1、新药安全药理学研究与进展,第二军医大学药物安全性评价中心 陆国才 袁伯俊,内 容,安全药理学：研究药物治疗范围内或治疗范围以上的剂量时，潜在不期望出现的对生理功能的不良影响次要药效学：主要研究药物非期望的、与治疗目的不相关的效应和作用机制,概 念,SFDA要求,核心组合试验(Core Battery) 中枢神经系统、心血管系统和呼吸系统研究追加的安全药理学研究（Follow-up） 对中枢神经系统、心血管系统和呼吸系统进行深入的研究 补充的安全药理学研究（Supplemental） 研究药物对三大系统以外器官(如泌尿系统、自主神经系统、胃肠道系统和其它器官组织)功能的影响,概 念,SFDA要求
2、,确定药物可能存在的非期望药效作用评价药物在毒理学和/或临床研究中所观察到的药物不良反应和/或病理生理作用研究所观察到的和/或推测的药物不良反应机制,目 的,SFDA要求,有助于了解新药的药理作用特点和机制有助于发现新药理作用，一药多用，药尽其用为毒理学研究打下基础，是各种毒性实验的必要补充,目 的,SFDA要求,贯穿在新药研究全过程中，可分阶段进行在药物进入临床试验前，应完成Core Battery Studies追加和/或补充的安全药理学研究可在申报生产前进行,阶段性,SFDA要求,内容要求,SFDA要求,神经系统 运动功能、行为改变、协调功能、感觉运动反射和体温等的变化(小鼠)心血管系统
3、 给药前后血压（包括收缩压、舒张压和平均压）、心电图（包括QT间期、PR间期、ST段和QRS波等）和心率等变化(Beagle犬)呼吸系统 呼吸频率和呼吸幅度(Beagle犬)根据情况增加其它系统或指标,1.动物选择 可采用不同类型动物(正常或模型，清醒或麻醉)。一般多用正常动物2.剂量途径 产生主要药效作用及以上的3个剂量，临床给药途径3.观察时间 药效学和药代动力学特性、受试动物、临床研究方案等因素选择观察时间点和观察时间,SFDA要求,有关问题,4.系统指标中枢神经系统心血管系统呼吸系统根据情况增加其它系统或指标5.体外研究 应确定受试物的浓度-效应关系。无明显影响作用时，应说明浓度选择范
4、围,SFDA要求,有关问题,中枢神经系统：对行为、学习记忆、神经生化、视觉、听觉和电生理等的影响心血管系统：对心输出量、心肌收缩作用、血管阻力等指标的检测呼吸系统：对气道阻力、肺动脉压力、血气分析等指标的检测,SFDA要求,追加安全药理学,泌尿系统：对肾功能的影响，如对尿量、比重、渗透压、pH、电解质平衡、蛋白质、细胞和血生化（如尿素氮、肌酐、蛋白质）等指标的检测自主神经系统：有关受体的结合、体内或体外对激动剂或拮抗剂的功能反应、对自主神经的直接刺激作用和对心血管反应、压力反射和心率等指标的检测,SFDA要求,补充安全药理学,胃肠系统：观察药物对胃肠系统的影响，如胃液分泌量和pH、胃肠损伤、胆
5、汁分泌、离体回肠收缩、胃液pH等指标的测定其它研究：潜在的依赖性、骨骼肌、免疫和内分泌功能等的影响,SFDA要求,补充安全药理学,体内血药浓度低，或其它组织器官分布很少的局部用药（如皮肤、眼科用药）只用于治疗晚期癌症病人的细胞毒类药物，在首次用于临床前可不做一般药理学研究，但不包括具有新作用机制的此类药物,SFDA要求,免做安全药理学,内 容,“Safety pharmacology includes the assessment of effects on vital functions, such as cardiovascular, central nervous system and
6、respiratory systems, and these should be evaluated prior to human exposure.” ICH M3: Nonclinical safety studies for the conduct of human clinical trials for pharmaceuticals. (July 1997)Value of safety pharmacology acknowledged by regulators and sponsors,ICH要求,ICH Guideline M3, 1997,“Safety pharmacol
7、ogy studies . investigate the potential for undesirable pharmacological activity in appropriate animal models and, where necessary, to incorporate particular monitoring for these activities in the toxicity studies and/or clinical studies.”“. these studies may allow for a mechanistically-based explan
8、ation of specific organ toxicities, which should be considered carefully with respect to human use and indication(s).”ICH S6 “Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals” (July 1997)Pharmacological activity in animals should be considered in toxicology and clinical studies
9、Mechanism-based understanding of toxicities (or pharmacological activities) should be carefully considered in the risk assessment,ICH要求,ICH Guideline S6, 1997,“This guidance was developed to help protect clinical trial participants and patients receiving marketed products from potential adverse effe
10、cts of pharmaceuticals, while avoiding unnecessary use of animals and other resources.”ICH S7A “Safety Pharmacology Studies for Human Pharmaceuticals” (July 2001)Safety pharmacology studies have the potential to reduce use of animals and other resources,ICH要求,ICH Guideline S7A, 2001,清醒动物-遥测系统,检测清醒状态
