常用中成药对细胞色素P450 3A4和P-糖蛋白的诱导研究
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摘要
由于中药制剂多长期和反复的用药,许多CYP450代谢酶和转运体活性可被诱导,临床常常引起药物相互作用,为了预防这类相互作用,我们拟考察常用中药对人肝脏中最主要的代谢酶CYP3A4和肠道中主要的药物转运体P-糖蛋白的诱导作用。
本文用待测药物与HepG2细胞共培养48小时后与CYP3A探针药物咪达唑仑共孵育,通过测定1-羟基咪达唑仑的生成速率,来比较药物刺激组和空白组的CYP3A4酶的相对活性,从而用于快速的预测药物的诱导作用。同时用real-time PCR法考察了筛选出的有诱导作用的药物刺激HepG2细胞48h后的CYP3A4的表达量的改变。我们用阳性诱导剂利福平对该筛选体系进行了检验,结果显示,利福平使HepG2细胞的CYP3A4酶活性增加1.97倍,使HepG2细胞中CYP3A4基因表达量增加2.3倍。进而我们考察了痰热清注射液等33种临床常用或者长期服用的中药制剂,结果发现麝香保心丸、肝苏颗粒和强肝胶囊对CYP3A4的酶活性影响较强,其中麝香保心丸的诱导作用最强。
为了进一步验证该筛选体系的可靠性,我们对麝香保心丸进行了体内和体外的考察,体外HepG2细胞结果显示利福平和麝香保心丸对HepG2细胞的CYP3A4酶活性和表达都呈现剂量依赖的诱导关系。经过6天连续灌胃给予麝香保心丸(6.75mg/kg)的Wistar大鼠的肝微粒体活性比空白组增加了1.57倍,CYP3A1和CYP3A2基因表达分别增加了1.40和2.31倍,说明该筛选体系可用于CYP3A4诱导作用的筛选。鉴于此,我们用HepG2细胞筛选体系进一步探索麝香保心丸诱导作用的活性成分,我们选取了麝香酮、睾丸酮、龙脑、肉桂醛、人参皂苷Rgl、人参皂苷Rf、人生皂苷Re、蟾毒灵、华蟾毒配基、酯蟾毒配基10种主要成分进行体外研究。结果显示,在麝香保心丸含量换算的剂量下,华蟾毒配基(0.03μg／mL)和蟾毒灵(0.01μg/mL)有一定的诱导作用,酶活性分别为空白组的1.14和1.58倍。当华蟾毒配基和蟾毒灵孵育浓度提高,诱导作用也增加。麝香酮曾经被报道对大鼠的CYP3A有诱导作用,但是在本课题的研究中,10-30μg/mL浓度的麝香酮与细胞孵育后显示诱导作用,在含量换算的浓度(0.31μg/mL)下与细胞共孵育48h对细胞的CYP3A4没有诱导作用,其他的单体在测试的浓度下都未显示出诱导作用。提示我们在麝香保心丸的诱导现象中,华蟾毒配基、酯蟾毒配基和蟾毒灵可能在麝香保心丸的诱导作用中起主要的作用,并且他们的结构很相似,很可能有协同的作用。因此在合用含有华蟾毒配基、酯蟾毒配基、蟾毒灵的药物与经过CYP3A代谢的药物时可能产生药物间的相互作用。
在本课题的研究中还使用Caco-2细胞Transwell模型考察了麝香保心丸对P-糖蛋白的诱导作用,首先我们用阳性诱导剂B[a]P对模型进行了评价,进而考察了麝香保心丸(30、20、10μg/mL)与Caco-2细胞单层共孵育72小时后罗丹明-123的转运率,结果显示麝香保心丸的诱导作用不明显,不具有统计学意义。然而Caco-2细胞与麝香保心丸(20、10μg/mL)共培养72小时后,细胞中MDR1基因表达分别为空白组的3.28和2.84倍。此外,经过6天连续灌胃给药麝香保心丸(6.75mg/kg)的Wistar大鼠十二指肠mdrla、mdrlb基因表达分别为空白组的1.54和2.88倍。
综上所述,我们建立了CYP3A4诱导的筛选体系,并用阳性药利福平对其进行了评价。同时用该筛选体系也评价了临床常用中药制剂,对临床药物的使用具有一定的指导意义。
The herbs were always used for long periods of time, many CYP450 metabolic enzymes and transporters activity can be induced. This kind of induction can lead to clinical drug-drug interactions. In order to prevent this interaction, the study on induction of the traditional Chinese medicines on liver metabolic enzymes CYP3A4 and the major drug transporters will help us to avoid the drug-drug interaction.
