1、人源P-糖蛋白高表达MDR1-MDCKII细胞模型的建立及应用 2、新型调血脂化合物IMM-H007的临床前药代动力学研究
|
摘要
中草药作为一种传统的疾病治疗手段已有数千年的历史,近年来由于现代医学的发展,主要作为补充和替代医疗手段在世界范围内广泛应用。研究表明,中草药对于慢性或全身严重性疾病如癌症和艾滋病的治疗有着独特的优势,所以在临床治疗中中草药通常和其他处方药物联合应用以增加疗效,但同时也忽略了药物-药物相互作用(DDI)发生的可能性。在DDI的发生机制中,代谢性DDI尤其是药物代谢酶和药物转运体介导的DDI是最重要的两个机制。在过去的几十年里,药物代谢酶介导的DDI是主要的研究重点,而药物转运体介导DDI的研究报道相对较少。近年来由于药物转运体介导DDI的临床报道日益增多,药物与转运体之间的相互作用成为现代医学研究的重点之一。
P-糖蛋白(P-glycoprotein, P-gp)分子量约170kD,属于ABC转运蛋白超家族,由位于7q21的基因MDR1编码。P-gp除了在肿瘤细胞高表达介导多药耐药外,也存在于正常组织如小肠、肝脏、肾脏及某些特殊生理屏障如血脑屏障的毛细血管内皮细胞上。P-gp主要功能是利用ATP水解提供的能量,将细胞内的有毒物质或者药物通过胃肠道、胆汁和尿液排出体外,发挥其抵抗外来毒性物质侵袭的作用。由于P-gp的外排作用能够影响药物的生物利用度、肝肾排泄率和组织分布,因此P-gp的诱导或抑制可能引起体内药物间相互作用,增加DDI发生的可能性。这对于一些治疗指数小的药物如华法林、地高辛等尤其重要。对于治疗窗狭窄的药物而言,体内药物暴露量的微小波动可能导致临床疗效和毒性作用的显著变化,从而导致不良反应的发生。
前期报道表明,多种传统中药材或中药材主要组分可影响P-gp的表达和活性。但由于人体口服中药后,真正进入体循环的中药有效成分常与中药原有成分不同,而这些中药有效成分和P-gp相互作用的文献资料相对较少。此外,不同实验室由于实验条件(环境、细胞类型和状态及给药剂量和给药时间)不同,同类研究可得出不同甚至是相反的实验结果。
人源P-gp高表达MDR1-MDCKⅡ细胞模型由于培养周期短,代与代之间均一性良好,常作为体外筛选P-gp底物和抑制剂的细胞模型,并可用于研究P-gp介导DDI发生的可能性。
本研究应用逆转录病毒感染MDCKⅡ细胞的方法建立人源P-gp高表达MDR1-MDCKⅡ细胞模型。应用此细胞模型研究了来源于25种临床常用中药的50种活性成分对P-gp活性的影响。此外,通过ATP酶活性测定初步探讨中药活性成分抑制P-gp的可能机制。应用分子对接技术分析中药活性成分和P-gp之间的构效关系。由于CYP3A4/5与P-gp有明显的底物和组织分布交叉性,因此进一步探讨具有P-gp抑制作用的中药活性成分对CYP3A4/5活性的影响。同时,应用大鼠体内模型评价活性成分与地高辛合用对药代动力学的影响,为临床DDI的发生提供有参考价值的实验依据。研究结果表明：
1.人源P-糖蛋白高表达MDR1-MDCKⅡ细胞模型的建立
1.1酶切实验和DNA测序结果表明,携带目的基因MDR1的P-gp表达质粒pHaMDRwt可以在大肠杆菌中正确扩增和富集。
1.2脂质体转染法介导pHaMDRwt质粒进入病毒包装细胞PA317,经秋水仙碱筛选后,可得到含有目的基因MDR1的高滴度逆转录病毒。病毒滴度经测定可达到8.8×10-5pfu/ml。
1.3逆转录病毒感染MDCKⅡ细胞,经秋水仙碱筛选后,可得到人源P-gp高表达MDR1-MDCKⅡ细胞模型。RT-PCR和western blot结果表明,MDR1-MDCKⅡ细胞株可稳定高表达外源基因MDR1。Rho123外排实验和地高辛双向转运实验测定结果表明转染细胞中外源基因MDR1功能表达良好,可作为体外筛选P-gp底物和抑制剂的细胞模型。
2.50种中药活性成分对P-糖蛋白和CYP3A4/5活性的影响及药物相互作用研究
2.1MDR1-MDCKⅡ和Caco-2田胞双向转运实验结果表明,大黄素、18β-甘草次酸、脱水穿心莲内酯和20(S)-人参皂苷F1(≤100μM)对P-gp有显著的抑制作用(>50%),而上述化合物的同分异构体／结构类似物大黄酚、18a-甘草次酸、穿心莲内酯和人参皂苷Rh1对P-gp的活性影响相对较弱。
2.2在MDR1-MDCKⅡ细胞中,对P-gp抑制活性最强的是大黄素(IC50=9.42μM)其次是18β-甘草次酸(IC50=21.78μM)、20(S)-人参皂苷F1(IC50=76.08μM)和脱水穿心莲内酯(IC50=77.80μM)。
2.3大黄素和脱水穿心莲内酯剂量依赖性激活P-gp ATPase舌性,Km和Vmax分别是48.61、29.09μM和71.29、38.45nmol/min/mg protein。高浓度(100μM)18p-甘草次酸和20(S)-人参皂苷F1对空白和维拉帕米激活的P-gp ATPase活性均有抑制作用。
2.418β-甘草次酸、脱水穿心莲内酯、20(S)-人参皂苷F1和人参皂苷Rh1显著抑制CYP3A的代谢活性,抑制率分别是44、41、23和15%。大黄素可以显著激活CYP3A的代谢活性,与空白对照组相比,CYP3A舌性增加了35%。
2.5分子对接研究初步阐明了中药活性成分和P-gp及CYP3A4之间的构效关系。结果表明,18-氢原子的差向异构(18β-甘草次酸)、氢键(大黄素)、双键(脱水穿心莲内酯)和糖基的取代位置(20(S)-人参皂苷F1)可能是化合物体现不同P-gp抑制活性的重要化学因素。与大黄酚、18a-甘草次酸、穿心莲内酯和人参皂苷Rhl相比,大黄素、18p-甘草次酸、脱水穿心莲内酯和20(S)-人参皂苷F1可以和P-gp形成更多的氢键作用,这可能是后者发挥更强活性的原因之一。此外,18p-甘草次酸和20(S)-人参皂苷F1均可和Arg212(CYP3A4与底物相互作用的重要氨基酸残基)形成一个较强的氢键,因此可能通过和咪达唑仑竞争性结合Arg212发挥CYP3A4抑制活性。而大黄素虽然和Arg212形成一个Pi键,但和CYP3A4之间的主要作用力是和Thr310形成一个较强的氢键,因此可能减少和咪达唑仑的竞争力而变构激活CYP3A4的活性。
2.6SD大鼠提前口服大黄素或18p-甘草次酸后,地高辛的AUC0-t和Cmax与空白对照组相比,分别升高了51%和58%。综上所述,本研究应用逆转录病毒感染MDCKⅡ细胞的方法构建了人源P-gp高表达MDR1-MDCKⅡ细胞模型,此模型稳定高表达外源基因MDR1,可作为体外筛选P-gp底物和抑制剂及研究化合物和P-gp相互作用的细胞模型。在选择研究的50种中药活性成分中,4种中药成分对P-gp有显著的抑制作用,分别为大黄素、18p-甘草次酸、脱水穿心莲内酯和20(S)-人参皂苷Fl。此外,具有P-gp抑制作用的中药活性成分可能同时抑制或激活CYP3A4/5的活性,因此临床联合用药时需注意DDI发生的可能性。上述研究为预测临床上由中药和其他处方药物合用引起相互作用的可能性提供科学、简便可靠的评价体系,并为指导临床合理用药提供有参考价值的实验依据。
IMM-H007是一种新型调血脂化合物,化学结构为腺苷类似物。药理学研究表明,IMM-H007(10μM)可抑制十八烯酸诱导的脂肪性变HepG2细胞内脂质的堆积。体内药效学研究发现,IMM-H007(2mg/kg)可降低高脂血症金黄地鼠升高的血清甘油三酯、总胆固醇、低密度脂蛋白和肝脏甘油三酯、总胆固醇水平。机制研究提示,IMM-H007可以上调十八烯酸诱导的脂肪病变HepG2细胞内或高脂血症金黄地鼠肝细胞内AMPK的磷酸化水平,是一种新型AMP-激活蛋白激酶(AMP-activated protein kinase, AMPK)激活剂。由此可见,IMM-H007作为一种与他汀类药物化学结构、作用靶点和代谢途径不同的新型调血脂化合物,有望成为临床预防和治疗心血管疾病的新药。
前期药代研究表明,IMM-H007通过酯酶而非CYP450代谢。IMM-H007在体内首先发生水解反应,终末水解产物M1可进入细胞,经磷酸化反应生成MP(M1的5’-磷酸化产物),或发生脱氧、脱核糖环反应或Ⅱ相加合反应。MP的结构与5’-磷酸腺苷(AMP)极其相似,而AMP是AMPK的有效激活剂,因此推测MP可能为IMM-H007激活靶酶AMPK的活性代谢物。
前期实验结果表明IMM-H007及代谢产物M1和MP在大鼠血浆和全血中的药代动力学特征明显不同,如MP在大鼠血浆内含量极低,主要存在于全血中。本论文初步探讨大鼠口服和静脉注射IMM-H007后,原型药及代谢产物M1和MP在大鼠全血内的药代动力学特点,并研究IMM-H007及M1在体外缓冲液和不同种属动物生物基质(血浆、全血、肝微粒体、肠道菌群)内的代谢稳定性,推测IMM-H007可能的代谢转化途径,为后期药代动力学研究提供有参考价值的依据。研究结果表明：
1.生物样品中IMM-H007及代谢产物M1和MP的HPLC-MS/MS分析方法的建立根据临床前药代动力学指导原则,建立了生物样品中IMM-H007及代谢产物M1和MP的HPLC-MS/MS分析方法。结果表明,各检测物质在大鼠全血中均未见明显基质和杂质干扰,IMM-H007、M1和MP分别在1～500、2～1000和10～5000ng/ml浓度范围内线性关系良好,最低定量限分别为1、2和10ng/ml。各待测物质日内和日间精密度相对标准差均小于15%,准确度为±5.7%。IMM-H007、M1和MP的低、中、高3个QC浓度的全血样品回收率分别为80～91.2%、107.4～108.7%和76.4～80.3%。该方法简便、可靠、灵敏度高、特异性强,可满足IMM-H007的临床前药代动力学研究。
2.大鼠口服和静脉注射IMM-H007的体内药代动力学研究
2.1雄雌大鼠单次口服IMM-H007后吸收较快,各剂量组给药后5min血中即可检测到原型药和代谢产物,给药后12～36h血药浓度接近检测限。各时间点原型药浓度均较低(多个时间点浓度接近检测限),达峰浓度与剂量不成比例,药时曲线呈现多峰现象。
2.2雄性大鼠口服不同剂量IMM-H007(50、50、450和900mg/kg)后,代谢产物M1的Cmax分别为37.52、61.13、82.69、141.18ng/ml, AUC0-t分别为160.47.328.71、799.57、1779.31h*μg/L, MRT0-t分别为3.18、3.93、7.5、11.3h；代谢产物MP的Cmax分别为88.2、153.46、204.95、309.42ng/ml, AUC0-t分别为362.71、676.26、2230.14、4536.37h*μg/L, MRT0-t分别为2.87、3.65、8.15、12.07h。代谢产物M1和MP的Cmax与AUC0-t均随给药剂量增加而递增,并与给药剂量呈正比,但两者的MRT0-t均随剂量增加而延长,提示代谢产物M1和MP在大鼠体内可能存在消除饱和现象。
2.3雌性大鼠口服不同剂量IMM-H007(50、150、450和900mg/kg)后,代谢产物M1的Cmax分别为37.78、210.72、256.89、325.77ng/ml,AUC0-t分别为151.87、854.34、1561.42、3334.16h*μg/L、MRT0-t分别为3.85、4.25、7.62、10.51h；代谢产物MP的Cmax分别为132.55.920.83、1214.01、1122.64ng/ml, AUC0-t分别为698.63、4000.82、8376.97、13900.82h*μg/L, MRT0-t分别为4.08、4.86、8.45、10.3h。代谢产物M1和MP的AUC0-t均随剂量增加而递增,呈现一定比例关系,但中、高剂量组M1和MP的Cmax与给药剂量不成比例,且两者的MRTo_t均随剂量增加而延长。上述结果提示代谢产物M1和MP在雌性大鼠体内可能存在吸收和消除饱和现象。
2.4雌性大鼠中、高剂量组代谢产物M1和MP的Cmax和AUCo-t均高于雄性大鼠,提示代谢产物M1和MP在大鼠体内的药代动力学存在一定的性别差异。
2.5IMM-H007的生物利用度以M1计算,雄鼠为3.04%,雌鼠为2.12%；以MP计算,雄鼠为5.61%,雌鼠为6.55%,无明显性别差异。
3. IMM-H007的体外代谢稳定性研究
3.1IMM-H007和代谢产物M1在人工胃液、人工肠液和Tris-HCl缓冲液中稳定性良好,4h后剩余量均高于85%。
3.2IMM-H007在不同种属动物(SD大鼠、C57小鼠、金黄地鼠、比格犬、食蟹猴、人)血浆中(温孵时间为2h)均不稳定,且呈现种属差异性,代谢稳定性结果为比格犬>人>食蟹猴>SD大鼠、C57小鼠、金黄地鼠。M1在体外不同种属动物血浆中均较稳定。体外温孵体系中无MP的生成。
3.3IMM-H007在不同种属动物(SD大鼠、C57小鼠、金黄地鼠、比格犬、食蟹猴、人)肝微粒中(温孵时间为2h)均不稳定,5min即减少90%以上,且IMM-H007减少程度不依赖NADPH。M1在体外不同种属动物肝微粒中均较稳定。体外温孵体系中无MP的生成。
3.4IMM-H007和M1在不同种属动物(SD大鼠、C57小鼠、金黄地鼠、比格犬、食蟹猴、人)全血中(温孵时间为2h)均不稳定,温孵体系中可检测到MP的生成。除比格犬外,其他动物全血中水解产物M1的生成随着原型药的减少均呈现先上升后下降趋势,而MP则一直呈现递增趋势。
3.5IMM-H007在体外大鼠肠道菌群中(温孵时间为8h)不稳定,可生成代谢产物M444、M402、M1、M344和M228。其中M444、M402和M1在体系中含量呈先上升后下降趋势；代谢产物M344和M228呈递增趋势。M1在体外大鼠肠道菌群中不稳定,可生成代谢产物M344和M228,其中M344含量呈先上升后下降趋势；M228呈递增趋势。
综上所述,本研究建立了生物样品中IMM-H007及代谢产物M1和MP的HPLC-MS/MS分析方法,该方法简便、可靠、灵敏度高、特异性强,可满足IMM-H007的临床前药代动力学研究。应用此分析方法初步探讨了大鼠口服和静脉注射IMM-H007后,原型药及代谢产物M1和MP在雌雄大鼠全血内的药代动力学特点。体外代谢稳定性结果表明IMM-H007和M1在体外不同种属动物全血和大鼠肠道菌群中均不稳定,可生成水解、脱氧、脱核糖环或磷酸加合产物。此研究为了解IMM-H007可能的代谢转化途径及后期药代动力学研究提供有参考价值的实验数据。
Herbal medicines have been traditionally used for the treatment of various diseases, including cold symptoms, pain, and inflammation for thousands of years. Currently, these medicines are still utilized worldwide as complementary or alternative medical ingredients with the expectation of promoting health and managing chronic or severe diseases such as cancer and HIV infection. Due to the common perception that herbal supplements are safe, they are often co-administered with conventional prescription drugs in clinical trial, raising the risk of herbal-drug interactions. Metabolic enzyme-mediated drug-drug interactions (DDI) have been extensively studied while studies on DDI related to transporters have been relatively limited, despite their importance in drug disposition.
