Study On The CYP3A Phenotyping In Human By Endogenous Cortisol Metabolism

ObjectiveThe present study was undertaken to demonstrate if the endogenous cortisol 0 to 24 hour AUC(AUCF)and 6β-hydroxylation clearance [Cl_m(6β)]can be used for in vivo CYP3A phenotyping.Method1.Baseline CYP3A activity evaluation101 healthy volunteers(50 male and 51 female)were orally administered midazolam(MDZ)7.5 mg.Blood samples were collected before and at 1,4,8,10,24 h after drug administration.Urine samples were collected at 0-4,4-8,8-10,10-24h time periods.All samples were stored at -20℃until analysis.The ratio of 1-hydroxymidazolam(1-OHMDZ)concentration to MDZ concentration at 1 h(MR)was used to reflect in vivo CYP3A activity.AUC_F was calculated from plasma cortisol(F)concentration at sampling time by trapezoid ruler.Cl_m(6β)was calculated by 24 hours accumulated excretion of 6β-OHF(X_0-24)to divide AUC_F.Plasma MDZ,1-OHMDZ and F concentration,and urine 6β-OHF concentration were determined by HPLC-UV.All statistic analysis were performed by using SPSS 12.0 statistics software.2.CYP3A activity variation evaluationA two phases cross over test design with a wash-out period of two weeks was adopted in this test.24 volunteers(12 male and 12 female) were randomly divided into two groups.250 mg clarithromycin tablet and placebo was given at 08:00 and 20:00 to the two groups for 4 days, respectively.On day 4,all subjects of group 2 were required to collect blood and urine samples as baseline CYP3A activity evaluation study for evaluating the affects of MDZ administration to cortisol level and metabolism.On day 5,the last dose of clarithromycin and placebo were given with a dose of 7.5mg MDZ,then the blood samples and urine samples were collected as the program of baseline CYP3A activity evaluation study.Blood and urine samples were stored at -20℃until analysis.MR,AUCF and Cl_m(6β)were calculated as the same method as baseline CYP3A activity evaluation study.Plasma MDZ,1-OHMDZ and F concentration,and urine F and 6β-OHF concentration were determined by HPLC-UV.All statistic analysis were performed by using SPSS 12.0 statistics software.Result1.Baseline CYP3A activity evaluationThere was no correlation between AUC_F and MR in all subjects and in female subjects.But there was a significant positive correlation (r=0.405,p=0.004)between AUC_F and MR in male subjects.No significant correlation was found between Cl_m(6β)and CYP3A activity(MR),in all subjects and in male and female subjects respectively.2.CYP3A activity variation evaluationCYP3A activity(MR)significantly decreased 75%after 4 days clarithromycin administration(p=0.000),AUCF increased 19%at the same time(p=0.040).There was no significant correlation between AUC_F and MR.Cl_m(6β)significantly decreased 54.2%(p=0.000)with the decrease of CYP3A activity,but also there was no significant correlation between Cl_m(6β)and MR.Conclusion1.If take the renal function and all other cortisol metabolites into full consideration,both AUC_F and Cl_m(6β)might be suitable indexes for baseline CYP3A phenotyping in vivo.2.AUC_F could not reflect the inhibitory effect of CYP3A activity accurately and quantitatively,it was not a suitable index for evaluation the changes of CYP3A activity.Cl_m(6β)could reflect the inhibitory effect of CYP3A activity,but it was not completely.The more precise indexes will be studied in our further researches.