利用Caco-2细胞模型对7-木糖-10-去乙酰基紫杉醇跨膜转运的研究
|
摘要
红豆杉(Taxus)的水提液在我国被用于治疗癌症,表明红豆杉(Taxus)的水提液中含有抗癌的活性物质。在寻找红豆杉(Taxus)中的抗癌的活性物质过程中,一种亲水性紫杉醇衍生物7-木糖-10-去乙酰基紫杉醇被筛选出来。然而在临床前药物动力学研究表明,口服7-木糖-10-去乙酰基紫杉醇后,产生了较低的血药浓度。为了研究较低血药浓度是否由于小肠的低吸收而产生的,我们首次利用Caco-2细胞模型对7-木糖-10-去乙酰基紫杉醇吸收进行了研究,研究结果如下：
1.通过测定Caco-2细胞单分子层的跨膜电阻来判断模型是否建立。在本实验的培养条件下,经过21天培养分化后,Caco-2细胞分化形成了具有细胞极性的单分子层结构,跨膜电阻大于300Ωcm2,符合作为药物小肠吸收转运的体外实验模型的要求。
2.采用MTT比色法研究了7-木糖-10-去乙酰基紫杉醇对Caco-2细胞的毒性。实验结果表明,小于20μM的药物对细胞没有毒性,而大于20μM的药物对细胞呈现毒性。
3.应用Caco-2细胞模型,对6个浓度(0.5μM、1μM、2μM、5μM、10μM、20μM)的7-木糖-10-去乙酰基紫杉醇,分别考察了从AP侧(顶侧)→BL侧(基底侧)和BL侧→AP侧的跨细胞转运特性,结果在180min内,6个浓度的转运随时间延长而呈线性。表明7-木糖-10-去乙酰基紫杉醇转运以被动扩散为主。
4.7-木糖-10-去乙酰基紫杉醇的Papp BL→AP由25.8±6.3×10-6cm/s变化到17.1±3.8×10-6cm/s,而Papp AP→BL则维持在16.3±6.3×10-6cm/s,表明7-木糖-10-去乙酰基紫杉醇从BL侧→AP侧的转运属于被动扩散加主动运输,而从AP侧→BL侧的转运属于被动扩散减主动运输。
5.AP侧加入7-木糖-10-去乙酰基紫杉醇而BL侧加入P-糖蛋白抑制剂维拉帕米和汉防己甲素之后,7-木糖-10-去乙酰基紫杉醇BL侧→AP侧流量减少50%,表明7-木糖-10-去乙酰基紫杉醇是P-糖蛋白的底物。
6.7-木糖-10-去乙酰基紫杉醇的主动运输KM值为93.4μM, Vmax为909.1pmol/hr/cm2,而紫杉醇的主动运输KM值为16.5μM, Vmax为1050 pmol/hr/cm2,表明7-木糖-10-去乙酰基紫杉醇吸收近似于紫杉醇(文献已报道)。
7.7-木糖-10-去乙酰基紫杉醇的Papp AP→BL维持在16.3±6.3×10-6cm/s,大于10.0×10-6cm/s,暗示7-木糖-10-去乙酰基紫杉醇具有良好的小肠吸收性。
总之,口服7-木糖-10-去乙酰基紫杉醇后,由于小肠对7-木糖-10-去乙酰基紫杉醇的高吸收,较低的血药浓度更可能是肝脏首次代谢而产生的。
Water decoctions from the leaves of Taxus are used in traditional Chinese medicine to treat cancer, indicating that water soluble constituents of this tree may indeed possess anticancer activity. In an effort to search for natural products from Taxus with improved pharmacological features compared to paclitaxel,7-xylosyl-10-deacetylpaclitaxel has been isolated and identified by our group. This is a naturally occurring xyloside, which has been shown to possess higher water solubility than paclitaxel. However, preliminary studies suggested that 7-xylosyl-10-deacetylpaclitaxel was not significantly absorbed after oral administration, and pharmacokinetic studies yielded low plasma concentrations of the active ingredient. It is necessary to investigate whether the observed low oral bioavailability is due to poor absorption. Therefore, we studied the transepithelial flux of 7-xylosyl-10-deacetylpaclitaxel using the human colonic cell line Caco-2, a well established model of human intestinal absorption, and the results showed that:
1. Caco-2 cell model was authenticated by determination of TEER of Caco-2 cell monolayers.The results indicated that in our laboratory, Caco-2 cells had reached integrated confluence and formed differentiated mono layers 21 days after seeding. TEER of the cells reached 300Ωcm2 and met the requirement of drug transport. So this model can be an in vitro model that investigated intestinal permeability and transport of 7-xylosyl-10-deacetylpaclitaxel.
2. Methyl thiazoly tetrazolium chromatometry was applied to study the cytotoxicity of 7-xylosyl-10-deacetylpaclitaxel in Caco-2 cells.The results showed that compared with the control group,drug solution concentration lower than 20μM had no toxicity to Caco-2 cell.while drug solution concentration higher than 20μM was toxical to Caco-2 cell.
3. The flux of 7-xylosyl-10-deacetylpaclitaxel (0.5-20μM) across the Caco-2 cell layer was linear with time for up to 3 hr. The data indicated that 7-xylosyl-10-deacetylpaclitaxel was primarily absorbed by passive diffusion across the Caco-2 cell membranes.
4. The Papp for BL to AP transport decreased from 25.8±6.3×10-6 cm/sec (mean±SE) at 0.5-2μM to 17.1±3.8×10-6 cm/sec at 5-20μM 7-xylosyl-10-deacetylpaclitaxel. The Papp for AP to BL transport was fairly constant (16.3±6.3×10-6) throughout the concentration range studied. Hence, the observed BL to AP flux was the result of passive diffusion plus active transport. The AP to BL flux may be the result of passive minus active transport.
5. Verapamil and tetrandrine reduced the efflux by about 50%. Concomitant with this inhibition we observed a about 2.5-fold increase in the AP to BL flux. It was shown that 7-xylosyl-10-deacetylpaclitaxel is a substrate of P-gp.
