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非临床评估对于确定一种实验药物是否可以作为潜在的治疗药物安全地在人体中进行试验非常重要。此类评估包括各种体外和体内研究,以评估安全性药理学、一般毒性、遗传毒性、致癌性以及发育和生殖毒性。良好实验室规范(GLP)为开展非临床研究提供了框架,遵守该规范将有助于确保所生成数据的质量和完整性。这反过来又会支持临床研究和未来上市许可的申请。再者,遵守GLP能准确重建最佳研究条件、保存高质量记录和报告。
1
良好实验室规范条例
在计划进行非临床安全性研究时,一个基本问题是需要考虑研究是否需要按照GLP法规进行。GLP是一个质量体系,对非临床研究的设计、执行、监控、记录、存档和报告的条件进行管理。GLP可确保研究结果的完整性,当监管机构审查研究结果时,可在一定程度上保证数据的准确性,并确保研究是按照明确规定的标准进行的。
需要符合GLP规范的非临床研究通常是在启动临床试验或药物获得上市批准之前为监管机构提供有关人体安全风险的重要信息的研究。这些研究包括对非人类试验系统(例如动物或细菌模型)进行试验药品给药以寻找不良反应的研究,以及某些类型的分析研究。应按照GLP进行的研究包括但不限于:
• 急性、亚慢性和慢性毒理学研究(一般毒理学研究);
• 遗传毒理学研究(体外和体内);
• 局部耐受性研究
• 致癌性研究
• 生殖和发育毒理学研究;
• 安全药理学研究(体外和体内);
• 皮肤、眼部、静脉和肌肉刺激性研究;
• 免疫毒性研究;
• 对从动物身上采集的生物样本进行分析研究;
• 剂量配方的分析研究--通常是试验品与载体或载体的混合物--用于将试验品注入试验系统;以及
• 对用于分析GLP研究样品的方法进行验证的研究。
GLP在美国的起源可以追溯到20世纪70年代,当时有大量报道称,在多个实验室进行杀虫剂和药品的动物安全性研究时,存在科学不端行为和数据造假问题。美国国会召开听证会对这些问题进行调查,1976年FDA提出了GLP法规,规定了如何进行非临床安全性研究的各个方面。1979年,FDA GLP法规成为法律,1981年,FDA发布了后续指南,回答了有关其解释和适用性的问题。1984年公布了修改建议,1987年,修订后的GLP最终规则成为美国法律(GLP Final Rule was enacted into US law),即21 CFR第58部分。
在美国之外,非临床研究的开展主要由经济合作与发展组织(OECD)制定的指导原则负责。经合组织于1981年制定了一套与FDA类似的GLP原则,后于1997年进行了修订。经合组织没有政府权力,因此其每个成员国都必须将GLP指南纳入法律,以便有效监督和执行GLP的遵守情况。作为GLP指南的配套文件,经合组织已出版了20多份文件,对GLP的特定方面及其应用和监测进行了解释;其中最新的文件于2022年发布。
01
数据互认(Mutual Acceptance of Data)
本着协调和减少重复研究的精神,经合组织理事会在1981-1997年期间发布了三项决定,这些决定是数据相互接受(MAD)系统的基础。在这一制度下,经合组织成员国按照GLP生成的非临床研究数据将被其他成员国的监管机构接受。1981年,最初的MAD决定发布,仅适用于经合组织成员国。1989年,制定了通过政府检查和安全审计来监督GLP合规性的程序,并规定成员国的GLP合规性应得到其他成员国的认可。最后,1997年决定,如果一个非经合组织(OECD)国家已经制定了经过验证的国家GLP计划,并符合某些标准,则可以加入MAD。
截至2022年底,以下国家已成为MAD体系下人类药品非临床数据的参与者:澳大利亚、奥地利、比利时、巴西、加拿大、捷克共和国、丹麦、爱沙尼亚、芬兰、法国、德国、希腊、匈牙利、印度、爱尔兰、以色列、意大利、日本、马来西亚、墨西哥、荷兰、新西兰、挪威、波兰、葡萄牙、新加坡、斯洛伐克、斯洛文尼亚、南非、南韩、西班牙、瑞典、瑞士、泰国、土耳其、美国和英国。这份国家名单仅适用于接受人用药品的非临床数据,可能不适用于兽药、化妆品、杀虫剂或食品添加剂等其他类别。智利、哥伦比亚、哥斯达黎加、冰岛、拉脱维亚、立陶宛和卢森堡是经合组织成员国,但由于缺乏国家GLP计划而被排除在MAD体系之外。
2
GLP概述
FDA和OECD指南中对书面GLP的组织和措辞可能有所不同,但两者涉及的非临床研究行为的各个方面是相同的。下文将对此进行简要概述。有关GLP内容的更多详情,请参阅FDA和OECD的官方网站,其中包含法规全文。
涉及的主要方面分为以下几类:
• 试验设施的组织、管理和人员
• 质量保证
• 设施
• 设备和试剂
• 测试系统
• 测试和控制物品
• 标准操作程序 (SOP)
• 研究方案和研究执行
• 报告研究结果
• 记录和材料的储存与保存
1
试验设施和组织人员(Test Facility Organization and Personnel)
测试或试验设施是启动和开展研究的主要地点。一项研究的所有阶段都在一个测试机构进行,这种情况并不罕见;但是,一项研究的不同阶段往往在不同的地点进行,这就是所谓的多地点或多中心研究。在多地点研究中,试验设施通常是研究负责人常住地,也是对试验系统使用试验药品的地方。例如,在进行多地点毒理学研究时,在试验设备内对大鼠使用试验药品,然后将大鼠的血液样本运送到另一个试验地点进行分析。
2
试验设施管理(Test Facility Management)
试验设施的管理部门负责确保GLP规定得到遵守,并确保整体运作和人员符合GLP要求。
试验设施管理的具体职责包括但不限于:
• 确保在试验场工作的人员经过培训、受过教育并具有相关经验;
• 为每项研究指定一名研究主任,并在必要时更换研究主任
• 确保设立质量保证单位(quality assurance unit,QAU);
• 确保质量保证组向研究主管报告任何偏离GLP的情况,并采取后续行动以确认已采取适当的纠正措施;
• 确保对所有试验品和对照品及其混合物进行适当的特征描述;
• 确保GLP研究期间有必要的人员、设备、材料和设施;
• 确保有经批准的方法和SOP供研究人员遵循;以及
• 确保进行多站点研究的各阶段试验站点符合GLP要求。
3
研究负责人(Study Director)
研究主管是一名合格的人员,负责监督从研究启动到研究记录存档的所有阶段。这包括监督所有的数据收集、研究程序和结果报告,并确保任何偏离GLP或研究方案的行为都被记录在案,同时对偏离行为的影响进行评估。研究主管通常被称为 "单点控制"(single point of control),一项非临床研究只能有一名研究主管。应避免指定其他人员担任 "后备研究主管 "或 "共同研究主管"(backup study director’ or‘co-study director’)。
研究主管负责批准方案或研究计划,并确保研究人员遵守研究方案、试验设施SOP和GLP规定。这一责任延伸到参与多点研究的任何试验场所。通过签署最终研究报告,研究负责人对研究数据和从结果中得出的结论的准确性以及研究的GLP合规性承担全部法律责任。后者通常记录在最终报告中的GLP合规性声明中。
4
主要研究人员/主要研究者(Principal Investigator)
研究主管可将研究某一特定阶段的监督工作委托给称为主要研究者的合格人员。这可能是因为该阶段的研究是在单独的试验场地进行的,或者是因为需要专业知识或培训来监督该阶段的研究。主要研究人员确保其所在阶段的研究按照GLP进行。
这些人也可称为主要研究者或贡献科学家,但他们在研究过程中的职责是相同的。值得注意的是,主要研究者的称谓仅出现在经合组织GLP指南中。
5
质量保证(Quality Assurance)
质量保证部是一个向试验机构管理层报告的个人或小组,负责确保设施、设备、人员、方法、实践、记录和控制都遵循GLP。具体做法如下:
对设施、流程和特定研究活动进行检查或审核。质量保证部的所有人员都必须熟悉研究程序,但绝不能直接参与研究的进行。质量保证部审查研究报告,以确保结果准确反映研究过程中产生的原始数据。
最终研究报告将包含一份由质量保证部人员签署的声明,其中列出接受检查的所有研究阶段和检查日期。质量保证部保存检测机构的非临床研究总日程表、研究方案副本和所有检查报。
6
设施(Facilities)
进行GLP研究时使用的设施应具有适当的规模、结构和位置,以满足研究类型的要求。设施的设计应为不同的研究活动提供独立的区域。应有足够的房间或区域确保隔离试验系统,如研究动物和可能含有生物危险材料的单个项目。应为诊断、治疗和控制提供适当的房间或区域,以确保试验系统不会出现不可接受的恶化。贮藏室或贮藏区,特别是贮藏和处理试验品的贮藏室或贮藏区,应与存放试验系统的贮藏室或贮藏区充分隔开,以防污染、虫蛀或变质。
7
设备和试剂(Equipment and Reagents)
用于生成、存储或检索数据以及控制环境因素的设备(包括计算机系统)应位置适当、设计合理、容量充足。设备应经过测试和校准,以确保使用该设备生成的数据准确无误。测试和校准方法应在书面SOP中说明,设备出现故障或失灵时应采取适当程序进行说明。研究中使用的所有试剂和材料均应明确标识,并适当标注有效期和储存要求。
8
测试系统(Test Systems)
