Iguratimod is a novel disease-modifying antirheumatic drug. A blue letter (safety advisory) for drug interaction between iguratimod and warfarin was issued by the Ministry of Health, Labour and Welfare of Japan in May 2013. Iguratimod may affect warfarin metabolism catalyzed by CYP. However, it is not clear whether iguratimod inhibits warfarin oxidation. This study was performed to investigate the effects of iguratimod on warfarin 7-hydroxylation with human liver microsomes (HLMs) and recombinant CYP enzymes. Iguratimod concentration-dependently inhibited R,S-warfarin 7-hydroxylase activity of HLMs with an IC 50 value of 15.2 µM. The inhibitory effect was examined with S-warfarin and R-warfarin to determine which enantiomer was more potently inhibited by iguratimod. Iguratimod potently inhibited the S-warfarin 7-hydroxylase activity of HLMs with an IC 50 value of 14.1 µM, but showed only slight inhibition of R-warfarin 7-hydroxylation. Furthermore, iguratimod inhibited the S-warfarin 7-hydroxylase activity of recombinant CYP2C9.1 (rCYP2C9.1) and rCYP2C9.3 in a concentration-dependent manner with IC 50 values of 10.8 and 20.1 µM, respectively. Kinetic analysis of the inhibition of S-warfarin 7-hydroxylation by iguratimod indicated competitive-type inhibition for HLMs and rCYP2C9.1 but mixed-type inhibition for rCYP2C9.3. The K i values for HLMs, rCYP2C9.1, and rCYP2C9.3 were 6.74, 4.23, and 14.2 µM, respectively. Iguratimod did not exert metabolism-dependent inhibition of S-warfarin 7-hydroxylation. These results indicated that iguratimod is a potent direct inhibitor of CYP2C9-mediated warfarin 7-hydroxylation and that its inhibitory effect on CYP2C9.1 was more sensitive than that on CYP2C9.3.Key words iguratimod; warfarin; CYP2C9; inhibition; drug-drug interaction; polymorphism Iguratimod is a novel disease-modifying antirheumatic drug, which has been used for the treatment of rheumatoid arthritis exclusively in Japan and China. It is notable that iguratimod exerts a beneficial effect in rheumatoid arthritis patients with inadequate response to methotrexate. 1,2) Iguratimod is extensively metabolized by the liver, and several metabolites of iguratimod have been characterized in plasma from healthy male subjects following oral administration of the drug 3) (Fig. 1). The major metabolites are a deformylated form (M1) and its N-acetylated form (M2). The other metabolites are phydroxylated forms in the 6-phenoxy groups of iguratimod, M1, and M2, which have been termed M4, M5, and M3, respectively. CYP is unlikely to be involved in the formation of M1 from iguratimod because M1 is formed regardless of recombinant CYP isoforms examined and the presence or absence of reduced nicotinamide adenine dinucleotide phosphate (NADPH). M1 was suggested to be metabolized by Nacetyltransferase to produce M2. On the other hand, iguratimod is metabolized by multiple isoforms, including CYP1A2, CYP2C9, and CYP3A4, to produce M4 and/or M5.When manufacturing approval was granted in Japan, the instructions indicated that care should be...