CYP2D亚家族基因在灵长类动物中的药物代谢及进化研究

CYP2D亚家族基因普遍存在于灵长类动物中,在药物代谢方面起着重要作用,但不同地域和种族的人的CYP2D酶对同一药物的代谢效果存在差异,非人灵长类动物的CYP2D酶代谢某些药物的效果比人类的更有效。此外,在漫长的进化过程中,灵长类动物CYP2D亚家族基因之间会经历频繁的基因复制或基因丢失,导致CYP2D亚家族基因数目存在差异,但尚未确定这种差异是否与物种的亲缘关系有关。本研究对CYP2D亚家族基因在灵长类动物中的药物代谢和进化研究方面进行综述,以期为实现个体化用药和精准性治疗提供一定的理论依据,并进一步了解CYP2D亚家族基因在灵长类中的进化情况。     

王琰:想做医药监管的眼睛

王琰博士，中国医学科学院药物研究所分析室、国家药物及代谢产物分析研究中心副研究员，药物质量控制及药物代谢研究组负责人。从事药物分析、新药质量控制、药物代谢等研究16年，负责或主要完成10余项新药研制和中药的质量研究课题。2002年入选北京市科技新星计划。     

P450酶的研究进展

肝是代谢药物的主要场所，而在代谢药物中起关键作用的酶是位于微粒体的细胞色素P-450系统，常称药酶或混合功能氧化酶，或单加氧酶。一般说来，许多药物经甘药酶代谢后生成低毒或无毒分子，随尿和胆汁排出，但亦有一些药物及其它化合物经肝药酶代谢激活成毒性更大的中间产物，引起毒性。因此，肝药酶代谢药物的分子机制及与毒理学的关系，是药理学基础理论研究的重要内容之一。     

醛氧化酶在药物代谢中的作用

目的介绍一种在药物代谢研究中日益重要的非CYP450氧化酶——醛氧化酶(aldehyde oxidase,简称AOX)的研究进展,为深入研究和了解该药物代谢酶提供参考。方法以65篇国内外相关文献为基础,进行分析、归纳和总结,阐明AOX在药物代谢过程中的作用。结果首先对AOX进行了简要介绍,接着描述了AOX的底物类型,不同实验动物的AOX基因(种属差异及对药物代谢和开发的影响),影响AOX活性的因素,AOX代谢决策树(筛选方法),最后对其在药物代谢中的作用进行了总结并展望未来可能的发展方向。结论鉴于AOX在药物代谢中的重要作用,需要研究者们从生物学、酶反应机理、底物专属性等方面更深入地研究AOX。     

食品安全与药物代谢分析研究评述

扼要介绍现代分析化学的发展,综述作者指导研究生近20年来在分离科学研究、食品安全分析、药物代谢分析等领域的研究进展,期望进一步开展食品组学和药物代谢组学分析研究.     

赤芍中芍药苷和芍药内酯苷的代谢及药动学研究进展

药物代谢是药物研发过程的重要环节,对于深入探讨药物的药效基础及在体内的代谢规律至关重要.芍药苷和芍药内酯苷是赤芍的主要活性成分,具有广泛的药理作用.文章通过查阅国内外文献,就其体内、体外代谢及药代动力学方面进行综述,介绍了芍药苷和芍药内酯苷体内、体外代谢机制,旨在为赤芍的研发和临床给药提供科学依据,同时也为其他药物的代谢研究提供参考.     

UPLC-MS法同时检测大鼠血浆中6种CYP450探针药物浓度

利用超高效液相色谱与三重四级杆质谱仪联用技术建立大鼠血浆中非那西丁、甲苯磺丁脲、奥美拉唑、睾酮、右美沙芬、氯唑沙宗6种常用CYP450探针药物浓度的检测方法,并以其药物代谢动力学参数考察所建方法的准确性。采用Waters ACQUITY-BEH C18柱(1.7μm,50 mm×2.5 mm),流动相为乙腈和0.1%甲酸,乙腈采用梯度洗脱,流速0.25 m L/min,柱温30℃,进样量5μL。电喷雾离子化,选择性离子监测。大鼠分为2组,分别灌胃给予6种底物与生理盐水,根据所建立的UPLC-MS法测定6种探针药物的血药浓度,并分析比较其药物代谢动力学参数。结果显示,6种物质的线性范围为5~1500 ng/m L,方法回收率为85%~110%,日内、日间RSD<10%,基质效应为88%~106%。试验所建方法准确快速,专属性强,样品前处理方便,且6种药物的主要药物代谢动力学参数与文献报道基本相符,可用于6种药物的同时检测及药物代谢动力学研究。     

