Effect Of Andrographolide On CYP450 In Rat Liver Microsomes Activity Both In Vivo And Vitro

OBJECTIVE 1. To develop HPLC methods for the determination of tolbutamide,omeprazole, phenacetin, midazolam, metoprolol and theirs metabolites in rat liver microsomes, respectively, for the evaluation of the activity of CYP 2C9,CYP 2C19,CYP 1A2,CYP 3A4 and CYP 2D6 in rat liver microsomes and study of the effect of andrographolide on CYP450 in vitro. 2. To develop a UPLC-MS method for simultaneous determination of tolbutamide, omeprazole, phenacetin,midazolam and metoprolol in rat plasma for study of the effect of andrographolide on the pharmacokinetics of the 5 probe drugs. METHODS 1. The analytical column was packed with Agilent ZORBAX SB-C18, the mobile phase consist of acetonitrile-water- 0.1% trifluoroacetic acid and the five substrate drugs tolbutamide, omeprazole, phenacetin, midazolam, metoprolol and their metabolites were detected respectively. The incubation was performed with the substrate drugs at 37 oC for a certain time, then the reaction was terminated. After the substrate drugs and its metabolites were extracted, the corresponding drugs was analyzed by HPLC. Then calculating the Km value near the substrate concentration, adding different concentration of andrographolide separately to the system, the half maximum inhibitory concentration(IC50) were calculated. 2. Twelve healthy male Sprague-Dawley(SD) rats were randomly divided into two groups: control group and andrographolide group, 6 rats in each group. The control group were intragastric administration with normal saline, dose of 5ml/kg, 1 times a day, for seven days. The andrographolide group were intragastric administration with andrographolide, dose of 10 mg/kg, 1 times a day, for seven days. On the seventh day, after administration for 30 min, a mix dose of the probe drugs were intragastric administration( 1 mg/kg of tolbutamide, 10 mg/kg of omeprazole, 5 mg/kg of phenacetin, 10 mg/kg of midazolam and 20 mg/kg of metoprolol). The 0.3 m L blood was obtained from tail vine at the different time points. Five probe drugs tolbutamide, omeprazole, phenacetin, midazolam and metoprolol were detected by UPLC–MS, the pharmacokinetic parameters(Cmax, CLz/F, Tmax, t1/2, AUC(0-t), AUC(0-∞)) were calculated by DAS 3.0 program and the differences between the two groups were compared using the SPSS 17.0 software, respectively. RESULTS 1. The andrographolide with the concentration of 100 μM could clearly inhibit the formation of CYP 2C9, CYP 2C19, CYP 1A2, CYP 3A4 and CYP 2D6 in rat liver microsomes. The half-maximal inhibitory concentration(IC50) values were 2.654 μM, 1.113 μM, 16.40 μM, 9.608 μM and 20.67 μM for tolbutamide, omeprazole, phenacetin, midazolam and metoprolol, respectively. 2. The andrographolide could inhibit the metabolism of the probe drugs tolbutamide, omeprazole, phenacetin, midazolam and metoprolol in rat plasma. comparing with the control group, The Cmax of tolbutamide increased and its clearance reduced, Other pharmacokinetic parameters were not statistically significant difference. The Cmax of omeprazole, phenacetin and metoprolol increased and other pharmacokinetic parameters were not statistically significant difference. The pharmacokinetic parameters of midazolam was not statistically significant difference. Conclusion: 1. The developed method of HPLC for determination of tolbutamide, omeprazole, phenacetin, midazolam, metoprolol and theirs metabolites in rat microsome respectively was accurate, specific, and was suitable for study of various substrate enzyme CYP 450 enzyme. 2. The developed method of UPLC-MS for simultaneous determination of tolbutamide, omeprazole, phenacetin, midazolam and metoprolol in rat plasma was accurate, specific, and was suitable for simultaneous determination of the 5 probe drugs and could be used to study of their pharmacokinetics. 3. The studies have shown that andrographolide could significantly inhibit the metabolism of CYP 450 both in vitro and vivo.Therefore, in clinical, when the andrographolide was co-administrated with some substrates of CYP 450, the effect on the substrate drugs should be considered and monitored.