CHI3L1

Issued 12,765,958 new sharesPriced at €2.35 per share, representing a 15.44% discount on the 5-day VWAPStrong investor demand, with the offering significantly oversubscribed€15 million anchored by a leading US institutional investorProceeds to fund clinical development and corporate initiatives MADRID and CAMBRIDGE, Mass., April 24, 2025 (GLOBE NEWSWIRE) -- Oryzon Genomics, S.A. (ISIN Code: ES0167733015, ORY), a clinical-stage biopharmaceutical company and a European leader in epigenetics, announced today the successful completion of a €30 million capital increase through the issuance of 12,765,958 new shares, priced at €2.35 per share. This pricing reflects a 15.44% discount to the 5-day volume-weighted average price (VWAP) of €2.779 per share prior to April 22, 2025, and a 19.38% discount to the April 22 closing price of €2.915. The offering, which was structured as straight equity with no warrants attached, attracted strong demand and was significantly oversubscribed. A US-based institutional investor anchored the round with a €15 million order, with the remaining demand filled by investors across the US, Europe, and Spain. The capital increase was capped at €30 million by the company’s Board of Directors. Maxim (U.S.), All-Invest (EU/UK), and Singular Bank (Spain) acted as bookrunners. Banco Sabadell, S.A. acted as the Settlement and Technical Pre-financing Agent for the Capital Increase, while Gómez-Acebo & Pombo Abogados, S.L.P. (Spanish law) and Gibson, Dunn & Crutcher LLP (U.S. law) served as legal advisors to the Company, and Loeb & Loeb LLP acted as U.S. legal counsel to Maxim Group LLC. The funds raised will be used to: Advance the clinical development of vafidemstat for the treatment of aggression in CNS disorders such as Borderline Personality Disorder (BPD) and Autism Spectrum Disorder (ASD)Expand ongoing clinical programs in oncology and hematologySupport CMC activities and preparations for Phase IIb-III trials, as well as other regulatory initiativesStrengthen the company's balance sheet to support future partnership or M&A negotiationsCover general administrative expenses and financial obligationsEnhance legal and audit readiness for a potential dual listing on Nasdaq Dr. Carlos Buesa, CEO of Oryzon, commented: “This financing marks a key milestone that enables us to continue progressing our clinical programs with vafidemstat in aggression-related CNS disorders and to further develop our oncology and hematology pipeline. It also reinforces our financial position, supporting strategic conversations with investors and potential partners.” About OryzonFounded in 2000 in Barcelona, Spain, Oryzon (ISIN Code: ES0167733015) is a clinical stage biopharmaceutical company and the European leader in epigenetics, with a strong focus on personalized medicine in CNS disorders and oncology. Oryzon’s team is composed of highly qualified professionals from the pharma industry located in Barcelona, Boston, and San Diego. Oryzon has an advanced clinical portfolio with two LSD1 inhibitors, vafidemstat in CNS (Phase III-ready) and iadademstat in oncology (Phase II). The company has other pipeline assets directed against other epigenetic targets like HDAC-6 where a clinical candidate ORY-4001, has been nominated for its possible development in CMT and ALS. In addition, Oryzon has a strong platform for biomarker identification and target validation for a variety of malignant and neurological diseases. For more information, visit www.oryzon.com About IadademstatIadademstat (ORY-1001) is a small oral molecule, which acts as a highly selective inhibitor of the epigenetic enzyme LSD1 and has a powerful differentiating effect in hematologic cancers (see Maes et al., Cancer Cell 2018 Mar 12; 33 (3): 495-511.e12.doi: 10.1016 / j.ccell.2018.02.002.). A