Study On The Interaction Of Danhong Injection And Aspirin Based On Human Liver Microsomal CYP450 Enzyme

Objective:The use of molecular docking,preliminary screening the main effect of Danhong injection(DHI)in composition and aspirin(Asp)and 7 kinds of human cytochrome CYP450(CYPs)isoforms between high affinity isoforms;The multiple choice reaction monitoring(MRM)mode was optimized by ultra performance liquid chromatography-four level rod time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS),and the incubation conditions were optimized,and the feasibility of the incubation system was verified;Further,we optimized the incubation system to study the effects of DHI and Asp alone and their combination on the activity of CYPs enzyme subtype,and to explore the metabolic interaction between DHI and Asp,so as to provide evidence for rational drug use in clinic.Method:1.By using Autodock 4.2 software,the main effect of DHI in components(salvianolic acid A and salvianolic acid B,Danshensu,rosmarinic acid,protocatechuic acid,protocatechuic acid,caffeic acid and hydroxysafflor yellow A),Asp and 7 kinds of CYPs isoforms(CYP1A2?CYP2C9?CYP2D6?CYP2B6?CYP2C19?CYP3A4?CYP2C8),the crystal protein(2HI4?4JNM?STFT?4D62Z?5UFG?4GQS?2NNJ)to simulate the docking,docking comprehensive simulation of the generation of the free energy and the inhibition constant(Ki)and sorting,screening and DHI,Asp and higher CYPs enzyme subtype.2.Taking a single CYPs enzyme subtype as the carrier and taking the corresponding probe product as the detection index,and using the MRM mode of UPLC-Q-TOF-MS/MS to determine the concentration of the probe product,we established 4 kinds of CYPs enzyme subtype in vitro incubation system.The conditions of each CYPs subtype were optimized(human liver microsomal protein concentration,incubation time in vitro,and concentration of probe product).Finally,the half inhibition constant(IC50)of the positive inhibitors of the 4 types of CYPs enzyme subtypes was calculated to verify the feasibility of establishing the incubating system.3.Using MRM model,different volume percentage of DHI was determined respectively(0.4%?0.8%.1.6%)and different doses of Asp(20?60?100 mg)monotherapy and combined administration(one of the drugs were pre incubated for a period of time after adding another drug)production of 4 kinds of CYPs isoforms corresponding probe products,differences in production compared with the single drug and combination probe of the product.Result:1.The 4 subtypes of CYP1A2?CYP2C9?CYP2D6 and CYP3A4 are highly compatible with the main effect components of DHI;2HI4 and salvianolic acid A?salvianolic acid B?Danshensu,rosmarinic acid?hydroxysafflor yellow A has lower free energy and the inhibition constant,the best combination of free energy were-34.42?-29.90?-3 3.12?-3 0.28?-27.35 KJ/mol,inhibition constants were 0.93?5.70?1.56?4.95?16.19?mol/L;4JNM and Salvianolic acid A and rosmarinic acid had lower free energy and the inhibition constant,the best combination of free energy were-25.62?-25.13 KJ/mol,and the inhibition constants were 16.20 and 30.03?mol/L;salvianolic acid A,Danshensu and rosmarinic acid had lower free energy and suppression one of the best constants,binding free energy were-27.85?-32.20?-29.90 KJ/mol,inhibition constants were 13.07?2.25?5.76 ?mol/L;4D6Z and salvianolic acid A,Danshensu,rosmarinic acid and hydroxysafflor yellow A has lower free energy and the inhibition constant.In the best combination of free energy were-28.35?-28.48?-30.11?-28.48 KJ/mol,inhibition constants were 10.63?10.19?5.28?10.22 ?mol/L;2.Chromatographic conditions:ACQUITY UPLC(?)HSS T3 Ci8(2.1 mm*100 mm,1.8 m selection)column;the mobile phase was A-B(0.1%formic acid water and acetonitrile,gradient elution):0?4 min,B 30%?100%;4?5.5 min,B 100%;5.5?7.5 min,B 100%?30%;column temperature:30?,the flow rate of 0.4 ml/min;Injection Volume:5?L;Mass spectrometry conditions:electrospray ionization(ESI),positive ion(negative ion)sensitivity model,capillary voltage is 2.5 kv(+),2.80kv(-),ion source temperature:120?,desolvation temperature is 550?,cone gas flow is 50 L/h,desolvation gas flow is 800 L/h,the MRM acquisition mode.In the incubation system of human liver microsomes,the acetonitrile containing internal standard(IS)was added to the system.After oscillating and cryogenic centrifugation,the production of probe products in CYPs enzyme subtype was determined by internal standard method.The methodological verification mainly includes:the test of the scale,the linear range,the precision,the accuracy,the matrix effect and the recovery rate.Using MRM model,the determination of human liver microsomes in vitro incubation system of acetaminophen,diclofenac,4-hydroxy dextrophan,1-hydroxymidazolam and IS generation method,linear relationship between the 4 probes product good(r>0.9941),intra day and inter day precision were less than 15%,the recovery of the probe product rate of within the range of 75?100%,to probe the product matrix effect in the range of 90?106%,does not affect the accuracy of quantitative,and good stability.Through the optimization of incubation condition,identified 4 CYPs isoforms of suitable human liver microsomes in vitro incubation system conditions are as follows:human liver microsomes in vitro incubation system volume was 200 L,human liver microsomal protein concentration(0.5?0.7?1.0?0.6 mg/mL)and in vitro