《药物的致癌性》课件

药物致癌性的研究

现状和动态第二军医大学药物平安性评价中心第二军医大学卫生毒理学教研室张天宝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2021年年发病数220万，2021年为300万。Age-adjustedCancerDeathRates,bySite,US,1930-20051957、1984、1999、2004年我国城市主要疾病死因构成比及死因顺位顺位1957198419992004死因比例(%)死因比例(%)死因比例(%)死因比例（%）1呼吸系统16.86心脏病22.65脑血管病22.28恶性肿瘤27.232传染病7.93脑血管病21.13恶性肿瘤21.66脑血管病18.473肺结核7.51恶性肿瘤21.11心脏病16.37心脏病17.234消化系统7.31呼吸系统8.79呼吸系统15.28呼吸系统12.775心脏病6.61消化系统4.32意外伤害6.52损伤中毒6.56特别严重的是,肿瘤是我国最正确劳动人口的首要死因，在35－59岁年龄人口中，所有年龄组的第一位死因都是肿瘤，只有到了60岁以后脑血管或心血管疾病才上升为第1位死因。Whatmaycausecancer?

Hereditarydisorders

ChemicalsVirusesChronicinflammation???WORLDHEALTHORGANIZATIONINTERNATIONALAGENCYFORRESEARCHONCANCERIARCMonographEvaluationsLYON,FRANCESlidecourtesyofV.Cogliano(IARC)IARC(2021)-Carcinogenictohumans(group1)–108agentsProbablycarcinogenictohumans(group2A)–66Possiblycarcinogenictohumans(group2B)–248Notclassifiableastoitscarcinogenicitytohumans(group3)–515Probablynotcarcinogenictohumans(group4)–1IARC:IARCGroup1–Carcinogenictohumans

Medicaldrugsandtreatments 24 Industrialprocesses 13 Infectiousagentsorprocesses 10 Physicalagents 10 Industrialchemicals 7 Inhaledparticulates 5 Metalsandinorganicsalts 5 Lifestylefactors(incl.herbalremedies) 7 Other 8Group2A–66

Medicaldrugsandtreatments12

ChemicalCarcinogenesis

inthe21stCenturyNewperceptionsofpreviouslyknowncarcinogens:

CombinedeffectsofmultipleexposuresExamples:AlcoholdrinkingandaflatoxinsAlcoholdrinkingandHBV/HBCAlcoholdrinkingandtobaccosmokingTobaccosmokingandasbestos/arsenic/radon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,说明在检测致癌物〔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.PerformanceofindividualgenotoxictestsindetectingrodentcarcinogensasanalyzedbyKirklandetal.(2005).

AmesMLNMNSensitivity(%)58.8 73.1 78.7Specificity(%)73.939.030.8Ames+MLAAmes+MNMLA+MNAmes+MLA+MNSensitivity(%)81.085.987.090.7Specificity(%)32.4 12.010.05.0PerformanceofsimultaneoustestingbatteriesofgenotoxictestsindetectingrodentcarcinogensasanalyzedInvitrogenotoxtesting: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AmesMLAAmesMNMN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药物致癌试验

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〕导致局部组织反响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开展成为肿瘤的过程。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

InitiatingEventCellProliferation(clonalexpansion)ProgressionCellProliferationCellProliferation

MalignancySecondMutatingEvent"N"MutatingEventInitiation

PromotionStagesofCarcinogenesisCellularandMolecularMechanismsinMultistageCarcinogenesis:

INITIATIONInitiatingeventinvolvescellulargenome–MUTATIONSTargetgenes:

-oncogenes/tumorsuppressorgenes

-signaltransduction

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

-increasedcellproliferation

-inhibitionofapoptosisNodirectstructuralalterationinDNAbyagentoritsmetabolitesCellularandMolecularMechanismsinMultistageCarcinogenesis:

PROGRESSIONIrreversibleenhancement/repressionofgeneexpressionComplexgeneticalterations(chromosomaltranslocations,deletions,geneamplifications,recombinations,etc.)Selectionofneoplasticcellsforoptimalgrowthgenotype/phenotypeinresponsetothecellularenvironment“Complex〞geneticchanges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相互作用的机制正常细胞遗传改变〔突变〕遗传改变的细胞癌细胞异倍体细胞表遗传干扰遗传毒性致癌物非遗传毒性致癌物遗传毒性和非遗传毒性致癌物在多阶段致癌中的区别遗传毒性和非遗传毒性致癌物作用机制的比较遗传毒性致癌物非遗传毒性致癌物遗传毒性测试阳性阴性多阶段致癌作用遗传改变表遗传改变第一阶段基因突变细胞增殖染色体畸变再生生长因子刺激过氧化物酶体增殖细胞间间隙连接通讯基因组印记以后阶段遗传或表遗传改变遗传或表遗传改变低剂量的剂量－反应关系无致癌性阈值线性剂量－反应曲线致癌性阈值S型剂量－反应曲线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根本的受检内容目录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/