RESUMO
The development of novel neuropharmaceuticals requires the evaluation of blood-brain barrier (BBB) permeability and toxicity. Recent studies have highlighted differences in the BBB among different species, with the most important differences involving the expression of P-glycoprotein (P-gp), multidrug resistance-associated proteins, transporters, and claudins. In addition, functional studies have shown that brain pharmacokinetics of P-glycoprotein substrates are different in humans and rodents. Therefore, human BBB models may be an important platform for initial drug screening before in vivo studies. This strategy might help to reduce costs in drug development and failures in clinical studies. We review the differences in the BBB among species, recent advances in the generation of human BBB models, and their applications in drug discovery and delivery.
Assuntos
Barreira Hematoencefálica , Descoberta de Drogas/métodos , Modelos Biológicos , Células-Tronco , Subfamília B de Transportador de Cassetes de Ligação de ATP , Animais , Transporte Biológico , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/fisiologia , Humanos , Camundongos , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
INTRODUCTION: By culturing Caco-2 cells according to a new and optimized protocol, it has been possible to accelerate the cell culture process in such a way that the cells can be used for experiments after only 6 days. The accelerated Caco-2 model has been compared to the traditional model (requiring 21-25 days of culture) in terms of tightness of the junctions, ability to rank chemical compounds for apparent permeability, active efflux and to discriminate P-gp substrates. METHODS AND RESULTS: In the new protocol, Caco-2 cells were cultured with the classical Caco-2 medium supplemented with puromycin. The initial cell seeding density was increased two times compared to the traditional procedure and the presence of a low concentration of puromycin in the culture medium reduced the Caco-2 permeability of mannitol. Bi-directional studies were performed with known P-gp substrates (rhodamine 123, digoxin and saquinavir) and with a total of 20 marketed drugs covering a wide range of physicochemical characteristics and therapeutic indications. Strong correlations were obtained between the apparent permeability in absorptive (Papp AâB) or secretory (Papp BâA) of the drugs in the accelerated model and in the traditional models and comparable efflux ratios were observed in the two studied models. DISCUSSION: The new protocol reduces costs for screening and leads to higher throughput compared to traditional Caco-2 cell models. This accelerated model provides short time-feedback to the drug design during the early stage of drug discovery.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Manitol/farmacocinética , Modelos Biológicos , Células CACO-2 , Permeabilidade da Membrana Celular , Custos e Análise de Custo , Digoxina/farmacologia , Desenho de Fármacos , Descoberta de Drogas , Ensaios de Triagem em Larga Escala/métodos , Humanos , Puromicina/química , Rodamina 123/farmacologia , Saquinavir/farmacologia , Fatores de TempoRESUMO
Toxicity to the central nervous system (CNS) is a key feature in the toxicological profile of compounds and there is a growing interest to use in vitro cell assays. The blood-brain barrier (BBB) is a highly restrictive barrier that preserves homeostasis within the brain microenvironment. By modelling the BBB it is possible to investigate whether a compound is likely to compromise its functionality, which would cause unwanted effects on brain cells. These investigations are usually performed using a single exposure to drugs, whereas CNS side effects usually result from repeated exposures. The main objective of this study was to adapt our established BBB model to the evaluation of repeated-dose toxicity at the BBB. Studies were undertaken within the European Predict-IV consortium to study the effect on BBB permeability of 12 selected drugs after 14 days of repeated treatment to a single pre-selected concentration. Compared to single exposure, a 100-fold lower colchicine concentration in 14 days repeated-dose treatment was toxic. This demonstrates the importance to evaluate the BBB toxicity in repeated-dose testing. Finally, the potentiating effects of cyclosporin A on the BBB toxicity of colchicine illustrate the possibility to use in vitro BBB models to make risk assessment of drug-drug interactions.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Toxicidade/métodos , Animais , Animais Recém-Nascidos , Barreira Hematoencefálica/metabolismo , Bovinos , Células Cultivadas , Técnicas de Cocultura , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Células Endoteliais , Neuroglia , Ratos , Ratos Sprague-DawleyRESUMO
ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).
