藥物的致癌性課件

1、藥物致癌性的研究現(xiàn)狀和動(dòng)態(tài),第二軍醫(yī)大學(xué)藥物安全性評(píng)價(jià)中心第二軍醫(yī)大學(xué)衛(wèi)生毒理學(xué)教研室張?zhí)鞂?藥物致癌性研究的必要性,腫瘤是一類嚴(yán)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(jù)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(zhǎng)19％， 死亡率增長(zhǎng)了18％。 我國(guó)惡性腫瘤(2000年)在各種死因中排在第二位

2、，城市已排在首位。每年新增腫瘤200萬人。據(jù)預(yù)測(cè)我國(guó)2010年年發(fā)病數(shù)220萬，2020年為300萬。,Age-adjusted Cancer Death Rates, by Site, US, 1930-2005,1957、1984、1999、2004年我國(guó)城市主要疾病死因構(gòu)成比及死因順位,,,,,特別嚴(yán)重的是,腫瘤是我國(guó)最佳勞動(dòng)人口的首要死因，在35－59歲年齡人口中，所有年齡組的第一位死因都是腫瘤，只有到了60歲以后腦血管或心血管

3、疾病才上升為第1位死因。,What may cause cancer ?,Hereditary disorders Chemicals Viruses Chronic inflammation ???,WORLD HEALTH ORGANIZATIONINTERNATIONAL AGENCY FOR RESEARCH ON CANCERIARC Monograph Evaluations

4、LYON, FRANCE,Slide courtesy of V. Cogliano (IARC),IARC (2009) - monographs.iarc.frCarcinogenic to humans (group 1) – 108 agentsProbably carcinogenic to humans (group 2A) – 66Possibly carc

5、inogenic to humans (group 2B) – 248Not classifiable as to its carcinogenicity to humans (group 3) – 515 Probably not carcinogenic to humans (group 4) – 1,,IARC:,IARC Group 1 – Carcinogenic to humans,Medica

6、l drugs and treatments24Industrial processes13Infectious agents or processes10Physical agents10Industrial chemicals 7Inhaled particulates 5Metals and inorganic salts 5

7、Lifestyle factors (incl. herbal remedies) 7Other 8,Group 2A – 66 Medical drugs and treatments 12,Chemical Carcinogenesis in the 21st Century,New perceptions of previously known carcinogens: Combined ef

8、fects of multiple exposuresExamples:Alcohol drinking and aflatoxinsAlcohol drinking and HBV/HBCAlcohol drinking and tobacco smokingTobacco smoking and asbestos/arsenic/radon,在研究藥物的潛在致癌作用中，致癌試驗(yàn)比現(xiàn)有遺傳毒性試驗(yàn)和系統(tǒng)暴露評(píng)價(jià)技術(shù)更有意義

9、。,致癌試驗(yàn)仍是目前評(píng)價(jià)藥物致癌作用最可靠和最有意義的方法,已評(píng)價(jià)的致癌物中有93%(515/554)至少在三項(xiàng)標(biāo)準(zhǔn)遺傳毒性試驗(yàn)中有一項(xiàng)呈陽(yáng)性, 表明在檢測(cè)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(yàn)中為陰性的物質(zhì)80% 以上有體外遺傳毒性陽(yáng)性的資料。,The European Centre for the Validation of Alternative Methods (ECVAM)

10、,A recent analysis of nearly 1000 chemicals for which data have been published has highlighted the strikingly imprecise nature of in vitro genetic toxicology tests in discriminating non-carcinogens from carcinogens. When

11、 the standard battery of two or three in vitro genotoxicity tests was performed, at least 80% of the 177 non-carcinogenic compounds tested gave a false positive result in at least one test. The false positive rate was h

12、ighest in mammalian cell tests such as those to detect chromosomal Aberrations or micronucleus in Chinese hamster cells, or Mutations in the mouse lymphoma assay. A similar outcome was obtained in analysis by the U.S. FD

