AQP1

A Shanghai-based startup quietly became the first in the world to start a clinical trial for a circular RNA therapy, a cutting-edge form of genetic medicine that could provide new ways to treat chronic diseases. Circular RNA is intended to strike a balance between short-lived mRNA therapies and permanent gene therapies. If it works, it could provide patients with a potentially cheaper and safer alternative, and there’s a race to get there first. Investors have poured $1.7 billion into three US companies looking to develop circular RNA treatments. But RiboX Therapeutics got to human trials first. Yet its announcement on March 9 went largely unnoticed , or unacknowledged, in the US. Since 2021, RiboX has kept a low profile. “They are intentionally very stealthy,” Mitchell Mutz, an entrepreneur partner at Vivo Capital, one of RiboX’s investors, told Endpoints News . “There’s a reason it’s flown under the radar: It’s not based in Cambridge, Massachusetts.” The pharmaceutical industry is increasingly looking to China as a source of new drugs that can compete on a global scale. And RiboX is among a growing number of companies that appear to be outpacing their US counterparts in pushing the needle on genetic technologies. “I don’t really see American biotech as competitive anymore,” said R. Alexander Wesselhoeft, a co-founder of the circular RNA startup Orna Therapeutics and currently director of RNA Therapeutics Core at Mass General Brigham. RiboX CEO Weiyi Zhang declined multiple interview requests. According to PitchBook and LinkedIn profiles of people associated with the company, RiboX has raised $110 million, but Mutz and Zhang would not comment on that figure. “Since day one of RiboX, I have decided to focus on science and program development, and stay below the radar as much and as long as we can,” Zhang wrote in an email. The Boehringer Ingelheim Venture Fund confirmed to Endpoints that it is also an investor in RiboX but didn’t respond to additional questions. Qiming Venture Partners includes the startup in its portfolio on its website. Eisai Innovation, Lilly Asia Ventures and Temasek are also investors in the startup, according to Mutz, who has been on the startup’s board since last summer. Eisai, Lilly and Qiming did not respond to requests for comment, and a spokesperson for Temasek would not confirm or deny if it was an investor. Moderna and other mRNA developers have said the messenger molecule can be used to deliver instructions for practically any therapeutic protein into the body. But the linear strings of mRNA used in Covid-19 vaccines are short-lived. Those quick bursts of protein expression work well enough for a vaccine, but have