Procarbazine Hydrochloride

点击上方的 行舟Drug ▲ 添加关注遗传毒性研究（Genotoxicity Study）是药物非临床安全性评价的重要内容，它与其他毒理学研究尤其是致癌性研究、生殖毒性研究有着密切的联系，是药物进入临床试验及上市的重要环节。拟用于人体的药物，应根据受试物拟用适应症和作用特点等因素考虑进行遗传毒性试验。前言遗传毒性试验是指用于检测通过不同机制直接或间接诱导遗传学损伤的受试物的体外和体内试验，这些试验能检测出DNA损伤及其损伤的固定。以基因突变、较大范围染色体损伤或重组形式出现的DNA损伤的固定，通常被认为是可遗传效应的基础，并且是恶性肿瘤多阶段发展过程的重要因素（恶性肿瘤发展变化是一个复杂的过程，遗传学改变可能仅在其中起部分作用）。染色体数目的改变也与肿瘤发生有关，并可提示生殖细胞出现非整倍体的可能性。在遗传毒性试验中呈阳性的化合物为潜在的人类致癌剂和/或致突变剂。由于在人体中已建立了某些致突变/遗传毒性化合物的暴露与致癌性之间的相关性，而对于遗传性疾病尚难以证明有类似的相关性，因此遗传毒性试验主要用于致癌性预测。但是，因为生殖细胞突变与人类疾病具有明确的相关性，所以也应同样重视化合物引起潜在可遗传性效应的风险。此外，遗传毒性试验结果可能对致癌性试验的结果分析有重要作用。因此，在药物开发的过程中，遗传毒性试验的目的是通过一系列试验来预测受试物是否有遗传毒性，在降低临床试验受试者和药品上市后使用人群的用药风险方面发挥重要作用。本指导原则重点阐述遗传毒性试验的基本原则，介绍标准试验组合方案，阐述体内外试验的基本原则，以及对试验结果的分析评价与追加研究策略。基本概念【适用范围】：适用于中药、天然药物和化学药物。【管理要求】：用于支持药品注册的单次给药毒性试验必须执行《药物非临床研究质量管理规范》（GLP）。【具体问题具体分析】遗传毒性试验的设计，应该在对受试物认知的基础上，遵循“具体问题具体分析”的原则。应根据受试物的结构特点、理化性质、已有的药理毒理研究信息等选择合理的试验方法，设计适宜的试验方案，并试验结果进行全面的分析与评价。【随机、对照、重复】遗传毒性试验应符合毒理学试验随机、对照、重复的基本原则。受试物中药、天然药物：受试物应采用能充分代表临床试验拟用样品和/或上市样品质量和安全性的样品。应采用工艺路线及关键工艺参数确定后的工艺制备，一般应为中试或中试以上规模的样品，否则应有充分的理由。化学药物：受试物应采用工艺相对稳定、纯度和杂质含量能反映临床试验拟用样品和/或上市样品质量和安全性的样品。受试物应注明名称、来源、批号、含量（或规格）、保存条件、有效期及配制方法等，并提供质量检验报告。试验中所用溶媒和/或辅料应标明名称、标准、批号、有效期、规格和生产单位等，并符合试验要求。应进行受试物样品分析，并提供样品分析报告。在药物研发的过程中，若受试物的工艺发生可能影响其安全性的变化，应进行相应的安全性研究。标准试验组合对药物而言，需对潜在的遗传毒性进行全面评价。遗传毒性试验方法有多种，根据试验检测的遗传终点，可将检测方法分为三大类，即基因突变、染色体畸变、DNA损伤；根据试验系统，可分为体内试验和体外试验。由于没有任何单一试验方法能检测出所有的与肿瘤发生相关的遗传毒性机制，因此，通常采用体外和体内试验组合的方法，以全面评估受试物的遗传毒性风险。这些试验相互补充，对结果进行判断时应综合考虑。1.标准试验组合应具备的特征标准试验组合应反映不同遗传终点，包括体内和体外试验，应包含以下内容：（1）应包含细菌回复突变试验（又称Ames试验）。该试验已证明能检出相关的遗传学改变和大部分啮齿类动物和人类的遗传毒性致癌剂。（2）应包含哺乳动物细胞体外和/或体内试验。哺乳动物细胞体外试验中，体外中期相染色体畸变试验、体外微核试验、体外小鼠淋巴瘤L5178Y细胞Tk基因突变试验（简称小鼠淋巴瘤细胞试验，MLA）已经过充分验证并广泛应用，且同样适合于检测染色体损伤。若实验室对这些方法已进行了充分的验证，当用于标准试验组合中时，这几个试验可互相替换。体内试验具有考虑到如吸收、分布、代谢、排泄等因素的优势，并且可检出体外试验无法检出的某些遗传毒性物质（注释1），因此标准试验组合应至少包含一项体内试验。可采用啮齿类动物造血细胞染色体损伤试验（包括骨髓或外周血红细胞微核试验、骨髓中期相细胞染色体畸变试验）或其他合适的体内试验。2.推荐的两种标准试验组合组合一：（1）一项细菌回复突变试验；（2）一项检测染色体损伤的体外细胞遗传学试验（体外中期相染色体畸变试验或体外微核试验），或一项体外小鼠淋巴瘤细胞Tk基因突变试验；（3）一项体内遗传毒性试验，通常为啮齿类动物造血细胞染色体损伤试验，用于检测微核或中期相细胞染色体畸变。组合二：（1）一项细菌回复突变试验；（2）采用两种不同组织进行的体内遗传毒性试验，通常为一项啮齿类动物造血细胞微核试验和第二项体内试验。以上两种试验组合同等适合（注释2），可根据受试物特点自主选择。体内试验可采用单次给药或重复给药的试验设计。如果试验设计科学合理，采用重复给药时可将遗传毒性终点指标整合入一般毒性试验中；当在体内评价一项以上的遗传毒性终点指标时，也可将它们整合在一项试验中。但是，整合的前提条件是试验设计（例如剂量、采样）对于重复给药试验是合适的，并且需要获得充分的支持信息。完成上述任何一种标准试验组合，若试验结果为阴性，通常可提示受试物不具有遗传毒性。对于标准试验组合结果为阳性的受试物，根据其治疗用途，可能需要进一步的试验。标准试验组合不包含为检测非整倍体而设计的特定试验。但是，检测中期相细胞染色体畸变的体外和体内试验可检测多种类型的染色体完整性方面的改变，微核试验具有检测某些非整倍体诱导剂的能力，小鼠淋巴瘤细胞试验也可检测染色体丢失（可能导致非整倍体）。建议采用标准试验组合并不意味着其他遗传毒性试验不合适，这些试验可作为标准试验组合以外的供选试验，用于对标准试验组合得到的遗传毒性试验结果的进一步研究。在某些情况下，标准试验组合中的一项或多项试验对于受试物不适合或因技术原因无法实施时，可采用其他经过验证的试验作为替代试验，但应提供充分的科学合理性及依据。附录部分简介标准试验组合中常用的几种试验方法，该部分内容只是基本原则，试验时应根据具体情况具体分析，合理设计。3.标准试验组合的调整标准试验组合在一些特殊情况下不适合，需要根据情况进行调整。（1）当受试物对细菌有高毒性时（如某些抗生素），仍应进行细菌回复突变试验，因为致突变性可能出现在较低的、毒性较小的浓度。同时，还应进行一项体外哺乳动物细胞试验，即采用标准试验组合一。（2）标准试验组合通常可检出具有遗传毒性作用警示结构的受试物（注释3），因为大部分“警示结构”被定义为与细菌诱变性有关。但是，对于具有某些特殊警示结构的化合物，需要对标准组合方案进行调整（注释4）。附加试验的选择或方案的调整取决于这些具有警示结构受试物的化学性质、已知活性和代谢信息。（3）某些特殊的受试物，如一些放射影像剂、抗酸铝合剂、一些吸入用药、一些皮肤或其他局部用药，毒代或药代动力学研究提示其不被全身吸收，因此在体内遗传毒性试验中无法到达靶组织（注释5）。对于这些受试物，体内试验（尤其是骨髓、血液、肝脏）难以提供有用的信息。在改变给药途径也不能提供足够的靶组织暴露，且对暴露量最高的组织无合适的遗传毒性试验的情况下，仅根据体外试验进行评价可能是合适的。某些情况下，采用接触部位评价遗传毒性作用可能也是合理的，尽管这些试验尚未广泛应用（注释6）。4.生殖细胞诱变剂的检测遗传毒性对生殖细胞的影响也极其重要。标准试验组合中不包含专门检测生殖细胞诱变剂的试验。但是，比较研究结果显示，从定性的角度，大多数生殖细胞诱变剂能在体细胞试验中检出，因此体内体细胞遗传毒性试验的阴性结果通常可提示受试物对生殖细胞无影响。体内体细胞试验结果为阳性时，在综合评价及指导用药时应关注受试物对生殖细胞的影响。试验基本要求遗传毒性的体内外试验方法较多，本指导原则仅讨论常用方法及需要重点关注的问题，试验时需具体情况具体分析。无论体外、体内试验，方法学均应经过充分验证。各实验室应建立历史背景对照数据库（包括阴性和阳性对照数据库）。1.体外试验基本要求1.1 细菌回复突变试验中采用的菌株细菌回复突变试验至少应采用5种菌株，包括用于检测组氨酸靶基因中鸟嘌呤-胞嘧啶（G-C）位点碱基置换或移码突变的4种组氨酸营养缺陷型鼠伤寒沙门氏菌（TA98；TA100；TA1535；TA1537/TA97/ TA97a），以及用于检测组氨酸或色氨酸基因中腺嘌呤-胸腺嘧啶（A-T）位点碱基置换与检测交联剂的鼠伤寒沙门氏菌TA102或埃希氏大肠杆菌WP2 uvrA或埃希氏大肠杆菌WP2 uvrA （pKM101）。1.2 体外试验中最高浓度的确定（注释7）体外试验中受试物的最高浓度主要取决于受试物对细菌/细胞的毒性和溶解度。1.2.1 最高浓度对不受溶解度或细胞毒性限制的受试物，细菌回复突变试验应达到的最高浓度为5mg/皿（液体受试物为5µl/皿），哺乳动物细胞试验为1mM 或0.5mg/ml（选用较低者）。1.2.2 要求达到的细胞毒性水平在遗传毒性体外试验中，某些遗传毒性致癌剂只有在检测浓度高达可产生一定程度的细胞毒性时才可检出，但毒性过高又会影响对相应的遗传终点进行恰当的评价。当哺乳动物细胞存活率很低时，一些遗传毒性以外的作用机制如细胞毒性（如与细胞凋亡、溶酶体释放核酸内切酶等有关的结果）会导致遗传毒性假阳性结果，这种情况常发生于受试物浓度达到毒性阈浓度时。鉴于以上情况，在体外细菌和哺乳动物细胞试验中，目前可接受以下的细胞毒性水平（浓度不应超过1.2.1中的规定）：（1）细菌回复突变试验中，进行评价的浓度应能显示明显的毒性，如回复突变菌落数目减少、背景菌苔减少或消失。（2）哺乳动物细胞体外遗传学试验中，最高浓度产生的细胞毒性应约为50%。（3）对于小鼠淋巴瘤细胞Tk基因突变试验，最高浓度产生的细胞毒性应为80%～90%。1.2.3 难溶受试物的检测用细菌和哺乳动物细胞遗传毒性试验检测某些受试物时，在不溶解的浓度范围内也能检测出剂量相关性的遗传毒性。建议采用以下策略检测相对不溶的受试物：（1）对于细菌回复突变试验，如果沉淀不干扰计数，应对产生沉淀的浓度进行计数，且最高浓度不超过5mg/皿或5µl/皿。当未观察到细菌毒性时，应以产生沉淀的最低浓度作为计数的最高浓度；当观察到剂量相关的细菌毒性或诱变性时，应按上述细胞毒性的要求来确定最高浓度。（2）对于哺乳动物细胞试验，若沉淀不干扰计数，最高浓度应是培养液中产生最少可见沉淀的最低浓度。应通过肉眼观察或镜检等方法来观察记录沉淀在培养过程中持续存在或培养过程中出现（至处理结束时）。1.3 体外试验的重现性体外试验应关注重现性。当采用新方法或试验出现非预期结果时，有必要进行重复试验。但是，当采用标准的、已广泛应用的常规体外试验方法时，若这些试验经过了充分验证且进行了有效的内部质量控制，得到明确的阳性或阴性结果，通常不需要重复试验。但是，若得到可疑结果，则需要进一步试验。2.体内试验基本要求2.1 检测染色体损伤的体内试验采用骨髓细胞分析染色体畸变或检测含微核的嗜多染红细胞的体内试验方法均可用于检测染色体断裂剂。由于细胞分裂后期的一个或多个染色体相对滞后也能形成微核，因此微核检测方法也能检测一些非整倍体诱导剂（注释8）。大鼠和小鼠均适用于骨髓微核试验。微核也可通过小鼠外周血中未成熟红细胞（如嗜多染红细胞）或大鼠血液新生网织红细胞测定（注释9）。同样，也可使用已证明了对检测断裂剂/非整倍体诱导剂具有足够灵敏度、来源于其他种属动物的骨髓或外周血的未成熟红细胞。除人工镜检方法外，若方法学经过了充分验证，也可采用自动化分析系统（如图像分析系统和流式细胞术）对微核进行检测。啮齿类动物给药后，取外周血淋巴细胞进行体外培养，也可用于分析染色体畸变（注释10）。2.2 其他体内遗传毒性试验第二项体内试验可作为第二种标准试验组合的一部分，并可用作追加试验以在评价体外或体内试验结果时提高证据权重（注释11）。体内组织和试验的选择应根据多种因素来确定，例如受试物可能的作用机制、体内代谢特征或者被认为是相关的暴露组织等信息。由于肝脏的暴露和代谢能力，肝脏是代表性的首选组织。第二种体内试验经常以评价DNA损伤为终点指标作为替代终点。目前，已有大量应用经验的试验包括：DNA链断裂试验如单细胞凝胶电泳试验（彗星试验）和碱洗脱试验、转基因小鼠体内突变试验、DNA共价结合试验。2.3 体内试验的给药途径一般情况下，给药途径应与临床拟用途径一致。若不一致，应说明理由。但是，为获得全身暴露，在适当时可进行调整，如局部给药的受试物。2.4 体内试验啮齿类动物性别的选用短期给药（通常是给药1～3次）的体内遗传毒性试验一般可单用雄性动物。若已有的毒性、代谢或暴露资料提示在所用动物种属上存在毒理学意义的性别差异，则应采用两种性别的动物。当遗传毒性试验整合在重复给药毒性试验中时，应对两种性别动物进行采样，如果毒性、代谢方面没有明显性别差异，可仅对单一性别进行评价。如果受试物拟专用于单一性别，可选用相应性别的动物进行试验。2.5 体内试验的剂量选择（注释7）通常应对有代表性的三个剂量水平进行分析检测。对于短期试验（通常是给药1～3次），推荐的最高剂量是：限度剂量2000mg/kg（若可耐受），或最大耐受量（为产生毒性的剂量，而基于同样的给药方案，比该剂量稍高即预期会出现死亡）。剂量选择时还应考虑骨髓红细胞生成的抑制。最高剂量之下的其他剂量一般剂量间距为约2～3倍。对于多次给药试验，分为两种情况：（1）当采用标准试验组合一时，若该试验整合在重复给药毒性试验中，如果该重复给药毒性试验符合支持人体临床试验的一个充分试验的标准，则通常认为对于遗传毒性评价剂量是合适的，该原则适用于体外哺乳动物细胞试验结果为阴性或不相关的阳性时。（2）当进行追加试验或当采用标准组合二时，应对多种因素进行评价以确定高剂量是否适合用于遗传毒性评价。毒性试验（尤其是大鼠试验）的高剂量需满足以下任何一条标准，才可用于遗传毒性评价：①最大给药量，基于受试物在溶剂中的理化性质。②对于给药14天或更长时间的试验，如果能耐受，限度剂量为1000 mg/kg。③最大可能暴露量，通过达到暴露峰值/稳态或受试物的蓄积来证明。若受试物的暴露量随给药时间增加而明显减少（如比起始暴露量减少≥50%），则不宜采用多次给药试验。④高剂量≥急性给药试验所采用高剂量的50%，即接近最小致死量。仅基于无毒性的暴露范围（高于临床暴露量的若干倍）来选择高剂量，不是充分合理的。对于具有血液或骨髓毒性的受试物，进行遗传毒性评价的剂量应在具有严重红细胞系毒性（如具有明显的嗜多染红细胞或网织红细胞抑制）的高剂量之下、间距不超过约2倍。试验的阶段性细菌回复突变试验可支持单次给药的临床试验；为支持多次给药的临床试验，应采用哺乳动物细胞进行一项评估染色体损伤试验；在Ⅱ期临床试验开始前完成完整的遗传毒性标准试验组合。但是，为了减少药物开发风险、保护受试者安全，建议在临床试验开始前完成遗传毒性标准试验组合。试验结果评价与追加研究策略遗传毒性研究是药物安全性评价与药物整体开发进程的一个重要组成部分，其最终目的在于预测受试物潜在的致癌性或其他遗传危害性。试验结果的分析与评价是试验的必要组成部分，应对研究结果进行科学和全面的分析与评价。在对遗传毒性试验结果进行评价时，应结合受试物的药学特点、药效学、药代动力学和其他毒理学研究的结果等信息进行综合分析。试验结果的评价最终应落实到临床试验受试者范围限定、风险效益评估以及必要防治措施的制定和应用上。遗传毒性试验组合检测的是主要通过直接的遗传损伤机制的致癌物质（如绝大多数已知的人类致癌剂），不适用于检测非遗传毒性致癌剂。每种试验系统均可能产生假阴性或假阳性结果。一些试验条件，如有限的体外代谢活化能力，可能导致体外试验出现假阴性结果。试验组合方法的设计是为了减少具有潜在遗传毒性的受试物产生假阴性结果的风险。另一方面，任何一项遗传毒性试验中的阳性结果并不一定能说明受试物对人类真正具有遗传毒性或致癌性的危险。在对体内外试验结果进行评价时，对阳性或阴性的结果均应予以充分考虑，尤其是在有疑问时。评价受试物的潜在遗传毒性时，应全面考虑各项试验结果、体内和体外试验方法的内在价值及其局限性，进行综合分析与评价。（一）体外试验结果的评价1.体外试验阳性结果1.1 体外细菌回复突变试验阳性结果的评价由于细菌回复突变试验的阳性结果提示DNA反应性，为评估对患者用药的潜在风险，需进行充分的追加试验以评价体内致突变和潜在致癌性，除非通过恰当的风险获益分析证明是合理的。细菌回复突变试验出现阳性结果，应考虑受试物的纯度，以确定阳性结果是否污染物所致。氨基酸（组氨酸或色氨酸）污染可能导致菌落数的升高而出现假阳性结果，因此细菌回复突变试验不适合检测可能会降解的肽类。一些特殊情况下，细菌回复突变试验阳性结果并不提示对人体有遗传毒性潜力，例如当发生细菌特异性代谢时（如通过细菌硝基还原酶活化）。1.2 体外哺乳动物细胞试验阳性结果的评价对于体外哺乳动物细胞试验阳性结果，应采用下述的证据权重法进行分析，必要时进行追加试验。例如（包括但不限于）：①阳性结果是否归因于体内不存在的条件（如pH值、渗透压、沉淀物）；②阳性结果仅发生于产生最高细胞毒性的浓度：在小鼠淋巴瘤细胞试验中阳性结果发生于相对总细胞生长率（Relative total growth，RTG）减少≥80%时；在体外细胞遗传学试验中阳性结果发生于细胞生长抑制≥50%时。对于以上情况，如果应用证据权重法分析提示缺乏潜在遗传毒性，可采用标准试验组合一，即一个体内试验即可。2.体外试验阴性结果对于体外试验阴性结果，在一些特殊情况下需考虑进行进一步的试验，例如（包括但不限于）：受试物的化学结构或已知代谢特征提示标准的体外代谢活化技术（如啮齿类动物肝脏S9）可能不适用；受试物的化学结构或已知活性提示采用其他试验方法或系统更合适。（二）体内试验结果的评价与体外试验相比，体内试验方法具有考虑到与人体应用相关的吸收、分布、排泄的优点，而且体内代谢相对于体外试验中的代谢系统更具有相关性。因此，体内试验在遗传毒性试验中具有更重要的意义。如果体外与体内试验的结果不一致，对其结果差异应采用具体问题具体分析的原则进行分析与评价，如代谢差异、受试物在体内快速和高效排泄等。1.体内试验阴性结果体内试验结果的意义与受试物在靶组织（注释5）中是否有足够的暴露直接相关，尤其是当体外试验为确定的阳性结果而体内试验结果为阴性时，或者是当未进行体外哺乳动物细胞试验时更为重要。受试物在靶组织中具有足够暴露的证据包括可疑组织的毒性，或下述的毒代动力学资料。如果受试物在靶组织中无法达到足够的暴露量，则常规的体内遗传毒性试验的意义较小。对于体外遗传毒性试验结果为阳性（或未进行）而体内试验结果为阴性的受试物，应采用以下任何一种方法来反映受试物在体内/靶组织的暴露水平：（1）细胞毒性对于细胞遗传学试验，可通过微核试验中各剂量组和各采样时间点所用组织（骨髓或外周血）中未成熟红细胞数与红细胞总数的比例的显著变化，或通过染色体畸变试验中有丝分裂指数的显著降低，来间接反映受试物的暴露水平。对其他体内遗传毒性试验，可通过肝脏或组织的毒性（如通过组织病理学检查或血液生化学指标）来间接反映受试物的暴露水平。（2）暴露通过测定血液或血浆中的受试物相关物质水平来反映受试物的体内暴露（注释12）；直接测定靶组织中的受试物相关物质；或通过放射自显影检测组织暴露水平。体内暴露的评估应采用与遗传毒性试验相同的动物种属、品系和给药途径，在最高剂量或其他相关剂量中进行。如果全身暴露与预期的临床暴露相似或更低，提示可能需要采用替代的方法，如采用不同的给药途径、采用具有更高暴露的不同种属、采用不同的组织或试验。若体外试验未显示受试物有潜在遗传毒性，可采用上述任何一种方法，也可用啮齿类动物吸收、分布、代谢和排泄试验结果来确定体内暴露水平。2.体内试验阳性结果体内遗传毒性试验也可能出现假阳性结果，例如：未给予任何遗传毒性物质，但由于干扰了红细胞生成而导致微核升高；DNA加合物数据应根据内源性加合物的已知背景水平进行解释分析；与毒性相关的间接作用可能影响DNA链断裂试验（如彗星试验和碱洗脱试验）的结果。因此评价遗传毒性数据时应考虑所有的毒理学和血液学发现（注释13）。与毒理学改变相关的间接作用可能有一个安全范围，且可能不具有临床相关性。（三）阳性结果追加研究策略1.对体外哺乳动物细胞试验阳性结果的追加以下讨论基于细菌回复突变试验结果为阴性。1.1 追加机制/体内试验体外哺乳动物细胞试验为阳性结果且无充分证据以排除生物学相关性时，建议进行追加试验以提供试验证据。可进行附加的体外试验或进行两种合适的体内试验，具体如下：（1）进行附加的体外试验。体外试验可为阳性结果缺乏生物学相关性提供机制信息，如小鼠淋巴瘤细胞试验中诱导染色体畸变或突变的受试物不是DNA损伤性物质的证据（如：除细菌回复突变试验外的其他突变/DNA损伤试验结果为阴性；化学结构上的考虑），或者体内可能不相关或可能具有阈值的间接机制的证据（如抑制DNA合成、仅在高浓度时产生活性氧簇等）。体外微核试验阳性结果的追加试验也可采用类似的试验，或者证据还可包含提示染色体丢失/非整倍体的已知机制、着丝点染色试验（注释14）提示染色体丢失。多倍体是体外染色体畸变试验中的一种常见现象。虽然非整倍体剂可诱导多倍体产生，但仅多倍体产生并不能提示受试物具有诱导非整倍体的潜力，而可能仅提示细胞周期紊乱；多倍体也常常与细胞毒性的升高有关。如果在体外试验中仅见多倍体，而未见结构上的染色体断裂，一个确保具有合适暴露的体内微核试验的阴性结果，通常可提供不具有潜在非整倍体诱导作用的充分证据。如果上述机制信息和证据权重分析支持受试物不具有相关的遗传毒性，仅需要一个具有合适暴露证据的体内试验，以确定受试物不具有遗传毒性作用。通常采用体内细胞遗传学试验，而且，当对潜在染色体丢失进行追加研究时要求进行体内微核试验。（2）进行两项合适的体内试验，通常采用不同的组织，并有支持暴露的证据。