Effects Of Gambogenic Acid On Activity Of Cytochrome P450 Isoforms In Rat Liver Microsomes In Vivo And In Vitro Using Cocktail Of Probe Substruates

1 Objective To investigate the influence of Gambogenic Acid on the activity of cytochrome P450 isoforms CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 in vivo and in vitro in rats by cocktail probe substrates method, which could provide a guide for the development and application of Gambogenic Acid.2 Methods In vitro study,Cocktail substrates solution was prepared by mixing six specific probe drugs of CYP isoforms CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4,namely theophylline, diclofenac sodium, omeprazole, dextromethorphan, chlorzoxazone,and midazolam respectively. Besides, we developed a HPLC-UV method for a Cocktail approach which can simultaneously determine the concentrations of 6 probe drugs in vitro in RLM. The incubation in vitro was divided into four groups: test group(gambogenic Acid group), inhibitor group(the corresponding specific inhibitor of cytochrome P450 isoforms group),control group(normal DMSO group),without CYP450 activity group(without NADPH regeneration system group) and each group followed three parallel samples. The concentrations of probe substrates in RLM in incubation were measured, thus the percent of control acticity of CYPs were obtained. Base on series concentrations of Gambogenic Acid and the percent of enzyme control acticity of CYPs,the inhibitory curves and corresponding IC50 values were calculated by Graph Pad Prism5.0. The IC50 value of Gambogenic Acid on each isoform was determined to assess the effects of Gambogenic Acid on CYP450 isoforms CYP1A2, CYP2C9, CYP2C19,CYP2D6, CYP2E1 and CYP3A4 in vitro.In vivo study, Cocktail substrates solution was prepared by mixing six specific probe drugs of CYP isoforms CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, namely theophylline, diclofenac sodium, omeprazole, dextromethorphan,chlorzoxazone, and midazolam respectively. Besides, we developed a HPLC-UV method which can simultaneously determine the concentrations of 6 probe drugs in vivo in plasma. The male rat was divided into test group and control group. They were injected with Gambogenic Acid through caudal veins at 2 mg?kg-1 once a day for seven days.control group were given a blank solvent in a volume of 1m L?kg-1 once a day for seven days. On the seven day, all rats were intraperitoneal injected after 30 min intravenously administration with single doses of the cocktail probe drugs, theophylline 20 mg?kg-1,diclofenac sodium 20 mg?kg-1,omeprazole 20 mg?kg-1,dextromethorphan 20 mg?kg-1,chlorzoxazone 20 mg?kg-1, and midazolam10 mg?kg-1.Blood samples were collected at 0.083, 0.17, 0.25, 0.5, 1, 2, 4, 8h. and plasma concentrations of the probe drugs were simultaneously determined. the main pharmacokinetic parameters were calculated by DAS 2.0. The effects Gambogenic Acid of on CYP450 isoforms CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were reflected by comparing the pharmacokinetic parameter of experimental and control groups.3 Results In vitro study: A HPLC method has been established which could simultaneously determine the concentrations of 6 probe drugs theophylline, diclofenac sodium,omeprazole, dextromethorphan, chlorzoxazone, and midazolam respectively in RLM incubation system, it could separate the six drugs effectively and the HPLC method was formally validated and showed good performances in terms of linearity, sensitivity,precision and accuracy. Incubation with the different concentration of Gambogenic Acid,the activity of six cytochrome P450 enzymes(CYP1A2, CYP2C9, CYP2C19, CYP2D6,CYP2E1, CYP3A4) originated from rat liver microsome have statistically difference comparing with each other, and with the concentration of Gambogenic Acid increased,the activity of enzymes showed a trend of lower. Gambogenic Acid IC50 values of CYP2C19, CYP2D6 and CYP3A4 were 4.180 ?mol·L-1, 45.6 ?mol·L-1, and 10.02?mol·L-1, and of CYP1A2, CYP2C9 and CYP2E1 were more than 100 ?mol·L-1,respectively.In vivo study: A HPLC method has been established which could simultaneously determine the concentrations of 6 probe drugs theophylline, diclofenac sodium,omeprazole, dextromethorphan, chlorzoxazone, and midazolam respectively in rat blood.it could separate the six drugs effectively and the HPLC method was formally validated and showed good performances in terms of linearity, sensitivity, precision and accuracy.Compared with the control group, there was no significant difference in the major pharmacokinetic parameters(t1/2?Tmax?Vd?MRT?CL?Cmax?AUC(0-t)and AUC(0- ?))of theophylline, diclofenac sodium, omeprazole, dextromethorphan, chlorzoxazone, and midazolam.4 Conclusion The inhibitory effects of Gambogenic Acid on the activities of CYP2C19, CYP3A4 are medium and weak, respectively, but no effect on the activities of CYP1A2,CYP2C9,CYP2D6 and CYP2E1. While in vivo, Gambogenic Acid had no significance differences on the activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1,CYP3A4.