13、A of an even larger database of chemicals.,Performance of individual genotoxic tests in detecting rodent carcinogens as analyzed by Kirkland et al. (2005).,,Performance of simultaneous testing batteries of genotoxic test

14、s in detecting rodent carcinogens as analyzed,In vitro genotox testing: the problem,…poor specificity,,大多數(shù)致癌物在組合試驗(yàn)中呈陽(yáng)性－－Good!,大多數(shù)非致癌物在組合試驗(yàn)中也呈陽(yáng)性－－Bad!,特異性,敏感性,Ames,MLA AmesMN MN,Indomethacin(吲哚美鋅) tested negative f

15、or in vivo cytogenetic assays in the regulatory tests, but was reported positive for the induction of DNA adducts in the literature. Halothane(氟烷) and pyrazinamide(吡嗪酰胺) were also in vivo positive for comet test in human

16、 lymphocytes and induction of sperm head abnormalities in mice, respectively, which are considered non-regulatory tests.,某些藥物是非遺傳毒性的致癌物---用遺傳毒性試驗(yàn)無法檢出,很多管理機(jī)構(gòu)都提出了致癌試驗(yàn)的要求,日本(1990)，如果臨床預(yù)期連續(xù)用藥6個(gè)月或更長(zhǎng)時(shí)間，則需要進(jìn)行致癌試驗(yàn)。盡管連續(xù)用藥少于6個(gè)月，如果

17、存在潛在致癌性因素，也可能需要進(jìn)行致癌試驗(yàn)。美國(guó)，一般藥物使用3個(gè)月或更長(zhǎng)時(shí)間，需要進(jìn)行致癌試驗(yàn)。歐洲，規(guī)定長(zhǎng)期應(yīng)用的藥物，即至少6個(gè)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(jìn)行致癌試驗(yàn).2010年04月01日我國(guó)SFDA制定發(fā)布了《藥物致癌試驗(yàn)必要性的技術(shù)指導(dǎo)原則》,藥物致癌試驗(yàn),2010年04月01日SFDA制定發(fā)布了《藥物致癌試驗(yàn)必要性的技術(shù)指導(dǎo)原則》 預(yù)期臨床用藥期至少連續(xù)6個(gè)月的藥物一般

18、應(yīng)進(jìn)行。 連續(xù)用藥沒有6個(gè)月，但以間歇的方式重復(fù)使用，如治療慢性和復(fù)發(fā)性疾病(包括過敏性鼻炎、抑郁癥和焦慮癥)，而需經(jīng)常間歇使用的藥物，一般也需進(jìn)行。 某些可能導(dǎo)致暴露時(shí)間延長(zhǎng)的釋藥系統(tǒng)，也應(yīng)考慮進(jìn)行致癌試驗(yàn)。,,,,存在潛在致癌的擔(dān)憂因素 1）已有證據(jù)顯示此類藥物具有與人類相關(guān)的潛在致癌性； 2）其構(gòu)效關(guān)系提示致癌的風(fēng)險(xiǎn)； 3）重復(fù)給藥毒性試驗(yàn)中有癌前病變的證據(jù)； 4）導(dǎo)致局部組織反應(yīng)或其它病理生

19、理變化的化合物或其代謝產(chǎn)物在組織內(nèi)長(zhǎng)期滯留 內(nèi)源性肽類、蛋白類物質(zhì)及其類似物 對(duì)于替代治療的內(nèi)源性物質(zhì)（濃度在生理水平），尤其是當(dāng)同類產(chǎn)品（如動(dòng)物胰島素、垂體來源的生長(zhǎng)激素和降鈣素）已有臨床使用經(jīng)驗(yàn)時(shí)，通常不需要進(jìn)行致癌試驗(yàn) 1）其生物活性與天然物質(zhì)明顯不同； 2）與天然物質(zhì)比較顯示修飾后結(jié)構(gòu)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(xué)致癌的過程及其機(jī)制,化學(xué)致癌 (che

