Pharmacology & TherapeuticsSoluble epoxide hydrolase as a therapeutic target for pain, inﬂammatory...

Pharmacology & Therapeutics xxx (2017) xxx–xxx

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Pharmacology & Therapeutics

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Soluble epoxide hydrolase as a therapeutic target for pain, inflammatoryand neurodegenerative diseases☆

Karen M. Wagner a, Cindy B. McReynolds a, William K. Schmidt b, Bruce D. Hammock a,⁎a Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California Davis, Davis, CA 95616, United Statesb EicOsis LLC, Davis, CA 95618, United States

Abbreviations: AD, Alzheimer's disease; ARA, arachidoCYP450, cytochrome P450; DHA, docosahexaenoic acidEETs, epoxyeicosatrienoic acids; EpFAs, epoxy-fatty areticulum stress; IBD, inflammatory bowel disease; LOsteroidal anti-Inflammatory drugs; NFĸB, nuclear factoractivated B cells; PD, Parkinson's disease; PGE2, prostaglhydrolase; sEHI, soluble epoxide hydrolase inhibitors; RO☆ Associate editor: Darryl Zeldin⁎ Corresponding author at: Department of Entomology

One Shields Ave., Davis, CA 95616, United States.E-mail address: [email protected] (B.D. Hamm

http://dx.doi.org/10.1016/j.pharmthera.2017.06.0060163-7258/© 2017 Published by Elsevier Inc.

Please cite this article as: Wagner, K.M., et adiseases, Pharmacology & Therapeutics (2017

a b s t r a c t
a r t i c l e i n f o
Keywords:
Eicosanoids are biologically active lipid signaling molecules derived from polyunsaturated fatty acids. Many ofthe actions of eicosanoidmetabolites formed by cyclooxygenase and lipoxygenase enzymes have been character-ized, however, the epoxy-fatty acids (EpFAs) formed by cytochrome P450 enzymes are newly described by com-parison. The EpFA metabolites modulate a diverse set of physiologic functions that include inflammation andnociception among others. Regulation of EpFAs occurs primarily via release, biosynthesis and enzymatic transfor-mation by the soluble epoxide hydrolase (sEH). Targeting sEHwith small molecule inhibitors has enabled obser-vation of the biological activity of the EpFAs in vivo in animal models, greatly contributing to the overallunderstanding of their role in the inflammatory response. Their role in modulating inflammation has been dem-onstrated in disease models including cardiovascular pathology and inflammatory pain, but extends to neuroin-flammation and neuroinflammatory disease. Moreover, while EpFAs demonstrate activity against inflammatorypain, interestingly, this action extends to blocking chronic neuropathic pain as well. This review outlines the roleof modulating sEH and the biological action of EpFAs in models of pain and inflammatory diseases.
© 2017 Published by Elsevier Inc.

Soluble epoxide hydrolase (sEH)Epoxy-fatty acids (EpFAs)Inflammatory painNeuropathic painDepressionAlzheimer's disease

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. sEH as a target for inflammatory diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. sEH as a target for neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. sEH as a target for pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

nic acid; COX, cyclooxygenase;; EPA, eicosapentaenoic acid;cids; ER Stress, endoplasmicX, lipoxygenase; NSAIDs, non-kappa-light-chain-enhancer ofandin E2; sEH, soluble epoxideS, reactive oxygen stress.

, University of California Davis,

ock).

l., Soluble epoxide hydrolase), http://dx.doi.org/10.1016/j

1. Introduction

Eicosanoids are a group of lipid mediators generated from arachi-donic acid (ARA) by activity of cyclooxygenases (COX), lipoxygenases(LOX) and cytochrome P450 (CYP450) enzymes. These fatty acid me-tabolites are implicated in critical biological processes throughout thebody in most cells, tissues and organs (Funk, 2001; Xu et al., 2016). Ei-cosanoids have been intensely investigated for their role in the inflam-matory response and more recently the complexity of the pro andanti-inflammatory as well as other non-inflammatory roles for thesemetabolites have been recognized (Dennis & Norris, 2015). Knowledgeof the complex signaling networks that the eicosanoids comprise now

as a therapeutic target for pain, inflammatory and neurodegenerative.pharmthera.2017.06.006

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extends to include the metabolites of other long chain polyunsaturatedacids (LC-PUFA) such as docosahexaenoic acid (DHA) andeicosapentaenoic acid (EPA) that are recognized to flow through thesame enzymatic pathways. Much is known about the bioactivity ofprostaglandinmetabolites of ARA, and similarly, the leukotrienemetab-olites have well described, potent biological action. There is less knownabout the metabolites of the CYP450s, the epoxy-fatty acids (EpFAs),though the body of knowledge regarding their bioactivity is growing.Moreover, when addressing the EpFAs specifically as a class of lipid me-diators, the epoxide metabolites of all LC-PUFAs can be included. It is inexamining the biology of this EpFA class that the importance of the sol-uble epoxide hydrolase (sEH) enzyme was revealed because it is amajor regulator of EpFA biology. Uncovering the physiologic role ofthe EpFAs has been greatly aided by the ability to inhibit the sEH en-zyme. Because the effects of maintaining EpFA titers in vivo has beenlargely beneficial, small molecule inhibitors of sEH (sEHI) have becomea novel approach to altering disease pathologies including cardiovascu-lar diseases, inflammation, neurodegenerative disorders and chronicpain among others.

1.1. EpFA biosynthesis and regulation

LC-PUFA are 14–26 long carbon chains with several double bondsimparting their polyunsaturated nature. The term “eicosa” refers to 20carbon length fatty acids formed mostly from 20:4(n-6) ARA which,along with the omega-3 metabolites of EPA (20:5, n-3) and longerchain DHA (22:6, n-3) fatty acids, are the major focus of this review.The CYP450 enzymes act on LC-PUFA to form EpFAs by epoxidation ofthe double bonds (Konkel & Schunck, 2011). Multiple regioisomers ofEpFAs are produced from theparent LC-PUFA depending on the locationof the epoxidized double bond. There is also a high degree ofenantiofacial selectivity (R/S regioisomer) conferred in this process(Spector, Fang, Snyder, & Weintraub, 2004). The epoxidizedmetabolites, epoxyeicosatrienoic acids (EETs) from omega-6 ARA,epoxyeicosatetraenoic acids (EEQs) from omega-3 EPA, andepoxydocosapentaenoic acids (EDPs) from omega-3 DHA are all classedas EpFAs and are principally anti-inflammatory eicosanoids (Morisseau

Fig. 1. Long chain polyunsaturated acidmetabolism through the CYP450 pathway. Arachidoniccytochrome P450 enzymes (CYP450) into the epoxy-fatty acids (EpFAs). For simplicity, themetEpFAs, the epoxyeicosatrienoic acids (EETs), are formed fromARA. Four individual regioisomerdepicted. In addition to the epoxides from LC-PUFA, any fatty acidswith an olefinic bondmay foring to yield the diol, in the case of ARAmetabolites are termed dihydroxyeicosatrienoic acids (Dpotent biologically active classes of EpFAs.

Please cite this article as: Wagner, K.M., et al., Soluble epoxide hydrolasediseases, Pharmacology & Therapeutics (2017), http://dx.doi.org/10.1016/j

et al., 2010). The relative contribution of different CYP450s to the totalproduction of the EpFAs will vary with substrate availability and con-centration. Also, the expression of the CYP450 monooxygenases thatproduce them vary depending on sex, species, organ and proportion ofthe regioisomer of epoxide they produce. However, both the CYP450sthat produce the EpFAs and the sEH that is their principal regulatory en-zyme are expressed at some level inmost tissues. This demonstrates thebiological relevance of these metabolites because all types of EpFAs aretransformed by the sEH into diols (Fig. 1) and in the case of EETs thediols are less active (Spector, 2009).

sEH (EC:3.3.2.10) is part of theα/β hydrolase fold super family and isa 120 kDhomodimer enzymewith a C-terminal hydrolase andN-termi-nal phosphatase (Beetham, Tian, & Hammock, 1993; Cronin et al.,2003). The phosphatase domain hydrolyzes phosphorylated lipidssuch as isoprenoid phosphates and lysophosphatidic acid that stimulatecell growth but far less is known about the biological role of this activity(Oguro & Imaoka, 2012; Oguro, Sakamoto, Suzuki, & Imaoka, 2009). TheC-terminal domain hydrolyzes the epoxides by addition of water to thethree membered oxirane ring (Spector, 2009). sEH expression is wellconserved among species from simple chordates to preclinical rodentsand all mammals tested to date indicating its fundamental role in biol-ogy (Harris & Hammock, 2013). sEH is widely distributed throughoutthe body with themost concentrated expression in the liver, kidney, in-testine and vasculature inmammals (Enayetallah, French, Thibodeau, &Grant, 2004). However, sEH is also found in the brain and in C57Bl/6mouse is observedmore strongly in the cortex, hippocampus, amygdalaand striatum (Marowsky, Burgener, Falck, Fritschy, & Arand, 2009). sEHexpression has been found in neurons along with the CYP450 enzymesthat produce EpFAs (Iliff, Wang, Zeldin, & Alkayed, 2009) and in astro-cytes including astrocytic end feet (Marowsky et al., 2009). In humannaïve brain, sEH is expressed in neurons, oligodendrocytes, astrocytesand ependymal cells (Sura, Sura, Enayetallah, & Grant, 2008).

