Epoxide Hydrolase Enzyme Research Articles

Abstract P238: High Fat Diet-induced Obesity Disrupts Vascular Homeostasis in Both Kidney and Retina: Role of Epoxyeicosatrienoic Acids

Obesity-induced vascular inflammation is considered an early and common pathological change for the development of microvascular complications including retinopathy and nephropathy. We have previously demonstrated that obesity down-regulated renal cytochrome P450 epoxygenase and decreased epoxyeicosatrienoic acids (EETs) levels in kidney as well as triggered retinal expression of the pro-inflammatory thioredoxin interacting protein (TXNIP) that coincided with vascular inflammation. The aim of this work is to examine the impact of obesity-induced reduction in EETs levels on TXNIP-inflammasome activation. Because EETs are quickly hydrolyzed by the soluble epoxide hydrolase (sEH) enzyme to inactive metabolites, we use sEH gene deleted mice (Ephx2 -/-) as a model with high EETs levels. WT and Ephx2 -/- were fed normal (14 % fat) or high fat diet (HFD, 60 % fat) for three months. HFD treatment down-regulated cyp2c44, the main epoxygenase for EETs production, and decreased EETs levels in kidney of obese mice and these changes were associated with podocyte loss and increased podocalyxin excretion as markers of glomerular injury. Although HFD treatment decreased EETs levels in Ephx2 -/- mice, it remained significantly higher than obese WT mice. HFD impaired endothelial function and induced TXNIP-inflammasome activation evident by increases in NOD-like receptor-3 (NLRP3) and interleukin-1β (IL-1β) in both retina and kidney. In parallel, HFD also increased leukostasis, retinal vascular permeability and development of acellular capillary, hall mark of ischemia in WT but not in TXNIP-/-. Restoring EETs levels in Ephx2 -/- mice improved endothelial function and was associated with decreased TXNIP-mediated inflammasome activation in the kidney of obese mice. Our data suggest that diet-induced obesity causes homeostatic imbalance by down-regulation of the anti-inflammatory epoxygenase/EETs levels and increasing the inflammatory TXNIP expression resulting in vascular injury in both retina and kidney.

Role of MAPKs and NF‐κB in the Development of Cellular Hypertrophy by 8‐ Hydroxyeicosatetraenoic acids in Human Ventricular Cardiomyocyte, RL‐14 Cell Line

Recent studies have established the role of mid-chain hydroxyeicosatetraenoic acids (HETEs) in the development of cardiovascular disease. Among these mid-chains, 8-HETE has been reported to have proliferator and pro-inflammatory action. However, whether 8-HETE can induce cardiac hypertrophy has never been investigated before. Therefore, the overall objective of the present study is to elucidate the potential hypertrophic effect of 8-HETE in the human ventricular cardiomyocytes, RL-14 cells, and to explore the mechanism(s) involved. Our results showed that 8-HETE induced cellular hypertrophy in RL-14 cells as evidence by the induction of cardiac hypertrophy markers ANP, BNP, α-MHC, and β-MHC in a concentration- and time-dependent manner and the increase in cell surface area. Mechanistically, 8-HETE was able to induce the binding activity of NF-κB to its responsive element and significantly induced the phosphorylation of ERK1/2. The induction of cellular hypertrophy was associated with a proportional increase in the formation of dihydroxyeicosatrienoic acids (DHETs) parallel to the increase of soluble epoxide hydrolase enzyme activity. Blocking the induction of NF-κB, ERK1/2 and sEH signaling pathways significantly inhibited 8-HETE-induced cellular hypertrophy. Our study provides the first evidence that 8-HETEs induce cellular hypertrophy in RL-14 cells through MAPKs and NF-κB dependent mechanism.

5-, 12- and 15-Hydroxyeicosatetraenoic acids induce cellular hypertrophy in the human ventricular cardiomyocyte, RL-14 cell line, through MAPK- and NF-κB-dependent mechanism.

Recent studies have established the role of mid-chain hydroxyeicosatetraenoic acids (HETEs) in the development of cardiovascular disease. Mid-chain HETEs have been reported to have vasoconstrictive and pro-inflammatory effects. However, whether mid-chain HETEs can induce cardiac hypertrophy remains unclear. Therefore, the overall objective of the present study was to elucidate the potential hypertrophic effect of mid-chain HETEs in the human ventricular cardiomyocytes, RL-14 cells, and to explore the mechanisms involved. For this purpose, RL-14 cells were treated with increasing concentrations of mid-chain HETEs (2.5, 5, 10 and 20µM). Thereafter, the cardiac hypertrophy markers and cell size were determined using real-time polymerase chain reaction and phase contrast imaging, respectively. Phosphorylated mitogen-activated protein kinase (MAPK) level and nuclear factor kappa B (NF-κB) binding activity were determined. Our results showed that mid-chain HETEs induced cellular hypertrophy in RL-14 cells as evidenced by the induction of cardiac hypertrophy markers, α- and β-myocin heavy chain and atrial and brain natriuretic peptide as well as the increase in cell size. Mechanistically, all mid-chain HETEs were able to induce the binding activity of NF-κB to its responsive element in a HETE-dependent manner, and they significantly induced the phosphorylation of ERK 1/2. The induction of cellular hypertrophy was associated with proportional increase in the formation of dihydroxyeicosatrienoic acids parallel to the increase of soluble epoxide hydrolase enzyme activity. In conclusion, our study provides the first evidence that mid-chain HETEs induce cellular hypertrophy in RL-14 cells through MAPK- and NF-κB-dependent mechanism.