FiM Phase I/IIa clinical trial with iadademstat in R/R AML patients demonstrated the safety and good tolerability of the drug and preliminary signs of antileukemic activity, including a CRi (see Salamero et al, J Clin Oncol, 2020, 38(36): 4260-4273. doi: 10.1200/JCO.19.03250). Iadademstat has shown encouraging safety and strong clinical activity in combination with azacitidine in a Phase IIa trial in elder 1L AML patients (ALICE trial) (see Salamero et al., ASH 2022 oral presentation & The Lancet Haematology, 2024, 11(7):e487-e498). Iadademstat is currently being evaluated in combination with gilteritinib in the ongoing Phase Ib FRIDA trial in patients with relapsed/refractory AML with FLT3 mutations, and in combination with azacitidine and venetoclax in 1L AML in an investigator-initiated study led by OHSU and in a trial sponsored by the U.S. National Cancer Institute (NCI) under the Cooperative Research and Development Agreement (CRADA) signed between Oryzon and the NCI to collaborate on further clinical development of iadademstat in different types of hematologic and solid cancers. Beyond hematological cancers, the inhibition of LSD1 has been proposed as a valid therapeutic approach in some solid tumors such as small cell lung cancer (SCLC), neuroendocrine tumors (NET), medulloblastoma and others. In a Phase IIa trial in combination with platinum/etoposide in second line ED-SCLC patients (CLEPSIDRA trial), preliminary activity and safety results have been reported (see Navarro et al., ESMO 2018 poster). Iadademstat is in a collaborative Phase II trial with the Fox Chase Cancer Center (FCCC) in combination with paclitaxel in R/R neuroendocrine carcinomas, and in a Phase I/II randomized trial in 1L ED-SCLC in combination with ICI sponsored by NCI and led by the Memorial Sloan Kettering Cancer Center. Oryzon is further expanding the clinical development of iadademstat through additional investigator-initiated studies. Iadademstat has orphan drug designation for SCLC in the US and for AML in the US and EU. About Vafidemstat Vafidemstat (ORY-2001) is an oral, CNS-optimized LSD1 inhibitor. The molecule acts on several levels: it reduces cognitive impairment, including memory loss and neuroinflammation, and at the same time has neuroprotective effects. In animal studies vafidemstat not only restores memory but reduces the exacerbated aggressiveness of SAMP8 mice, a model for accelerated aging and Alzheimer’s disease (AD), to normal levels and also reduces social avoidance and enhances sociability in murine models. In addition, vafidemstat exhibits fast, strong, and durable efficacy in several preclinical models of multiple sclerosis (MS). Oryzon has performed two Phase IIa clinical trials in aggressiveness in patients with different psychiatric disorders (REIMAGINE, see Ferrer et al, Psychiatry & Clin Neurosci, 2025, doi.org/10.1111/pcn.13800) and in aggressive/agitated patients with moderate or severe AD (REIMAGINE-AD), with positive clinical results reported in both. Additional finalized Phase IIa clinical trials with vafidemstat include the ETHERAL trial in patients with Mild to Moderate AD, where a significant reduction of the inflammatory biomarker YKL40 was observed after 6 and 12 months of treatment, and the pilot, small-scale SATEEN trial in Relapse-Remitting and Secondary Progressive MS, where anti-inflammatory activity was also observed. Vafidemstat has also been tested in a Phase II in severe Covid-19 patients (ESCAPE) assessing the capability of the drug to prevent ARDS, one of the most severe complications of the viral infection, where it showed significant anti-inflammatory effects in severe Covid-19 patients. Vafidemstat is currently advancing as a Phase III-ready asset in Borderline Personality disorder (BPD) following