仙茅苷在Caco-2细胞模型中跨膜转运特征研究

目的：采用Caco-2细胞Transwell模型研究仙茅苷的跨膜转运机制。方法：建立Caco-2细胞Transwell吸收模型,用聚酯碳酸酯膜培养Caco-2细胞21天,形成致密的单层细胞模型。研究浓度、时间、温度、细胞旁路转运及跨膜转运蛋白（P-糖蛋白,多药耐药蛋白,乳腺癌耐药蛋白）在仙茅苷跨膜转运中的作用。结果：仙茅苷在Caco-2细胞模型中的跨膜转运存在一定的浓度及时间依赖性,细胞旁路转运不参与其转运,仙茅苷的外排转运存在一定的能量依赖性,且由P-gp参与。结论：仙茅苷在Caco-2细胞模型上主要以主动转运方式作跨膜转运,且P-gp参与其跨膜转运。     

姜黄素在Caco-2细胞模型中的吸收机制研究

目的测定姜黄素透过Caco-2单细胞层的浓度,研究其吸收特征。方法用Caco-2细胞单层模型来考察时间、浓度、不同药物酮康唑、维拉帕米、胡椒碱对姜黄素吸收的影响。采用液相色谱-质谱联用(LC-MS)法,测定姜黄素浓度并计算其表观渗透系数(Papp)。结果不同浓度姜黄素在90 min之前Papp大小顺序为:180>240>300μg/ml;与对照组比较,胡椒碱,低浓度酮康唑,维拉帕米对姜黄素的吸收均呈不同程度的促进作用(P<0.05)。结论姜黄素的吸收具有明显的高浓度饱和现象,且不呈线性变化,初步判定其吸收为主动吸收机制;促进姜黄素吸收的原因可能与酮康唑抑制Ⅰ相代谢酶CYP450 3A4和1A2酶的活性;胡椒碱抑制姜黄素Ⅱ相代谢酶—葡萄糖醛酸苷酶;维拉帕米抑制药物外排性转运蛋白—P-糖蛋白的活性有关。     

Atorvastatin Transport in the Caco-2 Cell Model: Contributions of P-Glycoprotein and the Proton-Monocarboxylic Acid Co-Transporter

Purpose. The purpose of this study was to elucidate the mechanismsby which an HMG-CoA reductase inhibitor, atorvastatin (an organicacid with a pKa of 4.46), was transported in the secretory and absorptivedirections across Caco-2 cell monolayers. Methods. Caco-2 cells were grown on polycarbonate membrane insertsin 6-well Snapwell plates (Costar). The permeability of radiolabeledcompounds across Caco-2 cell monolayers was determined using aside-by-side diffusion apparatus (NaviCyte) and an automated liquidhandler (Hamilton Microlab 2200). The apical uptake of¹⁴C-atorvastatin was also determined in Caco-2 cells. Cyclosporin A (20 M) waspresent in the uptake media to block potential P-glycoprotein-mediatedatorvastatin efflux. Results. Polarized permeation of atorvastatin was observed with thebasolateral-to-apical (B-to-A) permeability being 7-fold greater thanthe A-to-B permeability (35.6 × 10^–6 and 4.9 × 10^–6 cm/s,respectively). The secretion of atorvastatin was a saturable process with anapparent K_m of 115 M. The B-to-A permeability of atorvastatin wassignificantly reduced by cyclosporin A (10 M), verapamil (100 M),and a P-glycoprotein specific monoclonal antibody, UIC2(10 g/ml)(43%, 25%, and 13%, respectively). Furthermore, both CsA andverapamil significantly increased the A-to-B permeability of atorvastatinby 60% however, UIC2 did not affect the A-to-B permeability ofatorvastatin. CsA uncompetitively inhibited the B-to-A flux ofatorvastatin with a K_i of 5 M. In addition, atorvastatin (100 M) significantlyinhibited the B-to-A permeability of vinblastine by 61%. The apicaluptake of atorvastatin increased 10.5-fold when the apical pH decreasedfrom pH 7.4 to pH 5.5 while the pH in the basolateral side wasfixed at pH 7.4. A proton ionophore, carbonylcyanidep-trifluoro-methoxyphenylhydrazone (FCCP) significantly decreased atorvastatinuptake. In addition, atorvastatin uptake was significantly inhibited bybenzoic acid, nicotinic acid, and acetic acid each at 20 mM (65%,14%, and 40%, respectively). Benzoic acid competitively inhibitedatorvastatin uptake with a K_i of 14 mM. Similarly, benzoic acid,nicotinic acid, and acetic acid significantly, inhibited the A-to-Bpermeability of atorvastatin by 71%, 21%, and 66%, respectively. Conclusion. This study demonstrated that atorvastatin was secretedacross the apical surface of Caco-2 cell monolayers viaP-glycoprotein-mediated efflux and transported across the apical membrane in theabsorptive direction via a H⁺-monocarboxylic acid cotransporter(MCT). In addition, this study provided the first evidence thatnegatively charged compounds, such as atorvastatin, can be a substrate forP-glycoprotein.     