为确保数据的质量,应制定和保持正确的测试系统储存、处理和存放程序。在首次施用或应用测试物品之前,测试系统应在适当的时间内适应测试环境。应记录和保存测试系统的来源、到达日期和到达时的状况。测试系统的维护方式必须避免污染其他测试系统和材料。应使用独特的、不可擦除的方法来识别单个试验对象,以确保数据收集的准确性。动物饲养容器或笼子的大小和尺寸必须符合试验设施所在地适用的动物福利规定。
9
试验品和对照品(Test and Control Articles)
试验品和对照品应根据特性、强度、纯度、成分或其他适当的特征来确定。物品的稳定性必须在研究开始前或在研究期间根据测试机构的标准操作程序加以确定。确保测试和对照物品具有适当的特性是测试机构管理层的责任。
GLP研究中使用的试验品应使用符合GLP的方法和程序进行表征和记录;然而,许多实验室和药品开发商使用符合良好生产规范 (GMP) 规定的程序来表征试验品。虽然这是一种常见的做法,在科学上也可能是合理的,但必须将其作为GLP偏差记录在案,研究负责人需要评估这种偏差对研究完整性的影响。
在研究过程中,必须定期分析试验品与载体(如剂量配方)的混合物(mixture),以确定配方中试验品的均匀性和浓度。混合物在研究使用条件下的稳定性,必须在研究开始前或在研究过程中根据试验设施的标准操作程序加以确定。
10
标准操作程序(Standard Operating Procedures)
标准操作程序是开展和再现GLP研究不可或缺的一部分。标准操作程序确保整个测试机构的所有研究人员始终如一地执行实验室程序和流程。所有实验室工作人员都应可随时查阅标准操作程序,并对标准操作程序进行强制审查和记录。测试机构管理层负责定期审核并根据需要更新标准操作程序的内容。
一般来说,研究方案概述了非临床研究期间要做的事情,而标准操作程序则描述了 如何执行这些程序。例如,研究方案会指示技术人员对研究动物进行静脉注射,而SOP则会规定使用何种规格的针头和注射器、合适的注射静脉、如何准备注射部位、约束动物的正确方法等。一套全面的标准操作程序应涉及试验设施运作的所有方面,包括保持符合GLP要求所需的所有流程和程序。
研究过程中出现的SOP偏差必须记录在案,研究负责人必须评估偏差对研究完整性的影响。
11
研究方案和研究的执行(Study Protocol and Conduct of the Study)
非临床研究中的所有程序都写在研究方案中。每项研究只能有一份方案(only one protocol per study)。因此,样本分析计划或为特定研究程序而生成的类似文件绝不能称为研究规程。研究主管在研究方案上签字并注明日期后,研究即告开始。在研究过程中,不可避免地需要对方案进行更改。计划中的更改和更改的原因将作为方案修正案(protocol amendments)发布。研究负责人签署修正案后,变更即生效。任何偏离方案的行为都必须记录在案,并由研究主管进行审查,以评估对研究的影响。
GLP的主要目标之一是制定数据记录标准,以确保非临床研究结果的准确性和可重复性。关于如何记录和维护数据的指导原则,长期以来一直用缩写ALCOA (Attributable, Legible, Contemporaneous, Original, and Accurate)来概括,即可归属、可辨认、同期、原始和准确。近年来,这一概念已扩展到电子数据,并增加了以下术语,称为ALCOA+:完整、一致、持久和可用(Complete, Consistent, Enduring, and Available)。
• Attributable可归属性--应明确谁在何时记录了数据。原始数据的所有更改、更改人和更改时间以及更改原因都应清楚地记录在案。对数据的更改不应掩盖原始条目。
• Legible清晰易读 - 记录的数据应清晰易读。手工记录的数据必须用墨水书写。如果自动系统打印的页面被视为原始数据,则所使用的墨水和纸张应不易变质。
• Contemporaneous同期 - 数据应在观察到时或观察后尽快记录。在事件发生数天或数周后记录的数据,其准确性可能会受到质疑。严禁倒录数据。
• Original原始数据--即使是以非常规的方式记录的数据,也需要保留首次记录的数据。例如,如果观察结果最初写在纸巾上,后来转录到纸上或电子条目中,则纸巾应作为原始数据保留在研究记录中。
• Accurate准确 - 原始数据应准确无误,没有错误和偏差。在以电子方式记录数据时,系统必须具有内置的准确性检查和验证控制功能。在此过程中,应定期校准测量设备。
• Complete完整 - 所有记录的数据都应有审计跟踪,以显示没有任何数据被删除或丢失;数据的任何更改都应有审计跟踪。
• Consistent 一致性 - 数据应按照特定的时间顺序保存,并标明日期和时间,以便于理解和审计。
• Enduring持久性 - 数据在记录后应可长期阅读;这一点对计算机化数据尤为重要,需要有相应的程序来保证数据的安全和完整。
• Available可用 - 数据必须在规定的保留期内随时都能方便地获取和阅读。
由于越来越多地使用计算机系统记录非临床研究数据,人们不禁要问,针对手工记录数据的GLP要求如何适用于电子数据。这些要求仍然完全相同,但在执行时必须适应不同的媒介。1997年,FDA颁布了相关法规(21 CFR第11部分),以确保电子记录和签名(electronic records and signatures) "可信、可靠,一般等同于纸质记录和在纸上执行的手写签名"。虽然从技术上讲,电子数据并不是GLP的一部分,但当电子数据成为GLP研究的一部分时,这些要求就有望得到遵守。
12
报告研究结果(Reporting Study Results)
每项由研究负责人签署方案的研究都必须有一份由研究负责人签署的最终报告。这包括已完成的研究和因故提前终止的研究。研究负责人签署最终报告的日期即为研究完成日期。
研究完成后,不得再收集数据或在报告中插入信息。如果出现影响已完成研究的结果或解释的新信息,则应编写最终报告修正案,由研究主管签字,并与原始最终报告一起保存。Figure 4-1列出了最终研究报告应包含的信息。大致如下:
(1)研究标识,包括测试机构和申办方的信息
(2)质量保证声明
(3)由研究负责人签署的GLP合规声明(不是GLP的要求,但却是常见做法)
(4)研究开始和完成日期
(5)试验药品和对照品的鉴定和特征描述
(6)有关试验系统的信息
(7)关于偏离GLP或研究方案或可能影响研究完整性或结果解释的任何其他情况的说明
(8)研究方法说明
(9)研究结果,包括总体结论
(10)主要研究人员提交的签名并注明日期的报告
关于保留和储存研究记录和材料的信息
作为药物开发过程的一部分,在申请临床试验研究和上市许可时,需要进行非临床或临床前研究,以评估药物的安全性。这些研究包括体外研究和体内研究。许多研究是在首次人体临床研究之前进行的,有些研究是与临床开发同时进行的,还有一些研究甚至是在药物获准上市后才需要进行的。
药物开发是一项全球性活动,因此有必要统一监管要求。20世纪80年代,欧洲率先成功开发了单一的药品市场(a single pharmaceutical market)。因此,1990年成立了国际人用药品技术要求协调理事会 (ICH),这是一个由国际卫生监管机构组成的联盟。
ICH发布了一整套指导文件,以帮助新药的非临床开发和单项研究的设计。ICH指南分为四类:质量、疗效、安全和多学科(Quality, Efficacy, Safety, and Multidisciplinary)。与非临床安全性测试和规划非临床开发计划有关的指南主要集中在安全性和多学科类别中。与GLP法规不同,ICH指南不具有法律约束力。不过,我们鼓励申办者遵循ICH指南,以确保以全球一致的方式开发安全有效的药物。ICH指南中描述的研究行为包括GLP和非GLP指南。ICH指南最初被称为 "三方 "指南,因为它是由美国、欧洲和日本三个国家的卫生监管机构代表共同制定的。图4-2列出了非临床安全性评价的目标。如下:
• 确定人体初始安全剂量和后续剂量递增方案
• 确定临床监测的安全参数
• 确定潜在的毒性靶器官以及这种毒性是否可逆
• 在无法或尚未对人体进行风险评估的情况下,确定潜在的人体健康风险(如致癌性、生殖毒性或发育毒性)
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研究药物类型(Investigational Drug Types)
随着药物产品种类的不断丰富和扩大,ICH发布了指导文件来指导其开发。在20世纪80年代中期公司开始生产生物制剂之前,化学或小分子药物是最常见的研发药物。随着药物开发的不断进步,基因疗法也变得越来越普遍,并产生了新的指导文件,如2021年的ICH S12。
虽然每类药品的非临床安全性评价途径一般相同,但每类药品所需的研究类型可能不同。可采用的研究类型包括:
• 安全药理学(心血管、呼吸、泌尿(如适用,一般为追加研究)和中枢神经系统)
• 药代动力学
• 一般毒性研究(单剂量和重复剂量)
• 局部耐受性
• 遗传毒性
• 致癌性
• 发育和生殖毒性
评估光毒性、免疫毒性、幼龄动物毒性和滥用责任的其他非临床研究应根据药物类别和目标人群的具体情况进行。
