Pharmacokinetic Interaction Between Methotrexate And Levetiracetam And Its Mechanism

Part one Determination of methotrexate and levetiracetam in biological samples based on UPLC-MS/MS TechnologyObjective:An UPLC-MS/MS method was established for the determination of methotrexate and levetiracetam in biological samples.Methods:Column:Waters UPLC ACQUITY HSS T3?100 mm×2.1 mm,1.8?m?;Column temperature:40?;Autosampler temperature:4?;The mobile phase:0.1%formic acid water?A?-Acetonitrile?B?,gradient elution;Flow rate:0.2 mL?min^-1;Injection volume:2?L;Ion source:ESI;Ion mode:MRM;Ion polarity:positive ions;Ion spray voltage source:5500 V;Ion source temperature:600?;EP:100 V;CXP:14 V;GS1:60 kPa;GS2:60 kPa;Methotrexate ion pair:m/z 445.4?308.3;Levetiracetam ion pair:m/z 171?126;Internal standard aminopterin ion pair:m/z 441.2?294.1.Plasma,urine,cell lysate,5%glucose samples were processed by methanol precipitation protein processing.Results:Both of the methods showed high specificity,and endogenous substances did not interfere with the determination.The parameters of methotrexateandlevetiracetamwereasfollows:thestandard curve,Y=0.0415X+0.0713,r=0.9970,Y=0.0784X+0.0263,r=0.9978;limit of quantification,0.5 ng mL^-11 and 0.5?g mL^-1;RSDs of inter-day and intra-day were all lower than 15%;the control samples of intra day and inter day RSD were less than 15%and relative recoveries were 85%-115%.matrix effect and extraction recovery could meet the requirements of the guidelines for the analysis of biological samples.Part twoInteraction between methotrexate and levetiracetam in rats and its mechanismObjective:Study on the interaction between methotrexate and levetiracetam in rats and related mechanisms.Methods:The oral absorption test,renal excretion test,reversed intestinal test,Caco-2 cell test and hepatorenal toxicity research were performed to explore the target and related mechanism of the interaction between methotrexate and levetiracetam.Results:After methotrexate combined with levetiracetam,the C_max,AUC_0-t-t and AUC_0-?of methotrexate increased,the CL and MRT decreased,and the differences were statistically significant;the CL and MRT of levetiracetam had a certain rising trend,C_max,AUC_0-t-t and AUC_0-?had a certain declining trend,and the differences were no statistically significant.The interaction occured after the combination of the two drugs;After the combination of the two drugs,the renal excretion of methotrexate increased significantly,and the renal excretion in levetiracetam decreased significantly;the concentration of methotrexate on the side of the reversed intestinal serosa was significantly increased,and the effect manner was time dependent;P-glycoprotein?P-gp?probe substrates?verapamil,digoxin?and levetiracetam increased the uptake of methotrexate in Caco-2 cells,and the effect of levetiracetam was dose dependent;The results of pathological section showed a significant increase in hepatic and renal toxicity in rats after the combination of the two drugs.Conclution:1.The UPLC-MS/MS method established in this study was accurate,efficient and simple for the determination of methotrexate and levetiracetam in biological samples.The result of methodology could meet the requirements of the guidelines for the analysis of biological samples.2.After methotrexate combined with levetiracetam in rats,the excretion of methotrexate in the intestinal tract decreased and the absorption of methotrexate increased.Methotrexate combined with levetiracetam caused methotrexate blood drug concentration increased and the kidney excretion increased,which lead to the aggravation of kidney damage in rats.The reason might be the P-gp competition between levetiracetam and methotrexate in the intestinum tenue of rats.It was suggested that methotrexate doses should be adjusted when methotrexate combined with levetiracetam in clinic,and blood concentration should be monitored if necessary.