions, the balance between the hydrophilic and lipophilic moieties in the drug, hydrophilic interactions and hydrogen bonding interactions are the key descriptors depicting drug permeability through the BBMEC. There were no clear differences between the molecular descriptors determining the in vitro permeability of drugs in the endothelial and epithelial cells. In conclusion, the predominance of passive permeability in in vitro setups may explain the similar in vitro-in vivo correlations previously obtained between endothelial and epithelial cell models. Therefore, the present results further support previous findings that epithelial cell models can be used instead of laborious primary endothelial cells as an in vitro BBB permeability model when passive transport is mainly being evaluated.

Evaluation of drug efflux transporter liabilities of darifenacin in cell culture models of the blood–brain and blood–ocular barriers

The objective of the present study was to evaluate drug efflux transporter interactions of darifenacin and examine the impact of such transporter interactions on darifenacin permeability in an in vitro model of the blood-brain barrier (BBB) and blood-ocular barrier (BOB). Cell membranes expressing human P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP) were examined for ATPase activity following darifenacin exposure (0-10 µM). Primary cultured bovine brain microvessel endothelial cells (BBMEC) and P-gp transfected Manin-Darby canine kidney epithelial cells (MDCKMDR1) were used to examine darifenacin permeability and drug efflux transporter responses. Concentration-dependent increases in ATPase activity was observed in P-gp membranes following darifenacin exposure. Both MRP and BCRP membrane preparations were unresponsive to darifenacin. Studies in both BBMEC and MDCKMDR1 monolayers confirmed a P-gp interaction for darifenacin and significantly greater efflux (basolateral to apical) permeability for darifenacin that was reduced by the P-gp inhibitor, elacridar. Darifenacin is a substrate for the P-gp drug efflux transporter present in both BBB and BOB. The P-gp drug efflux transporter liabilities of darifenacin may limit its penetration into brain and ocular tissue thereby reducing side effect potential.

Brain Pharmacokinetics of Two Prolyl Oligopeptidase Inhibitors, JTP-4819 and KYP-2047, in the Rat

Prolyl oligopeptidase (PREP) inhibitors are potential drug candidates for the treatment of neurological disorders, but little is known about their ability to cross the blood-brain barrier and to reach the target site. This study characterizes brain pharmacokinetics of two potent PREP inhibitors, JTP-4819 and KYP-2047. Firstly, the in vitro permeability (P(app) ) of JTP-4819 and KYP-2047 through a bovine brain microvessel endothelial cell monolayer was assessed. Then, the in vivo brain/blood ratio was determined for the total brain and plasma concentrations and also for the unbound extracellular drug concentrations after a single dose (50 μmol/kg i.p.). KYP-2047 had a significantly higher P(app) than JTP-4819. In vivo, KYP-2047 had higher total and unbound brain/blood ratios. KYP-2047 was equally distributed between the cortex, hippocampus and striatum. In the case of JTP-4819, the unbound brain extracellular concentrations could not be readily predicted from the unbound blood levels, probably because of its poor membrane penetration properties. KYP-2047 displayed a better ability to reach the intracellularly located brain PREP, and it inhibited this enzyme more effectively than JTP-4819 after an equimolar single dose. In conclusion, KYP-2047 showed better brain penetration characteristics than JTP-4819 both in vitro and in vivo. KYP-2047 is a brain-penetrating, potent and long-acting PREP inhibitor; thus, it represents a convenient pharmacological tool for assessing the potential of PREP as a drug target.

