川丁特罗体外吸收转运研究
摘要
本实验建立了Caco-2以及MDCK-MDR1和MDCK等细胞模型，并用跨膜电阻值、荧光黄透过量等评价了模型，证明模型得到的数值是真实可用的。而后利用这3种模型研究了川丁特罗的转运以及吸收上的一些性质。川丁特罗（2-(4-氨基-3-氯-5-三氟甲基苯基)-2-叔丁氨基乙醇，trantinterol），是沈阳药科大学程卯生教授在马布特罗的结构基础上，通过结构修饰，自主研制的治疗哮喘疾病的新型β2受体激动剂。
一、利用Caco-2细胞模型对川丁特罗的转运性质进行研究
建立Caco-2单层细胞模型，并通过生长曲线、跨膜电阻值（TEER）以及荧光黄的透过率的测定证明，本实验室所建立的Caco-2细胞模型可应用于药物转运的研究。
利用成功建立的Caco-2细胞模型研究川丁特罗转运性质，将维拉帕米抑制组作为阴性对照组。结果表明，川丁特罗在Caco-2细胞模型上的外排率为1.08；用维拉帕米抑制P-gp后，川丁特罗外排率为1.11。两者无显著性差异（P＞0.05），可见川丁特罗不是P-gp的底物，抑制P-gp后，不能显著改变川丁特罗的外排率。
二、利用MDCK和MDCK－MDR1细胞模型对川丁特罗的转运性质进行研究
建立MDCK和MDCK-MDR1体外药物转运细胞模型，通过跨膜电阻值（TEER）、荧光黄的表观渗透率（Papp）以及罗丹明123（Rho123）在细胞上的转运对模型进行评价。MDCK和MDCK-MDR1细胞的跨膜电阻均达到标准，荧光黄的表观渗透率均小于5%。用Rho123进行转运试验验证P-gp的表达量，MDCK-MDR1的外排率为5.16，说明P-gp的表达量很高。结果表明，MDCK和MDCK-MDR1细胞连接紧密，并且P-gp表达量较高，因此MDCK和MDCK-MDR1细胞模型成功建立了，各项指标符合规定，可以用于药物与P-gp相互作用的研究。
用MDCK和MDCK-MDR1细胞模型研究川丁特罗的双向转运。结果显示：川丁特罗在MDCK-MDR1和MDCK细胞上外排率极为相似，没有显著性差异，分别为1.2和1.05，说明川丁特罗不是P-gp的底物。
川丁特罗对Rho123在MDCK-MDR1细胞模型中转运的影响实验中，川丁特罗显著降低了Rho123的外排比率（P＜0.05）。这表明川丁特罗抑制了P-gp介导的Rho123外排，可能为P-gp的抑制剂。
在川丁特罗与P糖蛋白（P-gp）的ATP酶（ATPase）相互作用的研究中发现，川丁特罗对P-gp的抑制作用是通过抑制P-gp的ATPase活性实现的。
In this study, I established Caco-2, MDCK-MDR1and MDCK cellmodel, evaluated these cell model for transmembrane resistance value, fluorescentyellow Permeance to prove that the cell model is available. Then use these three kindsof models studies transportion and absorption of trantinterol. Used for trantinterol(2-(4-amino-3-chloro-5-(trifluoromethyl) phenyl)-2-tert aminoethanols, trantinterol), Shenyang PharmaceuticalUniversity, Professor Cheng Maosheng based on the Mabuterol structuredeveloped a new type of β2agonists.
1. Study the transport of trantinterol with Caco-2monolayer cell model
We established Caco-2monolayer cell model, and verificated it through thecell growth curve determination, the Lucifer yellow transmembrane resistancevalue, proofed the Caco-2cell model in this laboratory can be applied to thetransport study of drugs.
Caco-2cell model successfully established used for transportion study, andverapamil inhibition of the group as a negative control. The results show that theefflux ratio of trantintrol in the Caco-2cell model was1.08; negative control was1.11. There was no significant difference (P>0.05). After all trantintrol is not P-gpsubstrate.
