【作者】 王晓丹；

【导师】 曾苏；

【作者基本信息】 浙江大学 ， 药物分析学， 2009， 博士

【摘要】 花旗松素和落新妇苷同为二氢黄酮类化合物,是土茯苓、水红花子等中药的主要有效成分,同时两者也广泛存在于葡萄、柑橘等水果中。落新妇苷是花旗松素的鼠李糖苷。药理学研究表明花旗松素和落新妇苷具有显著的抗氧化、抗菌、抗病毒、抗肿瘤等作用,其中落新妇苷还具选择性免疫抑制作用。以落新妇苷为主要成分的土茯苓总苷提取物已被开发成为一种治疗免疫性疾病的新药。文献调研表明花旗松素和落新妇苷的药理作用已经非常明确,但两者的吸收、转运、分布、代谢和排泄（ADME）及动力学性质鲜有报道。本文采用体内外模型对花旗松素和落新妇苷的ADME及两者在大鼠体内的药物代谢动力学进行了研究,为两者的临床应用及其新药的研发提供理论和实验依据。1.花旗松素、落新妇苷在Caco-2细胞单层中的转运研究及对caco-2细胞中P-糖蛋白的影响采用Caco-2细胞单层作为模拟小肠药物吸收的体外模型,研究花旗松素和落新妇苷在小肠中可能的吸收和转运机制。花旗松素和落新妇苷在Caco-2细胞单层中的表观渗透系数均≤1×10^-6cm/s,提示两者口服后生物利用度较差。花旗松素和落新妇苷在Caco-2细胞单层中的转运存在显著的外排现象,花旗松素的外排可被多药耐药蛋白2（MRP2）抑制剂MK-571抑制,而落新妇苷的外排可被P-糖蛋白（P-gp）的经典抑制剂维拉帕米和GF 120918抑制,表明MRP2和P-gp分别参与落新妇苷和花旗松素在Caco-2细胞中的吸收和外排转运,提示外排转运蛋白可能是制约其口服生物利用的主要生理屏障因素之一。临床上同时服用MRP2或P-gp抑制剂时,可能会使花旗松素或落新妇苷的药物代谢动力学特征发生改变。采用Caco-2细胞作为研究药物在小肠吸收转运的体外模型,研究花旗松素、落新妇苷和转运蛋白P-glycoprotein（P-gp）之间的相互作用。以罗丹明123（R123）作为P-gp的探针底物,评价花旗松素和落新妇苷对P-gp转运功能的影响;采用WesternBlot、RT-PCR等分子生物学方法评价花旗松素和落新妇苷对Caco-2细胞中P-gp表达的影响。花旗松素和落新妇苷对Caco-2细胞单层中由P-gp介导的R123外排无影响,提示花旗松素和落新妇苷对P-gp无抑制作用。采用不同浓度的花旗松素和落新妇苷对Caco-2细胞进行药物处理24或36h,发现Caco-2细胞中P-gp在蛋白水平和mRNA水平的表达量均有所升高,提示花旗松素和落新妇苷对P-gp的表达有诱导作用,10μmol/L落新妇苷或100μmol/L花旗松素处理细胞36 h对P-gp表达的诱导作用最显著,且这种诱导作用的机制不是通过PXR通路的调节而实现的。P-gp蛋白的表达上调可能是机体限制外源物进入、保护机体不受损伤的一种调节机制。2.花旗松素在Caco-2细胞中的代谢采用HPLC和UPLC-MS研究花旗松素在Caco-2细胞中的主要代谢物,揭示花旗松素在小肠上皮细胞中可能发生的代谢过程。实验结果表明花旗松素经Caco-2细胞代谢后可生成4种主要代谢物,采用质谱、核磁共振等技术对代谢物进行鉴定,发现3’-O-甲基化花旗松素为花旗松素在Caco-2细胞中的主要代谢物,一种代谢物为木犀草素的同分异构体,还有两种分别为花旗松素和3’-O-甲基化花旗松素的同分异构体/差向异构体。3.花旗松素大鼠体内药物代谢动力学研究建立花旗松素及3’-O-甲基化花旗松素在大鼠血浆和尿中的UPLC-MS测定方法,测定大鼠口服与静脉注射花旗松素后血中药物代谢动力学及口服花旗松素后尿中花旗松素的排泄,同时还测定了血中、尿中的3’-O-甲基化花旗松素,并对尿中的其他可能存在的代谢物进行检测。花旗松素在大鼠体内的药物代谢动力学参数通过DAS 2.0软件计算获得。花旗松素口服吸收较差,在大鼠体内的绝对生物利用度仅为0.17%。口服给药组药物代谢动力学符合一室模型,静脉注射组药物代谢动力学符合三室模型。花旗松素口服后吸收较为迅速,在6～10 min即达T_max,提示其吸收发生在胃和小肠上部。口服给药后大鼠体内的药物代谢动力学为典型的非线性动力学过程,与花旗松素在体内发生代谢或生物转化相关的酶代谢过程饱和,或与药物吸收、排泄有关的载体转运过程饱和有关。研究发现,花旗松素在大鼠体内可以很快的发生Ⅱ相代谢。口服或尾静脉给药后3 min即可在血液中检测到3’-O-甲基化花旗松素,且3’-O-甲基化花旗松素的AUC趋势与花旗松素的AUC趋势相似,均在相同时间达到血药浓度峰值,首过效应非常明显,提示花旗松素在体内的生物转化速度非常快,甲基化反应主要在肝脏中发生。大鼠口服100mg/kg花旗松素后,花旗松素最大的尿排泄量发生在0～2h时间段内,而3’-O-甲基化花旗松素最大尿排泄量发生在7～10h时间段内,尿中花旗松素原型的排泄率为给药量的0.24%,以3’-O-甲基化代谢物形式排泄的花旗松素为给药量的0.32%。此外,尿中还检测到花旗松素和3’-O-甲基化花旗松素葡萄糖醛酸结合产物,未检测到硫酸酯结合物。4.落新妇苷大鼠体内药物代谢动力学研究目前落新妇苷体内外代谢的研究已有报道,大鼠口服落新妇苷后在尿中可检测到3’-O-甲基化落新妇苷。本实验建立了大鼠血浆中落新妇苷的UPLC-MS的测定方法,并用于研究落新妇苷在大鼠体内的药物代谢动力学过程,测定大鼠口服与静脉注射落新妇苷后的血中药物代谢动力学及考察同时给予P-gp抑制剂维拉帕米后落新妇苷的血药动力学的改变,并对大鼠血中可能存在的Ⅱ相代谢物进行检测。研究表明:落新妇苷在大鼠体内的绝对生物利用度仅为0.066%,属于口服吸收较差的化合物,与Caco-2细胞模型预测结果相符。大鼠口服给予不同剂量的落新妇苷后,体内药物代谢动力学均符合二室模型,达峰时间约为20 min,口服5和100 mg/kg落新妇苷的吸收半衰期约4 min,提示落新妇苷口服吸收发生在胃肠道上部,且吸收迅速;5mg/kg口服剂量给药时落新妇苷的吸收和消除均较为迅速;100 mg/kg口服剂量给药时落新妇苷吸收快但消除慢。同时口服维拉帕米后,落新妇苷的AUC增加2.57倍。实验结果提示维拉帕米对组织中P-gp外排功能的抑制可能是落新妇苷在大鼠体内“蓄积”而引起AUC增加的主要原因。大鼠静脉给予5mg/kg落新妇苷后,其药物代谢动力学行为符合二室模型。落新妇苷在大鼠体内的代谢物主要是3’-O-甲基化落新妇苷,与文献报道一致。试验中未发现相应的苷元或葡萄糖醛酸结合物等Ⅱ相代谢物。更多还原

