【作者】 张霞；

【导师】 蒋惠娣； 曾苏；

【作者基本信息】 浙江大学 ， 药学， 2011， 博士

【摘要】 狼毒为传统草药,在我国临床使用已有非常悠久的历史,广泛用于治疗水肿、恶性肿瘤、白血病、慢性气管炎、皮肤病、妇科病、肺结核、肠结核、淋巴结核等症。狼毒乙素(ECB)的化学名为2,4-dihydroxy-6-methoxy-3-methyl-acetophenone,新狼毒素A(NCA)的化学名为(2S,2’S,3S,3’S)-5,5’,7,7’-tetrahydroxy-2,2’ -bis (4-hydroxyphenyl)-3,3’-bichroman-4,4’ -dione。二者都是中药狼毒的主要有效成分,药理活性已有些报道。药理学研究表明狼毒乙素对耐药型和非耐药型结核杆菌具有明显抑制作用,是已上市药物结核灵片的主要有效成分之一。2,4-dihydroxy-6-methoxyl-methylene-3-methyl-acetophenone,一种狼毒乙素的结构类似物,最近被报导具强抗人宫颈癌细胞(Hela-60)活性,IC50值为95ng/mL。体外实验表明,新狼毒素A具有明显的抗前列腺癌细胞(LNCaP)活性。而狼毒乙素和新狼毒素A的药物代谢和动力学(DM/PK)至今尚未见研究。本文先从狼毒大戟与瑞香狼毒根中分离、制备狼毒乙素和新狼毒素A,再采用各种体内外实验模型对狼毒乙素和新狼毒素A的吸收、转运、代谢、排泄与动力学的性质进行了较系统的研究。本文的研究结果可为狼毒的临床应用及相关的新药研发提供理论和实验依据。1、新狼毒素A在MDCK和MDCK-MDR1细胞中的转运研究新狼毒素A在MDCK和MDCK-MDR1细胞上的转运实验表明,新狼毒素A在这两种细胞中的表观渗透系数均≤1×10-6cm/s,提示其口服后生物利用度较低。新狼毒素A在MDCK-MDR1细胞单层中的转运存在显著的外排现象,并可被P-糖蛋白(P-gp)的经典抑制剂维拉帕米所抑制,表明P-gp参与了新狼毒素A在MDCK-MDR1细胞中的外排转运,提示外排转运蛋白P-gp可能是制约其口服生物利用度的主要生理屏障因素之一。以罗丹明123(R123)作为P-gp的探针底物,评价新狼毒素A对P-gp转运功能的影响,结果表明100μmol/L的新狼毒素A对MDCK-MDR1细胞单层中由P-gp介导的R123外排有显著的抑制作用,提示新狼毒素A可能为P-gp抑制剂。另一方面,新狼毒素A对MDCK细胞具有比较明显的细胞毒性,但对MDCK-MDR1细胞的毒性显著减弱,可能是MDCK-MDR1细胞中高表达的外排蛋白P-gp参与了药物的外排,减少细胞内的蓄积,使毒性下降,也间接验证了新狼毒素A是P-gp底物的实验结果。2、狼毒乙素和新狼毒素A在大鼠肝微粒体和大鼠原代肝细胞中的代谢研究在β-NADPH存在下,狼毒乙素在大鼠肝微粒体中生成一个氧化代谢产物,质谱鉴定其结构是1-(2,4-dihydroxy-6-methoxy-3-methylphenyl)-2-hydroxyethanone,为单羟基化代谢物；在UDPGA存在下,狼毒乙素在大鼠肝微粒体中生成一个单取代葡萄糖醛酸苷,分离后经p-葡萄糖醛酸苷水解酶水解、质谱分析、核磁共振氢谱鉴定其结构为2-hydroxy-6-methoxy-3-methyl-acetophenone-4-O-β-glucuronide。孵育体系中同时加入β-NADPH和尿苷二磷酸葡萄糖醛酸(UDPGA)进行一相、二相级联代谢共孵育,结果超过80%的狼毒乙素均转化成单取代葡萄糖醛酸苷,提示狼毒乙素在大鼠体内主要经葡萄糖醛酸化代谢。新狼毒素A在大鼠肝微粒体中不发生氧化代谢,但在UDPGA存在下生成两个代谢产物,经葡萄糖醛酸苷水解酶水解和质谱分析,这两个代谢物均为单取代葡萄糖醛酸苷。狼毒乙素和新狼毒素A在大鼠肝微粒体中发生的葡萄糖醛酸化代谢及狼毒乙素发生的氧化代谢均符合米氏动力学方程。综合应用选择性化学诱导和抑制实验,进行筛选,大鼠肝微粒体中催化狼毒乙素代谢的酶主要是CYP3A, CYP2C11, CYP2C6和UGT1A6/9,其中UGT1A6的贡献更为显著；催化新狼毒素A葡萄糖醛酸化代谢的酶主要是UGT1A3/6/9。将新狼毒素A与大鼠原代肝细胞共孵育,没检测到代谢产物；将狼毒乙素与大鼠原代肝细胞共孵育,检测有痕量的两个代谢物,但相对肝微粒体,原代肝细胞的代谢活性要低很多。3、狼毒乙素和新狼毒素A的大鼠体内代谢、排泄和动力学研究狼毒乙素与新狼毒素A大鼠体内主要发生葡萄糖醛酸化代谢,代谢物分析均只检测到一个葡萄糖醛酸苷。狼毒乙素体内产生的葡萄糖醛酸苷跟体外产生的葡萄糖醛酸苷结构一致。从色谱保留时间判断,新狼毒素A体内产生的葡萄糖醛酸苷跟体外鼠肝微粒体产生的两个中的一个相对应,结构还在鉴定中。对大鼠口服狼毒乙素的动力学行为进行了初步研究,建立了血浆中狼毒乙素葡萄糖醛酸苷测定的UPLC-MS方法。结果显示,大鼠口服狼毒乙素后,在血浆中只明显检测到其单取代葡萄糖醛酸苷。该葡萄糖醛酸苷在大鼠体内的动力学行为符合二室模型,达峰时间约为4小时,提示大鼠口服狼毒乙素后吸收迅速。大鼠口服新狼毒素A后,血浆中没有检测到明显的代谢产物,原形药物的浓度也非常低,提示大鼠口服新狼毒素A的吸收很差,这与新狼毒素A是P-gp底物有关。4、狼毒乙素和新狼毒素A在人肝微粒体、HepG2细胞及人重组酶中的代谢研究狼毒乙素在人肝微粒体中的代谢结果与鼠肝微粒体中的一致,提示狼毒乙素在人体内主要发生葡萄糖醛酸化代谢。新狼毒素A在人肝微粒体中只生成了一个单取代葡萄糖醛酸苷。综合选择性化学抑制及人重组酶实验对这两个底物的代谢活性分析结果,可推测人肝微粒体中参与狼毒乙素代谢的主要酶系是UGT1A6/9、CYP3A4、CYP2C9,其中最主要的是CYP3A4与UGT1A6；参与新狼毒素A代谢的主要酶系是UGT1A3/6/9。狼毒乙素与HepG2细胞孵育时,仅检测到痕量的一相代谢物,没有发现二相代谢物；新狼毒素A与HepG2细胞孵育时,也没发现二相代谢物,提示HepG2细胞对这两个底物的代谢活性很低。更多还原

【Abstract】 Lang-du is a famous traditional herbal medicine and has long been used in China for the treatment of a wide range of ailments, including edema, malignant tumor, leukemia, chronic tracheitis, pulmonary tuberculosis, scrofula, intestinal tuberculosis, skin disease and gynecopathia. Ebracteolata compound B (ECB), chemically designated as 2,4-dihydroxy-6-methoxy-3-methyl-acetophenone and neochamaejasmin A (NCA), chemically designated (2S,2’S,3S,3’S)-5,5’,7,7’-tetrahydroxy-2,2’ - bis (4-hydroxyphenyl)- 3,3’-bichroman-4,4’ -dione, are primary active ingredients of this medicine. ECB was reported to exhibit significant antibacterial activities on both tuberculosis bacillus