阿立哌唑在Caco-2细胞单层模型中的跨膜转运

本文研究了阿立哌唑（aripiprazole）在Caco-2细胞模型中的跨膜转运特征。一种体外培养的人小肠上皮细胞模型 —— Caco-2细胞模型应用于阿立哌唑的跨膜转运研究。评价了时间、供给液浓度、pH值、温度及P-糖蛋白抑制剂对阿立哌唑跨膜转运的影响。采用高效液相色谱法检测药物浓度。结果表明阿立哌唑主要通过被动扩散的机制转运,同时兼有载体介导转运。阿立哌唑的转运量与时间、pH值、温度成正相关。表观渗透系数P_app值随供给液浓度升高而增大,10 μg·mL^-1时趋向饱和,之后随阿立哌唑浓度的增加而逐渐减小。P-糖蛋白抑制剂环孢菌素-A显著增加阿立哌唑的跨膜转运。     

Drug Absorption Limited by P-Glycoprotein-Mediated Secretory Drug Transport in Human Intestinal Epithelial Caco-2 Cell Layers

The hypothesis was tested that the operation of an ATP-dependent export pump localized at the apical (brush border) surface of the intestinal epithelium may limit substrate absorption kinetics. Human intestinal Caco-2 cell-layers display saturable secretion of vinblastine from basal to apical surfaces (K _m, 18.99 ± 5.55 µM; V _max, 1285.9 ± 281.2 pmol cm^–2 hr^–1) that is inhibited by verapamil, consistent with the expression of the ATP-dependent P-glycoprotein drug efflux pump at the apical brush border membrane. Inhibition of P-glycoprotein by a variety of modulators (verapamil, 1,9-dideoxyforskolin, nifedipine, and taxotere) is associated with an increased vinblastine absorptive permeability. Vinblastine absorption displayed a nonlinear dependence upon luminal (apical) vinblastine concentration, and vinblastine absorption increased markedly at concentrations where vinblastine secretory flux was saturated (>20 µM). Upon inhibition of P-glycoprotein by verapamil and 1,9-dideoxyforskolin, vinblastine absorption increased and was linearly dependent on vinblastine concentration. The limitation of P-glycoprotein substrate absorption by active ATP-dependent export via P-glycoprotein is discussed, together with the possibility that other classes of substrate may be substrates for different ATP-dependent export pumps.     

Predicting P-Glycoprotein Effects on Oral Absorption: Correlation of Transport in Caco-2 with Drug Pharmacokinetics in Wild-Type and mdr1a(-/-) Mice in Vivo