constrained their use as therapies for chronic diseases. Circular RNA provides an alternative. Several lab studies have shown that when the loose ends of mRNA are strung together, the resulting circular molecule is more stable. Several Cambridge-based companies — Orbital Therapeutics, Orna Therapeutics and Sail Biomedicines — have raised large amounts of capital to develop circular RNA therapies that allow cells to pump out therapeutic proteins for weeks instead of days. ​​ Sail Biomedicines , once viewed as “ Moderna 2.0 ,” is perhaps the biggest bet on circular RNA therapies with $750 million in funding. MIT spinout Orna Therapeutics is the US biotech that’s farthest along, with plans for clinical trials in 2026 using circular RNA for in vivo immune cell therapies. Sail and Orbital have not announced clinical trial plans yet. RiboX said that it treated the first patient with its circular RNA therapy in early March. The RNA encodes a water channel called human aquaporin 1. RiboX hopes the protein will increase saliva production and treat dry mouth in people whose salivary glands have been damaged from radiation used to treat head and neck cancer. A single dose of the therapy boosted saliva flow for roughly four weeks in animal models, according to the company’s press release. While many Chinese biotech companies often focus their initial clinical efforts in China, RiboX got FDA clearance to test its therapy in the US. The company has an office in Princeton, NJ to oversee the clinical trial , which is underway at the University of Iowa. Henry T. Hoffman, a professor of otolaryngology and radiation oncology at the university who is leading the study, declined an interview request and referred questions to RiboX. Mutz said that injecting circular RNA in or near the salivary glands helped overcome one of the biggest challenges for any RNA medicine: delivering it to the right part of the body. “They didn’t necessarily want to go to an organ that was hard to target,” Mutz said. “So there is something to be said for not trying to solve everything at once.” RiboX is based on the work of RNA biologist Ling-Ling Chen at the Shanghai Institute of Biochemistry and Cell Biology in China and lipid nanoparticle researcher Dan Peer at Tel Aviv University. Neither responded to interview requests. Zhang, the company’s CEO and co-founder, previously led Boehringer Ingelheim’s External Innovation Hub China in Beijing. “Because their academic founders have been doing this for a long time, they weren’t so far from having something clinical when they started,” Mutz said. And, he added, that the RiboX investment came at a “reasonable price” and not with a “super high valuation” like