| The blood brain barrier(BBB) is formed by the endothelium of brain microvessels endothelial cells(BMECs),under the inductive influence of associated cells especially astrocytes.It is the most important barrier involved in the regulation of molecules accessing the brain.Features that distinguish the brain endothelium from that of other organs include complex tight junctions,a low density of pinocytotic vesicles,and the expression of a number of specific uptake and effiux transport systems and metabolic enzymes.It is these properties that make BBB a regulatory interface that selectively limits drug delivery to the central nervous system(CNS).A relatively simple,widely applicable and robust method in vitro for predicting BBB permeability to CNS-acting drugs is an increasing need.The Madin-Darby canine kidney(MDCK) cell line is increasingly used as a substitute for more labor-intensive in vitro BBB models in drug passive permeability and membrane transport studies. MDCK-MDR1 cell line is generated from MDCK cell line.MDCK cell line was stably transfected with the human MDR1 gene leading to the polarized overexpression of P-glycoprotein(P-gp).The cells stably overexpress the human MDR1 gene and, thus,represent a specific model to study P-gp-mediated transport,since,in comparison,the expression of endogenous transporter in(wild-type) MDCK cells is negligible.The MDCK-MDR1 cell model may serve as a convenient tool to help guide drug discovery in terms of CNS penetration of new chemical entity(NCEs). The data suggest that the MDCK-MDR1 cell line could be used as a quick BBB model to aid drug discovery.It might be possible to use the MDCK-MDR1 cell line, which was identified as the most promising cell line,for qualitative predictions of brain distribution,and to distinguish between compounds that pass the BBB by passive diffusion and those that are substrates for active effiux by P-gp.Part 1 Establishment of high P-glycoprotein expression MDCK cell lineThis study established MDCK cell line which will stably express human P-gp and determined that it was an effective model to study drug transportation.The plasmid pcDNA3.1(+)/MDR1 was transfected into MDCK cell line using LipofectamineTM 2000 transfection reagent.Several stable transfected clones were obtained after selection with G418.Rhodamine 123(Rho123),a fluorescence probes substrate,was used to study the profiles of effiux and uptake to test P-gp activities,which was performed with MDCK and MDCK-MDR1 cells in an one-chamber system in the presence or absence of verapamil as inhibitor.The expression of P-gp mRNA and protein was detected by using RT-PCR and Western blot.This study measured the bidirectional transport of Rho123 across MDCK and MDCK-MDR1 monolayers in the absence or presence of four well-known P-gp inhibitors(Verapamil,CsA, Ketoconazole,Quinine).The RT-PCR and Western blot result indicated that there was high homo sapiens P-gp expression in transfected cells(MDCK-MDR1) comparing with controls.The Rho123 efflux ratios of MDCK and MDCK-MDR1 are 5.94 and 15.45,respectively.Several known P-gp inhibitors could significantly reduce the polarized effiux.In conclusion,the MDCK-MDR1 cell line developed is an effective model to study drug transportation in body.Part 2 MDCK-MDR1 cell model as fast permeability screen for the blood brain barrier drug1.BYYTs permeability across an in vitro model of the blood-brain barrierThe adequate distribution of BYYTs to the CNS,a series of novel central-acting cholinesterase inhibitors for the treatment of the symptoms of Alzheimer's disease,is critical for its effective use.But their permeability in terms of CNS penetration across the BBB was unknown.This study was undertaken to evaluate the in vitro BBB transport of BYYTs using MDCK and MDCK-MDR1 cell line models.Whether BYYTs were substrates or inhibitors of P-gp was determined by examining the bi-directional flux of them in MDCK and MDCK-MDR1 epithelial cell monolayers. Quantitation of BYYTs was performed by HPLC.The apparent permeabilitys(Papp) of BYYTs were determined in the