Study On The Influence Of Sophora Flavescens On Cytochrome P450 Isoforms By Cocktail Approach In Rats

Objective:To develop a HPLC method for a Cocktail approach which can simultaneously determine the metabolic activities of different CYP isoforms, and evaluate the effects of Sophora flavescens on the metabolic activities of CYP isoforms CYP1A2, CYP2C9, CYP2E1 and CYP3A4 in rats in vivo, thus providing a guide for clinical treatment of Sophora flavescens.Methods:Cocktail probe solution was prepared by mixing the specific probe drugs of CYP isoforms CYP1A2, CYP2C9, CYP2E1 and CYP3A4, namely caffeine, tolbutamide, chlorzoxazone and midazolam respectively. And we established a HPLC method for a Cocktail approach which can simultaneously determine the concentrations of 4 probe drugs in plasma. Rats were randomly divided into four groups:test group(Sophora flavescens group), control group, CYP450 induction group (Phenobarbitol Sodium group) and CYP450 inhibition group (Cimetidine group). Sophora flavescens were administered orally for five days at a dose of 100mg/kg body weight for Sophora flavescens group and saline were administered in the same way for control group at the same volume as test group. Phenobarbitol Sodium and Cimetidine injection were administered intraperitoneally for five days at a dose of 50mg/kg body weight for Phenobarbitol Sodium group and Cimetidine group respectively. On the sixth day, Cocktail solution was administered by caudal vein for all groups. The blood sample were taken from eyeball before administration and after administration at a series of time-points, the concentrations of probe drugs in plasma were measured by a HPLC method with UV detection, the pharmacokinetic parameters were calculated by DAS 2.0. The effects of Sophora flavescens on the activities of CYP450 isoforms CYP1A2, CYP2C9, CYP2E1 and CYP3A4 were judged indirectly by comparing the pharmacokinetic parameters of test group with those of control groups. Results:l.We developed a HPLC method which could simultaneously determine the concentrations of 4 probe drugs caffeine, tolbutamide, chlorzoxazone and midazolam in plasma, it could separate the four drugs effectively and the HPLC method was formally validated and showed good performances in terms of linearity, sensitivity, precision and accuracy.2.Comparing the pharmacokinetic parameters of Sophora flavescens group with control group. AUC and MRT of caffeine significantly decreased, while CL of caffeine significantly increased(P<0.01); AUC and MRTo-t of chlorzoxazone significantly decreased, while CL of caffeine significantly increased(P<0.05); AUC, MRT and t1/2 of tolbutamide significantly decreased, while CL of tolbutamide significantly increased(P<0.05); there were no statistically significant differences in AUC, MRT, t1/2 and CL of Midazolam(P>0.05).3.Comparing the pharmacokinetic parameters of Sophora flavescens group with Phenobarbitol Sodium group, AUC, MRT and t1/2 of caffeine of Sophora flavescens group were significantly higher than Phenobarbitol Sodium group(P<0.05). CL of caffeine was significantly lower than Phenobarbitol Sodium group(P<0.05); AUC and t1/2 of tolbutamide of Sophora flavescens group were significantly higher than Phenobarbitol Sodium group(P<0.05), CL of caffeine was significantly lower than Phenobarbitol Sodium group(P<0.05); there were no statistically significant differences in AUC, MRT, t1/2 and CL of chlorzoxazone between Sophora flavescens group and Phenobarbitol Sodium group(P>0.05).Conclusions:1. An efficient, fast and reliable analytical method was developed for simultaneous evaluation of the activities of four major human drug metabolising cytochrome P450 (1A2,2C9,2E1 and 3A4) with a Cocktail approach including four probe drugs, namely caffeine, tolbutamide, chlorzoxazone and midazolam. The HPLC method was formally validated and showed good performances in terms of linearity, sensitivity, precision and accuracy. Finally, the method was found suitable for the screening of these compounds in plasma samples. 2. Sophora flavescens can induce the metabolicactivities of CYP1A2 and CYP2C9, but the induction effect is weaker than Phenobarbitol Sodium; Sophora flavescens can induce the metabolicactivities of CYP2E1, and the induction effect has no significant differences with Phenobarbitol Sodium; Sophora flavescens has no influence on the metabolic activity of CYP3A4.