2. Transport of trantinterol in MDCK-MDR1cell monolayer model
We established MDCK and MDCK-MDR1monolayer cell model, andverificated it through the cell growth curve determination, the Lucifer yellowtransmembrane resistance value, the TEER and the Rho123transportion. MDCKand MDCK-MDR1cell model were successfully established. Lucifer yellow apparentpermeability was less than5%. The efflux rate of Rho123was5.16, indicatingthat P-gp expression is high. The results show that MDCK and MDCK-MDR1cellconnected tightly, and highly expressed P-gp. After all MDCK and MDCK-MDR1cell model was successfully established, all the indexes required, can be used for thestudy of interaction of drugs with P-gp.
Study the transportion of trantintrol in MDCK and MDCK-MDR1cell model. The results showed that: the efflux rate of trantinterol in MDCK-MDR1andMDCK cells are1.2and1.05, respectively. There is no significant difference, sotrantinterol is not a P-gp substrate.
The effect of trantinerol on the transport of Rho123in MDCK-MDR1cell model,trantinterol significantly reduce the efflux ratio of Rho123(P <0.05). In thistable, trantinterol inhibits P-gp-mediated Rho123efflux, may be P-gp inhibitor.
Interaction studies for trantinerol and ATPase (ATPase) on P-glycoprotein (P-gp),show that trantinterol inhibit P-gp achieved by inhibiting the ATPase activity of P-gp.
一、利用Caco-2细胞模型对川丁特罗的转运性质进行研究
建立Caco-2单层细胞模型，并通过生长曲线、跨膜电阻值（TEER）以及荧光黄的透过率的测定证明，本实验室所建立的Caco-2细胞模型可应用于药物转运的研究。
利用成功建立的Caco-2细胞模型研究川丁特罗转运性质，将维拉帕米抑制组作为阴性对照组。结果表明，川丁特罗在Caco-2细胞模型上的外排率为1.08；用维拉帕米抑制P-gp后，川丁特罗外排率为1.11。两者无显著性差异（P＞0.05），可见川丁特罗不是P-gp的底物，抑制P-gp后，不能显著改变川丁特罗的外排率。
二、利用MDCK和MDCK－MDR1细胞模型对川丁特罗的转运性质进行研究
建立MDCK和MDCK-MDR1体外药物转运细胞模型，通过跨膜电阻值（TEER）、荧光黄的表观渗透率（Papp）以及罗丹明123（Rho123）在细胞上的转运对模型进行评价。MDCK和MDCK-MDR1细胞的跨膜电阻均达到标准，荧光黄的表观渗透率均小于5%。用Rho123进行转运试验验证P-gp的表达量，MDCK-MDR1的外排率为5.16，说明P-gp的表达量很高。结果表明，MDCK和MDCK-MDR1细胞连接紧密，并且P-gp表达量较高，因此MDCK和MDCK-MDR1细胞模型成功建立了，各项指标符合规定，可以用于药物与P-gp相互作用的研究。
用MDCK和MDCK-MDR1细胞模型研究川丁特罗的双向转运。结果显示：川丁特罗在MDCK-MDR1和MDCK细胞上外排率极为相似，没有显著性差异，分别为1.2和1.05，说明川丁特罗不是P-gp的底物。
川丁特罗对Rho123在MDCK-MDR1细胞模型中转运的影响实验中，川丁特罗显著降低了Rho123的外排比率（P＜0.05）。这表明川丁特罗抑制了P-gp介导的Rho123外排，可能为P-gp的抑制剂。
在川丁特罗与P糖蛋白（P-gp）的ATP酶（ATPase）相互作用的研究中发现，川丁特罗对P-gp的抑制作用是通过抑制P-gp的ATPase活性实现的。
In this study, I established Caco-2, MDCK-MDR1and MDCK cellmodel, evaluated these cell model for transmembrane resistance value, fluorescentyellow Permeance to prove that the cell model is available. Then use these three kindsof models studies transportion and absorption of trantinterol. Used for trantinterol(2-(4-amino-3-chloro-5-(trifluoromethyl) phenyl)-2-tert aminoethanols, trantinterol), Shenyang PharmaceuticalUniversity, Professor Cheng Maosheng based on the Mabuterol structuredeveloped a new type of β2agonists.
