Monomethyl fumarate

DOI: 10.1021/jacs.4c05711 半胱氨酸（Cys）反应性共价弹头是共价药物和分子探针的设计中的"台柱子”，当前已经取得了不俗的成绩，然而，关于新的探索仍然是研发中的重点之一。尤其是当前共价抑制剂的发展已经是通过筛选具有亲电弹头的大型化合物库，以“亲电性优先“的设计、开发阶段，对于Cys反应性共价弹头的探索也是开发的重点。 近日，J Am Chem Soc上刊载了一篇非常实用性的研究性文章，报道了一种可塑性巯基反应性（STRP）方法，可与共价化合物竞争性与Cys反应，以比较灵敏的荧光检测做显示分析，非常便捷的探索大型化合物库与Cys残基的固有反应性。在实际的应用中，成功鉴定出一种新型Cys共价弹头：氮杂啶噁二唑，基于开环机制，选择性与Cys残基反应。 主要内容 01 可塑性巯基反应性分析的发展一种竞争性Cys残基反应性基团检测方法 02 高通量筛选Cys反应性基团应用：新型Cys共价弹头，噁二唑氮杂啶 03 总结与展望及往期相关精彩回顾 part.1 巯基反应性分析 竞争性检测Cys反应性试剂 半胱氨酸（Cys）残基相比于其它蛋白原氨基酸，其固有的亲核性，使其成为共价基团靶向的重要位点，在靶向药物、分子探针、交联剂和偶联剂等研究中起到重要作用。而探索Cys反应性化学片段也成为这些研发项目中的一大热点。 然而，除少数例外，当前FDA获批的药物中，Cys活性基团的适用很大程度上仅限于丙烯酰胺，探索高效、新型Cys共价弹头非常必要。 在共价小分子抑制剂的开发中，除了偶然发现，合理性设计多是以“亲和性优先”，先通过药化努力，优化获得高亲和力的非共价结合剂，再根据了解的结构信息，在合适的位置附加有限数量的“亲电弹头”以赋予这些亲和元件共价性。 近年来，利用化学蛋白质组学的无偏倚方法发现共价配体，使用多种亲电探索片段，广泛筛选细胞蛋白质组，以“亲电反应性优先”的方法扩大了可药物蛋白质组的范围，同时也颠倒了化学共价抑制剂发现的操作顺序，尤其是通过共价化合物库的直接高通量筛选。 然而，用于探索大型化合物库（分子数超过103以上）与Cys的反应活性的检测多是基于LC-MS或比色法，它们的可塑性和灵敏度都有限，这不利于高通量Cys共价弹头的探索。 本文就介绍了一种高灵敏度、高通量的策略，用以探索高通量化合物文库中与Cys反应性的亲电试剂：可塑性巯基反应性分析（STRP）试验。 这个检测策略启发于萤火虫荧光素：如下图A/B，D/L-半胱氨酸可与2-氰基-6-羟基苯并噻唑（CHBT）反应，生成D/L-萤火虫荧光素；而这种荧光素在它同源荧光素酶的作用下被氧化而发光。这种可塑性发光能力已经被广泛用于多种可塑性生物试验之中。 图片源自JACS. 受此启发，研究人员开发了一种竞争性试验，用以检测小分子与Cys的反应性活性，如上图。测试化合物首先与Cys共存，随后加入CHBT分子；具有Cys反应性的测试化合物消耗了Cys，无萤火虫荧光素产生，当加入荧光素酶时，不发光；没有Cys反应性的测试化合物组有可与CHBT反应的Cys，进而生成了萤火虫荧光素，在后续荧光素酶氧化下，发光。 因此，通过检测最终样品是否能够被荧光素酶诱导发光可知：发光，则测试化合物不具有Cys反应性（也可能是反应性不够？）；不发光，则测试化合物具有Cys反应性。 STRP竞争性检测方法如下流程图所示，共分为三步： 图片源自JACS. 步骤1：在多孔板的每孔中分别加入需要检测的化合物和D-Cys，室温共存孵育3小时（1.5-250 μM，TRIS缓冲液中）； 步骤2：每孔中加入适量CHBT，孵育30分钟，使没有Cys活性的样品孔内形成功能性荧光素； 步骤3：每孔中加入ATP-Mg2+和荧光素酶，使形成的萤火虫荧光素氧化发光而被检测到；低信号代表Cys活性化合物、高信号代表非Cys活性化合物。 通过探索，研究人员确定了D-Cys（1~10,000μM）、CHBT（1~1,000μM）、和荧光素（0.1~1,000μM）在实验条件下的可适用范围，最终产生的荧光信号的动态范围超过背景的104倍，如下图。值得注意的是，在筛选化合物的典型浓度区间（1~10μM），D-Cys的浓度可进行化学计量竞争，方便于应用。 图片源自JACS. 此外，研究人员还开发了对照试验检测，测试给定的Hit化合物是否可能通过与半胱氨酸反应以外的某种机制降低发光信号，如下图。对原有的操作顺序进行调整：顺序是D-Cys与CHBT孵育，形成D-荧光素；随后加入测试化合物、荧光素酶暴露、进行发光记录。在对照试验中降低发光信号的化合物可能是荧光素酶的抑制剂，或者该试验的非特异性调节剂，如聚集物。 图片源自JACS. 接下来，研究人员选择了文献报道的12种已知的Cys反应性试剂（下图B），作为一个“训练集”，对上述STRP分析试验进行准确性、和剂量依赖性测试。 图片源自JACS. 如上图B/C，结果表明，这12种试剂在STRP试验中： (1) 均能剂量依赖性地抑制发光信号; (2) 该分析可以准确预测两个具有既定反应性的相似分子中哪一个更具有反应性；如上图B/C中，富马酸二甲酯（化合物3）比富马酸单甲酯（化合物4）更具有效（分别为13.4μM、15.4μM）；碘乙酰胺（化合物10）比氯乙酰胺（化合物9）更有效（20.7μM、114.7μM）。 在对照试验中，仅有两个分子，化合物1和2在浓度小于250μM时显示出任何抑制活性，并且其IC50明显高于STRP试验。 总的说来，这些数据表明，STRP分析可以报告不同浓度范围内的各种亲电化学物质的半胱氨酸反应性。 应用于高通量筛选 part.2 新型Cys弹头噁二唑氮杂啶 2.1、高通量筛选：噁二唑氮杂啶弹头初发现 研究人员利用STRP分析策略对一个包含10,561个小分子的商业库进行高通量筛选，检测SRTP抑制活性。此外，这些小分子大部分缺乏常见的亲电试剂，如丙烯酰胺、氯乙酰胺等。筛选结果如下图A。 噁二唑氮杂啶弹头初发现：可以看出，红色标记的一系列氮杂啶类化合物（11个）发光信号很低，可能具有潜在的Cys反应活性。其中三个相关化合物如下图B，都含有噁二唑氮杂啶片段。 图片源自JACS. 如上图C、D，三个化合物在STRP试验中具有剂量依赖性反应性抑制活性，而在对照试验中均未显示出抑制活性。 Cys反应性机理：通过分析和试验验证，利用Boc2O酸酐捕获了噁二唑氮杂啶与Cys反应产物，如上图E。通过NMR鉴定（上图F），确定这类活性化合物与Cys反应是一种开环加成反应。 氨基酸选择性：此外，研究人员利用化合物15，对更多的具有亲核性残基的蛋白原氨基酸进行验证，如半胱氨酸、赖氨酸、丝氨酸、苏氨酸、组氨酸、和酪氨酸，仅有半胱氨酸（Cys）显示出与化合物15体外的反应产物，如上图G。 SAR研究，探究更广泛的氮杂啶类似物：研究人员又从24个商售的C3取代氮杂啶片段进行Cys反应性活性验证，仅发现其中3个（17、35、39）保留了一定的反应活性，如下图。其中，化合物39同样包含又噁二唑片段。 图片源自JACS 研究人员将化合物15与另一个不具有SRTP反应抑制活性的咪唑取代氮杂啶化合物19进行基于密度泛函理论（DFT）的计算，以获得噁二唑取代增加氮杂啶Cys反应性的潜在机制，如下图。 图片源自JACS 噁二唑有利氮杂啶开环的机理：如上图，研究人员认为氮杂啶开环反应是通过硫醇盐的协同SN2-样反应进攻氮杂啶环上的2-位碳而进行的。对比可知，化合物15的过渡态（A1-TS）比化合物19的过渡态（B1-TS）具有较低的能垒。而通过晶体结构可知，化合物15达到过渡态所需能量降低，是由于氮杂啶片段与噁二唑片段形成有利的分子内氢键相互作用，以及噁二唑的自然键轨道（NBO）对氮杂啶碳具有有利的NBO电荷。 2.2、氮杂噁二唑广泛参与整个蛋白质族的Cys靶向 体外试验获得了成功，那么，在活细胞中，针对蛋白质组的半胱氨酸残基，噁二唑氮杂啶弹头的共价配体作用如何呢？ 研究人员合成了化合物15的类似物，化合物41，在苯基芳环的对位以末端炔烃取代，用以连接亲和标签和富集研究，如下图A/B，化合物41在STRP中抑制活性与化合物15近似（34μM VS 45 μM），并在对照实验中没有活性。 图片源自JACS 上图C，在HEK293T细胞暴露于化合物41，显示化合物41仅标记活细胞中的Cys残基。 上图D，通过链霉亲和素富集和AP-MS/MS蛋白质组学分析相结合，鉴定出546个蛋白质在这些条件下具有统计学上的富集。 上图E，表明了约81%的蛋白靶标还没有药物化。 上图F，这配体蛋白对应于一系列蛋白质功能类别，如转运蛋白、酶、伴侣蛋白等。 上图G，其中一些特殊的蛋白类别，如RNA结合蛋白、泛素偶联机制和异构肽水解酶等。这些类别在最富集的蛋白质中非常具有代表性，如E3连接酶、半胱氨酸蛋白水解酶、和RNA结合物（图D）。 研究人员重点放在了两个高度富集的E3连接酶上：UBR7（泛素蛋白连接酶n-识别蛋白7）、UBE2O（泛素偶联酶E2 O）；以及两种高度富集的去泛素酶：USP11（泛素特异性肽酶11）、和UCHL1（泛素C-端水解酶L1）。 研究发现，适度的化合物41浓度（20μM），上述4种在细胞中过表达蛋白均可被强烈标记。 这些数据表明，噁二唑氮杂啶可以共价标记Cys蛋白质组的大部分，靶向许多尚未用小分子配体给药的蛋白质。 通过上图中H、I、J中的对比，表明，噁二唑氮杂啶于蛋白质组中相当独立的一类Cys结合，其反应性与其它亲电基团相当不一致。 2.3、应用：UCHL1激活子的发现 最后，研究人员通过表征含噁二唑氮杂啶片段的化合物可以调节共价配体修饰蛋白功能机制，来鉴定反应性基团的适用性。如化合物41与UCHL1的相互作用。UCHL1是一种硫醇肽酶，它从蛋白中切割C端结合的泛素分子，对维持神经细胞群至关重要。 化合物41可以在HEK293T细胞中，对过表达的FLAG标记的UCHL1靶标，以剂量依赖的方式共价标记。如下图A，将Flag标记的转基因C90或C125突变为丝氨酸或丙氨酸，只有在C152突变的情况下才取消标记。 下图B中显示，可以通过MS/MS检测到含有C152的打开的色氨酸加合物片段。 图片源自JACS 上图C中，可以看出UCHL1具有一个“交叉环”，在其运动时，可以进入蛋白质底物的活性位点。而C152就占据在活性位点交叉环的中间位置。这表明它的修饰可能会调节蛋白质的催化活性。 上图D中，增加化合物41浓度，反应效率将近增加了2倍（EC50=6.6μM）。 上图E中，研究还发现，从HEK293T细胞中分离出的化合物41（10μM）原位标记的Flag标记蛋白也显出增加的反应速率，不过程度较低。 上图F、G中，研究发现，化合物41对HEK293T细胞的处理，增加了myc标记的UCHL1进一步去泛素化Flag标记的SMAD2和ALK5的能力。 这些数据表明化合物41对C152的共价修饰在功能上增加了体外和细胞内UCHL1的异肽酶活性。 part.3 总结与展望 本文介绍的一种简单、便捷的可塑性Cys检测方法，用于小分子与Cys的内在反应性检测非常灵敏、准确。利用STRP分析，能够对Cys反应性进行无偏倚评估，准确地报告已知的Cys反应性亲电试剂在几种反应类型中的共价反应，如SNAr、Michael加成等。并且，这种检测策略适用于几个数量级的反应物浓度，产生可测量的发光信号，即使是弱的Cys反应活性分子，可能被适当地检测出来。对于高通量文库（如~104级）的Cys反应性确定非常便利。 声明：发表/转载本文仅仅是出于传播信息的需要，并不意味着代表本公众号观点或证实其内容的真实性。据此内容作出的任何判断，后果自负。若有侵权，告知必删！ 长按关注本公众号  粉丝群/投稿/授权/广告等 请联系公众号助手 觉得本文好看，请点这里↓