Assuntos
Redes Neurais de Computação , Testes de Toxicidade Aguda , Administração Oral , Alternativas aos Testes com Animais , Animais , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Sobrevivência Celular , Ensaio de Unidades Formadoras de Colônias , Simulação por Computador , Citocinas/metabolismo , Humanos , Absorção Intestinal , Dose Letal Mediana , Camundongos , Estresse Oxidativo , Ratos , Medição de RiscoRESUMO
1. The objective was to investigate the transport of an anticancer agent carboplatin across the blood-brain barrier in combination with hyperbaric oxygenation treatment. An in vitro well-validated model of bovine brain capillary endothelial cells was used. 2. A transendothelial transport of doxorubicin, a known P-glycoprotein substrate, was enhanced 1.5-fold by verapamil for 2-h incubation at 37 degrees C. A transendothelial permeability coefficient of carboplatin (1.29 x 10(-3)cm min-1) was also increased 1.8-fold by verapamil. 3. Under the hyperbaric oxygenation conditions (at 0.2 MPa for the first 10 min), the transendothelial transport for 2 h of doxorubicin and carboplatin were increased 1.3- to 1.8-fold by hyperbaric oxygenation, like the suppressive effects of verapamil on P-gp function, without increase of the transport of lucifer yellow, a non P-glycoprotein substrate. 4. Combination of hyperbaric oxygenation treatment and verapamil could not further increase the permeability coefficients of these drugs that were already enhanced by either treatment, implying the P-glycoprotein-mediated carboplatin efflux transport similarly as doxorubicin. 5. Together with our reported high efficacy of carboplatin combined with hyperbaric oxygenation therapy on brain tumours, the present results suggest that carboplatin could be transported by P-glycoprotein, but that this efflux mechanism would be reduced by the hyperbaric oxygenation with the consequences of clinical efficacy.
Assuntos
Antineoplásicos/farmacocinética , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar/fisiologia , Carboplatina/administração & dosagem , Carboplatina/farmacocinética , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Oxigenoterapia Hiperbárica , Animais , Antineoplásicos/administração & dosagem , Bovinos , Células Cultivadas , RatosRESUMO
The co-culture of bovine brain capillary endothelial cells and rat primary glial cells was established as an in vitro blood-brain barrier model to investigate the mechanisms by which the Gram-positive bacterial cell wall components lipoteichoic acid and muramyl dipeptide induced injury of blood-brain barrier structure and function. We found that highly purified lipoteichoic acid disrupted blood-brain barrier integrity in a concentration- and time-dependent manner indirectly, through glia activation. Low trans-endothelial electrical resistance and high permeability to fluorescein isothiocyanate-inulin observed in the presence of lipoteichoic acid-activated glial cells were potentiated by muramyl dipeptide and could be reversed only when glial cells were activated by lipoteichoic acid at 10 microg/ml but not with a higher lipoteichoic acid concentration (30 microg/ml). Immunocytochemistry analysis revealed no evident changes in the distribution of the cytoskeleton protein F-actin and tight junction proteins occludin and claudin after lipoteichoic acid treatment. However, the tight junction associated protein AHNAK clearly revealed the morphological alteration of the endothelial cells induced by lipoteichoic acid. Lipoteichoic acid-activated glial cells produced nitric oxide and pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-1beta) that contributed to lipoteichoic acid-induced blood-brain barrier disruption, since the direct treatment of the endothelial monolayer with tumor necrosis factor-alpha or interleukin-1beta increased blood-brain barrier permeability, whereas the pre-treatment of lipoteichoic acid-activated glial cells with antibodies against these two cytokines blocked lipoteichoic acid effects. Additionally, nitric oxide was also involved in blood-brain barrier damage, since the nitric oxide donor itself (diethylenetriamine-nitric oxide adduct) increased blood-brain barrier permeability and inducible nitric oxide synthase inhibitor (1400W) partially reversed lipoteichoic acid-induced trans-endothelial electrical resistance decrease.
