Buserelin Acetate

“ 点击蓝字 关注我们 郑珩 中国药科大学生命科学与技术学院副教授、博士生导师。中国药学会抗生素专业委员会委员、江苏省生物信息学专业委员会委员，《中国抗生素杂志》《药物生物技术》等杂志编委。主持国家重大新药创制专项、国家自然科学基金面上项目等，在 Nucleic Acids Res 等国内外核心期刊发表论文 80 多篇，获中国发明专利 10 项。主持完成“微生物发酵法生产天然型脱落酸”“微生物发酵法生产辅酶 Q10”“天然型茄尼醇制备技术”等课题，并实现工业化；参与“2 类新药螺旋藻多糖研制开发”“抗肿瘤 1 类新药安替安吉肽的研发”项目研发，已进入临床。获得教育部技术发明奖一等奖（2019 年）、教育部霍英东青年教师三等奖（2006 年）、中国药学会青年生物药物奖（2010 年）、江苏省六大人才高峰（2009 年）等；主讲的生物制药工艺学获国家级一流本科课程（2020 年），国家精品在线开放课程（2019 年）等，入选国家级教学团队（2010 年）。 抗癌肽的临床应用与研发进展 PPS  孙欣，刘艳超，郑珩 * （中国药科大学生命科学与技术学院，江苏 南京 211198） [ 摘要 ] 癌症是严重威胁人类生命健康的重大疾病之一。传统的癌症治疗方法包括化疗、放疗等多种方法，但均具有相应的副作用或局限性，特别是对化疗药物产生的多药耐药性是癌症治疗的一大阻碍。肽疗法的兴起为癌症治疗提供了新的思路，肽因其靶向性、低毒性和高选择性而成为一类有前途的抗癌剂。综述抗癌肽（anticancer peptides, ACPs）的抗癌机制、临床应用、研发历程和计算机辅助ACPs 设计，包括基于肽 - 蛋白质相互作用的设计和基于人工智能的设计，以期为新型 ACPs 类药物的设计研发提供参考。 癌症是当前人类过早死亡的主要原因，仅次于心血管疾病，且有超越心血管疾病成为大多数国家人类过早死亡的最主要原因的趋势 [1]。它具有 6 个特征，包括维持增殖信号、逃避生长抑制因子、抵抗细胞死亡、实现永生复制、诱导血管生成以及激活侵袭和转移 [2]。传统的癌症治疗方法包括化疗 [3]、放疗 [4]、免疫疗法 [5]、光动力疗法 [6]、光热疗法 [7] 等，但均具有相应的局限性。当前癌症治疗的主流疗法仍旧是化疗与放疗，但可能会伴随有严重的副作用。据报道，90% 接受化疗的癌症患者死亡是由于肿瘤的多药耐药性 [8]。由于与小分子化合物的结构差异，生物活性肽受到了广泛关注，并被认为是耐多药抗癌治疗的替代候选药物 [9-11]，抗癌肽（anticancer peptides，ACPs）便是一种新型抗癌治疗策略。 自从 1922 年，研究人员从动物胰腺中提取胰岛素治疗糖尿病，便将肽作为治疗药物引入医学治疗领域 [12]。肽代表一类独特的化合物，分子量介于小分子与蛋白质之间，有可能结合小分子难以靶向的蛋白质表面 [13]。在过去的几十年里，人们对肽用于临床治疗越来越感兴趣。肽疗法具有相对安全、选择性高、耐受性好、副作用少且生产成本低等优势 [14-15]，越来越多地被开发用于治疗许多疾病，包括糖尿病、骨质疏松症、人类免疫缺陷病毒（human immune deficiency virus，HIV）感染、慢性疼痛以及癌症等 [14, 16]。越来越多的肽类化合物进入临床前或临床研究，60 余种肽类药物在全球市场上获得批准 [13, 17]。本文将综述 ACPs 的作用机制及分类、临床应用和研发历程，并重点介绍 ACPs 的设计方法。 1 ACPs 的抗癌机制及分类 近年来，ACPs 因其对癌细胞的选择性和治疗特性而深受关注。癌症的特征是细胞增殖失控 [18]，糖酵解增加引起乳酸分泌导致癌细胞外酸化 [19]。健康细胞具有电中性，而癌细胞表面由于阴离子分子如磷脂酰丝氨酸、O-糖基化黏蛋白等的表达增加，癌细胞膜通常携带净负电荷 [20]。与癌细胞膜特征相反，ACPs 通常带有正净电荷 [21]，属于阳离子两亲性肽 [20]，肽与健康细胞膜之间相互作用的主要驱动力是疏水相互作用，而肽与癌细胞膜之间的相互作用是静电相互作用 [22]。 阳离子 ACPs 选择性地与癌细胞相互作用并穿透细胞膜 [23]，通过以下机制对癌细胞产生细胞毒性作用：1）膜溶解机制，大多数 ACPs 直接通过破坏细胞膜完整性来杀死癌细胞 [24-25]；2）诱导细胞凋亡或坏死，ACPs 的膜溶解作用方式不仅限于细胞膜，还可以扩展到线粒体膜的渗透和肿胀，导致细胞色素 c 的释放并诱导细胞凋亡 [26-27]；3）抑制肿瘤血管生成，某些 ACPs 可以通过干扰血管内皮生长因子（vascular endothelial growth factor，VEGF）与其受体的相互作用抑制新生血管的生成，从而抑制肿瘤生长和转移 [28-29]；4）与几种胞内蛋白质相互作用对抗癌细胞，某些 ACPs 被内化到癌细胞内，通过影响蛋白质-蛋白质相互作用（protein-protein interaction，PPI）或调节其构象变化来抑制蛋白激酶、蛋白酶或干扰功能蛋白发挥作用，从而抑制肿瘤的生长、侵袭和转移 [30-32]；5）激活免疫，某些ACPs 通过诱导细胞因子分泌、募集白细胞或刺激免疫细胞来发挥免疫调节功能，从而增强宿主抵抗癌细胞的能力 [33-35]。此外，肽还可以用作药物载体，通过肽的细胞渗透或细胞表面结合特性将化疗药物或细胞毒性剂递送到相应的靶标 [36]，从而实现抗癌治疗。 ACPs 最常见的分类方法是按照其结构划分，可以分为α-螺旋肽、β-折叠肽、无规卷曲肽以及环肽[21]。α-螺旋是 ACPs 最典型的结构类型，该类 ACPs 最丰富的来源是两栖动物的皮肤，从中分离得到的ACPs 包括 magainins，aureins，citropins 家族等 [37-39]。ACPs 的 α-螺旋结构中疏水残基的存在可以增强其抗癌性能 [40-41]。β-折叠 ACPs 中二硫键的形成对维持结构的稳定性至关重要，其结构比 α-螺旋 ACPs更复杂 [17]。一般来说，β-折叠 ACPs 的抗肿瘤活性低于 α-螺旋 ACPs，但 β-折叠 ACPs 对正常组织细胞的毒性较小 [21]。无规卷曲 ACPs 通常富含脯氨酸和甘氨酸，但是缺乏典型的二级结构 [21]。简单来说，环状 ACPs 带有跨越多个氨基酸残基的环结构，环状结构可以提高线性肽序列的细胞穿透能力，从而使环肽具有可调的刚性、稳定性及更好的药代动力学特性，因而备受关注 [42-43]。目前进入临床研究的ACPs 大多数是环状 ACPs，这些肽对癌细胞有很强的抑制作用 [44]。 2 ACPs 的临床应用 目前有越来越多的 ACPs 进入临床，DrugBank[45]中登记的在全球市场上获得批准的 ACPs 超过 20 种，表 1 列举了部分由美国食品药品监督管理局（Food and Drug Administration，FDA）、欧洲药品管理局（European Medicines Agency，EMA）和加拿大卫生部（Health Canada，HC）批准的 ACPs。 促 性 腺 激 素 释 放 激 素（gonadotropin-releasing hormone，GnRH）激动剂 triptorelin 是促黄体素释放激素（luteinizing hormone releasing hormone，LHRH）的合成十肽激动剂类似物。在前列腺癌一线疗法，如雄激素剥夺治疗（androgen deprivation therapy，ADT）中，GnRH 激动剂发挥着主导作用。特别是triptorelin，作为一线治疗激素，已被批准用于晚期前列腺癌的姑息性治疗 [46]。类似的 GnRH 激动剂还有 goserelin 和 buserelin[47]，长期给药可抑制垂体促性腺激素的分泌，从而降低睾酮（男性）和雌二醇（女性）的水平，可用于治疗激素反应性癌症，如前列腺癌或乳腺癌。 Leuprolide 是 GnRH 的人工合成九肽类似物，是 GnRH 受体的超级激动剂。与内源性十肽 GnRH不同，leuprolide 含有单个 D-氨基酸（D-亮氨酸）残基，其循环半衰期由 3 ~ 4 分钟延长至约 3 小时；leuprolide 能够与 GnRH 受体结合并诱导促性腺激素和性类固醇水平的下游调节，用于治疗前列腺癌、子宫平滑肌瘤等 [48]。Histrelin 与 leuprolide 类似，也是 GnRH 激动剂，用于晚期前列腺癌的姑息治疗 [49]。 Lanreotide是生长抑素的人工合成八肽类似物，可与生长抑素受体（somatostatin receptor，SSTR）特别是 SSTR-2 和 SSTR-5 结合，抑制大脑中生长激素的释放，已被批准用于治疗不可切除、中度分化良好、局部晚期或转移性胃肠胰神经内分泌肿瘤和肢端肥大症 [50]。 免疫调节剂 mifamurtide 是含有胞壁酰二肽（muramyl dipeptide，MDP）类似物的脂质体制剂，通过激活巨噬细胞和单核细胞发挥抗肿瘤活性，用于治疗手术切除后高级别、可切除、非转移性骨肉瘤[51]。 Carfilzomib 为蛋白酶体抑制剂，从化学结构上看，其也是一种改性的环氧四肽基和环氧菌素的类似物，可单独使用或与化疗方案联合使用，用于治疗复发或难治性多发性骨髓瘤患者 [52]。 放射性标记的生长抑素类似物 177Lu-DOTATATE属于新兴的治疗方法，称为肽受体放射性核素疗法（peptide receptor radionuclide therapy，PRRT）。该疗法使用携带放射性粒子的分子精准靶向肿瘤，这些粒子与肿瘤表达的特定受体结合，用于治疗SSTR 阳性胃肠胰神经内分泌肿瘤，包括成人前肠、中肠和后肠神经内分泌肿瘤 [53]。 二肽硼酸衍生物 bortezomib 属于蛋白酶体抑制剂，能可逆地抑制 26S 蛋白酶体。该蛋白酶体是一种降解泛氨酸蛋白的大型蛋白酶复合物。Bortezomib 通过阻断蛋白酶体对目标蛋白的水解作用而破坏各种细胞信号通路，从而导致细胞周期停滞、细胞凋亡和血管生成抑制 [54]。 