20、mical carcinogenesis) 化學(xué)物質(zhì)引起或增進(jìn)正常細(xì)胞發(fā)生惡性轉(zhuǎn)化并發(fā)展成為腫瘤的過程。具有這類作用的化學(xué)物質(zhì)稱為化學(xué)致癌物(chemical carcinogen).,化學(xué)致癌研究的重要?dú)v史事件,1761, J Hill 提出使用鼻煙可能會(huì)誘發(fā)鼻咽癌1775, P Pott 提出掃煙囪男童陰囊癌與煤煙過度暴露有關(guān)1895 , L Rehn 首次報(bào)道從事苯胺染料生產(chǎn)的工人發(fā)生膀胱癌1914, T Boveri

21、 提出惡性腫瘤起源于存在染色體異常的單個(gè)細(xì)胞（這就是著名的癌癥體細(xì)胞突變理論和腫瘤單細(xì)胞克隆起源學(xué)說）,1915, Yamagiwa, K Ichikawa 通過長(zhǎng)期給兔耳涂煤焦油成功地誘發(fā)皮膚癌（實(shí)驗(yàn)性化學(xué)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(dòng)(Initation)和促進(jìn)(Promo

22、tion)的概念, 根據(jù)多次使用巴豆油能促進(jìn)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; 1949 Foulds提出腫瘤演進(jìn)(Progression) 概念 1971-1981, C Peraino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-, A Balmain, 首次報(bào)道在化學(xué)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

23、種致癌物可使不同的癌基因活化和抑癌基因失活,Initiating Event,,,,,,,,,,,,,,,,,,Cell Proliferation(clonal expansion),,Progression,,,,Cell Proliferation,Cell Proliferation,,Malignancy,Second Mutating Event,"N" Mutating Event,Initiat

24、ion,Promotion,,Stages of Carcinogenesis,,,Cellular and Molecular Mechanisms in Multistage Carcinogenesis: INITIATION,Initiating event involves cellular genome – MUTATIONSTarget genes: - oncogenes/tumor suppressor gen

25、es - signal transduction - cell cycle/apoptosis regulators,“Simple” genetic changes,,Gentic and Epigenetic Models of The Cancer Initiation,,Epigenetically reprogrammed cells,Mutator phenotype cells,Endog

26、enous,Environmental,ALTERATIONS IN CELLULAR EPIGENOME,Normal cells,Cancer cells,,,,,,Clonal selection and expression of initiated cells,Mutator phenotype cells,Endogenous,Environmental,ACQUISITION OF ADDITIONAL RANDOM MU

27、TATIONS,Normal cells,Cancer cells,,,,,,,Cellular and Molecular Mechanisms in Multistage Carcinogenesis: PROMOTION,Reversible enhancement/repression of gene expression:- increased cell proliferation- inhibition of

28、apoptosisNo direct structural alteration in DNA by agent or its metabolites,Cellular and Molecular Mechanisms in Multistage Carcinogenesis: PROGRESSION,Irreversible enhancement/repression of gene expression Complex

29、genetic alterations (chromosomal translocations, deletions, gene amplifications, recombinations, etc.) Selection of neoplastic cells for optimal growth genotype/ phenotype in response to the cellular environment,“Compl

30、ex” genetic changes,致癌過程不同階段的特征,Initiation DNA modification, Mutation, Genotoxic One cell division necessary to lock in mutation Modification is not enough to produce cancer Nonreversible, Single

31、 treatment can induce mutationPromotion No direct DNA modification, Nongenotoxic, No direct mutation Multiple cell divisions necessary, Threshold Clonal expansion of the initiated cell population

32、Increase in cell proliferation or decrease in cell death (apoptosis) Reversible, Multiple treatments (prolonged treatment) necessaryProgression DNA modification, Genotoxic event? Mutation, chromosome disar

33、rangement , Irreversible Changes from preneoplasia to neoplasia benign/malignant Number of treatments needed with compound unknown,,,遺傳毒物對(duì)DNA和非DNA相互作用的機(jī)制,,,,,,,正常細(xì)胞,遺傳改變（突變）,,遺傳改變的細(xì)胞,癌細(xì)胞,異倍體細(xì)胞,,,,,,表遺傳干擾,,,,,,,,

