Influence Of Tanshinone ⅡA On Cytochrome P450Isoforms By Cocktail Approach In Rat Liver Microsomes In Vitro

Objective:To establish a HPLC approach for a Cocktail method which can simultaneously determine the metabolic activities of different CYP isoforms, optimize the rat liver microsomal (RLM) incubation conditions, develop a rat liver microsomal incubation system and evaluate the effects of Tanshinone IIA on the metabolic activities of CYP isoforms CYP1A2、CYP2D6、CYP2C19、CYP2C9、CYP2E1and CYP3A4in vitro in RLM, thus providing a guide for clinical treatment of Tanshinone IIA.Methods:Cocktail substrates solution was prepared by mixing six specific probe drugs of CYP isoforms CYP1A2、CYP2D6、CYP2C19、CYP2C9、CYP2E1and CYP3A4, namely caffeine、metoprolol、omeprazole、tolbutamide、chlorzoxazone and midazolam respectively. Besides, we developed a HPLC method for a Cocktail approach which can simultaneously determine the concentrations of6probe drugs in vitro in RLM. To establish the RLM in vitro incubation system via optimizing the microsomal protein concentration and incubation time, then study the influence of Tanshinone ⅡA on the six major CYPs in RLM.There were three groups:test group(Tanshinone ⅡA group), control group(normal saline group), without CYP450activity group(Without RLM group) and each group followed three parallel samples. The concentrations of probe substrates in RLM in incubates were quantified by a HPLC method with UV detection, thus the percent of control acticity of CYPs were obtained. Base on series concentrations of Tanshinone ⅡA and the percent of enzyme control acticity of CYPs, the inhibitory curves and corresponding IC50values were calculated by GraphPad Prism5.0. The effects of Tanshinone ⅡA on the activities of CYP450isoforms CYP1A2、CYP2D6、CYP2C19、CYP2C9. CYP2E1and CYP3A4in incubation system were judged indirectly by comparing the corresponding IC50values of test group with those of control groups.Results:1.A HPLC method has been established which could simultaneously determine the concentrations of6probe drugs caffeine, metoprolol, chlorzoxazone, omeprazole, tolbutamide 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.2.Tanshinone ⅡA at high concentrations (32and96μmol·L-1) has weak inhibitory effects on the metabolic activities of CYP1A2、CYP2D6、CYP2C19、 CYP2C9and CYP2E1in RLM incubation system in vitro(P<0.05), performance with IC50values were32、34.76、26.12、26.29and26.38μmol·L-1, while no significant inhibitory effects were observed for Tanshinone IIA at low concentrations (0.5、1、2、4、8、16μmo·L-1) on CYP1A2、CYP2D6、CYP2C19、CYP2C9、CYP2E1and CYP3A4enzyme activity in RLM (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(CYP1A2, CYP2D6, CYP2E1, CYP2C19and CYP2C9and3A4) with a Cocktail approach including six probe drugs, namely caffeine、metoprolol、 omeprazole、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 RLM samples.2. Tanshinone IIA has weak inhibitory effects on the metabolic activities of different CYP isoforms (CYP1A2、CYP2D6、CYP2C19、CYP2C9and CYP2E1) in RLM incubation system in vitro. Usually less prone to potential drug in clinic.