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Simultaneous inhibition of soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH) with a single small molecule represents a novel therapeutic approach in treating inflammatory pain, since both targets are involved in pain and inflammation processes. In this study using multi-target directed ligands methodology we designed and synthesized 7 quinolinyl-based dual sEH/FAAH inhibitors, using an optimized microwave-assisted Suzuki-Miyaura coupling reaction and tested their potency in human FAAH and human, rat, and mouse sEH inhibition assays. The structure-activity relationship study showed that quinolinyl moiety is well tolerated in the active sites of both enzymes, yielding several very potent dual sEH/FAAH inhibitors with the IC50 values in the low nanomolar range. The most potent dual inhibitor 4d was further evaluated in stability assay in human and rat plasma where it performed better than the standard Warfarin while in vivo study revealed that 1 mg/kg 4d can inhibit acute inflammatory pain in male rats to a similar degree as the traditional nonsteroidal anti-inflammatory drug ketoprofen (30 mg/kg) after intraperitoneal injection. ADMET prediction studies for this dual inhibitor show favorable pharmacokinetic properties which will guide the future in vivo evaluations.

Pain arising from trigeminal systems such as headache is common, debilitating, and current treatments (e.g., sumatriptan) are limited. New treatments that target novel mechanisms of action may be required to innovate both short- and long-term pain therapy. Fatty acid amide hydrolase and soluble epoxide hydrolase are two pain-related enzymes that regulate pain and inflammation via independent pathways. We have previously demonstrated that simultaneous inhibition of these enzymes using a novel dual inhibitor alleviates acute inflammatory pain in the hindpaw and does not depress wheel running in rats. Here, we expanded on these findings and performed structure-activity relationships of our lead compound, the 4-phenyl-thiazole-based dual inhibitor SW-17, to generate 18 analogs and tested them for their inhibition at both enzymes. Conversion of the sulfonamide group to a tertiary amine led to a general decrease in the potency for the sEH enzyme, while this change was well-tolerated at the FAAH enzyme yielding several strong inhibitors. Six selected inhibitors were evaluated in mouse and rat sEH inhibition assays and results showed a species difference, i.e. 4-phenyl-thiazole-based analogs are significantly less or not active in mouse sEH compared to human and rat enzymes. The most potent inhibitor, SW-17, was evaluated in a plasma stability assay in human and rat plasma and showed moderate stability. However, SW-17 did not alleviate orofacial inflammatory pain in female rats compared to the traditional anti-migraine agent sumatriptan. Although modification of 4-phenyl-thiazole-based dual inhibitor SW-17 changes potencies at both FAAH and sEH, these approaches may not produce antinociception against trigeminal pain. Key Words: polypharmacology, formalin, inflammation, enzyme inhibition, structure-activity relationship studies

Soluble epoxide hydrolase (sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding (88.6%) and better metabolic stability in vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0 g/kg. Inhibitor A20 also displayed robust analgesic effect in vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor (±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain.

Aflatoxin B1 (AFB1) contamination in food and feed leads to severe global health problems. Acting as the frontier immunological barrier, the intestinal mucosa is constantly challenged by exposure to foodborne toxins such as AFB1 via contaminated diets, but the detailed toxic mechanism and endogenous regulators of AFB1 toxicity are still unclear. Here, we showed that AFB1 disrupted intestinal immune function by suppressing macrophages, especially M2 macrophages, and antimicrobial peptide-secreting Paneth cells. Using an oxylipinomics approach, we identified that AFB1 immunotoxicity is associated with decreased epoxy fatty acids, notably epoxyeicosatrienoic acids, and increased soluble epoxide hydrolase (sEH) levels in the intestine. Furthermore, sEH deficiency or inhibition rescued the AFB1-compromised intestinal immunity by restoring M2 macrophages as well as Paneth cells and their-derived lysozyme and α-defensin-3 in mice. Altogether, our study demonstrates that AFB1 exposure impairs intestinal immunity, at least in part, in a sEH-mediated way. Moreover, the present study supports the potential application of pharmacological intervention by inhibiting the sEH enzyme in alleviating intestinal immunotoxicity and associated complications caused by AFB1 global contamination.

