摘要
遗传药理学研究发现,药物代谢酶和转运体活性的改变是导致临床上药物相互作用的主要机制,药物代谢酶和转运体的遗传多态性是药物相互作用个体差异的重要决定因素。近年来,由于天然中草药的使用越来越广泛,天然植物药与药物的相互作用受到越来越多的重视,对遗传药理学的深入研究将有助于我们根据患者的遗传特性来指导临床上的中西药合用,调整药物的使用方法,以达到减少药物不良反应及确保药物治疗作用的目的。
细胞色素P450(CYP450)酶系由基因超家族编码的酶蛋白所组成,参与许多内源性和外源性物质的生物转化。药物对细胞色素P450酶活性的影响,是导致药物相互作用的主要原因之一。细胞色素氧化酶CYP3A是一种重要的CYP450酶系,它在肝脏和肠道中含量丰富,占肝脏P450酶总量的25%,成人CYP3A主要有CYP3A4以及CYP3A5,临床常用药物的60%以上都通过CYP3A4和CYP3A5催化来完成氧化反应。CYP3A的活性高低影响许多药物的疗效以及患者对药物毒性的反应,同时还影响环境污染物致病的危险性。CYP3A表达增高可导致肿瘤化疗的失败。药物对CYP3A活性的影响是导致药物相互作用的主要原因之一。CYP3A基因表达存在显著的个体差异,目前认为,CYP3A5的基因突变是产生CYP3A酶活性差异的主要原因。CYP2C9酶是人类肝脏中重要的CYP2C亚家族同工酶,占人肝微粒体所有P450酶含量的20%左右,并代谢约10%的临床常用药物,如苯妥英,华法林,甲苯磺丁脲,格列吡嗪,格列本脲,托拉塞米,氯沙坦及许多非甾体抗炎药。CYP2C9基因具有遗传多态性,导致CYP2C9酶活性存在差异,这是药物代谢种族与个体差异的原因之一。具有功能意义的基因突变导致CYP2C9酶活性降低,可致使CYP2C9酶底物药物疗效下降或产生更多不良反应。较多的体内及体外试验研究表明,CYP2C9*2和CYP2C9*3会改变CYP2C9酶对某些底物如华法令,苯妥英等的催化活性,其代谢能力均明显降低。其中CYP2C9*3更为明显。许多体内体外研究表明,与野生型比较,CYP2C9*3(Ile359Leu)对甲苯磺丁脲、华法林等多种CYP2C9底物氧化活性显著下降,其结果是在体内延长药物的血浆消除半衰期,降低药物的清除率;在体外增高米氏常数(Km)值,降低最大反应速度(Vmax),酶活性显著降低。因此,携带CYP2C9突变等位基因的个体可能会出现代谢不全从而发生中毒或降低药物疗效。
P-糖蛋白是由MDR1基因编码的一种跨膜蛋白,是一种能量依赖型药物外排泵,转运大量结构不同的化合物,通常是亲脂性和两歧性分子的化合物。P-糖蛋白主要在肝脏和肠道及肾脏中表达,在药物的吸收,组织分布和排泄方面发挥重要的作用。肠道的P-糖蛋白可主动将口服药物泵出细胞外,抑制药物的吸收,使细胞内药物浓度降低,从而降低药物疗效。MDR1蛋白具有广泛的组织分布和底物特异性,尽管P-糖蛋白最首先在肿瘤细胞发现,后来观察到它表达在许多正常组织,如肠,肝脏,肾,胰腺,肾上腺,P-糖蛋白在血脑屏障,脉络丛也高表达,P-糖蛋白底物广泛,包括环孢霉素A,泰素,地塞米松,利多卡因,乙琥红霉素和蛋白酶抑制剂等。
Silymarin(水飞蓟素,益肝灵)是从菊科药用植物水飞蓟种子的种皮中提取出来的植物提取物,属于黄酮类物质,主要成分包括水飞蓟宾(silybin)、异水飞蓟宾(silydianin)、和silychristine等类黄酮物质,由于silybin是其中的主要成分,所以计算silymarin摩尔浓度的时候,都是以silybin的分子量为基数进行计算。黄酮类是一种广泛存在于水果、蔬菜和植物饮料中的化合物,在很多草药中也含有黄酮类物质。既往在美国人饮食中的研究表明,每天从食品中获得的黄酮类物质估计有1g左右,虽然此数据可能高估了摄攘俊ilymarin一直以来被用于保护肝脏和用于肝脏疾病的辅助治疗药物,同时它还具有其他广泛的作用,比如抗癌和预防胃溃疡的发生,治疗胆囊疾病、肝炎、肝硬化和黄疸它的护肝机理是通过阻碍有害毒素进入和帮助从肝细胞去除这些物质从而保护肝脏细胞。Silymarin一般最常用的用途为中和酒精对肝部的有害作用。组成silymarin的三种异构体(中文名为:水飞蓟宾,异水飞蓟宾、水飞蓟宁)中,水飞蓟宾(silybin)具有最强的活性,并且很大程度上是水飞蓟素所具有的特性的原因。临床常用的silymarin剂量为:用于肝部疾病和肝功能损伤时,研究认为应每天服用420-600mg。对于患有慢性肝部疾病的患者,silymarin往往作为长期治疗药物使用,所以此药物不可避免的与一些临床上往往长期服用的常用药物会发生合用现象。
过去的研究主要集中在该药的药效研究领域,而对该药的药动学和药物相互作用研究较少。药物对细胞色素P450酶和药物转运体活性的影响,是导致药物相互作用的主要原因之一。为了最大限度地减少因药物相互作用而导致的药物不良反应,指导临床合理用药,研究已知药物对细胞色素P450酶和药物转运体活性的影响具有重要的临床意义。
