Cav1.2

点击蓝字 关注我们 | 本文内容来源：征战AD 阿尔茨海默病 (AD) 是一种进行性的神经退行性疾病，其特征是认知功能逐渐下降，伴随大脑中β-淀粉样蛋白 (Aβ) 斑块的沉积和神经元损伤。近年来的研究表明，在AD早期，在神经元损伤之前，脑部血流 (CBF) 就已经出现下降，这种下降与认知功能的减退密切相关。 CBF的降低可能导致脑组织缺氧和营养供应不足，进而影响神经元的功能和生存。在AD的早期阶段，通过改善CBF可能有助于延缓疾病进展，保护认知功能。血管周细胞 (pericytes) 是包裹在脑毛细血管周围的特殊细胞，它们通过调节血管的收缩和舒张来控制CBF。在AD模型中，血管周细胞的异常收缩导致毛细血管血流阻力增加，CBF下降。因此，探索调节血管周细胞收缩的机制，寻找有效改善CBF的策略，对于AD的治疗具有重要的理论和实践意义。 基于此，Nils Korte 等研究学者在国际著名期刊 Nature Neuroscience 上发表题为 Inhibiting Ca2+ channels in Alzheimer’s disease model mice relaxes pericytes, improves cerebral blood flow and reduces immune cell stalling and hypoxia 的研究文章。旨在探究AD模型小鼠中血管周细胞收缩的调控机制，并研究钙离子通道阻断剂尼莫地平 (Nimodipine) 对血管周细胞收缩和CBF的影响。 通过活体双光子成像技术、激光多普勒流变测量和磁共振成像等先进技术，本研究揭示了L型电压门控钙通道 (CaVs) 在血管周细胞收缩中的关键作用，并发现尼莫地平能够通过阻断CaVs减轻血管周细胞的收缩，改善CBF，减少免疫细胞的停滞，并减轻脑组织缺氧。这些发现为AD的早期干预提供了新的潜在治疗策略，并为未来的临床研究和药物开发奠定了基础。 主要结果 1. L型钙通道 (CaVs) 和钙控氯通道TMEM16A调控血管周细胞张力： 实验数据显示，内皮素-1 (ET-1) 这类Gq蛋白偶联受体激动剂能够通过释放肌浆网中的钙离子，增加血管周细胞的钙离子浓度，并导致血管收缩。使用尼莫地平阻断CaVs或使用10bm阻断TMEM16A可以显著减轻这种钙离子浓度的上升和血管周细胞的收缩。通过双光子成像技术对NG2-CreERT2-GCaMP5g小鼠的大脑皮层切片进行观察，我们发现ET-1增加了血管周细胞的钙离子浓度，并在血管周细胞体处引起毛细血管收缩。此外，尼莫地平预处理能够减少ET-1引起的平滑肌细胞钙离子浓度上升，这与CaV1.2和TMEM16A在血管壁细胞中的高表达一致。研究模型展示了TMEM16A和CaVs如何控制血管周细胞的张力，这为开发针对AD早期阶段的治疗策略提供了新的见解。 2. CaVs在体内产生平滑肌细胞 (SMCs) 和肌源性张力： 通过在活体内使用激光多普勒流量测量和双光子成像技术，我们观察到，向野生型小鼠注入BBB可透过的CaV阻断剂尼莫地平后，CBF显著且持久地增加了28%。这种增加与血管直径的增加一致，包括皮质动脉、穿透动脉和1至3级分支顺序的毛细血管。尼莫地平引起的毛细血管扩张在血管周细胞体附近最为显著，并随着距离的增加而减少，表明血管周细胞介导了尼莫地平引起的毛细血管扩张。此外，我们同时测量了CBF和血压 (BP)，发现尼莫地平初始时会导致平均血压下降，但随着自动调节机制的恢复，CBF上升至尼莫地平处理前水平的25%以上，表明尼莫地平引起的局部血管阻力下降大于其对全身血压的降低效应。实验还证实了尼莫地平引起的血管壁细胞内钙离子浓度下降先于CBF的增加，并与血管舒张有关。因此，研究结果表明，CaVs在SMCs和1至3级分支顺序的血管周细胞中赋予了肌源性张力，这为理解CaVs在体内调控血管张力的作用提供了直接证据。 图1. CaVs在体内对SMCs和血管周细胞的调控作用 3. CaVs增强AD小鼠毛细血管床周细胞的收缩： 通过使用激光散斑成像技术，观察到AD小鼠的桶皮层毛细血管灌注显著减少。