基于MRGPRX2的盐酸青藤碱代谢物类过敏成分筛选分析基础研究

Sinomenine hydrochloride (SH) is a clinical drug with Chinese characteristics in treatment of rheumatoid arthritis. Whereas, adverse reactions cases of SH have been reported mainly because of its anaphylactoid reactions. MRGPRX2 has been recently identified as a key receptor in anaphylactoid reactions, and it is also an important target for SH-induced anaphylactoid reactions in our previous study. Due to complicated characteristics of SH metabolites, the material foundation of anaphylactoid components via MRGPRX2 is still undefined. Screening and analyzing the anaphylactoid components in SH metabolites may clarify the internal relationships between the material structures and allergenic effects, which is one of the hotspots in drug safety evaluation. This project will take MRGPRX2 as the study target, comprehensively utilize the experimental methods of biology and drug analysis to establish MRGPRX2/CMC coupled with LCMS-IT-TOF model, and effectively recognize, screen and identify the potential anaphylactoid components in SH metabolites. Furthermore, in vivo and in vitro pharmacological experiments will be applied to verify the dose-dependent manner of mast cells degranulation and mice anaphylactoid reactions treated by the SH metabolites. Through the competitive displacement and signal transduction analysis of ligand and receptor, we will preliminarily elucidate affinity relationships between anaphylactoid components and MRGPRX2. The completion of this project will establish a screen method for allergenic components in drug metabolites, and provide experimental evidence for the clinical safe use of SH. At the same time, we will provide new analytical methods and strategies for the material foundational research in drugs with allergic-like characteristics.

盐酸青藤碱（SH）是治疗类风湿性关节炎的中药特色药物，但其不良反应报道较多，以类过敏反应为主。MRGPRX2是近期发现的类过敏反应关键靶标，也是介导SH引发类过敏反应的关键受体。但SH代谢物结构多样复杂，通过MRGPRX2引发类过敏反应的物质基础尚不明确。筛选发现SH代谢物中的类过敏成分，可明确SH代谢物结构变化与致敏作用规律，是药物安全性研究的热点之一。本课题以MRGPRX2为靶标，建立MRGPRX2/CMC联用LCMS-IT-TOF模型，高效筛选-分析-鉴定SH代谢物中的类过敏成分；进一步通过体内和体外药理学实验评价其对肥大细胞的激活效应及致类过敏反应的量效关系；通过配体-受体竞争置换及信号转导分析，初步阐明其与MRGPRX2的亲和作用关系。项目的完成为SH的临床安全使用提供实验依据，同时建立药物代谢物类过敏成分筛选模型，为具有类过敏作用药物的物质基础研究提供新的分析方法和研究思路。

盐酸青藤碱（SH）是从青风藤中提取出的单体生物碱，其口服、注射制剂为治疗类风湿性关节炎的一线中药。SH具有明显的镇痛抗炎作用，但其可通过MRGPRX2靶点诱导肥大细胞脱颗粒引发类过敏反应，过敏样症状严重影响SH的临床应用。SH代谢物众多且结构母核类似，代谢产物在体内与原型药共同存在，SH代谢产物具有同样的抗炎活性及类过敏作用，可能与SH具有协同作用。本课题以MRGPRX2为靶标，建立MRGPRX2/CMC联用LCMS-IT-TOF模型，筛选发现SH代谢产物17-脱甲基青藤碱也同样具有引发类过敏反应的作用。利用CMC技术发现17-脱甲基青藤碱与MRGPRX2受体具有亲和作用，计算二者作用的平衡解离常数KD值为1.17×10-6 mol/L。采用MRGPRX2/HEK293细胞和人肥大细胞系LAD2发现17-脱甲基青藤碱可激活LAD2细胞释放β-氨基己糖苷酶、组胺、TNF-α等致敏介质，具有引发类过敏反应的潜在风险。利用C57bl/6小鼠发现，静脉注射17-脱甲基青藤碱可使小鼠出现体温下降的症状，皮下注射可引发皮肤红肿等过敏症状。而17-脱甲基藤碱在肥大细胞功能缺失的Kitw-sh/w-sh小鼠和MrgprB2敲除鼠中不可诱发类过敏反应，表明肥大细胞膜受体MRGPRX2/B2介导代谢产物17-脱甲基青藤碱引发类过敏症状。本研究首次提出研究盐酸青藤碱代谢产物引发类过敏反应的作用和机理，分析原型药与代谢产物引发过敏反应的物质基础，为提高疗效、降低不良反应提供支持，推动青藤碱类药物制剂安全、有效的临床应用，为中药安全可控研究提供新思路。