【Abstract】 Flavonoids are polyphenolic compounds present in many fruits,vegetables,and beverages.They have been strongly implicated as protectors in coronary heart disease and stroke as well as cancer.Astilbin and its aglycone,taxifolin,were isolated from many Chinese herbs,such as the rhizome of Smilax glabra,a Liliaceae plant, Dimorphandra mollis and Hypericum perforatum,and also have been found in many citrus fruits,especially grapefruit and orange.It has been reported that taxifolin and astilbin have many activities in anti-bacteria,anti-oxidative,anti-inflammation,as well as hepato-protection.Although astilbin and taxifolin provide many benefits to human health,the knowledge about the absorption,transport,metabolism,the interactions with drug-transporters in vivo and in vitro,and other pharmacokinetics profiles are still limited.The aims of this present study were to understand the drug metabolism and pharmacokinetic profiles of astilbin and taxifolin using in vivo and in vitro models.1.The transport of taxifolin and astilbin across the Caco-2 cell monolayers and the effects on the P-gp expressed in Caco-2 cellsThe aim of this study was to explore the potential absorption and transport mechanism of flavanonols astilbin and taxifolin across the Caco-2 cell monolayers,a model of human intestinal epithelial membrane.In the Caco-2 cells,these two flavanonols showed a low absorption.The apparent permeability coefficient(P_app) values of apical to basolateral direction for astilbin and taxifolin were less than 1×10^-6 cm/s.The efflux ratios of both compounds were much higher than 2.0.The efflux of taxifolin can be abolished in the presence of MK-571,the inhibitor of the multidrug resistance proterin 2（MRP2）,and the efflux of astilbin can be abolished in the presence of P-glycoprotein（P-gp） inhibitors verapamil and GF 120918.Those findings indicated that astilbin and taxifolin were transported by P-gp and MRP2,respectively.Astilbin and taxifolin may have low bioavailability after oral administration because the two efflux transporters participating in the absorption of the tow flavanonols.The drug-drug interactions should be considered when astilbin or taxifolin is co-administrated with the inhibitors of P-gp or MRP2 in the clinic.Using Rhodamine 123（R123） as the substrate probe of P-gp,we studied the effects of astilbin and taxifolin on the P-gp,the most important transporter in the intestine. Astilbin and taxifolin had no significant inhibition effects on the P-gp-mediated transport of R123 across the Caco-2 cell monolayers.Caco-2 cells exposed to astilbin or taxiolin for 36 h exhibited higher P-gp activity through up-regulating P-gp expression at protein and mRNA levels.These results indicated that astilbin and taxifolin may have effects on the regulation the expression of P-gp.The increased expression of P-gp in Caco-2 cells may serve as an adaptation