节点文献
人源CYP450s高表达体系和核受体介导的人源CYP3A4及MDR1诱导作用研究体系的构建和应用
【作者】 李越；
【作者基本信息】 中国协和医科大学 ， 药理学， 2010， 博士
【摘要】 随着人类基因组草图的完成和组合化学等的飞速发展,新药候选化合物的合成速度和来源明显增加。对候选化合物进行药效、药代和毒性早期评价是新药筛选的重要内容。药物代谢是新药研发过程中不可缺少的重要环节,每年都会有大量的候选药物因药代动力学参数和代谢特征不佳而被淘汰。因此,在药物设计及新药开发早期进行药物代谢评价能够降低候选药物的淘汰率,有助于获得安全、有效的治疗药物。体内外药物代谢评价体系亦日趋成熟,现已广泛应用于药物代谢酶的诱导与抑制、参与代谢的药酶种类、活性代谢物的生成等药物代谢特性方面的研究。上述研究还可为合并用药时药物-药物相互作用的预测提供重要依据。药物代谢评价方法可分为体内和体外两大类。相比体内研究,体外研究方法由于快速、简便、易行而受到广泛关注。目前常用的体外代谢评价体系主要包括基因重组酶系、亚细胞组分,细胞、组织切片等。基因重组酶系是指利用基因工程、细胞工程对人类药物代谢酶进行表达、纯化,获得较纯的单一药物代谢酶的方法,主要用于药物代谢途径、代谢酶类型和代谢产物鉴定、抑制作用、特异性底物和抑制剂研究,代谢性药物相互作用评价等。由于药物代谢酶存在种属差异,应用动物酶系预测新药药代特性具有一定的局限性,因此重组人源药物代谢酶系为评价基于药酶的药代特性提供了重要和理想的体外模型和有效的分析手段。细胞色素P450 (Cytochrome P450, CYP450)是人体内最重要的Ⅰ相药物代谢酶,主要催化氧化还原反应。CYP450酶系有3个基因家族(CYP1,CYP2,CYP3),参与药物代谢的CYP450s包括CYP3A4、CYP2D6、CYP2C9、CYP2C19、CYP2E1和CYP1A2,其中CYP3A4在药物及内源物代谢过程中发挥重要作用,临床上超过50%的药物均经此酶代谢。P-糖蛋白(P-glycoprotein, P-gp)是依赖ATP的膜转运蛋白之一,在肠、肝、肾脏等脏器均高度表达,可以影响部分药物吸收、分布和排泄等药动学过程。孕烷X受体(pregnane X receptor, PXR)和组成型雄甾烷受体(Constitutive androstane receptor, CAR)作为主要的核受体能够与RXRa形成异源二聚体,发挥调控CYP3A4和MDR1基因表达水平的作用。构建重组人源CYP450表达体系和PXR和CAR介导的人源CYP3A4和MDR1诱导作用研究体系可以为药物体外代谢和机制探讨提供有效的评价体系。黄芩苷、黄芩素、绿原酸和人参皂苷Rf是多种中草药中的有效成分,也广泛存在于蔬菜和水果中,具有抗肿瘤、抗炎、抗高血压和抗疲劳等药理作用。以往研究初步探讨了黄芩苷、黄芩素、绿原酸和人参皂苷Rf对CYP450s同工酶的诱导或抑制作用,但详细机制未见报道。本论文应用分子生物学和细胞生物学技术,建立人源CYP450s同工酶高表达体系和核受体介导的CYP3A4和MDR1诱导作用研究体系。再利用同工酶高表达体系初步探讨黄芩素和黄芩苷对CYP2E1重组酶的抑制作用；通过核受体介导的诱导体系探讨绿原酸、人参皂苷Rf,黄芩苷和黄芩素对CYP3A4和MDR1的诱导作用及其分子机制,不仅为体外药物代谢的研究提供实用的实验模型,也为上述药物的临床应用提供有参考价值的实验依据。1.人源CYP450s高表达体系的构建及应用1.1 CYP450氧化还原酶(Cytochrome P450 oxidoreductase, CYPOR)作为主要电子供体对CYP450同工酶活性的保持具有重要作用。本研究首先应用逆转录聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)扩增获得CYPOR编码序列基因片段,插入至pET-22b(+)原核表达载体后构建pET-22b(+)-CYPOR重组表达载体。重组质粒转化大肠埃希菌BL21,IPTG诱导表达后,成功获得CYPOR重组蛋白。经测定,重组CYPOR蛋白酶活性为1309.64nmol/mg·min,可作为工具酶用于后续研究。1.2杆状病毒-昆虫细胞表达系统(Bac-to-Bac)具有安全性好、表达水平高,可进行翻译后加工的特点,是一种非常理想的真核表达系统。本研究利用Bac-to-Bac系统建立了人源CYP450s高表达体系,获得了CYP2E1、CYP2D6和CYP3A4等3种重组同工酶。应用特异性底物测定表达产物酶活性,结果表明,CYP2E1重组酶活性最高；多种因素可对表达产物酶活性产生影响,条件优化是获得CYP2E1重组酶最佳表达量和最高酶活性的重要环节。具体而言,当MOI值为0.1时,CYP2E1重组酶活性最高,之后随着MOI值的增大而逐渐减小；重组病毒感染细胞72h时获得的CYP2E1酶活性显著高于48h、96h和120h；CYPOR和细胞色素b5(Cytochrome b5, CYb5)的浓度对重组酶活性也有很大影响,适量添加对酶的催化反应有利。CYP2D6和CYP3A4重组酶已经进行了MOI值、病毒感染时间和CYPOR、CYb5浓度等条件的优化,但更为适合的条件仍有待进一步探索。1.3应用已获得较高酶活性的CYP2E1重组酶初步研究了黄芩苷和黄芩素对CYP450同工酶的抑制作用。结果表明,黄芩苷的IC50为5.7021μM,而黄芩素的IC50>50μM。提示黄芩苷对CYP2E1具有较强的抑制作用,而黄芩素的抑制作用较弱。2.核受体介导的人源CYP3A4及MDR1诱导作用研究体系的构建和应用2.1本研究首先应用RT-PCR方法获得人源CYP3A4的启动子／增强子和视黄醛X受体(retinoid X receptorα, RXRα)基因片段,分别插入至pGL4.10和pCDNA3.1(-)表达载体中成功构建pGL4.10-CYP3A4启动子报告基因表达载体和pCDNA3.1(-)-RXRa真核表达载体。2.2 mRNA水平测定是基因转录调控研究的重要方面。本研究应用real time PCR方法考察了黄芩苷、黄芩素、绿原酸和人参皂苷Rf对CYP3A4和MDR1 mRNA水平的影响。结果显示,黄芩苷、黄芩素、绿原酸和人参皂苷Rf对CYP3A4和MDR1转录水平产生不同程度的诱导作用。黄芩素的诱导作用最强；绿原酸和人参皂苷Rf仅对CYP3A4有诱导作用；黄芩苷对两者则皆无明显影响。黄芩素、绿原酸和人参皂苷Rf还对CYP3A4 mRNA水平的影响具有一定的浓度依赖性,其浓度分别为20、0.1和10μM时对CYP3A4的诱导作用最强,为阴性对照的3.4倍、2.5倍和2倍。外,黄芩素、绿原酸和人参皂苷Rf对CYP3A4的蛋白表达和酶活性水平亦表现出一定的诱导作用。2.3 PXR和CAR是调控CYP3A4和MDR1基因转录水平的主要核受体。本研究应用报告基因检测和细胞转染技术建立了核受体介导的人源CYP3A4和MDR1诱导作用研究体系。并用PXR和CAR受体的典型配体-利福平和CITCO对该体系进行了证实。研究结果表明,利福平能够显著增强PXR受体介导的CYP3A4和MDR1基因转录；CITCO通过活化CAR受体途径上调了CYP3A4和MDR1基因转录水平。提示该研究体系成功建立,可用于药物对CYP3A4和MDR1诱导作用的研究。应用该体系,本研究考察了黄芩苷、黄芩素、绿原酸和人参皂苷Rf对CYP3A4和MDR1的诱导作用机制。结果显示,黄芩素可以显著增强CAR和PXR受体介导的CYP3A4和MDR1基因转录水平；绿原酸和人参皂苷Rf能够上调CAR受体介导的CYP3A4启动子活性,而PXR受体则较少参与,二者对MDR1启动子活性无明显影响。2.4 CYP3A4基因启动子反应元件-DR3和ER6以及MDR1基因启动子反应元件-DR4(Ⅰ)均能被CAR-RXRa和PXR-RXRa二聚体识别并发挥基因表达调控作用。