sEH Inhibitors Research Articles

Expression of soluble epoxide hydrolase in renal tubular epithelial cells regulates macrophage infiltration and polarization in IgA nephropathy.

Tubulointerstitial inflammatory cell infiltration and activation contribute to kidney inflammation and fibrosis. Epoxyeicosatrienoic acids (EETs), which are rapidly metabolized to dihydroxyeicosatrienoic acids by the soluble epoxide hydrolase (sEH), have multiple biological functions, including vasodilation, anti-inflammatory action, and others. Inhibition of sEH has been demonstrated to attenuate inflammation in many renal disease models. However, the relationship between sEH expression and macrophage polarization in the kidney remains unknown. In this study, we investigated the relationships between the level of sEH and clinical and pathological parameters in IgA nephropathy. The level of sEH expression positively correlated with proteinuria and infiltration of macrophages. sEH-positive tubules were found to be surrounded by macrophages. Furthermore, we found that incubation of immortalized human proximal tubular HK-2 cells with total urinary protein and overexpression of sEH promoted inflammatory factor production, which was associated with M1 polarization. We also exposed RAW264.7 mouse leukemic monocytes/macrophages to different HK-2 cell culture media conditioned by incubation with various substances affecting sEH amount or activity. We found that the upregulation of sEH promoted M1 polarization. However, pharmacological inhibition of sEH and supplementation with EETs reversed the conditioning effects of urinary proteins by inhibiting M1 polarization through the NF-κB pathway and stimulating M2 polarization through the phosphatidylinositol 3-kinase pathway. These data suggest that inhibition of sEH could be a new strategy to prevent the progression of inflammation and to attenuate renal tubulointerstitial fibrosis.

Lipidomic profiling reveals soluble epoxide hydrolase as a therapeutic target of obesity-induced colonic inflammation

Obesity is associated with enhanced colonic inflammation, which is a major risk factor for colorectal cancer. Considering the obesity epidemic in Western countries, it is important to identify novel therapeutic targets for obesity-induced colonic inflammation, to develop targeted strategies for prevention. Eicosanoids are endogenous lipid signaling molecules involved in regulating inflammation and immune responses. Using an LC-MS/MS-based lipidomics approach, we find that obesity-induced colonic inflammation is associated with increased expression of soluble epoxide hydrolase (sEH) and its eicosanoid metabolites, termed fatty acid diols, in colon tissue. Furthermore, we find that pharmacological inhibition or genetic ablation of sEH reduces colonic concentrations of fatty acid diols, attenuates obesity-induced colonic inflammation, and decreases obesity-induced activation of Wnt signaling in mice. Together, these results support that sEH could be a novel therapeutic target for obesity-induced colonic inflammation and associated diseases.

An inhibitor of soluble epoxide hydrolase ameliorates diabetes-induced learning and memory impairment in rats

BackgroundPharmacological inhibition of soluble epoxide hydrolase (sEH) enhances the synaptic function in the CNS and has a protective role in cognitive decline. We hypothesized that the sEH inhibitor TPPU might prevent the diabetes-induced decline in learning and memory which is associated with an alteration in the level of neurotransmitters and oxidative stress. MethodsType 1 diabetes was induced in rats and the animals were treated with TPPU for 8 weeks. The learning and memory functions were assessed by the Barnes maze and a step-down test. Indicators of oxidative stress, levels of neurotransmitters, and activity of acetylcholinesterase were measured in the discrete regions of the brain. ResultsOur results revealed that treatment with TPPU significantly improves learning and memory performance in diabetic rats along with decreasing the level of blood sugar. Moreover, treatment with TPPU significantly prevented the diabetes-induced alteration in levels of neurotransmitters, the activity of acetylcholinesterase and preserved anti-oxidant defence system. ConclusionInhibition of the sEH alleviates diabetes-induced decline in learning and memory.

The oxidized linoleic acid metabolite 12,13-DiHOME mediates thermal hyperalgesia during inflammatory pain

Eicosanoids play a crucial role in inflammatory pain. However, there is very little knowledge about the contribution of oxidized linoleic acid metabolites in inflammatory pain and peripheral sensitization. Here, we identify 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME), a cytochrome P450-derived linoleic acid metabolite, as crucial mediator of thermal hyperalgesia during inflammatory pain. We found 12,13-DiHOME in increased concentrations in peripheral nervous tissue during acute zymosan- and complete Freund's Adjuvant-induced inflammatory pain. 12,13-DiHOME causes calcium transients in sensory neurons and sensitizes the transient receptor potential vanilloid 1 (TRPV1)-mediated intracellular calcium increases via protein kinase C, subsequently leading to enhanced TRPV1-dependent CGRP-release from sensory neurons. Peripheral injection of 12,13-DiHOME in vivo causes TRPV1-dependent thermal pain hypersensitivity. Finally, application of the soluble epoxide hydrolase (sEH)-inhibitor TPPU reduces 12,13-DiHOME concentrations in nervous tissue and reduces zymosan- and CFA-induced thermal hyperalgesia in vivo. In conclusion, we identify a novel role for the lipid mediator 12,13-DiHOME in mediating thermal hyperalgesia during inflammatory pain and propose a novel mechanism that may explain the antihyperalgesic effects of sEH inhibitors in vivo.