基于以上研究背景,本课题旨在查明水飞蓟素对几种重要的CYP450酶和药物转运体在体外和人体内产生的影响及其临床意义。通过建立体外的细胞平台,在mRNA表达和蛋白表达水平层面探讨水飞蓟素对上述CYP450酶的影响;并进一步通过严格设计的临床实验,分别以咪达唑仑、洛沙坦和他林洛尔为探药,基于药物代谢酶和转运体的遗传多态性对其功能的影响,在整体药代动力学水平和药物相互作用层面探讨水飞蓟素制剂对相应CYP450酶和药物转运体活性的影响,为临床上药物相互作用及个体反应差异提供分子水平的解释,并为临床实施中西药合理运用和基因导向性个体化用药提供指导。本课题的主要研究结果如下:
1.在Chang Liver细胞系中观察水飞蓟素对CYP3A4,CYP2C9,CYP2C19,CYP2D6和CYP2E1 mRNA和蛋白表达的影响,发现水飞蓟素对CYP3A4 mRNA表达有明显的抑制作用,水飞蓟素处理Chang Liver细胞48小时能够显著减少CYP3A4 mRNA含量,并且呈浓度依赖性,在水飞蓟素处理24小时候也可以观察到CYP3A4 mRNA表达减少的趋势,并且在40μM处理组此差异具有统计学意义;水飞蓟素对CYP2C9 mRNA表达也呈现显著的抑制作用,水飞蓟素处理ChangLiver细胞24小时和48小时均能够使CYP2C9 mRNA含量显著减少,并且呈浓度依赖性,以48小时抑制作用表现更明显。对于CYP2C19、CYP2D6以及CYP2E1,不同浓度的水飞蓟素分别作用24和48小时则没有表现出明显的抑制或者诱导效应。水飞蓟素对CYP3A4和CYP2C9蛋白表达有明显的抑制作用,水飞蓟素处理Chang Liver细胞24小时和48小时均能够显著减少CYP3A4和CYP2C9蛋白含量,并且呈浓度依赖性,且在48小时抑制作用表现更明显。
2.以镇静安眠药咪达唑仑作为探药,在不同CYP3A5*3基因型个体中研究了水飞蓟素对人体CYP3A酶代谢活性的影响,表明水飞蓟素能够抑制CYP3A所介导的咪达唑仑羟化代谢。
3.用洛沙坦作为探药,研究了不同CYP2C9基因型个体中水飞蓟素对洛沙坦及其活性代谢产物E3174药代动力学的影响,发现洛沙坦转化为E3174的代谢率在应用水飞蓟素后显著降低,并且洛沙坦和E3174的AUC_((0-24)),AUC_((0-∞))和C_(max)的变化程度在CYP2C9不同基因型间有显著差异,表明水飞蓟素对人体CYP2C9酶活性有显著的抑制作用;
4.用他林洛尔作为探药,在不同MDR1基因型个体研究了水飞蓟素对人体P-糖蛋白转运体活性的影响,发现水飞蓟素显著增加了他林洛尔的血药浓度和口服利用度,提示水飞蓟素在人体内显著抑制了P-糖蛋白转运体活性;
总之,本项目从细胞到临床整体水平为水飞蓟素制剂的临床药物相互作用和反应个体差异提供了遗传分子水平的解释,为临床合理使用水飞蓟素和镇静安眠药咪达唑仑,降血压药物洛沙坦和β-受体阻滞药物他林洛尔以及其它相关药物提供了实验依据和理论指导。
Pharmacogenetics studies indicate that activity change of drug-metabolizing enzymes and transpoters is the major mechanism for clinically drug-drug interactions,and inherited variation in activities of drug-metabolizing enzymes and transporters results in interindividual differences in drug metabolism,disposition and efficacy.Recently,herbal medicines are more and more widely used by people around the world.The deepgoing study of pharmacogenetics will help us to choose right drug and adjust drug dose to avoid drug side effect and attain more drug efficacy.
Cytochrome P450s(CYP450) superfamily expressed widely in organisms are known to play an important role in the biotransformation of many endogenous and exogenous substances.Inhibition or induction of cytochrome P450 isozymes is one of the major causes for clinical drug-drug interactions. CYP3A enzymes are the most abundantly