荷叶代谢性药物相互作用及体内成分研究

【作者】 叶林虎；

【导师】 常琪；

【作者基本信息】 北京协和医学院 ， 生药学， 2014， 博士

【摘要】 细胞色素P450(cytochromeP450,CYP450)酶系统是机体重要的代谢酶,它参与了许多内源性和外源性物质的代谢。在药物的代谢过程中,CYP2C19、CYP2D6. CYP3A4、CYP2E1和CYP2C9是最主要的亚型,其参与了市场上90%左右药物的代谢。当两种药物合并使用时,一种药物对另一种药物的代谢酶产生抑制或诱导作用都可能产生药物相互作用,导致产生毒性或治疗失败,增加临床治疗的风险。在临床过程中,抑制作用的危害要远大于诱导作用的危害。抑制P450酶的活性可能会引起血药浓度升高而导致毒性作用,有时甚至会危及生命。很多药物因发生严重的代谢性药物相互作用而被撤出市场。中药是来自大自然的天然产物,其常被认为是“安全的,无毒副作用”的药物,从而受到人们的青睐,被广泛使用。然而,近年来的研究发现多种中(草)药及其制剂也会对P450酶产生抑制或诱导作用,从而引起代谢性药物相互作用。这种药物间的相互作用已经引起人们的关注。因此,对一些常用中药进行潜在药物相互作用研究,以预测其可能发生的药物相互作用,是保证临床用药安全有效的重要途径,具有极其重要的意义。荷叶(Nelumbinis Folium)为睡莲科植物Nelumbo nucifera Gaertn.的干燥叶,为药食两用的中药。其具有清暑化湿、升发清阳,凉血止血的功效,常用于暑热烦渴、暑湿泄泻、脾虚泄泻、血热吐衄、便血崩漏等症。现代药理研究表明,荷叶黄酮和／或生物碱成分具有降血脂,减肥,抗氧化,抗HIV,抗菌和降糖等多种生物活性。作为传统中药,荷叶是多种降脂复方制剂的重要组成部分；作为天然食用资源,目前已被开发成多种减肥茶或其他保健饮品。因此,荷叶及其制剂与其他药物,特别是降脂减肥类药物共同使用的几率较大。然而,到目前为止,有关荷叶及其制剂在使用过程中是否会对P450酶的活性产生影响,是否会导致药物相互作用发生的研究报道仍然缺乏。因此,本文将从P450酶方面来探讨荷叶潜在的药物相互作用。本研究采用液相色谱-质谱联用(LC-MS/MS)技术,建立体外、体内酶活性的评价方法。首先经过体外模型预测荷叶潜在的药物相互作用,然后再通过体内实验进行验证,最后再通过分析荷叶在大鼠体内的药物成分及其在组织中的分布,进一步的从物质基础方面来阐释荷叶潜在药物相互作用的发生原因。此外,还初步探讨了荷叶提取物对转运体P-糖蛋白(P-gp)功能的影响。本论文完成的研究工作主要有以下几个方面：1.以奥美拉唑、右美沙芬、睾丸酮、氯唑沙宗和甲苯磺丁脲分别作为CYP2C19、CYP2D6、CYP3A4、CYP2E1、CYP2C9的探针底物,将这5个探针底物与人肝微粒体共同孵育,并利用LC-MS/MS分析技术检测代谢产物的生成量,建立同时测定人肝微粒体P450酶5种亚型活性的cocktail探针底物体外评价方法；并运用所建立的方法研究12味常用中药(荷叶、酸枣仁、决明子、五味子、山楂、枸杞子、莲子心、人参、菊花、黄精、茯苓和金银花)对人肝微粒体P450同工酶活性的影响。研究结果显示,所建立的方法具有灵敏度高、特异性强、重现性好和准确度高的优点,能满足体外代谢性药物相互作用研究的要求。12味中药的水提取物对P450同工酶的抑制作用较弱或没有抑制作用；而乙醇提取物则有不同程度的抑制作用(除枸杞子外),而且这种抑制作用存在浓度依赖性。其中荷叶乙醇提取物对CYP2C19、CYP2D6、 CYP3A4、CYP2E1和CYP2C9半数抑制浓度IC50值分别为77.38、12.05、98.01、61.43和83.46μg/mL；菊花乙醇提取物对CYP2C9活性抑制的IC50值为45.25μg/mL;五味子乙醇提取物对CYP3A4和CYP2C19活性抑制的IC50值分别为47.24和64.42μg/mL。上述研究结果提示,日常服用这些中药时,尤其是在长期服用这些中药经乙醇提取后制备所得的制剂时,应注意其潜在的药物相互作用。2.采用cocktail探针底物法分别研究荷叶总黄酮和总生物碱及其主要化学成分对人肝微粒体P450酶各亚型活性的影响。结果表明荷叶总黄酮对肝微粒体P450酶活性的抑制作用相对较弱,其IC50值均在100μg/mL左右；总生物碱对P450酶活性的抑制作用则较强,其对CYP2C19、CYP2D6、CYP3A4、CYP2E1(?)CYP2C9的半数抑制浓度IC50值分别为40.79、0.96、44.87、63.84和52.58μg/mL,其中对CYP2D6同工酶的抑制作用最强。总生物碱中的主要成分荷叶碱、N-去甲荷叶碱和2-羟基-1-甲氧基阿朴啡通过竞争性抑制方式抑制CYP2D6酶的活性,其抑制常数Ki值分别为1.88、2.34和1.56μM。但这三者对CYP2D6酶活性的抑制不存在时间依赖性和NADPH依赖性的不可逆抑制作用。采用Caco-2细胞模型研究荷叶乙醇提取物、总黄酮和总生物碱对P-糖蛋白(P-gp)功能的影响。结果表明,荷叶乙醇提取物和总生物碱对P-gp存在浓度依赖性的抑制作用,在浓度为100μg/mL时,Caco-2细胞内的荧光强度明显增加,即Caco-2细胞对Rho-123的摄取量增加,有显著的抑制作用(P<0.05)；荷叶总黄酮对P-gP有一定的抑制作用,但与空白对照组相比较,无显著差异(P>0.05)。上述研究显示,荷叶对P450酶的活性有抑制作用,尤其是对CYP2D6亚型有较强的抑制作用；对P-gp功能也有一定的抑制作用。这些抑制作用主要与荷叶所含的生物碱成分有关。提示荷叶在与其他药物合并使用时,可能存在药物相互作用风险。3.采用右美沙芬和美托洛尔作为CYP2D6酶的探针底物,以LC-MS/MS技术为分析手段,研究荷叶总生物碱对这两种探针底物在体内的代谢产物右菲烷和α-羟基美托洛尔药代动力学的影响；给大鼠连续灌胃总生物碱(50mg/kg)7天,并于末次给药后2或12h时间点给大鼠静脉注射右美沙芬(5mg/kg)或美托洛尔(10mg/kg),观察其代谢产物血药浓度的变化。并通过蛋白质印迹法(western blot)研究总生物碱对大鼠体内CYP2D6酶蛋白表达水平的影响。结果表明,总生物碱能显著降低右菲烷和α-羟基美托洛尔的血药浓度。在末次给总生物碱后2或12h时间点给大鼠静脉注射右美沙芬或美托洛尔,总生物碱使右菲烷的AUCall从167.27h-ng/mL分别下降到43.13h-ng/mL(P<0.01)(给生物碱后2h时间点)和62.25h-ng/mL(P<0.05)(给生物碱后12h时间点)；使α-羟基美托洛尔的AUCall从347.68h·ng/mL下降到223.24h·ng/mL(P<0.05)(给生物碱后12h时间点)。