Hemin

“ 点击蓝字 关注我们 钱程根 博士，中国药科大学药学院副教授，博士生导师。江苏省优秀青年基金、江苏省双创博士、江苏省优秀博士论文和南京大学优秀博士论文获得者。从事药物制剂、生物功能材料及化学生物学等交叉领域的研究，近年主要关注肿瘤免疫治疗和阿尔茨海默病早期诊断和治疗研究。近年来在 Natl Sci Rev, PNAS, Sci Adv, Adv Mater, Adv Funct Mater, Angew Chem Int Ed Engl, ACS Nano, Nano Lett 等国际权威杂志共发表同行评议论文 43 篇，其中（共同）第一或通讯作者发表论文 14 篇（8 篇 IF ＞ 10），总被引 3 670 余次，单篇最高引用 450 次，H-index 为 31，申请中国发明专利 5 项。其中，发表在 Adv Mater, Angew Chem Int Ed Engl, Chem-A Eur J 的文章分别被选为封面文章或热点文章；目前有 3 篇文章入选 ESI 高被引论文。 肿瘤光动力治疗纳米制剂的研究进展PPS  李倪博文，汤迎琦，侯仁杰，高子然，钱程根 * （中国药科大学药学院药剂系 多靶标天然药物全国重点实验室，江苏 南京 210009） [摘要] 光动力疗法（photodynamic therapy，PDT）是一种非侵入性光化学疗法，它依赖于光敏剂在光照射下将光子能量转化为化学能，产生诱导细胞毒性的活性氧（reactive oxygen species，ROS），特别是用于肿瘤治疗。目前肿瘤 PDT 的临床应用受到肿瘤乏氧、还原性物质浓度高、免疫抑制、光穿透深度有限和肿瘤自救体系等肿瘤微环境因素的限制。随着纳米技术的发展，现在可以在纳米尺度上对 PDT 药物制剂进行精细化设计。以纳米技术增强 PDT 疗效为主题，对目前在增强氧气供应、改变肿瘤代谢和其他抗肿瘤疗法联用领域 PDT 纳米制剂的研究进展进行综述，为之后进一步开发符合临床需求的 PDT 纳米制剂提供参考。 近 20 年来，恶性肿瘤一直是我国人口的重要死亡诱因，改善肿瘤疗法顺应性，提高恶性肿瘤治愈率对提高全民生命健康水平具有重要意义 [1]。当前，我国肿瘤治疗的核心策略依旧聚焦于手术治疗，并辅以放射治疗与化学治疗，但这一系列疗法伴随的手术并发症、放化疗的不良反应以及肿瘤细胞的耐药性等问题，极大地削弱了肿瘤患者的生存质量 [2]。光动力疗法（photodynamic therapy，PDT）作为一种非侵入性治疗手段，正日益受到肿瘤治疗研究领域的青睐，相较于传统疗法，PDT 凭借其低毒性、微创性、经济性以及较低的耐药性等显著优势，展现了广阔的应用前景 [3]。PDT 主要通过产生活性氧（reactive oxygen species，ROS）引起肿瘤细胞DNA 突变、蛋白失活、氧化应激等反应最终诱导细胞凋亡 [4]。PDT 通过 2 种途径产生 ROS，光激发的光敏剂（Ⅰ型）直接与周围分子发生电子转移产生过氧化氢（H2O2）、羟基自由基（• OH）等 ROS，或是激发态光敏剂（Ⅱ型）和基态氧分子（3O2）发生能量交换产生反应性更强的激发单线态氧分子（1O2）。然而，PDT 产生 ROS 效果受到肿瘤微环境的制约，如肿瘤快速增殖耗氧阻碍 ROS 产生、肿瘤高浓度还原性谷胱甘肽（glutathione，GSH）还原ROS、及高间质压肿瘤抵抗 PDT 制剂递送等 [5]。纳米剂型以生物利用度高、比表面积大、体循环时间延长等特性，相较于传统剂型展现出显著优势。以纳米剂型为平台巧妙搭配药物并辅以合适的制剂设计可以设计大量新型 PDT 制剂来提高 PDT 抗肿瘤疗效（见图 1），这一策略不仅为克服 PDT 面临的微环境挑战提供了新思路，也为肿瘤治疗领域带来了革命性的进步。 1 改善肿瘤乏氧微环境增强光动力治疗 ROS 的浓度对 PDT 的抗肿瘤效果至关重要，但肿瘤的乏氧环境限制了 ROS 产生。目前的研究展示的有效改善肿瘤乏氧的策略主要从原位产氧、载体递氧、前药反应产生 ROS 等手段来提升 PDT 的ROS 生成效率；此外，研究还致力于开发新型Ⅰ型光敏剂，这类光敏剂在光激发下能够不依赖于高氧环境即可有效产生 ROS，从而降低了 PDT 对氧气的需求。本文涉及的光敏剂汇总于表 1。 1.1 分解过氧化氢增氧 1.1.1 递送 H2O2 酶  代肿瘤微环境中的 ROS 主要以H2O2 的形式存在，H2O2 酶（catalase，CAT）作为一种高效的催化剂，能降低 H2O2 分解的活化能，从而加速 H2O2 分解，CAT 和光敏剂的联合递送可解决小区域内 PDT 供氧不足问题。Xu 等 [6] 直接将 CAT 作为药物载体，用酰胺键偶联孟加拉玫瑰红（rose bengal，RB），通过控制 RB、CAT 比例可将 CATRB NPs 粒径保持在 100 nm 以下，该尺寸拥有对线粒体的高效渗透性，起到增氧 PDT 的作用，并直接损伤细胞“动力工厂”——线粒体。然而，CAT 的膜的不透过性和其相对较大的尺寸显著限制了 CAT 的效果，为此，Sim 等 [7] 通过超分子相互作用直接进行颗粒-酶偶联。即将 CAT 整合到未修饰的金属有机框架（metal organic framework，MOF）的表面，在 MOF 辅助下进入细胞，随后 CAT 与 MOF 缓慢脱离，释放出 CAT 以缓解细胞内的乏氧状态，从而增强 PDT 的疗效。为了增强靶向性，Zhang 等 [8]合成了可靶向膀胱癌细胞的纳米 DNA 适配体，将适配体和光敏剂二氢卟吩 e6（chlorin e6，Ce6）以及 CAT 连接，构建出 NT-CAT-Ce6 复合制剂。这种复合制剂能够在膀胱肿瘤病灶部位进行选择性增氧PDT，为膀胱癌的治疗提供了一种新的、更具针对性的策略。 1.1.2 纳米酶仿生 H2O2 酶  CAT 作为一种天然酶，对其稍剧烈处理可能导致失活，因此，开发模仿CAT 活性的非生物材料作为纳米酶，成为了实现高效催化能力的一条可行路径。Liang 等 [9] 以三氰基呋喃（tricyanofuran，TCF）为受体，以苯并噻唑（benzothiazole，BTH）/吲哚（indole，IND）为供体，同时合成了近红外七甲菁（heptamethine cyanines，Cy7），由于 BTH 相比 IND 氢键较少，氢键和分子间 π-π 相互作用的微调形成了松散的分子堆，聚集态的 BTH-Cy7-TCF 表现出更高的单线态氧产生量和竞争性光热转换效率，同时这种独特的分子堆积结构为 H2O2 提供了一个结合位点和催化中心，使其具有类似催化酶的活性，从而进一步提高了 PDT 的效率。 1.1.3 生物酶的联合作用 葡萄糖氧化酶（glucose oxidase，Gox）可专一性地氧化葡萄糖生成葡萄糖酸和 H2O2，为了进一步优化这一过程分解产氧效果，Jiang 等 [10] 以球形二氧化锰（manganese dioxide，MnO2）为基础合成介孔空心纳米粒 H-MnO2, 在其中装入 Gox 和 Ce6。这种 H-MnO2 纳米颗粒具有 pH 和 H2O2 响应性，能够控制封装在其内部的 Ce6 和Gox的释放，有效避免了它们在体液中的提前暴露。此外，Gox 与具有 CAT 样活性的 H-MnO2 之间发生的互催化反应，显著增强了氧依赖性 Ce6 介导的 PDT 效果，并协同 Gox 诱导的饥饿疗法，共同提升了治疗效果。为了加速 ROS 的生成效率，Yang 等 [11] 设计了一种自供氢离子和加速氧气生成效率的仿生杂化纳米酶，这一创新设计不仅实现了光激活下 ROS 信号的显著放大，还有效增强了乏氧肿瘤的治疗效果。 1.2 构建携氧载体 1.2.1 血红蛋白作为氧载体  人体中红细胞依靠血红蛋白（hemoglobin，Hb）和氧气的可逆结合运载氧气。Ding 等 [12] 受此启发合成了一种具有近红外荧光活性的半导体聚合物纳米颗粒（semiconducting polymer nanoparticle，SPN），并将 SPN 与 Hb 连接，同时包覆红细胞膜（red blood cell membrane，RBCM）。这层 RBCM 涂层赋予了 SPN-Hb 出色的生物相容性和长期血液循环能力，显著促进了 SPN 在肿瘤区域的富集。该 SPN 不仅可以用作 PDT 也可以用于荧光成像，实现了诊疗一体化，同时，通过与 Hb 的结合可作为氧载体缓解肿瘤缺氧，增强 PDT 疗效。基于 Hb 的研究有很多报道，但由于其载氧能力的局限性和结构的不稳定性，其实际应用仍面临一定挑战。 1.2.2 全氟化碳作为氧载体 全氟化碳（perfluocarbon，PFC）能够在给定的氧分压下保持相对较高的氧含量。凭借弱范德华相互作用，PFC 具有较好的水溶性。人体中 PFC 能够通过皮肤毛孔或呼吸作用排出体外，这赋予了它高度的生物相容性特质。Yang等[13] 开发了一种基于模块化设计和共组装纳米技术的无载体氧纳米罐，该纳米罐由新型氧化还原敏感前药（FSSP）和耗氧量抑制剂阿托伐醌（atovaquone，ATO）共同组装形成，其中 FSSP 结合了全氟己胺和焦磷烷 a，集成了氟化、响应和光动力功能。这种纳米罐能够在实现充分富氧的同时降低肿瘤内的耗氧量，从而彻底缓解肿瘤缺氧，并显示出卓越的携氧能力、良好的药代动力学以及可按需释放 ATO 的特性。Zhang 等 [14] 设计了一种可以光热控制的“氧蛋”，即将全氟化物内核封装在功能化双层聚合物外壳中。通过改良两步原位生长工艺，在聚合物外壳表面连接具有优异光热能量转换能力的近红外光热剂，实现热触发氧气释放，提供了一条可控和有效氧气释放的新途径。Yan 等 [15] 对 PFC 递氧的多模式应用进行了深入的探索，即将具有核磁成像性能的四氧化三铁加载到聚苯乙烯微球中，并用 PFC 填充剩余空间，在外部包裹聚多巴胺以防止 PFC 泄露，最后装载聚乙二醇（polyethylene glycol，PEG）改性的 Ce6。整个纳米体系可用于超声成像、核磁共振成像和荧光成像从而引导增强 PDT。 1.2.3 生物气囊泡作为氧载体 生物气囊泡（gas vesicle，GV）作为一种新型氧气载体具有缓解肿瘤缺氧的潜力。GV 是来源于藻类的，自然形成的充满气体的蛋白质壳隔室。Song 等 [16] 从小球藻中提取 GV 并在表面修饰一层脂质，形成 GV 脂质体。实验表明，GV 脂质体在缺氧溶液、缺氧细胞以及皮下肿瘤内均显著改善氧浓度，皮下肿瘤注射 GV并配合 PDT 可以观察到肿瘤细胞凋亡和坏死显著增强。此外，Yang 等 [17] 将光敏剂吲哚菁绿（indocyanine green，ICG）加载到载氧纳米气泡膜上，使得 ICG治疗效果稳定提升。尽管利用 GV 直接递氧仍可在一定程度上改善肿瘤乏氧，但存在半衰期短、载氧量低甚至可能发生溶血风险等问题。 1.3 反应产氧 除了通过分解肿瘤 H2O2 和开发氧气载体远程运送氧气外，直接在肿瘤部位反应产氧也是 PDT 供氧的选择之一。过氧化钙（calcium peroxide，CaO2）是一种遇水反应可生成氧气和氢氧化钙的金属过氧化物，Shen 等 [18] 把 CaO2 装在酸响应沸石咪唑骨架 -90（zeolitic imidazolate framework-90，ZIF-90）中覆以 Ce6-PEG 形成 CaO2@ZIF-Fe/Ce6@PEG，酸响应 ZIF-90 在肿瘤中分解，CaO2 反应产生氧气同时 CaO2 的酸性水解还产生副产物 H2O2，可补充肿瘤中 H2O2 不足。Sun 等 [19] 以 MOF 为基体封装光敏剂四苯基卟啉 [5，10，15，20-tetrakis(4-carboxyphenyl) porphyrin，H2TCPP]，随后在 MOF 表面沉积了纳米过氧化镁（magnesium peroxide，MgO2），并通过连接靶向肿瘤的 DNA 适配体，最终制成了 HfMOF-MgO2/DNA 纳米结构。DNA 适配体的介入很容易解决了 MgO2 在体内提前反应的问题，使 HfMOF-MgO2/DNA 在接近肿瘤部位再发挥作用。  细菌具备独特的肿瘤靶向能力，其中具有光合自养能力的光合细菌还可以利用类囊体膜上的叶绿素分子进行放氧光合作用。蓝藻，作为自然界中一类重要的光合细菌，为地球上生命的出现和进化过程提供重要的氧分子。基于蓝藻的析氧特性，联合光合细菌进行治疗是缓解肿瘤缺氧和促进 PDT 的有前途的方法。Qi 等 [20] 把黑磷纳米片（black phosphorus nanosheet，BP NS）和蓝藻细胞连接，利用蓝藻的光合作用产氧活性生产 ROS。Zhang 等 [21] 通过采用特制的 CP@ICG 系统，也实现了类似的效果。该系统不仅为光敏剂 ICG 提供了必要的氧气支持，而且 CP 在非光照条件下需要摄取肿瘤部位的葡萄糖等营养物质，以维持自身能量供应，从而有效抑制肿瘤生长，实现了长期的抗肿瘤效果。此外，Teng 等 [22] 将光合细菌与 Ce6 光敏剂和金纳米颗粒（Au NPs）光热剂相结合，得到的 Bac@Au-Ce6 可以有效靶向肿瘤组织，进一步增强 Au-Ce6 的肿瘤积累，其中光合细菌在光照射下持续产生氧气，显著改善肿瘤缺氧状态。 1.4 非氧依赖 PDT 肿瘤内部的缺氧环境对 ROS 的产生构成了直接限制，特别是对依赖于氧气的Ⅱ型光敏剂而言，其 ROS 产生效率受到了显著影响。相比之下，Ⅰ型光敏剂在产生 ROS 的过程中无需氧气，能够直接与水分子等底物发生反应，因此对氧气的需求相对较低。鉴于此，开发适宜的Ⅰ型 PDT 制剂，有望弥补当前以Ⅱ型光敏剂为主导的 PDT 制剂在面对肿瘤缺氧挑战时的不足，为 PDT 领域带来新的突破和发展机遇。  鉴于电荷转移是Ⅰ型 PDT 产生 ROS 的重要步骤，提高光敏剂周围电子丰度于提升Ⅰ型光敏剂效果而言至关重要。Chen 等 [23] 将碘离子引入 BODIPY 得到了激发态寿命更长的Ⅰ型光敏剂IBAB（iodine substituted butoxy-aza-BODIPY），同时将 IBAB 包裹在甲氧基-聚（乙二醇）[methoxypoly(ethyleneglycol)，mPEG]-b-聚 [2-（二异丙基氨基）乙基甲基丙烯酸酯 ][poly(2-(diisopropylamino) ethyl methacrylate，PPDA)] 中，mPEG-b-PPDA 不仅是 IBAB 的载体，其富电子环境在 IBAB 激发时可以提供电子给激发态的 IBAB 促进电荷分离，极大地提升了 IBAB 产生 ROS 效果。