Inhibition of Soluble Epoxide Hydrolase 2 Ameliorates Diabetic Keratopathy and Impaired Wound Healing in Mouse Corneas.

EPHX2 (encoding soluble epoxide hydrolase [sEH]) converts biologically active epoxyeicosatrienoic acids (EETs), anti-inflammatory and profibrinolytic effectors, into the less biologically active metabolites, dihydroxyeicostrienoic acids. We sought to characterize the expression and the function of EPHX2 in diabetic corneas and during wound healing. The expression of EPHX2 at both mRNA and protein levels, as well as sEH enzymatic activity, was markedly upregulated in the tissues/cells, including corneal epithelial cells as well as the retina of human type 2 and mouse type 1 (streptozotocin [STZ] induced) and/or type 2 diabetes. Ephx2 depletion had no detectable effects on STZ-induced hyperglycemia but prevented the development of tear deficiency. Ephx2-/- mice showed an acceleration of hyperglycemia-delayed epithelium wound healing. Moreover, inhibition of sEH increased the rate of epithelium wound closure and restored hyperglycemia-suppressed STAT3 activation and heme oxygenase-1 (HO-1) expression in the diabetic corneas. Treatment of diabetic corneas with cobalt protoporphyrin, a well-known HO-1 inducer, restored wound-induced HO-1 upregulation and accelerated delayed wound healing. Finally, Ephx2 depletion enhanced sensory innervation and regeneration in diabetic corneas at 1 month after epithelial debridement. Our data suggest that increased sEH activity may be a contributing factor for diabetic corneal complications; targeting sEH pharmacologically or supplementing EETs may represent a new, adjunctive therapy for treating diabetic keratopathy.

Suppression of Chemotherapy‐induced Cytokine/Eicosanoid Storm and Ovarian Tumor Growth by a Dual COX‐2/sEH Inhibitor

While chemotherapy remains a mainstay in cancer treatment, growing evidence indicates that it may stimulate tumor growth. Other cancer therapies including radiation, immunotherapy, and stem cell transplantation may also trigger the release of pro‐inflammatory and pro‐tumorigenic cytokines in the tumor stroma. We recently demonstrated that although cancer therapy reduces tumor burden by killing tumor cells, the resulting dead cells, or ‘debris', can promote tumor growth by stimulating the release of cytokines. Chemotherapy fails to generate remission in over 70% of patients with ovarian cancer, is rarely curative, and simultaneously creates tumor cell debris. Thus, cancer therapy is inherently a double‐edged sword and targeting a single cytokine or pathway may not prevent therapy‐induced cancer. Soluble epoxide hydrolase (sEH) inhibitors have been shown to stimulate the resolution of inflammation by promoting the synthesis of pro‐resolving mediators and counter‐regulating pro‐inflammatory cytokines. In this study, tumor cell debris was prepared in vitro by treating either murine or human ovarian tumor cells with first‐line platinum or taxane‐based cytotoxic chemotherapies used for treating ovarian cancer (e.g. cisplatin, carboplatin, or paclitaxel). Chemotherapy‐generated tumor cell debris stimulated ovarian tumor growth in both immunocompetent and immunocompromised hosts. Debris triggered macrophage production of a series of pro‐inflammatory and pro‐angiogenic cytokines. Further, LC‐MS‐MS–based oxylipin profiling of tumor cell debris generated by chemotherapy revealed a pathological release of tumor‐promoting bioactive lipids (‘eicosanoid storm’) including cyclooxygenase (COX)‐derived prostaglandins, lipoxygenase‐derived HETEs, CYP450‐derived DiHOMEs and EpOMEs. We hypothesize that dual COX‐2/sEH inhibition may be a novel modality in suppressing ovarian tumor growth by stimulating the natural debris‐clearing process. A dual COX‐2/sEH inhibitor, PTUPB, suppressed debris‐induced cytokines (e.g. TNFα, CCL2, CCL4, CXCL2, G‐CSF, ICAM‐1, MMP‐9, and PAI‐1) and eicosanoids (e.g. PGF2a, PGD2, and PGJ2). PTUPB also delayed the onset of debris‐stimulated tumor growth in an ovarian cancer (ID8) model, achieving sustained survival over 80 days post‐injection. It is imperative to overcome the predicament between killing tumor cells and the inherent tumor‐promoting activity of the debris. Thus, dual inhibition of COX‐2/sEH may be a novel approach in cancer therapy to suppress the therapy‐induced cytokine/eicosanoid storm and debris‐stimulated tumor growth.Support or Funding InformationNational Cancer Institute grant RO1 01CA170549‐02; ROCA148633‐01A4; NIEHS Superfund Research Program RO1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Potent Soluble Epoxide Hydrolase Inhibitors for Veterinarian Usage

