Hoechst Marion Roussel, Inc.

同写意20周年大会：中国医药创新未来之路！“意”同创新，穿越周期，赢取未来。7月中旬，苏州金鸡湖畔，让我们与“新药写意人”一起为中国医药创新“同写意”！ 安全药理学研究药物和生物制品对核心生理器官功能造成不良药效学影响的可能性。重点是研究这些产品类型（在非临床开发过程中可能以治疗浓度或高于治疗浓度给药）的药理学如何影响三个重要器官系统的功能--心血管（Cardiovascular CV）系统、呼吸系统和中枢神经系统（Central Nervous System，CNS）。对这三个重要器官系统的研究共同构成了安全药理学核心课程。次级器官系统包括胃肠道系统、肾脏系统、泌尿系统和自主神经系统以及其他系统等，在必要时也可进行评估。 不良药效学效应可能是由于受试产品的靶向一级药效学效应，可能与给药剂量、给药途径和频率以及全身暴露量有关，也可能是由于称为二级药效学的脱靶效应。安全药理学核心组合（core battery）包括的三个主要重要器官系统在生理上相互关联，这意味着在一个系统中出现的效应可能会在其他系统中引起适应性反应。因此，解读安全性药理学数据通常需要对器官功能的整体影响进行综合风险评估，并考虑目标临床人群、临床试验计划和安全系数。本章将讨论安全性药理学研究的时间安排，这与预期的患者人群相一致。 安全药理学已成为一门学科，在对新产品进行首次人体试验之前，通过开展核心组合研究来预测不良药效学风险。安全药理学研究有助于从效益风险较低的药品中筛选出安全性较高的药品，此类药品或很少或没有潜在的心血管、呼吸或中枢神经系统影响，或具有足够的安全系数。值得注意的是，如果在临床试验中出现不良反应，或者如果已上市产品被重新用于新的适应症、患者人群，或者以新的制剂或途径给药，而在此之前不需要进行安全药理学评估，那么处于开发后期的产品也可能需要进行安全药理学评估。 本章提供的信息主要基于美国食品药品管理局（FDA）、欧洲药品管理局 （EMA）和人用药品委员会（CHMP）的文件，这些文件讨论了国际人用药品技术要求协调理事会（ICH）关于人用药品安全药理学研究的安全 （S）指南7A。ICH S7A指导方针规定，所有人用药品（小分子、生物技术衍生药物或生物制品）在首次人体临床试验期间的用药安全性应达到合理水平。 因此，根据良好实验室规范（GLP）进行的安全性药理学研究的评估，在治疗范围及以上，人体首次用药预计不会对重要器官的生理功能产生任何不良的药效学影响。安全药理学研究的时间安排与FDA和EMA/CHMP就 ICH M3（R2）关于开展人体临床试验和药品上市许可的非临床安全性研究的指导意见相一致。目前，细胞和基因治疗产品或组织工程产品在安全药理学指南中没有专门论述。 本章还将讨论最近批准并实施的ICH E14/S7B Q&A指南，该指南概述了如何接受体外hERG（人类ether-a-go-go相关基因，human ether-a-go-go related gene）和体内QT间期研究数据（即：非临床、双阴性），均根据Q&A最佳实践建议进行，以支持两种临床场景（E14 Q&A 5.1和6.1），以申请豁免全面QT（TQT）研究。本章末尾提供了案例研究，以展示如何在实践中开展安全药理学研究。 1 安全药理学试验的一般原则和重要性 安全药理学指南于2000年首次制定，以应对抗组胺药物特非那定对心血管系统影响的不良报告。特非那定于1985年由Hoechst-Marion Roussel公司（现赛诺菲-安万特公司）引入市场，在美国以Seldane、英国以 Triludan和澳大利亚以 Teldane 等名称销售，用于治疗过敏性鼻炎。后来发现特非那定是钾离子通道（IKr 或 hERG）的强效抑制剂，会导致QT间期延长。这有可能发展为多形性室性心动过速，即Torsades de Pointes（TdP），字面意思是心电图等电位线周围的点或峰（R 波或 QRS 波群）扭曲。 在临床试验之前，特非那定的心脏毒性潜能尚未被发现，因为这种药物完全由肝酶细胞色素P450 3A4（CYP3A4）代谢，因此在血浆中的浓度未被检测到。此外，其代谢物特非那定羧酸盐（也称作非索非那定）对钾离子通道没有抑制作用，即使其浓度比特非那定的hERG IC50高30倍。然而，当患者患有影响 CYP3A4酶的肝病或正在服用抑制CYP3A410的其他药物时，原药特非那定的促心律失常潜能就会在高剂量下显现出来，因为这会导致代谢降低，从而使特非那定的血浆浓度升高，而非索非那定的浓度降低。 非索非那定的疗效与特非那定相似，但由于它只对hERG通道有轻微抑制作用，因此诱发心律失常的可能性要低得多。1996年，非索非那定以Allegra13的名称上市，用于治疗过敏性鼻炎和慢性荨麻疹等疾病，并被列入世界卫生组织的基本药物清单，而特非那定则因可能导致心律失常而于1997年退出市场。 因此，小分子或生物制品对所有三个核心器官系统(心血管、呼吸、中枢神经系统)的潜在不良影响必须在进行首次人体研究之前进行。虽然ICH S7A提供了核心组合研究的指导和收集的功能终点，但它没有概述研究设计本身。下一节将讨论体外和体内研究的安全药理学研究设计，并提供示例，可以根据评估中的药物或生物药物的药理学（或化合物类别）、途径和剂量、适应症和预期患者群体准确地应用或修改。 2 核心组合研究设计和终点 在测试新产品的安全药理学时，首先需要测试三个核心器官系统的潜在不良药效学效应：心血管系统、呼吸系统和中枢神经系统（CV system, the respiratory system, and the CNS）。 3 心血管系统 评估对心血管系统影响的研究类型包括： ▪ 体外研究（如心脏离子通道研究） ▪ 体外研究（如 Langendorff 技术） ▪ 体内研究（如啮齿和非啮齿动物模型遥测技术） 4 体外心血管研究 体外心血管检测大多涉及心脏离子通道的研究，法规要求进行hERG检测（Table 5-1）。其中一种检测方法是自动hERG检测，用于确定药物抑制 hERG 钾通道的潜力。在药物开发的早期阶段，使用单一浓度的供试品，这种检测法可作为筛选工具，在结构-活性关系研究过程中和确定先导化合物之前，识别出具有潜在hERG倾向的化合物。对发现(有倾向)的hERG试验进行后续跟进，使用剂量递增的供试品浓度来估算其IC50，即抑制生物效应50%所需的药物浓度。然后再按照GLP规定进行严格的手动膜片钳hERG试验。 最近发布的S7B Q&A要求跟进体外hERG试验。然后在过表达人心肌离子通道（S7B Q&A 2.1）的人类胚胎细胞或中国仓鼠卵巢细胞上进行实验因素的最佳实践，其中包括钾、钙和钠通道。ICH指南中有关实验因素的最佳实践问答要点包括： ▪ 在接近生理温度（35-37°C）下进行手动膜片钳实验； ▪ 测量心室动作电位电压方案； ▪ 监测过表达心脏通道细胞的健康状况； ▪ 记录密封电阻质量；以及 ▪ 报告 IC50 值和希尔系数 此外，经过验证的分析方法应支持浓度验证，并应将多非地利、昂丹司琼和莫西沙星这三种阳性对照品中的一种作为参照药物进行评估。关于后者，应进行重复评估，每次实验应使用两种或两种以上浓度的供试品，每种浓度至少包括四个细胞。 在考虑了血浆蛋白结合和使用血浆中的游离药物浓度后，计算出的IC50抑 制浓度的安全系数应大于预期临床暴露浓度的50倍。本章稍后提到的 "体外原发性心律失常综合分析"（CiPA）计划建议并纳入了对其他心脏离子通道的评估。 在评估肿瘤产品时，由于肿瘤产品的收益风险比可能高于其他药品，因此可以在不遵守GLP法规的情况下进行hERG和心脏离子通道抑制测定。对于生物制品或生物技术产品，靶点特异性通常很高（target specificity is usually very high），因此不需要但建议（not required but are recommended）进行体外 hERG 检测。在评估这些生物制品时，可将体内心血管终点整合到使用非侵入性遥测技术进行的重复给药毒理实验中（Table 5-2）。 5 体外朗根多夫技术 朗根多夫技术是一种体外试验，即用含氧缓冲克雷布斯溶液灌注动物离体心脏，在其中加入或不加入待测药物。其目的是观察药物对心电图参数、心率、左心室力学和冠状动脉血流估计值的影响。通常使用豚鼠或兔子心脏，因为它们的心脏具有IKr钾离子通道，而这是人类心室复极化（即心电图的QT间期）的主要心脏离子通道。研究表明，使用体外检测法，QT延长药物对人体的影响是一致的。将离体灌注豚鼠（啮齿动物）心脏测定法转化为大型动物QTc测定法，用于测定已知可延长QTc的药物和 13种已知对QTc间期无影响的药物的QTc变化。 实施该技术时，先移除动物的心脏，然后以逆行方式通过主动脉导入灌注液；这样就能通过冠状动脉输送含有载体或药物的富氧、营养灌注液。记录的终点包括从放置在心外膜表面的导联II配置电极上记录的心电图参数、心率、左心室收缩参数（如心室压力的最大上升率和最大下降率）以及平均冠脉流量。 通过检查心肌、平滑肌和冠状动脉血管内皮，可以检测试验药物对心肌功能（电活动和收缩活动）和冠状动脉血管的影响。尽管朗根多夫技术有一些局限性（如无法评估代谢物、分离超过4至6小时的心脏功能活力等），但它既可作为心血管疾病筛查试验提供快速信息，也可在需要对早期发现试验的结果（如心脏受体的受体结合试验或hERG结果）进行进一步检查时提供快速信息，因为该试验可生成心电图，用于评估间期（如：RR、PR、QRS、QRS）、 RR、PR、QRS、QT和计算出的QTc间期、心律和左心室压力曲线。 6 体内心血管研究 体内心血管研究包括通过手术植入遥测装置对自由活动（非啮齿类动物，如犬、非人灵长类或小型猪）动物的心血管系统进行评估。此类装置有多种生物电位和压力导管配置，也有用于测量呼吸功能的阻抗导线可供选择。有多种遥测装置可供选择，既可用于短期研究，也可用于长期研究，还可在多项研究中重复使用（有足够的洗脱期），通常以发射器的电池寿命为基础。植入式遥测使用血管内放置的实心心电图电极，具有高分辨率或低信噪比。这样就可以在采集后的分析过程中准确地在心电图间隔上放置信托标记。 其他选择包括非侵入性外套式体外遥测（JET），该方法将表面电极与小型植入式遥测装置配对，用于收集动脉血压（符合ICH S7A CV 研究要求），其中电气元件固定在外套口袋。Table 5-2列出了这些研究中收集的CV终点。使用内置仪器对动物进行遥测研究非常方便，因为动物可以自由移动，而且一旦手术植入并恢复，只要之前的测试材料已从动物体内清除，且研究之间有足够的洗脱期，动物就可以在多项CV试验中重复使用。 当植入的遥测设备同时记录ICH安全指南7A中规定的心血管和呼吸参数时，研究被称为心肺研究。独立的心血管或心肺功能研究通常在非啮齿类动物中进行，采用4 x 4拉丁方交叉设计，即在洗脱期（通常为3-7天）后，以随机方式给每只动物以对照品和低、中、高剂量的三个剂量水平的试验药物。该设计的统计效力优于平行或递增剂量设计，因为每只动物都是自己的对照组，而且每次试验都包括所有剂量水平，从而减轻了序列效应（表5-3）。这种设计使用的动物数量最少（总共四只），符合动物使用的3R原则--替代（replace）、优化（refine）、减少（reduce）--并可根据分组饲养选择或提高统计能力而进行修改。 半衰期较长的供试品(如寡核苷酸、单克隆抗体和生物制剂)洗脱期超过7天时，可采用平行研究设计。该设计由四组（每组4 - 6只）动物组成，可纳入独立遥测研究(安全药理学环境)，或者将CV安全药理评估整合到平行的重复给药毒理实验中（毒理学环境）。只有在暴露量没有性别差异的情况下，才可以使用雄性;否则，需同时使用雄性和雌性。恢复组通常只有对照组和高剂量组，因此使用4只动物，在低剂量组和中剂量组中没有恢复期动物。 考虑因素包括植入式遥测术的手术安排和植入成本。在某些情况下，基于总体方案考虑，逐步升级的研究设计也可以适用于动物依次接受载体对照、低、中、高剂量选择（表5-4）。安全药理学评估供试品的急性效应，因此建议在单次给药后第1天收集CV参数，如果将其整合到重复给药毒理实验中。如果供试品的动力学允许，当第1天的所有活动完成后，可以在第2天收集CV参数，因为第1天给药和所有其他活动（如毒代动力学采血、临床病理学和临床观察）会对CV参数造成不利影响。 心血管研究测量动脉血压（收缩压、舒张压和平均动脉压）、心率（次/分）、第二导联心电图参数（心电图间期和节律）和体温。定性心电图分析包括波形形态（P 波、QRS 波和 T 波）和任何心律失常的描述，定量测量包括心电图间期持续时间（RR、PR、QRS和QT）。应使用单独的心率校正方法对QT间期进行心率校正（QTc）。 CV遥测技术的优势在于，它通过收集每次心跳的数据来生成一个强大的数据集，而且从用药前至少1小时到用药后22小时都会对动脉血压进行连续监测。收集逐次心跳的CV数据可捕捉药效学效应（如果存在），包括起效、Cmax和恢复期。收集到的逐次心跳数据可分为1分钟数据，用于数据处理。可测量每次正常心跳的定量心电图间期（PR、QRS和QT），心率可表示为两个连续RR间期之间的间隔或每次血压脉冲的计数。心电图遥测研究按照GLP法规进行。 可在独立的心血管安全药理学研究中增加一个可选的毒代动力学阶段，在心血管阶段洗脱后在相同的动物中进行，采用重复拉丁方形设计，以剂量递增（低、中、高）的方式进行，并在两次剂量之间进行洗脱，或在心血管阶段洗脱后对所有四只动物一次给药（低、中、高）。毒代动力学研究也可在单独的一组动物中进行，在这种情况下，应进行单次毒代动力学数据收集，作为CV研究的一部分，用于确认浓度，但不用于Cmax，以避免在CV遥测数据收集期间干扰动物。也可考虑在CV监测期间增加一次毒代动力学数据采集。如果要收集数据，应计划在Tmax之后进行，以便将CV监测过程中的暴露量与完整的暴露概况联系起来。 7 呼吸系统评估 核心呼吸系统研究将呼吸频率、潮气量和每分钟呼吸量作为主要研究终点进行评估，因为它们不仅能捕捉到对肺力学的直接影响，还能捕捉到可能来自中枢神经系统呼吸中枢的间接呼吸影响（Table 5-2）。当确定或怀疑存在呼吸系统倾向时，可评估的其他终点包括氧饱和度和气管支气管树的气流（阻力、气流、气流持续时间）、肺运动和充盈的难易程度（顺应性、弹性）以及肺泡气体扩散。 大多数呼吸系统研究包括评估药物对通气模式的影响，有时还包括血氧饱和度。当预计会对呼吸系统产生直接影响时，可考虑采用核心组合以外的先进监测方法，如胸膜压力-容积环路，以量化支气管收缩可能导致的气道阻力变化。 啮齿类动物（大鼠比小鼠更常用）最常用于呼吸安全药理学研究。这些研究使用胸透法测量气道中的容积变化。在啮齿类动物中可考虑使用几种胸透仪配置，包括全身型、头外型和仅鼻型。所有这些配置都可用于测量呼吸频率和潮气量，但全身胸透的优点是不需要束缚动物。胸透法需要一个适应期，要么适应全身腔室，要么适应束缚期，通常在给药后4个小时才能收集数据。大多数小分子药物的典型研究设计是在独立的呼吸研究中进行一次给药，采用平行设计，分四组，每组至少八只动物，分别给予低、中或高剂量的供试品或对照品（Table 5-5）。为减少动物用量，啮齿动物呼吸试验也可采用拉丁方交叉设计。 这些研究中的基线数据应在首次给药前收集，给药后数据应在给药后4小时内立即收集。如果评估供试品的药代动力学需要更长的时间，则可使用全身胸透法收集啮齿动物用药后24小时内的额外时间点数据。 啮齿类动物的呼吸系统研究也可整合到重复给药毒理试验中，即指定研究动物的一个小组，并在给药第一周的指定一天（即第3天）用于测量呼吸系统终点（Table 5-6）。动物可以是单性别的，每组8只雌性动物，以避免超过胸透室的容纳量（中枢神经系统用6只雄性动物）；如果由于暴露量的性别差异则雌雄动物都需包括在内，则每种性别5只动物。 对于急性给药效应，在第1天进行安全药理学评估；如果对长效药物或生物制品进行评估，则在第1周和给药的最后一周进行安全药理学评估。不对恢复期动物进行评估。与独立的呼吸安全药理学研究类似，重复给药毒理试验中呼吸评估将在试验动物适当适应后使用胸透法，收集给药前至给药后4小时的数据。在非啮齿类动物研究中，主试验动物将使用配备呼吸电感胸透（JET-RIP）波段的非侵入性外套式体外遥测装置进行评估，或在动物体内通过外科手术植入心肺遥测装置。在重复给药毒理试验中，可选择在给药结束时和恢复期结束时收集数据。 在啮齿动物呼吸安全药理学研究中，提高灵敏度的关键因素是剂量。按升序（从对照组到高剂量组）和降序（从高剂量组到对照组）给每组一半的动物加药，这样所有的动物组都能暴露在给药室的技术人员面前，并有时间在胸透室中从加药期间的应激反应中恢复过来。 如心血管研究所示，对于持续时间较长的评估，建议在大动物（即非人灵长类和犬，小型猪较少）中进行心肺联合研究。研究期间测量的参数包括呼吸频率（呼吸频率）、潮气量、分钟量（呼吸频率与潮气量的乘积，也是总通气量的测量值）、吸气峰值流量、呼气峰值流量、增强暂停以及吸气和呼气时间。后续研究已经建立，并可根据需要对气道阻力、顺应性、肺动脉压、血气、血液 pH 值等进行评估。 8 中枢神经系统评估 药物对中枢神经系统的影响通常采用功能观察组合（Functional Observation Battery，FOB）或改良 Irwin 评估进行测量。中枢神经系统核心安全药理学试验可使用与毒理试验一致的啮齿动物物种单独进行，或将啮齿动物中枢神经系统安全药理学研究整合到重复给药毒理试验中（表5-5和5-6）。FOB和Irwin是符合ICH S7A要求的首选检测方法，但这些检测方法的主要问题在于其主观性和定性性，这一点与行为测试一样。如果有迹象或观察到对中枢神经系统的不良影响（如癫痫发作），则可能需要进行定量中枢神经系统安全性药理学后续研究，在这种情况下，可能需要进行脑电图（EEG）检查。 FOB和Irwin观察评估旨在评价运动活动、行为变化、协调性、感觉运动活动、感觉运动反射反应和体温。大多数小分子供试品的典型研究方法是单次给药、平行分组设计、独立的中枢神经系统研究，每组至少有六只大鼠（单一性别；如果没有暴露量性别差异，则为雄性），分低、中、高剂量给对照组或试验组。动物采用群居饲养，根据剂量组的分配情况，每个笼子最多可饲养三只动物。训练有素的中枢神经系统技术人员在进行FOB或改进的Irwin评估时，通常会对治疗方法设置盲区。数据应在给药前一天（即预处理）或给药当天（基线）收集。用药时间要错开，以便同一技术人员有时间在用药后的选定时间点进行FOB或改进的 Irwin 评估。 FOB评估与改进后的欧文评估之间的差异在于观察期间如何处理动物的细微差别。FOB通常会在Cmax前后和用药后24小时内进行评估，而改进后的Irwin可能会在Cmax前后和用药后5小时内进行多达五次观察，如果在用药后5小时仍检测到影响，则会在24小时内进行观察。 FOB和Irwin评估也可纳入重复给药毒理试验，以评估剂量范围、持续时间、行为可逆性和生理功能。根据目标人群的不同，研究中可使用不同年龄（新生儿、幼年、成年）或怀孕状态的啮齿类动物，每组六只主要动物（单一性别）或每种性别五只主要动物。如果将这些评估作为重复给药毒理试验的一部分来进行，通常会将雄性啮齿动物分配到中枢神经系统研究中，而将雌性啮齿动物分配到呼吸系统研究中（Table 5-6）。 同样，在对动物进行随机分组之前，首先要让动物适应实验室；这通常是毒理试验方案的一部分。在毒理试验的某一天安排进行FOB或Irwin评估，并在指定的基线时间点给动物注射对照物质和三个剂量水平的供试品，然后将动物放回笼子里；然后在选定的时间点观察动物，直至用药后24小时。在评估过程中，可交错给药，以便一名观察者在每个时间点收集和记录多个动物和处理组的所有参数。目的是确定动物可耐受且未观察到不良影响的最高剂量，以及确定对动物行为造成明显不良影响的最低剂量。 在Irwin评估中，包括几项观察： • 群养笼边观察，包括姿势、异常行为（如抽搐、颤抖、发声、刺激和刻板行为）；操作观察，如移出的难易程度、操作反应、体温、流涎、流泪或皮脂分泌； • 在开阔地带的运动活动，包括自发运动活动、步态异常、刻板行为、易激惹、排尿或叛逃；以及 • 自主神经活动评估，如触觉反应、惊吓反应、向右/睑/抓握反射、捏尾、热痛觉、共济失调、体温和瞳孔反应。 • 此外，还会记录临床症状和体重。 在进行FOB评估时可发现的症状包括： • 共济失调 • 反应性 • 催眠（对外界刺激缺乏反应）； • 被动（缺乏反应或不挣扎）； • 自发的运动活动（运动、饲养、梳理）； • 社会互动； • 刻板行为（重复、无目的的行为，如摇头、摇晃、啃咬、舔食）； • 死亡率； • 眼球突出（目测眼球突出）； • 步态（肌张力低下或肌张力亢进--肌肉无力导致的姿势歪斜）； • 抓握反射（通过有线操作测量抓握能力）； 握力 • Straub 尾部反应（尾部从水平线上抬起）； • 震颤（对立肌群交替收缩引起的不自主运动）； • 抽搐（自主肌肉剧烈的不自主收缩，可以是肌肉交替收缩和放松的阵挛性收缩，也可以是持续收缩的强直性收缩、痉挛样收缩或阵挛性和强直性混合收缩）； • 腹泻 • 流泪； • 瘙痒（毛发蓬乱）； • 上睑下垂（由于上眼睑下垂、瞳孔缩小而导致部分或全部闭眼）； • 直肠温度 • 流涎 • 排尿； • 角膜反射（眨眼反应）； • 直立反射（身体恢复正常直立姿势的能力）； • 松果体反射（缩回或抽动耳朵的反应）； • 夹尾巴；以及 • 热板反应 在ICH S7A中，通过FOB或Irwin评估筛选和监测候选新药对中枢神经系统的潜在不良影响，是评估中枢神经系统安全或人体安全风险的第一步，尽管这些测试取决于进行观察的人员的技能。可能需要综合各种终点来检测潜在的不良事件，而不仅仅是单个参数。有些终点不能直接转化为人体中枢神经系统不良事件，因此无法确定完全一致。 如果在FOB或Irwin试验、重复给药毒理试验或临床试验中发现了对中枢神经系统的不良影响，则可能需要进行后续研究（如认知和学习、恐惧和焦虑等）以及使用脑电图（EEG）进行癫痫发作责任评估，以进一步阐明中枢神经系统不良影响的性质和作用机制，然后再继续药物开发。在啮齿类动物中观察到的异常现象（如摇头、抓挠、过度梳理和舔食）往往发生在惊厥之前，可能是阵挛性或强直性的。 9 生物制品或生物技术衍生药品的安全药理学 ICH S6（R1）涵盖了生物制药（即生物制品或生物技术衍生药品）的安全药理学测试。 生物制品以其选择性（如抗体结合或受体配体）著称，预计不会影响CV或呼吸系统或中枢神经系统。然而，它们可能具有靶标放大药理学效应，或潜在的脱靶次生药理学活性，可能对构成安全药理学的重要器官产生不利影响。对于大多数生物制药（大分子或生物技术衍生产品）而言，根据ICH S6（R1），安全药理学终点被纳入重复给药毒理试验中。但是，如果生物制药对受体结合的选择性不高，代表一种新型或一流产品，或两者兼而有之，则可能需要进行独立或后续研究，以进行更广泛的安全药理学评估（即根据ICH S7A）。 对于在啮齿动物中具有活性的生物制品，中枢神经系统不良反应的安全药理学终点（即使用 FOB 或改进的Irwin评估）在单次给药或重复给药后进行评估；这取决于供试品的给药途径、给药方案。如果生物制品对非人灵长类动物和啮齿类动物都有活性，则可在啮齿类动物和非人灵长类动物中评估不良反应，但如果只对后者有活性，则只在非人灵长类动物重复给药试验中纳入安全性药理学终点。 在某些情况下，体外中枢神经系统安全性研究是在进行体内研究之前进行的，以指导选择安全药理学终点，并酌情纳入重复给药试验和随访研究（即根据ICH S6(R1)指南）。 10 肿瘤产品安全药理学 肿瘤产品的安全药理学已纳入ICH S9。对于专为晚期癌症设计的肿瘤产品，有必要加快候选药物的临床开发，以便患者能够及时获得这些药物。根据ICH S9，虽然无需进行独立的（即ICH S7A规定的）安全药理学研究，但可将对三个重要器官系统（心血管、呼吸和中枢神经系统）的不良影响评估纳入重复给药毒理试验中，或仅限于评估从大动物（如犬、非人灵长类或迷你猪）毒理学研究中收集的心电图（有科学依据）。 评估安全药理学核心组合中三个重要器官系统的重复给药毒理试验应按照GLP 法规进行，并进行详细的临床观察。在啮齿动物（通常是大鼠）中，重复给药毒理试验通常包括FOB或Irwin评估，28天毒理试验中一半的主试验动物（如10只雄性动物中的5只）在第1天接受评估，以及详细的呼吸系统观察（如10只雌性动物中的5只），给药组与对照组进行比较。 在非啮齿动物重复给药毒理试验中，除详细观察外，还要收集心电图和血液动力学测量数据（心率、血压和体温）；这些数据在基线（即给药前）、第1天（给药第一天）和给药最后一周（即第28天或第90天）收集，如果有必要，还包括在研究中期收集。给药组的测量结果将与对照组结果进行定量比较，并评估显著变化，将其与剂量水平、毒代动力学暴露量或两者相关联。如果在给药最后一周的评估中发现影响，一般会在恢复期增加CV、呼吸和中枢神经系统评估，以评估可逆性。 由于潜在的收益风险比更大，人们希望加快肿瘤产品的开发，监管部门不要求进行单独的安全药理学研究。由于安全药理学评估被整合到重复给药毒理试验中，因此患者安全问题可以得到充分解决，同时也符合ICH S9规定的动物使用3Rs要求。 在设计肿瘤产品的后续安全药理学研究时，需要考虑药理学和临床适应症，并根据具体情况进行此类研究（Table 5-7）。根据ICH S645和ICH S946，这些研究应予以精简，以减少动物数量，尤其是生物制品和肿瘤药物，而且还应根据具体情况对这些重要器官和其他器官进行补充试验（Figure 5-1）。 11 体外原发性心律失常综合测定 体外原发性心律失常综合分析法（CiPA）由FDA于2013年开发，旨在改进原发性心律失常标志物的评估，并提供更相关、可靠和全面的临床前体外心脏筛选工具。这一举措是基于某些药物（如维拉帕米）有可能阻断hERG通道，但在人体中不会导致心律失常。 顾名思义，CiPA包括对参与心室动作电位的心肌离子通道的全面评估，因此它的使用支持了对hERG评估的要求。CiPA的第一步是对转染hERG的细胞进行六种心脏离子通道的膜片钳检测：hERG、Nav1.5（晚期钠）、Cav1.2（钙）、KvLQT1（慢延迟整流钾电流；IKs）、Kir2.1（负责膜电位的内向整流钾通道；IK1）和Kv4.3（快速瞬时外向钾电流；Ito）。 这些膜片钳测定的数据随后被用于提供人体心室动作电位的计算机模拟模型，从而提供有关早期心脏潜在损伤的信息。驱动计算机模型的算法可以预测心室动作电位的变化（如延长）以及任何早期后极化的产生。早期后极化是导致心律失常风险的早期诱因。硅学模型，如O'Hara Rudy模型，是健康人心室动作电位的精确模型，能够评估延长QT间期的化合物，而这些化合物与前心律失常风险无关（如维拉帕米）。