Abstract 17368: The Novel P2X7 Receptor Antagonist SMW139 Inhibits Inflammasome Activation in STEMI Monocytes

Abstract

Introduction: The inflammasome and the downstream pro-inflammatory cytokine interleukin-1ß (IL-1ß) have emerged as critical modulators of atherosclerosis and acute coronary syndromes (ACS). It therefore represents an important therapeutic target to treat coronary artery disease. This study investigates the activation of the inflammasome in circulating monocytes of ACS patients, and the effectiveness of SMW139- a compound selected from a panel of over 70 novel P2X7 antagonists based upon its excellent pharmacokinetic properties- on modulating the downstream inflammatory signalling pathway. Hypothesis: The P2X7 antagonist SMW139 reduces inflammasome activation. Methods: In vitro: THP-1 monocytic cells were treated with interferon- γ and lipopolysaccharide for 3 hours, then pre-incubated with SMW139 (1-1000nM), prior to ATP stimulation to activate the inflammasome, and collection of supernatant for IL-1ß measurement. Ex vivo: Peripheral blood mononuclear cells (PBMCs) were collected from 19 STEMI patients at time of infarct, and 3 healthy controls. Monocytes were pre-incubated with SMW139 or DMSO control prior to ATP stimulation. IL-1ß and caspase-1 activity were measured by ELISA and fluorometric assay. Results: In vitro: Sigmoidal dose-response curve demonstrated a LogEC50 of -8.069±0.16. Ex vivo: High levels of detectable P2X7 expression in PBMCs was confirmed by flow cytometry. Baseline levels of secreted IL-1ß were significantly elevated in PBMCs from STEMI patients compared with healthy controls (STEMI: 8.2±0.6, Control: 4.2±0.1;p=0.02). There was a significant increase in IL-1ß secretion post ATP-induced inflammasome activation in STEMI PBMCs (53.8±9.4;p<0.0001) but not control (9.8±0.9). P2X7 inhibition significantly attenuated this ATP-induced IL-1ß secretion in STEMI cells (13.9±2.4;p<0.0001) but had no effect in controls (7.5±2.8). Similarly, P2X7 inhibition resulted in approximately 60% reduction of ATP-induced caspase-1 activity in STEMI cells (p=0.01). Conclusions: The P2X7 antagonist compound SMW139 effectively inhibits the inflammasome, causing a significant reduction in caspase-1 activity and IL-1ß cytokine levels, and holds promise as a novel therapy for patients with atherosclerosis and ACS.