completion of the global, randomized, double blind Phase IIb PORTICO trial (final data presented at ECNP-2024). Following receipt of the minutes from the End-of-Phase II meeting with the FDA to discuss PORTICO’s results, the company announced plans to move forward with a Phase III PORTICO-2 trial in agitation/aggression in BPD (FDA submission planned in 1H2025). Vafidemstat is also being investigated in a double-blind, randomized, placebo-controlled Phase IIb trial in negative symptoms of schizophrenia (EVOLUTION trial, recruitment ongoing). The company is also deploying a CNS precision medicine approach with vafidemstat in genetically-defined patient subpopulations of certain CNS disorders and is evaluating a clinical trial in Kabuki Syndrome patients. The company is also exploring the clinical development of vafidemstat in other neurodevelopmental syndromes. FORWARD-LOOKING STATEMENTS This communication contains, or may contain, forward-looking information and statements about Oryzon, including financial projections and estimates and their underlying assumptions, statements regarding plans, objectives, and expectations with respect to future operations, capital expenditures, synergies, products and services, and statements regarding future performance. Forward-looking statements are statements that are not historical facts and are generally identified by the words “expects,” “anticipates,” “believes,” “intends,” “estimates” and similar expressions. Although Oryzon believes that the expectations reflected in such forward-looking statements are reasonable, investors and holders of Oryzon shares are cautioned that forward-looking information and statements are subject to various risks and uncertainties, many of which are difficult to predict and generally beyond the control of Oryzon that could cause actual results and developments to differ materially from those expressed in, or implied or projected by, the forward-looking information and statements. These risks and uncertainties include those discussed or identified in the documents sent by Oryzon to the Spanish Comisión Nacional del Mercado de Valores (CNMV), which are accessible to the public. Forward-looking statements are not guarantees of future performance and have not been reviewed by the auditors of Oryzon. You are cautioned not to place undue reliance on the forward-looking statements, which speak only as of the date they were made. All subsequent oral or written forward-looking statements attributable to Oryzon or any of its members, directors, officers, employees, or any persons acting on its behalf are expressly qualified in their entirety by the cautionary statement above. All forward-looking statements included herein are based on information available to Oryzon on the date hereof. Except as required by applicable law, Oryzon does not undertake any obligation to publicly update or revise any forward‐looking statements, whether as a result of new information, future events, or otherwise. This document does not constitute an offer or invitation to purchase or subscribe shares in accordance with the provisions of Regulation (EU) 2017/1129 of the European Parliament and of the Council of 14 June 2017, and/or the restated text of the Securities Market Law, approved by Law 6/2023 of 17 March, and its implementing regulations. Nothing in this document constitutes investment advice. In addition, this document does not constitute an offer of purchase, sale or exchange, nor a request for an offer of purchase, sale or exchange of securities, nor a request for any vote or approval in any jurisdiction. The shares of Oryzon Genomics, S.A. may not be offered or sold in the United States of America except pursuant to an effective registration statement under the Securities Act of 1933 or pursuant to a valid exemption from registration.. Spain Oryzon IR & Media, Europe & US Patricia Cobo/Mario Cordera Emili Torrell Sandya von der Weid Atrevia Chief BD Officer LifeSci Advisors, LLC +34 91 564 07 25 +34 673 33 97 65 +34 93 515 1313 +41 78 680 05 38 pcobo@atrevia.com mcordera@atrevia.com etorrell@oryzon.com svonderweid@lifesciadvisors.com