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药品(Pharmaceuticals)
药品是小分子药物,也称为新化学实体/缩写NCE(以下简称小分子药物)。这些药物由化学合成,具有明确的结构,根据其大小(小于1 kDa),一般需要每天给药一次或两次。给药途径通常是口服,给药后通过新陈代谢降解。非临床研究项目非常广泛,需要对上述研究类型进行全面评估,包括发育和生殖毒性研究。
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生物技术药物产品(Biopharmaceuticals)
生物技术药物产品又称大分子药物、生物制品或生物治疗药物(以下简称生物制品)。这些药物通过生物合成(生物技术)制成,具有二级或三级复杂结构。它们包括多种药物类型,如单克隆抗体、疫苗、活体生物治疗药物(益生菌)、血液制品、基因治疗药物、人体细胞、组织以及基于细胞和组织的产品。根据其大小(>1 kDa),其通常可间隔 1 周或更长时间给药。其给药途径通常是肠外注射,给药后通过分解代谢或蛋白水解降解。基于动物模型的相关性和生物制剂的性质,对它们的非临床评估较为有限。不需要进行代谢研究;分子大小可防止与 DNA 发生相互作用,因此也不需要进行遗传毒性和致癌性研究。
个人注解:生物制品不需要代谢研究,以郭博士解释举例“以抗体或蛋白质药物为例,其主要代谢形式包括受体介导的酶降解和非特异性蛋白酶降解两种, 形成不具备活性和毒性的多肽和氨基酸,一般是不需要研究具体的代谢物的。”
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毒性试验用动物模型(Animal Models for Toxicity Testing)
动物研究是在特定的指导方针下进行的,这些指导方针符合美国的《动物福利法》和欧洲关于保护用于科学目的的动物的指令,并得到国际上的支持。使用动物的研究方案由进行研究的试验设施的机构动物护理和使用委员会或伦理审查委员会审查和批准。
根据ICH指南,应在两种哺乳动物(一种啮齿动物和一种非啮齿动物)中进行毒性研究。对于小分子药物的非临床评估,此类研究通常在大鼠和狗种属中进行。生物制品具有靶向性,需要使用相关物种进行安全性评估(use of relevant species for safety assessment)。相关物种是指药物因受体或表位的表达而具有药理活性,并能引起与人类相似的药理反应的物种(A relevant species is one in which the drug is pharmacologically active due to the expression of the receptor or an epitope and evokes a similar pharmacological response as that expected in humans)。不鼓励使用非相关物种,因为它们产生的结果不具代表性。
非人灵长类动物(Nonhuman primates)在基因组同源性和生理系统(如免疫系统)方面与人类相似,因此通常是这些研究的最相关物种。如果只有一种相关物种,使用单一物种进行毒性测试是可以接受的。如果没有相关物种,使用表达人类受体的转基因动物或使用替代药物(同源蛋白)可能是可以接受的替代方法。人类疾病的动物模型也可作为在未患病动物中进行毒性研究的一种可接受的替代方法。
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给药(Drug Administration)
在非临床研究中如何给药需要考虑几个因素,包括配方、途径、频率和剂量水平。在GLP毒性研究中对动物给药的药物应在GLP或GMP下生产,并如前所述(见本章 "试验和对照品 "部分)需充分表征(fully characterized)。非临床开发过程中使用的药物应能代表临床试验中使用的药物;然而,在首次进行GLP毒性研究时,临床药物制剂往往尚未完全优化。配方或生产工艺的改变需要有充分的文件证明(见第12章)。在研发后期为支持关键临床试验而进行的GLP毒性研究中,动物的用药剂量应能代表临床用药和可能上市的药物剂量。日后药物或生产工艺发生重大变化时,可能需要进行另一项动物研究,以证明安全性或比较药代动力学特性。
GLP非临床研究中使用的给药途径必须与临床试验中使用的相同。对于小分子药物来说,除了典型的口服给药途径(如通过药丸或片剂、胶囊或口服液)外,还可能有多种给药选择,包括静脉注射、皮肤(局部、皮下、皮内)、肌肉注射或针对特定身体部位(眼部、鼻内、鞘内、阴道或直肠),在人体给药前必须进行测试。
生物制剂更容易降解,因此选择也更有限,如静脉注射、皮下注射、皮内注射或肌肉注射。如决定改变临床给药方式,还需要进行额外的动物实验,以检查药物分布、药代动力学的变化,并确定安全性。
给药频率取决于药物类型和药代动力学参数(The frequency of dose administration is dependent upon the drug type and pharmacokinetic parameters.)。代谢和从血液循环中快速清除的小分子药物可能需要每天给药一次或更频繁。生物制品不会被代谢,半衰期较长,因此给药次数较少(每周一次或更少)(Biologics which are not metabolized, have a longer half-life, and therefore are dosed less frequently (once a week or less frequently))。
非临床研究对一系列剂量水平进行评估,以确定安全的起始剂量,并确定在临床试验中需要监测的潜在安全性参数。在毒性研究中,目的是确定无观察效应水平(NOEL,no observed effect level)或无观察不良反应水平(NOAEL,no observed adverse effect level),直至产生毒性的剂量水平或最大耐受剂量(MTD,maximum tolerated dose)。安全窗(safety margin)是由非临床研究中无观测不良效应水平(NOAEL)剂量与临床试验给药剂量之间的倍差决定的。在这种情况下,应提供剂量选择和安全系数的科学依据(scientific justification of the rationale)。
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毒性研究设计(Toxicity Study Design)
初始毒性研究一般只使用几只动物,可能不符合GLP标准,用于帮助选择GLP毒性研究的剂量水平。GLP毒性研究的设计和要求是标准化的。研究中使用三组或四组(代表低、中、高剂量水平范围)数量相等的雌雄动物。虽然统计分析要求每组有一定数量的动物,但在规划这些研究时应考虑3R原则--替换、减少和改进(replacement, reduction, and refinement)。
其中一个动物研究组将作为对照组,只用于给药制备剂量配方的对照品或载体,但该动物组在其他方面的条件与接受药物的动物组相同。其他组接受低、中或高剂量水平的评估。临终前评估(Antemortem evaluations)包括临床观察、食量、体重、临床病理学(如血液学、凝血学、尿液分析和临床化学)和眼科学(clinical observations, food consumption, body weight, clinical pathology (such as hematology, coagulation, urinalysis, and clinical chemistry) and ophthalmology。临终后评估(Postmortem evaluations)包括大体形态学、器官重量和标准组织清单的显微病理学(gross morphology, organ weights, and microscopic pathology of a standard list of tissues.)。
给药结束后,一部分动物会继续接受研究,在恢复期内接受与给药期间相同的评估。在研究期间,还将采集样本进行药代动力学分析,以便将药物效应与动物循环系统中的药物含量联系起来。
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非临床安全性评价(Nonclinical Safety Evaluation)
以下是不同类型的非临床安全性研究的简要概述,请参阅这些研究的各个章节了解更多细节。
(1)安全药理学研究(Safety Pharmacology Studies)
安全药理学研究检查药物对主要生理系统(中枢神经、心血管和呼吸系统)的功能影响,是临床试验研究申请在人体暴露之前的必要条件(见第5章)。对于小分子药物,第一项研究是体外研究,以检查其与hERG钾离子通道的相互作用。生物制品不进行此项研究,因为其体积太大,无法进入离子通道。体内研究可单独进行,如果是生物制品,则可纳入GLP重复剂量毒性研究。
(2)药代动力学研究(Pharmacokinetic Studies)