Final safety and efficacy results from the Phase 3 EVOLVE-MS-1 trial demonstrate decreases in disease activity and favorable tolerability for VUMERITY®(diroximel fumarate) consistent with previous assessments Patient-reported outcomes favored TYSABRI® (natalizumab) compared to Ocrevus® (ocrelizumab); new insights on subcutaneous administration one year after approval in Germany Data on interferon exposure during pregnancy showed that treatment with PLEGRIDY® (peginterferon beta-1a) or AVONEX® (interferon beta-1a) did not negatively impact child development CAMBRIDGE, Mass., Oct. 26, 2022 (GLOBE NEWSWIRE) -- Biogen Inc. (Nasdaq: BIIB) announced new data from its industry-leading portfolio of multiple sclerosis (MS) therapies being presented at the 38th Congress of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) meeting October 26-28, 2022. The presentations include the final safety and efficacy results of the VUMERITY® (diroximel fumarate) EVOLVE-MS-1 study, as well as a matching analysis comparing treatment with VUMERITY to Ponvory® (ponesimod) and Aubagio® (teriflunomide); patient-reported outcomes and an analysis of the proprietary StratifyJCV™ assay for TYSABRI® (natalizumab); a study evaluating treatment with TECFIDERA® (dimethyl fumarate) to prevent first clinical manifestation of MS for people with radiologically isolated syndrome; and studies assessing the impact of PLEGRIDY® (peginterferon beta-1a) and AVONEX® (interferon beta-1a) on pregnancy, breastfeeding and child development. “Presentations at this year’s ECTRIMS meeting highlight Biogen’s commitment to pursuing research that has a meaningful impact on patients,” said Maha Radhakrishnan, M.D., Chief Medical Officer at Biogen. “These new data provide patients and healthcare professionals with further insight on the safety and efficacy of Biogen’s robust MS portfolio to help inform treatment decisions throughout the lifelong MS journey, from the earliest phase of the disease to important milestones such as pregnancy.” Final Safety and Efficacy Results of VUMERITY EVOLVE-MS-1 Study Reported Presentations including data from EVOLVE-MS-1 and OPTIMUM clinical studies further support the safety and efficacy of VUMERITY: Final safety and efficacy results from EVOLVE-MS-1 demonstrate decreases in disease activity and favorable tolerability for VUMERITY in 1,057 patients over 96 weeks, in line with previous assessments. The reduction in annualized relapse rate (ARR) was 81.6%, the estimated proportion of patients who were relapse-free was 82.4%, and the estimated proportion with no evidence of disease activity (NEDA-3) was 41.1%. While 24.3% of patients discontinued treatment, discontinuation due to gastrointestinal adverse events (0.7%) and flushing (0.5%) were low. A matching-adjusted indirect comparison was done between VUMERITY (EVOLVE-MS-1) and Ponvory and Aubagio (OPTIMUM) clinical studies for ARR, 12-week confirmed disability progression (CDP), 24-week CDP, absence of gadolinium-enhancing (Gd+) T1 lesions and absence of new/enlarging T2 lesions. After weighting for cross-trial differences, VUMERITY was associated with a higher proportion of patients free of Gd+ T1 lesions and new/enlarging T2 lesions at the end of follow-up compared to Ponvory, with similar efficacy for ARR and 12- and 24-week CDP. VUMERITY had greater efficacy than Aubagio for all clinical and radiological outcome measures, except for 24-week CDP, in which there was similar efficacy. Studies Highlight Patient-Reported Outcomes for TYSABRI; Analysis Shows Impact of StratifyJCV Assay Two presentations highlight improvements in patient-reported outcomes following treatment with TYSABRI along with preference and satisfaction for the TYSABRI subcutaneous (SC) route of administration: The results of recent survey analysis, which included TYSABRI [n=52] and Ocrevus (ocrelizumab)-treated [n=92] relapsing-remitting multiple sclerosis (RRMS) patients aged 21 and older who