Hu 等 [24] 将芴取代的 BODIPY（D）和苝二酰亚胺（A）通过四氢键超分子相互作用组装成超分子光敏剂（DA），在光照射下 D 将电子转移到 A，导致电荷分离形成自由基离子对。该体系通过 2 种途径产生羟基自由基（• OH）：一方面，D 的阳离子自由基通过非氧依赖性方法直接氧化水生成羟基自由基（• OH）；另一方面，A 的阴离子自由基将电子转移到丙酮酸上以实现催化循环，利用丙酮酸氧化水分子产生 • OH。Hu 等 [24] 进一步提出了一种电子受体触发的光诱导电子转移（acceptor-triggered photoinduced electron transfer，a-PET）策略，侧重介绍了长波长激光对电子-空穴对的精细调控，通过结合基于非富勒烯支架的光敏剂和 A 这 2 个有机半导体分子来促进电子-空穴对的分离，显著促进Ⅰ型 PDT 途径产生丰富的 ROS。 2 调控肿瘤代谢增强光动力治疗 除上文提及的乏氧环境外，肿瘤还通过一系列特殊代谢机制来对抗 PDT 的效果。具体而言，肿瘤内部高浓度的 GSH 能够还原 ROS，从而降低 PDT 的杀伤力；同时，自噬途径也被肿瘤利用来拯救受损的细胞，进一步削弱 PDT 的疗效。此外，肿瘤乏氧环境还会促进异常血管的增生，这不仅加剧了肿瘤的恶性进展，还引发了高间质压，进而阻碍了 PDT 治疗制剂的有效吸收。因此，阻断乃至逆转这些代谢机制，对于提升 PDT 的治疗效果具有至关重要的意义。 2.1 阻断有氧呼吸 ATO 是呼吸链复合物Ⅲ抑制剂，Zhao 等 [25] 将 ATO 和 Ce6 通过疏水相互作用结合成纳米粒，显著提高了 ATO 和 Ce6 的溶解度和稳定性，还极大地增强了它们的细胞内摄取能力和肿瘤内部的渗透性。受益于 ATO 和 Ce6 协同机制，抑制有氧呼吸，从而减少耗氧，ATO-Ce6 纳米粒展现出更为强劲的抑制肿瘤生长的 PDT 效果。Ren 等 [26] 将阻断耗氧与供氧策略相结合，在脂质体中同时包埋 MnO2、ATO 以及光敏剂金丝桃素，为在不利环境中发展 PDT 提供了新策略。此外，二甲双胍（metformin，Met），这一常用于 2 型糖尿病治疗的药物，近年来被发现具有抑制呼吸链复合物 Ι 的作用，Sun等[27] 结合了减少耗氧与供氧的双重策略，制备了5'-鸟苷一磷酸（5'-guanosine monophosphate，5'GMP）水凝胶负载 Met 的体系，该系统在近红外光照射下能够局部释放 Met，同时通过将有 CAT 样活性的Hemin@mil88 和 Met 联合，有效提高了 ICG 发挥 PDT 作用时的局部氧气浓度。 2.2 下调谷胱甘肽  为维持细胞氧化还原平衡，肿瘤的高 ROS 必然带来高 GSH 的伴生。GSH 的还原性使其可以断裂二硫键、硫醚等化学键。Wang 等 [28] 联合 PEG和甲基丙烯酸吡啶基二硫化乙酯（pyridyl disulfide ethyl methacrylate，PyDSMA）合成两亲性嵌合物PEG-p，该嵌合物的亲脂 PyDSMA 端不仅用于缀合光敏剂，其独特的吡啶基二硫键更是成为消耗肿瘤内部过剩 GSH 的有力工具。Zhou 等 [29] 设计了一种包括上转换纳米晶体和负载 Ce6 的二硫键介孔有机硅的纳米复合材料 UCN/MON（upconverting nanocrystal/mesoporous organosilicon）。UCN/MON 内部的二硫键可以通过 GSH 的氧化还原反应降解。该纳米晶体不仅通过氧化还原触发的 Ce6 释放提高光敏剂的肿瘤选择性，还有效降低癌细胞中 GSH 的浓度，从而减少 ROS 的消耗。此外，GSH 消耗亦可通过高价金属离子还原实现，Zou 等 [30] 合成了可以在酸性肿瘤微环境中特异性释放铜离子（Cu2+）的 Cu/ZIF-8 纳米颗粒。这些纳米颗粒在GSH 的还原作用下会生成亚铜离子（Cu+），而 Cu+又能与过量的 H2O2 反应生成丰富的 • OH，为肿瘤治疗提供了新的 ROS 来源。 2.3 重塑肿瘤外基质 肿瘤部位脉管异常（如血管流速慢、血管渗漏、迂曲度高、结构不规则），细胞外基质致密和间质压力大，这些特征显著增加了 PDT 制剂和氧气通过血管进入肿瘤的难度。因此，降低间质压力和疏松基质是提升 PDT 疗效的重要途径。Liu 等 [31] 在胰腺肿瘤模型中使用基于纳米颗粒的 PDT，成功扰乱了肿瘤-基质串扰，抑制这种串扰可以持续松弛肿瘤外基质。松弛的基质不仅促进了后续治疗的肿瘤根除，还提高了药物的治疗效率。因此，PDT 可能成为结缔组织增生性癌症的有价值的个体辅助治疗手段。Deng 等 [32] 将一氧化氮（nitric oxide，NO）前药 S 亚硝基硫醇与α-环糊精/PEG 偶联合成了 GSH 敏感的 NO 输送剂，旨在实现多重治疗效果：首先 NO 前药的释放过程需要 GSH 的参与，确保了 NO 在肿瘤微环境中的特异性释放；同时，NO 本身具有氧化 GSH 的能力，进一步耗尽肿瘤细胞中的 GSH，增强 ROS 的杀伤效果；此外，NO 可以引起肿瘤血管的松弛，降低间质压力，从而提高肿瘤部位的氧气和药物蓄积；最后 NO 可以和 ROS 反应产生新的含氮自由基，进一步攻击肿瘤。 2.4 阻断肿瘤细胞自噬 自噬是细胞通过溶酶体清除自身成分的过程，根据溶酶体行为不同可分为以下 3 种类型：1）巨自噬，细胞募集蛋白形成大型自噬体，介导损伤成分进入溶酶体；2）微自噬，受损的细胞器自动和溶酶体结合，期间无大自噬体形成；3）分子伴侣介导自噬，用于水解错误折叠蛋白 [33]。自噬在 PDT 中是一把双刃剑：一方面，ROS 导致的细胞死亡需要自噬的参与；另一方面，肿瘤细胞可以通过自噬，特别是巨自噬，清除 ROS 造成的损伤成分，进而保护自身免受伤害。Xue 等 [34] 在测试新型光敏剂时发现，PDT 处理后细胞内的巨自噬蛋白表达明显增强，使用自噬抑制剂后，PDT 效果得到了显著增强。自噬保护在特定类型的肿瘤 PDT 耐受中起重要作用，阻断自噬往往可消除耐受。  氯喹（chloroquine，CQ）是一种成熟的疟疾治疗药物，作为一种弱碱，CQ 进入溶酶体后可以提升溶酶体的pH值，从而抑制了溶酶体水解自噬体过程。Dai 等 [35] 开发了仿生纳米材料 PCN-CQ@CCM，该材料以具有 PDT 作用的金属有机骨架材料 PCN-224 为载体，搭载自噬抑制剂 CQ，并在表面包被分离的口腔鳞状细胞癌细胞膜（carcinoma cell membrane，CCM）。由于癌细胞膜具有免疫逃避和同源靶向能力，PCN-CQ@CCM 可以逃避巨噬细胞吞噬作用并同源黏附在肿瘤细胞上，增强纳米材料在肿瘤微环境中的保留和吸收。CQ 通过有效抑制保护性自噬通量，进一步加剧了 ROS 致死途径。Xiao 等 [36] 开 发 了 一 种 共 封 装 盐 酸 多 西 环 素（doxycycline hydrochloride，doxy）和Ce6的脂质体纳米药物，其中，Doxy 可以诱导线粒体功能障碍，增加 ROS 生成，从而增强 PDT 的杀伤作用和免疫原性细胞死亡（immunogenic cell death，ICD）。同时，Doxy 通过有效的自噬抑制增加肿瘤细胞表面的主要组织相容性复合体 Ⅰ（major histocompatibility complexⅠ，MHC-Ⅰ）表达，从而提高抗原呈递和细胞毒性淋巴细胞的识别效率，增加肿瘤免疫原性。 3 光动力治疗的联合疗法 PDT 和其他疗法的联用具有很好的优势，例如可以重塑肿瘤免疫抑制环境以放大治疗效果，利用PDT 调控其他疗法协同作用，以及运用成熟技术增强 PDT 治疗等。 3.1 光动力治疗和免疫疗法 近年来，免疫检查点阻断疗法因其在多种肿瘤治疗中的广泛应用而备受关注。PDT 被认为可以通过诱导局部 ICD 来引发全身抗肿瘤反应。PDT 通过杀伤肿瘤细胞，暴露出肿瘤特异性抗原（tumor specific antigen，TSA）和损伤相关分子模式（damage-associated molecular pattern，DAMP）。DAMP 和 TSA 的暴露可以激活肿瘤浸润免疫细胞，诱导这些细胞发生全身免疫反应 [37]。然而，有研究表明，ICD 本身可能进一步恶化肿瘤的免疫抑制环境。具体来说，机体的免疫负反馈机制使得 DAMP 在诱导正向免疫反应的同时，增加了肿瘤免疫检查点的密度 [38]；而 TSA 本身也可能诱导免疫抑制性细胞的产生 [39]。因此，PDT 与免疫检查点阻断剂的联合使用可以逆转 ICD 引起的免疫抑制，有助于实现更有效和持久的抗肿瘤作用。 免疫细胞的抗癌活性受到细胞毒性 T 淋巴细胞相关蛋白 4（cytotoxic T lymphocyte-associated antigen-4，CTLA-4）和 PD-1/PD-L1 等免疫检查点的限制，近年来针对这些检查点开发的单抗对多种恶性肿瘤展现出治疗潜力。Zheng 等 [40] 引入了一种可引发 ICD 的光敏剂，该光敏剂会启动强烈的免疫级联反应，促进了肿瘤微环境中细胞毒性 T 淋巴细胞浸润的增加。Yu 等 [41] 通过 PDT 与光热疗法的自动切换，实现 ICD 期间损伤相关分子模式的顺序生成和释放。Yang 等 [42] 制备了含有小分子光敏剂的黏膜黏附性纳米颗粒，并与灭活悬浮肿瘤细胞直接共孵育。该制剂可以诱导癌细胞的强效免疫原性细胞死亡。随着药物的释放，可以激活安全而强大的肿瘤特异性免疫反应，从而有效抑制术后肿瘤复发和转移。  此外，PDT 可以触发巨噬细胞极化增强免疫效果。Liu 等 [43] 设计了一种嵌合肽工程自递送纳米药物，由光敏剂 Ce6，米喹莫特（R837）和 Toll 样受体激动剂组成，既可以作为光敏剂进行 PDT，还可以将 M2 型肿瘤相关巨噬细胞（tumor-associated macrophage，TAM）极化为 M1 型 TAM，与 ICD 联合激活 T 细胞抗肿瘤免疫。  分化决定簇抗原 47（duster of differentiation 47，CD47）作为细胞表面的“不要吃我”信号，可与巨噬细胞表面受体结合，使机体细胞免于被自体免疫攻击，肿瘤细胞通过高表达 CD47 来实现免疫逃逸。Xiao 等 [44] 制作了一种 pH 双响应胶束，这种胶束本身带负电，在肿瘤外层降解后释放 CD47 单抗并完成电荷翻转，正电部分促进制剂吸收并在溶酶体酸解释放 Ce6，这种双响应逐步释药的方式保护了 CD47 抗体不被降解 ，同时使 Ce6 和 CD47 抗体均在其最合适位置发挥作用。 3.2 光动力治疗和化疗联用   PDT 与化疗的联合使用，提高了治疗效果，尤其是在处理缺氧肿瘤时表现出显著的优势。化疗药物能够提高肿瘤细胞对 ROS 的敏感度，而在 PDT过程中产生的 ROS 不仅可以抑制细胞的药物外排活动，还能增强细胞对药物的摄取 [45]，从而提高了 PDT 在缺氧肿瘤中的疗效，减少了耐药性的发生，加强了癌症根除的效果。  部分化疗药物本身是没有生物毒性的药物前体，而在缺氧条件下它们的细胞毒性可被激活，这一特性使它们可以专一性地治疗缺氧肿瘤。替拉帕嗪（tirapazamine，TPZ）是一种可在低氧条件活化的化疗药，Ma 等 [46] 将 TPZ 连接二硬脂酰磷脂酰乙醇胺（1，2-distearoyl-sn-glycero-3-phosphoethanolamine，DSPE）-PEG形成 DSPE-PEG-TPZ，然后装载光敏剂 Ce6，最后封装进可溶性微针中进行递送。PDT 过程中产生的 ROS 会消耗肿瘤区域原本就稀缺的氧气，加速 TPZ 的活化，从而增强治疗效果。除了利用 PDT 加重缺氧条件来激活化疗前药外，PDT 产生的 ROS 本身也可以作为一种信号，触发化疗药物的释放。Wang 等[47] 合成了 GSH 反应性前药（BP），其中以 GSH 激活的 BODIPY 作为药物载体，抗肿瘤药物 7-乙基-10-羟基喜树碱（7-ethyl-10-hydroxycamptothecin，SN38）作为离去 基 团。BP 分 子 在 水 中 自 组 装 形 成 纳 米 前药，BODIPY 部分经 PDT 照射后产生 ROS 触发细胞氧化还原机制产生更高浓度 GSH，高浓度 GSH 亲核取代 BP 的 SN38 部分导致 SN38 的释放，同时使 BP 解聚，停止 ROS 的产生。缺氧肿瘤内含有丰富的硝基还原酶（nitrate reductase，NTR），这种酶在缺氧环境下活性更强。Yang 等 [48] 使用偶氮苯接头连接喜树碱（camptothecin，CPT）和 BODIPY 形成乏氧响应前药 CPT-BODIPY，前药继续组装成纳米粒用于肿瘤治疗，在光照条件下，纳米粒发挥 PDT 治疗，同时前药在乏氧条件下释放 CPT，用于杀伤肿瘤细胞，这种设计不仅提高了治疗效果，还能减少不良反应的发生。 3.3 光动力治疗和放射疗法的结合 PDT 依赖于光穿透皮肤来激活光敏剂，其常用的治疗波长主要集中在近红外至可见光范围内。然而，即便是在深层皮肤穿透方面表现优异的近红外光，其穿透能力也远不及放射疗法（radiation therapy，RT）中使用的高频电离辐射，如 X 射线。一些物质，如闪烁晶体，其具备吸收高频电磁波后发出较长波长可见荧光的能力 [49]。这一特性为将闪烁晶体纳米颗粒与 PDT 光敏剂结合使用提供了可能，利用 RT 产生的强大穿透力的高频辐射来激活深层组织中的 PDT，即所谓的 RT-PDT 疗法。Jiang 等 [50] 组建了一种双核树枝状 RT-PDT 平台，该平台以纳米闪烁体氟化钙（calcium fluoride，CaF2）为核心，外层覆盖由 PEG 修饰的聚酰胺（polyamidoamine，PAMAM）树枝状大分子。通过将光敏剂 RB 与 PAMAM 分子表面的官能团形成共价键连接，实现了药物在树枝状大分子表面和内部的有序装载。考虑到 PDT 可以激活肿瘤的血管生成机制，PAMAM 的剩余空间装载了抗血管生成药物舒尼替尼，旨在阻断肿瘤的新血管形成。实验结果显示，采用多次低剂量的 RT 辐照策略比单次大剂量辐照更为有效，这不仅提高了治疗效果，而且对于减轻患者的不良反应至关重要。 4 结语与展望 PDT 作为一种非侵入性疗法，凭借其具有低毒性、与化疗药物联合使用时无重叠毒性以及没有显著的不良反应（如纤维化）的优点，展现了在疾病联合治疗中的巨大潜力。基于 PDT 的精确疗法主要集中在发展新光敏剂、探究和理解新的光动力学机制、发展新纳米技术和成像工具以及肿瘤模型的集成和发现。随着肿瘤学研究的深入，肿瘤乏氧、高还原性物质和免疫抑制的环境限制了 PDT 的实际效果，然而，新型纳米光敏剂的出现对 PDT 过程更深层次的理解以及各种新纳米材料和技术的融合应用，极大地突破了肿瘤 PDT 临床前研究的障碍。这些进展不仅提升了 PDT的效果，还使得单一制剂能够同时达到增氧、改善免疫环境、逆转肿瘤代谢、实现药物的精准释放以及多种治疗方法的联合使用等多重目标。  自 PDT 首次被美国食品药品监督管理局（Food and Drug Administration，FDA）批准用于膀胱癌的治疗以来，该疗法已扩展到眼科、皮肤科及肿瘤学等多个领域 [4]。尽管 PDT 在临床上已被证实对于局部肿瘤（如前列腺癌、脑癌和膀胱癌）的微创治疗具有高度的时间和空间控制能力，并且不良反应较小，但在广泛癌症尤其是转移性疾病中的应用仍面临挑战。随着免疫疗法的兴起，PDT 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ACS Nano. 2021, 15(7): 11112- 11125. 美编排版：朱玲欣 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》2025年第 1 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