药代动力学在新药成药性转换医学研究中的作用

 全新分子实体作为新药的研发是一个长期的创新研究,是一个涉及多学科的系统工程。转换研究可以将新候选药物分子的基础实验室研究、临床前研究、临床评价和临床应用广泛联系在一起,建立新的开发模式,进而缩短研发周期和加快向临床推广速度。在全新研发过程和临床试验中,药物代谢和药物处置的属性特征(吸收、分布、代谢、排泄)是用以评价候选药物成药可能性的重要标准。因此,在整个全新的研发模式里,药代动力学研究对于全新分子实体在转换研究过程中的评估起着至关重要的作用。从实验室到临床,从临床到市场的转换研究方式对于提高效率是一种非常重要的策略,其中每一步都涉及大量的药代动力学研究;某一种化学实体成药可能性、早期失败、早期出局等问题可以在第一时间发现。因此,药代动力学评估在药物发现、设计、研发过程中对提高效率、控制成本并进一步获得安全有效的药物有重要价值。     

缺血性脑卒中治疗药物研究进展

本文以缺血性脑卒中为主要研究对象,论述了缺血性脑卒中的发病机制,综述了脑卒中治疗药物的临床使用现状和研究进展.笔者从溶栓药物、抗血小板聚集药物、降纤和抗凝药物以及神经保护药物等几个方面总结了近年来缺血性脑卒中治疗药物的研究概况和研究热点,分析了脑卒中治疗药物研究存在的问题,并展望了脑卒中药物治疗的发展方向.     

抗朊病毒药物筛选模型的研究进展

通过抗朊病毒药物筛选模型来筛选治疗以疯牛病为代表的朊病毒疾病的药物是目前国际上研究热点.结合作者正在进行的抗朊病毒药物筛选的研究,主要介绍了国际上流行的几种抗朊病毒药物筛选模型的原理与方法以及研究最新进展.     

人细胞色素P450氧化还原酶的克隆表达和抗体的制备

细胞色素P450还原酶(CPR)是人细胞色素P450酶系的电子供给者,对后者活性的发挥起到重要作用.本研究将从人胚胎cDNA文库中得到的人CPR的编码基因,连接入pT7450表达载体中,在大肠杆菌BL21(DE3)中高效表达并纯化.每升发酵液可得到纯化蛋白49 mg,占总蛋白含量的10%,以细胞色素C为底物检测酶活性,比活为65 u/mg.利用纯化的CPR作为抗原,常规免疫纯系大耳家兔,获得高效价、高特异性的抗血清,抗体滴度为1∶200 000(ELISA法),纯化后抗体应用于不同组织中CPR含量的评价,证明重组CPR及其抗体可用于细胞色素P450的体外药物代谢及细胞色素P450与CPR作用机理的研究.     

Odorant signal termination by olfactory UDP glucuronosyl transferase

The onset of olfactory transduction has been extensively studied, but considerably less is known about the molecular basis of olfactory signal termination. It has been suggested that the highly active cytochrome P450 monooxygenases of olfactory neuroepithelium are termination enzymes, a notion supported by the identification and molecular cloning of olfactory-specific cytochrome P450s (refs. 13-16). But as reactions catalysed by cytochrome P450 (refs 17, 18) often do not significantly alter volatility, lipophilicity or odour properties, cytochrome P450 may not be solely responsible for olfactory signal termination. In liver and other tissues, drug hydroxylation by cytochrome P450 is frequently followed by phase II biotransformation, for example by UDP glucuronosyl transferase (UGT), resulting in a major change of solubility and chemical properties. We report here the molecular cloning and expression of an olfactory-specific UGT. The olfactory enzyme, but not the one in liver microsomes, shows preference for odorants over standard UGT substrates. Furthermore, glucuronic acid conjugation abolishes the ability of odorants to stimulate olfactory adenylyl cyclase. This, together with the known broad spectrum of drug-detoxification enzymes, supports a role for olfactory UGT in terminating diverse odorant signals.     

