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Organophosphates (OPs), a class of phosphorus-containing chemicals that act by disrupting cholinergic transmission, include both toxic and fast-acting chemical warfare agents as well as less toxic but more easily accessible OP pesticides. The classical atropine/2-PAM antidote fails to protect against long-term symptoms following acute intoxication with OPs at levels that trigger status epilepticus. Acute OP intoxication also causes a robust neuroinflammatory response, which is implicated in the pathogenesis of long-term effects. In this study, we characterized the profiles of lipid mediators, important players in neuroinflammation, in the rat model of acute DFP intoxication. The profiles of lipid mediators were monitored in three different regions of the brain (cortex, hippocampus, and cerebellum) at 0, 1, 3, 7, 14, and 28 days post-exposure. The distribution pattern of lipid mediators was distinct in the three brain regions. In the cerebellum, the profile is dominated by LOX metabolites, while the lipid mediator profiles in cortex and hippocampus are dominated by COX metabolites followed by LOX and CYP 450 metabolites. Following acute DFP intoxication, most of the pro-inflammatory lipid mediators (e.g., PGD2 and PGE2) increased rapidly from day 1, while the concentrations of some anti-inflammatory lipid mediators (e.g. 14,15 EpETrE) decreased after DFP intoxication but recovered by day 14 post-exposure. The lipidomics results suggest two potential treatment targets: blocking the formation of prostaglandins by inhibiting COX and stabilizing the anti-inflammatory lipid mediators containing epoxides by inhibiting the enzyme soluble epoxide hydrolase (sEH).

BackgroundDiabetic nephropathy (DN) is the leading cause of renal failure, and podocyte dysfunction contributes to the pathogenesis of DN. Soluble epoxide hydrolase (sEH, encoded by Ephx2) is a conserved cytosolic enzyme whose inhibition has beneficial effects on renal function. The aim of this study is to investigate the contribution of sEH in podocytes to hyperglycemia-induced renal injury.Materials and methodsMice with podocyte-specific sEH disruption (pod-sEHKO) were generated, and alterations in kidney function were determined under normoglycemia, and high-fat diet (HFD)- and streptozotocin (STZ)-induced hyperglycemia.ResultssEH protein expression increased in murine kidneys under HFD- and STZ-induced hyperglycemia. sEH deficiency in podocytes preserved renal function and glucose control and mitigated hyperglycemia-induced renal injury. Also, podocyte sEH deficiency was associated with attenuated hyperglycemia-induced renal endoplasmic reticulum (ER) stress, inflammation and fibrosis, and enhanced autophagy. Moreover, these effects were recapitulated in immortalized murine podocytes treated with a selective sEH pharmacological inhibitor. Furthermore, pharmacological-induced elevation of ER stress or attenuation of autophagy in immortalized podocytes mitigated the protective effects of sEH inhibition.ConclusionsThese findings establish sEH in podocytes as a significant contributor to renal function under hyperglycemia.General significanceThese data suggest that sEH is a potential therapeutic target for podocytopathies.

The arachidonic acid cascade is arguably the most widely known biologic regulatory pathway. Decades after the seminal discoveries involving its cyclooxygenase and lipoxygenase branches, studies of this cascade remain an active area of research. The third and less widely known branch, the cytochrome P450 pathway leads to highly active oxygenated lipid mediators, epoxy fatty acids (EpFAs) and hydroxyeicosatetraenoic acids (HETEs), which are of similar potency to prostanoids and leukotrienes. Unlike the COX and LOX branches, no pharmaceuticals currently are marketed targeting the P450 branch. However, data support therapeutic benefits from modulating these regulatory lipid mediators. This is being approached by stabilizing or mimicking the EpFAs or even by altering the diet. These approaches lead to predominantly beneficial effects on a wide range of apparently unrelated states resulting in an enigma of how this small group of natural chemical mediators can have such diverse effects. EpFAs are degraded by soluble epoxide hydrolase (sEH) and stabilized by inhibiting this enzyme. In this review, we focus on interconnected aspects of reported mechanisms of action of EpFAs and inhibitors of soluble epoxide hydrolase (sEHI). The sEHI and EpFAs are commonly reported to maintain homeostasis under pathological conditions while remaining neutral under normal physiological conditions. Here we provide a conceptual framework for the unique and broad range of biological activities ascribed to epoxy fatty acids. We argue that their mechanism of action pivots on their ability to prevent mitochondrial dysfunction, to reduce subsequent ROS formation and to block resulting cellular signaling cascades, primarily the endoplasmic reticulum stress. By stabilizing the mitochondrial - ROS - ER stress axis, the range of activity of EpFAs and sEHI display an overlap with the disease conditions including diabetes, fibrosis, chronic pain, cardiovascular and neurodegenerative diseases, for which the above outlined mechanisms play key roles.