The veterinary pharmacopeia to treat pain and inflammation in companion animals is very limited. Currently, cyclooxygenase inhibitors (both NSAIDs and COXIBs) are the main class of drugs available to reduce pain and inflammation in cats, dogs and horses. However, these drugs often have insufficient efficacy and a great number of known adverse effects, underlying the need for novel pain medication for companion animals. Soluble epoxide hydrolase (sEH) inhibitors are a new class of anti‐inflammatory and analgesic drugs being tested in humans. They block the hydrolysis, and thus increase endogenous concentrations, of signaling molecules called epoxy‐fatty acids (EpFAs) that modulate inflammation and nociception. In addition to their use in human, sEH inhibitors may be a suitable alternative to cyclooxygenase inhibitors in companion animals. However, the compounds optimized for usage in primates and rodents are typically not sufficiently potent inhibitors of other animals' sEH for in vivo usage. Here, we screened a library of 2,300 inhibitors developed for the human enzyme with partially purified feline, canine and equine hepatic sEH to identify inhibitors that are broadly potent among species. Six candidate sEH inhibitors that are very potent across species (IC50 < 1nM for each enzyme tested) were identified. Their microsomal stability was then measured in hepatic extracts from cat, dog and horse, as well as their solubility in drug‐able solvents. Based on this work, trans‐4‐{4‐[3‐(4‐trifluoromethoxy‐phenyl)‐ureido]‐cyclohexyloxy}‐benzoic acid was selected for further testing in veterinary clinical trials of pain and inflammation in companion animals.Support or Funding InformationThis work was supported by the National Institute of Environmental Health Sciences grant numbers ES002710 and ES004699.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of pancreatic carcinoma growth through enhancing epoxide metabolic profile of ω‐3 polyunsaturated fatty acid by inhibiting soluble epoxide hydrolase

Anti‐inflammatory and anti‐carcinogenic effects of ω‐3 polyunsaturated fatty acid (PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), etc., are well known; but the mechanism/s remains unclear. Of the three metabolic pathways {Cyclooxygenase (COX), Lipooxygenase (LOX) and Cyto‐p450 (CYP)}, ω‐3 PUFAs are predominantly metabolized by CYP epoxygenase/s, leading to an accumulation of ω‐3 epoxy fatty acid (ω‐3 epoxides); and ω‐3 PUFAs are poor substrates of COX and LOX. However, under physiologic conditions, these ω‐3 epoxides are quickly inactivated by soluble epoxide hydrolase (sEH) to the diol products, and a sEH inhibitor appears crucial to stabilizing/enhancing these ω‐3 epoxides actions. In this study, using a transplantable murine PK03 pancreatic carcinoma model which carried mutant Kras gene, we have performed three animal experiments using Fat‐1 transgenic, sEH (−/−), and C57BL/6J mice subcutaneously implanted with a murine PK03 pancreatic carcinoma cells treated with or without sEH inhibitor and fed with either AIN‐76A or AIN‐76A diet supplemented with ω‐3 PUFAs. Aims of this study are to determine 1) if sEH gene knockout or inhibition stabilize the epoxide metabolites of ω‐3/ω‐6 PUFAs, 2) if sEH gene knockout or inhibition has more potent effects on inhibiting pancreatic cancer growth via enhancing the ω‐3 epoxide metabolites, and 3) potential molecular targets on mutant Kras mediated signaling, angiogenesis and inflammation. The significant findings were summarized as follows: in Fat‐1 mice treated with sEH inhibitor t‐AUCB or sEH (−/−) mice fed with AIN‐76A diet supplemented with ω‐3 PUFAs or C57BL/6J mice treated with sEH inhibitor and AIN‐76A diet supplemented with ω‐3 PUFAs, compared to fat‐1 mice without t‐AUCB treatment or sEH(−/−) mice fed with AIN‐76A diet, or C57BL/6J mice fed AIN‐76A diet, i) the size and tumor volume of xenograft PK03 pancreatic carcinoma was significantly inhibited ii) more glandular formation/well differentiation in the treated PK03 pancreatic carcinoma was observed by histopathologic analysis, iii) significant less inflammatory cell infiltrates and cell proliferation labeled immunohistochemically by anti‐myeloperoxidase and ki‐67 antibodies, as well as significant increased caspase‐3 labeled apoptosis and significant reduced intratumoral angiogenesis labeled by CD34 antibody were observed in the treated PK03 pancreatic carcinoma; iv) Western blot analysis showed a significant inhibition of mutant Kras‐activated signals including phosphorylated cRaf, MEK and ERK. Eicosanoic acid metabolic profiling analyzed by LC/MSMS method revealed a significant increase of the ratios of Epoxy to DiHDPE for DHA and EPA, and Epoxy/DiHOME for ARA and LA, as well as a significant increase of epoxy metabolites of DHA, EPA, ARA and LA. These results indicate that ω‐3 PUFAs supplementation together with sEH inhibition plays an important role in inhibiting pancreatic carcinogenesis mainly via enhancing the epoxy metabolites, and are highly potential approach for preventing pancreatic cancer.Support or Funding InformationSupported by NIH R01 DK107767 to Dr. Guang‐Yu Yang.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of sEH prevents high fructose‐induced impairement of myocardial oxygen comsumption in young rats