Background & Aims: Alcohol-associated liver disease (ALD) is a significant cause of liver-related morbidity and mortality worldwide and with limited therapies. Soluble epoxide hydrolase (sEH; Ephx2) is a largely cytosolic enzyme that is highly expressed in the liver and is implicated in hepatic function, but its role in ALD is mostly unexplored. Methods: To decipher the role of hepatic sEH in ALD, we generated mice with liver-specific sEH disruption (Alb-Cre; Ephx2fl/fl). Alb-Cre; Ephx2fl/fl and control (Ephx2fl/fl) mice were subjected to an ethanol challenge using the chronic plus binge model of ALD and hepatic injury, inflammation, and steatosis were evaluated under pair-fed and ethanol-fed states. In addition, we investigated the capacity of pharmacologic inhibition of sEH in the chronic plus binge mouse model. Results: We observed an increase of hepatic sEH in mice upon ethanol consumption, suggesting that dysregulated hepatic sEH expression might be involved in ALD. Alb-Cre; Ephx2fl/fl mice presented efficient deletion of hepatic sEH with corresponding attenuation in sEH activity and alteration in the lipid epoxide/diol ratio. Consistently, hepatic sEH deficiency ameliorated ethanol-induced hepatic injury, inflammation, and steatosis. In addition, targeted metabolomics identified lipid mediators that were impacted significantly by hepatic sEH deficiency. Moreover, hepatic sEH deficiency was associated with a significant attenuation of ethanol-induced hepatic endoplasmic reticulum and oxidative stress. Notably, pharmacologic inhibition of sEH recapitulated the effects of hepatic sEH deficiency and abrogated injury, inflammation, and steatosis caused by ethanol feeding. Conclusions: These findings elucidated a role for sEH in ALD and validated a pharmacologic inhibitor of this enzyme in a preclinical mouse model as a potential therapeutic approach.

The soluble epoxide hydrolase (sEH) is a potential target to treat cardiovascular, renal and neuronal diseases. A series of sEH inhibitors containing naturally occurring lipophilic groups (originating from camphor and fenchone) were developed. Inhibitory potency ranging from 0.7 nM to 6.47 μM was obtained. It was discovered that ureas derived from l-camphor were more active against sEH (2.3-fold average) than the corresponding analogues derived from d-camphor indicating enantiomeric preference of sEH. Ureas derived from fenchone possess lower activity against sEH (ca. 80-fold on average) than their camphor-derived analogs due to the specific structure of the lipophilic fragment and show less enantiomeric preference (1.75-fold on average). Moreover, fenchone-derived ureas show no consistency in enantiomeric preference. Endo/exo-form of compound L-3a derived from l-camphor is 4-fold more potent than the corresponding analogue prepared from d-camphor (IC50 = 0.7 nM vs 2.8 nM) making it the most promising sEH inhibitor among the tested series.

Epoxyeicosatrienoic acids (EETs) are formed from the metabolism of arachidonic acid by cytochrome P450s. EETs promote angiogenesis linked to tumor growth in various cancer models that is attenuated in vivo by cyclooxygenase 2 (COX-2) inhibitors. This study further defines a role for COX-2 in mediating endothelial EET metabolism promoting angiogenesis. Using human aortic endothelial cells (HAECs), we quantified 8,9-EET-induced tube formation and cell migration as indicators of angiogenic potential in the presence and absence of a COX-2 inducer [phorbol 12,13-dibutyrate (PDBu)]. The angiogenic response to 8,9-EET in the presence of PDBu was 3-fold that elicited by 8,9-EET stabilized with a soluble epoxide hydrolase inhibitor (t-TUCB). Contributing to this response was the COX-2 metabolite of 8,9-EET, the 11-hydroxy-8,9-EET (8,9,11-EHET), which exogenously enhanced angiogenic responses in HAECs at levels comparable to those elicited by vascular endothelial growth factor (VEGF). In contrast, the 15-hydroxy-8,9-EET isomer was also formed but inactive. The 8,9,11-EHET also promoted expression of the VEGF family of tyrosine kinase receptors. These results indicate that 8,9-EET-stimulated angiogenesis is enhanced by COX-2 metabolism in the endothelium through the formation of 8,9,11-EHET. This alternative pathway for the metabolism of 8,9-EET may be particularly important in regulating angiogenesis under circumstances in which COX-2 is induced, such as in cancer tumor growth and inflammation.

