基于整合网络毒理学和分子对接的补骨脂酚致肝毒性机制探讨

目的 基于网络毒理学和分子对接技术探究补骨脂酚致肝毒性的潜在机制。方法 通过TCM-BATMAN挖掘补骨脂酚靶点,DisGeNET挖掘肝毒性基因;Cytoscape 3.7.0分别构建补骨脂酚靶点和肝毒性基因蛋白互作关系（PPI）网络,并Merge取交集,运用软件中插件分析获得关键靶点,构建生物学总调控网络,并对其进行基因本体论（gene ontology,GO）和京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）富集分析;分子对接评价补骨脂酚与潜在关键靶点结合的亲和力。结果 共获得补骨脂酚靶点3 952个,肝毒性基因6 025个和交集基因2 221个。经分析,补骨脂酚主要作用于TP53、HSP90AA1、EP300等关键靶点,参与氧化应激反应与细胞增殖、分化、凋亡过程的调控,涉及多种酶调控等生物过程,主要通过PI3K-Akt、MAPK、细胞周期等信号通路引起肝毒性。补骨脂酚与EP300、HSP90AA1靶点的亲和力最强,能够与二者的结合口袋形成较强的疏水作用,total score值为8.858 2、7.217 8。结论 补骨脂酚致肝毒性具有多靶点、多途径的作用规律,主要通过PI3K-Akt、MAPK、细胞周期等信号通路引起肝毒性。     

甘草次酸的抗肝癌作用机制及其作为肝靶向配体的研究进展

目的:为甘草次酸作为肝靶向配体的开发应用提供参考。方法:以"甘草次酸""肝癌""肝靶向""给药系统""Glycyrrhetinic acid""Liver cancer""Hepatocellular carcinoma""Liver targeting""Drug delivery system"等为关键词,组合查询1990-2016年在Pub Med、Science Direct、中国知网、万方、泉方学术等数据库中的相关文献,从甘草次酸的抗肝癌药理活性、肝靶向作用机制以及作为肝靶向修饰配体的研究等方面进行综述。结果与结论:共检索到相关文献2 796篇,其中有效文献34篇。甘草次酸可通过多种途径抑制肝癌细胞的增殖。甘草次酸具有毒性低、易结合的特点,不仅可作为前药的靶向配体,而且其修饰的药物载体也能特异性地靶向至肝癌病变部位。但是,甘草次酸介导的靶向给药体系在肝疾病模型中能否达到理想的肝靶向作用以及如何避免损伤正常肝组织,仍需进一步的研究探索。     

政策、法规及治疗准则

美宣布人用药品临床试验数据标准格式、EMEA关注尽早发现肝毒性、对公司的审批激励可能加速靶向治疗研究、法国协作促进开发罕见病用药、印度进一步限制青霉素／6-APA进口、     

蛋白质组学技术在药物性肝损伤生物标志物研究中的应用

临床前药物性肝损伤的评价存在灵敏性低和特异性差的问题,常产生假阴性结果和出现意外的毒性,是导致药物终止开发甚至退市的重要原因之一。在药物肝毒性临床前研究中,可以利用蛋白质组学技术的快速、灵敏、高通量等优点,寻找和发现新的肝毒性生物标志物,使药物开发过程更为安全有效。本文对肝毒性生物标志物的研究现状,蛋白质组学在生物标志物的发现及验证等方面的技术发展进行了综述分析,并且着重归纳了该技术在中药致肝损伤方面的生物标志物研究中的应用。与传统评价方法相比,蛋白质组学技术对于发现新型肝潜在毒性生物标志物具有不可替代的优势。随着蛋白质组学技术结合其他组学技术的发展,其在药物肝毒性早期筛选、生物标志物的临床桥接等方面将取得突破性的进展。     

