Progress in Neurobiology 172 (2019) 23–39

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Progress in Neurobiology

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Review article Humble beginnings with big goals: Small molecule soluble epoxide inhibitors for treating CNS disorders T

Sydney Zarrielloa, Julian P. Tuazona, Sydney Coreya, Samantha Schimmela, Mira Rajania, ⁎⁎ ⁎ Anna Gorskya, Diego Incontria, Bruce D. Hammockb, , Cesar V. Borlongana, a Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, 12901 Bruce B Downs Blvd, Tampa, FL, 33612, United States b Department of Entomology & UCD Comprehensive Cancer Center, NIEHS-UCD Superfund Research Program, University of California – Davis, United States

ARTICLE INFO ABSTRACT

Keywords: Soluble (sEH) degrades epoxides of fatty acids including isomers Pharmacology (EETs), which are produced as metabolites of the cytochrome P450 branch of the pathway. Stroke EETs exert a variety of largely beneficial effects in the context of inflammation and vascular regulation. sEH TBI inhibition is shown to be therapeutic in several cardiovascular and renal disorders, as well as in peripheral Inflammation analgesia, via the increased availability of anti-inflammatory EETs. The success of sEH inhibitors in peripheral Preclinical studies systems suggests their potential in targeting inflammation in the central nervous system (CNS) disorders. Here, Clinical trials Druggability we describe the current roles of sEH in the pathology and treatment of CNS disorders such as stroke, traumatic brain injury, Parkinson’s disease, epilepsy, cognitive impairment, dementia and depression. In view of the robust anti-inflammatory effects of stem cells, we also outlined the potency of stem cell treatment and sEH inhibitors as a combination therapy for these CNS disorders. This review highlights the gaps in current knowledge about the pathologic and therapeutic roles of sEH in CNS disorders, which should guide future basic science research towards translational and clinical applications of sEH inhibitors for treatment of neurological diseases.

1. Introduction hydroxyeicosatrienoic acids (HETEs) and epoxyeicosatrienoic acid iso- mers (EETs). EETs are produced by the metabolic activity of CYP en- Three inflammatory pathways that produce eicosanoids derived zymes which among other metabolites produce epoxides from double from arachidonic acid (ARA) have been implicated in a variety of in- bonds (Capdevila et al., 2000). These compounds have been shown to flammation-plagued disorders such as stroke, , and renal exert potent, protective vasodilatory and anti-inflammatory effects on disease. The first pathway entails cyclooxygenase (COX) that the cardiovascular and renal systems (Spector et al., 2004). Moreover, are responsible for the conversion of ARA to prostaglandins and EETs also suppress hyperthermia, pathological fibrosis, the generation thromboxane (a potent vasoconstrictor). Both aspirin, prescribed for at- of reactive oxygen species, apoptosis, pain, and aggregation risk stroke patients, and non-steroidal anti-inflammatory drugs are COX (Zhang et al., 2007; Xu et al., 2013; Morisseau and Hammock, 2013; inhibitors (Vane, 1971). The second pathway consists of lipoxygenase Yang et al., 2015a; Harris and Hammock, 2013; Yang et al., 2015b) (LOX) enzymes that are required to produce pro-inflammatory leuko- Although the beneficial effects of EETs on the cardiovascular and renal trienes from ARA, and LOX inhibitors have been used therapeutically in systems have taken precedence over their roles in other systems, they seasonal allergies and asthma (Cegielska-Perun et al., 2016; Ribeiro assume an important role in central nervous system (CNS) signaling as et al., 2006). The third pathway is mediated by cytochrome P450 en- well (Iliff et al., 2010). While many of the effects of EETs on the CNS zymes (CYP) and results in two types of products: parallel their effects on peripheral systems, recent research reveals that

Abbreviations: ARA, arachidonic acid; COX, cyclooxygenase; LOX, lipoxygenase; CYP, cytochrome P450; CBF, cerebral blood flow; EET, epoxyeicosatrienoic acid; DHET, dihydroxyeicosatrienoic acid; EpFA, epoxy fatty acid; CNS, central nervous system; sEH, soluble epoxide hydrolase; sEHI, soluble epoxide hydrolase inhibitor; TBI, traumatic brain injury; PD, Parkinson’s disease; MCAO, middle cerebral artery occlusion ⁎ Corresponding author at: Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, 33612, United States. ⁎⁎ Corresponding author at: Department of Entomology & UCD Comprehensive Cancer Center, NIEHS-UCD Superfund Research Program, University of California – Davis, United States. E-mail addresses: [email protected] (B.D. Hammock), [email protected] (C.V. Borlongan). https://doi.org/10.1016/j.pneurobio.2018.11.001 Received 16 August 2018; Received in revised form 6 October 2018; Accepted 9 November 2018 Available online 14 November 2018 0301-0082/ © 2018 Elsevier Ltd. All rights reserved. S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39

Table 1 A summary of breakthroughs in the discovery and use of sEH. sEH reduction through deletion or the use of inhibitors demonstrates novel therapeutic appli- cations in CNS disorders. sEH inhibition results in an increase in EETs which aids in the treatment of such neurological disorders.

Major Studies Contributing to the Emergence of sEH into CNS Disorders

Study Discovery

Gill et al., 1974 Incubation of the juvenoid 1-(4’-ethylphenoxy)-6,7-epoxy-3,7-dimethyl-2-octene with rat liver microsomes forms diol metabolites and is the first description of sEH in mammals Hammock et al., 1976 Various experiments reveal sEH’s biochemical characteristics such as an isoelectric point of 4.9, a molecular weight of 150,000, and increased activity in kidney and liver soluble fractions relative to microsomal fractions Mumby and Hammock, 1979a,b In mouse livers, epoxide hydrase-mediated hydration rates change depending on the quantity of alkyl substitutions on the epoxide, exhibiting sEH substrate specificity for the first time Gill and Hammock, 1979 sEHs hydrate both cis- and trans- epoxymethyl stearates in mouse livers, especially in the cytosolic fraction; the first description of epoxy fatty acids as substrates Gill and Hammock, 1980 First comprehensive description of sEH, revealing that epoxide hydrase activity is present in various organs like the spleen, lung, colon, etc., but is highest in the liver and kidney; is higher in male mice compared to female mice, increases as mice age, does not require a , and is inhibited by inorganic ions. Ota and Hammock, 1980 sEH (cytosolic) activity, unlike microsomal enzymes, was overlooked by other researchers because assays with high pH were utilized, which minimizes sEH activity; sEH activity is lowest in rats compared to mice and guinea pigs, but rats are frequently used for epoxide hydrolase distribution investigations; and studies with styrene oxide led to the misconception that epoxide hydrolase activity requires membrane binding Hasegawa and Hammock, 1982 A spectrophotometric assay using substrate trans-stilbene oxide describes the measurement of mammalian cytosolic epoxide hydrolase activity Grant et al., 1993 Structural analysis of the isolated sEH coding sequence reveals the first characterization and transient expression of the cloned cDNA in cell culture Grant et al., 1994 Chromosomal location of murine sEH gene is determined at band D of 14, homologous to human 8, 13, and 14 Pinot et al., 1995 sEH activity in mice is higher in males than in females. sEH is under regulation by proliferators and hormones such as testosterone. sEH activity is also greater in the liver than the kidneys Draper and Hammock, 1999a,b sEH activity present in rat inflammatory cells is indistinguishable from rat liver cytosolic sEH Draper and Hammock, 1999a,b Zinc and other metals can act as inhibitors of sEH, suggesting a mechanism of down-regulation during inflammation Yu et al., 2000 EETs are involved in renal vasculature activity, thus the use of an sEH inhibitor to decrease EET hydrolysis is a novel therapy for regulating blood pressure Alkayed et al., 2002 Upregulation of EETs post ischemic stroke is an innate mechanism to protect the brain against future damage Morisseau et al., 2002 Lipophilicity controls the water solubility of sEH inhibitors, which limits their potency as regulators of blood pressure and inflammation Dorrance et al., 2005 AUDA, an inhibitor of sEH, reduces infarct size post ischemic stroke without affecting vascular structure Liu et al., 2005 Inhibition of sEH through AUDA enhances laminar flow PPAR-γ activity, which upregulates the anti-inflammatory effect demonstrating PPAR- γ influences EETs Schmelzer et al., 2005 sEH inhibitors reduce inflammation by reducing levels of proinflammatory cytokines and nitric oxide metabolites Zhang et al., 2007 sEHI AUDA-BE reduces infarct size (post ischemic stroke) and is neuroprotective by non-vascular mechanisms Zhang et al., 2008a,b sEH knockout mice, with EPHX2 gene deletion, demonstrate that sEH gene deletion protects against ischemic stroke via a vascular mechanism that reduces EET hydration Iliff et al., 2009 EETs aid in neurogenic and are produced by perivascular nerves Zhang et al., 2009 sEH in part explains sex-linked di fferences in blood flow and brain damage post ischemic stroke Sarkar et al., 2011 activity is impaired in Alzheimer’s disease Vanella et al., 2011 sEH inhibition, using siRNAs, decreases mesenchymal stem cell-derived adipocyte stem cell differentiation Fromel et al., 2012 Long-term sEH inhibition is detrimental to hematopoietic progenitor cell proliferation, mobilization, and vascular repair Shaik et al., 2013 sEHI t-AUCB reduces infarct volume, neuronal cell death, and behavioral deficits if administered during MCAO Strauss et al., 2013 sEH-KO mice display improved behavioral outcomes in comparison to wild-type mice post TBI Inceoglu et al., 2013 Mice treated with sEHIs are resistant to GABA-antagonist induced seizures Hung et al., 2015 sEH inhibitor AUDA attenuates inflammation and decreases seizure susceptibility in a rat model of temporal lobe epilepsy Li et al., 2015 EETs enhance hematopoietic stem and progenitor cell engraftment migration in mammals Qin et al., 2015 Tyrosine hydroxylase-positive cell death can be reduced by sEH deficiency or administration of 14,15-EET in a PD mouse model Liu et al., 2016 sEHIs 14,15-EET and AUDA cause suppressed astrogliosis, enhanced angiogenesis, decreased neural apoptosis, and reduced glial scar formation in vitro Ren et al., 2016 sEH inhibitor TPPU mitigates depressive symptoms in mice Zhang et al., 2017 sEH inhibition post OGD increases EET and VEGF levels, indicating that astrocytes may help control sEHI-induced neuroprotection Hung et al., 2017 sEH-KO mice and AUDA-treated mice display improved behavior, decreased brain edema, less brain tissue damage and apoptosis, and less BBB permeability post TBI Lakkappa et al., 2018 sEH inhibitor PTUPB provides neuroprotection in a Drosophila model of PD Ren et al., 2018 sEH inhibitor TPPU ameliorates reduction of DA, DOPAC, and HVA in a MPTP-induced mouse model of neurotoxicity

Table 1 Sydney Zarriello. Progress in Neurobiology.

