Preparation Of W3D Solid Dispersion And Its Activity Against Acetaminophen Induced Acute Liver Injury

Objective:In this study,pharmacokinetic research on the compound4-（5’-dimethylamino）-naphthalenesulfonyl-2（3H）-benzoxazolone（W3D）synthesized by our research group with high anti-inflammatory and analgesic activity was conducted to identify pharmacokinetic characteristics.Then pharmacy strategy was used to resolve the above problems,and the affect on acetaminophen（APAP）-induced acute liver injury（ALI）was investigated to evaluate its therapeutic effect.Methods:Part One:Content determination of Compound W3DHPLC method was performed on the Diamonsil C18 column（250 mm×4.6 mm,5μm）with the mobile phase consisted of methanol-water（75∶25）at a flow rate of 1.0m L·min^-1.The detection wavelength was set at 257 nm at 28℃.Part Two:Pharmacokinetic study of W3D in vivo1、HPLC method was performed on the Diamonsil C18 column（250 mm×4.6 mm,5μm）and Easy Guard C18（10×4.0 mm）guard column with the mobile phase consisted of methanol-water（75∶25）at a flow rate of 0.8 m L·min^-1.The detection wavelength was set at 257 nm at 25℃.After the methodological investigation,the content determination of W3D in plasma was established.2、SD rats were administrated with W3D using intravenous injection(30 mg·kg^-1)and oral administration(36 mg·kg^-1).Then the concentration of W3D in the plasma at different times were collected and measured to perform data analysis.Part Three:Preparation,quality evaluation and pharmacokinetic study of W3D solid dispersion1、The dissolution determination method in vitro was established using HPLC method.2、The preparation method,auxiliary materials,ratio of raw materials were screened to determine the best preparation process based on the dissolution rate in vitro;3、The quality of W3D solid dispersion was evaluated using mass fraction,saturated solubility,dissolution reproducibility,fourier infrared spectroscopy（FT-IR）,differential scanning calorimetry（DSC）and X-powder ray diffraction（XRD）methods.4、SD rats was administered with W3D solid dispersion at 9 mg·kg^-1and 4.5 mg·kg^-1,respectively.Then blood was collected at different times to measure the concentration of W3D,and data processing was performed to analysis the pharmacokinetic parameter.Part Four:Effect of W3D solid dispersion on APAP-induced acute liver injury in mice1、APAP-induced ALI mice were administered with positive drug,W3D and solid dispersion.The blood and liver tissues were collected 24 hours later,then the weight of liver was recorded to calculate liver index.2、The pathological changes in liver tissues were evaluated by HE staining;3、The content of ALT and AST in serum were detected by microplate method;IL-6and TNF-αin serum were measured by ELISA method;The SOD activity,the content of GSH and MDA in liver tissue were confirmed by microplate and TBA method.4、The expression of MD2 in liver tissue was assayed by immunohistochemical techniques;Results:Part One:Content determination of Compound W3DThe compound W3D could be completely separated under the above chromatographic conditions.The linear range was 1～100μg·m L^-1（R²=0.9999）.The precision and stability were in line with the requirement of pharmacopoeia.The contents of the three test products were all more than 99.5%.Part Two:Pharmacokinetic study of W3D in vivo1、The W3D could be separated with endogenous substances in plasma under the above chromatographic conditions.The method showed high separation degree,good specificity,high accuracy and recovery,good precision and stability,good dilution reliability,no residue which could be used to determine the content of W3D in plasma.2、The pharmacokinetic process of W3D in vivo was two-compartment model.The main pharmacokinetic parameters were listed as following.The area under the drug-time curve(AUC_（0-∞）),clearance rate,distribution half-life(t_1/2（α）)and elimination half-life(t_1/2（β）)were 716.442±108.259(μg·m L^-1)*min,0.043±0.006（L/min/kg）,15.668±3.301 min and 66.987±4.598 min when W3D was intravenous injected.When W3D was administered orally,the area under the drug-time curve(AUC_（0-∞）),clearance rate,distribution half-life(t_1/2（Ka）)and elimination half-life(t_1/2（β）)were 97.164±19.900(μg·m L^-1)*min,0.382±0.089（L/min/kg）,62.190±9.477 min and 69.306±0.071 min.Part Three:Preparation,quality evaluation and pharmacokinetic study of W3D solid dispersion1、W3D and the pharmaceutic adjuvant could be separated preferably under HPLC conditions above with good specificity,high recovery,good precision,and excellent stability;2、The PVPk30 was the optimal pharmaceutic adjuvant to prepare W3D solid dispersion with the proportion of materials 1:7 using the solvent method which showed the bested dissolution rate in vitro.3、The mass fraction of W3D solid dispersion were 12.25%,12.42%,and 12.48%,which were all consistent with the theoretical value（12.5%）.The saturated solubility in water was 54.83μg·m L^-1.There was no chemical interaction between the main drug and the pharmaceutic adjuvant,and W3D solid dispersion presented as amorphous powder.4、The pharmacokinetic process of W3D solid dispersion in vivo was two-compartment model.The main pharmacokinetic parameters were listed as following.The area under the drug-time curve(AUC_（0-∞）),clearance rate,distribution half-life(t_1/2（Ka）)and elimination half-life(t_1/2（β）)were 498.853±93.835(μg·m L^-1)*min,0.018±0.003（L/min/kg）,10.988±1.505 min and 69.668±0.648 min when W3D was administered orally about 9 mg·kg^-1.Meanwhile,the area under the drug-time curve(AUC_（0-∞）),clearance rate,distribution half-life(t_1/2（Ka）)and elimination half-life(t_1/2（β）)were 256.389±42.936(μg·m L^-1)*min,0.018±0.003（L/min/kg）,14.388±2.288 min and 68.281±1.085min when W3D was administered orally about 4.5 mg·kg^-1.Part Four:Effect of W3D solid dispersion on APAP-induced acute liver injury in miceW3D solid dispersion can dose-dependently reduce the liver index on APAP-induced ALI mice,and the necrosis,hemorrhage,and increased cytoplasmic eosinophilia were improved obviously.Meanwhile the expression of ALT,AST,TNF-αand IL-6 in serum were decreased significantly.In addition,GSH content,SOD activity in liver tissue were increased and MDA content was reduce in W3D solid dispersion treated group compared with model group.Conclusion:1、The pharmacokinetic process of W3D in vivo was two-compartment model.AUC_（0-∞）and K_awere significantly increased,and t_1/2（Ka）was reduced obviously in W3D solid dispersion treated groups which indicate the parameters could be enhanced when W3D was prepared as solid dispersion.2、Compound W3D showed determinate therapeutic effect on APAP-induced ALI mice which was better than the positive drug NAC.