本文關鍵詞： 靈芝 靈芝酸A 代謝產(chǎn)物 藥代動力學 轉運機制 藥物相互作用　出處：《北京協(xié)和醫(yī)學院》2017年博士論文　論文類型：學位論文

【摘要】：靈芝是擔子菌綱多孔菌科靈芝屬真菌赤芝(Ganoderma lucidum).和紫芝(Ganoderma sinense)的子實體,靈芝三萜和多糖是靈芝的兩種主要藥理活性組分。靈芝酸A(Ganodericacid A,GAA)是靈芝中含量最為豐富的三萜酸之一,普遍存在于靈芝屬植物中。研究報道顯示,GAA具有鎮(zhèn)痛、抗氧化、肝保護和抗癌等藥理活性。作為靈芝的主要活性成分,GAA單體的代謝、體內(nèi)藥代動力學特性和生物利用度仍鮮有報道。靈芝及其制劑在臨床及日常保健中被廣泛應用,常與其他處方藥物同時使用,其是否會和其他藥物發(fā)生藥物相互作用仍然缺乏相關的研究。本文以靈芝酸A為研究對象,從整體動物及細胞水平探討靈芝三萜在體內(nèi)的藥代動力學過程及吸收轉運機制;以靈芝提取物為研究對象,考察靈芝是否存在基于外排轉運體和細胞色素P450(CYP450)酶的潛在藥物相互作用,為靈芝的合理應用和新藥研發(fā)提供依據(jù)。本論文完成的研究工作主要有以下幾個方面:1.GAA代謝產(chǎn)物及代謝途徑研究采用HPLC-DAD-MS/MS技術,首次鑒定了 GAA的代謝產(chǎn)物,研究了其主要代謝產(chǎn)物的酶動力學特性。對靜脈給予大鼠GAA后采集到的生物樣品、以及GAA在大鼠和人肝微粒體孵育體系中的代謝產(chǎn)物進行鑒定,并對其主要代謝產(chǎn)物的酶動力學特性進行考察。從大鼠膽汁、血漿和尿液中共檢測鑒定了 37種代謝物;從體外大鼠肝微粒體(RLMs)孵育體系和人肝微粒體(HLMs)孵育體系中分別鑒定了與大鼠體內(nèi)相同的9和7種代謝物。研究提示GAA在人和大鼠體內(nèi)的代謝途徑具有相似性。GAA可以發(fā)生氧化還原羥基化的Ⅰ相代謝和葡萄糖醛酸化磺酸化的Ⅱ相代謝反應,其主要代謝位點為3、7、11、15、23位的羰基或羥基基團和12、20、28(29)位的碳原子。GAA的還原代謝產(chǎn)物在RLMs中的生成速率遠大于在HLMs中,且均由CYP3A參與代謝。2.GAA及其代謝產(chǎn)物體內(nèi)藥代動力學研究首先建立了靈敏的用于測定大鼠血漿、膽汁、尿液和腦微透析液中GAA濃度的UFLC-MS/MS分析方法。方法學考察顯示,該方法線性良好、靈敏可靠,具有低的檢測限(0.25 nmol/L)和定量限(2.00 nmol/L),精密度、準確度、萃取回收率、穩(wěn)定性等均能滿足生物樣品分析要求。用所建立的分析方法研究GAA及其主要代謝物靈芝酸C2(C1)、7β,11,15-trihydroxy-3,23-dioxo-lanost-8-en-26-oic acid(C2)、11,15-dihydroxy-3,7,23-trioxo-lanost-8-en-26-oicacid(C3)和靈芝酸B(C4)在大鼠體內(nèi)藥代動力學特性。代謝物濃度以GAA標準曲線進行定量。靜脈給予GAA(20mg/kg)后,原型藥在體內(nèi)逐漸消除,同時生成了豐富的代謝物C1-C4。各代謝物在5 min左右均達到Cmax值,分別為2.61、0.17、2.84和0.51 μmol/L,且C1、C2、C4在3-6h左右有明顯的重吸收峰。GAA 及其代謝物 C1-C4 的 t1/2 分別為 2.40、13.08、12.35、2.16 和 2.79h,并主要從膽汁中排出,0-24 h在膽汁中的累積排泄率分別為給藥劑量的21.37%、18.02%、2.22%、2.33%和0.70%。而GAA在尿液中的累積排泄率僅為2.59%,代謝物C1-C4的累積排泄率之和為0.076%�？诜o藥(20mg/kg)后,GAA原型藥和四個代謝物C1-C4在10-40 min出現(xiàn)第一個血藥濃度峰值,且在6-8 h左右均出現(xiàn)重吸收峰。GAA的口服生物利用度為8.68%。靜脈給藥方式下,GAA可快速通過血腦屏障(Tmax,0.25 h)進入腦組織,其血腦屏障透過率為2.96%,在腦透析液中未檢測到其代謝物C1-C4。3.GAA腸道吸收轉運機制研究采用Caco-2細胞模型,以UFLC-MS/MS分析方法首次研究了 GAA的腸吸收轉運機制。結果表明,GAA在不同濃度(25、50、100 μg/mL)下從腸腔側(AP)到基底側(BL)和從BL側到AP側的通透量隨濃度和時間的增加而相應增加,表觀滲透系數(shù)PappA→B為(4.30-4.99)×10-7cm/s,Papp B→A為(33.52-37.77)×10-7cm/s,外排率為 6.72-8.79。在 P-糖蛋白(P-gp)抑制劑維拉帕米,多藥耐藥蛋白(MRP)抑制劑MK571和乳腺癌耐藥蛋白(BCRP)抑制劑潘生丁的干預下,GAA的PappA→B值分別升高至5.79×10-7、12.14×10-7和4.62×10-7cm/s,PappB→A值分別降低至 30.17×10-7、23.55×10-7和 25.57×10-7cm/s,外排顯著降低。提示P-gp、MRP和BCRP均參與GAA的外排轉運,這可能是GAA 口服生物利用度低的主要原因。4.基于外排轉運體的靈芝潛在藥物相互作用研究通過Caco-2細胞攝取和跨膜轉運實驗,研究靈芝(Ganoderma lucidum)提取物及單體成分GAA對外排轉運體P-gp、MRP和BCRP功能的影響。細胞攝取實驗結果顯示,靈芝總提物(GLE)和靈芝三萜提取物(GLT)在濃度為100 μg/mL時,使P-gp底物羅丹明123(Rho)和MRP底物鈣黃綠素(Cal)的攝取量顯著增加,對P-gp和MRP功能表現(xiàn)出抑制作用;使BCRP的底物Hoechst 33342(Hoe)的攝取量顯著降低,對BCRP功能表現(xiàn)出誘導作用。靈芝多糖提取物(GLP)在實驗濃度下則對P-gp和MRP功能無顯著影響,在濃度為100 μg/mL時對BCRP表現(xiàn)出誘導作用。GAA在實驗濃度下對P-gp、MRP和BCRP功能均無顯著影響。Caco-2細胞轉運模型實驗中,用不同濃度GLE和GLT干預Rho的跨膜轉運時,Rho從AP到BL的通透量和Papp a→b值均顯著增加,從BL到AP的通透量和PappB→A值均顯著降低,且表現(xiàn)出劑量依賴性,提示GLE和GLT可以抑制P-gp的外排作用。用不同濃度GLE和GLT干預Cal的外排時,Cal在AP側的通透量表現(xiàn)出不同程度的降低,在Caco-2細胞單層膜中的累積量表現(xiàn)出不同程度的增加,提示GLE和GLT可以抑制MRP的外排作用。靈芝三萜是靈芝發(fā)揮外排轉運體抑制作用的主要活性成分。提示靈芝在與外排轉運體底物藥物合并使用時,可能存在藥物相互作用風險。5.基于CYP450酶的靈芝潛在藥物相互作用研究選擇臨床常用藥物非那西丁、奧美拉唑、右美沙芬、睪酮、甲苯磺丁脲、氯唑沙宗分別作為CYP1A2、CYP2C19、CYP2D6、CYP3A4、CYP2C9 和 CYP2E1 的探針底物,結合 UFLC-MS/MS 技術對底物代謝物進行檢測,以Cocktail探針底物肝微粒體代謝反應體外評價方法,對靈芝提取物和其主要成分GAA的CYP450酶抑制活性進行研究。結果顯示,在HLMs中,GLE對CYP2C19、2D6、3A4和2C9表現(xiàn)出微弱的抑制作用,IC50值分別為131.2、164.4、150.5和142.2 μg/mL。GLT的抑制作用略強于GLE,IC50值分別為102.5、116.1、136.4和82.2μg/mL。GLP對6種CYP450酶抑制作用均不明顯。在RLMs中,GLT對CYP2C9表現(xiàn)出微弱的抑制作用,IC50值為163.1μg/mL,而對其他酶活性無影響;GLE和GLP對6種CYP450酶均不產(chǎn)生抑制作用。單體成分GAA在1-50 μmol/L的濃度范圍內(nèi)對HLMs和RLMs中的CYP450酶均無抑制作用。