1. Study the transport of trantinterol with Caco-2monolayer cell model
We established Caco-2monolayer cell model, and verificated it through thecell growth curve determination, the Lucifer yellow transmembrane resistancevalue, proofed the Caco-2cell model in this laboratory can be applied to thetransport study of drugs.
Caco-2cell model successfully established used for transportion study, andverapamil inhibition of the group as a negative control. The results show that theefflux ratio of trantintrol in the Caco-2cell model was1.08; negative control was1.11. There was no significant difference (P>0.05). After all trantintrol is not P-gpsubstrate.
2. Transport of trantinterol in MDCK-MDR1cell monolayer model
We established MDCK and MDCK-MDR1monolayer cell model, andverificated it through the cell growth curve determination, the Lucifer yellowtransmembrane resistance value, the TEER and the Rho123transportion. MDCKand MDCK-MDR1cell model were successfully established. Lucifer yellow apparentpermeability was less than5%. The efflux rate of Rho123was5.16, indicatingthat P-gp expression is high. The results show that MDCK and MDCK-MDR1cellconnected tightly, and highly expressed P-gp. After all MDCK and MDCK-MDR1cell model was successfully established, all the indexes required, can be used for thestudy of interaction of drugs with P-gp.
Study the transportion of trantintrol in MDCK and MDCK-MDR1cell model. The results showed that: the efflux rate of trantinterol in MDCK-MDR1andMDCK cells are1.2and1.05, respectively. There is no significant difference, sotrantinterol is not a P-gp substrate.
The effect of trantinerol on the transport of Rho123in MDCK-MDR1cell model,trantinterol significantly reduce the efflux ratio of Rho123(P <0.05). In thistable, trantinterol inhibits P-gp-mediated Rho123efflux, may be P-gp inhibitor.
Interaction studies for trantinerol and ATPase (ATPase) on P-glycoprotein (P-gp),show that trantinterol inhibit P-gp achieved by inhibiting the ATPase activity of P-gp.
引文
[1]冯飞飞,张典瑞.口服药物吸收模型的研究进展[J].中国生化药物杂志,2009,30(5):351-353.
[2]陈伟薇,李俊,宋珏.药物肠吸收的研究方法进展[J].安徽医药,2009,13(4):349-352.
[3]金朝辉,徐珽,马音,唐尧.吸收促进剂研究进展概述[J].华西医学，2008，23（4）：940-941.
[4]Poelma FG, Tukker JJ. Evaluation of a chronically isolated internal loop in the ratfor the study of drug absorption kinetics [J]. J Pharm Sci.1987,76(6):433-436.
[5]祝诚诚，何新.药物肠道吸收研究方法[J].药物评价研究.2010,33(6):222-227.
[6]Braun A, H mmerle S, Suda K, et al. Cell cultures as tools in biopharmacy [J]. EurJ Pharm Sci.2000,11(2): S51-60.
[7]Hidalgo IJ, Raub TJ, Borchardt RT. Characterization of the human coloncarcinoma cell line (Caco-2) as a model system for intestinal epithelialpermeability [J]. Gastroenterology,1989,96(3):736-749.
[8]Troutman MD, Thakker DR. Novel experimental parameters to quantify themodulation of absorptive and secretory transport of compounds by P-glycoproteinin cell culture models of intestinal epithelium [J]. Pharm Res,2003,20(8):1210-1224.
[9]Artursson P, Karlsson J. Correlation between oral drug absorption in humans andapparent drug permeability coefficients in human intestinal epithelial (Caco-2)cells [J]. Biochem Biophys Res Commun,1991,175(3):880-885.
[10]赵静,梁爱华. Caco-2细胞模型及其在中药吸收转运研究中的应用[J].中国实验方剂学杂志,2009,15(5):79-83.
[11] Soldner A, Benet LZ, Mutschler E, et al. Active transport of the angiotensin-IIantagonist losartan and its main metabolite EXP3174across MDCK-MDR1andcaco-2cell monolayers [J]. Br J Pharmacol,2000,129(6):1235-1243.
