四氢帕马丁在MDCK-MDR1细胞系中的跨膜转运机制

目的:研究四氢帕马丁(tetrahydropalmatine,THP)在MDCK-MDR1单层细胞模型中的跨膜转运机制。方法:利用MDCK-MDR1单层细胞模型研究在有或无P-糖蛋白(P-gp)专属抑制剂维拉帕米时,THP双向转运特性,同时考察了温度、浓度对THP吸收的影响。采用HPLC法测定THP的含量,计算其表观渗透系数(Papp)。结果:当加入100μg/ml维拉帕米时,细胞单层顶端(apical,A)→基底端(basolateral,B)的Papp(A→B)增加,而B→A的Papp(B→A)显著降低。不加维拉帕米时外排率值为5.5,添加维拉帕米时外排率值为3.9。结论:THP在MDCK-MDR1单层细胞模型中的转运受到P-gp的外排作用,THP可能是P-gp的作用底物。THP跨MDCK-MDR1单层细胞转运具有温度依赖性和浓度饱和性。     

麦冬多糖MDG-1在Caco-2细胞模型中转运机制研究

目的研究麦冬多糖抗心肌缺血活性成分MDG-1在Caco-2细胞模型中转运机制。方法以Caco-2细胞作为转运研究模型,分别测定改变转运方向,使用P糖蛋白(P-gp)外排泵专属抑制剂维拉帕米(verapam il),以及改变给药浓度各种条件下,MDG-1的跨细胞转运情况。结果麦冬多糖MDG-1的分泌转运(BL-AP)的表观渗透系数Papp并未数倍于吸收转运(AP-BL),两者相近,同时P-gp抑制剂维拉帕米加入与否对麦冬多糖MDG-1转运没有影响;在考察的系列药物浓度范围内,MDG-1的转运随着药物浓度的增加而呈线性增加。结论麦冬多糖MDG-1在Caco-2细胞模型中的转运机制很可能是以被动扩散为主,并且以未降解的药物形式转运,无P-gp外排泵参与。     

麦冬多糖MDG-1在Caco-2细胞模型中转运机制研究

目的研究麦冬多糖抗心肌缺血活性成分MDG-1在Caco-2细胞模型中转运机制。方法以Caco-2细胞作为转运研究模型,分别测定改变转运方向,使用P糖蛋白(P-gp)外排泵专属抑制剂维拉帕米(verapam il),以及改变给药浓度各种条件下,MDG-1的跨细胞转运情况。结果麦冬多糖MDG-1的分泌转运(BL-AP)的表观渗透系数Papp并未数倍于吸收转运(AP-BL),两者相近,同时P-gp抑制剂维拉帕米加入与否对麦冬多糖MDG-1转运没有影响;在考察的系列药物浓度范围内,MDG-1的转运随着药物浓度的增加而呈线性增加。结论麦冬多糖MDG-1在Caco-2细胞模型中的转运机制很可能是以被动扩散为主,并且以未降解的药物形式转运,无P-gp外排泵参与。     

流式细胞仪筛选P-糖蛋白的底物或抑制剂

目的以Caco-2细胞模型筛选妥舒沙星、斯帕沙星、辛伐他汀是否为P-糖蛋白(P-gp)的底物。方法以Caco-2细胞为模型,以酮康唑、兰索拉唑为对照,用流式细胞仪测定妥舒沙星、斯帕沙星、辛伐他汀对Caco-2细胞表面的P-gp转运罗丹明-123的影响,经筛选得到可能为P-gp底物的药物后,再用药物转运实验,验证其是否为P-gp的底物。结果妥舒沙星和斯帕沙星存在明显抑制P-gp向细胞外转运罗丹明-123的作用;在加入P-gp抑制剂(酮康唑)后,妥舒沙星的PappBL→AP/PappAP→BL由10.03±0.47降至0.93±0.19,斯帕沙星PappBL→AP/PappAP→BL由2.54±0.12降至1.02±0.04。结论流式细胞仪联用罗丹明-123可简单有效地从多种药物中筛选出可能是P-gp底物的药物(如妥舒沙星和斯帕沙星)及转运机制等。     

芳香开窍化合物对MDCK-MDR1单层细胞透过性的影响(英文)

在中医理论体系中,芳香开窍类药物对于调节脑部的药物摄取具有重要的作用。对于此作用的分子机制研究目前仍然有限。MDCK-MDR1细胞是研究药物透过血脑屏障机制的一个理想体外模型,本文用稳定转染的方法建立了MDCK-MDR1细胞系并研究了几种重要的芳香药物对BBB透过性的作用。研究结果表明:我们发现芳香开窍化合物可以在MDCK-MDR1模型上增加FD4的透过性并减少Rho123的外排,说明这些化合物一方面可以打开细胞间的紧密联接,另一方面可以抑制P-gp的作用,从而影响药物吸收。此研究为阐明芳香化合物的作用机制提供了新的见解。     

