Effects Of Artificial Calculus Bovis On Activity Of Cytochrome P450 Isoforms In Rat Liver Microsomes In Vivo And In Vitro Using Cocktail Of Probe Substruates

Objective:Artificial Calculus Bovis is a substitute for Bovis Calculus,a traditional valuable animal medicine in China,which has the effect of clearing away heat and detoxification,resolving phlegm and calming shock.At present,it has formed a large number of proprietary Chinese medicine preparations with other drugs.While Artificial Calculus Bovis is widely used,serious adverse reactions caused by the combination of Chinese and Western medicines have gradually been paid attention to,especially the Compound Diclofenac Sodium Chlorphenamine Maleate preparation of Chinese and western medicine compound preparations?Artificial Calculus Bovis,diclofenac sodium and chlorpheniramine?Symptoms that cause hematuria are the most prominent,and patients have reported about a thousand cases of adverse reactions with hematuria after using the drug for a period of time.Further studies have shown that the mechanism of its interaction is caused by metabolic interaction,which is mainly mediated by cytochrome P450 enzymes.In recent years,there have been many reports about the interaction between Chinese medicine and western medicine to reduce the efficacy and even lead to toxic and side effects,which has aroused widespread concern.Through further research,it was found that the mechanism of its interaction is caused by metabolic interactions,the most of which are mediated by cytochrome P450 enzymes.To investigate the influence of Artificial Calculus Bovis on the activity of cytochrome P450 isoforms Cyp2e1,Cyp3a1,Cyp2d1,Cyp2c11,Cyp2c6,Cyp2c7,Cyp2b1 and Cyp1a2 in vivo and in vitro in rats by cocktail probe substrates method with LC-MS/MS,which could provide referencefor the combined application of Artificial Calculus Bovis with other Western medicine or traditional Chinese medicine.Methods:In vitro study:Rat liver microsome incubation experiment was carried out to establish an LC-MS/MS method to quantify the acetaminophen?Cyp1a2?,n-deethyl-amodiaquine?Cyp2c7?,dexorphane?Cyp2d1?,1-hydroxy-midazolam?Cyp3a1?,hydroxy-bupropion?Cyp2b1?,4'-hydroxy-mephenytoin?Cyp2c11?,6-hydroxy-chlorzoxazone?Cyp2e1?and 4-hydroxy-tolbutamide?Cyp2c6?corresponding to the specific substrate of CYP450 enzyme,and conduct a fully validation.Phenacetin/bupropion/amodiquine/chlorzoxazone/tolbutamide/mephenytoin/Dextromethorphan/midazolam were used as the specific substrates of CYP450 enzyme?Cyp1a2/Cyp2b1/Cyp2c7/Cyp2e1/Cyp2c6/Cyp2c11/Cyp2d1/Cyp3a1?to establish an in vitro evaluation system for the inhibition of rat liver microsomal CYP450 enzyme,and the corresponding CYP450 enzyme activity was characterized by the concentration of its corresponding metabolites.The reliability of the system was verified by examining the inhibitory potential of CYP450 enzymes of eight positive inhibitors??-naphthoflavone/thiotepa/quercetin/quinidine/Sulfaphenazole/Ticlopidine/ketoconazole/4-methylpyrazole?.To investigate the effect of Artificial Calculus Bovis extract on CYP450 enzyme subtypes,compare the Artificial Calculus Bovis with different concentrations in the incubation system as the experimental group and the control group without Artificial Calculus Bovis,and determine metabolites,analyze the effect of Artificial Calculus Bovis on enzyme subtypes.In vivo study:Establish an LC-MS/MS analysis method for the determination of metabolites corresponding to CYP450 enzyme-specific substrates in rats,and validate the method,including specificity,linear range,matrix effects,accuracy and precision,stability.12 male rats were randomly divided into two groups?n=6?:artificial calculus bovis group?10mg/kg?and control group.Then,the artificial calculus bovis extract were oral administration for consective 14 days while control group received 0.5%CMC-Na for 14days to rats.On the 15th day,rats in each group were injected with probe drugs via tail vein.Blood was collected from the eye canthus at the time point.The plasma concentration of eight metabolites in the plasma of the two groups of rats was determined by LC-MS/MS method.The main pharmacokinetic parameters of the metabolites were calculated using DAS2.0 software.By comparing the main pharmacokinetic parameters of the Artificial Calculus Bovis group and the blank group,the effects of Artificial Calculus Bovis on the metabolic activities of the enzymes Cyp2e1,Cyp3a1,Cyp2d1,Cyp2c11,Cyp2c6,Cyp2c7,Cyp2b1 and Cyp1a2 in the rat were evaluated.Results:In vitro study:An LC-MS/MS method with short detection period,high sensitivity,and simple operation,which can simultaneously determine eight probe substrate metabolites in liver microsomes in vitro,was established.The eight positive inhibitors in the evaluation system of in vitro incubation have significant inhibitory effects on their respective CYP450 enzymes,and the IC50 and FDA reference values measured are similar.The result showed that the artificial calculus bovis has different degrees of inhibitory effect on four CYP450 isoforms?Cyp2c6,Cyp2c11,Cyp2d1,Cyp3a1?.However,artificial calculus bovis had no significant influence on Cyp1a2,2b1,2c7 and 2e1.In vivo study:A LC-MS/MS method method has been established which could simultaneously determine the concentrations of 8 probe drugs metabolites acetaminophen,n-deethyl-amodiaquine,dexorphane,1-hydroxy-midazolam,hydroxy-bupropion,4'-hydroxy-mephenytoin,4-hydroxy-tolbutamide and 6-hydroxy-chlorzoxazone respectively in rat blood.The specificity and linear relationship are good,the accuracy and precision are high,and the stability and matrix effect meet the requirements of biological sample detection.Compared with the control group,the experimental group acetaminophen,N-desethyl amodiaquine,dextrorphan,1-hydroxy midazolam,hydroxylated bupropion,4-hydroxy tolbutamide and The main pharmacokinetic parameters t_1/2,Vd,MRT_?0-t?,MRT_?0^??,CL,AUC_?0^t?and AUC_?0^??of6-hydroxychlorozoxazone There is no significant difference.The Artificial Calculus Bovis extract had a significant effect on the pharmacokinetic parameters of4-hydroxymephenytoin,AUC_?0^t?increased significantly,and Vd decreased significantly.Conclusion:Artificial Calculus Bovis has different degrees of inhibition on four CYP450 isoforms?Cyp2c6,Cyp2c11,Cyp2d1,Cyp3a1?respectively,but no effect on the activities of Cyp1a2,2b1,2c7 and 2e1.While in vivo,artificial calculus bovis had induction on Cyp2c11,but no no effect on the activities of Cyp2e1?Cyp3a1?Cyp2d1?Cyp2c6?Cyp2c7?Cyp2b1 and Cyp1a2.