rCYP3A4孵育条件的优化及其中1’-羟基咪达唑仑含量测定  

Determination of 1'-Hydroxymidazolam in rCYP3A4 and Optimization of the Incubation in Vitro

作　　者：任秀华[1,2] 曹磊[2] 斯陆勤[2] 裘军[2] 李高[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院药学部 [2]华中科技大学同济医学院药学院,武汉430030

出　　处：《医药导报》2010年第4期418-421,共4页Herald of Medicine

基　　金：国家自然科学基金资助项目(基金编号:30572265)

摘　　要：目的建立rCYP3A4中1’-羟基咪达唑仑含量测定方法,并对rCYP3A4体外孵育条件进行优化。方法反相高效液相色谱法RP-HPLC测定rCYP3A4中1’-羟基咪达唑仑的浓度,用单因素实验法对其体外孵育条件进行优化,用线性Lineweaver-Burk双倒数作图法研究rCYP3A4酶促动力学。结果1’-羟基咪达唑仑在18.20-1820.00ng·mL^-1内线性关系良好;体外优化的孵育条件为5μmol·L-1咪达唑仑、10pmolrCYP3A4、孵育10min;rCYP3A4酶促动力学参数Km为0.96μmol·L^-1,Vmax为0.78pmol·min^-1。结论RP-HPLC法操作简便、灵敏、快速,适用于rCYP3A4中1’-羟基咪达唑仑的浓度测定,rCYP3A4孵育条件优化及其酶促动力学研究为研究经CYP3A4代谢的药物相互作用及其他物质对CYP3A4酶的影响提供理论依据。Objective To establish a HPLC method for determining 1＇-hydroxymidazolam and optimize the incubated conditions for rCYP3A4.Methods 1＇-hydroxymidazolam was determined by RP-HPLC.Single effect experimental design was used to optimize the incubated conditions and the enzyme kinetics was evaluated by graphical analysis with Lineweaver-Burk double reciprocal plots（1/velocity vs.1/[substrate]）.Results Calibration curves of 1＇-hydroxymidazolam were linear in the range of 18.20-1 820.00 ng·mL^-1（R2= 0.999 9）.The optimal incubated conditions were 5 μmol·L-1,10 pmol and 10 min for midazolam concentration,rCYP3A4 and incubation time,respectively.The enzymic kinetic parameters of rCYP3A4 were 0.96 μmol·L-1 for Km and 0.78 pmol·min-1 for Vmax.Conclusion The HPLC method is simple,accurate and rapid,suitable for the determination of 1＇-hydroxymidazolam in rCYP3A4.The incubated conditions of rCYP3A4 and its kinetics can be applied for study on the drug interaction metabolized by CYP3A and effects of others on it.

关 键 词：rCYP3A4 1’-羟基咪达唑仑 含量测定 酶促动力学 

分 类 号：R969[医药卫生—药理学]