Cell experiment:

Briefly, human Raji B cells are plated at a density of 0.5 × 104 cells/well in a 96-well plate and synchronized by incubation for 24 h in RPMI 1640 medium supplemented with 0.1-0.5% FBS. Synchronized cells are pretreated with Iguratimod or vehicle for 30 min prior to stimulation with macrophage migration inhibitory factor (MIF) for 24 h. At 20 h BrdU is added to cells and quantified using a BrdU Cell proliferation assay kit[3].

Animal experiment:

Mice[3]Endotoxemia is induced by intraperitoneal injection of LPS from E. coli O111:B4. In BALB/c animals, 5 mg/kg LPS is used as a lethal dose for survival experiments; animals are treated with Iguratimod (20 mg/kg i.p.) 0.5 h prior to LPS, 6 h after LPS, and then once daily for 3 days and monitored for survival over 2 weeks. In C57BL/6 animals, 20 mg/kg LPS is used as non-lethal dose for plasma cytokine experiments; animals are pretreated with Iguratimod (20 mg/kg i.p.) twice, one dose each at 2 and 0.5 h prior to LPS administration, and euthanized at 90 min post-LPS by CO2 asphyxiation with cervical dislocation. Blood is collected by cardiac puncture and allowed to clot 20 min at room temperature and 20 min at 4℃; sera are isolated by centrifugation at 300 × g for 10 min and stored at –20℃ for further analysis by TNFα ELISA (1:3 dilution)[3].

IC50: 2.0 (hepatocyte-stimulating activities) and 6.6 μg/ml (immunoreactivities) for IL-6 release.

Iguratimod is one of a series of 4H-1-benzopyran-4-ones which has potent anti-inflammatory, antipyretic and analgesic activity. Iguratimod also inhibits the production of tumour necrosis factor and interleukin-1 (IL-1), IL-6, IL-8.

In vitro: Iguratimod inhibited the release of immunoreactive IL-1 beta from human monocytic cell line stimulated with lipopolysaccharides (LPS) in a dose-dependent manner (0.3-30 μg/ml). Northern blotting analysis using LPS-stimulated THP-1 cells indicated that the inhibitory effect of Iguratimod on IL-1 beta production is caused by the suppression of IL-1 beta mRNA expression [1].

In vivo: Administration of Iguratimod did not inhibit the tumor growth, but resulted in attenuation of cachexia symptoms. Furthermore, Iguratimod decreased the serum levels of IL-6, and also reduced its gene expression in the tumor tissues. In addition, exogenously administered IL-6 nullified the suppressive effect of Iguratimod [2].

Clinical trial: A 52-week clinical study of iguratimod in 394 Japanese patients with rheumatoid arthritis to evaluate the long-term safety of the drug was conducted. Iguratimod was administered orally at a daily dose of 25 mg for the first 4 weeks and 50 mg for the subsequent 48 weeks. The cumulative incidence of adverse events for 100 weeks was 97.6%. The cumulative incidence of adverse reactions was 65.3%; unfavorable symptoms and signs accounted for 33.2% of the reactions, and abnormal laboratory data changes accounted for 50.4% [3].

References:
[1] Tanaka K, Aikawa Y, Kawasaki H, Asaoka K, Inaba T, Yoshida C.  Pharmacological studies on 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one (T-614), a novel antiinflammatory agent. 4th communication: inhibitory effect on the production of interleukin-1 and interleukin-6. J Pharmacobiodyn. 1992;15(11):649-55.
[2] Tanaka K, Urata N, Mikami M, Ogasawara M, Matsunaga T, Terashima N, Suzuki H.  Effect of iguratimod and other anti-rheumatic drugs on adenocarcinoma colon 26-induced cachexia in mice. Inflamm Res. 2007;56(1):17-23.
[3] Hara M, Abe T, Sugawara S, Mizushima Y, Hoshi K, Irimajiri S, Hashimoto H, Yoshino S, Matsui N, Nobunaga M.   Long-term safety study of iguratimod in patients with rheumatoid arthritis. Mod Rheumatol. 2007;17(1):10-6.