EETs have distinct functions in the brain, which may have anti-in- ascent into clinical research (Table 1) since their groundbreaking ca- flammatory effects. Some of these functions relevant to regulation of pacity to produce anti-hypertensive effects was discovered (Imig and inflammation include the modulation of angiogenesis, regulation of Hammock, 2009; Imig et al., 2002). The aforementioned anti-hy- cerebral blood flow (CBF), and mediation of neuroendocrine signaling pertensive effects were then attributed to the ability of sEHIs to inhibit (Iliff et al., 2010). the actions of hormone angiotensin-II (Jung et al., 2005). This finding Soluble epoxide hydrolase (sEH), first assayed (Mumby and not only bolstered previous research on the potential treatment of hy- Hammock, 1979a, b) and characterized (Gill and Hammock, 1979; pertension with sEHIs, but also elucidated the relationship between Grant et al., 1994) almost 40 years ago, is responsible for the de- sEHIs and the renal system. With the advent of more potent and bioa- gradation and conversion of EETs into dihydroxyeicosatrienoic acids vailable inhibitors, sEHIs entered clinical studies to evaluate their (DHETs), which have diminished and different biological effects com- safety and efficacy for health problems such as hypertension and type 2 pared to EETs (Spector et al., 2004; Fang et al., 2004). Furthermore, diabetes mellitus. In a phase IIa clinical trial, the sEHI AR9281 de- sEH inhibitors (sEHIs) harbor therapeutic potential by increasing the monstrated decent tolerability, rapid absorption, no severe side effects, availability of EETs and other epoxy fatty acids (EpFAs) (Shen, 2010; and a 90% inhibition rate of sEH activity in the blood (Chen et al., Yu et al., 2000). In the last two decades, sEHIs have enjoyed a rapid 2012). Additionally, combining sEHIs with the vasoconstrictor

24 S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39 urotensin II resulted in a larger increase in blood vessel flux in healthy possible involvement in regulating CBF, brain activity, and neurological patients and patients with heart failure, suggesting sEHIs’ potential to disease pathology (Sura et al., 2008). In fact, administering sEHIs de- treat heart failure (Tran et al., 2012). GSK2256294, another sEHI, was creases sEH activity and mitigates brain damage in stroke and TBI an- evaluated in both healthy males and obese smokers in a phase I clinical imal models (Zhang et al., 2007; Hung et al., 2017). This implies a trial, and was found to have no serious adverse effects, with contact neuroprotective effect upon sEH inhibition and advocates the devel- dermatitis and headaches as the most common side effects (Lazaar opment of potent sEHIs with favorable pharmacokinetic attributes as et al., 2016). The same sEHI was also studied in elderly males and fe- potential drug treatments for neurological diseases. males with similar results (Lazaar et al., 2016). Fortified by the ex- The unique distribution of sEH in the human body also reveals tensive research on hypertension and sEH, the putative neuroprotection sexually dimorphic expression. sEH activity is higher in post-puberty of sEHIs emerged into the field of ischemic stroke. sEH is abundantly male mice than in females (Gill and Hammock, 1980), suggesting dif- expressed in the cerebral cortex, striatum, hypothalamus, and brain ferential regulation by hormones such as testosterone (Pinot et al., stem, predominantly in neuronal cell bodies (Zhang et al., 2007; Sellers 1995) and estradiol (Koerner et al., 2008). Detailed research must be et al., 2005) but also in astrocytes and oligodendrocytes (Harris and conducted in the future to fully elucidate sex differences in sEH activity Hammock, 2013). Coinciding with the benefits of sEHIs in hypertension and distribution. This concept, along with the distinct impacts that and related cardiovascular disease, sEH activity may be present within specific neurological disorders have on the sexes, must be acknowl- the as well (Zhang et al., 2013a). Indeed, the inhibition of edged when developing and evaluating the effects of potential sEHIs. sEH attenuated inflammation and was associated with a smaller infarct size after middle cerebral artery occlusion (MCAO) in mice (Zhang 3. Absorption, distribution, metabolism, and excretion (ADME) of et al., 2007). Interestingly, sEH immunoreactivity was confirmed in sEHIs both non-vascularized and vascularized areas of the brain, suggesting that the beneficial actions of sEHIs may similarly comprise of vascular Maintaining simplicity of formulation and enhancing ADME prop- as well as non-vascular pathways. Due to the robust ability of sEHIs to erties of sEH inhibitors is crucial to bolstering their efficacy (Fig. 2) suppress the inflammatory response, sEHIs have become increasingly (Qiu et al., 2011). Ideal sEHIs maximize bioavailability and delay relevant in the possible treatment of CNS disorders (Fig. 1). Other elimination from the bloodstream as exhibited by longer half-lives, mechanisms by which sEHIs may have beneficial effects in the treat- higher maximum drug concentrations in the blood (Cmax), and larger ment of CNS disorders have also been unveiled, and they will be dis- area under the curve (AUC) values than earlier sEHIs such as 12-(3- cussed in detail below. adamantan-1-yl-ureido)-dodecanoic acid (AUDA) (Liu et al., 2009). In the present review, we seek to describe the current knowledge Additionally, lower melting points are beneficial for boosting absorp- surrounding the potential role of sEHIs in treatment for CNS disorders tion in the body (Chu and Yalkowsky, 2009) and higher solubility in such as stroke, traumatic brain injury (TBI), Parkinson’s disease (PD), water improves oral administration and bioavailability and other debilitating neurological conditions. Inflammation is a major (Chiamvimonvat et al., 2007; Kim et al., 2004). Furthermore, de- mediator of cell death in the diseases mentioned above, and we will creasing crystalline stability and increasing oil solubility enables sEHIs explore the anti-inflammatory mechanisms of sEHIs that serve as the to experience easier subcutaneous administration and dissolution in basis for combating these disorders. In addition, we hope to synthesize organic solvents used to transfer drugs (Chiamvimonvat et al., 2007). research conducted on the intersection of sEH inhibition and stem cell Initial sEHIs included dicyclohexylurea (DCU), a potent but highly treatment. The treatment of CNS diseases with stem cells has reached lipophilic compound with a high melting point. DCU is a byproduct of clinical trials, but to date, most of the reported findings relate to safety some industrial and laboratory synthesis procedures. However, DCU’s with limited efficacy and may benefit from combination therapy, such pharmacological utility is restricted as excessive lipophilicity in a as with sEHIs. While the anti-inflammatory effect of sEHIs observed in compound generates pharmacokinetic and formulation issues and hin- other body systems is a promising therapeutic tool for use in CNS dis- ders its specificity (Chiamvimonvat et al., 2007; Iliff and Alkayed, orders, unique properties of the brain and complex pathological pro- 2009). Adding polar functional groups to early sEHIs like DCU gener- cesses of each neurological disorder may hinder their swift integration ated a new line of potent, more water soluble sEHIs including AUDA into clinical practice. In this review, we will provide an outline of and its butyl ester (AUDA-BE) (Liu et al., 2009). Preclinical pharma- possible and current challenges sEHIs may face on their way into the cokinetics revealed that AUDA-BE, when injected intraperitoneally in clinic. We also hope to highlight the gaps in current scientific and mice, travels systemically to the brain, crosses the blood brain barrier translational knowledge to provide guidance for future clinical studies (BBB), and inhibits sEH in the brain (Zhang et al., 2007). sEH activity is geared towards advancing stem cell therapy and pharmacological sEHI inhibited by over 20% for 24 h, confirming AUDA-BE’s potency and treatment for neurological disorders. bioavailability (Zhang et al., 2007). AUDA-BE appears to exhibit su- perior bioavailability, but both AUDA and AUDA-BE are capable of 2. Distribution of sEH crossing the BBB and inhibiting sEH (Iliff and Alkayed, 2009). In- triguingly, AUDA-BE and AUDA’s other butyl and ester forms can be sEH is present in inflammatory cells and throughout multiple organs transmitted intraperitoneally, subcutaneously, or orally via food, while in mammals, including the liver and kidney (Chiamvimonvat et al., AUDA can be dosed orally via drinking water (Chiamvimonvat et al., 2007; Draper and Hammock, 1999a). It can be found in the en- 2007). Adamantane was used in many early sEHIs like AUDA because it dothelium and smooth muscle of vascular tissues, suggesting the en- could be detected at very low concentrations by liquid chromato- zyme’s potential connection to hypertension (Chiamvimonvat et al., graphy-mass spectrometry (LC-MS) and because it was rapidly meta- 2007). Within the cell, sEH is mainly located in the cytosol and per- bolized. AUDA compounds encounter swift metabolism via beta oxi- oxisomes, but this cytosolic/peroxisomal distribution changes with dation, hydroxylation of the adamantane, and subsequent excretion tissue type and physiological state (Chiamvimonvat et al., 2007). In- (Chiamvimonvat et al., 2007). They also have high melting points, low terestingly, sEH is also highly prevalent in the cerebral cortex and water solubility, and were designed as mimics of EETs (Chiamvimonvat striatum of the brain and typically resides in neuronal cell bodies, as- et al., 2007). Thus, they are also peroxisome proliferator-activated re- trocytes, oligodendrocytes, and the smooth muscle of cerebral blood ceptor-γ (PPAR-γ) agonists. vessels (Zhang et al., 2007; Sura et al., 2008). As stated above, sEH is Following AUDA came other sEHIs that were designed with stra- also found within the endothelium (Zhang et al., 2013a). These loca- tegic functional group placement, including 1-adamantan-1-yl-3-(5-(2- lization patterns, particularly sEH’s propensity for endothelial cells and (2-ethoxyethoxy)ethoxy)pentyl)urea (AEPU) (Qiu et al., 2011). Like smooth muscle cells of cerebrovasculature, implicate the enzyme’s many modern sEHIs, AEPU has a polar functional group located

25 S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39

Fig. 1. The effects of sEH inhibition on major CNS disorders. sEH inhibition prevents the typical breakdown of EETs and other EpFAs into DHETS or other corre- sponding 1,2-diols, facilitating the regenerative process of certain disorders. This treatment may be coupled with stem cell therapy in order to improve treatment outcomes.

Fig. 2. Soluble epoxide hydrolase inhibitor (sEHI) structures. The chemical structures of several sEHIs. These compounds inhibit sEH, whose three-dimensional structure is depicted on the right (PDB ID: 1CQZ; MMDB ID: 11526).