细胞色素P450与农药相互作用及其机理研究

综述了细胞色素P450的种类和功能多样性,介绍了细胞色素P450参与除草剂代谢及其作用机理．同时,简述了细胞色素P450 14DM与杀菌剂特异性作用的机制．报道了我们从抗除草剂的瑞士黑麦草中克隆CYP8181同源基因及其功能的研究,以及柑橘绿霉菌细胞色素P450 14DM基因的克隆表达．为深入研究杀菌剂作用细胞色素P450 14DM的机理从而设计以P450 14DM为特异性靶标的新型农药、以及进一步研究细胞色素P450酶系与除草剂代谢的关系打下了良好的基础．     

Docking and molecular dynamics studies on CYP2D6

Drug-metabolizing enzymes, also known as cytochrome P450s, are a superfamily of hemoglobin responsible for metabolizing more than 90% clinical drugs. Cytochrome P450 2D6 (CYP2D6) is a significant member of cytochrome P450s for the reason of metabolizing about 20% clinical drugs. In this paper, molecular docking and molecular dynamic simulations are used to investi- gate the active site of CYP2D6, roles of essential amino acids within the active site and time-dependent protein energy changes. The results suggest that amino acids Glu216, Asp301, Ser304 and Ala305 in the active site are likely to form hydrogen bonding interac-tions with substrates; the benzene ring of Phe120 and aromatic ring in the substrates form ∏-∏ interactions. In addition, molecular dynamics simulations prove that the catalytic conformation of CYP2D6 without ligands can be obtained by their own atomic fluctuations. The impact of ligands on protein system energy and large conformational shift is not very large. Cytochrome P450s is known for their genetic polymorphisms, which will result in severe adverse drug reactions. Ideally, we hope to use mo- lecular modeling to investigate the differences between the substrates of wild-type and mutants while they are bonded with drugs, and predict the drug metabolizing ability of mutants. Reduce the possibility for people taking drugs that they can not metabolize, therefore reduce the rate of adverse drug reactions, and eventually establish a platform of personalized drugs to largely benefit human health.     

Identification of the primary gene defect at the cytochrome P450 CYP2D locus

The mammalian cytochrome P450-dependent monooxygenase system is involved in the metabolism of drugs and chemical carcinogens. The role of these enzymes in toxicological response is exemplified by an autosomal recessive polymorphism at the cytochrome P450 CYP2D6 debrisoquine hydroxylase locus which results in the severely compromised metabolism of at least 25 drugs, and which in some cases can lead to life-threatening side-effects. In addition, this polymorphism, which affects 8-10% of the caucasian population, has been associated with altered susceptibility to lung and bladder cancer. Here we report the identification of the primary mutation responsible for this metabolic defect and the development of a simple DNA-based genetic assay to allow both the identification of most individuals at risk of drug side-effects and clarification of the conflicting reports on the association of this polymorphism with cancer susceptibility.     

Crystal structure of human cytochrome P450 2C9 with bound warfarin

Cytochrome P450 proteins (CYP450s) are membrane-associated haem proteins that metabolize physiologically important compounds in many species of microorganisms, plants and animals. Mammalian CYP450s recognize and metabolize diverse xenobiotics such as drug molecules, environmental compounds and pollutants. Human CYP450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing isoforms, and contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use. Polymorphic variants have also been reported for some CYP450 isoforms, which has implications for the efficacy of drugs in individuals, and for the co-administration of drugs. The molecular basis of drug recognition by human CYP450s, however, has remained elusive. Here we describe the crystal structure of a human CYP450, CYP2C9, both unliganded and in complex with the anti-coagulant drug warfarin. The structure defines unanticipated interactions between CYP2C9 and warfarin, and reveals a new binding pocket. The binding mode of warfarin suggests that CYP2C9 may undergo an allosteric mechanism during its function. The newly discovered binding pocket also suggests that CYP2C9 may simultaneously accommodate multiple ligands during its biological function, and provides a possible molecular basis for understanding complex drug-drug interactions.     

Characterization of the common genetic defect in humans deficient in debrisoquine metabolism

In population studies of individuals given the antihypertensive drug debrisoquine, two distinct phenotypes have been described: extensive metabolizers excrete 10-200 times more of the urinary metabolite 4-hydroxydebrisoquine than poor metabolizers. In family studies the poor-metabolizer phenotype behaves as an autosomal recessive trait with an incidence between 5% and 10% in the white population of Europe and North America, and extends to the deficient metabolism of more than 20 commonly prescribed drugs. Clinical studies have shown that such individuals are at high risk for the development of adverse side effects from these and probably many other drugs. Here we show that poor metabolizers have negligible amounts of the cytochrome P450 enzyme P450db1. We have cloned the human P450db1 complementary DNA and expressed it in mammalian cell culture. Furthermore, by directly cloning and sequencing cDNAs from several poor-metabolizer livers, we have identified three variant messenger RNAs that are products of mutant genes producing incorrectly spliced db1 pre-mRNA, providing a molecular explanation for one of man's most commonly defective genes (frequency of mutant alleles 35-43%).     

