8-异丙胺亚甲基橙皮素(IPHP)在Caco-2细胞模型上跨膜转运的研究

目的利用Caco-2细胞模型研究8-异丙胺亚甲基橙皮素(IPHP)在小肠吸收转运的机制。方法在Caco-2细胞模型上进行IPHP的跨膜转运实验,探讨药物浓度、p H、温度、P-gp抑制剂维拉帕米、MRP2抑制剂MK-571和丙磺舒对IPHP在体外细胞模型上跨膜转运的影响。结果 IPHP在Caco-2细胞模型上的转运具有一定的浓度依赖性,IPHP不同浓度从A侧到B侧的渗透系数Papp(AP-BL)(×10-5)分别为:(2.21±0.200)、(3.56±0.306)、(3.81±0.179)、(4.23±0.229)、(4.17±0.262)cm·s-1,B侧到A侧的渗透系数Papp(BL-AP)(×10-5)分别为:(3.57±0.209)、(4.51±0.113)、(4.97±0.229)、(5.24±0.550)、(5.07±0.557)cm·s-1,外排率分别为:1.61、1.26、1.3、1.23、1.21。温度和p H对其转运均有影响,而P-gp抑制剂对于IPHP的转运没有明显的影响,MRP2抑制剂在一定程度上增加了IPHP的转运量(P<0.05)。结论 IPHP在Caco-2细胞模型上的转运方式主要是被动扩散,且其转运不受P-gp外排蛋白影响,而外排蛋白MRP2可能参与了IPHP的外排转运。     

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。     

新抗癌活性物AG337在Caco-2细胞模型中经p-糖蛋白介导的跨细胞转运

目的研究AG337在Caco-2细胞模型中的转运机制.方法改变药物浓度和实验温度以及他用合适抑制剂,测定AG337的跨细胞转运速率及其在细胞内的累积量.结果AG337的跨细胞转运显示强烈的有向性,B→A转运(细胞绒毛而Apical→基底面Basolateral)大于A→B10倍以上,P-糖蛋白的专属抑制剂维拉帕米(Ver)可以消除这种有向性温度从37℃至4℃时B→A的转运速率下降50倍,而A→B下降不大.结论AG337在Caco-2模型中的跨细胞转运.受到P-糖蛋白强烈的外排作用.     

P-糖蛋白对番荔枝酰胺衍生物FLZ在Caco-2细胞模型中跨膜转运的影响

目的考察转运蛋白P-糖蛋白(P-glycoprotein,P-gp)对番荔枝酰胺衍生物FLZ体外跨血脑屏障(Blood-brain barrier,BBB)转运的影响。方法采用体外培养的Caco-2细胞建立体外BBB模型,研究1,5,10μM FLZ跨膜转运特性。并探讨加入P-gp抑制剂5μM zosuquidar后FLZ跨膜转运的表观渗透系数和外排率的变化。结果 FLZ在Caco-2细胞模型上显示出了极性转运特性,Papp(B~A)>Papp(A~B),并呈现出良好的剂量依赖关系。同时FLZ在Caco-2细胞的跨膜转运中也呈现外排现象,1,5,10μM FLZ的外排率ER值分别为2.56,3.67和5.06。P-gp抑制剂zosuquidar可以显著降低FLZ外排,增加转运。10μM FLZ的外排率由5.06降低为1.94,下降了2.6倍。结论 FLZ具有P-gp的底物特性,P-gp参与了FLZ在BBB跨膜转运中的外排。     

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。     

坎地沙坦与坎地沙坦酯在Caco-2细胞模型中的转运特性

目的 研究坎地沙坦(Cand)与坎地沙坦酯(Cil)在Caco-2细胞中的跨膜转运特征.方法 采用Caco-2细胞单层膜模型来考察药物的浓度、介质pH与P-gp抑制剂维拉帕米对Cand与Cil跨膜转运的影响,并比较两者双向跨膜转运的差异.结果 两者的吸收转运具有pH依赖性,分泌转运具有浓度依赖性,其分泌(BL-AP)方向转运均快于吸收(AP-BL)方向转运,且Cil的AP-BL方向转运比Cand快;在P-gp抑制剂维拉帕米存在下,两者的转运外排率显著下降(P<0.01).结论 Cil容易通过Caco-2细胞的单层膜,且外排蛋白参与了Cand与Cil的跨膜转运.     