CAMBRIDGE, Mass.--( BUSINESS WIRE )-- Alnylam Pharmaceuticals, Inc. (Nasdaq: ALNY), the leading RNAi therapeutics company, today reported its consolidated financial results for the fourth quarter and full year ended December 31, 2024 and reviewed recent business highlights. “ 2024 was another year of impressive execution for Alnylam, generating product revenues of over $1.6 billion, reflecting growth of 33% compared to 2023, and highlighting the strength of our base business in hATTR-PN and Rare in both the U.S. and international markets. We look forward to potential global launches of vutrisiran in ATTR-CM this year, which will mark an inflection point for our TTR franchise and put us on a path to achieve the financial guidance we’ve provided,” said Yvonne Greenstreet, MBChB, Chief Executive Officer of Alnylam. “ Furthermore, driven by our proven RNAi platform, we anticipate 2025 will bring major advancements in our pipeline and expect to have over 25 high-value programs in the clinic across diverse indications by the end of the year. We’ve seen a remarkable pace of progress toward our Alnylam P 5 x25 goals and believe we are well positioned for the next half of the decade to continue delivering sustainable innovation to patients.” Fourth Quarter 2024 and Recent Significant Business Highlights Commercial Performance Total TTR: ONPATTRO ® (patisiran) & AMVUTTRA ® (vutrisiran) Achieved global net product revenues for ONPATTRO and AMVUTTRA for the fourth quarter of $56 million and $287 million, respectively, and $343 million combined, representing 35% total TTR growth compared to Q4 2023, and full year 2024 revenues of $253 million and $970 million, respectively, and $1,223 million combined, representing 34% total TTR growth compared to full year 2023. Total Rare: GIVLAARI ® (givosiran) & OXLUMO ® (lumasiran) Achieved global net product revenues for GIVLAARI and OXLUMO for the fourth quarter of $65 million and $44 million, respectively, and $108 million combined, representing 18% total Rare growth compared to Q4 2023, and full year 2024 revenues of $256 million and $167 million, respectively, and $423 million combined, representing 29% total Rare growth compared to full year 2023. R&D Highlights Submitted a supplemental New Drug Application (sNDA) to the U.S. Food and Drug Administration (FDA) using a Priority Review Voucher for vutrisiran for the treatment of ATTR amyloidosis with cardiomyopathy. Parallel filings for regulatory approval have also been submitted in all major regions, including Europe and Japan. The FDA has accepted the sNDA and set an action goal date of March 23, 2025, under the Prescription Drug User Fee Act (PDUFA). Received United States Food & Drug Administration (FDA) Orphan Drug Designation for nucresiran (ALN-TTRsc04) and announced positive interim Phase 1 data in patients with ATTR amyloidosis. Announced positive initial results from the multiple dose portion of the Phase 1 study of mivelsiran in patients with Alzheimer’s disease. Initiated a Phase 1 study of ALN-HTT02 in adult patients with Huntington’s disease. Completed enrollment in the KARDIA-3 Phase 2 study of zilebesiran , and initiated a Phase 1 study of zilebesiran + REVERSIR for hypertension. Initiated Phase 1 studies of ALN-6400 for a bleeding disorder, and ALN-4324 for type 2 diabetes mellitus. Alnylam's partner, Regeneron Pharmaceuticals, reported positive updated Phase 3 data from an exploratory cohort in the ACCESS-1 trial investigating its first-in-class pozelimab and cemdisiran combination treatment compared to standard-of-care ravulizumab in patients with paroxysmal nocturnal hemoglobinuria (PNH) at the American Society of Hematology 2024 Annual Meeting. Upcoming Events Alnylam will host an R&D Day on February 25, 2025 at 9:00 am ET in New York City. This event, which will be webcast live on the Investors section of the Company’s website, www.alnylam.com , will showcase Alnylam’s robust pipeline comprising numerous multi-billion-dollar opportunities and highlight RNAi platform innovation driving the next era of growth. A replay will be available on the Company's website within 48 hours after the event. The PDUFA target action date for the sNDA for vutrisiran is March 23, 2025. Alnylam intends to initiate a Phase 3 study of nucresiran in patients with ATTR amyloidosis with cardiomyopathy in the first half of 2025. The PDUFA target action date for fitusiran , an investigational RNAi therapeutic in development for the treatment of hemophilia A and B, with or without inhibitors, is March 28, 2025. Fitusiran is being advanced by Alnylam’s partner Sanofi. Assuming approval, Alnylam is eligible to receive tiered royalties of between 15 and 30 percent on global net sales of fitusiran. Alnylam’s partner, Vir Biotechnology, expects to initiate a Phase 3 chronic hepatitis delta registrational study of elebsiran and to report functional cure results from a Phase 2 chronic hepatitis B study of elebsiran in 2025. Financial Highlights for the Fourth Quarter and Year End 2024 Three Months Ended December 31, Twelve Months Ended December 31, (In thousands) 2024 2023 2024 2023 Net product revenues $ 450,831 $ 346,288 $ 1,646,228 $ 1,241,474 Net revenues from collaborations 106,948 76,407 510,221 546,185 Royalty revenue 35,387 17,023 91,794 40,633 