如果无充分的证据或机制信息以排除相关的潜在遗传毒性，通常要求进行两项体内试验，需要采用合适的终点指标和合适的组织（通常是两种不同组织），且应在体内获得充分的暴露。终点指标充分合理并且证明有暴露的合适的体内试验的阴性结果，足以证明受试物不具有遗传毒性风险。1.2 依赖于S9的体外试验阳性结果的追加当阳性结果仅见于S9代谢活化条件下，首先应确认是否是代谢活化的原因而非其他一些不同条件（如，与非代谢活化培养条件下的≥10%血清浓度相比，S9混合物中血清浓度低或无血清）。因此追加策略的目的是确定体外结果与体内条件的相关性，通常采用肝脏体内试验（注释15）。2.对体内微核试验阳性结果的追加若体内微核升高，应对所有的毒理学资料进行评价，以确定是否是由于非遗传毒性作用所致或非遗传毒性作用是其中的一个作用因素。如果怀疑存在干扰红细胞生成作用或生理学的非特异性因素（如体温偏低或过高），进行一项体内染色体畸变试验更为合适。如果怀疑一个升高的结果，应进行研究以证明该升高是否是由于染色体丢失或染色体断裂所致（注释14）。有证据显示非整倍体诱导作用，如纺锤体抑制剂，具有非线性剂量反应关系。因此，可能可确定该作用是否有阈值暴露（低于该暴露下预期不会有染色体丢失），以及确定与临床暴露比较是否存在合适的安全范围。总之，受试物潜在遗传毒性的评价应考虑所有结果，并认识到体外与体内试验各自的内在价值及局限性。（四）与致癌性试验的肿瘤发现有关的追加遗传毒性试验在遗传毒性标准试验组合中呈阴性结果，但在致癌性试验中显示肿瘤发生率升高，而且无充分证据确定是非遗传毒性作用机制的受试物，应在合适的模型上进行附加的遗传毒性试验。为了帮助了解作用方式，附加试验可包括改变体外试验的代谢活化条件，或包括测定肿瘤诱导靶器官遗传学损伤的体内试验，如DNA链断裂试验（如彗星试验或碱洗脱试验）、DNA加合试验（如通过32P-后标记）、转基因突变诱导试验，或肿瘤相关基因遗传学改变的分子特征性分析。（五）综合分析与评价当遗传毒性试验结果为阳性时，对进入临床试验是否安全，应考虑所有的安全性资料，包括对所有遗传毒性资料的全面评价，以及拟进行的临床试验的性质。对于遗传毒性试验出现阳性结果、但不直接与DNA发生作用的受试物，不全都会带来明显的体内给药的风险。因此，当遗传毒性试验出现阳性结果时，建议提供有关遗传毒性机制的证据以及这种机制与预期体内暴露的相关性，或者通过试验排除为直接与DNA作用的机制，如证明受试物不使DNA烷化或DNA链断裂，并提供未观察到遗传毒性的剂量水平。若确认受试物可直接损伤DNA，在极特殊情况下，可能会被允许用于危及生命的疾病（如晚期癌症），但不能在健康受试者中使用。注释【注释1】：尽管只有少数但仍有一定数量的遗传毒性致癌剂能被骨髓染色体损伤试验检出，而在标准组合所列出的体外试验中却得到阴性、弱阳性或相互矛盾的结果。丙卡巴肼、对苯二酚、氨基甲酸乙酯、苯等致癌剂即为此类物质。【注释2】：虽然两种标准试验组合同等适合，但是某些受试物的特异性信息提示应首选其中一种组合。例如，如果动物上的全身暴露与预期临床暴露相当或更低，应进行体外试验即组合一；当预期在肝脏产生短暂的活性代谢物时，推荐采用包含有一项肝脏体内试验的组合二。【注释3】：某些具有遗传毒性警示结构的化合物被认为与致癌和/或致突变潜力有因果关系。警示结构包括烷化亲电子中心、不稳定过氧化物、芳香胺、偶氮结构、N-亚硝基基团、芳香硝基基团。【注释4】：对有特殊警示结构的几类化合物，如含有偶氮基团的分子、糖苷类、活化需要还原硝基的化合物如硝基咪唑类、代谢活化需要不同的啮齿类动物S9的化合物如非那西汀，为最适合检测遗传毒性，采用特殊的方案调整或附加试验是非常重要的。【注释5】：靶组织：此处特指体内试验的检测目标组织，如小鼠骨髓微核试验中的骨髓。【注释6】：采用皮肤和结肠进行诱导微核的体内试验已有一些经验，而且在这些组织中进行DNA损伤试验也是一种合适的替代方法。【注释7】：此处所指最高浓度的确定主要针对化学药物，因中药、天然药物所包含范围宽且情况复杂，在合适时可参考化学药物的最高浓度的确定原则进行设计，不合适时需根据具体情况进行合理的设计。【注释8】：虽然微核的形成源于受试物与纺锤体相互作用导致整条染色体分裂的滞后，但微核试验无法检测所有非整倍体诱导剂。更特异的非整倍体诱导检测方法之一是采用快速、灵敏的技术分析个体（啮齿类动物）分裂间期核中的染色体，如荧光原位杂交（FISH）方法。【注释9】：原则上，造血细胞的微核可采用任何动物种属的骨髓，以及循环系统中的含微核红细胞不会被脾脏清除的动物种属的外周血进行评价。在小鼠上，微核可通过血中的嗜多染红细胞进行测定，当小鼠持续给药约4周或更长时间时也可采用成熟红细胞进行测定。虽然大鼠可快速清除循环中的含微核红细胞，但是大鼠血液网织红细胞中可检测到由一系列断裂剂和非整倍体剂诱导的微核，因此大鼠外周血也可用于微核分析，但所采用方法学需确保能分析新生网织红细胞且方法学经过充分验证。【注释10】：在某些情况下，动物单次或多次给予受试物后取外周血淋巴细胞进行培养，可检测中期相染色体畸变，就如同可采用骨髓中期相细胞一样。因为循环中的淋巴细胞是不可复制的，所以该试验系统无法检测需要通过细胞复制才能显示出遗传毒性作用的受试物（如某些核苷类似物）。由于一些淋巴细胞寿命相对较长，原则上它们具有体内蓄积未修复DNA损伤的潜力，这使得细胞在体外受到刺激进行分裂时畸变率升高。体内淋巴细胞试验可用于断裂剂指征的追加研究，但是，对于造血细胞微核试验，采用其他组织（如肝脏）的试验通常可补充提供更多信息量，因为肝脏中药物和代谢物的暴露通常更高。【注释11】：在标准试验组合中包含第二项体内试验，是为了通过一种对受试物和/或其代谢物具有良好暴露的组织来确证受试物不具有遗传毒性。另外，一小部分被认为有遗传毒性的致癌剂在肝脏试验中结果为阳性，但在体内骨髓细胞遗传学试验中结果却为阴性。这些案例很可能反映了缺乏合适的代谢活性或缺乏活性中间体传递至骨髓造血细胞。【注释12】：骨髓是血液灌流性良好的组织，故血液或血浆中受试物相关物质的水平与骨髓中水平相似。无论何种给药途径，肝脏对于有全身暴露的受试物预期会有暴露。【注释13】：微核升高可发生于未给予任何遗传毒性物质时，而是由于红细胞生成的干扰（如再生障碍性贫血、髓外造血作用）、应激、体温偏低或体温过高所致。脾脏功能的改变可影响含微核细胞从血液中的清除，可导致循环中的含微核红细胞数量轻微升高。【注释14】：确定微核诱导是否主要由于染色体丢失或染色体断裂所致，试验应包括对体外或体内微核染色以确定着丝粒是否存在，例如，在着丝粒位置的DNA序列采用有探针的荧光原位杂交（FISH），或者采用抗着丝粒蛋白的标记抗体。如果大多数诱导的微核是着丝粒阳性，这提示有染色体丢失。但是，需要注意，强微管毒物如秋水仙碱和长春碱为一种特殊情况，它们虽然不会产生100%着丝粒阳性的微核，但典型的着丝粒阳性微核超过70%～80%，而在风险评估中却被公认为主要是非整倍体诱导剂。一种替代的方法是进行一项体外或体内中期相结构畸变试验，如果该试验结果是阴性，则提示微核诱导与染色体丢失有关。【注释15】：标准诱导的S9混合物比人S9具有更高的活化能力，并且缺乏Ⅱ相解毒能力，除非添加特异性的辅助因子。此外，在体外测试底物浓度很高时可发生非特异的代谢活化作用。采用人S9或其他人类相关的代谢活化系统进行遗传毒性试验是有用的。分析遗传毒性试验中培养物的代谢产物特性，与非临床种属（非诱导的微粒体或肝细胞，或体内）或来自于人体样本的已知代谢产物特性进行比较，也可有助于确定试验结果的相关性。而且，追加试验通常采用肝脏的体内试验。在S9存在条件下体外试验得到阳性结果的受试物，在体内可能不诱导遗传毒性，因为不形成代谢产物或形成极微量，或被代谢性解毒或快速排泄，这种情况可提示缺乏体内风险。【注释16】：小鼠淋巴瘤细胞试验为一种体外哺乳动物细胞Tk基因突变试验。除小鼠淋巴瘤L5178Y细胞外，人淋巴母细胞TK6细胞也可用于检测Tk基因位点突变。目前，TK6试验已纳入OECD指导原则，但是，对于该试验，目前积累的试验经验较少，且未进行充分的方法学验证。在该试验经过充分的方法学验证后才可用于药物注册目的。遗传毒性试验方法推荐的标准试验组合中的遗传毒性试验方法：注：以下方法中所提供的最高浓度和最高剂量设计原则主要针对化学药物，中药、天然药物由于情况复杂，应综合考虑多方面因素，不能简单套用该原则，试验时应根据具体情况进行合理的设计。（一）细菌回复突变试验（Bacterial reverse mutation test）1.菌株组氨酸营养缺陷型鼠伤寒沙门氏菌和/或色氨酸营养缺陷型埃希氏大肠杆菌，至少应包含下述五种菌株组合（除特殊说明外，均为鼠伤寒沙门氏菌）：（1）TA98；（2）TA100；（3）TA1535；（4）TA1537或TA97或TA97a；（5）TA102或大肠埃希杆菌WP2 uvrA或大肠埃希杆菌WP2 uvrA（pKM101）。菌株特性鉴定需符合要求，－80℃或液氮冻存备用。2.浓度至少应包含5个可用于结果分析的浓度。最高浓度主要取决于受试物对细菌的毒性和/或溶解度：（1）对于可溶性的无细菌毒性的受试物，推荐的最高浓度一般为5mg/皿（液体受试物为5µl/皿）。（2）对于可溶性的有细菌毒性的受试物，应根据细菌毒性情况确定最高浓度（详见正文1.2.2）。（3）对于难溶性的受试物，若未观察到细菌毒性，一般以最小沉淀浓度为评价的最高浓度；若观察到浓度相关性的细菌毒性或诱变性，则要求检测多个产生沉淀的浓度（详见正文1.2.3）。3.代谢活化一般采用诱导剂，如Aroclor 1254或苯巴比妥和β-萘黄酮联合诱导处理后的哺乳动物肝脏微粒体酶（S9）进行体外代谢活化试验，即在加S9和不加S9平行条件下测试。S9在S9混合液中的浓度一般为5%～30%（v/v）。4.对照代谢活化或非代谢活化条件下，均应设立平行阴性（空白对照和/或溶剂对照）和阳性对照组。阳性对照物应为已知的菌株特异性的阳性致突变剂。5.方法可采用标准平板掺入法或预培养法，受试物处理后48～72小时观察结果。每一浓度至少平行三皿。6.结果判定结果中应描述各浓度组细菌毒性大小和沉淀情况；结果表示为每皿的回复突变菌落数，并计算各组的均值和标准差。至少在一个菌株上，在有或无代谢活化条件下，受试物所诱发的回复突变菌落数出现浓度依赖性的增加和/或在一个或多个浓度组上出现可重复性的增加，可判定为阳性结果。结果判定时应首先考虑试验结果的生物学意义，统计学分析有助于结果的评价。（二）体外哺乳动物细胞染色体畸变试验（In vitro mammalian chromosomal aberration test）1.细胞可采用哺乳动物或人的细胞进行试验，如CHL细胞、CHO细胞、TK6细胞、人外周血淋巴细胞等，细胞系需定期检查核型和有无支原体污染等。-80℃或液氮冻存备用。2.浓度至少应包含3个可用于结果分析的浓度。最高浓度主要取决于受试物的细胞毒性和/或溶解度。（1）对于可溶性的无细胞毒性的受试物，推荐的最高浓度一般为1 mM或0.5 mg/ml（选用较低者）。（2）对于可溶性的有细胞毒性的受试物，应根据细胞毒性大小确定最高浓度。最高浓度所产生的细胞毒性应达到约50%但无需超过50%。对于哺乳动物细胞，可采用相对群体倍增数（Relative population doubling，RPD）和相对细胞增长数（Relative increase in cell count，RICC）的减少来反映细胞毒性；对培养的人外周血淋巴细胞，可通过有丝分裂指数（Mitotic Index，MI）的降低来反映细胞毒性。（3）对于难溶性的受试物，一般采用最小沉淀浓度为最高浓度。对最高浓度之下的其他浓度，对于细胞毒性小或无的受试物，一般浓度间距为2～3倍；对于有细胞毒性的受试物，所选择的浓度应覆盖产生细胞毒性的范围，并且包括具有中度或少/无细胞毒性的浓度；在一些情况下，如具有较陡峭的浓度反应关系，则可适当缩小浓度间距或将检测的浓度加至3个以上。每一浓度一般平行2皿；若能获得足够细胞数，可用单皿，尤其当超过3个检测浓度时。3.代谢活化一般采用诱导剂，如Aroclor 1254或苯巴比妥和β-萘黄酮联合诱导处理后的哺乳动物肝脏微粒体酶（S9）进行体外代谢活化试验，即在加S9和不加S9平行条件下测试。S9在试验介质中的终浓度一般为1%～10%（v/v）。4.对照代谢活化或非代谢活化条件下，均应设立平行阴性（空白对照和/或溶剂对照）和阳性对照组。阳性对照物应为已知的阳性诱变剂。5.方法处理及细胞收获时间：在代谢活化或非代谢活化条件下，受试物和细胞作用3～6小时，在1.5个正常细胞周期时收获细胞；在非代谢活化条件下，受试物和细胞还应持续作用至1.5个正常细胞周期时收获细胞。对于某些受试物，与细胞接触时间/收获细胞时间可能要大于1.5个正常细胞周期。读片分析：一般油镜下每种浓度至少观察300个分散良好的分裂中期相细胞，若观察到大量染色体畸变细胞，分析细胞数可相应减少。应分别记录各组含有结构畸变染色体的细胞数和畸变类型，染色单体型与染色体型畸变应分别记录并记录亚型（断裂、交换）。裂隙应单独记录，但不计入畸变率中。同时应单独记录多倍体和内复制等数目畸变，但不计入畸变率中。6.结果判定结果中应描述各浓度组细胞毒性大小和沉淀情况；结果表示为染色体结构畸变细胞的百分率。受试物在任一处理条件下至少一个浓度时染色体畸变率显著升高，升高具有浓度依赖性，且畸变率在阴性对照历史范围之外，可判定为阳性结果。结果判定时应首先考虑试验结果的生物学意义，统计学分析有助于结果的评价。如果结果不是明确的阳性或阴性结果，或者为了帮助确定结果的生物学意义，应对数据进行专家同行评议和/或进一步研究。分析更多的细胞（当可行时），或者通过改变试验条件进行重复试验（如改变试验浓度间距、改变代谢活化条件等）可能是有用的。应单独记录多倍体和内复制的发生率。多倍体数目的增加提示受试物可能会抑制有丝分裂或诱导染色体数目畸变。出现染色体内复制的细胞数增多提示受试物可能会影响细胞周期。（三）体外小鼠淋巴瘤细胞Tk基因突变试验（In vitro  mouse lymphoma cell Tk gene mutation assay，MLA）1.细胞通常采用小鼠淋巴瘤L5178Y tk+/- -3.7.2 C细胞（注释16）。细胞系需定期检查核型或有无支原体污染等，必要时进行自发突变细胞的清除。-80℃或液氮冻存备用。2.浓度至少应包含4个可用于结果分析的浓度。最高浓度主要取决于受试物的细胞毒性和/或溶解度：（1）对于可溶性的无细胞毒性的受试物，推荐的最高浓度一般为1mM或0.5mg/ml （选用较低者）。（2）对于可溶性的有细胞毒性的受试物，应根据细胞毒性大小确定最高浓度，最高浓度应能产生80%～90%的细胞毒性。通过相对总生长率（Relative total growth，RTG）来反映细胞毒性。（3）对于难溶性的受试物，若无细胞毒性，一般采用最小沉淀浓度作为最高浓度；即使细胞毒性出现在最小沉淀浓度以上，一般建议只检测1个沉淀浓度，因为沉淀可能产生干扰。对最高浓度之下的其他浓度，对于细胞毒性小或无的受试物，一般浓度间距为2～3倍；对于有细胞毒性的受试物，所选择的浓度应覆盖产生细胞毒性的范围，并且包括具有中度或少/无细胞毒性的浓度；在一些情况下，如受试物具有较陡峭的浓度反应关系，可适当缩小浓度间距或将检测的浓度增加至4个以上。3.代谢活化一般采用诱导剂，如Aroclor 1254或苯巴比妥和β-萘黄酮联合诱导处理后的哺乳动物肝脏微粒体酶（S9）进行体外代谢活化试验，即在加S9和不加S9平行条件下测试。S9在试验介质中的终浓度一般为1%～10%（v/v）。4.对照代谢活化或非代谢活化条件下，均应设立平行阴性（空白对照和/或溶剂对照）和阳性对照组。阳性对照物应为已知的阳性诱变剂。5.方法一般采用微孔法或软琼脂法进行试验。以微孔法为例：处理时间：在代谢活化或非代谢活化条件下，一般受试物与细胞作用3～4小时。如果作用3～4小时后结果为阴性，还需进行在非代谢活化条件下作用24小时的附加试验。突变表达期：受试物与细胞作用结束后，去除受试物，将细胞重悬于培养液中，一般L5178Y细胞的突变表达期为2天。分别在处理结束后及表达期结束后测定平板接种效率以评价细胞毒性。突变率测定：表达期结束后，将细胞接种于含有突变选择剂三氟胸苷（TFT）的96孔板中进行TFT抗性突变集落的测定。如果受试物出现阳性结果，需要对至少一个受试物浓度组（一般为最高浓度）和阴性、阳性对照组分别记录含有大、小集落的孔数；如果为阴性结果，仅需要对阴性和阳性对照组分别记录含有大、小集落的孔数。6.结果判定结果中应描述各浓度组细胞毒性大小和沉淀情况；结果表示为各浓度组的突变率（Mutant frequency，MF）。MLA试验中阴性对照组和阳性对照组必须符合以下标准才可判定MLA试验成立。阳性对照组的突变率、突变选择时的克隆率、悬液增长应满足以下条件：表1 MLA成立的阴性对照组标准参数软琼脂法微孔法突变率（MF）（35～140）×10-6（50～170）×10-6克隆率65%～120%65%～120%悬液增长8～32倍（3～4小时处理）32～180倍（24小时处理）8～32倍（3～4小时处理）32～180倍（24小时处理）阳性对照组应满足以下条件之一：（1）总突变率绝对增加，比自发背景突变率增加[即诱导MF（IMF）]至少300×10-6，至少40%的IMF应该在小菌落MF中。（2）与同期阴性对照组比较，小菌落MF增加至少150×10-6。MLA中突变率的增加具有生物学意义的评价标准为，在任何一种试验条件下，如果一个或多个浓度组的突变率增加超过总评价因子（Global evaluation factor，GEF）（GEF为诱导突变率，即高于同期阴性对照的突变率的增加。软琼脂法GEF为90×10-6，微孔法为126×10-6），且呈浓度依赖性，判定为阳性结果。如果在所有试验条件下各浓度组的突变率无浓度依赖性增加或突变率的增加未超过GEF，判定为阴性结果。如果出现明确的阳性或阴性结果，不需要重复试验。如果结果不是明确的阳性或阴性结果，或者为了帮助确定结果的生物学意义，应对数据进行专家同行评议和/或进一步研究。在重复试验中改变试验条件（如改变试验浓度间距、改变代谢活化条件和处理时间等）可能是有用的。（四）体外哺乳动物细胞微核试验（In vitro mammalian cell micronucleus test）1.细胞可采用哺乳动物或人的细胞进行试验，如CHL细胞、CHO细胞、TK6细胞、人外周血淋巴细胞等。细胞系需定期检查核型和有无支原体污染等。-80℃或液氮冻存备用。2.浓度至少应包含3个可用于结果分析的浓度。最高浓度主要取决于受试物的细胞毒性和/或溶解度：（1）对于可溶性的无细胞毒性的受试物，推荐的最高浓度一般为1mM或0.5mg/ml （选用较低者）。（2）对于可溶性的有细胞毒性的受试物，应根据细胞毒性大小确定最高浓度。最高浓度所产生的细胞毒性应达到约50%。无细胞松弛素B（Cyto B）时通过RICC、RPD的减少来反映细胞毒性，有细胞松弛素B时通过分裂阻滞增殖指数（cytokinesis-block proliferation index，CBPI）或复制指数（Replication index，RI）来反映细胞毒性。（3）对于难溶性的受试物，若无细胞毒性，一般采用最小沉淀浓度为最高浓度；若细胞毒性出现在最小沉淀浓度以上，一般建议只检测1个沉淀浓度，因为沉淀可能产生干扰。对最高浓度之下的其他浓度，对于细胞毒性小或无的受试物，一般浓度间距为2～3倍；对于有细胞毒性的受试物，所选择的浓度应覆盖产生细胞毒性的范围，并且包括具有中度或少/无细胞毒性的浓度；在一些情况下，如受试物具有较陡峭的浓度反应关系，则可适当缩小浓度间距或将检测的浓度增加至3个以上。每一浓度一般平行2皿；若能获得足够细胞数，可用单皿，尤其当受试物超过3个检测浓度。3.代谢活化一般采用诱导剂，如Aroclor 1254或苯巴比妥和β-萘黄酮联合诱导处理后的哺乳动物肝脏微粒体酶（S9）进行体外代谢活化试验，即在加S9和不加S9平行条件下测试。S9在试验介质中的终浓度一般为1%～10%（v/v）。4.对照代谢活化或非代谢活化条件下，均应设立平行阴性（空白对照和/或溶剂对照）和阳性对照组。阳性对照物应为已知的阳性诱变剂。5.方法处理及细胞收获时间：在代谢或非代谢活化的情况下，受试物和细胞作用3～6小时，加或不加细胞松弛素B（Cyto B）在1.5～2.0个正常细胞周期时收获细胞；在非代谢活化条件下，加或不加Cyto B受试物和细胞还应持续作用至1.5～2.0个正常细胞周期时收获细胞。对于某些受试物，与细胞接触时间/收获细胞时间可能要大于1.5个正常细胞周期。读片分析：每个浓度应至少观察2000个双核细胞（加Cyto B）或单核细胞（不加Cyto B），分析微核率。应至少观察500个细胞，加Cyto B时通过CBPI或RI来评价其细胞毒性，不加Cyto B时通过RICC或RPD来评价细胞毒性。也可采用已经过充分验证的自动化分析系统（如流式细胞术、激光扫描细胞计数仪、图像分析系统）对微核进行检测。6.结果判定结果中应描述各浓度组细胞毒性大小和沉淀情况；结果表示为微核率。受试物在任一处理条件下一个或多个浓度组微核率显著增加，增加具有浓度依赖性，且微核率在阴性对照历史范围之外，可判定为阳性结果。结果判定时应首先考虑试验结果的生物学意义，统计学分析有助于结果的评价。如果结果不是明确的阳性或阴性结果，或者为了帮助确定结果的生物学意义，应对数据进行专家同行评议和/或进一步研究。分析更多的细胞（当可行时），或者通过改变试验条件进行重复试验（如改变试验浓度间距、改变代谢活化条件等）可能是有用的。（五）哺乳动物体内微核试验（In vivo mammalian erythrocyte micronucleus test）1.动物骨髓试验通常采用小鼠和大鼠，如合适也可选用其他哺乳动物。微核也可通过小鼠外周血中未成熟（如嗜多染）红细胞或大鼠血液新生网织红细胞测定。