HC-030031 

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].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Rats^[2]
Male Sprague-Dawley rats (200-500 g) are used in all experiments. HC-030031 (100, 300 mg/kg) is used. For all experiments, HC-030031 is suspended in 0.5% Methylcellulose and the drug is dosed p.o. at a volume of 10 mL/kg. Naproxen (20 mg/kg) is dissolved in sterile water and dosed p.o. to serve as a positive comparator for the CFA experiment. Pregabalin (20 mg/kg) is dissolved in sterile water and dosed p.o. to serve as a positive comparator for the neuropathic pain experiment.
Mice^[3]
Adult male C57BL/6 mice (8-12 weeks old) are used. Mice are injected with a 30 µL emulsion of undiluted CFA into the medial left plantar hind paw. The vehicle control group is injected with 30 µL of sterile 0.9% saline solution. Two days after injection, at the peak of hypersensitivity, the magnitude of inflammation is measured at the midpoint of the hind paw using digital calipers (VWR). For one experiment, the membrane-impermeable sodium channel inhibitor lidocaine N-ethyl-bromide, also known as QX-314, (0.2% in saline; 30 µL) is injected with or without the TRPA1 agonist cinnamaldehyde (30 µM) into the left plantar hind paw 2 days post CFA injection. For another experiment, the TRPA1 antagonist HC-030031 (100 µg in 30 µL of 0.5% DMSO and 0.25% Tween-80 in PBS) is injected into the left plantar hind paw 2 days post CFA injection. Vehicle controls are injected with 30 µL 0.5% DMSO and 0.25% Tween-80 in PBS. All behavioral assays are completed between 1 and 4 hours following the QX-314, HC-030031 or vehicle injections.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. McNamara CR, et al. TRPA1 mediates formalin-induced pain. Proc Natl Acad Sci U S A. 2007 Aug 14;104(33):13525-30.  [Content Brief]

  [2]. Eid SR, et al. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity. Mol Pain. 2008 Oct 27;4:48.  [Content Brief]

  [3]. Lennertz RC, et al. TRPA1 mediates mechanical sensitization in nociceptors during inflammation. PLoS One. 2012;7(8):e43597.  [Content Brief]

  [4]. Miyake T, et al. Cold sensitivity of TRPA1 is unveiled by the prolyl hydroxylation blockade-induced sensitization to ROS. Nat Commun. 2016 Sep 15;7:12840.  [Content Brief]

  [5]. So K, et al. Hypoxia-induced sensitisation of TRPA1 in painful dysesthesia evoked by transient hindlimb ischemia/reperfusion in mice. Sci Rep. 2016 Mar 17;6:23261.  [Content Brief]