[Abstract]:Ganoderma lucidum is basidiomycetes Polyporaceae fungi of the genus Ganoderma lucidum Ganoderma lucidum (Ganoderma lucidum). (Ganoderma sinense) and Ganoderma fruiting bodies of Ganoderma lucidum three terpene and polysaccharide are two main pharmacological active components of Ganoderma lucidum. Ganoderic acid A (Ganodericacid A GAA) is one of three triterpene acids in Ganoderma lucidum rich most the content exists in the ganoderma genus. Research reports show that GAA has analgesic, antioxidant, hepatoprotective and anticancer activity of Ganoderma lucidum. As the main component, the metabolism of GAA monomer, pharmacokinetic characteristics and bioavailability of Ganoderma lucidum and its preparation is still rarely reported. It is widely applied in clinical and daily health care, often used in conjunction with other prescription drugs, it will occur and other drug drug interactions is still a lack of relevant research. In this paper, ganoderic acid A as the research object, from the whole animal and cellular level of three terpene in Ganoderma lucidum The in vivo pharmacokinetics and mechanism of absorption and transport; the Ganoderma lucidum extract of Ganoderma lucidum as the research object, the existence of efflux transporter and cytochrome P450 (CYP450) based on the interaction of the enzyme for potential drugs, the rational use of Ganoderma lucidum and new drug development. Provide the basis for the research in this thesis mainly includes the following aspects: study metabolites and metabolic pathways of 1.GAA using HPLC-DAD-MS/MS technology, first identified metabolites of GAA, enzyme kinetics of its main metabolites. The intravenous administration of rat GAA after collected biological samples, and GAA in rat and human liver microsomes incubated with metabolites in the system were identified, and the enzyme dynamic characteristics of the main metabolites were investigated. From rat bile, plasma and urine were detected and identified 37 metabolites from rat liver microsomes in vitro; (RLMs). The education system (HLMs) and human liver microsomes incubation system were identified with the rats the same 9 and 7 metabolites. Studies suggest that GAA metabolic pathways in rat and human body is similar to that of.GAA can undergo redox hydroxylation phase I metabolism and glucuronidation of sulfonated phase II metabolism the main metabolic reaction sites for 3,7,11,15,23 bits of the carbonyl or hydroxyl groups and 12,20,28 (29) generation rate reduction metabolites a carbon atom of.GAA in RLMs is much larger than that in HLMs, and CYP3A is involved in the metabolism of.2.GAA and