6. The apparent KM value of 93.4μM for the saturable process for 7-xylosyl-10-deacetylpaclitaxel was more than that previously reported for the P-gp substrate paclitaxel (16.5μM) in the Caco-2 cell system. The Vmax value of 909.1 pmol/hr/cm2 for 7-xylosyl-10-deacetylpaclitaxel was similar to that calculated for paclitaxel (1050 pmol/hr/cm2).This indicated that the active transport rate of 7-xylosyl-10-deacetylpaclitaxel was similar to that of paclitaxel.
7. This was 16.3×10-6 cm/sec for 7-xylosyl-10-deacetylpaclitaxel and only 4.4×10-6 cm/sec for paclitaxel. According to previous studies, a Papp value in Caco-2 cells of>10×10-6 cm/s suggest good drug absorption in vivo corresponding to 70-100% of the applied dose.
In summary, it is hypothesized that the low oral bioavailability of 7-xylosyl-10-deacetylpaclitaxel may be more dependent on presystemic metabolism in the liver than on lack of absorption.
1.通过测定Caco-2细胞单分子层的跨膜电阻来判断模型是否建立。在本实验的培养条件下,经过21天培养分化后,Caco-2细胞分化形成了具有细胞极性的单分子层结构,跨膜电阻大于300Ωcm2,符合作为药物小肠吸收转运的体外实验模型的要求。
2.采用MTT比色法研究了7-木糖-10-去乙酰基紫杉醇对Caco-2细胞的毒性。实验结果表明,小于20μM的药物对细胞没有毒性,而大于20μM的药物对细胞呈现毒性。
3.应用Caco-2细胞模型,对6个浓度(0.5μM、1μM、2μM、5μM、10μM、20μM)的7-木糖-10-去乙酰基紫杉醇,分别考察了从AP侧(顶侧)→BL侧(基底侧)和BL侧→AP侧的跨细胞转运特性,结果在180min内,6个浓度的转运随时间延长而呈线性。表明7-木糖-10-去乙酰基紫杉醇转运以被动扩散为主。
4.7-木糖-10-去乙酰基紫杉醇的Papp BL→AP由25.8±6.3×10-6cm/s变化到17.1±3.8×10-6cm/s,而Papp AP→BL则维持在16.3±6.3×10-6cm/s,表明7-木糖-10-去乙酰基紫杉醇从BL侧→AP侧的转运属于被动扩散加主动运输,而从AP侧→BL侧的转运属于被动扩散减主动运输。
5.AP侧加入7-木糖-10-去乙酰基紫杉醇而BL侧加入P-糖蛋白抑制剂维拉帕米和汉防己甲素之后,7-木糖-10-去乙酰基紫杉醇BL侧→AP侧流量减少50%,表明7-木糖-10-去乙酰基紫杉醇是P-糖蛋白的底物。
6.7-木糖-10-去乙酰基紫杉醇的主动运输KM值为93.4μM, Vmax为909.1pmol/hr/cm2,而紫杉醇的主动运输KM值为16.5μM, Vmax为1050 pmol/hr/cm2,表明7-木糖-10-去乙酰基紫杉醇吸收近似于紫杉醇(文献已报道)。
7.7-木糖-10-去乙酰基紫杉醇的Papp AP→BL维持在16.3±6.3×10-6cm/s,大于10.0×10-6cm/s,暗示7-木糖-10-去乙酰基紫杉醇具有良好的小肠吸收性。
总之,口服7-木糖-10-去乙酰基紫杉醇后,由于小肠对7-木糖-10-去乙酰基紫杉醇的高吸收,较低的血药浓度更可能是肝脏首次代谢而产生的。
Water decoctions from the leaves of Taxus are used in traditional Chinese medicine to treat cancer, indicating that water soluble constituents of this tree may indeed possess anticancer activity. In an effort to search for natural products from Taxus with improved pharmacological features compared to paclitaxel,7-xylosyl-10-deacetylpaclitaxel has been isolated and identified by our group. This is a naturally occurring xyloside, which has been shown to possess higher water solubility than paclitaxel. However, preliminary studies suggested that 7-xylosyl-10-deacetylpaclitaxel was not significantly absorbed after oral administration, and pharmacokinetic studies yielded low plasma concentrations of the active ingredient. It is necessary to investigate whether the observed low oral bioavailability is due to poor absorption. Therefore, we studied the transepithelial flux of 7-xylosyl-10-deacetylpaclitaxel using the human colonic cell line Caco-2, a well established model of human intestinal absorption, and the results showed that:
1. Caco-2 cell model was authenticated by determination of TEER of Caco-2 cell monolayers.The results indicated that in our laboratory, Caco-2 cells had reached integrated confluence and formed differentiated mono layers 21 days after seeding. TEER of the cells reached 300Ωcm2 and met the requirement of drug transport. So this model can be an in vitro model that investigated intestinal permeability and transport of 7-xylosyl-10-deacetylpaclitaxel.
2. Methyl thiazoly tetrazolium chromatometry was applied to study the cytotoxicity of 7-xylosyl-10-deacetylpaclitaxel in Caco-2 cells.The results showed that compared with the control group,drug solution concentration lower than 20μM had no toxicity to Caco-2 cell.while drug solution concentration higher than 20μM was toxical to Caco-2 cell.
3. The flux of 7-xylosyl-10-deacetylpaclitaxel (0.5-20μM) across the Caco-2 cell layer was linear with time for up to 3 hr. The data indicated that 7-xylosyl-10-deacetylpaclitaxel was primarily absorbed by passive diffusion across the Caco-2 cell membranes.
4. The Papp for BL to AP transport decreased from 25.8±6.3×10-6 cm/sec (mean±SE) at 0.5-2μM to 17.1±3.8×10-6 cm/sec at 5-20μM 7-xylosyl-10-deacetylpaclitaxel. The Papp for AP to BL transport was fairly constant (16.3±6.3×10-6) throughout the concentration range studied. Hence, the observed BL to AP flux was the result of passive diffusion plus active transport. The AP to BL flux may be the result of passive minus active transport.
5. Verapamil and tetrandrine reduced the efflux by about 50%. Concomitant with this inhibition we observed a about 2.5-fold increase in the AP to BL flux. It was shown that 7-xylosyl-10-deacetylpaclitaxel is a substrate of P-gp.
