Design and discovery of soluble epoxide hydrolase inhibitors for the treatment of cardiovascular diseases.

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Review

Design and discovery of soluble epoxide hydrolase inhibitors for the treatment of cardiovascular diseases

1.

Introduction

2.

ARA epoxidation

3.

Physiological properties of EETs

4.

Soluble epoxide hydrolase

5.

sEH inhibitory drugs

6.

Conclusion

7.

Expert opinion

Thomas Duflot, Clothilde Roche, Fabien Lamoureux, Dominique Guerrot & Jeremy Bellien† †

Institut de Biologie Clinique, CHU de Rouen, Service de Pharmacologie, Rouen, France

Introduction: Cardiovascular diseases are a leading cause of death in developed countries. Increasing evidence shows that the alteration in the normal functions of the vascular endothelium plays a major role in the development of cardiovascular diseases. However, specific agents designed to prevent endothelial dysfunction and related cardiovascular complications are still lacking. One emerging strategy is to increase the bioavailability of epoxyeicosatrienoic acids (EETs), synthesized by cytochrome P450 epoxygenases from arachidonic acid. EETs are endothelium-derived hyperpolarising and relaxing factors and display attractive anti-inflammatory and metabolic properties. Genetic polymorphism studies in humans, and experiments in animal models of diseases, have identified soluble epoxide hydrolase (sEH), the major enzyme involved in EET degradation, as a potential pharmacological target. Areas covered: This review presents EET pathway and its functions and summarises the data supporting the development of sEH inhibitors for the treatment of cardiovascular and metabolic diseases. Furthermore, the authors present the different chemical families of sEH inhibitors developed and their effects in animal models of cardiovascular and metabolic diseases. Expert opinion: Several generations of sEH inhibitors have now been designed to treat endothelial dysfunction and cardiovascular complications for a variety of diseases. The safety of these drugs remains to be carefully investigated, particularly in relation to carcinogenesis. The increasing knowledge of the biological role of each of the EET isomers and of their metabolites may improve their pharmacological profile. This, in turn, could potentially lead to the identification of new pharmacological agents that achieve the cellular effects needed without the deleterious side effects. Keywords: cardiovascular diseases, endothelium, enzymatic inhibitory drugs, epoxyeicosatrienoic acids, genetic polymorphisms, soluble epoxide hydrolase Expert Opin. Drug Discov. (2014) 9(3):229-243

1.

Introduction

Epoxyeicosatrienoic acids (EETs) are endogenous bioactive lipid mediators formed from arachidonic acid (ARA), a w-6 20-carbon polyunsaturated fatty acid, by cytochrome P450 (CYP450) epoxygenase enzymes, notably in the vascular endothelium [1-3]. Biological functions of EETs include vasodilation, increase in natriuresis and prevention of inflammation, apoptosis and thrombosis [1-3]. After their synthesis, EETs are rapidly incorporated into phospholipids of the cell membrane through an acyl-coenzyme A-dependent pathway, constituting a pool releasable by phospholipase A2, or are metabolised by soluble epoxide hydrolase

10.1517/17460441.2014.881354 © 2014 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X All rights reserved: reproduction in whole or in part not permitted

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Article highlights. . . .

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Endothelial dysfunction plays a key role in the development of cardiovascular diseases. Epoxyeicosatrienoic acids (EETs) are endothelial-derived eicosanoids with cardiovascular protective properties. New pharmacological agents inhibiting EET degradation by soluble epoxide hydrolase (sEH) are under active development. Inhibitors of sEH improve vascular and cardiac function in animal models of cardiovascular diseases and have recently moved into clinical trials. Clarifying the biological function of each EET isomer may help in developing specific modulators of this pathway.

This box summarises key points contained in the article.

(sEH) to dihydroxyeicosatrienoic acids (DHETs), with diminished or opposed effects to EETs [1-3]. Animal experiments and genetic polymorphism studies in humans have shown that sEH is a potential pharmacological target to treat cardiovascular diseases. The design and development of drugs inhibiting sEH, which enhance the biological effects of EETs, have stimulated experimental studies with promising results, especially in animal models of hypertension and metabolic diseases. Clinical trials have recently been initiated. However, the long-term safety profile of this new pharmacological class remains a matter of concern. In addition, given that the biological functions of the different EET isomers and their metabolites, the DHETs, may vary, the development of pharmacological agents more specifically affecting the EET pathway could be expected. This review presents the EET pathway and its biological functions, and summarises the data supporting the development of sEH inhibitors for the treatment of cardiovascular and metabolic diseases. In addition, potential pitfalls regarding the use of sEH inhibitors are discussed. 2.

ARA epoxidation

Free ARA can be metabolised by lipoxygenases (LOXs), cyclooxygenases (COXs) and CYP450s (Figure 1), which are a superfamily of cysteine-heme enzymes that mediate the oxidative metabolism of exogenous and endogenous molecules [1-3]. LOXs metabolise ARA to hydroperoxyeicosatetraenoic acids, which are subsequently converted to hydroxyeicosatetraenoic acids (HETEs), leukotrienes or lipoxins. COXs metabolise ARA to prostaglandin derivatives and thromboxane. CYP450 enzymes are epoxygenases (CYP1A, 2B, 2C, 2E and 2J subfamilies) that metabolise ARA to EETs, with the participation of reduced nicotinamide adenine dinucleotide phosphate or are hydroxylases (CYP 4A and 4F subfamilies) that metabolise ARA to 20-HETE, which is a potent constrictor of renal, cerebral, mesenteric and skeletal muscle arterioles [1-5]. 230

Accumulating evidence shows that CYP2C and 2J are the major enzymes involved in the synthesis of EETs, especially in human cardiovascular tissues [1-4]. The human CYP2C and 2J are located in the endoplasmic reticulum and add an epoxide across one of the four double bonds in ARA to produce four regioisomeric cis-EETs consisting in a mixture of two threo enantiomers (5R,6S)-EET, (5S,6R)-EET, (8R,9S)-EET, (8S,9R)-EET, (11R,12S)-EET, (11S,12R)-EET, (14R,15S)-EET and (14S,15R)-EET. The percentage of each enantiomer depends on the epoxygenase isoforms that are localised in different tissues. Interestingly, trans-substituted EETs have been shown to be synthesized, in particular, in red blood cells, probably through radical-driven reactions and exhibit similar or even higher vascular potencies than cis-EETs [6]. CYP450 epoxygenase isoforms vary between species. In particular, mice express CYP2C29, CYP2C38 and the more active CYP2C44 in blood vessels, whereas rats express CYP2C11 and CYP2C23. In human arteries and arterioles, CYP2C8 and CYP2C9 are the main CYP450 epoxygenases. The main product of CYP2C is 11,12-EET, identified as a major contributor to endotheliumderived hyperpolarising factor (EDHF) responses [1-3]. Of importance, in some circumstances, CYP2C can also produce reactive oxygen species, notably contributing to myocardial ischaemia-reperfusion injury [7,8]. Furthermore, the CYP2J2 is a main CYP450 epoxygenase isoform responsible for EET synthesis not only in the human heart, kidney, lung and pancreatic islets but also in circulating cells such as monocytes/ macrophages [1-3]. CYP2J2 produces preferentially 14,15-EET and particularly the 14,15(R,S)-EET enantiomer. 3.