26 S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39 approximately 8 Å away from its central carbonyl group necessary to maintain sEHI potency while simultaneously maximizing (Chiamvimonvat et al., 2007), allowing higher solubility in water and a availability in the brain. lower melting temperature, thereby making AEPU’s formulation more Most central pharmacophores in sEHIs are a urea, carbamate, or accessible. The inhibitor readily traverses membranes and possesses amide moiety (Chiamvimonvat et al., 2007). Modifying the secondary good bioavailability and effectiveness in vivo (Chiamvimonvat et al., pharmacophore by including hydroxyl, carbonyl, sulfonyl, ether, ester, 2007; Qiu et al., 2011). However, it was designed for transient activity carbamate, and other functional groups enhances the inhibitor’s phy- and quick metabolic breakdown (Chiamvimonvat et al., 2007). Other sical attributes, such as by decreasing melting temperature and in- new inhibitors, including t-AUCB (trans-4-(4-(3-adamantan-1-yl-ur- creasing binding to polar residues in the sEH catalytic tunnel eido)-cyclohexyloxy)-benzoic acid) and TPAU (1-tri- (Chiamvimonvat et al., 2007). Adding polar groups to inhibitors in the fluoromethoxyphenyl-3-(1-acetylpiperidin-4-yl) urea) have succeeded proper position may increase the likelihood of hydrogen bonding with earlier sEHIs with increased water solubility and abated metabolism catalytic sites on sEH and improve target selectivity and pharmacoki- due to their confined conformational structures (Qiu et al., 2011; Liu netic and physical aspects (Chiamvimonvat et al., 2007). For instance, et al., 2009). One compound of interest is 1-trifluoromethoxyphenyl-3- water solubility increases without compromising inhibitor efficacy (1-propionylpiperidine-4-yl) urea (TPPU), a potent, relatively easily- when polar functional groups are present on a linear alkyl chain at a formulated sEHI with sufficient solubility in water. It is currently the certain distance from the urea moiety (Kim et al., 2004). Thus, struc- most widely used sEHI in research. TPPU maintains its robust bioa- tural modifications of inhibitors can increase solubility in water or oil- vailability in the brain after intraperitoneal injection (Ulu et al., 2016) based solvents or lower melting points in order to increase bioavail- and oral administration (Ren et al., 2016), demonstrates vast systemic ability (Kim et al., 2004, 2007; Morisseau et al., 2002). Augmenting distribution to tissues, effectively crosses the BBB, experiences excellent bioavailability following oral administration and minimizing clearance absorption in the intestines, significantly inhibits sEH, possesses high from the bloodstream are current objectives driving the latest structural metabolic stability, and is effectively administered via drinking water alterations to sEHIs (Iliff and Alkayed, 2009). Of note, there has been a but must be formulated in a water miscible organic co-solvent (Ren breakthrough within the past 15 years in promising compounds pos- et al., 2016; Ostermann et al., 2015). Indeed, many sEHIs successfully sessing extensive half-lives and exceptional oral bioavailability pass the BBB and each new generation of inhibitors advances the pre- (Chiamvimonvat et al., 2007; Iliff and Alkayed, 2009). Pharmaceutical vious generation with improved physical characteristics like increased companies can potentially use these compounds as templates for de- water solubility. TPPU exhibits much greater stability, bioavailability, veloping highly effective sEHIs to treat a wide variety of diseases, in- half-life duration, and efficacy in clearing inflammation than t-AUCB, cluding neurological maladies like TBI and ischemic stroke (Iliff and appealing properties attributed to the substituted phenyl group at- Alkayed, 2009). In addition to developing sEHIs with slow metabolism tached to TPPU in contrast to the adamantyl group attached to t-AUCB and sufficient oral bioavailability, dual COX/sEH inhibitors are of (Liu et al., 2013). However, t-AUCB derivatives are broadly active on current interest. A COX-2/sEH inhibitor administered to rats shows a the sEH of many mammalian species while TPPU-like compounds are synergistic effect in treating lipopolysaccharide induced pain (Hwang most active on primate and rodent sEHs. These results further empha- et al., 2011). In fact, the compound shows an enhanced effect compared size the importance of sEHI structure and functional groups, and how to co-administration of both celecoxib (a COX-2 inhibitor) and t-AUCB these affect inhibitor efficacy and pharmacokinetic properties. It will be (Hwang et al., 2011). Furthermore, COX-2/sEH inhibition also sup- imperative to refine sEH structures with this in mind to develop opti- presses tumor growth by inhibiting angiogenesis, suggesting that mized drugs for treating diseases. creating future sEHIs that also contain the ability to inhibit COX may be While sEHIs like AUDA and TPPU can cross the BBB, their structure desirable in the treatment of certain diseases (Zhang et al., 2014a). has not been specifically optimized for BBB penetration. Lipid solubility There have been significant challenges faced in obtaining Food and is key to crossing the BBB (Banks, 2009), but excessive lipophilicity Drug Administration approval for sEHIs evaluated in completed clinical hinders sEHI formulation, creates pharmacokinetic issues trials (Table 2)(GlaxoSmithKline, 2014a, b). Despite the lack of serious (Chiamvimonvat et al., 2007), and can allow the sEHI to be taken from adverse effects caused by sEHI administration, some compounds, most the bloodstream by other tissues before it reaches the brain (Banks, notably GSK2256294, are on hold due to the rigorous standards that 2009). Thus, a compromise must be made to improve BBB penetration must be met for drug development pursuit. However, sEHIs are still while maintaining the inhibitor in the bloodstream (Banks, 2009). As undergoing evaluation for use in other disorders such as subarachnoid low molecular weight and lipid solubility facilitate easier crossing of hemorrhage (Oregon H and Science U, 2018) and diabetes mellitus, the BBB, it is possible that increasing hydrogen bonding capabilities or (Vanderbilt University Medical C, 2018). In addition, novel sEHIs may molecular weight by adding additional functional groups in some sEHIs be discovered based on previously developed pharmacophore models, may also prevent complete optimization of BBB crossing which have identified several new compounds from the Specs database (Chiamvimonvat et al., 2007; Banks, 2009). Thus, an ideal balance is capable of inhibiting sEH in extremely low concentrations

Table 2 A summary of completed and ongoing clinical trials involving sEHIs. sEHI treatment displays promise in a variety of disorders.

Clinical Trials Involving sEHIs

Trial, Year Complete Drug Disease Results

NCT00847899 (Chen et al., 2012) AR9281 Hypertension/Impaired Glucose No officially published results Intolerance NCT00654966 (Tran et al., 2012) AUDA Heart Failure AUDA reversed urotensin-II induced vasoconstriction in heart failure patients NCT01762774 (GlaxoSmithKline, 2014a) GSK2256294 COPD Reduction of high-density lipoprotein was observed after dose escalation in healthy males and male moderately obese smokers NCT02006537 (GlaxoSmithKline, 2014b) GSK2256294 Safety No serious adverse events occurred with 10 mg oral dose in healthy adult and elderly males and females NCT03318783 (Oregon H and Science U, 2018) GSK2256294 Subarachnoid Hemorrhage Recruiting NCT03486223 (Vanderbilt University Medical GSK2256294 Diabetes Mellitus and Metabolic Recruiting C, 2018) Disorders

27 S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39

(Waltenberger et al., 2016). This technique may be the forefront of drug resultingly increase and contribute to microglia deactivation and en- discovery concerning sEH inhibition in the coming years. Furthermore, hanced neuronal survival (Hung et al., 2017). EETs and other EpFAs company Eicosis is investigating the use of EC2506, a sEHI, as a have also been able to inhibit the expression of vascular cell adhesion treatment for diabetic peripheral neuropathy in a phase Ia clinical trial molecule-1 (VCAM-1), E-selectin, intercellular adhesion molecule 1 beginning in 2019 (Grant #R44ES025598). New sEHIs may be dis- (ICAM-1) and the nuclear translocation of nuclear factor kappa-light- covered naturally as well. Urea-based sEHIs have recently been isolated chain-enhancer of activated B cells (NF-κB) (Wagner et al., 2017). from the African plant Pentadiplandra brazzeana and shown to act as VCAM-1, E-selectin, and ICAM-1 are all cell adhesion molecules, so analgesics in a rat model (Kitamura et al., 2015). While some promising without them leukocytes and other immune system cells are unable to sEHIs are facing resistance in the receipt of FDA approval, other in- attach to injured tissue, and NF-κB helps regulate the immune response novative sEHIs are working towards translation to CNS disorders and by upregulating enzymes that contribute to inflammation (Wagner optimization to the human species, and this atmosphere is dynamic and et al., 2017). Leukocytes, one of the trademarks of inflammation, are evolving. reduced in the presence of EpFAs but the exact mechanism by which it occurs has not been fully elucidated (Iliff and Alkayed, 2009). 4. Inflammation and sEH In neurological diseases such as ischemic stroke, epilepsy, Alzheimer’s disease (AD), PD, and TBI, rudimentary research has begun Inflammation is a biological response to an injury that attempts to showing the positive effects of increased EET and other EpFA con- minimize functional and structural damage relating to the insult. The centrations following sEH inhibition. In ischemic stroke, EpFAs are response is characterized by the dilation of arterioles and capillaries, protective against cell death in a way independent of effects on CBF and increased permeability of microvasculature, and the invasion of leu- may be able to decrease the infarct size (Wagner et al., 2017; Bianco kocytes into injured tissue (Wagner et al., 2017). While inflammation et al., 2009; Matsumoto et al., 2014), complementing the vascular initially serves as a mechanism to resolve injuries, it becomes a problem mechanism of EpFAs. Epilepsy patients may be treated pre or post (and turns into a chronic condition) if it is not halted after a certain seizure with the anti-inflammatory properties of EpFAs, as anti-con- period of time (Wagner et al., 2017). Inflammation is a hallmark of vulsants are unable to prevent seizures (Wagner et al., 2017; Vito et al., many conditions, many of which involve the CNS. When affecting the 2014). Regarding AD and PD, EpFAs may be able to improve mi- CNS, inflammation (referred to as neuroinflammation) involves the tochondrial functioning and reduce neuroinflammation from oxidative recruitment of leukocytes and lymphocytes to the CNS and the activa- stress (Wagner et al., 2017). In TBI, EpFAs have the potential to de- tion of glial cells (including microglia, the phagocytes of the brain) crease brain edema due to their ability to decrease neuroinflammation (39). In some pathogenic processes, such as in stroke and TBI, in- (Hung et al., 2017). sEHIs’ ability to mount multi-pronged therapeutic flammation can also cause increased permeability of the BBB, allowing processes appeals to complex injuries, such as stroke and TBI char- harmful blood products to enter the brain parenchyma (Prakash and acterized by multiple cell death pathways, which likely cannot be ar- Carmichael, 2015). Neuroinflammation is a major cause of secondary rested by a drug designed to target a singular degenerative process (Iliff injury cascades, and it is primarily regulated by microglia and periph- and Alkayed, 2009). eral leukocytes (Hung et al., 2017). Once activated, microglia produce The ability of sEHIs to resolve inflammatory responses is promising, pro-inflammatory cytokines that result in cytotoxic effects if not regu- but more research is needed before they can be utilized clinically (Liu lated. This property of microglia makes them a prime contributor to et al., 2009). sEH is present in most tissues, so the systemic use of in- neuroinflammation (Hung et al., 2017). Due to its widespread and hibitors may have unintended effects outside of regulating inflamma- detrimental effects, treatment strategies have been designed to prevent tion (Bianco et al., 2009). Additionally, EETs and other EpFAs have or reverse neuroinflammation (Hung et al., 2017) been postulated to afford several functions in the brain, such as in- sEHIs may serve as therapeutic options when treating several dis- hibiting inflammation, and releasing peptide hormones, but the exact orders, some of which are neurodegenerative, due to their ability to pathway that EETs use in the brain remains poorly understood (Bianco prevent EET and other EpFA metabolism by sEH (Bianco et al., 2009). et al., 2009). The specific mechanism of sEHIs is also unknown, though EETs are made from ARA in the CYP reaction (Hung et al., 2017; Davis the inflammation-associated P38-MAPK is a potential target (Hung et al., 2017). The ARA network produces many inflammatory mediators et al., 2017). Much of the current literature involving sEH and sEHIs is that are implicated in numerous diseases, making the pathway another focused on cardiovascular and renal conditions. The inflammatory re- target of therapeutic intervention (Meng et al., 2015). EETs have many sponses in these conditions differ from neurodegenerative diseases due roles in the body, but they are quickly broken down and inactivated by to the presence of the blood brain barrier and a variety of CNS specific sEH, preventing them from utilizing their anti-inflammatory features cells. Before sEHIs can be translated to a clinical setting for treating CNS (Morisseau and Hammock, 2013). If sEH is inhibited, EET levels can disorders, additional studies are warranted to reveal how these mole- increase and exert anti-inflammatory actions. The EET increase is lim- cules directly affect the brain and if unintended side effects are pro- ited by a variety of other degradation pathways making it difficult to duced. increase EpFA to undesirable levels. Indeed, metal cations are shown to decrease in the serum during systemic inflammation, and they are 5. Stroke and sEH known natural endogenous inhibitors of sEH (Draper and Hammock, 1999b). This implies that sEH activity is increased during inflammation. Stroke, characterized by a loss of blood flow to the brain, remains a In fact, the increase is so dramatic that sEH message, protein, and leading cause of disability and death globally, with approximately catalytic activity can be used as a marker of tissue inflammation 800,000 stroke victims suffering each year in the United States alone (Matsumoto et al., 2014). Another way to decrease the concentration of (Benjamin et al., 2017). The extent of ischemic damage depends on the sEH is through genetic deletion, which has been shown to reduce severity of CBF reduction, which determines the extent of oxygen and neuronal death, apoptosis, brain edema, and BBB permeability fol- glucose deprivation from the cells. This hypoxic environment results in lowing TBI (Hung et al., 2017). extensive apoptosis and necrosis (Mehta et al., 2007). Subsequent ac- The exact method utilized by EETs to reduce inflammation is un- tivation of the tissue’s immune response initiates the upregulation of known, but it has been shown that in certain brain conditions, sEH pro-inflammatory cytokines (Chamorro et al., 2012), and a compro- increases in microglia while EETs and other EpFAs decrease. Microglia mised BBB allows for the rapid influx of neutrophils, macrophages, have a distinct role in neuroinflammation; they produce proin- leukocytes, B cells, and T cells to the stroke-damaged brain, further flammatory cytokines that cause neuronal damage and BBB disruption exacerbating neurodegeneration and aggravating the injury (Emsley (Hung et al., 2017). If sEH is inhibited in microglia, EpFAs will et al., 2003).

