MRP2和OAT1/3介导的大黄酸与甲氨蝶呤发生药物相互作用的药代动力学机制研究

Methotrexate, which is known as an antitumor drug through anti-metabolism, is extensively used in clinical in the treatment of rheumatoid arthritis and cancer chemotherapy. However, methotrexate is a highly toxic drug with a low therapeutic index. High-dose methotrexate is widely accepted to cause acute liver and kidney damage. Studies indicated that MRP2 and OAT1/3 involved in the hepatic and kidney disposition of methotrexate. Rhein is identified as a major metabolite of diacerein, a prodrug used in the treatment of osteoarthritis. In vivo, diacerein is completely converted to rhein before entering the systemic circulation. Previous reports suggested that rhein could inhibit the activity of MRP2 and OAT1/3. Methotrexate and diacerein are often used concomitantly in clinical practice for the treatment of early rheumatoid arthritis. However, it is not clear whether drug-drug interaction (DDI) mediated by transporters could occur when the two drugs used together, therefore, leading to unintended changes in liver and kidney toxicity. In this study, we employed transporter transfected cells, sandwich-cultured human and rat liver hepatocytes, in vivo rat pharmacokinetic study, in situ rat liver perfusion, immunohistochemistry and cell apoptosis detection techniques to clarify DDI mechanism mediated by transporters. The study would clarify whether rhein could influence the liver and kidney toxicity caused by methotrexate. Therefore, the dosage of methotrexate could be adjusted when co-administered with rhein. This study will provide theoretical basis for the safe and rational application of the two drugs in clinic.

甲氨蝶呤是抗叶酸类抗肿瘤药，在临床上广泛用于癌症化疗及类风湿性关节炎，但由于甲氨蝶呤治疗指数较窄，大剂量易引起急性肝、肾毒性。研究证实药物转运体MRP2和OAT1/3介导了甲氨蝶呤的肝、肾处置。大黄酸为免疫抑制药双醋瑞因活性形式，双醋瑞因在进入体循环之前几乎完全转化为大黄酸。前期研究表明大黄酸可抑制MRP2及OAT1/3活性。目前甲氨蝶呤与双醋瑞因在临床上合并用于治疗早期风湿性关节炎。联合用药过程中甲氨蝶呤与大黄酸是否会发生基于转运体的药物相互作用，从而影响甲氨蝶呤肝、肾毒性尚不明确。因此，本研究拟应用转运体转染细胞、大鼠/人”三明治”肝细胞、大鼠in vivo、肝灌流、大鼠/人肾切片、免疫组化、细胞凋亡检测等技术阐明两药合用后由转运体介导DDI的分子药代机制，明确大黄酸是否通过MRP2和OAT1/3影响甲氨蝶呤所致的肝、肾毒性，从而调整甲氨蝶呤的剂量，为两药临床安全、合理应用提供依据。

大黄酸被认为是治疗骨关节炎的前药双醋瑞因的主要代谢物。甲氨蝶呤（MTX）是一种治疗指数较低而毒性较高的药物。MTX和双醋瑞因在临床中被同时用于治疗类风湿关节炎。本研究一方面探讨了有机阴离子转运蛋白（OAT）介导的大黄酸对MTX体内分布以及其对MTX肾毒性的调节作用。研究显示给予大黄酸能显著增加MTX的大鼠全身暴露量。同样，大黄酸对大鼠肾切片和hOAT1/3-HEK293细胞中MTX的蓄积也有明显的抑制作用，表明OAT1和OAT3参与了上述两药在肾脏的药物相互作用（DDI）。同时大黄酸减轻了MTX对HOTA1/3-HEK93细胞以及大鼠肾脏的毒性。总之，大黄酸通过抑制OAT1和OAT3从而降低了MTX的肾脏消除，并减轻了MTX诱导的体内肾毒性。另一方面，本研究探讨了大黄酸是否能增强抗癌药物培美曲塞（PTX）治疗非小细胞肺癌（NSCLC）的作用，并阐明了其相关的分子机制。研究显示，大黄酸与PTX合用可明显增加大鼠体内PTX的暴露量，而这一现象很可能是通过抑制OATs介导的。此外，大黄酸可以在A549细胞中浓度依赖性地显著提高PTX的毒性。在流式细胞仪分析中，与单独使用PTX相比，同时使用大黄酸和PTX诱导细胞凋亡，其结果趋势与Bcl-2/Bax和Cl-Caspase3的蛋白表达一致。同时，单丹磺胺（MDC）染色实验表明，大黄酸与PTX联合作用可增强A549细胞的自噬作用。Western blot分析表明，大黄酸对PTX介导的自噬的协同作用可能与抑制PI3K-AKT-mTOR通路和提高p-AMPK和LC3-Ⅱ蛋白水平有关。因此，大黄酸可以通过调节A549细胞的PI3K-AKT-mTOR途径和Bcl-2蛋白家族，影响自噬和凋亡，从而增强PTX的抗肿瘤活性。这也说明，大黄酸作为一种候选药物与PTX联合应用有可能成为治疗人类肺癌的有效药物。