【作者】 李艺；

【导师】 蒋建东； 游雪甫； 宋丹青；

【作者基本信息】 北京协和医学院 ， 微生物与生化药学， 2010， 博士

【摘要】 小檗碱(Berberine, BBR)是从黄连等植物根茎中提取分离的异喹啉类天然抗腹泻药物,在我国临床使用已有数十年的历史。最近,我们发现BBR是一个作用机制与他汀类完全不同的新型降血脂药物,主要在转录后水平通过上调肝细胞低密度脂蛋白受体(LDLR)的基因表达,进而发挥降血脂作用。此外,我们还发现胰岛素受体(InsR)是BBR发挥降血糖作用的另一个关键靶点。提示,BBR显示出治疗代谢综合征(MS)的潜力。然而,BBR的代谢酶及其代谢产物的活性至今未知。本论文首先从药物代谢的角度,探讨BBR的代谢途径,寻找BBR经机体生物转化后的活性代谢产物及其代谢相关的肝脏CYP450 (cytochrome P450)酶,继而从活性代谢产物着手,开展前药的设计,以期获得具有自主知识产权、较高口服生物利用度的治疗MS候选化合物。为了研究BBR的Ⅰ相代谢产物,我们设计了一系列体内外实验。大鼠口服200mg/kg的BBR后,主要的Ⅰ相代谢产物有4个,分别是小檗红碱(berberrubine, M1)、唐松草分定(thalifendine,M2)、2，3-去亚甲基小檗碱(demethyleneberberine, M3)和药根碱(jatrorrhizine,M4)。对于BBR经人体肝药酶的代谢,我们分别从人肝微粒体和重组表达人源P450酶的昆虫微粒体的亚细胞水平,以及3种人肝细胞(人原代肝细胞、Bel-7402和HepG2)的细胞水平进行了考察,仅得到2种代谢产物,M2和M3。自大鼠的肝脏组织分别制备了肝匀浆、S9及微粒体,在体外模拟生理环境对BBR进行孵育,所得结果与人肝体外结果相似。我们认为,在当前构建的几种的肝脏体外代谢模型中,BBR的主要Ⅰ相代谢产物为M2和M3。为了进一步考察负责代谢BBR的肝脏P450酶,我们运用计算机小分子与生物大分子的对接手段预测了能够与BBR紧密结合的CYP450蛋白,按照对接评分由高到低分别为CYP2D6、CYP1A2、CYP3A4。接下来,将BBR与8种重组P450酶分别孵育,并结合化学抑制剂实验共同验证了酶筛结果。最后利用整体归一化法(total normalized rate, TNR)对负责代谢BBR的CYP450酶进行了相对贡献值的评估排序。对于M2的生成,3种酶的相对贡献值分别为CYP1A2:78.38%,CYP2D6:18.97%,CYP3A4: 2.65%;对于M3的生成,3种酶的相对贡献值分别为CYP3A4:38.43%, CYP1A2: 31.18%, CYP2D6:30.39%。为了深刻理解BBR在体内发挥药理作用的机制和物质基础,我们分别考察了BBR及其体内代谢产物M1-M4上调肝细胞LDLR mRNA的表达、上调InsR mRNA的表达及激活AMPK酶的活性,结果显示,代谢产物M1-M4的活性均表现出一定的活性,但却不及BBR母体本身,我们推测BBR进入体内后,与其代谢产物共同发挥调脂降糖的作用。在其4个代谢产物中M1的活性最高,虽然稍弱于BBR本身,但结构中裸露出的羟基(9-OH)使其通过制备前药而改善BBR的药代动力学特性、提高BBR的口服生物利用度成为可能。我们从活性代谢产物M1着手,在其9-OH引入脂溶性增加的载体,制备成M1前药。通过在大鼠血浆中的体外生物转化实验的筛选及动物体内降血脂活性的评价,从中筛选出生物利用度高、降血脂活性略强于或与BBR相当的酯类前药——M1棕榈酸酯(5g)。5g不仅拥有自主知识产权,还在BBR的基础上大大提高了生物利用度,安全性高、药效好,具有良好的应用前景,将BBR转化为具有创新性的新型代谢综合征治疗药物。综上所述,我们的研究包括了从亚细胞水平、细胞水平到动物整体水平,从生物学实验、计算机模拟到化学设计等一系列工作。研究表明,BBR在肝脏中发生了生物代谢反应,主要的Ⅰ相代谢酶为CYP2D6、CYP1A2和CYP3A4。BBR及其体内代谢产物M1-M4均具有调节细胞能量代谢活性,以BBR活性最强,M1次之,而设计合成M1前药可在一定程度上解决BBR口服生物利用度低的问题,是发现高效低毒的新型代谢综合征治疗药物的有效途径之一。更多还原

