Background/Purpose: Recent unpublished findings from our lab show that the extracellular enzyme sulfatase-2 (Sulf-2) facilitates pro-inflammatory TNF-α signaling which activates rheumatoid arthritis synovial fibroblasts (RASFs). The small molecule drug OKN-007 has been reported to inhibit Sulf-2 activity. OKN-007 demonstrated safety in human clinical trials at sustained plasma concentrations averaging 300 micromolar. In this study, we tested the efficacy of OKN-007 in reducing TNF-α-induced adhesion molecule and chemokine production by RASFs.

Methods: Experiments utilized primary human RASFs (n=5; age 30-61) from de-identified synovial tissues obtained under an IRB-approved protocol, from patients who met the ACR criteria for RA and were not treated with biologics. MTT viability assay was used to measure potential toxicity of OKN-007 [0-1000 µM] alone or with TNF-α for 24 h. To test effect of Sulf-2 inhibition on TNF-α induced proteins, RASFs were pre-treated with OKN-007 (0-1000 µM) for 12 h followed by TNF-α (20 ng/mL) stimulation for 24 h. RASFs not stimulated with TNF-α served as a negative control. Concentration of CCL2, CXCL5, CCL5, CXCL10, CXCL11 in cell supernatants were measured by ELISA. Expression of MAPK/NF-κB signaling and adhesion molecules (ICAM-1 and VCAM-1) were measured by densitometry on Western blots. The Sulf-2-inhibitory effects of OKN-007 were further validated by Sulf-2 small-interfering RNA (siRNA) method. Statistical value p< 0.05 was considered significant.

Results: OKN-007 alone at a dose range of 0-1000 µM did not significantly affect RASF viability (n=4). When combined with TNF-α, OKN-007 [500, 1000 µM] reduced the RASF viability by 22% and 29%, respectively (n=4; p< 0.05), suggesting sensitization of RASFs to TNF-α-induced apoptosis. Adhesion molecule expression was normalized to β-actin, accounting for any cell loss. OKN-007 [500, 1000 µM] decreased TNF-α-induced ICAM-1 by 20% and 36% and VCAM-1 by 23% and 35%, respectively (n=4; p< 0.05). OKN-007 (500-1000 µM) inhibited TNF-α-induced CCL2 (23-28%), CXCL5 (66-75%), CCL5 (~65%), CXCL10 (80-95%), and CXCL11 (43-65%) (n=4; p< 0.05). Further validation of the pharmacologic inhibition using Sulf-2-directed siRNA showed similar inhibition of TNF-α-induced chemokine production and adhesion molecule expression by human RASFs (n=4; p< 0.05). Evaluation of the signaling molecules suggest that OKN-007 suppressed TNF-α-induced signaling pathways to elicit its anti-inflammatory activity.

Conclusion: Our study in human RASFs suggests that pharmacologic inhibition of Sulf-2 with a safe, non-immunosuppressive compound may provide an adjunct therapeutic value with anti-TNF therapy for the treatment of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-extracellular-sulfatase-2-inhibitor-okn-007-abrogates-tnf-%ce%b1-induced-inflammatory-mediators-in-human-rheumatoid-arthritis-synovial-fibroblasts/