Presentation: Characterization of Anti-arthritic Effect of a Novel Small Molecule AP-1 Inhibitor T-5224. (2007)

1779 Characterization of Anti-arthritic Effect of a Novel Small Molecule AP-1 Inhibitor T-5224.

PURPOSE: Activator protein-1 (AP-1) is not only an important transcription factor responsible for arthritic joint destruction but also controls both inflammatory cytokines and matrix-degrading matrix metalloproteinases (MMPs). We previously designed and synthesized a small molecule c-Fos/AP-1 inhibitor T-5224 that inhibits development of type II collagen-induced arthritis (CIA) in mice almost completely (Aikawa Y, et al. Arthritis Rheum. 2006; 54(Suppl 9): S232). We here searched the molecular target of anti-arthritic effect of T-5224.
METHODS: Mouse CIA: CIA was induced in DBA/1J mice by the immunization with collagen type II twice on day 0 and 21. Efficacy was determined by arthritis score, X-ray examination and serum biochemical analysis. Antigen-specific Ab response: Mice were primed with NP-CGG/alum on day 0 and 70. T-5224 was administered from day 0, and sera were assayed on day 98 for anti-NP Ab. Synovial cell extension: A 20 × 20 mm cover slip, covered by human synovial adherent cells, was placed upside-down in a 6-well plate. Cells were cultured with IL-1β and/or T-5224 for 24 hr, and cell extension was assessed under phase-contrast microscope. Osteoclastogenesis: Macrophage-osteoclast precursor RAW264.7 cells were cultured with RANKL and/or T-5224, and the cells stained for TRAP and the lysate was blotted with anti-NFATc1 mAb.
RESULTS: To dissect the early pathologic events occurring in CIA, we administered T-5224 from day 21 until onset of arthritis (day 26). Quantification of major inflammatory cytokines in sera on day 27 revealed that IL-1β was the only inflammatory cytokine significantly inhibited by T-5224. The inhibition of arthritis and subsequent joint destruction was cancelled by exogenous IL-1β administered on day 27, indicating that IL-1β is primarily responsible for arthritis and T-5224 inhibits this IL-1β. The experiment also showed that T-5224 reduced the serum level of MMP-3 even at day 27, indicating that T-5224 also inhibits MMP which is under regulation of c-Fos/AP-1. While T-5224 did not inhibit antigen-specific secondary Ab response against NP residue in vivo, T-5224 completely inhibited in vitro pannus-like synovial extension and the expression of NFATc1, a signaling molecule right under c-Fos/AP-1, in osteoclast and its osteoclastogenesis under RANKL-stimulation dose-dependently.
CONCLUSIONS: T-5224 directly inhibited key molecules IL-1β and MMP-3. In addition to the inhibition of synovial cell and osteoclast in vitro, T-5224 totally prevented development of arthritic joint destruction, and thus the novel AP-1 inhibitor T-5224 seems promising as a new anti-rheumatic drug.

 T. Yamamoto, None; Y. Aikawa, None; A. Hashiramoto, None; K. Morimoto, None; H. Chaki, None; H. Narita, None; S. Hirono, None; S. Shiozawa, None.