and XDR-TB bacillus. Also, it is one major active component of the marketed drug "Jieheling Tablets". More interestingly, one homologous compound of ECB,2,4-dihydroxy-6-methoxyl-methylene-3-methylacetophenone, was recently demonstrated to possess obvious selective activity against human Hela-60 tumor cell with an IC50 value 95 ng/mL. NCA, was found to exhibit significant cytotoxicity in vitro against LNCaP cells. In this study, ECB and NCA were isolated and purified from the dried root of Euphorbia fischeriana and Stellera chamaejasme L. respectively and their absorption, transport, metabolism, excretion and pharmacokinetics profiles were investigated by using several in vitro and in vivo models.1. The transport of NCA across MDCK and MDCK-MDR1 cell monolayersIn the MDCK and MDCK-MDR1 monolayer cell cultures, NCA showed a low cell permeability. The apparent permeability coefficient (Papp) values of apical to basolateral direction were less than 1×10-6 cm/s. The efflux ratios in both cells were much higher than 2.0. This efflux can be inhibited obviously by verapamil which is the classical inhibitor of P-glycoprotein (P-gp). Those findings indicated that NCA was transported by P-gp. NCA may have low bioavailability after oral administration owing to the involvement of P-gp in the NCA transport. Using Rhodamine 123 (R123) as the probe substrate probe of P-gp, we studied the effects of NCA on the P-gp, the most important transporter in the intestine. NCA at 100μmol/L had significant inhibition effects on the P-gp-mediated transport of R123 across the MDCK-MDR1 cell monolayers, indicating that NCA was likely a P-gp inhibitor. On the other hand, NCA displayed significantly higher cytotoxicity to MDCK cells than to MDCK-MDR1 cells, likely because of the involvement of P-gp and decreasing the NCA accumulation in MDCK-MDR1 cells and the result confirmed indirectly that NCA was the substrate of P-gp.2. Metabolism of ECB and NCA studied in vitro with rat liver microsomes and hepatocytesOne monohydroxylation metabolite and one monoglucuronide were observed in rat liver microsomal incubates in the presence ofβ-NADPH or UDPGA, respectively. The monohydroxylation metabolite was determined by mass spectrometry to be 1-(2,4-dihydroxy-6-methoxy-3-methylphenyl)-2-hydroxyethanone, and monoglucuronide was determined by hydrolysis withβ-glucuronidase, mass spectrometry and 1HNMR to be 2-hydroxy-6-methoxy-3-methyl-acetophenone-4-O-β-glucuronide. But the mixed incubation of ECB with rat liver microsomes in the presence of bothβ-NADPH and UDPGA showed the monoglucuronide was the most major metabolite and more than 80% of ECB was converted into monoglucuronide, indicating glucuronidation was likely the major clearance pathway of ECB in rats.After NCA was incubated with rat liver microsomes, no oxidative metabolism was observed in the presence ofβ-NADPH. But in the presence of UDPGA, two metabolites were detected in the liver microsomal incubates and identified to be monoglucuronides by UPLC-MS analysis and hydrolysis usingβ-glucuronidase. The oxidative metabolism of ECB and the glucuronidation of ECB and NCA in rat liver microsomes all exhibited typical Michaelis-Menten patterns.The isoenzymes probably involved in ECB