incubation time(30?35?35?5 min),the final concentration of specific probes(40?79?17.38?23.68?0.12?mol/L selection).4 kinds of CYPs isoforms correspond to positive inhibitors of IC50 were 2.225?0.038?0.067?0.031 ?mol/L selection.3.Effects of single use and combined use on the activity of 4 kinds of enzymesDHI inhibited the activity of 4 kinds of CYPs enzymes,and the types of inhibition were all mixed.1)Compared with the blank group:The three volume percentage concentrations of DHI had significant inhibitory effects on the enzymes activity of CYP1A2?CYP2C9 and CYP3A4 enzymes,high volume percentage concentration on CYP2D6 isoforms also inhibited activity;The high volume percentage and Medium volume percentage concentration of DHI also inhibited the activity of CYP2D6 subtype;However,the low,medium and high doses of Asp had no significant effect on the 4 types of CYPs.2)The drug combination group(DHI + Asp)was compared with the blank group(preincubation of DHI)Different volume percentage of DHI combined with different doses of Asp could inhibit the activity of CYP1A2 enzyme subtype(p<0.05 or p<0.01);The combination of the high volume percentage concentration of DHI and 3 doses of aspirin inhibited the activity of the CYP2C9 subtype(p<0.05);The combination of medium and high volume percentage concentration of DHI and the three doses of Asp inhibited the activity of CYP2D6 subtype(P<0.05);The combination of the median volume percentage concentration of DHI and high dose of Asp inhibited the activity of the CYP3A4 subtype(P<0.05).3)The drug combination group(DHI + Asp)was compared with the blank group(preincubation of Asp):The three of dose groups of Asp were respectively combined with middle and high volume percentage of DHI to inhibit CYP1A2 enzyme subtype activity(p<0.05 or p<0.01);The low dose of Asp and DHI with high volume percentage concentration inhibited the activity of CYP2C9 enzyme subtype(p<0.01);The low dose of Asp and DHI(medium volume percentage concentration and high volume percentage concentration)inhibited the activity of CYP2D6 enzyme subtype(p<0.05 or p<0.01);The dose group in the middle dose of aspirin had a inhibitory effect on the subtype of CYP2D6 enzyme(p<0.05)after the combination of the high volume percentage concentration of DHI.4)The drug combination group(DHI + Asp)compared with the corresponding volume percentage of DHI group:CYP1 A2:There was no statistical difference between DHI and Asp group and DHI alone(p>0.05).CYP2C9:In addition to the medium volume percentage concentration of DHI and high dose of aspirin and corresponding DHI alone group compared with significant international(p<0.01),the rest were not statistically significant(p>0.05).CYP2D6:There was no statistical difference between DHI with different volume percentage concentration and different doses of Asp(p>0.05).CYP3A4:There was a significant difference in the combination of the median volume concentration group of DHI and the high dose of Asp(p<0.05).5)Comparison of the drug combination group(Asp + DHI)and the corresponding dose of Asp group:CYP1A2:Asp in low dose group,respectively with low volume percentage concentration of DHI combined,and the corresponding single group compared no significant difference(p>0.05),and high dose of Asp and 3 volume percentage concentration of DHI combined with inhibitory effect on CYP1A2 enzyme activity of both subtypes(p<0.05 or p<0.01).CYP2C9:Compared with the corresponding single group,low dose group,Asp and DHI in high volume percentage concentration after combination had significant difference(p<0.05 or p<0.01),high dose Asp group and the high percentage of volume concentration of DHI combined on CYP2C9 isoforms activity inhibition(p<0.01).CYP2D6:Compared with the corresponding single use group,there was a significant difference between the low and middle Asp group and the high volume percentage of DHI(p<0.01).CYP3A4:The three doses of Asp(low,medium and high)were compared with those of the high dose of DHI(p<0.05 or p<0.01).But Asp low,medium and high 3 dose groups were respectively combined with low and medium volume percentage of DHI,compared with the corresponding single use group,there was no significant difference(p>0.05).Conclusion:1.The four enzyme subtypes of CYP1A2?CYP2C9?CYP2D6 and CYP3A4 have higher affinity to DHI,so it provides a basis for selecting CYPs enzyme subtype in vitro incubation experiment.2.The establishment of the MRM model in UPLC-Q-TOF-MS/MS detection of human liver microsomes for acetaminophen,diclofenac,4-hydroxy dextrophan,1-hydroxymidazolam and IS(buspirone hydrochloride,repaglinide)concentration.The method is simple,fast,and with high sensitivity.In addition,an accurate and feasible Incubating System for human liver microsomes in vitro was established.3.Under common clinical dosage,DHI has inhibitory effect on human CYPs enzyme subtypes in vitro,while Asp has no effect on the subtypes of human CYPs enzymes.DHI alone and combined DHI + Asp had no significant effect on the activity of human CYPs enzyme subtype,while Asp alone and Asp+DHI had different effects on the activity of human CYPs enzyme subtype.To sum up,this experiment investigated the effect of DHI and Asp on the activity of CYPs enzyme subtype,and provided reference for further exploring the metabolic interaction between them and the scientific and rational combination of drugs.