Determination Of Apatinib And Dextromethorphan In Rats Plasma And Its Application To The Pharmacokinetics And Drug Interactions Study

Apatinib is a new oral small molecule angiogenesis inhibitor.It selectively acts on vascular endothelial growth factor receptor-2（VEGFR-2）,inhibits angiogenesis of tumors,and then inhibits the growth of tumors.It has high bioavailability in human body.It is mainly metabolized by CYP3A4/5and CYP2D6 enzymes,and has good safety and tolerance.A series of randomized controlled clinical trials have confirmed that it have a certain objective response rate and survival benefits.Dextromethorphan（DM）is the most widely used over-the-counter antitussive drug,mainly metabolized by CYP2D6 enzyme,and is the substrate of CYP2D6 enzyme.There may be a competitive relationship between the two agents with the metabolic enzymes.In this study,we established and validated two LC-MS/MS methods to determine the concentration of apatinib and DM in rat plasma,respectively.The interaction and pharmacokinetic effects of apatinib and DM were primarily studied to provide a basis for rational drug use in clinic.Part One Establishment of a method for determination of apatinib in rats plasma and the effect of dextromethorphan on the pharmacokinetics of apatinibObjective:To establish a LC-MS/MS method for the determination of apatinib in rats plasma,and to investigate the pharmacokinetic effects of DM on apatinib.Method:1.Determination of apatinib in rats plasma by LC-MS/MSChromatographic column:Kinetex C₈（2.10×100 mm,2.6μm）;Mobile phase:0.2%formic acid and 10 mM ammonium acetate aqueous solution（A）-methanol（B）,pH:3.21;Flow rate gradient elution programme:0.01-1.0 min,0.2 ml·min^-1;1.0-2.0 min,0.2-0.4 ml·min^-1;2.0-3.5 min,0.4 ml·min^-1;3.5-3.51 min,0.4-0.2 ml·min^-1;3.51-4.50 min,0.2 ml·min^-1,70%B.Ionization of analyte was chosen using the ESI technique with positive polarity and multiple reaction monitoring（MRM）mode.Quantitative ion pair of apatinib:m/z 398.2→211.9,quantitative ion pair of tinidazole（IS）:m/z248.0→121.1.Acetonitrile precipitation protein method was selected for plasma sample pretreatment.2.Effect of dextromethorphan on pharmacokinetic of apatinibEighteen male Sprague-Dawley（SD）rats were randomly divided into two groups by body weight:multiple dose of 10 mg/kg DM for 7 days and the control group.On the 8^th day,60 mg/kg apatinib was given in both groups.Before（0 h）and at 0.25,0.5,1,1.25,1.5,2,3,4,6,8 and 12 h post-dosing,the blood（0.5 ml）was collected from the intraocular canthus vein.The plasma concentration of apatinib was determined by LC-MS/MS.DAS 3.0non-compartment model was used to simulate the pharmacokinetic parameters.SPSS 21.0 was used to analyze the pharmacokinetic parameters of the two groups.P<0.05 showed that there was significant difference between the two groups.Results:1.Determination of apatinib in rat plasma by LC-MS/MSThe linear relationship of apatinib was good in the range of 5-1000 ng/ml.The correlation coefficients were all greater than 0.996,and the low limit of quantification（LLOQ）was 5 ng/ml.The intra-and inter-day RSD（%）of apatinib were less than 10.0%,and the accuracy was in the range of94.0%-108.0%.The matrix effect and recovery were in the range of98.0%-107.0%and 95.0%-104.0%,respectively.The stability under various conditions,residual effect and stability of plasma diluted 10 times were conformed to the validation guidelines for quantitative analysis of biological samples.2.Effect of DM on pharmacokinetic of apatinibThe C_last of the control group and the experimental group were23.45±19.63 ng/ml and 13.56±30.95 ng/ml,respectively.There was significant difference between the two groups（P<0.05）.There were no significant differences in other parameters,such as AUC_（0-t）,AUC_（0-∞）,MRT_（0-t）,MRT_（0-∞）,t_1/2,T_max,Vz/F,CLz/F and C_max.Part Two Establishment of a method for determination of dextromethorphan in rats plasma and the effect of apatinib on the pharmacokinetics of dextromethorphanObjective:To establish