had taken at least one prior disease modifying treatment (DMT), found more patients treated with TYSABRI versus Ocrevus reported improvements in disease activity (84.6% vs 59.8%), emotional symptoms (73.1% vs 35.9%), physical symptoms (69.2% vs 43.5%), cognitive symptoms (61.5% vs 32.6%) and social roles/activities (71.2% vs 35.9%). In addition, more patients treated with TYSABRI reported their DMT met or exceeded treatment expectations in comparison to those treated with Ocrevus (96.2% vs 72.8%). In the first interim analysis of 206 patients in the observational, prospective, multi-center SISTER study in Germany, the TYSABRI SC route of administration was preferred by individuals (89.6%; 163 patients) compared with intravenous administration; nearly all patients receiving SC treatment expressed satisfaction with their choice (98.7%; 156 patients) and the most frequent reasons for SC preference were shorter and more convenient administration. The TYSABRI SC administration is available in 26 countries and over 16,000 patients have been treated with SC therapy.1 “Given the recent introduction of natalizumab subcutaneous administration in Europe, these findings validate clinical study data with real-world insights,” said Prof. Ralf Gold, Ruhr-University Bochum, Bochum, Germany. “In our study, we found that nearly 90% of patients prefer subcutaneous administration given its shorter duration and convenience of dosing.” Separately, an analysis assessed utilization of Biogen’s StratifyJCV™ -- a proprietary antibody assay used to detect the presence of anti-JC virus (JCV) antibodies in serum and to quantify antibody index values, which are correlated to progressive multifocal leukoencephalopathy (PML) risk in patients treated with TYSABRI. More than 2 million StratifyJCV tests have been conducted worldwide. Aggregated data of the results of 845,498 StratifyJCV tests conducted from January 2015 to December 2021 showed a decrease in percentage of JCV-positive index results >1.5 in retests, from 15% in 2015 to 8% in 2021, demonstrating that healthcare professionals are using the assay to appropriately identify, monitor and manage patients on TYSABRI. Effect of TECFIDERA on Radiologically Isolated Syndrome Reported for the First Time Results of the Assessment of TECFIDERA in Radiologically Isolated Syndrome (ARISE) study, a placebo-controlled, multi-center, double-blinded clinical trial, will be presented in the late-breaking section. ARISE investigated the impact of therapeutic intervention in preventing the first clinical manifestation of MS for people with radiologically isolated syndrome (RIS). ARISE enrolled 87 patients who were randomized to TECFIDERA or placebo and treated for up to 96 weeks. Researchers found treatment with TECFIDERA resulted in an 82% risk reduction relative to placebo in the prevention of a first clinical event related to CNS demyelination. Impact of PLEGRIDYand AVONEXon Pregnancy, Breastfeeding and Child Development Two presentations assessed the impact of interferon exposure on pregnancy, breastfeeding and child development. Data from the PRIMA post-authorization safety study was consistent with results from previous studies showing that exposure to PLEGRIDY or AVONEX during pregnancy or lactation did not negatively impact child development or intrauterine growth. Further, a preliminary analysis of the German Multiple Sclerosis and Pregnancy Registry assessed child development in infants born to mothers with AVONEX or PLEGRIDY exposure during pregnancy, and treatment did not negatively impact children’s development. Title and Times of Data Presentations Featured at ECTRIMS: Natalizumab-Treated RRMS Patients with Prior DMT Use Report Better Outcomes, Treatment Satisfaction and Unique Benefits Than Similar Patients Treated with Ocrelizumab – EP0851 – October 26, 8 a.m. CET / 2 a.m. ET Disease Activity and Pregnancy Outcomes After Long-Term Exposure to Natalizumab During Pregnancy – 0039 – October 26, 2:56 p.m. CET / 8:56 a.m. ET SISTER – Subcutaneous: Non-Interventional, Observational, Prospective, German Multicentre, Open Label Study Over 12-Months for TYSABRI Patient Preference – Experience from Real-World – Preliminary Results of the 1st Interim Analysis – P365 – October 26, 8 a.m. CET / 2 a.m. ET Long-Term Effectiveness of Natalizumab for RRMS: Dutch and Global Interim Results from TYSABRI Observational Program – P373 – October 26, 8 a.m. CET / 2 