28 S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39

Despite the prevalence of stroke, treatment options are severely promotes a nonsignificant trend post-stroke towards enhanced CBF in limited. Thrombolytic therapy using tissue plasminogen activator (tPA) the reduced infarct region, but does not significantly alter CBF (Shaik remains the only treatment currently approved by the Food and Drug et al., 2013). Compounding this observation, sEH knockout (SEHKO) Administration, yet only 3–5% of stroke patients qualify for this therapy mice, with targeted EPHX2 gene deletion, show significantly higher due to strict requirements and a narrow administrative window rates of CBF, suggesting sEHIs may operate via vascular mechanisms (Adeoye et al., 2011). Attempts for therapeutic options for ischemic (Zhang et al., 2008b). Conversely, overexpression of sEH results in an stroke have largely failed in lab-to-clinic translation, possibly owing to impairment of the vasodilatory effects of sEHIs and a larger infarct size a narrow targeting approach to therapy. Focusing on a single molecular in mice (Zhang et al., 2013a). The explanation for this difference be- pathway among the complex ischemic cascade seems to be a misguided tween chemical sEHI and EPHX2 gene deletion remains unknown, al- treatment strategy. An alternative therapeutic approach focusing on an though the degree of inhibition appears as a contributing factor, in that agent that targets multiple mechanisms across the ischemic process may chemical sEH inhibition may not be sufficient to enhance CBF com- prove more effective (Iliff and Alkayed, 2009). By reducing en- pared to chronic loss of activity from gene deletion. This gap in doplasmic reticulum stress and its sequelae, sEH harnesses this ap- knowledge warrants future investigation into the various degrees of proach, and thus has emerged as a potential target for stroke treatment. inducing sEH inhibition, specifically focusing on CBF changes both re- The use of sEH in stroke therapy is based on early genetic studies gionally and collaterally. Clarifying the underlying vascular mechan- deciphering the connection between EPHX2 polymorphisms and sus- isms of sEHIs is crucial to understanding the profile of sEH and their ceptibility to cerebral . The Glu470Gly variant, corresponding capacity as therapeutic targets. with increased sEH activity, positively correlates with the incidence of Equally relevant to the regenerative capacity of sEHI pertains to the cerebral ischemia among African-Americans (Fornage et al., 2005). In drug’s protective capabilities in models that closely approximate the co- parallel, within a Chinese population an independent association exists morbid elements of stroke, such as hypertension. Following MCAO, the between variation of Arg287Gln, which results in decreased sEH ac- resulting infarct volume is larger in spontaneously hypertensive stroke- tivity, and a reduced risk of stroke (Zhang et al., 2008a). These findings prone rats (SHSPR) than in Wistar Kyoto (WKY) rats (Coyle and indicate the protective influence of EPHX2 gene polymorphisms on the Jokelainen, 1983), making these models relevant targets for sEH in- risk of ischemic injury, suggesting the significant role of sEH on the hibition therapy. AUDA still reduces infarct volume in SHSPRs fol- incidence and development of ischemic stroke to provide the basis for lowing MCAO, but this protection operates independently of vascular targeting sEH in stroke treatment. mechanisms (Dorrance et al., 2005). Chronic sEHI treatment provides The broad therapeutic potential of sEHIs may arise from the multi- vascular protection through reduced blood pressure and increases mi- faceted role of EETs on cardiovascular and cerebrovascular function. An crovessel density in SHSPR but not WKY rats, and generates neural upregulation of EET signaling following transient ischemia may serve as protection in both species (Simpkins et al., 2009).The varying neuro- a preconditioning stimulus to protect the brain against future damage protection of sEHIs on both SHRSP and WKY rats suggests its broad (Alkayed et al., 2002). These findings, along with the complex protec- therapeutic potential, applicable to a wide profile of models re- tive effects of EETs, suggest that this signaling pathway is activated by presentative of the human population. ischemic trauma to provide neuroprotection. To this end, both chemical While current studies suggest the neuroprotective potential of sEHIs sEH inhibition and gene deletion have been largely successful at pro- in treating ischemic stroke, there are still significant gaps in knowledge viding neuroprotection and reducing the infarct size following ischemic that must be examined for hopes of bringing this prospective therapy to stroke. Even a single low-dose administration of the t-AUCB at the time the clinic. As discussed previously, the cellular and biological me- of MCAO reduces infarct volume, improves behavioral outcome as ap- chanisms underlying sEHI neuroprotection in stroke remain unclear and proved by the Stroke Therapy Academic Industry Roundtable (STAIR) warrant investigation. Future studies should clarify the differential ef- criteria for rats, and dose-dependently decreases neuronal cell death fects between acute chemical sEHI and EPHX2 gene deletion on is- (Shaik et al., 2013). Moreover, a twofold elevation of the cumulative chemic mechanisms such as CBF regulation and inflammation. EETs-to-DHETs ratio in the brain cortex is recognized with even such Moreover, chronic sEHI investigations are significantly lacking which low dose administration (Shaik et al., 2013). in vitro, targeting multiple are needed to further shed light on the effects of various degrees of elements of neuroprotection with 14,15-EET, a substrate for sEH, and inhibition in stroke models. Additionally, future studies should in- AUDA suppresses astrogliosis, enhances angiogenesis, decreases neural vestigate the optimal timeline for sEH inhibition, as the progress of apoptosis, and reduces glial scar formation, confirming the broad pro- ischemic injury involves complex time-dependent processes such as the tective effects of sEH inhibition (Liu et al., 2016). sEHIs applied to inflammatory response. Despite the significant role of inflammation in cultured astrocytes following oxygen-glucose deprivation also increase exacerbating ischemic damage, studies investigating the anti-in- levels of EETs and vascular endothelial growth factor (VEGF), in- flammatory mechanisms of sEHI protection are severely limited. Of dicating the significant role of astrocytes as possibly mediating sEHI- note, the administration of an sEHI (TPPU) following focal ischemia at induced neuroprotection (Zhang et al., 2017). Furthermore, greater reperfusion and 24 h later reduces infarct size by 50% and suppresses ischemic damage is correlated with increased levels of sEH mRNA and inflammation, as measured by a decrease of IL-1β by 3.5 fold and tumor less EET production in the mouse endothelium after exposure to necrosis factor-alpha by 2.2 fold (Tu et al., 2018). A decrease in infarct oxygen-glucose deprivation (Gupta et al., 2012). Despite these docu- volume is achieved when sEHI is administered before and during mented efficacy data, the precise mechanisms of sEHIs as multi-target MCAO, as well as during reperfusion, likely by targeting the peak of the protection against ischemic injury remain unclear. Further studies are inflammatory response exacerbating stroke damage. The effects of sEHI needed in the field to examine the broad effects of sEHIs on stroke administration during post-ischemic reperfusion must be further ex- models in vivo and to elucidate the factors contributing to this ischemic amined on various models varying in ages and sex to confirm neuro- protection. protective effects across a wider patient pool. Because EETs are well known vasodilators (Iliff et al., 2009), a po- Evaluating various models in exploring the neuroprotective benefits tential mechanism of ischemic protection by sEHIs may be through the of sEHI remains critical to establish clinical significance. Sex differences preservation of CBF. In contrast, the reduction of infarct volume by pertaining to the distribution of sEH in the body have implications in AUDA-BE in ischemic mice reveals no change in regional CBF rates the field of stroke. Interestingly, SEHKO female mice do not exhibit (Zhang et al., 2007). While AUDA-treated rats display significantly reduced infarct volume consistent with gene deletion in male models, smaller infarct size, AUDA produces no significant effect on vascular possibly due to decreased sEH expression (Zhang et al., 2009). These structure and acts independently of blood pressure changes (Dorrance findings suggest sexually dimorphic expression of sEH might explain in et al., 2005). Interestingly, administration of t-AUCB at time of MCAO part the underlying differences in CBF and ischemic damage