AimsThis research was conducted to evaluate the hypothesis that gastric ulcers caused by the NSAID diclofenac sodium (DCF) can be prevented by the soluble epoxide hydrolase inhibitor TPPU.Main methodsMice were administered a single dose of 10, 30 or 100mg/kg of DCF. Once an ulcerative dose of DCF was chosen, mice were pretreated with TPPU for 7days at 0.1mg/kg to evaluate anti-ulcer effects of the sEH inhibitor on anatomy, histopathology, pH, inflammatory markers and epithelial apoptosis of stomachs.Key findingsDiclofenac caused ulceration of the stomach at a dose of 100mg/kg and a time post dose of 6h. Ulcers generated under these conditions were associated with a significant increase in the levels of TNF-α and IL-6 in serum and increased apoptosis compared to control mice. Pretreatment with TPPU resulted in a decrease of ulceration in mice treated with DCF with a significant decrease in the level of apoptosis, TNF-α and IL-6 in the serum in comparison to diclofenac-treated mice. TPPU did not affect the pH of the stomach, whereas omeprazole elevated the pH of the stomach as expected. A similar anti-ulcer effect was observed in sEH gene knockout mice treated with DCF.SignificanceThe sEH inhibitor TPPU decreases the NSAID-induced stomach ulcers.

Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid derived from the cytochrome P450 enzymes, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs but not their diols, have anti-inflammatory properties, and inhibition of sEH might provide protective effects against inflammatory bone loss. Thus, in the present study, we tested the selective sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), in a mouse model of periodontitis induced by infection with Aggregatibacter actinomycetemcomitans Oral treatment of wild-type mice with TPPU and sEH knockout (KO) animals showed reduced bone loss induced by A. actinomycetemcomitans This was associated with decreased expression of key osteoclastogenic molecules, receptor activator of nuclear factor-κB/RANK ligand/osteoprotegerin, and the chemokine monocyte chemotactic protein 1 in the gingival tissue without affecting bacterial counts. In addition, downstream kinases p38 and c-Jun N-terminal kinase known to be activated in response to inflammatory signals were abrogated after TPPU treatment or in sEH KO mice. Moreover, endoplasmic reticulum stress was elevated in periodontal disease but was abrogated after TPPU treatment and in sEH knockout mice. Together, these results demonstrated that sEH pharmacological inhibition may be of therapeutic value in periodontitis.