连续给予大鼠总生物碱7天后,大鼠体内CYP2D6酶蛋白的表达水平并无明显改变。上述研究结果提示,总生物碱在体内亦对大鼠CYP2D6酶有明显的抑制作用,与体外结果相吻合；但这种抑制作用并不影响CYP2D6蛋白的表达水平。4.采用LC-MS/MS分析方法,研究给大鼠灌胃荷叶总黄酮(50mg/kg)或总生物碱(50mg/kg)后的体内药物成分,以及大鼠静脉注射荷叶总生物碱(50mg/kg)后,其主要生物碱成分荷叶碱、N-去甲荷叶碱和2-羟基-1-甲氧基阿朴啡在大鼠组织中的分布情况。结果从大鼠血液和尿液中鉴定出37个黄酮成分,主要包括原型化合物和这些化合物与葡萄糖醛酸和硫酸等结合而形成的代谢产物,表明荷叶黄酮成分在体内的代谢途径主要为Ⅱ相代谢；从大鼠血液和尿液中鉴定出17个生物碱成分,包括生物碱原型化合物及其经氧化、甲基化等的代谢产物,和葡萄糖醛酸和硫酸等的结合物,表明荷叶生物碱在体内代谢广泛,其代谢途径包括Ⅰ相和Ⅱ相代谢。给大鼠静脉注射总生物碱后,三个主要生物碱成分荷叶碱、N-去甲荷叶碱和2-羟基-1-甲氧基阿朴啡能迅速(5min)分布到各组织,其中在肝脏、肺、脾脏和肾脏等组织中具有较高的浓度,并在体内维持较长时间。通过上述研究结果可初步推测荷叶对P450酶活性的抑制与其在体内的药物成分及其组织分布情况有关。总之,本文系统地研究了荷叶乙醇提取物、荷叶总黄酮和总生物碱及其主要化学成分对P450酶活性的影响,尤其是总生物碱对CYP2D6酶活性的抑制作用方面进行了深入研究。同时,对荷叶乙醇提取物及总黄酮和总生物碱对P-gp功能的影响进行了初步研究；最后,对给药后的大鼠进行体内药物成分分析和鉴定,并对总生物碱的主要成分在大鼠组织中的分布情况进行了研究。结果提示荷叶可通过抑制P450酶的活性或P-gp的功能,引起药物相互作用；而且,荷叶对P450酶的活性和P-gp功能的影响主要与其所含生物碱成分有关。本研究为荷叶在临床上的安全、合理应用提供了新信息；为荷叶成分的药理、毒理研究和开发利用提供了新思路。更多还原

【Abstract】 Cytochrome P450(CYP) is a major metabolizing enzyme system in the body and plays an important role in the metabolism of many endogenous and exogenous substances. Five P450isoenzymes, namely, CYP2C19, CYP2D6, CYP3A4, CYP2E1and CYP2C9are particularly important because they are responsible for the metabolism of approximately90%of the currently known therapeutic drugs. Drug-drug interactions are frequently occurrence when drugs are co-administered and one drug modifies the metabolic clearance of the other drugs by inhibition or induction of some P450isoenzymes. In general, inhibition is much more harmful than induction in clinic settings. The inhibition of P450activity may cause an increase in the blood concentrations of the drug that may lead to toxic symptoms and sometimes even fatal interactions. Many drugs have been withdrawn from the market due to their significant inhibition of some isoenzymes that resulted in serious clinical damages.Traditional Chinese medicines (TCMs) are often considered as safe and harmless natural products and are conventionally used with prescription drugs. However, many different side effects have been reported for some herbs and/or their formulations recently. Many TCMs have been reported to exhibit inhibitory or induction effects on the P450enzymes, this cause to herb-drug interactions (HDI) and have attracted much attention. Thus, it is extremely important to evaluate the potential HDI of the commonly used herbs for their safe and effective use.Nelumbinis Folium, the dried leaves of lotus (Nelumbo nucifera Gaertn), is utilized not only as a dietary staple but also as a TCM to treat sunstroke, assuage thirst, and cure both diarrhea and fever in China. Modern pharmacological studies have demonstrated that the flavonoids and/or alkaloids of the herb exhibit various pharmacological