药代动力学评价考察药物在给药后如何被吸收、分布、代谢和排泄(见第6章),通常将测量结果纳入毒性研究中。该分析研究动物体内的全身暴露量、与剂量水平的关系以及在研究过程中发生的情况。在某些情况下,可能需要对代谢物进行药代动力学分析。
(3)一般毒性研究(General Toxicity Studies)
一般毒性研究是小分子和大分子药物的主要安全性评估方法。这些研究评估的是一般毒性,即研究对动物健康各方面的影响,包括行为、体貌、体重、食量的变化、血液化学水平的改变以及器官和组织的微观损伤(including changes in behavior, physical appearance, body weight, food consumption, alterations in blood chemistry levels, and microscopic damage to organs and tissues.)。
急性毒性或单次剂量研究足以了解耐受性,而GLP重复剂量研究则为首次人体临床试验申请提供支持。此类安全性评估应说明剂量依赖性的特点,确定作为潜在药物靶点的器官或组织,检查不良反应与药物水平的关系,并在适当时检查潜在的可逆性。这类非临床研究有助于确定在随后的临床试验中需要监测的参数。首次GLP研究的时间长度(通常为2到13周)取决于临床适应症,在开始关键的三期临床试验之前,还需要进行第二次更长的GLP重复剂量研究(最长可达1年),对药物进行更长时间的剂量给药评估。
(4)局部耐受性研究(Local Tolerance Studies)
应使用拟用于临床的制剂并以相同的给药方式对局部耐受性进行评估。这一信息可从毒性研究中收集。这项评估的要求可能因提交临床试验研究申请的国家而异。
(5)遗传毒性研究(Genotoxicity Studies)
遗传毒性研究包括体外和体内试验,以确定药物是否会诱发遗传损伤(genetic damage)。由于生物制品不会直接与DNA或其他染色体物质发生作用,因此在测试新的生物制品时通常不需要进行这些研究。体外细菌反向突变试验(通常称为Ames 试验)通常足以支持临床试验研究申请的提交。在启动2期临床之前,需要进行一整套遗传毒性试验,通常包括两项附加研究:哺乳动物细胞体外染色体畸变试验和体内啮齿动物骨髓微核试验。还有其他各种类型的遗传毒性研究,可能需要根据小分子药物的类别进行研究。
(6)致癌性研究(Carcinogenicity Studies)
致癌性研究确定长期接触某种药物是否有可能导致恶性转化(cause malignant transformation)。致癌性研究的要求通常取决于适应症和药物类别。如果需要,致癌性研究可为上市许可申请提供支持(见第8章)。致癌性研究是在啮齿类动物中进行的长期研究(一般长达2年),通常在测试小分子药物时进行;评估生物制品时一般不需要这些研究。
(7)发育和生殖毒性研究(Developmental and Reproductive Toxicity Studies)
发育和生殖毒性研究可研究药物对生育能力、胚胎-胎儿生长以及围产期发育的潜在影响。进行这些研究的时间取决于临床适应症和预期用药人群(见第9 章)。如果相关物种为非人灵长类动物,则小分子药物和生物制品的相关此类研究有所不同。在获得对胚胎-胎儿发育影响的信息之前,将具有生育能力的女性纳入临床试验,需要进行适当的临床风险管理,如使用高效的避孕方法。一般来说,在将孕妇纳入临床试验之前,应先进行女性生殖毒性研究。
— 结论 —
在进行非临床研究时,遵守GLP非常重要,因为GLP有助于保证提交监管审查的数据的整体质量、完整性、准确性和可重复性。此外,无论药品是用于人类、动物还是环境,准确可靠的测试还有助于确保产品的安全性和有效性,且能达到预期目的。
个人拓展总结:FDA IND申请的非临床部分包含药理学和毒理学(体内或体外)研究的信息,根据这些研究,IND申请的申请人需得出结论,拟议进行的临床研究是合理安全的。IND申请中要求的动物和其他研究的种类、持续时间和范围将取决于拟议的临床研究的持续时间和性质。
药效:体外或体内的主要药效学研究是用于研究受试化合物与其预期治疗靶点相关的作用方式和作用,确定最小起效剂量,PK-PD关系等,这有助于后续非临床和临床试验的剂量选择。没有法规或指南明确规定动物模型数量。但要从科学性证明受试化合物对疾病模型有效。中美双报的项目,至少选择2-3个动物模型。
药代:应获得有关试验物的药代动力学(PK)(如吸收、分布、代谢和排泄)的进一步信息以及与潜在药物相互作用有关的体外生化信息。核心的安全药理学研究包括评估对心血管、中枢神经和呼吸系统的影响,一般应根据ICH S7A和S7B(参考文献5和6)进行。对于体外hERG试验,不是要求一定遵从GLP。根据ICH-S9的要求,为支持在晚期肿瘤患者中进行临床试验时,不要求进行单独的安全药理学研究,可以融合在重复毒性试验里。但需要关注融合的试验剂量是否能充分暴露可能的安全影响。
毒理学:为了支持临床开发各个阶段的安全性,应至少在两个动物物种(其中一个是非啮齿类动物)中进行研究,以评估拟议接触部位的急性、亚慢性和慢性毒性。研究期限应等于或长于临床试验中的拟议治疗期限,给药途径应与临床给药相当。急性毒性数据是通过在两个哺乳动物物种中使用临床和肠外给药途径的单剂量毒性研究获得的。然而,这种信息可以从适当的剂量递增研究或短期的剂量范围研究(DRF)中获得,这些研究在一般的毒性测试物种中确定了MTD。推荐的重复剂量毒性研究的持续时间通常与拟议的临床试验的持续时间、治疗指征和范围有关。原则上,在两种哺乳动物(一种非啮齿动物)中进行的动物毒性研究的持续时间应等于或超过人类临床试验的持续时间,直至重复剂量毒性研究的最大推荐持续时间。遗传毒性实验(基因突变的检测)通常被认为足以支持所有单剂量的临床开发试验。为支持多剂量临床开发试验,应完成一项能够在哺乳动物系统中检测染色体损伤的额外评估(参考文献10)。在启动2期临床试验之前,应完成完整的基因毒性测试(参考文献10)。如果出现阳性结果,应进行评估,然后可能进行额外的测试(参考文献10)。
遗传毒性实验一般Phase Ⅱ前:体外+体内标准组合(Core battery);而对于抗肿瘤药物IND阶段不是必须的。
生殖毒性实验,根据现行指南,Initial IND中美申报时可不提供生殖毒性试验相关内容;进入Ⅱ期临床时:Seg Ⅱ生殖毒性的预试验,进入Ⅲ期临床时:完成Ⅰ段生殖毒性和两种动物种属的Ⅱ段生殖毒性正式试验;NDA前完成围产期毒性试验。抗肿瘤药物:在上市前完成胚胎-胎仔毒性试验(Ⅱ段生殖毒性)。
申请人对临床适应症进行致癌性研究,以支持上市申请。只有在对致癌风险有重大担忧的情况下,才在临床阶段应提交研究致癌性结果以支持临床试验。对于临床给药时程 ≥ 6个月或治疗慢性复发性疾病而需要间歇使用的药物,需要考虑进行致癌性试验。抗癌药物通常不需要进行致癌性试验,但当抗癌药物较为有效并能明显延长生命的情况下,应考虑有关继发性肿瘤的问题。
关于FDA IND申请非临床研究详见推文“Drug RA | FDA IND: Pharmacology and Toxicology (PT) Information”。
英文原文
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Chapter 4. Principles of Good Laboratory Practice and Nonclinical Development
Nonclinical evaluation is of great importance in determining whether an experimental drug can be safely tested in humans as a potential therapeutic agent. Such evaluation includes a variety of in vitro and in vivo studies that assess safety pharmacology, general toxicity, genotoxicity, carcinogenicity, and developmental and reproductive toxicity. Compliance with good laboratory practice (GLP) regulations, which provide the framework for the conduct of nonclinical studies, will help ensure the quality and integrity of the data generated. This in turn will support applications for clinical studies and future marketing authorizations. GLP compliance also allows for the accurate reconstruction of optimal study conditions, quality recordkeeping, and reporting.