Anti-Angiogenic Effects of Resveratrol on Cerebral Angiogenesis

Angiogenesis is implicated in diseases of the central nervous system, and its modulation represents an attractive therapeutic strategy. This is the first report of the effects of resveratrol (trans-3,4',5-trihydroxystilbene) on cerebral angiogenesis, using an in vitro model. Processes associated with angiogenesis were studied in cerebral vascular endothelial cells, using the human brain endothelial cell line hCMEC/D3 and primary bovine brain microvessel EC (BBMEC). Comparisons were made to human umbilical vein EC (HUVEC). In cerebral cultures, resveratrol (24h) induced a dose dependent reduction in BrdU incorporation and cell numbers (MTT assay) between 10-100 µM, while lower doses (100 nM-5 µM) had no effect. Cell migration (scratch assay) was inhibited between 10-100 µM depending on cell type. Doses between 10-100 µM reduced average tubule length on Matrigel, while higher doses (50,100 µM) also inhibited process formation around explanted rat aortic rings (ex vivo assay). Cell cycle analysis in hCMEC/D3 (propidium iodide) showed reduced progression to the G2/M phase, with a maximal effect at 25 µM. Resveratrol did not induce apoptosis in hCMEC/D3, based on caspase-3 activity. Cytotoxicity (LDH release) was induced by resveratrol (50,100 µM) in hCMEC/D3 and HUVEC, peaking at ~20% in hCMEC/D3 and ~35% in HUVEC. Cytotoxic effects were not detected in BBMEC. Resveratrol (10-50 µM) inhibited phosphorylation of the serine/threonine kinase Akt, by Western blot (15 min, 1h) with a prolonged inhibition (24h) for 25 µM. In conclusion, this study shows inhibitory effects of resveratrol on cerebral angiogenesis, using an in vitro model. This is discussed in terms of dosage, in vivo equivalence and therapeutic potential.

N-Methyl Phenylalanine-Rich Peptides as Highly Versatile Blood−Brain Barrier Shuttles

Here we studied the capacity of N-MePhe-(N-MePhe)(3)-CONH(2), Cha-(N-MePhe)(3)-CONH(2), and 2Nal-(N-MePhe)(3)-CONH(2) to carry various drugs (cargos) in in vitro blood-brain barrier (BBB) models in order to determine the versatility of these peptides as BBB-shuttles for drug delivery to the brain. Using SPPS, the peptides were coupled to GABA, Nip, and ALA to examine their passive BBB permeation by means of PAMPA and their lipophilicity by IAMC. Unaided, these nonpermeating drugs alone did not cross the PAMPA barrier and the BBB passively; however, the peptides tested as potential BBB shuttles transferred them by passive transfer through the PAMPA phospholipid. The permeability of peptides that showed the highest permeability in PAMPA, and Ac-N-MePhe-(N-MePhe)(3)-CONH(2) as the parent peptide was also examined in bovine brain microvessel endothelial cells (BBMECs). These peptide-based BBB shuttles open up the possibility to overcome the formidable obstacle of the BBB, thereby achieving drug delivery to the brain.

Induction of P-glycoprotein expression and activity by ritonavir in bovine brain microvessel endothelial cells

Extended treatment with human immunodeficiency virus (HIV) protease inhibitors (HPIs) is standard in HIV/AIDS therapy. While these drugs have helped decrease the overall incidence of AIDS defining illnesses, the relative prevalence of HIV/AIDS dementia has increased. HPIs may cause induction of blood-brain barrier (BBB) drug transporters (P-glycoprotein; P-gp) and thereby limit entry of HPIs into brain tissue, increasing the probability that the brain could become an HIV sanctuary site. Using bovine brain microvessel endothelial cells (BMEC) as an in-vitro model of the BBB, the potential for the HIV protease inhibitor ritonavir to cause induction of P-gp activity and expression was examined. BMEC were isolated from fresh cow brain by enzymatic digest and density centrifugation. Primary culture BMEC were co-incubated with ritonavir or vehicle control for 120 h. Quantitative drug accumulation of rhodamine 123 (Rh123) and fluorescence microscopy were used as measures of P-gp activity. P-gp expression was assessed using quantitative Western blotting. Ritonavir decreased Rh123 cell accumulation and increased P-gp immunoreactive protein in a concentration-dependent manner. Fluorescent microscopy mirrored Rh123 quantitative studies. In BMEC pretreated with 30 microM ritonavir, Rh123 accumulation was decreased 40% and immunoreactive P-gp protein increased 2-fold. Collectively, a strong correlation between decreased Rh123 BMEC accumulation and increased P-gp immunoreactive protein was observed (Spearman r2 = 0.77, P < 0.0001). Thus extended exposure of BMEC to ritonavir caused a concentration-dependent increase in P-gp activity and expression. Similar findings may occur at the clinical level with prolonged HIV protease inhibitor use, giving insight into the central nervous system as an HIV sanctuary site and eventual development of HIV dementia.