a.m. ET Exploratory Magnetic Resonance Imaging Endpoints from NOVA: A Randomized Controlled Study of the Efficacy of 6-Week Dosing of Natalizumab vs Continued 4-Week Treatment for Multiple Sclerosis – P350 – October 26, 8 a.m. CET / 2 a.m. ET Interferon- or Peginterferon-Beta 1a Exposure During Pregnancy in Women with Multiple Sclerosis: Outcomes on Child Development – P075 – October 26, 8 a.m. CET / 2 a.m. ET and October 27, 1:05 p.m. CET / 7:05 a.m. ET StratifyJCV™ Serum Anti-JCV Antibody Assay for Natalizumab Patients: Unilabs Global Cohort Data Descriptive Analysis and Unilabs Customer Satisfaction Survey Results – P750 – October 26, 8 a.m. CET / 2 a.m. ET Matching-Adjusted Indirect Comparisons of Diroximel Fumarate, Ponesimod, and Teriflunomide for Relapsing Multiple Sclerosis – P708 – October 26, 8 a.m. CET / 2 a.m. ET Diroximel Fumarate in Patients with Relapsing-Remitting Multiple Sclerosis: Final Safety and Efficacy Results from the Phase 3 EVOLVE-MS-1 Study – P712 – October 26, 8 a.m. CET / 2 a.m. ET Interferon-Beta Exposure During Pregnancy and Breastfeeding: Impact on Birth Outcome and Child Development – Results from the Post-Authorisation Safety Study PRIMA – P478 – October 26, 8 a.m. CET / 2 a.m. ET Multi-Center, Randomized, Double-Blinded Assessment of Dimethyl Fumarate in Extending the Time to a First Clinical Demyelinating Event in Radiologically Isolated Syndrome (ARISE) – 0179 – October 28, 3:49 p.m. CET / 9:49 a.m. ET About VUMERITY® (diroximel fumarate) VUMERITY is an oral fumarate approved in the U.S. for the treatment of relapsing forms of multiple sclerosis in adults, including clinically isolated syndrome, relapsing-remitting disease and active secondary progressive disease. Once in the body, VUMERITY rapidly converts to monomethyl fumarate, the same active metabolite of dimethyl fumarate (TECFIDERA). VUMERITY is available in 15 countries, and more than 28,200 patients have been treated with it, representing more than 24,200 patient-years of exposure across clinical trial use and patients prescribed VUMERITY.2 VUMERITY is contraindicated in patients with a known hypersensitivity to diroximel fumarate, dimethyl fumarate or any of the excipients of VUMERITY and in patients taking dimethyl fumarate. VUMERITY can cause anaphylaxis and angioedema after the first dose or at any time during treatment. PML has occurred in patients with MS treated with dimethyl fumarate (which has the same active metabolite as VUMERITY). PML is an opportunistic viral infection of the brain caused by the JC virus (JCV) that typically only occurs in patients who are immunocompromised, and that usually leads to death or severe disability. A fatal case of PML occurred in a patient who received dimethyl fumarate for four years while enrolled in a clinical trial. Serious cases of herpes zoster have occurred with dimethyl fumarate, including disseminated herpes zoster, herpes zoster ophthalmicus, herpes zoster meningoencephalitis, and herpes zoster meningomyelitis. These events may occur at any time during treatment. Other serious opportunistic infections have occurred with dimethyl fumarate, including cases of serious viral (herpes simplex virus, West Nile virus, cytomegalovirus), fungal (Candida and Aspergillus), and bacterial (Nocardia, Listeria monocytogenes, Mycobacterium tuberculosis) infections. VUMERITY may decrease lymphocyte counts. In the MS placebo-controlled trials, mean lymphocyte counts decreased by approximately 30% during the first year of treatment with dimethyl fumarate and then remained stable. Four weeks after stopping dimethyl fumarate, mean lymphocyte counts increased but did not return to baseline. Clinically significant cases of liver injury have been reported in patients treated with dimethyl fumarate in the post-marketing setting. The onset has ranged from a few days to several months after initiation of treatment. Signs and symptoms of liver injury, including elevation of serum aminotransferases to greater than five-fold the upper limit of normal and elevation of total bilirubin to greater than two-fold the upper limit of normal have been observed. VUMERITY may cause flushing (e.g., warmth, redness, itching, and/or burning sensation). Forty percent of patients taking dimethyl fumarate reported flushing, which was mostly mild to moderate in severity. Three percent of patients discontinued dimethyl fumarate for flushing and <1% had serious flushing