and NCA metabolism in rat liver microsomes were identified by using selective chemical inhibition and induction, respectively. The results indicated CYP3A, CYP2C11, CYP2C6 and UGT1A6/9 (especially UGT1A6) were important catalytic enzymes in ECB metabolism and UGT1A3/6/9 were important catalytic enzymes in NCA glucuronidation.No metabolites were detected after NCA was incubated with primary cultured rat hepatocytes. Two metabolites were observed after ECB was incubated with primary cultured rat hepatocytes, but only in trace amounts. Compared with rat liver microsomes, the metabolic ability toward ECB and NCA is much low.3. Metabolism, excretion and pharmacokinetics of ECB and NCA in ratsOnly glucuronidation metabolism with one monoglucuronide of ECB and NCA were observed in rats. The monoglucuronide of ECB observed in rats was identical to that formed in rat liver microsomes. The monoglucuronide of NCA observed in rats was identical to one of two monoglucuronides formed in rat liver microsomes and its structure needed to be elucidatd.A sensitive ultra performance liquid chromatography-mass spectrometry (UPLC-MS) method has been developed and validated for the quantitation of ECB-glucuronide in plasma. This analytical method has been applied successfully to study pharmacokinetics of ECB in rats. The pharmacokinetic parameters of ECB-glucuronide were estimated in rats following a single-dose oral administration of 100 mg/kg. In rats, ECB was rapidly metabolized to its monoglucuronide and the glucuronide was the only drug-derived compound clearly detected in plasma. Two-compartment model was used to estimate ECB-glucuronide pharmacokinetics by using DAS software. The plasma profile of ECB-glucuronide was found to be rapid with an apparent tmax occurring at 4 h, indicating that ECB was rapidly absorbed in rats following an oral administration. In rats following an oral administration of NCA, no metabolites were evidently detected in plasma. Moreover, the parent drug NCA was observed in plasma only in trace amounts, indicating that the oral absorption of NCA in vivo may be poor. One reason leading to the poor oral absorption of NCA was probably the involvement of P-gp. 4. Metabolism of ECB and NCA studied in vitro with human liver microsomes, HepG2 Cells and recombinant human enzymesThe metabolic profile in human liver microsomes was consistent with that observed in rat liver microsomes and rats, indicating glucuronidation was probably the major metabolic pathway of ECB in humans.When NCA was incubated with human liver microsomes, only one monoglucuronide was observed.The catalytic isoenzymes probably involved in ECB and NCA metabolism in humans were identified by using selective chemical inhibition in human liver microsomes and recombinant human enzymes. The results indicated CYP3A4, UGT1A6/9, especially CYP3A4 and UGT1A6, were important catalytic enzymes in ECB metabolism. UGT1A3/6/9 were important catalytic enzymes in NCA glucuronidation metabolism.No metabolites were detected after NCA was incubated with HepG2 cells; No glucuronide and only trace monohydroxylation metabolite were observed after ECB was incubated with HepG2 cells, indicating the metabolic ability toward ECB and NCA is very low.更多还原