芦丁在Caco-2细胞模型中吸收和外排机制的研究

利用Caco-2细胞模型研究芦丁在小肠上皮的摄取、跨膜转运及外排动力学机制,评价孵育时间、芦丁浓度、p-糖蛋白抑制剂环孢素A和多药耐药相关蛋白抑制剂维拉帕米对芦丁的细胞摄取与转运的影响.结果表明,药物摄取量与孵育时间、药物浓度呈正相关,环孢素A和维拉帕米对芦丁的细胞摄取量无显著影响(P＞0.05).不同浓度药物从基底侧(basolateral,BL)到肠腔侧(Apical,AP)的表观渗透系数Papp,BL-AP与AP到BL的Papp,AP-BL比值均在0.5～1.5.试验结果提示芦丁是以被动扩散为主要转运方式被小肠上皮细胞摄取和转运,且不受外排蛋白外排作用的影响.     

P-糖蛋白抑制剂对蝙蝠葛碱跨膜转运的影响

目的 研究Caco-2细胞模型中P-糖蛋白抑制剂对蝙蝠葛碱跨膜转运的影响.方法 采用HPLC法测定转运液中蝙蝠葛碱的含量;采用Caco-2细胞模型双向转运实验,考察不同浓度维拉帕米、环孢素A和醋酸地塞米松3种p-糖蛋白抑制剂对蝙蝠葛碱跨膜转运的影响.结果 加入3种P-糖蛋白抑制剂后,蝙蝠葛碱的Papp(A-B)均有一定程度增加,而Papp(B-A)均有显著降低(P＜0.05),即均抑制了蝙蝠葛碱的外排转运.结论 外排转运体P-糖蛋白对蝙蝠葛碱有外排作用,蝙蝠葛碱可能为P-糖蛋白底物.     

MDCKII/MDCKII-BCRP细胞模型用于治疗胎儿快速性心律失常系列药物的跨胎盘转运机制的研究

研究治疗胎儿快速性心律失常的一系列药物在过表达乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)的马丁达比犬肾上皮细胞系MDCKII-BCRP单层细胞模型中的跨膜转运机制,筛选BCRP底物。利用MDCKII-BCRP和MDCKII单层细胞模型研究索他洛尔(sotalol)、普萘洛尔(propranolol)、普罗帕酮(propafenone)、普鲁卡因胺(procainamide)及氟卡尼(flecainide)的双向转运特性,采用HPLC或化学发光仪测定药物含量,计算其表观渗透系数(Papp)、外排率(RE)和净外排率(Rnet),将Rnet>1.5的药物进行细胞蓄积实验,考察药物浓度和BCRP抑制剂槲皮素对该药细胞内蓄积的影响。所选择的药物中,索他洛尔、普萘洛尔、普罗帕酮和普鲁卡因胺在两种细胞单层顶侧(apical,A)→基底侧(basolateral,B)的转运与B→A的转运之间无显著性差异,Rnet均小于1.5;氟卡尼浓度为20和5μmol·L-1时,Rnet分别为1.6和1.9。细胞蓄积实验证实氟卡尼在MDCKII、MDCKIIBCRP细胞内蓄积具有浓度依赖性,且MDCKII-BCRP细胞内的蓄积量明显低于MDCKII细胞;当同时在MDCKII-BCRP细胞内加入50μmol·L-1槲皮素时,氟卡尼在细胞中的蓄积量显著增加(P<0.05)。结果初步提示,索他洛尔、普萘洛尔、普罗帕酮和普鲁卡因胺可能不是BCRP底物;而氟卡尼可能是BCRP底物,因此母体用该药治疗胎儿快速性心律失常时,母体胎盘滋养层细胞膜上表达的BCRP极有可能会介导其外排,从而显著影响治疗效果。     

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.     

Specificity of doxorubicin versus rhodamine-123 in assessing P-glycoprotein functionality in the LLC-PK1, LLC-PK1:MDR1 and Caco-2 cell lines.

The LLC-PK1:MDR1, LLC-PK1 and Caco-2 cell lines were used to investigate whether rhodamine-123 or doxorubicin would be the preferred substrate to study P-glycoprotein (P-gp) functionality in vitro. Both rhodamine-123 and doxorubicin showed highly polarised transport in the Caco-2 cell line and the LLC-PK1:MDR1 cell line, indicating that P-gp is actively transporting these drugs. However, for rhodamine-123 polarised transport was also seen in the monolayers of the wild-type LLC-PK1 cell line, indicating the presence of another active transporter for this compound. Polarised transport of doxorubicin in the Caco-2 and the LLC-PK1:MDR1 cell lines could be inhibited by the P-gp inhibitors SDZ-PSC 833 (PSC 833), cyclosporin A (CsA), verapamil and quinine, but not by the inhibitors for the organic cation carrier systems cimetidine and tetraethylammonium (TEA). Polarised transport of rhodamine-123 in the Caco-2 cell line could only be inhibited by P-gp inhibitors. In the LLC-PK1:MDR1 and LLC-PK1 cell lines transport was also inhibited by inhibitors for the organic cation transport systems. In conclusion, rhodamine-123 is a substrate for both P-gp and the organic cation carrier systems in the kidney cell line. This indicates that rhodamine-123 is not selective enough to study P-gp functionality in cell systems were organic cation carrier systems are also present. Doxorubicin appears to be a more selective P-gp substrate and therefore more useful in studying P-gp functionality in vitro.     