AimWe designed a study to evaluate the cardioprotective effect of two soluble epoxide hydrolase (sEH) inhibitors, 1-(1-propanoylpiperidin-4-yl)-3-(4-trifluoromethoxy)phenyl)urea (TPPU) and trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]cyclohexyloxy}benzoic acid (t-TUCB), in ischemia-reperfusion (IR) model.MethodsCardioprotective effects of the sEH inhibitors were evaluated against IR-induced myocardial damage in hearts from normal, hypertensive, and diabetic rats using Langendorff's apparatus. In addition, the effect of sEH inhibitors on endothelial function was evaluated in vitro and ex vivo using isolated rat thoracic aorta.ResultsIschemia-reperfusion (IR) increased the myocardial damage in hearts from normal rats. IR-induced myocardial damage was augmented in hearts isolated from hypertensive and diabetic rats. Myocardial damage as evident from increase in the activities of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in heart perfusate was associated with significant decrease in the heart rate and developed tension, and increase in the resting tension in isolated heart. Both sEH inhibitors protected the heart in normal, hypertensive, and diabetic rats subjected to IR injury. The sEH inhibitor t-TUCB relaxed phenylephrine precontracted aorta from normal rats. Relaxant effect of acetylcholine (ACh) was reduced in aortas from diabetic and hypertensive rats compared to normal rats. Pretreatment of sEH inhibitors to diabetic and hypertensive rats increased relaxant effect of ACh on aortas isolated from these rats.ConclusionsProphylactic treatment with sEH inhibitors decreased myocardial damage due to IR, hypertension and diabetes, and decreased endothelial dysfunction created by diabetes and hypertension. Therefore, inhibitors of sEH are useful probes to study cardiovascular pathology, and inhibition of the sEH is a potential approach in the management of IR-induced cardiac damage and endothelial dysfunction-related cardiovascular disorders.

Tetramethylenedisulfotetramine (TETS, tetramine) is a formerly used and highly neurotoxic rodenticide. Its lethality, recent history of intentional use for mass poisoning, and the absence of a known antidote raise public health concerns. Therefore, rapid, high throughput, and sensitive methods for detection and quantification of TETS are critical. Instrumental analysis method such as GC/MS is sensitive but not rapid or high throughput. Therefore, an immunoassay selective to TETS was developed. The assay shows an IC50 of 4.5 ± 1.2 ng/mL, with a limit of detection of 0.2 ng/mL, comparable to GC/MS. Performance of the immunoassay was demonstrated by a recovery study using known concentrations of TETS spiked into buffer and human and mouse serum matrices giving recoveries in the range of 80-120%. The assay demonstrated good correlation in TETS recovery with established GC/MS analysis. The immunoassay was then used to quantify TETS concentration in the serum of mice exposed to 2× LD50 dose of TETS and to monitor kinetics of TETS clearance from blood over a short period of time. TETS concentration in the serum reached 150 ng/mL without significant change over 4 h post-treatment. Results obtained with the immunoassay had good correlation with GC/MS analysis. Overall, this immunoassay is an important tool to rapidly detect and quantify levels of TETS from biological samples with high sensitivity. The assay can be adapted to multiple formats including field or hospital use.

Recently, dibenzylurea-based potent soluble epoxide hydrolase (sEH) inhibitors were identified in Pentadiplandra brazzeana, a plant in the order Brassicales. In an effort to generalize the concept, we hypothesized that plants that produce benzyl glucosinolates and corresponding isothiocyanates also produce these dibenzylurea derivatives. Our overall aim here was to examine the occurrence of urea derivatives in Brassicales, hoping to find biologically active urea derivatives from plants. First, plants in the order Brassicales were analyzed for the presence of 1, 3-dibenzylurea (compound 1), showing that three additional plants in the order Brassicales produce the urea derivatives. Based on the hypothesis, three dibenzylurea derivatives with sEH inhibitory activity were isolated from maca (Lepidium meyenii) roots. Topical application of one of the identified compounds (compound 3, human sEH IC50 = 222 nM) effectively reduced pain in rat inflammatory pain model, and this compound was bioavailable after oral administration in mice. The biosynthetic pathway of these urea derivatives was investigated using papaya (Carica papaya) seed as a model system. Finally, a small collection of plants from the Brassicales order was grown, collected, extracted and screened for sEH inhibitory activity. Results show that several plants of the Brassicales order could be potential sources of urea-based sEH inhibitors.