活体双光子成像显示，在与年龄匹配的野生型 (WT) 小鼠相比，成熟AD小鼠的1至3级毛细血管血管腔直径在血管周细胞体处狭窄了21%。经尼莫地平处理后，AD小鼠的毛细血管直径增加了9.6%，CBF提高了51%。此外，尼莫地平引起的血管扩张先于CBF的增加，并且与血管舒张有关。数据显示，尼莫地平通过减少血管周细胞和SMCs的钙离子浓度来增加CBF，且这种效应独立于尼莫地平对其他细胞的影响。 在1至3级分支顺序的血管周细胞中，AD小鼠的钙离子瞬变频率显著增加，而尼莫地平治疗可大幅恢复这些细胞的钙离子瞬变频率至WT水平，并减少瞬变幅度。此外，尽管在AD小鼠中>3级毛细血管的血管周细胞体和过程的钙离子瞬变频率升高，尼莫地平并没有调节这些细胞的钙离子瞬变频率，但确实减少了瞬变幅度。研究结果证实，>3级血管周细胞能够响应缺血损伤而收缩，这可能在AD和缺血性脑皮质中具有重要意义，其中血管收缩剂如活性氧 (ROS) 的水平升高。 总之，研究结果表明，通过使用尼莫地平阻断CaVs，可以在AD小鼠中逆转早期CBF下降，为AD的早期干预提供了潜在的治疗策略。 图2. 血管周细胞在AD中的收缩作用及其对CBF的影响 图3. 中间和静脉端血管周细胞在AD小鼠中的增强收缩作用 4. 免疫细胞ROS提高AD小鼠的Ca2+水平，并降低CBF： 研究人员通过活体双光子成像技术观察到，在外源性给予过氧化氢 (H2O2) 以产生氧化应激时，血管周细胞的钙离子浓度显著上升，钙通道阻断剂尼莫地平可以减轻此现象。此外，利用NOX2的阻断剂GSK2795039和临床批准的抗氧化剂N-乙酰半胱氨酸 (NAC) 处理AD小鼠，可以显著增加CBF并降低血管周细胞和平滑肌细胞内的钙离子浓度。研究还发现，在AD小鼠中，与Aβ斑块相邻的小胶质细胞产生的ROS水平升高，而NAC预处理能够显著减少这种ROS的产生。此外，与WT小鼠相比，AD小鼠中小胶质细胞与血管周细胞之间的距离更近，这可能缩短了ROS扩散至血管并调节血管张力所需的距离。最后，通过活体成像技术，研究人员观察到AD小鼠小胶质细胞的累积像素监控（即小胶质细胞的活动）较WT小鼠显著减少，表明AD小鼠的小胶质细胞可能形态更不分支，与血管的交互作用减少。尽管小胶质细胞的监视活动减少，但AD小鼠的小胶质细胞/血管周围巨噬细胞体更接近血管，这可能影响血管周细胞的收缩。 综上所述，这些发现揭示了在AD小鼠中免疫细胞与血管周细胞之间的相互作用，以及这种相互作用如何通过影响钙离子浓度来调节脑血流，为未来AD的潜在治疗策略提供了新的见解。 图4. 免疫细胞ROS在AD小鼠中提高血管周细胞内钙离子浓度并减少脑血流 5. 静脉端周细胞介导AD小鼠白细胞停滞： 在AD小鼠模型中，血管周细胞诱导的毛细血管腔狭窄会导致血流在血管周细胞体附近停滞，特别是在毛细血管的静脉端。通过活体双光子成像技术，研究人员观察到AD小鼠的毛细血管阻塞百分比从WT小鼠的0.47%增加到5.2%。这些阻塞主要发生在远离动脉端的毛细血管分支，即毛细血管的静脉端，这里的毛细血管直径最小，在AD中进一步减小。研究人员还发现，循环中的单核细胞和中性粒细胞被招募到AD大脑中，并在毛细血管中引起阻塞。这些细胞比红细胞伸展性差且体积较大，导致它们在毛细血管中推动血管壁，可能增强了它们与内皮细胞上的ICAM-1和VCAM-1粘附分子的相互作用。使用尼莫地平处理后，AD小鼠的毛细血管灌注大部分恢复，停滞的白细胞和红细胞数量减少到低于WT小鼠的水平。 这些发现表明，尼莫地平通过多种方式增加AD小鼠的CBF：一是逆转毛细血管直径的减少，从而维持较低的血液粘度；二是减少随后由白细胞和红细胞引起的毛细血管阻塞。这些结果揭示了尼莫地平可能通过改善血管周细胞功能和减轻炎症反应来提高AD小鼠的CBF，为AD的潜在治疗提供了新的见解。 图5. CaV抑制剂减少AD小鼠血管周细胞引起的免疫细胞停滞 6. 