苯溴马隆代谢活化致特异质肝毒性机理研究

Gout is a form of inflammatory arthritis caused by elevation of blood urate levels (a condition known as hyperuricemia) which crystallize and deposit into joints and/or surrounding tissues. Benzbromarone (BBM) has been a quite useful drug for the treatment of gout. But BBM was reported to cause serious incidents of idiosyncratic liver injury (IDLI) which causes a serious threat to the life of the patient,the demand of understanding the detailed mechanisms of BBM-induced hepatotoxicity is in an urgent need. DILI is widely considered to be associate with metabolic activation. We hypothesize that BBM is biotransformed by P450 3A to epoxide-derived reactive intermediate(s) which adduct proteins and that the dysfunction of critical proteins, resulting from the protein modification, triggers the process of cell death. To probe the hypothesis, we will perform the following studies, including 1) establishing P450 3A induced or inhibited and Cyp3a gene knocked-out animal models; 2)investigating the sensitivity of animals to BBM, pharmacokinetics, the efficiency of metabolic activation of BBM in vitro, the depletion of GSH in liver and the ammount of reactive metabolites-protein adducts; 3) designing and synthesizing a series of BBM derivaties which will reduce or block the metabolic activation pathways as that of BBM,and comprehensively exploring their hepatotoxicity；4）evaluating the anti-gout activity of the derivatives. This application combines multiple disciplines that allows us to elucidate the mechanisms of BBM-induced idiosyncratic liver toxicity and to provide scientific foundation for clinic treatment of BBM poisoning as well as the research and development of new anti gout drugs with low toxicity and high efficiency.

痛风是由于尿酸盐沉积于关节所致，苯溴马隆（BBM）治疗痛风疗效显著但会引起严重的特异质肝毒性(IDLI)。由于IDLI的致毒机制复杂，BBM致IDLI机理尚不明确，因此厘清其毒性机制已刻不容缓。我们提出假说：BBM的IDLI与其代谢活化相关，BBM经P450 3A代谢生成环氧化的亲电反应性代谢物，与蛋白质共价结合引起关键蛋白功能丧失，导致细胞的损伤和死亡。本项目将运用P450 3A诱导和抑制及Cyp3a基因敲除动物模型；考察动物模型对BBM毒性敏感性、药代动力学行为改变、体内外代谢活化效率、肝脏GSH耗竭程度、反应性代谢物-肝脏蛋白质共价结合量；以阻断BBM环氧化代谢途径为目的设计合成系列BBM衍生物；评价BBM衍生物的体内外抗痛风活性。本项目将通过多学科交叉研究，阐明BBM的代谢活化和其IDLI的相关性，为临床BBM中毒治疗方案提供理论依据，为研发低毒有效抗痛风新药提供理论支持。

痛风是由于长期高尿酸血症导致尿酸盐沉积于关节所致，苯溴马隆（BBR）治疗痛风疗效显著，但会引起特异质肝毒性，因此厘清其毒性机制已刻不容缓。针对此问题，本项目从BBR的化学结构入手，围绕代谢活化、肝脏毒性及其药理活性，探讨了代谢活化在BBR引起的特异质肝毒性中的作用，获得了以下研究成果：（1）确定细胞色素P450 3A酶在BBR代谢活化致特异质肝毒过程中的角色，从药物代谢角度解释BBR的反应性代谢产物－代谢活化酶－特异质肝毒性之间的内在关联。即BBR在体内经P450 3A酶代谢发生环氧化反应，产生的环氧化反应性代谢物（epoxide）与蛋白质共价结合，造成肝内谷胱甘肽水平耗竭，继而引发肝毒性。（2）以减少乃至阻断BBR的环氧化代谢途径为目的设计并合成系列BBR衍生物，运用化学结构－代谢活化－毒效关系的研究，从毒理化学、酶动力学、蛋白质化学三个纬度解释BBR代谢活化与BBR特异质肝脏毒性的相关性。（3）成功建立了一套以 URAT1 为靶点、 hURAT1-MDCK细胞为载体、同位素稀释质谱法技术为检测手段的降尿酸活性检测方法，为促尿酸排泄药物的开发提供切实可行的评价方法。（4）在确定BBR的致毒机理后，通过评估BBR衍生物的抗痛风活性，得到了一个兼具低毒和良好药理活性的BBR衍生物（6-F-BBR），为临床BBR中毒治疗方案提供理论依据，为开发低毒有效抗痛风新药提供理论基础。截止目前，本项目共发表SCI论文11篇，培养博士研究生2名，硕士研究生2名。