一、Good Laboratory Practice Regulations
A fundamental question when a nonclinical safety study is being planned is whether the study needs to be conducted in compliance with GLP regulations. GLP is a quality system that governs the conditions under which nonclinical studies are designed, performed, monitored, recorded, archived, and reported. GLP ensures the integrity of the study results and that when a regulatory authority reviews the results that there is a level of assurance that the data are accurate and that the study was conducted according to well-defined standards.
The nonclinical studies that require GLP compliance are typically those that will provide regulatory authorities with critical information about human safety risks prior to initiation of clinical trials or prior to marketing approval of the drug. These include studies where the test article is administered to a nonhuman test system – whether that is an animal or bacterial model for example to look for adverse effects, as well as certain types of analytical studies. Studies that should be conducted in compliance with GLP include, but are not limited to:
• Acute, subchronic, and chronic toxicology studies (general toxicology studies);
•Genetic toxicology studies (in vitro and in vivo);
•Local tolerance studies;
•Carcinogenicity studies;
•Reproductive and developmental toxicology studies;
•Safety pharmacology studies (in vitro and in vivo);
•Dermal, ocular, venous, and muscle irritation studies;
•Immunotoxicity studies;
•Analytical studies of biological samples collected from animals;
•Analytical studies of the dose formulations – typically a mixture of the test article and a vehicle or carrier – that are used to administer the test article to the test system; and
•Studies to validate the methods that will be used to analyze samples from GLP studies.
The origins of GLP in the US can be traced back to the 1970s when there were well-publicized reports of scientific misconduct and falsification of data at multiple laboratories that conducted animal safety studies for pesticides and pharmaceuticals.1 Congressional hearings were convened to investigate these matters, and in 1976 the Food and Drug Administration (FDA) proposed GLP regulations that stipulated how all aspects of nonclinical safety studies should be conducted. These FDA GLP regulations became law in 1979 and in 1981 the FDA issued follow-up guidance that answered questions about their interpretation and applicability.2 Proposed changes were published in 1984, and the revised GLP Final Rule was enacted into US law in 1987 as 21 CFR Part 58.
Outside of the US, the conduct of nonclinical studies is covered primarily by guidance set out by the Organisation for Economic Cooperation and Development (OECD), an international organization of industrialized countries which at the time of writing has 38 members. The OECD developed a similar set of GLP principles to the FDA in 1981, which were later revised in 1997. The OECD does not have governmental authority so each of its member countries must enact the GLP guidance into law so that GLP compliance can be effectively monitored and enforced. As companions to the GLP guidance, the OECD has published over 20 documents that address interpretation of specific aspects of the GLPs and their application and monitoring; the most recent of these was released in 2022.
(1)Mutual Acceptance of Data
In the spirit of harmonization and to reduce duplications in conducting studies, the OECD Council issued three decisions over the span of 1981–1997 which are the basis for a Mutual Acceptance of Data (MAD) system. Under this system, nonclinical study data generated in compliance with GLPs in an OECD member country is to be accepted by regulatory authorities in other member countries. In 1981, the original MAD decision was issued, and applied only to OECD member countries. In 1989, procedures were established for monitoring GLP compliance through government inspections and safety audits and it was dictated that GLP compliance in a member country is to be recognized by other member countries. Finally, in 1997, it was decided that a non-OECD country can be included in MAD if that country has a verified national GLP program in place and meets certain criteria.
At the end of 2022, the following countries were participants under the MAD system for nonclinical data for human pharmaceuticals: Australia, Austria, Belgium, Brazil, Canada, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, India, Ireland, Israel, Italy, Japan, Malaysia, Mexico, Netherlands, New Zealand, Norway, Poland, Portugal, Singapore, Slovakia, Slovenia, South Africa, South Korea, Spain, Sweden, Switzerland, Thailand, Turkey, the US, and the UK.6 The majority of these countries are members of the OECD; the exceptions are Brazil, India, Malaysia, Singapore, South Africa, and Thailand which have verified national GLP programs that allow for their inclusion in the MAD system. This list of countries only applies to acceptance of nonclinical data for human pharmaceuticals and may not apply to other categories such as veterinary drugs, cosmetics, pesticides, or food additives. Chile, Colombia, Costa Rica, Iceland, Latvia, Lithuania, and Luxembourg are OECD member countries but are excluded from the MAD system because they lack national GLP programs.
二、Overview of GLPs
The organization and wording of the written GLPs may vary between the FDA and OECD guidance, but the aspects of nonclinical study conduct that are addressed are the same for both. These are briefly summarized in the following sections. For more detail on the content of the GLPs please refer to the official FDA and OECD websites that contain the full texts of the regulations.
The key aspects covered are divided into the following categories:
•Test facility organization, management and personnel
•Quality assurance
•Facilities
•Equipment and reagents
•Test systems
•Test and control articles
•Standard operating procedures (SOPs)
•Study protocol and performance of the study
•Reporting study results
•Storage and retention of records and materials
(1)Test Facility Organization and Personnel
The test or testing facility is the main location where the study is initiated and conducted. It is not unusual for all phases of a study to be conducted at a single test facility; however, it is often the case that different phases of a study are conducted at separate locations, and this is called a multisite or multicenter study. For a multisite study, the test facility is typically where the study director resides and where the test article is administered to the test system, and any ancillary location where a phase of the study is conducted is called a test site. For example, a multisite toxicology study is conducted in which the test article is dosed to rats at the test facility and blood samples from the rats are shipped to a separate test site for analysis.
(2)Test Facility Management
The management of the test facility is responsible for ensuring that GLP regulations are followed and that overall operations and personnel meet GLP compliance requirements.
Specific test facility management responsibilities include but are not limited to:
•Ensuring that personnel working at the site are qualified by training, education, and experience;
•Designating a study director for each study and replacing the study director if necessary
•Ensuring that there is a quality assurance unit (QAU);
•Ensuring that the QAU reports any deviations from the GLPs to the study director and follows up to confirm appropriate corrective actions have been made;
•Ensuring that all test and control articles and their mixtures have been appropriately characterized;
•Ensuring that the necessary personnel, equipment, materials, and facilities are available for use during GLP studies;
•Ensuring that approved methods and SOPs are available for study personnel to follow; and
•Ensure the GLP compliance of test sites that conduct phases of a multisite study.
(3)Study Director
The study director is a qualified individual who is responsible for overseeing all phases of the study’s conduct from its initiation through to the archival storage of the study’s records. This includes supervising all data collection, study procedures and reporting of results, and ensures any deviation from GLPs or the study protocol is documented, and the impact of the deviation is assessed. The study director is often referred to as the “single point of control” and there can only be one study director for a nonclinical study.
The designation of other individuals as ‘backup study director’ or‘co-study director’ should be avoided. The study director is responsible for approving the protocol or study plan as well as ensuring that the study personnel adhere to the study protocol, test facility SOPs, and GLP regulations. This responsibility extends to any test sites participating in a multisite study. By signing the final study report, the study director accepts full legal responsibility for accuracy of the study data and conclusions drawn from the results, as well as the study’s claim of GLP compliance. The latter is typically documented in a signed GLP compliance statement within the final report.
(4)Principal Investigator
The study director may delegate the oversight of a particular phase of a study to a qualified individual called a principal investigator. This may be warranted because the phase is being conducted at a separate test site or because specialized knowledge or training is needed to oversee that phase. The principal investigator ensures that their phase of the study was conducted according to GLP.
These individuals may also be called principal scientists or contributing scientists, but their responsibilities during the performance of the study are the same. Of note, the title of principal investigator only appears in the OECD GLP guidance.