由于大鼠脾脏可清除血液中的微核化红细胞，若采用大鼠外周血测定微核，需采用具有足够灵敏度的检测方法来测定新生网织红细胞中的微核。采用健康年轻性成熟动物，啮齿类动物给药起始年龄建议为6～10周龄。一般情况下雌性和雄性动物微核反应相似，故可采用一种性别动物，如雄性。若性别间存在明显的毒性或代谢方面的差异，则应采用两种性别的动物，每组可分析的动物数雌雄至少各5只。如果受试物拟专用于一种性别，可选用相应性别的动物进行试验。起始试验时，动物体重差异应在各性别平均体重的20%之内。2.剂量至少应设置3个剂量组。根据相关毒性试验或预试验的结果确定高剂量，高剂量应产生一定的毒性症状（如体重降低、造血系统细胞毒性）或骨髓毒性（如嗜多染红细胞占红细胞总数的比例降低超过50%）。如果受试物不引起毒性，给药时间<14天的推荐最高剂量为2000mg/kg/天，给药时间≥14天的推荐最高剂量为1000mg/kg/天。如果受试物能引起毒性，则最高剂量采用最大耐受量，且剂量应优选能覆盖产生最大毒性到少/无细胞毒性的剂量范围。当在所有剂量均观察到靶组织（骨髓）毒性时，建议在无毒性剂量下进一步研究。为更充分地研究剂量-反应曲线的形状可能需要增加剂量组。3.对照应设立平行阴性（空白对照和/或溶剂对照）和阳性对照组。阳性对照物应为已知的阳性诱变剂。4.方法给药方案：根据具体情况选择合适的给药方案，可采用单次给药（或一日内以不超过2～3小时间隔分多次给药）或多次给药（给药间隔为24 小时），首选多次给药。受试物的给药途径应尽可能与临床拟用途径相同，阴性对照物应与受试物给药途径一致，阳性对照物的给药途径可以不同于受试物。采样时间：采样时间根据靶组织中微核出现和消失的动力学特征确定。如果采用单次给药，应至少采样2次，骨髓采样时间应在给药后24～48小时内，外周血采样时间应在给药后36～72小时内。受试物第一个采样点应包括所有剂量组，第二个采样点可仅包括高剂量组。如果给药2次（约每24 小时给药一次），骨髓采样时间为末次给药后18～24 小时，外周血采样时间为末次药后36～48小时。如果给药3次及3次以上（约每24 小时给药一次），骨髓采样时间为末次给药24 小时内，外血周采样时间为末次给药后40小时内。读片分析：每只动物至少计数500个（骨髓）或2000个（外周血）红细胞以确定未成熟红细胞[嗜多染红细胞（PCE）或网织红细胞（RET）]占总红细胞[未成熟红细胞和正染红细胞（NCE）]的比例；每只动物至少计数4000个未成熟红细胞以测定未成熟红细胞的微核率。若阴性对照历史数据库的微核率背景值低于0.1%时，需要计数更多的未成熟红细胞。给药组未成熟红细胞占总红细胞的比例不应低于阴性/溶剂对照组的20%。如果给药时间在4周以上，可以直接计数4000个红细胞中的微核率。也可采用已经过充分验证的自动化分析系统（如图像分析系统、流式细胞术、激光扫描细胞计数仪）对微核进行检测。当计数CD71+未成熟细胞时，给药组未成熟细胞占总红细胞的比例不得低于阴性/溶剂对照组的5%。5.结果判定结果中应描述各剂量组的毒性大小，包括一般症状、死亡情况等，以及PCE/（PCE+NCE）或RET/（RET+NCE）的比例；结果表示为嗜多染细胞微核率（MNPCE）或网织红细胞微核率（MNRET）。受试物至少一个剂量组微核率显著升高，该增加在至少一个采样点具有剂量依赖性，且在阴性对照历史范围之外，可判定为阳性结果。如果结果不是明确的阳性或阴性，或者为了帮助确定结果的生物学意义（如弱的或边缘性增加），应对数据进行专家同行评议和/或进一步研究。有时候需要分析更多的细胞或者改变试验条件进行重复试验。（六）体内碱性彗星试验（In vivo mammalian alkaline comet assay）1.动物通常采用健康年轻性成熟的啮齿类动物，给药起始年龄6～10周龄。啮齿类动物种属选择应采用在毒性试验中使用的种属，致癌性试验中可致肿瘤的种属，或者与人类代谢最相关的种属。常采用大鼠，如果科学合理也可采用其他种属。彗星试验关于雌性动物的试验数据很少，参考其他体内遗传毒性试验，可采用一种性别进行试验，如雄性。若性别间存在明显的毒性或代谢方面的差异，则应采用两种性别的动物，可分析动物数每组雌雄至少各5只。如果受试物拟专用于一种性别，可采用相应性别的动物进行试验。2.剂量至少应设置3个剂量组。根据相关毒性试验或预试验的结果确定高剂量，可采用最大耐受量（MTD）、最大给药量、最大暴露量或限度剂量为最高剂量。对于低毒性化合物，给药时间<14天的推荐最高剂量为2000mg/kg/天，给药时间≥14天的推荐最高剂量为1000mg/kg/天。最高剂量之下的剂量以合适的剂量间距递减以研究剂量-反应关系，且剂量应优选能覆盖产生最大毒性到少/无毒性的剂量范围。当在所有剂量均观察到靶组织毒性，建议在无毒性剂量下进一步研究。为更充分地研究剂量-反应曲线的形状可能需要附加剂量组。3.对照应设立平行阴性（空白对照和/或溶剂对照）和阳性对照组。阳性对照物应为已知的阳性诱变剂。4.方法给药方案：可采用每天给药一次，给药2天或更多天。受试物的给药途径应尽可能与临床拟用途径相同，阴性对照物应与受试物给药途径一致，阳性对照物的给药途径可以不同于受试物。采样：采样时间对于彗星试验非常关键。采样时间由受试物达到靶组织中最大浓度所需的时间，以及诱导DNA链断裂但在这些断裂被清除、修复或细胞死亡之前来确定。彗星试验所能检测到的引起DNA断裂的一些损伤的持续时间可能非常短，因此，如果怀疑这种短暂的DNA损伤，应采取措施确保组织被尽早采样，以减少这种丢失。若可获得，应由药动学数据[如血浆或组织浓度达峰时间（Tmax）或多次给药后达到稳态浓度]来确定采样时间。若无药动学数据，采样时间在2次或更多次给药的末次给药后的2～6小时，或单次给药后的2～6小时和16～26小时两个时间点。若可获得，靶器官毒性作用表现也可用于选择合适的采样时间。应明确说明组织选择的合理性，组织选择应根据进行该项试验的理由、受试物已有的药动学（ADME）信息、遗传毒性、致癌性和其他毒性信息等来确定，考虑的重要因素包括受试物的给药途径、预测的组织分布和吸收、代谢的作用、可能的作用机制等。由于肝脏是外源性代谢的主要部位且常常高暴露于受试物和其代谢产物，在缺乏背景信息且未确定特殊关注组织的情况下，可选择肝脏作为研究组织。在一些情况下，可选择受试物直接接触部位进行研究。样品处理：样品处理过程极为关键，应严格控制试验条件，进行充分的方法学验证。取所选择组织，制备单细胞悬液，单细胞制备后尽快完成制片（理想的是1小时内）。所有玻片的裂解条件应保持恒定，低温（约2～8℃）避光裂解至少1小时（或过夜）。强碱条件下（pH≥13），解旋至少20分钟，在控制条件下进行电泳。电泳的电压应保持恒定，其他参数的变异应保持在狭窄和特定的范围内；解旋和电泳过程中应维持低温（通常为2～10℃）。读片分析：采用自动化或半自动化图像分析系统进行阅片，对彗星进行定量评价。细胞可分为三类：可评分细胞、不可评分细胞和刺猬样细胞（hedgehog）。对可评分细胞（具有清晰的头部和尾部，不干扰邻近细胞）的尾DNA百分率[% tail DNA，也称尾强度百分率（% tail intensity）]进行评价，来反映DNA链断裂。每个动物样本应至少对150个可评分细胞进行测定。刺猬样细胞是严重损伤的细胞，无法通过图像分析系统进行可靠测量，应单独评价刺猬样细胞。每个动物样本应至少对150个细胞进行观察并单独记录，计算刺猬样细胞百分率。5.结果判定结果中应描述各剂量组的毒性大小，包括一般症状，以及刺猬样细胞百分率。结果表示为各剂量组的尾DNA百分率（%tail DNA）（首选指标）、尾长或尾矩。除了遗传毒性外，靶组织毒性也可导致DNA迁移增加，故结果分析时需区分遗传毒性和细胞毒性。可通过组织病理学变化来反映细胞毒性，如炎症、细胞浸润、凋亡或坏死性变化与DNA迁移增加有关，血液生化学指标的改变（如AST、ALT等）也可提供细胞毒性的信息。受试物所诱发的尾DNA百分率与同期阴性对照组相比至少一个剂量组显著升高、升高具有剂量依赖性，且在阴性对照历史范围之外，可判定为阳性结果。如果结果不是明确的阳性或阴性，或者为了确定阳性结果的生物学意义，应进行专家同行评议和/或进一步研究，如分析更多的细胞（当可行时），或者采用优化的试验条件进行重复试验（如改变试验剂量间距、其他给药途径、其他采样时间或其他组织）。对于阴性结果，需提供支持靶组织暴露或毒性的直接或间接证据。为评价阳性或可疑结果的生物学意义，需提供靶组织细胞毒性信息。参考文献1.ICH. S2(R1)：Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use. 20112. ICH. M3 (R2): Non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals. 20093.FDA. Guidance for industry and review staff: Recommended approaches to integration of genetic toxicology study results. 20064.OECD. Guideline for testing of chemicals No.471： Bacterial reverse mutation test.19975.OECD. Guideline for testing of chemicals No.473：In Vitro mammalian chromosomal aberration test. 20166.OECD. Guideline for testing of chemicals No.474：Mammalian erythrocyte micronucleus test. 20167.OECD. Guideline for testing of chemicals No. 475：Mammalian bone marrow chromosome aberration test.  20168.OECD. Guideline for testing of chemicals No. 476: In vitro mammalian cell gene mutation tests using the Hprtand xprt genes. 20169.OECD. Guideline for testing of chemicals No. 487：In vitro mammalian cell micronucleus test. 201610.OECD. Guideline for testing of chemicals No. 488：Transgenic rodent somatic and germ cell gene mutation assays. 201311.OECD. Guideline for testing of chemicals No. 489：In vivo mammalian alkaline comet assay. 201612.OECD. Guideline for testing of chemicals No. 490：In vitro mammalian cell gene mutation tests using the thymidine kinase gene. 2016文章信息源于公众号药你懂点儿，登载该文章目的为更广泛的传递行业信息，不代表赞同其观点或对其真实性负责。文章版权归原作者及原出处所有，文章内容仅供参考。本网拥有对此声明的最终解释权，若无意侵犯版权，请联系小编删除。学如逆水行舟，不进则退；心似平原走马，易放难收。行舟Drug每日更新 欢迎订阅+医药大数据|行业动态|政策解读

Company to Feature 17 Company-Sponsored and Nine Collaborative Abstracts in Oncology and Hematology Hematology Presentations Include New Data from the Phase 3 and Continuation Trials Supporting the Recent U.S. FDA Approval of ADZYNMA (ADAMTS13, recombinant-krhn) Oncology Presentations Feature New Insights into Treatment of Newly Diagnosed Ph+ Acute Lymphoblastic Leukemia and Hodgkin Lymphoma OSAKA, Japan & CAMBRIDGE, Mass.--(BUSINESS WIRE)-- Takeda (TSE:4502/NYSE:TAK) today announced that it will present 17 company-sponsored abstracts at the 65th American Society of Hematology (ASH) Annual Meeting being held December 9-12, 2023 in San Diego. Takeda’s latest research focuses on improving treatment options for those living with hematologic diseases. Takeda’s presentations will include oral abstracts detailing new results from the first randomized, controlled, open-label, crossover Phase 3 trial in congenital thrombotic thrombocytopenic purpura (cTTP) as well as safety and efficacy data from the continuation trial. The totality of the evidence provided by an analysis of these trials supported the recent U.S. Food and Drug Administration (FDA) approval of ADZYNMA (ADAMTS13, recombinant-krhn) as the first and only FDA-approved therapy for the prophylactic and on-demand treatment of adult and pediatric patients with cTTP. Additional hematology oral sessions include topline Phase 1 data evaluating the safety and pharmacokinetics of investigational TAK-755 (recombinant ADAMTS13) in sickle cell disease and a meta-analysis of real-world evidence studies of the clinical outcomes for noninhibitor patients with hemophilia A. “Our aspiration is to deliver life-transforming medicines for difficult-to-diagnose, rare hematologic diseases with high unmet patient need,” said Björn Mellgård, M.D., Ph.D., vice president and global program lead of Rare Genetics and Hematology at Takeda. “Our continued research aims to improve long-term outcomes for patients suffering from these conditions. We look forward to presenting our latest trial results at ASH, a conference that cultivates an environment for scientific exchange that could one day lead to marked improvements in patient care.” Oncology presentations will include a subgroup analysis of the Phase 3 PhALLCON trial evaluating ICLUSIG® (ponatinib) versus imatinib plus reduced-intensity chemotherapy in adults with newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), a disease area for which there are currently no approved targeted treatments in the U.S. Additionally, the German Hodgkin Study Group will present data from the Phase 3 HD21 study comparing escalated doses of bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone (eBEACOPP) with ADCETRIS® (brentuximab vedotin) in combination with etoposide, cyclophosphamide, doxorubicin, dacarbazine and dexamethasone (BrECADD) in patients with newly diagnosed, advanced stage Hodgkin lymphoma. “We’re excited to share our latest oncology research at ASH this year, focusing on patients with difficult to treat blood cancers,” said Awny Farajallah, M.D., head of global medical affairs oncology at Takeda. “As part of our commitment to patients with limited or ineffective treatment options, we continue to evaluate new and expanded uses of our established medicines, including in lymphoma and leukemia, to enable even more patients to potentially benefit from these life-transforming therapies. We look forward to sharing these findings with the community.” A full list of company-sponsored abstracts, which include data in cTTP, sickle cell disease, hemophilia A, leukemia, lymphoma and multiple myeloma can be found here. ABOUT ADZYNMA ADZYNMA (ADAMTS13, recombinant-krhn) is a human recombinant “A disintegrin and metalloproteinase with thrombospondin motifs 13” ADAMTS13 (rADAMTS13) indicated for prophylactic or on-demand enzyme replacement therapy (ERT) in adult and pediatric patients with congenital thrombotic thrombocytopenic purpura (cTTP). ADZYNMA was previously granted Orphan Drug Designation (ODD) by the U.S. FDA for the treatment and prevention of TTP, including its acquired idiopathic and secondary forms, as well as Fast Track and Rare Pediatric Disease Designation. The U.S. FDA granted Takeda a Rare Pediatric