Study On The Metabolic Mechanisms And Toxicity Prediction For Xanthine Oxidoreductase Inhibitors

Xanthine oxidoreductase inhibitor can inhibit the production of uric acid effectively,with a significant effect on the treatment of hyperuricemia.The metabolism studies of xanthine oxidoreductase inhibitors can be used to not only explain their in-vivo conversion,but also predict the toxicity of them.In this thesis,pharmacodynamics and metabolomics studies were carried out on xanthine oxidoreductase inhibitors,and the toxicity of drugs and their metabolites was predicted by ProTox-II.It was of guiding significance for drug design and drug modification to evaluate efficacy and safety from the perspective of drug metabolism.The following results were achieved in the research of this thesis:(1)Pharmacodynamic evaluation of LS087.A hyperuricemia model was established by injecting hypoxanthine and potassium oxonate into SPF grade mice.The levels of serum urea nitrogen,creatinine and uric acid together with H&E,PAS,Masson staining of renal and liver were measured to evaluate the pharmacodynamics of LS087.The results showed that the serum uric acid levels of the febuxostat-treated group(P<0.001)and the LS087-treated group(P<0.001)was significantly lower than that of the model group.Febuxostat and LS087 could reduce the level of serum uric acid in hyperuricemia mice,with the equivalent effect.Serum urea nitrogen levels in the febuxostat-treated group(P<0.05)and LS087-treated group(P<0.01)were significantly lower than those in the model group.The effects of reducing the serum urea nitrogen level in hyperuricemia mice were equivalent between febuxostat and LS087.The serum creatinine in the model group was significantly higher than that of the control group(P<0.001).Compared with the model group,there was no significant difference between the febuxostat and LS087 treated groups.According to the histopathology and biochemical indicators of kidney,both febuxostat and LS087 can reduce uric acid,which can improve the renal tubular function of hyperuricemia mice to some extent.(2)Identification of in vivo metabolites of febuxostat and LS087.Oral administration of febuxostat and LS087 to rats in a single dose of 10 mg/kg was constructed to establish a model for the indentification of metabolites.A rapid and sensitive UHPLC-Q-TOF/MS method was established apply for the separation and determination of drugs and their metabolites in rat serum and urine.At the same time,standard data handler for structural identification of drugs and their metabolites was established on the basis of UHPLC-Q-TOF/MS method.The metabolites of febuxostat were predicted using Biotransformation Mass Defects software,and all the information such as molecular formulas and precise molecular weight was converted into a Personal Compound Database(PCD).The collected MS1 data could match with the possible metabolites in the PCD quickly.The possibility fragment of MS/MS was calculated using Molecular Structure Correlator(MSC).With the standard data handler,4 phase I and 2 phase II metabolites were discovered in the serum of the febuxostat-treated group,and 7 phase I and 3 phase II metabolites were discovered in the urine.Among those metabolites,M2,M5,M6 and M7 were not reported yet.At the same time,4 phase I metabolites and 4 phase II metabolites were discovered in the serum of rats in the LS087-treated group,and 5 phase I metabolites and 5 phase II metabolites in the urine.All metabolites of LS087 have not been reported.(3)Toxicity prediction of febuxostat,LS087 and their metabolites.The ProTox-II data platform was used to predict the toxicity of febuxostat,LS087 and their metabolites.It was found that febuxostat and all its metabolites were predicted to have hepatotoxicity.The results suggested that further toxicological investigation of febuxostat and its metabolites was needed.It is a guidance for the modification of febuxostat chemical structure and the synthesis of novel xanthine oxidoreductase inhibitors.Compared with febuxostat,LS087 and LS-M2,LS-M3,LS-M5,LS-M6,LS-M7,LS-M8 were predicted to have no potential hepatotoxicity.Even though LS-M1,LS-M4,LS-M9,and LS-M10 were predicted to have potential hepatotoxicity,the content of these four metabolites was very low,whose total amount was less than 1%.We believed that LS087 had a lower risk for treating hyperuricemia patients with liver damage compared with febuxostat.LS-M1 and LS-M8 were predicted to be potentially carcinogenic.And further studies are needed in the future to confirm the drug safety of LS087.However,since the total amount of LS-M1 and LS-M8 was less than 0.1%,we believed that LS087 may be a safe and effective xanthine oxidoreductase inhibitor compared to febuxostat.(4)Study on the metabolic mechanisms of febuxostat.Metabolomics was used to study the metabolic mechanism of febuxostat in hyperuricemia mouse model.By the principal component and hierarchical cluster analysis,the control,model and febuxostat-treated groups were featured by good clustering within the group,indicating that the model had good stability.They were completely separated between the groups,indicating a significant change in the serum metabolite profiles of the three groups of mice.16 potential biomarkers associated with hyperuricemia were identified by database matching.According to metabolic pathway analysis,when exogenous hypoxanthine was injected into the mouse,the metabolism of purine is over-activated,leading to the transformation of hypoxanthine into xanthine rapidly.However,influenced by febuxostat,the activity of XOR was inhibited and the transformation from hypoxanthine to xanthine was weakened.Therefore,the hypoxanthine in the model group was significantly lower than that of febuxostat-treated group.This process revealed the significant inhibitory ability of febuxostat on XOR during the transformation of hypoxanthine to xanthine.The results provided a basis for the elucidation of the mechanism of febuxostat and the development of xanthine oxidoreductase inhibitor drugs.