HC-030031 (TOSLAB 829227) is a potent TRPA1 inhibitor, which antagonizes AITC- and formalin-evoked calcium influx with IC50s of 6.2±0.2 and 5.3±0.2 μM, respectively.

Ca2+ influx (formalin-evoked):5.3±0.2 μM, Ca2+ influx (AITC-evoked):6.2±0.2 μM

Oral administration of HC-030031 (100 mg/kg) significantly reversed mechanical hypersensitivity in rat models of chronic inflammation or neuropathic pain, while local injection (100 μg) into the inflamed hind paw of mice attenuated mechanical, but not thermal, hypersensitivity.

HC-030031 rapidly inhibits both inward and outward currents induced by AITC or Formalin, and also suppresses TRPA1 activation caused by N-methylmaleimide and electrophilic prostaglandins.

The activity of specific TLR agonists is assessed using the secretory embryonic alkaline phosphatase (SEAP) reporter gene that is linked to NF-κB activation in response to TLR stimulation. Measurement of SEAP activity using the Quanti-blue substrate (InvivoGen) after TLR agonist treatment is carried out.

HC-030031 is prepared in DMSO and stored, and then diluted with appropriate medium (DMSO 0.4%) before use[2]. HEK-293 cells stably expressing human TRPA1 are plated into 384-well plates at a density of 20,000 cells/well 24 hours prior to assaying. On the day of assay, cells are loaded with 4 μM Fluo-4 dye and 0.08% pluronic acid for 1 hour at room temperature in assay buffer consisting of Hank's balanced salt solution supplemented with 20 mM HEPES, 2.5 mM probenecid, and 4% TR-40. Calcium influx assays are performed using the Fluorometric Imaging Plate Reader (FLIPR) TETRA. Concentration-response curves are generated for the TRPA1 agonists cinnamaldehyde and AITC prior to antagonist testing so EC60 concentrations could be determined. Titrations of HC-030031 are made from a DMSO stock solution and DMSO is kept to a constant of 0.4% in the assay. The antagonist is incubated with the cells for 10 minutes before the addition of an EC60 concentration of either cinnamaldehyde (18 μM) or AITC (6 μM) and calcium influx is monitored for an additional 10 minutes[2].

Animal Models: Male Sprague-Dawley rats. Formulation: 0.5% Methylcellulose. Dosages: 100,300 mg/kg. Administration: oral administration