Effects Of Ambroxol On The Distribution Of Voriconazole And Meropenem And Its Pharmacokinetics Comparision After Oral And Atomization Inalation Administration In Mice Lung

Part 1 Effects of different doses of ambroxol hydrochloride on the distribution of voriconazole in plasma and lung in miceObjective: To develop a HPLC method for the determination of voriconazole in mice plasma and lung,and investigate the effects of different doses of ambroxol hydrochloride on the distribution of voriconazole in plasma and lung of mice by comparing the pharmacokinetic parameters of voriconazole after different doses of ambroxol hydrochloride.Methods: Chromatographic separation was performed on a Diamonsil C₁₈ column（250 mm×4.6 mm,5 μm）with a mobile phase consisting of acetonitrile-0.01mol·L^-1 KH2PO4（triethylamine adjust pH to6.26）（47:53）at a flow rate of 1.0 mL·min^-1.The column temperature was set at 30°C,the detection wavelength was 254 nm and the injected volume was10 μL.Diltiazem hydrochloride was selected as the internal standard.Two hundred and forty male Kunming mice were randomly divided into control group and experimental group.The experimental group including high,medium and low three dose groups.Mice in the control group were orally administrated with voriconazole 66.67 mg·kg^-1.The rest of the three experimental groups were orally administrated with voriconazole 66.67mg·kg^-1 and at the same time add low(10 mg·kg^-1),medium(50 mg·kg^-1),large(150 mg·kg^-1)different doses of ambroxol respectively.Blood was sampled by removalling eye ball at different time points.Lung tissue homogenate were obtained after fully grind.Plasma samples were processed using the acetonitrile protein precipitation method and the lung tissue homogenate were extracted by ethyl acetate-methylene chloride（75:25）.The pharmacokinetic parameters were calculated with DAS 2.1.1 and statisticallyanalyzed with SPSS 21.0.Results: After the validation of this method,it showed that the voriconazole had good linear relationships in both plasma and lung tissue samples and the standard curve equations were Y₁=0.043 X₁-0.012（r=0.9996）(2⁶4 μg·mL^-1)and Y₂=0.303 X₂+0.119（r=0.9992）(0.4¹2.8μg·mL^-1),respectively.The average recovery range were 83.59%⁹4.80% and73.12%⁷7.67%,respectively.The RSD values of both intra-and inter-day precision were less than 15%.Plasma and lung tissue homogenate samples were stable when under freeze-thaw cycles,stored at-70℃ for 20 days and placed 8 h at room temperature after processed.The main pharmacokinetic parameters such as AUC_0-t,AUC_0-∞,t_1/2,T_max,CL,V,C_max in plasma and lung of the four groups had no significant differences（P>0.05）.Conclusions: The HPLC method is selective,accurate and precised.It can be used in the concentration determination of voriconazole in mice plasma and lung tissue.The distribution of voriconazole,both in plasma and lung,has no significant difference wether combining ambroxol hydrochloride or not.Part 2 The influence of different doses of ambroxol hydrochloride on the distribution of meropenem in the lung of miceObjective: To develop a LC-MS/MS method for determining the concentration of meropenem in mice lung,and study the influence of different doses of ambroxol hydrochloride on the distribution of meropenem in mice lung when they used in a combination.Methods: Chromatographic separation was performed on a Diamonsil C₁₈（150 mm×4.6 mm,5 μm）and the mobile phase consisted of acetonitrile-0.1% formic acid at a flow rate of 1.0 mL·min^-1,gradient elution,and ertapenem was selected as the internal standard.The mass spectrometer was operated with an electrospray ionization interface（ESI）,positive and negative ions were to switch.Quantification was performed to use multiple reactions monitoring（MRM）method with the ion transitions of m/z 384.1→141.2 for meropenem and m/z 474.4→265.2 for the IS（ertapenem）.144 mice were randomly divided into three groups: controlgroup,low dose group and high dose group.All the drugs were given through caudal vein.The low dose group and high dose group were injected ambroxol hydrochloride at different doses(9 mg·kg^-1 and 45 mg·kg^-1)respectively and the control group were injected saline in same quantity.Then all of mice were given meropenem(75 mg·kg^-1).The lung samples were collected at different points,and then made into lung tissue homogenate.Drug mass concentrations in lung were detected by LC-MS/MS method.The pharmacokinetic parameters were calculated by DAS 2.1.1 and statistically analyzed by SPSS21.0.Results: Meropenem had good linear relationships in lung tissue samples and the standard curve equations was Y=0.01507 X+0.01348（r=0.9997）(15.625²000 ng·mL^-1).The average recovery range was 98.05%¹02.54%,and the average matrix effect was 90.93%¹04.3%.After the validation of this method,it showed that precision,stability,dilution effect were all conform to the requirements of the determination of biological samples.The pharmacokinetic experiment results showed that AUC_0-t,AUC_0-∞,t_1/2,T_max,CL,V,C_max had no significant differences in mice lung of the three groups（P>0.05）.Conclusions: The LC-MS/MS method has a good performance in terms of accuracy,sensitivity and precision.It is suitable to determine the concentration of meropenem in mice lung.The experimental results show that different doses of ambroxol hydrochloride has no significant influence on the distribution of meropenem in mice lung.Part 3 Pharmacokinetics comparision of ambroxol hydrochloride in plasma and lung after oral and atomization inalation administration in miceObjective: To establish a LC-MS/MS method for the determination of ambroxol hydrochloride in mice plasma and lung,and study the pharmacokinetics of ambroxol hydrochloride in plasma and lung by the two ways of administrations.Methods: Chromatographic separation was carried out on a Diamonsil C₁₈ column（150 mm×4.6 mm,5 μm,Dikma Science,China）and which waskept a temperature at 35℃.The mobile phase consisting of acetonitrile-0.01%formic acid with 5mM ammonium acetate（37:64,v/v）was at a liquid flow rate of 1.0 mL·min^-1 and the sulfamethoxazole was selected as the internal standard.The mass spectrometer was operated in the positive electrospray ionization（ESI）mode.Quantification was performed to use multiple reactions monitoring（MRM）method with the ion transitions of m/z 379→263.7 with declustering potential（DP）57.16 and collision energy（CE）24.92 for ambroxol hydrochloride and m/z 254.2→155.8 with DP 73 and CE 22 for the IS.144 male Kunming mice were randomly divided into two groups,72 mice in each group.After given the same dose of ambroxol hydrochloride(4.5mg·kg^-1)by oral or atomization inhalation way respectively,the plasma and lung tissue were collected at different time points.All of the samples were processed with the acetonitrile protein precipitation method.The pharmacokinetics parameters were calculated by DAS 2.1.1 software and statistically analyzed by SPSS 21.0.Results: After oral administration,the mean C_maxvalues in plasma and lung were 138.57±52.21,361.48±103.54 ng·mL^-1 and the mean AUC_0-∞ values were 741.05±136.90,1482.41±427.25 ng·h·mL^-1,respectively.After atomization inalation administration,the mean C_max values were7419.67±3224.4,3415.33±941.94 ng·h·mL^-1 and the mean AUC_0-∞ values were 10113.16±2032.61,10153.26±1439.80 ng·h·mL^-1,respectively.The value of AUC_0-∞ and C_max in plasma and lung had significant differences between the two groups.Conclusions: A LC-MS/MS method is developed and validated for the determination of ambroxol hydrochloride in mice plasma and lung after oral and atomization inalation administration in mice.The results show that ambroxol hydrochloride can achieve a much higher concentration in plasma and lung by the atomization inalation administration,which indicate that atomization inalation may be a better treatment for lung diseases.