a LC-MS/MS method for the determination of DM in rats plasma,and to investigate the pharmacokinetic effects of apatinib on DM.Method:1.Determination of dextromethorphan in rats plasma by LC-MS/MSChromatographic column:Kinetex C₈（2.10×100 mm,2.6μm）;Mobile phase:0.1%formic acid（FA）and 10 mM ammonium acetate（AA）aqueous solution（A）-acetonitrile（B）,pH:3.48;Gradient elution programme:0.01-0.50 min,45%B;0.50-1.50 min,45%-80%B;1.50-3.50 min,80%B;3.50-3.51 min,80%-45%B;3.51-4.50 min,45%B.Ionization of analyte was chosen using the ESI technique with positive polarity and multiple reaction monitoring（MRM）mode.Quantitative ion pair of DM:m/z 272.1→147.1,quantitative ion pair of tinidazole（IS）:m/z248.0→121.1.Acetonitrile precipitation protein method was selected for plasma sample pretreatment.2.Effect of apatinib on pharmacokinetic of dextromethorphanThirty-four male SD rats were randomly divided into four groups by body weight:DM control group and experimental group（3 days）,8 rats in each group;DM control group and experimental group（7 days）,9 rats in each group.The dosage of apatinib was 40 mg/kg for 3 days or 7 days,and DM was given 15 mg/kg on the 4^th or 8^th day.Before（0 h）and at 0.25,0.5,0.75,1,1.33,1.66,2,3,4,6,8 and 12 h post-dosing,the blood（0.5 ml）was collected from the intraocular canthus vein.The plasma concentration of DM was determined by LC-MS/MS.DAS 3.0 non-compartment model was used to simulate the pharmacokinetic parameters.SPSS 21.0 was used to analyze the pharmacokinetic parameters of the two groups.P<0.05 showed that there was significant difference between the two groups.Results:1.Determiniation of dextromethorphan in rat plasma by LC-MS/MSThe linear relationship of DM was good in the range of 2-400 ng/ml.The correlation coefficients were all greater than 0.996,and LLOQ was 2 ng/ml.The intra-and inter-day RSD（%）of DM were less than 8.0%,and the accuracy was in the range of 93.0%-107.0%.The matrix effect and recovery were in the range of 86.0%-101.0%and 86.0%-94.0%,respectively.The stability under various conditions,residual effect and stability of plasma diluted 5 times were conformed to the validation guidelines for quantitative analysis of biological samples.2.Pharmacokinetic effects of apatinib on dextromethorphanThe pharmacokinetic parameters in the control group and the experimental group（3 days）were as follows:AUC_（0-t）were 678.60±281.19ng/ml·h and 293.56±67.84 ng/ml·h,AUC_（0-∞）were 699.27±289.72 ng/ml·h and 307.55±71.30 ng/ml·h,Vz/F were 76.18±69.88 L/kg and 179.20±85.40L/kg,CLz/F were 25.39±11.27 L/h/kg and 50.70±9.66 L/h/kg,respectively.There were significant differences between the two groups（P<0.05）.There were no statistical differences in other parameters,such as MRT_（0-t）,MRT_（0-∞）,C_last,t_1/2,T_maxax and C_max.The MRT_（0-t）and MRT_（0-∞）of the control group and the experimental group（7 days）were 2.15±0.31 h and 2.80±0.61 h,2.49±0.55 h and 3.35±1.13h,respectively.There were significant differences between the two groups（P<0.05）.There were no statistical differences in other parameters,including AUC_（0-t）,AUC_（0-∞）,C_last,t_1/2,T_max,Vz/F,CLz/F and C_max.Conclusion:1.LC-MS/MS methods have been established for the determination of apatinib and DM in rat plasma,respectively.Both of them are rapid,accurate,sensitive and specific.It is suitable for pharmacokinetic studies of apatinib and DM.2.By comparing the pharmacokinetic parameters of apatinib monotherapy group with those of DM coadministrated with apatinib group,it was found that DM significantly affected the C_last of apatinib in rats,but had no effect on other pharmacokinetic parameters.3.Compared with the control group,the main pharmacokinetic parameters of DM in rats treated with apatinib for 3 consecutive days were significantly different.AUC_（0-t）and AUC_（0-∞）significantly decreased,Vz/F and CLz/F increased.While for the group of DM in rats treated with apatinib for 7days,except for MRT_（0-t）and MRT_（0-∞）,the other pharmacokinetic parameters were no statistical differences.