PURPOSE: Cell-based permeability screens are widely used to identify drug-P-glycoprotein (PGP) interaction in vitro. However, their reliability in predicting the impact of PGP on human drug pharmacokinetics is poorly defined. The aim was to determine whether a quantitative relationship exists between PGP-mediated alterations in Caco-2 permeability and oral pharmacokinetics in mice. METHODS: Two indicators of drug efflux were measured in Caco-2 for a group of 10 compounds, the ratio of A-B and B-A transport (R9B-A/A-B)) and the ratio of A-B transport in the presence and absence of a PGP inhibitor, GF120918 (R(GF)). These data were correlated with ratios of oral plasma levels in either mdr1a(-/-) or mdr1a/1b(-/-) and wild-type mice (R(KO/WT in vivo)) calculated from literature data on these compounds. RESULTS: A significant, positive correlation (r2 = 0.8, p < 0.01) was observed between RGF and R(KO/WT in vivo). In contrast, R(B-A/A-B), a more commonly used in vitro measure, showed a much weaker correlation with in vivo data (r2 = 0.33, p = 0.11). A strong correlation with R(GF) was also observed after correction of in vivo data for PGP effects on IV clearance. CONCLUSION: The increase in A-B drug permeability following inhibition of PGP in Caco-2 allows a reasonable prediction of the likely in vivo impact that PGP will have on plasma drug levels after oral administration.     

HPLC考察白头翁汤4种标志成分在Caco-2细胞模型的转运特征

目的研究白头翁汤中标志成分秦皮甲素、秦皮乙素、小檗碱、白头翁皂苷B4在Caco-2细胞模型的转运特征及其机制。方法通过HPLC建立白头翁汤指纹图谱以及4种标志成分的同时定量分析方法,随后以Caco-2细胞模型和p-gP抑制剂维拉帕米考察复方标志成分的双向转运机制,通过HPLC检测药物浓度计算其表观渗透系数（Papp）。结果①秦皮甲素在顶端（AP）和底端（BL）双向转运大致相同,为被动转运机制;②秦皮乙素在AP侧转运大于BL侧,维拉帕米可抑制AP侧向BL侧转运,为主动转运机制;③小檗碱在AP侧和BL侧转运大致相同,对于BL-AP侧外排Papp值显著低于单体的文献报道值,在复方中可能存在化学成分抑制小檗碱BL侧向AP侧外流载体,促使外流减少,增加小檗碱的摄取;④白头翁皂苷B4紫外响应弱,未能检测出。结论白头翁汤中秦皮甲素、秦皮乙素、小檗碱可通过HPLC考察其在Caco-2细胞模型的转运特征。     

坤复康片中芍药内酯苷在Caco-2细胞模型中的吸收特征研究

目的 研究坤复康片中的芍药内酯苷在Caco-2细胞模型中的跨膜吸收特征,探讨人小肠对芍药内酯苷的吸收和转运。方法 研究了不同药物浓度、pH值、温度和抑制剂对芍药内酯苷在Transwell细胞培养板中从顶膜(apical,AP)到基底(basolateral,BL)的双向渗透吸收的影响。结果 芍药内酯苷以3种测试浓度给药时(5,20和100 μmol·L^-1),从AP到BL方向的表观渗透系数(P_app)值范围为(3.27~5.37)×10^-7 cm·s^-1。相反,从BL到AP,P_app值范围为(6.64~9.63)×10^-7 cm·s^-1。数据表明,芍药内酯苷转运具有pH和温度依赖性。芍药内酯苷的细胞通透性受多种抑制剂影响,包含Na⁺/K⁺离子通道抑制剂钒酸钠,多耐药蛋白抑制剂MK571和吲哚美辛及乳腺癌耐药蛋白抑制剂芹菜素。当坤复康溶液加入后,芍药内酯苷从AP到BL方向的P_app值显著增加。结论 芍药内酯苷在Caco-2细胞模型中的肠道吸收较差,其吸收机制可能涉及多种蛋白介导的主动转运。     

