白芷呋喃香豆素的体内外处置以及基于CYP酶的药物相互作用研究

发布时间：2017-12-26 16:23

本文关键词：白芷呋喃香豆素的体内外处置以及基于CYP酶的药物相互作用研究　出处：《中国人民解放军军事医学科学院》2017年博士论文　论文类型：学位论文

【摘要】：中药是中华医学的瑰宝,并逐渐受到全世界的关注和重视。随着中药的应用日益广泛以及多样化,中药与西药合用的不良或毒性反应有较多报道,中药与西药合用时发生的代谢性相互作用,是产生不良或毒性反应的原因之一。相较于化学药物,中药的活性成分复杂、活性成分在体内的代谢处置性质不清楚、以及活性成分与代谢酶或转运体的作用机制缺乏系统评价等因素,是中药-西药相互作用(herb-dru g interaction,HDI)研究的难点。因此,系统研究中药活性成分的体内外代谢处置性质、与代谢酶或转运体的作用及其机制,有助于全面认识中药药效与毒效的物质基础,理解中药多成分的配伍规律、以及药物合用发生相互作用的机制,对于临床安全有效用药和中药的现代化,具有重要意义。本课题以中药白芷为研究对象,通过评价9个白芷呋喃香豆素的体内外代谢处置性质、及其与细胞色素P450(cytochrome,CYP)酶的抑制或诱导作用,研究白芷呋喃香豆素基于代谢酶的药物相互作用及机制;并通过本课题的研究,探讨中药复杂成分HDI的研究策略和方法。白芷是应用广泛的传统中药,也是多个经典方剂的重要组分。呋喃香豆素是白白芷中的一大类活性成分,具有广泛的药理活性,在肝脏发生由CYP酶介导的广泛代谢。已有的研究表明,白芷与处方药物合用时,有发生HDI的风险,呋喃香豆素对CYP酶的抑制或诱导作用可能是白芷发生HDI的主要原因。但目前对白芷呋喃香豆素的体内外代谢处置性质及其与CYP酶相互作用的研究尚不系统,已有的研究多集中于欧前胡素等少数主要成分。为此,本课题旨在通过白芷呋喃香豆素吸收、分布、排泄和药代动力学过程的系统研究,阐明其体内外代谢处置特征,识别白芷给药后体内血循环和组织器官中的标志性呋喃香豆素成分。在此基础上,评价白芷呋喃香豆素对CYP酶的抑制或诱导作用,理解体内外代谢处置性质与药物相互作用的关系,深入研究CYP酶抑制作用机制,为白芷的临床合理用药和人体HDI预测,提供必要的数据支持和科学依据。同时,在本研究的实施过程中,摸索并探讨中药多组分药物相互作用研究的策略与方法。本课题的研究内容和结果总结如下:1..建立并验证了同时定量检测生物样品中欧前胡素(ⅠM)、异欧前胡:素(ⅡM)、水合氧化前胡素(OXYH)、佛手柑内酯(BER)、氧化前胡素(OXY)、花椒毒酚(XANT)、花椒毒素(XAN)、异茴芹内酯(IPIM)、补骨脂素(PSO)等9个呋喃香豆素的LC-MS/MS分析方法,方法灵敏、快速、可靠,可满足白芷呋喃香豆素体内外代谢处置的研究需求。2.大鼠口服白芷浸膏的血浆药代动力学结果显示,白芷呋喃香豆素在大鼠的吸收和消除均较快,药材中含量较高的ⅠM、ⅡM、OXYH和BER是大鼠血循环中的主要活性成分,代表了白芷呋喃香豆素90%以上的体内暴露量,是血浆暴露的标志性成分。排泄动力学结果显示,ⅠM、ⅡM、BER、XAN、IPIM和PSO的原型在尿、粪和胆汁中的回收率低于3%,表明其在体内发生广泛代谢,主要以产物的形式排泄。3.大鼠口服白芷浸膏后,呋喃香豆素在各组织中广泛分布;ⅠM、ⅡM、BER和OXYH是血浆和组织中的主要活性成分,占到总呋喃香豆素暴露量的90%以上,由此确认它们是白芷呋喃香豆素的体内暴露标志物。呋喃香豆素的肝脏分布显著高于血浆和其他组织,ⅠM、ⅡM和BER的肝脏和血浆暴露比值(K_p)分别为5.1、6.7和4.7,OXYH的K_p值为2.2。蛋白结合实验表明,ⅠM、ⅡM、BER和OXYH具有较高的血浆和组织蛋白结合率,其组织结合率高于血浆;经游离分数(fu)校正后,与组织总K_p值相比,组织和血浆的游离暴露比值(K_p,fu)显著降低,表明较高的蛋白结合是上述成分在组织高暴露的原因之一;ⅠM、ⅡM和BER的游离肝脏/血浆比值K_p,fu2.6,表明它们可特异性地分布于肝脏。4.采用悬浮培养的大鼠原代肝细胞模型,进一步探讨了 ⅠM、ⅡM、BER和OXYH在肝脏高水平分布的机制。实验表明,这些成分的肝细胞摄取是温度依赖的,37℃的摄取量显著高于4℃,提示它们的肝摄取可能存在主动转运机制;摄取动力学结果显示,ⅠM、ⅡM、BER和OXYH的肝细胞摄取同时涉及主动转运和被动扩散,其中ⅠM、ⅡM和BER以主动转运为主,主动摄取率(CLactive/CLuptake)高于70%;将呋喃香豆素在大鼠原代肝细胞中与摄取转运体的阳性抑制剂共孵育,摄取转运体Oatp的抑制剂可显著降低ⅠM和BER的肝细胞摄取,转运体Oat的抑制剂能明显抑制ⅡM的肝摄取,表明摄取转运体Oatp和Oat可能介导了 ⅠM、ⅡM和BER的主动转运过程,并在其肝脏特异性分布中发挥作用。5.给大鼠同时服用白芷和CYP1A2探针底物非那西丁,通过检测CYP1A2介导的非那西丁-O-脱乙基化反应,观察到单次口服白芷可显著提高非那西丁在大鼠的血浆暴露,白芷低剂量组(0.46 g/kg)和高剂量组(2.3 g/kg)的非那西丁血浆AUC分别为对照组血浆AUC的9和12倍,药物清除率下降,T1/2和MRT0-t延长;代谢产物对乙酰氨基酚的生成受到抑制,Cmax降低至对照组的14%-22%,达峰时间显著延迟,T1/2和MRT0-t延长;结果表明白芷单次给药可显著抑制大鼠CYP1A2酶的活性。6.采用混合探针底物法,评价9个白芷呋喃香豆素成分对人肝微粒体(HLM)和大鼠肝微粒体(RLM)主要CYP酶活性的抑制作用。结果表明,呋喃香豆素成分对HLM和RLM中多个CYP酶均有不同程度的抑制作用,其中对CYP1A2的抑制作用最强;ⅠM、ⅡIM、BER、OXY、XAN、IPIM 和 PSO 是人 CYP1A2的强抑制剂,IC50值在0.023-0.25 μmol/L之间;OXYH和XANT是中等抑制剂。进一步比较白芷提取液和9个呋喃香豆素混合溶液的CYP酶抑制作用,发现所测的9个呋喃香豆素成分可基本上代表中药白芷的CYP1A2抑制作用。由各呋喃香豆素单体在混合溶液中的含量分数和酶抑制IC50值,计算得到9个呋喃香豆素抑制CYP同工酶的整合IC50值;当CYP酶抑制作用分级为强或中等时,单体的整合IC50值与混合溶液的实测值相当,提示整合IC50值的方法可用于评价中药多组分的酶抑制作用。由酶抑制IC50值和大鼠肝脏浓度预测药物相互作用风险,ⅠM、ⅡM和BER对大鼠CYP1A2的[Ⅰ]/Ki值分别为19.6、4.02和2.0,提示它们在与CYP1A2底物药物合用时,具有较高的CYP1A2抑制相互作用风险,与大鼠体内实验结果相符。