events that led to hospitalization. The most common adverse events associated with dimethyl fumarate (incidence ≥10% and ≥2% more than placebo) were flushing, abdominal pain, diarrhea and nausea. A list of adverse events can be found in the full VUMERITY product labeling for each country where it is approved. Please click here for Important Safety Information and full Prescribing Information, including Patient Information for VUMERITY in the U.S., or visit your respective country’s product website. About TECFIDERA® (dimethyl fumarate) TECFIDERA is approved for the treatment of relapsing forms of multiple sclerosis (MS), including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease in adults. TECFIDERA has been shown to reduce the rate of MS relapses, slow the progression of disability and impact the number of MS brain lesions, while demonstrating a well-characterized safety pro people with relapsing forms of MS. TECFIDERA is approved in 69 countries, and more than 580,500 patients have been treated with it, representing more than 1.2 million patient-years of exposure across clinical trial use and patients prescribed TECFIDERA.3 TECFIDERA is contraindicated in patients with a known hypersensitivity to dimethyl fumarate or any of the excipients of TECFIDERA. TECFIDERA can cause anaphylaxis and angioedema after the first dose or at any time during treatment. PML has occurred in patients with MS treated with TECFIDERA. PML is an opportunistic viral infection of the brain caused by the JC virus (JCV) that typically only occurs in patients who are immunocompromised, and that usually leads to death or severe disability. A fatal case of PML occurred in a patient who received TECFIDERA for four years while enrolled in a clinical trial. Serious cases of herpes zoster have occurred with TECFIDERA, including disseminated herpes zoster, herpes zoster ophthalmicus, herpes zoster meningoencephalitis, and herpes zoster meningomyelitis. These events may occur at any time during treatment. Other serious opportunistic infections have occurred with TECFIDERA, including cases of serious viral (herpes simplex virus, West Nile virus, cytomegalovirus), fungal (Candida and Aspergillus), and bacterial (Nocardia, Listeria monocytogenes, Mycobacterium tuberculosis) infections. TECFIDERA may decrease lymphocyte counts. In the MS placebo-controlled trials, mean lymphocyte counts decreased by approximately 30% during the first year of treatment with TECFIDERA and then remained stable. Four weeks after stopping TECFIDERA, mean lymphocyte counts increased but did not return to baseline. Clinically significant cases of liver injury have been reported in patients treated with TECFIDERA in the post-marketing setting. The onset has ranged from a few days to several months after initiation of treatment. Signs and symptoms of liver injury, including elevation of serum aminotransferases to greater than five-fold the upper limit of normal and elevation of total bilirubin to greater than two-fold the upper limit of normal have been observed. TECFIDERA may cause flushing (e.g. warmth, redness, itching, and/or burning sensation). Forty percent of patients taking TECFIDERA reported flushing, which was mostly mild to moderate in severity. Three percent of patients discontinued TECFIDERA for flushing and <1% had serious flushing events that led to hospitalization. The most common adverse events associated with TECFIDERA (incidence ≥10% and ≥2% more than placebo) were flushing, abdominal pain, diarrhea and nausea. A list of adverse events can be found in the full TECFIDERA product labeling for each country where it is approved. Please click here for Important Safety Information and full Prescribing Information, including Patient Information for TECFIDERA in the U.S., or visit your respective country’s product website. About TYSABRI® (natalizumab) TYSABRI is approved in the U.S. as monotherapy for the treatment of relapsing forms of multiple sclerosis, including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease in adults. TYSABRI increases the risk of progressive multifocal leukoencephalopathy (PML). When initiating and continuing treatment with TYSABRI, physicians should consider whether the expected benefit of TYSABRI is sufficient to offset this risk. TYSABRI is approved in more than 80 countries, and over 248,000 people worldwide have been treated with TYSABRI, with more than 1 million patient-years of