BackgroundAutologous engineered skin substitutes comprised of keratinocytes, fibroblasts, and biopolymers can serve as an adjunctive treatment for excised burns. However, engineered skin lacks a vascular plexus at the time of grafting, leading to slower vascularization and reduced rates of engraftment compared with autograft. Hypothetically, vascularization of engineered skin grafts can be improved by treatment with proangiogenic agents at the time of grafting. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase (sEH). EETs have multiple biological activities and have been shown to promote angiogenesis. Inhibitors of sEH (sEHIs) represent attractive therapeutic agents because they increase endogenous EET levels. We investigated sEHI administration, alone or combined with EET treatment, for improved vascularization of engineered skin after grafting to mice.MethodsEngineered skin substitutes, prepared using primary human fibroblasts and keratinocytes, were grafted to full-thickness surgical wounds in immunodeficient mice. Mice were treated with the sEHI 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which was administered in drinking water throughout the study period, with or without topical EET treatment, and were compared with vehicle-treated controls. Vascularization was quantified by image analysis of CD31-positive areas in tissue sections.ResultsAt 2 weeks after grafting, significantly increased vascularization was observed in the TPPU and TPPU + EET groups compared with controls, with no evidence of toxicity.ConclusionsThe results suggest that sEH inhibition can increase vascularization of engineered skin grafts after transplantation, which may contribute to enhanced engraftment and improved treatment of full-thickness wounds.

ContextHypotension is one of the dose limiting side effects of benzodiazepines (BZDs), in particular of diazepam (DZP) which is still widely used in the clinic. Currently, only one FDA approved antidote exists for BZD overdose and novel approaches are needed to improve management of DZP overdose, dependency and withdrawal.ObjectiveHere, we hypothesized that increasing bioactive lipid mediators termed epoxy fatty acids (EpFAs) will prevent hypotension, as was shown previously in a murine model of LPS-induced hypotension. Therefore, we first characterized the time and dose dependent profile of DZP induced hypotension in mice, and then investigated the reversal of the hypotensive effect by inhibiting the soluble epoxide hydrolase (sEH), an enzyme that regulates the levels of EpFAs.Materials and methodsFollowing baseline systolic BP recording using tail cuffs, mice were administered a sEH inhibitor (TPPU) before DZP and BP was monitored. Blood and brain levels of DZP and TPPU were quantified to examine distribution and metabolism. Plasma EpFAs levels were quantified to determine TPPU target engagement.ResultsIn this murine model, DZP induced dose dependent hypotension which was more severe than midazolam. The temporal profile was consistent with the reported pharmacokinetics/pharmacodynamics of DZP. Treatment with TPPU reversed the hypotension resulting from high doses of DZP and decreased the sEH metabolites of EpFAs in the plasma demonstrating target engagement.Discussion and conclusionOverall, these findings demonstrate the similarity of a murine model of DZP induced hypotension to clinical observations in humans. Furthermore, we demonstrate that stabilization of EpFAs by inhibiting sEH is a novel approach to overcome DZP-induced hypotension and this beneficial effect can be enhanced by an omega three diet probably acting through epoxide metabolites of the fatty acids.

Proton pump inhibitors such as omeprazole (OME) reduce the severity of gastrointestinal (GI) ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs) but can also increase the chance of dysbiosis. The aim of this study was to test the hypothesis that preventive use of a soluble epoxide hydrolase inhibitor (sEHI) such as TPPU can decrease NSAID-induced ulcers by increasing anti-inflammatory epoxyeicosatrienoic acids (EETs). Dose- [10, 30, and 100 mg/kg, by mouth (PO)] and time-dependent (6 and 18 hours) ulcerative effects of diclofenac sodium (DCF, an NSAID) were studied in the small intestine of Swiss Webster mice. Dose-dependent effects of TPPU (0.001-0.1 mg/kg per day for 7 days, in drinking water) were evaluated in DCF-induced intestinal toxicity and compared with OME (20 mg/kg, PO). In addition, the effect of treatment was studied on levels of Hb in blood, EETs in plasma, inflammatory markers such as myeloperoxidase (MPO) in intestinal tissue homogenates, and tissue necrosis factor-α (TNF-α) in serum. DCF dose dependently induced ulcers that were associated with both a significant (P < 0.05) loss of Hb and an increase in the level of MPO and TNF-α, with severity of ulceration highest at 18 hours. Pretreatment with TPPU dose dependently prevented ulcer formation by DCF, increased the levels of epoxy fatty acids, including EETs, and TPPU's efficacy was comparable to OME. TPPU significantly (P < 0.05) reversed the effect of DCF on the level of Hb, MPO, and TNF-α Thus sEHI might be useful in the management of NSAID-induced ulcers.