种属差异比较可见,ⅠM、ⅡM、OXYH和BER对人CYP1A2的抑制作用强于大鼠,提示它们在人体具有CYP1A2酶抑制相互作用的高风险性。7.应用IC50漂移实验观察到,ⅡM、OXYH和BER对HLM中CYP1A2的抑制作用是时间依赖的,辅酶NADPH预孵组的抑制曲线明显向左偏移,IC50比值(-/+)大于4;而ⅠM的抑制作用是可逆性的。基于机制的抑制动力学实验进一步显示,ⅡM、OXYH和BER对CYP1A2的抑制作用是预孵育时间和浓度依赖的,为基于机制的抑制(MBI),产生半数最大失活速率的浓度(KI)分别为2.08、1.96 和 6.71 μmol/L,失活速率常数(kinact)分别为 0.07、0.04 和 0.07 min-1 对CYP1A2酶具有较强的灭活作用。8.白芷浸膏多次给药后,低剂量组和高剂量组使非那西丁的大鼠血浆暴露(AUC)分别下降45%和62%,清除率分别为对照组的1.76和2.65倍;代谢产物对乙酰氨基酚的达峰时间提前,高剂量组的Cmax增高,T1/2和MRT0-t缩短;表明白芷多次给药可能通过CYP1A2酶诱导作用显著加快非那西丁在大鼠体内的清除。9.大鼠肝脏CYP酶活性和mRNA表达水平的测定结果显示,白芷多次给药后,对大鼠肝脏CYP1A、CYP2B和CYP3A的酶活性和表达均有不同程度的诱导作用,其中,对CYP1A的诱导作用最强,并具有剂量依赖性;低剂量组和高剂量组CYP1A2的酶活性分别为对照组的1.9和2.2倍,CYP1A1的mRNA表达水平分别为对照组的8.4和43.5倍,CYP1A2的mRNA表达水平分别为对照组的2.7倍和16.8倍,显示白芷多次给药对大鼠CYP1A1的诱导作用强于CYP1A2。10.应用SimCypTM软件的PBPK建模和模拟预测功能,构建了 ⅠM、ⅡM、OXYH和BER的大鼠PBPK模型,并采用大鼠实测数据对模型进行校正和验证;在此基础上建立了上述成分的人体PBPK模型,预测了人体血浆药代动力学和药物相互作用潜能。ⅠM、ⅡM和BER可使健康人群的咖啡因、非那西丁和茶碱AUC分别提高至对照组的2.0、1.7和1.3倍,在严重吸烟人群中合用组和对照组的AUC比值略高于健康人群相应的AUC比值,表明白芷呋喃香豆素在健康人群和吸烟人群均有发生基于CYP1A2抑制的药物相互作用风险。本论文有如下的新发现或创新点:1.白芷药材中含量较高的ⅠM、ⅡM、OXYH和BER也是血浆和组织中的主要活性成分,可作为白芷呋喃香豆素的体内暴露标志物,用于药效与毒效评价,这一发现为白芷药理学和药物相互作用的物质基础研究,提供了重要的依据。2.ⅠM、ⅡM、OXYH和BER在大鼠肝脏的暴露水平显著高于血浆和其他组织;通过蛋白结合、大鼠原代肝细胞摄取和Caco-2跨膜转运等研究,发现摄取转运体Oatp和Oat介导的主动转运在ⅠM、ⅡM和BER的肝脏特异性分布中发挥重要作用,非特异性蛋白结合也是这些成分在肝脏高暴露的原因之一。3.给大鼠同服白芷和非那西丁的相互作用研究,揭示了白芷对CYP1A2酶的双向调控作用,白芷单次给药可强烈抑制大鼠CYP1A2酶,多次给药不仅逆转了单次给药的抑制作用,还可显著诱导CYP1A2的基因表达和酶活性,这一发现对白芷的临床合理用药具有重要的指导意义。4.建立并验证了整合IC50值的酶抑制活性评价方法,可用于中药多成分(强或中等抑制剂)酶抑制作用的整体评估,为多组分药物的相互作用研究提供了可行的体外评价方法,也为中药在人体的酶抑制相互作用预测提供了参考。5.ⅠM是人CYP1A2的可逆性抑制剂,ⅡM、OXYH和BER是CYP1A2基于机制的抑制剂,应用PBPK模型进行的人体预测显示,这些成分在临床与CYP1A2的底物药物合用时,存在一定的药物相互作用风险;预测结果为白芷的新药研发及其临床安全有效用药提供指导。
[Abstract]:Chinese medicine is a gem of Chinese medicine and has gradually attracted the attention and attention of the whole world. With the increasingly wide application and diversification of traditional Chinese medicine, there are many reports about adverse or toxic reactions of traditional Chinese medicine combined with western medicine. The metabolic interaction between Chinese medicine and Western medicine is one of the causes of adverse or toxic reactions. Compared with chemical drugs, traditional Chinese medicine active component complex, the active ingredient in vivo metabolism handling properties is not clear, as well as the active ingredient and metabolic enzyme or transporter mechanism lack of system evaluation and other factors, is a herbal drug interaction (herb-dru g interaction, HDI) the difficulties of the study. Therefore, the effect and mechanism of active ingredients of Chinese medicine on metabolism in vitro and in vivo disposition properties, and metabolic enzyme or transporter, contribute to the material basis for a comprehensive understanding of the efficacy of traditional Chinese medicine and toxic effects, compatibility rules, understanding of traditional Chinese medicine composition and drug