experience, based on clinical trials and prescription data.1 TYSABRI increases the risk of PML, a rare opportunistic viral infection of the brain which has been associated with death or severe disability. Risk factors that increase the risk of PML are the presence of anti-JC virus antibodies, prior immunosuppressant use and longer TYSABRI treatment duration. Patients who have all three risk factors have the highest risk of developing PML. These factors should be considered in the context of expected benefit when initiating and continuing treatment with TYSABRI. Healthcare professionals should monitor patients on TYSABRI for any new sign or symptom that may be suggestive of PML. TYSABRI dosing should be withheld immediately at the first sign or symptom suggestive of PML. TYSABRI is contraindicated in patients who have or have had PML and in patients who have had a hypersensitivity reaction to TYSABRI. TYSABRI also increases the risk of developing encephalitis and meningitis caused by herpes simplex and varicella zoster viruses, and serious, life-threatening and sometimes fatal cases have been reported in the post-marketing setting in MS patients receiving TYSABRI. Clinically significant liver injury, including acute liver failure requiring transplant, has also been reported in the post-marketing setting. Signs of liver injury, including markedly elevated serum hepatic enzymes and elevated total bilirubin, occurred as early as six days after the first dose; signs of liver injury have also been reported for the first time after multiple doses. Hypersensitivity reactions have occurred in patients receiving TYSABRI, including serious systemic reactions (e.g., anaphylaxis) which occurred at an incidence of <1%. The immune system effects of TYSABRI may increase the risk for infections. In Study MS1, certain types of infections—including pneumonias and urinary tract infections (including serious cases), gastroenteritis, vaginal infections, tooth infections, tonsillitis, and herpes infections—occurred more often in TYSABRI-treated patients than in placebo-treated patients. In clinical trials, TYSABRI was observed to induce increases in circulating lymphocytes, monocytes, eosinophils, basophils, and nucleated red blood cells. Observed changes persisted during TYSABRI exposure, but were reversible, returning to baseline levels usually within 16 weeks after the last dose. Cases of thrombocytopenia, including immune thrombocytopenic purpura, have been reported with the use of TYSABRI in the post-marketing setting. Cases of neonatal thrombocytopenia, at times associated with anemia, have been reported in newborns with in utero exposure to TYSABRI. The most common adverse reactions associated with TYSABRI (incidence ≥10% and ≥2% more than placebo) were headache, fatigue, infusion reactions, urinary tract infections, arthralgia, depression, pain in extremity, rash, gastroenteritis, and vaginitis. A list of adverse events can be found in the full TYSABRI product labeling for each country where it is approved. Please click here for Important Safety Information, including Boxed Warning, and full Prescribing Information, including Medication Guide for TYSABRI in the U.S., or visit your respective country’s product website. About PLEGRIDY® (peginterferon beta-1a) PLEGRIDY (peginterferon beta-1a) is indicated for the treatment of relapsing forms of multiple sclerosis, including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease in adults. PLEGRIDY is currently approved in over 60 countries including the U.S., Canada, Australia and Switzerland and across the European Union. Over 81,000 people worldwide have been treated with PLEGRIDY, with over 170,000 patient-years of experience, based on prescription data.4 Biogen continues to work toward making PLEGRIDY available in additional countries across the globe. PLEGRIDY is contraindicated in patients with a history of hypersensitivity to natural or recombinant interferon beta, or any other component of the formulation. Rare cases of severe hepatic injury, including cases of hepatic failure have been reported in patients taking PLEGRIDY. Symptoms of depression, suicidal ideation or psychosis, and cases of suicide, have been reported with increased frequency with patients receiving PLEGRIDY. Serious allergic reactions are rare complications of interferon beta; anaphylaxis has been reported with use of PLEGRIDY. Injection site reactions, including