尼莫地平改善AD小鼠的CBF并减少脑组织缺氧： 在AD小鼠模型中，为期1.5个月的尼莫地平治疗显著改善了小鼠的CBF，增加了皮层血管系统的毛细血管直径，并减少了血液在毛细血管中的停滞现象。此外，尼莫地平还显著降低了与Aβ斑块相关的缺氧细胞数量，表明其可能对改善AD小鼠的脑部缺氧状况具有积极作用。尽管长期尼莫地平治疗并没有调节血管内皮细胞的粘附分子表达，也没有影响FITC-右旋糖酐的渗出，但这些发现表明尼莫地平通过降低血管周细胞内的钙离子浓度，增加毛细血管直径，减少血液停滞和缺氧细胞数量，从而可能在AD早期治疗中发挥保护作用。然而，随着AD的进展，特别是在老年AD小鼠中，尼莫地平对CBF的改善作用可能会减少，这可能与血管周细胞的加速丧失和血脑屏障破坏有关。 上述结果表明，尼莫地平通过降低血管周细胞内的钙离子浓度，增加毛细血管直径，减少血液停滞和缺氧细胞数量，从而在AD早期治疗中可能发挥保护作用。 图6. 尼莫地平改善AD小鼠的脑血流并减少脑组织缺氧，逆转人类皮层组织中Aβ引起的血管周细胞收缩 7. 尼莫地平逆转人类皮质组织中Aβ引起的周细胞收缩： 在人类大脑皮层组织中，尼莫地平能够逆转由可溶性Aβ寡聚体引起的血管周细胞收缩。具体来说，研究发现在人工脑脊液 (aCSF) 中单独孵育的活体人脑皮层组织，与血管周细胞体相比，其周围的毛细血管直径显著扩张。而当加入Aβ寡聚体时，靠近血管周细胞体的毛细血管显著收缩，但在血管周细胞体远处的毛细血管直径没有显著变化。然而，在持续存在尼莫地平的情况下加入Aβ寡聚体，血管周细胞体附近的毛细血管直径与对照组相比没有显著差异。这表明在人类活体大脑组织中，Aβ引起的血管周细胞收缩是由L型钙通道（可能是在人血管周细胞中表达的CaV1.2亚型）的激活驱动的。 因此，尼莫地平有望在AD患者中增加毛细血管直径并减少缺氧，正如在小鼠模型中观察到的那样。 本研究揭示了尼莫地平这类药物可能成为改善AD早期脑血流减少的潜在治疗手段。通过在AD小鼠模型中使用尼莫地平，研究发现它能够显著增加毛细血管直径，减少血管周细胞收缩，改善脑血流，并减轻与AD相关的脑组织缺氧状况。此外，尼莫地平还能减少免疫细胞在毛细血管中的停滞，改善血液微循环。这些发现为AD的早期干预提供了新的治疗靶点，提示通过调节血管周细胞的功能，可能有助于延缓疾病进展，改善患者的认知功能。 科研启发： 本研究强调了血管周细胞在神经退行性疾病中的作用，并指出了L型钙通道 (CaVs) 和钙控氯通道TMEM16A作为治疗AD的新靶点。此外，研究还表明，通过降低血管周细胞内的钙离子浓度，可以减少血管周细胞的收缩，从而为开发新的药物提供了可能的分子机制。未来的研究可以进一步探索尼莫地平或其他CaVs阻断剂在临床上的应用，以及它们对AD病程的长期影响，为AD治疗提供更有效的策略。 喜鹊医药团队独家设计开发的创新化学药物MN-08，具有独特新颖的作用机理，既可通过拮抗NMDA受体抑制钙离子超载又能靶向NMDA受体释放NO扩张脑血管、改善脑血流，从而有效恢复脑部营养供给，起到治疗AD与血管性痴呆 (VD) 等神经退行性疾病的作用。临床1期研究证明MN-08具有良好的安全性与耐受性。目前，MN-08治疗中重度AD的2期临床试验正在进行，现有数据已初步显示出增加脑血流的积极效果，有望成为标本兼治的AD与VD的新一代治疗药物。 原文链接： 【1】Asllani, I. et al. Multivariate and univariate analysis of continuous arterial spin labeling perfusion MRI in Alzheimer’s disease. J. Cereb. Blood Flow Metab. 28, 725–736 (2008). 【2】Nortley, R. et al. Amyloid β oligomers constrict human capillaries in Alzheimer’s disease via signaling to pericytes. Science 365, eaav9518 (2019). 【3】https://doi.org/10.1038/s41593-024-01753-w 声明： 本文内容来源：征战AD。内容仅供参考，不构成任何投资及应用建议，亦不代表任何治疗指导及用药建议。如稿件涉及版权等问题，请与我司联系，我们将在第一时间协调给予处理或删除。

