长期毒性试验,目的意义 基本内容和要求 有关问题 Mechanic? Innovative ?,目的意义,毒性剂量 Toxic dosage 安全范围 Safety margin 毒性反应 Toxic reaction: 性质、程度、量毒关系、产生时间，达峰时间, 持续时间及反复产生毒性反应时间、迟发性、蓄积性、耐受性 毒性反应靶器官 Target organ 毒性反应是否可逆 Reversibility,Procedures,Animals Housing Test article Route Dosage and group Administration duration Observation,Procedures 1- animals,rodent non-rodent rat beagle dog mouse mongrel dog monkey,基本内容和要求 - 动物,二种动物，啮齿类首选大鼠，非啮齿类用犬(Beagle犬), 必要时用猴 动物应写明供应单位及邮编，品系及生产合格证号 大鼠6-8周龄，体重差异不超过平均体重的20。3个月以上可选5-6周龄大鼠 Beagle犬4-12月龄 各半,Procedures 2- housing,Diet, T, humidity, light, ventilation Rat: 5 /cage or separated， acclimation 1w Dog: TB test, anthelmintic acclimation: 1-2w / 1m,Procedures 3- test article,test article / solvent / excipient name, batch No. , source, specification, purity, preserve condition, etc same quality criterion,基本内容和要求 - 给药途径,与临床给药途径一致 混入饲料或饮水中灌服时 保证受试药分布均匀 在配制饲料或溶于饮水后仍旧稳定 计量准确 临床iv 时, 大鼠给药周期1月，可用ip代替，im或sc应变换注射部位 必要时改变给药途径,基本内容和要求 - 剂量及分组,三个给药组, 单位: mg(ml,IU) /kg 或 /m2 以不等浓度等容量给药 低剂量组-高于有效剂量，不出现毒性反应 中剂量组-产生轻微或中等度毒性反应 高剂量组-产生明显或严重的毒性反应，或个 别动物死亡 空白对照-溶媒或其它赋形剂，若溶媒或赋形 剂有毒性时则增加正常对照组,基本内容和要求 - 剂量及分组, 5 g/kg, iv 2g/kg仍无毒性反应和死亡或仅个别有毒性反应, 可只做一个50ACD(犬用30ACD)的剂量组,Procedures 6- ad. duration,Tab. Correspond duration of clinical ad. with animal ad. clinical ad. duration animal ad. duration 13d 14d 1w 1m 4w 3m 1m 6m duration 3m ad. 6 day weekly(mon. to sat. ),Procedures 7- Observations Clinical Examinations,rodent non-rodent signs of ill health +pupil , T behavioral changes breath, sialosis reaction to treatment vomiting Dead or moribund animal: necropsy within 1h,Procedures 7- Observations Hematology,Red blood cell count(RBC) Hemoglobin(Hb) Erythrocyte indices (MCV, MCH, MCHC) Hematocrit(Hct) Platlet count White blood cell count and percent differential Coagulation time(CT) Reticulocyte count, Bone marrow smear,Procedures 7- Observations Clinical chemistry,Alanine aminotransferase (ALT) （丙） Aspartate aminotransferase (AST)（草） Alkaline phosphatase (ALP) Total bilirubin（Tbill） Blood urea nitrogen（BUN） Creatinine（Cr） Total protein（TP） Albumin（Alb） Glucose (Glu) Total cholesterol （Tch）,Procedures 7- Observations Urinalysis,Color and appearance pH glucose ketones urobilinogen blood specific gravity nitrite protein bilirubin WBC microscopy of centrifuged deposit,Procedures 7- Observations Pathology,Organ weight assessment: heart liver thyroid lobes and spleen lung parathyroids kidneys brain (lobes weighed pituitary adrenals together) ovaries testes calculate organ weights relative to body weight , paired organs weigh together,Procedures 7- Observations Pathology,Histopathological examination: heart liver spleen lung kidney brain adrenals pancreas stomach duodenum jejunum ileum colon cecum（盲） pituitary thymus spinal cord prostate testes uterus ovaries optic nerve injection site bone and marrow(sternum) urinary bladder lymph nodes(mesenteric) thyroid lobes( and parathyroids),Procedures 7- Observations Pathology,tissues (c+h embedding dead+ sectioning target) staining examination,基本内容和要求 - 检测指标 心电图导联,大动物长期毒性必须做 主要观察指标：心率、P波电压、P-R间期、R波电压、QRS时间、ST段、T波电压和Q-T间期,Procedures 7- Observations Recovery,Observation : 1/31/2 animal, 24w Late toxic reaction and reversibility Items : same as treatment period Not reversible?,Procedures 7- Observations Frequency of lab. investigations,rat 3m Non-rodent last ad. interim twice pretreatment recovery end last ad. once every 1-1.5m recovery end last ad. recovery end,基本内容和要求 - 资料整理,首页写明: 负责者的姓名、职称、实验开始及结束日期、实验承担单位、研究室负责人和资料保存单位 试验指导者、课题负责人、病理学检查、血液生化指标测定、血液学指标测定及给药观察者 第二页书写试验摘要 第三页开始写正文、引言、材料与方法、结果 合理的统计图、表 有差异的指标应进行统计处理 应客观地描述尸检及组织病理学检查结果 对各种异常现象可用半定量的方式表述，揭示量-时关系,Problems,Pretest Dosage Animal & Housing Route Duration Observation Parameters Data handling Archives,Problems 1- pretest,What depend on? Overseas pre-clinical / clinical data Acute toxicity , pharmacodynamics and pharmacokinetics or intended clinical dose How long? 2w - 1m Recombinant article: antibody?,Problems 2- dosage,LD50 MTD MBS ACD 3m 6m 1/10 1 10Cmax 50100 30 50 1/50 1/3 5Cmax 3050 1525 1/100 1/10 Cmax 1020 510 half for dog and monkey,有关问题2-剂量,表 常用动物等效剂量折算表 项 目 人 犬 猴 兔 豚鼠 大鼠 小鼠 成年时体重(kg) 70 12 4 1.5 0.4 0.2 0.02 换算常数(k) 12.3 11.6 11.8 12 10.5 10.5 10 每公斤用药量折算(mg/kg) 1 1.5 2.2 3.1 4.3 5.4 11.