RESUMO
Deposition of amyloid-ß peptide (Aß(1-42)) is a hallmark of Alzheimer's disease. Clearance of Aß(1-42), across the blood-brain barrier (BBB), is mediated by ATP-binding Cassette (ABC) efflux transporters. Many therapeutic drugs inhibit ABC transporters, but little is known of the effect of therapeutic drugs on Aß(1-42) transport across BBB endothelial cells. The effects of selected, widely prescribed, therapeutic drugs on ABCB1, ABCC5 and ABCG2 activities were determined by measuring intracellular levels of calcein, GS-MF, and Hoechst 33342 respectively in primary porcine brain endothelial cells (PBECs). The ability of ABCB1, ABCC5 and ABCG2 to transport Aß(1-42) was determined using fluorescent Aß(1-42). The ability of the ABCB1, ABCC5 and ABCG2 inhibitor telmisartan to modify transcellular Aß(1-42) transport was investigated using PBEC monolayers housed in Transwell® inserts. Treatment of PBECs with ABC transporter inhibitory drugs (indomethacin, olanzapine, chlorpromazine, telmisartan, pantoprazole, quinidine, sulfasalazine and nefazodone) increased Aß(1-42) intracellular accumulation. Inhibition of ABCB1, ABCC5 and ABCG2 by telmisartan increased Aß(1-42) transport in the apical to basal direction and reduced its transport in basal to apical direction in PBEC monolayers. ABCB1, ABCC5 and ABCG2 mediate the efflux transport of Aß(1-42) in BBB endothelial cells. Inhibition of ABC transporters by therapeutic drugs, at plasma concentrations, could decrease Aß(1-42) clearance from brain, across BBB endothelial cells into blood, and potentially influence levels of the Aß(1-42) peptide within the brain.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Peptídeos beta-Amiloides/metabolismo , Células Endoteliais/metabolismo , Fragmentos de Peptídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Sobrevivência Celular , Células Cultivadas , Feminino , Masculino , Microvasos/citologia , Ratos , SuínosRESUMO
BACKGROUND: Deposition of amyloid-ß peptide (Aß(1-42)) within the brain is characteristic of Alzheimer's disease. Little is known of the effects of Aß(1-42) on blood-brain barrier (BBB) ATP-binding Cassette (ABC) efflux transporters which influence BBB permeability. The effects of Aß(1-42) on ABCB1, ABCC5 and ABCG2 activity and expression and pregnane X receptor (PXR) and constitutive androstane receptor (CAR) transcription factors expression were determined in primary porcine brain endothelial cells (PBECs). METHODS: The effect of Aß(1-42) on transporter activity was determined by measurement of intracellular accumulation of the fluorescent probes calcein (ABCB1), GS-MF (ABCC5) and Hoechst 33342 (ABCG2). Expression of transporters and transcription factors was assessed by Western blotting. RESULTS: Treatment of PBECs with Aß(1-42) significantly decreased activity of ABCB1 (Aß(1-42) at 10⯵g/ml, 25⯵g/ml and 50⯵g/ml), ABCC5 (Aß(1-42) at 25⯵g/ml and 50⯵g/ml) and ABCG2 (Aß(1-42) at 10⯵g/ml, 25⯵g/ml and 50⯵g/ml). Aß(1-42) also significantly decreased expression of ABCB1 (pâ¯<â¯0.05 at 25⯵g/ml and 50⯵g/ml), ABCG2 (pâ¯<â¯0.05 at 25⯵g/ml and pâ¯≤â¯0.001 at 50⯵g/ml), ABCC5 (pâ¯<â¯0.05 at 25⯵g/ml and 50⯵g/ml), PXR (pâ¯<â¯0.05 at 10⯵g/ml, 25⯵g/ml and 50⯵g/ml Aß(1-42)) and CAR (pâ¯<â¯0.05 at 25⯵g/ml and 50⯵g/ml Aß(1-42)). CONCLUSION: Aß(1-42) inhibits multiple ABC transporters and PXR and CAR in PBECs. GENERAL SIGNIFICANCE: Aß(1-42) reduces ABC transporter activity and expression in BBB endothelial cells and has the potential to influence BBB permeability characteristics.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Peptídeos beta-Amiloides/farmacologia , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , SuínosRESUMO
The first Workshop on Drug Delivery in Paediatric Brain Tumours was hosted in London by the charity Children with Cancer UK. The goals of the workshop were to break down the barriers to treating central nervous system (CNS) tumours in children, leading to new collaborations and further innovations in this under-represented and emotive field. These barriers include the physical delivery challenges presented by the blood-brain barrier, the underpinning reasons for the intractability of CNS cancers, and the practical difficulties of delivering cancer treatment to the brains of children. Novel techniques for overcoming these problems were discussed, new models brought forth, and experiences compared.
RESUMO
Central nervous system (CNS) drug disposition is dictated by a drug's physicochemical properties and its ability to permeate physiological barriers. The blood-brain barrier (BBB), blood-cerebrospinal fluid barrier and centrally located drug transporter proteins influence drug disposition within the central nervous system. Attainment of adequate brain-to-plasma and cerebrospinal fluid-to-plasma partitioning is important in determining the efficacy of centrally acting therapeutics. We have developed a physiologically-based pharmacokinetic model of the rat CNS which incorporates brain interstitial fluid (ISF), choroidal epithelial and total cerebrospinal fluid (CSF) compartments and accurately predicts CNS pharmacokinetics. The model yielded reasonable predictions of unbound brain-to-plasma partition ratio (Kpuu,brain) and CSF:plasma ratio (CSF:Plasmau) using a series of in vitro permeability and unbound fraction parameters. When using in vitro permeability data obtained from L-mdr1a cells to estimate rat in vivo permeability, the model successfully predicted, to within 4-fold, Kpuu,brain and CSF:Plasmau for 81.5% of compounds simulated. The model presented allows for simultaneous simulation and analysis of both brain biophase and CSF to accurately predict CNS pharmacokinetics from preclinical drug parameters routinely available during discovery and development pathways.
RESUMO
Whilst it is well documented that all components of the neurovascular unit contribute to the restrictive nature of the blood-brain barrier (BBB), astrocytes have been identified as the cellular component most likely to play an essential role in maintaining the barrier properties. The aim of this study was to examine the impact of the rat astrocyte cell line, CTX-TNA2, on the structural and functional characteristics of an in vitro BBB and determine the capacity of this astrocyte cell line to maintain the BBB phenotype. Co-culture of the CTX-TNA2 cells with primary porcine brain endothelial cells produced an in vitro BBB model which retains key features of the in vivo BBB. High transendothelial electrical resistances, comparable to those reported in vivo, were obtained. Ultrastructural analysis revealed distinct intercellular tight junction protein complexes and immunocytochemistry confirmed expression of the tight junction proteins ZO-1 and occludin. Western blotting and fluorescent tracer assays confirmed expression and functional activity of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) efflux transporters. Studies employing Alexa-fluor 555-conjugated human transferrin revealed temperature-sensitive internalisation indicating the BBB model retains functional receptor-mediated transferrin uptake. The findings of this study indicate that a robust BBB model has been produced and this is the first report of the inductive capacity of the CTX-TNA2 cell line. Since this in vitro BBB model possesses many key characteristics of the BBB in vivo it has the potential to be a valuable tool for the study of biochemical and physiological processes associated with the BBB.