针对DR4(Ⅰ)、DR3和ER6进行的EMSA研究可以更为深入的揭示诱导作用及其机制。研究显示,黄芩素可以明显增强CAR／RXRα二聚体与DR3、ER6／DR4(Ⅰ),PXR／RXRa二聚体与DR3的结合作用,而对DR4(Ⅰ)和ER6与PXR／RXRα二聚体的结合能力无显著影响。2.5大部分常用药物均经CYP3A代谢,包括新型免疫抑制剂他克莫司在内的多种药物亦是CYP3A和P-gp的共同底物；上述研究已经证实,黄芩素对CYP3A和P-gp均有诱导作用,因此当黄芩素制剂与上述药物合用时应考虑到药物间相互作用发生的可能性。本研究利用体内研究方法对此进行了探讨,结果显示,黄芩素多次给药可降低他克莫司血药浓度,其中峰浓度降低约57%,AUC减小约55%。综上所述,本研究构建了人源CYP450s同工酶高表达体系和核受体介导的CYP3A4和MDR1诱导作用研究体系。利用同工酶高表达体系获得的高活性重组CYP2E1同工酶研究证实,黄芩苷对CYP2E1具有一定的抑制作用,而黄芩素抑制作用较弱。本研究还利用核受体介导的CYP3A4和MDRl诱导作用研究体系考察了黄芩素、绿原酸和人参皂苷Rf对CYP3A4和MDR1的诱导作用及其机制。结果表明,黄芩素可以显著增强CAR和PXR受体介导的CYP3A4和MDR1基因转录水平；绿原酸和人参皂苷Rf能够上调CAR受体介导的CYP3A4启动子活性,而PXR受体则较少参与,二者对MDR1启动子活性无明显影响。人源化CYP450s同工酶高表达体系和核受体介导的人源CYP3A4和MDR1诱导作用研究体系的建立,为药代特性评价、代谢机制以及潜在的药物相互作用的研究提供科学、实用的体外模型。黄芩苷、黄芩素、绿原酸和人参皂苷Rf的研究结果也为预防药物相互作用、提高药物有效性和安全性提供了参考依据。
【Abstract】 With the explosive growth in the field of genomics and combinatorial chemistry, synthesizing candidate compounds have been accelerated greatly. It is an important part of new drug screening to evaluate pharmacodynamics, pharmacokinetics and toxicity of compound candidates. Drug metabolism is an indispensable part in the process of new drug research and development. Every year a large number of drug candidates are eliminated due to poor pharmacokinetic parameters and metabolic characteristics. Therefore, early evaluation can contribute to get safe and effective drugs, and to reduce the elimination rate during drug design and development.Evaluation of drug metabolism has become mature increasingly. Nowadays, it is widely used in the research of drug metabolism, including induction and inhibition of drug metabolizing enzyme, enzyme types involved in drug metabolism, active metabolites formation and so on.These studies will also provide important evidences to predict drug interactions during coadministration.Evaluation of drug metabolism include in vivo and in vitro. As simple and feasible methods, in vitro is widespread attention, compared to in vivo. Evaluation of metabolism in vitro includes recombinant enzymes, liver slices, liver cells.Recombinant enzyme is a method that can express and putify human drug metabolizing enzymes through genetic and cellular engineering to obtain pure, single drug metabolizing enzymes. They can be used to study the profile of drugs and xenobiotics metabolism, and analyze metabolites from drugs and xenobiotics catalyzed by single enzyme. The probe substrate and the selective inhibitor of the single metabolizing enzyme can be characterized with the recombinant enzymes, which provide information for the dtug interaction. Since drug metabolizing enzymes have species differences, application of animal or tissueit to predict characteristics of drug pharmacokineticsis is limit, and even lead to error messages. Recombinant enzymes can make up for shortcomings from animal or isolated tissue to some extent, avoid interference to results from other enzymes in the course of study, and increase relevance and credibility of research. Recombinant drug metaboliszing enzymes provide an important and desirable model in vitro and effective analytical tools for the evaluation of drug metabolism and the clinical rational application of drugsCytochrome P450 (CYP450), the most important I phase drug metabolizing enzymes, mainly catalyze the redox