P-glycoprotein (P-gp), a member of the ABC transporter superfamily, is encoded by the ABCB1/MDR1gene. It serves as a key factor in conferring the multi-drug resistance (MDR) phenotype to cancer cells in clinical trial. In addition, P-gp is highly expressed in the apical membrane of several pharmacologically important epithelial barriers such as the kidney, liver, intestine, and blood-brain barrier. P-gp could excrete xenobiotics such as cytotoxic compounds into the gastrointestinal tract, bile and urine. Thus the modulation of P-gp can affect the oral bioavailability, biliary or renal clearance, brain uptake of drugs and increase the risk of DDI. This is particularly concerning for drugs with narrow therapeutic indices such as warfarin and digoxin. Small changes in the systemic exposure of these drugs could result in substantial alterations in therapeutic effects and toxic outcomes.
Many commonly used traditional Chinese medicines (TCM) have been reported to interact with P-gp. However, the interaction profile is limited between P-gp and bioactive herbal constituents, which can effectively enter human systemic circulation after oral administration. In addition, controversial experimental results are also obtained in different laboratories due to the selection of cell strain, dosage and duration of drug exposure.
Due to the high expression of human P-gp and short culture time, MDR1-MDCKII cells transfected with the human MDR1gene have become an attractive model to study the DDI mediated via P-gp.
In the present study, MDR1-MDCKII cells were established by the transfection of a retrovirus carrying the human MDR1gene into parent MDCKII cells. The inhibitory effects of50major herbal constituents present in25commonly used TCM on P-gp were investigated in vitro and in vivo. The underlying inhibitory mechanism of herbal constituents was partially assessed using the ATPase activity assay. In addition, molecular docking analysis was performed to elucidate the structure-activity relationships of herbal compounds with P-gp. Due to the overlapping substrate specificities and tissue distribution of cytochrome P4503A4/5(CYP3A4/5) and P-gp, the herbal constituents that exhibited potential inhibition of P-gp were further examined for their effects on CYP3A4/5activity in human liver microsomes. The effects of a single dose of herbal inhibitors on the pharmacokinetics of digoxin were also evaluated in male SD rats. The results were summarized as follows:
1. Establishment of MDR1-MDCKII cells with high expression of human P-gp
1.1The results of restriction analysis and DNA sequencing showed the P-gp expressive vector pHaMDRwt carrying a human MDR1gene was accurately amplified and enriched in E.coli.
1.2Amphotropic virus was produced after the transfection of plasmid pHaMDRwt into PA317cells. Viral titer of up to8.8×10-5pfu/ml was observed in the culture of MDR1-PA317packaging cells.
1.3MDR1-MDCKII cells with high expression of human P-gp were established after MDCKII cells were infected with the retrovirus carrying a human MDR1gene and incubated with colchicine (60ng/ml) for two weeks. RT-PCR and western blotting analyses showed the expression of human P-gp was stable and high in MDR1-MDCKII cells. Moreover, the decreased accumulation of Rhol23and enhanced efflux of digoxin were observed in MDR1-MDCKII cells, indicating the high P-gp activity of MDR1-MDCKII cells.
2. Effects of50herbal constituents on P-gp and CYP3A4/5in vitro and in vivo: herb-drug interactions mediated via P-gp and CYP3A4/5
2.1Among50herbal constituents, emodin,18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and20(.S)-ginsenoside F1[20(S)-GF1](100μM or less, depending on their solubility and cytotoxicity) exhibited significant inhibition (>50%) on P-gp in MDR1-MDCKII and Caco-2cells. However, despite the structural similarity and/or identical molecular weight, the isomers or analogues of the4herbal constituents (chrysophanol,18a-GA, andrographolide, and ginsenoside Rh1) had no effect on P-gp function.
2.2Emodin was the strongest herbal inhibitor of P-gp (IC50=9.42μM) in MDR1-MDCKII cells, followed by180-GA (IC50=21.78μM),20(S)-GF1(IC50=76.08μM) and DAG (IC50=77.80μM).
2.3P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with Km and Vmax values of48.61,29.09μM and71.29,38.45nmol/min/mg protein, respectively. However,18β-GA and20(S)-GF1exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration (100μM).
2.418β-GA, DAG,20(S)-GF1and Rh1significantly inhibited CYP3A4/5activity (44,41,23and15%inhibition, respectively), while emodin increased the metabolic rate of midazolam by approximately35%compared to the control.
2.5Molecular docking analysis (CDOCKER) elucidated the mechanism for structure-activity relationships of herbal constituents with P-gp and CYP3A4. The results showed the isomerism of18-hydrogen atom (18β-GA), the existence of a hydroxyl (emodin) and double bond (DAG), and the position of glycosyl [20(S)-GF1] could be crucial chemical elements for the inhibition of P-gp. Due to these chemical elements, additional strong hydrogen bonds were formed between herbal inhibitors and P-gp, resulting in stronger inhibitory effects of herbal constituents for P-gp. In addition,18β-GA and20(S)-GF1may display their inhibition on CYP3A4by competitively binding with Arg212which was reported to be one of the key residues for the binding of compounds with CYP3A4, while emodin was able to bind more readily with another residue Thr310via a strong hydrogen bond, reducing the competition for this binding orientation/site and enabling activation to occur.
2.6Co-administration of digoxin with emodin increased the AUC of digoxin by55%while the Cmax of digoxin was increased by58%when18β-GA was pretreated in SD rats.
In conclusion, MDR1-MDCKII cells with high and stable expression of human P-gp were established in the present study, and it could be used as a valuable in vitro tool for the rapid screening of P-gp substrates/inhibitors and investigating the DDI mediated via P-gp. Among50herbal constituents, four constituents, including emodin,18β-GA, DAG and20(S)-GF1exhibited significant inhibition (>50%) on P-gp in vitro and/or in vivo. Moreover, dual inhibition of P-gp and CYP3A4/5activities could occur due to the overlapping substrate specificities between them. Our findings provided the basis for the reliable assessment of the potential risks of herb-drug interactions in humans and will provide useful information for the proper use of prescription drugs in combination with herbal medicines, which is particularly important for drugs with narrow therapeutic indices.
IMM-H007, an analog of adenosine, is a novel small molecule compound that could significantly improves lipid metabolism disorders in the hyperlipidemia animal models. The pharmacological studies showed that IMM-H007could inhibit cellular lipid accumulation in OLA-treated HepG2cells. Moreover, IMM-H007(2mg/kg per day and above) reduced elevated serum triglyceride, total cholesterol, low density lipoprotein cholesterol and hepatic cholesterol and triglyceride contents in HFD fed hamsters. The mechanism studies indicated that the activation of AMPK in OLA-induced steatosis of HepG2cells was up-regulated by the treatment with IMM-H007. The hepatic cellular AMPK phosphorylation was also up regulated by IMM-H007(6and18mg/kg) treatment in HFD fed hamsters. As a novel lipid metabolism regulator, IMM-H007has a differernt chemical structure, target and metabolic pathway from statins, and could be potentially used for patients who have adverse reactions to statins in clinical trial.
Previous studies showed that IMM-H007contained three acetyl groups, which were liable to be hydrolyzed by plasma/serum esterases rather than CYP450s in vitro and in vivo. IMM-H007could be rapidly hydrolyzed to M1, which was believed to be the active form of IMM-H007in vivo, after an oral administration of IMM-H007to rats. A further study indicated that M1was extensively metabolized in HepG2cells, and several metabolites were observed, including deoxidized product of Ml (M344), deribosyl derivative of M1(M228), glucuronide conjugate of M1, glucuronide conjugate of M344, glucuronide conjugate of M228, sulfate conjugate of Ml, phosphate conjugate of M1(MP) and et al. Because MP was an analog of AMP, which was proved to be an effective activator of AMPK, MP was likely the active form of IMM-H007to activate AMPK in HepG2cells.
Previous studies showed that the pharmacokinetic characteristics of IMM-H007and its major metabolites were varied between rat plasma and blood. For example, the concentration of MP was fairly low in rat plasma and far higher in rat blood. In the present study, the concentrations of IMM-H007, M1and MP in rat blood were detected after oral and intravenous dosing of IMM-H007to SD rats. Metabolic stabilities of IMM-H007and Ml were assessed in different biological systems such as simulated gastric/intestinal fluid, Tris-HCl buffer, and plasma, blood, liver microsomes of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans. The metabolic stability of IMM-H007in the gut microbiota of SD rats was also investigated in this study. The results were summarized as follows:
1. Simultaneous quantification of IMM-H007and its metabolites in rat blood using HPLC-MS/MS
Base on the guideline of preclinical pharmacokinetic study, the HPLC-MS/MS method for the quantitation of IMM-H007and its metabolites M1and MP in biological sample has been developed, which provided a simple, reliable, sensitive and specific assay for the preclinical pharmacokinetic study of IMM-H007in SD rats. Linear detection responses were obtained for IMM-H007, M1and MP ranging from1～500,2～1000and10～5000ng/ml, respectively. The intra-and inter-day precisions (R.S.D.%) were within15%for all analytes, while the deviation of assay accuracies was within±5.7%. The average recoveries of IMM-H007, M1and MP were80～91.2%,107.4～108.7%and76.4～80.3%, respectively.
2. Pharmacokinetic study of IMM-H007in SD rats
2.1IMM-H007and its metabolites M1and MP can be detected in rat blood5min after oral administration of IMM-H007(50,150,450and900mg/kg) to SD rats and the blood concentrations were close to the limit of quantitation (LLOQ) at12-36h postdose. The multiple peak concentration-time curves of IMM-H007were observed, and the concentration of parent drug IMM-H007was much lower than its metabolites in both male and female rats.
2.2After oral administration of IMM-H007(50,150,450and900mg/kg) to male rats, the average maximum concentration (Cmax) of M1were37.52,61.13,82.69and141.18ng/ml, AUC0-t were160.47,328.71,799.57and1779.31h*μg/L, MRT0-t were3.18,3.93,7.5and11.3h. The Cmax of MP in male rats were88.2,153.46,204.95and309.42ng/ml, AUC0-t were362.71,676.26,2230.14and4536.37h*μg/L, MRT0-t were 2.87,3.65,8.15and12.07h. Both Cmax and AUC0-t of Ml and MP were increased in a dose-dependent manner, but the MRT0-t was significantly delayed after oral administration of IMM-H007(450and900mg/kg) to male rats. The results indicated the saturation of elimination of M1and MP in male rats at high doses of IMM-H007(450and900mg/kg).
2.3After oral administration of IMM-H007(50,150,450and900mg/kg) to female rats, the Cmax of M1were37.78,210.72,256.89and325.77ng/ml, AUC0-t were151.87,854.34,1561.42and3334.16h*μg/L, MRT0-t were3.85,4.25,7.62and10.51h. The Cmax of MP in male rats were132.55,920.83,1214.01and1122.64ng/ml, AUC0-t were698.63,4000.82,8376.97and13900.82h*μg/L, MRT0-t were4.08,4.86,8.45and10.3h. The AUC0-t of M1and MP were increased in a dose-dependent manner, but the Cmax of M1and MP were not increased dose-dependently. In addition, the MRTo0-t was significantly delayed after oral administration of IMM-H007(450and900mg/kg) to female rats. The results indicated the saturation of absorption and elimination of M1and MP in female rats at high doses of IMM-H007(450and900mg/kg).
2.4Both Cmax and AUCo-t of Ml and MP were much higher in female rats than those in male rats after oral administration of IMM-H007(150,450and900mg/kg), indicating the gender difference between male and female rats.
2.5The oral bioavailability of M1was3.04%in male rats and2.12%in female rats after an oral administration of IMM-H007at50mg/kg; the oral bioavailability of MP was5.61%in male rats and6.55%in female rats after an oral administration of IMM-H007at50mg/kg.
3. In vitro metabolic stability of IMM-H007
3.1The concentrations of IMM-H007and M1did not change significantly after4h incubation with simulated gastric/intestinal fluid and Tris-HCl buffer. The remaing IMM-H007and M1were more than85%when the reaction was terminated with the addition of ice-cold acetonitrile.
3.2IMM-H007was unstable when incubated with animal plasma in vitro for2h. IMM-H007could be hydrolyzed to M1at different rate in the plasma of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans. M1was stable after2h incubation with animal plasma. Moreover, no MP was detected in the reaction mixture.
3.3IMM-H007was unstable when incubated with the liver microsomes of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans in vitro for2h. More than90%IMM-H007was metabolized in the first5min incubation and the reaction was not NADPH-dependent. M1was stable after2h incubation with liver microsomes. Moreover, no MP was detected in the reaction mixture.
3.4Both IMM-H007and M1were unstable when incubated with animal blood in vitro for2h. IMM-H007and M1showed different metabolic rates in the blood of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans. In addtion, high concentratin of MP was detected in the reaction mixture. The concentration profile of M1demonstrated an upward phase and a downward phase, suggesting onward metabolism to other products, while MP increased constantly until it reached a maximum level. Rates of increase and decrease of products differed by species.
3.5IMM-H007could be extensively metabolized into certain metabolites including M444, M402, M1,M344and M228by the gut microbiota of SD rats. Products M444, M402and M1appeared first, followed by M344and M228. Concentration profiles of M444, M402and M1demonstrated an upward phase and a downward phase, while M344and M228increased constantly until they reached the maximum levels. Products M344and M228also appeared when M1was incubated with the gut microbiota of SD rats for8h.
In conclusion, the HPLC-MS/MS method for the simultaneous quantitation of IMM-H007and its metabolites M1and MP in rat blood was developed in the present study. In addition, the pharmacokinetics of IMM-H007, M1and MP in rat blood were investigated after oral and intravenous dosing of IMM-H007to SD rats. The results of metabolic stabilities of IMM-H007and M1showed that IMM-H007and M1could be metabolized into several metabolites such as M444, M402, M1, M344, M228and MP by the blood of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys, humans and the gut microbiota of SD rats. Our findings will provide useful information for the further pharmacokinetic study of IMM-H007in other species and give insights to the possible metabolic pathway of IMM-H007in vivo.