(3R,5S,7as)-(3,5-Bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol, a novel neuroprotective agent.

Compounds that interact with microtubules, such as paclitaxel, have been shown to possess protective properties against beta-amyloid (Abeta) induced neurodegeneration associated with Alzheimer's disease. In this work, the novel agent (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol was investigated for effectiveness in protecting neurons against several toxic stimuli and its interaction with the microtubule network. Exposure of neuronal cultures to Abeta peptide in the presence of 5 nM (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol resulted in a 50% increase in survival. Neuronal cultures treated with other toxic stimuli such as staurosporine, thapsigargin, paraquat, and H(2)O(2) showed significantly enhanced survival in the presence of (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol. Microtubule binding and tubulin assembly studies revealed differences compared to paclitaxel but confirmed the interaction of (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol with microtubules. Furthermore, in vitro studies using bovine brain microvessel endothelial cells experiments suggest that (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol can readily cross the blood-brain barrier in a passive manner.

Quaternary Ammonium β-Cyclodextrin Nanoparticles for Enhancing Doxorubicin Permeability across the In Vitro Blood−Brain Barrier

This study describes novel quaternary ammonium beta-cyclodextrin (QAbetaCD) nanoparticles as drug delivery carriers for doxorubicin (DOX), a hydrophobic anticancer drug, across the blood-brain barrier (BBB). QAbetaCD nanoparticles show 65-88 nm hydrodynamic radii with controllable cationic properties by adjusting the incorporated amount of quaternary ammonium group in their structure. ATR-FTIR studies confirm the complexation between the QAbetaCD nanoparticles and DOX. QAbetaCD nanoparticles are not toxic to bovine brain microvessel endothelial cells (BBMVECs) at concentrations up to 500 microg x mL(-1). They also do not change the integrity of BBMVEC monolayers, an in vitro BBB model, including transendothelial electrical resistance value, Lucifer yellow permeability, tight junction protein occludin and ZO-1 expression and morphology, cholesterol extraction, and P-glycoprotein (P-gp) expression and efflux activity, at a concentration of 100 microg x mL(-1). Some QAbetaCD nanoparticles not only are twice as permeable as dextran (M(w) = 4000 g x mol(-1)) control, but also enhance DOX permeability across BBMVEC monolayers by 2.2 times. Confocal microscopy and flow cytometry measurements imply that the permeability of QAbetaCD nanoparticles across the in vitro BBB is probably due to endocytosis. DOX/QAbetaCD complexes kill U87 cells as effectively as DOX alone. However, QAbetaCD nanoparticles completely protect BBMVECs from cytotoxicity of DOX at 5 and 10 microM after 4 h incubation. The developed QAbetaCD nanoparticles have great potential in safely and effectively delivering DOX and other therapeutic agents across the BBB.

Effect of Pluronic P85 on Amino Acid Transport in Bovine Brain Microvessel Endothelial Cells

A synthetic amphiphilic block copolymer Pluronic P85 (P85) was shown to be among the most potent inhibitors of Pgp efflux system in the blood-brain barrier (BBB) and capable of enhancing delivery of Pgp substrates to the brain. The purpose of this work is to evaluate the effects of P85 on amino acid transport in BBB. Primary bovine brain microvessel endothelial cells (BBMEC) grown on membrane inserts were used as an in vitro BBB model. Expression of amino acid transporters, like large neutral amino acid transporter 1, cationic amino acid transporter 1, and small neutral amino acid transporter 1, were confirmed by reverse transcriptase polymerase chain reaction. Effects of P85 on amino acid transporters were examined using their substrates: (3)H-phenylalanine, (3)H-lysine, and (3)H-methylaminoisobutyric acid, respectively. BBMEC permeability studies were carried out in apical (AP) to basolateral (BL) and BL to AP directions. P85 added at the AP side had little, if any, effect on AP to BL ("blood to brain") transport for all examined amino acids in BBMEC monolayers. However, 0.1% P85 added at the BL side significantly increased the BL to AP transport of these substrates. Furthermore, the effective concentrations of P85 were also shown to induce plasma membrane depolarization and increase intracellular sodium concentration in BBMEC, which can contribute to the effects of the copolymer on the energy-dependent transport systems. All together, despite profound effects on transport system(s) at the brain side of cell monolayers, P85 had no effect on AP to BL transport of amino acids in brain microvessel endothelial cell model.