Physiological properties of EETs

EETs are important components of many intracellular signalling pathways in both cardiovascular and noncardiovascular tissues, although EET receptors still remain to be clearly identified. Significant CYP450 epoxygenase activity and EET production can be detected in endothelial cells and cardiomyocytes. The most important biological functions of EETs include vasodilatation, anti-inflammatory, thrombolytic, pro-angiogenic and anti-apoptotic properties. The most studied function of EETs is to act as vasodilators in various resistance and conduit arteries, including in humans in vivo [9]. In fact, EETs are synthesized in endothelial cells in response to the binding of agonists, such as acetylcholine or bradykinin, to their receptors or to the increase in shear stress. The four EET regioisomers are EDHFs causing hyperpolarisation and relaxation of vascular smooth muscle cells (VSMC). EETs diffuse out of the endothelial cells to activate large conductance calcium-activated potassium (BKCa) channels present in VSMC, directly promoting their hyperpolarisation [2]. In addition, EETs can also modulate the classical EDHF pathway by acting as autocrine factors to activate small or intermediate conductance calcium-activated potassium channels (SKCa and IKCa, respectively) located in endothelial cells [2]. Then, the

Expert Opin. Drug Discov. (2014) 9(3)

Design and discovery of sEH inhibitors for the treatment of cardiovascular diseases

Glycerophospholipids (PC, PE, PI, PS) Incorporation through an acyl-CoA dependent mechanism PLA2 Arachidonic acid O O

H

COXs PGG2

15-LOX CYP450 hydroxylases (CYP4A, 4F)

PGH2


20-HETE PGD2, PGE2, PGF2a, PGI2 TxA2, TxB2

CYP450 epoxygenases (CYP1A, 2B, 2C, 2E, 2J)

cis-(5R,6S)-EET cis-(5S,6R)-EET cis-(8R,9S)-EET cis-(8S,9R)-EET cis-(11R,12S)-EET cis-(11S,12R)-EET cis-(14R,15S)-EET cis-(14S,15R)-EET

12-LOX

15-HPETE

5-HETE

12-HPETE

15-HETE

LTA4

12-HETE

LXA4, LXB4

LTB4, LTC4, LTD4, LTE5 LXA4, LXB4

O

O 6

5-LOX

5 O

H

H H cis-(5R,6S)-EET Soluble epoxide hydrolase Threo-(5S,6S)-DHET Threo-(5R,6R)-DHET Threo-(8S,9S)-DHET Threo-(8R,9R)-DHET Threo-(11S,12S)-DHET Threo-(11R,12R)-DHET Threo-(14S,15S)-DHET Threo-(14R,15R)-DHET H

H OO HH

O O

H

Threo-(5S,6S)-DHET

Figure 1. Overview of arachidonic acid metabolism: arachidonic acid can be converted by COXs into prostaglandins and TxA2, TxB2 and by LOXs in HETEs and HPETEs as well as LTs and LXs. The third pathway of arachidonic acid metabolism is related to its conversion by CYP4A and CYP4F in 20-hydroxyeicosatetraenoic and by CYP450 epoxygenases into four EET regioisomers. EETs are then rapidly metabolised by soluble epoxide hydrolase to their corresponding DHETs. In addition, they can be incorporated into phosphoglycerols to form PC, PE, PI, PS through an acyl-coenzyme A-dependent pathway constituting a pool releasable by PLA2. COXs: Cyclooxygenases; DHETs: Dihydroxyeicosatrienoic acids; EET: Epoxyeicosatrienoic acid; HETEs: Hydroxyeicosatetraenoic; HPETEs: Hydroperoxyeicosatetraenoic acids; LOXs: Lipoxygenases; LTs: Leukotrienes; LXs: Leucotoxins; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PI: Phosphatidylinositol; PLA2: Phospholipase A2; PS: Phosphatidylserine; TxA2, TxB2: Thromboxanes.

hyperpolarisation is transmitted through the gap junction to the VSMC layer, or the potassium ions released from these channels activate inward rectifying potassium channels or the ouabain-sensitive Na+/K+ ATPase participating in VSMC hyperpolarisation [2]. At last, VSMC membrane hyperpolarisation leads to the reduction of the open-state probability of voltage-dependent calcium channels and to subsequent cell relaxation. Further, a close interaction between EET and nitric oxide (NO) pathways exists [10].

EETs increase endothelial NO-synthase (eNOS) expression and activity, thus promoting NO release in endothelial cells, whereas in contrast NO decreases CYP2C activity and thus EET synthesis [2]. The EET pathway, therefore, acts as a compensatory pathway when NO availability is compromised in some pathophysiological states to partially maintain a vasodilator response [2]. Of note, the vascular actions of EETs in the lung are totally contrary to those described in the systemic circulation. At this level, EETs induce a direct