its US competitors. There are very few public details on RiboX’s technology, but based on the company’s website and publications from RiboX founders, Wesselhoeft said that it looks like “the technology that everyone else is using, which is essentially MIT and Orna’s technology.” Zhang said in an email that the comparison to Orna’s technology was “false” and that the company’s “research and innovation are original, proprietary, and well protected,” she wrote. Mutz said that RiboX’s intellectual property with “predated” or “was differentiated enough” from Orna and other US companies for Vivo Capital to be comfortable with the investment. He also emphasized that RiboX’s ability to physically make circular RNA, a potential bottleneck in moving from the lab bench to the clinic, was where the startup “really shined.” “They had a circularization technology that gave them a good yield so that this would be scalable and manufacturable,” Mutz said. “Professors don’t often think about that stuff. And this is exactly where academic spinouts go to die. But the company always articulated this from day one.”

PRINCETON, NJ, USA I March 9, 2025 I RiboX Therapeutics Ltd. (RiboX), a pioneering biopharmaceutical company, focused on discovering and developing fully engineered circular RNA therapeutics, today announced that the first patient was dosed last week in its first-in-human (FIH) Phase I/IIa clinical trial (SPRINX-1) evaluating the safety and efficacy of RXRG001 in patients with radiation-induced-xerostomia (RIX) and hyposalivation. Xerostomia and hyposalivation are common side effects of radiation therapy in head and neck cancer (HNC) patients. Radiation therapy often results in damage to salivary glands and a significant decrease in saliva secretion, frequently leading to dry mouth, difficulties in chewing and swallowing, impaired oral health, and diminished quality of life. There is a significant unmet medical need to develop a new treatment for xerostomia and hyposalivation. “Patients suffering radiation-induced xerostomia face a need for new therapeutic options. RXRG001 introduces an innovative approach offering hope for my patients. We are proud to be the first site to treat a patient with RXRG001 and look forward to continuing to collaborate with RiboX and other investigative centers to rapidly enroll patients in this FIH trial. I am glad that our first patient is tolerating the investigational product well.”, commented Principal Investigator, Henry T. Hoffman, MD, MS, FACS, Professor, Otolaryngology and Editor, Iowa Head and Neck Protocols of University of Iowa. “We are privileged to work with Dr. Hoffman at University of Iowa and our other esteemed