Melphalan flufenamide 为肽药物偶联物，是由烷化剂 melphalan 和对氟-L-苯丙氨酸组成的亲脂性二肽的乙酯，具有潜在的抗肿瘤和抗血管生成活性，2021 年 2 月被 FDA 批准用于治疗复发或难治性多发性骨髓瘤 [55]，但由于其在Ⅲ期临床试验中数据显示总生存率下降而撤出市场 [56]。 3 ACPs 的研发历程 通过设计开发生物活性肽，可以解决常规癌症治疗药物的一些问题，包括水溶性差、缺乏靶点特异性、高细胞毒性以及低治疗指数。第 1 代 ACPs 主要包括源于天然产物的生物活性肽。如有研究表明，从香菇中提取出的肽可以诱导人肺癌细胞凋亡 [57]。从海葵中分离出的五肽通过促凋亡蛋白诱导细胞凋亡增加 [58]。无效或低活性的天然生物活性肽可以通过特殊功能修饰成新的肽，如将一些化学基团（如乙酰化或酰胺化）添加到天然肽中，可以增加阳离子特性和靶细胞特异性 [59]，基于此开发了第 2 代ACPs。此外，癌细胞靶标的发现可以提高癌症治疗的效果，从而避免损伤健康细胞。靶向肽可以特异性地直接结合和破坏癌细胞，但不影响健康细胞，如几种宫颈癌特异性结合肽（cervical cancer specific binding peptide，CSP）-GD 和 CSP-FT 等对宫颈癌细胞具有特异性靶向能力，结合位点主要位于细胞膜和细胞质上 [60]。 随着计算机科学的发展，计算机辅助药物设计（computer-aided drug design，CADD）和人工智 能 药 物 设 计（artificial intelligence drug design，AIDD）越来越多地被科学家们使用，为 ACPs 的研发带来了许多便捷。具体应用将在第 4 部分展开。 生物信息学、蛋白质组学和转录组学的最新进展揭示了一种被称作微肽的短肽，这类短肽在许多生物体的各种生物过程中发挥着重要的调节作用 [61]。基因组学研究表明，区别于传统的小的生物活性肽，微肽由隐藏在错误注释的非编码 RNA（non-coding RNA，ncRNA）中长度小于 300 nt 的小开放阅读框（small open reading frame，sORF）编码，通常是长链非编码 RNA（long noncoding RNA，lncRNA）和环状 RNA（circular RNA，circRNA）[62]。微肽在不同人类癌症的发展中起着重要作用，如与乳腺癌相关的微肽 CIP2A-BP，抑制 CIP2A 癌基因下游的PI3K/AKT/NFκB 通路，诱导基质金属蛋白酶（matrix metalloproteinase，MMP）-2、MMP-9 和转录因子Snail 表达水平降低，从而抑制癌症进展 [63]。微肽在调节肿瘤能量代谢、癌细胞上皮到间充质细胞转化、c-Myc 癌蛋白的稳定性以及肿瘤血管生成中发挥重要作用，因此可作为对癌症治疗有前途的候选药物、药物靶点或预测癌症患者预后的生物标志物 [64]。 4 计算机背景下的 ACPs 设计 4.1 CADD 在 ACPs 研发中的应用 肽的计算机辅助设计主要基于肽-蛋白质相互作 用（peptide-protein interaction，PepPI）， 包 括 2个步骤（见图 1）。首先要选取初始肽支架，这些支架在肽药物开发中起着关键作用。从天然蛋白质中可以找到许多表征良好的天然肽，并被证明能够保留其原始功能，如识别目标分子的能力。如精氨酸-甘氨酸-天冬氨酸（Arg-Gly-Asp，RGD）基序最初从纤连蛋白的细胞附着结构域中衍生出来，该结构域与膜结合受体蛋白整合素结合并激活细胞生长、分化、黏附和迁移 [65]；除了从天然蛋白质结构中获取初始肽支架外，人们还使用了噬菌体肽库筛选和基于计算机建模的设计方法选取初始肽支架，如 Jana 等 [66] 从由 14 种四肽组成的小肽库中筛选、设计合成了一种能够扰乱微管功能并导致癌细胞凋亡的新型四肽丝氨酸-亮氨酸-精氨酸-脯氨酸（Ser-Leu-Arg-Pro，SLRP）。 在选定初始肽支架后，需要对多肽和蛋白质进行对接。与 PPI 类似，可用的药物靶标的结构信息限制了 PepPIs 的预测准确性。很多研究都是利用来自结构数据库 [ 如蛋白质数据库（Protein Data Bank，PDB）[67]] 的现有信息来确定肽与蛋白质的相互作用。PDB 中肽-蛋白质结构数量的增加极大地促进了对接方法的开发和优化，从而能更准确地预测 PepPIs。肽-蛋白质的对接方法通常根据所获取的结构信息详尽程度，分为局部对接、全局对接和基于模板的对接。表 2 总结了几种肽-蛋白质对接方法的主要特点。 局部对接是最常用的策略，其在目标受体已解析的结构中搜索肽段在用户定义的结合位点上的潜在结合姿态。DynaDock[68]，Rosetta FlexPepDock[69]和 PepCrawler[70] 是最流行的方法，它们提供了不同的定义肽结合位点的方法。但并不是每个查询肽都有可用的骨架构象信息，在进行局部对接之前，允许获取近天然肽构象的采样方法变得十分重要。例如，Rosetta 软件中 FlexPepDock ab-initio（从头计算）[71] 是一种结合局部对接与从头折叠肽的方法，在去除受体结构的内部冲突后，将肽放入指定结合位点，并编译相应的肽骨架片段库，随后应用FlexPepDock ab-initio 先对肽骨架构象进行刚性低分辨率优化，然后对所有肽和受体侧链进行柔性高分辨率优化。 当没有关于结合位点的先验信息时，全局对接通常是首选方法。与搜索肽结合姿态的局部对接不同，全局对接方法还搜索靶蛋白处的肽结合位点。全局对接方法能够检索与肽的最终结合构象非常相似的结构片段。例如，ClusPro PeptiDock[72] 和 pepATTRACT[73]使用预定义的模板构象集合来查询序列线索，生成的肽段构象在之后的模拟中进行刚性对接。 除了局部对接与全局对接，还有一种基于模板的对接方法，也称作比较对接方法。这种方法使用已知结构作为模板支架，通过将查询肽段或目标蛋白的序列线索串联起来，构建相互作用复合物模型 [74]。随着 PBD 中肽 - 蛋白质结构数量的快速增长，模拟算法的开发效率大大提升，基于模板的对接逐渐被认为是肽-蛋白质对接的新类别。GalaxyPepDock[75]通过从实验确定的结构中查找模板来执行基于相似性的对接，并使用能量优化构建模型，以便更准确地预测相互作用复合体之间的结构。PepComposer[76]采用在单体蛋白质中发现的基序来构建其结构骨架，相较于从头设计的方法，该方法的优势在于所识别的骨架片段不仅具有化学上的三维立体结构，而且已经自然演化至能与蛋白质上特定的位点相契合。 4.2 AIDD 在 ACPs 研发中的应用 借助 AI 技术，科学家得以在体外实验前，精准预测并有效筛选出潜在的 ACPs 候选物。在肽设计领域，AIDD 的核心应用在于基于序列的计算方法，特别是机器学习与深度学习算法，它们能够快速预测肽的治疗潜力。通过机器学习构建的一个循环选择过程，结合对实验验证肽序列的深入分析，显著缩短了获取最优肽序列的时间周期 [77]。深度学习技术还可以从肽序列中抽取关键特征，进而实现对 ACPs 的有效预测 [78]。这些先进的 AI 手段，能够在成千上万乃至数百万计的候选肽中，迅速筛选并锁定那些具有抗肿瘤活性的潜在 ACPs。机器学习 [77]、深度学习 [78] 和混合学习 [79] 算法已用于开发、识别和预测 ACPs。表 3 中总结了一些 ACPs 预测工具[41,77-93]，展示了AI在该领域的广泛应用与显著成效。 ACPs 的活性会受到氨基酸组成及其结构的影响 [23]。基于序列的 ACPs 研发流程中，首先需要输入 ACPs 与非 ACPs 的数据集，再进行特征提取、分类及预测。数据集的质量和数量决定了机器学习或深度学习模型筛选 ACPs 的准确性。目前知名的ACPs 数据库主要是 CancerPPD[94]，此外还有一些抗菌肽数据库中也收录有部分 ACPs，如 DRAMP[95]。对现有 ACPs 数据库或数据集中氨基酸出现频率进行统计，结果表明 ACPs 中带正电荷的氨基酸及疏水氨基酸出现频率较高，而带负电荷氨基酸的出现频率相对较低，这一特点可为新型ACPs的设计提供参考[95]。 在肽药物开发与预测的研究中，常用的机器学习算法包括支持向量机（support vector machine，SVM）、朴素贝叶斯（naive Bayes，NB）、k-近邻算法（k-nearest neighbors，KNN）、人工神经网络（artificial neural network，ANN）、决策树（decision trees，DT）和随机森林（random forest，RF）等。作为经典的机器学习算法，SVM[96] 与 RF[97] 被广泛应用于构建 ACPs 预测模型，SVM 采用获得最大边距超平面（maximum-margin hyperplane）的策略，RF 是一种基于决策树的分类器，采用 bootstrap取样和随机特征选择。早期开发中 Tyagi 等 [98] 和Hajisharifi 等 [81] 分别使用了 200 个左右样本量的数据集，将 SVM 的计算算法应用于 ACPs 预测。此后，出现了越来越多的基于机器学习的预测模型，包括iACP-GAEnsC[84] 和 MLACP[77] 等， 特 别 是 iACP-GAEnsC 集成了多种分类算法并使用 3 种特征方式开发，预测准确率达 96.45%。 深度学习是机器学习的一种，其概念源于 ANN的研究。区别于浅层学习，深度学习强调了学习样本数据的内在规律和表示层次并明确了特征学习的重要性，常用的算法包括卷积神经网络（convolutional neural network，CNN）、递归神经网络（recurrent neural network，RNN）、 长 短 期 记 忆（long short-term memory，LSTM）等，并引入注意力机制。