Total revenues 593,166 439,718 2,248,243 1,828,292 Total operating costs and expenses 698,325 556,122 2,425,128 2,110,467 Loss from operations (105,159 ) (116,404 ) (176,885 ) (282,175 ) Total other expense, net (88,799 ) (21,283 ) (200,490 ) (151,342 ) Benefit from (provision for) income taxes 110,195 (183 ) 99,218 (6,725 ) Net loss $ (83,763 ) $ (137,870 ) $ (278,157 ) $ (440,242 ) Non-GAAP Operating (loss) income* $ (13,514 ) $ (74,410 ) $ 95,199 $ (60,495 ) Non-GAAP Net income (loss)* $ 8,048 $ (96,643 ) $ (3,051 ) $ (201,618 ) *For an explanation of our use of non-GAAP financial measures refer to the "Use of Non-GAAP Financial Measures" section later in this press release and for a reconciliation of each non-GAAP financial measure to the most comparable GAAP measures, see the table at the end of this press release. Net Product Revenues Three Months Ended December 31, Twelve Months Ended December 31, (In thousands) 2024 2023 2024 2023 ONPATTRO net product revenues $ 56,103 $ 79,006 $ 252,857 $ 354,546 AMVUTTRA net product revenues 286,510 175,254 970,450 557,838 Total TTR net product revenues 342,613 254,260 1,223,307 912,384 GIVLAARI net product revenues 64,645 59,298 255,871 219,251 OXLUMO net product revenues 43,573 32,730 167,050 109,839 Total Rare net product revenues 108,218 92,028 422,921 329,090 Total net product revenues $ 450,831 $ 346,288 $ 1,646,228 $ 1,241,474 Year over Year % Growth Three Months Ended December 31, 2024 Twelve Months Ended December 31, 2024 As Reported At CER* As Reported At CER* Total TTR net product revenues 35 % 34 % 34 % 34 % Total Rare net product revenues 18 % 17 % 29 % 28 % Total net product revenues 30 % 29 % 33 % 33 % * CER = Constant Exchange Rate, representing growth calculated as if the exchange rates had remained unchanged from those used in 2023. CER is a non-GAAP measure. For an explanation of our use of non-GAAP financial measures refer to the "Use of Non-GAAP Financial Measures" section later in this press release and for a reconciliation of each non-GAAP financial measure to the most comparable GAAP measures, see the table at the end of this press release. Net product revenues increased 30% and 33% at actual currency during the three and twelve months ended December 31, 2024, respectively, compared to the same periods in 2023, and 29% and 33% at CER, respectively. The increases are primarily due to growth from sales of AMVUTTRA driven by increased patient demand, partially offset by a decrease in sales of ONPATTRO due to patient switches to AMVUTTRA, as well as increased patients on GIVLAARI and OXLUMO therapies. Net Revenues from Collaborations Net revenues from collaborations increased $30.5 million during the three months ended December 31, 2024, as compared to the same period in 2023, primarily due to the timing of manufacturing activities under our collaboration with Regeneron, as well as revenue recognized under our license agreement with Novartis associated with the achievement of specified commercialization milestones. Net revenues from collaborations decreased by $36.0 million during the twelve months ended December 31, 2024, as compared to the same period in 2023, primarily due to differences in certain revenue items between 2023 and 2024. During 2023, we recognized $310.0 million of revenue from the upfront payment received from Roche in connection with execution of our zilebesiran collaboration. In comparison, during 2024, we recognized $185.0 million in revenues under our collaboration with Regeneron as we modified the collaboration in June 2024 and provided Regeneron with an exclusive license to develop, manufacture and commercialize cemdisiran as a monotherapy. Operating Expenses Three Months Ended December 31, Twelve Months Ended December 31, (In thousands) 2024 2023 2024 2023 Cost of goods sold $ 102,649 $ 71,975 $ 306,513 $ 268,216 Cost of goods sold as a percentage of net product revenues 22.8 % 20.8 % 18.6 % 21.6 % Cost of collaborations and royalties $ 168 $ 13,883 $ 16,857 $ 42,190 GAAP Research and development expenses $ 300,169 $ 272,141 $ 1,126,232 $ 1,004,415 Non-GAAP Research and development expenses $ 259,544 $ 253,056 $ 998,483 $ 907,142 GAAP Selling, general and administrative expenses $ 295,339 $ 198,123 $ 975,526 $ 795,646 Non-GAAP Selling, general and administrative expenses $ 244,319 $ 175,214 $ 831,191 $ 671,239 Cost of Goods Sold Cost of goods sold as a percentage of net product revenues increased during the three months ended December 31, 2024, as compared to the same period in the prior year, primarily due to higher royalty rates payable on net sales of AMVUTTRA. Cost of goods sold as a percentage of net product revenues decreased during the twelve months ended December 31, 2024, as compared to the same period in the prior year. Approximately 5.0% of the 21.6% of cost of goods sold as a percentage of net product revenues for the year ended December 31, 2023 was attributable to cancelled manufacturing commitments and the impairment of ONPATTRO inventory that had been manufactured for future demand associated with the use of ONPATTRO for the treatment of patients with ATTR amyloidosis with cardiomyopathy, for which we did not receive regulatory approval in the U.S. These one-time charges in 2023 did not recur in 2024, resulting in the decrease in cost of goods sold as a percentage of net product revenues in 2024, which was partially offset by higher volume and royalty rates payable on net sales of AMVUTTRA in 2024. Research & Development (R&D) Expenses GAAP and non-GAAP R&D expenses increased during the three and twelve months ended December 31, 2024, compared to the same periods in 2023, primarily due to increased costs associated with our preclinical activities as we develop our clinical pipeline of RNAi therapeutics targeting multiple tissues, increased clinical trial expenses associated with increased Phase 2 activities for the zilebesiran KARDIA-3 and mivelsiran cAPPRicorn-1 clinical trials, and increased employee compensation expenses. GAAP R&D expenses further increased