PUFAs are precursors to bioactive oxylipin metabolites that increase in the brain following CO2-induced hypercapnia/ischemia. It is not known whether the brain-dissection process and its duration also alter these metabolites. We applied CO2 with or without head-focused microwave fixation for 2 min to evaluate the effects of CO2-induced asphyxiation, dissection, and dissection time on brain oxylipin concentrations. Compared with head-focused microwave fixation (control), CO2 followed by microwave fixation prior to dissection increased oxylipins derived from lipoxygenase (LOX), 15-hydroxyprostaglandin dehydrogenase (PGDH), cytochrome P450 (CYP), and soluble epoxide hydrolase (sEH) enzymatic pathways. This effect was enhanced when the duration of postmortem ischemia was prolonged by 6.4 min prior to microwave fixation. Brains dissected from rats subjected to CO2 without microwave fixation showed greater increases in LOX, PGDH, CYP and sEH metabolites compared with all other groups, as well as increased cyclooxygenase metabolites. In nonmicrowave-irradiated brains, sEH metabolites and one CYP metabolite correlated positively and negatively with dissection time, respectively. This study presents new evidence that the dissection process and its duration increase brain oxylipin concentrations, and that this is preventable by microwave fixation. When microwave fixation is not available, lipidomic studies should account for dissection time to reduce these artifacts.

Lipoxygenase (LOX)-catalyzed oxidation of the essential fatty acid, linoleate, represents a vital step in construction of the mammalian epidermal permeability barrier. Analysis of epidermal lipids indicates that linoleate is converted to a trihydroxy derivative by hydrolysis of an epoxy-hydroxy precursor. We evaluated different epoxide hydrolase (EH) enzymes in the hydrolysis of skin-relevant fatty acid epoxides and compared the products to those of acid-catalyzed hydrolysis. In the absence of enzyme, exposure to pH 5 or pH 6 at 37°C for 30 min hydrolyzed fatty acid allylic epoxyalcohols to four trihydroxy products. By contrast, human soluble EH [sEH (EPHX2)] and human or murine epoxide hydrolase-3 [EH3 (EPHX3)] hydrolyzed cis or trans allylic epoxides to single diastereomers, identical to the major isomers detected in epidermis. Microsomal EH [mEH (EPHX1)] was inactive with these substrates. At low substrate concentrations (

There is an increased demand for comprehensive analysis of vitamin D metabolites. This is a major challenge, especially for 1α,25-dihydroxyvitamin D [1α,25(OH)2VitD], because it is biologically active at picomolar concentrations. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) was a revolutionary reagent in dramatically increasing sensitivity of all diene metabolites and allowing the routine analysis of the bioactive, but minor, vitamin D metabolites. A second generation of reagents used large fixed charge groups that increased sensitivity at the cost of a deterioration in chromatographic separation of the vitamin D derivatives. This precludes a survey of numerous vitamin D metabolites without redesigning the chromatographic system used. 2-Nitrosopyridine (PyrNO) demonstrates that one can improve ionization and gain higher sensitivity over PTAD. The resulting vitamin D derivatives facilitate high-resolution chromatographic separation of the major metabolites. Additionally, a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE) was developed to selectively extract 1α,25(OH)2VitD, while reducing 2- to 4-fold ion suppression compared with SPE alone. LLE-SPE followed by PyrNO derivatization and LC/MS/MS analysis is a promising new method for quantifying vitamin D metabolites in a smaller sample volume (100 µL of serum) than previously reported methods. The PyrNO derivatization method is based on the Diels-Alder reaction and thus is generally applicable to a variety diene analytes.

