High-throughput The Inhibition Potency Of Quyu Agents On The Activity Of Human Five Major Cytochrome P450 Enzymes Using An In Vitro Cocktail

Since traditional medicine and modern medicine have their own characteristics, traditional and modern medicines are generally combined, which is expected to yield synergistic action while avoiding untoward reactions. Recently, several serious adverse reactions were reported after co-using herbal medicine combined with western medicine, such as transplant rejection and bleeding or contraceptive failure after combination St John's wort with ciclosporin or oral contraceptive, which has drawn growing concern in clinical therapeutics. Further studies indicated that the mechanism of herb-drug interactions was metabolic interactions mediated by CYP450 enzymes. It is estimated that Quyu agents used for huoxue huayu in cardio-cerebrovascular diseases were the highest-selling medicines in China, and the investigated 18 Quyu agents are the most frequently used ones. Hence, we selected the 18 Quyu agents for in vitro study on CYP-mediated drug metabolism in HLMs using a validated cocktail method with LC-MS/MS, which is not only fast and efficient in determining the effects of herbs on five CYPs (1A2,2C9,2C19,2D6 and 3A4) at one time, but can also avoid interference.PART I Simultaneous determination of five metabolites of cytochrome P450 probe substrates by liquid chromatography tandem mass spectrometryObjectives:To development a liquid chromatography/tandem mass spectrometry method for rapid determination of five metabolites of cytochrome P450 (CYP) probe drugs including:acetaminophen (CYP1A2),4-hydroxytolbutamide (CYP2C9),5-hydroxy omeprazole (CYP2C19), dextrorphan (CYP2D6) and 1'-hydroxymidazolam (CYP3A4) in inactivation microsomal enzyme protein. Methods:Samples were precipitated with acetonitrile containing the internal standard. An aliquot (20μL) was injected into a CAPCELL PAK C18 column (MGⅢ,100 mm×2.0 mm ID,5μm) coupled with a Phenomenex C18 guard column (4.0 mm×3.0 mm,5μm), which were kept at room temperature. The flow rate was 0.3 mL/min. The mobile phase consisted of 0.02% formic acid in acetonitrile (A) and 0.02% formic acid in water (B). During the loading step (0-4 min), the solvent composition of mobile phase A was increased linearly from 5 to 70%, then reduced to 5% in 0.1 min. The injection column was then allowed to re-equilibrate at initial conditions. The complete run lasted for 7 min (4.4 min for the positive mode followed by 2.6 min for the negative mode). An API-3000 triple-quadrupole mass spectrometer, equipped with an electrospray ionization (ESI) source, was used for the mass analysis and detection. OHTB was operated in negative mode (-4,500 V) and other metabolites were monitored in positive mode (5,500 V). The multiple reaction monitoring (MRM) mode was chosen to quantify the metabolites of the probe substrates. Results:The method was validated over the concentration ranges 100-20000 nM for acetaminophen, 3.5-700 nM for 4-hydroxytolbutamide,3-600 nM for 5-hydroxyomeprazole,3-600 nM for dextrorphan, and 4-800 nM for 1'-hydroxymidazolam in inactivation microsomal enzyme protein. The intra- and inter-day precision were 2.99～10.62% and 2.84～10.90%, respectively, and the accuracy ranged from 95.71%～108.69% and 95.19～106.77%. The lower limit of quantification varied between 3 and 100 nM. Conclusions:The present method provides a robust, fast and sensitive analytical tool for the metabolites of "cocktail" probe drugs, and can be applied to simultaneously predict the inhibitory potency of compounds on five major CYP450 enzymes in vitro.PARTⅡPreparation of the human liver microsomes and determination of the protein contentObjectives:To prepare human hepatic microsomes and determined the microsomal protein content. Methods:Human liver microsomes were prepared using a differential centrifugation method as follows:the liver tissue was minced and rinsed in 1.15% KCl-50 mM Tris-HCl solution (pH 7.4). The tissue was weighed