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

口服给药（100 mg/kg）的HC-030031显著逆转慢性炎症或神经性疼痛大鼠模型中的机械超敏反应,而局部注射（100 μg）至发炎的小鼠后爪,减弱机械性而非热性超敏反应.

HC-030031可以快速阻断AITC或Formalin引起的内向和外向电流,也能抑制N-methylmaleimide和Electrophillic前列腺素引起的TRPA1激活。

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