CYP1A2*1F基因多态性的检测方法建立及其对阿戈美拉汀药代动力学的影响

Establishment of Detection Method for CYP1A2*1F and Its Effect on Agomelatine Pharmacokinetics

建立实时荧光定量PCR方法检测CYP1A2*1F基因多态性,并探讨CYP1A2*1F基因多态性对阿戈美拉汀药代动力学的影响。该方法采用扩增阻滞突变系统(ARMS)结合TaqMan探针技术(ARMS-TaqMan法)同时检测野生和突变两个反应体系,根据野生引物循环阈值(Ct_w)与突变引物循环阈值(Ct_m)的差值绝对值(△Ct)来判断CYP1A2*1F的基因型。同时进行口服阿戈美拉汀片25mg的药代动力学试验,采用液相色谱-串联质谱法测定血浆中阿戈美拉汀浓度,采用WinNonlin 6.3软件处理血浆中药物浓度-时间数据,非房室模型方法计算药代动力学参数T_(max)、C_(max)、AUC_(0→1)、AUC_(0→∞)和T_(1/2)。结果表明,在55名健康受试者中,CYP1A2*1F野生型共12例(21.8%),其△Ct值为9.89±0.56;杂合型22例(40.0%),其△Ct值为0.41±0.14;纯合突变型21例(38.2%),其△Ct值为6.85±0.32。统计分析显示,CYP1A2*1F3种基因型组间各药代动力学参数比较皆无显著性差异(P>0.05),但所建立的用于CYP1A2*1F基因多态性检测的ARMS-TaqMan法具有准确、操作简单、高通量等优势,便于进一步筛查基因多态性对阿戈美拉汀药代动力学的影响,适合临床应用。本方法检测原理可用于各种单碱基突变率的检测,具有广泛的应用前景。

A quantitative real-time PCR detection method for CYPl A2 * 1F gene polymorphism(SNP) was established,and the relationship between the SNPs and agomelatine pharmacokinetics was analyzed.By our method,both wild and mutation genes of CYP1A2 * 1F could be measured using TaqMan probe on the amplification refractory mutation system(ARMS).The CYP1A2 * 1F genotype was determined by delta cycle threshold(ΔCt),which was the absolute difference between wild primer cycle threshold(Ctw) and mutation primer cycle threshold(Ctm).At the same time,agomelatine pharmacokinetics was analyzed after an oral administration of 25 mg once daily.The plasma concentration of agomelatine was measured using liquid chromatography-tandem mass spectrometry technique,and the concentration-time data in plasma was analyzed by WinNonlin 6.3 software.Pharmacokinetic parameters,such as T_(max),C_(max),AUC_(0→t),AUC_(0→∞)and T_(1/2),were calculated by non-compartmental analysis.In this study,55 healthy male volunteers were enrolled.All cases were divided into three groups by their CYPl A2 * 1F genotypes,12 cases were wild-type(21.8%),whichACt was 9.89 ±0.56;22 cases were heterozygous(40.0%),which ACt was 0.41±0.14and21 cases were homozygous mutant(38.2%),whichACt was 6.85 ±0.32.Statistical analysis showed no significant difference among those three groups on agomelatine pharmacokinetics(P> 0.05).For the purpose of SNPs detection,the established ARMS-TaqMan system was an accurate,simple and high-throughput method for CYP1A2 * IF measurement.And it is also an available method to assess the effect of SNPs on drug pharmacokinetics,especially for clinical application.In a word,this method was a prospect for single base mutation detection in the future.