reactions. CYP450 include three gene families (CYP1, CYP2, CYP3), among which CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1 and CYP1A2 are mainly involved in drug metabolism. CYP3A4 is the most important metabolizing enzyme related to the biotransformation of endogenous and exogenous compounds including drugs, and metabolizesmore than 50% of the clinical drugs. P-glycoprotein (P-gp), one of ATP-dependent membrane transporter, is highly expressed in intestine, liver and kidney and can affect the absorption, distribution and excretion.Pregnane X receptor (PXR) and constitutive androstane receptor (CAR), the key nulear receptors, can interact with the retinoid X receptor a in response to structurally diverse compounds, and regulate the transcriptional activity of their target genes, such as CYP3A4 and multi-drug resistance 1 (MDR1). Construction of research system of PXR/CAR-mediated induction of CYP3A4 and MDR1 may provide effective evaluated system for the study of drug metabolism in vitro and mechanism.Baicalin, baicalein, chlorogenic acid, and ginsenoside Rf derived from herbal products are widely present in vegetables, fruit and herbal medicines with multiple beneficial effects, including anti-tumor, anti-inflammatory, anti-hypertensive and anti-fatigue. It is reported that the mentioned above compounds had induction or inhibition on some CYP450s isozymes, but the detailed molecular mechanisms underlying their action are not well explored.In the present study, molecular and cell biology methods were used to establish high expression system of human CYP450s isozymes and research system of nuclear receptors-mediated induction of CYP3A4 and MDR1. Then the inhibition of CYP2E1 recombinant enzyme by baicalein and baicalin was studied with the high expression system of human CYP450s isozymes. Moreover, the research system of nuclear receptors-mediated induction of CYP3A4 and MDR1 was also used to study the induction and molecular mechanism of CYP3A4 and MDR1 by baicalin, baicalein, chlorogenic acid and ginsenoside Rf. The present study will provide a good experimental model in vitro for research of drug metachnism in vitro, and also provide valuable experimental evidences for the clinical application of the above compounds.1. Construction and application of high expression system of human CYP450s isoenzymes1.1 As the primary electron donor, cytochrome P450 oxidoreductase (CYPOR) play an important role on the activity of CYP450 isoenzymes. The 2106bp in length cDNA fragment of CYPOR was amplified by RT-PCR, and inserted into prokaryotic expression vector pET-22b(+). Thus the recombinant expressive vector pET-22b(+)-CYPOR was constructed. After transformation into E.coli BL21with pET-22b(+)-CYPOR and induction with IPTG, recombinant target protein with Mr 75kD or so was expressed. Compared with negative control, CYPOR protein had higher enzymatic activity with 1309.64 nmol/mg-minute, and could be used for the following study.1.2 Baculovirus-insect cell (Bac-to-Bac) system is an ideal eukaryotic expression system due to good security, high expression and procession post-translation. The high expression system of CYP450s was established using Bac-to-Bac, through which some CYP450 isoenzymes such as CYP2E1, CYP2D6 and CYP3A4 were expressed. The results of