最后，通过硅学建模获得的膜片钳数据将被评估是否可转化为诱导多能干细胞衍生的心肌细胞（iPSc-CM）。 CiPA还包括使用人类诱导多能干细胞衍生的心肌细胞（hiPSC-CM）。利用多电极阵列技术对这些细胞进行评估，测量化合物的细胞外场电位，帮助确定对心血管电生理学有潜在不利影响的化合物。记录到的场电位波形的变化是心室动作电位变化的基础，可能表明hERG或其他离子通道（如钠通道Nav1.5，负责动作电位的上冲，或钙通道Cav1.2，负责动作电位的持续时间）受阻。 因此，场电位持续时间的延长可能导致心室动作电位的延长，并可能显示心电图上记录的QT间期的延长。药物引起的动作电位延长应与细胞间搏动的可变性引起的动作电位延长区分开来，并使用阳性和阴性对照化合物。如上所述，在CiPA期间还研究了除hERG之外的其他钾通道，如Kir2.1、Kv4.3、KCNQ1/KvLQT1_mink。 12 其他器官的安全药理学 根据治疗适应症和毒性研究中的不良反应结果，还可对核心组合以外的器官系统进行安全药理学研究。这包括肾脏或泌尿系统、胃肠道系统和自主神经系统等。此类研究的终点将取决于所测试的系统，但可能包括排尿量和电解质排泄量、胃肠道转运时间和胃排空、免疫细胞表型等免疫学参数、激素水平等内分泌参数以及正压性低血压。这些参数可用于评估这些其他器官系统的功能变化。 所有候选药物或生物制品都应在非临床药物开发期间或临床开发II期之前，根据具体情况，采用适合目的的研究设计，对这些其他器官系统进行必要的安全药理学测试。 • ICH E14/S7B Q&As - 临床 TQT 研究对非临床研究的需求 ICH E14指南和ICH S7B问答涵盖了临床TQT研究对非临床研究的需求。ICH S7B于2005年实施，旨在指导开展非临床安全药理学研究，评估新药在首次人体试验前延长心室复极化（QT间期）的可能性。同年，ICH E14获得通过，概述了开展临床研究的必要性，以评估这些新药对延迟心脏复极化潜能的影响。 FDA公布了2016年至2020年进行的TQT研究数量数据，发现32%的研究未能达到E14 Q&A 5.1的两倍高临床暴露阈值。此外，另有24%的临床研究采用了替代研究设计（其中 82% 用于肿瘤适应症），由于无法进行TQT研究，导致标注为  “QTc 影响较小"。为了减少临床TQT研究的数量，E14/S7B Q&As指导原则于2022年2月获得批准，2022年7月定稿，并于 2022 年8月实施。非临床双阴性结果（即 hERG和体内QT/QTc研究结果均为阴性），并结合临床E14 Q&A 5.1（当临床暴露量未达到2倍临床高水平时，支持TQT豁免）或E14 Q&A 6.1（支持 "延迟再极化导致促心律失常效应的可能性较低 "的依据）。 假设ICH S7B hERG和体内QT研究的结果显示心室复极化没有延迟，其将使监管机构和临床医生确信，实验药品不会对参与首次人体研究的健康志愿者造成风险要使用非临床 hERG和体 QT结果（双阴性）作为临床TQT研究的替代方法（E14 Q&A 5.1）或替代物（E14 Q&A 6.1），应具备以下条件： • 统一标准方案； • 实验条件遵循S7B Q&A 中概述的最佳实践（Q&A 2.1；hERG和3.1-3.5体内QT检测）；以及 • 监管审查报告的一致性。 S7B的体外hERG问答（用于膜片钳的问答2.1）提供了以下方面的指导： • 记录生理温度（35-37 °C）、应遵循的电压协议； • 记录 hERG 电流质量的测量方法； • 测量或计算的主要终点（如IC50、希尔系数和置信区间）；浓度验证；以及 • 阳性和阴性对照的使用。 S7B的体内QT检测问答（问答3.1至3.4）提供了以下方面的指导： • 种属选择与ICH S7B一致，选择与毒理试验一致的适当的、可自由活动的非啮齿动物物种； • 超过预期治疗浓度--倍数越高，对 E14 Q&A 6.1 中 QTc 检测灵敏度的依赖性越小（即 QTc 变异性的统计测量；以毫秒为单位检测 QTc 变化的最小显著差异，包括浓度 QTc 分析，E14 Q&A 6.1 要求，Q&A 5.1 建议）； • 使用个体动物心率校正方法--包括 QTc/RR 散点图，评估检测灵敏度和实验室历史灵敏度（最小可检测差异）。 只有在问答6.1中才要求检测QTc（最小显著性差异）与临床研究中测得的阈值之间变化的化验灵敏度。最佳做法是报告体内QT化验的QTc最小显著性差异，以支持问答5.1，但没有最小阈值，该值仅用作衡量研究中QTc的变异性。最后，S7B Q&A 3.5是报告结果时应考虑的要点。 因此，非临床双阴性数据现在可以通过首次人体监管审查，以ICH E14 Q&A 5.1或6.1作为原发性心律失常的非临床-临床风险评估。预计这将增加TQT豁免申请的数量，并减少所需的临床TQT研究数量。ICH S7B最佳实践（Q&A 2.2-2.5）使用心肌细胞和前心律失常模型（Q&A 4）对心室再极化进行了后续体外评估（参考ICH E14/S7B Q&A）。 13 付诸实践：安全药理学案例研究 案例研究5-1和案例研究5-2重点介绍了如何将本章提供的信息付诸实践。 案例研究 5-1 对具有纳摩尔hERG抑制作用的小分子应进行哪些安全性药理学研究？ • 研究背景 一种小分子（新分子实体）正被开发为治疗神经退行性疾病的全身用药。该药物将作为口服片剂，每天服用一次。在非临床开发过程中，该分子在非人灵长类动物体内表现出对hERG通道的纳摩尔抑制作用（IC50 <100 nM）。 • 解决方案 对于这种针对神经退行性疾病开发的小分子药物，需要进行核心的安全药理学研究。由于该分子是作为每日口服片剂开发的，因此药物开发计划包括两项CV研究，以解决重复给药问题，并将药代动力学与CV测量相关联。此外，本着3R的精神，大动物CV研究与通过遥测技术收集呼吸信息相结合。在中枢神经系统方面，将FOB纳入为期28天的啮齿动物重复毒理学研究。 初步心肺研究采用非GLP方法，使用递增剂量设计（药效剂量和高于首剂的两个剂量水平（2倍和6倍）），使用三只非新药遥测仪器的非人灵长类动物。关键的GLP心肺功能研究是使用未经药物测试的非人灵长类动物进行的，这些灵长类动物通过手术植入了遥测仪器。 研究采用平行分组设计，共分四组（每组三只动物）；第一组动物服用对照组（载体）药物，其他三组动物服用递增剂量的试验药物，最低有效剂量为第二组。最低有效剂量（第2组），以及比最低有效剂量高2倍和6倍的剂量（第3组和第4组）。该研究设计根据表5-4所示进行了修改。 非人灵长类动物每天用药一次，并在第-1、1、2、4和第7天收集CV和呼吸功能遥测数据。第8天，动物再服药一次，以便在第8天和第9天（服药后24小时）采血进行药代动力学分析。 这些心肺功能研究（包括非GLP和GLP）的结果表明，在每天一次、连续7天服用6倍有效剂量的情况下，该分子在体内没有心血管风险，因此支持其继续开发。 案例研究 5-2 对于有癫痫发作风险的小分子药物，应该进行哪些安全性药理学研究？ • 研究背景 一种需要每天口服两次的小分子钠通道阻断药物正在作为镇痛药进行开发。在开发过程中对大鼠和犬进行研究，发生惊厥的血浆暴露量高于患者可能出现的暴露量，犬的暴露量约为临床有效剂量的10倍，大鼠的暴露量约为临床有效剂量的45倍。 • 解决方案 需要进行适当的体外研究，以评估已观察到的抽搐的潜在机制。 可能需要进行犬脑电图研究，记录大脑的自发电活动，以便 • 确认中枢神经系统不良反应的性质，并确定抽搐是由于癫痫发作还是其他毒理效应（如晕厥或明显的运动障碍）引起； • 在观察到抽搐和其他中枢神经系统症状（如肌阵挛）时，确认血浆暴露量； • 确定可能是惊厥或癫痫发作前兆的临床表现，这些临床表现可作为临床试验的终点指标；以及 • 确认在未观察到惊厥的较低剂量下无脑电图异常。 脑电图研究通常包括视频脑电图和从用药前至少24小时到最后一次用药后至少24小时的连续记录。研究开始前72小时，将对所有犬进行连续脑电图记录和分析筛查，以评估是否存在异常脑电图形态，然后再分配到研究中。研究组的最小动物数为8人（通常为4雄4雌）。选择的剂量水平将包括尽可能高的脑电图 "未观察到效应水平 "（no observed effect level）和观察到抽搐的较高剂量水平，以确认中枢神经系统事件的性质。 研究设计可能具有适应性，一些研究组的剂量水平将根据之前实验组的结果进行选择。虽然这不是研究的主要目的，但研究还将确认惊厥是否具有自限性，并对治疗任何药物诱发的惊厥的紧急药物（如地西泮、苯妥英或丙泊酚）进行潜在评估。 当观察到严重的中枢神经系统临床症状时，将在计划外的时间点采集毒代动力学样本（包括重要的代谢物），以精确量化这些事件期间的血浆暴露量，并同时分析脑电图痕迹。重复给药的持续时间将超过重复剂量一般毒理学研究中诱发惊厥所需的持续时间。为避免速发型过敏症，将包括 1 个月的洗脱期。 鉴于大鼠脑电图研究的安全系数较高，而且犬的数据将说明这种责任的特征，因此可能不需要进行大鼠脑电图研究。 14 摘要和结论 多年来，安全药理学研究不断发展，以跟上不断变化的药物类型。尽管如此，安全药理学研究仍然侧重于在临床试验中首次给药前对潜在的不良药理作用进行评估。 安全药理学界已经产生了高质量的功能数据，用于评估药物的安全性，这些数据可以单独使用，也可以与重复给药毒理试验结合使用。过去二十年来的改进主要集中在最佳实践（如使用数字数据收集、适应和改进技术以降低压力）、致力于 3Rs 原则以减少动物和笼具的使用、单个动物心率校正、QTc/RR散点图等方面。 尽管如此，大多数这些细节的评估在整个行业中并不统一，也没有系统地纳入研究报告中。随着ICH S7B Q&As的发布，这些细节、统计测量和校正以及更多内容需要纳入最终报告中。为了实现这一单一目标，核心组合安全药理学研究一直侧重于首次人体试验，通常是单剂量和小样本量研究，并持续收集数据。根据所测试的药品或生物制药的药理学，可对研究进行修改，以适应后续研究中的其他设计。 在候选药物筛选阶段进行的各种安全药理学研究--体外hERG研究、CiPA计划中检测hERG阻滞和QT延长的能力，以及体内测试--避免了药物在临床开发后期因前心律失常而暂停。这就需要整合体外hERG和体内QT检测的非临床数据以及临床数据，以便对QT/QTc延长进行综合风险评估，如果在控制良好的非临床研究中未观察到hERG阻滞或QT/QTc延长，则有可能放弃或减少彻底的TQT临床研究。 对心血管系统的评估，单独的安全药理学研究是黄金标准，而不是整合在毒理试验中，但整合的安全药理学研究将继续用于小分子和大分子药物，以符合3Rs的要求，只要以严格和可解释的方式收集安全药理学终点，以阐明药物对结构和生理功能的影响。对于大多数CV研究而言，具有体温采集和呼吸功能的植入式遥测设备现在已被接受，而且比外套式遥测设备更为普遍，这为收集更可靠、更一致的心电图和呼吸波形铺平了道路，从而可进行更准确的分析，便于与毒性研究相结合。目前，S7A和S7B/E14指南中没有具体涉及细胞和基因治疗，因为它们正在迅速发展，但不久后可能也需要纳入指南范围。有必要继续评估安全药理学研究数据的转化效果，以减少临床试验中的失败。 英文版原文 （上下滑动查看更多）  Chapter 5 Safety Pharmacology Studies  Adverse pharmacodynamic effects may be due to the on-target primary pharmacology effects of the product being tested, which could be related to the given dose, the route and frequency of administration, and systemic exposures, or due to off-target effects termed as secondary pharmacology. The three primary vital organ systems included in the safety pharmacology core battery are physiologically correlated, meaning that effects seen in one system may induce an adaptative response in the others. As such, interpretation of safety pharmacology data typically requires an integrated risk assessment of the totality of effects on organ functions that considers the targeted clinical population, clinical trial plans, and safety margins. The timing of safety pharmacology studies, which are aligned with the intended patient populations, will be discussed in this chapter. Safety pharmacology has emerged as a discipline to predict risk of adverse pharmacodynamic liability3 through the conduct of core battery studies before performing the first-in-human studies with a new product. These have helped to separate out safer pharmaceuticals with either little or no potential for CV, respiratory, or CNS effects or which have adequate safety margins from those which have a less favorable benefit-risk profile. It should be noted that safety pharmacology assessments may also be required for products in the late stages of development if adverse effects arise in clinical trials or if a marketed product is being repurposed for a new indication, patient population, or administered in a new formulation or route which previously did not require safety pharmacology assessments. The information presented in this chapter is largely based on US Food and Drug Administration (FDA),1 European Medicines Agency (EMA), and Committee for Medicinal Products for Human Use (CHMP) documents that discuss the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Safety (S) guideline 7A on safety pharmacology studies for human pharmaceuticals. The ICH S7A guidance states that all human pharmaceuticals (small molecules, biotechnology-derived, or biologics) should be reasonably safe to be given to humans during first-in-human clinical trials. As such, the first dose in humans is not expected to have any undesirable pharmacodynamic effects on the physiological function of vital organs at the therapeutic range and above, as evaluated by safety pharmacology studies conducted in accordance with good laboratory practice (GLP) regulations.4 The timing of the safety pharmacology studies is aligned with the guidance from FDA and EMA/CHMP on ICH M3(R2) on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals.5,6 Currently, cell and gene therapy products or tissue-engineered products are not specially addressed in the Safety Pharmacology guidelines. This chapter also will discuss the more recently approved and implemented ICH E14/S7B Q&A guidance, which outlines the acceptance of in vitro hERG (human ether-a-go-go related gene) and in vivo QT interval study data (i.e., nonclinical, double negative), both conducted under the Q&A best practice recommendations in support of two clinical scenarios (E14 Q&A 5.1 and 6.1), to apply for a thorough QT (TQT) study waiver.7,8 Case studies are provided at the end of this chapter to demonstrate how safety pharmacology studies may be conducted in practice. 1.General Principles and Importance of Safety Pharmacology Testing Safety pharmacology guidelines were first developed in 2000 in response to the adverse reports of the effects of the antihistamine terfenadine on the CV system. Terfenadine, which had been introduced into the market by Hoechst-Marion Roussel (now Sanofi-Aventis) in 1985, was marketed as Seldane in the US, Triludan in the UK, and Teldane in Australia as a treatment for allergic rhinitis, among others.3 In the 1990s, however, some people taking terfenadine presented to the hospital with syncope, and in some instances, died. 3,9,10 It was later found that terfenadine is a potent inhibitor of the potassium ion channel (IKr or hERG) resulting in prolongation of the QT interval. 9-11 This had the potential to develop into a polymorphic ventricular tachycardia termed Torsades de Pointes (TdP), which literally means twisting of the points or peaks (R wave or QRS complex) around the ECG isoelectric line. The cardiotoxic potential of terfenadine was not detected prior to clinical trials because the drug was completely metabolized by the liver enzyme cytochrome P450 3A4 (CYP3A4), so levels went undetected in the plasma. Moreover, its metabolite terfenadine carboxylate (also called fexofenadine), did not inhibit the potassium ion channel even when levels were at 30 times higher concentrations than the hERG IC50 of terfenadine.11,13 The proarrhythmic potential of the prodrug terfenadine was revealed at very high doses, however, in patients with liver disease affecting the CYP3A4 enzyme or when patients were taking other medications that inhibited CYP3A410,13,14as this resulted in reduced metabolism and thus higher plasma levels of terfenadine and lower concentrations of fexofenadine. Fexofenadine had similar efficacy to terfenadine, but since it only slightly inhibited the hERG channel, it had much lower potential to induce arrhythmia. Fexofenadine was marketed in 1996 under the name Allegra13 for allergic rhinitis and chronic urticaria, among others, and was listed in the WHO Essential list of Medicines,15 while Terfenadine was withdrawn from the market in 1997 due to its potential to cause cardiac arrhythmia. Hence, the potential adverse effects of small molecules or biopharmaceuticals on all three core organ systems (CV, respiratory,CNS) must be conducted before performing first-in-human studies.1,2 While ICH S7A provides guidance on the conduct of the core battery studies and the functional endpoints to collect, it does not outline the study designs themselves. Safety pharmacology study designs for both in vitro and in vivo studies are discussed in the next section and are provided as examples which may be judiciously applied or modified according to the pharmacology (or class of compound), route and dose, indication and intended patient population of the pharmaceuticals or biopharmaceuticals under evaluation. 