使用深度学习开发的 ACPs 预测工具包括 DeepACP[91]与 ACPred-LAF[92] 等。DeepACP 结合了具有双向长短期记忆单元的 RNN 模型和氨基酸字符嵌入，展现了深度学习算法在肽预测方面的巨大潜力。 混合学习则是结合了深度学习与机器学习的方法，例如，在 iACP-DRLF[79] 的混合学习过程中，数据分割、嵌入和特征提取是由深度学习算法执行的，提取到的特征被输入到机器学习算法中进行分类。 5 结语与展望 ACPs 因其对癌细胞的选择性和治疗潜力越来越受到关注。ACPs 除了可以直接作为药物分子发挥治疗作用，还可以作为靶向分子与化疗药物或细胞毒性剂偶联发挥递送作用，最近兴起的微肽也显示出用于癌症治疗与诊断的潜力。随着 AI 工具的引入，ACPs 的开发也更加便捷，已有越来越多的 ACPs 进入临床。基于 AI 开发的结构预测工具 AlphaFold2，能够仅凭蛋白质序列就可精准预测其三维结构；AlphaFold3 的性能则进一步提升，其还能够预测蛋白质间的相互作用，以及蛋白质与小分子、DNA 之间的相互作用模式，从而促进基于PepPI 的多肽药物研发，这些进步极大地加速了药物靶标的识别和新药的研发进程；随着深度学习等算法的发展，AI 预测模型在计算速度和准确度方面都得到大幅度提升，可以在湿实验前进行快速筛选，从而促进新型 ACPs 的设计和研发。虽然当前癌症治疗的主流疗法仍然是化疗，但是随着 ACPs 研究的不断深入，未来 ACPs 治疗或许能成为癌症治疗的重要手段。 参考文献 ： [1] Bray F, Laversanne M, Weiderpass E, et al. 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Sci Rep, 2013, 3: 2984[2023-12-28]. http://doi.org/10.1038/srep02984. 美编排版：陈鑫茹 感谢您阅读《药学进展》微信平台原创好文，也欢迎各位读者转载、引用。本文选自《药学进展》2024年第 12 期。 《药学进展》杂志由国家教育部主管、中国药科大学和中国药学会共同主办，中国科技核心期刊（中国科技论文统计源期刊）。刊物以反映药学科研领域的新方法、新成果、新进展、新趋势为宗旨，以综述、评述、行业发展报告为特色，以药学学科进展、技术进展、新药研发各环节技术信息为重点，是一本专注于医药科技前沿与产业动态的专业媒体。 《药学进展》注重内容策划、加强组稿约稿、深度挖掘、分析药学信息资源、在药学学科进展、科研思路方法、靶点机制探讨、新药研发报告、临床用药分析、国际医药前沿等方面初具特色；特别是医药信息内容以科学前沿与国家战略需求相合，更加突出前瞻性、权威性、时效性、新颖性、系统性、实战性。根据最新统计数据，刊物篇均下载率连续三年蝉联我国医药期刊榜首，复合影响因子1.216，具有较高的影响力。 《药学进展》编委会由国家重大专项化学药总师陈凯先院士担任主编，编委由新药研发技术链政府监管部门、高校科研院所、制药企业、临床医院、CRO、金融资本及知识产权相关机构近两百位极具影响力的专家组成。 联系《药学进展》↓↓↓ 编辑部官网：pps.cpu.edu.cn； 邮箱：yxjz@163.com； 电话：025-83271227。 欢迎投稿、订阅！ 往期推荐 聚焦“兴药为民·2023生物医药创新融合发展大会”“兴药为民·2023生物医药创新融合发展大会”盛大启幕！院士专家齐聚杭城，绘就生物医药前沿赛道新蓝图“兴药强刊”青年学者论坛暨《药学进展》第二届青年编委会议成功召开“兴药为民·2023生物医药创新融合发展大会”路演专场圆满收官！校企合作新旅程已启航 我知道你在看哟

Press ReleaseParis, 30th July 2024 Net Sales down 9.6% year-on-year – Core EBITDA margin at 10.6%Accelerated commercial momentum with clients other than Sanofi, with Net Sales up 2.9%, excluding the Brindisi site, where production was temporarily suspendedEnhanced effectiveness across the entire organization, with sizeable improvements in industrial efficiencies€10 million Free Cash Flow before financing, driven by strengthened financial disciplineFY 2024 guidance confirmed " Our H1 actions set the foundations of our more agile, streamlined, and value-creating model. Over the past months, with the entire organization's support, we have made significant progress in deploying the four strategic pillars of our operational roadmap. Thanks to a comprehensive mitigation plan, we will resume shipment and production in Brindisi in the coming weeks." said Ludwig de Mot, Chief Executive Officer of EUROAPI. "Confident in the collective success of our plan, we are in advanced discussions with our stakeholders to finalize the financing of FOCUS-27 in the coming weeks, which will pave the way for long-term profitable growth.” €448.7 million in Net Sales, down by 9.6%1, shaped by the strong decrease in volumes from Sanofi and the suspension of production in Brindisi, which overshadowed the positive momentum of sales to clients other than Sanofi Brindisi GMP license reactivated mid-July; shipments and production to restart gradually during Q3 2024 Signature of a major CMO commercial phase contract with a global animal health company and a development and manufacturing agreement with Priothera, a biotechnology company specializing in oncology €47.6 million Core EBITDA, with a 10.6% Core EBITDA margin, down from 12.6% in H1 2023. Price increases and product mix, industrial efficiencies, and the impact of the revised commercial contractual clauses with Sanofi over the period partially offset the unfavorable fixed-cost absorption. €(1.4) million EBITDA, including €47.2 million exceptional costs related to the implementation of FOCUS-27. €(170.2) million Net Debt compared to €(171.0) million at the end of December 2023, with €10.0 million Free Cash Flow before financing, compared to €(111.2) million for the same period last year, driven notably by Controlled CAPEX (€61.3 million, of which 56% dedicated to growth) Improved Operating Working Capital thanks to a rigorous inventory reduction program and better cash collection €(34.8) million Net income, compared to €62.8 million in H1 2023 H1-2024 Key figures (in € millions)H1-2024H1-2023Net Sales448.7496.6Year-on-year change in %-9.6%+2.6%Gross profit98.097.0Gross Profit