探针药物法评价厚朴酚、和厚朴酚对大鼠肝微粒体中CYP450酶的抑制作用
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摘要
目的研究厚朴中的主要成分厚朴酚、和厚朴酚对CYP450酶的7种亚型酶活性的影响,预测可能的药物间相互作用,为中药的临床应用提供理论依据。方法采用Cocktail探针方法,将厚朴酚、和厚朴酚和CYP450酶7种亚型的特异性探针底物:甲苯磺丁脲(CYP2C)、香豆素(CYP2A6)、右美沙芬(CYP2D6)、氯唑沙宗(CYP2E1)、睾酮(CYP3A)、非那西丁(CYP1A2)、安非他酮(CYP2B6)与大鼠肝微粒体进行孵化反应,结合UPLC-MS/MS的多反应监测技术,测定对应的7种代谢产物(4-羟基甲苯磺丁脲、7-羟基香豆素、右啡烷、6-羟基氯唑沙宗、6β-羟基睾酮、对乙酰氨基酚和羟基丙酮)的峰面积,通过与对照组比较,确定厚朴酚、和厚朴酚对以上7种酶活性的影响,计算相应的IC50,评价是否有抑制作用。结果厚朴酚、和厚朴酚对大鼠肝微粒体中的4种亚型酶(CYP2C、CYP2D6、CYP2E1和CYP2B6)活性均有抑制作用,且随化合物浓度和预温育时间增加而增加机械抑制;另外3种亚型酶(CYP2A6、CYP3A4和CYP1A2)则不呈现此抑制规律。结论厚朴在与以上4种酶(CYP2C、CYP2D6、CYP2E1、CYP2B6)代谢的药物联合用药时,易产生药物相互作用,抑制药物代谢。本研究也为中药厚朴的临床应用以及新药研发提供重要的数据支持。
OBJECTIVE To study the effects of magnolol and honokiol on the 7 subtypes of CYP450 enzymes in Magnoliae Officinalis Cortex, and to predict the possible drug-drug interactions. To provide theoretical basis for the clinical application of traditional Chinese medicine. METHODS Used the Cocktail probe method, magnolol, honokiol and CYP450 enzyme specific substrates for 7 subtypes: Tolbutamide(CYP2C), Coumarin(CYP2A6), Dextromethorphan(CYP2D6), Chlorzoxazone(CYP2E1), Testosterone(CYP3A), Phenacetin(CYP1A2), Bupropion(CYP2B6) incubation reaction with rat liver microsomes, combined with UPLC-MS/MS multiple reaction monitoring technology, Determination of the peaks of the corresponding 7 metabolites(4-hydroxytoluidine, 7-hydroxycoumarin, dextrorphan, 6-hydroxy chlorzoxazone, 6-hydroxytestosterone, acetaminophen, and hydroxyacetone) area. By comparing with the control group, the effect of magnolol and honokiol on the activity of the above 7 enzymes was determined, and the corresponding IC50 was calculated to evaluate whether there is an inhibitory effect. RESULTS Magnolol and honokiol had inhibitory effects on the activity of 7 isoforms(CYP2C, CYP2D6, CYP2E1 and CYP2B6) in rat liver microsomes. And that was an increase in mechanical inhibition with increasing compound concentration and pre-incubation time. However the subtypes of enzymes(CYP2A6, CYP3A4 and CYP1A2) did not exhibit this inhibition pattern. CONCLUSION It is likely to inhibit drug metabolism and produce drug-drug interactions, during the combination of magnolia and drugs metabolized by 4 enzymes(CYP2C, CYP2D6, CYP2E1 and CYP2B6). This study also provides very important data support for the clinical application of Chinese herbal medicine Magnoliae Officinalis Cortex and the development of new drugs.
OBJECTIVE To study the effects of magnolol and honokiol on the 7 subtypes of CYP450 enzymes in Magnoliae Officinalis Cortex, and to predict the possible drug-drug interactions. To provide theoretical basis for the clinical application of traditional Chinese medicine. METHODS Used the Cocktail probe method, magnolol, honokiol and CYP450 enzyme specific substrates for 7 subtypes: Tolbutamide(CYP2C), Coumarin(CYP2A6), Dextromethorphan(CYP2D6), Chlorzoxazone(CYP2E1), Testosterone(CYP3A), Phenacetin(CYP1A2), Bupropion(CYP2B6) incubation reaction with rat liver microsomes, combined with UPLC-MS/MS multiple reaction monitoring technology, Determination of the peaks of the corresponding 7 metabolites(4-hydroxytoluidine, 7-hydroxycoumarin, dextrorphan, 6-hydroxy chlorzoxazone, 6-hydroxytestosterone, acetaminophen, and hydroxyacetone) area. By comparing with the control group, the effect of magnolol and honokiol on the activity of the above 7 enzymes was determined, and the corresponding IC50 was calculated to evaluate whether there is an inhibitory effect. RESULTS Magnolol and honokiol had inhibitory effects on the activity of 7 isoforms(CYP2C, CYP2D6, CYP2E1 and CYP2B6) in rat liver microsomes. And that was an increase in mechanical inhibition with increasing compound concentration and pre-incubation time. However the subtypes of enzymes(CYP2A6, CYP3A4 and CYP1A2) did not exhibit this inhibition pattern. CONCLUSION It is likely to inhibit drug metabolism and produce drug-drug interactions, during the combination of magnolia and drugs metabolized by 4 enzymes(CYP2C, CYP2D6, CYP2E1 and CYP2B6). This study also provides very important data support for the clinical application of Chinese herbal medicine Magnoliae Officinalis Cortex and the development of new drugs.