2.Core Battery Study Designs and Endpoints When testing the safety pharmacology of a new product, the potential adverse pharmacodynamic effects need to be tested first on the three core organ systems: the CV system, the respiratory system, and the CNS. 3.Cardiovascular System Types of studies that may be performed to assess the effects on the CV system include: • In vitro studies (e.g., cardiac ion channel studies) • Ex vivo studies (e.g., Langendorff technique) • In vivo studies (e.g., telemetry in rodent and non-rodent models) 4.In Vitro Cardiovascular Studies In vitro CV assays mostly involve the study of cardiac ion channels, with the regulatory requirement to conduct a hERG assay7,8,17 (Table 5-1). One such assay is the automated hERG assay to determine the potential of pharmaceuticals to inhibit the hERG potassium channel. Using a single concentration of the test article, this assay is used as a screening tool early in drug development to identify compounds with potential hERG liability during structure-activity relationship research efforts and before leadnomination.  Discovery hERG assays are followed up by using escalating dose concentrations of the test article to get an estimation of its IC50, which is the concentration of the drug that is required to inhibit a biological process by 50%. This is then followed by a rigorous manual patch clamp hERG assay in accordance with GLP regulations. The recently released S7B Q&As request following up the in vitro hERG assay. Best practices for experimental factors are then conducted on human embryonic cells or Chinese hamster ovary cells overexpressed with human cardiac ion channels (S7BQ&A 2.1)21,22, which includes potassium, calcium, and sodium channels. Highlights of the Q&A best practices for experimental factors set out in ICH guidance include: • Conducting manual patch clamp experiments at near physiological temperature (35-37°C); • Measuring the ventricular action potential voltage protocol; • Monitoring health of the cells overexpressing the cardiac channels; • Recording seal resistance quality; and • Reporting the IC50 value and Hill coefficient Moreover, a validated analytical method should support concentration verification, and one of the three positive controls –dofetilide, ondansetron, and moxifloxacin – should be evaluated as a reference drug. Regarding the latter, repeated evaluations should be done with two or more concentrations of the test article being used for each experiment and including at least four cells at each concentration. After consideration of protein binding and using free drug concentrations in plasma, the calculated IC50 concentration inhibition should have greater than 50-fold safety margin over that of anticipated clinical exposures. Assessment of additional cardiacion channels is recommended and included in the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, mentioned later in this chapter. When evaluating oncology products, hERG and cardiac ion channels inhibition assays can be conducted without compliance to GLP regulations because of the likely greater benefit to risk ratio of oncology products versus other pharmaceuticals. For biologics or biotechnology products, the target specificity is usually very high, and so in vitro hERG assays are not required but are recommended. When evaluating these biopharmaceuticals, CV endpoints can be incorporated in vivo in to repeat-dose toxicity studies conducted using non-invasive telemetry (Table 5-2). 5.Ex Vivo Langendorff Technique The Langendorff technique is an ex vivo assay in which the isolated heart of an animal is perfused with oxygenated, buffered Krebs solution, with and without the drug to be tested. The aim of this is to observe the effects of the drug on ECG parameters, heart rate, left ventricular mechanics, and estimates of coronary blood flow.26,27 Usually, the heart of a guinea pig or rabbit is used, as their hearts possess the IKr potassium ion channel, which is the primary cardiac ion channel responsible for ventricular repolarization (i.e., the QT interval of the ECG) in humans. Studies have demonstrated concordance to human effects of QT prolonging drugs using the ex-vivo assay. Translation of the isolated perfused guinea pig (rodent) heart assay to large animal QTc assay for determination of QTc changes with drugs known to prolong QTc and 13 drugs known to have no effect on the QTc interval was demonstrated. To perform the technique, the animal’s heart is removed, and the perfusate is introduced via the aorta in a retrograde fashion; this delivers the oxygenated, nutrient-rich perfusate containing either the vehicle or drug through the coronary arteries. Endpoints recorded include ECG parameters recorded from electrodes placed on the heart’s epicardial surface in a Lead II configuration, heart rate, left ventricular contractile parameters (e.g., the maximal rate of rise and maximum rate of fall in ventricular pressure), and mean coronary flow. The effect of the test drug on the function of the myocardium (electrical and contractile activity) and the coronary vessels can be examined by examining the myocardium, smooth musculature, and the endothelium of coronary vessels. Despite having some limitations (e.g., inability to assess metabolites, functional viability of the heart performance isolated for greater than 4 to 6 hours, etc.), the Langendorff technique can provide rapid information as both a CV screening assay and when further interrogation of results from earlier discovery assays (e.g. receptor binding assay on cardiac receptors or hERG results) is needed, as this assay generates an ECG for evaluation of intervals (e.g., RR, PR, QRS, QT and calculated QTc intervals), rhythm and left ventricular pressure curve. 6.In Vivo Cardiovascular Studies In vivo CV studies involve the evaluation of the CV system in free-moving (non-rodents like canine, non-human primates or minipig) animals via surgically implanted telemetry devices 30,31. Such devices are available with several biopotential and pressure catheter configurations as well as options for impedance leads for measurement of respiratory function 32,33. There are telemetry device options for use in both short and long term studies, as well as reuse of animals on multiple studies (with sufficient washout period), typically based on the battery life of the transmitter. Implanted telemetry, which uses solid tip ECG electrodes that are placed intravascularly, provides high resolution or low signal to noise ratio. This allows for accurate placement of fiduciary marks on the ECG intervals during post-acquisition analysis. Additional options include non-invasive jacketed external telemetry (JET), which pairs surface electrodes with a small, implanted telemetry device for collection of arterial blood pressure (required for compliance with ICH S7A CV studies) where the electrical components are secured in a jacket pocket.34 It should be noted that the small (rodent) telemetry device lacks the ability to collect continuous body temperature. CV endpoints collected in these studies are listed in Table 5-2. Telemetry studies using internally instrumented animals are convenient to perform, as the animals can move freely, and once surgically implanted and recovered, the animal can be re-used in several CV studies provided the previous test material has already cleared from their system and sufficient washout periods are allowed between studies. When the implanted telemetry device records both CV and respiratory parameters as set out in the ICH safety guideline 7A, the studies are termed cardio-respiratory studies. Stand-alone CV or cardio-respiratory studies are usually conducted in non-rodents in a 4 x 4 Latin square cross-over design, where each animal is dosed with the vehicle control and three dose levels of the test drug at low, mid, and high doses, in a random fashion following a washout period (typically 3-7 days). The statistical power of this design is superior to parallel or escalating dose designs as each animal serves as its own control, and any sequence effect is mitigated by the inclusion of all dose levels at each session (Table 5-3). This design uses the least number of animals (four in total) which is in keeping with the 3Rs of animal use – replace, refine, reduce – and can be modified for group housing options or to increase statistical power. Test articles with long half-lives (e.g., oligonucleotides, monoclonal antibodies, and biologics) where a washout period exceeds 7 days can use a parallel study design. This design consists of four groups of four to six animals that can be included in a stand-alone telemetry study (safety pharmacology environment), or alternatively, the CV safety pharmacology assessment can be incorporated in the parallel repeat dose toxicology study (toxicology environment). Males only can be used if there is no difference in exposures; otherwise, use both males and females. Recovery groups are often only in control and high-dose groups, so use four animals with no recovery animals in the low- and mid-dose groups. Considerations include scheduling of surgery for implanted telemetry and costs of implants. In some cases – and based on overall program considerations – escalating study designs can also be appropriate where animals receive a vehicle control, low-, mid-, and high-dose option sequentially (Table 5-4). Safety pharmacology evaluates the acute effects of the test article, hence the recommendation to collect CV parameters on Day 1 following a single dose if integrated in a repeat dose toxicology study. If the kinetics of the test article allow, CV parameters can be collected on Day 2 when all Day 1 activities are done because the excitement generated by Day 1 dosing and all the other activities, such as blood collection for toxicokinetics, clinical pathology, and clinical observations can have undue effects on CV parameters. CV studies measure arterial blood pressure (systolic, diastolic, and mean arterial pressure), heart rate (beats/min), Lead II ECG parameters (ECG intervals and rhythm) and body temperature. Qualitative ECG analysis includes a description of waveform morphology (for P, QRS, and T waves) and any arrhythmias, and quantitative measurements include ECG interval durations (RR,PR, QRS, and QT). The QT interval should be corrected for heart rate (QTc) using an individual heart rate correction method. The advantage of CV telemetry is that it generates a robust dataset by collecting data on every heartbeat and the arterial blood pressure is monitored continuously from at least 1-hour predose through to 22 hours postdose. Collecting beat-to-beat CV data captures pharmacodynamic effects, if present, at onset through Cmax and to recovery. The beat-to-beat data that is collected can be categorized into 1-minute means for data processing. Quantitative ECG intervals (PR, QRS, and QT) are measured for each normal heartbeat and heart rate can be expressed either as the interval between two consecutive RR intervals or from a count of each blood pressure pulse. CV telemetry studies are conducted according to GLP regulations. An optional toxicokinetic phase can be added to a stand-alone CV safety pharmacology study, in the same animals following a washout from the CV phase, as a repeat Latin square design, conducted as a dose escalation (low, mid and high) with washout between doses, or as a single dose (low, mid or high) administered once to all four animals after washout from the CV phase. The toxicokinetic study can also be performed in a separate group of animals and in this option, a single toxicokinetic data collection should be conducted as part of the CV study, for confirmatory concentration but not at Cmax to avoid disturbing the animals during CV telemetry data collection. The addition of a single toxicokinetic collection during CV monitoring can also be considered. When done, this collection is planned after Tmax to correlate exposure from the CV session with full exposure profile. 