effects, such as anti-hyperlipidemia, anti-obesity, anti-oxidant, anti-diabetic, anti-microbial, and anti-HIV activities. Currently, the herb is becoming more popular in China as a "tea drink"’or as a main ingredient of some herbal formulations, which implies that the herb and/or its products are now more likely to be concurrently administered with conventional medicines for losing body weight and reducing blood lipids. However, information on the leaf extract and its active compounds that cause HDI by inhibition of P450is limited. In the present study, we investigated the potential HDI of lotus leaves that mediated by P450through in vitro or in vivo models. Firsty, the inhibitory effects on the CYP activities in human liver microsomes were evaluated by in vitro model, and later were verified by in vivo model. Finally, the analysis of chemical constituents of lotus leaves in rats and their tissue distribution were investigated to interpret the mechanism of lotus leaves leading to the potential HDI. In addition, the P-gp function was preliminary investgated by Caco-2cell model. The main contents of this paper are as follows:1. By using LC-MS/MS techniques, an in vitro cocktail method was established for simultaneous determination of five major cytochrome P450isoenzymes activities by monitoring the metabolites of probe substrates, including omeprazole (CYP2C19), dextromethorphan (CYP2D6), testosterone (CYP3A4), chlorzoxazone (CYP2E1), tolbutamide (CYP2C9), in incubation human liver microsomes with mixed the five probe substrates. Inhibition activities of12TCMs including Nelumbinis Folium, Ziziphi Spinosae Semen, Cassiae Semen, Schisandrae Chinensis Fructus, Crataegi Fructus, Lycii Fructus, Nelumbinis Plumula, Ginseng Radix Et Rhizoma, Chrysanthemi Flos, Polygonati Rhizoma, Poria and Lonicerae Japonicae Flos on the five isoenzymes were evaluated using established method. The results indicated that the method possessed a good linearity and sensitivity, as well as the excellent precession and accuracy, and can be used for the high throughput screening of P450enzyme activities. In addition, aqueous extracts of12TCMs showed a weak or no inhibitory effects on P450enzymes, however, their ethanolic extracts possessed more potent and concentration-dependent inhibition effects (except for the Lycii Fructus). Among them, the lotus leaf ethanolic extract strongly inhibited the CYP2C19, CYP2D6, CYP3A4, CYP2E1and CYP2C9activities with IC50values of77.38,12.05,98.01,61.43and83.46μg/mL, respectively; chrysanthemum Flos ethanolic extract inhibited the CYP2C9activity with IC50value of45.25μg/mL; Schisandrae Chinensis Fructus ethanolic extract inhibited CYP3A4and CYP2C19activities with IC50values of47.24and64.42μg/mL, respectively. These results suggest that