Cytochrome P450 epoxygenase isozymes convert free arachidonic acid into eicosanoids named epoxyeicosatrienoic acids (EETs) that have roles in regulating inflammation. EETs are rapidly converted to dihydroxyeicosatrienoic acids (DiHETs) by soluble epoxide hydrolase (sEH). Little is known about the potential role of these metabolites in uveitis, but conversion of EETs to DiHETs could contribute to the inflammation. We tested a potent and orally available inhibitor of sEH for its ability to reduce ocular inflammation in a rabbit LPS-induced model of uveitis. Rabbits were treated by subcutaneous injection with the sEH inhibitor (UC1728, 3 mg/kg), or the vehicle control (PEG400) and uveitis was assessed at 6, 24 and 48 h post-intracameral LPS injection using a modified Hackett-McDonald scoring system. Eyes treated by intra-cameral injection of PBS, or by aseptic preparation served as further controls. Signs of inflammation in this model were mild and transient. Treatment with UC1728 did not significantly reduce inflammation compared to animals treated with the PEG400 vehicle. Blood levels of UC1728 were a thousand fold higher than the in vitro determined inhibitory potency (IC50) of the compound suggesting a significant degree of inhibition of sEH in the rabbit. The lack of efficacy suggests that sEH or its substrates the EETs may not be involved in mediating inflammation in this model of uveitis.

Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3mg/kg/day, p.o.) and OME (100mg/kg/day, p.o., 7 days)+TPPU (3mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE2 was monitored. While OME treatment by itself exhibited variable effects on PGE2 induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME+TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study.

Tetramethylenedisulfotetramine (TETS) is a potent convulsant GABAA receptor blocker. Mice receiving a lethal dose of TETS (0.15 mg/kg i.p.) are rescued from death by a high dose of diazepam (5 mg/kg i.p.) administered shortly after the second clonic seizure (∼20 min post-TETS). However, this high dose of diazepam significantly impairs blood pressure and mobility, and does not prevent TETS-induced neuroinflammation in the brain. We previously demonstrated that TETS alters synchronous Ca(2+) oscillations in primary mouse hippocampal neuronal cell cultures and that pretreatment with the combination of diazepam and allopregnanolone at concentrations having negligible effects individually prevents TETS effects on intracellular Ca(2+) dynamics. Here, we show that treatment with diazepam and allopregnanolone (0.1 μM) 20 min after TETS challenge normalizes synchronous Ca(2+) oscillations when added in combination but not when added singly. Similarly, doses (0.03-0.1 mg/kg i.p.) of diazepam and allopregnanolone that provide minimal protection when administered singly to TETS intoxicated mice increase survival from 10% to 90% when given in combination either 10 min prior to TETS or following the second clonic seizure. This therapeutic combination has negligible effects on blood pressure or mobility. Combined treatment with diazepam and allopregnanolone also decreases TETS-induced microglial activation. Diazepam and allopregnanolone have distinct actions as positive allosteric modulators of GABAA receptors that in combination enhance survival and mitigate neuropathology following TETS intoxication without the adverse side effects associated with high dose benzodiazepines. Combination therapy with a benzodiazepine and neurosteroid represents a novel neurotherapeutic strategy with potentially broad application.

