抗癌药物发展策略ppt课件.ppt

第十二章 抗癌药物发展策略,肿瘤研究的根本目的是降低死亡率及发病率癌预防药物的研究与开发抗癌药物的研究与开发,一、肿瘤化学预防药物的研究,肿瘤的化学预防指用化学药物预防肿瘤的发生,或使癌分化逆转,从而达到预防恶性肿瘤的目的化学预防药物抗始发剂(antiinitiation)抗促癌剂(antipromotor)抗演进剂(antiprogressor),(一)、肿瘤化学预防研究的方法及策略,1、化学预防药的筛选模型,2、化学预防药的筛选策略抗始发剂消除或避免与致癌物接触抑制致癌物的代谢活化或提高机体解毒酶系活性以减少致癌物的生成,抑制活性致癌物到达靶组织与DNA结合,减少DNA加合物的形成提高DNA修复能力,抗促癌剂抗氧化调节细胞信号传导的关键通路抗演进剂抑制血管形成,(二)、肿瘤化学预防药物,1、机制减少致癌物内源性合成的化合物减少或降低化学致癌物吸收的物质抑制活性致癌物与DNA结合的物质改变致癌物代谢或自由基,活性氧生成的物质抑制肿瘤形成的食物成分抑制促癌过程或细胞增殖的化合物,2、分类(1)、植物药及衍生物多酚黄酮与类黄酮芳香异硫氰酸酯有机硫化合物b-胡萝卜素蛋白酶抑制剂,(2)维生素A类化合物(3)维生素E(4)钙(5)硒(6)他莫昔芬(7)非甾体类抗炎药(8)抗癌乙丸,二、抗癌药物研究,1、抗癌药物筛选模型体外实验台盼蓝排斥试验四唑盐比色实验（MTT法）软琼脂克隆形成实验（Soft agar）,台盼蓝排斥试验原理：细胞损伤或死亡时，某些染料可穿透细胞膜，与解体的DNA结合，使其着色活细胞能阻止这类染料进入细胞内活细胞率（%）=活细胞总数/（活细胞总数+死细胞总数）X100,四唑盐比色实验（MTT法）原理：活细胞线粒体中的琥珀酸能使外源性的MTT还原为难溶的蓝紫色的结晶物（Formazan）并沉积在细胞中死细胞无此功能DMSO能溶解细胞中的紫色结晶物，在490nm检测其光吸收值，间接反映活细胞数量细胞存活率=实验组光吸收值/对照组吸收值X100%,软琼脂克隆形成实验（Soft agar）原理：单个细胞在体外持续增殖6代以上，其后代所组成的细胞群体称克隆或集落。每个克隆含50个以上的细胞，大小在0.3-1.0mm之间。通过计算克隆形成率，可对单个细胞的增殖潜力做定量分析。克隆形成率（%）=克隆数/接种细胞X100集落抑制率=（1-实验组集落形成率/对照组集落形成率）X100,实验药物配制：生理盐水、二甲基亚砜、培养基等实验药物浓度：10倍等量稀释，3-5个对数级浓度药物作用时间：短期法：1-3小时中期法：4-6小时长期法：2-3周,体内实验肿瘤抑制实验（荷瘤动物模型）肿瘤转移抑制实验（肿瘤转移动物模型）抗血管生成实验（鸡胚绒毛尿囊膜模型）,药物疗效评价：体外抑瘤实验合成化合物或植物提取物纯品的半数抑制浓度IC50 10ug/ml植物粗提物的IC50 20ug/ml并有细胞毒性的剂量依赖关系，最高抑制效应达80%以上,体内抑瘤实验实体瘤：抑瘤率=（1-实验组瘤重/对照组瘤重）X100%中草药抑制率30%合成药抑制率 40%腹水型：生命延长率=实验组存活天数/对照组存活天数-1）X100%非腹腔给药时生命延长率50%腹腔给药时生命延长率75%有统计学意义，重复3次，疗效稳定，则评定有一定抗肿瘤作用,临床实验 临床实验是一项决定治疗价值的研究，有其严谨性和目的性。应注意二方面的问题：它的最后结论必须由实验结果来支持为达到预期目标，实验必须是前瞻性的，并在严格的质量控制下进行,I期临床试验确定一个合适的剂量来供II期临床试验使用主要进行临床药代动力学研究：病人对药物的耐受性药物生物利用率药物生物转化血浆清除和排泄等,II期临床试验 找出对某药有效的肿瘤类型，并估算在特定病例群体中的有效率，同时注意疗效与计量及给药方案的关系，进一步观察药物的毒副作用。又分早期单药阶段和多药扩大临床实验阶段,III期临床试验又称随机有对照临床实验（randomized controlled trial，RCT）进一步考察新药的疗效，不良反应和适应症，以期在试生产期结束时对其安全有效性做出正确评价，为药政部门批准新药从试生产转位到正式生产提供科学依据。,2、抗癌药物研究肿瘤分化诱导剂克服肿瘤耐药的药物研究抗肿瘤侵袭与转移的药物抗肿瘤血管生成的药物信号传导阻滞剂端粒酶抑制剂细胞凋亡诱导剂,(一)、肿瘤分化诱导剂的研究,细胞分化异常和急性早幼粒细胞白血病血细胞的生成,白血病的形成,急性早幼粒细胞白血病(acute promyelocytic leukemic,APL)T(15;17)(q22;q21)PML-RARa 15号染色体上的早幼粒细胞白血病（PML）基因和17号染色体上的维甲酸受体a(RARa)基因易位，产生异常PML-RARa融合基因,4种变异型:t(11;17)(q23;q21)PLZF-RARat(5;17)(q35;q21)NPM-RARat(11;17)(q13;q21)NuMA-RARadup(17)(q11;q21)Stat5b-RARa,1、RARa的结构和功能,维甲酸(retinoic acid,RA),维甲酸受体(RAR),维甲酸受体（RAR），维甲类X受体（RXR）,RAR结构A/B:配体非依赖性反式激活调节区C:高度保守的DNA结合区,含2个C2C2锌指结构D:受体与共抑制因子相互作用的结构域E:与配体结合区,内源性转录激活区,蛋白二聚化形成的部位F:功能未知,功能配体(RA)缺乏时 RARa/RXRa异二聚体的转录沉默效应通过和一组共抑制因子形成复合物，再结合组蛋白去乙酰化酶（histone deacetylase,HDAC）。HDAC使核小体组蛋白的N-lys残基去乙酰化，并与带负电荷的DNA结合，保持染色体的致密卷曲结构从而抑制转录。生理浓度的ATRA存在时 RARa与ATRA结合，构象变化，共抑制物从RARa上解离，共激活因子复合物与RARa结合，组蛋白残基乙酰化，导致染色体结构舒展，激活转录。,2、PML的结构和功能,PMLthe gene associated with acute promyelacytic leukemia(APL)Initially termed myl,was identified by virtue of chromosomal translocation with the retinoic acid receptor a(RARa)resulting in 99%of all APL casesThe t(15;17)translocation generates two reciprocal fusion products:PML/RARa and RARa/PMLPML/RARa is able to heterodimer with PML and could,therefore,act as a potential dominant negative factor on the normal function of PML,(1)、structure,The PML locus is approximately 