the possible HDI between the herb ethanolic extracts and their preparations with conventional medicines should thus be taken into account.2. The inhibitory effects of the total flavonoids and total alkaloids of lotus leaves, as well as its main alkaloid compounds on the activities of human liver microsomal P450enzymes were investigated by the established cocktail probe substrate method. The results showed that the total flavonoids weakly inhibited P450enzyme activities, with IC50values around100μg/mL; while, the total alkaloids strongly inhibited the activities of CYP2C19, CYP2D6, CYP3A4, CYP2E1and CYP2C9with IC50value of40.79,0.96, 44.87,63.84and52.58μg/mL, respectively. The individual alkaloids, namely, nuciferine (NF), N-nornuciferine (N-NF), and2-hydroxy-l-methoxyaporphine (HMA) competitively inhibited CYP2D6activity with Ki values of1.88,2.34, and1.56μM, respectively. And the three alkaloids are not mechanism-based inhibitors of CYP2D6.The effects of lotus leaf ethanolic extract, total flavonoids and total alkaloids on the function of P-glycoprotein (P-gp) were investigated by using Caco-2cell model and a P-gp substrate Rhodamine-123(Rho-123). The result showed that the ethanolic extract and total alkaloids could significantly increase the uptake of Rho-123by Caco-2cells at the concentration of100μg/mL (P<0.05), this indicates that they can inhibit the function of P-gp. The total flavonoids showed a weak inhibition effect on P-gp, but no significant difference comparing with negative control (P>0.05).All these results suggest that lotus leaves have inhibitory effects on P450enzyme activities and P-gp function and the effects may probable related with its alkaloid components. There are potential drug interactions between total alkaloids and other therapeutic drugs.3. The in vivo inhibition effects of lotus leaf total alkaloids on CYP2D6activity was investigated in rats using dextromethorphan and metoprolol as probe substrates. After being pretreated with total alkaloids (50mg/kg) for continuous7days, the rats was intravenously administrated with dextromethorphan (5mg/kg) or metoprolol (10mg/kg) at2or12h time point of the last alkaloid dosing. The plasma concentrations of the substrate metabolites, dextrorphan and a-hydroxymetoprolol were determined by LC-MS/MS method, for comparing their pharmacokinetic properties between the treated and untreated rats, and protein expression of CYP2D6in rat liver was also determined by western blot. The results showed total alkaloids significantly decreased the plasma concentrations of the two probe metabolites, AUCall of dextrorphan was significantly decreased from167.27to43.13h-ng/mL (P<0.01)(given at2h after the last dose of alkaloids) and62.25h-ng/mL (P<0.05)(given at12h after the last dose of