口服药物吸收的细胞模型研究进展

多种细胞模型如最常用的Caco-2和MDCK细胞,以及新近开发的MDCK-MDR1、M细胞、药物溶出/Caco-2细胞组合、HT-29和TC7细胞等均可用于口服药物吸收的评价,但也存在一定的局限性。本文综述各类细胞模型的特点及其应用等相关研究进展。     

栀子苷与栀子柏皮汤中栀子苷在MDCK细胞跨膜转运模型上的转运研究

目的考察栀子苷与栀子柏皮汤中栀子苷在MDCK细胞跨膜转运模型上的转运特性。方法利用MTT实验筛选出栀子苷和栀子柏皮汤在MDCK细胞上的安全浓度,以MDCK细胞跨膜转运模型研究药物的双向转运,考察时间、药物浓度、P-糖蛋白(P-gp)抑制剂及EDTA对栀子苷转运的影响,然后比较相同浓度的栀子苷和栀子柏皮汤中栀子苷吸收转运的差异,采用HPLC法检测栀子苷的浓度,计算其表观渗透系数(Papp)。结果栀子苷在MDCK细胞模型上的转运量具有一定的时间和浓度依赖性,P-gp抑制剂对其转运没有明显影响,EDTA能够增加其跨膜转运量,各浓度栀子柏皮汤中栀子苷的吸收Papp分别为(8.96±0.35)×10-7cm·s-1,(8.95±0.38)×10-7cm·s-1和(9.16±0.30)×10-7cm·s-1、明显高于栀子苷单体的吸收Papp(5.85±0.44)×10-7cm·s-1、(6.88±0.38)×10-7cm·s-1和(6.31±0.19)×10-7cm·s-1(P<0.05)。结论栀子苷在MDCK细胞模型上表现为被动扩散,且不受P-gp的影响,可能有细胞旁路转运,栀子柏皮汤能促进栀子苷的吸收。     

绿原酸跨细胞转运机制研究

目的利用Caco-2和MDCK细胞单层模型研究绿原酸(chlorogenic acid,CGA)的跨细胞转运过程及其机制。方法①Caco-2和MDCK细胞单层模型建立:Caco-2、MDCK细胞分别按密度1×105、5×104个细胞/cm2接种到Milli-cell-CM culture plate inserts上培养,待细胞单层达到一定致密程度后进行透过实验。②透过实验:用M2e酶标仪测定CGA在不同方向、不同浓度下的跨细胞转运情况并计算累积透过量。结果在两种细胞模型上CGA均有不同程度的双向跨细胞转运(吸收和分泌),P-gp抑制剂维拉帕米能明显减少CGA的分泌。结论CGA跨细胞转运同时存在吸收和分泌的动力学过程。P-gp部分参与CGA的分泌机制。     

千层纸素A在Caco-2细胞模型中的吸收机制研究

目的研究千层纸素A在Caco-2细胞模型中的吸收机制。方法 MTT实验考察千层纸素A在Caco-2细胞中的安全浓度范围,再利用Caco-2细胞单层模型研究千层纸素A的双向转运机制,以转运量及表观渗透系数(Papp)为指标,考察时间、浓度、pH和P-gp抑制药维拉帕米对其吸收的影响。结果千层纸素A在Caco-2细胞模型中的转运与时间和浓度呈正相关;并受pH值影响,P-gp抑制药维拉帕米对其转运无影响,从单层细胞层顶端(AP)到基底端(BL)的转运与基底端到顶端的转运大致相同。结论千层纸素A在Caco-2细胞模型中的吸收是被动转运。     

P-糖蛋白介导药物相互作用的细胞模型研究概况

P-糖蛋白（P-glycoprotein，P-gP）是由多药耐药蛋白1（Multidrug resistance 1，MDR1）基因编码的能量（ATP）依赖性膜蛋白，可发挥外排泵作用，将细胞内的化合物逆浓度梯度转运至胞外。P-gp在正常人体肝、肾、肠道、胎盘、血脑屏障（BBB）、血睾屏障及淋巴细胞系和心脏内小动脉、毛细血管等部位均有表达。P—gp分子上有多个药物结合位点，故其底物范围非常广泛。由于P—gp底物和分布的广泛性，在合用药物时，可能发生竞争性或非竞争性的药物相互作用，影响药动学过程，引起临床疗效的改变或产生毒性。近年来，P—gp介导的药物相互作用是国内、外研究的热点。国内、外已普遍采用组织细胞模型作为P—gP介导的药物相互作用的筛选工具，常用的包括人类结肠癌单层细胞系Caco-2（Thehuman colon adenocarcinoma cell lines）、犬肾上皮细胞系MDCK（The madin darby canine kidney cell lines）、MDR1基因转染的MDR1-MDCK细胞等。本主要就这些细胞模型在研究P-gp介导的药物相互作用中的应用作一综述。     

Functional assessment of multiple P-glycoprotein (P-gp) probe substrates: influence of cell line and modulator concentration on P-gp activity.