Principal Investigators whose patients battle for their quality of life under the profound impact of radiation-induced xerostomia, in aftermath of their cancer treatment and survivorship,” shared Haifa Tyler, Head of Clinical Operations of RiboX, “It is an honor to initiate the first-ever circular RNA therapy clinical trial and now a pivotal moment in the dosing of our first patient.” “Today’s accomplishment marks an important milestone for both RiboX and most notably for xerostomia patients. We are grateful to our patients and investigators for their contribution to our clinical trials. RiboX is committed to introducing more novel treatments to patients around the world by leveraging our innovative circular RNA technology.” Yizhen Xu, MD, PhD, Chief Medical Officer of RiboX stated. About RXRG001 RXRG001 is a proprietary product developed using innovative circular RNA technology. In recent years, circular RNA has emerged as the next generation RNA therapy due to its high protein expression efficiency, low immunogenicity, and manufacture stability. RXRG001 consists of the circular RNA coding human aquaporin 1 (hAQP1, a water channel protein of cell membranes), encapsulated in lipid nanoparticles (LNPs). RXRG001 is anticipated to increase saliva production by restoring water permeability via overexpression of hAQP1, therefore it alleviates dry mouth symptoms.  Non-clinical studies have demonstrated a favorable risk and benefit profile of RXRG001 in animal models. For instance, a single administration of RXRG001 led to a significant increased salivary flow which was sustained for about four weeks. RXRG001 is the first-ever circular RNA therapy to receive FDA IND clearance back on October 25th, 2024. About RiboX Therapeutics RiboX is a globally operated biotech, dedicated to discovering and developing fully engineered circular RNA therapeutics, cofounded by Drs. Ling-Ling Chen, Dan Peer and Weiyi Zhang in 2021.  The company has operations in Princeton, NJ, USA, Rehovot, Israel and Shanghai, China. Circular RNA therapy is a novel modality, which is considered as the next generation RNA therapy. Compared to mRNA, circular RNA has a covalently closed single-stranded RNA structure that confers superior stability, lowers immunogenicity, and tunable protein expression, subsequently overcoming the current limitations of mRNA therapeutics and realizing the full potential of RNA medicines. RiboX’s pipeline spans various therapeutic areas, including radiation-induced xerostomia, monogenic rare diseases, autoimmune diseases, and metabolic diseases. RiboX is committed to pioneering innovative research in next-generation RNA therapies, with a vision to improve the quality of life for patients worldwide. For further details of the clinical trial, please visit: For further company information, please visit: https://www.riboxtx.com Media contact: bd@ribox-tx.com SOURCE: RiboX Therapeutics

