灵芝酸A药代动力学及灵芝潜在药物相互作用研究

发布时间：2018-02-10 19:37

本文关键词： 灵芝灵芝酸A 代谢产物药代动力学转运机制药物相互作用　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：灵芝是担子菌纲多孔菌科灵芝属真菌赤芝(Ganoderma lucidum).和紫芝(Ganoderma sinense)的子实体,灵芝三萜和多糖是灵芝的两种主要药理活性组分。灵芝酸A(Ganodericacid A,GAA)是灵芝中含量最为丰富的三萜酸之一,普遍存在于灵芝属植物中。研究报道显示,GAA具有镇痛、抗氧化、肝保护和抗癌等药理活性。作为灵芝的主要活性成分,GAA单体的代谢、体内药代动力学特性和生物利用度仍鲜有报道。灵芝及其制剂在临床及日常保健中被广泛应用,常与其他处方药物同时使用,其是否会和其他药物发生药物相互作用仍然缺乏相关的研究。本文以灵芝酸A为研究对象,从整体动物及细胞水平探讨灵芝三萜在体内的药代动力学过程及吸收转运机制;以灵芝提取物为研究对象,考察灵芝是否存在基于外排转运体和细胞色素P450(CYP450)酶的潜在药物相互作用,为灵芝的合理应用和新药研发提供依据。本论文完成的研究工作主要有以下几个方面:1.GAA代谢产物及代谢途径研究采用HPLC-DAD-MS/MS技术,首次鉴定了 GAA的代谢产物,研究了其主要代谢产物的酶动力学特性。对静脉给予大鼠GAA后采集到的生物样品、以及GAA在大鼠和人肝微粒体孵育体系中的代谢产物进行鉴定,并对其主要代谢产物的酶动力学特性进行考察。从大鼠胆汁、血浆和尿液中共检测鉴定了 37种代谢物;从体外大鼠肝微粒体(RLMs)孵育体系和人肝微粒体(HLMs)孵育体系中分别鉴定了与大鼠体内相同的9和7种代谢物。研究提示GAA在人和大鼠体内的代谢途径具有相似性。GAA可以发生氧化还原羟基化的Ⅰ相代谢和葡萄糖醛酸化磺酸化的Ⅱ相代谢反应,其主要代谢位点为3、7、11、15、23位的羰基或羟基基团和12、20、28(29)位的碳原子。GAA的还原代谢产物在RLMs中的生成速率远大于在HLMs中,且均由CYP3A参与代谢。2.GAA及其代谢产物体内药代动力学研究首先建立了灵敏的用于测定大鼠血浆、胆汁、尿液和脑微透析液中GAA浓度的UFLC-MS/MS分析方法。方法学考察显示,该方法线性良好、灵敏可靠,具有低的检测限(0.25 nmol/L)和定量限(2.00 nmol/L),精密度、准确度、萃取回收率、稳定性等均能满足生物样品分析要求。用所建立的分析方法研究GAA及其主要代谢物灵芝酸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)和灵芝酸B(C4)在大鼠体内药代动力学特性。代谢物浓度以GAA标准曲线进行定量。静脉给予GAA(20mg/kg)后,原型药在体内逐渐消除,同时生成了丰富的代谢物C1-C4。各代谢物在5 min左右均达到Cmax值,分别为2.61、0.17、2.84和0.51 μmol/L,且C1、C2、C4在3-6h左右有明显的重吸收峰。GAA 及其代谢物 C1-C4 的 t1/2 分别为 2.40、13.08、12.35、2.16 和 2.79h,并主要从胆汁中排出,0-24 h在胆汁中的累积排泄率分别为给药剂量的21.37%、18.02%、2.22%、2.33%和0.70%。而GAA在尿液中的累积排泄率仅为2.59%,代谢物C1-C4的累积排泄率之和为0.076%。口服给药(20mg/kg)后,GAA原型药和四个代谢物C1-C4在10-40 min出现第一个血药浓度峰值,且在6-8 h左右均出现重吸收峰。GAA的口服生物利用度为8.68%。静脉给药方式下,GAA可快速通过血脑屏障(Tmax,0.25 h)进入脑组织,其血脑屏障透过率为2.96%,在脑透析液中未检测到其代谢物C1-C4。