基于“化学-PD/PK”的附子质量标志物发现、确证及其调控毒效的分子机制研究

Fuzi (Aconiti Lateralis Radix Praeparata), showed excellent efficay and high toxicity, was commonly used in clinical Chinese medicine. The toxicity and efficacy (qualification assessment) indicators of Fuzi in the current Chinese Pharmacopoeia (CHP) are diester-diterpenoids alkaloids (DDAs) and monoester-diterpenoids alkaloids (MDAs), respectively. However, the physiological activities of Fuzi can’t be completely explained by the current Quality-Markers (Q-Markers), and the poisoning incidents happened frequently. Our previous research have demonstrated that metabolic enzymes and efflux transporters regulate the efficacy and toxicity of Fuzi by governing the exposure profile of Aconitum alkaloids in vivo. In addition, several new Aconitum alkaloids have been identified in our previous study. The pharmacodynamics (PD) and pharmacokinetics (PK) of these alkaloids are unclear. Therefore, we suppose that the Q-Markers of Fuzi can be categorized into two groups: Q-Markers in one group reflect the efficacy or toxicity of Fuzi, while Q-Markers in another group indicate both efficacy and toxicity. The properties of Q-Markers are determined by their exposure profiles and regulation mechanisms in vivo. Thus, in the current study, the Potential Q-Markers (PQ-Markers) of Fuzi will be discovered and screened by system analysis the constituents of Fuzi identify the metabolites of Fuzi in vivo. The “PD/PK” profiles of PQ-Markers will be characterized. Further more, Fuzi Q-Markers will be confirmed according to the relationship of the “PD/PK” profiles of PQ-Markers between the efficacy and toxicity of Fuzi. The detection method of Q-Markers will be developed. We will also elucidate the regulation mechanisms of the efficacy and toxicity of Q-Markers. Further, a new quality assessment standard of Fuzi will be produced and validated. We supposed that the outcome of this study will provide advanced concepts for CHP for developing new quality assessment standard of Fuzi.

附子是确有疗效常用有毒中药，现行药典质量（毒/效）控制指标分别是双/单酯链二萜乌头生物碱，但该标准未完全量化附子药效和控制毒性事件。我们前期发现，药物代谢酶和外排转运蛋白通过调控乌头生物碱体内暴露形式和量来调控附子毒效。我们也发现许多新乌头生物碱，其毒效（PD）和药代动力学（PK）均未清晰阐明。因此，我们认为控制附子毒效的质量标志物（Q-Markers）可分为两类：一类单纯控制药效或毒性；一类既控制药效又控制毒性，Q-Markers的属性取决于其体内暴露及调控机制。因此，本课题拟从附子全成分和代谢产物挖掘附子潜在质量标志物（PQ-Markers）；评价PQ-Markers“PD/PK”特征及其与附子毒效的相关性，进而确证Q-Markers成分，并建立其测定方法；同时阐明Q-Markers调控毒效的分子机制；形成附子新质量控制标准并对其进行验证。预期结果可为药典制定附子新质量标准提供依据。

附子是中医临床“补火助阳、回阳救逆、散寒止痛”要药，疗效显著且应用广泛。但附子毒性大，临床频繁出现中毒事件，提示现行的质量标志物并不能有效控制附子的毒效，附子中可能存在其它需要控制的成分，附子现行质量标志物体系亟待完善。本项目在国家自然科学基金面上项目的资助下，开展了基于“化学-PD/PK”的附子质量标志物发现、确证、及其调控毒效的分子机制研究。本项目发现了13个新的乌头生物碱和6个纯脂质生物碱，建立了同时定量附子中39种乌头生物碱的UHPLC-MS/MS方法，并基于附子药材体外化学成分和小鼠体内暴露分析，发掘了7个潜在质量标志物（PQ-Markers），分别是：尼奥灵、附子灵、宋果灵、塔拉萨敏、10-羟基中乌头碱、异塔拉定和16β-羟基心瓣雀翠碱。采用多元色谱和综合波谱法成功分离、富集、纯化并鉴定了尼奥灵、附子灵、宋果灵、塔拉萨敏、异塔拉定和16β-羟基心瓣雀翠碱6个PQ-Markers。采用动物和细胞模型对附子PQ-Markers的药效（PD）、毒性（TD）和药代动力学特征（PK）进行评价，确证了尼奥灵、附子灵、宋果灵和塔拉萨敏可作为附子药效Q-Markers，10-羟基中乌头碱可作为附子毒性Q-Markers。采用CYP3A4抑制小鼠模型和外排转运蛋白基因敲除小鼠模型证实CYP3A4代谢和MDR1外排是调控附子Q-Markers体内毒效及PK的关键因素。基于代谢组学技术，初步揭示了尼奥灵、附子灵、宋果灵和塔拉萨敏抗类风湿关节炎的机制。形成附子新质量控制技术1项，并应用于附子饮片的质量控制。以上研究成果为附子新质量标志物发现、附子饮片及含附子中成药质量控制、以及附子临床安全有效应用提供了重要参考依据。