Potent selective inhibitors of sEH were first described in the early1980′s as a mechanism to identify the biological importance of the en-zyme (Mullin & Hammock, 1982). The diols formed from sEH actiongenerally lack the activity of the epoxidized precursors however theyare dramatically more polar, move rapidly out of cells, and are easily

acid (ARA) and other long chain polyunsaturated fatty acids (LC-PUFA) aremetabolized byabolism of omega-6 ARA is depicted here as an example of LC-PUFAmetabolism. A class ofs can be formed by the epoxidation of any one of the four double bonds with the 14,15 EETrmepoxidizedmetabolites. The soluble epoxide hydrolase (sEH) addswater to the oxiraneHETs). This process is the same for omega-3 LC-PUFA including DHA and EPAwhich form



3K.M. Wagner et al. / Pharmacology & Therapeutics xxx (2017) xxx–xxx

conjugated and excreted (Greene, Newman, Williamson, & Hammock,2000). Yet, there is some evidence PUFA diols are chemoattractant formonocytes (Kundu et al., 2013) and that linoleic diols specifically actas lipokines in the regulation of brown adipose tissue and thermogene-sis (Lynes et al., 2017). There is noovert phenotypewith thewhole bodyknockout animals not subjected to physiologic stress (Spector & Kim,2015), however, knockout mice had lower survival following ischemicevents (Hutchens et al., 2008). In general, the biology of the knockoutanimals is mimicked by treatment with sEHI.

1.2. EpFA role in inflammation

Inflammation is the biological response to insult that includes car-diovascular dilation of arterioles and capillaries, increased permeabilityof the microvasculature, and leukocyte infiltration. The leukocyte infil-tration is typically in response to release of chemokines and cytokines(small cell signaling proteins), resulting in redness, heat and pain (Rot& von Andrian, 2004). Several hallmarks of inflammation have beenexploited as biomarkers of the condition including cytokines such as in-terleukins 1β and 6 (IL-1β, IL-6), tumor necrosis factor alpha (TNFα)and chemokines such as vascular cell adhesion molecule 1 (VCAM-1),intercellular adhesion molecule 1 (I-CAM 1), endothelial cell selectiveadhesion molecule (E-selectin), prostaglandins such as prostaglandinE2 (PGE2) and activation of nuclear factor kappa-light-chain-enhancerof activated B cells (NFĸB). TNFα is a cytokine which is a central media-tor of both acute phase reaction and systemic inflammation. VCAM-1 isactivated by TNFα via NFĸB andmediates chemotaxis of typicallymono-cytes and lymphocytes and is essential to their adhesion to and infiltra-tion of inflamed tissues. The inflammatory response removes an injuryor insult and resolves but when resolution fails it becomes a chroniccondition and chronic inflammation is related to numerous major dis-ease states.

Fig. 2. EpFAs block inflammation through several mechanisms. Regioisomers of the EET class ofthe adherence and infiltration of activated monocytes. EETs also reduce inflammation by blocblocks the increase of phospho-IκBα levels which activate NFĸB and thus inhibit NFĸB signalnitric oxide synthase (iNOS), lipoxygenase-5 (LOX-5), and cyclooxygenase-2 (COX-2, picturproduction of prostaglandin E2 (PGE2) which is a potent inflammatory agent. EETs also decreActivation of signal transducer and activator of transcription 3 (STAT3) and other nuclear recadditional anti-inflammatory mechanisms that have been described for EpFAs in blocking dow


EETs and other EpFAs are known to reduce inflammation throughmultiple mechanismswhich have been demonstrated in animalmodels(Fig. 2). One such mechanism of EET regioisomers in cardiovascular bi-ology is to inhibit VCAM-1, E-selectin and ICAM-1 expression (Node etal., 1999; Zhao et al., 2012). EETs also decrease TNFα secretion frommonocytic cells (Bystrom et al., 2011) andmay also inhibit their adher-ence (Node et al., 1999). Other mechanisms by which EETs reduce in-flammation include blocking the nuclear translocation of NFĸB(Bystrom et al., 2011; Fife et al., 2008; Node et al., 1999) which in turndownregulates several enzymes including calcium-insensitive nitricoxide synthase (iNOS), lipoxygenase-5 (LOX-5), and cyclooxygenase-2(COX-2) that are upregulated in inflammation (Schmelzer et al., 2005;Schmelzer et al., 2006). sEHI administration also blocked increases inphospho-IκBα levels which activate NFĸB and thus inhibited NFĸB sig-naling in amurinemodel (Xu et al., 2006). Activation of signal transduc-er and activator of transcription 3 (STAT3) (Williams, Bradley, Smith, &Foxwell, 2004) and other nuclear receptor activation such as peroxi-some proliferator activated receptor (PPAR) alpha and gamma are addi-tional mechanisms that have been described for EETs (Fang, 2006; Ng etal., 2007). In vivo sEH gene deletion and the resulting increase in EETslowered inflammatory gene expression and neutrophil recruitment,though these effects displayed someorgan specificity beingmore robustin lung (Deng et al., 2011). EpFAs derived from omega-3 LC-PUFA areless well described but recent studies demonstrate they also have gen-erally anti-inflammatory properties (Isobe & Arita, 2014; Morin, Sirois,Echave, Albadine, & Rousseau, 2010). However, it is critical that EpFAsand their regio and optical isomers be treated as distinct compounds.

Determining the mechanisms of EpFA action has been complicatedby the lack of a defined receptor. There has been a considerable effortin the last decade to identify a receptor, or more likely receptors, forthe EETswith little progress. The COX and LOX systems are perhaps bet-ter exploited because prostaglandins and leukotrienes have identified Gprotein coupled receptors (GPCR) and selective compounds for

EpFAs inhibit VCAM-1, E-selectin and ICAM-1 expression in endothelial cells which blocksking the nuclear translocation of NFĸB, and sEHI administration which elevates all EpFAs,ing. This results in the downregulation of several enzymes including calcium-insensitiveed) that are upregulated in inflammation. The downregulation of COX-2 also limits thease TNFα secretion from monocytic cells and inflammatory cytokines in several tissues.eptors such as peroxisome proliferator activated receptor (PPAR) alpha and gamma arenstream inflammation.




pharmacological agonism and antagonism. Although lacking a definedreceptor, G proteins have been implicated in the action of EETs in coro-nary smooth muscle (Li & Campbell, 1997) and EETs have been antago-nized with a synthetic antagonist (Gauthier et al., 2002; Gross et al.,2008). In cerebral artery smooth muscle cells, EETs bind to transient re-ceptor potential cation channel subfamily V member 4 (TRPV4) chan-nels (Earley, Heppner, Nelson, & Brayden, 2005; Vriens et al., 2005;Watanabe et al., 2003). However, there is evidence that EETs can acton more than one TRP channel (Loot & Fleming, 2011), and that theyhave effects that are independent of calcium signaling (reviewed inSudhahar, Shaw, and Imig (2010)). Thus, there aremultiple possible ac-tions of EpFAs and their mode of action may differ depending on com-pound, tissue type and receptor expression.

The sEHI reduce the severity of a variety health problem in animalmodels. In many cases inflammation could be seen as a commonmech-anism as introduced above, but in other cases it is hard to understandhow a singlemechanism could address such diverse illnesses as atrialfi-brillation and pancreatitis. It now appears that modulation of endoplas-mic reticulum stress and specifically the pathological axis frommitochondrial dysfunction through ROS generation and activation ofthe ER Stress pathway leading to cell damage is an event common tomany of the beneficial effects of EpFAs and sEHI.

The bioactivity of EpFAs is transient in vivo principally due to the ac-tion of sEH. The primary route of EpFA transformation to inactive diols isblocked by inhibiting the sEH enzyme to increase their residence timeand observe their bioactivity. Several commonly used sEHI are outlinedin Table 1 including their chemical structures (Table 1). Even with sEHinhibited or removed, EpFAs are metabolized at a somewhat slowerrate by beta oxidation or chain elongation, CYP450 oxidation, reincorpo-ration into glycerides and other pathways (Spector et al., 2004).Inhibiting sEH has demonstrated anti-inflammatory action in severalstudies using animal models (Liu, Tsai, et al., 2009; Liu, Yang, et al.,2010; Schmelzer et al., 2005). Anti-hyperalgesic activity in nociceptiveassays has also been correlated with increased concentration of EpFAsin vivo (Inceoglu et al., 2012). In addition the demonstrated bioactivityof EpFAs has been supported by advances made with the use of EET an-alogues in vivo as an alternative experimental strategy (Sudhahar et al.,2010).