Epoxy FAs (EpFAs) are important lipid mediators that are mainly metabolized by soluble epoxide hydrolase (sEH). Thus, sEH inhibition is a promising therapeutic target to treat numerous ailments. Several sEH polymorphisms result in amino acid substitutions and alter enzyme activity. K55R and R287Q are associated with inflammatory, cardiovascular, and metabolic diseases. R287Q seems to affect sEH activity through reducing formation of a catalytically active dimer. Thus, understanding how these SNPs affect the selectivity of sEH for substrates and inhibitors is of potential clinical importance. We investigated the selectivity of four sEH SNPs toward a series of EpFAs and inhibitors. We found that the SNPs alter the catalytic activity of the enzyme but do not alter the relative substrate and inhibitor selectivity. We also determined their dimer/monomer constants (KD/M). The WT sEH formed a very tight dimer, with a KD/M in the low picomolar range. Only R287Q resulted in a large change of the KD/M. However, human tissue concentrations of sEH suggest that it is always in its dimer form independently of the SNP. These results suggest that the different biologies associated with K55R and R287Q are not explained by alteration in dimer formation or substrate selectivity.

The metabolism of arachidonic acid (ARA) by cytochrome P450 ω/ω-1-hydroxylases leads to the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), which is an important lipid-signaling molecule involved in regulation of vascular tone, angiogenesis, and inflammation. Development of a simple method to prepare 20-HETE would greatly facilitate the investigation of its biological activities. The nonpathogenic yeast Starmerella bombicola has been shown to convert exogenously added arachidonic acid to 20-HETE via the biosynthetic pathway of sophorolipids; however, the yield was low. Here we demonstrate that genetic knockout of multifunctional enzyme type 2 (MFE-2), which is involved in the β-oxidation of fatty acids, significantly increases the yield of ARA conversion to 20-HETE and allows practical preparation of 20-HETE.

Monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis is a major event in inflammatory disease. Our prior studies have demonstrated that MCP-1-dependent chemotaxis requires release of arachidonic acid (AA) by activated cytosolic phospholipase A2 (cPLA2). Here we investigated the involvement of AA metabolites in chemotaxis. Neither cyclooxygenase nor lipoxygenase pathways were required, whereas pharmacologic inhibitors of both the cytochrome-P450 (CYP) and the soluble epoxide hydrolase (sEH) pathways blocked monocyte chemotaxis to MCP-1. To verify specificity, we demonstrated that the CYP and sEH products epoxyeiscosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs), respectively, restored chemotaxis in the presence of the inhibitors, indicating that sEH-derived products are essential for MCP-1-driven chemotaxis. Importantly, DHETs also rescued chemotaxis in cPLA2-deficient monocytes and monocytes with blocked Erk1/2 activity, because Erk controls cPLA2 activation. The in vitro findings regarding the involvement of CYP/sEH pathways were further validated in vivo using two complementary approaches measuring MCP-1-dependent chemotaxis in mice. These observations reveal the importance of sEH in MCP-1-regulated monocyte chemotaxis and may explain the observed therapeutic value of sEH inhibitors in treatment of inflammatory diseases, cardiovascular diseases, pain, and even carcinogenesis. Their effectiveness, often attributed to increasing EET levels, is probably influenced by the impairment of DHET formation and inhibition of chemotaxis.

