应用特异性探针药物分析双峰驼CYP2D6酶体外活性

Use Cocktail Probe Drug to Analyse the Activity of Bactrian Camel CYP2D6 Enzyme in vitro

动物CYP酶是代谢药物、外源性化学物质和内源性化合物的超家族酶系,其中CYP2D6亚酶虽然只占动物肝CYP酶系的2%,却承担着约20%左右的药物代谢。作者旨在较为系统地研究双峰驼CYP2D6酶的体外活性及其对特异性抑制剂的敏感性。采用差速离心法制备双峰驼肝微粒体,借助BCA法和CO还原差示光谱法测定肝微粒体蛋白浓度和CYP总酶含量。并在优化肝微粒体孵育体系及建立CYP2D6酶的特异性底物氢溴酸右美沙芬及其活性代谢产物去甲右美沙芬高效液相色谱法的基础上,研究双峰驼CYP2D6酶的动力学特征、底物代谢率及特异性抑制剂奎尼丁对该酶的抑制作用。结果表明,双峰驼肝微粒体蛋白浓度、CYP总酶含量均能满足试验要求。建立的测定酶底物和产物的HPLC法灵敏度高、稳定性好,且各成分分离完全、峰形良好、无干扰。双峰驼CYP2D6酶的Vmax值为(0.141 6±0.052 7)nmol·(min·mg)-1,Km值为(12.986 0±0.357 2)μmol·L-1。奎尼丁对双峰驼CYP2D6酶的半数抑制浓度IC50为(2.779 0±0.063 9)μmol·L-1。因此,该试验不仅为深入研究双峰驼CYP2D6酶体外活性成功建立并优化了反应体系,而且得出了双峰驼CYP2D6酶动力学参数和特异性抑制剂的IC50值,填补了该领域的空白。

Cytochrome P450 is a superfamily of enzymes responsible for the metabolism of drugs,xenobiotics and endogenous compounds.In them,the member of the CYP2D6 constitutes only about 2% of total hepatic CYPs,however,it is responsible for the metabolism of about 20% of commonly prescribed therapeutic compounds.The purpose of this research was to systematically study the activity of bactrian camel CYP2D6 enzyme and its sensitivity to the specific inhibitors in vitro.Liver microsomes of bactrian camels was prepared by modified differential centrifugation method and the protein concentration,total content of CYP enzyme were measured by BCA method and CO reduction method,respectively.Then the characteristics of Bactrian camelCYP2D6 enzyme kinetics,substrate metabolic rate and inhibitory effect of quinidine on the enzyme were studied respectively on the basis of optimization of liver microsomal system and the establishment of the HPLC method for testing the specific substrate dextromethorphan hydrobromide of CYP2D6 enzyme and its active metabolite dextrophan.The results showed that the both liver microsomal protein concentration and the total CYP enzyme content could meet the study requirements.The established HPLC method for determining the specific substrate of CYP2D6 enzyme and its metabolite was high sensitivity and stability,as well as all the peaks of each component were separated completely with no interference.The Vmaxvalue and Kmvalue of CYP2D6 enzyme were 0.141 6±0.052 7nmol·（min·mg）-1and 12.986 0±0.357 2μmol·L-1,respectively.The IC50 value of quinidine was 2.779 0±0.063 9μmol·L-1.Therefore,the incubation system of Bactrian camel liver microsomal was established and optimized successfully for the further studying of the CYP2D6 enzyme activities in vitro,and found out the kinetic parameters of Bactrian camel CYP2D6 enzyme and the specific inhibitor＇s IC50 value of the enzyme.Hence,the experimental results filled the blank in research area of CYP2D6 enzyme activities in vitro.