(5)Quality Assurance
The QAU is an individual or group that reports to test facility management and is responsible for ensuring that the facilities, equipment, personnel, methods, practices, records, and controls all follow GLP. This is accomplished through inspections or audits of facilities, processes, and study-specific activities. All QAU personnel need to be familiar with study procedures but should never be directly involved in the conduct of a study. The QAU reviews study reports to ensure that the results accurately reflect the raw data generated during the study. A final study report will contain a statement signed by QAU personnel that lists all phases of the study that were inspected and the dates of inspection. The QAU maintains the testing facility’s master schedule of nonclinical studies, copies of study protocols, and all inspection reports.
(6)Facilities
The facilities used during the conduct of a GLP study should be of a suitable size, construction, and location to meet the requirements for the type of study. Facilities should be designed to provide separate areas for different study activities. There should be sufficient rooms or areas to ensure isolation of test systems such as study animals and individual projects that may contain biohazardous materials. Suitable rooms or areas should be available for diagnosis, treatment, and control to ensure that there is no unacceptable deterioration in the test system. Storage rooms or areas, particularly those where test articles are stored and handled, should be separated adequately from rooms or areas housing test systems to protect against contamination, infestation, or deterioration.
(7)Equipment and Reagents
Equipment used for data generation, storage or retrieval and controlling environmental factors, including computerized systems, should be suitably located, appropriately designed, and have adequate capacity. Equipment should be tested and calibrated to ensure the accuracy of data generated using that equipment. Testing and calibration methods should be described in written SOPs as well as the procedures to be taken if equipment fails or malfunctions. All reagents and materials used during a study should be clearly identified and labeled appropriately with expiration date and storage requirements.
(8)Test Systems
Proper procedures for the storage, handling and housing conditions of test systems should be established and maintained to ensure data quality. Test systems should be acclimated to the test environment for an appropriate period before the first administration or application of the test article. Records of test system source, date of arrival, and condition on arrival should be logged and maintained. Test systems must be maintained in a manner that avoids contamination with other test systems and materials. Individual test subjects should be identified by a unique and indelible method to ensure accurate data collection. The size and dimensions of animal housing containers or cages need to comply with any applicable animal welfare regulations where the test facility is located.
(9)Test and Control Articles
Test and control articles should be characterized based on identity, strength, purity, composition, or other appropriate characteristics. The stability of the articles must be determined either before the study is initiated or during the study according to the test facility’s SOPs. Ensuring appropriate characterization of the test and control articles is a responsibility of the testing facility management.
Test articles used in GLP studies should be characterized and documented using GLP compliant methods and procedures; however, many laboratories and drug developers use procedures that comply with good manufacturing practice (GMP) regulations to characterize test articles. While this is a common practice and may be scientifically justifiable, it must be documented as a GLP deviation, and the study director needs to assess the impact of the deviation on the integrity of the study.
Mixtures of test article with a vehicle or carrier (such as the dose formulation) must be analyzed periodically over the course of a study for uniformity and concentration of the test article in the formulation. The stability of the mixture under the conditions of use on the study must be determined either before the study initiates or during the study according to test facility SOPs.
(10)Standard Operating Procedures
SOPs are an integral part of the conduct of and recreation of GLP studies. SOPs ensure that laboratory procedures and processes are consistently performed by all study personnel throughout the test facility. SOPs should be readily available to all laboratory staff, and a mandatory review of SOPs should be performed and documented. Test facility management is responsible for periodically reviewing and updating the content SOPs as needed.
In general, the study protocol outlines what is to be done during a nonclinical study, but SOPs describe how those procedures are to be performed. For example, the protocol would instruct a technician to administer an intravenous injection to a study animal, but SOPs would specify what size needle and syringe to use, the appropriate vein for the injection, how the injection site should be prepared, the proper method of restraint for the animal, and so on. A comprehensive set of SOPs should address all aspects of the functioning of a test facility including all processes and procedures needed to maintain GLP compliance.
Deviations from SOPs that occur during a study must be documented and the study director must assess the impact of the deviation on the integrity of the study.
(11)Study Protocol and Conduct of the Study
All of the procedures that will occur in a nonclinical study are written in a study protocol. There can be only one protocol per study. As such, sample analysis plans or similar documents that may be generated for study-specific procedures should never be titled as the study protocol. The study is initiated when the study director signs and dates the study protocol. It is inevitable that changes to the protocol will be needed over the course of a study. Planned changes and the reasons for these changes are issued as protocol amendments, and the changes are effective when the study director signs the amendment. Any deviations from the protocol must be documented and reviewed by the study director to assess the impact on the study.
One of the main objectives of GLP is to set standards for data recording to ensure that nonclinical study results are accurate and reproducible. The guiding principles for how data should be recorded and maintained have long been summarized by the acronym ALCOA – Attributable, Legible, Contemporaneous, Original, and Accurate. In recent years this concept has been expanded to be more inclusive of electronic data and is called ALCOA+ with the addition of the terms: Complete, Consistent, Enduring, and Available.
•Attributable – It should be clear who recorded the data and when. All changes to original data, who made the change and when, and the reason for change should be clearly documented. Changes to data should not obscure the original entry.
•Legible – The data should be recorded so that it can be clearly read. Hand-recorded data must be written in ink. If printed pages from automated systems are considered raw data, the ink and paper used should be resistant to deterioration.
•Contemporaneous – Data should be recorded at the time it is observed or as soon after as possible. The accuracy of data that are recorded days or weeks after the event occurred can be questionable. Backdating of data recording is strictly prohibited.
•Original – This first recording of the data needs to be maintained even if it is made in an unconventional way. For example, if an observation is initially written on a paper towel and later transcribed to paper or an electronic entry, the paper towel should be maintained in the study records as the raw data.
•Accurate – The raw data should be accurate and free of errors and bias. When recording data electronically, the system must have built-in accuracy checks and verification controls. Measurement equipment should be regularly calibrated as part of this process.
•Complete – All recorded data should have an audit trail to show nothing has been deleted or lost; there should be audit trails covering any changes made to the data.
•Consistent – The data should be saved consistently in a particular chronological order with date and time stamps for easy understanding and audit.
•Enduring – Data should be readable long after it has been recorded; this is particularly important for computerized data and processes need to be in place to keep data secure and intact.
•Available – Data must be easily accessible and readable at any time during the required retention period.
The increased use of computerized systems to record nonclinical study data prompted questions about how GLP requirements intended for hand-recorded data would apply to electronic data. The requirements remain exactly the same, but the implementation must be adapted to the medium. In 1997, FDA issued regulations (21 CFR Part 11) to ensure that electronic records and signatures are, “trustworthy, reliable, and generally equivalent to paper records and handwritten signature executed on paper.” While technically not a part of the GLPs, there is an expectation that these requirements will be followed when electronic data are part of a GLP study.
(12)Reporting Study Results
Each study with a study director-signed protocol must also have a study director-signed final report. This includes completed studies and studies that were terminated early for any reason. The date the study director signs the final report is the study completion date.
No further data may be collected, or information inserted into the report after study completion. In the event new information becomes available that affects a completed study’s outcome or interpretation, a final report amendment will be prepared, signed by the study director, and kept with the original final report. Information the final study report should contained are listed in Figure 4-1.
As part of the drug development process, nonclinical, or preclinical, studies are conducted to evaluate a drug’s safety profile as part of the applications for clinical trial research and marketing authorization. These studies consist of both in vitro and in vivo studies. Many are conducted prior to first-in-human clinical studies, some are conducted concurrently with clinical development, and others may even be required after a drug has been approved for marketing.
Drug development is a global activity and harmonization of regulatory requirements became necessary. This was pioneered in Europe in the 1980s with the successful move to develop a single pharmaceutical market. This led to the inception of the International
Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) in 1990, a consortium of international regulatory health authorities.
The ICH has published a comprehensive set of guidance documents to aid the nonclinical development of new drugs and the design of individual studies. The ICH guidelines are separated into four categories: Quality, Efficacy, Safety, and Multidisciplinary. The guidelines that pertain to nonclinical safety testing and planning a nonclinical development program are found mainly in the Safety and Multidisciplinary categories.
Unlike the GLP regulations, the ICH guidelines are non-legally binding. However, sponsors are encouraged to follow ICH guidelines to ensure the development of safe and effective medicines in a manner that is consistent worldwide. Conduct of the studies described in the ICH guidelines include both GLP and non-GLP guidance. The ICH guidelines were originally called “tripartite” because they were co-developed by regulatory health authority representatives from three countries – the US, Europe, and Japan. Goals of nonclinical safety evaluation are given in Figure 4-2.