Disease Priority Review Voucher for the approval of ADZYNMA. ADZYNMA has also been granted ODD by the European Medicines Agency (EMA) and Japan’s Ministry of Health, Labour and Welfare (MHLW) for the treatment of TTP. Important Safety Information ADZYNMA is contraindicated in patients who have experienced life-threatening hypersensitivity reactions to ADZYNMA or its components. Hypersensitivity Reactions: Allergic-type hypersensitivity, including anaphylactic reactions, may occur with ADZYNMA. Patients should be educated about early signs of hypersensitivity such as tachycardia, chest tightness, wheezing and/or acute respiratory distress, hypotension, generalized urticaria, pruritus, rhinoconjunctivitis, angioedema, lethargy, nausea, vomiting, paresthesia, and restlessness. If signs and symptoms of severe allergic reactions occur, immediately discontinue administration of ADZYNMA and provide appropriate supportive care. Immunogenicity: There is a potential for immunogenicity with ADZYNMA. Patients may develop neutralizing antibodies to ADAMTS13, which could potentially result in a decreased or lack of response to ADAMTS13. Patients may develop antibodies to host cell proteins which could potentially result in adverse reactions. There are no data on immunogenicity with ADZYNMA or to host cell proteins in previously untreated patients (subjects naïve to plasma-based products). Adverse Reactions: The most commonly observed adverse reactions (>5% of subjects) associated with ADZYNMA are headache, diarrhea, migraine, abdominal pain, nausea, upper respiratory tract infection, dizziness and vomiting. Use in Specific Populations: The safety of ADZYNMA for use during pregnancy has not been established in controlled clinical trials. Limited data are insufficient to inform a drug associated risk of adverse developmental outcomes. There is no information regarding the presence of ADZYNMA in human milk, its effects on milk production, or the breastfed infant. To report SUSPECTED ADVERSE REACTIONS, contact Takeda Pharmaceuticals U.S.A, Inc. at 1-877-TAKEDA-7 (1-877-825-3327) or FDA at 1-800-FDA-1088 or . Please see full Prescribing Information, including information for patients. About ICLUSIG® (ponatinib) tablets ICLUSIG is a kinase inhibitor targeting BCR::ABL1, an abnormal tyrosine kinase that is expressed in CML and Ph+ ALL. ICLUSIG is a targeted cancer medicine developed using a computational and structure-based drug-design platform, specifically designed to inhibit the activity of BCR::ABL1 and its mutations. ICLUSIG inhibits native BCR::ABL1, as well as all BCR::ABL1 treatment-resistant mutations, including the most resistant T315I mutation. This mutation has been associated with resistance to all other approved TKIs. ICLUSIG received full approval from the FDA in November 2016. In the U.S., ICLUSIG is indicated for the treatment of adult patients with chronic-phase (CP) CML with resistance or intolerance to at least two prior kinase inhibitors, accelerated-phase (AP) or blast-phase (BP) CML or Ph+ ALL for whom no other kinase inhibitor is indicated, T315I-positive CML (CP, AP or BP) or T315I-positive Ph+ ALL. ICLUSIG is not indicated and is not recommended for the treatment of patients with newly diagnosed CP-CML. IMPORTANT SAFETY INFORMATION WARNING: ARTERIAL OCCLUSIVE EVENTS, VENOUS THROMBOEMBOLIC EVENTS, HEART FAILURE, and HEPATOTOXICITY See full prescribing information for complete boxed warning. Arterial occlusive events (AOEs), including fatalities, have occurred in ICLUSIG-treated patients. AOEs included fatal myocardial infarction, stroke, stenosis of large arterial vessels of the brain, severe peripheral vascular disease, and the need for urgent revascularization procedures. Patients with and without cardiovascular risk factors, including patients age 50 years or younger, experienced these events. Monitor for evidence of AOEs. Interrupt or discontinue ICLUSIG based on severity. Consider benefit-risk to guide a decision to restart ICLUSIG. Venous thromboembolic events (VTEs) have occurred in ICLUSIG-treated patients. Monitor for evidence of VTEs. Interrupt or discontinue ICLUSIG based on severity. Heart failure, including fatalities, occurred in ICLUSIG-treated patients. Monitor for heart failure and manage patients as clinically indicated. Interrupt or discontinue ICLUSIG for new or worsening heart failure. Hepatotoxicity, liver failure and death have occurred in ICLUSIG-treated patients. Monitor liver function tests. Interrupt or discontinue ICLUSIG based on severity. WARNINGS AND PRECAUTIONS Arterial Occlusive Events (AOEs): AOEs, including fatalities, have occurred in patients who received ICLUSIG in OPTIC and PACE. These included cardiovascular, cerebrovascular, and peripheral vascular events. The incidence of AOEs in OPTIC (45 mgà15 mg) was 14% of 94 patients; 6% experienced Grade 3 or 4. In PACE, the incidence of AOEs was 26% of 449 patients; 14% experienced Grade 3 or 4. Fatal AOEs occurred in 2.1% of patients in OPTIC, and in 2% of patients in PACE. Some patients in PACE experienced recurrent or multisite vascular occlusion. Patients with and without cardiovascular risk factors, including patients age 50 years or younger, experienced these events. The most common risk factors observed with these events in PACE were history of hypertension, hypercholesterolemia, and non-ischemic cardiac disease. In OPTIC and PACE, AOEs were more frequent with increasing age. In OPTIC, patients with uncontrolled hypertension or diabetes and patients with clinically significant, uncontrolled, or active cardiovascular disease were excluded. In PACE, patients with uncontrolled hypertriglyceridemia and patients with clinically significant or active cardiovascular disease within the 3 months prior to the first dose of ICLUSIG were excluded. Consider whether the benefits of ICLUSIG are expected to exceed the risks. Monitor for evidence of AOEs. Interrupt, then resume at the same or decreased dose or discontinue ICLUSIG based on recurrence/severity. Consider benefit-risk to guide a decision to restart ICLUSIG. Venous Thromboembolic Events (VTEs): Serious or severe VTEs have occurred in patients who received ICLUSIG. In PACE, VTEs occurred in 6% of 449 patients including serious or severe (Grade 3 or 4) VTEs in 5.8% of patients. VTEs included deep venous thrombosis, pulmonary embolism, superficial thrombophlebitis, retinal vein occlusion, and retinal vein thrombosis with vision loss. The incidence was higher in patients with Ph+ ALL (9% of 32 patients) and BP-CML (10% of 62 patients). One of 94 patients in OPTIC experienced a VTE (Grade 1 retinal vein occlusion). Monitor for evidence of VTEs. Interrupt, then resume at the same or decreased dose or discontinue ICLUSIG based on recurrence/severity. Heart Failure: Fatal, serious or severe heart failure events have occurred in patients who received ICLUSIG. In PACE, heart failure occurred in 9% of 449 patients; 7% experienced serious or severe (Grade 3 or higher). Heart failure occurred in 13% of 94 patients in OPTIC; 1.1% experienced serious or severe (Grade 3 or 4). In PACE, the most frequently reported heart failure events (≥2%) were congestive cardiac failure (3.1%), decreased ejection fraction (2.9%), and cardiac failure (2%). In OPTIC, the most frequently reported heart failure events (>1 patient each) were left ventricular hypertrophy (3.2%) and BNP increased (3.2%). Monitor patients for signs or symptoms consistent with heart failure and manage heart failure as clinically indicated. Interrupt, then resume at reduced dose or discontinue ICLUSIG for new or worsening heart failure. Hepatotoxicity: ICLUSIG can cause hepatotoxicity, including liver failure and death. Fulminant hepatic failure leading to death occurred in 3 patients, with hepatic failure occurring within 1 week of starting ICLUSIG in one of these patients. These fatal cases occurred in patients with BP-CML or Ph+ ALL. Hepatotoxicity occurred in 28% of 94 patients in OPTIC and 32% of 449 patients in PACE. Grade 3 or 4 hepatotoxicity occurred in OPTIC (6% of 94 patients) and PACE (13% of 449 patients). The most frequent hepatotoxic events were elevations of ALT, AST, GGT, bilirubin, and alkaline phosphatase. Monitor liver function tests at baseline, then at least monthly or as clinically indicated. Interrupt, then resume at a reduced dose or discontinue ICLUSIG based on recurrence/severity. Hypertension: Serious or severe hypertension, including hypertensive crisis, has occurred in patients who received ICLUSIG. Patients may require urgent clinical intervention for hypertension associated with confusion, headache, chest pain, or shortness of breath. Monitor blood pressure at baseline and as clinically indicated and manage hypertension as clinically indicated. Interrupt, dose reduce, or stop ICLUSIG if hypertension is not medically controlled. For significant worsening, labile or treatment-resistant hypertension, interrupt ICLUSIG and consider evaluating for renal artery stenosis. Pancreatitis: Serious or severe pancreatitis has occurred in patients who received ICLUSIG. Elevations of lipase and amylase also occurred. In the majority of cases that led to dose modification or treatment discontinuation, pancreatitis