Margin21.8%19.5%EBITDA(1.4)52.1Core EBITDA47.662.5Core EBITDA Margin10.6%12.6%Net Income(34.8)62.8Basic EPS (in euros)(0.37)0.67 2024 outlook confirmed Between 8% and 11% decrease in 2024 Net Sales compared to 2023 on a comparable basis, with a second-half performance slightly exceeding that of the first half due to a phasing impact in CDMO. Core EBITDA margin objective expected between 4% and 7%. The positive impact in H1 of the revised contractual clauses with Sanofi will be lower in the second half. FOCUS-27 plan in action The execution of FOCUS-27 is on track, with several initiatives launched during the first half, and the discussions with the Revolving Credit Facility banking syndicate to finalize the financing announced on June 26, 2024, are in the advanced stage. Streamlined value-added portfolio List of the discontinued 13 APIs communicated to our clients; phase-out roadmap almost completedAccelerated momentum in Opiates, prompted by improving competitive positions Focused CDMO offer Solid and qualitative new late-stage projects with 3 marketed small molecules for large and mid-size pharma and food companies (including GMP intermediates). Increasing momentum in large molecules, with 5 early-stage projects in H1, mainly from large pharma companies, including 4 promising new oligonucleotides and 1 peptide-PMO conjugate project. Rationalized industrial footprint Ramp down of workshops initiated in Frankfurt (9 APIs to be discontinued), and Vertolaye (2 APIs to be discontinued)Inventory reduction program on track Haverhill divestment process well advanced; completion expected in 2025 Organizational transformation Headcount reduction plan initiated in Haverhill following the decrease in volumes of SevelamerContinued enhancement of the management teams across the group, including the reorganization of the Executive leadership team. H1-2024 Net Sales EUROAPI H1-2024 Net Sales reached €448.7 million, -9.6% versus H1 2023 and -9.3% at Constant Exchange Rates. Net sales per type of activity (in € millions)H1H1-2023ChangeAPI Solutions – Other clients168.6169.8-0.7%API Solutions – Sanofi163.7192.7-15.0%API Solutions332.4362.4-8.3%CDMO – Other clients72.382.7-12.7%CDMO – Sanofi44.151.4-14.2%CDMO116.4134.2-13.3%Net sales448.7496.6-9.6%Total Net Sales – Sanofi207.8244.1-14.9%Total Net Sales – Other clients 240.9252.5-4.6% API Solutions API Solutions' net sales decreased by 8.3% to €332.4 million. Sales to Sanofi decreased by 15.0% due to strong volume decrease, notably in Sevelamer, produced in Haverhill, and the suspension of production in Brindisi. H1 net sales include €29 million related to the revision of the historical MSA contractual clauses agreed with Sanofi in February 20242, primarily Buserelin’s3 stock clearance. Sales to Other Clients decreased by 0.7%, impacted by the temporary suspension of API production in Brindisi. Excluding Brindisi’s site, net sales would have grown 3.4%, driven by the cross-selling strategy (representing 9.1% of H1 API Solutions sales to Other Clients) and an increased customer base (22 new clients added in H1). CDMO CDMO sales decreased by 13.3% to €116.4 million. Sales to Sanofi decreased by 14.2% on the back of a strong H1 2023 performance, which was boosted by stock replenishment of Pristinamycin, an anti-infective produced exclusively for Sanofi in Elbeuf. H1 2024 sales benefited from the ramp-up of a commercial phase contract in Large Molecules.Sales to Other Clients decreased by 12.7% due to the temporary suspension of production in Brindisi, which affected a commercial phase contract. H1 2024 performance was also penalized by the downsizing of two large historical commercial phase contracts (approximately -10 M€), which more than offset the increase in revenue from new contracts. Excluding the impact of Brindisi, total CDMO sales to clients other than Sanofi would have grown by 1.8%, with increased revenues from early-stage projects. Throughout the semester, 14 new CDMO contracts were signed, and eight projects were completed, including two in late stage with Sanofi, and two projects were put on hold. (Number of CDMO projects)Phase 1 and earlierPhase 2Phase 3Commercial PhaseTotalLarge molecules742417Highly potent molecules1 12Biochemistry