In vitro activity: Pamapimod inhibited p38α and p38β enzymatic activity, with IC50 values of 14 and 480 nM, respectively. There was no activity against p38δ or p38γ isoforms. When profiled across 350 kinases, pamapimod bound only to four kinases in addition to p38. Cellular potency was assessed using phosphorylation of heat shock protein-27 and c-Jun as selective readouts for p38 and c-Jun NH2-terminal kinase (JNK), respectively. Pamapimod inhibited p38 (IC50, 0.06 μM), but inhibition of JNK was not detected.Pamapimod also inhibited lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) α production by monocytes, interleukin (IL)-1β production in human whole blood, and spontaneous TNFα production by synovial explants from RA patients. LPS- and TNFα-stimulated production of TNFα and IL-6 in rodents also was inhibited by pamapimod.

Kinase Assay: Pamapimod is a novel p38 mitogen-activated protein kinase inhibitor. Pamapimod inhibited p38α and p38β enzymatic activity, with IC50 values of 14 and 480 nM, respectively. Pamapimod is p38 inhibitor with IC50 of 0.06μM in THP-1 cell. IC50: 14 and 480 nM (p38α and p38β enzymatic activity, respectively.) IC50:0.06μM (THP-1 cell).

Cell Assay: THP-1 cells, growing in log phase, were collected by centrifugation and resuspended in RPMI 1640 containing 5.5×10-5 M 2-mercaptoethanol and 10% fetal bovine serum to a final cell concentration of 2.5×106 cells/ml. Dilutions of pamapimod were predispensed in 25 μl aliquots (before addition of cells) into round-bottom 96-well plates. The starting concentration was 100 μM in 5% dimethyl sulfoxide, and six half-log serial dilutions were made. After the addition of 200 μl of cell suspension and 25 μl of 5 μg/ml LPS in medium, the final dimethyl sulfoxide concentration was 0.5%. Compounds were diluted an additional 10-fold, and the final LPS concentration was 500 ng/ml. The cell suspensions and compound dilutions were combined and incubated for 30 min at 37°C in a 5% CO2 humidified atmosphere, before the addition of LPS (or medium for non-LPS control samples). After the addition of LPS, plates were incubated for 2 h, followed by centrifugation to pellet cells. Cell supernatants were stored at 4°C until analysis for TNF-α content. TNF-α levels were determined by ELISA. Cytokine concentrations were determined.