【Abstract】 Berberine (BBR), an isoquinoline natural product isolated from herbs such as Coptis chinensis, has been used in China for several decades as an anti-diarrhea drug. Our previous studies showed that BBR was a novel LDLR-up-regulator and cholesterol-lowering agent with a mechanism on the post-transcriptional level, which is distinct from that of statins. In addition, we also found that InsR was another key target for its hypoglycemic effect. These results indicate that BBR was a potential drug for the treatment of metabolic syndrome (MS). However, the metabolic enzymes of BBR and bioactivities of BBR metabolites are still unknown. To address this issue, the present study was performed to investigate the active metabolites of BBR and to identify the CYP450 (cytochrome P450) isoenzymes that are responsible for the production of BBR metabolites. Next, we take the active metabolite as a parent compound for the pro-drug study, in order to obtain effective candidate compounds for MS, with independent intellectual property and better bioavailability.We have designed a series of in vitro and in vivo experiments to study the I-phase metabolites. In the animal experiment, we used 200 mg/kg of BBR to treat the Wistar rats orally. We identified 4 metabolites, M1 (berberrubine), M2 (thalifendine), M3 (demethyleneberberine) and M4 (jatrorrhizine). For the in vitro studies, we investigated respectively in the rat liver homogenate, S9, liver microsomes, human liver microsomes, insect microsomes expressing human P450, primary hepatocyte, and Bel-7402 and HepG2 cell line. We found metbolites M2 and M3 of BBR in all the in vitro systems.In order to further identify P450 isozymes responsible for BBR’s metabolism, we firstly used computer assistant docking software, by which we found that CYP2D6, CYP1A2 and CYP3A4 were the top three. Subsequently, two biological experiments, recombinant P450 incubation and inhibitory assay were developed to verify the screening P450 isozymes. Next, we ranked these P450 isozymes with the method of total normalized rate (TNR). For BBR to M2, CYP1A2, CYP2D6 and CYP3A4 are responsible for 78.38%, 18.97%and 2.65%of total transformation, respectively. And for M3 formation, CYP3A4 for 38.43%, CYP1A2 for 31.18%and CYP2D6 for 30.39%, respectively.To deeply understand the mechanisms and the chemical entities of BBR’s multiple effects, we studied the activities of LDLR mRNA and InsR mRNA up-regulating, AMPK acivating in BBR and its metabolites M1-M4. M1-M4 remained bioactivities, and BBR in its original form showed the highest activity among these chemical entities. We speculated that BBR combined its major metabolites M1-M4 in modulating cellular energy metabolism in vivo. Besides, among the metabolites, Ml showed the best activity, very close to BBR. And the exposed 9-OH in Ml was better convenient for synthesizing pro-drugs, which could improve the bioavailability of BBR.Therefore, we semi-synthesized a series of M1 pro-drugs by 9-OH modification. By screening the plasma hydrolysis behavior and animal anti-hyperlipidemic experiment, we found 5g (Ml palmitate) for the first time which transformed BBR to a novel MS drug with better bioavailability and biactivity.In summary, evidences from subcellular, cellular level to animal; from biological study, computer technology, to chemical design all prove that BBR was bio-metabolized in vivo. It was metabolized at least through the action of human hepatocyte CYP2D6, CYP1A2 and CYP3A4. The phase I metabolites of BBR remained to be active in on the targets with significantly reduced potency. Designing pro-drugs of BBR’s active metabolite M1 was one of the most effective approaches to improve BBR’s bioavailability and discover novel MS drugs which are effective and low toxic independently.更多还原