药物的致癌性课件

药物致癌性的研究

现状和动态药物致癌性研究的必要性肿瘤是一类严重影响人类健康和生命的疾病肿瘤已成为人类死亡的第1或2位原因，每年约有700万人死于癌症。据2003年WHO资料,目前每年新增肿瘤1000万人，其中男性530万人，与1990年相比，全球癌症患者发病率增长19％，死亡率增长了18％。

我国恶性肿瘤(2000年)在各种死因中排在第二位，城市已排在首位。每年新增肿瘤200万人。据预测我国2010年年发病数220万，2020年为300万。Age-adjustedCancerDeathRates,bySite,US,1930-2005Whatmaycausecancer?

Hereditarydisorders

ChemicalsVirusesChronicinflammation???WORLDHEALTHORGANIZATIONINTERNATIONALAGENCYFORRESEARCHONCANCERIARCMonographEvaluationsLYON,FRANCESlidecourtesyofV.Cogliano(IARC)IARC(2009)-monographs.iarc.frCarcinogenictohumans(group1)–108agentsProbablycarcinogenictohumans(group2A)–66Possiblycarcinogenictohumans(group2B)–248Notclassifiableastoitscarcinogenicitytohumans

(group3)–515

Probablynotcarcinogenictohumans(group4)–1IARC:ChemicalCarcinogenesis

inthe21stCenturyNewperceptionsofpreviouslyknowncarcinogens:

CombinedeffectsofmultipleexposuresExamples:AlcoholdrinkingandaflatoxinsAlcoholdrinkingandHBV/HBCAlcoholdrinkingandtobaccosmokingTobaccosmokingandasbestos/arsenic/radon

在研究药物的潜在致癌作用中，致癌试验比现有遗传毒性试验和系统暴露评价技术更有意义。致癌试验仍是目前评价药物致癌作用最可靠和最有意义的方法

已评价的致癌物中有93%(515/554)至少在三项标准遗传毒性试验中有一项呈阳性,表明在检测致癌物（敏感性）是成功的；然而鉴定非致癌物的能力（特异性）较差，183种在大、小鼠致癌试验中为阴性的物质80%以上有体外遗传毒性阳性的资料。TheEuropeanCentrefortheValidationofAlternativeMethods(ECVAM)Arecentanalysisofnearly1000chemicalsforwhichdatahavebeenpublishedhashighlightedthestrikinglyimprecisenatureofinvitrogenetictoxicologytestsindiscriminatingnon-carcinogensfromcarcinogens.Whenthestandardbatteryoftwoorthreeinvitrogenotoxicitytestswasperformed,atleast80%ofthe177non-carcinogeniccompoundstestedgaveafalsepositiveresultinatleastonetest.ThefalsepositiveratewashighestinmammaliancelltestssuchasthosetodetectchromosomalAberrationsormicronucleusinChinesehamstercells,orMutationsinthemouselymphomaassay.AsimilaroutcomewasobtainedinanalysisbytheU.S.FDAofanevenlargerdatabaseofchemicals.Invitrogenotoxtesting:theproblem

…poorspecificity大多数致癌物在组合试验中呈阳性－－Good!大多数非致癌物在组合试验中也呈阳性－－Bad!特异性敏感性AmesMLAAmesMNMNIndomethacin(吲哚美锌)testednegativeforinvivocytogeneticassaysintheregulatorytests,butwasreportedpositivefortheinductionofDNAadductsintheliterature.Halothane(氟烷)andpyrazinamide(吡嗪酰胺)werealsoinvivopositiveforcomettestinhumanlymphocytesandinductionofspermheadabnormalitiesinmice,respectively,whichareconsiderednon-regulatorytests.某些药物是非遗传毒性的致癌物---用遗传毒性试验无法检出很多管理机构都提出了致癌试验的要求日本(1990)，如果临床预期连续用药6个月或更长时间，则需要进行致癌试验。尽管连续用药少于6个月，如果存在潜在致癌性因素，也可能需要进行致癌试验。美国，一般药物使用3个月或更长时间，需要进行致癌试验。欧洲，规定长期应用的药物，即至少6个月的连续用药，或频繁的间歇性用药以致总的暴露量与前者相似的药物需要进行致癌试验.2010年04月01日我国SFDA制定发布了《药物致癌试验必要性的技术指导原则》

