P63 Therapeutic potential of CLM-022, an inhibitor of NLRP3 inflammasome-mediated pyroptosis, for acute and chronic inflammatory liver diseases

<h3></h3> Inflammation and hepatocyte cell death are common elements in the pathogenesis of most chronic liver diseases leading to cirrhosis and liver failure. Pyroptosis is a lytic form of inflammatory regulated cell death that plays a complex role in liver disease. When moderate, pyroptosis contributes to the clearance of damaged cells, but when excessive, intensifies inflammatory responses, contributing to organ dysfunctions and disease aggravation. Pyroptosis is regulated by inflammasomes which are intracellular multiprotein complexes expressed in both parenchymal and non-parenchymal cells of the liver. In response to danger signals such as PAMPs, inflammasomes activate caspase-1, release IL-1β and IL-18 (Ribeiro et Szabo 2022). The aim of this work was to characterize the mechanism of action of investigational drug CLM-022 and to evaluate its activity on inflammasome-driven inflammation and pyroptosis. The in vitro activity of CLM-022 was assessed in an inflammasome-induced model of LPS-primed and nigericin stimulated THP1 macrophages. NLRP3 complex assembly, cleaved-Gasdermin D (GSDMD), IL1-β release and pyroptotic cell death were assessed by western blot, HTRF and LDH release, respectively. Efficacy of CLM-022 on cytokine inhibition was assessed in LPS-stimulated human whole blood cells. The in vivo efficacy of a single administration of CLM-022 was evaluated in an acute liver injury and inflammation mouse model, triggered by galactosamine (GalN) and lipopolysaccharide (LPS) and in an endotoxemia rat model, induced by LPS injection. CLM-022 treatment blocks NLRP3 complex assembly, reflected by the loss of oligomers. CLM-022 strongly inhibits the NLRP3 inflammasome dependent pyroptotic cell death in THP1 human macrophages (inhibition of IL-1β production, LDH release and GSDMD cleavage). In a whole human blood assay, CLM-022 strongly inhibits IL-1β, IL-6 and TNF-α production when added concomitantly and after LPS stimulation, (IC50 ~135nM-121nM, 363nM-456nM, 283nM-380nM, in co and post treatment, respectively). In vivo, CLM-022 lowers circulating cytokines and improves hepatic functions (monitored with ASAT and ALAT) in the GalN/LPS mouse and in the LPS-induced endotoxemia rat models. The mechanism of action of CLM-022 involves the inhibition of NLRP3 complex assembly and downstream pyroptosis, inhibition of human blood cytokine release, reduction of circulating cytokines in vivo and improvement of hepatic function. These results demonstrate a therapeutic potential of investigational drug CLM-022 for the treatment of acute and chronic inflammatory late-stage liver diseases, including ACLF.