New “Decision to grant” communications in Europe and JapanMADRID and CAMBRIDGE, Mass., April 21, 2025 (GLOBE NEWSWIRE) -- Oryzon Genomics, S.A. (ISIN Code: ES0167733015, ORY), a clinical-stage biopharmaceutical company and a European leader in epigenetics, announced today that the European Patent Office has issued a “Decision to grant” communication for its European patent application EP20712563.4 relating to vafidemstat. The patent, titled “Methods of treating Attention Deficit Hyperactivity Disorder using KDM1A inhibitors such as the compound vafidemstat”, covers the use Oryzon’s LSD1 inhibitor, vafidemstat, currently in clinical development for psychiatric disorders. A “Decision to grant” is a formal communication from a Patent Office indicating that the patent application has met all requirements for issuance as a patent. Once formally granted, this European patent will remain in force until at least 2040, excluding any potential patent term extension. A counterpart patent has already been granted in Japan, and additional applications are pending in other countries. Oryzon also announced that it has received a “Decision to grant” from the Japanese Patent Office for its patent application JP2022-514201, titled “Methods of treating autism spectrum disorder”. This will be the first granted patent in this patent family, with applications pending in other countries. This Japanese patent, once issued, will also remain in force until at least 2040, not including potential term extension. “These new patent allowances in Europe and Japan complement the ample portfolio we’ve already secured for vafidemstat,” said Neus Virgili, Oryzon’s Chief IP Officer. “They further strengthen our global IP position and support the compound’s commercial potential across multiple CNS indications and our competitive position.” Oryzon’s patent porfolio for vafidemstat in CNS also includes two additional patent families:. One covers the treatment of aggression and social withdrawal, with patents already granted or allowed in Europe, Australia, Hong Kong, Korea, Malaysia, the Philippines, and Russia, and patent applications pending in other countries. These patents will remain in force until at least 2038, excluding any potential patent term extensions, which may provide additional years of protection.The second patent family relates to the treatment of borderline personality disorder, with allowed or granted patents in Europe, Japan, Mexico, Russia, Singapore and South Africa, and patent applications pending in additional countries. These patents will remain in force until at least 2040, excluding any potential patent term extensions, which may provide additional years of protection. About OryzonFounded in 2000 in Barcelona, Spain, Oryzon (ISIN Code: ES0167733015) is a clinical stage biopharmaceutical company and the European leader in epigenetics, with a strong focus on personalized medicine in CNS disorders and oncology. Oryzon’s team is composed of highly qualified professionals from the pharma industry located in Barcelona, Boston, and San Diego. Oryzon has an advanced clinical portfolio with two LSD1 inhibitors, vafidemstat in CNS (Phase III-ready) and iadademstat in oncology (Phase II). The company has other pipeline assets directed against other epigenetic targets like HDAC-6 where a clinical candidate ORY-4001, has been nominated for its possible development in CMT and ALS. In addition, Oryzon has a strong platform