High fructose diet increases tissue oxidative stress and has been shown to be detrimental to cardiac function. Epoxyeicosainoic acids (EETs) are metabolites of arachidonic acid, which possess cardioprotective properties and are metabolized by soluble epoxide hydrolase (sEH). Therefore, inhibition of sEH is proven to be beneficial to cardiovascular function. The specific impact of a high fructose diet and the role of EETs/sEH in the regulation of cardiac function in the adolescent population have not been studied. To test the hypothesis that sEH inhibition attenuates the adverse action of fructose consumption on the heart of young rats, 4 week old rats were fed a normal diet (as control; ND) or a high fructose diet (HFD) for 30 days, without or with concomitant administration of TPPU, a specific sEH inhibitor, by oral‐gavage (1mg/kg body weight: HFD‐TPPU).MethodsCardiac output was analyzed by echocardiography and blood pressure was recorded using tail‐cuff measurements. Myocardial oxygen consumption (MV̇o2) was measured in freshly isolated left ventricular tissue from all groups of rats under control conditions and following incubation of tissue with the nitric oxide (NO) synthase inhibitor Nω‐nitro‐l‐arginine methyl ester (L‐NAME; 10−4M), or NADPH oxidase inhibitor (Nox), apocynin (10−4M) for 1 hour. Cytosolic and mitochondrial superoxide levels were determined by HPLC‐fluorescence detection via incubation of myocardial tissues with dihydroethidium (DHE) or Mitosox. Nox 2 expression was measured via western blot analysis.ResultsFructose feeding significantly impaired MV̇o2 compared to hearts of ND rats, as indicated by the loss of NO‐mediated regulation of MV̇o2. Incubation of myocardial tissue isolated from the ND group with L‐NAME yielded the same pattern as fructose feeding, further confirming that the NO‐mediated response is compromised with high fructose consumption. The treatment of HFD rats with TPPU normalized their MV̇o2 to the level of ND rats. Furthermore, incubation of myocardial tissues isolated from HFD rats with apocynin elicited normalized MV̇o2, comparable to that observed in HFD‐TPPU rats. In addition, mitochondrial superoxide and Nox 2 expression were significantly increased in myocardial tissue of HFD rats compared to ND and HFD‐TPPU rats. Thus, we conclude that 1) high fructose‐induced increase in oxidative stress is responsible for impaired NO‐mediated regulation of MV̇o2. 2) Inhibition of sEH reversed the oxidative stress‐induced impairment of MV̇o2 via perhaps, increases in tissue levels of antioxidant EETs.Support or Funding InformationThis work was supported by HL070653 and HL129797This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pharmacological inhibition or genetic ablation of soluble epoxide hydrolase attenuates obesity‐induced nonalcoholic fatty liver disease

The prevalence of nonalcoholic fatty liver disease (NAFLD) in western countries is 20–30%; it is of critical importance to identify new therapeutic target, in order to reduce the risks of NAFLD and associated diseases. Here, we report that the expression of soluble epoxide hydrolase (sEH) is increased in liver tissues of a high‐fat diet‐induced NAFLD model; in addition, pharmacological inhibition or genetic ablation of sEH attenuates high‐fat diet‐induced NAFLD, suggesting that sEH could be a novel therapeutic target of NAFLD. We find that after 8‐week dietary feeding of high‐fat diet (60%cal), the expression of sEH is dramatically increased in liver. To explore the roles of sEH in obesity‐induced NAFLD, we test whether pharmacological inhibition or genetic ablation of sEH affects the development of NAFLD. We find that oral administration of TPPU and t‐TUCB, which are two different sEH inhibitors, attenuates high‐fat diet‐induced fat accumulation in liver (assess by liver triglyceride analysis, oil red o staining and H&E histology staining), and reduces expressions of pro‐inflammatory genes in liver (Mcp‐1 and Tnf‐α). Similar results are observed in sEH knockout mice. Regarding the mechanisms, we find that pharmacological inhibition or genetic ablation of sEH suppresses the expression of Srebf1, Ppar‐γ, Cd36 and Dgat2, which play critical roles in lipid synthesis; and increases the expression of Ppar‐α, regulating fatty acid β‐oxidation. Together, these results support that sEH could be a promising therapeutic target for NAFLD, suppressing fat accumulation and inflammation in liver. This could lead to rapid human translations, because the pharmacological inhibitors of sEH are being evaluated in human clinical trials targeting multiple disorders.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipidomic Profiling Reveals Soluble Epoxide Hydrolase As a Therapeutic Target of Obesity‐induced Colonic Inflammation

Obesity is associated with enhanced colonic inflammation, which is a major risk factor of colorectal cancer. Considering the obesity epidemic in United States, there is an urgent need to identify novel therapeutic targets for obesity‐induced colonic inflammation. Eicosanoids are endogenous lipid signaling molecules involved in regulating inflammation and immune responses. Using a LC‐MS/MS‐based lipidomics approach, we found that obesity‐induced colonic inflammation is associated with increased expression of soluble epoxide hydrolase (sEH) and its eicosanoid metabolites, termed fatty acid diols, in colon tissue. To study the functional role of sEH, we treated mice with sEH inhibitors, TPPU or t‐TUCB, in high fat diet (HFD)‐induce obesity model, and found that pharmacological inhibition of sEH attenuated obesity‐induced colonic inflammation. Administration of sEH inhibitors diminished obesity‐induced infiltration of immune cells and expressions of pro‐inflammatory cytokines in colon tissue. Furthermore, we examined the sEH−/− mice in obesity model, and found genetic ablation of sEH reduced colonic concentrations of fatty acid diols and decreased the expressions of pro‐inflammatory cytokines. Finally, HFD treatment increased expression of phosphorylated GSK3β in colon tissue, while such effect was attenuated in sEH−/− mice, or by treatment with sEH inhibitors, suggesting that pharmacological inhibition and genetic ablation of sEH attenuated HFD‐induced activation of Wnt signaling in colon. Together, these results support that sEH could be a novel therapeutic target for obesity‐induced colonic inflammation and associated diseases.Support or Funding InformationThis work was supported by USDA NIFA grants 2016‐67017‐24423 and 2014‐67017‐21762, NIEHS grant R01 ES002710 and NIEHS Superfund Research Program P42 ES004699, and National Natural Science Foundation of China (NSFC) grants 81702832, 81672938 and 81470588.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Soluble Epoxide Hydrolase/Phosphodiesterase Dual Inhibitor is Efficacious Against Inflammatory Pain