存在潜在致癌的担忧因素

1）已有证据显示此类药物具有与人类相关的潜在致癌性；

2）其构效关系提示致癌的风险；

3）重复给药毒性试验中有癌前病变的证据；

4）导致局部组织反应或其它病理生理变化的化合物或其代谢产物在组织内长期滞留内源性肽类、蛋白类物质及其类似物

对于替代治疗的内源性物质（浓度在生理水平），尤其是当同类产品（如动物胰岛素、垂体来源的生长激素和降钙素）已有临床使用经验时，通常不需要进行致癌试验1）其生物活性与天然物质明显不同；2）与天然物质比较显示修饰后结构发生明显改变3）药物的暴露量超过了血液或组织中的正常水平

化学致癌的过程及其机制化学致癌(chemicalcarcinogenesis)化学物质引起或增进正常细胞发生恶性转化并发展成为肿瘤的过程。具有这类作用的化学物质称为化学致癌物(chemicalcarcinogen).化学致癌研究的重要历史事件

1761,JHill提出使用鼻烟可能会诱发鼻咽癌1775,PPott提出扫烟囱男童阴囊癌与煤烟过度暴露有关1895,LRehn首次报道从事苯胺染料生产的工人发生膀胱癌1914,TBoveri提出恶性肿瘤起源于存在染色体异常的单个细胞（这就是著名的癌症体细胞突变理论和肿瘤单细胞克隆起源学说）

1915,Yamagiwa,KIchikawa通过长期给兔耳涂煤焦油成功地诱发皮肤癌（实验性化学致癌研究的开端）1932--1938，先后给雄性小鼠注射雌激素诱发乳腺癌、通过慢性饲喂偶氮染料O－氨基偶氮甲苯诱发出大鼠肝癌、使用β－萘胺诱发狗膀胱癌成功

1941-1944,首次提出启动(Initation)和促进(Promotion)的概念,

根据多次使用巴豆油能促进苯并芘诱发小鼠皮肤癌提出小鼠皮肤癌两阶段致癌模型;1949Foulds提出肿瘤演进(Progression)概念

1971-1981,CPeraino等,发现小鼠皮肤癌两阶段致癌理论同样适用于大鼠肝癌的发生情况；随后建立了适用于各种脏器肿瘤的多阶段癌变理论1984-,ABalmain,首次报道在化学致癌物诱发的小鼠皮肤乳头状瘤中的c-Ha-ras基因被激活；随后发现多种致癌物可使不同的癌基因活化和抑癌基因失活CellularandMolecularMechanismsinMultistageCarcinogenesis:

INITIATIONInitiatingeventinvolvescellulargenome–MUTATIONSTargetgenes:

-oncogenes/tumorsuppressorgenes

-signaltransduction

-cellcycle/apoptosisregulators“Simple”geneticchangesGenticandEpigeneticModelsofTheCancerInitiationEpigeneticallyreprogrammedcellsMutatorphenotypecellsEndogenousEnvironmentalALTERATIONSINCELLULAREPIGENOMENormalcellsCancercellsClonalselectionandexpressionofinitiatedcellsMutatorphenotypecellsEndogenousEnvironmentalACQUISITIONOFADDITIONALRANDOMMUTATIONSNormalcellsCancercellsCellularandMolecularMechanismsinMultistageCarcinogenesis:PROMOTIONReversibleenhancement/repressionofgeneexpression:

-increasedcellproliferation

-inhibitionofapoptosisNodirectstructuralalterationinDNAbyagentoritsmetabolites致癌过程不同阶段的特征InitiationDNAmodification,Mutation,GenotoxicOnecelldivisionnecessarytolockinmutationModificationisnotenoughtoproducecancerNonreversible,SingletreatmentcaninducemutationPromotionNodirectDNAmodification,Nongenotoxic,NodirectmutationMultiplecelldivisionsnecessary,ThresholdClonalexpansionoftheinitiatedcellpopulationIncreaseincellproliferationordecreaseincelldeath(apoptosis)Reversible,Multipletreatments(prolongedtreatment)necessaryProgression

DNAmodification,Genotoxicevent?Mutation,chromosomedisarrangement,IrreversibleChangesfrompreneoplasiatoneoplasiabenign/malignantNumberoftreatmentsneededwithcompoundunknown遗传毒物对DNA和非DNA相互作用的机制化学致癌的生物学特征致癌物多数具有遗传毒性，遗传毒性致癌物尽管化学结构和性质不尽相同，但有一共同的特点，即皆为亲电子剂。致癌作用依赖于化学致癌物的剂量,大剂量的致癌物可增强肿瘤的发生,缩短潜伏期.肿瘤的产生取决于化学致癌物的总剂量。同时暴露于几种致癌物，可发生联合作用。致癌作用的充分表达需相当长的时间,无论致癌物的剂量和性质如何，在肿瘤形成前，总有一个潜伏期。在细胞恶变以前，细胞存在着多阶段的癌前病变。