for biomarker identification and target validation for a variety of malignant and neurological diseases. For more information, visit www.oryzon.com About Vafidemstat Vafidemstat (ORY-2001) is an oral, CNS-optimized LSD1 inhibitor. The molecule acts on several levels: it reduces cognitive impairment, including memory loss and neuroinflammation, and at the same time has neuroprotective effects. In animal studies vafidemstat not only restores memory but reduces the exacerbated aggressiveness of SAMP8 mice, a model for accelerated aging and Alzheimer’s disease (AD), to normal levels and also reduces social avoidance and enhances sociability in murine models. In addition, vafidemstat exhibits fast, strong, and durable efficacy in several preclinical models of multiple sclerosis (MS). Oryzon has performed two Phase IIa clinical trials in aggressiveness in patients with different psychiatric disorders (REIMAGINE, see Ferrer et al, Psychiatry & Clin Neurosci, 2025, doi.org/10.1111/pcn.13800) and in aggressive/agitated patients with moderate or severe AD (REIMAGINE-AD), with positive clinical results reported in both. Additional finalized Phase IIa clinical trials with vafidemstat include the ETHERAL trial in patients with Mild to Moderate AD, where a significant reduction of the inflammatory biomarker YKL40 was observed after 6 and 12 months of treatment, and the pilot, small-scale SATEEN trial in Relapse-Remitting and Secondary Progressive MS, where anti-inflammatory activity was also observed. Vafidemstat has also been tested in a Phase II in severe Covid-19 patients (ESCAPE) assessing the capability of the drug to prevent ARDS, one of the most severe complications of the viral infection, where it showed significant anti-inflammatory effects in severe Covid-19 patients. Vafidemstat is currently advancing as a Phase III-ready asset in Borderline Personality disorder (BPD) following completion of the global, randomized, double blind Phase IIb PORTICO trial (final data presented at ECNP-2024). Following receipt of the minutes from the End-of-Phase II meeting with the FDA to discuss PORTICO’s results, the company announced plans to move forward with a Phase III PORTICO-2 trial in agitation/aggression in BPD (FDA submission planned in 1H2025). Vafidemstat is also being investigated in a double-blind, randomized, placebo-controlled Phase IIb trial in negative symptoms of schizophrenia (EVOLUTION trial, recruitment ongoing). The company is also deploying a CNS precision medicine approach with vafidemstat in genetically-defined patient subpopulations of certain CNS disorders and is evaluating a clinical trial in Kabuki Syndrome patients. The company is also exploring the clinical development of vafidemstat in other neurodevelopmental syndromes. FORWARD-LOOKING STATEMENTS This communication contains, or may contain, forward-looking information and statements about Oryzon, including financial projections and estimates and their underlying assumptions, statements regarding plans, objectives, and expectations with respect to future operations, capital expenditures, synergies, products and services, and statements regarding future performance. Forward-looking statements are statements that are not historical facts and are generally identified by the words “expects,” “anticipates,” “believes,” “intends,” “estimates” and similar expressions. Although Oryzon believes that the expectations reflected in such forward-looking statements are reasonable, investors and holders of Oryzon shares are cautioned that forward-looking information and statements are subject to various risks and uncertainties, many of which are difficult to predict and generally beyond the control of Oryzon that could cause actual results and developments to differ materially from those