(13)Investigational Drug Types
As the types of drug products continue to diversify and expand, ICH guidance documents have been published to guide their development. Chemical or small molecule drugs were the most common in development until the mid-1980s when companies started to produce biologics. Continuing in the progress of drug development, gene therapy has become more prevalent and resulted in new guidance documents such as ICH S12 in 2021.
While the nonclinical safety evaluation pathway is generally the same for each drug product class, the types of studies required in each of these categories may differ. Types of studies that may be used include:
• Safety pharmacology (cardiovascular, respiratory, renal, and central nervous systems)
• Pharmacokinetics
• General toxicity studies (single and repeat-dose)
• Local tolerance
• Genotoxicity
• Carcinogenicity
• Developmental and reproductive toxicity
Other nonclinical studies to evaluate phototoxicity, immunotoxicity juvenile animal toxicity, and abuse liability should be conducted on a case-by-case basis that is dictated by the class of drug and the target population.
(14)Pharmaceuticals
Pharmaceuticals are small molecule drugs, also known as a new chemical entity or by the abbreviation NCE (herein referred to as small molecules). These drugs are chemically synthesized with a well-defined structure and based on their size (<1 kDa), they generally need to be administered once or twice daily. The route of administration is typically oral and after administration they degrade via metabolism. The nonclinical program is extensive and a full evaluation of the aforementioned study type categories is required including developmental and reproductive toxicity studies.
(15)Biopharmaceuticals
Biopharmaceuticals are also known as large molecules, biologics or biotherapeutics (herein referred to as biologics). These drugs are made via biological synthesis (biotechnology) and are complex with secondary or tertiary structures. They include a wide variety of drug types such as monoclonal antibodies, vaccines, live biotherapeutics (probiotics), blood products, gene therapies, human cells, tissues, and cellular and tissue-based products. Based on their size (>1 kDa) they typically can be administered at intervals of 1 week or longer. Their route of administration is typically by parenteral injection and after administration they degrade via catabolism or proteolysis. Their nonclinical assessment is more limited based upon the relevance of animal models and the nature of biologics. Metabolism studies are not required; the molecule size prevents interaction with DNA and therefore genotoxicity and carcinogenicity studies are also not required.
(16)Animal Models for Toxicity Testing
Animal studies are conducted under specific guidelines that comply with the Animal Welfare Act in the US and defined in the European Directive on the protection of animals used for scientific purposes and upheld internationally.12-14 Study protocols using animals are reviewed and approved by the Institutional Animal Care and Use Committee or Ethical Review Committee of the test facility where the study is conducted.
Based upon ICH guidance, toxicity studies should be conducted in two mammalian species (one rodent and one nonrodent). For the nonclinical evaluation of small molecules such studies are typically conducted in rats and dogs. Biologics are target-specific and require the use of relevant species for safety assessment. A relevant species is one in which the drug is pharmacologically active due to the expression of the receptor or an epitope and evokes a similar pharmacological response as that expected in humans. Use of a non-relevant species is discouraged as they produce non-representative results. Nonhuman primates are generally the most relevant species for these studies based upon their similarity to humans in terms of genome homology and physiological systems, such as the immune system. Using a single species for toxicity testing is acceptable when there is only one relevant species. When no relevant species exists, use of a transgenic animal expressing the human receptor, or the use of a surrogate drug (homologous protein) may be acceptable alternatives. An animal model of the human disease may also be an acceptable alternative to a toxicity study in non-diseased animals.
(17)Drug Administration
Several factors need to be considered for how the drug will be administered in a nonclinical study including formulation, route, frequency, and dose level. The drug administered to animals in a GLP toxicity study is expected to be manufactured under GLP or GMP and fully characterized as described earlier (see under ‘Test and Control Articles’ in this chapter). The drug used during nonclinical development should be representative of what will be used in clinical trials; however, it is often the case that the clinical drug formulation has not been fully optimized at the time of the first GLP toxicity study. Changes in formulation or manufacturing process need to be well documented (see Chapter 12). In GLP toxicity studies that occur later in development to support the pivotal clinical trial(s), the animals should be dosed with drug that is representative of what is used in the clinic and what will potentially be marketed. Substantial changes in drug or manufacturing processes, at a later date, may result in the necessity of another animal study to either demonstrate safety or compare pharmacokinetic properties.
The route of drug administration used in a GLP nonclinical study is required to be identical to what will be used in the clinical trial. For a small molecule there may be a variety of dosing options beyond the typical route of oral administration (e.g. via a pill
or tablet, capsule or liquid) including intravenous infusion, dermal (topical, subcutaneous, intradermal), intramuscular, or targeted to specific body areas (ocular, intranasal, intrathecal, vaginal, or rectal) that must be tested before administration in humans. Biologics are more easily degradable and therefore the options are more limited such as intravenous infusion, subcutaneous, intradermal, or intramuscular. A decision to change the clinical mode of administration requires additional animal studies to examine the change in drug distribution, pharmacokinetics, and determine safety.
The frequency of dose administration is dependent upon the drug type and pharmacokinetic parameters. Small molecule drugs that are metabolized and cleared rapidly from the circulation may need to be administered once daily or more frequently. Biologics which are not metabolized, have a longer half-life, and therefore are dosed less frequently (once a week or less frequently).
A range of dose levels are evaluated in nonclinical studies to determine a safe starting dose as well as identify potential safety parameters to monitor in clinical trials. In toxicity studies, the aim is to identify a no observed effect level (NOEL) or no observed adverse effect level (NOAEL) up to a dose level that produces toxicity or a maximum tolerated dose (MTD).10 A safety margin is determined by the fold-difference between the NOAEL dose level in the nonclinical study and what will be administered in the clinical trial. Some drug classes produce little or no toxicity and it may not be possible to define a MTD. In these cases, a scientific justification of the rationale for the dose selection and safety margin should be provided.
(18)Toxicity Study Design
An initial toxicity study generally uses only a few animals, may be non-GLP compliant, and is used to help select the dose levels for the GLP toxicity study. The GLP toxicity study design and requirements are standardized. Three or four groups (representing ranges of low, medium, and high dose levels) of equal numbers of male and female animals are used in the study. While statistical analysis requires a certain number of animals per group, the principles of the 3Rs – replacement, reduction, and refinement– should be considered when planning these studies.16 One of the study groups will be a control group that receives only the control article or vehicle used for preparing the dose formulation, but the animals are otherwise subjected to the identical conditions as the animals receiving the drug. The other groups receive low, medium, or high dose levels for evaluation. Antemortem evaluations include clinical observations, food consumption, body weight, clinical pathology (such as hematology, coagulation, urinalysis, and clinical chemistry) and ophthalmology. Postmortem evaluations include gross morphology, organ weights, and microscopic pathology of a standard list of tissues. Upon completion of dosing, a subset of animals remains on study for a recovery period during which they undergo the same evaluation that was conducted during drug administration. During the study, samples are also collected for pharmacokinetic analysis to correlate drug effects with the amount of drug present in their circulatory system.
(19)Nonclinical Safety Evaluation
A brief overview of the different types of nonclinical safety studies follows, please see the individual chapters on these studies for more detail.
•Safety Pharmacology Studies
Safety pharmacology studies examine the functional effect of the drug on the major physiological systems (central nervous, cardiovascular, and respiratory systems) and are required for the clinical trial research application, prior to human exposure (see Chapter
5). For small molecule drugs, the first study is an in vitro study to examine the interaction with the hERG potassium ion channel which plays an important role in repolarization of the myocardium. This study is not conducted with biologics as they are too large to fit in the ion channel. In vivo studies may be stand alone or if a biologic drug, included in GLP repeat-dose toxicity studies.
•Pharmacokinetic Studies
Pharmacokinetic evaluation looks at how the drug is absorbed, distributed, metabolized, and excreted after administration (see Chapter 6) and measurements are normally integrated into the toxicity studies. The analysis examines the systemic exposure achieved in animals, the relationship to dose level, and what happens over the time course of the study. In some cases, a pharmacokinetic analysis of metabolites may be required.