Assuntos
Barreira Hematoencefálica/fisiologia , Córtex Cerebral/irrigação sanguínea , Citocinas/fisiologia , Endotélio Vascular/fisiologia , Lipopolissacarídeos/farmacologia , Neuroglia/fisiologia , Óxido Nítrico/fisiologia , Ácidos Teicoicos/farmacologia , Actinas/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Capilares , Bovinos , Permeabilidade da Membrana Celular , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Endotélio Vascular/efeitos dos fármacos , Bactérias Gram-Positivas/química , Lipopolissacarídeos/isolamento & purificação , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Ácidos Teicoicos/isolamento & purificação , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/fisiologiaRESUMO
The aim of the present study was to identify a model for the blood-brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood-brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brain(conc.)/Plasma(conc.)) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood-brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r2 values were seen in the bovine brain endothelial cells (r2=0.43) and MDCKwt (r2=0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells (r2=0.74), MDCKwt (r2=0.65) and Caco-2 (r2=0.86). By plotting in vivo Papp values against logDpH7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood-brain barrier by passive diffusion, (2) compounds crossing the blood-brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood-brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. Papp and Pe values obtained using the different in vitro models were also plotted against logDpH7.4 and compared to the plot obtained when in vivo Papp values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between Pe or Papp for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood-brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of "batteries" of in vitro tests are likely to be necessary in order to improve in vitro-in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.
Assuntos
Barreira Hematoencefálica/citologia , Células Cultivadas/metabolismo , Endotélio Vascular/citologia , Modelos Biológicos , Xenobióticos/farmacocinética , Animais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Bovinos , Cães , Endotélio Vascular/metabolismo , Humanos , Camundongos , Permeabilidade , Ratos , Reprodutibilidade dos TestesRESUMO
Association between doxorubicin (DOX) and gamma-cyclodextrin (gamma-CD) or hydroxypropyl-gamma-CD (HP-gamma-CD) has been examined to increase the delivery of this antitumoral agent to the brain. The stoichiometry and the stability constant of gamma-CD or HP-gamma-CD and DOX complexes were determined in physiological medium by UV-visible spectroscopy. By using an in vitro model of the blood-brain barrier (BBB), endothelial permeability and toxicity toward the brain capillary endothelial cells of DOX, gamma-CD, and HP-gamma-CD were performed. For each CD, endothelial permeability was relatively low and a disruption of the BBB occurred at 20 microM, 20 mM, and 50 mM DOX, gamma-CD, and HP-gamma-CD, respectively. Increasing amounts of CDs were added to a fixed DOX concentration. Addition of gamma-CD or HP-gamma-CD, up to 15 and 35 mM, respectively, decreased the DOX delivery, probably due to the low complex penetration across the BBB and the decrease in free DOX concentration. Higher CD concentrations increased the DOX delivery to the brain, but this effect is due to a loss of BBB integrity. In contrast to what was observed on Caco-2 cell model with various drugs, CDs are not able to increase the delivery of DOX across our in vitro model of BBB.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Doxorrubicina/metabolismo , gama-Ciclodextrinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Capilares/citologia , Capilares/efeitos dos fármacos , Capilares/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Técnicas de Cocultura , Condutividade Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Imunofluorescência , Microscopia de Fluorescência , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Piperidinas/farmacologia , Ratos , Espectrofotometria Ultravioleta , Triazinas/farmacologiaRESUMO
PURPOSE: The objective of the current study was to investigate whether blood-brain barrier (BBB) permeability studies in vitro could be accelerated by running several compounds together in the same experiment. METHODS: To address this question, we compared the transport of six compounds run separately with the results of the same compounds run together (cocktails). RESULTS: The study clearly demonstrated that the outcome of the experiments were totally different depending on the strategy used. Furthermore, the study highlights the importance of having the resistance to drug transport offered by filters without cells under control, as the filter membrane itself can be the rate-limiting step for some compounds; in addition, there is always a potential risk of interactions between molecules in cocktails as well as drug-drug interaction at the level of BBB transporters. In this study, the presence of several P-glycoprotein substrates in the drug cocktail was found to cause breakdown of the BBB. CONCLUSIONS: The results demonstrate that unless a strategy that involves running several compounds in the same experiment is properly validated, the results are of little predictive value.