expressed in, or implied or projected by, the forward-looking information and statements. These risks and uncertainties include those discussed or identified in the documents sent by Oryzon to the Spanish Comisión Nacional del Mercado de Valores (CNMV), which are accessible to the public. Forward-looking statements are not guarantees of future performance and have not been reviewed by the auditors of Oryzon. You are cautioned not to place undue reliance on the forward-looking statements, which speak only as of the date they were made. All subsequent oral or written forward-looking statements attributable to Oryzon or any of its members, directors, officers, employees, or any persons acting on its behalf are expressly qualified in their entirety by the cautionary statement above. All forward-looking statements included herein are based on information available to Oryzon on the date hereof. Except as required by applicable law, Oryzon does not undertake any obligation to publicly update or revise any forward‐looking statements, whether as a result of new information, future events, or otherwise. This document does not constitute an offer or invitation to purchase or subscribe shares in accordance with the provisions of Regulation (EU) 2017/1129 of the European Parliament and of the Council of 14 June 2017, and/or the restated text of the Securities Market Law, approved by Law 6/2023 of 17 March, and its implementing regulations. Nothing in this document constitutes investment advice. In addition, this document does not constitute an offer of purchase, sale or exchange, nor a request for an offer of purchase, sale or exchange of securities, nor a request for any vote or approval in any jurisdiction. The shares of Oryzon Genomics, S.A. may not be offered or sold in the United States of America except pursuant to an effective registration statement under the Securities Act of 1933 or pursuant to a valid exemption from registration.. Spain Oryzon IR & Media, Europe & USPatricia Cobo/Mario Cordera Emili Torrell Sandya von der Weid Atrevia Chief BD Officer LifeSci Advisors, LLC +34 91 564 07 25 +34 673 33 97 65 +34 93 515 1313 +41 78 680 05 38 pcobo@atrevia.com mcordera@atrevia.com etorrell@oryzon.com svonderweid@lifesciadvisors.com

关注小药说药，一起成长！ 前言 阿尔茨海默病（AD）是导致老年人认知障碍的最常见疾病。然而，即使在专门的记忆诊所，临床医生也很难将AD患者与其他类型的痴呆症（如额颞叶痴呆症、帕金森病（PD）相关痴呆症和血管性痴呆症）区分开来，导致AD患者的误诊率超过30%。AD的神经病理学诊断的关键病理是神经元中淀粉样蛋白-β（Aβ）的沉积和细胞内高磷酸化tau蛋白（p-tau）的聚集。因此，开发易于获得、可靠且具有成本效益的诊断工具对于AD的诊断至关重要，尤其是对于早期患者。 由中科大施炯教授、福建医科大学附属第一医院欧启水教授和福建医科大学附属协和医院陈晓春教授牵头，中华医学会神经病学分会痴呆与认知障碍学组和中华医学会检验医学分会共同制定的《阿尔茨海默病血液生物标志物——临床应用指南》，于2024年10月发表在《Medicine Plus》。 由于临床症状滞后于神经病理变化的时间长达一二十年，因此在认知障碍显现之前，大脑中可能已经存在大量的Aβ沉积物。因此AD神经病理变化的早期发现对于这些疾病修饰疗法（DMT）取得有效的临床疗效至关重要。最近的研究发现，血液中可以检测到诊断AD的生物标志物，如Aβ和p-tau，并且血液生物标志物水平的变化与脑病理变化具有一致性，表明血液生物标志物在AD临床诊断中具有可行性。中华医学会神经病学分会痴呆与认知障碍学组、中华医学会检验医学分会的专家们汇聚一堂，讨论了近年来的临床研究进展和国际共识，共同制定了本指南，以指导我国AD血液生物标志物的临床应用。 基于脑脊髓液和影像学生物标志物的AD临床诊断 在脑脊液中可以检测到Aβ和p-tau。在AD患者中，CSF中的Aβ42与Aβ40的比例会显著降低。结合淀粉样蛋白靶向正电子发射计算机断层扫描（PET）发现，脑脊液中Aβ的变化与脑内淀粉样蛋白沉积之间存在很强的相关性，脑脊液中的Aβ42或Aβ42/Aβ40在预测脑内Aβ沉积方面具有很高的准确性。翻译后修饰的tau蛋白，如几种磷酸化tau蛋白（包括p-tau181、p-tau205、p-tau217和p-tau231），仅在AD患者的CSF中显示出显著变化。与p-tau181相比，P-tau 217可以更好地区分AD痴呆与其他类型的痴呆，而微管结合区tau（MTBR tau）的水平与tau-PET结果和认知功能的相关性最强。国外已批准了几种Aβ PET示踪剂，如氟替美托。最近，氟贝他苯也获得了中国国家医药产品管理局（NMPA）的临床试验批准。总之，目前基于CSF和PET检测的成熟方法对AD病理具有良好的诊断能力。 综上所述，目前基于CSF和PET检测的成熟方法对AD病理具有良好的诊断能力，但由于侵入性（通过腰椎穿刺采集CSF）和PET成像探针的放射性等因素，这些检测方法的临床应用受到限制。 AD血液生物标志物的研究进展 血液中Aβ的检测 大脑中的Aβ可通过血脑屏障释放到血液中。目前，电化学发光技术、单分子免疫检测技术和免疫沉淀质谱法（IP-MS）在内的多种检测方法都可以检测血液中的Aβ。电化学发光技术和单分子免疫检测技术是在传统夹心检测法基础上的升级版。电化学发光技术在微孔板上捕获抗体，而单分子免疫检测技术则通过磁珠偶联检测抗体。与电化学发光技术和单分子免疫检测技术不同，IP-MS通过免疫沉淀方法富集样品中的目标蛋白质，并使用传统的蛋白质质谱技术检测样品浓度。由于使用的抗体或方法不同，这些检测方法检测淀粉样蛋白水平的能力也不同。在一项对检测血浆中Aβ的八种方法进行的头对头比较研究中发现，基于IP-MS的方法在预测CSF Aβ42/ Aβ40的异常变化方面表现最佳，其AUC为0.86。 