The soluble epoxide hydrolase (sEH) is the principal regulator of epoxy fatty‐acids (EpFA) transforming them in to the less active diols. Inhibitors of sEH block this activity and maintain levels of EpFA in vivo which has demonstrated potent analgesia in several preclinical models of acute and chronic pain. However, sEH inhibitors have no observable action in the absence of a pain state [1]. It was therefore hypothesized that an additional factor was present in the painful state that was required for the antinociceptive action of the EpFA [2]. Investigation began with cyclic adenosine monophosphate (cAMP) and experiments demonstrated this was active but also that increasing cAMP with phosphodiesterase inhibitors increased EpFA as well. Phosphodiesterase enzymes hydrolyze the cyclic nucleotide into adenosine monophosphate altering its secondary messenger capacity. This led to the design and synthesis of several dual inhibitors of sEH and phosphodiesterase.Multi‐target ligands are designed to improve safety and efficacy in treatment of diseases. The use of drug combinations cannot be assumed to be safe given drug‐drug interactions. Additionally, the prediction of pharmacodynamic and pharmacokinetic relationships is less complicated with a single entity. The multi‐target ligands are selected to improve therapeutic efficacy by hitting multiple targets in physiological disorders. Thus, increasing the EpFA by halting their degradation and at the same time potentiating their activity with an increase in cAMP was the goal with synthesis of the dual inhibitor. We present the biochemical characterization as well as the in vivo efficacy in an inflammatory pain model for the selected dual inhibitor of sEH and PDE enzymes. The IC50 of RBH61 for sEH determined on the sEH human recombinant enzyme is 2.1 ± 0.5 nM. This is paired with a IC50 in the PDE assay of 8.1 ± 0.05 nM in human embryonic kidney (HEK293) cells. Evaluation of the dual inhibitor in a model of inflammatory pain in rats demonstrates its antinociceptive efficacy.Support or Funding InformationThis work was supported, in part, by National Institutes of Health grants: NIEHS R01 ES002710 and NIEHS/Superfund Research Program P42 ES004699, NINDS U54 NS079202 and Grants NIEHS T32ES007059, NIH 5T32DC008072‐05 and 4T32HL086350‐09.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of a Novel Inhibitor of Soluble Epoxide Hydrolase and Role in Ocular Neovascularization

ObjectiveOcular neovascularization causes blindness in diseases such as proliferative diabetic retinopathy (PDR) and wet age‐related macular degeneration (AMD). Today, the effort to treat wet AMD is hampered by patients who are resistant and refractory to the current standard of care, anti‐vascular endothelial growth factor (VEGF) therapies. Therefore, identification of novel therapeutic targets and inhibitors is desirable to address the unmet needs in antiangiogenic treatment. Previously, we have identified soluble epoxide hydrolase (sEH) as a binding target of an antiangiogenic small molecule, SH‐11037. Here, we aimed to further investigate sEH expression in the eye and the inhibition of its activity by SH‐11037.MethodsThe expression and localization of sEH in retinal sections of laser‐induced choroidal neovascularization (L‐CNV) mice were evaluated by immunohistochemistry. The sEH enzymatic activity in ocular tissue homogenates of L‐CNV and control mice was assayed using trans‐stilbene oxide (t‐SO) as substrate. The sEH inhibition by SH‐11037 and mode of inhibition was assessed by recombinant sEH activity assay using a fluorogenic substrate, PHOME. Docking and molecular dynamics simulations were performed to predict the binding mode of SH‐11037 within the active site of sEH. In addition, intravitreal injections of sEH substrate 19,20‐epoxy docosapentaenoic acid (EDP) and product 19,20‐dihydroxy docosapentaenoic acids (DHDP) were given to L‐CNV mice, and effects were assessed by imaging and choroidal flatmount immunofluorescence.ResultssEH was upregulated in the rod photoreceptors in L‐CNV mice compared to control mice and it did not co‐localize with other retinal cell type markers. Correspondingly, sEH activity was increased in L‐CNV eyes and was normalized by SH‐11037 or a known sEH inhibitor. In a recombinant enzyme assay, SH‐11037 inhibited sEH activity in a dose‐dependent manner, with IC50 = 0.15 μM. Enzyme kinetics analysis demonstrated that increasing concentrations of SH‐11037 decreased Vmax and increased KM, revealing SH‐11037 as a mixed‐type inhibitor of sEH, with Ki = 1.73 ± 0.45 μM. Docking of SH‐11037 to sEH showed an energetically favorable binding mode with the ligand spanning the active site of the enzyme and interacting with key catalytic residues. Intravitreal 19,20‐EDP reduced CNV lesion volume compared to vehicle and 19,20‐DHDP, confirming antiangiogenic properties of this sEH substrate.ConclusionOur studies confirm the relevance of sEH for ocular neovascularization and reveal the mechanism for SH‐11037's inhibition of sEH. Further optimization of SH‐11037's structure could yield more potent, novel sEH inhibitors.Support or Funding InformationNIH/NEI R01EY025641, Retina Research Foundation, Research to Prevent Blindness, Inc.