P-糖蛋白(P-glycoprotein, P-gp)分子量约170kD,属于ABC转运蛋白超家族,由位于7q21的基因MDR1编码。P-gp除了在肿瘤细胞高表达介导多药耐药外,也存在于正常组织如小肠、肝脏、肾脏及某些特殊生理屏障如血脑屏障的毛细血管内皮细胞上。P-gp主要功能是利用ATP水解提供的能量,将细胞内的有毒物质或者药物通过胃肠道、胆汁和尿液排出体外,发挥其抵抗外来毒性物质侵袭的作用。由于P-gp的外排作用能够影响药物的生物利用度、肝肾排泄率和组织分布,因此P-gp的诱导或抑制可能引起体内药物间相互作用,增加DDI发生的可能性。这对于一些治疗指数小的药物如华法林、地高辛等尤其重要。对于治疗窗狭窄的药物而言,体内药物暴露量的微小波动可能导致临床疗效和毒性作用的显著变化,从而导致不良反应的发生。
前期报道表明,多种传统中药材或中药材主要组分可影响P-gp的表达和活性。但由于人体口服中药后,真正进入体循环的中药有效成分常与中药原有成分不同,而这些中药有效成分和P-gp相互作用的文献资料相对较少。此外,不同实验室由于实验条件(环境、细胞类型和状态及给药剂量和给药时间)不同,同类研究可得出不同甚至是相反的实验结果。
人源P-gp高表达MDR1-MDCKⅡ细胞模型由于培养周期短,代与代之间均一性良好,常作为体外筛选P-gp底物和抑制剂的细胞模型,并可用于研究P-gp介导DDI发生的可能性。
本研究应用逆转录病毒感染MDCKⅡ细胞的方法建立人源P-gp高表达MDR1-MDCKⅡ细胞模型。应用此细胞模型研究了来源于25种临床常用中药的50种活性成分对P-gp活性的影响。此外,通过ATP酶活性测定初步探讨中药活性成分抑制P-gp的可能机制。应用分子对接技术分析中药活性成分和P-gp之间的构效关系。由于CYP3A4/5与P-gp有明显的底物和组织分布交叉性,因此进一步探讨具有P-gp抑制作用的中药活性成分对CYP3A4/5活性的影响。同时,应用大鼠体内模型评价活性成分与地高辛合用对药代动力学的影响,为临床DDI的发生提供有参考价值的实验依据。研究结果表明：
1.人源P-糖蛋白高表达MDR1-MDCKⅡ细胞模型的建立
1.1酶切实验和DNA测序结果表明,携带目的基因MDR1的P-gp表达质粒pHaMDRwt可以在大肠杆菌中正确扩增和富集。
1.2脂质体转染法介导pHaMDRwt质粒进入病毒包装细胞PA317,经秋水仙碱筛选后,可得到含有目的基因MDR1的高滴度逆转录病毒。病毒滴度经测定可达到8.8×10-5pfu/ml。
1.3逆转录病毒感染MDCKⅡ细胞,经秋水仙碱筛选后,可得到人源P-gp高表达MDR1-MDCKⅡ细胞模型。RT-PCR和western blot结果表明,MDR1-MDCKⅡ细胞株可稳定高表达外源基因MDR1。Rho123外排实验和地高辛双向转运实验测定结果表明转染细胞中外源基因MDR1功能表达良好,可作为体外筛选P-gp底物和抑制剂的细胞模型。
2.50种中药活性成分对P-糖蛋白和CYP3A4/5活性的影响及药物相互作用研究
2.1MDR1-MDCKⅡ和Caco-2田胞双向转运实验结果表明,大黄素、18β-甘草次酸、脱水穿心莲内酯和20(S)-人参皂苷F1(≤100μM)对P-gp有显著的抑制作用(>50%),而上述化合物的同分异构体／结构类似物大黄酚、18a-甘草次酸、穿心莲内酯和人参皂苷Rh1对P-gp的活性影响相对较弱。
2.2在MDR1-MDCKⅡ细胞中,对P-gp抑制活性最强的是大黄素(IC50=9.42μM)其次是18β-甘草次酸(IC50=21.78μM)、20(S)-人参皂苷F1(IC50=76.08μM)和脱水穿心莲内酯(IC50=77.80μM)。
2.3大黄素和脱水穿心莲内酯剂量依赖性激活P-gp ATPase舌性,Km和Vmax分别是48.61、29.09μM和71.29、38.45nmol/min/mg protein。高浓度(100μM)18p-甘草次酸和20(S)-人参皂苷F1对空白和维拉帕米激活的P-gp ATPase活性均有抑制作用。
2.418β-甘草次酸、脱水穿心莲内酯、20(S)-人参皂苷F1和人参皂苷Rh1显著抑制CYP3A的代谢活性,抑制率分别是44、41、23和15%。大黄素可以显著激活CYP3A的代谢活性,与空白对照组相比,CYP3A舌性增加了35%。
2.5分子对接研究初步阐明了中药活性成分和P-gp及CYP3A4之间的构效关系。结果表明,18-氢原子的差向异构(18β-甘草次酸)、氢键(大黄素)、双键(脱水穿心莲内酯)和糖基的取代位置(20(S)-人参皂苷F1)可能是化合物体现不同P-gp抑制活性的重要化学因素。与大黄酚、18a-甘草次酸、穿心莲内酯和人参皂苷Rhl相比,大黄素、18p-甘草次酸、脱水穿心莲内酯和20(S)-人参皂苷F1可以和P-gp形成更多的氢键作用,这可能是后者发挥更强活性的原因之一。此外,18p-甘草次酸和20(S)-人参皂苷F1均可和Arg212(CYP3A4与底物相互作用的重要氨基酸残基)形成一个较强的氢键,因此可能通过和咪达唑仑竞争性结合Arg212发挥CYP3A4抑制活性。而大黄素虽然和Arg212形成一个Pi键,但和CYP3A4之间的主要作用力是和Thr310形成一个较强的氢键,因此可能减少和咪达唑仑的竞争力而变构激活CYP3A4的活性。
2.6SD大鼠提前口服大黄素或18p-甘草次酸后,地高辛的AUC0-t和Cmax与空白对照组相比,分别升高了51%和58%。综上所述,本研究应用逆转录病毒感染MDCKⅡ细胞的方法构建了人源P-gp高表达MDR1-MDCKⅡ细胞模型,此模型稳定高表达外源基因MDR1,可作为体外筛选P-gp底物和抑制剂及研究化合物和P-gp相互作用的细胞模型。在选择研究的50种中药活性成分中,4种中药成分对P-gp有显著的抑制作用,分别为大黄素、18p-甘草次酸、脱水穿心莲内酯和20(S)-人参皂苷Fl。此外,具有P-gp抑制作用的中药活性成分可能同时抑制或激活CYP3A4/5的活性,因此临床联合用药时需注意DDI发生的可能性。上述研究为预测临床上由中药和其他处方药物合用引起相互作用的可能性提供科学、简便可靠的评价体系,并为指导临床合理用药提供有参考价值的实验依据。
IMM-H007是一种新型调血脂化合物,化学结构为腺苷类似物。药理学研究表明,IMM-H007(10μM)可抑制十八烯酸诱导的脂肪性变HepG2细胞内脂质的堆积。体内药效学研究发现,IMM-H007(2mg/kg)可降低高脂血症金黄地鼠升高的血清甘油三酯、总胆固醇、低密度脂蛋白和肝脏甘油三酯、总胆固醇水平。机制研究提示,IMM-H007可以上调十八烯酸诱导的脂肪病变HepG2细胞内或高脂血症金黄地鼠肝细胞内AMPK的磷酸化水平,是一种新型AMP-激活蛋白激酶(AMP-activated protein kinase, AMPK)激活剂。由此可见,IMM-H007作为一种与他汀类药物化学结构、作用靶点和代谢途径不同的新型调血脂化合物,有望成为临床预防和治疗心血管疾病的新药。
前期药代研究表明,IMM-H007通过酯酶而非CYP450代谢。IMM-H007在体内首先发生水解反应,终末水解产物M1可进入细胞,经磷酸化反应生成MP(M1的5’-磷酸化产物),或发生脱氧、脱核糖环反应或Ⅱ相加合反应。MP的结构与5’-磷酸腺苷(AMP)极其相似,而AMP是AMPK的有效激活剂,因此推测MP可能为IMM-H007激活靶酶AMPK的活性代谢物。
前期实验结果表明IMM-H007及代谢产物M1和MP在大鼠血浆和全血中的药代动力学特征明显不同,如MP在大鼠血浆内含量极低,主要存在于全血中。本论文初步探讨大鼠口服和静脉注射IMM-H007后,原型药及代谢产物M1和MP在大鼠全血内的药代动力学特点,并研究IMM-H007及M1在体外缓冲液和不同种属动物生物基质(血浆、全血、肝微粒体、肠道菌群)内的代谢稳定性,推测IMM-H007可能的代谢转化途径,为后期药代动力学研究提供有参考价值的依据。研究结果表明：
1.生物样品中IMM-H007及代谢产物M1和MP的HPLC-MS/MS分析方法的建立根据临床前药代动力学指导原则,建立了生物样品中IMM-H007及代谢产物M1和MP的HPLC-MS/MS分析方法。结果表明,各检测物质在大鼠全血中均未见明显基质和杂质干扰,IMM-H007、M1和MP分别在1～500、2～1000和10～5000ng/ml浓度范围内线性关系良好,最低定量限分别为1、2和10ng/ml。各待测物质日内和日间精密度相对标准差均小于15%,准确度为±5.7%。IMM-H007、M1和MP的低、中、高3个QC浓度的全血样品回收率分别为80～91.2%、107.4～108.7%和76.4～80.3%。该方法简便、可靠、灵敏度高、特异性强,可满足IMM-H007的临床前药代动力学研究。
2.大鼠口服和静脉注射IMM-H007的体内药代动力学研究
2.1雄雌大鼠单次口服IMM-H007后吸收较快,各剂量组给药后5min血中即可检测到原型药和代谢产物,给药后12～36h血药浓度接近检测限。各时间点原型药浓度均较低(多个时间点浓度接近检测限),达峰浓度与剂量不成比例,药时曲线呈现多峰现象。
2.2雄性大鼠口服不同剂量IMM-H007(50、50、450和900mg/kg)后,代谢产物M1的Cmax分别为37.52、61.13、82.69、141.18ng/ml, AUC0-t分别为160.47.328.71、799.57、1779.31h*μg/L, MRT0-t分别为3.18、3.93、7.5、11.3h；代谢产物MP的Cmax分别为88.2、153.46、204.95、309.42ng/ml, AUC0-t分别为362.71、676.26、2230.14、4536.37h*μg/L, MRT0-t分别为2.87、3.65、8.15、12.07h。代谢产物M1和MP的Cmax与AUC0-t均随给药剂量增加而递增,并与给药剂量呈正比,但两者的MRT0-t均随剂量增加而延长,提示代谢产物M1和MP在大鼠体内可能存在消除饱和现象。
2.3雌性大鼠口服不同剂量IMM-H007(50、150、450和900mg/kg)后,代谢产物M1的Cmax分别为37.78、210.72、256.89、325.77ng/ml,AUC0-t分别为151.87、854.34、1561.42、3334.16h*μg/L、MRT0-t分别为3.85、4.25、7.62、10.51h；代谢产物MP的Cmax分别为132.55.920.83、1214.01、1122.64ng/ml, AUC0-t分别为698.63、4000.82、8376.97、13900.82h*μg/L, MRT0-t分别为4.08、4.86、8.45、10.3h。代谢产物M1和MP的AUC0-t均随剂量增加而递增,呈现一定比例关系,但中、高剂量组M1和MP的Cmax与给药剂量不成比例,且两者的MRTo_t均随剂量增加而延长。上述结果提示代谢产物M1和MP在雌性大鼠体内可能存在吸收和消除饱和现象。
2.4雌性大鼠中、高剂量组代谢产物M1和MP的Cmax和AUCo-t均高于雄性大鼠,提示代谢产物M1和MP在大鼠体内的药代动力学存在一定的性别差异。
2.5IMM-H007的生物利用度以M1计算,雄鼠为3.04%,雌鼠为2.12%；以MP计算,雄鼠为5.61%,雌鼠为6.55%,无明显性别差异。
3. IMM-H007的体外代谢稳定性研究
3.1IMM-H007和代谢产物M1在人工胃液、人工肠液和Tris-HCl缓冲液中稳定性良好,4h后剩余量均高于85%。
3.2IMM-H007在不同种属动物(SD大鼠、C57小鼠、金黄地鼠、比格犬、食蟹猴、人)血浆中(温孵时间为2h)均不稳定,且呈现种属差异性,代谢稳定性结果为比格犬>人>食蟹猴>SD大鼠、C57小鼠、金黄地鼠。M1在体外不同种属动物血浆中均较稳定。体外温孵体系中无MP的生成。
3.3IMM-H007在不同种属动物(SD大鼠、C57小鼠、金黄地鼠、比格犬、食蟹猴、人)肝微粒中(温孵时间为2h)均不稳定,5min即减少90%以上,且IMM-H007减少程度不依赖NADPH。M1在体外不同种属动物肝微粒中均较稳定。体外温孵体系中无MP的生成。
3.4IMM-H007和M1在不同种属动物(SD大鼠、C57小鼠、金黄地鼠、比格犬、食蟹猴、人)全血中(温孵时间为2h)均不稳定,温孵体系中可检测到MP的生成。除比格犬外,其他动物全血中水解产物M1的生成随着原型药的减少均呈现先上升后下降趋势,而MP则一直呈现递增趋势。
3.5IMM-H007在体外大鼠肠道菌群中(温孵时间为8h)不稳定,可生成代谢产物M444、M402、M1、M344和M228。其中M444、M402和M1在体系中含量呈先上升后下降趋势；代谢产物M344和M228呈递增趋势。M1在体外大鼠肠道菌群中不稳定,可生成代谢产物M344和M228,其中M344含量呈先上升后下降趋势；M228呈递增趋势。
综上所述,本研究建立了生物样品中IMM-H007及代谢产物M1和MP的HPLC-MS/MS分析方法,该方法简便、可靠、灵敏度高、特异性强,可满足IMM-H007的临床前药代动力学研究。应用此分析方法初步探讨了大鼠口服和静脉注射IMM-H007后,原型药及代谢产物M1和MP在雌雄大鼠全血内的药代动力学特点。体外代谢稳定性结果表明IMM-H007和M1在体外不同种属动物全血和大鼠肠道菌群中均不稳定,可生成水解、脱氧、脱核糖环或磷酸加合产物。此研究为了解IMM-H007可能的代谢转化途径及后期药代动力学研究提供有参考价值的实验数据。
Herbal medicines have been traditionally used for the treatment of various diseases, including cold symptoms, pain, and inflammation for thousands of years. Currently, these medicines are still utilized worldwide as complementary or alternative medical ingredients with the expectation of promoting health and managing chronic or severe diseases such as cancer and HIV infection. Due to the common perception that herbal supplements are safe, they are often co-administered with conventional prescription drugs in clinical trial, raising the risk of herbal-drug interactions. Metabolic enzyme-mediated drug-drug interactions (DDI) have been extensively studied while studies on DDI related to transporters have been relatively limited, despite their importance in drug disposition.
P-glycoprotein (P-gp), a member of the ABC transporter superfamily, is encoded by the ABCB1/MDR1gene. It serves as a key factor in conferring the multi-drug resistance (MDR) phenotype to cancer cells in clinical trial. In addition, P-gp is highly expressed in the apical membrane of several pharmacologically important epithelial barriers such as the kidney, liver, intestine, and blood-brain barrier. P-gp could excrete xenobiotics such as cytotoxic compounds into the gastrointestinal tract, bile and urine. Thus the modulation of P-gp can affect the oral bioavailability, biliary or renal clearance, brain uptake of drugs and increase the risk of DDI. This is particularly concerning for drugs with narrow therapeutic indices such as warfarin and digoxin. Small changes in the systemic exposure of these drugs could result in substantial alterations in therapeutic effects and toxic outcomes.
Many commonly used traditional Chinese medicines (TCM) have been reported to interact with P-gp. However, the interaction profile is limited between P-gp and bioactive herbal constituents, which can effectively enter human systemic circulation after oral administration. In addition, controversial experimental results are also obtained in different laboratories due to the selection of cell strain, dosage and duration of drug exposure.
Due to the high expression of human P-gp and short culture time, MDR1-MDCKII cells transfected with the human MDR1gene have become an attractive model to study the DDI mediated via P-gp.
In the present study, MDR1-MDCKII cells were established by the transfection of a retrovirus carrying the human MDR1gene into parent MDCKII cells. The inhibitory effects of50major herbal constituents present in25commonly used TCM on P-gp were investigated in vitro and in vivo. The underlying inhibitory mechanism of herbal constituents was partially assessed using the ATPase activity assay. In addition, molecular docking analysis was performed to elucidate the structure-activity relationships of herbal compounds with P-gp. Due to the overlapping substrate specificities and tissue distribution of cytochrome P4503A4/5(CYP3A4/5) and P-gp, the herbal constituents that exhibited potential inhibition of P-gp were further examined for their effects on CYP3A4/5activity in human liver microsomes. The effects of a single dose of herbal inhibitors on the pharmacokinetics of digoxin were also evaluated in male SD rats. The results were summarized as follows:
1. Establishment of MDR1-MDCKII cells with high expression of human P-gp
1.1The results of restriction analysis and DNA sequencing showed the P-gp expressive vector pHaMDRwt carrying a human MDR1gene was accurately amplified and enriched in E.coli.
1.2Amphotropic virus was produced after the transfection of plasmid pHaMDRwt into PA317cells. Viral titer of up to8.8×10-5pfu/ml was observed in the culture of MDR1-PA317packaging cells.
1.3MDR1-MDCKII cells with high expression of human P-gp were established after MDCKII cells were infected with the retrovirus carrying a human MDR1gene and incubated with colchicine (60ng/ml) for two weeks. RT-PCR and western blotting analyses showed the expression of human P-gp was stable and high in MDR1-MDCKII cells. Moreover, the decreased accumulation of Rhol23and enhanced efflux of digoxin were observed in MDR1-MDCKII cells, indicating the high P-gp activity of MDR1-MDCKII cells.
2. Effects of50herbal constituents on P-gp and CYP3A4/5in vitro and in vivo: herb-drug interactions mediated via P-gp and CYP3A4/5
2.1Among50herbal constituents, emodin,18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and20(.S)-ginsenoside F1[20(S)-GF1](100μM or less, depending on their solubility and cytotoxicity) exhibited significant inhibition (>50%) on P-gp in MDR1-MDCKII and Caco-2cells. However, despite the structural similarity and/or identical molecular weight, the isomers or analogues of the4herbal constituents (chrysophanol,18a-GA, andrographolide, and ginsenoside Rh1) had no effect on P-gp function.
2.2Emodin was the strongest herbal inhibitor of P-gp (IC50=9.42μM) in MDR1-MDCKII cells, followed by180-GA (IC50=21.78μM),20(S)-GF1(IC50=76.08μM) and DAG (IC50=77.80μM).
