The soluble epoxide hydrolase (sEH) plays a significant role in the biosynthesis of inf lammation mediators as well as xenobiotic transformations. Herein, we report the discovery of substituted ureas and carbamates as potent inhibitors of sEH. Some of these selective, competitive tightbinding inhibitors with nanomolar K i values interacted stoichiometrically with the homogenous recombinant murine and human sEHs. These inhibitors enhance cytotoxicity of transstilbene oxide, which is active as the epoxide, but reduce cytotoxicity of leukotoxin, which is activated by epoxide hydrolase to its toxic diol. They also reduce toxicity of leukotoxin in vivo in mice and prevent symptoms suggestive of acute respiratory distress syndrome. These potent inhibitors may be valuable tools for testing hypotheses of involvement of diol and epoxide lipids in chemical mediation in vitro or in vivo systems.Epoxide hydrolases (EH; E.C.3.3.2.3) catalyze the hydrolysis of epoxides or arene oxides to their corresponding diols by the addition of water (1). In mammals, the hepatic microsomal and soluble epoxide hydrolase forms are known to complement each other in detoxifying a wide array of mutagenic, toxic, and carcinogenic, xenobiotic epoxides (2, 3). Soluble EH (sEH) is also involved in the metabolism of arachidonic (4) and linoleic (5) acid epoxides. Arachidonate epoxides and diols are elevated in association with pregnancy-induced hypertension and modulate vascular permeability in the heart and kidneys (6). Diols derived from epoxy-linoleate (leukotoxin) perturb membrane permeability and calcium homeostasis (5), resulting in inflammation modulated by nitric oxide synthase and endothelin-1 (7, 8). Micromolar concentrations of leukotoxin reported in association with inflammation and hypoxia (9) depress mitochondrial respiration in vitro (10) and cause mammalian cardiopulmonary toxicity in vivo (7,11,12). Leukotoxin toxicity presents symptoms suggestive of multiple organ failure and acute respiratory distress syndrome (9). In both cellular and organismal models, leukotoxin-mediated toxicity depends on epoxide hydrolysis (5).The bioactivity of these epoxide hydrolysis products and their association with inflammation suggest that inhibition of vicinal-dihydroxylipid biosynthesis may have therapeutic value, making sEH a promising pharmacological target. Previously described selective sEH inhibitors, substituted chalcone oxides (as compound 1 in Table 1), and phenylglycidols (13,14) are epoxides that are hydrolyzed slowly by the target enzyme. Inhibition stems from an electronically stabilized covalent intermediate that results in low turnover and transient inhibition (15). Moreover, these compounds are relatively unstable, particularly in the presence of glutathione (13), making them of limited use in vivo. We describe herein the discovery of new potent and stable inhibitors of soluble EH and their application to both in vitro and in vivo models. MATERIALS AND METHODSSynthesis. Compounds 2, 3, 7-9, and 11-18 were obtained from Aldri...

Leukotoxin is a linoleic acic oxide produced by leukocytes and has been associated with the multiple organ failure and adult respiratory distress syndrome seen in some severe burn patients. Leukotoxin has been reported to be toxic when injected into animals intravenously. Herein, we report that this lipid is not directly cytotoxic in at least two in vitro systems. Using a baculovirus expression system we demonstrate that leukotoxin is only cytotoxic in the presence of epoxide hydrolases. In addition, it is the diol metabolite that proves toxic to pulmonary alveolar epithelial cells, suggesting a critical role for the diol in leukotoxin-associated respiratory disease. In vivo data also support the toxicity of leukotoxin diol. For the first time we demonstrate that soluble epoxide hydrolase can bioactivate epoxides to diols that are apparently cytotoxic. Thus leukotoxin should be regarded as a protoxin corresponding to the more toxic diol. This clearly has implications for designing new clinical interventions.

Soluble epoxide hydrolase (sEH) is suggested to alter the mode of action and increase the toxic potency of fatty acid epoxides. To characterize the structural features necessary for sEH-dependent epoxy fatty acid toxicity, 75 aliphatic compounds were assayed for cytotoxicity in the presence and absence of sEH. Three groups of aliphatic epoxide-diol pairs were described by their observed differential toxicity. Group I compounds were typified by terminal epoxides whose toxicity was reduced in the presence of sEH. Group II compounds were toxic in either their epoxide or diol form, but toxicity was unaffected by sEH. Group III compounds exhibited sEH-dependent toxicity and were therefore used to investigate the structural elements required for cytotoxicity in this study. The optimal structure for group III compounds appeared to be a fatty acid 18-20 atoms long (e.g., a carbon backbone plus a terminal heteroatom) with an epoxide positioned between C-7 and C-12. In the absence of sEH, replacement of epoxides with a vicinal diol was required for toxicity. While diol stereochemistry was unimportant, vicinal diol-induced toxicity exhibited fewer positional constraints to toxicity than sEH-dependent epoxide toxicity. Tested fatty acids and esters with neither an epoxide nor a vicinal diol were not toxic. These data support the hypothesis that long-chain epoxy fatty acid methyl esters are potential pro-toxins metabolized by sEH to more toxic diols. Furthermore, our results suggest that the endogenous compounds, leukotoxin methyl ester, 9,10(Z)-epoxyoctadec-12(Z)-enoic acid methyl ester, and isoleukotoxin methyl ester, 12, 13(Z)-epoxyoctadec-9(Z)-enoic acid methyl ester, are structurally optimized to elicit the observed effect.

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Closure of small- and medium-sized PDA with the Nit-Occlud PDA is effective and safe when compared with OPC.