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constriction of VSMC, likely by a transient receptor potential C6 channel-dependent pathway [11]. Moreover, EETs play an important role in the modulation of cardiac and vascular inflammatory processes. In particular, EETs decrease the expression of vascular cell adhesion molecule-1 and decrease leucocyte adhesion to the vascular wall in response to pro-inflammatory signals, through the inhibition of nuclear factor kB (NF-kB) and IkB kinase [12], thus preventing their rolling and migration across the endothelium. The anti-inflammatory effects of EETs are also mediated by the activation of subfamilies of nuclear PPARs and decreased expression of inducible COX-2 [2]. Furthermore, 11,12-EET, but not 5,6-EET, 8,9-EET and 14,15-EET, presents antimigratory and anti-proliferative effects on cultured VSMC, by a mechanism involving intracellular cyclic adenosine monophosphate and downstream protein kinase A [13,14]. In addition, EETs are known to be involved in a wide range of cellular effects leading to angiogenesis. In particular, some studies demonstrated that EETs are second messengers of the VEGF signalling pathway [15]. Moreover, 5,6-EET and 8,9-EET contribute to endothelial cell migration and tube formation by promoting extracellular matrix degradation, a necessary prerequisite for the angiogenic process [16]. Other signalling pathways involved in the regulation of angiogenesis by EETs include the activation of eNOS, ERK kinase/MAPK and phosphatidylinositol 3-kinase (PI3K) [2]. Regarding the regulation of hemostasis, EETs can be stored in red blood cells and released from these stores to participate in vasodilatation, platelet aggregation inhibition and inflammation resolution [1-3]. Finally, EETs also have the potential to enhance the expression and release of fibrinolytic enzymes in endothelial cells and notably tissue plasminogen activator, including in vivo in humans [17,18]. In contrast, the physiological role of EETs in the regulation of cardiac function remains unclear, although their protective action during cardiac ischaemia-reperfusion injury is undisputable. EETs were reported not to alter contractility in normal isolated guinea pig heart [19], although they act on many cardiomyocyte membrane channels. In particular, EETs open both sodium and ATP-sensitive potassium channels in rat ventricular myocytes [19]. Moreover, EETs inhibit porcine L-type calcium channels reconstituted in artificial cell membrane [20] but activate these channels in rat cardiomyocytes [21], thus emphasizing the complex and divergent effects of EETs on cardiac function regarding the experimental models used. In addition to playing a key role in cardiovascular homeostasis, EETs possess various metabolic effects. In the pancreas, 5,6-EET promotes the release of insulin, whereas 8,9-EET, 11,12-EET and 14,15-EET may exert an opposite effect on glycaemia by inducing glucagon release [22]. Moreover, EETs modulate insulin sensitivity, notably by decreasing the phosphorylation of c-Jun N-terminal kinase and Ser312 on the insulin receptor substrate-1 in hepatocytes [23]. In the same manner, EETs activate PPARs and thus may modulate lipogenesis through an upregulation of 232

the expression of various enzymes involved in the transport, binding and metabolism of fatty acids or triglycerides, and in b-oxidation processes [1-3]. 4.

Soluble epoxide hydrolase

sEH structure The sEH, with the microsomal epoxide hydrolase (mEH), belongs to an a/b hydrolase fold family of enzymes. EPHX1 and EPHX2 genes transcription and translation result in the mEH and the sEH proteins, respectively [1]. EPHX2, which has been cloned and characterised in 1993 [24], is a 60 kb gene, including 19 exons with lengths from 27 to 265 bp, which encode 555 amino acids [24]. EPHX2 is located in chromosomal region 8p21-p12 in humans. The mammalian sEH is a ubiquitously expressed homodimeric enzyme, consisting of two 62 kDa monomers arranged in an anti-parallel fashion [25], localised in the cytosol and in peroxisomes. Importantly, sEH is a bifunctional enzyme with two distinct structural domains (Figure 2) [24,26,27]. The epoxide hydrolase activity of human sEH is located in the 35 kDa C-terminal domain (from amino acid 235 to amino acid 555, EPHX2 gene 5¢/3¢ nucleotidic position: nt 25751 -- 60842) [25] and has a high homology with the bacterial haloalkane dehalogenase enzyme, whereas the phosphatase activity similar to haloacid dehalogenase enzyme is located in the 25 kDa N-terminal domain (from amino acid 1 to amino acid 224, EPHX2 gene 5¢/3¢ nucleotidic position : nt 1 -- 25720). Hydrolase and phosphatase domains are linked by a proline-rich linker segment (from amino acid 225 to amino acid 234, EPHX2 gene 5¢/3¢ nucleotidic position: nt 25721 -- 25750) [26,28-30]. The structure of sEH is composed of a domain-swapped dimer in which the hydrolase domain of one monomer binds to the phosphatase domain of the opposite monomer [31]. The Arg287 residue plays a major role in determining human sEH quaternary structure [28]. Indeed, an inter-monomer ionic salt bridge interaction between the Glu254 and Arg287 is required to ensure dimerisation and activity of the sEH, although a combination of two mutations Glu254Arg/Arg287Glu may reverse the salt bridge and rescue both dimerisation and activity [31]. Asp333, His523 and Asp495 form a catalytic triad needed for the hydrolase activity of sEH [32]. This activity was shown to be highest in the liver, followed by the kidney, and to a lesser extent in other extrahepatic tissues. 4.1

sEH enzymatic activities The a/b hydrolase fold family of enzymes hydrolyses epoxides in their corresponding diols. Although the mEH is described to be a key enzyme in the metabolism of environmental contaminants and prefers mono- and cis-disubstituted epoxides as substrates, the sEH is the main enzyme involved in the metabolism of EETs [25]. Other EET metabolising pathways include b-oxidation, chain elongation and metabolism by COX for 5,6-EET. After metabolisation by sEH, the cis-EETs formed 4.2



R103C

R287Q

K55R


C154Y

N-terminal phosphatase domain C-terminal epoxide hydrolase domain

Figure 2. Crystal structure of soluble epoxide hydrolase monomer is shown. The main sites of single nucleotide polymorphism with demonstrated clinical impact are annotated and represented in balls and sticks. Modified from the RCSB PDB (www.rcsb.com) of PDB ID 1S8O: Ref. [26]