injection site necrosis, can occur with the use of interferon beta, including PLEGRIDY; injection site abscesses and cellulitis have been reported in the post-marketing setting with use of PLEGRIDY. Some cases required treatment with hospitalization for surgical drainage and intravenous antibiotics. Cases of congestive heart failure, cardiomyopathy and cardiomyopathy with congestive heart failure have been reported in patients without known predisposition. Decreased peripheral blood counts have been reported from post-marketing experience; monitor patients for infections, bleeding and anemia; monitor complete blood cell counts, differential white cell counts, and platelet counts. Cases of thrombotic microangiopathy, including thrombotic thrombocytopenic purpura and hemolytic uremic syndrome, some fatal, have been reported several weeks to years after starting interferon beta products. Autoimmune disorders of multiple target organs including idiopathic thrombocytopenia, hyper- and hypothyroidism, and autoimmune hepatitis have been reported. Seizures have been reported in patients using PLEGRIDY. The most common adverse reactions in clinical trials of subcutaneous PLEGRIDY (incidence ≥10% and at least 2% more frequent on PLEGRIDY than on placebo) were injection site erythema, influenza-like illness, pyrexia, headache, myalgia, chills, injection site pain, asthenia, injection site pruritus, and arthralgia. A list of adverse events can be found in the full PLEGRIDY product labeling for each country where it is approved. Please click here for Important Safety Information and full Prescribing Information, including Medication Guide for PLEGRIDY in the U.S., or visit your respective country’s product website. About AVONEX® (interferon beta-1a) AVONEX is indicated for the treatment of relapsing forms of multiple sclerosis, including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease in adults. AVONEX is approved in over 90 countries and over 625,000 people worldwide have been treated with AVONEX, with over 2.8 million patient-years of experience, based on prescription data.5 Avonex is contraindicated in patients with a history of hypersensitivity to natural or recombinant interferon beta, or any other component of the formulation. Symptoms of depression, suicidal ideation or psychosis, and cases of suicide, have been reported with increased frequency with patients receiving AVONEX. Rare cases of severe hepatic injury, including cases of hepatic failure have been reported in patients taking AVONEX. Anaphylaxis has been reported as a rare complication of AVONEX use; other allergic reactions have included dyspnea, orolingual edema, skin rash and urticaria. Injection site reactions, including injection site necrosis, can occur with the use of interferon beta, including AVONEX. Injection site abscesses and cellulitis and injection site necrosis have been reported in the post-marketing setting with use of AVONEX. Some cases required treatment with hospitalization for surgical drainage and intravenous antibiotics. Cases of congestive heart failure, cardiomyopathy and cardiomyopathy with congestive heart failure have been reported in patients without known predisposition. Decreased peripheral blood counts have been reported from post-marketing experience. Cases of thrombotic microangiopathy (TMA), including thrombotic thrombocytopenic purpura and hemolytic uremic syndrome, some fatal, have been reported several weeks to years after starting interferon beta products. Seizures have been temporally associated with the use of beta interferons in clinical trials and post-marketing safety surveillance. Post-marketing reports of autoimmune disorders of multiple target organs in AVONEX-treated patients including idiopathic thrombocytopenia, hyper- and hypothyroidism, and rare cases of autoimmune hepatitis. Routine periodic blood chemistry, hematology, liver function and thyroid function tests are recommended. The most commonly reported adverse reactions (at least 5% more frequent on AVONEX than on placebo) were flu-like symptoms. A list of adverse events can be found in the full AVONEX product labeling for each country where it is approved. Please click here for Important Safety Information and full Prescribing Information, including Medication Guide for AVONEX in the U.S., or visit your respective country’s product website. About Biogen As pioneers in neuroscience, Biogen