Caco-2细胞对酸枣仁皂苷A的跨膜转运

目的研究酸枣仁皂苷A（Jujuboside A,JuA）在Caco-2细胞的跨膜转运特性。方法采用体外培养的Caco-2细胞单层模型,考察时间、介质pH值、药物浓度、抑制剂对JuA在Caco-2细胞上转运的影响。结果 Caco-2细胞转运JuA呈时间及浓度依赖性;在pH为5.0～8.0范围内,Caco-2细胞对JuA的转运不受pH值的影响;P-糖蛋白（P-glycoprotein,P-gp）抑制剂维拉帕米（Verapamil,Ver）对Caco-2细胞转运JuA无影响;线粒体呼吸链复合体Ⅳ抑制剂叠氮化钠（Sodium azide）对Caco-2细胞转运JuA有抑制作用;JuA的AP-BL侧的Papp与BL-AP侧的Papp的两组均数比较无统计学意义。结论 JuA不是P-gp的底物,其跨膜转运是被动转运与主动转运共同参与的过程。     

矢车菊黄素在Caco-2细胞模型中的吸收机制研究

目的 研究矢车菊黄素（centaureidin）在Caco-2细胞单层模型中的吸收机制。方法 以HPLC分析矢车菊黄素浓度,用Caco-2 细胞单层模型评价吸收时间、药物浓度、介质pH值、抑制剂等对矢车菊黄素吸收的影响,研究矢车菊黄素的吸收机制,计算表观渗透系数（apparent permeability coefficient, Papp）。结果 药物的吸收与药物浓度和吸收时间正相关;弱酸性介质条件下有利于药物的吸收;2,4-二硝基酚（DNP）对药物吸收无影响,但异博定（verapamil）可增加药物的吸收;从肠腔侧到基底侧的转运小于基底侧到肠腔侧的转运。结论 矢车菊黄素在Caco-2细胞模型中的吸收主要是被动转运,受P-糖蛋白的外排作用。     

Profound Effect of Plasma Protein Binding on the Polarized Transport of Furosemide and Verapamil in the Caco-2 Model

Pharmaceutical Research -     

Transport Characteristics of Peptidomimetics. Effect of the Pyrrolinone Bioisostere on Transport Across Caco-2 Cell Monolayers

Purpose. To compare the permeation characteristics of amide bond-containing HIV-1 protease inhibitors and their pyrrolinone-containing counterparts across Caco-2 cell monolayers, a model of the intestinal mucosa. Methods. Transepithelial transport and cellular uptake of three pairs of amide bond-containing and pyrrolinone-based peptidomimetics were assessed in the presence and absence of cyclosporin A using the Caco-2 cell culture model. The potential of the peptidomimetics to interact with biological membranes was estimated by IAM chromatography. Results. In the absence of cyclosporin A, apical (AP) to basolateral (BL) flux of all compounds studied was less than the flux determined in the opposite direction (i.e., BL-to-AP). The ratio of the apparent permeability coefficients (P_app) calculated for the BL-to-AP and AP-to-BL transport (P_BLAP/P_APBL) varied between 1.7 and 36.2. When individual pairs were compared, P_BLAP/P_APBL ratios of the pyrrolinone-containing compounds were 1.5 to 11.5 times greater than those determined for the amide bond-containing analogs. Addition of 25 M cyclosporin A to the transport buffer reduced the P_BLAP /P_APBL ratios for all protease inhibitors to a value close to unity. Under these conditions, the amide bond-containing peptidomimetics were at least 1.6 to 2.8 times more able to permeate Caco-2 cell monolayers than were the pyrrolinone-containing compounds. The intrinsic uptake characteristics into Caco-2 cells determined in the presence of 25 M cyclosporin A were slightly greater for the amide bond-containing protease inhibitors than for the pyrrolinone-containing analogs. These uptake results are consistent with the transepithelial transport results determined across this in vitro model of the intestinal mucosa. Conclusions. The amide bond-containing and pyrrolinone-based peptidomimetics are substrates for apically polarized efflux systems present in Caco-2 cell monolayers. The intrinsic permeabilities of the amide bond-containing protease inhibitors are slightly greater than the intrinsic permeabilities of the pyrrolinone-based analogs through Caco-2 cell monolayers.     

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.     