Protein Conjugation with Amphiphilic Block Copolymers for Enhanced Cellular Delivery

Modification of a model protein, horseradish peroxidase (HRP), with amphiphilic block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic), was previously shown to enhance the transport of this protein across the blood-brain barrier in vivo and brain microvessel endothelial cells in vitro. This work develops procedures for synthesis and characterization of HRP with Pluronic copolymers, having different lengths of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks. Four monoamine Pluronic derivatives (L81, P85, L121, P123) were synthesized and successfully conjugated to a model protein, HRP, via biodegradable or nondegradable linkers (dithiobis(succinimidyl propionate) (DSP), dimethyl 3,3'-dithiobispropionimidate (DTBP), and disuccinimidyl propionate (DSS)). The conjugation was confirmed by HRP amino group titration, matrix-assisted laser desorption/ionization-time of flight spectroscopy, and cation-exchange chromatography. HRP conjugates containing an average of one to two Pluronic moieties and retaining in most cases over 70% of the activity were synthesized. Increased cellular uptake of these conjugates was demonstrated using the Mardin-Derby canine kidney cell line and primary bovine brain microvessel endothelial cells. The optimal modifications included Pluronic L81 and P85. These copolymers have shorter PPO chains compared to Pluronic P123 and L121, which were less efficient. There was little if any dependence of the uptake on the length of the hydrophilic PEO block for the optimal modifications. The proposed modifications may be used to increase cellular uptake of other proteins.

393 POSTER The multidrug transporter MRP4/ABCC4 is a powerful marker of poor prognosis in neuroblastoma and a target for therapeutic suppression

Multidrug resistance-associated protein (MRP) actively transports a broad range of anionic compounds out of the cell. To date, six different homologues of MRP (i.e. MRP1–MRP6) have been identified. The current study examines the expression of the various MRP homologues in both primary cultured bovine brain microvessel endothelial cells (BBMEC) and the capillary-enriched fraction from bovine brain homogenates. RT–PCR analysis demonstrated the presence of MRP1, MRP4, MRP5 and MRP6 in both BBMEC and the capillary-enriched fractions of brain homogenates. While low levels of MRP3 were detected in the BBMEC, it was not observed in the capillary-enriched fraction. In addition, RT–PCR and Western blot studies indicated an absence of MRP2 expression in both blood–brain barrier preparations. The presence of several different MRP homologues in the brain microvessel endothelial cells may be important in controlling the permeability of the blood–brain barrier to organic anions.

COMPARISON OF DRUG EFFLUX TRANSPORT KINETICS IN VARIOUS BLOOD-BRAIN BARRIER MODELS

The present study quantitatively compared the drug efflux transport kinetics of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and its fluorescent metabolite 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in various blood-brain barrier (BBB) models. BCECF-AM was exposed to freshly isolated bovine brain microvessels (BBM), primary cultured bovine brain microvessel endothelial cells (BBMEC), and MDCK-MDR1 cells for 30 min in the presence or absence of the P-glycoprotein (P-gp) inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). P-gp transport kinetics were determined indirectly by calculating the difference in BCECF accumulation when P-gp was functional and completely inhibited by GF120918 (3.2 microM). Multidrug resistance-associated protein (MRP) transport kinetics were determined by measuring the amount of BCECF transported out of the cell over time. For P-gp-related transport, Km values for BCECF-AM were approximately the same in all three models (around 2 microM), whereas the Vmax was 4-fold greater in the BBM than in the BBMEC or MDCKII-MDR1 cells. For MRP-related transport, Km values for BCECF varied widely among the three BBB models with a rank order of MDCKII-MDR1 < BBMEC < BBM. Like P-gp, the Vmax of BCECF for MRP-related transport was overwhelmingly higher in the BBM compared with the cultured cells. Because differences in the expression of P-gp, MRP5, and MRP6 were observed in the various BBB models using reverse transcription-polymerase chain reaction techniques, the disparity in transport kinetics between the BBB models may be linked to variations in the amount or type of drug efflux transporters expressed in each model. The present study introduces a method of quantitatively evaluating drug efflux transport kinetics in the BBB.