29 S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39 experienced between males and females. Along this line of investiga- gene deletion of EPHX2 shows significant promise in attenuating TBI- tion, sEH gene deletion, but not the t-AUCB, reduces infarct size in induced functional and neurological deficits, discrepant results are reproductively senescent female mice, yet neither method decreases observed between SEHKO individuals and chemical sEHIs. In stroke infarct size in young mice (Zuloaga et al., 2014). This indicates that models, t-AUCB administration at time of MCAO does not significantly sEHI protection against cerebral ischemia may be altered by age and alter CBF (Shaik et al., 2013), while SEHKO mice show markedly higher gender. Since in mice, sEH levels are profoundly influenced by both, the CBF rates (Zhang et al., 2008b). Moreover, gene deletion of sEH, but not lack of data evaluating female and/or aged models in sEH inhibition for t-AUCB, reduces infarct size in female mice following stroke (Zuloaga ischemic stroke should direct future research to investigate the effects et al., 2014). The challenges observed in sEH stroke models are of in- of this inhibition in various models emulating a representative stroke terest in TBI due to the overlap of injury mechanism. This warrants the population. Recognizing the therapeutic potential of sEHIs involves a need for investigating the effects of chemical sEHI administration and multi-target approach to ischemic stroke suggests that the complexity of sEH gene deletion on neurodegeneration and inflammation in TBI this therapy will require further research to clarify the factors of sEH- models, to better understand the optimal extent of inhibition. The ap- mediated neuroprotection. propriate dosage of sEHIs also represents an area of future direction because of the profile of patient suffering from TBI. The incidence of 6. Traumatic brain injury and sEH TBI is especially prevalent in athletes (Tagge et al., 2018) and military combat settings, with more than 300,000 US service members since Operating under similar mechanisms as in stroke, sEH as a ther- 2000 sustaining a TBI (Helmick et al., 2015). These TBI victims re- apeutic target for traumatic brain injury (TBI) is much less documented. present a younger and healthier patient pool compared to those suf- TBI affects unique groups of people in disproportionate rates, such as fering from stroke, given the pertinent association to co-morbidity military personnel and incarcerated individuals. About 12% of military factors such as aging and obesity. Therefore, the appropriate sEHI personnel (Schneiderman et al., 2008) and 60.25% of incarcerated therapeutic threshold dosages may be lower for TBI patients than persons (Shiroma et al., 2012) have a history of TBI. TBI, characterized stroke, but still achieve a better prognosis. In the same vein, while by a blow or penetration to the head, induces primary damage to the stroke and TBI are highly unpredictable diseases, the occurrence of TBI brain tissue, followed by cerebral inflammation representing a major in soldiers and athletes is likely higher than at-risk stroke patients, secondary cell death pathway (McConeghy et al., 2012). EET and other suggesting a unique opportunity for prophylactic sEHIs for TBI. Ad- EpFA levels in the brain increase following TBI (Boezaart and Kadieva, ditionally, penetrating brain injury (PBI), comprising all TBIs which are 1992; Hung et al., 2017). Exogenous administration of 14,15-EET and not caused by a blunt mechanism, represents an area of future direction AUDA promotes angiogenesis, reduces neuroinflammation, suppresses for sEHI therapy. Treatment for such open head wounds have shifted astrogliosis, and improves behavioral outcome in ischemic stroke from aggressive removal of deep fragments to antibiotic prophylactic models (Liu et al., 2016). Moreover, administration of TPPU sig- treatment to improve neurological outcomes (Kazim et al., 2011). This nificantly reduces inflammation, as evaluated by a decrease of pro-in- represents an underexplored opportunity for delivery of sEHIs to pre- flammatory cytokine TNF-α by 2.2 fold and of IL-1β by 3.5 fold (Tu vent subsequent damage by anti-inflammatory and neuroprotective et al., 2018). This capacity of sEHIs to attenuate inflammation and mechanisms. Although inhibition of sEH is gaining ground in the field promote neuroprotection suggests the therapeutic promise of sEHIs in of TBI therapy, further research is needed to ascertain the therapeutic treating TBI in addition to stroke. Targeting sEH in TBI models reveals potential of these inhibitors. that SEHKO mice outperform wild-type mice on the beam walk, in- dicating improved motor coordination both pre- and post-TBI (Strauss 7. Parkinson’s disease and sEH et al., 2013). Interestingly, SEHKO mice exhibit minor learning deficits in the Morris water maze independent of TBI (Strauss et al., 2013). PD is one of the most prevalent neurodegenerative diseases, second Sham-operated SEHKO mice also express an unexpected injured phe- only to AD (Ren et al., 2018). Within the next 20 years, the number of notype in the water maze assessing working memory, resembling si- patients with PD is predicted to double (Chen, 2010). PD is responsible milar deficits as brain-injured mice (Strauss et al., 2013). A valid con- for the progressive deterioration of normal movement and balance sideration for these unexpected findings is the role of the EPHX2 gene capabilities along with other nonmotor symptoms (Chen, 2010). Over in both epoxide hydrolase and lipid phosphatase activity (Newman the course of PD, dopaminergic neurons of the substantia nigra die et al., 2003), possibly explaining the impaired behavioral phenotypes prematurely and Lewy bodies (abnormal protein aggregates of α-sy- associated with SEHKO mice. Further studies investigating SEHKO mice nuclein) accumulate in the brain (Kalia and Lang, 2015). Both motor and selective sEH inhibition are needed to confirm the role of sEH in deficits characteristic of PD including speech impediments, dyskinesia, memory consolidation and motor coordination following TBI. Ex- and nonmotor impairments such as psychosis are unable to be suffi- amining discrete brain regions (e.g., hippocampus) to evaluate memory ciently treated with current medical or surgical approaches (Kalia and deficits commonly associated with TBI would enhance our knowledge Lang, 2015). Current medical options attempt to treat PD by increasing of the broader potential of sEHIs. Moreover, gene deletion of sEH sig- dopamine (DA) concentrations or through the stimulation of DA re- nificantly ameliorates TBI-induced brain tissue damage and neurolo- ceptors. These approaches are likely not satisfactory due to their limited gical deficits, as shown by reduced neuronal cell death, brain edema, targeting of the basal ganglia and DA as opposed to a wider range of the BBB permeability, apoptosis, matrix metalloproteinase 9 activity, and neurotransmitters involved in PD (Kalia and Lang, 2015). Other non- neutrophil infiltration (Hung et al., 2017). SEHKO mice also exhibit an pharmacological treatment options such as physiotherapy are being increase in anti-inflammatory M2 microglia/macrophages and a de- examined; however, these preliminary findings have yet to yield ac- crease pro-inflammatory M1 microglia/macrophages in the injured cepted treatment options in humans (Bloem et al., 2015). Though cer- cortex, as well reduced levels of cytokines IL-1β, IL-6, MIP-2, and MCP- tain treatments may alleviate symptoms of PD, there is currently no 1, suggesting the anti-inflammatory effect of sEH deletion in TBI (Hung cure and the trigger for the brain death characteristic of the disease is et al., 2017). Pharmacological inhibition by AUDA also attenuates not well understood, restricting treatment to the later phases of the neuroinflammation, brain edema, and apoptosis following TBI (Hung disease (Borlongan, 2018). et al., 2017). Both sEH gene deletion and pharmacological inhibition by sEH levels are elevated in the regions of the brain that correlate with sEHIs indicate functional benefits of targeting sEH in TBI. dopaminergic death in both humans and animal models of PD (Ren While these limited studies suggest the neuroprotective and anti- et al., 2018). The expression of sEH is also indicative of striatal α-sy- inflammatory potential of sEH inhibition, significant gaps in knowledge nuclein phosphorylation (Ren et al., 2018). Because PD may not display must be addressed to validate its therapeutic promise for TBI. Although symptoms until up to 80% of striatal DA has already been lost, a means