7.Respiratory System Assessment Core respiratory studies include measures of the respiratory rate, tidal volume, and minute volume as the major study endpoints assessed because they capture direct effects on lung mechanics but also indirect respiratory effects which may originate from CNS respiratory centers, based on ICH S7A guidance 1,2 (Table 5-2). Additional endpoints that can be assessed when a respiratory liability is identified or suspected are oxygen saturation and airflow to the tracheobronchial tree (resistance, airflow, flow durations), the ease of lung motion and filling (compliance, elasticity), and alveolar gas diffusion.1,2,40 Visual observations of the respiratory rate are not sufficient to meet ICH S7A guidance critera1,2 and respiratory parameters should be quantified with fully characterized methodologies. Most respiratory studies include evaluation of the effects of pharmaceuticals on ventilation patterns and in some cases blood oxygen saturation. When direct effects on the respiratory system are expected, advanced monitoring methodologies beyond the core battery may be considered such as pressure-volume loops of pleural pressures to quantify changes in airway resistance which can be caused by bronchoconstriction. Rodent species (rats are used more than mice) are most often used for respiratory safety pharmacology studies. These studies use plethysmography to measure changes in volume in the airways.40 Several plethysmograph configurations can be considered for use in rodents including whole body, head-out, and nose-only. All these configurations can be used to measure the respiratory rate and tidal volume, but the whole-body plethysmography has the advantage of not requiring the animals to be restrained. Plethysmography requires an acclimation period either to the whole-body chamber or to the restraint period typically for up to 4 hours postdose for data collection. A typical study design for most small molecules is a single administration in a stand-alone respiratory study, using a parallel design with four groups of animals of at least eight animals per group which are given a single low, mid or high dose of the test substance or a control substance (Table 5-5). To reduce the use of animals, the rodent respiratory study can also be conducted using the Latin square cross over design. Baseline data in these studies should be collected before the first dose is given and postdose data collected immediately post dose up to 4 hours later. If the assessment of the test article’s pharmacokinetics requires a longer duration, whole-body plethysmography can be used to collect data at additional timepoints up to 24 hours postdose in the rodent. Respiratory studies in rodents can also be incorporated in repeat-dose toxicity studies whereby a subset of the study animals is assigned and a designated day during the first week of dosing (i.e., Day 3) is reserved to measure respiratory endpoints (Table 5-6). Animals could either be single-sex, with eight (8) females per group to keep from exceeding the plethysmography chamber (and 6 males used for CNS) or 5 per sex if both sexes are included due to sex differences in exposures. Safety pharmacology assessments are conducted on Day 1 for acute dosing effects or during Week 1 and last week of dosing if conducting assessments of long-acting pharmaceuticals or biologics. No assessments on the recovery animals are done. Similar to a stand-alone respiratory safety pharmacology study, a respiratory evaluation within a repeat-dose toxicity study will use plethysmography following appropriate acclimation of the study animals, with data collected pre- to 4 hours postdose. In non-rodent studies, the main study animals will be evaluated using non-invasive jacketed external telemetry equipped with respiratory inductance plethysmography (JET-RIP) bands, or in animals that have a cardio-respiratory telemetry device surgically implanted. Optional data collections can be performed at the end of dosing for repeat-dose toxicity studies and at the end of recovery period. A key component to improve sensitivity in a rodent respiratorynsafety pharmacology study is dosing. Dosing half the cohort of animals per group in ascending order (from control to high dose) and the other half in descending order (from high dose to control) will allow all animal groups to be exposed to the technicians in the room administering the dose and have time in the plethysmography chamber to recover from the stress of being handled during dosing. As indicated for CV studies, and for longer durations of assessment, a combined cardio-respiratory study in a large animal (i.e., non-human primates and dog, less so in minipig) is recommended. Parameters measured during the study can include breathing frequency (respiratory rate), tidal volume, minute volume (the product of respiratory rate and tidal volume and the measure of total ventilation), peak inspiratory flow, peak expiratory flow, enhanced pause, and inspiration and expiration times. Follow-up studies are established and may be performed to evaluate airway resistance, compliance, pulmonary arterial pressure, blood gases, blood pH, among others as warranted. 8.Central Nervous System Evaluation The effects of pharmaceuticals on the CNS are usually measured using a functional observation battery (FOB)41 or modified Irwin42 assessment. CNS core safety pharmacology studies can be conducted as standalone using a rodent species consistent with the toxicology studies, or rodent CNS safety pharmacology studies are also often incorporated in repeat-dose toxicity studies (Tables 5-5 and 5-6). FOB and Irwin are good first assays to comply with ICH S7A requirements, but the main concern with these assays is their subjective and qualitative nature, as might be expected with behavioral testing. Follow-up safety pharmacology studies with quantitative CNS measures may need to be performed if there are signs or observations of adverse effects on the CNS, such as seizure liabilities, in which case, an electroencephalogram (EEG) may be necessary. FOB and Irwin observational assessments aim to evaluate motor activity, behavioral changes, coordination, sensory-motoractivity , sensory-motor reflexes responses, and body temperature. A typical study for investigating most small molecule test articles is a single administration, parallel group design, stand-alone CNS study, with four groups of at least six rats per group (single sex; males if no exposure differences are anticipated) which are administered the control or test substance at low, mid and high doses. Animals are socially housed, with up to three animals per cage based on their dose group allocation. The trained CNS technician conducting the FOB or modified Irwin assessment is typically blinded to the treatment. Data should be collected prior to dose administration, either the day before (i.e., pretreatment) or on the day of dosing (baseline). Dosing is staggered to allow time for the same technician to conduct the FOB or modified Irwin assessments at select time points postdose. The differences between the FOB and modified Irwin assessments are the subtleties in how the animals are handled during the observational battery. The FOB typically will have an assessment around Cmax, and 24 hours postdose, whereas the modified Irwin may have up to five observations up to and including Cmax to 5 hours postdose, or 24 hours if effects are still detected at 5 hours postdose. The FOB and Irwin assessments also can be incorporated in repeat-dose toxicity studies to assess dose range, duration, reversibility of behavior, and physiological functions. Depending on the intended human population, rodents of different ages (neonatal, juvenile, adult) or pregnancy status can be used in the study, with six main animals (single sex) per group or five main animals per sex. If conducting these assessments as part of repeat dose toxicology studies, males are typically assigned to the CNS study while the females are assigned to the respiratory study (Tables 5-6). Again, animals are first acclimatized to the experimental room prior to being randomized and distributed in groups; this is usually done as part of the toxicity study protocol. A particular day within the toxicity study is scheduled for the FOB or Irwin assessments to be carried out, and the animals are dosed with the control substance and three dose levels of the test article at a designated baseline time point and then placed back in their home cage; the animals are then observed at selected time points up to 24 hours postdose. Dosing can be staggered during the evaluations so that a single observer can collect and record all parameters from several animals and treatment groups at each timepoint. The objective is to determine the highest dose that is tolerated with no observed adverse effect and determine the lowest dose that causes noticeable adverse effect on the behavior of the animals. In the Irwin assessment, several observations are included: · Socially housed home cage observations which include posture, unusual behaviors such as convulsion, shivering, vocalization, stimulation, and stereotypy; · Handling observations, such as ease of removal, handling reactivity, body tone, salivation, lacrimation or piloerection; ·  Motor activity in open field including spontaneous motor activity, gait abnormalities, stereotypic behavior, irritability, piloerection, urination or defection; and · Autonomic activity assessment such as touch response, startle response, righting/palpebral/ grasping reflexes, tail pinch, thermal nociception, ataxia, body temperature and pupil response. · Clinical signs and body weights are also recorded. Observations that can be identified during FOB assessments include: · Ataxia; · Reactivity; · Catalepsy (lack of response to external stimuli); · Passivity (lack of response or absence of struggle); · Spontaneous motor activity (locomotor, rearing, grooming); · Social interaction; · Stereotypy (repetitive, purposeless behavior such as head bobbing, shaking, gnawing, licking); · Mortality; · Exophthalmia (protrusion of the eyeball assessed visually); · Gait (hypotonic or hypertonic- splayed posture to muscle weakness); · Grasping reflex (measured by wired maneuver which measures ability to grasp); · Grip strength; · Straub tail reaction (elevation of tail from horizontal); · Tremor (involuntary movements caused by alternating contractions of opposing muscle groups); · Convulsions (violent involuntary contractions of voluntary muscles which can be clonic with alternating contraction and relaxation of muscles, or tonic, which are sustained contractions, cramp-like or mixed clonic and tonic); · Diarrhea; · Lacrimation; · Piloerection (ruffling of fur); · Ptosis (partial or full eye closures due to drooping of upper eyelids, pupil size); · Rectal temperature;  Salivation; · Urination; · Corneal reflex (eye blink response); · Righting reflex (ability to rest or the body to its normal upright position); · Pineal reflex (response to retract or twitch the ear); · Tail pinch; and · Hot plate response In ICH S7A, screening and monitoring for potential adverse CNS effects by new pharmaceutical candidates with FOB or Irwin assessments is the first step in assessing CNS safety liabilities or risk to human safety, even though these tests are dependent on the skills of the person conducting the observational battery. The conglomeration of endpoints may need to be integrated to detect potential adverse events and not just individual parameters. Some endpoints do not translate directly to CNS adverse events in humans, and complete concordance cannot be ascertained. If an adverse effect on CNS is identified during an FOB or Irwin test, during a repeat-dose toxicity study or in clinical trials, follow-up studies (e.g., cognition and learning, fear and anxiety, etc.) and seizure liability assessments using electroencephalogram (EEG) may be needed to elucidate further the nature and mechanism of action of adverse CNS effect before continuing drug development. Observations in rodents such as sterotypies (i.e., head bobbing, scratching, excessive grooming and licking) often precede convulsions, and could be clonic or tonic. 