Assuntos
Barreira Hematoencefálica/fisiologia , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos/métodos , Preparações Farmacêuticas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico Ativo , Células Cultivadas , Desenho de Fármacos , Endotélio Vascular/metabolismo , Microscopia de Fluorescência , Neuroglia/metabolismo , Ratos , SacaroseRESUMO
Cyclodextrins (CDs) can be envisaged to cure some diseases related to the brain, but the behavior of these compounds toward the blood-brain barrier (BBB) remains largely unexplored to envisage such clinical applications. To fulfill this gap, the toxicity and endothelial permeability for native, methylated, and hydroxypropylated alpha-, beta-, and gamma-CDs have been studied on an in vitro model of BBB. As shown by the endothelial permeability for sucrose and immunofluorescence stainings, the native CDs are the most toxic CDs (alpha- > beta- > gamma-CD). Whereas the chemical modification of beta-CD did not affect the toxicity of this CD, differences are observed for the alpha- and gamma-CD. To determine the origin of toxicity, lipid effluxes on the brain capillary endothelial cells were performed in the presence of native CDs. It was found that alpha-CD removed phospholipids and that beta-CD extracted phospholipids and cholesterol. gamma-CD was less lipid-selective than the other CDs. Finally, the endothelial permeability of each CD has been determined. Surprisingly, no structure/permeability relationship has been observed according to the nature and chemical modifications of CDs.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , alfa-Ciclodextrinas/farmacologia , beta-Ciclodextrinas/farmacologia , gama-Ciclodextrinas/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Permeabilidade/efeitos dos fármacos , RatosRESUMO
Tianeptine is an antidepressant with proven clinical efficacy and effects on hippocampal plasticity. Hypoxia increased lactate dehydrogenase (LDH) release from cortical neuronal cultures, and tianeptine (1, 10 and 100 microM) inhibited LDH release as efficiently as the N-methyl-D-aspartate (NMDA) antagonist, MK-801. However, tianeptine did not block apoptosis in cultured cortical neurones caused by NMDA, but reduced apoptosis when interleukin-1beta (IL-1beta) was included with NMDA. In 5-day old mice, intracerebral injection of ibotenate induced reproducible lesions in cortex and white matter. Lesion size was markedly reduced by co-administration of MK-801 (1 mg/kg i.p.) but neither by tianeptine or its enantiomers administered acutely (1, 3 or 10 mg/kg i.p.) nor by tianeptine administered chronically (10 mg/kg i.p. for 5 days). Chronic administration of IL-1beta (10 ng/kg i.p. for 5 days) prior to ibotenate injection exacerbated lesion size in cortex and white matter, and this exacerbation was prevented by chronic pre-treatment with tianeptine (10 mg/kg i.p.) or by acute administration of tianeptine (10 mg/kg i.p.) concomitantly with ibotenate. Thus tianeptine has neuroprotective effects against hypoxia in tissue culture and against the deleterious effects of cytokines in cortex and white matter, but not against NMDA receptor-mediated excitotoxicity.