resolved within 2 weeks. Monitor serum lipase every 2 weeks for the first 2 months and then monthly thereafter or as clinically indicated. Consider additional serum lipase monitoring in patients with a history of pancreatitis or alcohol abuse. Interrupt, then resume at the same or reduced dose or discontinue ICLUSIG based on severity. Evaluate for pancreatitis when lipase elevation is accompanied by abdominal symptoms. Increased Toxicity in Newly Diagnosed Chronic Phase CML: In a prospective randomized clinical trial in the first line treatment of newly diagnosed patients with CP-CML, single agent ICLUSIG 45 mg once daily increased the risk of serious adverse reactions 2-fold compared to single agent imatinib 400 mg once daily. The median exposure to treatment was less than 6 months. The trial was halted for safety. Arterial and venous thrombosis and occlusions occurred at least twice as frequently in the ICLUSIG arm compared to the imatinib arm. Compared to imatinib-treated patients, ICLUSIG-treated patients exhibited a greater incidence of myelosuppression, pancreatitis, hepatotoxicity, cardiac failure, hypertension, and skin and subcutaneous tissue disorders. ICLUSIG is not indicated and is not recommended for the treatment of patients with newly diagnosed CP-CML. Neuropathy: Peripheral and cranial neuropathy occurred in patients in OPTIC and PACE. Some of these events in PACE were Grade 3 or 4. Monitor patients for symptoms of neuropathy, such as hypoesthesia, hyperesthesia, paresthesia, discomfort, a burning sensation, neuropathic pain or weakness. Interrupt, then resume at the same or reduced dose or discontinue ICLUSIG based on recurrence/severity. Ocular Toxicity: Serious or severe ocular toxicity leading to blindness or blurred vision have occurred in ICLUSIG-treated patients. The most frequent ocular toxicities occurring in OPTIC and PACE were dry eye, blurred vision, and eye pain. Retinal toxicities included age-related macular degeneration, macular edema, retinal vein occlusion, retinal hemorrhage, and vitreous floaters. Conduct comprehensive eye exams at baseline and periodically during treatment. Hemorrhage: Fatal and serious hemorrhage events have occurred in patients who received ICLUSIG. Fatal hemorrhages occurred in PACE and serious hemorrhages occurred in OPTIC and PACE. In PACE, the incidence of serious bleeding events was higher in patients with AP-CML, BP-CML, and Ph+ ALL. Gastrointestinal hemorrhage and subdural hematoma were the most frequently reported serious hemorrhages. Events often occurred in patients with Grade 4 thrombocytopenia. Monitor for hemorrhage and manage patients as clinically indicated. Interrupt, then resume at the same or reduced dose or discontinue ICLUSIG based on recurrence/severity. Fluid Retention: Fatal and serious fluid retention events have occurred in patients who received ICLUSIG. In PACE, one instance of brain edema was fatal and serious events included pleural effusion, pericardial effusion, and angioedema. Monitor for fluid retention and manage patients as clinically indicated. Interrupt, then resume at the same or reduced dose or discontinue ICLUSIG based on recurrence/severity. Cardiac Arrhythmias: Cardiac arrhythmias, including ventricular and atrial arrhythmias, occurred in patients in OPTIC and PACE. For some patients, events were serious or severe (Grade 3 or 4) and led to hospitalization. Monitor for signs and symptoms suggestive of slow heart rate (fainting, dizziness) or rapid heart rate (chest pain, palpitations or dizziness) and manage patients as clinically indicated. Interrupt, then resume at the same or reduced dose or discontinue ICLUSIG based on recurrence/severity. Myelosuppression: Grade 3 or 4 events of neutropenia, thrombocytopenia, and anemia occurred in patients in OPTIC and PACE. The incidence of myelosuppression was greater in patients with AP-CML, BP-CML, and Ph+ ALL than in patients with CP-CML. Obtain complete blood counts every 2 weeks for the first 3 months and then monthly or as clinically indicated. If ANC less than 1 x 109/L or platelets less than 50 x 109/L, interrupt ICLUSIG until ANC at least 1.5 x 109/L and platelets at least 75 x 109/L, then resume at same or reduced dose. Tumor Lysis Syndrome (TLS): Serious TLS was reported in ICLUSIG-treated patients in OPTIC and PACE. Ensure adequate hydration and treat high uric acid levels prior to initiating ICLUSIG. Reversible Posterior Leukoencephalopathy Syndrome (RPLS): RPLS (also known as Posterior Reversible Encephalopathy Syndrome) has been reported in patients who received ICLUSIG. Patients may present with neurological signs and symptoms, visual disturbances, and hypertension. Diagnosis is made with supportive findings on magnetic resonance imaging (MRI) of the brain. Interrupt ICLUSIG until resolution. The safety of resumption of ICLUSIG in patients upon resolution of RPLS is unknown. Impaired Wound Healing and Gastrointestinal Perforation: Impaired wound healing occurred in patients receiving ICLUSIG. Withhold ICLUSIG for at least 1 week prior to elective surgery. Do not administer for at least 2 weeks following major surgery and until adequate wound healing. The safety of resumption of ICLUSIG after resolution of wound healing complications has not been established. Gastrointestinal perforation or fistula occurred in patients receiving ICLUSIG. Permanently discontinue in patients with gastrointestinal perforation. Embryo-Fetal Toxicity: Based on its mechanism of action and findings from animal studies, ICLUSIG can cause fetal harm when administered to a pregnant woman Advise pregnant women of the potential risk to the fetus. Advise females of reproductive potential to use effective contraception during treatment with ICLUSIG and for 3 weeks after the last dose. ADVERSE REACTIONS The most common (>20%) adverse reactions are rash and related conditions, arthralgia, abdominal pain, headache, constipation, dry skin, hypertension, fatigue, fluid retention and edema, pyrexia, nausea, pancreatitis/lipase elevation, hemorrhage, anemia, hepatic dysfunction and AOEs. The most common Grade 3 or 4 laboratory abnormalities (>20%) are platelet count decreased, neutrophil cell count decreased, and white blood cell decreased. To report SUSPECTED ADVERSE REACTIONS, contact Takeda Pharmaceuticals at 1-844-817-6468 or FDA at 1-800-FDA-1088 or . DRUG INTERACTIONS Strong CYP3A Inhibitors: Avoid coadministration or reduce ICLUSIG dose if coadministration cannot be avoided. Strong CYP3A Inducers: Avoid coadministration. USE IN SPECIFIC POPULATIONS Lactation: Advise women not to breastfeed during treatment with ICLUSIG and for 6 days following last dose Females and Males of Reproductive Potential: Verify pregnancy status of females of reproductive potential prior to initiating ICLUSIG. Ponatinib may impair fertility in females, and it is not known if these effects are reversible. Pre-existing Hepatic Impairment: Reduce the starting dose of ICLUSIG to 30mg orally once daily for patients with pre-existing hepatic impairment as these patients are more likely to experience adverse reactions compared to patients with normal hepatic function. Prescribing Information About ADCETRIS® (brentuximab vedotin) ADCETRIS is an antibody-drug conjugate (ADC) comprising an anti-CD30 monoclonal antibody attached by a protease-cleavable linker to a microtubule disrupting agent, monomethyl auristatin E (MMAE), utilizing Seagen’s proprietary technology. The ADC employs a linker system that is designed to be stable in the bloodstream but to release MMAE upon internalization into CD30-positive tumor cells. ADCETRIS injection for intravenous infusion has received FDA approval for six indications in adult patients with: (1) previously untreated systemic anaplastic large cell lymphoma (sALCL) or other CD30-expressing peripheral T-cell lymphomas (PTCL), including angioimmunoblastic T-cell lymphoma and PTCL not otherwise specified, in combination with cyclophosphamide, doxorubicin, and prednisone, (2) previously untreated Stage III or IV classical Hodgkin lymphoma (cHL), in combination with doxorubicin, vinblastine, and dacarbazine, (3) cHL at high risk of relapse or progression as post-autologous hematopoietic stem cell transplantation (auto-HSCT) consolidation, (4) cHL after failure of auto-HSCT or failure of at least two prior multi-agent chemotherapy regimens in patients who are not auto-HSCT candidates, (5) sALCL after failure of at least one prior multi-agent chemotherapy regimen, and (6) primary cutaneous anaplastic large cell lymphoma (pcALCL) or CD30-expressing mycosis fungoides (MF) who have received prior systemic therapy. ADCETRIS has also received FDA approval for the treatment of pediatric patients 2 years and older with previously untreated high risk classical Hodgkin lymphoma (cHL), in combination with doxorubicin, vincristine, etoposide, prednisone, and cyclophosphamide. Health Canada granted ADCETRIS approval with conditions for relapsed or refractory Hodgkin lymphoma and sALCL in 2013, and non-conditional approval for post-autologous stem cell transplantation (ASCT) consolidation treatment of Hodgkin lymphoma patients at increased risk of relapse or progression in 2017, adults with pcALCL or