3.GAA肠道吸收转运机制研究采用Caco-2细胞模型,以UFLC-MS/MS分析方法首次研究了 GAA的肠吸收转运机制。结果表明,GAA在不同浓度(25、50、100 μg/mL)下从肠腔侧(AP)到基底侧(BL)和从BL侧到AP侧的通透量随浓度和时间的增加而相应增加,表观渗透系数PappA→B为(4.30-4.99)×10-7cm/s,Papp B→A为(33.52-37.77)×10-7cm/s,外排率为 6.72-8.79。在 P-糖蛋白(P-gp)抑制剂维拉帕米,多药耐药蛋白(MRP)抑制剂MK571和乳腺癌耐药蛋白(BCRP)抑制剂潘生丁的干预下,GAA的PappA→B值分别升高至5.79×10-7、12.14×10-7和4.62×10-7cm/s,PappB→A值分别降低至 30.17×10-7、23.55×10-7和 25.57×10-7cm/s,外排显著降低。提示P-gp、MRP和BCRP均参与GAA的外排转运,这可能是GAA 口服生物利用度低的主要原因。4.基于外排转运体的灵芝潜在药物相互作用研究通过Caco-2细胞摄取和跨膜转运实验,研究灵芝(Ganoderma lucidum)提取物及单体成分GAA对外排转运体P-gp、MRP和BCRP功能的影响。细胞摄取实验结果显示,灵芝总提物(GLE)和灵芝三萜提取物(GLT)在浓度为100 μg/mL时,使P-gp底物罗丹明123(Rho)和MRP底物钙黄绿素(Cal)的摄取量显著增加,对P-gp和MRP功能表现出抑制作用;使BCRP的底物Hoechst 33342(Hoe)的摄取量显著降低,对BCRP功能表现出诱导作用。灵芝多糖提取物(GLP)在实验浓度下则对P-gp和MRP功能无显著影响,在浓度为100 μg/mL时对BCRP表现出诱导作用。GAA在实验浓度下对P-gp、MRP和BCRP功能均无显著影响。Caco-2细胞转运模型实验中,用不同浓度GLE和GLT干预Rho的跨膜转运时,Rho从AP到BL的通透量和Papp a→b值均显著增加,从BL到AP的通透量和PappB→A值均显著降低,且表现出剂量依赖性,提示GLE和GLT可以抑制P-gp的外排作用。用不同浓度GLE和GLT干预Cal的外排时,Cal在AP侧的通透量表现出不同程度的降低,在Caco-2细胞单层膜中的累积量表现出不同程度的增加,提示GLE和GLT可以抑制MRP的外排作用。灵芝三萜是灵芝发挥外排转运体抑制作用的主要活性成分。提示灵芝在与外排转运体底物药物合并使用时,可能存在药物相互作用风险。5.基于CYP450酶的灵芝潜在药物相互作用研究选择临床常用药物非那西丁、奥美拉唑、右美沙芬、睾酮、甲苯磺丁脲、氯唑沙宗分别作为CYP1A2、CYP2C19、CYP2D6、CYP3A4、CYP2C9 和 CYP2E1 的探针底物,结合 UFLC-MS/MS 技术对底物代谢物进行检测,以Cocktail探针底物肝微粒体代谢反应体外评价方法,对灵芝提取物和其主要成分GAA的CYP450酶抑制活性进行研究。结果显示,在HLMs中,GLE对CYP2C19、2D6、3A4和2C9表现出微弱的抑制作用,IC50值分别为131.2、164.4、150.5和142.2 μg/mL。GLT的抑制作用略强于GLE,IC50值分别为102.5、116.1、136.4和82.2μg/mL。GLP对6种CYP450酶抑制作用均不明显。在RLMs中,GLT对CYP2C9表现出微弱的抑制作用,IC50值为163.1μg/mL,而对其他酶活性无影响;GLE和GLP对6种CYP450酶均不产生抑制作用。单体成分GAA在1-50 μmol/L的浓度范围内对HLMs和RLMs中的CYP450酶均无抑制作用。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

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