35Kb in length and contains nine exons.The human PML gene undergoes alternative splicing to generate multiple transcripts encoding at least thirteen different PML isoforms.All PML isoforms share the N-terminal amino acid region,but differ in their central and c-terminal portions.,P:proline-rich regionThe RBCC motifR:ring finger motif(zinc-binding domain)B1,B2:B-boxes(two cys/his-rich region)Helical coiled coil regionNLS(nuclear localization signal)S/P(acidic Ser-Pro rich region)C-t:c-terminal,(2)、regulation of PML expression,Cellular stresstypeI/II interferons(IFNs),(3)、PML-NB,PML is localized to specific nuclear matrix-associated subdomains called PML-NB(the PML nuclear body)This structure is also known by a variety of names,including ND10(nuclear domain 10),Kremer(Kr)bodies,PODs(PLM oncogenic domains)and PML bodies,PML is required for the proper formation and integrity of the NBSUMOylation of PML is critical in maintaining the structure of this nuclear compartmentPML is covalently modified by a ubiquitin-like protein called PML interacting clone-1(PIC1)also known as SUMO-1 or SentrinSUMO-1 may function in inhibiting ubiquitin conjugation of target proteins and therefore increasing the concentration and/or stability of PML within the nuclear bodies,(4)、functions,Control of cell proliferation,apoptosis,transcription regulation and tumorigenesis,Growth and tumor suppression,Apoptosis,Transcriptional regulationTranscriptional repressionTranscriptional co-activatorWhere various PML isoforms might have different transcriptional roles,3、PML-RARa,T(15;17)translocationPML-RARa fusion genePML-RARa fusion protein(PML:ring finger,the B boxes and the coiled-coil regionsRARa:B-F domain),PML-RARa can interfere with the transcription function of RARa,impairing myeloid differentiationPML-RARa can also form heterodimeric complexes with PML,which delocalize PML from the NB resulting in the disruption of this structure,interfere with PML function and other NB components,4、诱导分化治疗的发展历史,1960Pierce等最早发现小鼠睾丸畸胎瘤细胞可自发分化为良性或正常细胞1971Friend等报告DMSO诱导小鼠红白血病细胞分化为正色性幼红细胞1978 Sadchs最早提出分化治疗概念1980Breitman 发现维甲酸能够诱导HL-60和其他一些人体白血病细胞的分化1983 13-顺式维甲酸(13-CRA)可诱导APL病人完全缓解1987 上海血液所发现ATRA和三尖杉能诱导HL-60细胞分化,体外诱导APL细胞分化,5、分化治疗的成功模型全反式维甲酸治疗APL,1987 上海血液所 6例完全缓解1988 24例 23例缓解1990 90例 76例(ATRA)67例缓解 14例(ATRA+化疗)10例缓解,1991年以来用ATRA治疗APL的完全缓解率年 份 作 者 方 案 病例数 CR率（）1991 陈子吉等 ATRA 50 941992 中国APL研究协作组 ATRA 放疗 544 841993 上海APL研究协作组 ATRA 91 81.3 ATRA 化疗 16 751993 Fenanx等 ATRA 54 911994 Warrell等 ATRA 79 861995 Kanamarn等 ATRA 化疗 109 891995 tollman等 ATRA 164 67,(1)、维甲酸诱导APL细胞分化的分子机制,体外模型NB4细胞系NB4细胞是法国Lanotte博士于1989年从一例复发APL患者的骨髓细胞中分离得到的第一株人早幼粒细胞白血病细胞系其他NB4细胞BN4-R1NB4-R2,(2)、RA诱导APL细胞分化机制,PML-RARa融合蛋白是APL发病的主要原因PML-RARa与RXR形成异二聚体,以显性负的方式干扰野生型RAR,RXR及其他核受体的正常生理功能PML-RARa与PML作用,干扰PML正常定位和生理功能PML-RARa形成二聚体,竞争性与靶基因启动子RARE结合,影响基因的转录表达,RA诱导APL分化机制RA使PML-RARa蛋白降解,释放RXR,使PODs结构恢复正常PML发挥其抑制细胞生长和诱导分化的能力RAR/RXR等其他核受体信号通路发挥正常功能,使细胞走向分化,ATRA与PML-RARa的作用无配体或配体浓度低时,PML-RARa与共抑制因子复合物紧密结合,使靶基因上核小体组蛋白处于去乙酰化状态,阻止分化相关基因的表达药理浓度的ATRA与PML-RARa结合,改变融合蛋白的空间结构,与共抑制因子分离,并与具组蛋白乙酰化活性的共激活因子结合,使组蛋白乙酰化,导致染色体结构松散,启动靶基因的转录,3、ATRA治疗的缺陷,维甲酸综合症(retinoic