CD30-expressing MF who have had prior systemic therapy in 2018, for previously untreated Stage IV Hodgkin lymphoma in combination with doxorubicin, vinblastine, and dacarbazine in 2019, and for previously untreated adult patients with sALCL, peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS) or angioimmunoblastic T-cell lymphoma (AITL), whose tumors express CD30, in combination with cyclophosphamide, doxorubicin, prednisone in 2019. ADCETRIS received conditional marketing authorization from the European Commission in October 2012, and the specific obligations of the conditional marketing authorization were fulfilled in May 2022. The approved indications in the European Union are: (1) for the treatment of adult patients with previously untreated CD30-positive Stage III & IV Hodgkin lymphoma in combination with doxorubicin, vinblastine and dacarbazine (AVD), (2) for the treatment of adult patients with CD30-positive Hodgkin lymphoma at increased risk of relapse or progression following ASCT, (3) for the treatment of adult patients with relapsed or refractory CD30-positive Hodgkin lymphoma following ASCT, or following at least two prior therapies when ASCT or multi-agent chemotherapy is not a treatment option, (4) for the treatment of adult patients with relapsed or refractory sALCL, (5) for the treatment of adult patients with previously untreated sALCL in combination with CHP and (6) for the treatment of adult patients with CD30-positive cutaneous T-cell lymphoma (CTCL) after at least one prior systemic therapy. ADCETRIS has received marketing authorization by regulatory authorities in more than 70 countries for relapsed or refractory Hodgkin lymphoma and sALCL. See Important Safety Information below. ADCETRIS is being evaluated broadly in more than 70 clinical trials, including a Phase 3 study in first-line Hodgkin lymphoma (ECHELON-1) and another Phase 3 study in first-line CD30-positive peripheral T-cell lymphomas (ECHELON-2), as well as trials in many additional types of CD30-positive malignancies. Seagen and Takeda are jointly developing ADCETRIS. Under the terms of the collaboration agreement, Seagen has U.S. and Canadian commercialization rights and Takeda has rights to commercialize ADCETRIS in the rest of the world. Seagen and Takeda are funding joint development costs for ADCETRIS on a 50:50 basis, except in Japan where Takeda is solely responsible for development costs. ADCETRIS (brentuximab vedotin) Important Safety Information (European Union) Please refer to Summary of Product Characteristics (SmPC) before prescribing. Contraindications ADCETRIS is contraindicated for patients with hypersensitivity to brentuximab vedotin and its excipients. In addition, combined use of ADCETRIS with bleomycin causes pulmonary toxicity. Special Warnings and Precautions Progressive multifocal leukoencephalopathy (PML): John Cunningham virus (JCV) reactivation resulting in progressive multifocal leukoencephalopathy (PML) and death can occur in patients treated with ADCETRIS. PML has been reported in patients who received ADCETRIS after receiving multiple prior chemotherapy regimens. PML is a rare demyelinating disease of the central nervous system that results from reactivation of latent JCV and is often fatal. Closely monitor patients for new or worsening neurological, cognitive, or behavioral signs or symptoms, which may be suggestive of PML. Suggested evaluation of PML includes neurology consultation, gadolinium-enhanced magnetic resonance imaging of the brain, and cerebrospinal fluid analysis for JCV DNA by polymerase chain reaction or a brain biopsy with evidence of JCV. A negative JCV PCR does not exclude PML. Additional follow-up and evaluation may be warranted if no alternative diagnosis can be established. Hold dosing for any suspected case of PML and permanently discontinue ADCETRIS if a diagnosis of PML is confirmed. Be alert to PML symptoms that the patient may not notice (e.g., cognitive, neurological, or psychiatric symptoms). Pancreatitis: Acute pancreatitis has been observed in patients treated with ADCETRIS. Fatal outcomes have been reported. Closely monitor patients for new or worsening abdominal pain, which may be suggestive of acute pancreatitis. Patient evaluation may include physical examination, laboratory evaluation for serum amylase and serum lipase, and abdominal imaging, such as ultrasound and other appropriate diagnostic measures. Hold ADCETRIS for any suspected case of acute pancreatitis. ADCETRIS should be discontinued if a diagnosis of acute pancreatitis is confirmed. Pulmonary Toxicity: Cases of pulmonary toxicity, some with fatal outcomes, including pneumonitis, interstitial lung disease, and acute respiratory distress syndrome (ARDS), have been reported in patients receiving ADCETRIS. Although a causal association with ADCETRIS has not been established, the risk of pulmonary toxicity cannot be ruled out. Promptly evaluate and treat new or worsening pulmonary symptoms (e.g., cough, dyspnea) appropriately. Consider holding dosing during evaluation and until symptomatic improvement. Serious infections and opportunistic infections: Serious infections such as pneumonia, staphylococcal bacteremia, sepsis/septic shock (including fatal outcomes), and herpes zoster, cytomegalovirus (CMV) (reactivation) and opportunistic infections such as Pneumocystis jiroveci pneumonia and oral candidiasis have been reported in patients treated with ADCETRIS. Patients should be carefully monitored during treatment for the emergence of possible serious and opportunistic infections. Infusion-related reactions (IRR): Immediate and delayed IRR, as well as anaphylaxis, have been reported with ADCETRIS. Carefully monitor patients during and after an infusion. If anaphylaxis occurs, immediately and permanently discontinue administration of ADCETRIS and administer appropriate medical therapy. If an IRR occurs, interrupt the infusion and institute appropriate medical management. The infusion may be restarted at a slower rate after symptom resolution. Patients who have experienced a prior IRR should be premedicated for subsequent infusions. IRRs are more frequent and more severe in patients with antibodies to ADCETRIS. Tumor lysis syndrome (TLS): TLS has been reported with ADCETRIS. Patients with rapidly proliferating tumor and high tumor burden are at risk of TLS. Monitor these patients closely and manage according to best medical practice. Peripheral neuropathy (PN): ADCETRIS treatment may cause PN, both sensory and motor. ADCETRIS-induced PN is typically an effect of cumulative exposure to ADCETRIS and is reversible in most cases. Monitor patients for symptoms of neuropathy, such as hypoesthesia, hyperesthesia, paresthesia, discomfort, a burning sensation, neuropathic pain, or weakness. Patients experiencing new or worsening PN may require a delay and a dose reduction or discontinuation of ADCETRIS. Hematological toxicities: Grade 3 or Grade 4 anemia, thrombocytopenia, and prolonged (equal to or greater than one week) Grade 3 or Grade 4 neutropenia can occur with ADCETRIS. Monitor complete blood counts prior to administration of each dose. Febrile neutropenia: Febrile neutropenia has been reported with ADCETRIS. Complete blood counts should be monitored prior to administration of each dose of treatment. Closely monitor patients for fever and manage according to best medical practice if febrile neutropenia develops. When ADCETRIS is administered in combination with AVD or CHP, primary prophylaxis with G-CSF is recommended for all patients beginning with the first dose. Severe cutaneous adverse reactions (SCARs): Cases of SCARs, including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS) have been reported with ADCETRIS. Fatal outcomes have been reported for SJS and TEN. If SJS, TEN or DRESS occur, ADCETRIS should be discontinued and appropriate medical therapy should be administered. Gastrointestinal (GI) Complications: GI complications, some with fatal outcomes, including intestinal obstruction, ileus, enterocolitis, neutropenic colitis, erosion, ulcer, perforation and haemorrhage, have been reported with ADCETRIS. Promptly evaluate and treat patients if new or worsening GI symptoms occur. Hepatotoxicity: Elevations in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) have been reported with ADCETRIS. Serious cases of hepatotoxicity, including fatal outcomes, have also occurred. Pre-existing liver disease, comorbidities, and concomitant medications may also increase the risk. Test liver function prior to treatment initiation and routinely monitor during treatment. Patients experiencing hepatotoxicity may require a delay, dose modification, or discontinuation of ADCETRIS. Hyperglycemia: Hyperglycemia has been reported during trials in patients with an elevated body mass index (BMI) with or without a history of diabetes mellitus. Closely monitor serum glucose for patients who experience an event of hyperglycemia. Administer anti-diabetic treatment as appropriate. Infusion site extravasation: Extravasation during intravenous infusion has occurred. Given the possibility of extravasation, it is recommended to closely monitor the infusion site for possible infiltration during drug administration. Renal and