Despite intensive effort and resulting gains in understanding the mechanisms underlying neuropathic pain, limited success in therapeutic approaches have been attained. A recently identified, nonchannel, nonneurotransmitter therapeutic target for pain is the enzyme soluble epoxide hydrolase (sEH). The sEH degrades natural analgesic lipid mediators, epoxy fatty acids (EpFAs), therefore its inhibition stabilizes these bioactive mediators. Here we demonstrate the effects of EpFAs on diabetes induced neuropathic pain and define a previously unknown mechanism of pain, regulated by endoplasmic reticulum (ER) stress. The activation of ER stress is first quantified in the peripheral nervous system of type I diabetic rats. We demonstrate that both pain and markers of ER stress are reversed by a chemical chaperone. Next, we identify the EpFAs as upstream modulators of ER stress pathways. Chemical inducers of ER stress invariably lead to pain behavior that is reversed by a chemical chaperone and an inhibitor of sEH. The rapid occurrence of pain behavior with inducers, equally rapid reversal by blockers and natural incidence of ER stress in diabetic peripheral nervous system (PNS) argue for a major role of the ER stress pathways in regulating the excitability of the nociceptive system. Understanding the role of ER stress in generation and maintenance of pain opens routes to exploit this system for therapeutic purposes.

We need better medicines to control acute and chronic pain. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) catalyze the deactivating hydrolysis of two classes of bioactive lipid mediators--fatty acid ethanolamides (FAEs) and epoxidized fatty acids (EpFAs), respectively--which are biogenetically distinct but share the ability to attenuate pain responses and inflammation. In these experiments, we evaluated the antihyperalgesic activity of small-molecule inhibitors of FAAH and sEH, administered alone or in combination, in two pain models: carrageenan-induced hyperalgesia in mice and streptozocin-induced allodynia in rats. When administered separately, the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea (TPPU) and the peripherally restricted FAAH inhibitor URB937 were highly active in the two models. The combination TPPU plus URB937 was markedly synergistic, as assessed using isobolographic analyses. The results of these experiments reveal the existence of a possible functional crosstalk between FAEs and EpFAs in regulating pain responses. Additionally, the results suggest that combinations of sEH and FAAH inhibitors might be exploited therapeutically to achieve greater analgesic efficacy.

Niacin is effective in treating dyslipidemias but causes cutaneous vasodilation or flushing, a side effect that limits its clinical use. Blocking prostaglandins in humans reduces but does not consistently eliminate flushing, indicating additional mechanisms may contribute to flushing. The transient receptor potential vanilloid 1 (TRPV1) channel, when activated, causes cutaneous vasodilation and undergoes tachyphylaxis similar to that seen with niacin. Using a murine model, early phase niacin-induced flushing was examined and TRPV1 channel involvement demonstrated using pharmacologic blockade, desensitization, and genetic knockouts (TRPV1 KO). The TRPV1 antagonist AMG9810 reduced the magnitude of the initial and secondary peaks and the rapidity of the vasodilatory response (slope). TRPV1 desensitization by chronic capsaicin reduced the initial peak and slope. TRPV1 KO mice had a lower initial peak, secondary peak, and slope compared with wild-type mice. Chronic niacin reduced the initial peak, secondary peak, and slope in wild-type mice but had no effect in knockout mice. Furthermore, chronic niacin diminished the response to capsaicin in wild-type mice. Overall, these data demonstrate an important role for TRPV1 channels in niacin-induced flushing, both in the acute response and with chronic administration. That niacin-induced flushing is a complex cascade of events, which should inform pharmacological intervention against this side effect.

We describe here three urea-based soluble epoxide hydrolase (sEH) inhibitors from the root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl) urea (MMU) is the most abundant (42.3 μg/g dry root weight). All of the ureas were chemically synthesized, and their inhibitory activity toward recombinant human and recombinant rat sEH was measured. The most potent compound, MMU, showed an IC50 of 92 nM via fluorescent assay and a Ki of 54 nM via radioactivity-based assay on human sEH. MMU effectively reduced inflammatory pain in a rat nociceptive pain assay. These compounds are among the most potent sEH inhibitors derived from natural sources. Moreover, inhibition of sEH by these compounds may mechanistically explain some of the therapeutic effects of P. brazzeana.

Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABAAR) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABAAR positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15mg/kg, ip). Administration of a high dose of diazepam (5mg/kg, ip) immediately following the second clonic seizure (approximately 20min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABAAR antagonists. The sEH inhibitor TUPS (1mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5mg/kg, ip) and TUPS (1mg/kg, ip, starting 1h after diazepam and repeated every 24h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication.

Lipid derived mediators contribute to inflammation and the sensing of pain. The contributions of omega-6 derived prostanoids in enhancing inflammation and pain sensation are well known. Less well explored are the opposing anti-inflammatory and analgesic effects of the omega-6 derived epoxyeicosatrienoic acids. Far less has been described about the epoxidized metabolites derived from omega-3 long chain fatty acids. The epoxide metabolites are turned over rapidly with enzymatic hydrolysis by the soluble epoxide hydrolase being the major elimination pathway. Despite this, the overall understanding of the role of lipid mediators in the pathology of chronic pain is growing. Here, we review the role of long chain fatty acids and their metabolites in alleviating both acute and chronic pain conditions. We focus specifically on the epoxidized metabolites of omega-6 and omega-3 long chain fatty acids as well as a novel strategy to modulate their activity in vivo.

UnlabelledNeuropathic pain is currently an insufficiently treated clinical condition. There remains a critical need for efficacious therapies without severe side effects to treat the uniquely persistent and tonic pain of neuropathy. Inhibitors of the soluble epoxide hydrolase (sEH) enzyme that stabilize endogenous epoxy fatty acids have demonstrated antihyperalgesia in clinical chronic inflammatory pain and modeled neuropathic pain. Recently, the conditioned place preference assay has been used to evaluate the tonic nature of neuropathy in several animal models. The current experiments use the conditioned place preference assay alongside withdrawal thresholds to investigate the antihyperalgesic efficacy of sEH inhibitors in a murine model of diabetic neuropathy. Here, the sEH inhibitor trans-4-[4-(3-trifluoromethoxyphenyl-1-ureido)-cyclohexyloxy]-benzoic acid (t-TUCB) at 10 mg/kg induced a robust place preference in diabetic neuropathic mice representative of pain relief. Importantly, this effect was absent both in control mice and in sEH-knockout mice at the same dose, indicating that t-TUCB is not positively reinforcing or rewarding. When compared to gabapentin, t-TUCB elicited a similar degree of withdrawal threshold improvement without the same degree of spontaneous locomotion decline in neuropathic mice. Overall, these experiments show that inhibiting the sEH enzyme attenuates chronic pain and offers an alternative to current side-effect-limited therapies to meet this clinical need.PerspectiveThese experiments demonstrate antihyperalgesia in a murine chronic pain model mediated by inhibiting the sEH enzyme. The results of this study indicate that inhibiting the sEH is a promising alternative for blocking chronic pain.

Epoxyeicosatrienoic acids (EETs), lipid mediators produced by cytochrome P450 epoxygenases, regulate inflammation, angiogenesis, and vascular tone. Despite pleiotropic effects on cells, the role of these epoxyeicosanoids in normal organ and tissue regeneration remains unknown. EETs are produced predominantly in the endothelium. Normal organ and tissue regeneration require an active paracrine role of the microvascular endothelium, which in turn depends on angiogenic growth factors. Thus, we hypothesize that endothelial cells stimulate organ and tissue regeneration via production of bioactive EETs. To determine whether endothelial-derived EETs affect physiologic tissue growth in vivo, we used genetic and pharmacological tools to manipulate endogenous EET levels. We show that endothelial-derived EETs play a critical role in accelerating tissue growth in vivo, including liver regeneration, kidney compensatory growth, lung compensatory growth, wound healing, corneal neovascularization, and retinal vascularization. Administration of synthetic EETs recapitulated these results, whereas lowering EET levels, either genetically or pharmacologically, delayed tissue regeneration, demonstrating that pharmacological modulation of EETs can affect normal organ and tissue growth. We also show that soluble epoxide hydrolase inhibitors, which elevate endogenous EET levels, promote liver and lung regeneration. Thus, our observations indicate a central role for EETs in organ and tissue regeneration and their contribution to tissue homeostasis.