30 S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39 for earlier diagnosis would be highly progressive in broadening the paramount to the disease process in other CNS disorders, has been search for treatment options (Marsden, 1990). Peripheral detection of linked to epileptic pathogenesis (Vezzani et al., 2013; D’Ambrosio et al., elevated levels of sEH in the gut may be possible prior to dopaminergic 2013). Due to its role in inflammation, sEH has become an attractive death, potentially providing an early indicator of PD (Borlongan, 2018; target for therapeutic intervention in epilepsy. AUDA treatment in rats Marsden, 1990). subjected to induced temporal lobe status epilepticus and the in- The detrimental effects of sEH are well documented (Ren et al., flammatory response decreases expression of inflammatory cytokines 2018; Qin et al., 2015). In a mouse model of PD, both a deficiency in IL-1β and IL-6 in the hippocampus (Hung et al., 2015). The ratio of sEH and the administration of sEH substrate, 14,15-EET, reduces tyr- EETs to DHETs is also increased in the AUDA treated group, suggesting osine hydroxylase-positive cell death (Qin et al., 2015). The deletion of that the inhibition of sEH slowed the degradation of EETs. Notably, the sEH gene also protects against neurotoxicity in a PD mouse model treatment with AUDA successfully increases seizure induction (Ren et al., 2018). Although the inhibition of this gene does not provide threshold, and decreases seizure susceptibility (Hung et al., 2015). This a current treatment approach for PD patients, it does allow future observation suggests that inhibition of sEH produces therapeutic, anti- medical therapies to target this enzyme as method for slowing the inflammatory effects in the brain as well as in peripheral systems. progression of PD. Although inflammation is key to epilepsy pathology, other findings Furthermore, the inhibition of sEH may also provide a treatment for suggest that sEHIs may confer protection via other mechanisms. Mice PD (Ren et al., 2018). The clinical utility of sEH inhibition is recognized administered sEHIs and exogenous EETs are resistant to gamma-ami- in various heart and lung diseases in asthma patients and smokers, as nobutyric acid (GABA) antagonist seizure induction (Inceoglu et al., well as in the brain, discussed here (Ono et al., 2014; Yang et al., 2013). It is hypothesized that CYP is involved with GABA signaling, 2017a). sEH inhibition likely reduces both oxidative stress and neu- which is a key epileptic pathway (Inceoglu et al., 2013). However, these roinflammation through the elevation of endogenous EETs and other mice remain susceptible to seizures induced by other means, as evi- EpFAs (Qin et al., 2015; Lakkappa et al., 2018). An inhibitor of sEH and denced by the failure of sEHI 1-(1-methanesulfonyl-piperidin-4-yl)-3- COX-2, PTUPB provides significant neuroprotective activity in a model (4-trifluoromethoxy-phenyl)-urea (TUPS) to halt seizures produced by of PD using Drosophila melanogaster (Lakkappa et al., 2018). TPPU, powerful convulsant tetramethylenedisulfotetramine (TETS) when ad- another inhibitor of sEH, significantly alleviates the reduction of DA, ministered after the seizure onset, but is effective when initiated prior 3,4-dihydroxyphenylacetic acid and homovanillic acid post-MPTP-in- to the seizure (Vito et al., 2014). In addition, TUPS is effective at re- duced neurotoxicity in a mouse model (Ren et al., 2016, 2018). Because ducing TETS-induced cell death and neuroinflammation when ad- the degeneration of dopaminergic neurons is one of the main char- ministered in combination with GABA-A receptor agonist diazepam acteristics of PD, sEH could ameliorate the progression of the disease (Vito et al., 2014). These results suggest that EETs offer therapeutic (Kalia and Lang, 2015). In tandem, its potential to slow PD progression ability by modulating excitatory signaling in addition to attenuating suggests that TPPU would likely be more effective if its administration inflammation. EETs’ effects on neuronal excitability are supported by began earlier in the course of the disease, prior to the onset of symp- an additional finding that EET isomer 11,12 EET hyperpolarizes CA1 toms. Future trials examining the efficacy of such sEH inhibition pyramidal neurons in the hippocampus by inducing increased G-pro- treatments beginning at various stages of PD would guide the transla- tein-activated K+ conductance (Mule et al., 2017). tional application of this drug to the clinic. The practical benefits of The applications of sEHIs in epilepsy hold promise, but clinical TPPU indicate that the repeated oral delivery of TPPU significantly translation is lacking. In a study of 20 patients with temporal lobe reduces neurotoxicity in the striatum of mice (Ren et al., 2018). Indeed, epilepsy, sEH enzyme levels are increased compared to the control orally administered TPPU demonstrates the ability to suppress apop- group (Ahmedov et al., 2017). Furthermore, seizure duration and fre- tosis in PARK2 neurons, further demonstrating the negative effects of quency correlate with sEH level (Ahmedov et al., 2017). This suggests sEH and the benefits of its inhibition (Ren et al., 2018). As noted above, that there is a niche for sEHI intervention in humans, but more studies EETs have previously been used in cases of cerebral stroke, cardiac must be conducted to better elucidate the role of sEH in epilepsy. failure, and hypertension to ameliorate inflammation, caspase activa- tion, and apoptosis, among other damaging processes (Lakkappa et al., 8.2. Cognitive impairment and dementia 2016). Inhibition of sEH has the potential to increase the half-life of endogenous EETs. EETs may aid in neuroprotection in models of PD Pathologic manifestations of cognitive decline and dementia im- through mechanisms similar to their cytoprotective properties in plicate sEH's important role in these debilitating neurodegenerative models of other disorders such as stroke, TBI, and epilepsy (discussed diseases. An estimated 13.9% of people over the age of 71 suffer from later) (Lakkappa et al., 2016). Inhibition of sEH in PD has yet to be the cognitive decline, making it a major public health concern clinically verified. in vivo animal models lend support to both the sig- (Plassman et al., 2007). Causes of dementia include AD, vascular pa- nificant detrimental implications of elevated sEH levels and the ame- thology, accumulation of Lewy bodies, and others. Based on the docu- liorating effect of reducing the expression levels of the enzyme. It is mented pathological effects of EETs on vasculature, sEH has been critical to further study the use of elevated sEH levels in the brain as a identified as a potential therapeutic target for vascular cognitive de- biomarker of early PD detection and its treatment with sEH inhibition cline. sEH has also been recently identified to have detrimental con- prior to onset of symptomatic loss of striatal DA and neuronal death. sequences in AD, and the relevant research will be discussed below. Moreover, the use of sEH inhibitors such as TPPU and EETs should be Accumulation of beta amyloid protein aggregates (Aβ) is a hallmark evaluated at different stages of PD in animal models and clinical trials of AD and a strong initiator of cytotoxicity. Recently, a pathological link to determine whether they are viable as treatment options for patients between Aβ and the formation of epoxides from CYP P450 is detected in with preexisting and progressive stages of PD. cerebral microsomes prepared from brain tissues of Sprague-Dawley rats that show a 30% decrease in 14,15 EET production when cultured 8. Other neurological disorders and sEH with Aβ. These findings suggest that EpFA production is impaired in AD (Sarkar et al., 2011). While limited data support the relationship be- 8.1. Epilepsy tween sEH and AD, this study serves as a platform for future in- vestigation. In addition to stroke, PD, and TBI, sEH has been implicated in The role of sEH in vascular cognitive impairment (VCI) is notable in epilepsy within the last five years. Epilepsy is defined as recurrent individuals diagnosed with VCI that showed increased DHET levels in seizures characterized by extensive, synchronized excitatory signaling brain tissues post-mortem, suggesting upregulated activity of sEH by neurons (Stafstrom and Carmant, 2015). Neuroinflammation, (Nelson et al., 2014). Moreover, increased sEH immunoreactivity in the

31 S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39 cerebrovascular endothelium is observed adjacent to hyperintense particularly in westernized countries with poor diets consisting of white matter lesions (WMH), which are characteristic of VCI. The simple carbohydrates and trans-fats (Bliss and Whiteside, 2018). The R287Q sEH polymorphism is also associated with increased WMH vo- estimated prevalence of obesity in the United States is 36.5% of adults lume (Nelson et al., 2014). Interestingly, there is an association be- (Ogden et al., 2015). Obesity is a major risk factor for a variety of severe tween WMH and psychiatric diseases that involve cognitive impairment health conditions such as diabetes mellitus and cancer due to an in- (Swardfager et al., 2018). This implies that there is an intimate re- creased chronic inflammatory state (Garg et al., 2014). While there are lationship between cell signaling, cerebrovasculature, and behavior, many factors influencing weight, such as diet, genetics, and physical and that sEH may represent a common denominator when investigating activity, the brain-gut axis plays an especially important role in endo- possible treatment options for these disorders. These results suggest crine signaling and microbiome interaction (Bliss and Whiteside, 2018). that sEH may represent a viable target for the treatment of cognitive sEH inhibition represents a promising treatment for cardiovascular impairment in humans, though more in vivo and in vitro research must diseases, discussed in detail above. There is accumulating evidence that be conducted to establish a causative relationship. sEH may contribute to obesity-related disorders as type II diabetes mellitus, hypertension, and cardiomyopathy (Huang et al., 2016). 8.3. Depression In rats, there is an observed post-prandial decrease in sEH, marked by oxylipin (Yang et al., 2017b). This decrease in sEH activity is ad- Depression is the most common psychiatric disorder, and can cause ditive with increased dietary potassium, which is shown to decrease risk crippling symptoms such as fatigue, suicidality, lack of appetite, and of stroke (Yang et al., 2017b; D’Elia et al., 2011). However, rats treated anhedonia. To date, treatment options for depression are relatively with antibiotics to eradicate gut flora do not show the attenuation of limited to selective-serotonin reuptake inhibitors (SSRIs), selective- sEH activity seen in non-treated rats (Yang et al., 2017b). This in- norepinephrine reuptake inhibitors (SNRIs), and counseling formation suggests that sufficient dietary potassium may induce in- (Hashimoto, 2016). Even with the availability of SSRIs and SNRIs, hibition of sEH modulated by the gut microbiome, and altogether may many still do not respond to pharmacological intervention. Inflamma- be protective against cerebrovascular accident. In rats fed a high-fat tion, like in other diseases affecting the brain, could have a feasible and diet for 10 weeks, CYP-derived EET production is significantly de- damaging role in depression as well, as suggested by Zunszain, Hepgul, creased compared to rats with a normal diet (Wang et al., 2003). Mice and Pariante (Zunszain et al., 2013). This paves the way for the notion treated with sEHIs for 5 weeks demonstrate a 32% decrease in caloric that EETs may have advantageous effects on depressive symptoms due intake after being fed a high-fat high-fructose diet prior to the sEHI to their anti-inflammatory properties. administration, leading to weight loss (do Carmo et al., 2012). The Seasonal major depression is accompanied by increased sEH derived same mice also show decreased leptin levels after sEHI treatment (do oxylipins in patients during the winter, suggesting increased sEH ac- Carmo et al., 2012). Furthermore, colonic inflammation associated with tivity (Hennebelle et al., 2017). Although the clinical significance of obesity is correlated with increased levels of sEH in mice, and its ge- this study is limited by the small sample size (n = 15), these results netic deletion is associated with decreased inflammation (Wang et al., offer venues for more studies investigating the use of sEH derived 2018). Therefore, diet, a major contributing factor to obesity, is shown oxylipins as potential biomarkers for depression. Furthermore, TPPU to influence the production of EETs and activity of sEH. As discussed administration is associated with ameliorated depressive symptoms in thoroughly above, increased availability of EETs/sEH inhibition may be social defeat stress models in mice (Ren et al., 2016). SEHKO mice and protective in stroke. The established interaction between the gut mi- TPPU-treated mice also display greater resilience to social defeat stress, crobiome (Galland, 2014) and brain, due to the findings that sEH ac- which corresponds to upregulated brain-derived neurotrophic factor tivity may be partially dependent on gut bacteria modulation, may be of signaling via the TrkB receptor (Ren et al., 2016), (Wu et al., 2017). interest in future research concerning sEH in obesity and obesity-re- This provides further evidence for sEH inhibition’s integral role in de- lated disorders. In summary, sEH inhibition may promote a healthy gut, pression pathology and its potential use as an antidepressant. More which not only has beneficial effects on the gastrointestinal tract and studies must be executed to uncover the exact mechanisms by which obesity, but also on the brain. sEH is involved in depression. 10. sEHI therapeutic effects in other non-CNS organs 8.4. Subarachnoid hemorrhage sEHIs exhibit therapeutic effects in other non-CNS organs, such as Subarachnoid hemorrhage has a similar mechanism of injury as the the liver, heart, and kidney (Harris et al., 2015; Sirish et al., 2013; Kim disorders mentioned above, with acute inflammation serving as a major et al., 2014). Particularly, sEHIs mitigate tissue fibrosis in several or- bad actor in poor outcomes (Miller et al., 2014). Therefore, sequestra- gans such as the liver, as TPPU reduces collagen deposition, in- tion of the inflammatory response in subarachnoid hemorrhage may be flammation, and endoplasmic reticulum stress in the livers of mice beneficial, and this may be mediated by an increased level of EETs. In given carbon tetrachloride (Harris et al., 2015). TPPU also attenuates SEHKO mice, less edema is observed in white matter tracts after sub- heart remodeling associated with cardiac fibrosis by improving heart arachnoid hemorrhage-induction compared to wild-type mice (Siler function, decreasing collagen deposition, reducing cardiac fibroblasts, et al., 2015a). SEHKO mice are also found to express less VCAM-1 after and preventing cardiomyocyte hypertrophy (Sirish et al., 2013). Ad- subarachnoid hemorrhage, which is a marker of damaged endothelium ditionally, sEHIs reduce myocardial infarct sizes after heart ischemia (Siler et al., 2015a). This may be the reason for the reduction in edema and preclude cardiac hypertrophy and arrhythmia via EET-related in these mice. 14,15-EET is also shown to be protective against delayed pathways in animal models (Gross et al., 2008; Seubert et al., 2006). cerebral ischemia in mice subjected to subarachnoid hemorrhage (Siler Similarly, inhibiting sEH decreases inflammation, cell death, and oxi- et al., 2015b). As described above, there is one clinical trial that has dative stress, and precludes renal fibrosis in mouse kidneys (Kim et al., been launched, aiming to evaluate the effects of GSK2256294 (Oregon 2014). Moreover, sEHIs protect against hypertension-related damage to H and Science U, 2018). Although the use of sEHIs as treatment for vascular and glomerular structures in the kidney and reduce collagen subarachnoid hemorrhage is still in its rudimentary phase of clinical type IV expression (Zhao et al., 2004; Imig et al., 2012). In fact, ad- evaluation, preclinical data are supportive. ministering AUDA prevents hypertension and type 2 diabetes-asso- ciated renal damage by decreasing urinary albumin and MCP-1 excre- 9. Obesity and sEH tion and the infiltration of monocytes/macrophages in the kidney (Olearczyk et al., 2009). Thus, sEHIs are capable of protecting the heart Obesity is an increasingly common public health problem, and kidney from cardiovascular diseases such as hypertension (Imig