2.3P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with Km and Vmax values of48.61,29.09μM and71.29,38.45nmol/min/mg protein, respectively. However,18β-GA and20(S)-GF1exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration (100μM).
2.418β-GA, DAG,20(S)-GF1and Rh1significantly inhibited CYP3A4/5activity (44,41,23and15%inhibition, respectively), while emodin increased the metabolic rate of midazolam by approximately35%compared to the control.
2.5Molecular docking analysis (CDOCKER) elucidated the mechanism for structure-activity relationships of herbal constituents with P-gp and CYP3A4. The results showed the isomerism of18-hydrogen atom (18β-GA), the existence of a hydroxyl (emodin) and double bond (DAG), and the position of glycosyl [20(S)-GF1] could be crucial chemical elements for the inhibition of P-gp. Due to these chemical elements, additional strong hydrogen bonds were formed between herbal inhibitors and P-gp, resulting in stronger inhibitory effects of herbal constituents for P-gp. In addition,18β-GA and20(S)-GF1may display their inhibition on CYP3A4by competitively binding with Arg212which was reported to be one of the key residues for the binding of compounds with CYP3A4, while emodin was able to bind more readily with another residue Thr310via a strong hydrogen bond, reducing the competition for this binding orientation/site and enabling activation to occur.
2.6Co-administration of digoxin with emodin increased the AUC of digoxin by55%while the Cmax of digoxin was increased by58%when18β-GA was pretreated in SD rats.
In conclusion, MDR1-MDCKII cells with high and stable expression of human P-gp were established in the present study, and it could be used as a valuable in vitro tool for the rapid screening of P-gp substrates/inhibitors and investigating the DDI mediated via P-gp. Among50herbal constituents, four constituents, including emodin,18β-GA, DAG and20(S)-GF1exhibited significant inhibition (>50%) on P-gp in vitro and/or in vivo. Moreover, dual inhibition of P-gp and CYP3A4/5activities could occur due to the overlapping substrate specificities between them. Our findings provided the basis for the reliable assessment of the potential risks of herb-drug interactions in humans and will provide useful information for the proper use of prescription drugs in combination with herbal medicines, which is particularly important for drugs with narrow therapeutic indices.
IMM-H007, an analog of adenosine, is a novel small molecule compound that could significantly improves lipid metabolism disorders in the hyperlipidemia animal models. The pharmacological studies showed that IMM-H007could inhibit cellular lipid accumulation in OLA-treated HepG2cells. Moreover, IMM-H007(2mg/kg per day and above) reduced elevated serum triglyceride, total cholesterol, low density lipoprotein cholesterol and hepatic cholesterol and triglyceride contents in HFD fed hamsters. The mechanism studies indicated that the activation of AMPK in OLA-induced steatosis of HepG2cells was up-regulated by the treatment with IMM-H007. The hepatic cellular AMPK phosphorylation was also up regulated by IMM-H007(6and18mg/kg) treatment in HFD fed hamsters. As a novel lipid metabolism regulator, IMM-H007has a differernt chemical structure, target and metabolic pathway from statins, and could be potentially used for patients who have adverse reactions to statins in clinical trial.
Previous studies showed that IMM-H007contained three acetyl groups, which were liable to be hydrolyzed by plasma/serum esterases rather than CYP450s in vitro and in vivo. IMM-H007could be rapidly hydrolyzed to M1, which was believed to be the active form of IMM-H007in vivo, after an oral administration of IMM-H007to rats. A further study indicated that M1was extensively metabolized in HepG2cells, and several metabolites were observed, including deoxidized product of Ml (M344), deribosyl derivative of M1(M228), glucuronide conjugate of M1, glucuronide conjugate of M344, glucuronide conjugate of M228, sulfate conjugate of Ml, phosphate conjugate of M1(MP) and et al. Because MP was an analog of AMP, which was proved to be an effective activator of AMPK, MP was likely the active form of IMM-H007to activate AMPK in HepG2cells.
Previous studies showed that the pharmacokinetic characteristics of IMM-H007and its major metabolites were varied between rat plasma and blood. For example, the concentration of MP was fairly low in rat plasma and far higher in rat blood. In the present study, the concentrations of IMM-H007, M1and MP in rat blood were detected after oral and intravenous dosing of IMM-H007to SD rats. Metabolic stabilities of IMM-H007and Ml were assessed in different biological systems such as simulated gastric/intestinal fluid, Tris-HCl buffer, and plasma, blood, liver microsomes of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans. The metabolic stability of IMM-H007in the gut microbiota of SD rats was also investigated in this study. The results were summarized as follows:
1. Simultaneous quantification of IMM-H007and its metabolites in rat blood using HPLC-MS/MS
Base on the guideline of preclinical pharmacokinetic study, the HPLC-MS/MS method for the quantitation of IMM-H007and its metabolites M1and MP in biological sample has been developed, which provided a simple, reliable, sensitive and specific assay for the preclinical pharmacokinetic study of IMM-H007in SD rats. Linear detection responses were obtained for IMM-H007, M1and MP ranging from1～500,2～1000and10～5000ng/ml, respectively. The intra-and inter-day precisions (R.S.D.%) were within15%for all analytes, while the deviation of assay accuracies was within±5.7%. The average recoveries of IMM-H007, M1and MP were80～91.2%,107.4～108.7%and76.4～80.3%, respectively.
2. Pharmacokinetic study of IMM-H007in SD rats
2.1IMM-H007and its metabolites M1and MP can be detected in rat blood5min after oral administration of IMM-H007(50,150,450and900mg/kg) to SD rats and the blood concentrations were close to the limit of quantitation (LLOQ) at12-36h postdose. The multiple peak concentration-time curves of IMM-H007were observed, and the concentration of parent drug IMM-H007was much lower than its metabolites in both male and female rats.
2.2After oral administration of IMM-H007(50,150,450and900mg/kg) to male rats, the average maximum concentration (Cmax) of M1were37.52,61.13,82.69and141.18ng/ml, AUC0-t were160.47,328.71,799.57and1779.31h*μg/L, MRT0-t were3.18,3.93,7.5and11.3h. The Cmax of MP in male rats were88.2,153.46,204.95and309.42ng/ml, AUC0-t were362.71,676.26,2230.14and4536.37h*μg/L, MRT0-t were 2.87,3.65,8.15and12.07h. Both Cmax and AUC0-t of Ml and MP were increased in a dose-dependent manner, but the MRT0-t was significantly delayed after oral administration of IMM-H007(450and900mg/kg) to male rats. The results indicated the saturation of elimination of M1and MP in male rats at high doses of IMM-H007(450and900mg/kg).
2.3After oral administration of IMM-H007(50,150,450and900mg/kg) to female rats, the Cmax of M1were37.78,210.72,256.89and325.77ng/ml, AUC0-t were151.87,854.34,1561.42and3334.16h*μg/L, MRT0-t were3.85,4.25,7.62and10.51h. The Cmax of MP in male rats were132.55,920.83,1214.01and1122.64ng/ml, AUC0-t were698.63,4000.82,8376.97and13900.82h*μg/L, MRT0-t were4.08,4.86,8.45and10.3h. The AUC0-t of M1and MP were increased in a dose-dependent manner, but the Cmax of M1and MP were not increased dose-dependently. In addition, the MRTo0-t was significantly delayed after oral administration of IMM-H007(450and900mg/kg) to female rats. The results indicated the saturation of absorption and elimination of M1and MP in female rats at high doses of IMM-H007(450and900mg/kg).
2.4Both Cmax and AUCo-t of Ml and MP were much higher in female rats than those in male rats after oral administration of IMM-H007(150,450and900mg/kg), indicating the gender difference between male and female rats.
2.5The oral bioavailability of M1was3.04%in male rats and2.12%in female rats after an oral administration of IMM-H007at50mg/kg; the oral bioavailability of MP was5.61%in male rats and6.55%in female rats after an oral administration of IMM-H007at50mg/kg.
3. In vitro metabolic stability of IMM-H007
3.1The concentrations of IMM-H007and M1did not change significantly after4h incubation with simulated gastric/intestinal fluid and Tris-HCl buffer. The remaing IMM-H007and M1were more than85%when the reaction was terminated with the addition of ice-cold acetonitrile.
3.2IMM-H007was unstable when incubated with animal plasma in vitro for2h. IMM-H007could be hydrolyzed to M1at different rate in the plasma of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans. M1was stable after2h incubation with animal plasma. Moreover, no MP was detected in the reaction mixture.
3.3IMM-H007was unstable when incubated with the liver microsomes of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans in vitro for2h. More than90%IMM-H007was metabolized in the first5min incubation and the reaction was not NADPH-dependent. M1was stable after2h incubation with liver microsomes. Moreover, no MP was detected in the reaction mixture.
3.4Both IMM-H007and M1were unstable when incubated with animal blood in vitro for2h. IMM-H007and M1showed different metabolic rates in the blood of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys and humans. In addtion, high concentratin of MP was detected in the reaction mixture. The concentration profile of M1demonstrated an upward phase and a downward phase, suggesting onward metabolism to other products, while MP increased constantly until it reached a maximum level. Rates of increase and decrease of products differed by species.
3.5IMM-H007could be extensively metabolized into certain metabolites including M444, M402, M1,M344and M228by the gut microbiota of SD rats. Products M444, M402and M1appeared first, followed by M344and M228. Concentration profiles of M444, M402and M1demonstrated an upward phase and a downward phase, while M344and M228increased constantly until they reached the maximum levels. Products M344and M228also appeared when M1was incubated with the gut microbiota of SD rats for8h.
In conclusion, the HPLC-MS/MS method for the simultaneous quantitation of IMM-H007and its metabolites M1and MP in rat blood was developed in the present study. In addition, the pharmacokinetics of IMM-H007, M1and MP in rat blood were investigated after oral and intravenous dosing of IMM-H007to SD rats. The results of metabolic stabilities of IMM-H007and M1showed that IMM-H007and M1could be metabolized into several metabolites such as M444, M402, M1, M344, M228and MP by the blood of SD rats, C57mice, golden hamsters, beagle dogs, cyno monkeys, humans and the gut microbiota of SD rats. Our findings will provide useful information for the further pharmacokinetic study of IMM-H007in other species and give insights to the possible metabolic pathway of IMM-H007in vivo.
引文
[1]Zhang QH, L. Y. Quantitative prediction of in vivo drug-drug interactions based on in vitro inhibition or/and induction data for CYP3A4 [J]. Yao Xue Xue Bao 2010 45 (8):952-959.
[2]Zhou, S.; Gao, Y.; Jiang, W.; Huang, M.; Xu, A.; Paxton, J. W. Interactions of herbs with cytochrome P450 [J]. Drug Metab Rev 2003,35 (1):35-98.
[3]Thiebaut, F.; Tsuruo, T.; Hamada, H.; Gottesman, M. M; Pastan, I.; Willingham, M. C. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues [J]. Proc Natl Acad Sci U S A 1987,84 (21):7735-8.
[4]Borst, P.; Elferink, R. O. Mammalian ABC transporters in health and disease [J]. Annu Rev Biochem 2002,71:537-92.
[5]Ho, R. H.; Kim, R. B. Transporters and drug therapy:implications for drug disposition and disease [J]. Clin Pharmacol Ther 2005,78 (3):260-77.
[6]van den Bout-van den Beukel, C. J.; Koopmans, P. P.; van der Ven, A. J.; De Smet, P. A.; Burger, D. M. Possible drug-metabolism interactions of medicinal herbs with antiretroviral agents [J]. Drug Metab Rev 2006,38 (3):477-514.
[7]Hartel U, V. E. Use and acceptance of classical natural and alternative medicine in Germany--findings of a representative population-based survey [J]. Forsch Komplementarmed Klass Naturheilkd 2004,11 (6):327-334.
[8]Kaufman, D. W.; Kelly, J. P.; Rosenberg, L.; Anderson, T. E.; Mitchell, A. A. Recent patterns of medication use in the ambulatory adult population of the United States:the Slone survey [J]. JAMA 2002,287 (3):337-44.
[9]Deferme, S.; Kamuhabwa, A.; Nshimo, C.; de Witte, P.; Augustijns, P. Screening of Tanzanian plant extracts for their potential inhibitory effect on P-glycoprotein mediated efflux [J]. Phytotherapy Research 2003,17(5):459-464.
[10]van den Bout-van den Beukel, C. J. P.; Koopmans, P. P.; van der Ven, A. J. A. M.; De Smet, P. A. G. M.; Burger, D. M. Possible Drug-Metabolism Interactions of Medicinal Herbs with Antiretroviral Agents [J]. Drug Metabolism Reviews 2006,38 (3):477-514.
[11]Qi FH, W. Z., Cai PP, Zhao L, Gao JJ, Kokudo N, Li AY, Han JQ, Tang W. Traditional Chinese medicine and related active compounds:A review of their role on hepatitis B virus infection [J]. Drug Discov Ther.2013,7(6):212-224.
[12]Durr, D.; Stieger, B.; Kullak-Ublick, G. A.; Rentsch, K. M.; Steinert, H. C; Meier, P. J.; Fattinger, K. St John's Wort induces intestinal P-glycoprotein/MDR1 and intestinal and hepatic CYP3A4 [J]. Clin Pharmacol Ther 2000,68 (6):598-604.
[13]Molnar, J.; Szabo, D.; Pusztai, R.; Mucsi, I.; Berek, L.; Ocsovszki, L; Kawata, E.; Shoyama, Y. Membrane associated antitumor effects of crocine-, ginsenoside-and cannabinoid derivates [J]. Anticancer Res 2000,20 (2A):861-7.
[14]Tawab, M. A.; Bahr, U.; Karas, M.; Wurglics, M.; Schubert-Zsilavecz, M. Degradation of ginsenosides in humans after oral administration [J]. Drug Me tab Dispos 2003,31 (8):1065-71.
[15]Yamamura, Y.; Kawakami, J.; Santa, T.; Kotaki, H.; Uchino, K.; Sawada, Y.; Tanaka, N.; Iga, T. Pharmacokinetic profile of glycyrrhizin in healthy volunteers by a new high-performance liquid chromatographic method [J]. J Pharm Sci 1992,81 (10):1042-6.
[16]Ploeger, B.; Mensinga, T.; Sips, A.; Seinen, W.; Meulenbelt, J.; DeJongh, J. The pharmacokinetics of glycyrrhizic acid evaluated by physiologically based pharmacokinetic modeling [J]. Drug Metab Rev 2001,33 (2):125-47.
[17]Zou, Q.; Wei, P.; Li, J.; Ge, Z. X.; Ouyang, P. Simultaneous determination of 18alpha-and 18beta-glycyrrhetic acid in human plasma by LC-ESI-MS and its application to pharmacokinetics [J]. Biomed Chromatogr 2009,23 (1):54-62.
[18]Yoshida, N.; Koizumi, M.; Adachi, I.; Kawakami, J. Inhibition of P-glycoprotein-mediated transport by terpenoids contained in herbal medicines and natural products [J]. Food Chem Toxicol 2006,44 (12):2033-9.
[19]Hou, Y. C.; Lin, S. P.; Chao, P. D. Liquorice reduced cyclosporine bioavailability by activating P-glycoprotein and CYP 3A [J]. Food Chem 2012,135 (4):2307-12.
[20]Li, J.; Wang, Y.; Hidalgo, I. J. Kinetic analysis of human and canine P-glycoprotein-mediated drug transport in MDR1-MDCK cell model:Approaches to reduce false-negative substrate classification [J]. J Pharm Sci 2013,102 (9): 3436-46.
[21]Maier, P.; von Kalle, C.; Laufs, S. Retroviral vectors for gene therapy [J]. Future Microbiology 2010,5 (10):1507-1523.
[22]Wandel, C.; Kim, R. B.; Kajiji, S.; Guengerich, P.; Wilkinson, G. R.; Wood, A. J. P-glycoprotein and cytochrome P-450 3A inhibition:dissociation of inhibitory potencies [J]. Cancer Res 1999,59 (16):3944-8.
[23]Kuteykin-Teplyakov, K.; Luna-Tortos, C.; Ambroziak, K.; Loscher, W. Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport [J]. Br J Pharmacol 2010,760(6):1453-63.
[24]Balimane, P. V.; Marino, A.; Chong, S. P-gp inhibition potential in cell-based models:which "calculation" method is the most accurate? [J]. A APS J 2008,10 (4): 577-86.