[12]刘瑶,俞春娜,曾苏. P-糖蛋白高表达马丁达比狗肾上皮细胞系的建立[J].中国药学杂志.2009,44(21):1608-1613.
[13]刘瑶,曾苏. MDCK-MDR1细胞模型及其在药物透过研究中的应用进展[J].药学学报,2008,43(6):559-564.
[14]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.
[15] Liang E, Chessic K, Yazdanian M. Evaluation of an accelerated Caco-2cellpermeability model [J]. J Pharm Sci,2000,89(3):336-345.
[16]胡晓渝,姚彤炜,曾苏. Caco-2细胞系及其在药物吸收、代谢中的应用[J].中国现代应用药学杂志,2002,19(2):88-90.
[17] Behrens I, Kamm W, Dantzig AH, Kissel T.2004.Variation of peptidetransporter (PepT1and HPT1) expression in Caco-2cells as a function of cellorigin [J]. J Pharm Sci93:1743–1754.
[18] Richardson JCW, Scalera V, Simmons NL.1981.Identification of two strains ofMDCK cells which resemble separate nephron tubule segments [J]. BiochimBiophys Acta673:26–36.
[19] Yu H, Cook TJ, Sinko PJ.1997. Evidence for diminished functional expressionof intestinal transporters in Caco-2cell monolayers at high passages [J]. PharmRes14:757–762.
[20] Lu S, Gough AW, Bobrowski WF, Stewart BH.1996. Transport properties are notaltered across Caco-2cells with heightened TEER despite underlyingphysiological and ultrastructural changes [J].J Pharm Sci85:270–273.
[21] Lentz KA, Hayashi J, Lucisano LJ, Polli JE.2000.Development of a more rapid,reduced serum culture system for Caco-2monolayers and application to thebiopharmaceutics classification system [J]. Int J Pharm200:41–51.
[22] Bravo SA, Nielsen CU, Amstrup J, Frokjaer S,Bodin B.2004. In-depthevaluation of Gly-Sar transport parameters as a function of culture time in theCaco-2cell model [J]. Eur J Pharm Sci21:77–86.
[23] Anderle P, Niederer E, Rubas W, Hilgendorf C, Spahn-Langguth H,Wunderli-Allenspach H, Merkle HP, Langguth P.1998. P-glycoprotein (P-gp)mediated efflux in Caco-2cell monolayers: The influence of culturing conditionsand drug exposure on P-gp expression levels [J]. J Pharm Sci87:757–762.
[24] D’Souza VM, Shertzer HG, Menon AG, Pauletti GM.2003. High glucoseconcentration in isotonic media alters Caco-2cell permeability[J]. AAPSPharmSci5: article24.
[25] D’Souza VM, Buckley DJ, Buckley AR, Pauletti GM.2003. Extracellularglucose concentration alters functional activity of intestinal oligopeptidetransporter (PepT-1) in Caco-2cells[J]. J Pharm Sci92:594–603.
[26] DeMarco VG, Li N, Thomas J, West CM, Neu J.2003. Glutamine and barrierfunction in cultured Caco-2epithelial cell monolayers[J]. J Nutr133:2176–2179.
[27] Bestwick CS, Milne L.2001. Alteration of culture regime modifies antioxidantdefenses independent of intracellular reactive oxygen levels and resistance tosevere oxidative stress within confluent Caco-2‘‘intestinal cells’’[J]. Dig DisSci46:417–423.
[28] Ward RK, Mungall S, Carter J, Clothier RH.1997.Evaluation of tissue cultureinsert membrane compatibility in the fluorescein leakage assay [J]. Toxicol InVitro11:761–768.
[29] Adson A, Burton PS, Raub TJ, Barshun CL, Audus KL, Ho NFH.1995. Passivediffusion of weak organic electrolytes across Caco-2cellmonolayers:Uncoupling the contributions of hydrodynamic, transcellular andparacellular barriers[J]. J Pharm Sci84:1197–1204.
[30] Wilson G.1990. Cell culture techniques for the study of drug transport [J]. Eur JDrug Metab Pharmacokinet15:159–163.
[31]钟大放.以加权最小二乘法建立生物分析标准曲线的若干问题[J].药物分析杂志,1996,16(5):343-346.