32 S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39 and Hammock, 2009). Furthermore, sEHIs’ anti-inflammatory proper- 2017), which are well-documented disorders with neuroinflammation ties and ability to mitigate systemic inflammation (Schmelzer et al., as a major secondary cause of cell death. In similar fashion, stem cell 2005) may also prevent additional inflammation-induced damage to therapy in these disorders dampens inflammation (Neal et al., 2018; these organs resulting from the actions of pro-inflammatory molecules Stonesifer et al., 2017; Acosta et al., 2015b; Lozano et al., 2015; Acosta and pathways following tissue insult (Harris et al., 2008). et al., 2014; Lippert et al., 2018; Rao et al., 2017). sEHIs suppress in- flammation by reducing proinflammatory lipid mediators (Schmelzer 11. Stem cells and sEH et al., 2005), but an equally potent anti-inflammatory mechanism is the activation of the EET-induced PPAR-γ pathway (Liu et al., 2005), which The anti-inflammatory properties of sEHIs in CNS disorders can be inhibits the NF-κB pathway and reduces VCAM-1 expression (Morisseau enhanced by similar anti-inflammatory and regenerative characteristics and Hammock, 2013; Liu et al., 2005; Shen and Hammock, 2012). of stem cell therapy. Although the mechanisms of stem cell therapeutic Conversely, PPAR-α and PPAR-γ agonists are potent inducers of the sEH techniques remain not well understood, even short-lived stem cell en- enzyme (De Taeye et al., 2010). In parallel, the anti-inflammatory graftment may serve as key regenerative process towards direct re- properties of stem cells (Stonesifer et al., 2017; Acosta et al., 2015b; placement for damaged or dead cells (Rolfe and Sun, 2015; Borlongan Kim et al., 2010), acting likely via reduction of ICAM-1 is also seen in et al., 2004; Lee et al., 2017). A concern of sEHI use is that it may stem cell transplants in experimental stroke (Cheng et al., 2018). Al- reduce endogenous stem cell proliferation (Fromel et al., 2012), and together, the postulated mechanism mediating inflammation in CNS this may be amended through the engraftment of exogenous stem cells disorders reveals equally common cellular and molecular targets to to maintain neurogenesis. Additionally, transplanted stem cells can probe stem cells and sEHI interactions. Furthermore, the recognition of provide indirect aid by secreting a variety of therapeutic molecules to sEH-mediated inflammation and the poor stem cell survival following stimulate the repair of dying brain cells (Blandini et al., 2010; Park transplantation due to inflammation (Sullivan et al., 2015)offers novel et al., 2008; Lee et al., 2017) with anti-inflammatory factors among insights for the use of sEHIs to enhance stem cell therapeutic effects. these secreted factors as highly potent for affording regeneration (Chen sEHIs and stem cells share many signaling pathways, which may et al., 2003; Drago et al., 2013; Blais et al., 2013). Similarly, sEHIs explain their similarities in the resulting functional outcomes. The Wnt exhibit anti-inflammatory properties (Harris and Hammock, 2013; Liu and PI3K/AKT signaling pathways are involved in both sEH and stem et al., 2005), making them prime candidates for combination therapy cells (Fromel et al., 2012; Chen et al., 2013; Takahashi et al., 2005; Van with stem cells. In tandem, stem cells can advance sEHI applications by Camp et al., 2014). Interestingly, both the PI3K/AKT and AKT/GSK3β compensating for the deteriorating endogenous cells through reduction pathways are regulated by EETs for promoting angiogenesis (Qu et al., of inflammation-plagued CNS disorders. 2015) and endothelial progenitor cell (EPC) function (Guo et al., 2018), sEH overexpression in CNS disorders characterized by protein ag- respectively. In addition, the EET-mediated activation of the PI3K/AKT gregate-mediated inflammation may benefit from a combination of sEH pathway provides protection after cerebral ischemia/ inhibition and stem cell therapy. In PD patients, sEH levels are elevated (Qu et al., 2015). With this in mind, the idea that sEHIs enhance stem and centralized around inflamed areas associated with α-synuclein cell therapeutic effects via PI3K/AKT or AKT/GSK3β pathway should aggregation (Borlongan, 2018), a pathological hallmark of the disease. be further explored to fully understand the benefits of sEHI on stem cell While α-synuclein aggregation in PD is primarily observed in the survival towards optimizing an sEH-based stem cell combination brainstem and cortex, such aberrant protein accumulation can also be therapy. detected peripherally. Thus, sEH, a mediator in the accumulation of α- sEHIs and stem cells both enhance the vasculature via the PPAR-γ synuclein, may be detected outside the CNS as well. If altered sEH levels pathway, an established regulator of angiogenesis. EETs are en- can be assayed peripherally, then early diagnosis and intervention are dogenous activators of PPAR-γ (Morisseau and Hammock, 2013; Harris possible to prevent extensive dopaminergic neuron loss in PD, whose and Hammock, 2013; Liu et al., 2005), which can modulate EPC symptoms are not recognized until 80% of the dopaminergic neurons function (Xu et al., 2013; Guo et al., 2018). Moreover, sEHIs increase are depleted. Interestingly, sEH inhibition reduces neurotoxicity in cell the expression of VEGF and hypoxia-inducible factor-1α (HIF-1α) via and animal models (Ren et al., 2018), and the treatment may be en- PPAR-γ (Xu et al., 2013), which are secreted by stem cells (Stonesifer hanced by the addition of stem cells (Borlongan, 2018). While sEH et al., 2017) and are neuroprotective for CNS disorders (Greenberg and activity in plasma is very low, the activity and protein can be monitored Jin, 2013). Furthermore, MSCs can synthesize EETs (Kim et al., 2010), in peripheral blood cells. Because endogenous stem cells from PD pa- and in turn EETs are able to enhance stem cell migration following tients show increased concentrations of sEH, cell replacement with transplantation (Li et al., 2015). Equally convincing evidence also show exogenous stem cells containing lower levels of sEH represents a viable that, sEH and EETs can inhibit hematopoietic progenitor cell pro- method for promoting brain regeneration (Borlongan, 2018). Interest- liferation and MSC-derived adipocyte stem cell differentiation (Fromel ingly, similar to PD, a pathological link between inflammation and et al., 2012; Kim et al., 2010; Vanella et al., 2011), suggesting that a protein aggregation accompanies other CNS disorders; notably mutant much more careful assessment is necessary when contemplating sEH huntingtin in HD (Luo et al., 2018), platelet-leukocyte aggregations in inhibition and stem cell combination therapy. Along this cautionary stroke (Tao et al., 2016), increased α-synuclein in TBI (Acosta et al., note, both the NF-κB and Wnt signaling pathways are involved in 2015a), increased amyloid and tau in dementia (Femminella et al., cancer stem cells (Van Camp et al., 2014; Rinkenbaugh and Baldwin, 2018), depositions prion protein upregulation in clinical depression 2016; Reya et al., 2001). Supporting this, sEH expression is found to be (Beckman and Linden, 2016), and spectrin aggregate formations in increased in many different neoplastic tissues (Harris and Hammock, epilepsy (Syrbe et al., 2017). Cognizant of these overlapping patholo- 2013). Translational research efforts evaluating sEHIs’ role in reducing gies, combination therapy of stem cells and sEHI stands as potentially the potential tumorigenic risk of stem cell therapy while maximizing efficacious strategy to treat CNS disorders plagued by neuroinflamma- their therapeutic effects (Stonesifer et al., 2017; Reya et al., 2001) will tion and proteinopathy. guide not just the efficacy, but also the safety of sEHI and stem cell sEH inhibition may harbor an anti-inflammatory environment in combined therapy. CNS disorders due to the intrinsic properties of EETs (Zhang et al., 2007; Harris and Hammock, 2013; Yang et al., 2015b), and this ther- 12. Synergistic effects of sEHIs combined with other compounds apeutic pathway may be enhanced by stem cell adjunctive therapy (Fig. 3). Indeed, sEHIs have been studied in the context of stroke (Zhang While benefits of sEHIs are typically associated with EETs, which et al., 2007; Shaik et al., 2013; Shen and Hammock, 2012), epilepsy are anti-inflammatory and promote vasodilation (Imig and Hammock, (Inceoglu et al., 2013), PD (Lakkappa et al., 2018), and TBI (Hung et al., 2009), other EpFAs are also active and exert detrimental or beneficial