2. sEH as a target for inflammatory diseases

Eicosanoids play a fundamental role in inflammation, and classicalpharmaceutical approaches have focused on blocking the formation ofmetabolites or antagonising their receptor mediated action. This is thecase with most non-steroidal anti-inflammatory drugs (NSAIDs) andleukotriene receptor antogonists. However, while formally describedas inflammatory, the plietropic effects of the eicosanoids are nowmore deeply appreciated, and it is understood that many of the side ef-fects of these therapies are due to blocking their production. More re-cently, attention has been focused on alternative eicosanoid ordocosanoid metabolites that are anti-inflammatory or pro-resolving in-cluding the EpFAs (for a review of specialized pro-resolving mediators(SPMs) see Chiang & Serhan, 2017). Even compounds considered aspredominantly proinflammatory seem to regulate a series of eventsleading to resolution of inflammation. Targeting the sEH is a novel strat-egy in this scheme because inhibiting sEH sustains the endogenousEpFAs to attain their biological effects. As mentioned above, otherdegredation routes for the EpFA exist such as β-oxidation (Spector etal., 2004). Thus, inhibiting sEH is unlikely to build a large or chronic in-crease in EpFAs, enabling the beneficial effects of theirmodulationwith-out a large side effect profile.

2.1. Inflammatory bowel disease (IBD) and chronic peptic ulcer

EpFAs are also active against inflammatory disorders of the gas-trointestinal tract. Both pharmacological inhibition and gene


ablation of sEH were investigated for their ability to reduce chronicactive inflammatory bowel disease (Zhang et al., 2012). In thisstudy using a genetically engineered IL-10 null mouse model ofIBD, both approaches of limiting sEH activity lowered thenumber of ulcers and transmural inflammation. Quantitative realtime PCR demonstrated an increase in inflammatory cytokines andchemokines including IFN-γ, TNF-α, MCP-1 and VCAM-1 in IL-10null mice compared to double IL-10/sEH null and sEHI reduced IFN-γ, MCP-1 and VCAM-1 mRNA. Western blot analysis also showedthat phosphorylated NFĸB was downregulated and oxylipin analysisrevealed increased EET to diol ratios, and a decrease in both leukotri-ene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE)metabo-lites (Zhang et al., 2012).

The anti-inflammatory efficacy and physiological improvementfound in the IBDmodel are unique to sEH inhibition because the clin-ical use of NSAIDs including COX-2 selective inhibitors exacerbatesIBD (Berg et al., 2002; Kaufmann & Taubin, 1987; Reuter, Asfaha,Buret, Sharkey, & Wallace, 1996). In a later study it was determinedthat sEH gene deletion decreased adenocarcinoma tumors in the IL-10 null mousemodel of IBD (Zhang, Liao, et al., 2013). In this context,the absence of sEH is anti-inflammatory and therefore limits thetransition of IBD to tumor formation in the bowel. However, therole of EpFAs in tumor formation is still poorly understood and likelycomplex. EETs have demonstrated angiogenic activity which is animportant contribution to their regulatory role in hyperemia andmodulation of neurovascular coupling in the cerebral vasculature.Additionally, their angiogenic activity may be useful in improvingwound repair mechanisms (Sander, et al., 2011; Sander, et al.,2013). Angiogenesis is a critical biological process but pathologicalangiogenesis can lead among other things to enhanced tumorgrowth. However, in some preclinical cancer models, particularlywith solid tumors, sEHI can increase tumor growth. For example,with a solid tumor a 10× therapeutic dose of a sEHI for 10 weeks re-sulted in slightly increased tumor growth, angiogenesis and metas-tasis (Panigrahy et al., 2012). In addition to the effects of inhibitingsEH, when EETs were increased using other techniques including ge-netic manipulation, multi-organ metastasis and tumor dormancy es-cape were observed in mice. An interesting enigma is that, in Lewislung xenographs, high doses of sEHI led to angiogenesis and tumorgrowth but, if sEHI were given with celecoxib (a NSAID) or anomega-3 LC-PUFA, sEHI demonstrated dramatically reduced angio-genesis and tumor growth in both lung and breast tumor xenographs(Zhang, Panigrahy, et al., 2013; Zhang et al., 2014). A partial hypoth-esis to explain this is that an angiogenic metabolite of 8,9-EET can beformed by COX enzymes but the metabolite does not form in thepresence of COX inhibitors or competing omega-3 lipids (Rand etal., 2017). The EDPs which cannot form an analogous angiogenic me-tabolite are inherently antiangiogenic and the EETs, in the presenceof celecoxib, are as well. At this point the proangiogenic effects ofsEHI appear minor and the antiangiogenic effects in the presence ofcelecoxib or an omega-3 dietary supplement appear major. Thus,the homeostatic balance of angiogenesis in relation the EpFAsneeds to be further investigated for effects in cancer biology. In con-trast, the role of sEH inhibition and EpFA in blocking inflammation inthe bowel and the subsequent reduction of adenocarcinoma appearsmore robust. In addition to genetically induced models of IBD, sEHinhibition has also demonstrated positive results in models ofNSAID-induced intestinal ulcer. Diclofenac induced ulcers were re-duced by pretreatment of sEHI with efficacy comparable to protonpump inhibitors but at a far lower dose (Goswami et al., 2016). Theeffect on ulcer correlated with increased EpFAs of both omega-6and omega-3 classes. Furthermore, the efficacy of sEHI in this studysuggests that reported beneficial effects of PUFA for ulcer relief(Pineda-Pena, Jimenez-Andrade, Castaneda-Hernandez, & Chavez-Pina, 2012) are potentially mediated by the EpFA metabolites. Italso appears that part of the efficacy of proton pump inhibitors



Table 1Commonly used sEH inhibitors.

TPPU, UC17701-Trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea

Most commonly used sEHI in recentpublications. High activity on theprimate sEH, good activity with rodentsEH and often poor activity on sEH ofother species. High oral availability andgood PK-ADME with PK data availablein many species. Centrally active inmice. Lipophilic and high meltingrequiring a long dissolution time inwater and careful formulation (Inceogluet al., 2013; Rose et al., 2010).

APAU, UC1153, AR92811-(1-Acetypiperidin-4-yl)-3-adamantanylurea

No toxicity at high doses in humanPhase I and II trials. No clinically usefulefficacy in a human hypertension trial.Short half-life in man and rodents. PKdata available in multiple species.Surprisingly high water solubility andrapid dissolution. Potent inhibitor ofrodent sEH, less active on primate sEHand poor activity in many other species.Poor target occupancy with human sEH(Chen, Whitcomb, et al., 2011).

t-AUCB, UC1471trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid

Good potency on sEH from a variety ofspecies. PK-ADME known in multiplespecies. Half-life is longer than AEPUbut shorter than TPPU. Good watersolubility off-sets lower potency andshorter half-life compared to TPPU(Hwang et al., 2007; Shaik et al., 2013).

t-TUCB, UC1728trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid

Good potency on sEH from a variety ofspecies. PK-ADME known in multiplespecies. Longer half-life than t-AUCBabove (Hwang et al., 2007; Liu, Tsai, etal., 2009).

AEPU, UC9501-Adamantanyl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl]}urea

Moderate potency on sEH from a widevariety of species. Short half-life butbioactive metabolites in mice increaseefficacy. Very water soluble anddissolved directly in water. Most watersoluble of the potent sEHI (Liu et al.,2015).

AUDA, UC70012-(3-adamantan-1-yl-ureido) dodecanoic acid

Moderate potency on sEH from a widevariety of species. Short half-life,commercially available (A. N. Simpkinset al., 2009).

GSK 2256294A(1R,3S)-N-(4-cyano-2-(trifluoromethyl)benzyl)-3-((4-methyl-6-(methylamino)-1,3,5-triazin-2-yl)amino)cyclohexanecarboxamide.

Tested in human trials. Good PK-ADMEin human. High melting with poorwater solubility. Careful formulation isneeded. Activity on multiple species notreported(Lazaar et al., 2016; Podolin et al.,2013).

Sorafenib4-(4-(3-(4-chloro-3- 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methylpyridine-2-carboxamide

Potent inhibitor of the human androdent sEH. A registered drug to treatcancer possibly as a Raf-1 or pan kinaseinhibitor. Poor solubility needingcomplex formulation. Numerous sideeffects at high doses (Liu, Park, et al.,2009).

Regorafenib4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]-3-fluorophenoxy]-N-methylpyridine-2-carboxamide

Roughly an order of magnitude morepotent on the human sEH thanSorafenib. Similar side effect spectrumand physical properties (Hwang et al.,2013).

