In a recent study published in Scientific Reports, researchers described the preclinical profile of JT002, a novel and orally bioavailable small molecule inhibitor of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome.
The NLRP3 inflammasome constitutes an intracellular complex of multiple proteins that promote caspase1 autocatalysis and the consequent maturation and production of the proinflammatory cytokines interleukin (IL)1 and IL18.
The pathophysiology of several autoimmune disorders and inflammatory diseases, including atherosclerosis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), gout, severe asthma, multiple sclerosis, non-alcoholic steatohepatitis (NASH), and Alzheimer's disease (AD), is linked to persistent NLRP3 activation.
About the study
In the present study, researchers discovered and characterized JT002, and reported its potential in managing autoimmune and inflammatory diseases.
The team investigated JT002 efficacy in vivo after oral dosing in a murine model of cryopyrin-associated periodic syndromes (CAPS) and two models of neutrophilic inflammation in the airways, in addition to the in vivo pharmacodynamic and pharmacokinetic profiles of the small molecule NLRP3 inhibitor. Male Sprague-Dawley rats and Beagle dogs were administered JT002 intravenously and orally for pharmacokinetic evaluation.
The potency and selectivity of JT002 in inhibiting NLRP3-mediated pathways were evaluated in various cell-based assays using lipopolysaccharide (LPS) as the primary activator and secondary activators such as cholesterol crystals (CHC), adenosine triphosphate (ATP), and monosodium urate (MSU) crystals and nigericin.
In addition, the team investigated the effect of JT002 on inhibiting the alternative pathway of NLRP3 activation using only LPS stimulation of human whole blood.
The potency of JT002 was evaluated in LPS-primed mouse bone-marrow-derived macrophages (mBMDMs) by determining interleukin (IL)-1β concentrations following stimulation with nigericin, ATP, MSU, or CHC crystals. Human IL-1α, TNF-α, IL-1β, and murine IL-1β levels were determined using fluorescence and enzyme-linked immunosorbent assays (ELISA).
Intracellular caspase-1 (Casp1) activity in nigericin- and LPS-activated human peripheral blood mononuclear cells (PBMCs) was evaluated. Next, the team investigated whether JT002 blocked ASC complex assembly by immunostaining and imaging analyses.
The effects of JT002 treatment on hepatic and systemic inflammation and liver fibrosis were evaluated in vivo using a murine Nlrp3 knock-in (KI) model of human Muckle-Wells syndrome (MWS). Pro-fibrotic and pro-inflammatory gene expression in the liver was assessed among JT002-treated animals.
The effects of two doses of JT002 were evaluated in ovalbumin (OVA)-challenged, OVA-sensitized, and iTreg-transferred cluster of differentiation 8-alpha (CD8α)−/− mice. JT002 was administered orally to the murine animals once every day on days 23-29 before airway hyperresponsiveness (AHR) and airway inflammation assessments on day 30.0 in a model of interleukin-17-based neutrophilic asthma.
Female C57BL6/J mice were used to investigate the effects of one- or two-dose orally administered JT002 in a model of ozone-induced neutrophilic inflammation in the airways. Ribonucleic acid (RNA) was isolated from the cells and subjected to a quantitative polymerase chain reaction (qPCR). In addition, Western blot tests and immunofluorescence analyses were performed.
JT002 selectively and potently inhibited pro-inflammatory cytokine generation and caspase 1-triggered pyroptosis using various stimuli.
Orally administered JT002 showed excellent target involvement in vivo at therapeutically relevant concentrations, prevented weight loss and improved fibrotic and inflammatory endpoints in the MWS model. The engagement of the target translated into the efficacy of the small molecule inhibitor in vivo.
Further, JT002 administration significantly lowered airway neutrophilia and AHR in the murine animals. In human whole blood, JT002 was three times more potent in inhibiting IL-1β produced via the alternative pathway as compared to the canonical pathway.
Daily 30 mg/kg JT002 administration to Nlrp3 A350V amino acid substitution-expressing mice attenuated inflammation and liver fibrosis but maintained total leukocyte counts and alanine transaminase (ALT) levels in the normal range.
JT002 was effective in acute as well as chronic models. The study findings indicated that NLRP3 activation was critical for ozone-induced inflammation in the airways and interleukin-17-induced neutrophilic asthma, underpinning that NLRP3 could be targeted with JT002 for therapeutic benefits in managing steroid-resistant and severe neutrophilic asthma.
Furthermore, oral 25 to 30 mg per kg of JT002 administered daily was effective in preventing the progression of disease in the MWS model and the two murine asthma models.
However, 25 mg/kg of orally administered JT002 did not result in complete NLRP3 inhibition, as indicated by the absence of interleukin-1β inhibition at 24 hours post-dosing.
In rats, the sodium salt of JT002 was readily absorbed after oral administration, showing 73 to 100% bioavailability, and attained a maximal serological concentration (Cmax) of 16.0 µg/mL within 30 minutes of one 5.0 mg dose. In dogs, a Cmax of 4 µg/mL was attained 60 minutes after one mg oral dose. JT002 significantly reduced serological total immunoglobulin E (IgE) and anti-OVA IgE titers.
Genes associated with fibrosis such as CTGF (connective tissue growth factor) and Acta2 (alpha-smooth muscle actin) were considerably downregulated in the hepatic tissues of JT002-treated animals. In addition, JT002 considerably downregulated the Pycard (ASC), myeloperoxidase (MPO), Casp1, and IL-1β genes.
The team observed considerable reductions in inflammation, collagen content, neutrophil recruitment, and alpha-smooth muscle actin (α-SMA) staining in JT002-treated mice.
Based on the study findings, JT002, a potent NLRP3-targeted molecule, has the potential to improve the treatment of autoimmune and inflammatory disorders and can aid in developing clinical candidates for NLRP3-related diseases.