Fexofenadine transport in Caco-2 cells: inhibition with verapamil and ritonavir

This study investigated fexofenadine (FXD) transport and the inhibition of FXD transport in Caco-2 cell monolayer transwells, using rhodamine 123 (RH123) transport as a positive control. FXD transport from the basolateral (B) to apical (A) compartment was fivefold higher than A to B transport. FXD transport was linear with respect to time (up to 270 min) and concentration (up to 300 microm). Similar results were seen with the positive control RH123. Ritonavir (100 PM) and verapamil (100 microm) reduced transport of FXD and RH123 by more than 80%, whereas transport was not inhibited by 100 m indomethacin or 2 mM probenecid. This suggests predominantly P-glycoprotein (P-gp)-mediated transport as opposed to transport by multidrug resistance protein. In concentration-response experiments, FXD transport was inhibited by verapamil and ritonavir with IC50 values of 6.5 microm and 5.4 microm, respectively. Results from this in vitro study demonstrate differential transport of FXD across Caco-2 cell monolayers and inhibition of FXD transport by established P-gp inhibitors. Thefindings support the use of FXD as an index or probe compound to reflect P-gp activity in vivo.     

MDCK-MDR1细胞模型及其在药物透过研究中的应用进展

本文介绍了MDCK-MDR1细胞系的特点、模型的建立，药物在其细胞模型上吸收转运的研究进展。概述了国内外关于利用MDCK-MDR1细胞系作为模型进行药物筛选、药物相互作用和研究药物吸收转运机制等方面的内容。MDCK-MDR1细胞系高表达P-糖蛋白(P-glycoprotein，P-gp)，且P-gp呈极性分布，因而它可以作为药物转运模型快速筛选P-gp底物和抑制剂，以及肠道、血脑屏障、肾脏的体外模型。     

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.     

Transport characteristics of rutin deca (H-) sulfonate sodium across Caco-2 cell monolayers

The aim of this study was to explore potential transport mechanisms of rutin deca (H-) sulfonate sodium (RDS) across Caco-2 cell monolayers. As an in-vitro model of human intestinal epithelial membrane, Caco-2 cells were utilized to evaluate the transepithelial transport characteristics of this hydrophilic macromolecular compound. Bi-directional transport study of RDS demonstrated that the apparent permeability (P(app)) in the secretory direction was 1.4 approximately 4.5-fold greater than the corresponding absorptive P(app) at concentrations in the range 50.0 approximately 2,000 microM. The transport of RDS was shown to be concentration, temperature and pH dependent. In the presence of ciclosporin and verapamil, potent inhibitors of P-glycoprotein (P-gp)/MRP2, the absorptive transport was enhanced and secretory efflux was diminished. RDS significantly reduced the efflux ratio of the P-gp substrate rhodamine-123 in a fashion indicative of P-gp activity suppression, while rhodamine-123 competitively inhibited the polarized transport of the compound. In conclusion, the results indicated that RDS was likely a substrate of P-gp. Several efflux transporters, including P-gp, participated in the absorption and efflux of RDS and they might play significant roles in limiting the oral absorption of the compound. These observations offered important information for the pharmacokinetics of RDS.     

Role of P-glycoprotein-mediated secretion in absorptive drug permeability: An approach using passive membrane permeability and affinity to P-glycoprotein.