Comparison of the Transport Characteristics of D- and L-Methionine in a Human Intestinal Epithelial Model (Caco-2) and in a Perfused Rat Intestinal Model

Absorption mechanisms of L- and D-methionine (MET) in an in vitro cultured human intestinal epithelial cell model (Caco-2) and an in situ perfused rat intestinal model were investigated to determine if the kinetic characteristics of absorption are comparable in these two popular absorption models. The results indicate that the transport of L- and D-MET were concentration-dependent in both model systems, and displayed comparable K_m values. The K_m value for L-MET is 1.34 mM in the Caco-2 model and 3.6 mM in the perfused rat intestinal model, while the K_m value for D-MET is 1.79 mM in the Caco-2 model and 2.87 mM in the perfused rat intestinal model. Although the J_max values were not comparable because of significant methodology differences, the J_max values for L-MET were always higher than that for D-MET. In addition, transport of L- and D-MET across the Caco-2 cell monolayers were also inhibited by 10 mM Phe and Lys while MeAIB, Pro and Glu were generally ineffective. Similar results were also observed with these inhibitors in the perfused rat intestinal model with the exception that a combination of Pro and Glu stimulated the uptake of L-MET. In conclusion, the transport characteristics of L- and D-MET are comparable in both model systems.     

蛇床子素及其制剂在Caco-2细胞模型中转运机制的研究

I摘要】目的：研究蛇床子素及其制剂在Caco-2细胞模型中的摄取、转运机制。方法：建立Caco-2细胞单层模型,研究蛇床子素、蛇床予素β-环糊精包合物及其包合物分散片的摄取和跨膜转运,考察其时间、温度、浓度、吸收促进剂和抑制剂对药物摄取及跨膜吸收的影响,并比较原料药与制剂吸收过程的差异。结果：Caco-2细胞对蛇床子素及其制剂溶液的摄取与时间、温度、浓度均呈正相关,p-糖蛋白抑制剂（CyA）与能量抑制剂（NaN3）对摄取无显著性影响,其摄取量的大小依次为分散片〉包合物〉原料药;转运实验中,随浓度和温度的增加,蛇床子素及其制刑的溶液在Caco-2细胞中的转运量均增加,而只r与Rt比值无明显变化,P-糖蛋白抑制剂（CyA）、能量抑制剂（NaN,）、细胞内吞抑制剂．氧化苯砷（oxophenylarsine）7L胞旁路转运促进剂一去氧胆酸钠（sodiumdeoxycholate,SDCh）对蛇床子素的转运无影响,而去氧胆酸钠对包合物和分散片的转运有明显的影响,3种制刑AP—BL方向上的Papp为分散片〉包合物〉原料药。结论：蛇床子素主要以被动扩散的方式被吸收,包合物和分散片中的药物以被动转运为主,少部分以细胞旁路转运途径被细胞吸收;难溶性药物经包合物可促进其吸收,制剂辅料对药物的吸收有一定的促进作用。     

人参皂苷Rb_3在Caco-2单细胞层模型上的吸收特征研究

目的:研究人参皂苷Rb3在Caco-2单细胞层模型上的吸收特征。方法:人参皂苷Rb3细胞样品经高速离心后取上清液,以乙腈-1mmo·lL-1甲酸铵水溶液(34∶66)为流动相,以人参皂苷Rg2为内标,采用电喷雾离子源(ESI),在负离子条件下以多重离子反应监测模式(MRM)检测。检测离子:m/z1077.7→m/z783.4(人参皂苷Rb3),m/z783.6→m/z475.1(人参皂苷Rg2)。测定透过Caco-2单细胞层的人参皂苷Rb3浓度并计算其表观渗透系数(Papp)。结果:人参皂苷Rb3的线性范围为50～2000ng·mL-1,日间、日内精密度均小于15%。Papp从基顶侧(AP)到基底侧(BL)为3.22×10-6cm·s-1,从BL侧到AP侧为6.0×10-6cm·s-1,其P(BL-AP)/P(AP-BL)比值为1.86。结论:本方法简单、灵敏度高,可用于研究人参皂苷Rb3在Caco-2单细胞层模型上的吸收特征。     