[32] Pade V, Stavchansky S.1997. Estimation of the relative contribution of thetranscellular and paracellular pathway to the transport of passively absorbeddrugs in the Caco-2cell culture model [J].Pharm Res14:1210–1215.
[33] Tamihide MATSUNAGA,Eiji KOSE,Sachiyo YASUDA. Determination ofP-Glycoprotein ATPase Activity Using Luciferase[J]. Biol. Pharm. Bull.29(3)560—564(2006)
[34] Yoshiyuki SHIRASAKA,Yuko ONISHI,Aki SAKURAI, Evaluation of HumanP-Glycoprotein (MDR1/ABCB1) ATPase ActivityAssay Method by Comparingwith in Vitro Transport Measurements:Michaelis–Menten Kinetic Analysis toEstimate the Affinity of P-Glycoprotein to Drugs[J]. Biol. Pharm. Bull.29(12)2465—2471(2006)
[35] Luker GD, Piea CM, Kumar AS, et al. Effects of cholesterol and enantiomeric
cholesterol on P-glycoprotein localization and function in low density membrane
domains [J]. Biochemistry,2000,:39(29):8692.
[2]陈伟薇,李俊,宋珏.药物肠吸收的研究方法进展[J].安徽医药,2009,13(4):349-352.
[3]金朝辉,徐珽,马音,唐尧.吸收促进剂研究进展概述[J].华西医学，2008，23（4）：940-941.
[4]Poelma FG, Tukker JJ. Evaluation of a chronically isolated internal loop in the ratfor the study of drug absorption kinetics [J]. J Pharm Sci.1987,76(6):433-436.
[5]祝诚诚，何新.药物肠道吸收研究方法[J].药物评价研究.2010,33(6):222-227.
[6]Braun A, H mmerle S, Suda K, et al. Cell cultures as tools in biopharmacy [J]. EurJ Pharm Sci.2000,11(2): S51-60.
[7]Hidalgo IJ, Raub TJ, Borchardt RT. Characterization of the human coloncarcinoma cell line (Caco-2) as a model system for intestinal epithelialpermeability [J]. Gastroenterology,1989,96(3):736-749.
[8]Troutman MD, Thakker DR. Novel experimental parameters to quantify themodulation of absorptive and secretory transport of compounds by P-glycoproteinin cell culture models of intestinal epithelium [J]. Pharm Res,2003,20(8):1210-1224.
[9]Artursson P, Karlsson J. Correlation between oral drug absorption in humans andapparent drug permeability coefficients in human intestinal epithelial (Caco-2)cells [J]. Biochem Biophys Res Commun,1991,175(3):880-885.
[10]赵静,梁爱华. Caco-2细胞模型及其在中药吸收转运研究中的应用[J].中国实验方剂学杂志,2009,15(5):79-83.
[11] Soldner A, Benet LZ, Mutschler E, et al. Active transport of the angiotensin-IIantagonist losartan and its main metabolite EXP3174across MDCK-MDR1andcaco-2cell monolayers [J]. Br J Pharmacol,2000,129(6):1235-1243.
[12]刘瑶,俞春娜,曾苏. P-糖蛋白高表达马丁达比狗肾上皮细胞系的建立[J].中国药学杂志.2009,44(21):1608-1613.
[13]刘瑶,曾苏. MDCK-MDR1细胞模型及其在药物透过研究中的应用进展[J].药学学报,2008,43(6):559-564.
[14]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.
[15] Liang E, Chessic K, Yazdanian M. Evaluation of an accelerated Caco-2cellpermeability model [J]. J Pharm Sci,2000,89(3):336-345.
[16]胡晓渝,姚彤炜,曾苏. Caco-2细胞系及其在药物吸收、代谢中的应用[J].中国现代应用药学杂志,2002,19(2):88-90.
[17] Behrens I, Kamm W, Dantzig AH, Kissel T.2004.Variation of peptidetransporter (PepT1and HPT1) expression in Caco-2cells as a function of cellorigin [J]. J Pharm Sci93:1743–1754.