It has been shown in vivo and in vitro that P-glycoprotein (P-gp) may be able to influence the permeability of its substrates across biological membranes. However, the quantitative contribution of the secretion process mediated by P-gp on the overall permeability of membranes has not been determined yet. In particular, observations need to be clarified in which substrates showing high affinity to P-glycoprotein, e.g., verapamil, apparently do not seem to be greatly influenced by P-gp in their permeability and consequently also with respect to their extent of GI-absorption after oral administration, whereas weaker substrates of P-gp, e.g., talinolol, have clearly shown P-gp-related absorption phenomena such as nonlinear intestinal permeability and bioavailability. Experiments with Caco-2 cell monolayers and mathematical simulations based on a mechanistic permeation model should aid in clarifying the underlying mechanism for these observations and quantifying the influence of passive membrane permeability and affinity to P-gp to the overall transmembrane drug flux. In addition, the concentration range of drug at which P-glycoprotein-mediated transport across the biological membrane is relevant should be examined. The permeability of various drugs in Caco-2 monolayers was determined experimentally and modeled using a combination of passive absorptive membrane permeability and a Michaelis-Menten-type transport process in the secretory direction. The passive permeabilities were experimentally obtained for the apical and basolateral membrane by efflux experiments using Caco-2 monolayers in the presence of a P-gp inhibitor. The Michaelis-Menten parameters were determined by a newly developed radioligand-binding assay for the quantification of drug affinity to P-gp. The model was able to accurately simulate the permeability of P-glycoprotein substrates, with differing passive membrane permeabilities and P-glycoprotein affinities. Using the outlined approach, permeability vs donor-concentration profiles were calculated, and the relative contribution of passive and active transport processes to the overall membrane permeability was evaluated. A model is presented to quantitatively describe and predict direction-dependent drug fluxes in Caco-2 monolayers by knowing the affinity of a compound to the exsorptive transporter P-gp and its passive membrane permeability. It was shown that a combination of high P-gp affinity with good passive membrane permeability, e.g., in the case of verapamil, will readily compensate for the P-gp-mediated reduction of intestinal permeability, resulting in a narrow range in which the permeability depends on the apical drug concentration. On the other hand, the permeability of compounds with low passive membrane permeability (e. g., talinolol) might be affected over a wide concentration range despite low affinity to P-gp.     

Increase in doxorubicin cytotoxicity by carvedilol inhibition of P-glycoprotein activity

Acquired resistance to chemotherapy is a major problem during cancer treatment. One mechanism for drug resistance is overexpression of the MDR1 (multidrug resistance) gene encoding for the transmembrane efflux pump, P-glycoprotein (P-gp). The calcium channel blocker verapamil has been shown to reverse cellular drug resistance by inhibiting P-gp drug efflux. This study evaluated whether the new antihypertensive drug carvedilol influenced doxorubicin (Dox) cytotoxicity and P-gp activity in a P-gp-expressing cell line compared to a non-expressing subline. Verapamil (10 micromol/L), and even more markedly, carvedilol (10 micromol/L) increased cellular uptake of P-gp-transported calcein of a P-gp-expressing breast cancer cell line (Hs578T-Dox). In the subline (Hs578T) not expressing P-gp, no effects of carvedilol or verapamil on calcein uptake were seen. Carvedilol and verapamil (10 micromol/L) reduced the LD50 (dose which results in the death of half the number of cells) of the Hs578T-Dox subline from 200 mg/L to approx. 10 mg/L Dox, whereas the LD50 of the Hs578T subline was only marginally affected. Carvedilol (10 micromol/L) reduced P-gp activity approximately twice as effectively as verapamil at an equimolar concentration. Carvedilol did not affect pyrogallol cytotoxicity and pyrogallol was without effect on calcein accumulation of the Hs578T-Dox cell line, indicating the lack of antioxidative properties affecting P-gp activity and associated toxicity of the drug. The results suggest that carvedilol has the clinical potential to reverse tumour MDR involving the efflux protein P-gp.     

Involvement of P-Glycoprotein in the Transport of Saquinavir and Indinavir in Rat Brain Microvessel Endothelial and Microglia Cell Lines

PURPOSE: Membrane-bound efflux transporters, such as P-glycoprotein (P-gp), may limit the brain entry and distribution of HIV-1 protease inhibitors and be in part responsible for HIV-1-associated dementia treatment failure. The purpose of this study was to characterize the transport properties of saquinavir and indinavir in a brain microvessel endothelial cell line and in microglia, the immune cells of the brain and primary HIV-1 cellular target. METHODS: Biochemical and transport studies were performed in an immortalized rat brain endothelial cell line (RBE4), a rat microglia cell line (MLS-9), and a P-gp overexpressing Chinese hamster ovary cell line (CHRC5). RESULTS: Western blot analysis using the P-gp monoclonal antibody C219 detected a single band at approximately 170 to 180 kDa (a size previously reported for P-gp) in all cell lines. Cellular accumulation of [14C]saquinavir and [3H]indinavir by RBE4, MLS-9, and CHRC5 monolayers was significantly enhanced in the presence of P-gp inhibitors, HIV-1 protease inhibitors, the ATPase inhibitor sodium azide, and the ATP depleting agent 2',4'-dinitrophenol respectively. [14C]Saquinavir and [3H]indinavir efflux from both cell systems was rapid and significantly reduced in the presence of PSC833. CONCLUSIONS: These results provide evidence for P-gp mediated transport of saquinavir and indinavir in RBE4 and MLS-9 and suggest that this transporter can restrict, at least in part, the permeation of HIV-1 protease inhibitors at both the brain barrier site and in brain parenchyma.