Study On PK And PD Interaction Between Xuebijing Injection And Azithromycin In Inflammatory State

Clinical studies have showed that the combination of Xue Bijing injection（XBJ）and azithromycin（AZM）in the treatment of children with mycoplasma pneumonia could significantly improve the clinical symptoms and shorten the course of treatment,and its efficacy was significantly better than that of AZM alone.From this,we speculated there may be synergistic effect when the two drugs used in combination.Based on this,this paper discussed the pharmacokinetic and pharmacodynamic interactions of XBJ combined with AZM in the treatment of pneumonia,and explained the rationality and scientific connotation of the compatibility of Chinese and Western medicines.First of all,we compared the differences in blood drug concentration and PK pharmacokinetic parameters of lung target tissues between single and combination of XBJ and AZM in mice with pneumonia model.Then,we further determined the effects of single or combination of the two drugs on pharmacokinetic parameters of RAW264.7 cells stimulated by LPS.Finally,the inhibition of NF-κB was measured with high content imagine system to evaluate anti-inflammatory efficacy of two drugs,so as to prove whether the synergistic effect of two drugs was indeed present.Objective:1.To analyze pharmacokinetic characteristics and lung tissue distribution of XBJ and AZM in mice with pneumonia by liquid chromatography-mass spectrometry（LC-MS/MS）.2.To define pharmacokinetic interactions of RAW264.7 cells after combination of two drugs by using the method of cell dynamics.3.To evaluate synergistic effect of XBJ and AZM on the anti-inflammatory effect of RAW264.7 cells by high content imagine system.Methods:1.Determination of the content of XBJ and AZM in plasma and lung tissue in mice with pneumonia:Plasma samples and lung tissue samples were removed by methanol precipitation,and then supernatant was taken after centrifugation,BEH C18（2.1×100 mm,1.7μm）was used as the analytical column,with 0.1%formic acid water and 0.1%formic acid acetonitrile as the mobile phase,and the gradient elution was carried out at a flow rate of 0.3m L/min.MRM monitoring was performed on two main pharmacodynamic components,paeoniflorin（PE,525.3→449.1）,hydroxyl safflower yellow A（HSYA,611.0→491.3）by electrospray（ESI）ionization in negative ion mode,and AZM（749.6→591.3）in positive ion mode.2.Cell dynamics:The study of cell dynamics began with RAW264.7 cell resuscitation,passage and culture,and then the concentration of XBJ and AZM were determined by CCK-8assay.When the cells were stimulated to make them in an inflammatory state by LPS,the cells were broken after incubation with drug for a certain period of time.Finally,the concentration of HSYA,PE and AZM in cells were determined by LC-MS/MS.3.Evaluation of synergistic anti-inflammatory effect:The inflammatory model of RAW264.7 cells was established by LPS.After incubation with drug,the nucleation of NF-κB was detected by high content system after cell fixation,holing,sealing,primary antibody,secondary antibody and hoechst staining.And then synergistic effect on anti-inflammatory effects was evaluated by Compu Syn software.Results:1.Results of pharmacokinetics in mice with pneumonia:Compared with single dose group,the plasma exposure of XBJ combined with AZM was significantly increased.The pharmacokinetic parameters of AZM have changed as follows,half-life(t_{1/2 alone}=11.54±3.34 h,t_{1/2 combination}=14.11±2.65 h)was significantly prolonged,area under the drug-time curve（AUC）increased by 73%,and the clearance rate（CL）in vivo was significantly reduced by 41.6%.While the pharmacokinetic parameters of HSYA and PE were changed respectively,AUC of HSYA increased by 26%,CL was significantly reduced by 17%,and the AUC of PE increased by 71%,CL was significantly reduced by 40%.2.Lung target tissue distribution results:The drug tissue concentration also increased significantly after the combination of XBJ and AZM,and AZM exposure in lung tissue was significantly higher in 0-192 h than that of single dose group,and the concentrations of HSYA and PE in the combination group were also significantly increased within 0 h-12 h.3.Results of cell dynamics experiment:Combination of XBJ（dilution 50,100,200 times）and AZM（2μg/m L）can significantly promote the uptake of AZM and increase the concentration in cells.4.The results of intracellular fluorescence intensity by the high-content test showed that compared with the model group,the AZM（0.5,1,2,5,10μg/m L）group,the XBJ（dilution 20,50,100,200 times）group and the dexamethasone（DEX:4μg/m L）group all showed reduced intracellular fluorescence intensity,indicating that NF-κB was inhibited.Compared to the treatment group with AZM or XBJ alone,the inhibitory effect of AZM and XBJ on NF-κB was significantly stronger in the combination group.It indicates that the combination of AZM and XBJ has a certain synergistic effect on the anti-inflammatory effect.Conclusion:1.Pharmacokinetic interactions occurred after the combination of XBJ and AZM,and the pharmacokinetic parameters of each component changed significantly,increasing exposure of the drug in plasma and lung tissue.2.After combination of XBJ and AZM,the pharmacokinetic behavior of AZM at the cellular level changed,increasing the uptake of AZM by macrophages.3.In the inflammatory state,XBJ combined with AZM has occurred pharmacodynamic interaction,suggesting a synergistic effect on the NF-κB inflammatory pathway.