Transepithelial Transport of Diphenhydramine Across Monolayers of the Human Intestinal Epithelial Cell Line Caco-2

Purpose. The transepithelial transport characteristics of theantihistamine, diphenhydramine, were studied in human intestinal Caco-2 cellmonolayers to elucidate the mechanisms of its intestinal absorption. Methods. The transepithelial transport and the cellular accumulationof diphenhydramine were measured using Caco-2 cell monolayersgrown in Transwell chambers. Results. The transepithelial transport of diphenhydramine from theapical to basolateral side was saturable, and the flux and cellularaccumulation of diphenhydramine were dependent on the apicalextracellular pH (pH 7.4 > 6.5 > 5.5). Transport and accumulation ofdiphenhydramine from the apical side were inhibited by anotherantihistamine, chlorpheniramine, while typical substrates for the renal organiccation transport system such as tetraethylammonium, cimetidine andguanidine had no effect. The transepithelial transport and cellularaccumulation of diphenhydramine from the basolateral side were alsopH-dependent and inhibited by chlorpheniramine. In addition, intracellulardiphenhydramine preloaded was preferentially effluxed to the apicalside, suggesting the involvement of the secretory pathway indiphenhydramine transport. Furthermore, diphenhydramine uptake from boththe apical and basolateral sides was stimulated by preloadingmonolayers with chlorpheniramine (trans-stimulation effect). Conclusions. Transepithelial transport of diphenhydramine acrossCaco-2 cells is mediated by pH-dependent, specific transport systemsthat exist in both the apical and basolateral membranes.     

Age-dependent Expression of P-Glycoprotein gp17O in Caco-2 Cell Monolayers

Purpose. To determine whether the expression and activity of the P-glycoprotein (P-GP) drug efflux pump vary with the culture age of Caco-2 cell monolayers. Methods. Caco-2 cell monolayers were grown for 3–27 days on tissue culture-treated Transwells. P-GP efflux function was determined by measuring transmonolayer fluxes of cyclosporin A (CsA) and verapamil, while P-GP expression level was evaluated by Western blot analysis using monoclonal antibody C219. Results. The apparent permeability coefficient (Papp) of CsA (0.5 µM) in the basolateral-to-apical (B A) direction increased with culture age and was higher than the apical-to-basolateral (A B) direction at all times. Net secretory Papp significantly increased from day 17 onward compared to that observed during day 3 through 13. Verapamil (100 µM) significantly inhibited CsA transport in the B A direction from day 17 to 27, while elevating CsA transport in the A B direction from day 6 to 27. Interestingly, the Papp of verapamil (0.5 µM) in the B A direction was significantly higher than in the A B direction from day 6 to 27, rendering increases in net secretory Papp of verapamil with culture age. Western analysis revealed that P-GP expression level was in the order of 4 weeks 1 week > 3 weeks > 2 weeks at equal loading of cell proteins. Conclusions. P-GP is continuously expressed throughout the culture period, but it may not be fully functional at an early age. Caco-2 cell monolayers of day 17 to 27 appear to be a good model to evaluate the functional role of P-GP in drug efflux.     

LC-MS测定雷公藤甲素及其在Caco-2细胞上的转运特征

目的：建立体外模拟体内肠道细胞的Caco-2细胞Transwell模型,以此研究雷公藤甲素在Caco-2细胞模型上的跨膜转运特征。方法：采用聚酯碳酸酯膜连续培养Caco-2细胞21天,形成致密的单层细胞模型。然后对影响雷公藤甲素在Caco-2细胞模型上转运特征的因素包括浓度、时间及跨膜转运蛋白（P-糖蛋白,多药耐药蛋白,乳腺癌耐药蛋白）进行考察;同时采用LC-MS对溶液中的雷公藤甲素的含量进行测定。结果：雷公藤甲素主要以主动转运的方式进行吸收,且随着时间和药物浓度的增加,转运量明显增加。结论：雷公藤甲素在Caco-2细胞上转运存在一定的浓度及时间依赖性,且P-gp介导雷公藤甲素在Caco-2细胞上转运。