In Vitro and In Vivo Metabolism of Soluble Epoxide Hydrolase Inhibitor 1‐ Trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl) Urea (TPPU)

Epoxy fatty acids (EpFAs) are highly potent lipid mediators, formed by cytochrome P450 (CYP450) from polyunsaturated fatty acids. In living organisms, they will be rapidly metabolized by soluble epoxide hydrolase (sEH) to these 1,2‐diol metabolites. A still growing body of evidence suggests that the stabilization of EpFAs by inhibition of sEH has effect of anti‐inflammatory, analgestic, antihypertension and so on. Therefore, sEH inhibitors of high potency are designed for targeting sEH for disease treatment. Among them, 1‐trifluoromethoxyphenyl‐3‐ (1‐propionylpiperidine‐4‐yl)urea (TPPU) of high potency (IC50 of HsEH, 0.6 nM) is the most promising drug. However, the drug metabolism of TPPU has not been studied. Nowadays, drug metabolism has become essential consideration for the safe and efficacious use of drug in human. Therefore, we in this study reported the comprehensive metabolic profiling of TPPU by in vitro and in vivo studies. A developed sensitive liquid chromatography–mass spectrometry (LC‐MS) method was used for identify all these phase I and phase II metabolites of TPPU. Our results indicated three major metabolic pathways of TPPU from phase I metabolism, and one from phase II metabolism.Support or Funding InformationNIEHS grant R01ES002710 and NIEHS Superfund Research Program P42 ES004699 (to B.D.H.), the West Coast Metabolomics Center at UC Davis (NIH/NIDDKU24DK097154) (to B.D.H.) and NIH grant R01 AG051618 (to J. O.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibitor of Soluble Epoxide Hydrolase Attenuates Decline in Learning and Memory of Diabetic Rats

Epoxyeicosatrienoic acids (EETs) are epoxide derivatives formed from arachidonic acid and are neuroprotectant. These bioactive lipids are degraded by soluble epoxide hydrolase (sEH). Pharmacological inhibition of sEH enhances the synaptic function in the central nervous system and has a protective role in the age‐related cognitive decline. We have evaluated the hypothesis that the sEH inhibitor TPPU might prevent a diabetes‐induced decline in learning and memory which is associated with alteration in the level of neurotransmitter and oxidative stress. Type 1 diabetes (streptozotocin 52 mg/kg, i.p.) was induced in rats and the animals were treated with TPPU (0.1 and 0.3 mg/kg body weight; p.o.) for 8 weeks. The learning and memory functions were assessed by Barnes maze and step down test. Level of oxidative stress indicators (reduced glutathione and malondialdehyde) and neurotransmitters (γ‐Aminobutyric acid, norepinephrine, and dopamine), and activity of acetylcholinesterase and were measured in the discrete regions of the brain. Our results revealed that treatment with TPPU which penetrates the blood‐brain barrier significantly improved learning and memory performance in diabetic rats with a parallel decrease in the level of blood sugar. Moreover, treatment with TPPU significantly minimized the diabetes‐induced alteration in levels of neurotransmitters and activity of acetylcholinesterase. In addition, TPPU protected anti‐oxidant defense system. Based on our experimental findings, it can be concluded that the inhibition of sEH is a viable strategy for retrieving diabetes‐induced decline in learning and memory.Support or Funding InformationThis study was partially funded by the National Institute of Environmental Health Sciences (NIEHS) [Grant R01 ES002710], NIEHS Superfund Research Program [Grant P42 ES004699], West Coast Central Comprehensive Metabolomics Center [Grant U24 DK097154] to BDH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Structural‐Activity Features of Long Chain Polyunsaturated Fatty Acid Epoxides in Angiogenesis