enzyme activity determination demonstrated that CYP2E1 had higher enzyme activity. The expression and activity of CYP2E1 recombinant enzyme closely related to many factors, thus optimization was an important aspect to obtain the highest expression and activity of recombinant enzymes. Specifically, when the MOI value was 0.1, the activity of CYP2E1 recombinant enzyme was highest, and then gradually decreased with the MOI value increasing. In comparision with 48h,96h and 120h, CYP2E1 enzyme activity was highest at 72h when Sf9 cells were infected with recombinant virus. Since the concentrationof CYPOR and CYb5 had a great effect on the CYP2E1 enzyme activity, the appropriate concentration should be selected to increase enzyme catalytic activity. Some conditions such as MOI value, infection time, and the concentration of CYPOR and CYb5 have been optimized to improve the activity of CYP2D6 and CYP3A4 recombinant enzymes, but more suitable factors need to be further studied.1.3 The inhibition of activity of CYP2E1 by baicalin and baicalein was studied using CYP2E1 recombinant enzyme with high activity. The results showed that baicalin had the most potent inhibition with an IC50 value of 5.702μM, while the IC50 value of baicalein was over 50μM. The results indicated that baicalin had a stronger inhibition on CYP2E1, while the inhibition by baicalein was weak.2. Construction and application of research system of nuclear receptors-mediated induction of human CYP3A4 and MDR12.1 The DNA fragment of CYP3A4 promoter/enhancer and retinoid X receptor (RXRa) gene were amplified by RT-PCR, and inserted into expression vectors pGL4.10 and pCDNA3.1(-), respectively. Thus pGL4.10-CYP3A4 promoter reporter gene expression vector and pCDNA3.1(-)-RXRa eukaryotic expression vector were constructed successfully.2.2 The effects of baicalin, baicalein, chlorogenic acid, and ginsenoside Rf on the mRNA expression of CYP3A4 and MDR1 were investigated using real time PCR because transcriptional regulation is the key mechanism for gene expression and modulation. As a result, it was different that baicalin, baicalein, chlorogenic acid and ginsenoside Rf showed induction on CYP3A4 and MDR1 at the level of transcription. Baicalein was found to be the strongest inducer of CYP3A4 and MDR1 among the four bioactive ingredients, while chlorogenic acid and ginsenoside Rf only showed induction of CYP3A4. However, baicalin had no effect on either CYP3A4 or MDR1 gene expression. Baicalein, chlorogenic acid, and ginsenoside Rf were observed to up-regulate the mRNA level of CYP3A4 in a dose-dependent manner, and the maximal induction was achieved at the concentration of 20,0.1 and 10μM, respectively. Furthermore, CYP3A4 activity and protein levels were also increased by baicalein-/chlorogenic acid-/ginsenoside Rf.2.3 PXR and CAR are considered to be the key transcriptional regulators of CYP3A4 and MDR1. The research system of nuclear receptors-mediated induction of CYP3A4 and MDR1 was established by reportor gene assay with cotransfection, and was confirmed with rifampin and CITCO which were the typical ligands of pregnane X receptor (PXR) and constitutive Androstane receptor (CAR), respectively. As a result, rifampin significantly enhanced PXR-mediated CYP3A4 and MDR1 gene transcription, while CITCO