Hepatic Impairment: There is limited experience in patients with renal and hepatic impairment. Available data indicate that MMAE clearance might be affected by severe renal impairment, hepatic impairment, and by low serum albumin concentrations. CD30+ CTCL: The size of the treatment effect in CD30 + CTCL subtypes other than mycosis fungoides (MF) and primary cutaneous anaplastic large cell lymphoma (pcALCL) is not clear due to lack of high level evidence. In two single arm phase II studies of ADCETRIS, disease activity has been shown in the subtypes Sézary syndrome (SS), lymphomatoid papulosis (LyP) and mixed CTCL histology. These data suggest that efficacy and safety can be extrapolated to other CTCL CD30+ subtypes. Carefully consider the benefit-risk per patient and use with caution in other CD30+ CTCL patient types. Sodium content in excipients: This medicinal product contains 13.2 mg sodium per vial, equivalent to 0.7% of the WHO recommended maximum daily intake of 2 g sodium for an adult. Traceability: In order to improve the traceability of biological medicinal products, the name and the batch number of the administered product should be clearly recorded. INTERACTIONS Patients who are receiving a strong CYP3A4 and P-gp inhibitor, concomitantly with ADCETRIS may have an increased risk of neutropenia. If neutropenia develops, refer to dosing recommendations for neutropenia (see SmPC section 4.2). Co-administration of ADCETRIS with a CYP3A4 inducer did not alter the plasma exposure of ADCETRIS, but it appeared to reduce plasma concentrations of MMAE metabolites that could be assayed. ADCETRIS is not expected to alter the exposure to drugs that are metabolized by CYP3A4 enzymes. PREGNANCY: Advise women of childbearing potential to use two methods of effective contraception during treatment with ADCETRIS and until 6 months after treatment. There are no data from the use of ADCETRIS in pregnant women, although studies in animals have shown reproductive toxicity. Do not use ADCETRIS during pregnancy unless the benefit to the mother outweighs the potential risks to the fetus. LACTATION (breast-feeding): There are no data as to whether ADCETRIS or its metabolites are excreted in human milk, therefore a risk to the newborn/infant cannot be excluded. A decision should be made whether to discontinue breast-feeding or to discontinue/abstain from this therapy, taking into account a potential risk of breast-feeding for the child and the benefit of therapy for the woman. FERTILITY: In non-clinical studies, brentuximab vedotin treatment has resulted in testicular toxicity, and may alter male fertility. MMAE has been shown to have anagenic properties. Therefore, men being treated with this medicine are advised to have sperm samples frozen and stored before treatment. Men being treated with this medicine are advised not to father a child during treatment and for up to 6 months following the last dose. Effects on ability to drive and use machines: ADCETRIS may have a moderate influence on the ability to drive and use machines. UNDESIRABLE EFFECTS Monotherapy: The most frequent adverse reactions (≥10%) were infections, peripheral sensory neuropathy, nausea, fatigue, diarrhea, pyrexia, upper respiratory tract infection, neutropenia, rash, cough, vomiting, arthralgia, peripheral motor neuropathy, infusion-related reactions, pruritus, constipation, dyspnea, weight decreased, myalgia and abdominal pain. Serious adverse drug reactions occurred in 12% of patients. The frequency of unique serious adverse drug reactions was ≤1%. Adverse events led to treatment discontinuation in 24% of patients. Combination Therapy: In the studies of ADCETRIS as combination therapy in 662 patients with previously untreated advanced HL (C25003) and 223 patients with previously untreated CD30+ PTCL, the most common adverse reactions (≥ 10%) were: infections, neutropenia, peripheral sensory neuropathy, nausea, constipation, vomiting, diarrhea, fatigue, pyrexia, alopecia, anemia, weight decreased, stomatitis, febrile neutropenia, abdominal pain, decreased appetite, insomnia, bone pain, rash, cough, dyspnea, arthralgia, myalgia, back pain, peripheral motor neuropathy, upper respiratory tract infection, and dizziness. In patients receiving ADCETRIS combination therapy, serious adverse reactions occurred in 34% of patients. Serious adverse reactions occurring in ≥ 3% of patients included febrile neutropenia (15%), pyrexia (5%), and neutropenia (3%). Adverse events led to treatment discontinuation in 10% of patients. ADCETRIS (brentuximab vedotin) for injection U.S. Important Safety Information BOXED WARNING PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY (PML): JC virus infection resulting in PML and death can occur in ADCETRIS-treated patients. Contraindication ADCETRIS concomitant with bleomycin due to pulmonary toxicity (e.g., interstitial infiltration and/or inflammation). Warnings and Precautions Peripheral neuropathy (PN): ADCETRIS causes PN that is predominantly sensory. Cases of motor PN have also been reported. ADCETRIS-induced PN is cumulative. Monitor for symptoms such as hypoesthesia, hyperesthesia, paresthesia, discomfort, a burning sensation, neuropathic pain, or weakness. Institute dose modifications accordingly. Anaphylaxis and infusion reactions: Infusion-related reactions (IRR), including anaphylaxis, have occurred with ADCETRIS. Monitor patients during infusion. If an IRR occurs, interrupt the infusion and institute appropriate medical management. If anaphylaxis occurs, immediately and permanently discontinue the infusion and administer appropriate medical therapy. Premedicate patients with a prior IRR before subsequent infusions. Premedication may include acetaminophen, an antihistamine, and a corticosteroid. Hematologic toxicities: Fatal and serious cases of febrile neutropenia have been reported with ADCETRIS. Prolonged (≥1 week) severe neutropenia and Grade 3 or 4 thrombocytopenia or anemia can occur with ADCETRIS. Administer G-CSF primary prophylaxis beginning with Cycle 1 for patients who receive ADCETRIS in combination with chemotherapy for previously untreated Stage III/IV cHL or previously untreated PTCL. Monitor complete blood counts prior to each ADCETRIS dose. Monitor more frequently for patients with Grade 3 or 4 neutropenia. Monitor patients for fever. If Grade 3 or 4 neutropenia develops, consider dose delays, reductions, discontinuation, or G-CSF prophylaxis with subsequent doses. Serious infections and opportunistic infections: Infections such as pneumonia, bacteremia, and sepsis or septic shock (including fatal outcomes) have been reported in ADCETRIS-treated patients. Closely monitor patients during treatment for bacterial, fungal, or viral infections. Tumor lysis syndrome: Closely monitor patients with rapidly proliferating tumor and high tumor burden. Increased toxicity in the presence of severe renal impairment: The frequency of ≥Grade 3 adverse reactions and deaths was greater in patients with severe renal impairment compared to patients with normal renal function. Avoid use in patients with severe renal impairment. Increased toxicity in the presence of moderate or severe hepatic impairment: The frequency of ≥Grade 3 adverse reactions and deaths was greater in patients with moderate or severe hepatic impairment compared to patients with normal hepatic function. Avoid use in patients with moderate or severe hepatic impairment. Hepatotoxicity: Fatal and serious cases have occurred in ADCETRIS-treated patients. Cases were consistent with hepatocellular injury, including elevations of transaminases and/or bilirubin, and occurred after the first ADCETRIS dose or rechallenge. Preexisting liver disease, elevated baseline liver enzymes, and concomitant medications may increase the risk. Monitor liver enzymes and bilirubin. Patients with new, worsening, or recurrent hepatotoxicity may require a delay, change in dose, or discontinuation of ADCETRIS. PML: Fatal cases of JC virus infection resulting in PML have been reported in ADCETRIS-treated patients. First onset of symptoms occurred at various times from initiation of ADCETRIS, with some cases occurring within 3 months of initial exposure. In addition to ADCETRIS therapy, other possible contributory factors include prior therapies and underlying disease that may cause immunosuppression. Consider PML diagnosis in patients with new-onset signs and symptoms of central nervous system abnormalities. Hold ADCETRIS if PML is suspected and discontinue ADCETRIS if PML is confirmed. Pulmonary toxicity: Fatal and serious events of noninfectious pulmonary toxicity, including pneumonitis, interstitial lung disease, and acute respiratory distress syndrome, have been reported. Monitor patients for signs and symptoms, including cough and dyspnea. In the event of new or worsening pulmonary symptoms, hold ADCETRIS dosing during evaluation and until symptomatic improvement. Serious dermatologic reactions: Fatal and serious cases of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) have been reported with ADCETRIS. If SJS or TEN occurs, discontinue ADCETRIS and administer appropriate medical therapy. Gastrointestinal (GI) complications: Fatal and serious cases of acute pancreatitis