The cytochrome P450‐derived fatty acid epoxides are bioactive lipids with regulatory roles in a wide range of homeostatic and pathophysiological events. These lipid mediators are metabolized efficiently by the mammalian soluble epoxide hydrolase enzyme (sEH) to corresponding 1,2‐diols. Inhibition of sEH has been shown to be beneficial to human health via increasing concentrations of a variety of short‐lived epoxylipids in vivo (2). Identifying the most potent polyunsaturated fatty acid (PUFA) epoxide metabolites will provide insight on the effects of sEH inhibition. Recent studies have shown that the omega‐6 epoxyeicosatrienoic acids (EETs) from arachidonic acid are pro‐angiogenic, while the omega‐3, docosahexaenoic acid (DHA)‐derived epoxydocosapentaenoic acids (EDPs) inhibit angiogenesis, tumor growth, and metastasis (1). Given these findings, we are interested in exploring the roles of various long chain PUFA epoxides in angiogenesis. We tested a library of omega‐3 and omega‐6 epoxides in a newly developed high‐throughput tube formation assay and found their effects are regiospecific and stereospecific; Of note, the 19(S),20(R)‐EDP stereoisomer was a less effective inhibitor of endothelial cell tube formation than its respective racemic mixture. Here, we report the angiogenic activities of diverse omega‐3 and omega‐6 fatty acid epoxides that could be present in mammals from the diet, including the less explored epoxides from both omega‐3 and omega‐6 docosapentaenoic acid (DPA). We will discuss the structure‐activity features of the most potent epoxide species and also present methodology to synthesize and purify the regioisomers and stereoisomers of each series of epoxides. Ultimately, understanding activities of sEH substrates in specific regulatory environments will guide development of targeted sEH modulators that promote the beneficial effects of PUFA metabolites in human health and disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Soluble Epoxide Hydrolase Attenuates Food Allergen‐induced Eosinophil Recruitment and Gastrointestinal Inflammation

The prevalence of food allergies is on the rise, affecting nearly 4–6% of the population in the US and impacting their daily life in various ways. Although present in the normal gastrointestinal (GI) mucosa under homeostatic conditions, eosinophils are recruited to the intestinal mucosa in increased numbers in food allergy and contribute to inflammation. Soluble epoxide hydrolase (sEH) is known to play a pro‐inflammatory role during inflammation by metabolizing anti‐inflammatory epoxyeicosatrienoic acids (EETs) to pro‐inflammatory diols. In the current study, the role of sEH in a murine model of food allergy was investigated and the potential therapeutic effect of a highly selective sEH inhibitor (t‐TUCB) was evaluated. Oral exposure of mice on a soy‐free diet to soy protein isolate (SPI) induced expression of intestinal sEH, increased circulating total and antigen‐specific IgE levels and caused significant weight loss. Additionally, SPI induced GI inflammation characterized by increased recruitment of eosinophils and mast cells, elevated eotaxin‐1 levels, mucus hypersecretion and decreased epithelial junction protein expression. Administration of t‐TUCB to SPI‐challenged mice not only inhibited IgE levels and prevented SPI‐induced weight loss but also significantly decreased eosinophilia, mast cell recruitment and mucus secretion relative to untreated mice while SPI‐induced loss of junction protein expression was prevented to variable levels. Inflammatory mediators such as TNF‐α and eotaxin‐1 were found to induce sEH expression in eosinophils. Treatment of eosinophils with t‐TUCB significantly inhibited eosinophil migration, an effect which was mirrored by treatment with 11,12‐EET, by inhibiting intracellular signaling events such as ERK (1/2) activation and eotaxin‐1‐induced calcium flux. These studies suggest that sEH induced by soy proteins promotes allergic responses including eosinophilic GI inflammation and that inhibition of sEH can attenuate these allergen‐induced responses.Support or Funding InformationSupport was provided in part by National Institute of Environmental Health Sciences and National Institute of Diabetes and Digestive and Kidney Diseases to B.D.H. and by the University of Minnesota College of Veterinary Medicine Research Office, Minnesota Agricultural Experiment Station, and General Ag Research Funds to R.J.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of soluble epoxide hydrolase attenuates the kidney injury caused by ischemia/reperfusion in a murine model of acute kidney injury involved in GSK‐3β phosphorylation

Acute kidney injury (AKI) causes severe morbidity and mortality for which new therapeutic strategies are needed. Inhibition of soluble epoxide hydrolase (sEH) has been reported previously to attenuate the kidney injury in cisplatin‐caused AKI. However, the effect of sEH inhibition on ischemia/reperfusion (I/R)‐caused‐AKI remains unknown. In addition, the mechanism underlying the benefit of sEH inhibition to AKI has not been well understood. Here we report that epoxyeicosatrienoic acids (EETs) are the major mediators responsible for the beneficial effect of sEH inhibition in I/R‐caused AKI. Inhibition of sEH resulted in a significant increase in plasma level of 14(15)‐EET. Administration of both 14(15)‐EET alone and TPPU (a potent sEH inhibitor) significantly prolonged the survival of AKI mice. Co‐administration of 14(15)‐EET with TPPU enhanced such effect in survival. Administration of 14(15)‐EET with or without TPPU to AKI mice significantly attenuated the kidney injury, which was supported by the histological analysis of renal tissue, plasma levels of creatinine and urea nitrogen, and renal levels of NGAL. Furthermore, 14(15)‐EET significantly reversed the I/R‐caused reduction in glycogen synthase kinase 3β (GSK3β) phosphorylation in murine kidney, dose‐dependently inhibited the hypoxia/reoxygenation (H/R)‐caused apoptosis of murine renal tubular epithelial cells (mRTECs), and reversed the H/R‐caused reduction in GSK3β phosphorylation in mRTECs. Therefore, inhibition of sEH attenuates kidney injury via, at least partially, 14(15)‐EET modulating the apoptosis of RTECs and GSK3β phosphorylation in mRTECs. This study provides AKI patients with promising therapeutic strategies.Support or Funding InformationThis study was supported in part by NSFC grants 81470588 and 81100090, as well as NIEHS Grant (R01 ES02710), NIEHS Superfund Grant (P42 ES04699), NIH/NHLBI grant (R01 HL59699‐06A1), and a Translational Technology Grant from the UC Davis Medical Center. K.S.S.L. is supported by NIEHS Grant R00 ES024806.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Soluble Epoxide Hydrolase by t‐TUCB Ameliorated Liver Injury in a Chronic‐Binge Ethanol Administration Mouse Model

