藥物的致癌性課件

藥物致癌性的研究

現(xiàn)狀和動態(tài)藥物致癌性研究的必要性腫瘤是一類嚴重影響人類健康和生命的疾病腫瘤已成為人類死亡的第1或2位原因，每年約有700萬人死于癌癥。據2003年WHO資料,目前每年新增腫瘤1000萬人，其中男性530萬人，與1990年相比，全球癌癥患者發(fā)病率增長19％，死亡率增長了18％。

我國惡性腫瘤(2000年)在各種死因中排在第二位，城市已排在首位。每年新增腫瘤200萬人。據預測我國2010年年發(fā)病數220萬，2020年為300萬。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

在研究藥物的潛在致癌作用中，致癌試驗比現(xiàn)有遺傳毒性試驗和系統(tǒng)暴露評價技術更有意義。致癌試驗仍是目前評價藥物致癌作用最可靠和最有意義的方法

已評價的致癌物中有93%(515/554)至少在三項標準遺傳毒性試驗中有一項呈陽性,表明在檢測致癌物（敏感性）是成功的；然而鑒定非致癌物的能力（特異性）較差，183種在大、小鼠致癌試驗中為陰性的物質80%以上有體外遺傳毒性陽性的資料。TheEuropeanCentrefortheValidationofAlternativeMethods(ECVAM)Arecentanalysisofnearly1000chemicalsforwhichdatahavebeenpublishedhashighlightedthestrikinglyimprecisenatureofinvitrogenetictoxicologytestsindiscriminatingnon-carcinogensfromcarcinogens.Whenthestandardbatteryoftwoorthreeinvitrogenotoxicitytestswasperformed,atleast80%ofthe177non-carcinogeniccompoundstestedgaveafalsepositiveresultinatleastonetest.ThefalsepositiveratewashighestinmammaliancelltestssuchasthosetodetectchromosomalAberrationsormicronucleusinChinesehamstercells,orMutationsinthemouselymphomaassay.AsimilaroutcomewasobtainedinanalysisbytheU.S.FDAofanevenlargerdatabaseofchemicals.Invitrogenotoxtesting:theproblem

…poorspecificity大多數致癌物在組合試驗中呈陽性－－Good!大多數非致癌物在組合試驗中也呈陽性－－Bad!特異性敏感性AmesMLAAmesMNMNIndomethacin(吲哚美鋅)testednegativeforinvivocytogeneticassaysintheregulatorytests,butwasreportedpositivefortheinductionofDNAadductsintheliterature.Halothane(氟烷)andpyrazinamide(吡嗪酰胺)werealsoinvivopositiveforcomettestinhumanlymphocytesandinductionofspermheadabnormalitiesinmice,respectively,whichareconsiderednon-regulatorytests.某些藥物是非遺傳毒性的致癌物---用遺傳毒性試驗無法檢出很多管理機構都提出了致癌試驗的要求日本(1990)，如果臨床預期連續(xù)用藥6個月或更長時間，則需要進行致癌試驗。盡管連續(xù)用藥少于6個月，如果存在潛在致癌性因素，也可能需要進行致癌試驗。美國，一般藥物使用3個月或更長時間，需要進行致癌試驗。歐洲，規(guī)定長期應用的藥物，即至少6個月的連續(xù)用藥，或頻繁的間歇性用藥以致總的暴露量與前者相似的藥物需要進行致癌試驗.2010年04月01日我國SFDA制定發(fā)布了《藥物致癌試驗必要性的技術指導原則》

存在潛在致癌的擔憂因素

1）已有證據顯示此類藥物具有與人類相關的潛在致癌性；

2）其構效關系提示致癌的風險；

3）重復給藥毒性試驗中有癌前病變的證據；

4）導致局部組織反應或其它病理生理變化的化合物或其代謝產物在組織內長期滯留內源性肽類、蛋白類物質及其類似物

對于替代治療的內源性物質（濃度在生理水平），尤其是當同類產品（如動物胰島素、垂體來源的生長激素和降鈣素）已有臨床使用經驗時，通常不需要進行致癌試驗1）其生物活性與天然物質明顯不同；2）與天然物質比較顯示修飾后結構發(fā)生明顯改變3）藥物的暴露量超過了血液或組織中的正常水平

化學致癌的過程及其機制化學致癌(chemicalcarcinogenesis)化學物質引起或增進正常細胞發(fā)生惡性轉化并發(fā)展成為腫瘤的過程。具有這類作用的化學物質稱為化學致癌物(chemicalcarcinogen).化學致癌研究的重要歷史事件