from ARA epoxidation each result in two erythro (S,S or R,R) DHET enantiomers (Figure 1) [1-3]. However, the hydrolase activity of sEH is not restricted to EETs and various lipid epoxides involved in the regulation of cardiovascular function are also metabolised by sEH. In particular, the linoleic acid derivatives of epoxyoctadecenoic acids are converted to the pro-inflammatory dihydroxyoctadecenoic acids, and inhibition of this metabolic pathway may contribute to the beneficial effects of sEH inhibitors. In addition, fatty acid epoxides, notably the epoxides of w-3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), formed from the action of CYP450 epoxygenases on DHA and EPA, are metabolised by sEH [1-3,33,34]. The conversion of EETs into DHETs by sEH is regioselective and the lowest rates of hydrolysis are observed for epoxides close to the carboxylic acid. Thus, 14,15-EET is the preferred substrate of sEH with the highest Vm and lowest Km, followed by 11,12-EET, 8,9-EET and 5,6-EET [1-3,33,34]. In addition, it has been reported that the favourite sEH substrates are trans-substituted compared to cis-substituted epoxides and that w-3 lipid epoxides are better substrates than w-6 lipid epoxides [34]. In contrast to the hydrolase domain, the natural substrates as well as the biological functions of the phosphatase domain of sEH remain poorly understood at this time, but some cholesterol precursors and lysophosphatidic acid are dephosphorylated by this domain in vitro [35]. sEH in physiology and pathophysiology Many biological functions appear to be regulated by sEH. First, sEH plays a major role in the regulation of vascular resistance to blood flow and thus blood pressure, by 4.3

hydrolysing vasoactive linoleate and arachidonate epoxides and, in particular, the vasodilators and natriuretic EETs. In this respect, the increase in EET bioavailability in the first sEH knockout mouse colony, created by disruption of exon one by a neo-cassette, was associated with a decreased blood pressure [36]. However, this decrease was only detected in males and this may be related to the fact that estrogens downregulate the expression and activity of sEH [37]. More surprisingly, other groups generating independent sEH knockout mice did not retrieve these findings [38]. Furthermore, experimental data reported that in spontaneously hypertensive rats (SHRs) and angiotensin II-dependent hypertensive animals the sEH expression is increased, enhancing renal and vascular hydrolysis of EETs to DHETs [39-42]. Regarding the mechanisms involved, angiotensin II upregulates sEH expression both at the mRNA and the protein levels, by stimulating the sEH promoter activity by the activator protein-1 (AP-1) pathway through the binding of c-Jun or c-Fos, which are liberated after the fixation of angiotensin II on angiotensin type 1 receptor, on these AP-1 sites [43]. Accordingly, patients with renovascular hypertension have a decrease in plasma EET/DHET ratio, suggesting an increase in sEH activity [44]. In addition, the impairment of endotheliumdependent flow-mediated dilation of conduit arteries in patients with essential hypertension is notably due to a complete loss of EET release [9]. Furthermore, sEH may accelerate the development of heart failure, since angiotensin II-induced increase in sEH expression is also observed in the heart, contributing to the development of cardiac remodelling by the suppression of the anti-hypertrophic


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effect of EETs, mediated through NF-kB inhibition [45]. In this respect, a role for an altered EET pathway in the development of heart failure in the absence of hypertension has recently been also put forward [46]. Indeed, genetically selected rats with an upregulation of the EPHX2 transcript and sEH protein expression, promoting the decrease in EET availability, develop cardiac hypertrophy and dysfunction [46]. Furthermore, sEH may play a critical role in the maintenance of glucose and lipid homeostasis. In a model of obesity and insulin resistance, the Zucker rat, sEH expression is upregulated in renal arteries, contributing to alteration of endothelium-dependent dilation [47]. In addition, sEH may favour the transition between insulin resistance and type 2 diabetes. In fact, the total adipose sEH activity is increased in mice fed with a high-fat diet, altering the inhibitory action of EETs on adipocyte differentiation [47]. Additionally, total plasma cholesterol was 25% lower in sEH knockout male mice compared with sEH wild-type male mice, but no difference was observed between knockout and wild-type females [48]. Thus, an increased activity of sEH may favour the development of atherosclerosis through a deregulation of cholesterol metabolism and by modifying the ratio between the antiinflammatory epoxides and pro-inflammatory diols. In fact, sEH appears to be an important regulator of cholesterol levels through both its phosphatase and hydrolase domains [48]. In sEH D9A-mutant HepG2 cells, possessing only a hydrolase activity, total cholesterol was decreased but sEH D335S-mutant HepG2 cells, possessing only a phosphatase activity, have a 40% increase in cholesterol compared with the wild-type mutant [48]. This can be explained by the fact that the hydrolase domain downregulates hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase expression, whereas the phosphatase domain overexpresses HMG-CoA reductase and hydrolyses intermediates of the cholesterol biosynthesis pathway, involved in the regulation of various nuclear receptors controlling cholesterol biosynthesis, such as liver X receptor and PPAR-a [48]. Other functions of sEH include the regulation of blood flow and neurotransmitter release in the brain [49] and the control of the development of the reproductive system and fertility in male and female [50]. In addition, in the lungs and pulmonary arteries, sEH is expressed in the endothelium and VSMC of the alveolar capillaries and is increased in bronchi of asthmatic humans, and in fact is also a critical pharmacological target for the treatment of inflammatory lung diseases [51,52].

EPHX2 genetic polymorphisms and cardiovascular diseases

4.4

It has been suggested, before 1990s, that a genetic factor may influence human sEH enzymatic activity [53]. In fact, EPHX2 is a highly polymorphic gene with > 100 single nucleotide 234

polymorphisms (SNPs), and non-synonymous SNPs resulting in amino acid substitutions have been reported to influence sEH activity [28,54-56]. The SNPs Lys55Arg (rs41507953), Cys154Tyr (rs57699806), Arg287Gln (rs751141) and the Arg103Cys/Arg287Gln (combination of the two SNPs rs17057255 and rs751141) significantly alter epoxide hydrolase activity [56]. The rs41507953 and rs57699806 polymorphisms result in a decrease in phosphatase activity [28], whereas rs751141 and rs17057255 have been associated with a higher phosphatase activity [54], compared to the most frequent allele (wild-type allele, NG_012064) (Figure 2) [26,27]. Such genetic polymorphisms of sEH have been linked to cardiovascular diseases, although some controversies exist notably related to the ethnic origin of the subjects explored (Table 1) [55-60]. In particular, two frequent SNPs have been well studied: . rs41507953: the Lys55Arg (K55R) mutation was