一、原因分析 1.包材影响 注射剂的pH值升高可能与包装材料有关，特别是安瓿等玻璃容器。某些包材可能会释放OH-离子，导致成品中OH-浓度增加，从而引起pH值升高。不同厂家的安瓿对pH的影响存在差异，可以通过在安瓿中灌入注射用水并灭菌，观察灭菌前后pH值的变化来进行初步筛选。 注射剂中可能影响pH值的包材成分主要包括以下几类： l玻璃包装容器中的无机盐：玻璃包装容器中常见的无机盐成分，如Si、Na、K、Li、Al、Ba、Ca、Mg、B、Fe、Zn、Mn、Cd、Ti、Co、Cr、Pb、As、Sb等，这些离子在特定条件下可能会迁移进入注射液中，从而影响其pH值。 l聚丙烯和聚环烯烃（COP）注射器：研究表明，COP注射器在不同pH值下表现不同，尤其在高pH值（如pH 12）时，可能会显著增加某些物质的浓度，这可能会影响注射液的pH值。 l硅油（PDMS）：硅油常用于注射器的塞子和主体，以实现塞子沿主体平滑滑动。虽然硅油的毒性较低，但在某些pH条件下，其浓度变化可能对注射液的pH产生影响。 l橡胶塞：橡胶塞也可能影响药液的pH值，尤其是在制备过程或储存过程中，可能会导致药品pH的变化。 2.制剂工艺的影响 l灭菌过程：高压灭菌时的高温可能导致玻璃容器中的弱碱性成分分解释放到注射用水中，从而引起pH值升高。 这一现象在多项研究中得到了证实。例如，一项研究指出，在高压灭菌时，安瓿玻璃中的弱碱性成分会分解，导致灭菌注射用水的pH值升高。此外，青霉素瓶中注射用水的生产过程中也观察到类似的问题，即玻璃瓶内壁的氧化物与注射用水反应，导致pH值和电导率上升。 l缓冲剂的选择和使用：在注射剂中使用的缓冲剂，如碳酸氢钠等，可能在制备或储存过程中与空气中的二氧化碳反应，导致pH值升高。 碳酸氢钠在潮湿空气中会缓慢分解，释放出二氧化碳，但这通常不会导致溶液pH值的显著升高，反而可能因为碳酸氢钠的减少而略微降低pH值（尽管这种变化可能很小）。 然而，在实际应用中，更常见的是碳酸氢钠溶液吸收空气中的二氧化碳，导致碳酸氢根离子（HCO₃⁻）浓度的增加，从而可能使溶液的pH值升高。这是因为二氧化碳溶于水形成碳酸（H₂CO₃），碳酸进一步电离产生氢离子（H⁺）和碳酸氢根离子（HCO₃⁻），但在碳酸氢钠溶液中，氢离子会被碳酸氢根离子中和，形成碳酸根离子（尽管这个过程中的电离和中和反应是复杂的，但总体上可以理解为碳酸氢钠溶液吸收了二氧化碳后，其碱性增强，pH值升高）。 3.溶媒和附加剂 注射剂中的溶媒和附加剂也可能影响pH值。例如，使用某些缓冲溶液或pH调节剂（如盐酸、氢氧化钠等）时，如果添加不当，可能会导致pH值升高。 在注射剂制备中，溶媒和附加剂的选择对药物的pH值有显著影响。以下几种溶媒和附加剂最常导致pH值升高： ① 葡萄糖注射液：葡萄糖注射液的pH值通常在3.2到6.5之间，虽然其酸性范围较广，但某些情况下仍可能导致pH值升高。 ② 氯化钠注射液：0.9%氯化钠注射液的pH值范围为4.5到7.0，属于中性偏碱性，因此在某些情况下可能使pH值升高。 ③ 碳酸氢钠：作为pH调节剂，碳酸氢钠常用于提高溶液的pH值，以增加药物的稳定性和溶解度。 ④ 氢氧化钠：同样作为pH调节剂，氢氧化钠可以显著提高溶液的pH值，常用于需要碱性环境的药物制备。 ⑤ 表面活性剂：如聚山梨酯类（吐温类）、泊洛沙姆（普朗尼克）等，虽然主要用于增溶、润湿和乳化，但在某些配方中也可能间接影响pH值。 ⑥ 抗氧剂：例如硫代硫酸钠，这种碱性抗氧剂在调节pH值方面也有一定作用。 4.环境因素 环境中的水分渗透到注射剂中，也可能导致pH值的变化。例如，胶塞在干燥后仍可能含有残余水分，这些水分随时间推移可能会进入冻干块中，从而影响pH值。 5.药物配伍 注射剂与其他药物配伍时，由于pH值的差异，可能会引起药物的溶解度变化、化学稳定性降低，甚至产生沉淀。二、解决办法 1.选择合适的包材 在生产过程中，应选择对pH值影响较小的包材，并定期检测包材对pH值的影响，以确保成品的pH值稳定。 选择对pH值影响较小的注射剂包材需要考虑以下几个方面： 根据国家食品药品监督管理局《关于加强药用玻璃包装注射剂药品监督管理的通知》的规定，生物制品、偏酸偏碱及对pH敏感的注射剂由于风险较高，包材选择时应采用双1级药用玻璃或其他适宜的包材。为规范和指导化学药品注射剂与药用玻璃包装容器相容性研究工作，保障试验研究的质量，国家食品药品监督管理局与2015年7月制定发布了《化学药品注射剂与药用玻璃包装容器相容性研究技术指导原则（试行）》，旨在为注射剂药品更加合理地选择药用玻璃包材及评价药用玻璃包材的稳定性给出具有实际操作意义的指导性意见。这种玻璃具有较好的化学稳定性和耐水性，能够减少与药品成分的相互作用，从而保持药品的pH值稳定。 对于一些特殊药品，如喹诺酮类注射液，由于其含有卤素元素，对玻璃的要求较高。若处理不当，玻璃中的元素会迁移到药液中，导致pH值升高。因此，可以在玻璃内壁进行覆膜处理，以降低玻璃表面的碱度，减少药液与包材的相互作用。 在选择包材时，应进行充分的相容性研究，确保所选包材不会与药品发生化学反应，影响药品质量。例如，《化学药品注射剂与药用玻璃包装容器相容性研究技术指导原则（试行）》提供了相关的研究方法和标准。 2.对包材进行预处理 在灭菌前对玻璃容器进行预处理，如煮沸或浸泡在特定的溶液中，以去除或减少其中的弱碱性成分，是一种常见的实验室操作步骤。这种预处理的主要目的是去除玻璃器面的皿表游离碱性物质，这些物质可能会影响实验结果或灭菌效果。 根据多项证据，新购入的玻璃器皿通常含有游离碱性物质，因此需要进行预处理。例如，可以将新购的玻璃器皿浸泡在2%的盐酸溶液中数小时，以中和这些游离碱性物质。之后，用自来水冲洗干净，再用蒸馏水冲洗多次，以确保没有残留的酸性物质。 此外，对于反复使用的玻璃器皿，如平皿、试管等，在高压灭菌前也需要进行类似的预处理步骤。