Assuntos
Antidepressivos Tricíclicos/farmacologia , Citocinas/metabolismo , Fármacos Neuroprotetores/farmacologia , Tiazepinas/farmacologia , Animais , Animais Recém-Nascidos , Antidepressivos Tricíclicos/química , Encéfalo/metabolismo , Encéfalo/patologia , Hipóxia Celular , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios , Hipóxia/metabolismo , Hipóxia/patologia , Ácido Ibotênico , Interleucina-1/farmacologia , L-Lactato Desidrogenase/metabolismo , Camundongos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Estereoisomerismo , Tiazepinas/químicaRESUMO
Antioxidant 8-alkylamino-1,4-benzoxazines, (R,S)-(3-tert-butyl-8-phenylethylamino-3,4-dihydro-2H-1,4-benzoxazin-5-yl) (phenyl) methanone (S 24429) and (R,S)-(3-cyclopentyl-8-benzylamino-3,4-dihydro-2H-1,4-benzoxazin-5-yl) (phenyl) methanone (S 24718), were prepared according to a two-step one-pot electrochemical procedure. These compounds had been selected from a previous study of structure/activity. Both compounds (1-100 microM) prevented the fall in ATP levels caused by 24 h of hypoxia in astrocytes. Both compounds (1 and 10 mg/kg i.p.) were powerful neuroprotective agents in protecting against the lesions induced by 15 microg S-bromo-willardiine injected into the cortex or white matter of 5-day old mice pups. In contrast, exifone, an antioxidant compound, was inactive at these doses. S 24429 and S 24718 appear to be novel neuroprotective agents, which are effective in a model of brain damage mimicking the lesions underlying cerebral palsy.
Assuntos
Alanina/análogos & derivados , Antioxidantes/farmacologia , Fármacos Neuroprotetores/farmacologia , Oxazinas/farmacologia , Trifosfato de Adenosina/metabolismo , Alanina/administração & dosagem , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Benzofenonas/farmacologia , Benzoxazinas , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Relação Dose-Resposta a Droga , Camundongos , Oxigênio/farmacologia , RatosRESUMO
Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation, which accumulates in the brain during neurodegenerative disorders. Prior to determining Lf function in pathological brain tissues, we investigated its transport through the blood-brain barrier (BBB) in inflammatory conditions. For this purpose, we used a reconstituted BBB model consisting of the coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes in the presence of tumor necrosis factor-alpha (TNF-alpha). As TNF-alpha can be either synthesized by brain glial cells or present in circulating blood, BBCECs were exposed to this cytokine at their luminal or abluminal side. We have been able to demonstrate that in the presence of TNF-alpha, whatever the type of exposure, BBCECs were activated and Lf transport through the activated BBCECs was markedly increased. Lf was recovered intact at the abluminal side of the cells, suggesting that increased Lf accumulation may occur in immune-mediated pathophysiology. This process was transient as 20 h later, cells were in a resting state and Lf transendothelial traffic was back to normal. The enhancement of Lf transcytosis seems not to involve the up-regulation of the Lf receptor but rather an increase in the rate of transendothelial transport.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Lactoferrina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Transporte Biológico/efeitos dos fármacos , Bovinos , Técnicas de Cocultura , Selectina E/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Inflamação , Molécula 1 de Adesão Intercelular/metabolismo , Ratos , Estimulação QuímicaRESUMO
A cell culture model of the blood-brain barrier (BBB) consisting of a coculture of bovine brain capillary endothelial cells and rat astrocytes has been used to examine the ability of 60-nm nanoparticles with different physicochemical characteristics to cross the BBB. Neutral, anionic, and cationic nanoparticles were made from crosslinked malto-dextrins derivatized or not (neutral) with phosphates (anionic), quaternary ammoniums (cationic) ligands. Then, these particles were coated or not with a lipid bilayer made of dipalmitoyl phosphatidyl choline and cholesterol. Lipid coating of ionically charged nanoparticles was able to increase BBB crossing 3- or 4-fold compared with uncoated particles, whereas coating of neutral particles did not significantly alter their permeation characteristics across the endothelial cell monolayer. Lipid-coated nanoparticles were nontoxic toward BBB integrity, and crossed the BBB by transcytosis without any degradation. Furthermore, a 27-fold increase in albumin transport was observed when albumin had previously been loaded in the cationic lipid-coated nanoparticles. The influence of red blood cells was studied; a marked inhibition of the transport was observed, probably due to strong interaction between nanoparticles and red blood cells.