during the three and twelve months ended December 31, 2024, compared to the same periods in 2023, due to higher stock-based compensation expense in 2024. Selling, General & Administrative (SG&A) Expenses GAAP and non-GAAP SG&A expenses increased during the three and twelve months ended December 31, 2024, compared to the same periods in 2023, primarily due to higher costs associated with marketing investments to promote our TTR therapies and prepare for the potential launch of AMVUTTRA for the treatment of ATTR amyloidosis with cardiomyopathy and increased employee compensation expenses. Benefit from (provision for) income taxes During the year ended December 31, 2024, we recorded a net benefit from income taxes of $99.2 million. This is primarily comprised of $106.8 million of foreign deferred benefit, partially offset by $1.6 million of domestic state current provision and $5.9 million of foreign current provision. Other Financial Highlights Cash, cash equivalents and marketable securities were $2.69 billion as of December 31, 2024, as compared to $2.44 billion as of December 31, 2023, with the increase primarily due to improved operating performance and increased net proceeds from the issuance of common stock in connection with stock option exercises. A reconciliation of our GAAP to non-GAAP results for the three and twelve months ended December 31, 2024 and 2023, is included in the tables of this press release. 2025 Financial Guidance Our full-year 2025 financial guidance is summarized below: Total TTR net product revenues (ONPATTRO, AMVUTTRA) 1 $1,600 million - $1,725 million Total Rare net product revenues (GIVLAARI, OXLUMO) $450 million - $525 million Total net product revenues $2,050 million - $2,250 million Net product revenues growth vs. 2024 at currency exchange rates as of December 31, 2024 2 25% - 37% Net product revenues growth vs. 2024 at constant exchange rates 3 26% - 39% Net revenues from collaborations and royalties 4 $650 million - $750 million Non-GAAP R&D and SG&A expenses 5 $2,100 million - $2,200 million Non-GAAP Operating income 5 Achieve profitability 1 Assumes U.S. sNDA approval of AMVUTTRA for ATTR-CM by the March 23, 2025 PDUFA action date and approvals and launches in Germany and Japan in the second half of 2025 2 Full-year 2025 guidance utilizing currency exchange rates as of December 31, 2024: 1 EUR = 1.04 USD and 1 USD = 157 JPY 3 Representing growth calculated as if the exchange rates had remained unchanged from those used in 2024, which is a non-GAAP financial measure 4 Collaboration revenues assume achievement of $300 million milestone from Roche for initiation of a Phase 3 cardiovascular outcomes trial for zilebesiran. Royalty revenues assume approval of fitusiran by Sanofi by the March 28, 2025 PDUFA action date 5 Primarily excludes $270-$330 million of stock-based compensation expense from estimated GAAP R&D and SG&A expenses Use of Non-GAAP Financial Measures This press release contains non-GAAP financial measures, including adjustments to exclude certain non-cash items and non-recurring transactions or events outside the ordinary course of the Company’s business. These measures are not in accordance with, or an alternative to, GAAP, and may be different from non-GAAP financial measures used by other companies. The items included in GAAP presentations but excluded for purposes of determining non-GAAP financial measures for the periods presented in this press release are stock-based compensation expenses and realized and unrealized gains and losses on marketable equity securities. The Company has excluded the impact of stock-based compensation expense, which may fluctuate from period to period based on factors including the variability associated with performance-based grants for stock options and restricted stock units and changes in the Company’s stock price, which impacts the fair value of these awards. The Company has excluded the impact of the realized and unrealized gains and losses on marketable equity securities because the Company does not believe these adjustments accurately reflect the performance of the Company’s ongoing operations for the period in which such gains or losses are reported, as their sole purpose is to adjust amounts on the balance sheet. Percentage changes in revenue growth at CER are presented excluding the impact of changes in foreign currency exchange rates for investors to understand the underlying business performance. The current period’s foreign currency revenue values are converted into U.S. dollars using the average exchange rates from the prior period. The Company believes the presentation of non-GAAP financial measures provides useful information to management and investors regarding the Company’s financial condition and results of operations. When GAAP financial measures are viewed in conjunction with non-GAAP financial measures, investors are provided with a more meaningful understanding of the Company’s ongoing operating performance and are better able to compare the Company’s performance between periods. Non-GAAP financial measures are not intended to be considered in isolation or as a substitute for GAAP financial measures. A reconciliation between GAAP and non-GAAP measures is provided later in this press release. Conference Call Information Management will provide an update on the Company and discuss fourth quarter and full year 2024 results as well as expectations for the future via conference call on Thursday, February 13, 2025 at 8:30 am ET. A live audio webcast of the call will be available on the Investors section of the Company’s website at www.alnylam.com/events . An archived webcast will be available on the Alnylam website approximately two hours after the event. About ONPATTRO ® (patisiran) ONPATTRO is an RNAi therapeutic that is approved in the United States and Canada for the treatment of adults with hATTR amyloidosis with polyneuropathy. ONPATTRO is also approved in the European Union, Switzerland and Brazil for the treatment of hATTR amyloidosis in adults with Stage 1 or Stage 