In our study, the HepG2 cells were incubated with test drug for 48 h in vitro. The activity of CYP3A was measured by the quantification of 1-hydroxymidazolam formation using a liquid chromatographic-tandem mass spectrometry method. Meanwhile, the levels of mRNA of CYP3A were determined by real-time PCR. We used the positive inducter to evaluate our model. Then, the inductive effects of about thirty three Chinese traditional patent medicines on CYP3A were evaluated respectively in HepG2 cells. The result indicated that the Shexiang baoxin pill (SBP) can induce the CYP3A4 enzyme activity strongly.
To further verify the reliability of the model, in vitro in HepG2 cells and in vivo in rats approaches were used to estimate the inductive effect of SBP on CYP3A. SBP increased mRNA expression and enzyme activity of CYP3A in a dose-dependent manner in HepG2 cells. As well as the microsomal activity and the expression of CYP3A in rats were both up-regulated in the SBP pretreated rats. Furthermore, we took advantage of the HepG2 cells to further identify its ten main components (muscone, testosterone, ginsenoside Rf, ginsenoside Rgl, ginsenoside Re, borneol, bufalin, cinobufagin, resibufogenin, cinnamaldehyde) as a potential inducer of CYP3A. The real-time PCR results showed that enzyme activity of CYP3A in HepG2 cells treated with bufalin (0.01μg/mL), cinobufagin (0.03μg/mL) increased 1.58-fold and 1.14-fold respectively. Muscone at higher concentration (10μg/mL) increased the enzyme activity and mRNA expression. Thus, bufalin, cinobufagin, resibufogenin might play the key role in the enzyme-inducing effect of SBP. In conclusion, there may be potential drug-drug interactions during long-term clinical application of the drug which contain the bufalin, cinobufagin, resibufogenin, when substrates of CYP3A4 are co-administered.
In this study, Caco-2 cell monolayers were treated with SBP (30,20, 10μg/mL) for 72 h to investigate the relationship between the potential affects of SBP on P-gp. After SBP treatment, the ratio of the apparent permeability coefficients (Papp) of rhodamine-123 (a substrate of P-gp) efflux is similar to the control, but an increase in mRNA expression of MDR1 (P-gp) was confirmed by real-time PCR. Furthermore, we investigate the effects of SBP on gene expression on message levels of mdr1a and mdr1b in small intestine of the rats treated orally with SBP (6.75mg/kg) for 6 days. The results of real-time PCR showed that the levels of mdr1a and mdr1b were increased 1.54 and 2.88-fold respectively.
In summary, we established a model for screening the inducer. We used the positive inducter to evaluate our model. Meanwhile, the inductive effects of Chinese traditional patent medicines were evaluated by the models. The deepgoing study of drug-drug interaction will help us to choose right drug and adjust drug dose to avoid drug side effect and attain more drug efficacy.