1761,JHill提出使用鼻煙可能會誘發(fā)鼻咽癌1775,PPott提出掃煙囪男童陰囊癌與煤煙過度暴露有關1895,LRehn首次報道從事苯胺染料生產的工人發(fā)生膀胱癌1914,TBoveri提出惡性腫瘤起源于存在染色體異常的單個細胞（這就是著名的癌癥體細胞突變理論和腫瘤單細胞克隆起源學說）

1915,Yamagiwa,KIchikawa通過長期給兔耳涂煤焦油成功地誘發(fā)皮膚癌（實驗性化學致癌研究的開端）1932--1938，先后給雄性小鼠注射雌激素誘發(fā)乳腺癌、通過慢性飼喂偶氮染料O－氨基偶氮甲苯誘發(fā)出大鼠肝癌、使用β－萘胺誘發(fā)狗膀胱癌成功

1941-1944,首次提出啟動(Initation)和促進(Promotion)的概念,

根據多次使用巴豆油能促進苯并芘誘發(fā)小鼠皮膚癌提出小鼠皮膚癌兩階段致癌模型;1949Foulds提出腫瘤演進(Progression)概念

1971-1981,CPeraino等,發(fā)現(xiàn)小鼠皮膚癌兩階段致癌理論同樣適用于大鼠肝癌的發(fā)生情況；隨后建立了適用于各種臟器腫瘤的多階段癌變理論1984-,ABalmain,首次報道在化學致癌物誘發(fā)的小鼠皮膚乳頭狀瘤中的c-Ha-ras基因被激活；隨后發(fā)現(xiàn)多種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致癌過程不同階段的特征InitiationDNAmodification,Mutation,GenotoxicOnecelldivisionnecessarytolockinmutationModificationisnotenoughtoproducecancerNonreversible,SingletreatmentcaninducemutationPromotionNodirectDNAmodification,Nongenotoxic,NodirectmutationMultiplecelldivisionsnecessary,ThresholdClonalexpansionoftheinitiatedcellpopulationIncreaseincellproliferationordecreaseincelldeath(apoptosis)Reversible,Multipletreatments(prolongedtreatment)necessaryProgression

DNAmodification,Genotoxicevent?Mutation,chromosomedisarrangement,IrreversibleChangesfrompreneoplasiatoneoplasiabenign/malignantNumberoftreatmentsneededwithcompoundunknown遺傳毒物對DNA和非DNA相互作用的機制化學致癌的生物學特征致癌物多數具有遺傳毒性，遺傳毒性致癌物盡管化學結構和性質不盡相同，但有一共同的特點，即皆為親電子劑。致癌作用依賴于化學致癌物的劑量,大劑量的致癌物可增強腫瘤的發(fā)生,縮短潛伏期.腫瘤的產生取決于化學致癌物的總劑量。同時暴露于幾種致癌物，可發(fā)生聯(lián)合作用。致癌作用的充分表達需相當長的時間,無論致癌物的劑量和性質如何，在腫瘤形成前，總有一個潛伏期。在細胞惡變以前，細胞存在著多階段的癌前病變。

致癌作用所引起的細胞變化可傳到子細胞

致癌作用可被非致癌因子所修飾

細胞增生是細胞癌變過程的重要階段

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藥物致癌性評價方法StipulatedRationaleforChoosingaShort-orMedium-TermTestSystemasSupplementtoOne2-YearBioassay?Themechanismofcarcinogenesisinthemodelshouldmostlikelyberelevanttohumans,andthereforetheuseofthemodelshouldbeapplicabletohumanriskassessment.?Theuseofthemodelshouldsupplementthe2-yearcarcinogenicitystudyanditshouldprovideadditionalinformationthatisnotreadilyavailablefromthe2-yearstudy.?Animalwelfare,animalnumbers,andoveralleconomyofthecarcinogenicevaluationprocessshouldbeconsidered.Two-yearCarcinogenesis“Bioassay”ProtocolCurrentGlobalCarcinogenicityStudyRequirementsStandardTissueListKidneyUrinarybladderAortaHeartTracheaLungsLiverGallbladderPancreasFatSalivaryglandSpleenCervicallymphnodeMesentericlymphnodeThymusTongueEsophagusStomachDuodenumJejunumIleumCecumColonMammaryglandSkinSkeletalmuscleSciaticnerveParathyroidThyroidAdrenalglandPituitaryProstateSeminalvesiclesTestesEpididymidesOvariesOviductsUterinehornsUterinebodyCervixVaginaBrainSpinalcordSternumRib/boneEyesHarderianglandsBMsmearNaresClitoral/preputialglandZymbal’sglandGrosslesions美國毒性病理學會（STP）建議致癌試驗進行組織病理學檢查的最基本的受檢內容目錄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