34、,,,遺傳毒性致癌物,非遺傳毒性致癌物,遺傳毒性和非遺傳毒性致癌物在多階段致癌中的區(qū)別,遺傳毒性和非遺傳毒性致癌物作用機(jī)制的比較,化學(xué)致癌的生物學(xué)特征致癌物多數(shù)具有遺傳毒性，遺傳毒性致癌物盡管化學(xué)結(jié)構(gòu)和性質(zhì)不盡相同，但有一共同的特點(diǎn)，即皆為親電子劑。,,致癌作用依賴于化學(xué)致癌物的劑量,大劑量的致癌物可增強(qiáng)腫瘤的發(fā)生, 縮短潛伏期.腫瘤的產(chǎn)生取決于化學(xué)致癌物的總劑量。同時(shí)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(dá)需相當(dāng)長(zhǎng)的時(shí)

35、間, 無論致癌物的劑量和性質(zhì)如何，在腫瘤形成前，總有一個(gè)潛伏期。在細(xì)胞惡變以前，細(xì)胞存在著多階段的癌前病變。,致癌作用所引起的細(xì)胞變化可傳到子細(xì)胞 致癌作用可被非致癌因子所修飾 細(xì)胞增生是細(xì)胞癌變過程的重要階段,The development of cancer is a multi-step process,“Initiation” forms an early adenoma“Promotion” leads to a la

36、te adenoma“Progression” leads first to a cancer in situ, then on to … “Malignant conversion,” which leads to true a carcinomaThis set of processes often takes YEARS,Genotoxic carcinogens,increase tumour frequency in a

37、nimal cancer bioassaypositive results from in vitro and in vivo genotoxicity testseither direct-acting or indirectly acting genotoxic carcinogens,Non-genotoxic carcinogens,usually act as tumor promoterspositive in can

38、cer bioassay in animals, but negative in genotoxicity tests The mechanism of carcinogenicity may includethe chronic injury and regenerationhormonal mechanismsincrease in the cell proliferation or decrease in the cell

39、 death in target organ,The concept of a “complete” vs. an “incomplete” carcinogen,When one foreign chemical is suffient to cause cancer, either as a direct or indirect carcinogen, it is said to be completeWhen it requir

40、es a tumor promoter to cause cancer, it is an incomplete carcinogenPromotors are compounds that induce cells, like the mutated cancer initiator cell, to grow and divide, making more,ICH Guideline S1B onTesting for Carc

41、inogenicity of Pharmaceuticals,choosing one 2-year rodent carcinogenicity study (rat) plus one other study that supplements the 2-year study and providing additional information that is not readily available from the 2-y

42、ear study: either (1) a short- or medium-term in vivo rodent test system or (2) a 2-year carcinogenicity study in a second rodent species (mouse).,the short- or medium-term models was intended to focus on the use

43、of in vivo models providing insight into carcinogenic endpoints such as initiation–promotion rodent models and models of carcinogenesis using transgenic or neonatal rodents,藥物致癌性評(píng)價(jià)方法,Stipulated Rationale for Choosing a S

44、hort- or Medium-Term Test System as Supplement to One 2- Year Bioassay,? The mechanism of carcinogenesis in the model should most likely be relevant to humans, and therefore the use of the model should be applicable to h

45、uman risk assessment.? The use of the model should supplement the 2-yearcarcinogenicity study and it should provide additional information that is not readily available from the 2-yearstudy.? Animal welfare, animal n

46、umbers, and overall economy of the carcinogenic evaluation process should be considered.,Two-year Carcinogenesis “Bioassay” Protocol,Current Global Carcinogenicity Study Requirements,Standard Tissue List,Kidney

47、 Urinary bladder AortaHeart Trachea LungsLiver Gallbladder

48、 PancreasFat Salivary gland SpleenCervical lymph node Mesenteric lymph node ThymusTongue Esophagus