6. The apparent KM value of 93.4μM for the saturable process for 7-xylosyl-10-deacetylpaclitaxel was more than that previously reported for the P-gp substrate paclitaxel (16.5μM) in the Caco-2 cell system. The Vmax value of 909.1 pmol/hr/cm2 for 7-xylosyl-10-deacetylpaclitaxel was similar to that calculated for paclitaxel (1050 pmol/hr/cm2).This indicated that the active transport rate of 7-xylosyl-10-deacetylpaclitaxel was similar to that of paclitaxel.
7. This was 16.3×10-6 cm/sec for 7-xylosyl-10-deacetylpaclitaxel and only 4.4×10-6 cm/sec for paclitaxel. According to previous studies, a Papp value in Caco-2 cells of>10×10-6 cm/s suggest good drug absorption in vivo corresponding to 70-100% of the applied dose.
In summary, it is hypothesized that the low oral bioavailability of 7-xylosyl-10-deacetylpaclitaxel may be more dependent on presystemic metabolism in the liver than on lack of absorption.
引文
[1]Stenberg P, Begrstrom CA,Luthmna K,et al.The oretical predictions of drug absorption in drug discovery and development [J]. Clin Phmaracokinet,2002,41(11):877-899.
[2]Bruan A,Hammerle S,Suda K,et al.Cell cultures as tools in biopharmacy.Eur J Phmar Sci.2000; 11 (Suppl2):S51-60
[3]Yee S.In vitro permeabiliy across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth.Pharm Res.1997;14(6):763-765
[4]Gan LSL,Thakker DR.Applications of the Caco-2 mode in the design and development of orally active drugs:Elucidation of biochemical and physical barriers posed by the intestinal epithelium.Adv Drug Deliv Rev 1997;23(1):77-98
[5]Garberg P,Eriksson P,Schipper N,et al.Automated absorption assessment using Caco-2 cells cultured on both sides of polycarbonate membranes.Pharm Res.1999:16(3):441-5
[6]Markowska M,Oberle R,Juzwin S.Optimizing Caco-2 cell monolayers to increase throughput in drug intestinal absorption analysis.JPharmacol Toxicol Metllod,2001,46(1):51-55
[7]Hilgers A R,ConradiR A,Burton P S.Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa.Pharm Res,1990,7(9):902-910
[8]Artusson P,Karlsson J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells[J].Biochem Biophys Res Commum,1991,175(3):880-885
[9]Nellans HN.Paracellular intestinal transport:Modulation of absorption[J].Adv.Drug Deliv.Rev,1991,7:339-364
[10]Lennemas H,Nilsson D,Aquilonius SM,et al.The effect of L-leucin on the absorption of levodopa,studied by regional jejeunal perfusion in man[J].Br.J.Clin. Pharmacol, 1993,35:243-250
[11]Phung BV,Warnery A,Scherman D,et al.Interaction of pristinamycin I with P-glycoprotein in human intestinal epithelial cells[J].Eur.J.Pharmacol,1995,288:187-192
[12]Lazorova L,Artursson P,Engstrom A,et al.Transport of an influenza virus vaccine formulation(iscom)in human intestinal epithelial(Caco-2) cells[J].Am.J.Physiol,1996,270:6554-6564
[13]L.Gan LS,Thankker DR.Applications of the Caco-2 model in the design and development of orally active drugs:elucidation of biochemical and physical barriers posed by the intestinal epithelium[J].Adv Drug Delivery Rev,1997,23(1-3):77-98
[14]Kaeko M,Sumie S.UniqueUptake,and Transport of Isoflavone Aglycones by Human Iniestinal Caco-2Cells:Compaison of lsoflavonoids and Flavonoids.The Journal of Nutrilion,2002,132:1956-1961
[15]Obermeier MT,Chong S,Dando SA.Predrugs of BMS-183920:metabolism and permeability considerations[J].J pharm Sci,1996,85(8):828-833
[16]Jeanelle B,Dan B.In vitro digestion and lactase etreatment influence uptake of quereetin and quereetin glueoside by theCaco-2 cellmonolayer.TheJournal of,Nutrition,2005,135:1-11
[17]Yan Liu,Ming Hu.Absorption and Metabolism of Flavonoids in the Caco-2 Cel lCulture Model and a Perused Rat Intestinal Model.Drug Metabolism and Disposition,2002,30(4):370-377
[18]Raymond PG,Darrell RV C.Iron Uptake is Enhaneed in Caco-2 Cell Monolayers by Cysteine and Redueed Cysteiny 1Glyeine.The Journal of Nutrition,1997,127(4):642-647
[19]沙先谊,方晓玲,吴云娟.9-硝基喜树碱在Caco-2细胞模型中的体外摄取、转运及外 排动力学[J].药学学报,2004,39(10)：839-843
[20]Hovggard L,rondsted H.Drug delivery studies in Caco-2 onolayers.Ⅳ.Absorption enhancer effects of cyclodextrins[J].Pharm Res,1995,12(9):1328-1332
[21]62.Ginski MJ,Olli JE.Prediction of dissolution absorption relationships from a dissolution/Caco-2 system[J].Int J Pharm,1999,177(1):17-125
[22]D'Agostino L,pignata S.Polymine uptake by human colon carcinoma cell line Caco-2 [J].Digestion,1990,46(suppl 2):352-359
[23]Tomon M,Tenenhouse HS,Jones G.Expression of 25-hydroxyvitamin D3-24-hydroxylase activity in Caco-2 cells.An in vitro model of intestinal vitamin D metabolism [J].Endocrinology,1990,126(6):2868-2875
[24]Chen JU,Hu M,Zhu YP,et al.The study of kinetic model for the frug efflux from a human intestinal epithelial membrane model system [J].Acta Acad Med Shanghai(in chinese),1996,23(4):247-251