IntroductionCytochrome P450 epoxygenases are important enzymes involved in the conversion of polyunsaturated fatty acids (PUFAs) into epoxides, which have been shown to have beneficial effects in a number of inflammation‐related diseases. However, the in vivo half‐lives of these epoxides is short, due to the action of soluble epoxide hydrolase (sEH), an enzyme responsible for converting epoxides into their bioactive dihydroxy metabolites. sEH inhibitors, such as trans‐4‐{4‐[3‐(4‐trifluoromethoxyphenyl)‐ureido] cyclohexyloxy} benzoic acid (t‐TUCB), are being explored as a therapy for inflammation‐related diseases. Alcoholic liver disease (ALD) is one such disease, characterized by hepatic steatosis, inflammation and injury. Alcoholic hepatitis (AH) is a life threatening condition with a one‐year survival rate of less than 50%, highlighting the urgent need for improved therapies.HypothesisInhibition of sEH by t‐TUCB will attenuate chronic‐binge ethanol (EtOH)‐induced liver injury in a mouse model of AH.Materials and MethodsC57BL/6 male mice were fed all liquid diets containing 5% ethanol (n=13), 5% EtOH plus 5 μg/ml t‐TUCB (n=15), or pair‐fed with isocaloric maltodextrin (n=15) for 10 days, followed by a single oral EtOH gavage (5 g/kg). Mice were euthanized 9h later and blood and liver tissue were collected. Plasma alanine aminotransferase (ALT) levels were measured as a marker of liver injury. Formalin‐fixed, paraffin‐embedded liver sections were prepared and stained with haemotoxylin and eosin, terminal deoxynucelotidyl transferase dUTP nick end labeling (TUNEL), and chloroacetate esterase (CAE) for histological evaluation of liver damage, hepatic apoptosis and neutrophil accumulation, respectively. Liver RNA was isolated for determination of gene expression changes related to pro‐inflammatory and ER stress pathways.ResultsCompared to control animals, chronic‐binge EtOH administration resulted in liver injury, which was significantly attenuated by inhibition of sEH. Plasma ALT levels were significantly lower in the EtOH+t‐TUCB group compared to the EtOH‐administered group (102+27 vs 355+114, U/L, respectively, p < 0.05). In addition, a significant decrease in hepatocyte apoptosis (TUNEL‐positive cells) was observed in the EtOH+t‐TUCB group compared to EtOH group. Hepatic steatosis and inflammation (CAE‐positive cells) were similar in EtOH and EtOH+t‐TUCB treated animals. t‐TUCB treatment did not affect expression of genes related to ER stress (Chop and Xbp1) or inflammation (Pai1), suggesting that other ER stress or inflammation‐related genes or other pathways are necessary for the beneficial effect of sEH inhibition in ALD.ConclusionsThese data demonstrate the ability of sEH inhibition to attenuate chronic‐binge‐EtOH induced liver injury (as shown by decreased plasma ALT activity) in a mouse model mimicking human AH. The molecular mechanisms underlying this effect need further investigation.Support or Funding InformationSupported by grants from NIH and Veterans AdministrationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Key Role of Soluble Epoxide Hydrolase in the Pathogenesis of Parkinson's Disease

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder. Although there are many medications available to treat the symptoms in PD patients, these medications cannot reverse the progress of the disease. The soluble epoxide hydrolase (sEH) plays a key role in the inflammation which is involved in the pathogenesis of PD. The present study was undertaken to examine whether sEH plays a role in the pathogenesis of PD. MPTP (1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine)‐induced dopaminergic neurotoxicity in the striatum and substance nigra (SNr) of mice could be attenuated by subsequent repeated administration of TPPU, a potent sEH inhibitor. Furthermore, deletion of sEH gene protected against MPTP‐induced neurotoxicity in the striatum and SNr. Moreover, the treatment with TPPU or deletion of sEH gene attenuated and protected the MPTP‐induced ER stress and oxidative stress in the striatum. In addition, expression of sEH protein in the striatum from MPTP‐treated mice and patients with dementia of Lewy body (DLB) was higher than controls. Overexpression of sEH after AAV injection into the striatum significantly enhanced MPTP‐induced neurotoxicity in this region. Interestingly, expression of sEH mRNA in the neuron from iPS cells from PD patients was higher than that of healthy control subjects, and treatment with TPPU could protect against neurotoxicity in the neurons from iPS cells of PD patients. The present results suggest that sEH plays a key role in the pathogenesis of PD, and that epoxy fatty acids, their mimics as well as sEH inhibitors could be potential therapeutic or prophylactic drugs for PD.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.