expressed cytochrome P450 enzymes in liver and intestine and play an essential role in the oxidative biotransformation of endogenous compounds and exogenous chemicals, including more than 60%of medically relevant drugs,such as antitumor drugs. CYP3A4 and CYP3A5 are the predominant functional forms of human CYP3A enzyme in adult.The activity of CYP3A influences curative effect of drugs and the patients' reaction to the toxicity of many medicines.In the meantime,it also has a relationship with the risk that the pollution of the environment leads to disease.The high expression of CYP3A can cause the failure of tumor chemotherapy.Effects of drugs on the activity of CYP3A is one of the main causes of drug-drug interaction.Polymorphic CYP3A5 expression in the adult liver and small intestine is strongly correlated with a single nucleotide polymorphism(6986A＞G),which,located in the intron 3 of CYP3A5,could cause alternative splicing and protein truncation resulting in the absence of CYP3A5 protein or seriously decreasing activity of CYP3A.CYP2C9 is a polymorphic enzyme that is responsible for the metabolism of many clinically useful drugs,such as S-warfarin,tolbutamide,phenytoin,glipizide,losartan and numerous non-steroidal anti-inflammatory drugs.Several allelic variants of CYP2C9 have been identified,with the most important mutations being CYP2C9~*2 and CYP2C9~*3.The CYP2C9~*3 variant is well studied and has been reported to show decreased metabolic activity for many drug substrates of the enzyme,so the therapeutic effects of its substrate drugs can be reduced or more adverse reactions should occur.The change of CYP2C9 activity is a significant factor of racial differences and individual differences in clinical medication and drug interactions.
P-glycoprotein,an ATP-dependent efflux transporter coded by multidrug resistence gene(MDR1;ABCB1) and widely located at human normal tissues such as liver,kidney,small intestine and brain,plays the key role in drug absorption,distribution and elimination.Also,P-glycoprotein inhibitors are very important for reversement of multiple resistences of antitumor drugs.
