Gentiopicroside Human Hepatic Cytochrome Cyp450 Enzyme

Gentiopicroside(GE) is a kind of traditional Chinese medicine,which was developed independently by Biomedicine Key Laboratory of Shaanxi Province, has passed theⅠ,Ⅱclinical trial. The results of clinical trials claim that gentiopicroside has a good effect on liver jaundice. To find the potential drug interations and apply to clinic, we examined the effect of GE on human liver CYP450 isozymes in vitro.The first part:To evaluate induction potential of gentiopicroside on 2 major cytochrome P450 (CYP) 1A2 and 3A4 activities in healthy adult human primary hepatocyte. METHODS:The activity of CYP1A2 will be determined by analyzing the production of phenacetin demethylation. The activity of CYP3A4 will be determined by analyzing the production of 6β-hydroxylation of testosterone.The indction of gentiopicroside on CYP450 isoforms will be evaluated by monitoring the increase of the formation of metabolites of the probe substrates for CYP450 isoforms. The retention time of acetaminophen is 2.05min, and the rentention time of 6β-hydroxytestosterone is 2.6min. Human primary cultured hepatocytes were pre-incubated with gentiopicroside (50, 500μg.mL-1), selective chemical inducers (omeprazole or rifampcin) at 37℃with 5% CO2 for 2 days, and then incubated with probe substrates of CYP 1A2 (phenacetin 100μM), and 3A4 (testosterone 50μM) for 60 minutes. The metabolites(acetaminophen and 6β-hydroxytestosterone) were determined by the developed and validated LC-MS/MS methods. The results prompted that GE maybe induce CYP 1A2 in higher dosage. The second part:The study was conducted to evaluate the inhibitive potential of gentiopicroside on cytochrome P450 (CYP) 1A2,2A6,2C9,2C19,2D6,2E1 and 3A4 in human liver microsomes. Establish the LC-MS/MS methods to determine the metabolites of probe substrates of CYP450 isoforms respectively. The probe substrates of CYP450S and their metabolites are as follows. CYP1A2:phenacetin/ acetaminophen; CYP2A6:coumarin/7-hydroxycoumarin; CYP2C9:tolbutamide/ 4-hydroxylbutamide; CYP2C19:S-mephenytoin/4-hydroxymephenytoin; CYP2D6: dextromethorphan/dextrophan; CYP2E1:chlorzoxazone/6-hydroxychlorzoxazone; CYP3A4:testosterone/6β-hydroxytestosterone. The human liver microsomes were pre-incubated with gentiopicroside (1,5,10,50,100,500, 1000μg.mL-1) at 37℃with 5% CO2 for 15minutes, and then incubated with probe substrates of CYP1A2,2A6, 2C9,2C19,2D6,2E1 and 3A4 in presence of NADPH and the cofactor of cytochrome P450 oxidases at 37℃for 30 minutes. The formations of metabolites of CYP450 isoforms were measured by LC-MS/MS. GE can inhibit CYP2A6,2C19 and 2E1.