and defense mechanism in limiting the entry of xenobiotics into the body.2.The metabolism of taxifolin in Caco-2 cellsThe aim of this study was to find out the possible metabolism of taxifolin in human intestine using Caco-2 cells in vitro.After adding the taxifolin into the culture medium and incubating with Caco-2 cells for 48 h,we found four new compounds in the culture medium and Ml was the main metabolite.Using HPLC,MS and NMR,we prepared the metabolites and identified the structure of Mlas 3’-O-methyltaxifolin. 3.The pharmacokinetic studies of taxifolin in ratsIn this study,a sensitive ultra performance liquid chromatography-mass spectrometry （UPLC-MS） method has been developed and validated for the quantification of taxifolin and 3’-O-methyltaxifolin in rat plasma and urine.The present methods were successfully applied to the estimation of the pharmacokinetic parameters of taxifolin following intravenous and oral administration to rats.The absolute bioavailability of taxifolin was 0.17%.And the possible metabolites in rat plasma and urine were detected, especially 3’-O-methyltaxifolin,the main metabolite in rat plasma.Using DAS 2.0 software,it was found that the best fit pharmacokinetic model was single compartment model with weight of 1/C² for oral administration,and three compartments model with weight of 1/C for intravenous administration.The area under the curve（AUC） and C_max were non-proportional to the dose of oral administration ranged from 10 mg/kg to 100 mg/kg.T_max and t_1/2 were depended on the doses of oral administration.Those results indicated the absorption of taxifolin in rat by oral administration was a typical non-linear process.The biotransformation of taxifolin in rat was so fast that the metabolites can be detected at the first sampling time for both oral administration and intravenous administration.The results were helpful for studying the kinetics of taxifolin,and allowed us to make better understanding of the metabolism mechanism of taxifolin in human.4.The pharmacokinetic studies of astilbin in ratsIn this study,a sensitive UPLC-MS method has been developed and validated for the quantification of astilbin in rat plasma and was successfully applied to the estimation of the pharmacokinetic parameters of astilbin following intravenous and oral administration to rats.Using DAS 2.0 software,it was found that the best fit pharmacokinetic model was two compartments model with weight of 1/C² for both oral administration and intravenous administration.The absolute bioavailability of astilbin was 0.066%indicating that the oral absorption of astilbin in vivo may be poor.The AUC of astilbin in the rats with co-administration with verapamil was increased 2.57 times indicating that the P-gp plays an important role in the pharmacokinetics of astilbin in rats.更多还原