Compounds known to modulate P-glycoprotein (P-gp) activity were evaluated in cell monolayers expressing P-gp for their effects on the secretory transport of P-gp substrates paclitaxel, vinblastine, and digoxin. Paclitaxel has been proposed to selectively interact with a binding site on P-gp that is distinct from the vinblastine and digoxin-binding site. Using Madin-Darby canine kidney (MDCK)-multidrug resistance-1 (MDR1), MDCK-wild-type (WT), and Caco-2 cell monolayers, the basal-to-apical (BL-AP) apparent permeability (Papp) of [3H]paclitaxel, [3H]vinblastine, and [3H]digoxin in the presence of various concentrations of a series of structurally diverse P-gp substrates and modulators of P-gp function were determined. MDCK-WT cell monolayers demonstrated active secretory transport of all P-gp substrate probes, although the sensitivity to inhibition by verapamil was lower than that demonstrated in MDCK-MDR1 cell monolayers. When evaluated as competitive inhibitors, several known P-gp substrates had no effect or only a slight modulatory effect on the BL-AP Papp of all probe substrates in MDCK-MDR1 cells. The secretory transport of P-gp substrates in MDCK-WT cells was more sensitive to inhibition by known P-gp modulators compared with MDCK-MDR1 cells. Low concentrations of ketoconazole (1-3 microM) activated the BL-AP Papp of [3H]vinblastine and [3H]digoxin in MDCK-MDR1 cells but not in MDCK-WT or Caco-2 cells. Determination of secretory transport in P-gp expressing cell monolayers, such as MDCK-MDR1 and Caco-2, may be complicated by substrate cooperativity and allosteric binding, which may result in the activation of P-gp. In addition, expression of other efflux transporters in these cell lines introduces additional complexity in distinguishing which transporter is responsible for substrate recognition and transport.     

Rapid identification of P-glycoprotein substrates and inhibitors.

Identifying molecules that interact with P-glycoprotein (P-gp) is important for drug discovery but is also generally reliant on time-consuming in vitro and in vivo studies. As an alternative approach, the current study applied pharmacophore models and database screening to rapidly retrieve molecules that bind as substrates or inhibitors for P-gp from commercial databases and then confirmed their affinity as inhibitors in vitro. Seven molecules (acitretin, cholecalciferol, misoprostol, nafcillin, repaglinide, salmeterol, and telmisartan) with no published details for P-gp affinity, one positive control inhibitor (miconazole), and two negative control molecules (phenelzine and zonisamide) were selected for testing. The MDCK-MDR1 in vitro cell model was used to confirm their inhibitory effect on [3H]digoxin transport, and the ATPase assay was used as an additional in vitro tool to indicate P-gp activation. All seven test drugs were confirmed to have P-gp affinity. Additionally, our experimental results provided plausible explanations for the published pharmacokinetic profiles of the tested drugs and their classification according to the biopharmaceutics and drug disposition classification system. In this study, we showed the successful application of pharmacophore models to accurately predict P-gp binding, which holds promise to anticipate drug-drug interactions from screening drug databases and a priori prediction of novel P-gp inhibitors or substrates.     

Are MDCK Cells Transfected with the Human MDR1 Gene a Good Model of the Human Intestinal Mucosa?