11、下、自由活动动物的安全药理学指标 节省动物符合动物福利要求适合不同给药方式连续动态、长时间检测指标,ICH要求,4 x 4拉丁方实验设计,ICH要求,4 x 8拉丁方实验设计,ICH要求,Drug to increase gastrointestinal (GI) motilityOver 5.4 million prescriptions in 1996 (US)34 patients TdP, 23 long QT (1993-1996) Cardiotoxicity reported when used with Ketoconazole and/or ErythromycinCase
12、report of 8-year old girlintensive care,ICH要求,Cisapride Story,ICH要求,Drug Interactions Leading to QT,Drug to increase gastrointestinal (GI) motilityOver 5.4 million prescriptions in 1996 (US)34 patients TdP, 23 long QT 1993-1996 Cardiotoxicity reported when used with Ketoconazole and/or ErythromycinC
13、ase report of 8-year old girlintensive care4 deaths and 16 resuscitated from 1993 to 1996Drug withdrawn from US market July, 2000,ICH要求,Cisapride Story,FDA recommended removal of Terfenadine containing products from the market in 01/1997FDA issued warning letter to Doctors about cardiac safety of Ci
14、sapridePost-marketing surveillance became more important Medwatch reporting systemSafety pharmacology guidances were approved to look at cardiovascular, respiratory and CNS side-effects of drug candidates ICH S7A and S7BScreening for QT prolongation is now an integral part of drug development (lead
15、optimization to clinical),ICH要求,Regulatory Response,“The study results can be used to elucidate the mechanism of action and, when considered with other information, estimate risk for delayed ventricular repolarization and QT interval prolongation in humans.”ICH S7B: Nonclinical Evaluation of the Pot
16、ential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals (October 2005)Mechanism of action can be a goal of studiesUse data with other data to estimate risk for QT prolongation in humans,ICH要求,ICH Guideline S7B, 2005,Bazett: QTc = QT / RR 0.5 Fridericia: QTc
17、= QT / RR 0.33,ICH要求,QTc Interval,心肌动作电位时的离子通道,ICH要求,IKr: 快速延迟整流K+电流,QT/QTc Prolongation,IKr Inhibition,Delayed Ventricular Repolarization,Arrhythmia,ADDITIONAL RISK FACTORS,IKr inhibitory potency: can help predict risk of QT/QTc prolongation is a biomarker for increased risk of Ventricular Arrhythm
18、ias is not a biomarker for Torsade de Pointes,ICH要求,IKr &TdP,ICH要求,Early after-depolarization（早后去极化） TdP,Drug Class Date WithdrawnTerfenadineAntihistamineFeb 1998SertindoleAntipsychoticDec 1998AstemizoleAntihistamineJun 1999Grepafloxacin AntibioticNov 1999CisaprideGI ProkineticJuly 2000,ICH要求,Drugs
19、Withdrawn for TdP risk,采用离体动物或人体心脏细胞、培养心脏细胞系或克隆人离子通道的异种表达体系测定离子流在离体心脏标本进行动作电位参数测定，或在麻醉动物中进行能体现动作电位周期的特异性电生理参数检测测定清醒或麻醉动物的ECG参数在离体心脏标本或动物进行致心律失常作用测定,QT间期延长非临床研究方法,ICH要求,内 容,2009,趋势展望,安全药理学政策法规不断完善,趋势展望,QT间期研究策略,体外电生理IKr研究采用原代或表达的IKr通道蛋白(如由hERG编码的蛋白)评价药物对离子电流的影响体内对QT的研究从体内测定心室复极化，如QT间期检测结果进行评价化学/药理的分类