引文
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[11]HANAPI N A,AZIZI J,ISMAIL S,et al.Evaluation of selected malaysian medicinal plants on phase I drug metabolizing enzyme,CYP2C9,CYP2D6 and CYP3A4 activities in vitro[J].Int JPharmacol,2010,6(4):490-495.
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[13]CAI T,ZHANG L,WANG H,et al.Assisted inhibition effect of acetylcholinesterase with n-octylphosphonic acid and application in high sensitive detection of organophosphorous pesticides by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry[J].Anal Chim Acta,2011,706(2):291-296.
[14]OTTEN J N,HINGORANI G P,HARTLEY D P,et al.An in vitro,high throughput,seven CYP cocktail inhibition assay for the evaluation of new chemical entities using LC-MS/MS[J].Drug Metab Lett,2011,5(1):17-24.
[15]刘彦卿,洪燕君,曾苏.代谢性药物-药物相互作用的研究进展[J].浙江大学学报,2009,38(2):215-224.
[16]SUN M,TANG Y,DING T,et al.Inhibitory effects of celastrol on rat liver cytochrome P450 1A2,2C11,2D6,2E1and 3A2 activity[J].Fitoterapia,2014(92):1-8.
[17]CI X Y,LI W,CUI T,ET AL.Establishment and application of a model for cytochrome P450 enzyme induction[J].Chin JNew Drugs(中国新药杂志),2018,27(6):688-694.
[2]WATANABE A,NAKAMURA K,OKUDAIRA N,et al.Risk assessment for drug-drug interaction caused by metabolism-based inhibition of CYP3A using automated in vitro assay systems and its application in the early drug discovery process[J].Drug Metab Dispos,2007,35(7):1232-1238.
[3]NISHIYA Y,HAGIHARA K,ITO T,et al.Mechanism-based inhibition of human cytochrome P450 2B6 by ticlopidine,clopidogrel,and the thiolactone metabolite of prasugrel[J].Drug Metab Dispos,2009,37(3):589-593.
[4]马瑞芳,韩国柱.中药对细胞色素P450 2E1影响的研究进展[J].中草药,2009,40(11):1841-1844.
[5]QU Y Q,LIANG R,SUN D G,et al.Inhibitory effect of diethylstilbestrol on cytochrome P450 3A4 and cytochrome P450 2C9 in human liver microsomes[J].Chin J Pharmacol Toxicol(中国药理学与毒理学杂志),2011,25(2):170-175.
[6]RENDIC S,DI CARLO F J.Human cytochrome P450enzymes:A status report summarizing their reactions,substrates,inducers,and inhibitors[J].Drug Metab Rev,1997,29(1/2):413-580.
[7]潘柳萌.细胞色素CYP1A2、CYP3A4介导的硫杂蒽酮类光引发剂体外代谢[D].杭州:浙江大学,2018.
[8]MUROYAMA A,FUJITA A,LV C,et al.Magnolol protects against MPTP/MPP+-Induced toxicity via inhibition of oxidative stress in vivo and in vitro models of parkinson’s disease[J].Parkinson’s Dis,2012,2012(4):985157.Doi:10.1155/2012/985157.
[9]ZUO G Y,ZHANG X J,HAN J.In vitro Synergism of magnolol and honokiol in combination with antibacterial agents against clinical isolates of methicillin-resistant staphylococcus aureus(MRSA)[J].BMC Complement Altern Med,2015(15):425.Doi:10.1186/s12906-015-0938-3.
[10]肖娟.厚朴酚与和厚朴酚对大鼠肝脏CYP450酶活性的影响及抑制类型研究[D].武汉:湖北大学,2012.
[11]HANAPI N A,AZIZI J,ISMAIL S,et al.Evaluation of selected malaysian medicinal plants on phase I drug metabolizing enzyme,CYP2C9,CYP2D6 and CYP3A4 activities in vitro[J].Int JPharmacol,2010,6(4):490-495.
[12]DE BOCK L,BOUSSERY K,COLIN P.Development and validation of a fast and sensitive UPLC-MS/MS method for the quantification of six probe metabolites for the in vitro determination of cytochrome P450 activity[J].Talanta,2011(89):209-216.
[13]CAI T,ZHANG L,WANG H,et al.Assisted inhibition effect of acetylcholinesterase with n-octylphosphonic acid and application in high sensitive detection of organophosphorous pesticides by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry[J].Anal Chim Acta,2011,706(2):291-296.
[14]OTTEN J N,HINGORANI G P,HARTLEY D P,et al.An in vitro,high throughput,seven CYP cocktail inhibition assay for the evaluation of new chemical entities using LC-MS/MS[J].Drug Metab Lett,2011,5(1):17-24.
[15]刘彦卿,洪燕君,曾苏.代谢性药物-药物相互作用的研究进展[J].浙江大学学报,2009,38(2):215-224.
[16]SUN M,TANG Y,DING T,et al.Inhibitory effects of celastrol on rat liver cytochrome P450 1A2,2C11,2D6,2E1and 3A2 activity[J].Fitoterapia,2014(92):1-8.
[17]CI X Y,LI W,CUI T,ET AL.Establishment and application of a model for cytochrome P450 enzyme induction[J].Chin JNew Drugs(中国新药杂志),2018,27(6):688-694.