化学发光法用于芳香酯酶抑制剂体外筛选(英文)

A Chemiluminescent Method for In-vitro Screeningof Arylesterase Inhibitors

在前期的研究中,我们将9-(4-氯苯氧羰基)-10-甲基吖啶酯三氟甲基磺酸盐(CPOCMA)用于测定血清芳香酯酶活性,取得满意结果.在此基础上,本文以CPOCMA为底物,建立化学发光法评估药物对芳香酯活性影响的新方法.以硝酸甘油为模型药物,比较了化学发光法与UV方法的一致性.并将此法应用于评价三种抗炎药吲哚美辛、阿司匹林和乙酰氨基酚对芳香酯酶活性的影响.药物的加入使血清催化CPOCMA水解的动力学减缓,这表明这些药物均为抑制剂.吲哚美辛、阿司匹林和乙酰氨基酚表现出的IC50值分别为0.254、0.564和0.656 mmol/L,抑制常数ki分别为0.154、1.38和2.98 mmol/L.加入药物后的Lineweaver-Burk曲线表明这三种药物对PON的抑制均为竞争性抑制.根据加入药物后的动力学曲线,其IC50值、抑制常数和米氏常数的变化均表明这三种药物的抑制能力大小顺序:吲哚美辛>阿司匹林>乙酰氨基酚.CPOCMA可以作为PON底物体外评价药物对PON的抑制能力和抑制机理.UV法不适合评价紫外吸收峰与UV法的检测波长重叠的药物,而新建立的化学发光法对这类药物的筛选有独特优势.

In our previous work, the 9-（4-chlorophenyloxycarbonyl）-10-methylacridinium triflate ester （CPOCMA） was used as a chemiluminescent substrate for determination of serum arylesterase activity successfully. Based on CPOCMA, a chemiluminescent method was developed for assessing drugs＇ effect on this enzyme activity. The method was first validated with a UV method based on phenyl acetate by using trinitroglycerine as a model drug. The inhibitory effects of drugs were then exemplified by three anti-inflammatory drugs （including indometacin, aspirin and acetaminophen）. It was observed that the serum-mediated CPOCMA hydrolysis slowed down due to addition of the drugs individually. It means that all the three drugs were PON inhibitors. The IC50 values of indometacin, aspirin and acetaminophen were 0.254, 0.564 and 0.656 mmol/L, respectively; and their average inhibitory constants were 0.154, 1.38, and 2.98 mmol/L, respectively. Competitive inhibitory type was observed for all the three drugs by plotting the Lineweaver-Burk curves. According to the kinetics of the hydrolysis, IC50 values, inhibitory constants, and Michaelis constants, the inhibitory abilities of the three drugs were ranked as： indometacin〉aspirin〉acetaminophen. This chemiluminescent method is especially valuable for evaluation of those drugs which the UV method cannot work for.