its metabolic products in vitro in pharmacokinetic studies was first established for the determination of high sensitive the concentration of GAA in plasma, bile, urine and brain Microdialysate UFLC-MS/MS analysis method. The influences of display method, this method has good linearity, sensitivity and reliability, has a low detection limit (0.25 nmol/L) and limit of quantification (2 nmol/L) , precision, accuracy, recovery, stability can meet the requirements of biological sample analysis. By analyzing the research of GAA and its metabolites ganoderic acid C2 (C1), 7 beta, 11,15-trihydroxy-3,23-dioxo-lanost-8-en-26-oic acid (C2), 11,15-dihydroxy-3,7,23-trioxo-lanost-8-en-26-oicacid (C3) and ganoderic acid B (C4) in vivo in rats pharmacokinetic characteristics. Metabolite concentrations were quantified using the GAA standard curve. Intravenous GAA (20mg/kg), the prototype drug gradually eliminate the in vivo metabolite of C1-C4. rich metabolite reached Cmax value at about 5 min is obtained at the same time, respectively 2.61,0.17,2.84 and 0.51 mol/L, and C1, C2, C4 have obvious weight the absorption peak of.GAA and its metabolite C1-C4 t1/2 were 2.40,13.08,12.35,2.16 and 2.79h in 3-6h, and mainly from the bile discharge, 0-24 h in the bile of the cumulative excretion rate Don't let the dosage of 21.37%, 18.02%, 2.22%, 2.33% and 0.70%. and GAA cumulative excretion in the urine was only 2.59%, the cumulative excretion of metabolites C1-C4 and oral administration of 0.076%. (20mg/kg), GAA drug prototype and four metabolites C1-C4 first peak blood concentration in 10-40 min and, at about 6-8 h appeared heavy absorption peak of.GAA by the oral bioavailability is 8.68%. intravenous administration, GAA can quickly through the blood-brain barrier (Tmax, 0.25 h) into the brain tissue, the blood brain barrier permeability is 2.96%, in brain dialysate was not detected in the metabolism of C1-C4.3.GAA study on intestinal absorption mechanism using Caco-2 cell model, GAA method was studied for the first time the intestinal absorption mechanism by UFLC-MS/MS analysis. The results showed that GAA in different concentration (25,50100 g/mL) from the intestinal lumen to the basolateral side (AP) (BL) and permeability from BL side to side with the concentration of AP Increased and time and the corresponding increase in the apparent permeability coefficient PappA, B (4.30-4.99) * 10-7cm/s, Papp B, A (33.52-37.77) * 10-7cm/s, the efflux rate of 6.72-8.79. in the P- glycoprotein (P-gp) inhibitor Vera Pammy, multidrug resistance protein (MRP) inhibitor MK571 and breast cancer resistance protein (BCRP under the intervention of Pan Shengding) inhibitor, GAA PappA, B values were increased to 5.79 * 10-7,12.14 * 10-7 and 4.62 * 10-7cm/s, PappB, A values were reduced to 30.17 * 10-7,23.55 * 10-7 and 25.57 * 10-7cm/s, suggesting that P-gp efflux was significantly reduced. MRP, and BCRP were involved in GAA efflux transporters, this GAA may be the main reason of low oral bioavailability of Ganoderma lucidum.4. potential