(continued on next page)


Please cite this article as: Wagner, K.M., et al., Soluble epoxide hydrolase as a therapeutic target for pain, inflammatory and neurodegenerativediseases, Pharmacology & Therapeutics (2017), http://dx.doi.org/10.1016/j.pharmthera.2017.06.006


Triclocarban3-(4-chlorophenyl)-1-(3,4-dichlorphenyl)urea or 3,4,4′-trichlorocarbanilide

A reported anti-microbial used in somesolid soaps. Approved for topical useonly. Selective inhibitor of the humansEH and of similar potency to APAUabove on the human sEH. Poorly activein other species. Short half-life in vivofollowing topical application. Rapidlyforms N-glucuronides (Schebb et al.,2012).

For full data sheets on the above compounds, contact authors.


such as omeprazol may be due to a reduction in inflammation causedby a dramatic increase in EpFA (Goswami et al., 2016).

2.2. Destructive bone diseases

2.2.1. ArthritisRecently, an sEHI was evaluated in a randomized, blind preclinical

study in aged dogs with natural osteoarthritic pain compared to vehiclecontrol and is first reported on here. The pain was evaluated with aquestionnaire adapted from the canine brief pain inventory (CBPI).The CBPI was originally developed for owners to assess the pain levelof their pets with existing painful conditions. The questionnaire usedfor this experiment had 12 total questions to assess pain based on activ-ities such as initiation ofmovement, walking, climbing stairs or jumpingfor a reward scoredwith a scale of 0 (not due to pain), 1 (not able to de-termine), and 2 (obviously due to pain) for the first 11 questions. The12th question regarding overall pain was scored on a scale from 0 (nopain) to 4 (unwilling to participate). The dogs were assessed for fiveconsecutive days prior to treatment to establish a baseline measure-ment of their pain. Treatments were with 5 mg/kg of the sEHI t-TUCB(Hwang, Tsai, Liu, Morisseau, & Hammock, 2007) in an oral capsule orplacebo capsule for 5 days. Scores are reported as the percent of maxi-mum possible pain score (score = observed / maximum ∗ 100, maxi-mum = 26 for the entire questionnaire). The 5 days of baselinemeasurements were pooled to compare 5 days of treatment with t-TUCBor placebo. This study revealed that in naturally occurring arthritissEH inhibition was effective in blocking pain (Fig. 3). The sEHI

Fig. 3. sEH inhibition and EpFAs block natural arthritic pain. Male and female beagle dogs(ages 8–14 years) with naturally occurring osteoarthritis were administered the sEHI t-TUCB at 5 mg/kg orally in a capsule or placebo control for five days (n = 7 dogs/group).The results are presented as an average of 5 days of testing pre-treatment (pooledbaseline) compared to an average of 5 days of testing under treatment (placebo or t-TUCB). The sEHI significantly lowered pain scores compared to both pooled baseline andplacebo control (p ≤ 0.001).


significantly lowered pain scores compared to both the pooled vehicleand placebo control (Kruskal-Wallis One Way Analysis of Variance onRanks, H = 21.438 with 2 degrees of freedom, p ≤ 0.001, n = 7 dogs/group). Importantly the results in dogs reported here represent efficacyagainst naturally occurring arthritis and not an induced model. Whilethe study is limited due to the number of dogs naturally presenting ar-thritis and therefore the absence of a positive control, the result of sEHItreatmentwas statistically significant and biologicallymeaningful by in-creasing activity in thedogs. This efficacy in a chronic condition ismean-ingful, particularly in dogs, because they are highly sensitive to the sideeffects of NSAIDs which can be lethal (Mathews, 2000). Inhibition ofsEH has been suggested as an anti-inflammatory strategy for chronicuse in arthritis over NSAIDs and steroidals that have dose limiting sideeffects (Pillarisetti & Khanna, 2012). Schmelzer et al. demonstratedthat sEHI and the resulting increased EETs transcriptionally down regu-late a number of enzymes involved in mediation of inflammation in-cluding induced COX-2 (Schmelzer et al., 2005). sEHI have also beenfound by a number of studies to synergize with NSAIDs. Since sEHI re-duce hypertension which is often associated with long term use ofNSAIDs, as well as gastrointestinal erosion (Goswami et al., 2016) andNSAIDs mediated cardiovascular side effects, (Liu, Li, et al., 2010;Schmelzer et al., 2006) sEHI may act as safeners of NSAIDs when thetwo are combined.

2.2.2. OsteoporosisOsteoporosis differs from arthritis in the amount of asymptomatic

bone resorption often resulting in fragility fractures (Rachner, Khosla,&Hofbauer, 2011). In a recent study, EETs blocked bone loss by effectingosteoclast differentiation in vitro via downregulation of ROS. In addi-tion, EETs administration also reduced bone loss in ovariectomizedmice as a model of estrogen deficiency-induced osteoporosis (Guan,Zhao, Cao, Chen, & Xiao, 2015). In these experiments EETs decreasedROS release, TNFα levels and NFĸB activation. These results supportthe observation that sEHI inhibition has broad efficacy including lyticbone disease.

2.2.3. Chronic periodontitisSome of the available data in periodontitis is focused on the role of

eicosanoids in general. A recent study examined the oxylipins fromhuman periodontitis samples and found inflammatory markers includ-ing increased prostaglandins and leukotrienes (Huang et al., 2014). EETsand diols were also measured in this study but did not exhibit as muchchange. In a preclinical model of periodontitis in mouse, both sEHI andsEH null mice showed reduced bone loss when exposed to A.actinomycetemcomitans. In these animals, the chief osteoclastogenicmolecules RANK/RANKL/OPG and the chemokine MCP-1 were down-regulated and downstream inflammatory JNK and p38 kinase signalingwas abated. In addition, this study demonstrated that ER Stress was up-regulated with periodontal disease but was blocked by both sEHI ad-ministration and in the sEH knockout mice (Trindade da Silva et al.,2017). Thus, given the role of sEHI and EpFAs action against inflamma-tion combined with the effects on other bone loss conditions, sEH inhi-bition may be a useful strategy to combat periodontitis.




2.3. Sepsis

Sepsis or endotoxemia in humans is a diverse syndrome of physio-logical (i.e. systemic arterial hypotension) and immunological re-sponses to microbial infection that include increased circulating levelsof cytokines TNFα and IL-6 and high mobility group protein B1(HMGB1).Historically, therapeutic approaches using antibodies to com-bat sepsis have had poor results (reviewed in Fink et al. (Fink, 2014))and blunting of the innate immune response shows more promise.The approach of using sEHI to block the inflammatory response inmodeled sepsis prevented lipopolysaccharide (LPS) induced death, re-duced pro-inflammatory cytokines, chemokines and prostaglandins(Schmelzer et al., 2005) and restored systolic blood pressure to nearnormal levels while increasing EETs to diol ratios (Liu, Tsai, et al.,2009). One can envision sEHI as muting the cytokine storm and speed-ing resolution. The level of linoleic acid diols in the plasma may be avaluable marker for the onset of sepsis. Since they cause the may becausative as well (Moghaddam et al., 1997; Slim et al., 2001). Thus, al-though animal models of sepsis are inadequate to completely mirrorthe human immune response in this condition, they remain a criticalpath to the development of new therapeutic strategies and sEH inhibi-tion shows great promise. The difficulty of designing a clinical trial forsepsis is due to its often unpredictable and rapid progression as wellas defining a profitable clinical path makes sepsis a difficult target. Pos-sibly treating other conditions leading to a cytokine storm such as viralinfections or the severe inflammation initiated bymodern immunother-apy such as CAR-T for cancer represent more reasonable paths foragents as sEHI that can moderate the cytokine release syndrome(DeFrancesco, 2014).

2.4. Cardiovascular disease

The largest literature base on the anti-inflammatory properties ofEpFAs is in the field of cardiovascular physiology and pathology. Bythe early 1980s there was a shift in cardiovascular research from afocus solely on the description of anatomic structures (e.g. the endothe-lium of blood vessels) to their actual function (Furchgott & Zawadzki,1980). The action of aspirin had been elucidated by John Vane whoshared the 1982 Nobel Prize in Physiology or Medicine with laureatesSune Bergstrom and Bengt Samuelson who described the prostaglandinlipid metabolites. As the biological activity of select prostaglandins invasculature was further elucidated (Moncada & Vane, 1979) a series ofpapers on the sEH enzyme was published (Gill & Hammock, 1980). Itwas later found that sEH is a master regulatory enzyme of the endoge-nous EETs which were revealed to have vasodilatory effects (Singer &Peach, 1983). The effect of EETs on the modulation of vascular tone ismore thoroughly reviewed in Sudhahar et al. (2010). In the 1990s therelationship between atherosclerosis and inflammation was recognized(Boring, Gosling, Cleary, & Charo, 1998). This underscored the impor-tance of EpFAs action in the cardiovasculature and implied that theanti-inflammatory properties may be a vital part of their antihyperten-sive action (Libby, Ridker, &Maseri, 2002). Thiswas investigated in pre-clinical models where CYP2J2 epoxygenase overexpression and directEETs administration inhibitedVCAM-1 inducedwith TNFα independentof hyperpolarizing effects (Node et al., 1999). Anti-inflammatory effectsincluding inhibiting activation of NFĸB, cellular adhesion molecules, cy-tokine expression, and neutrophil infiltration were demonstrated invivo in mice overexpressing CYP2J2, CYP2C8 and sEH knockouts(Deng et al., 2011). Additionally, an EET analog decreased inflammationand oxidative stress which attenuated renal injury in Dahl-salt sensitiverats (Hye Khan et al., 2013) and EpFA action, demonstrated by anenriched omega-3 diet combined with administration of an sEH inhibi-tor, decreased renal inflammation in an angiotensin- II model of hyper-tension (Ulu et al., 2013).