致癌作用所引起的细胞变化可传到子细胞

致癌作用可被非致癌因子所修饰

细胞增生是细胞癌变过程的重要阶段

Thedevelopmentofcancerisa

multi-stepprocess

“Initiation”formsanearlyadenoma“Promotion”leadstoalateadenoma“Progression”leadsfirsttoacancerinsitu,thenonto…“Malignantconversion,”whichleadstotrueacarcinomaThissetofprocessesoftentakesYEARSGenotoxiccarcinogensincreasetumourfrequencyinanimalcancerbioassaypositiveresultsfrominvitroandinvivogenotoxicitytestseitherdirect-actingorindirectlyactinggenotoxiccarcinogensNon-genotoxiccarcinogensusuallyactastumorpromoterspositiveincancerbioassayinanimals,butnegativeingenotoxicitytestsThemechanismofcarcinogenicitymayincludethechronicinjuryandregenerationhormonalmechanismsincreaseinthecellproliferationordecreaseinthecelldeathintargetorganTheconceptofa“complete”vs.an“incomplete”carcinogenWhenoneforeignchemicalissuffienttocausecancer,eitherasadirectorindirectcarcinogen,itissaidtobecompleteWhenitrequiresatumorpromotertocausecancer,itisanincompletecarcinogenPromotorsarecompoundsthatinducecells,likethemutatedcancerinitiatorcell,togrowanddivide,makingmoreFifteenexamplekeyeventsrepresentingdiversecarcinogenicmodesofactionDNAreactivity(covalentbinding)GenemutationChromosomalbreakageAneuploidyEnzyme-mediatedeffectsonDNAdamageorrepairEpigeneticeffectsCellsignaling:nuclearreceptor-mediatedCellsignaling:otherthannuclearreceptor-mediatedImmuneresponsemodulationInflammationCytotoxicityandcompensatorycellproliferationMitogenicityChronicmetabolicorphysiologicoverloadNutrientdeficiencyrelatedInterferencewithintercellularcommunicationICHGuidelineS1BonTestingforCarcinogenicityofPharmaceuticalschoosingone2-yearrodentcarcinogenicitystudy(rat)

plusoneotherstudythatsupplementsthe2-yearstudyandprovidingadditionalinformationthatisnotreadilyavailablefromthe2-yearstudy:either(1)ashort-ormedium-terminvivorodenttestsystem