found in ~ 15% of an inter-ethnic population [58] and is associated with a higher hydrolase:phosphatase ratio, suggesting that this SNP enhances epoxide hydrolase activity and decreases phosphatase activity [28]. According to the expected decrease in EET bioavailability, the incidence of coronary artery diseases appears to be higher in subjects with the Lys55Arg mutation [58]. In addition, white Americans, but not black Americans, holding the Lys55Arg mutation have a reduced endothelium-dependent dilation of resistance arteries [59]. . rs751141: the Arg287Gln (R287Q) mutation is present in 10 -- 15% of the general population [60]. This SNP is associated with a lower hydrolase activity [28], an altered subcellular localisation phenotype, and a higher phosphatase activity in vivo [48]. Moreover, rs751141 leads to sEH structural changes, altering the inter-monomer ionic salt bridge interaction and reducing sEH stability. Surprisingly, despite the expected increase in EET bioavailability, this SNP was reported to be associated with an increased risk of coronary artery calcification in blacks but not in non-Hispanic whites [57]. In addition, no association between the Arg287Gln and coronary heart disease was noticed [57]. This may be related to the impact of this SNP on the phosphatase activity, which may be hypothetically important in the control of glucose and lipid homeostasis. In support of this hypothesis, the R287Q mutation was also associated with insulin resistance in patients with type 2 diabetes [61]. Moreover, plasma cholesterol and triglyceride levels were significantly increased in patients with familial hypercholesterolemia presenting the R287Q mutation [62]. Of note, it has been proposed that a gene--gene interaction between rs751141 and a mutation localised on the low-density lipoprotein (LDL) receptor gene may cause its failure [62]. Finally, in contrast to the Lys55Arg mutation, black Americans, but




Table 1. Main single nucleotide polymorphisms of sEH and impact in cardiovascular diseases. Localisation

Amino acid mutation

Exon 2 on 19

K55R/ Lys55Arg

Intron 2

X

Exon 3 on 19

R103C/ Arg103Cys

Exon 4 on 19

C154Y/ Cys154Tyr

Exon 8 on 19

R287Q/ Arg287Gln

Intron 11

X

Unknown

Intron 12 Intron 16 3¢ UTR region

X X X

Unknown Unknown Unknown

Impact on cardiovascular diseases in vivo

Impact on sEH activity

Higher hydrolase activity and lower phosphatase activity Unknown Higher hydrolase activity and higher phosphatase activity Higher hydrolase activity and lower phosphatase activity Lower hydrolase activity and higher phosphatase activity

Higher risk of coronary heart disease in Caucasians [58,59] Higher risk of coronary and carotid calcified plaques in African Americans [60] Unknown Unknown Higher risk of coronary artery calcification in African Americans but not in white Americans [57] Higher risk of insulin resistance [61] Higher plasma cholesterol and triglycerides [62] Increased endothelium-dependent dilation in African Americans [59] Higher risk of coronary calcified plaques in African Americans and Caucasians [60] Higher risk of ischaemic stroke in white Europeans [65] Higher risk of ischaemic stroke in white Europeans [65] Lesser risk of kidney allograft dysfunction [63]

sEH: Soluble epoxide hydrolase.

not white Americans, holding the Arg287Gln mutation have an increased vasodilator function of resistance arteries [59]. Another frequent mutation located in the 3¢ untranslated region (UTR) was associated with impaired kidney function in kidney transplant recipients [63]. In fact, the absence of this SNP is believed to be predictive of impaired kidney function [63]. To date, the possibility of the presence of other unidentified functional variants in linkage disequilibrium cannot be ruled out, as well as the effect of variations in other yet unidentified genes adjacent to the EPHX2 gene. In addition, ‘non-coding’ SNPs located in intron sequences, which may play a role in sEH activity and/or expression through mRNA or other mechanisms [64], have been linked with cardiovascular diseases, in particular ischaemic stroke [65], carotid and coronary artery calcified plaques [57], as well as carotid artery intima-media thickening in participants of the Diabetic Heart Study [60]. 5.

hydrolase activity of the C-terminal domain of the enzyme without affecting the N-terminal phosphatase domain [66,67]. The first sEH inhibitors were chalcone oxides and trans-3-phenylglycidols, which are structurally similar [68]. Then, researches led to new classes of inhibitors, the urea derivatives [67] and more recently the 4-substituted benzoxazolone and the sulphoxides. Physicochemical properties of the sEH inhibitor families 5.1.1 Trans-3-phenylglycidols 5.1

These derivatives are enantioselective slow-binding inhibitors of sEH, except those without a hydroxyl group, or with a blocked hydroxyl group, that have an IC50 in the micromolar range and that poorly affect the mEH [69]. The (2S,3S)3-(4-nitrophenyl)glycidol, is 750-fold more potent that the (2R,3R)-enantiomer, and the (2R,3R)-1-benzoyloxy2,3-epoxy-3-(4-nitrophenyl)propane is also a much better inhibitor than the (2S,3S)-enantiomer [69].

sEH inhibitory drugs Chalcone oxides Substituted chalcone oxides are more potent inhibitors than trans-3-phenylglycidols. The mechanism of action occurs through a covalent enzyme-inhibitor intermediate, which is electronically stabilised but these compounds are unstable in the presence of glutathione [69]. Tests on murine and human recombinant sEH demonstrated that the inhibitory potency of chalcone oxides is increased with n-pentyl, n-phenyl and n-propyl groups, which have optimal steric constraints. Moreover, the presence of hydrophobic moieties at position 5.1.2

Based on the above-described results obtained in animals, and on linkage of polymorphisms in human diseases, it is now well established that the increase in EET bioavailability by sEH inhibition may be useful for the treatment or the prevention of cardiovascular diseases. Since the early 2000s, pharmacological inhibitors of sEH have been under active development (Figure 3) and major advances in their potency and pharmacokinetic properties, allowing oral administration, have been made. Importantly, these agents inhibit the epoxide


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Urea derivatives:

trans-phenylglycidiols: (2S,3S)-3-(4-nitrophenyl) glycidol

DCU

Chalcone oxides: 4-PCO

CDU


CUDA

AUDA 4-substitued benzoxazolone: benzoxazolinonyl p-methyl benzoate

APAU

AEPU Sulfoxides: Fulvestrant t-AUCB

TPPU

Figure 3. Inhibitors of the hydrolase domain of soluble epoxide hydrolase are shown. 4-PCO: 4-phenylchalcone-oxide; AEPU: 1-adamantanyl-3-(5-(2-(2-ethoxyethoxy)ethoxy)pentyl)urea; APAU: 1-(1-acetypiperidin-4-yl)-3-adamantanylurea; AUDA: N-adamantanyl-N’-dodecanoic acid; CDU: N-cyclohexyl-N’-dodecylurea; CUDA: N-cyclohexyl-N’-dodecanoic acid urea; DCU: N,N’-dicyclohexylurea; t-AUCB: Trans-4[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid; TPPU: 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea.