[18] Richardson JCW, Scalera V, Simmons NL.1981.Identification of two strains ofMDCK cells which resemble separate nephron tubule segments [J]. BiochimBiophys Acta673:26–36.
[19] Yu H, Cook TJ, Sinko PJ.1997. Evidence for diminished functional expressionof intestinal transporters in Caco-2cell monolayers at high passages [J]. PharmRes14:757–762.
[20] Lu S, Gough AW, Bobrowski WF, Stewart BH.1996. Transport properties are notaltered across Caco-2cells with heightened TEER despite underlyingphysiological and ultrastructural changes [J].J Pharm Sci85:270–273.
[21] Lentz KA, Hayashi J, Lucisano LJ, Polli JE.2000.Development of a more rapid,reduced serum culture system for Caco-2monolayers and application to thebiopharmaceutics classification system [J]. Int J Pharm200:41–51.
[22] Bravo SA, Nielsen CU, Amstrup J, Frokjaer S,Bodin B.2004. In-depthevaluation of Gly-Sar transport parameters as a function of culture time in theCaco-2cell model [J]. Eur J Pharm Sci21:77–86.
[23] Anderle P, Niederer E, Rubas W, Hilgendorf C, Spahn-Langguth H,Wunderli-Allenspach H, Merkle HP, Langguth P.1998. P-glycoprotein (P-gp)mediated efflux in Caco-2cell monolayers: The influence of culturing conditionsand drug exposure on P-gp expression levels [J]. J Pharm Sci87:757–762.
[24] D’Souza VM, Shertzer HG, Menon AG, Pauletti GM.2003. High glucoseconcentration in isotonic media alters Caco-2cell permeability[J]. AAPSPharmSci5: article24.
[25] D’Souza VM, Buckley DJ, Buckley AR, Pauletti GM.2003. Extracellularglucose concentration alters functional activity of intestinal oligopeptidetransporter (PepT-1) in Caco-2cells[J]. J Pharm Sci92:594–603.
[26] DeMarco VG, Li N, Thomas J, West CM, Neu J.2003. Glutamine and barrierfunction in cultured Caco-2epithelial cell monolayers[J]. J Nutr133:2176–2179.
[27] Bestwick CS, Milne L.2001. Alteration of culture regime modifies antioxidantdefenses independent of intracellular reactive oxygen levels and resistance tosevere oxidative stress within confluent Caco-2‘‘intestinal cells’’[J]. Dig DisSci46:417–423.
[28] Ward RK, Mungall S, Carter J, Clothier RH.1997.Evaluation of tissue cultureinsert membrane compatibility in the fluorescein leakage assay [J]. Toxicol InVitro11:761–768.
[29] Adson A, Burton PS, Raub TJ, Barshun CL, Audus KL, Ho NFH.1995. Passivediffusion of weak organic electrolytes across Caco-2cellmonolayers:Uncoupling the contributions of hydrodynamic, transcellular andparacellular barriers[J]. J Pharm Sci84:1197–1204.
[30] Wilson G.1990. Cell culture techniques for the study of drug transport [J]. Eur JDrug Metab Pharmacokinet15:159–163.
[31]钟大放.以加权最小二乘法建立生物分析标准曲线的若干问题[J].药物分析杂志,1996,16(5):343-346.
[32] Pade V, Stavchansky S.1997. Estimation of the relative contribution of thetranscellular and paracellular pathway to the transport of passively absorbeddrugs in the Caco-2cell culture model [J].Pharm Res14:1210–1215.
[33] Tamihide MATSUNAGA,Eiji KOSE,Sachiyo YASUDA. Determination ofP-Glycoprotein ATPase Activity Using Luciferase[J]. Biol. Pharm. Bull.29(3)560—564(2006)
[34] Yoshiyuki SHIRASAKA,Yuko ONISHI,Aki SAKURAI, Evaluation of HumanP-Glycoprotein (MDR1/ABCB1) ATPase ActivityAssay Method by Comparingwith in Vitro Transport Measurements:Michaelis–Menten Kinetic Analysis toEstimate the Affinity of P-Glycoprotein to Drugs[J]. Biol. Pharm. Bull.29(12)2465—2471(2006)
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