The main mechanisms of this anti-inflammatory action in thecardiovasculature are via endothelial cells and monocytes and several


putative receptors including PPAR, GPCRs and TRP channels. Shearstress was demonstrated to attenuate inflammation through PPARgamma mediated signaling (Liu et al., 2005). A radiolabeled EET analogwas used to determine that EETs have the capacity to bind to a cell sur-face receptor (Chen, Falck, et al., 2011) and this may occur through a Gprotein (Li & Campbell, 1997). TRPV4 channels are active in regulatingvascular tone and this is one of the suggested mechanisms of EETs ac-tion in vasodilation (Sonkusare et al., 2012; Vriens et al., 2005;Watanabe et al., 2003). However, it should be cautioned that the actionof EETs in the cardiovasculature is complex and a single regioisomer canact selectively in different vascular beds and tissues (Hercule et al.,2009; Quilley & McGiff, 2000). Additionally, there is some evidence ofEET action directly at the TRP channels (Watanabe et al., 2003), butthere is also evidence that EETs act via increased translocation of otherTRPC receptors to the cell surface (Fleming et al., 2007). There is alsosome evidence of TRPA1 activity in excitable cells with one regioisomer(Brenneis et al., 2011). Overall the EET-TRP axis (Loot & Fleming, 2011)is incompletely described in vasculature, and it is even more poorly un-derstood in the central nervous system. Despite this incomplete mech-anistic knowledge, evidence inmultiplemodels across different types ofbiology and in several species demonstrates the anti-inflammatory ac-tion of EpFAs. The bioactivity of the EpFA points out the potential oftargeting sEH to modulate inflammation.

3. sEH as a target for neurodegenerative diseases

With the general population ageing, the incidences of neurodegen-erative diseases and chronic inflammatory conditions are both on therise. Thus, there is an urgent need for new approaches to mitigateneuro-inflammation. The role of the EpFAs in regulating inflammatoryconditions particularly in the brain is a potential target and inhibitingsEH as a strategy to sustain their biological activity is a novel approachwith great promise. Here we outline the role of EpFA and sEH in severalmodeled neurological diseases with an inflammatory component.

3.1. Stroke

EpFAs, the EETs in particular, are known to be protective against celldeath in ischemic stroke (Alkayed et al., 1996; Iliff et al., 2010; Simpkinset al., 2009). Importantly, evidence about sexually dimorphic gene ex-pression and stroke outcomes demonstrate the protective effects oflowering sEH enzyme and increasing EpFA producing CYP450s(Alkayed et al., 1996; Gupta, Davis, Nelson, Young, & Alkayed, 2012;Koerner et al., 2007; Zhang, Otsuka, et al., 2008). A sEH genetic polymor-phism in humanshas been linked to stroke risk aswell as cardiovasculardisease (Fornage et al., 2005) and notably a G860A sEH polymorphismthat reduces sEH activity and not CYP2J2 gene expression had a protec-tive effect in nonsmoker stroke in China (Zhang, Ding, et al., 2008).Overexpression of sEHwith a K55R polymorphism in Swedishmen cor-related with hypertension and increased ischemic stroke risk (Fava etal., 2010). In addition to expression of sEH in endothelial cells, sEH isexpressed in astrocytes, oligodendrocytes, and neurons in humanbrain (Sura et al., 2008). In mice, sEH expression is found in astrocytesand neurons and has been characterized in brain regions providingbackground for preclinical models of stroke and other neurological dis-orders (Marowsky et al., 2009; Zhang et al., 2007). In rat, neurons andastrocytes produce EETs (Alkayed et al., 1996; Amruthesh, Falck, &Ellis, 1992) suggesting there is a fundamental regulation of EpFA bioac-tivity in the cells of the brain. As an example, it has been demonstratedthat EETs are involved in neurovascular coupling (Farr & David, 2011;Higashimori, Blanco, Tuniki, Falck, & Filosa, 2010). Inhibiting sEH hasalso demonstrated protective effects against ischemic brain damage in-dependent of effects on cerebral blood flow (Iliff et al., 2009; Zhang etal., 2007). It is important to note that using sEHI as a strategy hasoften been favored over exogenous EETs administration in these studiesbecause EETs are quickly metabolized (Imig, Simpkins, Renic, & Harder,




2011). Thus, these results reflect on the elevation of potentially severalclasses of EpFAs.

3.2. Seizure

Early attention for treating epilepsy was focused on anticonvulsantswhich regulate ion channels and affect mediator proteins to control sei-zures. While this approach greatly improved the quality of life for epi-leptics, they did little prevent seizures. Current research is focused onneuroinflammation and neural plasticity as potential targets to preventrecurrent seizures. The observation that blocking leukocyte infiltrationblocked epileptogenesis clarified the role of inflammation in initiatingepilepsy (Fabene et al., 2008). However, inflammation is also a conse-quence of seizure and plays a role in the development of epilepsy afterthe first seizure event (Vezzani, French, Bartfai, & Baram, 2011). Thus,preventing neuroinflammation that promotes seizures or treating neu-roinflammation post seizure event are both current therapeutic targets.

The literature on the benefits of EpFAs for seizure reveals there is asubstantial release of PUFA in the seizure events and neuroinflamma-tion occurs secondary to them (Bazan, Birkle, Tang, & Reddy, 1986).An approach to mitigate the seizures and subsequent neuroinflamma-tion by using sEHI, sEH gene ablation and direct administration ofEETs to the brain demonstrated delayed onset of GABA mediated sei-zure (Inceoglu et al., 2013). Furthermore, sEHI are efficacious in bothchemical and electrical models of temporal lobe epilepsy. In pilocarpineinduced status epilepticus (SE), sEHI increased EETs and EET/DHET ra-tios, reduced neuroinflammation, aswell as seizure frequency and dura-tion (Hung et al., 2015). In the same study, sEHI increased seizure-induction thresholds in epileptogenesis induced by electricalbasolateral amygdala (BLA) kindling. In addition to controlling neuroin-flammation, sEH inhibition has been shown to influence synaptic trans-mission and neuroplasticity byupregulatingNMDAreceptors (Wu et al.,2015). These data suggest that increasing EpFA levels by inhibiting sEHhas potential to halt seizures by reducing neuroinflammation and in-creasing neural plasticity.

3.3. Alzheimer's disease

There is consensus in the current literature that a large componentof the pathogenesis of Alzheimer's disease (AD) is neuroinflammation(Wyss-Coray & Rogers, 2012). The pathogenesis of AD is also relatedto oxidative stress and mitochondrial dysfunction (Sultana &Butterfield, 2010). Thesemechanisms are viewed as either contributingto the formation of or subsequently adding to the damage caused by β-amyloid plaques and tau-protein tangle deposition (Bronzuoli,Iacomino, Steardo, & Scuderi, 2016; Minter, Taylor, & Crack, 2016). Ithas also been demonstrated that PUFA concentrations decline withage in vivo and much study has been made of supplementation inhumans (Dacks, Shineman, & Fillit, 2013; Freund-Levi et al., 2006). Ithas been hypothesized that the anti-inflammatory benefits of fish oil,which contains high levels of omega-3 fatty acids, are beneficial in lim-iting the damage of neurological inflammation leading to amyloidplaque formation (Barberger-Gateau et al., 2002; Fiala et al., 2015). Un-fortunately, the results have been mixed with little improvement seenwith omega-3 fatty acids for established dementia but some improve-ment for mild cognitive impairment (MCI) as a precursor to AD(Boudrault, Bazinet, & Ma, 2009). These inconsistent results might bedue, in part, to poor characterization of dietary components or highlevels of lipid peroxide in the omega-3 LC-PUFAused. It has been recent-ly observed that this may relate to the role of APOE4 and delivery ofDHA to the brain (Yassine et al., 2017). Despite this, omega-3 supple-mentation is still regarded as a meritorious strategy given that it iswell tolerated and has minimal side effects.

There are opposing biological effects of omega-6 versus omega-3 LC-PUFAs (Schmitz & Ecker, 2008), but there are opposing effects withinonly the omega-6 derived metabolites as well. Several of the omega-6


generated prostaglandins have been implicated in contributing to in-flammation including in the brain. Lowering amyloid precursor proteinlevels and limiting production of pro-inflammatory mediators by usingNSAIDs to block COX metabolite production has been newly suggestedas a strategy to protect against AD (Herbst-Robinson et al., 2015). How-ever, this type of pharmacological intervention has been limited by thelack of central nervous system (CNS) penetration in addition to thewell-known gastrointestinal and hematological side effects of NSAIDs.