4 strengthened inhibition, while neither the charge state nor the hydrophobicity of the 4-position appeared to influence the inhibitory potency of the chalcone oxides examined [69]. Urea derivatives This family is a recent subclass of selective and stable sEH inhibitors with improved bioavailability compared with chalcone oxides and trans-3-phenylglycidols. Ureas are potent inhibitors of both the rodent and human sEH with IC50 in the low nanomolar range for the last compounds [69-71]. N, N¢-dicyclohexylurea (DCU) was the first urea used in vivo to demonstrate potential beneficial cardiovascular actions of sEH inhibitors [39], although it did not have the physical properties necessary for pharmaceutical formulation. Then, one cyclohexyl group has been substituted by an alkyl chain to form N-cyclohexyl-N¢-dodecylurea (CDU), but the use of this drug is also limited due to its rapid metabolisation in hepatic microsomes, through a multiple step pathway, 5.1.3

236

including CYP450 hydroxylases, alcohol dehydrogenase, microsomal CYP450-related alcohol oxygenase and aldehyde dehydrogenase, to a more water-soluble carboxylic derivative with a partial maintaining of sEH inhibitor activity [72]. To increase water solubility and allow oral administration, a carboxylic acid has been added at the end of the hydrocarbon chain [73] to form the N-cyclohexyl-N¢-dodecanoic acid urea (CUDA). Next, a substitution of the second cyclohexyl by an adamantanyl group in N-adamantanyl-N¢-dodecanoic acid urea (AUDA) and 1-adamantanyl-3-(5-(2-(2ethoxyethoxy)ethoxy)pentyl)urea (AEPU) further improved water solubility and melting points than DCU and CDU, but these compounds are still rapidly metabolised because of the alkyl chain. To eliminate the possibility of b-oxidation, the alkyl chain was replaced by a conformationally restricted molecule, such as piperidine heterocycle or phenyl group, to form 1-(1-acetypiperidin-4-yl)-3-adamantanylurea (APAU), which was the first sEH inhibitor to be used in clinical studies,




or cis- and trans-4[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB, t-AUCB) [1]. APAU and AUCB possess improved metabolic activity compared to the older drugs with an alkyl chain [74]. Finally, since the adamantanyl group has been reported to be metabolically unstable, the replacement of the adamantanyl by a 4-trifluoro-methoxyphenyl group, as in 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea, improves pharmacokinetics of urea-based derivatives, affording a better IC50, half-life time and maximum blood concentration with a slower absorption, and could thus be a better clinical candidate than APAU [1,74]. The putative mechanism of action of these drugs is that the urea carbonyl oxygen forms hydrogen bonds with the hydroxyl groups of Tyr381 and Tyr465 and interacts with Gln382 to stabilise the partial negative charge on the epoxide oxygen. Moreover, the two amino groups of the urea derivatives could interact with the Asp333 carboxylate side chain and prevent the nucleophilic attack of sEH on EETs [75]. Indeed, incorporation of polar functional group into one of the alkyl chains of 1,3-disubstitued urea derivatives resulted in potent sEH inhibitors with improved physical properties [70]. Thus, 1,3-disubstitued ureas interact stoichiometrically with purified recombinant sEH and are tightbinding inhibitors with nanomolar Ki values on hydrolase activity [67].

bonds with two key catalytic residues (Tyr383 and 466) in a similar manner as the way the carbonyl of urea inhibitors binds at the active site of sEH [77,78]. Fulvestrant is the first sEH inhibitor that contains a sulphoxide as central pharmacophore and displays similar potency to that of the urea/amidecontaining inhibitors [77]. As for benzoxazolone, this could rapidly give new promising sulphoxide-containing compounds because fulvestrant is already used in humans, for the treatment of hormone receptor-positive metastatic breast cancer [79]. However, the fact that amide and urea form a stronger network of interactions with sEH than the fulvestrant seems to indicate that groups around the sulphoxide could be of interest to substitute on urea derivatives such as the fluorinated alkyl chain [79]. Indeed, fluorinated alkyl chain of fulvestrant occupies the smaller of the two lipophilic pockets in the sEH active site, whereas the steroid part of the molecule is directed towards the large deep pocket that opens towards solvent. The aromatic cyclopentyl ring of fulvestrant is at the entrance of the catalytic tunnel with the hydroxyl group on this ring facing outward towards the solvent when the hydroxyl group on the aromatic ring is pointing towards the amide nitrogen of Asn472. Moreover, it has been demonstrated that replacing aryl groups by alkyl groups in sulphoxide compounds make them more potent inhibitors [77].

5.1.4

4-substituted benzoxazolone These agents are promising sEH inhibitors, allowing oral administration [76]. Benzoxazolone analogues were synthesized and evaluated as sEH inhibitors in vitro [76]. Several groups on 4-benzoxazolone have been tested to improve water solubility and bioavailability. First, benzoate and besilate were introduced at 4-position to strengthen the skeleton structure of the benzoxazolone. Then, the substitution by glycosyl and amino acid allowed the formation of bioactive compounds, and amino acid-substitution with a phenyl, a pyrrolidine or a sulfhydryl group further increased the inhibitory activity of 4-substitued benzoxazolone against sEH [76]. Moreover, substitution with a meta-electron withdrawing or a para-electron donor increases the inhibitory effect of the 4-substitued benzoxazolone in the low micromolar range [76]. Finally, the development of this new class of sEH inhibitors, in particular regarding their pharmacokinetic properties and toxicity, could be facilitated because benzoxazolones are already used in humans as antibacterial and anti-inflammatory drugs [76].