also upregulated the transcription level of CYP3A4 and MDR1 genes through the activation of CAR receptor. The results suggested that the the research system of nuclear receptors-mediated induction of CYP3A4 and MDR1 was constructed successfully, and can be used for drugs induction of CYP3A4 and MDR1 in vitro. The molecular mechanisms of induction of CYP3A4 and MDR1 by baicalein, chlorogenic acid and ginsenoside Rf were investigated through the construced research system. The results showed that baicalein significantly induced the expression of CYP3A4 and MDR1 mRNA by activating PXR and CAR. Chlorogenic acid and ginsenoside Rf induced CAR but not PXR-mediated CYP3A4 expression, and demonstrated effects on MDR1 via neither the CAR nor PXR pathways.2.5 The response elements of the CYP3A4 promoter consist of two copies of the DR3 and ER6, and DR4 is an important response element in the MDR1 promoter. All of these elements could be recognized by CAR and PXR after heterodimerization with RXR. Baicalein significantly increased the binding of CAR/RXRa heterodimers to the DR3 and ER6 response elements of CYP3A4, and to the DR4(I) response element of MDR1. However, no effect on the binding of PXR/RXRa heterodimers to the DR4(I) and ER6 elements of MDR1 and CYP3A4 except DR3 sequences was observed.2.6 It is reported that most common used oral drugs were metabolized by CYP3A in human and most of them including tacrolimus, a new type of immunosuppressive drug, were also cosubstrates of both CYP3A and P-gp. Since baicalein had induction on CYP3A and P-gp, coadministration interaction should be concerned between those drugs and pharmaceutics from baicalein. Our results showed that the blood concentration of tacrolimus was decreased (Cmax reduced by 57%, AUC reduced by 55%) by multiple dosing of baicalein. As a result, the dosage of tacrolimus should be increased to maintain effective plasma concentration and avoid the occurring of organ rejection when this drug was used in combination with baicalein.In conclusion, the high expression system of CYP450s isoenzymes and research system of nuclear receptors-mediated induction of CYP3A4 and MDR1 were established in the present study, which provide a good experimental model in vitro for research of the induction/inhibition and related mechanisms of drug metabolizing enzymes and transporter proteins, and drug interactions. The inhibition of activity of CYP2E1 by baicalin and baicalein was studied using CYP2E1 recombinant enzyme with high activity. The results indicated that baicalin had a stronger inhibition on CYP2E1, while the inhibition by baicalein was weak. Moreover, the research system of nuclear receptors-mediated induction of CYP3A4 and MDR1 was also used to study the induction and molecular mechanism of CYP3A4 and MDR1 by baicalin, baicalein, chlorogenic acid and ginsenoside Rf. The results showed that baicalein significantly induced the expression of CYP3A4 and MDR1 mRNA by activating PXR and CAR. Chlorogenic acid and ginsenoside Rf induced CAR but not PXR-mediated CYP3A4 expression, and demonstrated effects on MDR1 via neither the CAR nor PXR pathways.The results of baicalin, baicalein, chlorogenic acid and ginsenoside Rf also provide a reference for prevention of drug interactions and improving drug efficacy and safety.