The EETs, on the other hand, demonstrated interaction with mito-chondria in the presence of β-amyloid. Select regioisomers of EETswere able to reduce mitochondrial membrane depolarization and frag-mentation improving cellular respiration (Sarkar et al., 2014). The 14,15EET regioisomer and sEHI both increased cell viability in modeled reac-tive oxygen stress induced by hydrogen peroxide challenge. A CYP450inhibitor, miconazole, decreased cell viability in co-cultured astrocytesand dopaminergic neurons (Terashvili, Sarkar, Van Nostrand, Falck, &Harder, 2012). β-amyloid also reduced EET synthesis in hippocampalastrocytes and neurons (Sarkar, Narayanan, & Harder, 2011), thus,supporting the levels of EETs by inhibiting sEH holds great promise byimproving mitochondrial function in this pathological condition. Morerecently molecular mechanisms of this action have been revealed inhuman embryonic kidney 293 (HEK293)/Tau cells activated by hydro-gen peroxide (Yao, Tang, Liu, &Wang, 2016). Cell viabilitywas increasedwith sEHI corresponding to a lower phosphorylation of tau protein, up-regulation of p-AKT and greater GSK-3β phosphorylation. Related to ADthe investigation into the relationship of EpFAs and sEH, EETs specifical-ly, in vascular cognitive impairment in humans revealed increasedDHETs in VCI patients (Nelson et al., 2014). EETs and sEHI have also in-creased axonal outgrowth in primary cultures of cortical, sympatheticand sensory neurons from rat (Abdu et al., 2010). Thus, both EpFA ad-ministration and sEH inhibition are viable strategies in blocking delete-rious events contributing to AD.

3.4. Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder that hasboth genetic and environmental etiologies. There is substantial evidencefor reactive oxygen species and oxidative stress in the damage that oc-curs to dopaminergic signaling neurons that give rise to the motor dys-function characteristic of the disease (Dauer & Przedborski, 2003). PD isthe second leading age related neurodegenerative disease after AD andthe hallmarks of the disease are oxidative stress,mitochondrial dysfunc-tion, and abnormal protein aggregation such as alpha-synuclein in Lewybodies (Dauer & Przedborski, 2003; Halbach, Schober, & Krieglstein,2004). More recently the role of secondary neuroinflammation relatedto oxidative stress has been investigated as the route to neuronaldeath in PD (Hirsch & Hunot, 2009; Hirsch, Vyas, & Hunot, 2012). In ad-dition to the anti-inflammatory effect of EpFAs, EETs have demonstratedeffects in preventing mitochondrial dysfunction (Liu et al., 2011). Anearly investigation of allylic human sEH polymorphism and risk of de-veloping PD was inconclusive (Farin et al., 2001), however, preclinicaltesting has demonstrated efficacy of both sEHI and sEH gene knockoutagainst MPTP-induced Parkinsonism (Qin et al., 2015). In this model,sEH deficiency and inhibition prevented dopamine (tyrosine hydroxy-lase-positive) neuronal loss and improvedmotor performance. Further-more 14,15 EET administration protected dopaminergic neurons inmice treated with MPTP. Thus, because of the role neuroinflammationand mitochondrial dysfunction play in PD and the efficacy in preclinicalmodels, it is hypothesized that sEH inhibitionmay have potential bene-fit in this condition in humans (Lakkappa, Krishnamurthy, Hammock,Velmurugan, & Bharath, 2016).

3.5. Depression

There is ample evidence that LC-PUFAs have a role in depression (Linet al., 2012; Martins, Bentsen, & Puri, 2012). It is also understood that




inflammation can be an integral part of depression. The rate limiting en-zyme indoleamine 2,3-dioxygenase (IDO) that metabolizes tryptophanand thereby limits serotonin production is under regulation of inflam-matory cytokines (O'Connor et al., 2009; Oxenkrug, 2010). In addition,increased inflammatory cytokines have been observed in human clini-cal patients as well as in post mortem brains from individuals withmajor depressive disorder (Dean, Tawadros, Scarr, & Gibbons, 2010;Haapakoski, Mathieu, Ebmeier, Alenius, & Kivimaki, 2015; Strawbridgeet al., 2015). This is the background rationale for the use of inflammato-ry agents to induce depressive states in preclinical models (Frenois etal., 2007; Fu et al., 2010; Miller, Maletic, & Raison, 2009).

Recently, to exploit the anti-inflammatory properties of EpFAs, theeffects of sEH inhibition and genetic ablation have been demonstratedin depression models. Use of the sEHI lowered TNFα in LPS treatedbut not control mice (Ren et al., 2016). These experiments also investi-gated the effects of 14,15 EET and sEHI on neuronal outgrowth in PC12cells. Other antidepressants have an effect onneuronal plasticity and theresults of elevating EpFAs in either manner showed increased NGF-in-duced neurite outgrowth in these cells (Ren et al., 2016). In behavioraltests, sEHI administered both prophylactically and therapeutically re-sulted in less inactivity in LPS induced depression in mice. sEH inhibi-tion was also effective against social defeat stress in these studies. sEHinhibition did not alter body weight in control mice but increased su-crose preference in social defeatmice. sEH gene deletion conferred resil-ience to social defeat stress with sEH knockout mice exhibiting similarsocial interaction time to non-stressed wild type controls. Importantly,when brain-derived neurotrophic factor - tropomyosin receptor kinaseB (BDNF-TrkB) signalingwas investigated for its role in themodeled de-pression, it was revealed that sEH knockout mice have higher BDNFlevels in several brain regions compared towild type controls. Addition-ally, though all groups had similar tissue levels of TrkB, there was ahigher p-TrkB to TrkB protein ratio in sEH null mice as well as synapto-genesis markers (Glutamate receptor subunit GluA1 and post synapticdensity protein PSD-95) by Western blot. Thus, these mechanismsmay be involved in the resilience to social stress in sEH null mice.

This study also quantified sEH protein levels whichwere higher in inboth murine induced stress models compared to controls. This resultparalleled with increases in post mortem human brain (parietal cortex)samples from depressed, bi-polar or schizophrenic humans examinedfor sEH protein levels. An important finding in this work that meritshighlighting is the rapid onset of anti-depressive action of sEHI in bothmodels of depression. This is noteworthy because pre-clinically aswell as clinically, current anti-depressant therapies take several weeksto be at full effect (Gaynes et al., 2009; Krishnan & Nestler, 2010). Thisrapid anti-depressive action is without any observable side effects un-like ketaminewhich is now known to have rapid effects against depres-sion but has major psychotomimetic side effects and abuse potential(Kirby, 2015). Thus, it is now suggested that sEHI may be a promisingnew strategy to combat depression (Hashimoto, 2016).

Depression is the mental disorder with the highest numbers ofeffected individuals world-wide but there are other mental conditionsthat reveal similar changes in sEH and EpFA biology. Increased sEH ex-pression has been observedwith anorexia nervosa in human clinical pa-tients whichmay be associated with depression and anxiety (Scott-VanZeeland et al., 2014; Shih et al., 2016). Additionally, there may be a roleneuroinflammation plays in the pathogenesis of anxiety, post-traumaticstress disorder and obsessive compulsive disorder (Furtado & Katzman,2015) and thus an opportunity for sEH inhibition to improve mentalhealth as well as physical ailment is very broad.

4. sEH as a target for pain

The sEHI and EpFAs have demonstrated greater potency thanNSAIDs and synergism with inhibitors of both COX and LOX enzymesin reducing inflammation. It is therefore not surprising that this efficacyextends to pain, one of the hallmarks of inflammation. Pain is a complex


signaling network that stems from noxious insult or tissue injury andrelease of inflammatory mediators such as cytokines, ions, bradykinins,prostaglandins and leukotrienes amongothers. These act on nociceptorsdirectly and drive action potentials signaling the sensation of pain(Woolf & Ma, 2007). Typically, pain is a signal to avoid further damageuntil the crisis resolves. However, there are situations where the alter-ation to nociceptors allows pain to persist beyond the inflammation orinsult, and this signaling in the absence of stimulation is a pathologicalpain termed neuropathy.

The mechanisms driving neuropathic pain are still poorly under-stood and perhaps therefore also poorly treated. New approaches areurgently needed.While there aremore available approaches to treat in-flammatory pain, most of these therapies have dose or use limiting sideeffects and new approaches are desirable. Herewe outline targeting thesEH as novel strategy with the unique property of being efficaciousagainst both inflammatory and neuropathic pain.

