“代谢-转运互作”介导槲皮素及其活性代谢物Q3GA调控CsA药动学的分子机制

Quercetin (Que) and cyclosporine A (CsA) are frequently administered simultaneously to ameliorate CsA-induced chronic hepatotoxicity and nephrotoxicity and to improve clinical efficacy in solid organ transplant recipients. However, Que does not exist in the form of prototype in vivo. Our previous research demonstrated that quercetin-3-O-glucuronide (Q3GA), the major circulating active metabolite of Que, had many overlaps with CsA in the key disposal target proteins (drug-metabolizing enzymes and drug transporters), suggesting the existence of complex interactions. Based on the single metabolic enzyme or transporter and Que prototype, therefore, there are obvious defects to explore the pharmacokinetic mechanism of CsA combination. Recently, the model of “metabolism-transport interplay” in enterocyte and hepatocyte has been established to provide a new opportunity for the study of the complex interrelationship between drugs. We hereby put forward a hypothesis that Que and Q3GA can significantly alter the pharmacokinetics of CsA mediated by the "metabolism-transport interplay". In the whole human and animal studies, in situ/ex vivo, cellular level, focusing on the key targets of metabolism and transport of CsA, the current proposed project will develop specific metabolism-transport interplay model by applying key technologies (gene transfection and RNA interference). Importantly, this study will analyze the detailed role mechanisms of the key drug-metabolizing enzymes and drug transporters in enterocyte and hepatocyte. Finally, from the overall perspective of metabolism-transport interplay, we will take a deep insight into the effect of Que and its metabolic activation product Q3GA on the disposition of CsA and clarify the molecular mechanisms of the interplay. The ultimate purpose of the study is to provide new ideas and experimental foundations for further refine the decision-making strategy of CsA in combination with other drugs, as well as implementing precision therapy of CsA.

器官移植受者临床常联用槲皮素（Que）防治环孢素（CsA）肝肾毒性和增强疗效。但Que在体内并非以原型形式存在，我们前期发现其主要活性代谢物槲皮素-3-O-葡萄糖醛酸苷（Q3GA）与CsA彼此关键处置靶点蛋白（代谢酶和转运体）有诸多重叠，提示存在复杂交互作用。基于单一代谢酶或转运体和Que原型物探讨联合用药时CsA药动学机制有明显缺陷。新近构建的肠肝“代谢−转运互作”模型为研究药物间复杂互作关系提供新契机，我们假设通过代谢−转运互作机制，Que、Q3GA显著影响CsA药动学。本项目拟从整体、在体/离体和细胞水平，针对CsA代谢转运关键靶点，应用基因转染、RNAi等关键技术构建特异代谢−转运互作模型，在逐一剖析肠肝关键代谢酶与转运体作用基础上，从代谢-转运整体互作角度深入研究阐明Que及其代谢活化产物Q3GA对CsA处置影响和互作分子机制，将为细化CsA联合用药方案、实现精准治疗提供新思路。

器官移植受者临床常联用槲皮素（Que）防治免疫抑制剂如他克莫司（TAC）或环孢素（CsA）肝肾毒性和增强疗效。Que及其活性代谢物Q3GA与TAC/CsA处置的关键代谢酶和转运体上有诸多重叠，提示其体内药物处置过程存在交互作用。我们用新近构建的肠道“代谢−转运互作”模型研究Que/Q3GA与TAC/CsA复杂药动学相互作用。我们通过“动物-离体组织-细胞”三个层面进行验证。首先我们使用P-gp和CYP3A4的抑制剂，单独给予CsA或联合给予不同剂量Que/Q3GA，验证了大鼠体内CsA的药动学曲线及Que、Q3GA 对 CsA 药动学的作用确实被“P-gp-CYP3A4互作”影响。肝微粒体和离体肠壁灌流系统明确了Que、Q3GA 对 CsA 药动学作用的关键酶和肠区。接下来我们构建了CYP3A4、P-gp双稳转MDCK细胞系，通过添加CYP3A4和P-gp特异性抑制剂，验证P-gp/CYP3A4单独存在以及两者同时存在时，TAC透膜能力以及不同浓度Que/Q3GA对TAC透膜能力的影响，从细胞水平明确了P-gp-CYP3A4互作会减低彼此对TAC的代谢转运能力，当低剂量QUE/Q3GA与TAC联用时，首先抑制了P-gp对药物的转运能力，P-gp-CYP3A4互作被取消，CYP3A4对TAC代谢能力变强。当Que/Q3GA浓度增大时，其对CYP3A4的抑制作用变强，减少了TAC的代谢增加了血中TAC浓度。我们的结论提示基于单一代谢酶或转运体和Que原型物探讨联合用药时CsA药动学机制并不准确，从细胞或转基因动物层面构建“代谢−转运互作”模型能更好的揭示药物的体内ADME以及药物-药物相互作用机制。同时，在拓展试验中，我们明确了肠道菌群对CsA在体内ADME过程的作用及机制，探讨了通过万古霉素抑制肠道βGUS水解酶逆转TAC诱发的新发糖尿病的作用及机制。

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