Pharmacokinetics And PK-PD Model Of Xian-Xiong-Gu-Kang In Osteoarthritic Rats By UPLC-MS

Objective The pharmacokinetics(PK)and pharmacodynamics(PD)of Xian-Xiong-Gu-Kang(XXGK)were studied,and PK was associated with PD to establish the PK-PD model,so as to further reveal the pharmacodynamic substance basis and mechanism of Xian-Xiong-Gu-Kang in the treatment of osteoarthritis(OA).Methods①Literature research was conducted on the active ingredients of XXGK,combined with the effects of each ingredient,to determin the pharmacodynamic components and effect indexes of OA.② OA in rats after surgery for cruciate ligament-meniscus injury.Hematoxylin and eosin(H&E)and safranin O(SO)staining were performed on the rats knee sections to observe the pathological changes and evaluate the OA model.③The plasma of OA rats after oral administration of XXGK was analyzed to study the blood components,and to optimaze the analysis conditions.④Using UPLC and quadrupole linear ion trap mass spectrometer(Qtrap-MS/MS),to develop and valitate a multi-components quantification method,and simultaneously determine the contents of the blood components in XXGK extract.⑤Taking the blood components of XXGK as the analysis targets,the pharmacokinetics of XXGK in pathological animals was studied by the developed method.⑥Enzyme linked immunosorbent assay(ELISA)was used to study the pharmacodynamics of XXGK.⑦Utilizing different methods for modeling,the PK-PD model was studied.By comparing the results of different PK-PD models,we tried to reveal the pharmacodynamic substance basis and mechanism of XXGK for OA.Results ①XXGK associated with OA efficacy components including icarrin,epimedin A,epimedin B,epimedin C,icariside Ⅰ,icariside Ⅱ,icaritin,ferulic acid,ligustrazine,senkyunolide A,senkyunolide I,senkyunolide H,ligustilide,oleanolic acid,cinnamic acid and chlorogenic acid.Interleukin-1β(IL-1β),tumor necrosisfactor-α(TNF-α),matrix metalloproteinase-3(MMP-3),MMP-13 and nitric oxide(NO)were closely related to the efficacy of these 16 components and OA.②The OA rat model was successfully established.Relative to the normal group,cartilage layers,including the cartilage surface,of the OA rats were seriously damaged,while chondrocytes were significantly reduced.③After XXGK administration,the main components detected in OA rat plasma were chlorogenic acid,ferulic acid,senkyunolide Ⅰ,epimedin A,epimedin B,epimedin C,icariin,cinnamic acid，icariside Ⅱ,senkyunolide A and ligustilide.④A UPLC-QTRAP-MS/MS method for simultaneous quantification of eleven components of XXGK in OA rat plasma was successfully developed.An ACQUITY UPLCTM BEH C18 column(2.1 mm×100 mm,1.7 μm)was employed to separate the samples at 30℃,at a flow rate of 0.3 mL/min with acetonitrile(A)and 0.05%(v/v)formic acid aqueous solution(B)as mobile phase.The elution was set as follows:0～20 minutes,10%～90%(A);20～21 min,90%～10%(A);21～25 min,10%(A).Injection volume=5 μL.The MRM(multiple reaction monitoring)in ESI+and ESI-(positive and negative electrospray ionization)modes was employed to detect and identify all compounds.⑤Methodological verification results showed that this method had good specificity,linearity,accuracy and precision,high extraction recovery and no obvious matrix effect,and good stability under general experimental conditions.The contents of the 11 components in XXGK extract ranged from 103.15 to 2569.12 μg/mL.⑥PK data showed that after single administration,the 11 components were absorbed rapidly in the body,the peak time in vivo was 0.12～0.67 h,the half-life was 2.70-8.77 h,and the max concentration was 8.78～830.61 ng/mL.PD data showed that the concentrations of TNF-α,MMP-3,MMP-13 and NO in plasma of rats were significantly decreased,with statistical significance,and the efficacy lagged.IL-1βconcentration showed a decreasing trend,and there was no statistical significance.It is suggested that the target of XXGK may be related to TNF-α,MMP-3,MMP-13 and NO.⑦The single-component PK-PD model showed that the PK-PD model of TNF-α,MMP-13 and NO fitted well with the 11 components,indicating a strong correlation.Integrated PK curves obtained by weighting coefficients of AUC0-∞ and EC50 and principal component analysis(PCA)were basically the same.The fitting results with different PD indicated that the drug components had stronger binding ability to TNF-αand MMP-13 receptor,suggesting that XXGK may mainly act by inhibiting the overexpression of TNF-α and MMP-13.The fitting of integrated PK(∑PK)and integrated Pd(∑PD)obtained by PCA was better,indicating that the two can better represent the overall change and effect of XXGK.PK-PD model fitted by artificial neural network had good predictive ability,which can predict the effect after administration.Conclusion The developed UPLC-QTRAP-MS/MS method was suitable for simultaneous quantitative analysis of XXGK components and for study of PK.The PK-PD models showed that TNF-α and MMP-13 had better correlation with each component and ∑PK,suggesting that XXGK might act mainly by inhibiting the expression of TNF-α and MMP-13.∑PK and ∑PD were more representative of the overall change and effect of XXGK.