Ofirnoflast suppresses heme-induced inflammation, representing a novel therapeutic strategy for hemolytic anemias

Abstract

Abstract Background: Hemolytic anemias, such as sickle cell disease, are characterized by accelerated red blood cell destruction, which results in the release of cell-free heme. This free heme acts as a damage-associated molecular pattern, triggering activation of the NLRP3 inflammasome in immune and endothelial cells. Activation of the inflammasome leads to caspase-1-mediated cleavage of pro-inflammatory cytokines, vascular inflammation, and pyroptotic cell death. Inhibition of the NLRP3 inflammasome has been shown to mitigate inflammation and cell death in preclinical models of hemolytic anemia. Ofirnoflast (HT-6184) is a novel, orally bioavailable small molecule that selectively inhibits NLRP3 inflammasome activation by allosteric modulation of Nek7, a critical scaffolding protein for inflammasome assembly. We investigated the efficacy of Ofirnoflast in a human monocytic cell model of heme-induced NLRP3 activation. Objective: To assess the efficacy of Ofirnoflast in suppressing heme-driven NLRP3 inflammasome activation and pyroptosis in human THP-1 cells, supporting its therapeutic utility in hemolytic anemias. Methods: THP-1 monocytes were differentiated into macrophage-like cells using PMA (100,000 cells/well; 48h treatment). After PMA differentiation, cells were rested in RPMI with 10% FBS with no PMA. After resting, cells were switched to serum-free media to eliminate interference with heme activation. Cells were pre-treated with Ofirnoflast for 1 hour before LPS priming (250 ng/mL, 3h), followed by heme stimulation (50 µM, 1h). IL-1β secretion was quantified by ELISA. Intracellular cleaved IL-1β and ASC speck formation were evaluated by confocal microscopy. Results: Heme stimulation led to robust NLRP3 activation, evidenced by elevated IL-1β secretion, intracellular cleaved IL-1β, and ASC speck formation. Ofirnoflast pre-treatment resulted in a dose-dependent reduction of IL-1β secretion (IC₅₀ = 123 nM), achieving a maximal inhibition of 89.9% at 1 µM. Confocal imaging mirrored these findings, showing marked reductions in cleaved IL-1β and ASC speck formation, indicating effective blockade of inflammasome activity. Conclusion: Ofirnoflast potently inhibits heme-induced activation of the NLRP3 inflammasome in vitro, reducing both inflammatory cytokine release and pyroptotic markers. These findings support continued investigation of Ofirnoflast as a promising therapeutic approach in sickle cell disease and other hemolytic anemias characterized by NLRP3-mediated inflammation.