9.Safety Pharmacology for Biologics or Biotechnology-derived Pharmaceuticals Safety pharmacology testing for biopharmaceuticals (i.e., biologics or biotechnology-derived pharmaceuticals) is covered by ICH S6(R1). Biopharmaceuticals are known for their selectivity (like antibody binding or ligands for receptors), and they are not expected to affect the CV or respiratory systems, or the CNS. However, they may have on-target exaggerated pharmacology, or potential off-target secondary pharmacology activity that may contribute to adverse effects on the vital organs that make up safety pharmacology. For the majority of biopharmaceuticals (large molecules or biotechnology-derived products), safety pharmacology endpoints are incorporated in the repeat dose toxicology studies according to ICH S6(R1).45 If, however, biopharmaceuticals are not highly selective for receptor binding, represents a novel or first-in-class product, or both, then stand-alone or follow-on studies for more extensive safety pharmacology evaluations (i.e., as per ICH S7A) may be necessary. For biopharmaceutical products which are active in rodents, safety pharmacological endpoints for CNS adverse effects (i.e., using FOB or modified Irwin assessments) are evaluated after a single dose or repeated dose administration; this depends on the test article’s route. of administration, dosing regimen. If the biopharmaceutical is active in non-human primates and rodents then adverse effects can be evaluated in both rodents and non-human primates, but if it is only active in the latter, then safety pharmacology endpoints are only incorporated into non-human primate repeat-dose studies. In some cases, in vitro CNS safety studies are conducted before in vivo studies are carried out,44 guiding the selection of safety pharmacology endpoints to include in repeat-dose studies and follow-up studies as appropriate (i.e., as per ICH S6(R1) guidance). 10.Safety Pharmacology for Oncology Products Safety pharmacology for oncology products is covered by ICH S9. For oncology products that are designed for late-stage cancers, there is a need to accelerate the clinical development of the drug candidate so they can be accessible to patients without undue delay. While no stand-alone (i.e., per ICH S7A) safety pharmacology studies are necessary, the assessment of adverse effects on the three vital organ systems (CV, respiratory, and CNS) can be incorporated in repeat-dose toxicity studies or limited to the assessment of ECGs (with scientific justification) collected from a large animal (e.g., dog, non-human primate, or minipig) toxicology study, according to ICH S9. Repeat-dose toxicity studies evaluating the three vital organ systems from the safety pharmacology core battery should be conducted in accordance with GLP regulations and with detailed clinical observations. In rodents (usually rats), repeat-dose studies usually incorporate the FOB or Irwin assessments, with half of the main study animals in the 28-day toxicity study (e.g., five of 10 males) undergoing assessment on Day 1, as well as detailed respiratory observations (e.g., five of 10 females), with treated groups being compared with the vehicle control group. In a non-rodent repeat-dose toxicity study, ECG and hemodynamic measurements (heart rate, blood pressure, and temperature) are collected in addition to detailed observations; these are collected at baseline (i.e., predose), on Day 1 (the first day of dosing) and during the last week of dosing (i.e., Day 28 or Day 90), including interim collection mid-study if warranted. Measurements from treated groups will be compared quantitatively with the control vehicle group and significant changes will be assessed and correlated with dose levels, toxicokinetic exposures, or both. If effects are identified during evaluations on the last week of dosing, CV, respiratory and CNS evaluations will generally be added to the recovery period to assess reversibility. The desire to accelerate the development of oncology products due to the potentially greater benefit to risk ratio is aligned with the lack of regulatory requirement for stand-alone pharmacology studies. As safety pharmacology assessments are incorporated in repeat-dose toxicology studies, patient safety can be adequately addressed while also adhering to the 3Rs of animal use according to ICH S9. When designing follow-up safety pharmacology studies for oncology products, the pharmacology and the clinical indication need to be considered and such studies are performed on a caseto-case basis (Table 5-7). They should be streamlined to reduce the number of animals especially for biopharmaceuticals and oncology drugs according to ICH S645 and ICH S946 and supplemental tests on these vital organs as well as other organs should also be done on a case-by-case basis (Figure 5-1). 11.Comprehensive in vitro Proarrhythmia Assay The Comprehensive in vitro Proarrhythmia Assay (CiPA)47,48 was developed in 2013 by the FDA with the goal of improving the assessment of proarrhythmic markers and to provide more relevant, reliable and comprehensive preclinical in vitro cardiac screening tools. This initiative was based around the potential for some pharmaceuticals (e.g., verapamil) to block the hERG channel however not to be proarrhythmic in humans. As its name suggests, CiPA includes an assessment of comprehensive list of cardiac ion channels that are involved in the ventricular action potential, and as such its use supports the requirement for hERG evaluation.49,50 The first step of CiPA is to conduct patch clamp assays on hERG transfected cells for six cardiac ion channels: hERG, Nav1.5 (late sodium), Cav1.2 (calcium), KvLQT1 (slow delayed rectifying potassium current; IKs), Kir2.1 (inward rectifying potassium channel responsible for membrane potential; IK1) and Kv4.3 (fast transient outward potassium current; Ito). The data from these patch clamp assays are then used to supply computer-simulated models of the human ventricular action potential that can provide information on potential early cardiac liabilities. The algorithms driving the computerized models can predict changes in the ventricular action potential (e.g., prolongation) as well as generation of any early afterdepolarizations. Early afterdepolarizations are the early triggers of proarrhythmic risk. The in silico models, such as the O’Hara Rudy model, which is an accurate model for healthy human ventricular action potential, are capable of assessing compounds that prolong the QT interval that is not associated with proarrhythmic risk (e.g., verapamil).51 Lastly, the patch clamp data through in silico modeling are assessed for translation against induced pluripotent stem cells-derived cardiomyocytes (iPSc-CM). CiPA also includes the use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). These cells are assessed using multi-electrode array technology to measure the extracellular field potential of compounds, helping to identify those with potentially adverse effects on CV electrophysiology. Changes in the recorded field potential waveforms underlie changes in the ventricular action potential and could indicate blockage of the hERG or other ion channels, such as the sodium channel Nav1.5, which is responsible for the upstroke of the action potential or calcium channel Cav1.2, which is responsible for the duration of the actional potential. Hence a prolonged field potential duration could result in the prolongation of ventricular action potential and could indicate a prolongation of the QT interval recorded on the ECG. Drug-induced action potential prolongation should be distinguished from prolongation arising from the variability of cell-to-cell beating with the use of positive and negative control compounds.55 As indicated above, other potassium channels in addition to hERG, like Kir2.1, Kv4.3, KCNQ1/KvLQT1_mink) are also investigated during CiPA. 12.Safety Pharmacology for Other Organs Safety pharmacology studies for organ systems other than those included in the core battery can also be evaluated depending on the therapeutic indication and adverse findings in the toxicity studies. This includes the renal or urinary systems, the gastrointestinal system, and the autonomic nervous system, among others. Endpoints of such studies will depend on the system being tested, but may include the volume of urine excreted and electrolyte excretion, gastrointestinal transit time and gastric emptying, immunological parameters such as immune cell phenotyping, endocrine parameters such as hormone levels, and orthostatic hypotension. These parameters could be used to assess functional changes in these other organ systems. All pharmaceutical or biopharmaceutical drug candidates should be tested for safety pharmacology of these other organ systems as necessary, and on a case-by-case basis, with a fit-forpurpose study design, either during nonclinical drug development or before Phase 2 of clinical development. ICH E14/S7B Q&As - The Need for Nonclinical Studies for Clinical TQT Studies The need for nonclinical studies for clinical TQT studies is covered by ICH guidance E14 and the ICH S7B Q&As. ICH S7B was implemented in 2005 to guide the conduct of nonclinical safety pharmacology studies assessing the potential of new agents to prolong ventricular repolarization (QT interval) before first-in-human studies. That same year, ICH E14 was adopted, outlining the need to conduct a clinical study to assess the effects of these new agents on the potential to delay cardiac repolarization. The FDA has published data on the number of TQT studies conducted from 2016 through 2020 and found that 32% of studies failed to reach the E14 Q&A 5.1 threshold of 2X high clinical exposures. Additionally, another 24% of clinical studies had alternate study designs (82% of these were for oncology