molecules derived from fermentation2 68Complex chemical synthesis molecules9772346Total191193473 Net Sales per type of molecule (in € million)H1-2024H1-2023ChangeLarge molecules58.835.0+67.9%Highly potent molecules47.143.7+7.8%Biochemistry molecules derived from fermentation43.785.5-48.8%Complex chemical synthesis molecules299.1332.4-10.0%Net Sales448.7496.6-9.6% Large molecules increased by 67.9% to €58.8 million, driven by the stock clearance of Buserelin, and the ramp-up of a commercial phase contract in Large Molecules with Sanofi. Highly potent molecules were up 7.8% to €47.1 million on the back of a low comparison base in H1 2023, which was impacted by the suspension of Prostaglandins’ production.Biochemistry molecules derived from fermentation decreased 48.8% to €43.7 million. While H1 2023 performance was boosted by stock replenishment of Pristinamycin, H1 2024 was impacted by the temporary suspension of API production in Brindisi. Complex chemical synthesis molecules decreased by 10.0% to €299.1 million, mainly impacted by the decreasing volume demand from Sanofi. Financial performance (in € million)H1-2024H1-2023Net Sales448.7496.6Other revenues2.21.9Gross profit98.097.0Gross Profit Margin21.8%19.5%EBITDA(1.4)52.1Non-recurring costs49.010.4Core EBITDA47.662.5Core EBITDA Margin10.6%12.6%Operating Income(33.4)16.0Finance revenues/costs(8.1)(3.3)Income before tax(41.5)12.6Income tax expense6.750.1Net income/(loss)(34.8)62.8EPS (in euros)(0.37)0.67Average number of shares outstanding (in millions)94.393.9Fully diluted EPS (in euros)(0.37)0.66Average number of shares after dilution (in millions)95.995.5 Gross profit was €98.0 million, compared to €97.0 million in H1 2023, with the gross profit margin up 231 basis points year-on-year to 21.8%. The exceptional impact of Buserelin’s stock clearance, positive price and mix effect, lower energy and raw materials prices, and improved industrial performance more than offset unfavorable fixed-cost absorption as we sold in H1 24 products manufactured at the peak level of the past 18 months inflation cycle. Core EBITDA amounted to €47.6 million, down 23.8% compared to €62.5 million in H1 2023. The core EBITDA margin was 10.6%, compared to 12.6% in H1 2023. The increase in OPEX was mostly driven by the company's transformation and reorganization. EBITDA was €(1.4) million compared to €52.1 million in H1 2023. The 49.0 million non-recurring costs include €47.2 million in exceptional items4, of which €33.8 million of idle costs5 linked to the execution of FOCUS-27, including the ramp-down of two workshops in Frankfurt started in H1 2024 and reduced inventories in Vertolaye €9.0 million of internal and external costs related to the transformation of the company €4.4 million of employee-related expenses, including redundancy plans. Key components of the change in Core EBITDA marginH124/H123 in percentage points (rounded figures)H1 2023 Core EBITDA margin12.6%Volume-0.0 ptsPrice and Mix+3.0 ptsImpact of Buserelin’s stock clearance+2.1 ptsIndustrial performance+2.4 ptsEnergy and Raw Materials+1.2 ptsUnfavorable fixed cost absorption-5.5 ptsOther Gross Margin impacts -1.9 ptsOPEX -2.9 ptsBrindisi site, including the impact of the suspension of production-0.3 ptsH1 2024 Core EBITDA margin10.6% Operating Income was €(33.4) million compared to €16.0 million in H1 2023. Financial income was €(8.1) million, compared with €(3.3) million in H1 2023, due notably to the increase in interest rates and the full drawdown of the RCF. Income before tax was €(41.5) million6. Net income was €(34.8) million in H1 2024. Net Debt Position and Cash Flow (in € million)30 June 2024Net cash/(Debt) position – December 2023(171.0)Cash Flow from Operating activities71.2Of which change in Working Capital66.1 (Increase)/decrease in inventories 4.1 (Increase)/decrease in trade receivables 40.0 Increase/(decrease) in trade payables (16.2) Other current assets and liabilities 38.3Cash Flow from Investing Activities(61.3)of which CAPEX(61.3)Cash Flow from Financing activities(8.3)Exchange rate((0.9)Net Cash/(Debt) position – June 2024(170.2) The decrease in Net Debt position, €(170.2) million compared to a €(171.0) million at the end of December 2023, was