Pharmacological Analysis of NLRP3 Inflammasome Inhibitor Sodium [(1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl][(1-methyl-1H-pyrazol-4-yl)({[(2S)-oxolan-2-yl]methyl})sulfamoyl]azanide in Cellular and Mouse Models of Inflammation Provides a Translational Framework.

Interleukin (IL)-1ß is an apex proinflammatory cytokine produced in response to tissue injury and infection. The output of IL-1ß from monocytes and macrophages is regulated not only by transcription and translation but also post-translationally. Release of the active cytokine requires activation of inflammasomes, which couple IL-1ß post-translational proteolysis with pyroptosis. Among inflammasome platforms, NOD-like receptor pyrin domain-containing protein 3 (NLRP3) is implicated in the pathogenesis of numerous human disorders in which disease-specific danger-associated molecular patterns (DAMPS) are positioned to drive its activation. As a promising therapeutic target, numerous candidate NLRP3-targeting therapeutics have been described and demonstrated to provide benefits in the context of animal disease models. While showing benefits, published preclinical studies have not explored dose-response relationships within the context of the models. Here, the preclinical pharmacology of a new chemical entity, [(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl][(1-methyl-1H-pyrazol-4-yl)({[(2S)-oxolan-2-yl]methyl})sulfamoyl]azanide (NT-0249), is detailed, establishing its potency and selectivity as an NLRP3 inhibitor. NT-0249 also is evaluated in two acute in vivo mouse challenge models where pharmacodynamic/pharmacokinetic relationships align well with in vitro blood potency assessments. The therapeutic utility of NT-0249 is established in a mouse model of cryopyrin-associated periodic syndrome (CAPS). In this model, mice express a human gain-of-function NLRP3 allele and develop chronic and progressive IL-1ß-dependent autoinflammatory disease. NT-0249 dose-dependently reduced multiple inflammatory biomarkers in this model. Significantly, NT-0249 decreased mature IL-1ß levels in tissue homogenates, confirming in vivo target engagement. Our findings highlight not only the pharmacological attributes of NT-0249 but also provide insight into the extent of target suppression that will be required to achieve clinical benefit.

A novel mutation of IL1RN in the deficiency of interleukin-1 receptor antagonist syndrome: description of two unrelated cases from Brazil.

OBJECTIVE: Monogenic autoinflammatory diseases are disorders of Mendelian inheritance that are characterized by mutations in genes that regulate innate immunity and whose typical features are systemic inflammation without high-titer autoantibodies or antigen-specific T cells. Skin and bone inflammation in the newborn period have been described in 3 of these autoinflammatory disorders: neonatal-onset multisystem inflammatory disease, Majeed syndrome, and deficiency of interleukin-1 (IL-1) receptor antagonist (DIRA) syndrome. This study was undertaken to present the characteristics of the DIRA syndrome in 2 cases from Brazil, and describe a novel mutation in IL1RN. METHODS: Two unrelated Brazilian patients were evaluated for the clinical signs and symptoms of these 3 disorders, and peripheral blood samples were assessed for mutations in NLRP3, LPIN2, and IL1RN by DNA resequencing analysis. A mutation in IL1RN that encodes a mutant protein was identified, and the expression and function of this mutant protein were compared to those of the wild-type protein. RESULTS: Both patients presented with pustular dermatitis resembling generalized pustular psoriasis, recurrent multifocal aseptic osteomyelitis, and elevation in the levels of acute-phase reactants, all of which are features most consistent with the DIRA syndrome. Chronic lung disease was observed in 1 of the patients, and jugular venous thrombosis was observed in the other patient. Both patients showed a partial response to corticosteroid therapy, and 1 patient experienced an initial improvement of dermatitis with the use of acitretin. Both patients were homozygous for a novel 15-bp (in-frame) deletion on the IL1RN gene. The mutated protein expressed in vitro had no affinity with the IL-1 receptor, and stimulation of the patients' cells with recombinant human IL-1α or IL-1ß led to oversecretion of proinflammatory cytokines, similar to the findings obtained in previously reported patients. CONCLUSION: The presence of the same homozygous novel mutation in IL1RN in 2 unrelated Brazilian patients suggests that this genetic variant may be a founder mutation that has been introduced in the Brazilian population.

Native IL-32 is released from intestinal epithelial cells via a non-classical secretory pathway as a membrane-associated protein.

Although IL-32 has been shown to be induced under various pathological conditions, a detailed understanding of native IL-32 intracellular distribution and mechanism of release from cells has not been reported. We examined the expression of IL-32 in the intestinal epithelial cell line HT-29 following TNFα and IFNγ co-stimulation. The subcellular localization of induced IL-32 was associated with the membrane of lipid droplet-like structures and vacuolar structures that co-localized with markers of endosomes and lysosomes. Prolonged co-stimulation resulted in cell death and appearance of IL-32 in the culture medium. IL-32 released from co-stimulated HT-29 cells was found in a detergent-sensitive particulate fraction, and in a step density gradient the IL-32 particulate was buoyant, suggesting association with a membrane-bound vesicle. Upon Triton X-114 partitioning, most of the IL-32 partitioned to the detergent phase, suggesting hydrophobic characteristics. When IL-32-containing vesicles were subjected to protease K treatment, a protease resistant ∼12kDa fragment was generated from ∼24kDa IL-32. We propose that under these conditions, native IL-32 is released via a non-classical secretory route perhaps involving multi-vesicular bodies and exosomes. Demonstration of membrane association for both intracellular and released IL-32 suggests this unique cytokine may have a complex biosynthetic pathway and mechanism of action.