sd-0006 and Arthritis--Rheumatoid

Tumor necrosis factor alpha (TNFalpha), a pro-inflammatory factor, plays an important role in many inflammatory diseases. Inhibition of p38 is being pursued as a pharmaceutical treatment to reduce TNFalpha release. Since a variety of cytokines and factors may exist at different amounts in patients, we explored how differences in the cytokine environment impact p38 inhibitor potency. Cytokine co-stimulation with LPS was compared against LPS stimulation alone. In both differentiated U937 cells and peripheral monocytes, GM-CSF co-stimulation with LPS increased TNFalpha release and led to an increased residual TNFalpha levels with p38 inhibitor. Adding MEK inhibitor in the presence of p38 inhibitor further reduced TNFalpha release suggesting that the ERK pathway plays a role in GM-CSF induced reduction of the p38 inhibitor potency. When cells were stimulated with different concentrations of LPS and GM-CSF, the minimal TNFalpha level obtained by MEK inhibitor was not dependent on the stimulation condition; while it was dependent on GM-CSF level for p38 inhibitor. TNFalpha release in the presence of combinations of p38 and MEK inhibitors under different stimulation conditions was measured. A linear model was created using the initial relative ERK and p38 phosphorylation levels and p38 and MEK inhibitor concentrations to accurately predict released TNFalpha level, suggesting these four parameters are sufficient to predict TNFalpha levels. We then used the model to show that with same TNFalpha levels, higher ERK pathway activity reduces p38 inhibitor potency. These results suggest that p38 inhibitor will be a more potent anti-TNFalpha therapy for patients with low ERK pathway activity.

Topics: Arthritis, Rheumatoid; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Macrophage Activation; Monocytes; p38 Mitogen-Activated Protein Kinases; Pyrazoles; Pyrimidines; Signal Transduction; Tumor Necrosis Factor-alpha; U937 Cells

In this report, we show that apoptosis signal-regulating kinase 1(-/-) (ASK1 KO) mice were resistant to inflammatory arthritis induced in the K/BxN serum transfer model of rheumatoid arthritis (RA). The p38 inhibitor, SD-0006 was administered to wild type (WT) mice as a comparator. Both ASK1 KO and p38 inhibition resulted in marked attenuation of edema, cartilage damage, bone resorption, and general inflammatory responses. Transcriptional profiling of mRNA prepared from paw tissue demonstrated that the production of many proinflammatory genes including cytokines, chemokines, and extracellular matrix degradative enzymes were maintained at basal levels by either ASK1 KO or prophylactic p38 MAPK inhibition. In the mouse whole blood (MWB) assay, tumor necrosis factor-α (TNF-α)-induced KC and CCL2 levels and also LPS-induced interleukin-6 (IL-6), CCL2, and KC levels in MWB from ASK1 KO were significantly lower than those from WT. Furthermore, both p38 and JNK were activated by TNF-α in human synovial fibroblasts isolated from RA patients (RASF). SD-0006 or SP600125, a JNK inhibitor, partially blocked the elevation of IL-6 production in RASF following stimulation with TNF-α. In contrast, dual inhibition with both p38/JNK inhibitors almost completely abolished TNF-α-induced IL-6 production from these cells. Ablation of ASK1 expression in RASF using siRNA for ASK1 resulted in inhibition of TNF-α-induced IL-6 and PGE(2) production. This study is the first to suggest that ASK1 is critical for the development of RA and that ASK1 may be involved in the production of proinflammatory mediators in response to TNF-α stimulation in the RA joint.

Topics: Animals; Apoptosis; Arthritis; Arthritis, Rheumatoid; Cells, Cultured; Dinoprostone; Female; Fibroblasts; Gene Expression Profiling; Humans; Interleukin-6; MAP Kinase Kinase Kinase 5; Mice; Mice, Inbred C57BL; Mice, Knockout; Pyrazoles; Pyrimidines; Tumor Necrosis Factor-alpha