notably driven by Working Capital, including the improvement in inventories. Improved cash collection drove the change in Trade receivables, with DSO down to 53 compared to 56 in December 2023 and 70 in H1 2023. Other current assets and liabilities include a €27 million variation in VAT tax reimbursement. Capex reached €(61.3) million (13.7% of Net Sales), of which 56% were dedicated to growth projects, including increased capacities for Peptides and Oligonucleotides, Vitamin B12, and Prostaglandins. Free Cash Flow before financing activities was €10.0 million, compared to €(111.2) million at the end of June 2023.7 (in € million)30 June 202431 December 2023Bank Cash Balances282.834.5Revolving Credit Facilities(453.0)8(205.5)Net Debt Position(170.2)(171.0) Net Debt to Core EBITDA restated for IFRS 16 was 2.38x, below the RCF covenant of 4.0x.ESG Roadmap EUROAPI ESG roadmap is on track to achieve its 2030 goals and has recently achieved: 100% of its sites certified ISO14001 (environment management) and ISO50001 (energy management). CDP score B, which indicates that the group is addressing the environmental impacts of its business and ensures good environmental management. Main H1 2024 events On January 25, 2024, EUROAPI announced that it had initiated a pivotal collaboration with SpiroChem, a leading Contract Research Organization (CRO).On March 14, 2024, EUROAPI announced the pause of API production at the Brindisi site in Italy after identifying quality control deficiencies and decided to suspend its full-year 2024 guidance.On May 23, 2024, EUROAPI announced a Contract Manufacturing Organization (CMO) agreement with a global animal health company to supply key veterinary product. The total contract value is expected to range between €130 and 150 million over 2025-2029.On June 6, 2024, EUROAPI received official notification from the European Commission that it had been selected as one of the 13 companies eligible to share up to EUR 1 billion in total public funding under the Important Project of Common European Interest (IPCEI) dedicated to the pharmaceutical sector.On June 18, 2024, EUROAPI announced the implementation of a 5-year development and manufacturing agreement with Priothera, a biotechnology company specializing in treating hematological malignancies and improving CAR-T cell therapies.On June 26, 2024, EUROAPI detailed the FOCUS-27 strategy and announced its target to generate €75 million to €80 million annual run-rate incremental Core EBITDA by the end of 2027. More detail on EUROAPI’s website: EUROAPI is moving ahead with its FOCUS-27 plan, setting the foundations for future profitable growth | EUROAPIannounced that the Brindisi site will progressively resume shipments and production during Q3 2024. Glossary and definition of non-GAAP indicators EBITDA and Core EBITDA EBITDA corresponds to operating income (loss) restated for depreciation and amortization and net impairment of intangible assets and property, plant and equipment.Core EBITDA thus corresponds to EBITDA restated for restructuring costs and similar items (excluding depreciation and write-downs), allocations net of reversals of unutilized provisions for environmental risks, and other items not representative of the Group’s current operating performance or related to the effects of acquisitions or disposals. Cash Flow before Financing activities Cash Flow before Financing activities corresponds to the sum of Cash Flow from Operating Activities and Cash Flow from Investing Activities as presented in the consolidated statement of Cash Flow. Months on Hand (MOH) Net Inventory value at the of the period divided by Net Sales New clients Clients representing at least €50 thousands of net sales on the year. Cross Selling Selling a different product to an existing client that is already buying one or several products from EUROAPI. Early-stage and Late-stage projects Early-stage: pre-clinical, phase 1, and phase 2Late-stage: phase3, in validation, and commercial Presentation of H1-2024 results An analysts’ conference call will be held by EUROAPI’s management tomorrow (31 July 2024) at 8:30 a.m. CET via an audio webcast (live and replay), and the results presentation will be available on the corporate