have been reported. Other fatal and serious GI complications include perforation, hemorrhage, erosion, ulcer, intestinal obstruction, enterocolitis, neutropenic colitis, and ileus. Lymphoma with preexisting GI involvement may increase the risk of perforation. In the event of new or worsening GI symptoms, including severe abdominal pain, perform a prompt diagnostic evaluation and treat appropriately. Hyperglycemia: Serious cases, such as new-onset hyperglycemia, exacerbation of pre-existing diabetes mellitus, and ketoacidosis (including fatal outcomes) have been reported with ADCETRIS. Hyperglycemia occurred more frequently in patients with high body mass index or diabetes. Monitor serum glucose and if hyperglycemia develops, administer anti-hyperglycemic medications as clinically indicated. Embryo-fetal toxicity: Based on the mechanism of action and animal studies, ADCETRIS can cause fetal harm. Advise females of reproductive potential of the potential risk to the fetus, and to avoid pregnancy during ADCETRIS treatment and for at least 6 months after the final dose of ADCETRIS. Most Common (≥20% in any study) Adverse Reactions Peripheral neuropathy, fatigue, nausea, diarrhea, neutropenia, upper respiratory tract infection, pyrexia, constipation, vomiting, alopecia, decreased weight, abdominal pain, anemia, stomatitis, lymphopenia, and mucositis. Drug Interactions Concomitant use of strong CYP3A4 inhibitors or inducers has the potential to affect the exposure to monomethyl auristatin E (MMAE). Use in Specific Populations Moderate or severe hepatic impairment or severe renal impairment: MMAE exposure and adverse reactions are increased. Avoid use. Advise males with female sexual partners of reproductive potential to use effective contraception during ADCETRIS treatment and for at least 6 months after the final dose of ADCETRIS. Advise patients to report pregnancy immediately and avoid breastfeeding while receiving ADCETRIS. Please see the full Prescribing Information, including BOXED WARNING, for ADCETRIS here. Prescribing Information Takeda’s Commitment to Hematology Takeda is a leader in hemophilia with the longest heritage and market-leading portfolio, backed by established safety and efficacy profiles with decades of real-world experience. We have 70+ years driving innovation for patients and a broad portfolio of [nine therapies across more than 10 indications]. Our experience as a leader in hematology means we are well prepared to meet today’s needs as we pursue future developments in the care of blood disorders. Together with the hematology community, we are raising expectations for the future, including earlier diagnosis, earlier and full protection against bleeds, and more personalized patient care. Takeda’s Commitment to Oncology At Takeda Oncology, we are united by our aspiration to cure cancer and motivated every day to work harder for patients with limited or ineffective treatment options. Our agile structure and deep in-house expertise are complemented by a network of partnerships that optimize our ability to research, develop and deliver transformative medicines to people living with cancer. Building on decades of leadership in oncology and a portfolio of approved medicines for hematologic cancers and solid tumors, we are advancing a cutting-edge pipeline focused on the power of innate immunity. With inspiration from patients and innovation from everywhere, our goal is to introduce new classes of immunotherapies that can lead to deep, durable responses so that more patients can benefit from – and have access to – innovative medicines. About Takeda Takeda is focused on creating better health for people and a brighter future for the world. We aim to discover and deliver life-transforming treatments in our core therapeutic and business areas, including gastrointestinal and inflammation, rare diseases, plasma-derived therapies, oncology, neuroscience and vaccines. Together with our partners, we aim to improve the patient experience and advance a new frontier of treatment options through our dynamic and diverse pipeline. As a leading values-based, R&D-driven biopharmaceutical company headquartered in Japan, we are guided by our commitment to patients, our people and the planet. Our employees in approximately 80 countries and regions are driven by our purpose and are grounded in the values that have defined us for more than two centuries. For more information, visit . Important Notice For the purposes of this notice, “press release” means this document, any oral presentation, any question-and-answer session and any written or oral material discussed or distributed by Takeda Pharmaceutical Company Limited (“Takeda”) regarding this release. This press release (including any oral briefing and any question-and-answer in connection with it) is not intended to, and does not constitute, represent or form part of any offer, invitation or solicitation of any offer to purchase, otherwise acquire, subscribe for, exchange, sell or otherwise dispose of, any securities or the solicitation of any vote or approval in any jurisdiction. No shares or other securities are being offered to the public by means of this press release. No offering of securities shall be made in the United States except pursuant to registration under the U.S. Securities Act of 1933, as amended, or an exemption therefrom. This press release is being given (together with any further information which may be provided to the recipient) on the condition that it is for use by the recipient for information purposes only (and not for the evaluation of any investment, acquisition, disposal or any other transaction). Any failure to comply with these restrictions may constitute a violation of applicable securities laws. The companies in which Takeda directly and indirectly owns investments are separate entities. In this press release, “Takeda” is sometimes used for convenience where references are made to Takeda and its subsidiaries in general. Likewise, the words “we,” “us” and “our” are also used to refer to subsidiaries in general or to those who work for them. These expressions are also used where no useful purpose is served by identifying the particular company or companies. Forward-Looking Statements This press release and any materials distributed in connection with this press release may contain forward-looking statements, beliefs or opinions regarding Takeda’s future business, future position and results of operations, including estimates, forecasts, targets and plans for Takeda. Without limitation, forward-looking statements often include words such as “targets,” “plans,” “believes,” “hopes,” “continues,” “expects,” “aims,” “intends,” “ensures,” “will,” “may,” “should,” “would,” “could,” “anticipates,” “estimates,” “projects” or similar expressions or the negative thereof. These forward-looking statements are based on assumptions about many important factors, including the following, which could cause actual results to differ materially from those expressed or implied by the forward-looking statements: the economic circumstances surrounding Takeda’s global business, including general economic conditions in Japan and the United States; competitive pressures and developments; changes to applicable laws and regulations, including global health care reforms; challenges inherent in new product development, including uncertainty of clinical success and decisions of regulatory authorities and the timing thereof; uncertainty of commercial success for new and existing products; manufacturing difficulties or delays; fluctuations in interest and currency exchange rates; claims or concerns regarding the safety or efficacy of marketed products or product candidates; the impact of health crises, like the novel coronavirus pandemic, on Takeda and its customers and suppliers, including foreign governments in countries in which Takeda operates, or on other facets of its business; the timing and impact of post-merger integration efforts with acquired companies; the ability to divest assets that are not core to Takeda’s operations and the timing of any such divestment(s); and other factors identified in Takeda’s most recent Annual Report on Form 20-F and Takeda’s other reports filed with the U.S. Securities and Exchange Commission, available on Takeda’s website at: or at . Takeda does not undertake to update any of the forward-looking statements contained in this press release or any other forward-looking statements it may make, except as required by law or stock exchange rule. Past performance is not an indicator of future results and the results or statements of Takeda in this press release may not be indicative of, and are not an estimate, forecast, guarantee or projection of Takeda’s future results. Medical Information This press release contains information about products that may not be available in all countries, or may be available under different trademarks, for different indications, in different dosages, or in different strengths. Nothing contained herein should be considered a solicitation, promotion or advertisement for any prescription drugs including the ones under development. ADZYNMA is a trademark of Takeda Pharmaceuticals International AG.