药物的致癌性

药物致癌性的研究现状和动态,第二军医大学药物安全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2010年年发病数220万，2020年为300万。,Age-adjusted Cancer Death Rates, by Site, US, 1930-2005,1957、1984、1999、2004年我国城市主要疾病死因构成比及死因顺位,特别严重的是,肿瘤是我国最佳劳动人口的首要死因，在3559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。,What may cause cancer ?,Hereditary disorders Chemicals Viruses Chronic inflammation ?,WORLD HEALTH ORGANIZATIONINTERNATIONAL AGENCY FOR RESEARCH ON CANCERIARC Monograph EvaluationsLYON, FRANCE,Slide courtesy of V. Cogliano (IARC),IARC (2009) - monographs.iarc.frCarcinogenic to humans (group 1) 108 agentsProbably carcinogenic to humans (group 2A) 66Possibly carcinogenic 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,Medical drugs and treatments24Industrial processes13Infectious agents or processes10Physical agents10Industrial chemicals 7Inhaled particulates 5Metals and inorganic salts 5Lifestyle 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 effects of multiple exposuresExamples:Alcohol drinking and aflatoxinsAlcohol drinking and HBV/HBCAlcohol drinking and tobacco smokingTobacco smoking and asbestos/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。,The European Centre for the Validation of Alternative Methods (ECVAM),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 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 highest 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. FDA 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 tests in detecting rodent carcinogens as analyzed,In vitro genotox testing: the problem,poor specificity,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,Ames,MLA AmesMN MN,Indomethacin(吲哚美锌) tested negative for 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 lymphocytes and induction of sperm head abnormalities in mice, respectively, which are considered non-regulatory tests.,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,药物致癌试验,2010年04月01日SFDA制定发布了药物致癌试验必要性的技术指导原则 预期临床用药期至少连续6个月的药物一般应进行。 连续用药没有6个月，但以间歇的方式重复使用，如治疗慢性和复发性疾病(包括过敏性鼻炎、抑郁症和焦虑症)，而需经常间歇使用的药物，一般也需进行。 某些可能导致暴露时间延长的释药系统，也应考虑进行致癌试验。,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 (chemical carcinogenesis) 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(chemical carcinogen).,化学致癌研究的重要历史事件,1761, J Hill 提出使用鼻烟可能会诱发鼻咽癌1775, P Pott 提出扫烟囱男童阴囊癌与煤烟过度暴露有关1895 , L Rehn 首次报道从事苯胺染料生产的工人发生膀胱癌1914, T Boveri 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, K Ichikawa 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; 1949 Foulds提出肿瘤演进(Progression) 概念 1971-1981, C Peraino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-, A Balmain, 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,Initiating Event,Cell Proliferation(clonal expansion),Progression,Cell Proliferation,Cell Proliferation,Malignancy,Second Mutating Event,N Mutating Event,Initiation,Promotion,Stages of Carcinogenesis,Cellular and Molecular Mechanisms in Multistage Carcinogenesis: INITIATION,Initiating event involves cellular genome MUTATIONSTarget genes: - oncogenes/tumor suppressor genes - signal transduction - cell cycle/apoptosis regulators,“Simple” genetic changes,Gentic and Epigenetic Models of The Cancer Initiation,Epigenetically reprogrammed cells,Mutator phenotype cells,Endogenous,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 MUTATIONS,Normal cells,Cancer cells,Cellular and Molecular Mechanisms in Multistage Carcinogenesis: PROMOTION,Reversible enhancement/repression of gene expression:- increased cell proliferation- inhibition of 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 genetic alterations (chromosomal translocations, deletions, gene amplifications, recombinations, etc.) Selection of neoplastic cells for optimal growth genotype/ phenotype in response to the cellular environment,“Complex” genetic changes,致癌过程不同阶段的特征,Initiation DNA modification, Mutation, Genotoxic One cell division necessary to lock in mutation Modification is not enough to produce cancer Nonreversible, Single treatment can induce mutationPromotion No direct DNA modification, Nongenotoxic, No direct mutation Multiple cell divisions necessary, Threshold Clonal expansion of the initiated cell population Increase in cell proliferation or decrease in cell death (apoptosis) Reversible, Multiple treatments (prolonged treatment) necessaryProgression DNA modification, Genotoxic event? Mutation, chromosome disarrangement , Irreversible Changes from preneoplasia to neoplasia benign/malignant Number of treatments needed with compound unknown,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,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,The development of cancer is a multi-step process,“Initiation” forms an early adenoma“Promotion” leads to a late 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 animal 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 cancer 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 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 requires 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 Carcinogenicity 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-year 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 of in vivo models providing insight into carcinogenic endpoints such as initiationpromotion rodent models and models of carcinogenesis using transgenic or neonatal rodents,药物致癌性评价方法,Stipulated Rationale for Choosing a Short- 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 human 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 numbers, 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 Urinary bladder AortaHeart Trachea LungsLiver Gallbladder PancreasFat Salivary gland SpleenCervical lymph node Mesenteric lymph node ThymusTongue Esophagus StomachDuodenum Jejunum IleumCecum Colon Mammary glandSkin Skeletal muscle Sciatic nerveParathyroid Thyroid Adrenal glandPituitary Prostate Seminal vesiclesTestes Epididymides OvariesOviducts Uterine horns Uterine bodyCervix Vagina BrainSpinal cord Sternum Rib/boneEyes Harderian glands BM smearNares Clitoral/preputial gland Zymbal s glandGross lesions,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 - Bearing Animals in Control Groups from Rodent Studies,Source : J. K. Haseman (unpublished summary 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; 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 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 potential 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 hours 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 assay 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 specificity 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 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 equivalent 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 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 females 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 birth. 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 of up to 100 and 200 l, 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. 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 positive control.,Tg.AC (v-Ha-ras) Transgenic Mouse,89 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 DMSO. TPA (12-o-tetradecanoylphorbol-13-acetate) was the positive control compound26 weeks,Hemizygous p53 +/ Knockout Mouse6 to 10 weeks old, Genotype analysis was recommended prior to assignment to dose groupsGroups of 15 mice/sex/group, Three dose levels, a vehicle and a positive control were used. Two additional groups of 15 wild-type mice/sex/group received the vehicle and the high dosage of the test compound.p-Cresidine at 400 mg/kg/day by gavage in corn oil (10ml/kg), or benzene at 100 mg/kg/day by gavage in corn oil (5 ml/kg) were recommended as positive controls.18 to 24 month bioassay,car