叠鞘石斛与药典品种石斛酚含量对比研究

目的:建立叠鞘石斛与药典收载品种金钗石斛,鼓槌石斛中石斛酚的高效液相色谱含量测定方法。方法:DiamonsilCl8(150mm×4.6mm,5μm)色谱柱,乙腈-0.5%冰醋酸(35:65)为流动相,检测波长280nm,流速1mg.mL-1,柱温30℃。结果:石斛酚在0.01228~0.2456mg.mL-1(r=0.9999)范围内与峰面积线性关系良好,平均回收率为100.39%,RSD为2.17%。结论:本方法操作简单、快捷、重现性好、准确率高,可用于上述三种石斛中石斛酚的含量测定。     

应用高内涵分析技术进行药物肝毒性风险评估的研究

目的研究尝试应用高内涵分析技术建立药物肝毒性高内涵毒性评价指数测定的标准方法。方法采用Tox Insight药物肝毒性检测试剂盒,利用对多靶点的特异性染色,结合高内涵活细胞成像分析平台考察4种药物对人源性肝癌细胞Hep G2的细胞毒性作用,包括对该细胞的细胞数、核DNA含量、细胞质内谷胱甘肽降低水平、活性氧水平、线粒体膜电位的改变等5个指标进行综合分析,拟对受试样品的肝毒性进行评估。结果氟西汀和罗格列酮的肝毒性评估结果为阴性;甲芬那酸和噻氯匹定的肝毒性评估结果为阳性,提示具有肝毒性风险,且甲芬那酸的肝毒性风险比噻氯匹定的高。结论应用高内涵分析技术评估药物肝毒性的方法基于活细胞、多参数、实时、高通量,能够实现药物多种生物活性、毒性的早期、快速检测,并且灵敏稳定,简便可行,可尝试用于对药物进行体外肝毒性的安全风险评估。     

补骨脂酚的体外肝微粒体代谢及代谢减毒作用的种属比较

目的研究补骨脂酚在人、比格犬和大鼠肝微粒体的体外代谢动力学及种属差异,评价不同种属肝微粒体对补骨脂酚人肾近曲小管上皮细胞(HK-2)的减毒作用。方法应用MTT法检测HK-2细胞的存活率来评价补骨脂酚对HK-2细胞的毒性作用以及不同种属肝微粒体对其毒性的影响。应用HPLC法分析补骨脂酚在3个种属肝微粒体孵育液中的剩余浓度,研究其在肝微粒体中的代谢稳定性和代谢动力学。结果在3个种属肝微粒体分别作用下,补骨脂酚的HK-2细胞毒性明显降低。补骨脂酚在人肝微粒体中的代谢最慢,在大鼠肝微粒体中的代谢最快,人、犬和大鼠的代谢动力学参数Km、Vmax、T21和CLint分别为:(81.66±3.41),(89.35±4.32)和(31.89±2.60)μmol.L-1;(0.47±0.01),(0.57±0.01)和(1.88±0.09)μmol.L-1.min-1;(44.14±1.13),(53.31±0.29)和(6.79±0.39)min;(39.38±1.04),(65.16±0.35)和(365.92±20.01)ml.min-1.kg-1。结论肝微粒体降低补骨脂酚的HK-2细胞毒性,这一减毒作用与补骨脂酚经肝微粒体酶的代谢相关。补骨脂酚的体外肝代谢动力学性质存在着一定的种属差异;犬肝微粒体对高浓度补骨脂酚所致的HK-2细胞毒性的降低作用要弱于人和大鼠肝微粒体。     

石斛多糖抗氧化活性研究

目的研究石斛多糖的体内、体外抗氧化活性。方法①体外抗氧化实验：用化学反应法检测石斛多糖对邻苯三酚自氧化反应产生的超氧阴离子和Fenton反应产生的羟自由基的清除作用;②体内抗氧化实验：昆明种小鼠随机分为空白对照组、小剂量组、中剂量组和大剂量组,每组10只。研究石斛多糖对小鼠血清及肝组织中SOD、GSH-Px、MDA的影响。结果石斛多糖具有清除羟基自由基和超氧阴离子自由基的作用,可显著提高小鼠血清和肝组织中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活力,降低丙二醛（MDA）含量。结论石斛多糖具有明显的抗氧化活性,为进一步开发石斛药材提供依据。     