20、可诱导人QT间期延长作用的药物如抗精神病类药物，组胺H-1受体拮抗剂、氟喹酮类,QT间期研究策略,趋势展望,追加试验离体心脏 麻醉动物 重复给药试验动物种属和性别的选择用代谢性诱导剂或用拮抗剂用阳性药和参比化合物其他通道的抑制作用在多个时间点测定电生理参数模拟病理状况和心律失常综合风险评估,QT间期研究策略,趋势展望,计算机模拟 (in silico)干细胞定向分化为心肌细胞下丘脑病理切片HERG通道体外筛选新方法荧光筛选(测量膜电位)结合试验 (标记的HERG通道阻滞剂，HTS)斑马鱼HTS结合药代/药效，研究毒理学生物标志，预测临床,安全药理学研究新技术新方法,趋势展望,计算机模拟 (in
21、 silico)干细胞定向分化为心肌细胞下丘脑病理切片HERG通道体外筛选新方法荧光筛选(测量膜电位)结合试验 (标记的HERG通道阻滞剂，HTS)斑马鱼HTS结合药代/药效，研究毒理学生物标志，预测临床,安全药理学研究新技术新方法,趋势展望,PART I MUTAGENESIS AND MUTANTSHighly Efficient ENU Mutagenesis in ZebrafishProduction of Pseudotyped Retrovirus and the Generation of Proviral Transgenic ZebrafishUndertaking a S
22、uccessful Gynogenetic Haploid Screen in ZebrafishCryopreservation and In Vitro Fertilization at the Zebrafish International Resource Center,计算机模拟 (in silico)干细胞定向分化为心肌细胞下丘脑病理切片HERG通道体外筛选新方法荧光筛选(测量膜电位)结合试验 (标记的HERG通道阻滞剂，HTS)斑马鱼HTS结合药代/药效，研究毒理学生物标志，预测临床,安全药理学研究新技术新方法,趋势展望,PART II TRANSGENESISTransient
23、 and Stable Transgenesis Using Tol2 Transposon VectorsAnalysis of Genes and Genome by the Tol2-Mediated Gene and Enhancer Trap MethodsBacterial Artificial Chromosome Transgenesis for ZebrafishSimple and Efficient Transgenesis with Meganuclease Constructs in Zebrafish,计算机模拟 (in silico)干细胞定向分化为心肌细胞下丘脑
24、病理切片HERG通道体外筛选新方法荧光筛选(测量膜电位)结合试验 (标记的HERG通道阻滞剂，HTS)斑马鱼HTS结合药代/药效，研究毒理学生物标志，预测临床,安全药理学研究新技术新方法,趋势展望,PART III TISSUE-SPECIFIC MANIPULATIONSNitroreductase-Mediated Cell Ablation in Transgenic Zebrafish EmbryosFocal Electroporation in Zebrafish Embryos and LarvaeTissue Micromanipulation in Zebrafish Emb
25、ryos,计算机模拟 (in silico)干细胞定向分化为心肌细胞下丘脑病理切片HERG通道体外筛选新方法荧光筛选(测量膜电位)结合试验 (标记的HERG通道阻滞剂，HTS)斑马鱼HTS结合药代/药效，研究毒理学生物标志，预测临床,安全药理学研究新技术新方法,趋势展望,PART IV ANALYZING GENE EXPRESSIONZebrafish Spotted-Microarray for Genome-Wide Expression Profiling Experiments. Part I: Array Printing and HybridizationPart II : Da
26、ta Acquisition and AnalysisValidating microRNA Target Transcripts Using Zebrafish Assays,计算机模拟 (in silico)干细胞定向分化为心肌细胞下丘脑病理切片HERG通道体外筛选新方法荧光筛选(测量膜电位)结合试验 (标记的HERG通道阻滞剂，HTS)斑马鱼HTS结合药代/药效，研究毒理学生物标志，预测临床,安全药理学研究新技术新方法,趋势展望,PART V IMAGINGMultiple Embryo Time-Lapse Imaging of Zebrafish Development Live C
27、ell Imaging of Zebrafish LeukocytesImaging Zebrafish Embryos by Two-Photon Excitation Time-Lapse MicroscopyMorphological Analysis of the Zebrafish Digestive SystemIn Toto Imaging of Embryogenesis with Confocal Time-Lapse Microscopy,High sensitivityHigh concentration in myocardium after myocardial in
28、juryRapid release for early diagnosisLong half-life in blood for late diagnosisHigh specificityAbsent in non-myocardial tissuesNot detectable in blood of non-diseased subjects,生物标志物,Ideal Cardiac Biomarker,Analytical characteristicsMeasurable by cost-effective assaySimple to performRapid turnaround timeSufficient precision and truenessClinical characteristicsAbility to influence therapyAbility to improve patient outcome,生物标志物,Ideal Cardiac Biomarker,Biomarkers of cardiac disease,生物标志物,趋势展望,小 结,New, Robust, Reliable, Reproducible, Predictive assay,谢谢!,