alkaloids), respectively; and AUCall of a-hydroxy-metoprolol were also significantly decreased from347.68to223.24h-ng/mL (P<0.05)(given at2h after the last dose of alkaloids). The total alkaloids had no effect on the protein expression of CYP2D6in rat liver. These results suggest that the total alkaloids have significant inhibition on rat CYP2D6enzyme, which was consistent with the in vitro results.4. Based on developed LC-MS/MS methods, the in vivo components of lotus leaf extracts were identified after oral administration of total flavonoids and total alkaloids to rats. In addition, the tissue distribution of alkaloids was also studied after intravenous administration to rats. A total of37flavonoid compounds, including parent flavonoids and their glucuronidation and sulfation metabolites, were identified in rat plasma and urine. This indicates that flavonoids are mainly biotransformed by phase II metabolism pathway. A total of18alkaloids were identified, including parent alkaloids and their oxidation, methylation, glucuronidation and sulfation metabolites, were also identified in rat plasma and urine. This indicates that the alkaloids were biotransformed by both phase I and phase II metabolism pathways. The studies on tissue distribution of alkaloids showed that NF, N-NF and HMA could quickly (within5min) distribute to various tissues, especially in the liver, lungs, spleens and kidneys with a relative high concentrations, and could remained in the body for a relative long period. It is speculated that the inhibitory effects of lotus leaves on P450enzyme activities are likely to relate to these in vivo components.In conclusion, the present study provides systematic investigations on the in vitro inhibitory effects of lotus leaf ethanolic extracts, and its main two fractions total flavonoids and total alkaloids on P450enzyme activities, particularly the CYP2D6isoenzyme, which was further confirmed by an in vivo study. Meanwhile, the effect of lotus leaf ethanolic extract and the two fractions on the function of P-gp was preliminary evaluated by using a Caco-2cell model. In addition, the in vivo components of lotus leave flavonoids and alkaloids, as well as the tissue distribution of alkaloids in rats were also investigated. The results reveals that the lotus leaves can inhibit the activities of P450enzymes, especially for CYP2D6, and inhibit P-gp function. These effects are mainly related to the alkaloids existed in lotus leaves, this might cause the potential HDI and should pay much attentions when used in clinic. The present studies provide new information for safe and appropriate use of lotus leaves, and also provide ideas for further investigation of pharmacology and toxicology, and further development and utilization of the herb.更多还原