本文用待测药物与HepG2细胞共培养48小时后与CYP3A探针药物咪达唑仑共孵育,通过测定1-羟基咪达唑仑的生成速率,来比较药物刺激组和空白组的CYP3A4酶的相对活性,从而用于快速的预测药物的诱导作用。同时用real-time PCR法考察了筛选出的有诱导作用的药物刺激HepG2细胞48h后的CYP3A4的表达量的改变。我们用阳性诱导剂利福平对该筛选体系进行了检验,结果显示,利福平使HepG2细胞的CYP3A4酶活性增加1.97倍,使HepG2细胞中CYP3A4基因表达量增加2.3倍。进而我们考察了痰热清注射液等33种临床常用或者长期服用的中药制剂,结果发现麝香保心丸、肝苏颗粒和强肝胶囊对CYP3A4的酶活性影响较强,其中麝香保心丸的诱导作用最强。
为了进一步验证该筛选体系的可靠性,我们对麝香保心丸进行了体内和体外的考察,体外HepG2细胞结果显示利福平和麝香保心丸对HepG2细胞的CYP3A4酶活性和表达都呈现剂量依赖的诱导关系。经过6天连续灌胃给予麝香保心丸(6.75mg/kg)的Wistar大鼠的肝微粒体活性比空白组增加了1.57倍,CYP3A1和CYP3A2基因表达分别增加了1.40和2.31倍,说明该筛选体系可用于CYP3A4诱导作用的筛选。鉴于此,我们用HepG2细胞筛选体系进一步探索麝香保心丸诱导作用的活性成分,我们选取了麝香酮、睾丸酮、龙脑、肉桂醛、人参皂苷Rgl、人参皂苷Rf、人生皂苷Re、蟾毒灵、华蟾毒配基、酯蟾毒配基10种主要成分进行体外研究。结果显示,在麝香保心丸含量换算的剂量下,华蟾毒配基(0.03μg／mL)和蟾毒灵(0.01μg/mL)有一定的诱导作用,酶活性分别为空白组的1.14和1.58倍。当华蟾毒配基和蟾毒灵孵育浓度提高,诱导作用也增加。麝香酮曾经被报道对大鼠的CYP3A有诱导作用,但是在本课题的研究中,10-30μg/mL浓度的麝香酮与细胞孵育后显示诱导作用,在含量换算的浓度(0.31μg/mL)下与细胞共孵育48h对细胞的CYP3A4没有诱导作用,其他的单体在测试的浓度下都未显示出诱导作用。提示我们在麝香保心丸的诱导现象中,华蟾毒配基、酯蟾毒配基和蟾毒灵可能在麝香保心丸的诱导作用中起主要的作用,并且他们的结构很相似,很可能有协同的作用。因此在合用含有华蟾毒配基、酯蟾毒配基、蟾毒灵的药物与经过CYP3A代谢的药物时可能产生药物间的相互作用。
在本课题的研究中还使用Caco-2细胞Transwell模型考察了麝香保心丸对P-糖蛋白的诱导作用,首先我们用阳性诱导剂B[a]P对模型进行了评价,进而考察了麝香保心丸(30、20、10μg/mL)与Caco-2细胞单层共孵育72小时后罗丹明-123的转运率,结果显示麝香保心丸的诱导作用不明显,不具有统计学意义。然而Caco-2细胞与麝香保心丸(20、10μg/mL)共培养72小时后,细胞中MDR1基因表达分别为空白组的3.28和2.84倍。此外,经过6天连续灌胃给药麝香保心丸(6.75mg/kg)的Wistar大鼠十二指肠mdrla、mdrlb基因表达分别为空白组的1.54和2.88倍。
综上所述,我们建立了CYP3A4诱导的筛选体系,并用阳性药利福平对其进行了评价。同时用该筛选体系也评价了临床常用中药制剂,对临床药物的使用具有一定的指导意义。
The herbs were always used for long periods of time, many CYP450 metabolic enzymes and transporters activity can be induced. This kind of induction can lead to clinical drug-drug interactions. In order to prevent this interaction, the study on induction of the traditional Chinese medicines on liver metabolic enzymes CYP3A4 and the major drug transporters will help us to avoid the drug-drug interaction.
In our study, the HepG2 cells were incubated with test drug for 48 h in vitro. The activity of CYP3A was measured by the quantification of 1-hydroxymidazolam formation using a liquid chromatographic-tandem mass spectrometry method. Meanwhile, the levels of mRNA of CYP3A were determined by real-time PCR. We used the positive inducter to evaluate our model. Then, the inductive effects of about thirty three Chinese traditional patent medicines on CYP3A were evaluated respectively in HepG2 cells. The result indicated that the Shexiang baoxin pill (SBP) can induce the CYP3A4 enzyme activity strongly.