2 polyneuropathy, and in Japan for the treatment of hATTR amyloidosis with polyneuropathy. ONPATTRO is an intravenously administered RNAi therapeutic targeting transthyretin (TTR). It is designed to target and silence TTR messenger RNA, thereby reducing the production of TTR protein before it is made. Reducing the pathogenic protein leads to a reduction in amyloid deposits in tissues. For more information about ONPATTRO, including full Prescribing Information , visit ONPATTRO.com . About AMVUTTRA ® (vutrisiran) AMVUTTRA ® (vutrisiran) is an RNAi therapeutic that delivers rapid knockdown of mutant and wild-type transthyretin (TTR), addressing the underlying cause of transthyretin (ATTR) amyloidosis. Administered quarterly via subcutaneous injection, AMVUTTRA is approved and marketed in more than 15 countries for the treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis (hATTR-PN) in adults. Vutrisiran is also in development for the treatment of ATTR amyloidosis with cardiomyopathy (ATTR-CM), which encompasses both wild-type and hereditary forms of the disease. For more information about AMVUTTRA, including the full U.S. Prescribing Information , visit AMVUTTRA.com . About GIVLAARI ® (givosiran) GIVLAARI (givosiran) is an RNAi therapeutic targeting aminolevulinic acid synthase 1 (ALAS1) approved in the United States and Brazil for the treatment of adults with acute hepatic porphyria (AHP). GIVLAARI is also approved in the European Union for the treatment of AHP in adults and adolescents aged 12 years and older. In the pivotal study, GIVLAARI was shown to significantly reduce the rate of porphyria attacks that required hospitalizations, urgent healthcare visits or intravenous hemin administration at home compared to placebo. GIVLAARI is Alnylam’s first commercially available therapeutic based on its Enhanced Stabilization Chemistry ESC-GalNAc conjugate technology to increase potency and durability. GIVLAARI is administered via subcutaneous injection once monthly at a dose based on actual body weight and should be administered by a healthcare professional. GIVLAARI works by specifically reducing elevated levels of ALAS1 messenger RNA (mRNA), leading to reduction of toxins associated with attacks and other disease manifestations of AHP. For more information about GIVLAARI, including the full U.S. Prescribing Information , visit GIVLAARI.com . About OXLUMO ® (lumasiran) OXLUMO (lumasiran) is an RNAi therapeutic targeting hydroxyacid oxidase 1 (HAO1). HAO1 encodes glycolate oxidase (GO). Thus, by silencing HAO1 and depleting the GO enzyme, OXLUMO inhibits production of oxalate – the metabolite that directly contributes to the pathophysiology of PH1. OXLUMO utilizes Alnylam’s Enhanced Stabilization Chemistry (ESC)-GalNAc-conjugate technology, which enables subcutaneous dosing with increased potency and durability and a wide therapeutic index. OXLUMO has received regulatory approvals from the U.S. Food and Drug Administration (FDA) for the treatment of primary hyperoxaluria type 1 (PH1) to lower urinary and plasma oxalate levels in pediatric and adult patients and from the European Medicines Agency (EMA) for the treatment of PH1 in all age groups. In the pivotal ILLUMINATE-A study, OXLUMO was shown to significantly reduce levels of urinary oxalate relative to placebo, with the majority of patients reaching normal or near-normal levels. In the ILLUMINATE-B pediatric Phase 3 study, OXLUMO demonstrated an efficacy and safety profile consistent to that observed in ILLUMINATE-A. In the ILLUMINATE-C study, OXLUMO resulted in substantial reductions in plasma oxalate in patients with advanced PH1. Across all three studies, injection site reactions (ISRs) were the most common drug-related adverse reaction. OXLUMO is administered via subcutaneous injection once monthly for three months, then once quarterly beginning one month after the last loading dose at a dose based on actual body weight. For patients who weigh less than 10 kg, ongoing dosing remains monthly. OXLUMO should be administered by a healthcare professional. For more information about OXLUMO, including the full U.S. Prescribing Information , visit OXLUMO.com . About LNP Technology Alnylam has licenses to Arbutus Biopharma lipid nanoparticle (LNP) intellectual property for use in RNAi therapeutic products using LNP technology. About RNAi RNAi (RNA interference) is a natural cellular process of gene silencing that represents one of the most promising and rapidly advancing frontiers in biology and drug development today. Its discovery has been heralded as “ a major scientific breakthrough that happens once every decade or so,” and was recognized with the award of the 2006 Nobel Prize for Physiology or Medicine. By harnessing the natural biological process of RNAi occurring in our cells, a new class of medicines known as RNAi therapeutics is now a reality. Small interfering RNA (siRNA), the molecules that mediate RNAi and comprise Alnylam’s RNAi therapeutic platform, function upstream of today’s medicines by potently silencing messenger RNA (mRNA) – the genetic precursors – that encode for disease-causing or disease pathway proteins, thus preventing them from being made. This revolutionary approach has transformed the care of patients with genetic and other diseases. About Alnylam Pharmaceuticals Alnylam Pharmaceuticals (Nasdaq: ALNY) has led the translation of RNA interference (RNAi) into a whole new class of innovative medicines for people afflicted with rare and prevalent diseases with unmet need. Based on Nobel Prize-winning science, RNAi therapeutics represent a powerful, clinically validated approach yielding transformative medicines. Since its founding in 2002, Alnylam has led the RNAi revolution and continues to deliver on a bold vision to turn scientific possibility into reality. Alnylam’s commercial RNAi therapeutic products are ONPATTRO ® (patisiran), AMVUTTRA ® (vutrisiran), GIVLAARI ® (givosiran), OXLUMO ® (lumasiran), and Leqvio ® (inclisiran), which is being developed and commercialized by Alnylam’s