1000 - Anti-Inflammatory Profile of AS2444697, A Novel Interleukin-1 Receptor-Associated Kinase-4 Inhibitor

Monday, November 7, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
Junko Imanishi, Takeshi Ishikawa, Emiko Imamura, Hidekazu Mizuhara, Haruna Iwaoka, Ball Evelyn, Hiroshi Inami, Tsuyoshi Mizutani, Junko Watanabe, Hiroyuki Usuda, Shinya Nagashima, Tomonori Ito, Toru Kontani, Yasuaki Shimizu and Seitaro Mutoh, Astellas Pharma Inc., Tsukuba, Japan
Presentation Number: 1000

Background/Purpose:Toll-like receptors (TLRs) function in the innate immune response by recognizing pathogen-associated molecular patterns or host-derived “danger signals” produced during tissue injury or inflammation. Interleukin-1 (IL-1) receptor-associated kinase-4 (IRAK-4) is known to be a pivotal mediator on the TLR/IL-1R signaling pathway, as both IRAK-4-knockout mice and IRAK-4-deficient human patients exhibit functional defects in this pathway. Observations of increased expression of TLRs and TLR ligands in patients with rheumatoid arthritis (RA) suggest that these receptors may contribute to RA pathogenesis. Here, we report the pharmacological profile of AS2444697, a novel IRAK-4 inhibitor.

Method:In vitro IRAK-4 inhibitory activities of AS2444697 were evaluated using recombinant human and rat IRAK-4 enzymes and synthetic substrates (FGL ARF SRF AGS SPS QSS MVA RTQ TVR GTL A). IL-1β-mediated IRAK-1 degradation in A549 (human alveolar basal epithelial cell line) was assessed with western blot analyses using anti-IRAK-1 antibody (Cell Signaling Technology). The effect of AS2444697 on IL-1β-, TNF-α- and TLR ligands- (LPS for TLR-4, peptidoglycan for TLR-2, imiquimod for TLR-7) stimulated cytokine production were examined using A549 and two primary cells, PBMCs (Peripheral Blood Mononuclear Cells from healthy volunteers) and HFLS-RA (Human Fibroblast-Like Synoviocytes-Rheumatoid Arthritis from RA patient). In vivo anti-inflammatory activity was evaluated using lipopolysaccharide (LPS) induced cytokine production and adjuvant-induced arthritis (AIA) in rats.


AS2444697 inhibited human and rat IRAK-4 with the same IC50 values of 21 nM. It also suppressed IL-1β-mediated IRAK-1 degradation in A549 in a concentration-dependent manner with a range of 10 nM ` 10 μM. It also inhibited IL-1 (1 ng/mL) and TNFα (3 ng/mL) stimulated IL-6 production from A549 with IC50 of 250 nM and 3900 nM, respectively. AS2444697 inhibited LPS- (0.01 ng/mL) induced TNF and IL-6 production from PBMCs, with IC50 values of 47 and 59 nM, respectively. In addition, AS2444697 was also effective on IL-6 production from HFLS-RA stimulated with IL-1β 1 pg/mL and TNFα 10 pg/mL (IC50: 170 and 380 nM respectively). AS2444697 inhibited TNFα production from LPS (3 ng /animal) injected rat dose dependently with an ED50 of 3.4 mg/kg. Further, prophylactic administration of AS2444697 ameliorated paw swelling in AIA rats with an ED50 of 4.5 mg/kg (p.o. b.i.d).

Conclusion: AS2444697 inhibited not only TLR, IL-1β but also TNFα pathway. These results suggest that AS2444697 will an attractive agent for the treatment of RA.

Keywords: kinase

Disclosure: J. Imanishi, Astellas Pharma Inc., 3 ; T. Ishikawa, Astellas Pharma Inc., 3 ; E. Imamura, Astellas Pharma Inc., 3 ; H. Mizuhara, Astellas Pharma Inc., 3 ; H. Iwaoka, Astellas Pharma Inc., 3 ; B. Evelyn, Astellas Pharma Inc., 3 ; H. Inami, Astellas Pharma Inc., 3 ; T. Mizutani, Astellas Pharma Inc., 3 ; J. Watanabe, Astellas Pharma Inc., 3 ; H. Usuda, Astellas Pharma Inc., 3 ; S. Nagashima, Astellas Pharma Inc., 3 ; T. Ito, Astellas Pharma Inc., 3 ; T. Kontani, Astellas Pharma Inc., 3 ; Y. Shimizu, Astellas Pharma Inc, 3 ; S. Mutoh, Astellas Pharma Inc., 9 .