To further verify the reliability of the model, in vitro in HepG2 cells and in vivo in rats approaches were used to estimate the inductive effect of SBP on CYP3A. SBP increased mRNA expression and enzyme activity of CYP3A in a dose-dependent manner in HepG2 cells. As well as the microsomal activity and the expression of CYP3A in rats were both up-regulated in the SBP pretreated rats. Furthermore, we took advantage of the HepG2 cells to further identify its ten main components (muscone, testosterone, ginsenoside Rf, ginsenoside Rgl, ginsenoside Re, borneol, bufalin, cinobufagin, resibufogenin, cinnamaldehyde) as a potential inducer of CYP3A. The real-time PCR results showed that enzyme activity of CYP3A in HepG2 cells treated with bufalin (0.01μg/mL), cinobufagin (0.03μg/mL) increased 1.58-fold and 1.14-fold respectively. Muscone at higher concentration (10μg/mL) increased the enzyme activity and mRNA expression. Thus, bufalin, cinobufagin, resibufogenin might play the key role in the enzyme-inducing effect of SBP. In conclusion, there may be potential drug-drug interactions during long-term clinical application of the drug which contain the bufalin, cinobufagin, resibufogenin, when substrates of CYP3A4 are co-administered.
In this study, Caco-2 cell monolayers were treated with SBP (30,20, 10μg/mL) for 72 h to investigate the relationship between the potential affects of SBP on P-gp. After SBP treatment, the ratio of the apparent permeability coefficients (Papp) of rhodamine-123 (a substrate of P-gp) efflux is similar to the control, but an increase in mRNA expression of MDR1 (P-gp) was confirmed by real-time PCR. Furthermore, we investigate the effects of SBP on gene expression on message levels of mdr1a and mdr1b in small intestine of the rats treated orally with SBP (6.75mg/kg) for 6 days. The results of real-time PCR showed that the levels of mdr1a and mdr1b were increased 1.54 and 2.88-fold respectively.
In summary, we established a model for screening the inducer. We used the positive inducter to evaluate our model. Meanwhile, the inductive effects of Chinese traditional patent medicines were evaluated by the models. The deepgoing study of drug-drug interaction will help us to choose right drug and adjust drug dose to avoid drug side effect and attain more drug efficacy.
引文
[1]S. Rendic, Summary of information on human CYP enzymes:human P450 metabolism data, Drug Metab Rev 2002 34(1-2):83-448.
[2]Y. Deng, H.C. Bi, L.Z. Zhao, X.D. Wang, J. Chen, Z.M. Ou, L. Ding, L.J. Xu, S. Guan, X. Chen, S.F. Zhou, M. Huang, Induction of cytochrome P450 3A by the Ginkgo biloba extract and bilobalides in human and rat primary hepatocytes, Drug Metab Lett 2008 2(1): 60-66.
[3]A.H. Schinkel, J.W. Jonker, Mammalian drug efflux transporters of the ATP binding cassette (ABC) family:an overview, Adv Drug Deliv Rev 55(1) (2003) 3-29.
[4]A. Ayrton, P. Morgan, Role of transport proteins in drug absorption, distribution and excretion, Xenobiotica 2001 31(8-9):469-497.
[5]A. Collett, J. Tanianis-Hughes, G. Warhurst, Rapid induction of P-glycoprotein expression by high permeability compounds in colonic cells in vitro:a possible source of transporter mediated drug interactions?, Biochem Pharmacol 2004 68(4):783-790.
[6]L. Laitinen, E. Takala, H. Vuorela, P. Vuorela, A.M. Kaukonen, M. Marvola, Anthranoid laxatives influence the absorption of poorly permeable drugs in human intestinal cell culture model (Caco-2), Eur J Pharm Biopharm 2007 66(1):135-145.
[7]T. Mitin, L.L. Von Moltke, M.H. Court, D.J. Greenblatt, Levothyroxine up-regulates P-glycoprotein independent of the pregnane X receptor, Drug Metab Dispos 2004 32(8): 779-782.