Tetramethylenedisulfotetramine (tetramine; TETS) is a potent convulsant poison that is considered to be a chemical threat agent. To provide a basis for the investigation of antidotes for TETS-induced seizures, we characterized the convulsant activity of TETS in mice and rats when administered by the intraperitoneal, intravenous, oral, and intraventricular routes as a single acute dose and with repeated sublethal doses. In mice, parenteral and oral TETS caused immobility, myoclonic body jerks, clonic seizures of the forelimbs and/or hindlimbs, tonic seizures, and death. The CD₅₀ values for clonic and tonic seizures after oral administration were 0.11 and 0.22 mg/kg, respectively. Intraventricular administration of TETS (5-100 μg) in rats also caused clonic-tonic seizures and death. In mice, repeated sublethal doses of TETS at intervals of 2, 24, and 48 h failed to result in the development of persistent enhanced seizure responsivity ("kindling") as was observed with repeated pentylenetetrazol treatment. In mice, sublethal doses of TETS that produced clonic seizures did not cause observable structural brain damage as assessed with routine histology and Fluoro-Jade B staining 7 days after treatment. However, 1 to 3 days after a single convulsant dose of TETS the expression of glial fibrillary acidic protein, an astrocyte marker, and ionized calcium binding adaptor molecule 1, a microglia marker, were markedly increased in cortex and hippocampus. Although TETS doses that are compatible with survival are not associated with overt evidence of cellular injury or neurodegeneration, there is transient reactive astrocytosis and microglial activation, indicating that brain inflammatory responses are provoked.

Soluble epoxide hydrolase (Ephx2, sEH) is a bifunctional enzyme with COOH-terminal hydrolase and [NH.sub.2]-terminal phosphatase activities. sEH converts epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), and the phosphatase activity is suggested to be involved in cholesterol metabolism. EETs participate in a wide range of biological functions, including regulation of vascular tone, renal tubular transport, cardiac contractility, and inflammation. Inhibition of sEH is a potential approach for enhancing the biological activity of EETs. Therefore, disruption of sEH activity is becoming an attractive therapeutic target for both cardiovascular and inflammatory diseases. To define the physiological role of sEH, we characterized a knockout mouse colony lacking expression of the Ephx2 gene. Lack of sEH enzyme is characterized by elevation of EET to DHET ratios in both the linoleate and arachidonate series in plasma and tissues of both female and male mice. In male mice, this lack of expression was also associated with decreased plasma testosterone levels, sperm count, and testicular size. However, this genotype was still able to sire litters. Plasma cholesterol levels also declined in this genotype. Behavior tests such as anxiety-like behavior and hedonic response were also examined in Ephx2-null and WT mice, as all can be related to hormonal changes. Null mice showed a level of anxiety with a decreased hedonic response. In conclusion, this study provides a broad biochemical, physiological, and behavioral characterization of the Ephx2-null mouse colony and suggests a mechanism by which sEH and its substrates may regulate circulating levels of testosterone through cholesterol biosynthesis and metabolism. cholesterol; fertility

Study is conducted to demonstrate the three different homozygous mutations such as p.S156P, p.R104X and p.V206SfsX117 in the fibroblast growth factor 3 (FGF3) gene affected members of these families, cosegregating with the autosomal recessive transmission as a completely penetrant phenotype. Findings show the involvement of FGF3 mutations in a human malformation syndrome.