33 S. Zarriello et al. Progress in Neurobiology 172 (2019) 23–39

Fig. 3. The potential mechanisms by which sEH inhibition and stem cell therapy may be synergistic. Beneficial properties of each treatment may be enhanced when used in combination, improving outcomes of CNS diseases. effects (Wagner et al., 2017). For instance, sEH metabolizes linoleic synergistic effects such as increased antinociception and reduced COX epoxides to linoleic diols, which demonstrate toxicity, modulate ther- enzyme expression, which leads to decreased pain and inflammation mogenesis and brown adipose tissue, and cause vascular permeability while avoiding detrimental thrombotic cardiovascular side effects as- and sepsis (Zhang et al., 2014b; Wagner et al., 2017). Epoxyeicosa- sociated with NSAIDs (Schmelzer et al., 2006). Indeed, co-administra- trienoic acids (EEQs) from (EPA) are implicated tion of aspirin and t-AUCB enables a lower dose of aspirin to be used, in anti-inflammation and various organs such as the lungs and uterus achieving the same anti-inflammatory benefits as a higher dose while (Morin et al., 2010). Epoxydocosapentaenoic acids (EDPs) from doc- minimizing the risk of blood clotting and gastrointestinal side effects osahexaenoic acid (DHA) are often more active than EETs, are highly (Liu et al., 2010). Administering the COX-2 inhibitor (coxib) celcecoxib present in the retina and brain (Zhang et al., 2014b), and possess potent together with t-AUCB reduces inflammation, increases eicosanoids like anti-angiogenic (Zhang et al., 2013b) and vasodilatory (Zhang et al., EETs that provide cardiovascular protection, and inhibits tumor an- 2014b) properties. In particular, 17,18-EEQ and 19,20-EDP decrease giogenesis to preclude tumors from growing and metastasizing in an- autophagy and ER stress in obese mice (Lopez-Vicario et al., 2015) and imal cancer models (Zhang et al., 2014a). Similarly, t-AUCB boosts 5- inhibit sEH metabolism more effectively than other omega-3 regioi- lipoxygenase (5-LOX) activation protein (FLAP) inhibitor MK886- somers (Zhang et al., 2014b). Interestingly, EEQs and EDPs may exhibit mediated anti-inflammation and lipoxygenase inhibition, apparent by a greater increase in vasodilation and decrease in pain and inflamma- the reduction in the pro-inflammatory 5-LOX metabolites such as leu- tion than EETs (Morisseau et al., 2010). Of note, omega-3 rich diets kotriene and 5-HETE in a murine model (Liu et al., 2010). Furthermore, promote anti-inflammation and raise plasma and tissue concentrations adding omeprazole, a CYP 450 inducer, together with TPPU has a of EEQ and EDPs in mammals, as these compounds are derived from combined effect which increases pain reduction, P450 activity, and EET omega-3 fatty acids (Arnold et al., 2010). Moreover, sEHIs are more levels (Goswami et al., 2015). Combining PPAR-γ agonists and sEHIs efficient in most assays if the animal’s diet is high in omega-3 fatty acids reduces glomerular injury in the kidneys of hypertensive obese rats and lacks omega-6 fatty acids (Lopez-Vicario et al., 2015; Arnold et al., significantly more than the PPAR-γ agonist or sEHI alone (Imig et al., 2010). sEHIs exhibit beneficial synergistic effects such as increased 2012). Of note, administering both sEHIs and phosphodiesterase in- anti-inflammation when applied with other compounds. Combining hibitors has a stronger effect on decreasing pain and elevating the sEHIs with a diet rich in omega-3 polyunsaturated fatty acids (PUFAs) plasma ratio of epoxy/dihydroxy fatty acids (Inceoglu et al., 2011). mitigates hypertension induced by angiotensin-II, increases EPA and With these favorable synergistic effects, it is possible that combining DHA epoxides in the kidney, decreases inflammation, and inhibits COX sEHIs and stem cells may augment the latter’s anti-inflammatory and LOX metabolic pathways compared to a diet rich in PUFAs and no characteristics and may exhibit other beneficial effects. administration of sEHIs (Ulu et al., 2013). Moreover, using sEHIs in Administration of both sEHIs and exogenous EETs have beneficial tandem with nonsteroidal anti-inflammatory drugs (NSAIDs) exerts effects on the brain and other body systems, as described above.

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Inhibition of sEH seems to be a more important objective than ad- Currently, stem cell therapies for CNS disorders such as PD, HD, ministering EETs themselves. EEQs and EDPs may have more anti-in- stroke, TBI, and epilepsy are in clinical trials. Optimizing the delivery of flammatory properties than EETs, the availability of which would be exogenous stem cells and the ability to stimulate endogenous stem cells, increased by sEH inhibition and not by EET administration (Morisseau and overcoming risk factors including tumorigenesis, are avenues to et al., 2010). The unique distribution of sEH in the brain also presents a test the adjunctive therapeutic potential of sEHIs. CNS disorders are therapeutic challenge. This supports the inhibition of sEH as a target, so often characterized by chronic inflammation and neuronal death. sEHIs that effects take place where the enzyme works, rather than the can reduce neuroinflammation by upregulating EETs and other EpFAs transplant of EETs. EETs are also susceptible to stomach acid de- and promoting angiogenesis, thus alleviating some symptoms of CNS gradation, beta oxidation, and hydroxylation (Fang et al., 2001). disorders, but also enhancing stem cell grafts’ functional effects. In However, AUDA shows significantly more activity in the dilation of combination, sEH inhibition can reduce inflammation while the trans- mesenteric vessels than other sEHIs, and this has been attributed to its plantation of exogenous stem cells can provide a neuroprotective effect. ability to mimic epoxides (EETs and EpFAs) (Olearczyk et al., 2006). Elucidating the exact mechanisms by which sEH inhibition and stem Therefore, there is evidence that an ideal therapeutic regimen may cell therapy interact represents gaps in our knowledge that when re- entail the use of both EETs and sEHIs. solved can allow a safe and effective combination therapy of sEHI and stem cells. 13. Conclusion In conclusion, sEHIs offer many attractive therapeutic features that may aid in treating a variety of CNS disorders. This review outlined the sEH is an enzyme that has been linked to many pathological con- most studied concepts, as well as highlighted the novelty of sEHI use in ditions within the last 20 years, ranging from cardiovascular disease to specific CNS disorders. It is possible that a majority of sEHI action neurological disease. Its association with CNS disorders has been more comes from preserving EPA and DHA epoxides more than ARA (EETs) recently established, and inhibition of sEH shows robust therapeutic and blocking formation of linoleate diol (leukotoxin). Future basic potential. Some of the applications of sEHIs include the attenuation of science directions may uncover innovative mechanisms by which sEHIs inflammation, regulation of blood flow, and protection of neurons from can exert therapeutic effects, thereby guiding translational efforts to- excitotoxicity. However, many more mechanisms could be uncovered wards realizing their full clinical potential for treating brain maladies. and further investigation of sEH’s involvement in CNS disorders is warranted. In addition to discovering other methods by which sEHIs Acknowledgements may be beneficial to the progression of neurological diseases, the pharmacological properties of such drugs must be optimized. The en- CVB is funded by NIH NINDSR01NS102395. BDH is funded by NIH visioned safe and effective sEHI will likely possess druggable properties NIEHSR01ES002710 and NIH NIEHS Superfund ProgramP42ES004699. that include sufficient water solubility, a half-life justifying once a day dosing, and the capacity to cross the BBB. Although some sEHIs are References known to cross the BBB, there has not been a systematic process to optimize CNS exposure of sEHIs. Formulating and manufacturing Acosta, S.A., Tajiri, N., Shinozuka, K., Ishikawa, H., Sanberg, P.R., Sanchez-Ramos, J., compounds that are tailored toward these properties will be crucial to et al., 2014. Combination therapy of human umbilical cord blood cells and granu- locyte colony stimulating factor reduces histopathological and motor impairments in the success of sEHIs for treating CNS disorders. an experimental model of chronic traumatic brain injury. PLoS One 9 (3), e90953. The neuroprotection of sEH inhibition in neurological disorders has Acosta, S.A., Tajiri, N., de la Pena, I., Bastawrous, M., Sanberg, P.R., Kaneko, Y., et al., been documented in stroke, as evidenced by reduced infarct size ac- 2015a. Alpha-synuclein as a pathological link between chronic traumatic brain injury fl and Parkinson’s disease. J. Cell. Physiol. 230 (5), 1024–1032. companied by dampened in ammatory response likely by decreasing Acosta, S.A., Tajiri, N., Hoover, J., Kaneko, Y., Borlongan, C.V., 2015b. Intravenous bone pro-inflammatory cytokine secretion. sEHIs may also improve outcomes marrow stem cell grafts preferentially migrate to spleen and abrogate chronic in- by modulating CBF, although equally compelling evidence suggest non- flammation in Stroke. Stroke. 46 (9), 2616–2627. CBF mechanisms. Future studies must address deficits in current Adeoye, O., Hornung, R., Khatri, P., Kleindorfer, D., 2011. Recombinant tissue-type plasminogen activator use for ischemic stroke in the United States: a doubling of knowledge on stroke relating to vascular involvement, clinical differ- treatment rates over the course of 5 years. Stroke 42 (7), 1952–1955. ences between pharmacological inhibition of sEH and gene deletion, Ahmedov, M.L., Kemerdere, R., Baran, O., Inal, B.B., Gumus, A., Coskun, C., et al., 2017. and sex differences that may result in differed treatment. While sEH Tissue expressions of soluble human epoxide Hydrolase-2 enzyme in patients with temporal lobe epilepsy. World Neurosurg. 106, 46–50. inhibition is thought to operate under similar mechanisms in TBI Alkayed, N.J., Goyagi, T., Joh, H.D., Klaus, J., Harder, D.R., Traystman, R.J., et al., 2002. treatment, many more investigations must be pursued to identify the Neuroprotection and P450 2C11 upregulation after experimental transient ischemic exact targets of sEH-derived pathology in TBI. attack. Stroke 33 (6), 1677–1684. Arnold, C., Markovic, M., Blossey, K., Wallukat, G., Fischer, R., Dechend, R., et al., 2010. The sEH represents a highly unique and innovative target for po- Arachidonic acid-metabolizing cytochrome P450 enzymes are targets of {omega}-3 tential treatment in PD. Studies have shown that sEH levels in both the fatty acids. J. Biol. Chem. 285 (43), 32720–32733. brain and gut may be a possible PD pathology indicator well before Banks, W.A., 2009. Characteristics of compounds that cross the blood-brain barrier. BMC Neurol. 9 (Suppl 1), S3. symptoms arise, highlighting its importance in the early stages of the Beckman, D., Linden, R., 2016. A roadmap for investigating the role of the prion protein disease. sEH inhibition has also been associated with decreased loss of in depression associated with neurodegenerative disease. 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