[25]Dantzig AH,Duckworth DC,Tabas LB.Transport mechanisms responsible for the absorption of loracarbef,cefixime and cefuroxime axetil into human intestinal Caco-2 cells[J].Biochem Biophys Acta,1994,1191(1):7-13
[26]Hidalgo IJ,Borchardt RT.Transport of a large neutral amino acid(phenylalanine)in a human intestinal epithelial cell line:Caco-2[J].Biochem Biophys Acta,1990,1028 (1):25-30
[27]Mason JB,Shoda R,Haskell M,et al.Carrier affinity as a mechanism for the pH-dependence of folate transport in the small intestine[J].Biochem Biophys Acta,1990,1024(2):331-335
[28]Meunier V,Bourrie M,Berger Y.The human intestinal epithelial cell line Caco-2 pharmacological and pharmacokinetic applications[J].Cell Biol Toxicol,1995,11(3-4):187-194
[29]Rosenberg DW.Regulation of cytochrome P450 in cultured human colonic cells[J]. Arch Biochem Biophys,1993,300(1):186-192
[30]Tse CM,Levine SA,yun CH.Cloning and expression of a rabbit cDNA encoding a serum-activated ethylisopropylamiloride resistant epithelial Na+/H+ exchanger isoform (NHE-2)[J].J Biol Chem,1993,268(16):11917-11924
[31]谢海棠,王广基,赵小辰,等.Caco-2细胞对人参皂苷Rg3的摄取及代谢研究[J].中国临床药理学与治疗学,2004,9(3)：257-260
[32]Bae EA,Han MJ,Choo MK,et al.Metabolism of 20(S)-and 20(R)-ginsenoside Rg3 by human intestinal bacteria and its relation to in vitro biological activities[J].BiolPharm Bull,2002,25(1):58-63
[33]Artursson P.Epithelial transport of drugs in cell culture Ⅰ:A model for studying the passive diffusion of drugs over intestinal absorptive(Caco-2)cells.J Pharm Sci,1990,79(2):476-482
[34]Woll J S,Lee C H,Shim C K,Hwang S J. Enhanced oral bioavailability of paclitaxel by coadministration of the P-glycoprotein inhibitor KR30031.Pharm Res,2003,20(1):24-30
[35]Menon R M,Barr W H.Transporters involved in apica land basolateral uptake of ceftibuten into Caco-2 cells.Biopharm Drug Dispos,2002,23(8):317-32
[36]Liang E,Chessic K,YaZdanian M.Evaluation of an aceelerated Caco-2 Permeability model.J Pharm Sci,2000,89(3):336-345
[37]林晓,徐德生,冯怡.药物的肠吸收与处置研究进展.中国临床药理学杂志,2004,20(1)：72-75
[38]Patel N H,Rothenberg M L.Multidrug resistance in cancer chemotherapy.Invest New Drugs,1994,12(l):1-13
[39]鲁小笋.肠道P-糖蛋白和细胞色素P4503A对口服活性成分吸收的联合作用.国外医学-药学分册,2004,31(2)：108-11
[40]曾苏.药物代谢学.杭州：浙江大学出版社,2004,66-80
[41]Walgren R A,Kamaky K J,Lindenmayer G E,Walle T.Effiux of dietary flavonoid quercetin 4'β-glucoside across human intestinal Caco-2 cell monolayers by apical multidrug resistance-associated protein-2.J Pharmacol ExP Ther,2000,294(3):830-836
[42]Takara K,Tsujimoto M,Ohnishi N,et al.Effects of continuous exposure to digoxin on MDR1 function and expression in Caco-2 cells[J].Pharmacol,2003,55(5):675-681
[43]Oitate M,Nakaki R,Koyabu N.Transcellular transport of genistein,a soybean-derived isoflavonem across human colon carcinoma cell line(Caco-2)[J].Biopharm Drug Dispos,2001,22(1):23-29
[44]Kohle C,Badary 0 A,Nill K,Bock-Hennig B S,Bock K W.Serotonin lueuronidation by Ah receptor and oxidative stress-indueible human UDP-glucuronosyltransferase (UGT) 1A6 in Caco-2 cells.Biochem Pharmacol,2005,69(9):1397-1402
[45]Woo JS,Lee CH,Shim CK,et al.Enhanced oral bioavailability of paclitaxel by coadminiatration of the P-glycoprotein inhibitor KR30031[J].Pharm Res,2003,20 (1):24-30
[46]Maeng HJ,Yoo HJ,Kim IW.P-glycoprotein-mediated transport of berberine across Caco-2 cell monolayers[J].J Pharm Sci,2002,91(12):2614-2621
[47]Hollman PCH,De Vries JHM,Van Leauwen SD,et al.Absorption of dietary quercetin glyconides and quercetin in healthy ileostomy volunteers[J].Am J Clin Nutr,1995, 62:1276-1282
[48]Manach C,Morand C,Texier O,et al.Quercetin metabolites in plasma of rats fed diets containing rutin or quercetin[J].J Nutr 1995,125:1911-1922
[49]杨建波,姚佳,杨洁.药物体外ADME筛选模型[J].药学进展,2004,28(4)：163-167
[50]杨海涛,王广基.Caco-2单层细胞模型及其在药学中的应用[J].药学学报,2000,35(10)：797-800
[51]蒋学华,贾运涛,袁媛,等.Caco-2细胞模型在口服药物吸收过程研究中的应用[J].中国药学杂志,2002,37(5)：325-327
[52]关溯Caco-2细胞模型-药物吸收研究的有效“工具”[J].中国药理学报,2004,20(6)：609-614
[53]Lucas H. Arch Pharm.1856,95:145
[54]Chiang HC, et al. Chem Commun.1967,1201
[55]Woods WC,et al. J Am Chem Soc.1968,90:552
[56]Della Casa de Marcano DP,et al. Ibid.1969,1282
[57]Della Casa de Marcano DP,et al. Ibid.1970,1381-1382
[58]Della Casa de Marcano DP,et al. Chem Commun.1970,216.
[59]W ani MC, Taylor HL, Wall ME, et al. Plant antitumor agents, Ⅵ. The isolation and structure of taxol, a novel antileukemia and antitumor agent from Taxus brevifolia. J Am Chem Soc,1971,93:2325～2327
[60]Diergarten K, Dreps A. Taxol:A new antineoplastic agent. Onkologie,1993,16(5):329～ 337
[61]Rowinsky EK, Donehower RC. Paclitaxel(taxol). N Engl J Med,1995,332(15):1004～ 1014
[62]Oliver SJ, Banguerigo ML, Brahn E. Suppression of collagen-induced arthritis using an angiogenesis inhibitor, AGM-1470, and a microtubule stabilizer, taxol.Cell Immunol,1994, 157(1):291-299.