or(2)a2-yearcarcinogenicitystudyinasecondrodentspecies(mouse).theshort-ormedium-termmodelswasintendedtofocusontheuseofinvivomodelsprovidinginsightintocarcinogenicendpointssuchasinitiation–promotionrodentmodelsandmodelsofcarcinogenesisusingtransgenicorneonatalrodents药物致癌性评价方法StipulatedRationaleforChoosingaShort-orMedium-TermTestSystemasSupplementtoOne2-YearBioassay•Themechanismofcarcinogenesisinthemodelshouldmostlikelyberelevanttohumans,andthereforetheuseofthemodelshouldbeapplicabletohumanriskassessment.•Theuseofthemodelshouldsupplementthe2-yearcarcinogenicitystudyanditshouldprovideadditionalinformationthatisnotreadilyavailablefromthe2-yearstudy.•Animalwelfare,animalnumbers,andoveralleconomyofthecarcinogenicevaluationprocessshouldbeconsidered.Two-yearCarcinogenesis“Bioassay”ProtocolCurrentGlobalCarcinogenicityStudyRequirementsStandardTissueListKidneyUrinarybladderAortaHeartTracheaLungsLiverGallbladderPancreasFatSalivaryglandSpleenCervicallymphnodeMesentericlymphnodeThymusTongueEsophagusStomachDuodenumJejunumIleumCecumColonMammaryglandSkinSkeletalmuscleSciaticnerveParathyroidThyroidAdrenalglandPituitaryProstateSeminalvesiclesTestesEpididymidesOvariesOviductsUterinehornsUterinebodyCervixVaginaBrainSpinalcordSternumRib/boneEyesHarderianglandsBMsmearNaresClitoral/preputialglandZymbal’sglandGrosslesions美国毒性病理学会（STP）建议致癌试验进行组织病理学检查的最基本的受检内容目录Tumor-BearingAnimalsinControlGroupsfromRodentStudiesSource:J.K.Haseman(unpublishedsummaryofU.S.NTPdata).ComparativePercentIncidenceofPertinentNeoplasiainDifferentStrainsofRatsandMice(104WeeksOld)Note:F344,Fischer244rats;S-D,Sprague–Dawleyrats;B6C3F1,mice,(C57BL/6N+C3H/HeN)F1;CD-1,1CRCr:CD-1mice;NA,nonapplicable;theaveragenumberusedbyspecies/strain/genderwasinexcessof750animalsPreclinicalapproachesforassessingcarcinogenicpotentialTumorigenicityinhumans,nonhumanprimatesandrodentsSpontaneoustumorratesinthebreederandcontrolanimalsTheNeonatalMousePietraetal.(1959).Theneonatalmouseisoneofthealternativeinvivomodels,fordetectingthecarcinogenicpotentialofpharmaceuticals.ThisisinagreementwiththesuggestionsofICH,whichallowstheuseofonealternativestudyinplaceofoneofthe2-yearcarcinogenicitystudies.Whentreatmentbeginswithinthefirst24hoursoflife,thestudydesignisdescribedas“newbornmouse”.“neonatalmouse”includestestitemadministrationatdifferenttimepointsfrombirthtothreeweeksofage.Fujii(1991)reportedthattheneonatalmouseassayshowedasensitivityof85%andapositivepredictionrateof96%comparedtotheresultsoftheadultmouse2-yearcarcinogenicitystudy.Flammangetal.(1997)consideredthismodeltohavehighsensitivityandspecificitytodetectgenotoxiccarcinogensaswellaspresentingadvantagessuchasreducedtestarticlerequirements,decreasedanimalnumbersandcostsandareducedcompletiontime.Itdoesnotrespondtochemicalsactingviaepigeneticmechanisms.

McClainetal.(2001)reportedthatneonatalmicehavebeenshowntohaveareducedtimefortumorinduction,ahighermultiplicityofinducedtumors,alowerspontaneoustumorrateandanequivalentorhighersensitivitytocarcinogenswhencomparedtoadultmice.Thismodelalsorespondstoawiderangeofstructurallydissimilargenotoxiccompounds.Additionally,theneonatalmousepossessesthemajorityofthephaseIandIIbiotransformationliverenzymesinvolvedintheprocessesofactivationanddetoxificationofcarcinogensfromdifferentchemicalclasses.CD-1mice10to12weeksofage.Micewerecagedwith5femalespermaleandexaminedeachdayforthepresenceofavaginalcopulationplug.Femaleswereisolateduntildelivery,6litterswith4neonates/sex/litterwereassignedtoeachgroupduringthefirstweekafterbirth.Threeorfourdoselevels,avehicleandapositivecontrolwereused.

Groupsconsistedof24animals/sex/group.Theyweredosedonthebasisoftheiraveragebodyweight,ondays8and15ofage,usingdosevolumesofupto100and200μl,respectively.Doselevelswereselectedonthebasisoftheresultsobtainedindoserangefindingstudies,inwhichtheMTDortheMFD(MaximumFeasibleDose)forneonatalmice,weredetermined.Thepupswereweanedaround22daysofage,housed4/sex/cageandthenmaintaineduntil1yearofage,whentheyweresacrificed.DEN(diethylnitrosamine)atadosageof2mg/kgdissolvedinwaterwasusedasthepositivecontrol.Tg.AC(v-Ha-ras)TransgenicMouse8–9weeksoldGroupsof15mice/sex/dosewererandomlyassignedtothestudygroups.Thevehiclesusedfordrugsandpositivecontrolagentswereacetone,ethanolorDMSO.TPA(12-o-tetradecanoylphorbol-13-acetate)wasthepositivecontrolcompound26weeksHemizygousp53+/–KnockoutMouse6to10weeksold,GenotypeanalysiswasrecommendedpriortoassignmenttodosegroupsGroupsof15mice/sex/group,Threedoselevels,avehicleandapositivecontrolwereused.Twoadditionalgroupsof15wild-typemice/sex/groupreceivedthevehicleandthehighdosageofthetestcompound.p-Cresidineat400mg/kg/daybygavageincornoil(10ml/kg),orbenzeneat100mg/kg/daybygavageincornoil(5ml/kg)wererecommendedaspositivecontrols.18