indications) where no TQT study could be conducted, therefore resulting in a label of “no large QTc effects.” In an effort to reduce the number of clinical TQT studies, E14/S7B Q&As were approved in February 2022, finalized in July 2022, and were implemented in August 2022.7,8 This guidance provides the opportunity to review the double negative nonclinical results (i.e., both hERG and in vivo QT/QTc studies are negative) in combination with clinical E14 Q&A 5.1 (in support of TQT waiver when clinical exposure does not reach 2x high clinical) or E14 Q&A 6.1 (to support labeling of “low likelihood of proarrhythmic effects due to delayed repolarization”). Assuming the results from ICH S7B hERG and in vivo QT studies show no delay to ventricular repolarization, they will give confidence to regulators and clinicians that the tested compounds do not pose a risk to healthy volunteers participating in first-inhuman studies. In order to use the nonclinical hERG and in vivo QT results (double negative) as an alternative (E14 Q&A 5.1) or as a substitute (E14 Q&A 6.1) for a clinical TQT study, there should be: · Alignment of standard protocols; · Experimental conditions that follow the best practices as outlined in S7B Q&As (Q&A 2.1; hERG and 3.1-3.5 in vivo QT assay); and · Consistency of reports for regulatory review. The in vitro hERG Q&As of S7B (Q&A 2.1 for patch clamp) provide guidance on:65 · Recording temperature (35-37 °C), voltage protocol to follow; · Measures of recording hERG current quality; ·  Primary endpoints to measure or calculate (e.g., IC50, Hill coefficient, and confidence intervals); Concentration verification; and · Use of positive and negative controls. The in vivo QT assay Q&As of S7B (Q&A 3.1 to 3.4) provide guidance on: · Species selection consistent with ICH S7B for appropriate, freely moving, non-rodent species consistent with toxicology studies; · Exceeding anticipated therapeutic concentrations – the higher the multiple, the less reliance on assay sensitivity for QTc under E14 Q&A 6.1 (i.e., statistical measure of QTc variability; least significant difference to detect changes in QTc in milliseconds, inclusion of concentration QTc analysis, required for E14 Q&A 6.1 and recommended for Q&A 5.1); · Use of an individual animal heart rate correction method -including QTc/RR scatter plots, assessing assay sensitivity and laboratory historical sensitivity (minimal detectable difference). The assay sensitivity to detect changes in QTc (least significant difference) to the threshold measured in clinical studies is only required under Q&A 6.1. It is best practice to report the least significant difference for QTc for the in vivo QT assay in support of Q&A 5.1, however, there is no minimal threshold and the value is used only as a measure of variability for QTc within a study. Finally, S7B Q&A 3.5 are points to consider for reporting the results. Thus, the nonclinical double negative data can now be advanced past first-in-human regulatory review for consideration with ICH E14 Q&As 5.1 or 6.1 as the nonclinical-clinical risk assessment for proarrhythmia. This is anticipated to increase submissions for TQT waivers and reduce the number of required clinical TQT studies. Follow-up in vitro assessments on ventricular repolarization are included in the ICH S7B best practice(Q&A 2.2-2.5) using cardiomyocytes and  proarrhythmia models (Q&A 4) (ICH E14/S7B Q&A reference). 13.Summary and Conclusion Safety pharmacology studies has evolved over the years to keep up with the changing modalities of pharmaceuticals. Nonetheless, they have remained focused on the assessment of potential adverse pharmacological effects prior to the first human dose being given in clinical trials. The safety pharmacology community has produced high quality functional data to assess drug safety, either as stand alone or in combination with repeat dose toxicology studies. The refinements over the past two decades have focused on best practices (e.g., using digital data collection, acclimation and refinement techniques to lower stress), and a commitment to the 3Rs principles to reduce the use of animals and social housing, individual animal heart rate correction, QTc/RR scatter plots, among others. That said, the assessment of most of these details is not standardized across the industry and are not systematically included in study reports. With the publication of the ICH S7B Q&As, those details, statistical measures and corrections and more need to be in the final reports. Towards this singular goal, core battery safety pharmacology studies have been focused on first-in-human studies, usually single dose and small sample size studies, with continuous data collection. Based on the pharmacology of the pharmaceutical or biopharmaceutical being tested, studies may be modified to accommodate other designs in follow-up studies. The conduct of various safety pharmacology studies – in vitro hERG studies, the ability to detect a hERG block and QT prolongation in the CiPA initiative, in addition to in vivo testing– which are undertaken at the screening stage of drug candidate selection, has staved off drug attrition due to proarrhythmia at the later stages of clinical development. This necessitated integrating the in vitro hERG and in vivo QT assay nonclinical data as well as clinical data for integrated risk assessments of QT/QTc prolongation, and the potential waiver and reduction of thorough TQT clinical studies if there is no observed hERG block or QT/QTc prolongation in well-controlled nonclinical studies. For CV assessments, stand-alone safety pharmacology studies are the gold standard rather than combination toxicology studies, but combination safety pharmacology studies will continue to be used for both small and large molecules, in keeping with the 3Rs and as long as safety pharmacology endpoints are collected in a rigorous and interpretable manner to elucidate effects of the pharmaceutical on both structural and physiological functions. For most CV studies, implanted telemetry devices with body temperature collection and respiratory capabilities are now acceptable and more prevalent than jacketed telemetry, paving the way to collect more reliable and consistent ECG and respiratory waveforms, leading to more accurate analysis that facilitate combination with toxicity studies. Currently, cell and gene therapy are not specifically addressed in the S7A and S7B/E14 guidance as they are rapidly evolving, but soon they may need to be included in the scope of the guidelines as well. There is a need to continue to assess the effects of translating the data garnered from the safety pharmacology studies to limit failures during clinical trials. Case Study 5-1 What safety pharmacology studies should be conducted for a small molecule that has nanomolar inhibition of hERG? • Background A small molecule (new molecular entity) is being developed as a systemic drug for neurodegenerative disease. The intention is that it will be administered as an oral tablet to be taken once daily. During nonclinical development, the molecule exhibited nanomolar inhibition of the hERG channel (IC50 <100 nM) in nonhuman primates. • Solution The core battery of safety pharmacology studies is required for this small molecule being developed for a neurodegenerative indication. As the molecule was being developed as a daily oral tablet, the drug development plan accounted for two CV studies to address repeat-dosing and correlate pharmacokinetics with CV measurements. In addition, in the spirit of the 3Rs, the large animal CV study was combined with respiratory collection via telemetry. For the CNS, FOB was integrated into the 28-day rodent repeat toxicology study. The preliminary cardio-respiratory study was conducted non-GLP using an escalating dose design (at pharmacologically efficacious dose and two dose levels (2X and 6X) above the first dose using three non-naïve telemetry instrumented nonhuman primates. The pivotal GLP cardio-respiratory study was conducted using drug-naïve nonhuman primates, which were surgically implanted with a telemetry device. A parallel group study design was used, with four groups of animals (three animals per group); animals in Group 1 were given a control (vehicle) substance and the animals in the other three groups received escalating doses of the test drug, at the lowest efficacious dose (Group 2), and at 2X and 6X above the lowest efficacious dose (Groups 3 and 4, respectively). This study design is modified from that shown in Table 5-4). Nonhuman primates were dosed once daily, and telemetry data for CV and respiratory function were collected on Days -1, 1, 2, 4 and Day 7. On Day 8, the animals were dosed one more time to enable blood collection for pharmacokinetic analysis on Days 8 and 9 (24 hours postdose). The results of these cardio-respiratory studies (both non-GLP and GLP) indicated no in vivo CV risk of the molecule at 6X the efficacious dose when administered once-daily for 7 days, supporting its continued development. Case Study 5-2 What safety pharmacology studies should be conducted for a small molecule that has a seizure liability? • Background A small molecule sodium-blocking drug that needs twice-daily oral administration is under development as an analgesic. Rat and dog studies have been used in its development but convulsions have been observed. The convulsions occurred at plasma exposures that are estimated to be higher than those likely to be seen in patients, at approximately 10X the efficacious clinical dose in dogs and 45X of the efficacious clinical dose in rats. • Solution Appropriate in vitro studies need to be conducted to evaluate potential mechanisms for the convulsions that have been observed. A dog EEG study which records spontaneous electrical activity of the brain68,69 will likely be required to: · Confirm the nature of the adverse CNS effects and establish if the convulsions were due to seizure or another toxicological effects (e.g., syncope or overt movement disorder); · Confirm the plasma exposure when the convulsion and other CNS signs (e.g., myoclonus) are observed; · Identify clinical signs that may be a precursor to the convulsions or seizures and that could be used as stopping criteria in clinical trials; and · Confirm the absence of EEG abnormalities at lower doses where no convulsions were observed. The EEG study will typically involve video-EEG68-70 and continuous recording from at least 24 hours prior to dosing until at least 24 hours after the last dose. All dogs will be screened with continuous EEG recorded and analyzed for 72 hours prior to study start to evaluate for the presence of abnormal EEG morphologies prior to assignment on study. The minimum group size is eight (typically four males and four females). The dose levels will be selected to include an EEG ‘no observed effect level’ ideally as high as possible and a higher dose level at which convulsions were observed to confirm the nature of the CNS events. The study design may be adaptative, with dose levels for some study groups being selected based on the outcome from the results with the previous experimental groups. Although not a primary objective, the study will also confirm if convulsions are self-limiting and potentially evaluate emergency drugs (e.g., diazepam, phenytoin, or propofol) to treat any drug-induced seizures. Toxicokinetic sample collections (including important metabolites) will be used at unscheduled timepoints when severe CNS clinical signs are observed to precisely quantify plasma exposure during those events and with concurrent analysis of EEG traces. The duration of repeat administration will be selected to exceed the duration that was required to induce convulsions in the repeated dose general toxicology study. To avoid tachyphylaxis, a 1 month wash-out period will be included. A rat EEG study will likely not be required given the higher safety margins in this species and the data from the dog that will characterize this liability. Chapter 6 Pharmacokinetic and Toxicokinetic Studies The goal of preclinical development is to generate sufficient data to allow an estimation of the benefit/risk profile of a new drug product prior to its use in patients. In general,the benefit(s) are ascertained from a combination of discovery phase studies in cells (i.e., in vitro) or animal-based (i.e., in vivo) models, and from reports in the scientific literature. The risk(s) are determined through a battery of nonclinical safety assessments. Pharmacokinetic (PK) and toxicokinetic (TK) studies – often simplified as a description of ‘what the body does to the drug’– are invaluable for this process as they provide a quantitative connection between the dose administered, and the amount of drug that is subsequently present internally at various times following dosing. When paired with a specific assessment of benefit (e.g., a reduction in cholesterol) or toxicity (e.g., cardiovascular dysfunction, such as evidence of QT interval prolongation on an electrocardiogram, ECG), this information can ultimately be used to develop and justify a clinical dosing regimen that maximizes the benefits of the drug, while minimizing its risks. This chapter will start with a brief overview of PK/TK itself, including an introduction to drug disposition and several other key background concepts. This will be followed by a discussion of the specific global regulatory recommendations/requirements pertaining to the conduct and interpretation of PK/TK analyses, largely from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use(ICH). Finally, the chapter will end with a description of how the results of PK and TK analyses should be presented in marketing and pre-marketing submissions to global regulatory bodies. 参考文献：（上下滑动查看更多） All references verified 16 February 2023. 1. US Food and Drug Administration. Guidance for Industry: S7A safety pharmacology studies for human pharmaceuticals. Current as of July 2001. https://www.fda.gov/media/72033/download 2. International Council for Harmonisation. ICH harmonized tripartite guideline. Safety pharmacology studies for human pharmaceuticals. S7A. Current as of 8 November 2000. https://database.ich.org/sites/ default/files/S7A_Guideline.pdf 3. Pugsley MK, Authier S, Curtis MJ. Principles of safety pharmacology. British J Pharmacol. 2008;154(7):1382-99. https://bpspubs. onlinelibrary.wiley.com/doi/full/10.1038/bjp.2008.280 4. 21 CFR Part 58: Good Laboratory Practice. Current as of 24 October 2022. https://www.ecfr.gov/current/title-21/chapter-I/subchapter-A/part-58 5. 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FDA website. https://www.fda.gov/media/71990/download. Accessed 20October 2022. 声明：原英文原文版权归美国法规事务协会（RAPS）所有，本译文供参考，如有任何建议，请联系我们。 近期直播推荐:

导读       前段时间，一位资深教授向我们推荐这本书《生命科技启示录——捕捉下一只独角兽》，并将书籍的作者柳达先生推荐给我们。柳达先生是华润正大生命科学基金董事总经理，是知名的医药投资专家。他在闲暇之余撰写投资心得，最后汇集成书。该书记录了”生物科技在生命科学创新领域中浓墨重彩的一笔”。希望本书里面的内容，能对国内当下生机勃勃的生物医药热潮下有所启迪和帮助。柳达（知名医药投资专家）|撰文01前言现代制药行业有超过100年历史，第一个行业公认的里程碑药品是德国拜耳的阿司匹林。1899年3月6日，拜耳获得阿司匹林的注册商标，至今拜耳阿司匹林依然在美国、中国、欧洲畅销，其325毫克及81毫克的剂型针对不同适应症，用于止痛、降温及心梗预防等。阿司匹林是少有的经历百年依然不衰的药品。在20世纪前半叶，有一些经典药品出现并且被广泛使用，例如：盘尼西林、阿莫西林、华法林等。在20世纪后半叶，伴随着一系列“重磅炸弹”药物的问世，化学药品的黄金时代到来。泰胃美（Tagamet）的上市，开创先河，标志着现代化学制药进入高速发展阶段。立普妥（Lipitor）的上市，并且在2004年成为全球首个年销售额超过100亿美元的药品，标志着化学药顶峰的来临，立普妥的年度销售峰值（Peak Sales）超过130亿美元，成为行业公认最赚钱的专利药。2011年11月，随着其在美国市场的专利到期由于仿制药在市面上以极低的价格出售，立普妥销售大幅度下滑，跌出全球前十，这一标志性时刻意味着化学药黄金时代的结束。以“重磅炸弹”药品为主线是研究化学药范式比较好的维度。在参考李杰教授的著作《“重磅炸弹”药物：医药工业兴衰录》的同时，笔者结合在美国药学院学习及作为临床药剂师的经验，整理了这部分内容。这里并没有面面俱到覆盖全部的治疗领域，例如：计划生育药品（Birth ControlPills）、治疗儿童多动症（Attention Deficit Hyperactivity Disorder，ADHD）的药品、抗生素等。本章按照治疗领域，归纳经典案例，主要提及重要的科学家、药品主要作用原理（Mechanism of Action）及临床贡献、涉及的制药公司。标题按照突破性机理、适应症（Indication）及同类最优（Best-inclass）或同类第一（First-in-class）药品的结构呈现。02经典案例一：H2阻断剂及消化性胃溃疡，Zantac1986年，泰胃美的年销售额超过10亿美元，是全球第一个“重磅炸弹”药品。泰胃美（Tagamet）是治疗消化性溃疡药品，由史克公司在英国的JamesBlack团队发现，在此之前，史克是默默无名的美国制药企业，在泰胃美成功之后，升格为全球知名制药公司之列，按照销售金额，位居第九位。泰胃美属于H2受体阻抗剂（H2 blocker），用于减少胃酸分泌，治疗疾病为十二指肠及胃溃疡、酸反流性烧心、消化不良等。在泰胃美出现前，消化性溃疡的治疗方案主要是休息、清淡饮食、服用抗酸剂等。患者因为溃疡复发而需接受手术。而根据Lancet发表的一项研究，泰胃美上市以后，美国溃疡手术的数量比此前下降30%。世界卫生组织（WHO）也将泰胃美列入世界最基本药物之一。鉴于泰胃美的专利在1994年到期，公司在1980年后半期就开始启动临床试验，寻求美国FDA批准非处方药（Over-the-counter，OTC）版本。这一保护利润的策略，日后被其他制药企业效仿。泰胃美给患者带来巨大福祉，在商业上也取得非凡成绩，可是它有两个短处，一是药品的半衰期（Half-life）只有2个小时，意味着每天需要服用四次，对于患者而言，用药依从性（Medication adherence）较差。另一个问题是部分患者会产生轻度皮疹的副作用。英国公司葛兰素制药（GSK.US）研制了善胃得（Zantac），并于1983年上市销售。虽然同为H2受体阻抗剂，但善胃得的效价强度（Potency）更高，因此半衰期为3个小时，每天只需服用两次，并且副作用较少。鉴于以上优势，以及高超的定价策略：葛兰素将善胃得的价格定得高于泰胃美50%，并且将额外收入投资在市场营销。产生结果是，1986年善胃得的销售超过20亿美元。鉴于H2 Blocker的革命性发现，以及泰胃美及善胃得的辉煌业绩，其他制药公司纷纷进入这个领域，其中有三家获得成功：日本山之内（Yamanouchi）、美国礼来（EliLilly，LLY.US）和日本帝国（Teikoku）。山之内研发出世界上第三个H2 Blocker—Pepcid。1988年，美国礼来制药（Eli Lilly）在美国上市了第四个H2 Blocker—Axid。泰胃美、Pepcid专利分别于1994年及2020年到期，善胃得及Axid的专利也都于2002年到期；专利到期后，这四家公司都以降低药品剂量的方式，将仿制药（Generic drug）以通用名形式在非处方药市场（OTC）上广泛销售。03经典案例二：质子泵抑制剂及GERD，Losec胃酸对于消化蛋白质及脂肪不可或缺，食物经胃酸降解后进入小肠，营养物质被吸收，不可降解物被排出体外。胃酸不足会引起各种不适，但是胃酸过多会引起烧心（Heartburn）及溃疡（Ulcer）。患者一直采用非处方药的抗酸剂产品，例如：AlkaSeltzer、Maalox、Mylanta、Pepto-Bismol、Tums以及Gaviscon等。这些产品大多含有简单的无机硷作为药物中的活性成分（Activeingredient），通过中和胃酸，可以在几分钟内缓解症状。这些药品疗效时间短，一般维持大约几个小时左右。由于胃酸持续出现，这些OTC产品的药效治标不治本。1990年代初，质子泵抑制剂（Proton-PumpInhibitor）洛赛克的出现，是一个里程碑，改变了这个领域的格局。其药理作用为不可逆地抑制胃壁细胞上的氢/钾离子ATP酶（H+/K+ATPase），比H2Blocker更加强效。First-in-Class药品，包括：洛赛克（Losec）、耐信（Nexium）以及其他me-too药品，例如：普托平（Prevacid）、潘妥洛克（Protonix）和波利特（Pariet），可以有效地阻止胃酸的大量分泌，接近90%的患者可以获得完全缓解。这些药品半效期较长，每天只需服用一次。图1：质子泵抑制剂的作用机制9质子抑制剂的研究与开发过程，再次证明创新药品的基础科学研究及商业合作的全球特徵。需要强调的是，这一领域的进步是无数科学家、临床医生坚持梦想、相信直觉，以及运气的综合结果。几个关键科学家对于这一全新领域做出非凡的贡献：1、GeorgeSachs10的课题组证实：氢/钾离子ATP酶是运输胃酸通过黏膜和胃壁细胞的质子泵；2、制药公司AstraHassle11的药化学家们对于分子结构做出巨大贡献，最终洛赛克问世。1988年，瑞典药政局批准药品用于治疗十二指肠溃疡及反流性食道炎。洛赛克为胃溃疡治疗带来一场革命，在1999年销售达到60亿美元。虽然洛赛克非常有效，但并不是适用于全部患者，这主要是因为它具备与众不同的生物利用度（Bioavailability）。1987年，AstraHassle开始研究更好生物利用度的后备化合物，最终开发出耐信，2000年及2001年在瑞典和美国获批。而当时洛赛克的美国专利即将到期。随着洛赛克的巨大成功，其他制药企业也纷纷加快me-too药物的开发，其中几个成功的药品包括：1、1984年，史克与德国Byk Gulden公司达成协定，共同开发质子泵抑制剂，最终发现泮托拉唑（Pantoprazole），由于这个产品的水溶性（Watersolubility）很好，水中稳定性高，得以成为世界上第一个上市的用于特护病人给药的质子泵抑制剂，于1994年被美国FDA批准上市；2、由日本武田公司（Takeda Chemical Industries）发现的普托平；3、日本卫材（Eisai）发现的Aciphex。图2：各质子抑制剂历来的化学结构发展及开发年份质子泵抑制剂被广泛使用，其中洛赛克等被降低剂量，转换成OTC销售。2003年，美国市场质子剂抑制剂的销售额为135亿美元，仅次于降胆固醇的他汀类药物（Statins），数百万患者从中受益。2005年，西澳大利亚的病理学家Robin Warren及医生Barry Marshall发现幽门螺旋杆菌在胃炎及消化性溃疡中扮演的角色，并获得诺贝尔生理学或医学奖。基于这个发现，治疗幽门螺旋菌的一线标准治疗方案得以诞生，这一重大发现将胃溃疡治疗又推进一步。04经典案例三：Anti-histamines及过敏治疗，Claritin过敏症是美国第六大慢性疾病，患者超过5,000万，每年因此产生的医疗费用超过180亿美元。过敏症主要由花粉、食物、尘螨、动物等引起，春天是由花粉引起的过敏高峰。另外，因为海鲜、花生等食物引起的过敏也非常普遍。过敏的症状主要包括：瘙痒、肿胀、皮肤发红、打喷嚏、流鼻涕和流眼泪。科学家们一直在探索过敏的机理，寻求治疗方案。世界上第一个抗组胺药（Anti-histamine）是由瑞士科学家Daniel Bovet于1937年发现，抗组胺药Phenbenzamine及Pyrilamine分别于1942年及1944年上市。Bovet也因此于1957年获得诺贝尔生理学或医学奖。第一代抗组胺药主要的作用机理为阻断H1受体（H1receptor）。但由于亲脂性较高，这类药品可以比较容易地通过血脑屏障（Blood-BrainBarrier，BBB）进入大脑，阻断大脑内的受体，导致比较强烈的中枢神经系统（Central Nervous System，CNS）副作用，例如：瞌睡、镇静、眩晕、记忆损失等。作为广泛认知并且至今依然大量使用的第一代抗组胺药品Benadryl，是由美国辛辛那提大学化学教授George Rieveschl于1940年代初发明。这个药品还被用于其他适应症（例如：晕车、晕船、止吐等），知名的品牌为Dramamine。位于新泽西州的先灵葆雅（Schering-Plough）公司，在2009年被默沙东收购之前，就有发现、生产、销售抗组胺药。第一个产品Trimeton因其瞌睡的副作用小于市场上其他的同类药品，而被广泛使用于感冒及过敏。先灵葆雅的化学家FrankVillani在第一代产品上稍加改善，开发出Piriton，这种药半衰期更长，每天只需服用一次，获得了巨大成功，至今这个药的有效成分Chlorphenamine依然被广泛使用在OTC的感冒及过敏药中。第二代抗组胺药的CNS不良反应明显减少，主要是因为它们可较少地穿透血脑屏障。第一个产品为Hoechst Marion Roussel的Seldane，1985年在美国上市，是当年最畅销的处方药。这个药品由AlbertKerr发现，是首批直接通过电视广告向消费者推销的药品之一，反响空前。先灵葆雅的Villani团队在1970年发现氯雷他定（Loratadine），在1980年申请专利，这类阻胺药具有较小或者没有镇静反应。1993年，Loratadine终于获得美国FDA批准，商品名为Claritin。1997年，FDA放宽对于处方药的广告规定。先灵葆雅在1998、1999年花费3.22亿美元用于市场推广，Claritin的年销售额由1997年的14亿美元，增长至2000年的26亿美元。为了解决开瑞坦专利到期而导致的专利断崖，先灵葆雅开发了Clarinex，于2002年上市销售，但这个产品的疗效并没有优越很多。Claritin及Clarinex一举将先灵葆雅推入世界一流制药公司的行列。05经典案例四：血液稀释药（Blood thinner），波立维（Plavix）血液主要由红细胞、白细胞、血小板及血浆组成。红细胞的作用是运输氧气到各个器官，白细胞的主要作用是抵御细菌及病毒的侵入，血小板是黏而少的组成部分，占大约1%，促进血液凝固。失去凝血功能的人患有血友病（Hemophilia），这类患者需要定期注射凝血因子。然而，如果血小板过多，会引起血液黏稠，导致血栓等心脑血管疾病。经常长途飞行的旅客，由于长时间局限于狭窄空间久坐不动；或者一些需要长期站立工作的职业（例如：药剂师），都容易患有深部静脉血栓（Deep Vein Thrombosis）。“栓塞”是指凝血块在体内通过循环系统迁移而导致阻塞，是非常危险、致命的症状。血液稀释药是预防及治疗血栓及栓塞的首选药物。1916年，科学家们在美国约翰霍普金斯大学发现肝素（Heparin），是世界上发现最早、至今依然被广泛使用的经典药品之一。Heparin是一种天然糖胺聚糖抗凝血剂，为医院常备药品，用于血液透析、血管手术、器官移植等，同时用于支架手术，挽救了数以百万计患者生命。Heparin基本上需要在医院诊所内注射用药，由于全球众多药厂生产，因此没有形成“重磅炸弹”产品。华法林（Warfarin）为另一款经典的临床抗血栓药物，与Heparin不同，可以口服用药。其作用原理为通过抑制维生素K依赖的凝血因数（II、VII、IX、X）的活化发挥抗凝作用，被广泛应用于临床抗凝治疗。虽然其价格低廉、且为口服，但存在较大的用药疗效及安全问题。许多食物、药物以及疾病都可能影响华法林的药效，为保证治疗的安全性及有效性，病人常常需要频繁抽血监测并对剂量进行调整。有鉴于华法林的较多瑕疵，药厂研发出新型口服抗凝血剂（Novel Oral Anticoagulants，NOACs），这类药物不需经常检测副作用，因此在一些适应症的治疗上正慢慢取代华法林。但同时，这类药物的价格为华法林的近30倍。目前市场上主要的NOACs药物为BMS和Pfizer的Eliquis，以及Johnson & Johnson和Bayer的Xarelto，在2020年全球畅销药物中排名第四及第十，全年销售额为91.7亿美元及69.3亿美元。Eliquis的销量甚至超过了BMS的肿瘤免疫疗法畅销药Opdivo。德国拜耳阿司匹林（Aspirin）由化学家Felix Hoffmann合成得出，至今已超越百年，依然被用于止痛、解热、抗凝，是制药历史上经典的药品之一。它的作用机理直到1971年才得以清晰的阐明。John Vane发现，阿司匹林通过抑制前列腺素合成酶（Cyclooxygenase）而起效，解释了其抗血小板、解热和抗炎等作用。Vane凭藉这一发现获得1982年的诺贝尔生理学及医学奖。1985年，时任美国卫生部长向公众宣佈，一日一片81毫克的阿司匹林有助于预防二次心肌梗死。从此，阿司匹林成为家庭药箱的必备品。法国的制药巨人赛诺菲（Sanofi）是后起之秀，1973年才成立。公司的化学家Jean-Pierre Maffrand凭藉两个血液稀释药Ticlid和Plavix使Sanofi成为世界一流的制药企业。1978年，Ticlid获批上市。1993年，Sanofi选择与美国的百时美施贵宝（Bristol-MyersSquibb，BMS）合作，在美国进行氯吡格雷（Clopidogrel）的临床III期开发、市场推广与销售。Clopidogrel与阿司匹林的作用机理不同，可选择性阻断血小板上的ADP受体，从而抑制血小板凝聚。一份大规模的临床试验CAPRIE结果证明，Clopidogrel比阿司匹林更加有效，并且对于肠胃的副作用更小。1997年4月，Sanofi及BMS递交在美国上市申请，被授予“优先审评”。1997年11月，FDA批准Clopidogrel以商品名Plavix在美国上市，批准适用症为预防和治疗因血小板高聚集引起的心、脑及其他动脉循环障碍疾病。在获批后的一年内，仅在美国就有超过300万的患者服用Plavix。日本的第一三共（Daiichi Sankyo）通过修饰Clopidogrel开发出Effient25，并于2009年在美国上市，这一药品是Me-too药，它比Clopidogrel作用更强，起效更快，但是安全性较差，出血事件频率较高。阿斯利康的倍林达（Brilinta）对于血小板聚集抑制活性是可逆的，且无需经过肝脏代谢活化，于2011年获批在美国上市。06经典案例五：止痛、降温、消炎类及COX-2抑制剂，Celebrex人类有记录历史，就有关于疼痛的描述，并且疼痛经常伴随发烧（Fever）及发炎（Inflammation）。有两类药品被视为经典，其中一类是非类固醇抗发炎药（Non-Steroidal Anti-Inflammatory Drugs，NSAIDs），这类中的代表是阿司匹林（Aspirin）、布洛芬（Ibuprofen）等。阿司匹林的起源是柳树树皮，可以追溯到公元前1500年。1897年，德国拜耳（Bayer）化学家Felix Hoffmann将其制备合成。早期的阿司匹林对于胃肠道有一定的副作用，后期通过肠溶剂型（Enteric Coating），已经基本解决这一问题。布洛芬被使用在经痛，也是儿童感冒OTC药品中常见的活性成分。这类的其他药品还包括了Naproxen26、Indomethacin、Diflunisal及DiclofenacSodium。另一类经典止痛降温药品为乙酰氨基酚（Acetaminophen/Paracetamol），商品名为泰诺（Tylenol），至今其作用机制还未完全明了，这个药品在美国医院内基本每个病人都会配置。目前，acetaminophen是感冒药中主要的活性成分之一，同时也具备滴剂（Drop）或者口服液（Syrup）等剂型给儿童使用。鉴于用药过量（Overdosing）对于肝有损伤的副作用，FDA建议成人每天的摄取量不超过4克。在制药行业历史上，美国强生制药（Johnson & Johnson）对泰诺安全意外事件的处理，被行业视为经典的危机公共关系处理案例，也大大提升了强生制药的品牌形象。环氧化酶（COX-2）抑制剂的出现，改变了这个领域的行业格局。两个代表药品为美国辉瑞的西乐葆及美国默沙东的万络，它们相继成为重磅产品，但是结局各有不同。环氧化酶包括两种亚类型（COX-1及COX-2），COX-1保护胃黏膜及维持血管扩张；COX-2主要分布在有炎症的细胞和组织中，在急性炎症反应中被启动。因此，选择性抑制COX-2的抗炎药更为安全有效。美国孟山都（Monsanto）公司的Phillip Needleman领衔发现世界上第一个COX-2抑制剂Celecoxib。1998年，美国Pharmacia公司收购了孟山都的药物板块，1999年开始与辉瑞合作，将药品以商品名西乐葆（Celebrex）上市。1999年，美国默沙东公司的Prasit领衔团队研发出世界上第二个COX-2抑制剂Vioxx（万络）。这两个药品的适应症包括骨关节炎症（Osteoarthritis）及风湿性关节炎（Rheumatoid Arthritis）。