acid syndrome,RAS),又称白细胞增多综合症白细胞升高血管通透性增高一些细胞因子升高,维甲酸耐药性诱导性血浆药物浓度降低胞质RA结合蛋白和细胞色素P450氧化酶增高,导致胞质游离ATRA减少P-糖蛋白的作用,使ATRA不能在胞内积聚PML-RARa融合蛋白的RARa在配体结合区发生突变,无法与RA结合,导致APL对ATRA耐药在不同的APL患者中存在长型,短型和变异型PML-RARa融合蛋白,导致患者对药理浓度的RA治疗反应差,使APL患者产生ATRA原发耐药,(2)、诱导分化剂,RA及其衍生物细胞因子:G-CSF:促进粒系细胞增殖分化成中性粒细胞 GM-CSF:促进粒细胞和巨噬细胞集落的形成IFN-a:促进造血祖细胞的分化抗肿瘤药物:阿糖胞苷,抗肿瘤抗生素其他:DMSO,TPA,VD3,As2O3,(3)、问题与展望,细胞分化机制的研究诱导分化剂的发展方向:高效,低毒,抗耐药诱导分化治疗实体瘤,改造RA化合物第一代维甲酸类化合物:改造极性基团形成的衍生物第二代维甲酸类化合物:改造环己烯结构形成的衍生物第三代维甲酸类化合物:改造多烯链形成的衍生物从天然药物中挖掘有效的诱导剂,(二)、克服肿瘤耐药的药物研究,多药耐药性(multidrug-resistant,MDR)指肿瘤细胞对一种抗肿瘤药物产生耐药性的同时,对结构和作用机制不同的抗肿瘤药物产生交叉耐药性,In 1950,Burchenal JH et al,first discoveried drug resistance in mouse leukemic cells that acquired resistance to 4-amino-N10-methyl-pteroylglutanic acidIn1986,the multidrug resistance gene(MDR1)was cloned and recognized as the first molecule to explain the multidrug resistance phenomenonSeveral other candidate genes have been isolated and characterized such MRP1 and cMOAT,1、molecular structure related to drug resistance,(1)、ATP-binding cassette transporters(ABC superfamily)48human ABC genes have been identifiedThere are seven subfamilies classified as ABC transporters(ABCA through ABCG)that are expressed in both normal and malignant cells,The ABC superfamily is among the largest and most widespread protein superfamilies known and its members are reasonable for the active transport of a wide variety of compounds across biological membranes including phospholipids,irons,peptides,steroids,polysaccharides,amino acids,organic anions,drugs and other xenobiotics,There are involved in the transport of many substances,including the excretion of toxins from the liver,kidneys,and gastrointestinal tract,and they limit permeation of toxins to critical structures,such as the brain,placenta,and testis.,P-glycoprotein(P-gp),encoded by MDR11280 amino acid,170KD12 transmembrane domains and two intracellular ATPase sitesTwo TM domainTwo ABD domain,P-gp is phosphorylated by an energy-rich ATP molecule,results in its conformation changeA site is uncovered in the peptide chain of great affinity to the compound to be transportedAfter the molecule is bounded,it is transferred to the opposite membrane side,dephosphorylated,followed by further conformation changes to liberate the transported moleculeA return to the conformation able to bind drug again is achieved by hydrolysis of ATP at the second binding site,Multidrug resistance-associated protein(MRP),ABC transporter subfamily C(ABCC)Designated C,was originally cloned from a multidrug resistant human small cell lung cancer cell line,MRP1/ABCC1190KDAn ATP-dependent efflux of several natural product type drugsProtecting tissues from toxin induced damageLocalized to the basolateral membrane of epithelial cells in most tissues with the possible exception of the placenta and therefore pumps its substrate into the interstitial space rather