Assuntos
Barreira Hematoencefálica/fisiologia , Microesferas , Animais , Astrócitos/metabolismo , Bovinos , Células Cultivadas , Técnicas de Cocultura , Endotélio/citologia , Endotélio/metabolismo , Lipossomos , Microscopia de Fluorescência , Fosfolipídeos/metabolismo , Polissacarídeos , RatosRESUMO
Tetanus neurotoxin reaches the CNS by axonal retrograde transport and thus becomes inaccessible to current treatments. A possible strategy to improve current therapy for tetanus disease would be the vectorization of Fab'2 fragments, allowing their delivery into the CNS. The purpose of this study was to investigate whether after cationization anti-tetanus Fab'2 fragments are able to cross the blood-brain barrier, the first obstacle to CNS delivery. We used primary cocultures of bovine brain capillary endothelial cells and newborn rat astrocytes as an in vitro model to study the binding and transport of cationized Fab'2 (cFab'2) fragments across the brain endothelium. We first show that cationization does not alter Fab'2 affinity for tetanus toxin. Then we demonstrate that after cationization Fab'2 fragments are able to bind to the negative charges on the surface of endothelial cells and subsequently to be transported across the endothelial cell monolayer without any modification of affinity. Finally, using fluorescence microscopy, we show that cFab'2 fragments are transported through endocytotic vesicles. The present study demonstrates that cationization allows Fab'2 directed against tetanus toxin to be transported through brain endothelium by adsorptive-mediated transcytosis. We suggest that this vectorization way could be a promising delivery strategy for carrying anti-tetanic immunoglobulin fragments across the blood-brain barrier to improve tetanus treatment.
Assuntos
Barreira Hematoencefálica/fisiologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Biológicos , Tétano/imunologia , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Transporte Biológico , Encéfalo/irrigação sanguínea , Cátions , Bovinos , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/metabolismo , Radioisótopos do Iodo , Microscopia de Fluorescência , RatosRESUMO
Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein mainly located in intracellular vesicles and secreted in biological fluids. In previous works, we demonstrated that CyPB interacts with T lymphocytes and enhances in vitro cellular incorporation and activity of CsA. In addition to its immunosuppressive activity, CsA is able to promote regeneration of damaged peripheral nerves. However, the crossing of the drug from plasma to neural tissue is restricted by the relative impermeability of the blood-brain barrier. To know whether CyPB might also participate in the delivery of CsA into the brain, we have analyzed the interactions of CyPB with brain capillary endothelial cells. First, we demonstrated that CyPB binds to two types of binding sites present at the surface of capillary endothelial cells from various species of tissues. The first type of binding sites (K(D) = 300 nM; number of sites = 3 x 10(6)) is related to interactions with negatively charged compounds such as proteoglycans. The second type of binding sites, approximately 50,000 per cell, exhibits a higher affinity for CyPB (K(D) = 15 nM) and is involved in an endocytosis process, indicating it might correspond to a functional receptor. Finally, the use of an in vitro model of blood-brain barrier allowed us to demonstrate that CyPB is transcytosed by a receptor-mediated pathway (flux = 16.5 fmol/cm2/h). In these conditions, CyPB did not significantly modify the passage of CsA, indicating that it is unlikely to provide a pathway for CsA brain delivery.