concentration 10,50 and 100μmol/L.The effects of four BYYTs(1-200μmol/L) on the bidirectional transport of 4μmol/L Rho123 were also examined.The transepithelial transport of BYYTs did not differ between the two cell lines,suggesting that BYYTs are not substrates for P-gp and they exhibit bi-directional transport with a Papp(A-B) value in the range of 0.94-2.41×10-5cm/sec. However,BYYTs were found to have an inhibitory effect on the P-gp-mediated transport of Rho123 with an IC50 value of 40μ.mol/L-54μmol/L.Thereby it is unlikely that multidrug resistance(MDR) function affects BYYTs absorption and brain distribution and they have high brain uptake potential.The pharmacophoric moieties of BYYTs interact with the binding site of P-gp probably is 5,6-dimethoxy-inden-l-one.2.Gastrodin and its aglycone permeability across an in vitro model of the blood-brain barrierThe objectives of this study were to evaluate the ability of gastrodin(GAS) and gastrodigenin(HBA) to enter the CNS and whether it was P-gp-mediated effiux in BBB.We determined the MDCK and MDCK-MDR1 cell permeation characteristics of two drugs.This study examined GAS and HBA permeability across the BBB,using in vitro models.The cellular permeation characteristics of GAS and HBA in the concentration 10,50 and 100μmol/L were measured by HPLC using MDCK and MDCK-MDR1 cells.The effects of two inhibitors of P-gp(verapamil and CsA) on the bidirectional transport of 10.0μmol/L GASμmol/L were also examined. Intracellular GAS and HBA accumulation was investigated using the two cell lines. The transepithelial transport of HBA did not differ between the two cell lines,and it exhibited bi-directional transport with a Papp(A-B) value in the range of 2.06-2.83×10-5 cm/sec.GAS displayed polarized transport,with the net effiux ratio of 1.92,1.53 and 1.39.It showed absorptive permeability coefficients values rang from 1.63-1.83×10-7 cm/sec.Uptake of GAS in MDCK-MDR1 was 5 times lower than in wild-type MDCK cells.The effiux of the GAS could be reversed by verapamil and CsA(two inhibitors for P-gp).These in vitro results suggested that HBA was not a substrate for P-gp and it had fairly high BBB uptake potential.However,it presets direct evidence that GAS is effiuxed by P-gp and have lower permeability which limit penetration into the CNS and contribute to its extremely poor brain uptake.3.Rochs permeability across an in vitro model of the blood-brain barrierTo evaluate whether the five kinds of Roch compounds were P-gp-mediated effiux in BBB,we examined their MDCK and MDCK-MDR1 cell permeation characteristics. Transport studies were performed in MDCK and MDCK-MDR1 cell monolayers 5 days after seeding.The cellular permeation characteristics of Rochs were measured by HPLC using MDCK cells and MDCK-MDRlcells grown onto microporous membranes.The apparent permeabilitys(Papp) of Rochs were determined in the concentration of 10,50 and 100μmol/L.The effects of two inhibitors of P-gp (verapamil and CsA) on the bidirectional transport of 1.0μmol/L Roch-2 and 10.0μmol/L Roch-5 were also examined.The transepithelial transport of Roch-1, Roch-3 and Roch-4 did not differ between the two cell lines,and they exhibited bi-directional transport with a Papp(A-B) value in the range of 0.37-2.63×10-5 cm/sec. Roch-2 and Roch-5 displayed polarized transport,with the Papp(B-A) being 2.47-to 5.18-fold and 1.29-to 4.01-fold higher than the Papp(A-B) on MDCK-MDR1 cells.And their net effiux ratios are higher than 2.Roch-2 showed absorptive permeability coefficients values ranging from 0.55-1.06×10-5 cm/sec,while Roch-5 was 0.89-1.33×10-6 cm/sec.The effiux of the Roch-2 and Roch-5 could be inhibited by verapamil and CsA(two inhibitors for P-gp).This study clearly indicated that Roch-1, Roch-3 and Roch-4 had high brain uptake potential.However the fact that Roch-2 and Roch-5 are two good substrates for P-gp suggests that their permeation across BBB would be significantly restricted.Roch-2 is predicted to have moderately permeation and Roch-5 has poor brain uptake.
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