[25]Cao, J.; Chen, X.; Liang, J.; Yu, X. Q.; Xu, A. L.; Chan, E.; Wei, D.; Huang, M; Wen, J. Y.; Yu, X. Y.; Li, X. T.; Sheu, F. S.; Zhou, S. F. Role of P-glycoprotein in the intestinal absorption of glabridin, an active flavonoid from the root of Glycyrrhiza glabra [J]. Drug Me tab Dispos 2007,35 (4):539-53.
[26]Yamaoka, K.; Nakagawa, T.; Uno, T. Application of Akaike's information criterion (AIC) in the evaluation of linear pharmacokinetic equations [J]. J Pharmacokinet Biopharm 1978,6(2):165-75.
[27]Gibbs, M. A.; Thummel, K. E.; Shen, D. D.; Kunze, K. L. Inhibition of cytochrome P-450 3A (CYP3A) in human intestinal and liver microsomes:comparison of Ki values and impact of CYP3A5 expression [J]. Drug Metab Dispos 1999,27 (2): 180-7.
[28]Zha, W.; Wang, G.; Xu, W.; Liu, X.; Wang, Y.; Zha, B. S.; Shi, J.; Zhao, Q.; Gerk, P. M; Studer, E.; Hylemon, P. B.; Pandak, W. M., Jr.; Zhou, H. Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages [J]. PLoS ONE 2013,8 (1):e54349.
[29]Dolghih, E.; Bryant, C; Renslo, A. R.; Jacobson, M. P. Predicting binding to p-glycoprotein by flexible receptor docking [J]. PLoS Comput Biol 2011,7 (6): el 002083.
[30]Sugiyama, M.; Fujita, K.; Murayama, N.; Akiyama, Y.; Yamazaki, H.; Sasaki, Y. Sorafenib and sunitinib, two anticancer drugs, inhibit CYP3A4-mediated and activate CY3A5-mediated midazolam 1'-hydroxylation [J]. Drug Metab Dispos 2011, 39 (5):757-62.
[31]Varma, M. V.; Panchagnula, R. Enhanced oral paclitaxel absorption with vitamin E-TPGS:effect on solubility and permeability in vitro, in situ and in vivo [J]. Eur J Pharm Sci 2005,25 (4-5):445-53.
[32]Sugimoto, H.; Matsumoto, S.; Tachibana, M.; Niwa, S.; Hirabayashi, H.; Amano, N.; Moriwaki, T. Establishment of in vitro P-glycoprotein inhibition assay and its exclusion criteria to assess the risk of drug-drug interaction at the drug discovery stage [J]. J Pharm Sci 2011,100 (9):4013-23.
[33]Yamashita, S.; Tanaka, Y.; Endoh, Y.; Taki, Y.; Sakane, T.; Nadai, T.; Sezaki, H. Analysis of drug permeation across Caco-2 monolayer:implication for predicting in vivo drug absorption [J]. Pharm Res 1997,14 (4):486-91.
[34]Polli, J. W.; Wring, S. A.; Humphreys, J. E.; Huang, L.; Morgan, J. B.; Webster, L. O.; Serabjit-Singh, C. S. Rational use of in vitro P-glycoprotein assays in drug discovery [J]. J Pharmacol Exp Ther 2001,299 (2):620-8.
[35]Keubler, A.; Weiss, J.; Haefeli, W. E.; Mikus, G.; Burhenne, J. Drug interaction of efavirenz and midazolam:efavirenz activates the CYP3A-mediated midazolam l'-hydroxylation in vitro [J]. Drug Metab Dispos 2012,40 (6):1178-82.
[36]Olukoga, A.; Donaldson, D. Historical perspectives on health. The history of liquorice:the plant, its extract, cultivation, commercialisation and etymology [J]. JR Soc Promot Health 1998,118 (5):300-4.
[37]Zhang, J.; Zhou, F.; Wu, X.; Gu, Y.; Ai, H.; Zheng, Y.; Li, Y.; Zhang, X.; Hao, G.; Sun, J.; Peng, Y.; Wang, G.20(S)-ginsenoside Rh2 noncompetitively inhibits P-glycoprotein in vitro and in vivo:a case for herb-drug interactions [J]. Drug Metab Dispos 2010,38 (12):2179-87.
[38]Izzo, A. A.; Ernst, E. Interactions between herbal medicines and prescribed drugs:an updated systematic review [J]. Drugs 2009,69 (13):1777-98.
[39]Kock, K.; Xie, Y.; Hawke, R. L.; Oberlies, N. H.; Brouwer, K. L. Interaction of silymarin flavonolignans with organic anion-transporting polypeptides [J]. Drug Metab Dispos 2013,41 (5):958-65.
[40]Choi, R. J.; Ngoc, T. M.; Bae, K.; Cho, H. J.; Kim, D. D.; Chun, J.; Khan, S.; Kim, Y. S. Anti-inflammatory properties of anthraquinones and their relationship with the regulation of P-glycoprotein function and expression [J]. Eur J Pharm Sci 2013,48 (1-2):272-81.
[41]Martin, C.; Berridge, G.; Higgins, C. F.; Mistry, P.; Charlton, P.; Callaghan, R. Communication between multiple drug binding sites on P-glycoprotein [J]. Mol Pharmacol 2000,58 (3):624-32.
[42]Wang, R. B.; Kuo, C. L.; Lien, L. L.; Lien, E. J. Structure-activity relationship: analyses of p-glycoprotein substrates and inhibitors [J]. J Clin Pharm Ther 2003,28 (3):203-28.
[43]Teng, Z.; Yuan, C; Zhang, F.; Huan, M.; Cao, W.; Li, K.; Yang, J.; Cao, D.; Zhou, S.; Mei, Q. Intestinal absorption and first-pass metabolism of polyphenol compounds in rat and their transport dynamics in Caco-2 cells [J]. PLoS ONE 2012,7 (1): e29647.
[44]Dantzig, A. H.; Shepard, R. L.; Cao, J.; Law, K. L.; Ehlhardt, W. J.; Baughman, T. M.; Bumol, T. F.; Starling, J. J. Reversal of P-glycoprotein-mediated multidrug resistance by a potent cyclopropyldibenzosuberane modulator, LY335979 [J]. Cancer Res 1996,56 (18):4171-9.
[45]Thomas, H.; Coley, H. M. Overcoming multidrug resistance in cancer:an update on the clinical strategy of inhibiting p-glycoprotein [J]. Cancer Control 2003,10 (2): 159-65.
[46]Bohari, M. H.; Sastry, G. N. FDA approved drugs complexed to their targets: evaluating pose prediction accuracy of docking protocols [J]. Journal of Molecular Modeling 2012,18 (9):4263-74.
[47]Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.; Harrell, P. M.; Trinh, Y. T.; Zhang, Q.; Urbatsch, I. L.; Chang, G. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding [J]. Science 2009,323 (5922):1718-22.
[48]Jabeen, I.; Pleban, K.; Rinner, U.; Chiba, P.; Ecker, G. F. Structure-activity relationships, ligand efficiency, and lipophilic efficiency profiles of benzophenone-type inhibitors of the multidrug transporter P-glycoprotein [J]. J Med Chem 2012,55 (7):3261-73.
[49]Jiao, W.; Dong, W.; Li, Z.; Deng, M.; Lu, R. Lathyrane diterpenes from Euphorbia lathyris as modulators of multidrug resistance and their crystal structures [J]. Bioorg Med Chem 2009,17 (13):4786-92.
[50]Jin, M. S.; Oldham, M. L.; Zhang, Q.; Chen, J. Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans [J]. Nature 2012,490 (7421): 566-9.
[51]Zhou, S. F. Structure, function and regulation of P-glycoprotein and its clinical relevance in drug disposition [J]. Xenobiotica 2008,38 (7-8):802-32.
[52]Hummel, M. A.; Gannett, P. M.; Aguilar, J. S.; Tracy, T. S. Effector-mediated alteration of substrate orientation in cytochrome P450 2C9 [J]. Biochemistry 2004, 43 (22):7207-14.
[53]Atkins, W. M. Current views on the fundamental mechanisms of cytochrome P450 allosterism [J]. Expert Opin Drug Metab Toxicol 2006,2 (4):573-9.
[54]Mudra DR, D. K., Desai PV. In silico, in vitro and in situ models to assess interplay between CYP3A and P-gp [J]. Curr Drug Metab.2011 12 (8):750-773.
[55]Ekroos M, S. T. Structural basis for ligand promiscuity in cytochrome P4503A4. [J]. Proc Natl Acad Sci U S A.200612 (103):13682-13687.
[56]Kenworthy, K. E.; Clarke, S. E.; Andrews, J.; Houston, J. B. Multisite kinetic models for CYP3A4:simultaneous activation and inhibition of diazepam and testosterone metabolism [J]. Drug Metab Dispos 2001,29 (12):1644-51.
[57]Okada, Y.; Murayama, N.; Yanagida, C.; Shimizu, M.; Guengerich, F. P.; Yamazaki, H. Drug interactions of thalidomide with midazolam and cyclosporine A: heterotropic cooperativity of human cytochrome P450 3A5 [J]. Drug Metab Dispos 2009,37(1):18-23.
[58]Hummel, M. A.; Locuson, C. W.; Gannett, P. M.; Rock, D. A.; Mosher, C. M.; Rettie, A. E.; Tracy, T. S. CYP2C9 genotype-dependent effects on in vitro drug-drug interactions:switching of benzbromarone effect from inhibition to activation in the CYP2C9.3 variant [J]. Mol Pharmacol 2005,68 (3):644-51.
[59]Grandvuinet, A. S.; Vestergaard, H. T.; Rapin, N.; Steffansen, B. Intestinal transporters for endogenic and pharmaceutical organic anions:the challenges of deriving in-vitro kinetic parameters for the prediction of clinically relevant drug-drug interactions [J]. JPharm Pharmacol 2012,64 (11):1523-48.
[60]Boocock, D. J.; Faust, G. E. S.; Patel, K. R.; Schinas, A. M.; Brown, V. A.; Ducharme, M. P.; Booth, T. D.; Crowell, J. A.; Perloff, M.; Gescher, A. J.; Steward, W. P.; Brenner, D. E. Phase I Dose Escalation Pharmacokinetic Study in Healthy Volunteers of Resveratrol, a Potential Cancer Chemopreventive Agent [J]. Cancer Epidemiology Biomarkers & Prevention 2007,16(6):1246-1252.
[61]Hong X, H. T., Mi SQ, Ye SM, Ou WP, Wang NS. Pharmacokinetics of Chuanxinlian tablet in healthy Chinese volunteers [J].2007.
[62]Howes J, W. M., Huang.L, Howes LG Long-term pharmacokinetics of an extract of isoflavones from red clover (Trifolium pratense) [J]. J Altern Complement Med. 20025(2):135-142.
[63]Hu, Z.; Yang, J.; Cheng, C,; Huang, Y.; Du, F.; Wang, F.; Niu, W.; Xu, F.; Jiang, R.; Gao, X.; Li, C. Combinatorial Metabolism Notably Affects Human Systemic Exposure to Ginsenosides from Orally Administered Extract of Panax notoginseng Roots (Sanqi) [J]. Drug Metabolism and Disposition 2013,41 (7):1457-1469.
[64]Li, H.; Ren, P.; Huang, X.; Tang, W. F.; Wei, H. Q. [Simultaneous determination and pharmacokinetic studies of ferulic acid and paeoniflorin in human serum by high performance liquid chromatography after oral administration of Modified Xiao-yao Decoction] [J].Zhong Xi Yi Jie He Xue Bao 2008,6 (11):1178-83.
[65]Panossian, A.; Hovhannisyan, A.; Mamikonyan, G.; Abrahamian, H.; Hambardzumyan, E.; Gabrielian, E.; Goukasova, G.; Wikman, G.; Wagner, H. Pharmacokinetic and oral bioavailability of andrographolide from Andrographis paniculata fixed combination Kan Jang in rats and human [J]. Phytomedicine 2000,7 (5):351-364.
[66]Qin, F.; Huang, X.; Zhang, H.-M.; Ren, P. Pharmacokinetic comparison of puerarin after oral administration of Jiawei-Xiaoyao-San to healthy volunteers and patients with functional dyspepsia:influence of disease state [J]. Journal of Pharmacy and Pharmacology 2009,61 (1):125-129.
[67]Shen, T.; Xu, H.; Weng, W.; Zhang, J. Single-and Multiple-Dose Pharmacokinetics of a Novel Tetramethylpyrazine Reservoir-Type Transdermal Patch versus Tetramethylpyrazine Phosphate Oral Tablets in Healthy Normal Volunteers, and in Vitro-in Vivo Correlation [J]. Biological and Pharmaceutical Bulletin 2013,36 (6): 931-937.
[68]Xu, M; Yin, J.; Xie, L.; Zhang, J.; Zou, C; Zou, J.; Liu, F.; Ju, W.; Li, P. Pharmacokinetics and tolerance of toal astragalosides after intravenous infusion of astragalosides injection in healthy Chinese volunteers [J]. Phytomedicine 2013,20 (12):1105-1111.
[69]Zhang, X.-L.; Xu, H.-R.; Chen, W.-L.; Chu, N.-N.; Li, X.-N.; Liu, G.-Y.; Yu, C. Matrine determination and pharmacokinetics in human plasma using LC/MS/MS [J]. Journal of Chromatography B 2009,877 (27):3253-3256.
[70]Wu, X.-I.; Hang, T.-j.; Shen, J.-p.; Zhang, Y.-d. Determination and pharmacokinetic study of oxymatrine and its metabolite matrine in human plasma by liquid chromatography tandem mass spectrometry [J]. Journal of Pharmaceutical and Biomedical Analysis 2006,41 (3):918-924.
[71]Jukema, J. W.; Cannon, C. P.; de Craen, A. J.; Westendorp, R. G.; Trompet, S. The controversies of statin therapy:weighing the evidence [J]. J Am Coll Cardiol 2012, 60(10):875-81.
[72]McGovern, P. G.; Pankow, J. S.; Shahar, E.; Doliszny, K. M.; Folsom, A. R.; Blackburn, H.; Luepker, R. V. Recent trends in acute coronary heart disease--mortality, morbidity, medical care, and risk factors. The Minnesota Heart Survey Investigators [J]. N Engl J Med 1996,334 (14):884-90.
[73]Alberton, M.; Wu, P.; Druyts, E.; Briel, M.; Mills, E. J. Adverse events associated with individual statin treatments for cardiovascular disease:an indirect comparison meta-analysis [J]. QJM2012,105 (2):145-57.
[74]Rendic S, D. C. F. Human cytochrome P450 enzymes:A status report summarizing their reactions, substrates, inducers and inhibitors [J]. Drug Me tab Rev 1997,29 (1-2):413-580.
[75]Chatzizisis, Y. S.; Koskinas, K. C; Misirli, G.; Vaklavas, C; Hatzitolios, A.; Giannoglou, G. D. Risk factors and drug interactions predisposing to statin-induced myopathy:implications for risk assessment, prevention and treatment [J]. Drug Saf 2010,33 (3):171-87.
[76]Wang, Y.; Wang, R.; Gao, X.; Hong, B.; Gao, S. A practical VEP-based brain-computer interface [J]. IEEE Trans Neural Syst Rehabil Eng 2006,14 (2): 234-9.
[77]Ding, J.; Sperling, G.; Srinivasan, R. Attentional modulation of SSVEP power depends on the network tagged by the flicker frequency [J]. Cereb Cortex 2006,16 (7):1016-29.
[78]Hardie, D. G. Role of AMP-activated protein kinase in the metabolic syndrome and in heart disease [J]. FEBS Lett 2008,582 (1):81-9.
[79]Viollet, B.; Guigas, B.; Leclerc, J.; Hebrard, S.; Lantier, L.; Mounier, R.; Andreelli, F.; Foretz, M. AMP-activated protein kinase in the regulation of hepatic energy metabolism:from physiology to therapeutic perspectives [J]. Acta Physiol (Oxf) 2009,196(1):81-98.
[80]Lin, H. Z.; Yang, S. Q.; Chuckaree, C; Kuhajda, F.; Ronnet, G.; Diehl, A. M. Metformin reverses fatty liver disease in obese, leptin-deficient mice [J]. Nat Med 2000,6(9):998-1003.
[81]Bugianesi, E.; Gentilcore, E.; Manini, R.; Natale, S.; Vanni, E.; Villanova, N.; David, E.; Rizzetto, M.; Marchesini, G. A randomized controlled trial of metformin versus vitamin E or prescriptive diet in nonalcoholic fatty liver disease [J]. Am J Gastroenterol 2005,100 (5):1082-90.