例如，可以先用肥皂水煮沸，再用蒸馏水冲洗干净。这一步骤有助于去除器皿表面的油脂和其他有机物，从而提高灭菌效果。 煮沸法也是一种常用的预处理方法，尤其适用于不耐高温高压的玻璃器皿。煮沸可以有效地杀死大部分细菌和病毒，但需要注意的是，玻璃器皿在煮沸过程中应完全浸没在水中，并逐渐加热以避免因骤热而导致玻璃器皿破裂。 在进行灭菌之前对玻璃容器进行适当的预处理是确保实验结果准确性和灭菌效果的重步骤要。通过使用酸性溶液浸泡和煮沸等方法，可以有效去除玻璃器皿表面的杂质和游离碱性物质，为后续的灭菌操作打下良好的基础。 3.优化高压灭菌的条件 高温可能会对热敏感材料造成损害。因此，可以尝试将灭菌温度降低至接近121°C，这样既能保证有效的灭菌效果，又能减少对玻璃容器中弱碱性成分的热分解风险。 对于一些热敏感的材料，可以考虑使用过滤灭菌或其他非热灭菌方法，以避免高温对材料的破坏。 4.为了防止碳酸氢钠等缓冲剂在制备或储存过程中与空气中的二氧化碳反应导致pH值升高，可以采取以下措施： ① 密封储存：将注射剂密封储存在干燥、阴凉、避光的环境中，以减少与空气中二氧化碳的接触。 ② 使用惰性气体：在制备过程中，可以使用惰性气体（如氮气）对注射剂进行充气，以排除空气中的二氧化碳。 ③ 控制湿度和温度：保持适宜的湿度和温度条件，以减少碳酸氢钠的分解和与二氧化碳的反应。 ④ 选择合适的缓冲剂：根据药物的性质和稳定性要求，选择合适的缓冲剂类型和浓度，以确保注射剂的pH值在适宜的范围内。 5.优化溶媒和附加剂的选择 在制备注射剂时，应选择合适的溶媒和附加剂，并严格控制其用量。例如，使用磷酸盐缓冲液、硼酸盐缓冲液等来调节pH值，同时避免使用可能导致pH值升高的附加剂。 在注射剂制备过程中，如何有效避免因溶媒和附加剂使用不当而导致的pH值异常，从以下几点进行考虑： ① 选择合适的溶媒：根据药物的性质选择适宜的溶媒是关键。例如，青霉素的稳定pH为6.0-6.8，因此不宜用含葡萄糖的输液做溶媒，而应使用生理盐水（pH 4.5-7.0）。奥美拉唑则需要专用溶媒以保持其稳定性。 ② 严格遵循药品说明书：药品说明书通常会明确指出推荐使用的溶媒类型及其用量。例如，某些抗菌药物只能用盐水或糖水配制，因为这些溶媒的pH值与药物本身的pH相近，有助于保持药物的稳定性。 ③ 注意溶媒的pH范围：不同溶媒的pH范围可能有所不同。例如，《中华人民共和国药典》规定葡萄糖注射液的pH范围为3.2～6.5，而0.9%氯化钠注射液的pH范围为4.5～7.0。制药企业在生产时通常会控制在特定范围内，如葡萄糖注射液通常控制在4左右，氯化钠注射液通常控制在6左右。 ④ 监测和调整pH值：在制备过程中，可以通过加入碳酸氢钠等调节剂来调节溶液的pH值，以确保其在适宜范围内。例如，奥美拉唑粉针的配制溶媒为酸性(pH4.5)，而重组溶液的pH在8.8-9.2时最稳定。 ⑤ 考虑患者病情和特殊需求：对于糖尿病患者或心衰、肾功能不全等需要水钠限制的患者，应优先选择葡萄糖注射液作为溶媒，并控制氯化钠注射液的摄入量和输液速度。 ⑥ 避免使用非专用溶媒：非专用溶媒可能导致溶液pH值偏离正常范围，从而增加不良反应的风险。例如，右雷佐生使用非专用溶媒溶解后，其配伍液的pH值会显著降低至3.1左右，而使用专用溶媒溶解后，pH值可保持在4.7左右。 6.控制环境条件 在生产过程中，应尽量减少环境水分对注射剂的影响，例如通过改善生产环境的湿度控制和定期检查设备密封性。 要有效减少注射剂中胶塞的残余水分，可以采取以下几种措施： ① 高温烘干：将胶塞在105°C至120°C的温度下进行高温烘干2-4小时，这不仅能够去除胶塞表面的水分，还能去除大部分内部的结晶水，从而降低水分迁移的可能性。 ② 氮气充填：在胶塞烘干后，充填氮气并压塞，这样可以防止瓶中高真空将胶塞中的水分吸进瓶中，从而避免水分增加。 ③ 清洗和灭菌：采用全自动湿法气冲胶塞清洗机对胶塞进行批量清洗，并通过目测观察确认清洗后的胶塞表面无明显水滴，从而确保其含水量符合要求。此外，清洗后的胶塞需要进行灭菌和干燥处理，以进一步降低水分含量。 ④ 真空干燥和热空气干燥：蒸汽灭菌后的胶塞可以通过真空干燥8小时或在110℃的循环热空气中干燥8小时来降低其含水量。研究表明，经过真空干燥和热空气干燥处理的胶塞在存储过程中水分增长较慢。 ⑤ 选择合适的胶塞材料和配方：不同配方的胶塞其残留水分含量不同，选择合适的胶塞材料和配方可以有效减少水分迁移。例如，丁基橡胶塞（无氯化）的残留水分含量较低。 ⑥ 控制储存条件：在储存过程中，应尽量控制温度和湿度，以减缓水分的吸收和迁移。例如，在较低温度（如5℃）下储存可以显著延长水分达到平衡状态的时间。 7.合理配伍 在注射剂与其他药物配伍使用时，应仔细考虑药物间的pH值差异，避免配伍不当导致的pH变化和药物降解。三、小结 在注射剂的研发过程中，pH值的变化是一个关键因素，因为它直接影响药物的稳定性、溶解度以及临床应用的安全性和有效性。本文作者结合文献报道和自身经历，从注射剂研发过程中pH升高原因及解决办法两大方面进行了探讨，旨在为相关研究人员和生产人员提供思路。在注射剂的pH研究和生产过程中，研究人员和生产人员需要考虑多个方面以确保产品的质量和安全性。本文不足之处，还望广大同行批评指正！ 参考文献 1.Karin Larmené-Beld, Wijnsma, R. , Kuiper, A. , Berkel, S. V. , & Frijlink, H. . (2021). Science- and risk-based strategy to qualify prefillable autoclavable syringes as primary packaging material. European Journal of Hospital Pharmacy, ejhpharm-2020-002333. 2.高玲, 何海涛, 刘文学, 郑科, 王强, & 蒋德席. (2021). 稳定小容量玻璃容器装液体制剂的pH值和/或电导率的方法. CN202010548647.3. 3.张亚兰,陈艳,石可欣,等.注射剂一致性评价关键技术要点[J].流程工业, 2020(4):5. 4.《药用玻璃包装容器影响药品质量的相关问题浅析》 5.《化学药品注射剂与药用玻璃包装容器相容性研究技术指导原则（试行）》 ------------THE END------------