Cytochrome P450 Monooxygenase Immobilization as a Model of Herbicide Metabolism in vitro

To investigate herbicide metabolism in vitro by cytochrome P450 with stable enzymatic activity, cy-tochrome P450 is immobilized in silk fibroin. The enzymatic activity of immobilized cytochrome P450 is maintained above 80% after repeated batch experiments for 10 times. Moreover, the enzymatic activity of immobilized cytochrome P450 is kept as relatively high as 73.8% after storage for two months at 4℃. In addition, immobilization can improve the temperature and pH stability of cytochrome P450. Immobilized cytochrome P450 has the similar affinity Km values for herbicide chlorsulfuron and triasulfuron as the free cytochrome P450. In the case of chlorosulfuron, affinity Km value is 53μmol/L for free cytochrome P450,and 63μmol/L for immobilized cytochrome P450, respectively. In the case of triasulfuron affinity, Km value is 36μmol/L for free cytochrome P450, and 44μmol/L for immobilized cytochrome P450, respec-tivily. Immobilized cytochrome P450 will be convenient, rapid, stable and continuous for herbicide metabolism in micro-bioreactor in vitro.     

Metabolism of fibrates by cytochrome P450s and UDP-glycosyltransferases in rat and human liver microsomes

Fibrates are widely used for the treatment of dyslipidemia.However,the contributions of the phase I and phase II metabolic pathways to the clearance of fibrates are unclear.In this study,we investigated the metabolism of gemfibrozil(Gem) ,clofibric acid(CA) ,fenofibric acid(FA) and bezafibrate(Beza) by cytochrome P450s(P450s) and UDP-glycosyltransferases(UGTs) using a substrate depletion approach.We also compared the metabolic characteristics of rat liver microsomes(RLM) and human liver microsomes(HLM) .The intrinsic clearance rates mediated by P450s,UGTs and both were 172 ± 22,643 ± 26,798 ± 103 μL min-1 mg-1,respectively,for Gem and 43 ± 11,88 ± 12,119 ± 15 μL min-1 mg-1,respectively,for CA in RLM.The fractions metabolized by P450s and UGTs in RLM were 22% and 81% for Gem,36% and 74% for CA.The P450-and UGT-mediated depletion rates for Gem were 303 and 1607 nmol min-1 mg-1 in RLM versus 86 and 243 nmol min-1 mg-1 in HLM.The corre-sponding rates for CA were 1.1 and 1.7 nmol min-1 mg-1 in RLM versus 0.025 and 0.038 nmol min-1 mg-1 in HLM.Accordingly,both P450s and UGTs substantially contribute to the clearance of Gem and CA,with UGTs playing a greater role.To avoid un-der-estimating the impact of these pathways,it is necessary to measure NADPH-and UDPGA-dependent metabolism.Although the fractions of these two pathways in RLM and HLM were similar,the depletion rate of Gem and CA in RLM was higher than that in HLM.The metabolism of FA and Beza by P450s and UGTs was too low to calculate intrinsic clearance in both RLM and HLM.These results indicate that fibrates are metabolized via similar pathways in rats and humans,and it is applicable to use RLM to predict the clearance of fibrates in human.     

Trifluoromethylation of arenes and heteroarenes by means of photoredox catalysis

Modern drug discovery relies on the continual development of synthetic methodology to address the many challenges associated with the design of new pharmaceutical agents. One such challenge arises from the enzymatic metabolism of drugs in vivo by cytochrome P450 oxidases, which use single-electron oxidative mechanisms to rapidly modify small molecules to facilitate their excretion. A commonly used synthetic strategy to protect against in vivo metabolism involves the incorporation of electron-withdrawing functionality, such as the trifluoromethyl (CF(3)) group, into drug candidates. The CF(3) group enjoys a privileged role in the realm of medicinal chemistry because its incorporation into small molecules often enhances efficacy by promoting electrostatic interactions with targets, improving cellular membrane permeability, and increasing robustness towards oxidative metabolism of the drug. Although common pharmacophores often bear CF(3) motifs in an aromatic system, access to such analogues typically requires the incorporation of the CF(3) group, or a surrogate moiety, at the start of a multi-step synthetic sequence. Here we report a mild, operationally simple strategy for the direct trifluoromethylation of unactivated arenes and heteroarenes through a radical-mediated mechanism using commercial photocatalysts and a household light bulb. We demonstrate the broad utility of this transformation through addition of CF(3) to a number of heteroaromatic and aromatic systems. The benefit to medicinal chemistry and applicability to late-stage drug development is also shown through examples of the direct trifluoromethylation of widely prescribed pharmaceutical agents.