[82]Lian, Z.; Li, Y.; Gao, J.; Qu, K.; Li, J.; Hao, L.; Wu, S.; Zhu, H. A novel AMPK activator, WS070117, improves lip id metabolism discords in hamsters and HepG2 cells [J]. Lipids Health Dis 2011,10:67.
[83]刘茵.WS070117药代动力学和作用机制研究[D].北京：中国医学科学院北京协和医学院博士论文,2012.
[84]Wang, Z.; Wang, X.; Qu, K.; Zhu, P.; Guo, N.; Zhang, R.; Abliz, Z.; Yu, H.; Zhu, H. Binding of cordycepin monophosphate to AMP-activated protein kinase and its effect on AMP-activated protein kinase activation [J]. Chem Biol Drug Des 2010,76 (4):340-4.
[85]Guo, P.; Kai, Q.; Gao, J.; Lian, Z. Q.; Wu, C. M.; Wu, C. A.; Zhu, H. B. Cordycepin prevents hyperlipidemia in hamsters fed a high-fat diet via activation of AMP-activated protein kinase [J]. J Pharmacol Sci 2010,113 (4):395-403.
[86]Liu, Y.; He, J.; Abliz, Z.; Zhu, H. In vitro stability and metabolism of O2', O3', 05'-tri-acetyl-N6-(3-hydroxylaniline) adenosine in rat, dog and human plasma: Chemical hydrolysis and role of plasma esterases [J]. Xenobiotica 2011,41 (7): 549-560.
[87]Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones, B. C; MacIntyre, F.; Rance, D. J.; Wastall, P. The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data [J]. J Pharmacol Exp Ther 1997,283 (1): 46-58.
[88]Lu, A. Y.; Wang, R. W.; Lin, J. H. Cytochrome P450 in vitro reaction phenotyping: a re-evaluation of approaches used for P450 isoform identification [J]. Drug Metab Dispos2003,31 (4):345-50.
[89]Tucker, G. T.; Houston, J. B.; Huang, S. M. Optimizing drug development:strategies to assess drug metabolism/transporter interaction potential--toward a consensus [J]. Pharm Res 2001,18(8):1071-80.
[90]Di, L. The role of drug metabolizing enzymes in clearance [J]. Expert Opin Drug Metab Toxicol 2014,10 (3):379-93.
[1]Ho RH, Kim RB. Transporters and drug therapy:implications for drug disposition and disease [J]. Clin Pharmacol Ther,2005,78:260-277.
[2]Niemi M. Role of OATP transporters in the disposition of drugs [J]. Pharmacogenomics,2007,8:787-802.
[3]Jacquemin E, Hagenbuch B, Stieger B, et al. Expression cloning of a rat liver Na(+)-independent organic anion transporter [J]. Proc. Natl. Acad. Sci.,1994,91:133-137.
[4]Smith NF, Figg WD, Sparreboom A. Role of the liver-specific transporters OATP1B1 and OATP1B3 in governing drug elimination [J]. Expert Opin. Drug Metab. Toxicol.,2005,1:429-445
[5]Deng JW, Song IS, Shin HJ, et al. The effect of SLCO1B1*15 on the disposition of pravastatin and pitavastatin is substrate dependent:the contribution of transporting activity changes by SLCO1B1*15[J]. Pharmacogenet. Genomics,2008,18:424-433
[6]Sissung TM, Baum CE, Kirkland CT, et al. Pharmacogenetics of Membrane Transporters:An Update on Current Approaches [J]. Mol Biotechnol,2010,44: 152-167
[7]Abe T, Unno M, Onogawa T, et al. LST-2, a human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers [J]. Gastroenterology,2001,120:1689-1699.
[8]Meier-Abt F, Mokrab Y, Mizuguchi K. Organic anion transporting polypeptides of the OATP/SLCO superfamily:identification of new members in nonmammalian species, comparative modeling and a potential transport mode [J]. J Membr Biol, 2005,208:213-227.
[9]Mahagita C, Grassl SM, Piyachaturawat P, et al. Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport [J]. Am J Physiol Gastrointest Liver Physiol,2007,293:G271-G278
[10]Ismair MG, Stieger B, Cattori V, et al. Hepatic uptake of cholecystokinin octapeptide by organicanion-transporting polypeptides OATP4 and OATP8 of rat and human liver [J]. Gastroenterology,2001,121:1185-1190.
[11]Kalliokoski A, Niemi M. Impact of OATP transporters on pharmacokinetics [J]. Br. J. Pharmacol.,2009,158:693-705
[12]Vavricka SR, Van Montfoort J, Ha HR, et al. Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver [J]. Hepatology,2002, 36:164-172
[13]Meier-Abt F, Faulstich H, Hagenbuch B. Identification of phalloidin uptake systems of rat and human liver [J]. Biochim Biophys Acta.,2004,1664:64-69
[14]Fischer WJ, Altheimer S, Cattori V, et al. Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin [J]. Toxicol Appl Pharmacol.,2005,203:257-263
[15]Smith NF, Acharya MR, Desai N, et al. Identification of OATP1B3 as a high-affinity hepatocellular transporter of paclitaxel [J]. Cancer Biol Ther.,2005b,4:815-818
[16]Hu S, Franke RM, Filipski KK, et al. Interaction of imatinib with human organic ion carriers [J]. Clin Cancer Res.,2008,14:3141-3148
[17]Abe T, Unno M, Onogawa T, et al. LST-2, a human liver-specific organic anion transporter determines methotrexate sensitivity in gastrointestinal cancers [J]. Gastroenterology,2001,120:1689-1699
[18]Smith NF, Marsh S, Scott-Horton TJ, et al. Variants in the SLCO1B3 gene: interethnic distribution and association with paclitaxel pharmacokinetics [J]. Clin Pharmacol.,2007,81:76-82
[19]amaguchi H, Kobayashi M, Okada M, et al. Rapid screening of antineoplastic candidates for the human organic anion transporter OATP1B3 substrates using fluorescent probes [J]. Cancer Lett.,2008,260:163-169
[20]Shimizu M, Fuse K, Okudaira K, et al. Contribution of OATP (organic anion-transporting polypeptide) family transporters to the hepatic uptake of fexofenadine [J]. Drug Metab Dispos.2005,33:1477-1481
[21]Treiber A, Schneiter R, Hausler S, et al. Bosentan is a substrate of human OATP1B1 and OATP1B3:inhibition of hepatic uptake as the common mechanism of its interactions with cyclosporin A, rifampicin, and sildenafil [J]. Drug Metab Dispos., 2007,35:1400-1407
[22]Liu L, Cui Y, Chung AY, et al. Vectorial transport of enalapril by Oatp1a1/Mrp2 and OATP1B1 and OATP1B3/MRP2 in rat and human livers [J]. J Pharmacol Exp Ther., 2006,318:395-402
[23]Nakagomi-Hagihara R, Nakai D, Kawai K, et al. OATP1B1, OATP1B3, and MRP2 are involved in hepatobiliary transport of olmesartan, a novel angiotensin Ⅱ blocker [J]. Drug Metab Dispos.,2006,34:862-869
[24]Ishiguro N, Maeda K, Kishimoto W, et al. Predominant contribution of OATP1B3 to the hepatic uptake of telmisartan, an angiotensin Ⅱ receptor antagonist, in humans [J]. Drug Metab Dispos.,2006,34:1109-1115
[25]Yamashiro W, Maeda K, Hirouchi M, et al. Involvement of transporters in the hepatic uptake and biliary excretion of valsartan, a selective antagonist of the angiotensin Ⅱ AT1-receptor, in humans [J]. Drug Metab Dispos.,2006,34: 1247-1254
[26]Kullak-Ublick GA, Ismair MG, Stieger B, et al. Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver [J]. Gastro-enterology,2001,120:525-533.
[27]Kopplow K, Letschert K, Konig J, et al. Human hepatobiliary transport of organic anions analyzed by quadrupletransfected cells [J]. Mol Pharmacol.,2005,68: 1031-1038
[28]Hirano M, Maeda K, Shitara Y, et al. Drug-drug interaction between pitavastatin and various drugs via OATP1B1 [J]. Drug Metab Dispos.,2006,34:1229-1236
[29]Seithel A, Eberl S, Singer K, et al. The influence of macrolide antibiotics on the uptake of organic nions and drugs mediated by OATP1B1 and OATP1B3 [J]. Drug Metab Dispos.,2007,35:779-786
[30]Ho RH, Tirona RG, Leake BF, et al. Drug and bile acid transporters in rosuvastatin hepatic uptake:function, expression, and pharmacogenetics [J]. Gastroenterology, 2006b,130:1793-1806
[31]Vavricka SR, Van Montfoort J, Ha HR, et al. Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver [J]. Hepatology,2002, 36:164-172
[32]Nozawa T, Sugiura S, Nakajima M, et al. Involvement of organic anion transporting poly-peptides in the transport of troglitazone sulfate:implications for understanding troglitazone hepatotoxicity [J]. Drug Metab Dispos.,2004,32:291-294
[33]Letschert K, Faulstich H, Keller D, et al. Molecular characterization and inhibition of amanitin uptake into human hepatocytes [J].Toxicol Sci.,2006,91:140-149
[34]Annaertl P, Yel ZW, Stieger B, et al. Interaction of HIV protease inhibitors with OATP1B1,1B3, and 2B1[J]. Xenobiotica,2010,40:163-176
[35]Kimoto E, Chupka J, Xiao Y, et al. Characterization of digoxin uptake in sandwich-cultured human hepatocytes [J]. Drug Metab. Dispos.,2011,39:47-53.
[36]Le Vee M, Jouan E, Moreau A, et al. Regulation of drug transporter mRNA expression by interferon-y in primary human hepatocytes [J]. Fundam Clin Pharmacol,2011,25:99-103.
[37]Herfindal L, Myhren L, Kleppe R., et al. Nostocyclopeptide-M1:A potent, nontoxic inhibitor of the hepatocyte drug transporters OATP1B3 and 1B1 [J]. Mol Pharm., 2011,8:360-367
[38]Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/ SLC21 family:phylogenetic classification as OATP/SLCO superfamily, new nomenclature and molecular/functional properties[J]. Pflugers Arch,2004,447: 653-665
[39]Konig J, Seithel A, Gradhand U, et al. Pharmacogenomics of human OATP transporters [J]. Naunyn Schmiedebergs Arch Pharmacol,2006,372:432-443
[40]Seithel A, Glaeser H, Fromm MF, et al. The functional consequences of genetic variations in transporter genes encoding human organic anion-transporting polypeptide family members [J]. Expert Opin. Drug Metab. Toxicol,2008,4:51-64.
[41]Smith NF, Marsh S, Scott-Horton TJ, et al. Variants in the SLCO1B3 gene: interethnic distribution and association with paclitaxel pharmacokinetics [J]. Clin Pharmacol Ther,2007,81:76-82.
[42]Tsujimoto M, Hirata S, Dan Y, et al. Polymorphisms and linkage disequilibrium of the OATP8 (OATP1B3) gene in Japanese participants [J]. Drug Metab Pharmacokinet,2006,21:165-169.
[43]Laitinen A, Niemi M. Frequencies of Single-Nucleotide Polymorphisms of SLCO1A2, SLCO1B3 and SLCO2B1 Genes in a Finnish Population [J]. Basic Clin Pharmacol Toxicol,2011,108:9-13
[44]Letschert K, Keppler D, Konig J. Mutations in the SLCO1B3 gene affecting the substrate specificity of the hepatocellular uptake transporter OATP1B3 (OATP8) [J]. Pharmacogenetics,2004,14:441-452.
[45]Hamada A, Sissung T, Price DK, et al. Effect of SLCO1B3 haplotype on testosterone transport and clinical outcome in Caucasian patients with androgen-independent prostatic cancer [J]. Clin. Cancer Res.,2008,14:3312-3318
[46]Sharifi N, Hamada A, Sissung T, et al. Epub 2008 Jun 4. A polymorphism in a transporter of testosterone is a determinant of androgen independence in prostate cancer [J]. BJU Int,2008,102:617-621.
[47]Muto M, Onogawa T, Suzuki T, et al. Human liverspecific organic anion transporter-2 is a potent prognostic factor for human breast carcinoma [J]. Cancer Sci,2007,98:1570-1576.
[48]Lee W, Belkhiri A., Lockhart AC, et al. Overexpression of OATP1B3 confers apoptotic resistance in colon cancer [J]. Cancer Res.,2008,68:10315-10323.
[49]Narita M, Hatano E, Arizono S, et al. Expression of OATP1B3 determines uptake of Gd-EOB-DTPA in hepatocellular carcinoma [J]. J Gastroenterol.,2009,44:793-798.
[50]Orans J, Teotico DG, Redinbo MR.The nuclear xenobiotic receptor pregane X receptonrecent insights and new challenges [J]. Mol Endocrinol.,2005,19: 2891-2900.
[51]Cui X, Thomas A, Montgomery D, et al. Rat PXR reporter-gene activity correlates with the induction of CYP3A in rat precision-cut liver slices[J].Comb Chem High Throughput Screen.2005,8:341-346.
[52]Meyer zu Schwabedissen HE, Kim RB. Hepatic OATP1B transporters and nuclear receptors PXR and CAR:interplay, regulation of drug disposition genes, and single nucleotide polymorphisms [J]. Mol Pharm,2009,6:1644-1661.
[2]Zhou, S.; Gao, Y.; Jiang, W.; Huang, M.; Xu, A.; Paxton, J. W. Interactions of herbs with cytochrome P450 [J]. Drug Metab Rev 2003,35 (1):35-98.
[3]Thiebaut, F.; Tsuruo, T.; Hamada, H.; Gottesman, M. M; Pastan, I.; Willingham, M. C. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues [J]. Proc Natl Acad Sci U S A 1987,84 (21):7735-8.
[4]Borst, P.; Elferink, R. O. Mammalian ABC transporters in health and disease [J]. Annu Rev Biochem 2002,71:537-92.
[5]Ho, R. H.; Kim, R. B. Transporters and drug therapy:implications for drug disposition and disease [J]. Clin Pharmacol Ther 2005,78 (3):260-77.
[6]van den Bout-van den Beukel, C. J.; Koopmans, P. P.; van der Ven, A. J.; De Smet, P. A.; Burger, D. M. Possible drug-metabolism interactions of medicinal herbs with antiretroviral agents [J]. Drug Metab Rev 2006,38 (3):477-514.
[7]Hartel U, V. E. Use and acceptance of classical natural and alternative medicine in Germany--findings of a representative population-based survey [J]. Forsch Komplementarmed Klass Naturheilkd 2004,11 (6):327-334.
[8]Kaufman, D. W.; Kelly, J. P.; Rosenberg, L.; Anderson, T. E.; Mitchell, A. A. Recent patterns of medication use in the ambulatory adult population of the United States:the Slone survey [J]. JAMA 2002,287 (3):337-44.
[9]Deferme, S.; Kamuhabwa, A.; Nshimo, C.; de Witte, P.; Augustijns, P. Screening of Tanzanian plant extracts for their potential inhibitory effect on P-glycoprotein mediated efflux [J]. Phytotherapy Research 2003,17(5):459-464.
[10]van den Bout-van den Beukel, C. J. P.; Koopmans, P. P.; van der Ven, A. J. A. M.; De Smet, P. A. G. M.; Burger, D. M. Possible Drug-Metabolism Interactions of Medicinal Herbs with Antiretroviral Agents [J]. Drug Metabolism Reviews 2006,38 (3):477-514.
[11]Qi FH, W. Z., Cai PP, Zhao L, Gao JJ, Kokudo N, Li AY, Han JQ, Tang W. Traditional Chinese medicine and related active compounds:A review of their role on hepatitis B virus infection [J]. Drug Discov Ther.2013,7(6):212-224.
[12]Durr, D.; Stieger, B.; Kullak-Ublick, G. A.; Rentsch, K. M.; Steinert, H. C; Meier, P. J.; Fattinger, K. St John's Wort induces intestinal P-glycoprotein/MDR1 and intestinal and hepatic CYP3A4 [J]. Clin Pharmacol Ther 2000,68 (6):598-604.
[13]Molnar, J.; Szabo, D.; Pusztai, R.; Mucsi, I.; Berek, L.; Ocsovszki, L; Kawata, E.; Shoyama, Y. Membrane associated antitumor effects of crocine-, ginsenoside-and cannabinoid derivates [J]. Anticancer Res 2000,20 (2A):861-7.
