本文關(guān)鍵詞：白芷呋喃香豆素的體內(nèi)外處置以及基于CYP酶的藥物相互作用研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 白芷 呋喃香豆素 代謝處置 中藥藥物相互作用 基于生理的藥代動力學(xué)模型

【摘要】：中藥是中華醫(yī)學(xué)的瑰寶,并逐漸受到全世界的關(guān)注和重視。隨著中藥的應(yīng)用日益廣泛以及多樣化,中藥與西藥合用的不良或毒性反應(yīng)有較多報道,中藥與西藥合用時發(fā)生的代謝性相互作用,是產(chǎn)生不良或毒性反應(yīng)的原因之一。相較于化學(xué)藥物,中藥的活性成分復(fù)雜、活性成分在體內(nèi)的代謝處置性質(zhì)不清楚、以及活性成分與代謝酶或轉(zhuǎn)運體的作用機制缺乏系統(tǒng)評價等因素,是中藥-西藥相互作用(herb-dru g interaction,HDI)研究的難點。因此,系統(tǒng)研究中藥活性成分的體內(nèi)外代謝處置性質(zhì)、與代謝酶或轉(zhuǎn)運體的作用及其機制,有助于全面認識中藥藥效與毒效的物質(zhì)基礎(chǔ),理解中藥多成分的配伍規(guī)律、以及藥物合用發(fā)生相互作用的機制,對于臨床安全有效用藥和中藥的現(xiàn)代化,具有重要意義。本課題以中藥白芷為研究對象,通過評價9個白芷呋喃香豆素的體內(nèi)外代謝處置性質(zhì)、及其與細胞色素P450(cytochrome,CYP)酶的抑制或誘導(dǎo)作用,研究白芷呋喃香豆素基于代謝酶的藥物相互作用及機制;并通過本課題的研究,探討中藥復(fù)雜成分HDI的研究策略和方法。白芷是應(yīng)用廣泛的傳統(tǒng)中藥,也是多個經(jīng)典方劑的重要組分。呋喃香豆素是白白芷中的一大類活性成分,具有廣泛的藥理活性,在肝臟發(fā)生由CYP酶介導(dǎo)的廣泛代謝。已有的研究表明,白芷與處方藥物合用時,有發(fā)生HDI的風險,呋喃香豆素對CYP酶的抑制或誘導(dǎo)作用可能是白芷發(fā)生HDI的主要原因。但目前對白芷呋喃香豆素的體內(nèi)外代謝處置性質(zhì)及其與CYP酶相互作用的研究尚不系統(tǒng),已有的研究多集中于歐前胡素等少數(shù)主要成分。為此,本課題旨在通過白芷呋喃香豆素吸收、分布、排泄和藥代動力學(xué)過程的系統(tǒng)研究,闡明其體內(nèi)外代謝處置特征,識別白芷給藥后體內(nèi)血循環(huán)和組織器官中的標志性呋喃香豆素成分。在此基礎(chǔ)上,評價白芷呋喃香豆素對CYP酶的抑制或誘導(dǎo)作用,理解體內(nèi)外代謝處置性質(zhì)與藥物相互作用的關(guān)系,深入研究CYP酶抑制作用機制,為白芷的臨床合理用藥和人體HDI預(yù)測,提供必要的數(shù)據(jù)支持和科學(xué)依據(jù)。同時,在本研究的實施過程中,摸索并探討中藥多組分藥物相互作用研究的策略與方法。本課題的研究內(nèi)容和結(jié)果總結(jié)如下:1..建立并驗證了同時定量檢測生物樣品中歐前胡素(ⅠM)、異歐前胡:素(ⅡM)、水合氧化前胡素(OXYH)、佛手柑內(nèi)酯(BER)、氧化前胡素(OXY)、花椒毒酚(XANT)、花椒毒素(XAN)、異茴芹內(nèi)酯(IPIM)、補骨脂素(PSO)等9個呋喃香豆素的LC-MS/MS分析方法,方法靈敏、快速、可靠,可滿足白芷呋喃香豆素體內(nèi)外代謝處置的研究需求。2.大鼠口服白芷浸膏的血漿藥代動力學(xué)結(jié)果顯示,白芷呋喃香豆素在大鼠的吸收和消除均較快,藥材中含量較高的ⅠM、ⅡM、OXYH和BER是大鼠血循環(huán)中的主要活性成分,代表了 白芷呋喃香豆素90%以上的體內(nèi)暴露量,是血漿暴露的標志性成分。排泄動力學(xué)結(jié)果顯示,ⅠM、ⅡM、BER、XAN、IPIM和PSO的原型在尿、糞和膽汁中的回收率低于3%,表明其在體內(nèi)發(fā)生廣泛代謝,主要以產(chǎn)物的形式排泄。3.大鼠口服白芷浸膏后,呋喃香豆素在各組織中廣泛分布;ⅠM、ⅡM、BER和OXYH是血漿和組織中的主要活性成分,占到總呋喃香豆素暴露量的90%以上,由此確認它們是白芷呋喃香豆素的體內(nèi)暴露標志物。