discovers, develops, and delivers worldwide innovative therapies for people living with serious neurological diseases as well as related therapeutic adjacencies. One of the world’s first global biotechnology companies, Biogen was founded in 1978 by Charles Weissmann, Heinz Schaller, Sir Kenneth Murray, and Nobel Prize winners Walter Gilbert and Phillip Sharp. Today, Biogen has a leading portfolio of medicines to treat multiple sclerosis, has introduced the first approved treatment for spinal muscular atrophy, and developed the first and only approved treatment to address a defining pathology of Alzheimer’s disease. Biogen is also commercializing biosimilars and focusing on advancing one of the industry’s most diversified pipelines in neuroscience that will transform the standard of care for patients in several areas of high unmet need. In 2020, Biogen launched a bold 20-year, $250 million initiative to address the deeply interrelated issues of climate, health, and equity. Healthy Climate, Healthy Lives™ aims to eliminate fossil fuels across the company’s operations, build collaborations with renowned institutions to advance the science to improve human health outcomes, and support underserved communities. We routinely post information that may be important to investors on our website at . Follow us on social media - Twitter, LinkedIn, Facebook, YouTube. Biogen Safe Harbor This news release contains forward-looking statements, including statements made pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995, relating to the potential benefits, safety and efficacy of TYSABRI and VUMERITY; the results of certain real-world data; clinical trials and data readouts and presentations; the identification and treatment of MS; our research and development program for the treatment of MS; and the potential of our commercial business, including TYSABRI and VUMERITY. These forward-looking statements may be identified by words such as “aim,” “anticipate,” “believe,” “could,” “estimate,” “expect,” “forecast,” “goal,” “intend,” “may,” “plan,” “possible,” “potential,” “will,” “would” and other words and terms of similar meaning. Drug development and commercialization involve a high degree of risk, and only a small number of research and development programs result in commercialization of a product. You should not place undue reliance on these statements or the scientific data presented. These statements involve risks and uncertainties that could cause actual results to differ materially from those reflected in such statements, including without limitation the occurrence of adverse safety events and/or unexpected concerns that may arise from additional data or analysis; risks of unexpected costs or delays; failure to protect and enforce our data, intellectual property and other proprietary rights and uncertainties relating to intellectual property claims and challenges; product liability claims; third party collaboration risks; and the direct and indirect impacts of the ongoing COVID-19 pandemic on our business, results of operations and financial condition. The foregoing sets forth many, but not all, of the factors that could cause actual results to differ from our expectations in any forward-looking statement. Investors should consider this cautionary statement as well as the risk factors identified in our most recent annual or quarterly report and in other reports we have filed with the U.S. Securities and Exchange Commission. These statements are based on our current beliefs and expectations and speak only as of the date of this news release. We do not undertake any obligation to publicly update any forward-looking statements, whether as a result of new information, future developments or otherwise. References: Combined post-marketing data based on prescriptions and clinical trials exposure to TYSABRI as of July 31, 2022. TYSABRI subcutaneous administration exposure as of September 30, 2022. Combined post-marketing data based on prescriptions and clinical trials exposure to VUMERITY as of June 30, 2022. Combined post-marketing data based on prescriptions and clinical trials exposure to TECFIDERA as of June 30, 2022. Post-marketing data based on PLEGRIDY prescriptions as of June 30, 2022. Post-marketing data based on AVONEX prescriptions as of June 30, 2022. MEDIA CONTACT: Allison Parks + 1 248 229 4461 public.affairs@biogen.com INVESTOR CONTACT: Mike Hencke +1 781 464 2442 IR@biogen.com