虽然检测血液中的Aβ42/Aβ40可以在一定程度上预测大脑淀粉样病理的变化，但与脑脊液相比，血液中Aβ42/Aβ40的变化幅度要小得多。中国老龄化与神经退行性疾病倡议（CANDI）临床队列研究发现，AD患者脑脊液中的 Aβ42/Aβ40为0.05，而认知功能正常者为0.09，变化幅度超过40%。然而，在血液中，AD患者和正常人的Aβ42/Aβ40分别为0.05和0.06，幅度仅相差10%左右。这使得很难使用血液Aβ42/Aβ40作为诊断AD的生物标志物。淀粉样蛋白在大脑中的沉积发生在认知障碍发生的许多年之前，导致血液中Aβ42/Aβ40甚至会在认知发生明显变化之前就发生了变化。这有助于在认知正常的老年人群中识别出大脑中存在淀粉样蛋白病理变化但认知正常的个体，这些个体在未来可能有患AD的风险。因此，建立标准化、稳定的血液Aβ检测系统对诊断AD仍具有重要意义。 血液中磷酸化tau蛋白的检测 相对于血浆中Aβ的水平，血浆中磷酸化tau蛋白的水平较低，AD患者血浆中某些位点的磷酸化tau蛋白低于1 pg/mL。近年来，涌现出了多种新技术和合适的抗体对，可以使用单分子免疫检测技术、电化学发光技术和IP-MS方法检测血浆中的各种磷酸化tau蛋白（如p-tau181、p-tau217和p-tau231）。血浆中的这些磷酸化tau蛋白，与AD的病理特征（CSF中Aβ42/Aβ40比值的异常变化、脑淀粉样蛋白沉积和tau蛋白神经纤维缠结）密切相关。相比之下，在其他与tau蛋白有关的神经退行性疾病中，如进行性核上性麻痹和额颞叶痴呆，血浆中磷酸化tau蛋白的水平没有明显变化。因此，血浆中p-tau蛋白的存在可表明患者是否患有AD病变。 与血浆中Aβ42/Aβ40比值的微小变化不同，AD患者血浆中磷酸化tau蛋白的水平增加了2-4倍。在CANDI研究中，AD患者血浆中p-tau181的水平是认知功能正常者的两倍。p-tau217可在认知障碍的早期阶段区分Aβ阳性和Aβ阴性人群。不同形式的磷酸化tau蛋白在不同疾病阶段的变化幅度不同，因此对大脑病理变化的预测价值也不同。使用IP-MS方法同时检测血浆磷酸化tau蛋白的变化时发现，p-tau217、p-tau231和p-tau205比其他形式的磷酸化tau蛋白对脑病理学的预测能力更准确。例如，p-tau231出现在淀粉样蛋白开始积累的阶段，但在淀粉样蛋白积累后，变化变得不那么显著。这可能是由于磷酸化位点出现的时间点不同造成的。因此，在临床应用中使用不同形式的磷酸化tau蛋白进行早期筛查或疾病病理评估时，需要进行进一步的评估。 血液中的神经胶质纤维酸性蛋白（GFAP） 神经退行性疾病患者（包括AD在内）大脑内星形胶质细胞通常处于异常激活的病理状态，表现为GFAP阳性星形胶质细胞的显著增加。这些活化的星形胶质细胞参与了神经元损伤过程以及与神经退行性疾病相关的病理过程，是一种神经炎症。尽管作为星形胶质细胞特征标志物的GFAP在许多神经退行性疾病患者的脑脊液中的水平发生了变化，但其在血液中的变化却与Aβ病理学密切相关。与血浆中的p-tau181相似，GFAP也能预测大脑中的淀粉样蛋白沉积。此外，已经发现，在Aβ阳性的认知正常个体中，血液中GFAP水平高的个体脑中tau病理的积累更快，这表明血液GFAP具有预测认知正常个体中tau病理的潜力。与GFAP相反，几丁质酶-3样蛋白1（YKL-40）在AD患者中没有表现出特定的变化，并且不是AD星形胶质细胞激活的特定标志物。重要的是，血浆GFAP蛋白的浓度很高，在AD患者中超过100 pg/mL，这使得开发基于血液的GFAP诊断系统很容易。 血液中神经变性的标志物 虽然AD患者都有淀粉样蛋白和神经纤维缠结病变，但神经损伤或认知障碍的程度各不相同，因此需要能准确反映大脑神经退行性变化的标志物。与脑脊液中许多与神经损伤密切相关的标志物不同，目前还缺乏合适的血液中神经损伤标志物，尤其是AD特异性神经损伤标志物。神经丝蛋白轻链（NFL）在各种神经退行性疾病（如肌萎缩性脊髓侧索硬化症、多发性硬化症和人类免疫缺陷病毒感染患者）的血液中都有显著变化。与健康人相比，AD患者血液中的NFL有所增加，尤其是在AD晚期。在一项针对家族性阿尔茨海默病的队列研究中发现，这些阿尔茨海默病患者的血液NFL含量约在临床发病前10年逐渐升高。在患有淀粉样脑血管疾病的患者中，血浆NFL的变化最为明显。此外，研究发现，血浆NFL水平与年龄和脑血管疾病以及其他年龄相关的并发症密切相关。因此，在使用血浆NFL评估AD患者神经损伤程度或评价临床药物试验和治疗效果时，需要考虑更多因素。 中枢神经系统来源的tau蛋白约占血液中蛋白质的20%，导致血浆T-tau水平和CSF T-tau水平之间的相关性较弱。最近的研究结果表明，血清脑源性tau蛋白和CSF脑源性tau蛋白之间具有高度相关性，但是血清T-tau蛋白和CSF T-tau蛋白之间没有相关性。这表明，脑源性tau蛋白可能是一种潜在的基于血液的生物标志物，用于预测AD神经退行性变化。 瞬时受体电位通道6（TRPC6）是一种非选择性阳离子通道蛋白，在神经元的存活、树突的发育和神经网络的建立中发挥着重要作用。TRPC6与淀粉样前体蛋白（APP）结合后，可阻断γ-分泌酶对APP分裂位点的识别，从而特异性地减少Aβ的生成。最近在中国进行的一项多中心临床试验（NMPA，20200005）（其中包括734名正常对照组和366名经Aβ-PET验证的AD患者）发现，与对照组相比，AD患者血液中TRPC6 mRNA水平显著降低。TRPC6的降低与AD患者认知功能的衰退、脑内Aβ沉积和脑萎缩有关。因此，TRPC6 mRNA水平有望作为早期AD的血液生物标志物，为早期药物治疗提供依据。 外泌体中的Aβ和tau在AD中的应用 外泌体是直径为30-150纳米的小囊泡，由多囊泡体与细胞膜融合后由细胞主动分泌到细胞外空间。在中枢神经系统中，神经元和神经胶质细胞都能分泌外泌体，而且外泌体具有细胞特异性。研究发现，Aβ和tau蛋白可以以外泌体的形式分泌，而且在淀粉样斑块中发现了Alix等外泌体标志物，这表明外泌体可能与AD的发生有关。外泌体作为一种新型的生物标志物载体系统，为神经退行性疾病的诊断和治疗提供了新的思路和方法。虽然目前还存在许多挑战，但随着技术和研究的不断发展，希望未来外泌体能更好地用于这些疾病的预防和治疗。 血液生物标志物在AD临床诊断中的应用 目前，AD生物标志物的临床使用标准（包括2013年的Aβ PET标准和2018年基于CSF AD生物标志物的使用标准）表明，这些生物标志物可用于区分AD痴呆和非AD痴呆。然而，这些生物标志物是否可用于检测认知正常个体或主观认知障碍患者的AD风险仍存在争议。AD血液生物标志物因其便利性，在结合临床和其他检测结果后，可帮助医生筛查和评估患者发生AD的可能性，从而优化AD的临床诊断流程。因此，尽管它们目前还不能取代CSF或PET成像检测，但它们将成为AD临床诊断的重要补充。 如前所述，血浆Aβ42/Aβ40的降幅非常小（约10%），因此其临床诊断应用非常有限。使用IP-MS或单分子免疫检测等技术检测Aβ的稳定性有待进一步提高，这也限制了其临床应用。在认知功能正常的脑淀粉样病理个体中，血浆Aβ42/Aβ40会下降；在预测轻度认知障碍的进展时，血浆Aβ42/Aβ40可提高磷酸化tau蛋白的预测能力。因此，建立一种灵敏、稳定的血浆Aβ42/Aβ40检测方法有助于临床诊断。然而，由于各种因素（如Aβ42和Aβ40缺乏标准化的样本采集、血样中脂质含量的影响、缺乏老年人的各种合并症对血液中Aβ水平明确的影响效果），导致目前的Aβ检测方法难以在临床中应用。未来需要开发标准化的Aβ参考材料，建立稳定的Aβ检测系统，并在大型多中心临床队列和社区人群中评估影响血液中Aβ水平的因素。 在AD相关MCI患者或AD患者的血浆中，多个亚型的磷酸化tau蛋白显著增加，因此可以有效预测AD在大脑中的特征性病理变化，从而有助于将AD痴呆与其他神经退行性疾病区分开来。最近的研究表明，使用IP-MS等更稳定、更灵敏的技术可以提高血液中p-tau蛋白的预测能力，使其达到与CSF生物标志物相似的水平。虽然与p-tau217相比，AD患者血浆中p-tau181的变化较小，出现的时间也较晚，但由于p-tau181的含量是p-tau217的10多倍，因此其检测更容易。   推荐意见： （1）血液Aβ42/Aβ40比值检测对诊断AD痴呆症的预测价值中等，可用作早期筛查工具（IIa类推荐，A级证据）。 （2）血液p-tau181和p-tau217与AD源性MCI患者或AD患者的AD病理学高度相关（I类推荐，A级证据）。 （3）血液GFAP有助于区分AD痴呆与非AD痴呆（I类推荐，A级证据）。 （4）血液NFL虽然对AD诊断的特异性不够，但可用于评估神经退行性变，帮助监测AD病理的进展（IIb类推荐，A级证据）。 AD血液生物标志物在早期AD筛查中的潜在应用 对AD进行早期干预是DMT（疾病修饰治疗）治疗AD的关键，因此尽早对AD病理进行准确而经济的筛查至关重要。建立以AD血液生物标志物检测系统可以大大降低早期筛查的成本。