5.2

Sulphoxides Sulphoxides represent a new class of competitive sEH inhibitors and among them, fulvestrant was found to have a low nanomolar Ki for the human sEH [77]. Fulvestrant binds in the hydrolase catalytic pocket of sEH by an interaction between the sulphur atom of sulphoxide and Asp335, and this is quite similar to the bond observed between the amino groups of the urea and the Asp335 residue [77,78]. The oxygen atom of the sulphoxide pharmacophore makes hydrogen

Increasing experimental studies have demonstrated the beneficial impact of sEH inhibitors on cardiovascular abnormalities in a variety of animal models (Table 2) [2]. In accordance with the vasodilator and natriuretic properties of EETs, it was first demonstrated that a single dose of the urea sEH inhibitor DCU induced a profound but transient decrease in blood pressure in SHR [39]. The blood pressure-lowering properties of various sEH inhibitors were then reported in other models, with a more marked impact in angiotensin II-dependent hypertension [41,42]. Independently from the decrease in blood pressure, sEH inhibition with AUDA reverses the endothelial vasomotor dysfunction, notably in coronary arteries [42]. This aspect is particularly important because endothelial dysfunction, which is a major contributor to the increased cardiovascular morbidity and mortality of essential hypertensive patients, is not always prevented by available antihypertensive therapies [2,9]. Moreover, cardiac hypertrophy and dysfunction were prevented by sEH inhibitors in these models, and this may further contribute to the beneficial impact of this pharmacological strategy during hypertension [42,80]. Cardiac protection with the sEH inhibitors AUDA and t-AUCB was also observed during myocardial ischaemia-reperfusion injury [81,82], and this may be notably mediated by the activation of PI3K/Akt pathway and ATP-dependent potassium channels by EETs, promoting myocyte survival [2]. Importantly, these effects also probably contribute to the prevention and treatment of heart failure induced by myocardial

5.1.5

Effects of sEH inhibitors in cardiovascular and metabolic diseases


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Table 2. Main effects of sEH inhibitors in animal models of cardiovascular diseases. Target Kidney


Heart

Pancreas

Vessels

Effects of sEH inhibition

Models

Prevent renal damage

Murine models of salt-sensitive and angiotensin II-dependent hypertension and streptozotocin-induced type 1 diabetes [41,73,90,92], 5/6 nephrectomy model in mice [94]

Enhance albuminuria and do not prevent glomerulosclerosis and tubulointerstitial damage Reverse the endothelial vasomotor dysfunction Prevent cardiac hypertrophy and dysfunction Induce cardiac protection during myocardial ischaemia-reperfusion injury Prevent and treat heart failure induced by myocardial infarction Improve glucose homeostasis by increasing insulin release and sensitivity Prevent b-cell loss and dysfunction Decrease blood pressure

Mice with renovascular hypertension and deoxycorticosterone acetate (DOCA)-salt rats [42,80] Angiotensin II-infused mice, DOCA-salt rats and mice fed with high-fat diet-induced type 2 diabetes [42,80-82,89] Definitive coronary occlusion in mice [83,84] Streptozotocin-induced type 1 diabetes and mice fed with high-fat diet [85,87], Streptozotocin-induced type 1 diabetes [85,86], Spontaneously hypertensive rats, angiotensin II-infused mice, renal stenosis in mice, DOCA-salt rats [38,40-42] Angiotensin II-dependent hypertension and high-fat diet-induced type 2 diabetic mice [42,80,87,89] Apolipoprotein E-deficient mice infused with angiotensin II [95,96]

Abrogate endothelial dysfunction in aortic and mesenteric arteries Prevent the development of vascular remodelling

sEH: Soluble epoxide hydrolase.

infarction with AUDA, AEPU and t-AUCB [83,84]. Regarding cardiometabolic diseases, the previously described effects of EETs on insulin sensitivity and release, as well as their protective cardiovascular properties, make pharmacological inhibition of sEH attractive in diabetes. Accordingly, it was shown that oral administration of various sEH inhibitors exerts a hypoglycaemic action in streptozotocin-induced type 1 diabetes [85,86] and in models of type 2 diabetes [87,88]. The improvement in glucose homeostasis is not always associated with the prevention of insulin resistance and appears to vary between different experimental conditions [86,88]. Interestingly, a preventive effect of t-AUCB on b-cell loss and dysfunction has been observed [85,86]. In addition to these metabolic effects, sEH inhibition prevents the cardiovascular complications associated with diabetes, which are major causes of morbidity and mortality in patients. In particular, t-AUCB and APAU abrogates endothelial dysfunction in aortic and mesenteric arteries in animal models of type 2 diabetes [87,89]. In addition, echocardiographic experiments suggest that sEH inhibition may be useful in preventing cardiac hypertrophy and diastolic dysfunction in mice fed with high-fat diet-induced type 2 diabetes, through anti-inflammatory and anti-fibrotic effects [89]. Finally, controversial results have been reported regarding the impact of sEH inhibition on the kidney. On one hand, sEH inhibitors prevent renal damage in animals with saltsensitive and angiotensin II-dependent hypertension [73,90,91] and in streptozotocin-induced type 1 diabetes [92]. Interestingly, direct effects of sEH inhibitors are expected since these agents provide protection against renal damage even in experimental conditions where blood pressure or glucose were not improved [90,93]. On the other hand, in the mouse 238

5/6 nephrectomy model that mimics progressive human renal failure, c-AUCB further enhanced albuminuria and did not prevent glomerulosclerosis and tubulointerstitial damage, possibly because of a shift of ARA metabolism to the LOX pro-inflammatory pathway [94]. Additionally, sEH inhibitors may help in preventing the development of vascular remodelling and atherosclerosis in cardiovascular and metabolic diseases, not only through the improvement in endothelial function but also through the anti-inflammatory effects of EETs, and in decreasing circulating LDL cholesterol, as shown in apolipoprotein E-deficient mice infused with angiotensin II [95,96]. Globally, these experimental data give strong evidence that sEH inhibitors not only improve endothelial function and prevent cardiovascular damage but also exert beneficial actions on metabolic abnormalities. Thus, these agents may be particularly useful in humans to prevent and treat these diseases, and clinical trials have recently begun to address these promising issues. 6.

Conclusion

The inhibitors of sEH are a new pharmacological class increasing the bioavailability of epoxyeicosanoids, notably EETs, which are crucial endothelial vasodilators with powerful anti-inflammatory properties. Several generations of sEH inhibitors have been successively developed. Experimental studies have demonstrated promising beneficial effects of sEH inhibitors in animal models of various diseases. Some sEH inhibitors are now moving up into clinical trials to investigate their safety and confirm their interest in the prevention and treatment of cardiometabolic diseases in humans, with



the expected goal to reduce cardiovascular morbidity and mortality.


7.

