Determination Of Avitinib In Rat Plasma And Its Effect On CYP450 Enzyme Subtype Activity By UPLC-MS/MS

Objective:avitinib is China's first independently developed third-generation EGFR inhibitor,which is mainly used to treat non-small cell lung cancer,and is resistant to the use of the first-generation EGFR targeted drugs.In this experiment,avitinib was determined by UPLC-MS/MS to observe the changes of the concentration.Through the methodological investigation,condition optimization and other methods,explore the bioavailability of avitinib and in rats.To explore the effect of avitinib on the metabolic activity of CYP450 enzymes in vivo and in vitro with detection of CYP450 enzyme probes?dextromethorphan,bupropion hydrochloride,phenacetin,chlorzoxazone,tolbutamide and midazolam?and their metabolites by UPLC-MS/MS methods.Methods:?1?An ACQUITY UPLC BEH C18?50 mm×2.1 mm,1.7?m?column was used to separate the analytes.Acetonitrile and water?containing 0.1%formic acid?were chosen as the mobile phase.The analytes were quantified by multiple reaction monitoring mode with positive electrospray ionization.?2?Twelve healthy male SD rats were randomly selected and divided into two groups,with 6 rats in each group,and were given avitinib at the same time.The oral group was given 10 mg/kg orally;the other 6 rats were given intravenously 1 mg/kg by sublingual vein.Fasting for 12 hours before medication,and blood samples were collected at 0.083,0.25,0.5,1,1.5,3,4,6,8,12 and 24 hours after medication.The UPLC-MS/MS method was established to detect the plasma concentration of avitinib in rats,the methodological system was established and the conditions were optimized.Pharmacokinetic parameters and statistical analysis were obtained using DAS 3.0 software and SPSS 17.0 software.The bioavailability of avitinib in rats was determined to provide a basis for clinical safe and reasonable medication.?3?To establish a rat liver microsomal incubation system with six probe drugs include phenacetin,bupropion,dextromethorphan,tolbutamide,chlorzoxazone and midazolam as substrates and avitinib as inhibitor.After the optimization of the conditions,the 200?L incubation system was selected.Among them,5-10?L rat liver microsomes,2?L probe substrate mixture and 100 mM potassium phosphate buffer?PH=7.4?,Then add 1 to 100?M concentration gradient solution of avitinib to the system,place it in a 37°C water bath and shake for 5 min,then add NADPH?1 mM?to start the reaction,remove it after 30 min Add ice-cold acetonitrile to the ice bath to precipitate protein.After full vortexing,centrifuge for 10 min at 13000 rpm,2?L of supernatant was used for injection.The drug metabolism was detected by UPLC-MS/MS,and IC50 values were calculated to investigate the in vitro metabolism of avitinib.?4?Twelve healthy male SD rats were randomly divided into an experimental group and a control group with 6 rats in each group.The experimental group rats were orally administrated with avitinib at the dosage of 10 mg/kg for 1 week;the control group rats were administrated with CMC-Na?carboxymethylcellulose sodium?for1 week.The two groups of rats were given a mixed dose of 10 mg/kg of 6 kinds of probe drugs on the day of the experiment,and 0.083,0.25,0.5,1,1.5,2,3,4,6,8,12,and 24 h were taken The blood sample was placed in a 1.5 mL centrifuge tube at a centrifugal speed of 4000 rpm.After centrifugation for 10 min,the plasma was stored at-80°C.Conditional optimization was established by establishing a methodology,and at the same time,the plasma concentration of probe drugs and their metabolites in rat plasma was detected by UPLC-MS/MS method,and avitinib and probe were investigated by analyzing the kinetic parameters of plasma Chinese drugs-drug interactions in rats.Results:?1?The analysis showed that avitinib in plasma had a good linear relationship within a certain concentration range.After a methodological investigation,the interday and intraday precision?RSD?activities were in the range of 8.28%and 6.29%;the interday and intraday accuracy?RE?were in the range of 96.32%and 97.24%;the extraction recovery rate between 85.71%-89.26%,it shows that the sample has better extraction recovery rate under this method;the matrix effect is between 92.149%-104.24%,indicating that the detection of the sample is not affected by the matrix;the mixed sample is in the following four Conditions:6 h at room temperature,24 h at 4°C,2 weeks at?80°C,and good stability after repeated freezing and thawing 3 times.After analyzing the pharmacokinetic parameters,the bioavailability was calculated to be 30.81%.?2?Through the incubation experiment in vitro,the concentrations of six kinds of probe substrates were determined:phenacetin,bupropion,dextromethorphan,tolbutamide,chlorzoxazone and midazolam solution were 40?M,20?M,10?M,100?M,20?M and 5?M,respectively.In the concentration range of 1-100?M,the drug avitinib showed different inhibitory effects on six probe drugs.The IC50 values are as follows:bupropion 6.39?M,phenacetin 15.79?M,chlorzoxazone 23.15?M,midazolam 27.64?M,tolbutamide 42.18?M,dextromethorphan44.39?M.?3?Through the in vivo experiment in rats,it was found that avitinib could induce the probe drug.A comparative analysis of the experimental group and the control group showed that the AUC_?0-t?of the phenacetin experimental group was 76.7%lower than that of the control group,and the clearance rate of CLz/F was 4.3 times that of the control group;the AUC of the bupropion experimental group was 88%less than that of the control group,and 52%less than that of the control group,and 52%less than that of the control group,while the AUC?0-t?of the bupropion experimental group was 87.1%lower than that of the control group.The AUC?0-t?of dextromethorphan experimental group decreased by 86.7%compared with the control group,and the AUC?0-t?of tolbutamide in the experimental group decreased by 87.5%compared with the control group.The AUC_?0-t?in the chlorzoxazone experimental group decreased by 41.7%compared with the control group and 25.8%by 25.8%compared with the control group.The AUC?0-t?of midazolam experimental group decreased by 79.3%compared with the control group,and the clearance rate of CLz/F increased by 64%compared with the control group.For T_max,except tolbutamide is about 5 h,the other five probe drugs are all less than 0.5 h,especially bupropion and midazolam at 0.29 h.In the tolbutamide group and dextromethorphan group,the T_1/2 was 8.96 h and 5.04 h respectively,while that of other probe drugs was between0.89 h to 3.05 h.To sum up,avitinib could induce the six probe drugs in different degrees,and the results in vivo were opposite to those in vitro.Conclusion:?1?A sensitive and reliable UPLC-MS/MS method was established to determine the concentration of avitinib in rat plasma.Through analysis of the results of this study,avitinib has low bioavailability after oral administration.When clinical medication is used,it is necessary to pay attention to the mode of administration and timely medication monitoring.?2?Through the analysis of the results of this study,we believe that avitinib has different degrees of inhibitory effects on probe drugs in vitro in rats,with bupropion and phenacetin having the most severe inhibitory effects.There is a risk of liver damage from long-term use of avitinib.At the same time,avitinib has an inducing effect in rats.When patients use avitinib and drugs metabolized by CYP450 enzymes,the dosage should be adjusted.