在类风湿关节炎治疗中来氟米特与甲氨蝶呤的生理药代-毒效（PBPK-TD）机制研究

Rheumatoid arthritis (RA) is s a chronic, systemic inflammatory disorder that primarily affects joints and it is hard to stop the progress of the disease. In clinical practice, the general therapy strategy is the combination treatment with leflunomide (LEF) and methotrexate (MTX) for patients with active RA. However, this often lead to seriously adverse reactions such as liver injury. And the mechanism of LEF and MTX combination induced hepatotoxicity has not been clarified systematically..According to our preliminary work by proteomics and other assays, we have revealed the impact of RA (early stage) to liver functions, and the roles of CYP in LEF liver toxicity. Species differences was identified in LEF’s toxicity. The objective of the present study is to explore the mechanism of LEF-MTX induced liver injury from pharmacokinetics and toxicodynamics, respectively. Humanized animals plus RA model will be employed to bridge species differences; sandwich-cultured hepatocytes, precision-cut liver slices and other in vitro assays will be utilized to identify molecule mechanisms of liver toxicity in different RA progression status. Proteomics analysis and other approaches will help to find and verify the critical regulation pathways of the hepatotoxicity after LEF and MTX treatment. Finally, a physiology-based pharmaracokinetic-toxidynamic model (PBPK-TD) will be established and provide a a mechanistic approach to individualization according to RA status, and clinical trial designs such as those involving enrichment have also enabled individualization.

来氟米特（LEF）和甲氨蝶呤（MTX）联合用药是类风湿性关节炎（Rheumatoid arthritis, RA）治疗的经典策略。然而这一措施使肝损伤风险显著增加，但是其诱导机制不明。.我们前期通过蛋白质组学等手段，初步确认了RA对于肝脏功能的影响和CYP在LEF肝毒性中的作用，发现LEF的肝毒性有明显种属差异。本课题拟从药代和毒效两个方面来发掘LEF和MTX联合用药的毒性机制。将采用人源化动物和RA等动物模型相结合的手段弥合种属差异；通过三明治原代肝细胞培养、精密肝切片等离体模型辅助研究RA病理进程对于LEF和MTX暴露量和肝胆分布等的影响和内在机制；结合蛋白质组学技术，考察LEF和MTX合用对肝脏中关键的信号调节通路的影响，揭示毒性机制。在此基础上，建立并验证具有生理意义的PBPK-TD模型（PBPK-TD），通过根据RA进程的合理剂量调整，实现临床个体化合理用药。

甲氨蝶呤（MTX）和来氟米特（LEF）是临床上最广泛应用的两个治疗类风湿性关节炎（RA）的药物。在临床治疗中发现，两者合用能够显著能够产生肝脏毒性，而且合用后毒性较单用明显增强。本课题着眼于RA的病理状态，MTX处置等因素对药物肝脏毒性的影响，结合动物病理模型及敲除模型，药代动力学研究，生物化学，组学等多种层次的研究手段和方法，深入探索MTX和LEF合用后产生肝脏毒性的分子机制，阐明临床现象。.在课题开始阶段，我们利用健康大鼠和RA病理造模大鼠模型，系统考察了生理以及炎症情况下，MTX和LEF的肝脏毒性。数据表明，在大鼠离体模型中，MTX和LEF的毒性表现与临床不相符，后续通过大鼠整体动物实验，初步断定LEF可能通过影响肝脏中MTX的代谢转运而影响MTX的体内处置以及毒性。.在此基础上，后续采用小鼠整体动物实验考察LEF和MTX的毒性关系。我们发现LEF能够显著增加MTX和其毒性代谢产物7OH MTX的血药浓度以及肝肾蓄积。但是LEF仅仅能够微弱诱导肝脏醛氧化酶（AOX），增加 7-OH MTX生成。与此同时，LEF显著下调肝脏胆管侧外排转运体MRP2，导致MTX和7-OH MTX的胆道清除减少，在肝脏蓄积增多。同时，来氟米也显著上调血窦侧外排转运体MRP3，导致两者的血药浓度增加，并使得肾脏代偿性蓄积。进一步的机制研究发现，LEF主要通过激活PPAR alpha调控MRP2和MRP3的表达和功能。通过人肝原代细胞蛋白质组学研究，我们发现LEF对PPAR alpha的激活作用在小鼠和人上是一致的。综上所述，LEF对MTX体内处置的影响部分解释了两者合用增强肝脏毒性的原因主要是通过LEF对MTX肝肾分布的影响造成的，和代谢关系较弱。本课题研究成果也提示了PPAR alpha受体激动剂有可能在临床上使用时对MRPs底物药物可能产生影响。