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DOP67 CKD-506, a selective histone deacetylase (HDAC) 6 inhibitor, ameliorates colitis through regulation of NF-kB and AP-1 signalling pathway

J. Shin1, N. Ha1, D. Bae1, Y.J. Lee1, Y.I. Choi1, K.H. Ryu1, G.S. Youn2, J. Park2

1Department of Non-clinical Study, Chong Kun Dang Pharm. Research Institute, Youngin-si, Korea Republic of, 2Department of Biomedical Science, Hallym University, Chuncheon, Korea Republic of

Background

HDAC6 is a stress-inducible gene and highly expressed in pathological conditions as well as inflammatory bowel disease. Immuno-modulatory functions of HDAC6 inhibitors are well established and proposed therapeutic effects for autoimmune diseases through regulation of Treg cell function and inflammation. Moreover, HDAC6 inhibitors regulate inflammatory cytokines and chemokines, neutrophil activities, and epithelial regeneration in colitis models. CKD-506, a potent and selective oral HDAC6 inhibitor, is generally safe and well-tolerated in human, and is now investigating the efficacy in patients with rheumatoid arthritis. Herein, for future investigation with IBD, we identified molecular action mechanisms of CKD-506 involved in anti-colitis effects.

Methods

Mouse peritoneal macrophages or Raw264.7 cells were transfected with HDAC6 overexpression plasmid or empty vector as control. Cells were cultured in the presence or absence of 0.03~3 μM CKD-506, and the expression and production of inflammatory mediators were determined by RT–PCR and ELISA respectively. For reporter assays, Raw264.7 cells were transfected with pNF-kB-luc or pAP-1-luc plasmid and luciferase activity in cell lysates was determined by a luminometer. Signalling molecules in HDAC6 overexpressed cells were checked by immunoblot analysis. For the efficacy test of CKD-506, we used DSS-, TNBS-, Piroxicam (IL-10−/−)-, and adaptive T-cell transfer (RAG1−/−)-mediated colitis animal models. Colitis animals were treated with 1 to 100 mg/kg of CKD-506 and analysed disease activities and inflammatory mediators.

Results

In vivo, CKD-506 strongly inhibited disease activities in DSS-, TNBS-, Piroxicam-, and adaptive T-cell transfer-mediated colitis. In the chemical-induced colitis model, the expression of cell adhesion molecules and chemokines such as IP-10 and also infiltration of immune cells to colon tissues were reduced in CKD-506 treated mice. In vitro, HDAC6 overexpression strongly induced ROS and NADPH oxidase activity in Raw264.7 cells and CKD-506 significantly and dose-dependently inhibited HDAC6-mediated ROS and NADPH activity. Moreover, CKD-506 inhibited the production of pro-inflammatory cytokines and chemokines which are up-regulated in HDAC6 overexpressed mouse peritoneal macrophages or Raw264.7 cells. In promoter assay, HDAC6 overexpression highly induced NF-kB and AP-1 activity and CKD-506 strongly and dose-dependently inhibited both signalling pathways.

Conclusion

These data provide insight that CKD-506, a selective HDAC6 inhibitor, has anti-inflammatory and anti-colitis effects through regulation of NF-kB and AP-1 signalling pathway. Therefore, CKD-506 may provide beneficial effects in patients with Crohn’s disease and ulcerative colitis.

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