[14]Tawab, M. A.; Bahr, U.; Karas, M.; Wurglics, M.; Schubert-Zsilavecz, M. Degradation of ginsenosides in humans after oral administration [J]. Drug Me tab Dispos 2003,31 (8):1065-71.
[15]Yamamura, Y.; Kawakami, J.; Santa, T.; Kotaki, H.; Uchino, K.; Sawada, Y.; Tanaka, N.; Iga, T. Pharmacokinetic profile of glycyrrhizin in healthy volunteers by a new high-performance liquid chromatographic method [J]. J Pharm Sci 1992,81 (10):1042-6.
[16]Ploeger, B.; Mensinga, T.; Sips, A.; Seinen, W.; Meulenbelt, J.; DeJongh, J. The pharmacokinetics of glycyrrhizic acid evaluated by physiologically based pharmacokinetic modeling [J]. Drug Metab Rev 2001,33 (2):125-47.
[17]Zou, Q.; Wei, P.; Li, J.; Ge, Z. X.; Ouyang, P. Simultaneous determination of 18alpha-and 18beta-glycyrrhetic acid in human plasma by LC-ESI-MS and its application to pharmacokinetics [J]. Biomed Chromatogr 2009,23 (1):54-62.
[18]Yoshida, N.; Koizumi, M.; Adachi, I.; Kawakami, J. Inhibition of P-glycoprotein-mediated transport by terpenoids contained in herbal medicines and natural products [J]. Food Chem Toxicol 2006,44 (12):2033-9.
[19]Hou, Y. C.; Lin, S. P.; Chao, P. D. Liquorice reduced cyclosporine bioavailability by activating P-glycoprotein and CYP 3A [J]. Food Chem 2012,135 (4):2307-12.
[20]Li, J.; Wang, Y.; Hidalgo, I. J. Kinetic analysis of human and canine P-glycoprotein-mediated drug transport in MDR1-MDCK cell model:Approaches to reduce false-negative substrate classification [J]. J Pharm Sci 2013,102 (9): 3436-46.
[21]Maier, P.; von Kalle, C.; Laufs, S. Retroviral vectors for gene therapy [J]. Future Microbiology 2010,5 (10):1507-1523.
[22]Wandel, C.; Kim, R. B.; Kajiji, S.; Guengerich, P.; Wilkinson, G. R.; Wood, A. J. P-glycoprotein and cytochrome P-450 3A inhibition:dissociation of inhibitory potencies [J]. Cancer Res 1999,59 (16):3944-8.
[23]Kuteykin-Teplyakov, K.; Luna-Tortos, C.; Ambroziak, K.; Loscher, W. Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport [J]. Br J Pharmacol 2010,760(6):1453-63.
[24]Balimane, P. V.; Marino, A.; Chong, S. P-gp inhibition potential in cell-based models:which "calculation" method is the most accurate? [J]. A APS J 2008,10 (4): 577-86.
[25]Cao, J.; Chen, X.; Liang, J.; Yu, X. Q.; Xu, A. L.; Chan, E.; Wei, D.; Huang, M; Wen, J. Y.; Yu, X. Y.; Li, X. T.; Sheu, F. S.; Zhou, S. F. Role of P-glycoprotein in the intestinal absorption of glabridin, an active flavonoid from the root of Glycyrrhiza glabra [J]. Drug Me tab Dispos 2007,35 (4):539-53.
[26]Yamaoka, K.; Nakagawa, T.; Uno, T. Application of Akaike's information criterion (AIC) in the evaluation of linear pharmacokinetic equations [J]. J Pharmacokinet Biopharm 1978,6(2):165-75.
[27]Gibbs, M. A.; Thummel, K. E.; Shen, D. D.; Kunze, K. L. Inhibition of cytochrome P-450 3A (CYP3A) in human intestinal and liver microsomes:comparison of Ki values and impact of CYP3A5 expression [J]. Drug Metab Dispos 1999,27 (2): 180-7.
[28]Zha, W.; Wang, G.; Xu, W.; Liu, X.; Wang, Y.; Zha, B. S.; Shi, J.; Zhao, Q.; Gerk, P. M; Studer, E.; Hylemon, P. B.; Pandak, W. M., Jr.; Zhou, H. Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages [J]. PLoS ONE 2013,8 (1):e54349.
[29]Dolghih, E.; Bryant, C; Renslo, A. R.; Jacobson, M. P. Predicting binding to p-glycoprotein by flexible receptor docking [J]. PLoS Comput Biol 2011,7 (6): el 002083.
[30]Sugiyama, M.; Fujita, K.; Murayama, N.; Akiyama, Y.; Yamazaki, H.; Sasaki, Y. Sorafenib and sunitinib, two anticancer drugs, inhibit CYP3A4-mediated and activate CY3A5-mediated midazolam 1'-hydroxylation [J]. Drug Metab Dispos 2011, 39 (5):757-62.
[31]Varma, M. V.; Panchagnula, R. Enhanced oral paclitaxel absorption with vitamin E-TPGS:effect on solubility and permeability in vitro, in situ and in vivo [J]. Eur J Pharm Sci 2005,25 (4-5):445-53.
[32]Sugimoto, H.; Matsumoto, S.; Tachibana, M.; Niwa, S.; Hirabayashi, H.; Amano, N.; Moriwaki, T. Establishment of in vitro P-glycoprotein inhibition assay and its exclusion criteria to assess the risk of drug-drug interaction at the drug discovery stage [J]. J Pharm Sci 2011,100 (9):4013-23.
[33]Yamashita, S.; Tanaka, Y.; Endoh, Y.; Taki, Y.; Sakane, T.; Nadai, T.; Sezaki, H. Analysis of drug permeation across Caco-2 monolayer:implication for predicting in vivo drug absorption [J]. Pharm Res 1997,14 (4):486-91.
[34]Polli, J. W.; Wring, S. A.; Humphreys, J. E.; Huang, L.; Morgan, J. B.; Webster, L. O.; Serabjit-Singh, C. S. Rational use of in vitro P-glycoprotein assays in drug discovery [J]. J Pharmacol Exp Ther 2001,299 (2):620-8.
[35]Keubler, A.; Weiss, J.; Haefeli, W. E.; Mikus, G.; Burhenne, J. Drug interaction of efavirenz and midazolam:efavirenz activates the CYP3A-mediated midazolam l'-hydroxylation in vitro [J]. Drug Metab Dispos 2012,40 (6):1178-82.
[36]Olukoga, A.; Donaldson, D. Historical perspectives on health. The history of liquorice:the plant, its extract, cultivation, commercialisation and etymology [J]. JR Soc Promot Health 1998,118 (5):300-4.
[37]Zhang, J.; Zhou, F.; Wu, X.; Gu, Y.; Ai, H.; Zheng, Y.; Li, Y.; Zhang, X.; Hao, G.; Sun, J.; Peng, Y.; Wang, G.20(S)-ginsenoside Rh2 noncompetitively inhibits P-glycoprotein in vitro and in vivo:a case for herb-drug interactions [J]. Drug Metab Dispos 2010,38 (12):2179-87.
[38]Izzo, A. A.; Ernst, E. Interactions between herbal medicines and prescribed drugs:an updated systematic review [J]. Drugs 2009,69 (13):1777-98.
[39]Kock, K.; Xie, Y.; Hawke, R. L.; Oberlies, N. H.; Brouwer, K. L. Interaction of silymarin flavonolignans with organic anion-transporting polypeptides [J]. Drug Metab Dispos 2013,41 (5):958-65.
[40]Choi, R. J.; Ngoc, T. M.; Bae, K.; Cho, H. J.; Kim, D. D.; Chun, J.; Khan, S.; Kim, Y. S. Anti-inflammatory properties of anthraquinones and their relationship with the regulation of P-glycoprotein function and expression [J]. Eur J Pharm Sci 2013,48 (1-2):272-81.
[41]Martin, C.; Berridge, G.; Higgins, C. F.; Mistry, P.; Charlton, P.; Callaghan, R. Communication between multiple drug binding sites on P-glycoprotein [J]. Mol Pharmacol 2000,58 (3):624-32.
[42]Wang, R. B.; Kuo, C. L.; Lien, L. L.; Lien, E. J. Structure-activity relationship: analyses of p-glycoprotein substrates and inhibitors [J]. J Clin Pharm Ther 2003,28 (3):203-28.
[43]Teng, Z.; Yuan, C; Zhang, F.; Huan, M.; Cao, W.; Li, K.; Yang, J.; Cao, D.; Zhou, S.; Mei, Q. Intestinal absorption and first-pass metabolism of polyphenol compounds in rat and their transport dynamics in Caco-2 cells [J]. PLoS ONE 2012,7 (1): e29647.
[44]Dantzig, A. H.; Shepard, R. L.; Cao, J.; Law, K. L.; Ehlhardt, W. J.; Baughman, T. M.; Bumol, T. F.; Starling, J. J. Reversal of P-glycoprotein-mediated multidrug resistance by a potent cyclopropyldibenzosuberane modulator, LY335979 [J]. Cancer Res 1996,56 (18):4171-9.
[45]Thomas, H.; Coley, H. M. Overcoming multidrug resistance in cancer:an update on the clinical strategy of inhibiting p-glycoprotein [J]. Cancer Control 2003,10 (2): 159-65.
[46]Bohari, M. H.; Sastry, G. N. FDA approved drugs complexed to their targets: evaluating pose prediction accuracy of docking protocols [J]. Journal of Molecular Modeling 2012,18 (9):4263-74.
[47]Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.; Harrell, P. M.; Trinh, Y. T.; Zhang, Q.; Urbatsch, I. L.; Chang, G. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding [J]. Science 2009,323 (5922):1718-22.
[48]Jabeen, I.; Pleban, K.; Rinner, U.; Chiba, P.; Ecker, G. F. Structure-activity relationships, ligand efficiency, and lipophilic efficiency profiles of benzophenone-type inhibitors of the multidrug transporter P-glycoprotein [J]. J Med Chem 2012,55 (7):3261-73.
[49]Jiao, W.; Dong, W.; Li, Z.; Deng, M.; Lu, R. Lathyrane diterpenes from Euphorbia lathyris as modulators of multidrug resistance and their crystal structures [J]. Bioorg Med Chem 2009,17 (13):4786-92.
[50]Jin, M. S.; Oldham, M. L.; Zhang, Q.; Chen, J. Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans [J]. Nature 2012,490 (7421): 566-9.
[51]Zhou, S. F. Structure, function and regulation of P-glycoprotein and its clinical relevance in drug disposition [J]. Xenobiotica 2008,38 (7-8):802-32.
[52]Hummel, M. A.; Gannett, P. M.; Aguilar, J. S.; Tracy, T. S. Effector-mediated alteration of substrate orientation in cytochrome P450 2C9 [J]. Biochemistry 2004, 43 (22):7207-14.
[53]Atkins, W. M. Current views on the fundamental mechanisms of cytochrome P450 allosterism [J]. Expert Opin Drug Metab Toxicol 2006,2 (4):573-9.
[54]Mudra DR, D. K., Desai PV. In silico, in vitro and in situ models to assess interplay between CYP3A and P-gp [J]. Curr Drug Metab.2011 12 (8):750-773.
[55]Ekroos M, S. T. Structural basis for ligand promiscuity in cytochrome P4503A4. [J]. Proc Natl Acad Sci U S A.200612 (103):13682-13687.
[56]Kenworthy, K. E.; Clarke, S. E.; Andrews, J.; Houston, J. B. Multisite kinetic models for CYP3A4:simultaneous activation and inhibition of diazepam and testosterone metabolism [J]. Drug Metab Dispos 2001,29 (12):1644-51.
[57]Okada, Y.; Murayama, N.; Yanagida, C.; Shimizu, M.; Guengerich, F. P.; Yamazaki, H. Drug interactions of thalidomide with midazolam and cyclosporine A: heterotropic cooperativity of human cytochrome P450 3A5 [J]. Drug Metab Dispos 2009,37(1):18-23.
[58]Hummel, M. A.; Locuson, C. W.; Gannett, P. M.; Rock, D. A.; Mosher, C. M.; Rettie, A. E.; Tracy, T. S. CYP2C9 genotype-dependent effects on in vitro drug-drug interactions:switching of benzbromarone effect from inhibition to activation in the CYP2C9.3 variant [J]. Mol Pharmacol 2005,68 (3):644-51.
[59]Grandvuinet, A. S.; Vestergaard, H. T.; Rapin, N.; Steffansen, B. Intestinal transporters for endogenic and pharmaceutical organic anions:the challenges of deriving in-vitro kinetic parameters for the prediction of clinically relevant drug-drug interactions [J]. JPharm Pharmacol 2012,64 (11):1523-48.
[60]Boocock, D. J.; Faust, G. E. S.; Patel, K. R.; Schinas, A. M.; Brown, V. A.; Ducharme, M. P.; Booth, T. D.; Crowell, J. A.; Perloff, M.; Gescher, A. J.; Steward, W. P.; Brenner, D. E. Phase I Dose Escalation Pharmacokinetic Study in Healthy Volunteers of Resveratrol, a Potential Cancer Chemopreventive Agent [J]. Cancer Epidemiology Biomarkers & Prevention 2007,16(6):1246-1252.
[61]Hong X, H. T., Mi SQ, Ye SM, Ou WP, Wang NS. Pharmacokinetics of Chuanxinlian tablet in healthy Chinese volunteers [J].2007.
[62]Howes J, W. M., Huang.L, Howes LG Long-term pharmacokinetics of an extract of isoflavones from red clover (Trifolium pratense) [J]. J Altern Complement Med. 20025(2):135-142.
[63]Hu, Z.; Yang, J.; Cheng, C,; Huang, Y.; Du, F.; Wang, F.; Niu, W.; Xu, F.; Jiang, R.; Gao, X.; Li, C. Combinatorial Metabolism Notably Affects Human Systemic Exposure to Ginsenosides from Orally Administered Extract of Panax notoginseng Roots (Sanqi) [J]. Drug Metabolism and Disposition 2013,41 (7):1457-1469.
[64]Li, H.; Ren, P.; Huang, X.; Tang, W. F.; Wei, H. Q. [Simultaneous determination and pharmacokinetic studies of ferulic acid and paeoniflorin in human serum by high performance liquid chromatography after oral administration of Modified Xiao-yao Decoction] [J].Zhong Xi Yi Jie He Xue Bao 2008,6 (11):1178-83.
[65]Panossian, A.; Hovhannisyan, A.; Mamikonyan, G.; Abrahamian, H.; Hambardzumyan, E.; Gabrielian, E.; Goukasova, G.; Wikman, G.; Wagner, H. Pharmacokinetic and oral bioavailability of andrographolide from Andrographis paniculata fixed combination Kan Jang in rats and human [J]. Phytomedicine 2000,7 (5):351-364.
[66]Qin, F.; Huang, X.; Zhang, H.-M.; Ren, P. Pharmacokinetic comparison of puerarin after oral administration of Jiawei-Xiaoyao-San to healthy volunteers and patients with functional dyspepsia:influence of disease state [J]. Journal of Pharmacy and Pharmacology 2009,61 (1):125-129.
[67]Shen, T.; Xu, H.; Weng, W.; Zhang, J. Single-and Multiple-Dose Pharmacokinetics of a Novel Tetramethylpyrazine Reservoir-Type Transdermal Patch versus Tetramethylpyrazine Phosphate Oral Tablets in Healthy Normal Volunteers, and in Vitro-in Vivo Correlation [J]. Biological and Pharmaceutical Bulletin 2013,36 (6): 931-937.
[68]Xu, M; Yin, J.; Xie, L.; Zhang, J.; Zou, C; Zou, J.; Liu, F.; Ju, W.; Li, P. Pharmacokinetics and tolerance of toal astragalosides after intravenous infusion of astragalosides injection in healthy Chinese volunteers [J]. Phytomedicine 2013,20 (12):1105-1111.
[69]Zhang, X.-L.; Xu, H.-R.; Chen, W.-L.; Chu, N.-N.; Li, X.-N.; Liu, G.-Y.; Yu, C. Matrine determination and pharmacokinetics in human plasma using LC/MS/MS [J]. Journal of Chromatography B 2009,877 (27):3253-3256.