Silymarin is a purified extract from the seeds of milk thistle,which has been used for over 2,000 years as one of the most popular herbs worldwide.It is reported that a standardized extract of milk thistle contains at least 70-80% silymarin,that consists predominantly of up to 60%of silibinin,but also contains isosilibinin,silycristin,silydianin,and other closely related avonolignans.Silymarin is used mainly for supportive treatment of alcoholic liver disease,chronic hepatitis and cirrhosis.Milk thistle ranks among the top-selling botanical supplements in the United States and is easily available as an over-the-counter herbal remedy in China.Milk thistle has become one of the most commonly used medical remedies all over the world,and its opportunities applied with other drugs are also increasing.
Up to date,although a lot of studies on silymarin pharmacological actions have been done.However,there is little report and study on metabolite and metabolic mechanism of silymarin.In addition,the inhibitive and inducing effects of silymarin on other drugs are not very clear.Inhibition or induction of cytochrome P450 isozymes and drug transporters is one of the major causes for clinical drug-drug interactions.To investigate medicines effect on CYP450s activities has important clinical significance,it helps with clinical rational administration and reducing the drug adverse reaction inducing by drug interactions.Research has found that silymarin could obviously inhibit a variety of CYP450 isozymes in mice and human liver microsomes.However, silymarin's effects on cytochrome P450s expression in human cell lines and in vivo have not been clarified.
Based on above information,our study was aimed to find effects of herbal medicine silymarin on some important CYP450 enzymes and drug transporters in vitro and in vivo.By setting up cell-based platform in vitro,the effects of silymarin on CYP450 enzymes have been explored at mRNA and protein level. Furthermore,in strictly designed clinical experiments,using midazolam, losartan,and talinolol as the substrate drugs,the potential impact of silymarin on corresponding CYP450 enzymes and drug transporters based on their common genetic polymorphisms have been clarified accociated with drug pharmacokinetics and herb-drug interactions.
The present series of studies have found that:
1.Silymarin can significantly inhibit the expression of CYP3A4 mRNA after Chang Liver cells were treated for 48 hours,and the inhibition effect is concentration dependent.We can also find a decline of CYP3A4 mRNA level after 24-hour silymarin treatment,which is of statistic significance at concentration of 40μM;Silymarin can also significantly inhibit the expression of CYP2C9 mRNA after Chang Liver cells were treated for both 24 and 48 hours.The inhibition effect is concentration dependent and more significant for 48 hours.Silymarin's effects on CYP2C19,CYP2D6 and CYP2E1 were not observed.Silymarin can significantly inhibit the expression of CYP3A4 and CYP2C9 proteins after Chang Liver cells were treated for both 24 and 48 hours,and the inhibition effect is concentration dependent for 48 hours.
2.420 mg daily administration of silymarin for 14 days significantly inhibits the metabolism of midazolam to 1-OH midazolam in healthy volunteers,with no interaction differing in individuals with different CYP3A5 genotypes.
3.420 mg daily administration of silymarin for 14 days significantly inhibits the metabolism of losartan to E-3174,with the magnitude of the interaction differing in individuals with different CYP2C9 genotypes. indicating that Silymarin can significantly inhibits CYP2C9 activity in humans;
4.420 mg daily administration of silymarin for 14 days increases the oral bioavailability of talinolol,and the increased plasma concentration of talinolol caused by silymarin co-administration may partially due to inhibition of intestinal talinolol efflux through P-gp.However,the interaction is not differing in individuals with different MDR1 genotypes. The present study has provided molecular mechanism for the herb-drug interactions and interindividual variations involving silymarin both in vitro and in vivo,provides experimental and theoretic guidance for combination use of silymarin formulations with sedative and hypnagogue midazolam, hypotensive drug losartan andβ-receptor antagonist talinolol as well as similar drugs.
引文
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