Epoxide hydrolases are a small superfamily of enzymes important for the detoxification of chemically reactive xenobiotic epoxides and for the processing of endogenous epoxides that act as signaling molecules. Here, we report the identification of two human epoxide hydrolases: EH3 and EH4. They share 45% sequence identity, thus representing a new family of mammalian epoxide hydrolases. Quantitative RT-PCR from mouse tissue indicates strongest EH3 expression in lung, skin, and upper gastrointestinal tract. The recombinant enzyme shows a high turnover number with 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET), as well as 9,10-epoxyoctadec-11-enoic acid (leukotoxin). It is inhibited by a subclass of N,N’-disubstituted urea derivatives, including 12-(3-adamantan-1-yl-ureido)-dodecanoic acid, 1-cyclohexyl-3-dodecylurea, and 1-(1-acetylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea, compounds so far believed to be selective inhibitors of mammalian soluble epoxide hydrolase (sEH). Its sensitivity to this subset of sEH inhibitors may have implications on the pharmacologic profile of these compounds. This is particularly relevant because sEH is a potential drug target, and clinical trials are under way exploring the value of sEH inhibitors in the treatment of hypertension and diabetes type II.

Beneficial physiological effects of long-chain n-3 polyunsaturated fatty acids are widely accepted but the mechanism(s) by which these fatty acids act remains unclear. Herein, we report the presence, distribution, and regulation of the levels of n-3 epoxy-fatty acids by soluble epoxide hydrolase (sEH) and a direct antinociceptive role of n-3 epoxy-fatty acids, specifically those originating from docosahexaenoic acid (DHA). The monoepoxides of the C18:1 to C22:6 fatty acids in both the n-6 and n-3 series were prepared and the individual regioisomers purified. The kinetic constants of the hydrolysis of the pure regioisomers by sEH were measured. Surprisingly, the best substrates are the mid-chain DHA epoxides. We also demonstrate that the DHA epoxides are present in considerable amounts in the rat central nervous system. Furthermore, using an animal model of pain associated with inflammation, we show that DHA epoxides, but neither the parent fatty acid nor the corresponding diols, selectively modulate nociceptive pathophysiology. Our findings support an important function of epoxy-fatty acids in the n-3 series in modulating nociceptive signaling. Consequently, the DHA and eicosapentaenoic acid epoxides may be responsible for some of the beneficial effects associated with dietary n-3 fatty acid intake.

We previously established that proteinuria alters the apolipoprotein content of lipoproteins. This study was conducted to establish whether proteinuria also alters the concentrations of oxidized lipids within lipoprotein density fractions. To this end, we induced passive Heymann nephritis in Sprague Dawley rats and measured an array of alkaline-stable oxylipids in VLDL, LDL, and HDL particles. Proteinuria increased the total oxylipid amounts in the HDL and VLDL fractions. More importantly, these levels were increased when expressed per unit lipoprotein protein, indicating that the oxidized lipid load per particle was increased. Epoxides and diols increased ∼2-fold in HDL and ∼5-fold in VLDL, whereas LDL showed ∼2-fold decreases. The hydroxyeicosatetraenoic acids and hydroxyoctadecadienoic acids (HODEs) increased >4-fold in HDL and >20-fold in VLDL, whereas LDL showed ∼2-fold decreases in the HODEs. Therefore, nephrotic syndrome alters the lipoprotein oxylipid composition independently of an increase in total lipoprotein levels. These proteinuria-induced changes may be associated with the cardiovascular risk of lipoprotein oxidation.

A method for the simultaneous quantification of urinary linoleic and arachidonic acid derived epoxides and diols, as well as the arachidonate omega hydroxylated product has been developed. The method employs negative mode electrospray ionization and HPLC with tandem mass spectroscopy for quantification. Odd chain length saturated epoxy and dihydroxy fatty acids are used as analytical surrogates resulting in linear calibrations (r 2 ⩾ 0.9995). Standard addition analyses showed that matrix effects do not prevent these surrogates from yielding reliable quantitative results. Using 4 ml urine aliquots at a final extract volume of 100 μl and injecting 10 μl, method detection limits and limits of quantification were ⩽0.5 and 1.5 nM, respectively. The sensitivity for dihydroxy lipids was from 3- to 10-fold greater than the corresponding epoxy fatty acid. Shot to shot run times of 31 min were achieved.Rodent and human urine analyses indicated the method sensitivity is sufficient for general research applications. In addition, diurnal fluctuations in linoleate and arachidonate derived metabolites were observed in human subjects.