interaction mechanism, for the safe and effective clinical medication and traditional Chinese medicine modernization that is important. This topic in Angelica dahurica as the research object, through the evaluation of 9 Angelica Furanocoumarin metabolism in vitro and in vivo properties, and the disposal of cytochrome P450 (cytochrome, CYP) enzyme inducing or inhibiting effect of angelica, furanocoumarins based on drug metabolizing enzyme interactions and mechanisms; and through this study, study strategy and the method of HDI complex ingredients of traditional Chinese medicine. Radix Angelicae dahuricae is a widely used traditional Chinese medicine, and it is also an important component of many classical prescriptions. Furanolazin is a major active component in dahurica dahurica and has extensive pharmacological activity. It is widely metabolized by CYP enzyme in the liver. Previous studies have shown that when Angelica dahurica and prescription drugs are used together, there is a risk of HDI. The inhibition or induction of furfuryl coumarin on CYP enzyme may be the main cause of HDI in Angelica dahurica. But now the research in vivo and in vitro metabolism properties of Angelica disposal furanocoumarins and CYP enzyme interaction is not systematic, the existing research focused on imperatorin and a few main components. Therefore, the aim of this study is to elucidate the characteristics of metabolic disposal in vivo and in vitro through systematic study of absorption, distribution, excretion and pharmacokinetics of furofcoumarin in Angelica dahurica, and identify the labeled furan coumarin components in blood circulation and tissues of Angelica dahurica. On the basis of evaluating the inhibitory Furanocoumarin to CYP enzyme or the induction, understand the relation between in vitro and in vivo metabolism handling properties and drug interaction, in-depth study of CYP enzyme inhibition mechanism, prediction for Angelica clinical rational use of drugs and human HDI, provide necessary data support and scientific basis. At the same time, in the process of the implementation of this study, we explored and explored the strategies and methods of the research on the interaction of multiple components of traditional Chinese medicine. The main research contents and results are summarized as follows: 1.. Is established and verified at the same time, the quantitative detection of biological samples of imperatorin (I M) and ISO imperatorin: (II M), oxypeucedanin hydrate (OXYH), bergapten (BER), oxypeucedanin (OXY), Pepper Virus phenol (XANT), Xanthotoxin (XAN), isopimpinellin (IPIM), psoralen (PSO) 9 furanocoumarins LC-MS/MS analysis method, the method is sensitive, rapid and reliable, and can meet the needs of Angelica furanocoumarins in vivo metabolism disposal. 