Influence Of P-glycoprotein On Brucine Transport At The In Vitro Blood-brain Barrier

OBJECTIVETo understand whether brucine is the substrate of P-glycoprotein (P-gp), and furthermore the influence of P-gp on brucine transport at the in vitro blood-brain barrier (BBB).METHODS1 Establishment and evaluation of the in vitro blood-brain barrierAn in vitro BBB model was established, comprising a co-culture of brain microvessel endothelial cell (BMEC) and astrocyte (As) from the same genus. The BMEC was characterized on the basis of cell morphology, the functional expression of factorⅧand P-gp. The As was characterized on the basis of cell morphology and the functional expression of glial fibrillary acidic protein (GFAP). The BBB was evaluated on the basis of cell morphology, electron microscope observation, leakage experiment, transendothelial electrical resistance (TEER) mensuration, and the permeability of fluorescein sodium and 125Ⅰ-albumin(125Ⅰ-ALB).2 In vitro P-gp affinity for brucineThe in vitro P-gp affinity for brucine can be investigated employing the P-gp ATPase analytical method. The activity of ATPase was catalyzed by P-gp, which was induced by its substrate. The P-gp membrane was co-incubated with brucine of a series concentrations of 0.01-1000μmol·L-1. ATPase activity can be quantified by measuring concentrations of inorganic phosphate (Pi) that results from ATP hydrolysis to adenosine diphosphate (ADP) during the energy-dependant P-gp drug transport process. Michaelis-Menten equation was used in order to obtain the Vmax, Km'Vmax/Km.3 Influence of P-gp on brucine transport at the in vitro blood-brain barrierMTT (diphenyltetrazolium bromide) assay was used to find the non-cytotoxicity dosage of brucine (0.05-500μmol·L-1) and verapamile (0.1-100μmol·L-1) in BMEC and As cells. The dosage at which the survival rate of the cell is above 90% was considered as the highest non-cytotoxic dosage.UPLC-MS/MS method for determination of brucine was developed and validated.The effects of duration, drug concentration, P-gp inhibitor (verapamile) on brucine transport were detected using in vitro BBB model. Rhodamine 123 (Rh 123) was used as the positive control. The amount of the bi-direction transportation of brucine was analysed with UPLC-MS/MS, and the apparent permeability coefficient (Papp) and excretion ratio (ER) were obtained.RESULTS1 Establishment and evaluation of the in vitro blood-brain barrierThe BMEC displayed characteristic endothelial cell morphology and the expression of factorⅧand P-gp. The As displayed characteristic astrocyte morphology and expressed GFAP. The morphologies of the co-cultured BMEC and As were similar with morphologies when cultured alone. Co-culture with astrocytes increased TEER ((283.78±18.85)Ω·cm2) and decreased paracellular transport, for Papp of fluorescein sodium and 125Ⅰ-ALB was (10.36±0.86)×10-6 cm·s-1 and (6.00±0.78)×10-6 cm·s-1, respectively. Besides, expression of the tight junction was demonstrated in the endothelial cells of the BBB. All the results showed that the in vitro BBB model was established.2 In vitro P-gp affinity for brucineBrucine (Vmax/Km:1.6) stimulates P-glycoprotein ATPase activity to some degree in a time and dose dependent fashion, and have a lower affinity for P-gp than verapamile (Vmax/Km:2.9).3 Influence of P-gp on brucine transport at the in vitro blood-brain barrierThe 24h MTT experiment showed that the highest non-cytotoxic dosage of brucine for BMEC and As was 500μmol·L-1 and 50μmol·L-1, respectively. Verapamile at concentrations of 0.01-100μmol·L-1 was found to be non-cytotoxic towards the two cells. The concentrations of 0.05,5,50μmol·L-1 for brucine and 50μmol·L-1 for verapamile were chosed for the transport experiment.The results of transport experiment showed that: In the absence of P-glycoprotein inhibitor, verapamile, the degree of the brucine transport from basal-to-apical was higher than the one from apical-to-basal at three concentrations (P<0.05), and the ER were 1.32,1.56 and 1.54, respectively. However, when verapamile was added, the ER of three concentrations were decreased (P<0.05). P-glycoprotein may influence the transportation of brucine to some degree.CONCLUSION1. An in vitro BBB model was established, comprising a co-culture of BMEC and As from the same genus. It retains the characteristics of BBB in vivo, including cell morphology, ultrastructure and restrictive permeability.2. Brucine has a moderate affinity to P-gp. P-gp may influence the transportation of brucine to some degree. Further research should be done at various levels for the identification of brucine as the substrate of P-gp.