[63]Pouvelle B, farley PJ, Long CA, et al. Taxol arrests the development of blood-stage plasmodium faciparum in vitro and plasmodium chabaudi adimi in malaria-infected mice. J clin Invest,1994,1:413-417
[64]Burke WJ, Raghu G, Strong R. Taxio protects against calcium-mediate death of differentiated rat pheochromocytoma cells. Life Sci,1994,55(16):313-319.
[65]Wood DDL, Miao SYP, Pilayo JC, et al. Taxol inhibits progression of congenital polycytic kidney disease. Nature,1994,368(6473):750-753
[66]Shuler ML, Hirasuna TJ, Willard DM. Kinetic of taxol production by tissue ulture.Second National Cancer Institute Workshop on Taxol and Taxus. AlexandriaVirginia USA, Mark Center,1992,9:23-24
[67]Sharma A, Straubinger RM. Novel taxol formulation:Preparation andcharacterization of taxol-containing liposomes. Pharm Res,1994,11(16):889-896
[68]Kingston DGI, Samaranake G, Ivey CA. The Chemistry of taxol, A clinically useful anticancer agent. J Nat Prod,1990,53(1):1-2
[69]Lataste H,Senilh V,Wright M,Guenard D and Potier P. Relationships between the Structures of Taxol and Baccatin Ⅲ Derivatives and Their in Vitro Action on the Disassembly of Mammalian Brain and Physarum Amebal Microtubules[J]. Proceedings of the National Academy of Sciences of the United States of America,1984,81(13):4090-4094
[70]Mandai T,Okumoto H,Oshitari T,Nakanishi K,Mikuni K,Hara K,Hara K,Iwatani W,Amano T,Nakamura K and Tsuchiya Y. Synthesis and Biological Evaluation of Water Soluble Taxoids Bearing Sugar Moieties[J]. Heterocycles,2001,54(2):561-566
[71]Mandai T,Okumoto H,Hara K,Mikuni K,Hara K and Hamada H. Preparation of Taxoid Derivatives as Anticancer Agents with Increased Solubility[P]. JP:421092,1998
[72]Bandai T,Okumoto H,Mikuni K,Hara K and Hamada H. Preparation of Taxoid Derivatives as Anticancer Agents with Improved Solubility[P]. JP:731474,1997
[73]Hara K,Mikuni K,Bandai T,Okumoto H and Hamada H. Glycosylated Taxoid Derivatives as Water-soluble Anticancer Agents and Their Manufacture with Transglycosidase[P]. JP: 618755,1997
[74]Michel P. Carbohydrate Derivatives of Paclitaxel and Docetaxel, Method for Producing Same and Uses There of[P]. USP:569854,2004
[75]Biedler JL,Riehm H. Cellular Resistance to Actinomycin D in Chinese Hamster Vells in Vitro:Cross-Resistance,Radioautographic,and Cytogenetic Studies. Cancer Research,1 970,30(4):1174-1184
[76]Juliano RL,Ling V A Surface Glycoprotein Modulating Drug Permeability in Chinese Hamster Ovary Cell Mutants. Biochimica and Biophysica Acta,1976,455(1):152-162.
[77]Marzolini C,Paus E,Buclin T,et al. Polymorphisms in Human MDR1 (P-Glycoprotein):Recent Advances and Clinical Relevance. Clinical Pharmacology and Therapeutics,2004,75(1):13-33
[78]Ceckova-Novotna M,Pavek P,Stand F. P-glycoprotein in the Placenta:Expression,Localization,Regulation and Function. Reprod Toxicol,2006 22(3):400-410
[79]Schwab M,Eichelbaum M,Fromm MF. Genetic Polymorphisms of the Human MDR1 Drug Transporter. Annual Review of Pharmacology and Toxicology,2003,43:285-307.
[80]Gottesman M M, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter [J]. Ann Rev Biochem,1993,62:385-427
[81]高贤钧.中草药钙通道阻滞剂的研究与展望.中西医结合杂志,1990,10(7)：425
[82]立彦,王自正,俞腾飞等.人参皂甙Rb1对耐长春新碱的急性早幼粒白血病(HL60/VCR)细胞多药耐药逆转的实验研究.放射免疫学杂志,2005,18(5)：362.365
[83]史曦凯,张翼军.人参皂苷单体Rb1对多药耐药细胞系K562/HHT细胞内柔红霉素浓度的影响.重庆医学,2001,30(6)：507-509
[84]敖忠芳,夏薇.汉防己甲素逆转白血病细胞耐药的研究.中华血液学杂志,1995,16(5)：235-237
[85]Ferry D R, Traunecker H, Kerr D J. Clinical trials of P-glycoprotein reversal in solid tumours [J]. Eur J Cancer,1996,32a:1070-1081
[86]Jiang S, Zu Y, Fu Y, Zhang Y and Efferth T. Activation of the mitochondriadriven pathway of apoptosis in human PC-3 prostate cancer cells by a novel hydrophilic paclitaxel derivative,7-xylosyl-10-deacetylpaclitaxel.International Journal of Oncology 2008; 33(1): 103-111
[87]杨海涛,王广基.Caco-2单层细胞模型及其在药学中的应用[J].药学学报,2000,35(10)：797-800
[88]Markowska M,Oberle R,Juzwin S,et al.Optimizing Caco-2 cell monolayers to increase throughput in drug intestinal absorption analysis.J Pharmacol Toxicol Methods.2001; 46(1):51-5
[89]Mosmnm T.Rapid colorimetric assay for cellular growth and surival application to proliferation and cytotoxicity assays.J Immunol Method,1983,65(1-2):55-63
[90]Sleller H.Mechanisms and genes of cellular suicide. Science,1995,267(10):1445-1449
[91]杨涛,吴宗群.抗肿瘤紫杉醇的实验与临床应用进展[J].大连医科大学学报,2007,29(2)：197-199
[92]RaymondP.GCaco-2 Cell Ferritin Formation Prediets Nonradiolabeled Food Iron Availabillty in an InVitro Digestio/Caco-2 Cell Culture Model.The Journal of Nutrition,1998,128(9):1555-1561