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). 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深度聚焦类器官与3D培养论坛，OTC2024论坛合作详询：王晨 180 1628 8769Timothy 综合征 （TS）是一种严重的罕见遗传病，死亡率非常高，它是由CACNA1C基因外显子8A中的c.1216G>A致病性突变引起，这个heterozygous点突变导致 L-type钙通道蛋白Cav1.2的p.G406R的错义变异。TS影响多个器官系统，是自闭症谱系障碍 (ASD) 和癫痫症中最具致病性的遗传病因之一。CACNA1C的常见变异也与其他神经精神疾病密切相关，包括精神分裂症、双相情感障碍和注意力缺陷多动障碍，这表明Cav1.2也是神经精神疾病的关键易感因素。迄今为止，一型TS既无动物模型报道，也没有药物开发的尝试。2024年4月24号，斯坦福大学 Sergiu Pasca团队在Nature上发表了封面文章 Antisense oligonucleotide therapeutic approach for Timothy syndrome，应用类脑器官进行罕见病基因治疗药物开发。Cover image: Gheorghe Popa团队通过靶向CACNA1C exon 8A的反义寡核苷酸 ASO来筛选ASO，发现了至少三个ASO可以时间和剂量依赖的方式有效抑制exon 8A 的剪接。当这些ASO进入TS类脑神经元， ASO不仅纠正了Cav1.2延迟失活和去极化诱导的钙异常升高，而且还修正了forebrain assembloid中发现的皮质中间神经元迁移缺陷。为了验证ASO能不能通过在体内给药，给药后是否有治疗效果，他们利用了最近开发的类脑移植模型（Revah,Gore and Kelley et al. Nature 2022）。在这个系统中，皮层类脑移植到新生免疫缺陷无胸腺大鼠的大脑皮层中，可以逐渐发育为成熟的人类神经元，并且可以整合到大鼠感觉和动机相关的神经回路中控制大鼠行为。令人惊喜的是，将ASO鞘内注射到移植有TS皮层类脑的大鼠体内可以有效降低exon 8A，并且可以纠正TS去极化诱导的钙信号缺陷。这项研究发现了TS疾病突变可以直接影响RNA剪接来正向增加本身的突变蛋白；这是应用类脑和类脑移植进行神经系统药物研发的首创研究，先在体外培养的类脑中进行前期基因治疗的ASO筛选，再将有前景的ASO通过鞘内注射进入移植后的含有人类神经元的类脑，这对于目前尚没有老鼠疾病模型的TS药物开发具有重大意义；虽然TS是一种罕见神经疾病，但是世界范围内仍有足够病人申请进行临床试验。另外，虽然这项工作主要重点在开发治疗Timothy 综合征，但是该研究建立了从疾病致病机理，药物开发筛选，体内体外验证的pipeline。用类脑人类神经元进行体内体外药物开发可以补充动物模型的很多不足，尤其是类脑移植，未来不仅仅局限于ASO，还非常适合AAV病毒和其他小分子或者蛋白药物的开发，更可以应用到很多其他神经系统疾病。该研究通讯作者Sergiu Pasca是斯坦福大学医学院精神病学与行为科学系教授，第一作者为Xiaoyu Chen陈晓雨为斯坦福大学博士后，共同第一作者Fikri Birey现为埃默里大学人类遗传学系助理教授。原文链接：https://www.nature.com/articles/s41586-024-07310-6END深度聚焦类器官与3D培养论坛，OTC2024论坛合作详询：王晨 180 1628 8769戳“阅读原文”立即领取限量免费参会名额!