丁香酸和柠檬苦素对小鼠肝脏CYP450酶的影响

目的 研究丁香酸和柠檬苦素对小鼠肝脏细胞色素P450主要亚型mRNA及蛋白表达水平的影响.方法 C57BL/6小鼠随机分为空白对照组、丁香酸组、柠檬苦素组和苯巴比妥组,连续灌胃给药2周,末次给药后处死,提取小鼠肝脏总RNA及肝微粒体,荧光定量聚合酶链式反应(PCR)技术和蛋白质免疫印迹(Western blot)测定CYP450酶主要亚型mRNA和蛋白表达水平.结果 在mRNA水平上,丁香酸对Cyp1a2、Cyp2c37、Cyp2d9 mRNA表达没有明显作用,柠檬苦素对Cyp1a2 mRNA的表达有显著诱导作用;在蛋白水平上,丁香酸对CYP1A1、CYP1A2、CYP3A、CYP2D和CYP2E1蛋白的表达有明显的诱导作用,柠檬苦素对CYP1A1、CYP1A2、CYP2A、CYP2D和CYP2E1有显著的诱导,对CYP2B和CYP2C蛋白表达产生抑制作用.结论 丁香酸和柠檬苦素对细胞色素P450主要亚型均具有不同程度的诱导和抑制作用.     

四种石斛的薄层鉴别及多糖含量测定

目的研究美花石斛、金钗石斛、鼓槌石斛和流苏石斛四种石斛的理化鉴别,并测定其石斛多糖含量差异。方法采用薄层色谱法对四种石斛进行鉴别研究,硫酸-苯酚法测定四种石斛的多糖含量。结果鼓槌石斛和毛兰素对照品的斑点Rf值及颜色相同,其他3种石斛无此成分;四种石斛中仅流苏石斛无石斛酚对照品的斑点;四种石斛的多糖含量分别为24.02%、13.48%、6.55%、8.55%。结论薄层色谱法能有效的鉴别出美花石斛、金钗石斛、鼓槌石斛和流苏石斛;四种石斛中的多糖含量有较明显的差异。     

Gigantol isolated from the whole plants of Cymbidium goeringii inhibits the LPS-induced iNOS and COX-2 expression via NF-kappaB inactivation in RAW 264.7 macrophages cells

During our efforts to find bioactive natural products with anti-inflammatory activity, we isolated gigantol from the whole plants of Cymbidium goeringii (Orchidaceae) by activity-guided chromatographic fractionation. Gigantol was found to have potent inhibitory effects on LPS-induced nitric oxide (NO) and prostaglandin E (2) (PGE (2)) production in RAW 264.7 cells. Consistent with these findings, gigantol suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in RAW 264.7 cells in a concentration-dependent manner. Our data also indicate that gigantol is a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) release and influenced the mRNA expression levels of these cytokines in a dose-dependent manner. Furthermore, a reporter gene assay for nuclear factor kappa B (NF-kappaB) and an electromobility shift assay (EMSA) demonstrated that gigantol effectively inhibited the activation of NF-kappaB, which is necessary for the expression of iNOS, COX-2, TNF-alpha, IL-1beta and IL-6. Thus, our studies suggest that gigantol inhibits LPS-induced iNOS and COX-2 expression by blocking NF- kappaB activation.     

Greater than the sum of its parts: combining models for useful ADMET prediction

In silico ADMET (absorption, distribution, metabolism, excretion, and toxicity) models are important tools in combating late-stage attrition in the drug discovery process. This work shows how ADMET models can be combined to tailor predictions depending on one's needs. We demonstrate how the judicious use of data and considered combination of predictions can produce models that provide truly useful answers. This approach is illustrated with the prediction of hERG channel blocking and cytochrome P450 2D6 inhibition, where combination of two predictive models (with >80% of compounds correctly predicted) resulted in models with even better predictive values (with >90% of compounds correctly predicted for those classes of interest).     