drug efflux transporter based on the interaction between the Caco-2 cell uptake and the transmembrane transport experiments of Ganoderma lucidum (Ganoderma lucidum) extracts and monomer composition of GAA efflux transporter P-gp, MRP The influence and the function of BCRP. Cell uptake results showed that Ganoderma lucidum extract (GLE) and three (GLT) of Ganoderma lucidum terpenoid extract at the concentration of 100 g/mL, the P-gp substrate 123 (Rho) and Luo Danming MRP (Cal) substrate calcein uptake increased significantly, the P-gp and MRP functions the inhibitory effect of the BCRP Hoechst 33342 substrate; (Hoe) the intake significantly decreased, the function of BCRP showed induction effect. Polysaccharide extracts of Ganoderma (GLP) at the concentration of P-gp and MRP had no significant effect on the concentration of 100 g/mL of BCRP showed the induction of.GAA in the concentration of P-gp, MRP and BCRP have no significant effect on.Caco-2 cells in model experiment, with the transmembrane transport of different concentrations of GLE and GLT intervention Rho, Rho from AP to BL and Papp a, B permeability values were significantly increased, from BL to AP and PappB, A transparent the values were significantly decreased, and the Showed dose dependent, suggesting that GLE and GLT can inhibit the activity of P-gp. With different concentrations of GLE and GLT intervention Cal efflux, Cal permeability in AP side showed different degrees of reduction, accumulation in Caco-2 cell monolayer showed different degrees of increase, in GLE and GLT can suppress the activity of MRP. The three is the main active ingredient of Ganoderma lucidum triterpenoids inhibition effect of Ganoderma lucidum play efflux transporter. In Ganoderma lucidum and efflux transporter substrates with drug use, there may be a risk of drug interactions.5. potential drug omeprazole Ganoderma CYP450 enzyme interaction of clinically used drugs acetophenetidin based on that, dextromethorphan, testosterone, tolbutamide chlorzoxazone, respectively as CYP1A2, CYP2C19, CYP2D6, CYP3A4, CYP2C9 and CYP2E1 probe substrate, combined with the technology of UFLC-MS/MS substrate metabolism into line with detection. Cocktail probe substrate liver microsomal metabolism reaction in vitro evaluation methods, CYP450 enzyme of Ganoderma lucidum extract and its main component of GAA inhibitory activity were studied. The results showed that in HLMs, GLE of CYP2C19,2D6,3A4 and 2C9 showed weak inhibition, IC50 was inhibition of 131.2164.4150.5 and 142.2 g/mL.GLT is stronger than GLE, IC50 the values were 102.5116.1136.4 and 82.2 g/mL.GLP to 6 CYP450 enzyme inhibition was not obvious. In RLMs, GLT showed a weak inhibitory effect on CYP2C9, IC50 value is 163.1 g/mL, but have no effect on other enzyme activity; GLE and GLP of 6 kinds of CYP450 enzymes was not inhibited. Monomer the component GAA in the concentration range of 1-50 mol/L CYP450 enzyme on HLMs and RLMs were not inhibited.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R285.5