partner, Novartis. Alnylam has a deep pipeline of investigational medicines, including multiple product candidates that are in late-stage development. Alnylam is executing on its “ Alnylam P 5 x25 ” strategy to deliver transformative medicines in both rare and common diseases benefiting patients around the world through sustainable innovation and exceptional financial performance, resulting in a leading biotech profile. Alnylam is headquartered in Cambridge, MA. For more information about our people, science and pipeline, please visit www.alnylam.com and engage with us on X (formerly Twitter) at @Alnylam , or on LinkedIn , Facebook , or Instagram . Alnylam Forward Looking Statements This press release contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933 and Section 21E of the Securities Exchange Act of 1934. All statements other than historical statements of fact regarding Alnylam’s expectations, beliefs, goals, plans or prospects including, without limitation, statements regarding Alnylam’s expectations regarding the potential approval and launch of AMVUTTRA for the treatment of ATTR amyloidosis with cardiomyopathy in the U.S. in early 2025 and in other countries later in 2025; Alnylam’s advancement towards its “Alnylam P 5 x25” goals and positioning to deliver sustainable innovation to patients for the next half of the decade; the potential for Alnylam to advance its research and development programs, including the number of high-value programs Alnylam will have in the clinic by the end of 2025 and the timing of Alnylam’s initiation of a Phase 3 clinical trial of nucresiran in ATTR-CM; the advancement of fitusiran through regulatory review and approval and Alnylam’s receipt of any royalties on sales of fitusiran; and Alnylam’s projected commercial and financial performance, including the expected range of net product revenues and net revenues from collaborations and royalties for 2025 and the expected range of aggregate annual GAAP and non-GAAP R&D and SG&A expenses for 2025, should be considered forward-looking statements. Actual results and future plans may differ materially from those indicated by these forward-looking statements as a result of various important risks, uncertainties and other factors, including, without limitation, risks and uncertainties relating to: Alnylam’s ability to successfully execute on its “ Alnylam P 5 x25 ” strategy; Alnylam’s ability to discover and develop novel drug candidates and delivery approaches and successfully demonstrate the efficacy and safety of its product candidates; the pre-clinical and clinical results for Alnylam’s product candidates, including vutrisiran, zilebesiran, nucresiran and mivelsiran; actions or advice of regulatory agencies and Alnylam’s ability to obtain and maintain regulatory approval for its product candidates, including vutrisiran, as well as favorable pricing and reimbursement; successfully launching, marketing and selling Alnylam’s approved products globally; delays, interruptions or failures in the manufacture and supply of Alnylam’s product candidates or its marketed products; obtaining, maintaining and protecting intellectual property; Alnylam’s ability to successfully expand the approved indications for AMVUTTRA in the future; Alnylam’s ability to manage its growth and operating expenses through disciplined investment in operations and its ability to achieve a self-sustainable financial profile; Alnylam’s ability to maintain strategic business collaborations; Alnylam’s dependence on third parties for the development and commercialization of certain products, including Roche, Novartis, Sanofi, Regeneron and Vir; the outcome of litigation; the risk of future government investigations; and unexpected expenditures; as well as those risks and uncertainties more fully discussed in the “Risk Factors” filed with Alnylam’s most recent Quarterly Report on Form 10-Q filed with the Securities and Exchange Commission (SEC), as may be updated from time to time in other filings that Alnylam makes with the SEC. In addition, any forward-looking statements represent Alnylam’s views only as of today and should not be relied upon as representing its views as of any subsequent date. Alnylam explicitly disclaims any obligation, except to the extent required by law, to update any forward-looking statements. This release discusses investigational RNAi therapeutics and uses of previously approved RNAi therapeutics in development and is not intended to convey conclusions about efficacy or safety as to those investigational therapeutics or uses. Vutrisiran has not been approved by any regulatory agency for the treatment of ATTR amyloidosis with cardiomyopathy. No conclusions can or should be drawn regarding its safety or effectiveness in treating cardiomyopathy in this population. There is no guarantee that any investigational therapeutics or expanded uses of commercial products will successfully complete clinical development or gain health authority approval. ALNYLAM PHARMACEUTICALS, INC. CONSOLIDATED BALANCE SHEETS (In thousands, except per share amounts) December 31, 2024 December 31, 2023 ASSETS Current assets: Cash and cash equivalents $ 966,428 $ 812,688 Marketable debt securities 1,719,920 1,615,516 Marketable equity securities 8,156 11,178 Accounts receivable, net 405,308 327,787 Inventory 78,509 89,146 Prepaid expenses and other current assets 116,964 126,382 Total current assets 3,295,285 2,982,697 Property, plant and equipment, net 502,784 526,057 Operating lease right-of-use assets 191,148 199,732 Deferred tax assets 116,863 10,101 Restricted investments 68,593 49,391 Other assets 65,310 61,902 Total assets $ 4,239,983 $ 3,829,880 LIABILITIES AND STOCKHOLDERS’ EQUITY (DEFICIT) Current liabilities: Accounts payable $ 88,415 $ 55,519 Accrued expenses 887,472 713,013 Operating lease liabilities 41,886 41,510 Deferred revenue 55,481 102,753 Liability related to the sale of future royalties 113,018 54,991 Total current liabilities 1,186,272 967,786 Operating lease liabilities, net of current portion 229,541 243,101 