Xanthine oxidoreductase (XOR) inhibitors, allopurinol, febuxostat, and topiroxostat are hyperuricemia and gout therapeutic drugs, which were supposed to be same. However, it was likely to be different effects in recent clinical and animal study. The activity of allopurinol is its metabolites, oxipurinol, while the others are themselves. Those XOR inhibitors have different modes of action in XOR inhibition. The XOR inhibitory activity in with or without human serum albumin (HSA) and HSA binding assay were evaluated, the constant was also calculated according to the scatchard and Klotz plot analysis. Moreover, the plasma XOR inhibitory activity was simulated based on the pharmacokinetics in repeated dose study. The XOR activity in the model increased with the S9 fraction. IC50 values of oxipurinol, febuxostat and topiroxostat were 244±50 nM, 20.5±2.8 nM and 1.26±0.25 nM, respectively. Febuxostat dose-dependently weakened the XOR inhibitory activity by HSA, whereas oxipurinol and topiroxostat had little impact on HSA. Additionally, the binding constant (K) to HSA in febuxostat and topiroxostat were 8.07× 104 M-1 and 1.38× 104 M-1, respectively, and the binding mode to HSA of the both drugs were nonspecific. Furthermore, XOR inhibitory activity of the lowest level in febuxostat (20 mg once a day) and topiroxostat (80mg twice a day) was estimated 61.4% and 86.8%, respectively, suggesting that the dose of twice a day was desirable to serially suppress the XOR activity in the blood. Albumin impacted on plasma XOR activity of febuxostat, not topiroxostat. Therefore, topiroxostat bound highly efficient to XOR, resulting in the strongest inhibitor of plasma XOR.

Abbreviations: CV: Coefficient of Variation; LC: Liquid Chromatography; LLOQ: Lower Limit of Quantification; SD: Standard Deviation; RE; Relative Error; TQMS: Triple Quadrupole Mass Spectrometry; UA: Uric Acid; XDH: Xanthine Dehydrogenase; XO: Xanthine Oxidase; XOR: Xanthine Oxidoreductase; PK/PD: Pharmacokinetics/Pharmacodynamics; ALT: Alanine Transaminase; AST: Aspartate Transaminase; eGFR: estimated Glomerular Filtration Rate; HSA: Human Serum Albumin.