自动化酶反应新方法建立并应用于待测化合物对CYP酶的抑制情况研究

发布时间：2019-02-14 08:28

【摘要】：细胞色素P450(Cytochrome P450 proteins,CYPs)是一类混合功能氧化酶和血红素酶系统,广泛存在于动物、植物、微生物体内,参与代谢各类化学物,包括药物、农药、致癌物质、除草剂、食品添加剂及其它类型的化合物的。据报道,人体内CYPs参与了90%以上药物代谢反应[1]。然而当化合物对其活性具有抑制作用时,可能导致其它化合物的代谢受阻,造成药物相互作用,96%的代谢性药物相互作用是由CYPs介导的,而由抑制导致的药物相互作用占代谢性药物相互作用的70%[1]。在新药研发进程中,确定负责各候选药物代谢的特定CYPs亚型,以及该候选药物是否会有抑制或诱导CYPs的可能是非常重要的。体外利用人肝微粒体及探针底物(probe substrate)法的反应模型是评价药物对混合人肝微粒蛋白(HLM)中CYPs影响状况的最常用的方法之一。一直以来,该反应模型都是通过人工玻璃管孵育完成。受限于操作者的科研经验及试验操作规范、熟练程度、甚至情绪变化,人工玻璃管孵育法在精准度方面存在一定的缺陷,并且人工玻璃孵育法难以实现高通量化研究。本研究中,建立了一套基于Tecan RSP 100/8 Genesis series液体处理工作站及IKA KS 130小型振荡摇床定制改造而来,用于评价候选药物对CYPs抑制情况的自动微型化平台。优化反应条件,使用96孔板进行全自动化的孵育操作,所有的试剂通过Tecan液体处理工作站的针头移送到96孔板的指定孔位,该工作站能够处理大批量试剂及化合物的稀释过程,同时振荡摇床为酶反应提供了所必须的恒温及振荡环境。反应终止后,建立快速化LC-MS/MS分析方法对代谢产物进行分析。本研究建立了两套自动化孵育程序,一套用于同时执行单一浓度多化合物对多种CYPs抑制情况的初步评价;另一套用户同时执行多个浓度多个化合物对单一CYP抑制情况的评价,即可用于半数抑制剂量(IC50)的计算。设定前者初步评价程序可以帮助判断是否有进行完整IC50值测算操作的必要。每种CYPs都加入其特异性的阳性参考抑制剂,并测算阳性参考抑制剂IC50值,结果均在文献参考范围内,一定程度上证明了这套体系的正确性。最后本研究使用此套自动化系统研究了大黄4种主要成分对CYPs的抑制作用,研究结果表明大黄酚对2E1,大黄酸对2C19、2E1,大黄素对2C8、2C19,芦荟大黄素对2B6、2C19有较强烈的抑制作用。对于三种最主要的CYPs亚型3A4/5、2D6、2C9,本研究进行了其IC50值的测定,其中大黄素对3A4/5、2D6抑制IC50值分别为15.82 mol/L、12.68 mol/L;大黄素酚对3A4/5的IC50值为18.40 mol/L;大黄酸对2C9的IC50值为24.17 mol/L,其余组别测得IC50值均大于25.00 mol/L。为大黄药用价值进一步开发过程中,中药配伍造成可能的药物相互作用或食药相互作用风险提供参考。
[Abstract]:Cytochrome P450 proteins,CYPs (Cytochrome P450 proteins,CYPs) is a kind of mixed functional oxidase and heme enzyme system, which widely exists in animals, plants and microorganisms, and is involved in the metabolism of various chemicals, including drugs, pesticides, carcinogens, herbicides, and herbicides. Food additives and other types of compounds. CYPs is reported to be involved in more than 90% of drug metabolic reactions [1]. However, when compounds inhibit their activity, it may lead to the inhibition of metabolism of other compounds, resulting in drug interactions, 96% of which are mediated by CYPs. Drug interactions due to inhibition account for 70% of metabolic drug interactions [1]. In the process of new drug development, it is important to determine the specific CYPs subtypes responsible for the metabolism of each candidate drug and whether the candidate drug may inhibit or induce CYPs. (probe substrate) reaction model using human liver microsomes and probe substrates in vitro is one of the most commonly used methods to evaluate the effect of drugs on CYPs in (HLM) of mixed human hepatic microsomes. All along, the model has been incubated with artificial glass tubes. Limited by the operator's scientific research experience and operating standard, proficiency and even emotional changes, the artificial glass tube incubation method has some defects in precision, and the artificial glass incubation method is difficult to achieve high-throughput research. In this study, an automatic miniaturization platform was established based on Tecan RSP 100 / 8 Genesis series liquid processing workstation and IKA KS 130 small oscillatory shaking bed to evaluate the inhibition of candidate drugs to CYPs. Optimization of reaction conditions, the use of 96 hole plate for full automatic incubation operation, all reagents are transferred through the needle of the Tecan liquid treatment workstation to the designated hole position of the 96 hole plate. The workstation can handle the dilution process of a large quantity of reagents and compounds. At the same time, oscillating shaking table provides the necessary constant temperature and oscillating environment for enzyme reaction. After the reaction was terminated, a rapid LC-MS/MS analysis method was established for the analysis of metabolites. In this study, two sets of automated incubation procedures were established. One set was used to perform a preliminary evaluation of the inhibition of multiple CYPs by a single concentration of multiple compounds. Another set of users simultaneously performed multiple concentrations of multiple compounds to evaluate the inhibition of a single CYP, which can be used to calculate the half inhibitor dose (IC50). Setting up a preliminary evaluation program for the former can help to determine whether there is a need for a complete IC50 calculation operation. Each CYPs was added with its specific positive reference inhibitors, and the IC50 value of the positive reference inhibitors was calculated. The results were all within the scope of references, which to some extent proved the correctness of the system. Finally, the inhibitory effects of four main components of rhubarb on CYPs were studied by using this automatic system. The results showed that rhubarb phenols had a strong inhibitory effect on 2E1, Rhein on 2C19O2E1, emodin on 2C8C19 and aloe emodin on 2B6O2C19. In this study, the IC50 values of three major CYPs subtypes 3A4 / 5D6D6D2C9 were determined. The inhibitory IC50 value of emodin on 3A4/5 was 15.82 mol/L,12.68 mol/L;. The IC50 value of emodin on 3A4/5 was 18.40 mol/L;. The IC50 value of Rhein to 2C9 is 24.17 mol/L,. The IC50 value of other groups is more than 25.00 mol/L.. In the further development of the medicinal value of rhubarb, the possible drug interaction or the risk of food and drug interaction caused by the compatibility of traditional Chinese medicine were provided.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q559.9;R96

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