[93]RouquayrolM,GaucherB,RoeheD.Transepithelial transport of prodrugs of the HIV Protease inhibitors saquinavir,indinavir and nelfinavir acrossCaco-2 cell monolayers. Pharm Res,2002,19(11):1701-1712
[94]Shougang Jiang,Yuangang Zu,Yu Zhang, Yuejie Fu, Thomas Efferth,Zhuo Wang,Jingtao Wang.Transport of a hydrophilic paclitaxel derivative,7-xylosyl-10-deacetylpaclitaxel by human intestinal epithelial Caco-2 cells Plant Medica
[2]Bruan A,Hammerle S,Suda K,et al.Cell cultures as tools in biopharmacy.Eur J Phmar Sci.2000; 11 (Suppl2):S51-60
[3]Yee S.In vitro permeabiliy across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth.Pharm Res.1997;14(6):763-765
[4]Gan LSL,Thakker DR.Applications of the Caco-2 mode in the design and development of orally active drugs:Elucidation of biochemical and physical barriers posed by the intestinal epithelium.Adv Drug Deliv Rev 1997;23(1):77-98
[5]Garberg P,Eriksson P,Schipper N,et al.Automated absorption assessment using Caco-2 cells cultured on both sides of polycarbonate membranes.Pharm Res.1999:16(3):441-5
[6]Markowska M,Oberle R,Juzwin S.Optimizing Caco-2 cell monolayers to increase throughput in drug intestinal absorption analysis.JPharmacol Toxicol Metllod,2001,46(1):51-55
[7]Hilgers A R,ConradiR A,Burton P S.Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa.Pharm Res,1990,7(9):902-910
[8]Artusson P,Karlsson J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells[J].Biochem Biophys Res Commum,1991,175(3):880-885
[9]Nellans HN.Paracellular intestinal transport:Modulation of absorption[J].Adv.Drug Deliv.Rev,1991,7:339-364
[10]Lennemas H,Nilsson D,Aquilonius SM,et al.The effect of L-leucin on the absorption of levodopa,studied by regional jejeunal perfusion in man[J].Br.J.Clin. Pharmacol, 1993,35:243-250
[11]Phung BV,Warnery A,Scherman D,et al.Interaction of pristinamycin I with P-glycoprotein in human intestinal epithelial cells[J].Eur.J.Pharmacol,1995,288:187-192
[12]Lazorova L,Artursson P,Engstrom A,et al.Transport of an influenza virus vaccine formulation(iscom)in human intestinal epithelial(Caco-2) cells[J].Am.J.Physiol,1996,270:6554-6564
[13]L.Gan LS,Thankker DR.Applications of the Caco-2 model in the design and development of orally active drugs:elucidation of biochemical and physical barriers posed by the intestinal epithelium[J].Adv Drug Delivery Rev,1997,23(1-3):77-98
[14]Kaeko M,Sumie S.UniqueUptake,and Transport of Isoflavone Aglycones by Human Iniestinal Caco-2Cells:Compaison of lsoflavonoids and Flavonoids.The Journal of Nutrilion,2002,132:1956-1961
[15]Obermeier MT,Chong S,Dando SA.Predrugs of BMS-183920:metabolism and permeability considerations[J].J pharm Sci,1996,85(8):828-833
[16]Jeanelle B,Dan B.In vitro digestion and lactase etreatment influence uptake of quereetin and quereetin glueoside by theCaco-2 cellmonolayer.TheJournal of,Nutrition,2005,135:1-11
[17]Yan Liu,Ming Hu.Absorption and Metabolism of Flavonoids in the Caco-2 Cel lCulture Model and a Perused Rat Intestinal Model.Drug Metabolism and Disposition,2002,30(4):370-377
[18]Raymond PG,Darrell RV C.Iron Uptake is Enhaneed in Caco-2 Cell Monolayers by Cysteine and Redueed Cysteiny 1Glyeine.The Journal of Nutrition,1997,127(4):642-647
[19]沙先谊,方晓玲,吴云娟.9-硝基喜树碱在Caco-2细胞模型中的体外摄取、转运及外 排动力学[J].药学学报,2004,39(10)：839-843
[20]Hovggard L,rondsted H.Drug delivery studies in Caco-2 onolayers.Ⅳ.Absorption enhancer effects of cyclodextrins[J].Pharm Res,1995,12(9):1328-1332
[21]62.Ginski MJ,Olli JE.Prediction of dissolution absorption relationships from a dissolution/Caco-2 system[J].Int J Pharm,1999,177(1):17-125
[22]D'Agostino L,pignata S.Polymine uptake by human colon carcinoma cell line Caco-2 [J].Digestion,1990,46(suppl 2):352-359
[23]Tomon M,Tenenhouse HS,Jones G.Expression of 25-hydroxyvitamin D3-24-hydroxylase activity in Caco-2 cells.An in vitro model of intestinal vitamin D metabolism [J].Endocrinology,1990,126(6):2868-2875
[24]Chen JU,Hu M,Zhu YP,et al.The study of kinetic model for the frug efflux from a human intestinal epithelial membrane model system [J].Acta Acad Med Shanghai(in chinese),1996,23(4):247-251
[25]Dantzig AH,Duckworth DC,Tabas LB.Transport mechanisms responsible for the absorption of loracarbef,cefixime and cefuroxime axetil into human intestinal Caco-2 cells[J].Biochem Biophys Acta,1994,1191(1):7-13
[26]Hidalgo IJ,Borchardt RT.Transport of a large neutral amino acid(phenylalanine)in a human intestinal epithelial cell line:Caco-2[J].Biochem Biophys Acta,1990,1028 (1):25-30
[27]Mason JB,Shoda R,Haskell M,et al.Carrier affinity as a mechanism for the pH-dependence of folate transport in the small intestine[J].Biochem Biophys Acta,1990,1024(2):331-335