4.1. Inflammatory pain

Targeting sEH for pain relief arose from initial investigations of theanti-inflammatory roles of sEHI. In studies that investigated LPS inducedsepsis a sophisticated mass spectrometry analysis revealed thatinhibiting sEH altered not only the EETs and diol metabolite levels, butalso several other metabolites in the COX and LOX metabolism path-ways of the ARA cascade (Schmelzer et al., 2005). In particular, targetingthe sEH with small molecule inhibitors lowered levels of PGE2, a potentinflammation mediator and algogen, in addition to NO and other cyto-kines. This finding was groundbreaking because it demonstrated thatstabilizing endogenous bioactive lipids was a novel strategy to limit in-flammation compared to other enzyme inhibitors that blocked produc-tion of inflammatory metabolites. The observed shift in otherprostaglandin metabolites and decrease in COX-2 protein levels withinhibiting sEH was the basis for hypothesizing that sEH inhibitionwould limit inflammatory painwhichwas investigated in several subse-quent studies (Inceoglu et al., 2006; Schmelzer et al., 2006).

sEHI were first tested in a model of inflammatory pain for their abil-ity to synergize with COX-2 selective NSAIDs due to the previously ob-served effect of sEHI suppressing the COX-2 enzyme (Schmelzer et al.,2006). This resulted in synergistic decreases in PGE2, reduced COX-2 ex-pression and increased thermal withdrawal latencies in mice. Impor-tantly, these improvements occurred without an observable change inthe prostacyclin to thromboxane ratio. Changes in the homeostatic bal-ance of these COX metabolites are suspected to initiate thromboticevents that are adverse side effects of COX-2 selective NSAIDs(Fitzgerald, 2004).

The sEHI were then tested as single administrations to determine ifthey were anti-hyperalgesic by themselves. Topical administration oftwo different sEHI was effective in increasing both thermal withdrawallatencies andmechanical thresholds in an LPS inflammatory painmodelin rat (Inceoglu et al., 2006). This study also investigated the direct top-ical administration of EETs which revealed the EpFA metabolites wereanti-hyperalgesic by increasing thermal withdrawal latencies againstLPS pain. Interestingly EETs demonstrated a slight pro-nociceptive effectin naïve rats compared to the sEHI which had no effect in the thermalassay. The anti-hyperalgesic action was demonstrated to have a multi-factorialmechanism. Interestingly, in this study COX-2 expression levelsin spinal cord did not correlate with decreased pain behavior but sEHIelicited anti-hyperalgesia which distinguishes them from glucocorti-coids which repress inducible COX-2 to produce anti-hyperalgesia(Brostjan, Anrather, Csizmadia, Natarajan, & Winkler, 1997; Inceogluet al., 2008). To further explore this, EETs were screened against apanel of cell receptors and were found to bind the translocator protein(TSPO) which transports cholesterol through mitochondrial mem-branes. The TPSO cooperates with steroidogenic acute regulatory pro-tein (StARD1) which is upregulated by EETs (Inceoglu et al., 2008;Miller, 2007; Wang et al., 2006). It was evident from these experiments



Fig. 4. sEH inhibition and EpFAs block chronic neuropathic pain. The use of surgicalmodelsof neuropathic pain allows the testing of the chronic nature of the developed pain state. Inthe chronic constriction injury (CCI) model a 3.0 mg/kg oral gavage dose of the sEHI TPPUwas effective in male and female rats against neuropathy assessed with a von Frey assay.TPPU treatmentwashighly significant compared to PEG300 vehicle control inmales (**p b

0.001) and was also significant in females compared to vehicle control in females (*p b

0.010).


that the activity of sEH inhibition depends on a cellular factor present ininflammation that has a role in activating the TSPO/StARD1 pathway;this was identified as elevated cyclic adenosine monophosphate(cAMP).

Individual regioisomers of the EpFAs have also been examined foranti-hyperalgesic activity (Morisseau et al., 2010). This study demon-strated that all classes of EpFAs were efficacious against inflammatorypain, and that the EpFAhad higher activity than the parent LC-PUFA. Im-portantly, the DHA derived EDPs appear to be more potent anti-hyperalgesics than the other classes. This merits using sEHI as a strategybecause it elevates the levels of several EpFA as opposed to single ad-ministering of one type of epoxidized metabolite or an epoxide mimic.

Importantly, sEHI can also block the pain produced by PGE2 admin-istration (Inceoglu et al., 2011). This was observed in examining thepain dependence of sEHI action which revealed several pieces of infor-mation. First, sEHI are distinct from NSAIDs and steroids which act up-stream of PGE2 and prevent its production, and second, that sEHIactivity depends on the amount of pain present and does not alterpain thresholds in the absence of a painful condition. Because the actionof sEHI was previously hypothesized to require the presence of cAMP, aphosphodiesterase inhibitor (PDEI) used to block the inactivation ofcAMP was co-administered with sEHI in this pain model. This strategyresulted in an enhanced response compared to single administrationof the sEHI or PDEI. Oxylipin analysis revealed that PDEI increasedEpFAs which may contribute to their action in the CNS (Inceoglu et al.,2011). Thus, the EpFAs are analgesic in several models of induced in-flammatory pain and the anti-hyperalgesia relates to direct administra-tion of the EpFA but also to sEH inhibition; both producing anti-hyperalgesic activity only in active pain states.

4.2. Neuropathic pain

The initial investigation of sEHI efficacy in neuropathywas intendedto compare a painmodel with relatively low COX-2 induction to inflam-matory pain. Itwas a great surprisewhen sEH inhibition blocked diabet-ic neuropathy, a chronic pain model that was proposed as the negativecontrol model (Inceoglu et al., 2008). This was surprising because mostNSAIDs that block inflammation have little efficacy against neuropathicpain (Gore, Dukes, Rowbotham, Tai, & Leslie, 2007;Wagner et al., 2013).

Exploring this efficacy against diabetic neuropathy in a preclinicalmodel revealed that there were dose dependent improvements in me-chanical pain thresholds and that the sEHI outperformed the standardof care gabapentin (Inceoglu et al., 2012). This anti-hyperalgesiawas in-dependent of changes in glucose tolerance, insulin tolerance and glu-cose stimulated insulin secretion. Interestingly, the sEH enzymeactivity was increased although the synthetic capacity to form EETswas similar between diabetic and control rats. There is debate as tothe relevance of testing evoked responses to stimulus in a chronic neu-ropathy that has the hallmarks of pain in the absence of stimulation andtonic pain. Therefore, the conditioned place preference paradigm wasused to further examine the efficacy of the sEHI in diabetic neuropathicpain. This assay combines compound administration with environmen-tal cues during training to assess an acquired place preference associat-ed with pain relief. Studies employing this technique demonstrated thedose dependent efficacy of the sEHI, and importantly, that as potent an-algesics, the sEHI lacked rewarding effects in the absence of pain(Wagner, Yang, Inceoglu, & Hammock, 2014). Importantly, direct ad-ministration of EpFAs also induced a conditioned response in diabeticmice (Wagner, Lee, Yang, & Hammock, 2016). Further investigation ina naturally occurring Type I diabetic mouse model (Akita) demonstrat-ed the efficacy of the sEHI (Wagner et al., 2017). Use of the Akita miceshowed the sEHI are effective against diabetic neuropathy and more-over that sEH activity correlates with disease severity in this strain.

Recently the chronic constriction injury (CCI) model (Bennett & Xie,1988) was used as another paradigm to test the efficacy of sEHI againstchronic pain. Surgical models of neuropathic pain allow the testing of


the chronic nature of the developed pain state in contrast to acute noci-ceptive pain and offer an alternative to chemical agents that may altermetabolism. Both male and female adult Sprague Dawley ratsunderwent the surgical ligature with 4 loose ligatures of the sciaticnerve. Several weeks after surgery when the incision had healed andthe neuropathy due to the ligatures had developed, the rats weredosed by oral gavage with vehicle (PEG300) or 3 mg/kg of the sEHITPPU (Rose et al., 2010) and testedwith the von Frey assay for their sen-sitivity to mechanical touch. An electronic aesthesiometer attached to arigid tip probe was used to measure the response of both the ipsilateral(Fig. 4) and contralateral (not shown) hind paws. The points depicted inthe graph represent the average score and standard error of mean for agroup of rats that were assayed each 3–5 times per time point with a 1-minute interval between repeated stimulation. The TPPU treatmentwashighly significant in males (p b 0.001) and females (p b 0.010) com-pared to their respective vehicle controls (Two Way ANOVA, Holm-Sidak method post hoc, n = 3/group). The time course demonstratedthat there is both long lasting efficacy and rapid action relieving painby 30min after oral gavage. The males also had a significantly more ro-bust response (pb 0.001) than the females possibly due to thehigher di-morphic sEH expression observed in male versus female rodents (Gill &Hammock, 1980;Wagner et al., 2017). Overall, the sEHIwas an effectivestrategy in both male and female CCI neuropathic rats which supportsearlier data in streptozocin induced diabetes in rat and both inducedand natural diabetes in mouse (Inceoglu et al., 2012; Wagner et al.,2014, 2017).