Expert opinion

Several generations of sEH inhibitors with improved potency and physicochemical properties have been designed, allowing oral administration for the latest agents. This new pharmacological class exhibits attractive metabolic and blood pressurelowering properties, and very potent effects against target organ damage, endothelial/vascular dysfunction, cardiac hypertrophy and dysfunction and renal dysfunction in various animal models of cardiometabolic diseases. AEPU (AR9281) was the first sEH inhibitor to be tested in a Phase I clinical trial with a satisfactory safety profile, but pharmacokinetic studies suggest that a thrice-daily dosing regimen is necessary to effectively maintain sEH inhibition during 24 h [97]. Whether these poor pharmacokinetic properties or the variable effect of sEH inhibitors on insulin sensitivity may have contributed to the failure of the Phase IIA performed in patients with mild-to-moderate hypertension and impaired glucose tolerance (NCT00847899) remains unclear. More recently, the new sEH inhibitor from GlaxoSmithKline GSK2256294, (1R,2S)-cis-N-{[4-cyano-2-(trifluoromethyl) phenyl]methyl}-3-{[4-methyl-6-(methylamino)-1,3,5-triazin2-yl]amino}cyclohexanecarboxamide, an amide derivative, entered a Phase I trial to assess its safety, tolerability, pharmacokinetics and pharmacodynamics, including measures of endothelial function, in healthy volunteers and adult male moderately obese smokers (NCT01762774) [98]. At this time, it is necessary to point out that the safety of sEH inhibitors needs to be carefully investigated, because the increase in EET availability during a long-term period in humans may be associated with detrimental effects. In particular, due to the pro-angiogenic and anti-apoptotic effects of EETs, sEH inhibitors may potentiate tumourogenesis and metastasis, as suggested in some mouse models of cancer [99]. However, this aspect needs to be thoroughly investigated because the association between sEH and cancer development remains elusive. In fact, sEH protein expression is decreased in human renal and hepatic tumours, arguing for its role in cancer development, but in contrast upregulated in other tumour types [99]. In addition, sEH inhibitors also increase the bioavailability of potential anticancer molecules, such as

DHA epoxides, which may counteract the deleterious action of EETs at this level [100]. Further, inhibiting sEH may be detrimental at the level of the pulmonary circulation because EETs promote pulmonary vasoconstriction and remodelling [2,11]. However, the N-terminal phosphatase domain, which activity is normally not altered by sEH inhibitors, may play a more important role than the C-terminal hydrolase domain in the long-term regulation of pulmonary tone and morphology [101]. At last, whether ARA may bypass to more detrimental metabolising pathways, such as the formation of 20-HETE or leukotrienes [94], during the long-term use of sEH inhibitors, must be carefully evaluated. In this context, the optimisation of chemical properties of the sEH inhibitors and drug formulation may not only lead to improvement in their pharmacokinetic properties but may also help in targeting specific organs, using cyclodextrins or liposomes, and achieving the cellular effects needed, thus avoiding detrimental side effects. In the same way, the specific biological role of each EET regioisomer may be clearly identified, and the development of dual-activity EET analogues/ sEH inhibitors, allowing to increase one or more EET regioisomers [102], may contribute to improve the specificity of this pharmacological approach. Future researches should also investigate the exact role of the EET metabolites DHETs that may be involved in the control of coronary vasomotor tone and metabolic homeostasis [2,103] and the decrease of which may, therefore, limit sEH inhibitors efficacy. At last, the determination of genetic polymorphisms of EPHX2 affecting sEH activity may help in identifying individuals in which using sEH inhibitors would be particularly efficient in preventing or treating cardiovascular diseases.

Acknowledgments The authors thank I Segalas-Milazzo (Research Institute in Fine Organic Chemistry, Mont Saint Aignan, France) for her help in the presentation of the crystal structure of the sEH monomer.

Declaration of interest The authors declare that they have no conflict of interest and have received no payment in the preparation of their manuscript.


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Affiliation Thomas Duflot1,3, Clothilde Roche3, Fabien Lamoureux1, Dominique Guerrot2,3 & Jeremy Bellien†1,3 † Author for correspondence 1 Rouen University Hospital, Department of Pharmacology, Rouen, France 2 Rouen University Hospital, Department of Nephrology, Rouen, France 3 University of Rouen, Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) U1096, Institute for Research and Innovation in Biomedicine, Rouen Cedex, 76031, France Tel: +33 2 32 88 90 30; Fax: +33 2 32 88 91 16; E-mail: [email protected]

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Identification of potent inhibitors of the chicken soluble epoxide hydrolase.

Peripheral FAAH and soluble epoxide hydrolase inhibitors are synergistically antinociceptive.

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors.

Design, synthesis and biological evaluation of 4-benzamidobenzoic Acid hydrazide derivatives as novel soluble epoxide hydrolase inhibitors.

Structure-activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase.

Occurrence of urea-based soluble epoxide hydrolase inhibitors from the plants in the order Brassicales.

18F-FNDP for PET Imaging of Soluble Epoxide Hydrolase.

In vitro and in vivo metabolism of N-adamantyl substituted urea-based soluble epoxide hydrolase inhibitors.

Rational Design of Potent and Selective Inhibitors of an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa.

1,3-Disubstituted and 1,3,3-trisubstituted adamantyl-ureas with isoxazole as soluble epoxide hydrolase inhibitors.

Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening.

Disrupting Dimerization Translocates Soluble Epoxide Hydrolase to Peroxisomes.

Soluble Epoxide Hydrolase Deficiency or Inhibition Attenuates MPTP-Induced Parkinsonism.

Reperfusion Injury in Soluble Epoxide Hydrolase-Deficient Mice.

Soluble epoxide hydrolase inhibitory and anti-inflammatory components from the leaves of Eucommia ulmoides Oliver (duzhong).

Soluble epoxide hydrolase as an anti-inflammatory target of the thrombolytic stroke drug SMTP-7.

Inhibition of soluble epoxide hydrolase in mice promotes reverse cholesterol transport and regression of atherosclerosis.

Opposite effects of gene deficiency and pharmacological inhibition of soluble epoxide hydrolase on cardiac fibrosis.

Membrane topology of epoxide hydrolase.

Effect of a Soluble Epoxide Hydrolase Inhibitor, UC1728, on LPS-Induced Uveitis in the Rabbit.

Pharmacokinetics, pharmacodynamics and adverse event profile of GSK2256294, a novel soluble epoxide hydrolase inhibitor.

Increased epoxyeicosatrienoic acids and reduced soluble epoxide hydrolase expression in the preeclamptic placenta.

Genetic deletion of soluble epoxide hydrolase attenuates inflammation and fibrosis in experimental obstructive nephropathy.

Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa.