Pharmacological profile and efficiency in vivo of diflapolin, the first dual inhibitor of 5-lipoxygenase-activating protein and soluble epoxide hydrolase

Arachidonic acid (AA) is metabolized to diverse bioactive lipid mediators. Whereas the 5-lipoxygenase-activating protein (FLAP) facilitates AA conversion by 5-lipoxygenase (5-LOX) to pro-inflammatory leukotrienes (LTs), the soluble Epoxide Hydrolase (sEH) degrades anti-inflammatory epoxyeicosatrienoic acids (EETs). Accordingly, dual FLAP/sEH inhibition might be advantageous drugs for intervention of inflammation. We present the in vivo pharmacological profile and efficiency of N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N'-(3,4-dichlorophenyl)urea (diflapolin) that dually targets FLAP and sEH. Diflapolin inhibited 5-LOX product formation in intact human monocytes and neutrophils with IC₅₀ = 30 and 170 nM, respectively, and suppressed the activity of isolated sEH (IC₅₀ = 20 nM). Characteristic for FLAP inhibitors, diflapolin (I) failed to inhibit isolated 5-LOX, (II) blocked 5-LOX product formation in HEK cells only when 5-LOX/FLAP was co-expressed, (III) lost potency in intact cells when exogenous AA was supplied, and (IV) prevented 5-LOX/FLAP complex assembly in leukocytes. Diflapolin showed target specificity, as other enzymes related to AA metabolism (i.e., COX1/2, 12/15-LOX, LTA₄H, LTC₄S, mPGES₁, and cPLA₂) were not inhibited. In the zymosan-induced mouse peritonitis model, diflapolin impaired vascular permeability, inhibited cysteinyl-LTs and LTB₄ formation, and suppressed neutrophil infiltration. Diflapolin is a highly active dual FLAP/sEH inhibitor in vitro and in vivo with target specificity to treat inflammation-related diseases.