Generation of a set of simple, interpretable ADMET rules of thumb

A set of simple, consistent structure-property guides have been determined from an analysis of a number of key ADMET assays run within GSK: solubility, permeability, bioavailability, volume of distribution, plasma protein binding, CNS penetration, brain tissue binding, P-gp efflux, hERG inhibition, and cytochrome P450 1A2/2C9/2C19/2D6/3A4 inhibition. The rules have been formulated using molecular properties that chemists intuitively know how to alter in a molecule, namely, molecular weight, logP, and ionization state. The rules supplement the more predictive black-box models available to us by clearly illustrating the key underlying trends, which are in line with reports in the literature. It is clear from the analyses reported herein that almost all ADMET parameters deteriorate with either increasing molecular weight, logP, or both, with ionization state playing either a beneficial or detrimental affect depending on the parameter in question. This study re-emphasizes the need to focus on a lower molecular weight and logP area of physicochemical property space to obtain improved ADMET parameters.     

基于非布司他生理药动学模型预测潜在药物相互作用

目的：建立非布司他在健康人体内的生理药动学（PBPK）模型,计算人体内不同部位的药物浓度,预测非布司他对基于CYP2C8和CYP2D6介导代谢的药物的潜在相互作用（DDIs）。方法：通过文献收集和ADMET Predictor^TM软件预测获得非布司他建模的理化性质参数、生物药剂学参数、人体生理参数等;同时收集不同给药剂量非布司他在人体内的血药浓度（PK）数据,建立并验证PBPK模型。采用PBPK模型计算非布司他在肠道上皮细胞浓度、血浆达峰浓度、肝脏达峰浓度、肝脏入口游离药物浓度,预测非布司他与基于CYP2C8和CYP2D6代谢酶代谢药物的DDIs。结果：PBPK模型预测非布司他不同剂量下的药-时曲线与实测值拟合良好。预测的非布司他不同剂量下药动学参数（AUC,C_max,T_max）与实测值相近,倍数误差<2,模型准确可靠。DDIs预测结果显示采用肝脏达峰药物浓度与肝入口游离药物浓度来代替酶活性位点抑制剂浓度作出的DDIs预测结果更与临床结果相符合,即非布司他在健康人群日服用80 mg剂量下,基本不会对由CYP2C8和CYP2D6酶代谢的药物产生明显的DDIs。结论：所建立的PBPK模型可较好预测非布司他的体内药时曲线与机体不同部位的药物浓度,且能准确预测药物可能的相互作用。     

Two novel bibenzyls from Dendrobium trigonopus

Two novel bibenzyl trigonopols A (1) and B (2), together with seven known compounds, gigantol (3), tristin (4), moscatin (5), hircinol (6), naringenin (7), 3-(4-hydroxy-3-methoxyphenyl)-2-propen-1-ol (8), and ( - )-syringaresinol (9), have been isolated from the stems of Dendrobium trigonopus, of which compounds 6, 8, and 9 were isolated for the first time from this species. The structures of two new compounds were elucidated as threo-22-(17-hydroxyl-9-(3-hydroxyl-4-methoxy-phenethyl)-13,16,18-trimethoxy-21H-benzo[c]chromen-21-yl)ethane-22, 23-diol (1) and 9-(4-hydroxyl-3-methoxy-phenethyl)-17-(21-hydroxyl-20-methoxy-phenyl)chroman-11, 16-diol (2) on the basis of spectroscopic methods. Trigonopol A was found to exhibit antiplatelet aggregation activity in vitro with 67.55% inhibitory ration at 1.4337 x 10(- 3) M.     