通过检测血液生物标志物，可以确定AD的低、中、高风险，并决定哪些人需要进一步检查，如CSF或PET扫描。血液生物标志物筛查可以减少不必要的CSF或PET扫描，确保真正需要的患者得到及时诊断和治疗，这对临床实践非常重要。 如果需要在AD的治疗窗口期进一步干预（在发病前进行治疗），就需要在认知正常的个体中筛选出脑内已有淀粉样病变并有发病风险的高危人群。在这种情况下，基于AD血液生物标志物检测系统的优势就凸显出来了。在60岁或以上有认知障碍的人群中，只有不到30%的人脑中存在淀粉样病变。使用PET或 CSF筛查无症状AD患者的生物标志物需要大量的人力和物力。A4研究是首个针对临床前AD的III期临床试验，历时三年半，进行了4000多次Aβ PET扫描，招募了1169名符合研究标准的参与者。因此，在目前针对临床前AD的抗Aβ免疫疗法临床试验中，血液的生物标志物已被用于AD的筛查。 血液生物标志物的优势在于对认知正常和轻度认知障碍人群进行筛查。如果单独使用血液生物标志物，其预测脑部病变的能力需要超过90%，才具有临床可行性。目前，血液生物标志物还不能准确预测疾病的未来发展，因此有必要建立一套全面的血液生物标志物筛查系统。此外，大脑中存在淀粉样病变的老年人在5-10年甚至一生中临床症状都不会进一步恶化。因此，需要对AD高危人群进行筛查，以减少对非AD高危人群进行不必要的治疗。此外，对于副作用较小的干预性临床试验，如针对AD的生活方式干预临床试验，可使用血液的生物标志物进行临床筛查。 推荐意见： （5）血液Aβ42/Aβ40比值可用于AD高危人群的早期筛查（IIa类推荐，A级证据）。 （6）血液p-tau181和p-tau217可用于AD高危人群的早期筛查（I类推荐，A级证据）。 （7）高危人群在经过预筛查AD病理后，需要通过CSF Aβ42/40比值、p-tau181或p-tau217检测，或Aβ PET确认AD特异性病变（I类推荐，A级证据）。 血液生物标志物在预测AD疾病进展中的应用 在纵向研究中，许多与AD相关的生物标志物，如Aβ42/Aβ40、p-tau181、p-tau217、p-tau231、GFAP和NFL，都与疾病进展密切相关。在家族性AD患者和家族性AD基因突变携带者中，血浆NFL在认知障碍发生前10年开始出现显著变化，并随着病理发展而逐渐增加 。这可能与NFL反映神经退行性疾病过程的能力有关。同时，血浆p-tau和GFAP也会随着疾病的进展而显著增加，但与NFL不同的是，它们只与AD患者的疾病进展密切相关。此外，使用Aβ抗体进行的临床试验显示，血液中p-tau和GFAP蛋白明显减少，且大脑淀粉样病理却有所改善。这进一步表明，AD血液生物标志物可用于预测疾病进展或观察药物的治疗效果。 然而，在利用血液生物标志物预测疾病进展的临床应用中，可能需要根据不同情况选择不同的生物标志物。例如，血浆NFL在家族性AD患者中显示出显著的早期变化，但在散发性AD患者中只有在痴呆阶段才会显著增加。此外，在抗 Aβ治疗的临床研究中，已观察到CSF中NFL的变化，但血浆中却没有。这可能是因为血浆中的NFL受多种因素的影响。此外，在AD的不同阶段，不同磷酸化 tau蛋白的变化模式也不同，如p-tau217随疾病进展的变化程度最高，与脑萎缩和认知能力下降的相关性最强。 推荐意见： （8）血液p-tau181、p-tau217、GFAP 和NFL可用于评估AD疾病进展（IIa级推荐，A级证据）。 血液生物标志物与其他生物标志物的结合使用 如上所述，不同的AD血液生物标志物在各种情况下可能存在差异，而且AD病理的复杂性使得仅使用单一的血液生物标志物很难准确评估大脑病理和预测疾病进展。因此，可能需要结合多种因素来提高准确性。CANDI队列研究发现，无论是Aβ42/Aβ40还是p-tau181，如果与载脂蛋白E（APOE）ε4基因或APOE ε4蛋白检测相结合，都能显著提高预测脑淀粉样蛋白病理的能力，尤其是在轻度认知障碍阶段或临床前阶段。对于MCI个体，血液生物标志物的组合也可以准确预测脑淀粉样蛋白沉积。在痴呆阶段，血浆p-tau181和p-tau217可以很好地预测脑淀粉样蛋白病理变化，这可能是因为AD痴呆患者脑内已经积累了足够多的p-tau蛋白。因此，即使结合基因型或Aβ42/Aβ40，也很难进一步提高其预测能力。如果痴呆症患者的p-tau水平正常，NFL检测可帮助诊断非AD痴呆症，如额颞叶痴呆症。 尽管p-tau 181或p-tau 217与NFL或血浆Aβ42/Aβ40的组合对轻度认知障碍或痴呆个体的预测能力的改善有限，但与脑结构成像的组合，例如AD患者的特征性海马萎缩，可以进一步提高脑淀粉样蛋白病理的诊断能力。一项研究发现，结合p-tau 217和三项简单的认知测试在预测未来AD方面比临床医生更准确，达到了使用CSF标志物预测疾病进展的水平。关于多种血液生物标志物的使用，或者将血液生物标志物与其他用于诊断或预测AD的测试相结合，可能需要进一步研究以建立临床使用的标准方案。 推荐意见： （9）APOE基因型检测与AD血液生物标志物相结合，可用于诊断AD源性MCI或临床前AD人群（IIa类推荐，A级证据）。 （10）脑结构成像结合AD血液生物标志物可用于诊断AD源性MCI或临床前AD人群（IIb类推荐，B级证据）。 AD血液生物标志物在临床实践中的应用标准 为了验证AD血液生物标志物在临床诊断中的准确性和可靠性，应使用经批准的检查方法，如CSF Aβ42/Aβ40或Aβ PET作为参考标准。此外，由于tau PET 也能反映脑内tau蛋白纤维缠结的病理变化，因此tau PET示踪剂也被批准用于临床使用，并可作为血液生物标志物诊断的参考标准。 除了建立血液生物标志物的参考标准外，建立血液样本采集的标准化程序也很重要。Aβ蛋白和tau蛋白都容易聚集并粘附在采集管壁上，因此样本采集是准确检测AD血液生物标志物的关键。建立统一的外周血采集和处理方案并推广使用，是标准化检测最重要的准备工作。目前，研究发现，不同类型的血样采集管（血浆应使用乙二胺四乙酸（EDTA）抗凝管采集）、样本采集与离心之间的时间间隔、离心机的参数、样本转移管的类型（推荐使用低附着力的聚丙烯管）、每支样本转移管的容量以及冻融循环的次数（建议控制在三次以内）都会影响检测结果。目前已开发出多种AD血液生物标志物检测系统。这些系统在投入临床使用前应经过严格的性能验证，以科学、严谨地评估其临床价值。此外，不同的检测系统有不同的参考范围和临界值，因此各医院应建立自己的参考范围，并对试剂盒提供的临界值进行验证；不同实验室应积极探索促进结果互通的方法。 此外，研究还发现不同种族群体之间的生物标志物水平存在差异。与白种人相比，拉美人或黑人血浆中的Aβ和p-tau水平存在差异。目前，东西方人群之间的具体差异尚不明确，因此有必要设计统一的纳入标准和样本处理标准，并开展头对头研究，以确定这些人群之间AD血液生物标志物的具体差异。生活方式、环境因素和合并症的变化也可能导致血液中生物标志物水平的变化。低体重指数（BMI）、心血管疾病和慢性肾脏疾病会增加血浆中p-tau217的水平。此外，NFL与年龄密切相关，但AD患者血浆中的NFL水平还可能受到其他疾病因素的影响，如脑血管疾病和周围神经系统疾病。因此，需要对不同人群进行大规模临床试验，以明确这些生理性变化对血液生物标志物水平的影响，从而科学可靠地解释结果，提高生物标志物的诊断价值。 结果与展望 血液生物标志物已经在AD临床试验设计中发挥了一定的作用，加快了临床试验的进展，降低了经济成本。今后，应加大力度推广血液生物标志物的应用，确定血液生物标志物的诊断阈值，进一步明确其临床意义。为提高血液生物标志物的诊断价值，应努力建立稳定的检测系统，控制分析前变量，并进行质量控制。同时，无论是使用多种血液生物标志物组合诊断或预测AD，还是使用血液生物标志物与其他AD风险因素相结合诊断或预测AD，都需要建立一个便捷高效的预测模型，以帮助医生在收到检测结果后迅速做出临床决策。此外，目前针对AD血液生物标志物的研究主要集中在Aβ和p-tau等与AD病理密切相关的生物标志物上，缺乏真正与神经元和突触损伤相关的生物标志物。因此，在AD血液生物标志物的临床实践中仍有许多问题亟待解决，希望随着未来更多高质量的临床研究的开展，AD血液生物标志物的临床应用水平能够得到进一步提高。本指南不具有法律效力，仅供医生参考。 参考文献： 1.Blood-based biomarkers of Alzheimer’s disease—A guideline for clinical use. Medicine Plus Volume 1, Issue 4, December 2024, 100057 公众号内回复“ADC”或扫描下方图片中的二维码免费下载《抗体偶联药物：从基础到临床》的PDF格式电子书！ 公众号已建立“小药说药专业交流群”微信行业交流群以及读者交流群，扫描下方小编二维码加入，入行业群请主动告知姓名、工作单位和职务。