[70]Wu, X.-I.; Hang, T.-j.; Shen, J.-p.; Zhang, Y.-d. Determination and pharmacokinetic study of oxymatrine and its metabolite matrine in human plasma by liquid chromatography tandem mass spectrometry [J]. Journal of Pharmaceutical and Biomedical Analysis 2006,41 (3):918-924.
[71]Jukema, J. W.; Cannon, C. P.; de Craen, A. J.; Westendorp, R. G.; Trompet, S. The controversies of statin therapy:weighing the evidence [J]. J Am Coll Cardiol 2012, 60(10):875-81.
[72]McGovern, P. G.; Pankow, J. S.; Shahar, E.; Doliszny, K. M.; Folsom, A. R.; Blackburn, H.; Luepker, R. V. Recent trends in acute coronary heart disease--mortality, morbidity, medical care, and risk factors. The Minnesota Heart Survey Investigators [J]. N Engl J Med 1996,334 (14):884-90.
[73]Alberton, M.; Wu, P.; Druyts, E.; Briel, M.; Mills, E. J. Adverse events associated with individual statin treatments for cardiovascular disease:an indirect comparison meta-analysis [J]. QJM2012,105 (2):145-57.
[74]Rendic S, D. C. F. Human cytochrome P450 enzymes:A status report summarizing their reactions, substrates, inducers and inhibitors [J]. Drug Me tab Rev 1997,29 (1-2):413-580.
[75]Chatzizisis, Y. S.; Koskinas, K. C; Misirli, G.; Vaklavas, C; Hatzitolios, A.; Giannoglou, G. D. Risk factors and drug interactions predisposing to statin-induced myopathy:implications for risk assessment, prevention and treatment [J]. Drug Saf 2010,33 (3):171-87.
[76]Wang, Y.; Wang, R.; Gao, X.; Hong, B.; Gao, S. A practical VEP-based brain-computer interface [J]. IEEE Trans Neural Syst Rehabil Eng 2006,14 (2): 234-9.
[77]Ding, J.; Sperling, G.; Srinivasan, R. Attentional modulation of SSVEP power depends on the network tagged by the flicker frequency [J]. Cereb Cortex 2006,16 (7):1016-29.
[78]Hardie, D. G. Role of AMP-activated protein kinase in the metabolic syndrome and in heart disease [J]. FEBS Lett 2008,582 (1):81-9.
[79]Viollet, B.; Guigas, B.; Leclerc, J.; Hebrard, S.; Lantier, L.; Mounier, R.; Andreelli, F.; Foretz, M. AMP-activated protein kinase in the regulation of hepatic energy metabolism:from physiology to therapeutic perspectives [J]. Acta Physiol (Oxf) 2009,196(1):81-98.
[80]Lin, H. Z.; Yang, S. Q.; Chuckaree, C; Kuhajda, F.; Ronnet, G.; Diehl, A. M. Metformin reverses fatty liver disease in obese, leptin-deficient mice [J]. Nat Med 2000,6(9):998-1003.
[81]Bugianesi, E.; Gentilcore, E.; Manini, R.; Natale, S.; Vanni, E.; Villanova, N.; David, E.; Rizzetto, M.; Marchesini, G. A randomized controlled trial of metformin versus vitamin E or prescriptive diet in nonalcoholic fatty liver disease [J]. Am J Gastroenterol 2005,100 (5):1082-90.
[82]Lian, Z.; Li, Y.; Gao, J.; Qu, K.; Li, J.; Hao, L.; Wu, S.; Zhu, H. A novel AMPK activator, WS070117, improves lip id metabolism discords in hamsters and HepG2 cells [J]. Lipids Health Dis 2011,10:67.
[83]刘茵.WS070117药代动力学和作用机制研究[D].北京：中国医学科学院北京协和医学院博士论文,2012.
[84]Wang, Z.; Wang, X.; Qu, K.; Zhu, P.; Guo, N.; Zhang, R.; Abliz, Z.; Yu, H.; Zhu, H. Binding of cordycepin monophosphate to AMP-activated protein kinase and its effect on AMP-activated protein kinase activation [J]. Chem Biol Drug Des 2010,76 (4):340-4.
[85]Guo, P.; Kai, Q.; Gao, J.; Lian, Z. Q.; Wu, C. M.; Wu, C. A.; Zhu, H. B. Cordycepin prevents hyperlipidemia in hamsters fed a high-fat diet via activation of AMP-activated protein kinase [J]. J Pharmacol Sci 2010,113 (4):395-403.
[86]Liu, Y.; He, J.; Abliz, Z.; Zhu, H. In vitro stability and metabolism of O2', O3', 05'-tri-acetyl-N6-(3-hydroxylaniline) adenosine in rat, dog and human plasma: Chemical hydrolysis and role of plasma esterases [J]. Xenobiotica 2011,41 (7): 549-560.
[87]Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones, B. C; MacIntyre, F.; Rance, D. J.; Wastall, P. The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data [J]. J Pharmacol Exp Ther 1997,283 (1): 46-58.
[88]Lu, A. Y.; Wang, R. W.; Lin, J. H. Cytochrome P450 in vitro reaction phenotyping: a re-evaluation of approaches used for P450 isoform identification [J]. Drug Metab Dispos2003,31 (4):345-50.
[89]Tucker, G. T.; Houston, J. B.; Huang, S. M. Optimizing drug development:strategies to assess drug metabolism/transporter interaction potential--toward a consensus [J]. Pharm Res 2001,18(8):1071-80.
[90]Di, L. The role of drug metabolizing enzymes in clearance [J]. Expert Opin Drug Metab Toxicol 2014,10 (3):379-93.
[1]Ho RH, Kim RB. Transporters and drug therapy:implications for drug disposition and disease [J]. Clin Pharmacol Ther,2005,78:260-277.
[2]Niemi M. Role of OATP transporters in the disposition of drugs [J]. Pharmacogenomics,2007,8:787-802.
[3]Jacquemin E, Hagenbuch B, Stieger B, et al. Expression cloning of a rat liver Na(+)-independent organic anion transporter [J]. Proc. Natl. Acad. Sci.,1994,91:133-137.
[4]Smith NF, Figg WD, Sparreboom A. Role of the liver-specific transporters OATP1B1 and OATP1B3 in governing drug elimination [J]. Expert Opin. Drug Metab. Toxicol.,2005,1:429-445
[5]Deng JW, Song IS, Shin HJ, et al. The effect of SLCO1B1*15 on the disposition of pravastatin and pitavastatin is substrate dependent:the contribution of transporting activity changes by SLCO1B1*15[J]. Pharmacogenet. Genomics,2008,18:424-433
[6]Sissung TM, Baum CE, Kirkland CT, et al. Pharmacogenetics of Membrane Transporters:An Update on Current Approaches [J]. Mol Biotechnol,2010,44: 152-167
[7]Abe T, Unno M, Onogawa T, et al. LST-2, a human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers [J]. Gastroenterology,2001,120:1689-1699.
[8]Meier-Abt F, Mokrab Y, Mizuguchi K. Organic anion transporting polypeptides of the OATP/SLCO superfamily:identification of new members in nonmammalian species, comparative modeling and a potential transport mode [J]. J Membr Biol, 2005,208:213-227.
[9]Mahagita C, Grassl SM, Piyachaturawat P, et al. Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport [J]. Am J Physiol Gastrointest Liver Physiol,2007,293:G271-G278
[10]Ismair MG, Stieger B, Cattori V, et al. Hepatic uptake of cholecystokinin octapeptide by organicanion-transporting polypeptides OATP4 and OATP8 of rat and human liver [J]. Gastroenterology,2001,121:1185-1190.
[11]Kalliokoski A, Niemi M. Impact of OATP transporters on pharmacokinetics [J]. Br. J. Pharmacol.,2009,158:693-705
[12]Vavricka SR, Van Montfoort J, Ha HR, et al. Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver [J]. Hepatology,2002, 36:164-172
[13]Meier-Abt F, Faulstich H, Hagenbuch B. Identification of phalloidin uptake systems of rat and human liver [J]. Biochim Biophys Acta.,2004,1664:64-69
[14]Fischer WJ, Altheimer S, Cattori V, et al. Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin [J]. Toxicol Appl Pharmacol.,2005,203:257-263
[15]Smith NF, Acharya MR, Desai N, et al. Identification of OATP1B3 as a high-affinity hepatocellular transporter of paclitaxel [J]. Cancer Biol Ther.,2005b,4:815-818
[16]Hu S, Franke RM, Filipski KK, et al. Interaction of imatinib with human organic ion carriers [J]. Clin Cancer Res.,2008,14:3141-3148
[17]Abe T, Unno M, Onogawa T, et al. LST-2, a human liver-specific organic anion transporter determines methotrexate sensitivity in gastrointestinal cancers [J]. Gastroenterology,2001,120:1689-1699
[18]Smith NF, Marsh S, Scott-Horton TJ, et al. Variants in the SLCO1B3 gene: interethnic distribution and association with paclitaxel pharmacokinetics [J]. Clin Pharmacol.,2007,81:76-82
[19]amaguchi H, Kobayashi M, Okada M, et al. Rapid screening of antineoplastic candidates for the human organic anion transporter OATP1B3 substrates using fluorescent probes [J]. Cancer Lett.,2008,260:163-169
[20]Shimizu M, Fuse K, Okudaira K, et al. Contribution of OATP (organic anion-transporting polypeptide) family transporters to the hepatic uptake of fexofenadine [J]. Drug Metab Dispos.2005,33:1477-1481
[21]Treiber A, Schneiter R, Hausler S, et al. Bosentan is a substrate of human OATP1B1 and OATP1B3:inhibition of hepatic uptake as the common mechanism of its interactions with cyclosporin A, rifampicin, and sildenafil [J]. Drug Metab Dispos., 2007,35:1400-1407
[22]Liu L, Cui Y, Chung AY, et al. Vectorial transport of enalapril by Oatp1a1/Mrp2 and OATP1B1 and OATP1B3/MRP2 in rat and human livers [J]. J Pharmacol Exp Ther., 2006,318:395-402
[23]Nakagomi-Hagihara R, Nakai D, Kawai K, et al. OATP1B1, OATP1B3, and MRP2 are involved in hepatobiliary transport of olmesartan, a novel angiotensin Ⅱ blocker [J]. Drug Metab Dispos.,2006,34:862-869
[24]Ishiguro N, Maeda K, Kishimoto W, et al. Predominant contribution of OATP1B3 to the hepatic uptake of telmisartan, an angiotensin Ⅱ receptor antagonist, in humans [J]. Drug Metab Dispos.,2006,34:1109-1115
[25]Yamashiro W, Maeda K, Hirouchi M, et al. Involvement of transporters in the hepatic uptake and biliary excretion of valsartan, a selective antagonist of the angiotensin Ⅱ AT1-receptor, in humans [J]. Drug Metab Dispos.,2006,34: 1247-1254
[26]Kullak-Ublick GA, Ismair MG, Stieger B, et al. Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver [J]. Gastro-enterology,2001,120:525-533.
[27]Kopplow K, Letschert K, Konig J, et al. Human hepatobiliary transport of organic anions analyzed by quadrupletransfected cells [J]. Mol Pharmacol.,2005,68: 1031-1038
[28]Hirano M, Maeda K, Shitara Y, et al. Drug-drug interaction between pitavastatin and various drugs via OATP1B1 [J]. Drug Metab Dispos.,2006,34:1229-1236
[29]Seithel A, Eberl S, Singer K, et al. The influence of macrolide antibiotics on the uptake of organic nions and drugs mediated by OATP1B1 and OATP1B3 [J]. Drug Metab Dispos.,2007,35:779-786
[30]Ho RH, Tirona RG, Leake BF, et al. Drug and bile acid transporters in rosuvastatin hepatic uptake:function, expression, and pharmacogenetics [J]. Gastroenterology, 2006b,130:1793-1806
[31]Vavricka SR, Van Montfoort J, Ha HR, et al. Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver [J]. Hepatology,2002, 36:164-172
[32]Nozawa T, Sugiura S, Nakajima M, et al. Involvement of organic anion transporting poly-peptides in the transport of troglitazone sulfate:implications for understanding troglitazone hepatotoxicity [J]. Drug Metab Dispos.,2004,32:291-294
[33]Letschert K, Faulstich H, Keller D, et al. Molecular characterization and inhibition of amanitin uptake into human hepatocytes [J].Toxicol Sci.,2006,91:140-149
[34]Annaertl P, Yel ZW, Stieger B, et al. Interaction of HIV protease inhibitors with OATP1B1,1B3, and 2B1[J]. Xenobiotica,2010,40:163-176
[35]Kimoto E, Chupka J, Xiao Y, et al. Characterization of digoxin uptake in sandwich-cultured human hepatocytes [J]. Drug Metab. Dispos.,2011,39:47-53.
[36]Le Vee M, Jouan E, Moreau A, et al. Regulation of drug transporter mRNA expression by interferon-y in primary human hepatocytes [J]. Fundam Clin Pharmacol,2011,25:99-103.
[37]Herfindal L, Myhren L, Kleppe R., et al. Nostocyclopeptide-M1:A potent, nontoxic inhibitor of the hepatocyte drug transporters OATP1B3 and 1B1 [J]. Mol Pharm., 2011,8:360-367
[38]Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/ SLC21 family:phylogenetic classification as OATP/SLCO superfamily, new nomenclature and molecular/functional properties[J]. Pflugers Arch,2004,447: 653-665
[39]Konig J, Seithel A, Gradhand U, et al. Pharmacogenomics of human OATP transporters [J]. Naunyn Schmiedebergs Arch Pharmacol,2006,372:432-443
[40]Seithel A, Glaeser H, Fromm MF, et al. The functional consequences of genetic variations in transporter genes encoding human organic anion-transporting polypeptide family members [J]. Expert Opin. Drug Metab. Toxicol,2008,4:51-64.
[41]Smith NF, Marsh S, Scott-Horton TJ, et al. Variants in the SLCO1B3 gene: interethnic distribution and association with paclitaxel pharmacokinetics [J]. Clin Pharmacol Ther,2007,81:76-82.
[42]Tsujimoto M, Hirata S, Dan Y, et al. Polymorphisms and linkage disequilibrium of the OATP8 (OATP1B3) gene in Japanese participants [J]. Drug Metab Pharmacokinet,2006,21:165-169.
[43]Laitinen A, Niemi M. Frequencies of Single-Nucleotide Polymorphisms of SLCO1A2, SLCO1B3 and SLCO2B1 Genes in a Finnish Population [J]. Basic Clin Pharmacol Toxicol,2011,108:9-13
[44]Letschert K, Keppler D, Konig J. Mutations in the SLCO1B3 gene affecting the substrate specificity of the hepatocellular uptake transporter OATP1B3 (OATP8) [J]. Pharmacogenetics,2004,14:441-452.
[45]Hamada A, Sissung T, Price DK, et al. Effect of SLCO1B3 haplotype on testosterone transport and clinical outcome in Caucasian patients with androgen-independent prostatic cancer [J]. Clin. Cancer Res.,2008,14:3312-3318
[46]Sharifi N, Hamada A, Sissung T, et al. Epub 2008 Jun 4. A polymorphism in a transporter of testosterone is a determinant of androgen independence in prostate cancer [J]. BJU Int,2008,102:617-621.
[47]Muto M, Onogawa T, Suzuki T, et al. Human liverspecific organic anion transporter-2 is a potent prognostic factor for human breast carcinoma [J]. Cancer Sci,2007,98:1570-1576.
[48]Lee W, Belkhiri A., Lockhart AC, et al. Overexpression of OATP1B3 confers apoptotic resistance in colon cancer [J]. Cancer Res.,2008,68:10315-10323.
[49]Narita M, Hatano E, Arizono S, et al. Expression of OATP1B3 determines uptake of Gd-EOB-DTPA in hepatocellular carcinoma [J]. J Gastroenterol.,2009,44:793-798.
[50]Orans J, Teotico DG, Redinbo MR.The nuclear xenobiotic receptor pregane X receptonrecent insights and new challenges [J]. Mol Endocrinol.,2005,19: 2891-2900.
[51]Cui X, Thomas A, Montgomery D, et al. Rat PXR reporter-gene activity correlates with the induction of CYP3A in rat precision-cut liver slices[J].Comb Chem High Throughput Screen.2005,8:341-346.
[52]Meyer zu Schwabedissen HE, Kim RB. Hepatic OATP1B transporters and nuclear receptors PXR and CAR:interplay, regulation of drug disposition genes, and single nucleotide polymorphisms [J]. Mol Pharm,2009,6:1644-1661.