2003年，西乐葆的后续产品Bextra获批上市，由辉瑞及Pharmacia公司合作市场推广。同一年，辉瑞制药收购Pharmacia，获得COX-2抑制剂产品线。2004年，默沙东的VIGOR28临床试验中结果显示，与服用Naproxen的患者相比，服用Vioxx的患者心机梗死的发病率增加5倍。2004年9月30日，公司自愿从市场上召回Vioxx，资本市场反应剧烈，数月内默沙东市值下跌300亿美元，这对于公司的声望是巨大打击。一直到生物制药PD-1研发的意外成功，公司才再现雄风。2005年，由于潜在的心肌梗死及严重皮肤过敏等副作用，辉瑞也自愿撤回Bextra。目前，全球市场在COX-2抑制剂领域仅存辉瑞制药的Celebrex。即便如此，在治疗自身免疫系统的生物制药出现之前，COX-2抑制剂在关节炎治疗领域独领风骚。07经典案例六：SSRIs与抗抑郁类药品，「百忧解」Prozac抑郁是在美国非常普遍的一种精神疾病。在中国，由于严重缺乏治疗精神疾病的专科医生，估计有大量未经过诊断的病人。1950年代，三环类抗抑郁药（Tricyclic Antidepressants，TCAs）是最常用的药品类别，在一定程度上缓解症状，但是副作用比较大，例如：瞌睡、便秘、视力模糊，更为严重的是，如果剂量超过10倍，容易引发心律失常而导致死亡。另一类比较常用的药品为单胺氧化酶抑制剂（Monoamine oxidase inhibitors，MAOIs），对于治疗抑郁有一定帮助，但是容易产生同许多药品的互相作用（Drug interaction）。出身香港的华裔科学家汪大卫（David Wong）在美国礼来（Eli Lilly）所带领的课题组研制了影响深远的名药百忧解（Prozac）。百忧解属于全新药品类别：选择性血清素再吸收抑制剂（Selective Serotonin Receptor Inhibitors，SSRIs），同传统的三环类抗抑郁药相比，具备以下优点：1、副作用小，尤其是对于中枢神经系统；2、药效稳定、半衰期长，每天服用一次即可；3、还可以用于神经性贪食症或者强迫症。FDA在1988年正式批准上市，鉴于它的有效性及安全性，百忧解迅速成为重磅炸弹。这个突破性药品甚至被评为20世纪最伟大的发明之一。在2003年专利保护期截止前，销售峰值23亿美元，占礼来制药全部收入的三分之一。这个药品使礼来制药成为精神类领域的巨人，此后礼来制药又开发了欣百达（Cymbalta）、再普乐（Zyprexa）、择思达（Strattera）。在这一领域的另一个畅销药品是英国GSK的Paxil，一度在美国畅销成为「重磅炸弹药品」，但是2003年专利到期后，由于价格低廉的仿制药出现，其销量大幅下降。美国惠氏制药（Wyeth）的怡诺思（Effexor）于1993年上市，销售峰值在2008年达到39亿美元，专利到期后，在2009年销售仅5.2亿美元。另一类治疗抑郁及强迫症的一线用药是辉瑞的复甦乐（Zoloft），销售峰值达到33亿美元，2006年专利到期后，销售也跌至5亿多美元。08经典案例七：GABA及抗癫痫药品，Neurontin癫痫（Epilepsy），俗称「羊癫风」，是大脑神经元突发性异常放电，导致短暂大脑功能障碍的一种慢性疾病。全球大约有1%的人口患有癫痫，到目前为止，并没有根治的方法。药物为控制癫痫症状的主要方法，目的在于减少或防止发作。控制癫痫的第一个有效药品是苯巴比妥（Phenobarbital），由Emil Fischer于1903年合成，但却有催眠的副作用，这个药品至今依然在医院被广泛使用于镇静。制药企业Parke-Davis的大仑丁（Dilantin）在1939年上市，是第一个结构与巴比妥相似，但没有催眠副作用的抗癫痫药品，具有划时代的意义。但这个药物有可能会带来白血球减少的副作用，增加感染风险。Parke-Davis通过一系列在抗癫痫领域的后续产品奠定了领导地位。作为一个新的作用机理，加巴喷丁（Gabapentin），商品名为Neurontin，于1993年被美国FDA批准辅助控制癫痫，并且于1994年上市销售，后续更是在临床被常用于缓解糖尿病或带状疱疹引起的神经痛。2001年销售额到达17.5亿美元，于2003年达到销售峰值30亿美元，其中主要原因是由于辉瑞制药对其off-label use的推广。09经典案例八：HMG-CoA还原酶抑制剂及治疗高血脂药品，Lipitor高血脂症指血液中脂类物质过多，使血液黏稠度增高，这些脂类会在血管壁内膜沉积，逐渐形成小块，阻塞血管，导致血流变慢或者断流。根据发生的部位不同，会引起冠心病、脑中风、肾动脉硬化，并产生一系列的诱发疾病。血脂主要由胆固醇、甘油三脂及类脂组成，与临床有比较密切关系的主要是胆固醇及甘油三脂。根据不同的作用机理，治疗高血脂的药物种类比较多，但是全球公认最有效的是他汀类药物（Statins），作用机理为抑制HMG-CoA还原酶（HMGCoA reductase），从而降低血液中的低密度脂蛋白—胆固醇（LDL-C）。1987年，美国得克萨斯大学的科学家Michael Brown及Joseph Goldstein因为对于胆固醇调节机理的研究，获得诺贝尔医学及生理学奖。多项大规模临床试验结果显示，Statins可针对性减少胆固醇的生成，对于减少动脉粥样硬化性心血管疾病有显著作用。Statins的历史可以追溯到1976年，日本科学家AkiraEndo从桔青霉提取了美伐他汀（Mevastatin）。1979年，默沙东的科学家也另从土曲霉素中提取了洛伐他汀（Lovastatin）。这两种为天然他汀。1987年，默沙东的Zocor被美国FDA批准上市，这是第一个半合成他汀，为First-in-class药品。此后，美国BMS及日本第一三共制药研发了me-too药品Pravastatin。1993年，瑞士诺华制药研发出氟伐他汀（Fluvastatin），是第一个全合成他汀。1996年，美国Warner Lambert公司的立普妥（Lipitor）被批准上市，后来公司被辉瑞制药收购。这款药物也为全合成他汀，凭借辉瑞制药强大的市场推广能力，以及优于同类药品的疗效，Lipitor成为超级重磅炸弹，在2011年专利到期之前，累计销售1,250亿美元，是人类历史上第一个销售超过1,000亿美元的药品。2003年，英国/瑞典阿斯利康的可定（Crestor）及日本兴和的力清之（Livalo）分别在美国与日本上市，它们的药物相互作用较少，半衰期长，可以在一天内任意时间服用，其中力清之的生物利用度非常高。可定在2015年全球销售峰值到达50亿美元，2016年仿制药上市后，销售额大幅下降。图3：各他汀类药物的化学结构34在他汀类药物风光20年后，前蛋白转化酶枯草溶菌素9（PCSK9）于2003年被首次报导。从分子机理阐明到相应药物上市仅用了10几年时间，是又一类划时代的药品。临床试验结果表明，PCSK9抑制剂可以非常明显的降低血液中的LDL-C水平。2015年7月及8月，美国安进（Amgen）研发的Repatha获得EMA及FDA批准上市。2015年7月及9月，法国赛诺菲及美国再生元（REGN.US）联合开发的Praluent也分别获得FDA及EMA批准上市。与他汀类是化学合成的药品不同，PCSK9是单克隆抗体药物（Monoclonal antibody），具有靶向性强、副作用小的特点，但是由于生物制药品的制作成本较高，药品定价较高，销售不及预期。虽然如此，在治疗指南中，针对他汀不耐受患者或者服用大量他汀后、LDL-C水平依然较高患者，推荐使用PCSK9抑制剂或者联合他汀使用。10经典案例九：呼吸系统疾病及「一代名药」Advair呼吸系统疾病在全球高踞前列，主要包括慢性阻塞性肺病、哮喘，及过敏性鼻炎。近20年以来，全球COPD的发病率整体下降，这与各国政府加强对于COPD的教育及疾病管理密切相关。超过90%的死亡发生在低等收入或者高收入国家。在吸烟率及人口老龄化较高的国家，发病率有提升的趋势。哮喘多发生在幼儿及青少年，发病率较高，每万人中患者超过55人。相对于COPD，哮喘的死亡率较低，合适的病情管理及用药可以比较好的控制病情。过敏性鼻炎近年来呈明显上升趋势，与环境、季节、过敏原有明显关系。临床治疗主要以吸入制剂为主，患者一般需要在其疾病发展过程持续日常使用吸入制剂。吸入制剂的四种主要剂型包括雾化剂、干粉剂、气雾剂、鼻喷剂，其中干粉制剂携带方便，是最普及的剂型。纵观呼吸制剂发展历史，英国的葛兰素史克、英国/瑞典的阿斯利康以及德国的勃林格殷格翰（Boehringer-Ingelheim，BI）占据绝对领先及主导的地位。1969年，葛兰素史克的第一款哮喘药品Ventolin上市。1961年、1975年及1980年，勃林格殷格翰的Alupent、Atrovent及Berodual分别上市。在上世纪90年代，阿斯利康的Pulmicort、葛兰素史克的BeconaseAQ及Flovent是最常使用的药品。这些药品都具有单一有效药物成分，其辅助的呼吸器械装置也相对简单。葛兰素史克的Advair Diskus是最常见的治疗COPD及哮喘的药品，长期位列全球销量前十名。这款呼吸制剂混合了两种药效的药物，并使用了独特的呼吸装置。呼吸制剂是药品及器械的组合，并且对于呼吸装置的技术要求非常高。因此，尽管2011年AdvairDiskus的化合物专利到期、2013年FDA出了仿制指南、2016年呼吸装置的专利保护到期，直到2019年1月才有仿制产品在美国上市。2017年，葛兰素史克的首款三合一吸入制剂全再乐（Trelegy Ellipta）被FDA批准上市。呼吸制剂正从单剂、双剂，向三合一有效制剂方向发展。但是，全球销量最好的产品依然是相对早期的成熟产品Advair、Spiriva及Symbicort。11经典案例十：PDE5i及勃起功能障碍，Viagra1998年3月，万艾可（Viagra）在美国上市，被称为「蓝药丸」，是制药史上的里程碑之一。在此之前，勃起功能障碍（Erectile Dysfunction，ED）并没有被广泛关注，一方面是因为涉及个人隐私，难以启齿；另一方面是因为没有比较好的治疗方法。那时，辉瑞制药在研发治疗肺动脉高血压药物的过程中，偶然发现该药的副作用可对病者的性生活有改善，因此对阴茎海绵体平滑肌的作用展开了研究，并研发出万艾可，迅速获得商业上的成功，是制药行业典型的「无心插柳柳成荫」案例。辉瑞制药依靠其强大的、行业内一流的市场推广及销售能力，利用铺天盖地的广告，使患者、医生及公众认为勃起功能性障碍是人类正常疾病的一部分，可以治疗、可以改善婚姻及生活质量，甚至是人的基本权利的一部分。辉瑞制药成功说服美国政府给低收入人群的联邦医疗补助（Medicaid）报销此药。ED是指持续性的不能获得或者不能维持勃起以获得满意的性生活，时间超过3个月。ED的流行性病学难度比较大，根据美国马萨诸塞州的2019年调研，40-70岁男性患病率为52%；在中国，一项针对11个城市的ED调查研究表明，40岁以上男性为高发人群，发病率超过40%。ED目前无法根治，需要长期治疗。在口服药品出现之前，主要的治疗方法包括真空负压勃起装置、阴茎海绵体药物注射疗法、手术治疗。这些疗法在欧洲发达国家的使用率不高（大约20-30%），在中国更低。ED的病因比较复杂，主要有心理因素（焦虑、紧张、悲伤等精神状态）、心血管疾病（高血压、高血脂、糖尿病）及忧郁症等疾病所致的神经问题、本身睾丸发育问题或者受伤、以及药物副作用影响。万艾可属于5型磷酸二脂酶抑制剂（PDE5i），对于环磷酸鸟苷（cGMP）的水解起拮抗作用，可以维持阴茎海绵血管舒张，保持勃起状态。在PDE5i领域一共有三个药品，美国辉瑞的万艾可（Viagra）、德国拜耳的艾力达（Levitra），以及美国礼来的希爱力（Cialis）。Cialis具备起效时间较短（30分钟），持续时间长（36个小时），以及不受适度饮酒的影响等优势。因此，目前Cialis的全球市场份额已经超越Viagra。注：本文节选自由香港商务印书馆出版的《生命科技投资启示录·捕捉下一只独角兽》，文章已获出版社及作者授权。（欢迎转发，请联系：shuisongye）作者简介柳达柳达先生是一位药剂师，但更是一位投资者，双重身份的结合令他眼光独到。独角兽是科学家的梦想，也是投资者的追求。它的诞生，要有好的初心、好的研发团队、好的领军人物、好的投资者，以及好的政策环境。独角兽，是人心中的祥瑞。遇见它，对研发团队而言，意味着梦想成真；对投资者而言，则意味着巨大的回报；对于人类社会，它的意义显然要大得多。因为每一个生命科技独角兽的诞生，都是点亮人类战胜不治之症征途的一个火把。这麽一个一个的点着，点着，前赴后继，最终成为光明大道。推荐阅读卖了8年的PD-1，还能保持20%的增长率，它是怎么做到的？这款药被2022版上海市新冠病毒感染诊疗规范推荐，临床价值在哪里？罗氏2022，营收近700亿美元！新增长抵消新冠影响点击这里，报名创新药BD高阶研讨会【上海站】！

Seven biopharma companies are under new leadership. This week's Movers & Shakers highlights these new chief executive officers who aim to guide their companies into the future. Gamida Cell Abigail L. Jenkins will become the new president and chief executive officer of Boston-based Gamida Cell Ltd. She succeeds Julian Adams, who is retiring. Adams will continue to serve as a member of the board of directors. In a brief statement, Jenkins said she will focus on bringing omidubicel to market if approved. Omidubicel is an allogeneic hematopoietic stem cell therapy for cancer patients. "I am excited to lead Gamida Cell as we work to fulfill our mission of creating cures for blood cancers and serious hematologic diseases," she said in a statement. Jenkins brings more than two decades of leadership experience to her new role. Previously, she held the position of chief commercial and business officer at Lyndra Therapeutics. There, she formed and helmed commercial, business development, corporate strategy and portfolio management across multiple therapeutic areas. Before that, Jenkins served as chief commercial officer and U.S. business head at Aquinox Pharmaceuticals. Additionally, she held senior commercial and business development positions at Relypsa, Actavis, Pfizer and Medimmune/AstraZeneca. Adhera Therapeutics Effective Sept. 30, Zahed Subhan will take over as CEO of Louisiana-based Adhera. Subhan will also serve as chairman of the company's board of directors. Subhan has been serving as a board member since 2021. He brings more than 35 years of experience in research, development and commercialization. Over the course of his career, Subhan has held roles of increasing responsibility at Sanofi, GlaxoSmithKline and Dupont Pharmaceuticals. Current CEO Andrew Kucharchuk will transition into the role of chief operating officer. He will oversee daily operations at the company. Viracta Therapeutics Mark Rothera, the former CEO of Silence Therapeutics, will assume leadership of San Diego-based Viracta Therapeutics. He replaces Ivor Royston, who will remain a board of directors member. Viracta is a precision oncology company aimed at virus-associated malignancies, including Epstein-Barr-associated cancers. Rothera has a long history in the biopharmaceutical industry. Prior to his role as CEO of RNA-focused Silence Therapeutics, he served as CEO of Orchard Therapeutics. Before Orchard, Rothera served as CCO of PTC Therapeutics, where he helped launch two rare disease therapies. Previously, he served as global president of Aegerion Pharmaceuticals Inc. and vice president and general manager of commercial operations at Shire Human Genetic Therapies. "I am thrilled to be joining Viracta at this exciting time, as the company progresses Nana-val across multiple indications with the ambition of representing a tumor-agnostic approach to Epstein-Barr virus-associated cancers," Rothera said in a statement. Gain Therapeutics Matthias Alder, currently COO of Maryland-based Gain Therapeutics, will take over as CEO. Alder succeeds Eric Richman, who has served in the role since July 2020 and led the company through its initial public offering. Richman will continue to serve as a board member and senior advisor to the company. Since joining Gain as COO in 2021, Alder has been instrumental in building the company's corporate and business development strategy. Before Gain, he served as chief business officer and head of U.S. business operations at Autolus Therapeutics. Before that, he held senior roles in business development, licensing, and legal affairs at Sucampo Pharmaceuticals Cytos Biotechnology AG, and Micromet, Inc. "Gain is poised for success as we focus on our continued execution against established strategic objectives, including advancing our two lead programs in Parkinson's and Gaucher disease and expanding the application of our proprietary computational drug discovery platform through collaborations and strategic partnerships," Alder said in a statement. Septerna After serving as interim CEO, Jeffrey Finer has been tapped as the full-time head of Bay Area-based Septerna, a company that launched earlier this year with a $100 million Series A. A co-founder of Septerna, Finer has helped create and launch multiple new biotech companies, including Maze Therapeutics and Ambys Medicines. He also held leadership roles at Theravance Biopharma, Five Prime Therapeutics, and Cytokinetics. Finer serves on the boards of Strateos, Ambys, and Maze. NorthX Biologics Helena Strigård was named the new CEO of Sweden's NorthX Biologics. She joins the company from her role as director general of SwedenBio, the nation's life science industry organization. Strigård lead NorthX's continued expansion in developing and producing advanced biologics. She assumes her role on Sept. 30. Ted Fjällman, the current CEO of NorthX, will continue to support the company as a member of the board. Scholar Rock The Scholar Rock Board of Directors appointed Jay Backstrom as CEO-elect. Backstrom will work closely with founding and Interim CEO Nagesh Mahanthappa before officially assuming the role on Oct. 20. Backstrom most recently served as executive vice president of research and development at Acceleron Pharma, acquired by Merck last year. Prior to Acceleron, Backstrom served as the chief medical officer and head of global regulatory affairs for Celgene. Before Celgene, he was vice president of global medical and safety at Pharmion Corporation. Early in his career, he held industry roles at Quintiles, Hoechst Marion Roussel, and Marion Merrell Dow. "I am excited about the potential of Scholar Rock's spinal muscular atrophy and oncology programs, where we have near-term opportunities to develop transformative therapies for patients through a highly differentiated platform targeting growth factors like TGFβ," Backstrom said in a statement.