【相似文獻】

相關期刊論文 前10條

1 胡明,賴琪,蒲劍,劉一飛;美國藥物相互作用計算機審查系統(tǒng)的發(fā)展與應用概況[J];中國醫(yī)院藥學雜志;2001年01期

2 ;藥物相互作用(續(xù)表)[J];中國鄉(xiāng)村醫(yī)藥;2001年12期

3 張芳,牛其昌;引起不良理化變化的藥物相互作用(一)[J];中國臨床醫(yī)生;2001年03期

4 盧艷青;藥物相互作用及其臨床意義[J];廣東微量元素科學;2002年04期

5 馮曉燕;淺談藥物相互作用[J];醫(yī)學理論與實踐;2004年08期

6 Carol Ukens ,周怡;研究認為應杜絕嚴重的藥物相互作用[J];中國處方藥;2004年11期

7 Kelly Dowhower Karpa;于麗;;藥物相互作用帶來的困擾[J];中國處方藥;2006年05期

8 張玉霞;;分析藥物相互作用方法的進展[J];中國水運(理論版);2007年05期

9 柯元南;;藥物相互作用和用藥安全[J];中華老年心腦血管病雜志;2007年09期

10 劉亞賢;;藥物相互作用及其對治療的影響淺析[J];臨床藥物治療雜志;2008年04期

相關會議論文 前10條

1 戴體俊;;藥物相互作用的概念和定量分析[A];第十二屆全國數(shù)學藥理學術大會論文集[C];2009年

2 戴體俊;;藥物相互作用的概念和定量分析[A];中國藥理學會第十次全國學術會議�？痆C];2009年

3 劉高峰;;中藥代謝與藥物相互作用[A];第四屆全國藥學服務與研究學術論壇論文集[C];2011年

4 吳榮榮;魏振滿;;藥物相互作用研究[A];中國成人醫(yī)藥教育論壇（2009）[C];2009年

5 鄭青山;孫瑞元;;藥物相互作用動力學:分析模型與電腦類比[A];海峽兩岸三地藥理學學術報告會論文匯編[C];2001年

6 曾蘇;;代謝性藥物相互作用[A];2005年浙江省合理用藥與藥品不良反應監(jiān)測學術研討會專題報告集[C];2005年

7 王娟;;藥物相互作用[A];廣東省藥學會送學下鄉(xiāng)地市藥學人員繼續(xù)教育學習班講義[C];2005年

8 王益平;項青青;;氨基糖苷類抗生素對腎毒及其它藥物相互作用[A];2013年浙江省醫(yī)院藥學學術年會會議報告與論文匯編[C];2013年

9 袁秉祥;;心血管藥物相互作用與復方藥品研發(fā)[A];第八屆海峽兩岸心血管科學研討會論文集[C];2011年

10 袁秉祥;;基于藥物相互作用理論和多因素多水平分析技術的復方藥品研發(fā)[A];中國藥理學會第十一次全國學術會議專刊[C];2011年

相關重要報紙文章 前10條

1 美國德克薩斯大學圣安東尼奧醫(yī)學院 李俊旭;如何進行藥物相互作用研究[N];醫(yī)藥經(jīng)濟報;2009年