呋喃香豆素的肝臟分布顯著高于血漿和其他組織,ⅠM、ⅡM和BER的肝臟和血漿暴露比值(K_p)分別為5.1、6.7和4.7,OXYH的K_p值為2.2。蛋白結(jié)合實驗表明,ⅠM、ⅡM、BER和OXYH具有較高的血漿和組織蛋白結(jié)合率,其組織結(jié)合率高于血漿;經(jīng)游離分數(shù)(fu)校正后,與組織總K_p值相比,組織和血漿的游離暴露比值(K_p,fu)顯著降低,表明較高的蛋白結(jié)合是上述成分在組織高暴露的原因之一;ⅠM、ⅡM和BER的游離肝臟/血漿比值K_p,fu2.6,表明它們可特異性地分布于肝臟。4.采用懸浮培養(yǎng)的大鼠原代肝細胞模型,進一步探討了 ⅠM、ⅡM、BER和OXYH在肝臟高水平分布的機制。實驗表明,這些成分的肝細胞攝取是溫度依賴的,37℃的攝取量顯著高于4℃,提示它們的肝攝取可能存在主動轉(zhuǎn)運機制;攝取動力學(xué)結(jié)果顯示,ⅠM、ⅡM、BER和OXYH的肝細胞攝取同時涉及主動轉(zhuǎn)運和被動擴散,其中ⅠM、ⅡM和BER以主動轉(zhuǎn)運為主,主動攝取率(CLactive/CLuptake)高于70%;將呋喃香豆素在大鼠原代肝細胞中與攝取轉(zhuǎn)運體的陽性抑制劑共孵育,攝取轉(zhuǎn)運體Oatp的抑制劑可顯著降低ⅠM和BER的肝細胞攝取,轉(zhuǎn)運體Oat的抑制劑能明顯抑制ⅡM的肝攝取,表明攝取轉(zhuǎn)運體Oatp和Oat可能介導(dǎo)了 ⅠM、ⅡM和BER的主動轉(zhuǎn)運過程,并在其肝臟特異性分布中發(fā)揮作用。5.給大鼠同時服用白芷和CYP1A2探針底物非那西丁,通過檢測CYP1A2介導(dǎo)的非那西丁-O-脫乙基化反應(yīng),觀察到單次口服白芷可顯著提高非那西丁在大鼠的血漿暴露,白芷低劑量組(0.46 g/kg)和高劑量組(2.3 g/kg)的非那西丁血漿AUC分別為對照組血漿AUC的9和12倍,藥物清除率下降,T1/2和MRT0-t延長;代謝產(chǎn)物對乙酰氨基酚的生成受到抑制,Cmax降低至對照組的14%-22%,達峰時間顯著延遲,T1/2和MRT0-t延長;結(jié)果表明白芷單次給藥可顯著抑制大鼠CYP1A2酶的活性。6.采用混合探針底物法,評價9個白芷呋喃香豆素成分對人肝微粒體(HLM)和大鼠肝微粒體(RLM)主要CYP酶活性的抑制作用。結(jié)果表明,呋喃香豆素成分對HLM和RLM中多個CYP酶均有不同程度的抑制作用,其中對CYP1A2的抑制作用最強;ⅠM、ⅡIM、BER、OXY、XAN、IPIM 和 PSO 是人 CYP1A2的強抑制劑,IC50值在0.023-0.25 μmol/L之間;OXYH和XANT是中等抑制劑。進一步比較白芷提取液和9個呋喃香豆素混合溶液的CYP酶抑制作用,發(fā)現(xiàn)所測的9個呋喃香豆素成分可基本上代表中藥白芷的CYP1A2抑制作用。由各呋喃香豆素單體在混合溶液中的含量分數(shù)和酶抑制IC50值,計算得到9個呋喃香豆素抑制CYP同工酶的整合IC50值;當CYP酶抑制作用分級為強或中等時,單體的整合IC50值與混合溶液的實測值相當,提示整合IC50值的方法可用于評價中藥多組分的酶抑制作用。由酶抑制IC50值和大鼠肝臟濃度預(yù)測藥物相互作用風險,ⅠM、ⅡM和BER對大鼠CYP1A2的[Ⅰ]/Ki值分別為19.6、4.02和2.0,提示它們在與CYP1A2底物藥物合用時,具有較高的CYP1A2抑制相互作用風險,與大鼠體內(nèi)實驗結(jié)果相符。種屬差異比較可見,ⅠM、ⅡM、OXYH和BER對人CYP1A2的抑制作用強于大鼠,提示它們在人體具有CYP1A2酶抑制相互作用的高風險性。7.應(yīng)用IC50漂移實驗觀察到,ⅡM、OXYH和BER對HLM中CYP1A2的抑制作用是時間依賴的,輔酶NADPH預(yù)孵組的抑制曲線明顯向左偏移,IC50比值(-/+)大于4;而ⅠM的抑制作用是可逆性的�；跈C制的抑制動力學(xué)實驗進一步顯示,ⅡM、OXYH和BER對CYP1A2的抑制作用是預(yù)孵育時間和濃度依賴的,為基于機制的抑制(MBI),產(chǎn)生半數(shù)最大失活速率的濃度(KI)分別為2.08、1.96 和 6.71 μmol/L,失活速率常數(shù)(kinact)分別為 0.07、0.04 和 0.07 min-1 對CYP1A2酶具有較強的滅活作用。