Purpose. To investigate whether Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) are a good model of the human intestinal mucosa. Methods. P-glycoprotein (P-gp) expression in Caco-2 cells was compared with P-gp expression in MDCK wild- type (MDCK-WT) and MDCK-MDR1 cells using Western blotting methods. The polarized efflux activities of P-gp(s) in MDCK-MDR1 cells, MDCK-WT cells, and Caco-2 cells were compared using digoxin as a substrate. Apparent Michaelis-Menten constants (K _M,V _max) for the efflux of vinblastine in these three cell lines were determined. Apparent inhibition constants (K _I) of known substrates/inhibitors of P-gp were determined by measuring their effects on the efflux of digoxin in Caco-2 or MDCK-MDR1 cell monolayers. Results. MDCK-MDR1 cells expressed higher levels of P-gp compared to Caco-2 and MDCK-WT cells, as estimated by Western blots. Two isoforms of P-gp were expressed in Caco-2 and MDCK cells migrating with molecular weights of 150 kDa and 170 kDa. In MDCK-MDR1 cells, the 150 kDa isoforms appeared to be overexpressed. The MDCK-MDR1 cells exhibited higher polarized efflux of [³H]-digoxin than did Caco-2 and MDCK-WT cells. K _M values of vinblastine in Caco-2, MDCK-WT, and MDCK-MDR1 cells were 89.2 ± 26.1, 24.5 ± 1.1, and 252.8 ± 134.7 M, respectively, whereas V _max values were 1.77 ± 0.22, 0.42 ± 0.01, and 2.43 ± 0.86 pmolcm^–2s^–1, respectively. Known P-gp substrates/inhibitors showed, in general, lower K _I values for inhibition of digoxin efflux in Caco-2 cells than in MDCK-MDR1 cells. Conclusions. These data suggest that the MDCK-MDR1 cells overexpress the 150 kDa isoform of P-gp. MDCK-MDR1 cells are a useful model for screening the P-gp substrate activity of drugs and drug candidates. However, the apparent kinetics constants and affinities of substrates determined in the MDCK-MDR1 cell model may be different than the values obtained in Caco-2 cells. These differences in substrate activity could result from differences in the relative expression levels of total P-gp in Caco-2 and MDCK-MDR1 cells and/or differences in the partitioning of substrates into these two cell membrane bilayers.     

Bidirectional transport of rhodamine 123 and Hoechst 33342, fluorescence probes of the binding sites on P-glycoprotein, across MDCK-MDR1 cell monolayers

The bidirectional permeation characteristics of rhodamine 123 and Hoechst 33342, fluorescence probes of the binding sites on P-glycoprotein (P-gp), across monolayers of MDCK cells transfected with the human MDR1 gene (MDCK-MDR1) were investigated. The ratios of the apparent permeability coefficients (P(app)) of rhodamine 123 and Hoechst 33342 flux measured in the basolateral (BL) to apical (AP) direction versus the flux in the AP-to-BL direction (P(app BL-to-AP)/P(app AP-to-BL)) were 115 and 177, respectively. The P-gp inhibitor GF-120918 could significantly reduce the polarized efflux of both rhodamine 123 and Hoechst 33342. Rhodamine 123 appeared to "stimulate" the polarized efflux of Hoechst 33342 across MDCK-MDR1 cell monolayers. In contrast, Hoechst 33342 partially inhibited the polarized efflux of rhodamine 123 across these cell monolayers whereas daunorubicin partially inhibited the polarized efflux of both rhodamine 123 and Hoechst 33342. The uptake characteristics of rhodamine 123 and Hoechst 33342 in MDCK-MDR1 cells were measured in the absence and presence of GF-120918 and known P-gp substrates (Hoechst 33342, rhodamine 123, and daunorubicin). The uptake of rhodamine 123 and Hoechst 33342 in MDCK-MDR1 cells was enhanced more than twofold by inclusion of GF-120918 (2 microM) in the incubation medium. Daunorubicin (160 microM) increased the relative fluorescence unit (RFU) values of cytoplasm-associated rhodamine 123 by up to 30%. However, daunorubicin (40 microM) and rhodamine 123 (5 microM) decreased the RFU values of cell membrane-associated Hoechst 33342 by 70% and 40%, respectively. To further explore what appears to be a "stimulatory" effect of daunorubicin and rhodamine 123 on the uptake of Hoechst 33342 and a stimulatory effect of daunorubicin on Hoechst 33342 transport across cell monolayer, uptake of Hoechst 33342 into liposomes in the presence and absence of GF-120918, daunorubicin, and rhodamine 123 was determined. GF-120918 exhibited no effect on the RFU values of liposome-associated Hoechst 33342. In contrast, rhodamine 123 and daunorubicin decreased the fluorescence of liposome-associated Hoechst 33342 suggesting these molecules were either quenching the fluorescence of this chemical probe or displacing it from the lipid bilayer. In conclusion, these bidirectional transport data indicate that rhodamine 123 and Hoechst 33342 are excellent substrates of P-gp in MDCK-MDR1 cells. The ability of Hoechst 33342 to partially inhibit the polarized efflux of rhodamine 123 is consistent with these substrates binding to the same site on P-gp. In contrast, the ability of rhodamine 123 to apparently "stimulate" the efflux of Hoechst 33342 in both the transport and uptake experiments suggests the substrates might bind to different sites on P-gp. However, experimental results using liposomes suggested that this "stimulation" phenomenon by rhodamine 123 on Hoechst 33342 uptake and efflux might simply be an artifact. Thus, the use of Hoechst 33342 to probe the binding sites on a membrane-bound protein such as P-gp might be problematic.     