[28]Meunier V,Bourrie M,Berger Y.The human intestinal epithelial cell line Caco-2 pharmacological and pharmacokinetic applications[J].Cell Biol Toxicol,1995,11(3-4):187-194
[29]Rosenberg DW.Regulation of cytochrome P450 in cultured human colonic cells[J]. Arch Biochem Biophys,1993,300(1):186-192
[30]Tse CM,Levine SA,yun CH.Cloning and expression of a rabbit cDNA encoding a serum-activated ethylisopropylamiloride resistant epithelial Na+/H+ exchanger isoform (NHE-2)[J].J Biol Chem,1993,268(16):11917-11924
[31]谢海棠,王广基,赵小辰,等.Caco-2细胞对人参皂苷Rg3的摄取及代谢研究[J].中国临床药理学与治疗学,2004,9(3)：257-260
[32]Bae EA,Han MJ,Choo MK,et al.Metabolism of 20(S)-and 20(R)-ginsenoside Rg3 by human intestinal bacteria and its relation to in vitro biological activities[J].BiolPharm Bull,2002,25(1):58-63
[33]Artursson P.Epithelial transport of drugs in cell culture Ⅰ:A model for studying the passive diffusion of drugs over intestinal absorptive(Caco-2)cells.J Pharm Sci,1990,79(2):476-482
[34]Woll J S,Lee C H,Shim C K,Hwang S J. Enhanced oral bioavailability of paclitaxel by coadministration of the P-glycoprotein inhibitor KR30031.Pharm Res,2003,20(1):24-30
[35]Menon R M,Barr W H.Transporters involved in apica land basolateral uptake of ceftibuten into Caco-2 cells.Biopharm Drug Dispos,2002,23(8):317-32
[36]Liang E,Chessic K,YaZdanian M.Evaluation of an aceelerated Caco-2 Permeability model.J Pharm Sci,2000,89(3):336-345
[37]林晓,徐德生,冯怡.药物的肠吸收与处置研究进展.中国临床药理学杂志,2004,20(1)：72-75
[38]Patel N H,Rothenberg M L.Multidrug resistance in cancer chemotherapy.Invest New Drugs,1994,12(l):1-13
[39]鲁小笋.肠道P-糖蛋白和细胞色素P4503A对口服活性成分吸收的联合作用.国外医学-药学分册,2004,31(2)：108-11
[40]曾苏.药物代谢学.杭州：浙江大学出版社,2004,66-80
[41]Walgren R A,Kamaky K J,Lindenmayer G E,Walle T.Effiux of dietary flavonoid quercetin 4'β-glucoside across human intestinal Caco-2 cell monolayers by apical multidrug resistance-associated protein-2.J Pharmacol ExP Ther,2000,294(3):830-836
[42]Takara K,Tsujimoto M,Ohnishi N,et al.Effects of continuous exposure to digoxin on MDR1 function and expression in Caco-2 cells[J].Pharmacol,2003,55(5):675-681
[43]Oitate M,Nakaki R,Koyabu N.Transcellular transport of genistein,a soybean-derived isoflavonem across human colon carcinoma cell line(Caco-2)[J].Biopharm Drug Dispos,2001,22(1):23-29
[44]Kohle C,Badary 0 A,Nill K,Bock-Hennig B S,Bock K W.Serotonin lueuronidation by Ah receptor and oxidative stress-indueible human UDP-glucuronosyltransferase (UGT) 1A6 in Caco-2 cells.Biochem Pharmacol,2005,69(9):1397-1402
[45]Woo JS,Lee CH,Shim CK,et al.Enhanced oral bioavailability of paclitaxel by coadminiatration of the P-glycoprotein inhibitor KR30031[J].Pharm Res,2003,20 (1):24-30
[46]Maeng HJ,Yoo HJ,Kim IW.P-glycoprotein-mediated transport of berberine across Caco-2 cell monolayers[J].J Pharm Sci,2002,91(12):2614-2621
[47]Hollman PCH,De Vries JHM,Van Leauwen SD,et al.Absorption of dietary quercetin glyconides and quercetin in healthy ileostomy volunteers[J].Am J Clin Nutr,1995, 62:1276-1282
[48]Manach C,Morand C,Texier O,et al.Quercetin metabolites in plasma of rats fed diets containing rutin or quercetin[J].J Nutr 1995,125:1911-1922
[49]杨建波,姚佳,杨洁.药物体外ADME筛选模型[J].药学进展,2004,28(4)：163-167
[50]杨海涛,王广基.Caco-2单层细胞模型及其在药学中的应用[J].药学学报,2000,35(10)：797-800
[51]蒋学华,贾运涛,袁媛,等.Caco-2细胞模型在口服药物吸收过程研究中的应用[J].中国药学杂志,2002,37(5)：325-327
[52]关溯Caco-2细胞模型-药物吸收研究的有效“工具”[J].中国药理学报,2004,20(6)：609-614
[53]Lucas H. Arch Pharm.1856,95:145
[54]Chiang HC, et al. Chem Commun.1967,1201
[55]Woods WC,et al. J Am Chem Soc.1968,90:552
[56]Della Casa de Marcano DP,et al. Ibid.1969,1282
[57]Della Casa de Marcano DP,et al. Ibid.1970,1381-1382
[58]Della Casa de Marcano DP,et al. Chem Commun.1970,216.
[59]W ani MC, Taylor HL, Wall ME, et al. Plant antitumor agents, Ⅵ. The isolation and structure of taxol, a novel antileukemia and antitumor agent from Taxus brevifolia. J Am Chem Soc,1971,93:2325～2327
[60]Diergarten K, Dreps A. Taxol:A new antineoplastic agent. Onkologie,1993,16(5):329～ 337
[61]Rowinsky EK, Donehower RC. Paclitaxel(taxol). N Engl J Med,1995,332(15):1004～ 1014
[62]Oliver SJ, Banguerigo ML, Brahn E. Suppression of collagen-induced arthritis using an angiogenesis inhibitor, AGM-1470, and a microtubule stabilizer, taxol.Cell Immunol,1994, 157(1):291-299.
[63]Pouvelle B, farley PJ, Long CA, et al. Taxol arrests the development of blood-stage plasmodium faciparum in vitro and plasmodium chabaudi adimi in malaria-infected mice. J clin Invest,1994,1:413-417
[64]Burke WJ, Raghu G, Strong R. Taxio protects against calcium-mediate death of differentiated rat pheochromocytoma cells. Life Sci,1994,55(16):313-319.