website 2024 Half year results | EUROAPI Financial agenda (all dates to be confirmed) 11 February 2025: FY 2024 Results21 May 2025: 2025 AGM29 July 2025: H1 2025 results About EUROAPIEUROAPI is focused on reinventing active ingredient solutions to sustainably meet customers’ and patients’ needs around the world. We are a leading player in active pharmaceutical ingredients with approximately 200 products in our portfolio, offering a large span of technologies while developing innovative molecules through our Contract Development and Manufacturing Organization (CDMO) activities. Taking action for health by enabling access to essential therapies inspires our 3,650 people every day. With strong research and development capabilities and six manufacturing sites, all located in Europe, EUROAPI ensures API manufacturing of the highest quality to supply customers in more than 80 countries. EUROAPI is listed on Euronext Paris; ISIN: FR0014008VX5; ticker: EAPI). Find out more at www.euroapi.com and follow us on LinkedIn. Media Relations contact:Laurence BollackTel.: +33 (0)6 81 86 80 19mr@euroapi.com Investor Relations contacts:Sophie Palliez-CapianTel.: +33 (0)6 87 89 33 51Sophie.palliez@euroapi.com Camille RicotierTel: +33 (0)6 43 29 93 79Camille.ricotier@euroapi.com Forward-Looking StatementsCertain information contained in this press release is forward looking and not historical data. These forward-looking statements are based on opinions, projections and current assumptions including, but not limited to, assumptions concerning the Group’s current and future strategy, financial and non-financial future results and the environment in which the Group operates, as well as events, operations, future services or product development and potential. Forward-looking statements are generally identified by the words “expects”, “anticipates”, “believes”, “intends”, “estimates”, “plans” and similar expressions. Forward looking statements and information do not constitute guarantees of future performances, and are subject to known or unknown risks, uncertainties and other factors, a large number of which are difficult to predict and generally outside the control of the Group, which could cause actual results, performances or achievements, or the results of the sector or other events, to differ materially from those described or suggested by these forward-looking statements. These risks and uncertainties include those that are indicated and detailed in Chapter 3 “Risk factors” of the Universal Registration Document filed with the French Financial Markets Authority (Autorité des marchés financiers, AMF) on April 5, 2024. These forward-looking statements are given only as of the date of this press release and the Group expressly declines any obligation or commitment to publish updates or corrections of the forward-looking statements included in this press release in order to reflect any change affecting the forecasts or events, conditions or circumstances on which these forward-looking statements are based. Appendix Consolidated Income Statement (in € millions)30-Jun-2430-Jun-23Net sales448.7 496.6 Other revenues2.2 1.9 Cost of sales(352.9)(401.4)Gross profit98.0 97.0 Selling and distribution expenses(18.7)(21.3)Research and development expenses(13.9)(13.3)Administrative and general expenses(46.6)(42.8)Other operating income1.3 0.7 Impairment of assets(3.9) Restructuring costs and similar items(49.7)(4.3)Operating income(33.4) 16.0 Financial expenses(11.2)(5.3)Financial income3.1 1.9 Income/(loss) before tax(41.5) 12.6 Income tax expense6.7 50.1 Net income/(loss)(34.8) 62.8 Number of shares outstandings94.393.9EPS(0.37)0.67Diluted number of shares 95.995.5EPS diluted(0.37)0.66 Consolidated Balance Sheet (in € millions)30-Jun-2431-Dec-23Goodwill4.6 4.6 Property, plant and equipment469.0 468.9Right-of-use assets35.2 37.2Intangible assets38.9 34.2Other non-current assets6.7 9.0Deferred tax assets85.7 79.2Non-current assets640.2633.1Inventories638.7 644.8Trade receivables176.7 216.3Other current assets57.5 83.7Cash and cash equivalents282.8 34.5Current assets1,155.8979.3Total assets1,795.91,612.4(in € millions)30-Jun-2431-Dec-23Equity attributable to owners of the parent889.5927.7Total