A comparison of the effects of p-glycoprotein inhibitors on the blood-brain barrier permeation of cyclic prodrugs of an opioid peptide (DADLE)

The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the blood-brain barrier (BBB) permeation of cyclic prodrugs of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). The BBB permeation characteristics of these prodrugs and DADLE were determined using an in situ perfused rat brain model and in vitro cell culture model (MDCK-MDR1 cells) of the BBB. The activities of P-gp in these models were characterized using a known substrate (quinidine) and known inhibitors [cyclosporine A (CyA), GF-120918, PSC-833] of P-gp. Cyclic peptide prodrugs exhibited very poor permeation in both models. Inclusion of GF-120918, CyA, or PSC-833 in the brain perfusion medium or the cell culture medium significantly increased the permeation of these cyclic prodrugs. The order of potency of these P-gp inhibitors, as measured using the cyclic prodrugs as substrates, was, by in vitro MDCK-MDR1 cells: GF-120918 = CyA >or= PSC-833; and by in situ rat brain perfusion: GF-120918 > CyA = PSC-833. In conclusion, P-gp in the BBB is the major factor restricting the brain permeation of these cyclic prodrugs. MDCK-MDR1 cells can predict the order of potencies of the investigated P-gp inhibitors to enhance the rat BBB permeation of quinidine and the cyclic prodrugs.     

左旋紫草素肠道转运及Caco-2细胞转运特性

目的研究左旋紫草素肠道及Caco-2细胞转运特征及其机制。方法应用翻转肠囊法和Caco-2细胞模型考察时间、浓度对左旋紫草素转运吸收特性的影响,应用P-糖蛋白抑制剂维拉帕米对左旋紫草素转运吸收机制进行研究,采用HPLC法测定左旋紫草素的浓度,计算其表观渗透系数(Papp)。结果在Caco-2细胞模型,随浓度增加和时间延长,左旋紫草素的累积转运量逐渐增加;加用维拉帕米后,使AP侧到BL侧的表观渗透系数Papp(AP→BL)显著增加,而从BL侧到AP侧的表观渗透系数Papp(BL→AP)显著降低。在翻转肠囊模型,100μmol·L-1左旋紫草素中加入维拉帕米后Papp显著增加。结论左旋紫草素的转运存在被动转运和主动转运2种形式,P糖蛋白参与主动转运过程;该药经肠道吸收中等,加入维拉帕米可能促进吸收。     

Evaluation of P-glycoprotein-mediated renal drug interactions in an MDR1-MDCK model

STUDY OBJECTIVE: To evaluate P-glycoprotein (P-gp)-mediated renal drug interactions in an in vitro model of tubular secretion. DESIGN: In vitro experiment. SETTING: University-affiliated pharmacokinetics laboratory. CELL LINES: Madin-Darby canine kidney (MDCK), multidrug-resistant-1 (MDR1)-MDCK, and human colon carcinoma (Caco-2) cells. INTERVENTION: Transepithelial transport (basolateral-to-apical and apical-to-basolateral) of cimetidine was assessed in the absence and presence of various concentrations of the P-gp inhibitors itraconazole and PSC-833 in a renal P-gp cell culture model (MDR1-MDCK). MEASUREMENTS AND MAIN RESULTS: Apparent permeability of cimetidine was characterized, and level of P-gp expression was determined by Western blot analysis, in MDCK (wild type), MDR1-MDCK, and Caco-2 cells (for relative comparison). In the presence of PSC-833, cimetidine's apparent permeability value for basolateral-to-apical transport decreased from 2.96 to 1.15 x 10(-6) cm/second, coupled with a decrease in efflux ratio from 2.36 to 1.80. The effect of itraconazole was concentration dependent, with cimetidine's apparent permeability value for basolateral-to-apical transport decreasing from 3.96 to 1.92 x 10(-6) cm/second (p < 0.05), resulting in a 50% decrease in efflux ratio. Expression of P-gp was negligible in MDCK (wild-type) cells, but high-level expression was confirmed in both MDR1-MDCK and Caco-2 cells. CONCLUSION: P-glycoprotein plays a significant role in the renal tubular secretion of organic cations such as cimetidine, and the high level of P-gp expression in MDR1-MDCK cells makes this a well-suited model for evaluating mechanisms of renal drug interactions.