•General Toxicity Studies
General toxicity studies are the main safety assessment for both small and large molecule drugs. These studies evaluate general toxicity in that they look for effects on all aspects of an animal’s health including changes in behavior, physical appearance, body weight, food consumption, alterations in blood chemistry levels, and microscopic damage to organs and tissues. Acute toxicity or single dose studies are sufficient to access tolerability while a GLP repeat-dose study is conducted to support the application for the first in-human clinical trial. This safety assessment should characterize dose dependence, identify organs or tissues that are potential drug targets, examine the relationship of an adverse effect to drug levels and, when appropriate, potential reversibility. This type of nonclinical study helps to determine the parameters to monitor in a subsequent clinical trial. The length of the first GLP study (usually from 2 to 13 weeks) is dependent upon the clinical indication and a second, longer GLP repeat-dose study (up to 1 year) that evaluates a drug over a longer period of dose administration is required prior to starting a pivotal Phase 3 clinical trial.
•Local Tolerance Studies
Local tolerance should be evaluated using the formulation intended for use in the clinic and by the same mode of administration. This information may be collected from the toxicity studies. The requirements for this evaluation may differ based on the country
where the clinical trial research application is filed.
•Genotoxicity Studies
Genotoxicity studies include in vitro and in vivo tests to ascertain whether a drug induces genetic damage. These studies are typically not required when testing a new biologic agent because biologics do not interact directly with DNA or other chromosomal material. An in vitro bacterial reverse mutation assay (often called the Ames test) is often sufficient to support filing of the clinical trial research application. A complete genotoxicity test battery is required prior to initiating Phase 2 trials and usually includes two additional studies: an in vitro chromosome aberration test in mammalian cells and an in vivo rodent bone marrow micronucleus test. There are a variety of other types of genotoxicity studies, and these may be required based on the class of the small molecule drug.
•Carcinogenicity Studies
Carcinogenicity studies determine if exposure to a drug over a long period of time has the potential to cause malignant transformation. The requirement for carcinogenicity studies is most often dependent upon the indication and drug class. If required, these studies support the marketing authorization application (see Chapter 8). These are long-term (generally up to 2 years) studies that are performed in rodents and are more commonly undertaken when testing small molecule drugs; they are generally not required when evaluating a biologic agent.
•Developmental and Reproductive Toxicity Studies
Developmental and reproduction toxicity studies may investigate the potential effects of a drug on fertility, embryo-fetal growth, and pre- and post-natal development. The timing of when these studies are conducted depends on the clinical indication and the population intended to receive the drug (see Chapter 9). These studies differ for small molecule and biologics programs if the relevant species is a nonhuman primate. Including women of child-bearing potential in clinical trials before acquiring information on effects on embryo-fetal development, requires appropriate clinical risk management such as the use of highly effective methods of contraception. In general, female reproduction toxicity studies should be conducted before including pregnant women in clinical trials.
三、Conclusion
Compliance with GLP is important when conducting a nonclinical study as it will help guarantee the overall quality, integrity, accuracy, and reproducibility of data that are submitted for regulatory review. Moreover, accurate and reliable testing will help ensure products are both safe and effective for their intended purpose, whether that is for human, animal, or environmental use.
参考文献:(上下滑动查看更多)
All references verified 10 February 2023.
1.Baldeshwiler AM. History of FDA good laboratory practices. Qual Assur J. 2003;7(3):157-161.
2.Food and Drug Administration. Guidance for Industry, Good Laboratory Practices Questions and Answers. Current as of July 2007. Accessed 29 November 2022. http://www.fda.gov/downloads/ICECI/
EnforcementActions/BioresearchMonitoring/ucm133748.pdf
3.21 CFR Part 58, Good Laboratory Practice for Nonclinical Laboratory Studies, Final Rule, 1987. Current as of 20 July 2022. Accessed 29 November 2022. https://www.accessdata.fda.gov/scripts/cdrh/ cfdocs/cfcfr/CFRSearch.cfm?CFRPart=58
4.Organisation for Economic Cooperation and Development. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring. No. 1 OECD Principles of Good Laboratory Practice. Accessed 29 November 2022. https://www-oecd-org.libproxy1.nus.edu.sg/officialdocuments/publicdisplaydocumentpdf/?cote=env/mc/chem(98)17&doclanguage=en
5.Organisation for Economic Cooperation and Development. OECD Series on Principles of Good Laboratory Practice (GLP) and Compli ance Monitoring. Accessed 29 November 2022.
https://www-oecd-org.libproxy1.nus.edu.sg/chemicalsafety/testing/oecdseriesonprinciplesofgoodl
boratorypracticeglpandcompliancemonitoring.htm
6.Organisation for Economic Cooperation and Development. National GLP Compliance Monitoring Programmes which participate in MAD (status and contact information). Current as of 17 November 2022. Accessed 29 November 2022. https://www-oecd-org.libproxy1.nus.edu.sg/chemicalsafety/testing/contact-points-working-group-on-good-laboratory-practice.htm
7.21 CFR Part 11, Electronic Records; Electronic Signatures. Current as of 20 July 2022. Accessed 29 November 2022. https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=11
8.International Council for Harmonisation. History. Accessed 29 November 2022. https://www.ich.org/page/history
9.International Council for Harmonisation. ICH Harmonised Guideline. Nonclinical Biodistribution Considerations for Gene Therapy Products S12. Issued 3 June 2021. Accessed 29 November 2022. https://database.ich.org/sites/default/files/ICH_S12_Step2_Draft Guideline_2021_0603.pdf
10.International Council for Harmonisation. ICH Harmonised Tripartite Guideline. Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals M3 (R2). Current as of 11 June 2009. Accessed 29 November 2022. https://database.ich.org/sites/default/files/M3_R2__Guideline.pdf
11.International Council for Harmonisation. ICH Harmonised Tripartite Guideline. Preclinical Safety Evaluation of Biotechnology Derived Pharmaceuticals S6 (R1). Current as of 12 June 2011. Accessed 29 November 2022. https://database.ich.org/sites/default/files/S6_R1_Guideline_0.pdf
12.National Research Council. Guide for the Care and Use of Laboratory Animals: 8th ed. The National Academies Press; 2011. Verified 27 October 2022. https://doi-org.libproxy1.nus.edu.sg/10.17226/12910.
13.9 CFR Parts 1, 2, and 3, Animal and Animal Products. Current as of 28 November 2022. Accessed 29 November 2022. https://www.ecfr.gov/current/title-9
14.European Parliament, Council of the European Union. Regulation(EU) 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union. Published 26 June 2019. Accessed 27 October 2022. https://eur-lex.europa.eu/legal-content/ EN/TXT/?uri=CELEX%3A32010L0063&qid=1669066899741
15.Prior H, Haworth R, Labram B, et al. Justification for species selection for pharmaceutical toxicity studies. Toxicology Research 2020;9(6):758-770. Verified 27 October 2022. https://doi-org.libproxy1.nus.edu.sg/10.1093/ toxres/tfaa081
16.Hubrech RC, Carter E. The 3Rs and human experimental technique: implementing change. Animals 2019;9(10):754-763. Verified 27 October 2022. https://doi-org.libproxy1.nus.edu.sg/10.3390/ani9100754
17.International Council for Harmonisation. ICH Harmonised Tripartite Guideline. Safety Pharmacology Studies for Human Pharmaceuticals S7A. Current as of 8 November 2000. Accessed 29 November 2022. https://database.ich.org/sites/default/files/S7A_Guideline.pdf
18.International Council for Harmonisation. ICH Harmonised Tripartite Guideline. Note for Guidance on Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies S3a. Current as of 27 October 1994. Accessed 29 November 2022. https://database.ich.org/sites/default/files/S3A_Guideline.pdf
19.International Council for Harmonisation. ICH Harmonised Tripartite Guideline. Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use S2(R1). Current as of 9 November 2011. Accessed 29 November 2022. https://database.ich. org/sites/default/files/S2%28R1%29%20Guideline.pdf
20.International Council for Harmonisation. ICH Harmonised Tripartite Guideline. Nonclinical Evaluation for Anticancer Pharmaceuticals S9. Current as of 29 October 2009. Accessed 29 November 2022. https://database.ich.org/sites/default/files/S9_Guideline.pdf
21.International Council for Harmonisation. ICH Harmonised Tripartite Guideline. Guideline on the Need for Carcinogenicity Studies of Pharmaceuticals S1A. Current as of 29 November 1995. Accessed 29 November 2022. https://database.ich.org/sites/default/files/S1A%20 Guideline.pdf
22.International Council for Harmonisation. ICH Harmonised Guideline. Detection of Reproductive and Developmental Toxicity for Human Pharmaceuticals S5(R3). Current as of 18 February 2020. Accessed 29 November 2022. https://database.ich.org/sites/default/files/S5R3_Step4_Guideline_2020_0218_1.pdf
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