Objective: To find previously unknown properties of ML3000, a competitive inhibitor of the cyclooxygenase (COX) and the lipoxygenase (LO) pathway.
Methods: Gene expression of ML3000 treated and untreated rheumatoid arthritis synovial fibroblasts (RASF) were measured with Affymetrix gene arrays. Downregulation of chemokine (C-X-C motif) ligands CXCL9, CXCL10 and CXCL11 was verified with Real-time PCR, CXCL10 protein levels were determined with ELISA. RASF were treated with the COX inhibitor naproxen, the 5-LO inhibitor BWA4C and the 5-lipoxygenase-activating protein (FLAP) inhibitor MK886, and consecutive changes in CXCL10 protein levels measured. 5-LO expression was determined by PCR and Western blot.
Results: In synovial fibroblasts and monocyte-derived macrophages ML3000 inhibited the TNF induced expression of CXCL9, CXCL10 and CXCL11, which are all ligands of the chemokine receptor CXCR3. No effect was observed in monocytes. Whereas inhibition of the COX pathway or the FLAP protein showed no effect, blockade of 5-LO significantly downregulated CXCL10 protein levels. 5-LO mRNA was detected in monocytes and in monocyte-derived macrophages. All tested cell types expressed 5-LO protein.
Conclusion: ML3000 effectively downregulates CXCR3 ligands. This study confirms that a thorough analysis of the impact of a drug on its target cells cannot only reveal unexpected properties of a substance, but also helps to understand the underlying molecular mechanisms. Accordingly, our data provide the basis for further clinical studies testing the application of ML3000 in diseases like rheumatoid arthritis or multiple sclerosis.
- CXCR3 ligands
- cyclooxygenase/lipoxygenase inhibitor
- rheumatoid arthritis