2. rat oral angelica extract the plasma pharmacokinetic results showed that Angelica furanocoumarins in rats of absorption and elimination were fast, high content of Chinese herbal medicine of M, M, OXYH II and BER are the main active ingredients of rats in blood circulation, representing 90% of the angelica furanocoumarins in vivo exposure that is a symbol of the composition of plasma exposure. The results of excretion kinetics showed that the recovery rate of M, M, BER, XAN, IPIM and PSO in urine, feces and bile was less than 3%, indicating that they were extensively metabolized in vivo, mainly excreted in the form of products. 3. rat oral angelica extract, furanocoumarins are widely distributed in various tissues; I M and I M, BER and OXYH are the main active components in plasma and tissue, accounted for more than 90% of the total furanocoumarins exposure, so that they are Angelica furanocoumarins in vivo exposure markers. The liver distribution of furofcoumarin was significantly higher than that in plasma and other tissues. The exposure ratio (K_p) of liver and plasma of I M, II M and BER were 5.1, 6.7 and 4.7, respectively, and the K_p value of OXYH was 2.2. Protein binding assay showed that plasma and tissue protein I, II, BER and M M OXYH has a higher binding rate, the tissue binding rate is higher than the plasma free fraction; (Fu) after correction, compared with the total K_p value, the ratio of tissue and plasma free exposure (K_p, Fu) was significantly lower, show high protein binding is one of the reasons for the composition of high exposure to the organization; I M and I M and BER free liver / plasma ratio of K_p, fu2.6, show that they specifically distributed in liver. 4. the mechanism of the high level distribution of I M, II M, BER and OXYH in the liver was further investigated by using the primary rat hepatocyte model in suspension culture. Experimental results show that the hepatic uptake of these components is temperature dependent, intake of 37 DEG C was significantly higher than 4 DEG C, suggesting that their hepatic uptake may be an active transport mechanism; uptake kinetics showed that the hepatic uptake of M, M, BER I and OXYH II was also involved in active transport and passive diffusion, which I M M and BER II, active transport, active uptake rate (CLactive/CLuptake) higher than 70%; the positive inhibitor furanocoumarins in primary rat hepatocyte and uptake transporter co incubation, Oatp uptake transporter inhibitors can significantly reduce the hepatic uptake of M and BER, Oat can significantly inhibit the transporter inhibitor II M hepatic uptake showed that uptake transporter Oatp and Oat may be mediated by the active transport process of M, M and BER II, and play in the liver specific distribution
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285

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