Two novel bibenzyls from Dendrobium trigonopus

Two novel bibenzyl trigonopols A (1) and B (2), together with seven known compounds, gigantol (3), tristin (4), moscatin (5), hircinol (6), naringenin (7), 3-(4-hydroxy-3-methoxyphenyl)-2-propen-1-ol (8), and ( - )-syringaresinol (9), have been isolated from the stems of Dendrobium trigonopus, of which compounds 6, 8, and 9 were isolated for the first time from this species. The structures of two new compounds were elucidated as threo-22-(17-hydroxyl-9-(3-hydroxyl-4-methoxy-phenethyl)-13,16,18-trimethoxy-21H-benzo[c]chromen-21-yl)ethane-22, 23-diol (1) and 9-(4-hydroxyl-3-methoxy-phenethyl)-17-(21-hydroxyl-20-methoxy-phenyl)chroman-11, 16-diol (2) on the basis of spectroscopic methods. Trigonopol A was found to exhibit antiplatelet aggregation activity in vitro with 67.55% inhibitory ration at 1.4337 x 10(- 3) M.     

Maximizing the outcome of early ADMET models: strategies to win the drug-hunting battles?

Despite substantial changes in the drug discovery paradigm leveraged from the advancement of early ADMET technologies, an open debate remains on how full the return on investment is, along with where to balance risks to costs of lost opportunities in the clinic. Here, the recent advancement of ADMET tools, the areas where they seem to work and where their application and connection with physiology in man remain challenging are briefly reviewed. While the 'more is better' type of 'box-checking' profiling strategy is no longer viable, the key to success lies in an intelligent integration of existing in silico, in vitro and in vivo ADMET data to help generate and test hypotheses that are critical for projecting the benefits and risks of a drug candidate in the clinic. The improvement of in silico, in vitro and in vivo correlations (ISIVIVC) and best utilization of early ADMET data are far more critical and urgent than expanding capacity and portfolio in leveraging ADMET to win the drug-hunting battles in the post-genome era.     

Toward in silico structure-based ADMET prediction in drug discovery

Quantitative structure-activity relationship (QSAR) methods and related approaches have been used to investigate the molecular features that influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. As the three-dimensional structures of several major ADMET proteins become available, structure-based (docking-scoring) computations can be carried out to complement or to go beyond QSAR studies. Applying docking-scoring methods to ADMET proteins is a challenging process because they usually have a large and flexible binding cavity; however, promising results relating to metabolizing enzymes have been reported. After reviewing current trends in the field we applied structure-based methods in the context of receptor flexibility in a case study involving the phase II metabolizing sulfotransferases. Overall, the explored concepts and results suggested that structure-based ADMET profiling will probably join the mainstream during the coming years.     

ADMET considerations for phosphodiesterase-5 inhibitors

Introduction: ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiling is an important aspect of all drug developments. The pharmaceutical industry must always consider ADMET properties in order to optimize drug candidates and to introduce new formulations against existing marketed drugs. Consequently, candidate drug development may be halted early in the discovery phase or during the more costly drug development process because of their poor ADMET properties. Areas covered: The main focus of this article is ADMET profiling, pharmacokinetic (PK) drug interactions, mechanisms and possible adverse drug reactions (ADRs) for approved phosphodiesterase-5 inhibitors (PDE5Is). The authors also look at the efficacy and non-erectogenic benefits of current PDE5Is, which are widely used by patients with erectile dysfunction (ED). The authors also discuss other unapproved PDE5Is such as aildenafil and udenafil, which are currently in use in clinical trials. Expert opinion: The authors believe that the enhancing effect of PDE5Is on the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway means that PDE5Is could be used to treat various conditions. An important issue in their development is 'cross-talk' between PDE5 and other PDEs and thus their specificity for other PDEs. But while it might be difficult to achieve the ideal ADMET profile, it should not necessarily prevent further development of a lead PDE5I. The risk assessment of PDE5Is, with respect to their ADMET properties, is therefore very important for predicting drug-drug interactions, possible side effects, ADRs and its future clinical applications.