Assuntos
Barreira Hematoencefálica/fisiologia , Ciclofilinas , Endotélio Vascular/metabolismo , Imunofilinas/metabolismo , Receptores de Peptídeos/fisiologia , Animais , Apêndice/irrigação sanguínea , Astrócitos/fisiologia , Transporte Biológico , Encéfalo/irrigação sanguínea , Capilares , Bovinos , Linhagem Celular , Técnicas de Cocultura , Ciclosporina/metabolismo , Humanos , Radioisótopos do Iodo , Cinética , Peptidilprolil Isomerase , Veias UmbilicaisRESUMO
Since endothelial cells (ECs) play a key role in immune defense mechanisms and in immunopathology, we investigated whether the intravascular helminth parasite Schistosoma mansoni could interact with and activate resting ECs in vitro. Microscopic analysis revealed that the lung-stage schistosomula specifically attached to microvascular ECs. This adherence was associated to active cellular processes involving actin filament formation. Since variation of permeability of cultured capillary brain ECs is a good marker for endothelial activation, the transendothelial passage of a low-molecular-weight molecule (inulin) on monolayers of bovine brain capillary ECs (BBCEC) was measured in response to parasites. Schistosomula induced a dramatic decrease in transendothelial permeability, a characteristic marker for the generation of an anti-inflammatory phenotype to ECs. This paracellular barrier enhancing effect on endothelial monolayers was due to a soluble substance(s) (below 1 kDa in size) secreted from S. mansoni schistosomula and not by mechanisms associated to adherence between parasites and ECs. The reinforcement of the endothelial barrier function was accompanied by an elevation of intracellular concentration of cyclic AMP (cAMP). The use of specific kinase inhibitors confirms that schistosomula activate ECs through a cAMP/protein kinase A pathway that leads to an increased phosphorylation of the myosin light-chain kinase. These combined findings suggest that the secretory/excretory products from schistosomula possess anti-inflammatory factor(s) that signal host microvascular endothelium. The immunological consequences of such activation are discussed.
Assuntos
Endotélio Vascular/fisiopatologia , Endotélio Vascular/parasitologia , Schistosoma mansoni/parasitologia , Esquistossomose mansoni/parasitologia , Animais , Permeabilidade Capilar , Bovinos , Comunicação Celular , Células Cultivadas , Inflamação , Schistosoma mansoni/fisiologia , Esquistossomose mansoni/fisiopatologia , Transdução de SinaisRESUMO
Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (Kd = 37.5 nM; n = 90,000/cell) and low (Kd = 2 microM; n = 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport.
Assuntos
Endocitose , Lactoferrina/metabolismo , Animais , Membrana Basal/metabolismo , Barreira Hematoencefálica , Bovinos , Linhagem Celular , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Ligação ProteicaRESUMO
The passage of either unesterified docosahexaenoic acid (DHA) or lysophosphatidylcholine-containing DHA (lysoPC-DHA) through an in vitro model of the blood-brain barrier was investigated. The model was constituted by a brain capillary endothelial cell monolayer set over the medium of an astrocyte culture. Cells were incubated for 4 h with a medium devoid of serum, then the endothelial cell medium was replaced by the same medium containing labeled DHA or lysoPC-DHA and incubations were performed for 2 h. DHA uptake by cells and its transfer to the lower medium (astrocyte medium when they were present) were measured. When the lower medium from preincubation and astrocytes were maintained during incubation, the passage of lysoPC-DHA was higher than that of unesterified DHA. The passage of both forms decreased when astrocytes were removed. The preference for lysoPC-DHA was not seen when the lower medium from preincubation was replaced by fresh medium, and was reversed when albumin was added to the lower medium. A preferential lysoPC-DHA passage also occurred after 2 h with brain endothelial cells cultured without astrocytes but not with aortic endothelial cells cultured and incubated under the same conditions. Altogether, these results suggest that the blood-brain barrier cells released components favoring the DHA transfer and exhibit a preference for lysoPC-DHA.
Assuntos
Barreira Hematoencefálica/fisiologia , Ácidos Docosa-Hexaenoicos/farmacocinética , Lisofosfatidilcolinas/farmacocinética , Animais , Aorta/citologia , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/irrigação sanguínea , Capilares/citologia , Permeabilidade Capilar/fisiologia , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Bicamadas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Ratos , TrítioRESUMO
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.