[8]R. Venkataramanan, V. Ramachandran, B.J. Komoroski, S. Zhang, P.L. Schiff, S.C. Strom, Milk thistle, a herbal supplement, decreases the activity of CYP3A4 and uridine diphosphoglucuronosyl transferase in human hepatocyte cultures, Drug Metab Dispos 2000 28(11):1270-1273.
[9]M. Zhu, K.W. Chan, L.S. Ng, Q. Chang, S. Chang, R.C. Li, Possible influences of ginseng on the pharmacokinetics and pharmacodynamics of warfarin in rats, J Pharm Pharmacol 1999 51(2):175-180.
[10]S. Harmsen, A.S. Koster, J.H. Beijnen, J.H. Schellens, I. Meijerman, Comparison of two immortalized human cell lines to study nuclear receptor-mediated CYP3A4 induction, Drug Metab Dispos 2008 36(6):1166-1171.
[11]K.A. Youdim, C.A. Tyman, B.C. Jones, R. Hyland, Induction of cytochrome P450: assessment in an immortalized human hepatocyte cell line (Fa2N4) using a novel higher throughput cocktail assay, Drug Metab Dispos 2007 35(2):275-282.
[12]L. Vignati, E. Turlizzi, S. Monaci, P. Grossi, R. Kanter, M. Monshouwer, An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics, Toxicology 2005 216(2-3):154-167.
[13]K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 2001 25(4):402-408.
[14]L.D. Murphy, C.E. Herzog, J.B. Rudick, A.T. Fojo, S.E. Bates, Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression, Biochemistry 1990 29(45): 10351-10356.
[15]H.G. Niesters, Quantitation of viral load using real-time amplification techniques, Methods 2001 25(4):419-429.
[16]M. Ogino, K. Nagata, Y. Yamazoe, Selective suppressions of human CYP3A forms, CYP3A5 and CYP3A7, by troglitazone in HepG2 cells, Drug Metab Pharmacokinet 2002 17(1):42-46.
[17]W.M. Westerink, W.G. Schoonen, Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells, Toxicol In Vitro 2007 21(8):1581-1591.
[18]N.J. Hewitt, P. Hewitt, Phase Ⅰ and Ⅱ enzyme characterization of two sources of HepG2 cell lines, Xenobiotica 2004 34(3):243-256.
[19]G. Elizondo, I.M. Medina-Diaz, Induction of CYP3A4 by lalpha,25-dyhydroxyvitamin D3 in HepG2 cells, Life Sci 2003 73(2):141-149.
[20]T. Mosmann, Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays, J Immunol Methods 1983 65(1-2):55-63.
[21]S. Li, X.H. Miao, [Induction of CYP3A4 by lalpha,25-dyhydroxyvitamin D3 in HepG2 cells], Zhonghua Gan Zang Bing Za Zhi 2008 16(3):220-223.
[22]孙敏捷,盛星,胡一桥,Caco-2细胞单层模型的建立与验证,中国药理学杂志200641(18)：1431-1434.
[23]M.T. Donato, A.M. Bassi, M.J. Gomez-Lechon, S. Penco, E. Herrero, D. Adamo, J.V. Castell, M. Ferro, Evaluation of the xenobiotic biotransformation capability of six rodent hepatoma cell lines in comparison with rat hepatocytes, In Vitro Cell Dev Biol Anim 1994 30A(9):574-580.
[2]Y. Deng, H.C. Bi, L.Z. Zhao, X.D. Wang, J. Chen, Z.M. Ou, L. Ding, L.J. Xu, S. Guan, X. Chen, S.F. Zhou, M. Huang, Induction of cytochrome P450 3A by the Ginkgo biloba extract and bilobalides in human and rat primary hepatocytes, Drug Metab Lett 2008 2(1): 60-66.
[3]A.H. Schinkel, J.W. Jonker, Mammalian drug efflux transporters of the ATP binding cassette (ABC) family:an overview, Adv Drug Deliv Rev 55(1) (2003) 3-29.