49、 StomachDuodenum Jejunum IleumCecum Colon Mammary glandSkin Skeletal

50、muscle Sciatic nerveParathyroid Thyroid Adrenal glandPituitary Prostate Seminal v

51、esiclesTestes Epididymides OvariesOviducts Uterine horns Uterine bodyCervix Vagina

52、 BrainSpinal cord Sternum Rib/boneEyes Harderian glands BM smearNares

53、 Clitoral/preputial gland Zymbal ’ s glandGross lesions,美國(guó)毒性病理學(xué)會(huì)（STP）建議致癌試驗(yàn)進(jìn)行組織病理學(xué)檢查的最基本的受檢內(nèi)容目錄,Tumor - Bearing Animals in Control Groups from Rodent Studies,Source : J. K. Haseman (unpublished summary

54、 of U.S. NTP data).,Comparative Percent Incidence of Pertinent Neoplasia in Different Strains of Rats and Mice (104 Weeks Old),Note : F344, Fischer 244 rats; S- D, Sprague– Dawley rats; B6C3F1, mice, (C57BL/6N+C3H/HeN)F1

55、; CD- 1, 1CRCr: CD- 1 mice; NA, nonapplicable; the average number used by species/strain/gender was in excess of 750 animals,Preclinical approaches for assessing carcinogenic potential,Tumorigenicity in humans, nonhuman

56、primates and rodents,Spontaneous tumor rates in the breeder and control animals,The Neonatal Mouse,Pietra et al. (1959).The neonatal mouse is one of the alternative in vivo models, for detecting the carcinogenic potenti

57、al of pharmaceuticals. This is in agreement with the suggestions of ICH, which allows the use of one alternative study in place of one of the 2-year carcinogenicity studies.When treatment begins within the first 24 hour

58、s of life, the study design is described as “newbornmouse”. “neonatal mouse” includes test item administration at different timepoints from birth to three weeks of age.Fujii (1991) reported that the neonatal mouse assa

59、y showed a sensitivity of 85% and a positive prediction rate of 96% compared to the results of the adult mouse 2-year carcinogenicity study.,Flammang et al. (1997) considered this model to have high sensitivity and speci

60、ficity to detect genotoxic carcinogens as well as presenting advantages such as reduced test article requirements, decreased animal numbers and costs and a reduced completion time. It does not respond to chemicals acting

61、 via epigenetic mechanisms. McClain et al. (2001) reported that neonatal mice have been shown to have a reduced time for tumor induction, a higher multiplicity of induced tumors, a lower spontaneous tumor rate and an eq

62、uivalent or higher sensitivity to carcinogens when compared to adult mice. This model also responds to a wide range of structurally dissimilar genotoxic compounds. Additionally, the neonatal mouse possesses the majority

63、of the phase I and II biotransformation liver enzymes involved in the processes of activation and detoxification of carcinogens from different chemical classes.,CD-1 mice 10 to 12 weeks of age. Mice were caged with 5 fem

64、ales per male and examined each day for the presence of a vaginal copulation plug. Females were isolated until delivery, 6 litters with 4 neonates /sex/ litter were assigned to each group during the first week after birt

65、h. Three or four dose levels, a vehicle and a positive control were used. Groups consisted of 24 animals/sex/group.They were dosed on the basis of their average bodyweight, on days 8 and 15 of age, using dose volumes

66、of up to 100 and 200 μl, respectively. Dose levels were selected on the basis of the results obtained in dose range finding studies, in which the MTD or the MFD (Maximum Feasible Dose) for neonatal mice, were determined.

67、 The pups were weaned around 22 days of age, housed 4/sex/cage and then maintained until 1 year of age, when they were sacrificed. DEN (diethylnitrosamine) at a dosage of 2 mg/kg dissolved in water was used as the posit

68、ive control.,,,,Tg.AC (v-Ha-ras) Transgenic Mouse,8–9 weeks oldGroups of 15 mice/sex/dose were randomly assigned to the study groups.The vehicles used for drugs and positive control agents were acetone, ethanol or