and then homogenized with the same buffer (1:3, w/v) in a homogenizer. The homogenate was centrifuged at approximately 10 000 g for 30 min at 4℃. The supernatant was transferred to fresh centrifuge tubes and then centrifuged at approximately 105 000 g for 60 min at 4℃. The final microsomal pellet was resuspended in 0.1 M phosphate buffer, pH 7.4 containing 20% glycerol and stored at-80℃until use. Microsomal protein content was determined using the Bradford method. The activity of CYPs were measured by determination the metabolites of probes with LC-MS/MS after incubation. Results:Microsomal protein content determined by Bradford method was 67.2 and 33.4 mg/mL for two batches. There were metabolites of five specific probes after incubation. Conclusions:This method to prepare the hepatic microsomes is convenient. The microsomes can be stored at-80℃for long time. PARTⅢHigh-throughput the inhibition potency of Quyu agents on the activity of human five major cytochrome P450 enzymes using an in vitro cocktailObjectives:Validation the in vitro cocktail method and determination the inhibition potency and IC50 of Quyu agents on the activity of human five major cytochrome P450 enzymes using the in vitro cocktail. Methods:The incubation system contained cocktail probes substrates (35/170/10/10/4μM for PN/TB/OPZ/DM/MDZ, respectively, equal to their respective Km values), HLM, MgCl2(10 mM)-K2HPO4, The final volume was 200μL. The metabolites of five CYP probe substrates (ACE/OHTB/OHOPZ/DX/OHMDZ) were analyzed by LC/MS/MS. Five specific inhibitors:a-Naphthoflavone (CYP1A2), Sulfaphenazole (CYP2C9), Fluconazol (CYP2C19), Quinidine (CYP2D6) and Ketoconazole (CYP3A4) or Quyu agents were added into the incubation system, the metabolites of five CYP probe substrates were analyzed by LC/MS/MS. Inhibition was expressed as the percentage of control and calculated by metabolites with medicine added to the metabolites with no medicine added. Data were presented as mean±SD of n=3 replicates. IC50 values were calculated in a sigmoidal dose-response model and a two-sample t-test was used to test the effect of estimated concentration of herbal preparations on CYPs via GraphPad Prism 5.0 software. Results:The IC50 values of five specific inhibitors were in less than three times of literatures.1) Predicted concentration of tested medicines:①comprising major components of danshen extracts:The inhibition of DZY on five tested CYPs were stronger than positive inhibitors (1μM); XSP significantly inhibit CYP1A2, CYP2D6, CYP2C19 and CYP3A4; DSP, XKP, and FDP significantly inhibited CYP1A2;②comprising major components of saponins:XZY significantly attenuated the activity of CYP1A2 and CYP3A4, but activated CYP2C9; DXJ activated CYP1A2 and CYP2D6; XTP and XSJ activated CYP2C9 and CYP3A4, respectively;③comprising more than five herbs:XZJ significantly inhibited CYP1A2, CYP2C19 and CYP2D6, but acitivated CYP2C9; NXJ attenuated the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4; SBW inhibited CYP1A2 and TXJ activated CYP1A2;④YXP, SJW and SFT did not effect the five tested CYPs.2) IC50 values:①comprising major components of danshen extracts:The IC50 values of DZY to five CYPs were very small; The IC50 values of XKP to CYP1A2, CYP2C9, CYP2C19 and CYP2D6,XSP to CYP1A2 and CYP2D6, FDP to CYP1A2 were smaller than 100μg/mL;②comprising major components of saponins:The IC50 values of XZY to CYP2C9, CYP2C19 and CYP3A4 were small; XSR significantly activated the activity of CYP3A4(227.75% at concentration 1mg/mL);③omprising more than five herbs:The IC50 values of these medicines to five tested CYPs were more than 100μg/mL;④The IC50 values of SJW to CYP1A2, CYP2C19 and CYP3A4 were smaller than 100μg/mL; The IC50 values of these YXP and SFT to five tested CYPs were more than 100μg/mL. Conclusions:The results indicated that the formulas, comprising major components of danshen extracts, such as DZY, XKP, XSP may lead to herb-drug interactions and CYP1A2 was found the most susceptible enzyme of the five CYP450 tested.