[4]A. Ayrton, P. Morgan, Role of transport proteins in drug absorption, distribution and excretion, Xenobiotica 2001 31(8-9):469-497.
[5]A. Collett, J. Tanianis-Hughes, G. Warhurst, Rapid induction of P-glycoprotein expression by high permeability compounds in colonic cells in vitro:a possible source of transporter mediated drug interactions?, Biochem Pharmacol 2004 68(4):783-790.
[6]L. Laitinen, E. Takala, H. Vuorela, P. Vuorela, A.M. Kaukonen, M. Marvola, Anthranoid laxatives influence the absorption of poorly permeable drugs in human intestinal cell culture model (Caco-2), Eur J Pharm Biopharm 2007 66(1):135-145.
[7]T. Mitin, L.L. Von Moltke, M.H. Court, D.J. Greenblatt, Levothyroxine up-regulates P-glycoprotein independent of the pregnane X receptor, Drug Metab Dispos 2004 32(8): 779-782.
[8]R. Venkataramanan, V. Ramachandran, B.J. Komoroski, S. Zhang, P.L. Schiff, S.C. Strom, Milk thistle, a herbal supplement, decreases the activity of CYP3A4 and uridine diphosphoglucuronosyl transferase in human hepatocyte cultures, Drug Metab Dispos 2000 28(11):1270-1273.
[9]M. Zhu, K.W. Chan, L.S. Ng, Q. Chang, S. Chang, R.C. Li, Possible influences of ginseng on the pharmacokinetics and pharmacodynamics of warfarin in rats, J Pharm Pharmacol 1999 51(2):175-180.
[10]S. Harmsen, A.S. Koster, J.H. Beijnen, J.H. Schellens, I. Meijerman, Comparison of two immortalized human cell lines to study nuclear receptor-mediated CYP3A4 induction, Drug Metab Dispos 2008 36(6):1166-1171.
[11]K.A. Youdim, C.A. Tyman, B.C. Jones, R. Hyland, Induction of cytochrome P450: assessment in an immortalized human hepatocyte cell line (Fa2N4) using a novel higher throughput cocktail assay, Drug Metab Dispos 2007 35(2):275-282.
[12]L. Vignati, E. Turlizzi, S. Monaci, P. Grossi, R. Kanter, M. Monshouwer, An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics, Toxicology 2005 216(2-3):154-167.
[13]K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 2001 25(4):402-408.
[14]L.D. Murphy, C.E. Herzog, J.B. Rudick, A.T. Fojo, S.E. Bates, Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression, Biochemistry 1990 29(45): 10351-10356.
[15]H.G. Niesters, Quantitation of viral load using real-time amplification techniques, Methods 2001 25(4):419-429.
[16]M. Ogino, K. Nagata, Y. Yamazoe, Selective suppressions of human CYP3A forms, CYP3A5 and CYP3A7, by troglitazone in HepG2 cells, Drug Metab Pharmacokinet 2002 17(1):42-46.
[17]W.M. Westerink, W.G. Schoonen, Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells, Toxicol In Vitro 2007 21(8):1581-1591.
[18]N.J. Hewitt, P. Hewitt, Phase Ⅰ and Ⅱ enzyme characterization of two sources of HepG2 cell lines, Xenobiotica 2004 34(3):243-256.
[19]G. Elizondo, I.M. Medina-Diaz, Induction of CYP3A4 by lalpha,25-dyhydroxyvitamin D3 in HepG2 cells, Life Sci 2003 73(2):141-149.
[20]T. Mosmann, Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays, J Immunol Methods 1983 65(1-2):55-63.
[21]S. Li, X.H. Miao, [Induction of CYP3A4 by lalpha,25-dyhydroxyvitamin D3 in HepG2 cells], Zhonghua Gan Zang Bing Za Zhi 2008 16(3):220-223.
[22]孙敏捷,盛星,胡一桥,Caco-2细胞单层模型的建立与验证,中国药理学杂志200641(18)：1431-1434.
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