CMI-392 

5-lipoxygenese activity in cell lysate is determined as follows: 0.1 mL reactions consisting of buffer, test compound (CMI-392 in DMSO), and an amount of cell lysate that will convert 15% of [¹⁴C]AA substrate mix to oxygenated products are incubated (20 min, room temperature). A substrate mix containing [¹⁴C]AA is added and incubated further (5 min, 37°C). The reaction is terminated by adding 0.2 mL of an organic extraction solution containing triphenylphosphine, followed by microcentrifugation. The organic phase (50 μL) is spotted onto silica gel TLC plates. The plates are developed in ethyl ether/acetic acid (100:0.1) (25 min, room temperature). Plates are exposed to film for 36 h. The film is developed and scanned using a densitometer, and the peak areas of AA and its products are calculated^[1].

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

Mice: Acute TPA-induced ear edema in mice is determined by topically applying TPA to the ears of mice. Mice are sacrificed after 6 h and the ear punch biopsies are weighed. Chronic TPA-induced ear edema in mice is determined by topically applying TPA once a day every 2 days for a total of 10 days. CMI-392 is topically administered twice daily on the last 3 days of the experiment. Mice are then sacrificed and the ear punch biopsies are weighed. Biopsies are homogenized and MPO content is determined via spectrophotometric assay^[1].

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

• [1]. Cai X, et al. (+/-)-trans-2-[3-methoxy-4-(4-chlorophenylthioethoxy)-5-(N-methyl-N- hydroxyureidyl)methylphenyl]-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran (CMI-392), a potent dual 5-lipoxygenase inhibitor and platelet-activating factor receptor antagonist  [Content Brief]