than excreting them into the bile,urine or gutIts level in the lung,testes and kidney are relatively high,MRP2/ABCC2/cMOAT(canalicular multispecific organic anion transporter)Found in apical membrane of polarized cellsExpressed mainly in the liver,kidney,and gutAssociated with bilirubin glucuronide transporter,MRP3Localized to basolateral membranesExpressed in the liver,kidney,gut,pancreas and prostateInvolved in the efflux of organic anions from liver to the blood in the presence of biliary obstruction,MRP4MRP4 mRNA is expressed most aboundantly in the prostate and at low levels in several other normal tissues including the lungMRP5MRP5 mRNA is widely expressed in many tissues with the highest levels found in skeletal muscle,heart and various segment of the brainMRP4/5:transport nucleoside analogsMRP6Lipophilic anion pump with a wide spectrum of chemotherapy resistance,Functionsprotection from environmental chemicalsResistance to heavy metal oxyanions Protection from anti-cancer agents the only convincing evident of a link to chemical drug resistance for the MRP family is associated with MRP1,(2)、thiols,Reduced glutathione S and its metabolic enzymesGSH-tripeptide of glycine,glutamate and cysteineReact with toxic endogeneous and exogeneous substrates,including free radicals and anticancer agentsGST(glutathione S-transferase)conjugates many different bilogical active and potential carcinogenic compounds to GSHAcquired resistence to these agents is frequently accompanied by an elevation in the GSH contend and in GST activity,MRP1 and cMOAT were identified as candidate molecules of GSU-conjugate transporters,Metallothionein(MT)MT is a cysteine-rich low molecule thiol that has a high affinity for heavy metalsThe resistant mechanism includes not only a chelating affinity with heavy metals but also an inactivation capacity to superoxide generated by anticancer drugs,(3)、topoisomerase,DNA topoisomerases are nuclear enzymes that alter the topological(conformational)state of DNA through DNA strand cleavage,strand passage and religationTopoisomerases bind to double-stranded DNA and induce single-strand breaks(topo I)or double-strand break(topo II)to uncoil the hypercoiled DNA,Anti-drugMutated topoisomerase proteinDecreased expression of topoisomerase and decreased protein activity,2、多药耐药性药物多药耐药性的逆转药物钙拮抗剂抗激素类化合物环胞菌素A及衍生物GSH耗竭剂,抗肿瘤多药耐药药物抗MDR相关肿瘤药物的结构修饰物破坏MDR机制的抗MDR药诱导caspases非依赖性凋亡的抗MDR药干扰神经酰胺代谢的抗MDR药机制未明的抗MDR药,What is the molecular mechanism for the promiscurity of multidrug efflux pumps and how do they bind and transport multiple structuraly unrelated substrates?What is the natural physiological role of multidrug efflux pumps?Do they serve to protect cells from endogeneous and exogenous toxic compounds or do they have other physiologically relevant substrates and only transport toxic compounds fortuitously or opportunisitically?Why are there so many multidrug pumps?,（三）抗癌药物发展策略和方向,发展策略：针对机制的抗癌药物筛选,发展方向：肿瘤分化诱导剂克服肿瘤耐药的药物研究抗肿瘤侵袭与转移的药物抗肿瘤血管生成的药物信号传导阻滞剂端粒酶抑制剂细胞凋亡诱导剂,