基于代谢靶点、毒性靶点的中药蟾酥配伍作用规律和潜在相互作用的预测研究

Although Chinese medicine "Chansu" exhibited significant bioactivities, it usually had the cardiotoxicities and unsatisfactory therapeutic window, which resulting in its clinic application as Chinese herbal compound. Therefore, it is very important and necessary to elucidate its compatibility rule and rationality in clinic. On the basis of our preliminary investigation and clinic usage of Chansu, we intend to study the influences of other Chinese medicine in the combination, to ADME/Tox parameters of bufadienolides, according to their metabolism and cardiotoxicity targets. These findings would reveal the compatibility rule and rationality of TCM Chansu clearly. Firstly, PK parameters of bufadienolides between alone and combination were compared by the in vivo metabolism methods, and these experimental data could indicate which Chinese herbs may affect the in vivo metabolism of Chasu significantly. And then the kinetic parameters related to the interaction, were measured using the in vitro metabolism methods of ADME, which providing the vital compatibility regulations of Chansu in the combination with major chemical constituents of other herbal medicines, on the basis of metabolic targets of Chansu. Similarly, we used the in vivo pharmacodynamics approaches such as myocardial infusion, to investigate the influence of combinating herbal medicines for the toxicity of Chansu, and then molecular biological approaches were applied to reveal the interaction mechanism on the level of cell. Based on the previous data, we would summarize the qualitative and quantitative mode, according to major metabolism or cardiotoxicity targets, to predict the possible drug-drug interaction by using computational chemistry and molecular docking methods. All of these results would indicate that the compatibility rule and rationality of toxic medicine Chansu on the basis of the molecular targets of metabolism and toxicity, and give the vital and useful guidance for the safe and reasonable usage of Chansu and its related preparations.

中药蟾酥药理活性显著但具有心脏毒性,治疗窗窄,临床常以复方使用。因此，阐明蟾酥配伍作用规律和合理性就显得尤为重要。根据前期研究和临床复方用药情况，本项目拟基于代谢/毒性靶点,研究常见配伍中药对于蟾毒甾烯ADME/Tox属性的影响，揭示蟾酥配伍作用规律与合理性。通过体内代谢手段，揭示配伍中药对蟾毒甾烯的药代动力学影响，再结合ADME体外研究手段，测定相关代谢动力学参数，从代谢靶点角度诠释配伍成分对蟾毒甾烯代谢的影响规律。同样，通过心脏灌流等整体药效学手段，考察配伍中药对于蟾酥心脏毒性的影响；再利用原代细胞(Na+-K+ ATP酶)，基于毒性靶点揭示配伍成分对于其毒性的影响规律。在此基础上,利用计算化学手段根据关键的代谢/毒性靶点,构建结构与配伍作用的定性、定量构效关系模型，预测存在的潜在相互作用。最终基于代谢/毒性靶点科学揭示毒性中药蟾酥的配伍规律和合理性，指导蟾酥及其复方的安全合理应用。

蟾酥作为一种常用中药，在抗肿瘤、镇痛方面具有良好疗效，但其主要成分蟾蜍甾烯的代谢属性和抗肿瘤作用机制以及配伍规律目前尚不明晰。因此，本项目以中药蟾酥主要成分蟾蜍甾烯类成分为研究对象，首先研究其关键的代谢靶点（I和II代谢酶）的归属与表征，同时建立代谢靶点表征的新方法，揭示部分中药成分通过代谢靶点对中药蟾酥主要成分代谢的影响，同时利用分子生物学和药理学手段，研究代谢稳定性优良的成分的抗肿瘤作用机制。我们成功的揭示了蟾酥主要成分的代谢酶分别为CYP3A4、UGT1A4、SULT2A1、GLUA，代谢反应类型主要有1,5羟基化、葡萄糖醛酸化、磺酸化、糖苷水解，计算了其主要代谢反应动力学等参数，并通过计算化学角度诠释了代谢靶点和蟾蜍甾烯的作用规律，包括关键作用的氨基酸残基和关键结构作用域。同时采用化学生物学手段建立起蟾蜍甾烯关键代谢靶点的荧光/非荧光小分子探针（CYP3A4、UGT1A4、SULT2A1、GLUA等），并构建其这些代谢酶靶点调控的高通量筛选方法，并用于配伍中药成分的抑制活性筛选，发现了系列的配伍规律。例如黄酮类成分（尤其是异戊烯基黄酮类）可以通过抑制CYP3A4酶，从而影响蟾酥成分的代谢，并通过计算化学手段，构建了结构预测模型，揭示成分之间配伍的合理性，指导临床合理用药。再如发现天然甾体类成分可以选择性的强效抑制UGT1A4酶，从而影响UGT1A4对于蟾蜍甾烯类物质的清除，同样通过结构与抑制动力学分析，阐明了天然甾体类成分对于UGT1A4的代谢抑制规律，并构建预测模型等。此外我们采用蛋白组学等技术、解释了蟾酥中代谢稳定性最好的日蟾毒它灵的抗肺癌、骨髓癌活性与机制，其可以作为C-myc、Hsp90的抑制剂阻断肿瘤的发生与发展。本研究主要从代谢角度入手，揭示了中药蟾酥的代谢规律和配伍使用规律，有力的指导了中药蟾酥及其相关制剂的合理使用与开发。