Deferred revenue, net of current portion — 188,175 Convertible debt 1,024,621 1,020,776 Liability related to the sale of future royalties, net of current portion 1,334,353 1,322,248 Other liabilities 398,108 308,438 Total liabilities 4,172,895 4,050,524 Stockholders’ equity (deficit): Preferred stock, $0.01 par value per share, 5,000 shares authorized and no shares issued and outstanding as of December 31, 2024 and December 31, 2023 — — Common stock, $0.01 par value per share, 250,000 shares authorized as of December 31, 2024 and December 31, 2023, respectively; 129,294 shares issued and outstanding as of December 31, 2024; 125,794 shares issued and outstanding as of December 31, 2023 1,293 1,259 Additional paid-in capital 7,388,061 6,811,063 Accumulated other comprehensive loss (34,518 ) (23,375 ) Accumulated deficit (7,287,748 ) (7,009,591 ) Total stockholders’ equity (deficit) 67,088 (220,644 ) Total liabilities and stockholders’ equity (deficit) $ 4,239,983 $ 3,829,880 This selected financial information should be read in conjunction with the consolidated financial statements and notes thereto included in Alnylam’s Annual Report on Form 10-K which includes the audited financial statements for the year ended December 31, 2024. ALNYLAM PHARMACEUTICALS, INC. CONSOLIDATED STATEMENTS OF OPERATIONS (In thousands, except per share amounts) Three Months Ended December 31, Twelve Months Ended December 31, 2024 2023 2024 2023 (Unaudited) (Unaudited) Revenues: Net product revenues $ 450,831 $ 346,288 $ 1,646,228 $ 1,241,474 Net revenues from collaborations 106,948 76,407 510,221 546,185 Royalty revenue 35,387 17,023 91,794 40,633 Total revenues 593,166 439,718 2,248,243 1,828,292 Operating costs and expenses: Cost of goods sold 102,649 71,975 306,513 268,216 Cost of collaborations and royalties 168 13,883 16,857 42,190 Research and development 300,169 272,141 1,126,232 1,004,415 Selling, general and administrative 295,339 198,123 975,526 795,646 Total operating costs and expenses 698,325 556,122 2,425,128 2,110,467 Loss from operations (105,159 ) (116,404 ) (176,885 ) (282,175 ) Other (expense) income: Interest expense (38,971 ) (31,338 ) (141,858 ) (121,221 ) Interest income 31,019 30,406 121,992 95,561 Other expense, net (80,847 ) (20,351 ) (180,624 ) (125,682 ) Total other expense, net (88,799 ) (21,283 ) (200,490 ) (151,342 ) Loss before income taxes (193,958 ) (137,687 ) (377,375 ) (433,517 ) Benefit from (provision for) income taxes 110,195 (183 ) 99,218 (6,725 ) Net loss $ (83,763 ) $ (137,870 ) $ (278,157 ) $ (440,242 ) Net loss per common share — basic and diluted $ (0.65 ) $ (1.10 ) $ (2.18 ) $ (3.52 ) Weighted-average common shares used to compute basic and diluted net loss per common share 129,116 125,613 127,651 124,906 ALNYLAM PHARMACEUTICALS, INC. RECONCILIATION OF SELECTED GAAP MEASURES TO NON-GAAP MEASURES (In thousands, except per share amounts) (Unaudited) Three Months Ended December 31, Twelve Months Ended December 31, 2024 2023 2024 2023 Reconciliation of GAAP to Non-GAAP research and development: GAAP Research and development expenses $ 300,169 $ 272,141 $ 1,126,232 $ 1,004,415 Less: Stock-based compensation expenses (40,625 ) (19,085 ) (127,749 ) (97,273 ) Non-GAAP Research and development expenses $ 259,544 $ 253,056 $ 998,483 $ 907,142 Reconciliation of GAAP to Non-GAAP selling, general and administrative: GAAP Selling, general and administrative expenses $ 295,339 $ 198,123 $ 975,526 $ 795,646 Less: Stock-based compensation expenses (51,020 ) (22,909 ) (144,335 ) (124,407 ) Non-GAAP Selling, general and administrative expenses $ 244,319 $ 175,214 $ 831,191 $ 671,239 Reconciliation of GAAP to Non-GAAP operating (loss) income: GAAP Loss from operations $ (105,159 ) $ (116,404 ) $ (176,885 ) $ (282,175 ) Add: Stock-based compensation expenses 91,645 41,994 272,084 221,680 Non-GAAP Operating (loss) income $ (13,514 ) $ (74,410 ) $ 95,199 $ (60,495 ) Reconciliation of GAAP to Non-GAAP other expense, net: GAAP Total other expense, net $ (88,799 ) $ (21,283 ) $ (200,490 ) $ (151,342 ) Add (Less): Realized and unrealized losses (gains) on marketable equity securities 166 (767 ) 3,022 16,944 Non-GAAP Other expense, net $ (88,633 ) $ (22,050 ) $ (197,468 ) $ (134,398 ) Reconciliation of GAAP to Non-GAAP net income (loss): GAAP Net loss $ (83,763 ) $ (137,870 ) $ (278,157 ) $ (440,242 ) Add: Stock-based compensation expenses 91,645 41,994 272,084 221,680 Add (Less): Realized and unrealized losses (gains) on marketable equity securities 166 (767 ) 3,022 16,944 Non-GAAP Net income (loss) $ 8,048 $ (96,643 ) $ (3,051 ) $ (201,618 ) Reconciliation of GAAP to Non-GAAP net loss per common share-basic and diluted: GAAP Net loss per common share - basic and diluted $ (0.65 ) $ (1.10 ) $ (2.18 ) $ (3.52 ) Add: Stock-based compensation expenses 0.71 0.33 2.13 1.77 Add (Less): Realized and unrealized losses (gains) on marketable equity securities — (0.01 ) 0.02 0.14 Non-GAAP Net loss per common share - basic and diluted $ 0.06 $ (0.77 ) $ (0.02 ) $ (1.61 ) Please note that the figures presented above may not sum exactly due to rounding ALNYLAM PHARMACEUTICALS, INC. RECONCILIATION OF GAAP TO NON-GAAP PRODUCT REVENUE GROWTH AT CONSTANT EXCHANGE RATE December 31, 2024 Three Months Ended Twelve Months Ended Total TTR net product revenue growth*, as reported 35 % 34 % Add: Impact of foreign currency translation (1 ) — Total TTR net product revenue growth at constant exchange rate 34 % 34 % Total Rare net product revenue growth*, as reported 18 % 29 % Add: Impact of foreign currency translation (1 ) (1 ) Total Rare net product revenue growth at constant exchange rate 17 % 28 % Total net product revenue growth*, as reported 30 % 33 % Add: Impact of foreign currency translation (1 ) — Total net product revenue growth at constant exchange rate 29 % 33 % Total revenue growth*, as reported 35 % 23 % Add: Impact of foreign currency translation (1 ) — Total revenue growth at constant exchange rate 34 % 23 % *As compared to the three and twelve months ended December 31, 2023, respectively.