2 本報記者 吳若琪;藥物分合的科學解析[N];中國醫(yī)藥報;2011年

3 衛(wèi)生部北京醫(yī)院藥劑科 劉治軍;重視藥物相互作用 減少藥源性損害[N];中國醫(yī)藥報;2011年

4 劉元江;網(wǎng)上看藥物相互作用[N];健康報;2004年

5 一文;何謂藥物相互作用？[N];醫(yī)藥經(jīng)濟報;2002年

6 孟剛;老年人用藥要抓主要矛盾謹防藥物相互作用[N];中國消費者報;2007年

7 北京大學腫瘤醫(yī)院藥師 楊銳;合并用藥與藥物相互作用該如何平衡[N];中國醫(yī)藥報;2012年

8 湖南省獸藥飼料監(jiān)察所 肖安東 研究員;優(yōu)質(zhì)獸藥必備的因素[N];中國畜牧獸醫(yī)報;2006年

9 陸志城 石葦;部分植物及機體所需營養(yǎng)物質(zhì)微量元素金屬鹽類可能存在的藥物相互作用[N];醫(yī)藥經(jīng)濟報;2003年

10 陸志城 石葦;部分植物及機體所需營養(yǎng)物質(zhì)微量元素金屬鹽類可能存在的藥物相互作用[N];醫(yī)藥經(jīng)濟報;2003年

相關博士學位論文 前2條

1 曹方瑞;靈芝酸A藥代動力學及靈芝潛在藥物相互作用研究[D];北京協(xié)和醫(yī)學院;2017年

2 高怡文;人CYP2C8多態(tài)性功能及CYP2C8基因依賴性藥物相互作用的體外研究[D];西北大學;2010年

相關碩士學位論文 前10條

1 徐亞飛;基于抑制CYP450酶的代謝性藥物—藥物相互作用體外研究及實例報告[D];延邊大學;2015年

2 丁林松;基于序列的蛋白質(zhì)—藥物相互作用預測研究[D];南京理工大學;2015年

3 畢凱;基于集成學習的藥物相互作用信息抽取系統(tǒng)的研究與實現(xiàn)[D];西北農(nóng)林科技大學;2016年

4 王海峰;自動化酶反應新方法建立并應用于待測化合物對CYP酶的抑制情況研究[D];華南理工大學;2015年

5 徐云婷;甜菊醇�；咸烟侨┧峤Y合物介導的藥物相互作用機制研究[D];蘇州大學;2016年

6 趙鵬姚;藥物相互作用的網(wǎng)絡藥理學分析與預測[D];北京交通大學;2016年

7 邱婷婷;我院部分老年住院患者基于CYP450代謝的用藥合理性分析及實例報告[D];廣西醫(yī)科大學;2016年

8 劉萍;對合理用藥中藥物相互作用的現(xiàn)狀及對策研究[D];沈陽藥科大學;2008年

9 曲衍清;體外預測己烯雌酚引起藥物—藥物相互作用的可能性[D];大連醫(yī)科大學;2011年

10 徐佳;（5R）-5-羥基雷公藤內(nèi)酯醇的吸收機制和與甲氨蝶呤聯(lián)合用藥的藥物—藥物相互作用研究[D];華東理工大學;2014年

，

本文編號：1501277