8.白芷浸膏多次給藥后,低劑量組和高劑量組使非那西丁的大鼠血漿暴露(AUC)分別下降45%和62%,清除率分別為對照組的1.76和2.65倍;代謝產(chǎn)物對乙酰氨基酚的達峰時間提前,高劑量組的Cmax增高,T1/2和MRT0-t縮短;表明白芷多次給藥可能通過CYP1A2酶誘導(dǎo)作用顯著加快非那西丁在大鼠體內(nèi)的清除。9.大鼠肝臟CYP酶活性和mRNA表達水平的測定結(jié)果顯示,白芷多次給藥后,對大鼠肝臟CYP1A、CYP2B和CYP3A的酶活性和表達均有不同程度的誘導(dǎo)作用,其中,對CYP1A的誘導(dǎo)作用最強,并具有劑量依賴性;低劑量組和高劑量組CYP1A2的酶活性分別為對照組的1.9和2.2倍,CYP1A1的mRNA表達水平分別為對照組的8.4和43.5倍,CYP1A2的mRNA表達水平分別為對照組的2.7倍和16.8倍,顯示白芷多次給藥對大鼠CYP1A1的誘導(dǎo)作用強于CYP1A2。10.應(yīng)用SimCypTM軟件的PBPK建模和模擬預(yù)測功能,構(gòu)建了 ⅠM、ⅡM、OXYH和BER的大鼠PBPK模型,并采用大鼠實測數(shù)據(jù)對模型進行校正和驗證;在此基礎(chǔ)上建立了上述成分的人體PBPK模型,預(yù)測了人體血漿藥代動力學(xué)和藥物相互作用潛能。ⅠM、ⅡM和BER可使健康人群的咖啡因、非那西丁和茶堿AUC分別提高至對照組的2.0、1.7和1.3倍,在嚴重吸煙人群中合用組和對照組的AUC比值略高于健康人群相應(yīng)的AUC比值,表明白芷呋喃香豆素在健康人群和吸煙人群均有發(fā)生基于CYP1A2抑制的藥物相互作用風險。本論文有如下的新發(fā)現(xiàn)或創(chuàng)新點:1.白芷藥材中含量較高的ⅠM、ⅡM、OXYH和BER也是血漿和組織中的主要活性成分,可作為白芷呋喃香豆素的體內(nèi)暴露標志物,用于藥效與毒效評價,這一發(fā)現(xiàn)為白芷藥理學(xué)和藥物相互作用的物質(zhì)基礎(chǔ)研究,提供了重要的依據(jù)。2.ⅠM、ⅡM、OXYH和BER在大鼠肝臟的暴露水平顯著高于血漿和其他組織;通過蛋白結(jié)合、大鼠原代肝細胞攝取和Caco-2跨膜轉(zhuǎn)運等研究,發(fā)現(xiàn)攝取轉(zhuǎn)運體Oatp和Oat介導(dǎo)的主動轉(zhuǎn)運在ⅠM、ⅡM和BER的肝臟特異性分布中發(fā)揮重要作用,非特異性蛋白結(jié)合也是這些成分在肝臟高暴露的原因之一。3.給大鼠同服白芷和非那西丁的相互作用研究,揭示了白芷對CYP1A2酶的雙向調(diào)控作用,白芷單次給藥可強烈抑制大鼠CYP1A2酶,多次給藥不僅逆轉(zhuǎn)了單次給藥的抑制作用,還可顯著誘導(dǎo)CYP1A2的基因表達和酶活性,這一發(fā)現(xiàn)對白芷的臨床合理用藥具有重要的指導(dǎo)意義。4.建立并驗證了整合IC50值的酶抑制活性評價方法,可用于中藥多成分(強或中等抑制劑)酶抑制作用的整體評估,為多組分藥物的相互作用研究提供了可行的體外評價方法,也為中藥在人體的酶抑制相互作用預(yù)測提供了參考。5.ⅠM是人CYP1A2的可逆性抑制劑,ⅡM、OXYH和BER是CYP1A2基于機制的抑制劑,應(yīng)用PBPK模型進行的人體預(yù)測顯示,這些成分在臨床與CYP1A2的底物藥物合用時,存在一定的藥物相互作用風險;預(yù)測結(jié)果為白芷的新藥研發(fā)及其臨床安全有效用藥提供指導(dǎo)。
[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
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R285

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