[65]Wood DDL, Miao SYP, Pilayo JC, et al. Taxol inhibits progression of congenital polycytic kidney disease. Nature,1994,368(6473):750-753
[66]Shuler ML, Hirasuna TJ, Willard DM. Kinetic of taxol production by tissue ulture.Second National Cancer Institute Workshop on Taxol and Taxus. AlexandriaVirginia USA, Mark Center,1992,9:23-24
[67]Sharma A, Straubinger RM. Novel taxol formulation:Preparation andcharacterization of taxol-containing liposomes. Pharm Res,1994,11(16):889-896
[68]Kingston DGI, Samaranake G, Ivey CA. The Chemistry of taxol, A clinically useful anticancer agent. J Nat Prod,1990,53(1):1-2
[69]Lataste H,Senilh V,Wright M,Guenard D and Potier P. Relationships between the Structures of Taxol and Baccatin Ⅲ Derivatives and Their in Vitro Action on the Disassembly of Mammalian Brain and Physarum Amebal Microtubules[J]. Proceedings of the National Academy of Sciences of the United States of America,1984,81(13):4090-4094
[70]Mandai T,Okumoto H,Oshitari T,Nakanishi K,Mikuni K,Hara K,Hara K,Iwatani W,Amano T,Nakamura K and Tsuchiya Y. Synthesis and Biological Evaluation of Water Soluble Taxoids Bearing Sugar Moieties[J]. Heterocycles,2001,54(2):561-566
[71]Mandai T,Okumoto H,Hara K,Mikuni K,Hara K and Hamada H. Preparation of Taxoid Derivatives as Anticancer Agents with Increased Solubility[P]. JP:421092,1998
[72]Bandai T,Okumoto H,Mikuni K,Hara K and Hamada H. Preparation of Taxoid Derivatives as Anticancer Agents with Improved Solubility[P]. JP:731474,1997
[73]Hara K,Mikuni K,Bandai T,Okumoto H and Hamada H. Glycosylated Taxoid Derivatives as Water-soluble Anticancer Agents and Their Manufacture with Transglycosidase[P]. JP: 618755,1997
[74]Michel P. Carbohydrate Derivatives of Paclitaxel and Docetaxel, Method for Producing Same and Uses There of[P]. USP:569854,2004
[75]Biedler JL,Riehm H. Cellular Resistance to Actinomycin D in Chinese Hamster Vells in Vitro:Cross-Resistance,Radioautographic,and Cytogenetic Studies. Cancer Research,1 970,30(4):1174-1184
[76]Juliano RL,Ling V A Surface Glycoprotein Modulating Drug Permeability in Chinese Hamster Ovary Cell Mutants. Biochimica and Biophysica Acta,1976,455(1):152-162.
[77]Marzolini C,Paus E,Buclin T,et al. Polymorphisms in Human MDR1 (P-Glycoprotein):Recent Advances and Clinical Relevance. Clinical Pharmacology and Therapeutics,2004,75(1):13-33
[78]Ceckova-Novotna M,Pavek P,Stand F. P-glycoprotein in the Placenta:Expression,Localization,Regulation and Function. Reprod Toxicol,2006 22(3):400-410
[79]Schwab M,Eichelbaum M,Fromm MF. Genetic Polymorphisms of the Human MDR1 Drug Transporter. Annual Review of Pharmacology and Toxicology,2003,43:285-307.
[80]Gottesman M M, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter [J]. Ann Rev Biochem,1993,62:385-427
[81]高贤钧.中草药钙通道阻滞剂的研究与展望.中西医结合杂志,1990,10(7)：425
[82]立彦,王自正,俞腾飞等.人参皂甙Rb1对耐长春新碱的急性早幼粒白血病(HL60/VCR)细胞多药耐药逆转的实验研究.放射免疫学杂志,2005,18(5)：362.365
[83]史曦凯,张翼军.人参皂苷单体Rb1对多药耐药细胞系K562/HHT细胞内柔红霉素浓度的影响.重庆医学,2001,30(6)：507-509
[84]敖忠芳,夏薇.汉防己甲素逆转白血病细胞耐药的研究.中华血液学杂志,1995,16(5)：235-237
[85]Ferry D R, Traunecker H, Kerr D J. Clinical trials of P-glycoprotein reversal in solid tumours [J]. Eur J Cancer,1996,32a:1070-1081
[86]Jiang S, Zu Y, Fu Y, Zhang Y and Efferth T. Activation of the mitochondriadriven pathway of apoptosis in human PC-3 prostate cancer cells by a novel hydrophilic paclitaxel derivative,7-xylosyl-10-deacetylpaclitaxel.International Journal of Oncology 2008; 33(1): 103-111
[87]杨海涛,王广基.Caco-2单层细胞模型及其在药学中的应用[J].药学学报,2000,35(10)：797-800
[88]Markowska M,Oberle R,Juzwin S,et al.Optimizing Caco-2 cell monolayers to increase throughput in drug intestinal absorption analysis.J Pharmacol Toxicol Methods.2001; 46(1):51-5
[89]Mosmnm T.Rapid colorimetric assay for cellular growth and surival application to proliferation and cytotoxicity assays.J Immunol Method,1983,65(1-2):55-63
[90]Sleller H.Mechanisms and genes of cellular suicide. Science,1995,267(10):1445-1449
[91]杨涛,吴宗群.抗肿瘤紫杉醇的实验与临床应用进展[J].大连医科大学学报,2007,29(2)：197-199
[92]RaymondP.GCaco-2 Cell Ferritin Formation Prediets Nonradiolabeled Food Iron Availabillty in an InVitro Digestio/Caco-2 Cell Culture Model.The Journal of Nutrition,1998,128(9):1555-1561
[93]RouquayrolM,GaucherB,RoeheD.Transepithelial transport of prodrugs of the HIV Protease inhibitors saquinavir,indinavir and nelfinavir acrossCaco-2 cell monolayers. Pharm Res,2002,19(11):1701-1712
[94]Shougang Jiang,Yuangang Zu,Yu Zhang, Yuejie Fu, Thomas Efferth,Zhuo Wang,Jingtao Wang.Transport of a hydrophilic paclitaxel derivative,7-xylosyl-10-deacetylpaclitaxel by human intestinal epithelial Caco-2 cells Plant Medica