Adding to the evidence in preclinical species, sEH inhibition is suc-cessful in blocking neuropathic pain in veterinary patients presentingwith pathological painful disease. The sEHI t-TUCB was used to treat se-vere equine laminitis, a condition that often requires humane euthana-sia of the horses. The first use of sEHI in this condition was concurrentwith failing standard of care therapy and allowed the horse to stand,eat and later recover (Guedes et al., 2013). Subsequent studies havedemonstrated the continued success of sEHI as a strategy to combatthis condition (Guedes et al., 2017). Thus, sEH inhibition is active againstsevere neuropathic pain under conditions where standard of care ther-apies have failed and has broken the species barrier.

The efficacy of EpFA mediated analgesia in neuropathic pain re-vealed another independent mechanism of action. Diabetic rats wereassessed for activation of ER Stressmarkers and the effect of sEH inhibi-tion (Inceoglu et al., 2015). ER Stress occurs when the homeostatic




protein folding and trafficking in the cell is overwhelmed or unbalancedand leads to the unfolded protein response (UPR) and often to apoptosis(Hotamisligil, 2010). Investigation into the beginning pathogenesis ofdiabetes has long implicatedmisfolded proteins and ER Stress as precur-sors to the UPR and pancreatic beta islet apoptosis (Kozutsumi, Segal,Normington, Gething, & Sambrook, 1988; Oyadomari, Araki, & Mori,2002). ER Stress is regulated by three main membrane associated sen-sors, protein kinase R-like endoplasmic reticulum kinase (PERK), inosi-tol-requiring enzyme 1α (IRE1α) and activating transcription factor 6(ATF6) which induce the UPR when activated (Fig. 5). Administrationof sEHI to diabetic rats was able to suppress these markers in skin andspinal cord and elicit anti-hyperalgesic effects in the animals (Inceogluet al., 2015).

The role of ER Stress in the pathogenesis of diabetes is wellestablished but there are alsomany links between ER Stress and inflam-mation including the activation of NFĸB pathway, ROS production and c-Jun N-terminal kinase (JNK) signaling (Hotamisligil, 2010). In addition,there is evidence that the cellular dysfunction of ER Stress has conse-quences for neurodegeneration and progression of diseases such as AD(Lin, Walter, & Yen, 2008; Riederer, Leuba, Vernay, & Riederer, 2011;Sin & Nollen, 2015). Thus, protecting against the trio of ER Stress, ROSand mitochondrial dysfunction is the unifying action that allows thesEHI to be beneficial in a wider variety of pathological conditions.

4.3. Potential side effects of targeting sEH

Importantly, the analgesic efficacy of sEH inhibition occurs withoutthe common side effects of NSAIDs or narcotic analgesics. The well de-scribed gastrointestinal (GI) ulceration produced by NSAID use is not

Fig. 5. sEH inhibition and EpFAs block Endoplasmic Reticulum Stress (ER Stress). A variety ofproteins, high glucose as in diabetes, or reactive oxygen species (ROS) which can be contributER Stress pathway and stabilize mitochondria (not shown). The three key protein sensors of(ATF6) and PKR-like endoplasmic reticulum-resident kinase (PERK). Downstream of IRE1αrelease the active NH2-terminal domain ATF6(N) and both enter the nucleus as transcription f2 (eIF2α), ATF4 activation and transcription of C/EBP homologous protein (CHOP) a major pexcessive, prolonged, or insufficiently neutralized and is initiated through downstream pathwa single EET regioisomer is depicted on the right. ARA released by phospholipase A2 from the pmetabolized by the sEH into a less active 14,15 DHET. Inhibiting sEH maintains the EpFAs wand ATF6(N) expression. In addition to this action, sEHI also normalize phospho-p38 and phodoes not lead to changes in ER Stress pathways.


trivial given the significant rate of hospitalization and death associatedwith GI bleeding, particularly in elderly patients (Lazzaroni & BianchiPorro, 2004; Shah & Mehta, 2012). There are also cardiovascular effectsof NSAIDs including the selective COX-2 inhibitors that were thought tospare the GI side effects (Lazzaroni & Bianchi Porro, 2004). Interestingly,it has been demonstrated that the anti-inflammatory action of sEH inhi-bition does not have GI side effects, in fact asmentioned previously, sEHinhibition can mitigate GI ulceration caused by NSAIDs (Goswami et al.,2016). Inhibiting the sEH does not alter the thromboxane to prostacy-clin ratio in vivo (Schmelzer et al., 2005) or clotting time (Liu, Yang, etal., 2010; Schmelzer et al., 2006) and thus are not anticipated to havethe cardiovascular side effects associated with NSAIDs. The other in-tense side effects of NSAIDs including induced hypersensitivity,NSAID-exacerbated cutaneous disease, respiratory disease and/or in-duced urticaria/angioedema have not been observed with sEH inhibi-tion in preclinical models, though specific tests for these reactionshave not been reported on.

It has also been demonstrated that the analgesia mediated by sEH in-hibition does not exhibit the hallmarks of narcotic pain therapies. Typicalopioid narcotics cause tolerance, withdrawal and addiction as on-targetside effects and also have severe off-target side effects of respiratory de-pression and constipation. Recent studies of sEH inhibition in chronicpain states have used an operant conditioned preference assay to notonly assess pain relief but also the addictive potential of sEHI mediatedanalgesia (Wagner et al., 2014). This work demonstrated inhibiting sEHdid not produce reward seeking behavior (a conditioned place prefer-ence) in naïve and sEH null mice. Additionally, direct administration theEpFAs demonstrated both analgesic efficacy against neuropathic pain aswell as a lack of rewarding side effects (Wagner et al., 2016).

biological signals can influence the ER Stress pathway such as unfolded and miss foldeded by mitochondrial dysfunction and other sources. EETs reduce the effects of ROS on thethe ER Stress are inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6X-box binding protein-1 when spliced (XBP1s) is activated, similarly ATF6 is cleaved toactors. Phosphorylated PERK results in the phosphorylation of eukaryotic initiation factorarticipant in genes involved in apoptosis. Apoptotic responses occur when ER Stress isays such as ER-associated protein degradation (ERAD) and CHOP. The enzymatic flow ofhospholipid bilayer is acted on by CYP450 epoxygenase to form 14,15 EET and would be

hich block phosphorylation of PERK, eIF2α, and IRE1α and significantly decrease XBP1sspho-JNK, kinase mediators of neuropathic pain. However, in healthy rats, sEH inhibition




5. Conclusion

Inhibiting sEH stabilizes endogenous EpFAs that have demonstratedbeneficial effects in regulating inflammation including in neurologicaldiseases in addition to combatting chronic and inflammatory pain inpreclinical models. sEH inhibitors have been optimized as experimentaltools in the past several decades. Thesemolecules designed as transitionstate mimics of sEH have improved in potency to the single nanomolarrange. There are commercially available sEH inhibitors (AUDA, t-AUCBand TPPU) as well as published methods for their synthesis and alarge body of literature describing the biological effects of their use inexperimental models. In particular, the analgesia mediated by sEHI inpreclinical models is novel because it is non-narcotic, out performsNSAIDs without their side effects and has advantages for conditionswith inflammatory and hypertensive comorbidities. Additionally, theability of EpFAs to reduce neuroinflammation has great potential to in-tervene in the progression of neurodegenerative diseases. Overall, therobust analgesia in both inflammatory pain and chronic painful condi-tions typically refractory to most therapies offers promise for a new ap-proach to alleviate pain. Building on the efficacy of sEHI against pain andinflammation in preclinicalmodels and clinical veterinary patients, sEHIare being developed for use in humans. Previous clinical trials with sEHinhibitors such as GSK2256294 have demonstrated that sEH inhibitorshave the potential for broad application and targeting sEH is well toler-ated. Optimized inhibitors of sEH are currently being advanced to theclinic for the treatment of diabetic neuropathy.

Conflict of interest statement

The University of California holds patents on the sEH inhibitors usedin this study as well as their use to treat inflammation, inflammatorypain, and neuropathic pain. BD Hammock and CB McReynolds are co-founders and KM Wagner and WK Schmidt are employees of EicOsisL.L.C., a startup company advancing sEH inhibitors into the clinic.

Acknowledgments

This work was supported by the National Institute of EnvironmentalHealth Sciences (NIEHS) Grant R01 ES002710, NIEHS SuperfundResearch ProgramP42 ES004699, National Institute of Neurological Dis-orders and Stroke (NINDS) U54 NS079202-01 and Grants NIEHST32ES007059, NIH 5T32DC008072-05 and 4T32HL086350-09 (toK.W.). Partial support for clinical developmental of sEH inhibitors forhuman medicine comes from the NIEHS SBIR Program R44ES025598and the NIH NINDS Blueprint Neurotherapeutics NetworkUH2NS094258. The content is solely the responsibility of the authorsand does not necessarily represent the official views of the National In-stitutes of Health.

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Pharmacology & TherapeuticsSoluble epoxide hydrolase as a therapeutic target for pain, inﬂammatory...

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