Vol. 303, Issue 1, 104-109, October 2002

Biological Activity of a Novel Nonpeptide Antagonist to the Interleukin-6 Receptor 20S,21-Epoxy-resibufogenin-3-formate

Masahiko Hayashi, Mun-Chual Rho , Akiko Fukami, Akiko Enomoto, Shinobu Nonaka, Yoshino Sekiguchi, Tadashi Yanagisawa, Ayano Yamashita, Toshihiko Nogawa, Yoshiaki Kamano and Kanki Komiyama

The Kitasato Institute, Tokyo, Japan (M.H., M.-C.R., A.F., A.E., S.N., Y.S., K.K.); Cardiovascular Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea (M.-C.R.); Department of Applied Biochemistry, Utsunomiya University, Utsunomiya, Japan (T.Y.); and Faculty of Science, Kanagawa University, Hiratsuka, Japan (A.Y., T.N., Y.K.)

Interleukin (IL)-6 is a key mediator in the regulation and coordination of the immune response and participates in pathogenesis of cancer cachexia, autoimmune disease, and postmenopausal osteoporosis. In the course of a screening program aimed at IL-6 inhibitor from natural products, we isolated 20S,21-epoxy-resibufogenin-3-formate (ERBF) from bufadienolide and examined the effect of ERBF on activities of various cytokines. ERBF dose dependently suppressed IL-6 activity and caused a parallel rightward shift of dose-response curves to IL-6 at concentrations of 0.03 to 10 ng/ml. Analysis of data yields a pA₂ of 5.12 and a slope of 0.99. Selectivity of ERBF on activity of cytokines was examined using cytokine-dependent cell lines. ERBF did not affect IL-2-dependent growth of CTLL-2 cells, IL-3-dependent growth of Baf3 cells, or tumor necrosis factor (TNF)-induced growth suppression in TNF-sensitive L929 cells. ERBF also did not affect IL-4-stimulated expression of FcR II receptor (CD23) in U-937 cells, the IL-8-induced chemotaxis of human neutrophils, or nerve growth factor-stimulated neuronal differentiation in PC-12 cells. In contrast, ERBF dose dependently suppressed IL-6-induced neuronal differentiation in PC-12 cells. Furthermore, ERBF suppressed only IL-6-induced osteoclast formation without affecting osteoclast formation induced by IL-11, leukemia inhibitory factor, and 1,25-dihydroxyvitamin D₃. In receptor binding assay, unbound (free) IL-6 was increased in a dose-dependent manner by pretreatment with ERBF on IL-6 receptor (IL-6R), suggesting that ERBF suppresses binding of IL-6 to IL-6R. These results clearly indicate that ERBF is a novel specific small molecule to show IL-6 receptor antagonist activity.