equity889.5927.7Non-current lease liabilities14.515.5Provisions155.3158.6Other non-current liabilities0.00.0Deferred tax liabilities0.81.6Non-current liabilities170.6175.7Trade payables143.6159.6Other current liabilities135.1139.3Current lease liabilities4.34.6Short-term debt and other financial liabilities452.8205.4Current liabilities735.9508.9Total equity and liabilities1,795.91,612.4 Consolidated Statements of Cash Flow (in € millions)30-Jun-2430-Jun-23Net income / (loss)(34.8)(62.8)Depreciation & amortization 32.0 36.1 Net change in current & deferred taxes(6.7)(50.1)Other profit or loss items with no cash effect and reclass of interest 14.7 8.2 Operating cash flow before changes in working capital 5.1 56.9 (Increase)/decrease in inventories 4.1 (66.0)(Increase)/decrease in trade receivables 40.0 30.1 Increase/(decrease) in trade payables(16.2)(49.0)Net change in other current assets and other current liabilities 38.3 (10.3)Net cash provided by operating activities 71.2 (38.2) Acquisitions of property, plant and equipment and intangible assets(61.3)(73.1)Acquisition of shares on consolidated entities, net of cash acquired - Sales/Acquisitions to/from Group entities 0.0 - Net cash (used in) investing activities(61.3)(73.1)Capital increases - - Repayment of lease liabilities(2.7)(4.6)Net change in short-term debt 246.0 100.0 Finance costs paid(4.1)(2.0)Acquisitions and disposals of treasury shares(0.0)(0.2)Other net cash flow arising from financing activities(0.3)(0.0) Net cash provided by financing activities 238.8 93.2 Impact of exchange rates on cash and cash equivalents(0.5) 0.6 Net change in cash and cash equivalents 248.3 (17.5) Cash and cash equivalents at beginning of period 34.5 74.5 Cash and cash equivalents at end of period 282.8 57.0 Reconciliation of Consolidated Operating Income (EBIT) to restated Core EBITDA (in € millions)30-Jun-2430-Jun-23Operating income(33.4) 16.0 Depreciation and amortization 32.0 36.1 EBITDA(1.4) 52.1 Restructuring costs and similar items (excluding depreciation and amortization) 47.2 4.3 Of which idle costs33.8 Of which employee-related expenses4.4 Of which internal and external costs related to FOCUS-27, and transformation9.04.3Allocations net of reversals of unutilized provisions for environmental risks(0.2)(0.3)Other 2.0 6.3 Core EBITDA 47.6 62.5 Core EBITDA10.6%12.6% Idle costs are mainly affecting Vertolaye, with the inventory reduction program being the main business factor, as well as the discontinuation of 2 APIs, Frankfurt, with the decision to discontinue 9 APIs (the ramp down of two workshops has already started in 2024). Employee-related expenses of €4.4 million include the impact of the redundancy plan announced in EUROAPI UK and severance payments in connection with the renewal of the Executive leadership teams. Restructuring costs correspond to expenses incurred in connection with the transformation or reorganization of the EUROAPI Group’s operations and support functions. These costs include collective redundancy plans, compensation awarded to third parties for the early termination of contracts, commitments made in connection with transformation and reorganization decisions, and idle costs related to the temporary shutdown of sites or production lines associated with such programs.They also include accelerated depreciation charges arising from closures of production facilities (including leased facilities), and losses on any resulting asset disposals.In addition, restructuring costs and similar items comprise expenses (both internal and external)incurred in connection with FOCUS-27. 1 All comments in this press release are made compared to H1 2023 figures unless stated otherwise2 Full-year expected impact: €38 million (Regulated Agreement approved by the May 22nd AGM)3 Buserelin is a large molecule used primarily in the treatment of prostate cancer and endometriosis.4 See appendix page 13 5 Under-activity triggered by the implementation of FOCUS-27 6 H1 2023 income tax included €46.8m deferred tax income related to the revaluation of EUROAPI’s Hungarian assets.7 See detailed in Consolidated Cash Flow Statement page 128 RCF: €451 million + €2 million accrued interests Attachment EUROAPI - Press release - July 30, 2024