Epoxyeicosatrienoic acids, hypertension, and kidney injury.

Kidney disease afflicts 33 million in the United States, and chronic kidney disease (CKD) accounts for >$60 billion in Medicare costs.1,2 Hypertension afflicts 75 million in the United States, and significant portions of those patients develop CKD and progress to end-stage renal disease.1–5 Interestingly, resistant hypertension which is defined as uncontrolled hypertension, despite 3 antihypertensive medication classes, increases the risk for cardiovascular diseases and end-stage renal disease.6 These recent findings in the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) highlights the fact that current treatments only slow the loss of kidney function, or have no benefit at all.5,6 New therapeutic approaches are urgently needed. Development of drugs to increase a novel class of fatty acids, epoxyeicosatrienoic acids (EETs), represents a unique approach to treat hypertension and kidney disease. EETs are generated from the substrate arachidonic acid by cytochrome P450 (CYP) epoxygenase enzymes.7,8 There are 4 regioisomeric EETs formed: 5,6-EET; 8,9-EET; 11,12-EET; and 14,15-EET. These regioisomeric EETs are further metabolized to less active or inactive diols by the soluble epoxide hydrolase (sEH; Ephx2 ) enzyme. For clarity, EETs will be used as a general term and regioisomers mentioned when actions can be attributed to a specific regioisomeric EET. In the majority of circumstances, the primary EETs evaluated for cardiovascular and renal function have been 11,12-EET and 14,15-EET.7 Once formed, EETs act in an autocrine or paracrine manner to elicit biological responses. Vascular endothelial and renal epithelial cells are major sites for EET production.7,8 This localized EET generation aligns with the biological actions and contribution of EETs to cardiovascular and renal function. Prominent biological actions of EETs include their role as endothelial-derived hyperpolarizing factors and regulation of tubular sodium reabsorption by inhibiting epithelial …

Soluble Epoxide Hydrolase Inhibition Is Antinociceptive in a Mouse Model of Diabetic Neuropathy

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

Design, Synthesis and Biological Evaluation of Some Oxadiazole Derivatives as Novel Amide-Based Inhibitors of Soluble Epoxide Hydrolase

Soluble epoxide hydrolase (sEH) enzyme plays an important role in the metabolism of endogenous chemical mediators which are involved in the regulation of blood pressure and inflammation. Although the most reported potent sEH inhibitors are urea derivatives, these compounds have limited pharmacokinetic profile. In order to improve physicochemical properties, besides having favorable potency, amide non-urea derivatives with oxadiazole ring as a novel secondary pharmacophore against sEH enzyme were developed. Most of the novel compounds with appropriate physical properties, had comparable in vitro sEH inhibitory activity to 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a potent urea-based sEH inhibitor. The IC50 value of the most potent compound (15c) was 0.43 nM.

The soluble epoxide hydrolase as a novel target for chronic neuropathic pain (659.15)

Neuropathic pain remains an unmet clinical need. In addition to nerve injury cases of neuropathy diabetes is increasing worldwide and diabetic neuropathy, the most frequent secondary condition to diabetes, will increase with it. Recent investigations reveal lipids as signaling mediators and their functional significance in nociception is receiving attention. Notably, there is evidence of antihyperalgesia mediated by epoxy fatty acids (EpFAs), metabolites generated by cytochrome P450 epoxygenase in an arm of the arachidonic acid cascade. These endogenous lipid mediators are subsequently degraded by the soluble epoxide hydrolase (sEH) enzyme to their mostly inactive diols. Metabolomic studies show that administration of sEH inhibitors (sEHI) stabilize EpFA to diol ratios in vivo and lead to decreased inflammation and pain related behavior in inflammatory pain models. Unexpectedly, the sEHI are also active against a model of type I diabetic neuropathy where the role of inflammation is minimal. The experiments here in rat and mouse demonstrate that potent sEHI mediate antihyperalgesia in both the sciatic nerve ligation (SNL) neuropathic pain and diabetic neuropathic pain (DNP) models. While mechanical withdrawal thresholds were used to assess both the SNL and DNP modeled pain, there are limitations to using acute stimulus reflex responses to assess chronic neuropathy. Therefore, the conditioned place preference (CPP) assay was used to evaluate the tonic nature of chronic pain and to address these limitations. Here, the CPP assay alongside withdrawal thresholds demonstrates the dose dependent antihyperalgesic efficacy of sEHI in the murine DNP model. Importantly, the CPP effect was absent both in control mice and in sEH knockout mice indicating the sEHI is not positively reinforcing or rewarding. Overall, these experiments indicate that inhibiting the sEH attenuates neuropathic pain and offers an alternative to current therapies to meet this clinical need.Grant Funding Source: Supported by R01 ES002710, P42 ES004699, R21AR062866

N-Palmitoylethanolamide protects the kidney from hypertensive injury in spontaneously hypertensive rats via inhibition of oxidative stress

Hypertension is an important risk factor for kidney failure and renal events in the general population. Palmitoylethanolamide (PEA) is a member of the fatty acid ethanolamine family with profound analgesic and anti-inflammatory effects, resulting from its ability to activate peroxisome proliferator activated receptor (PPAR)α. A role for this nuclear receptor has been addressed in cardiovascular system and PPARα ligands have been shown to protect against inflammatory damage especially resulting from angiotensin II hypertension.In this study, we demonstrated that PEA significantly reduced blood pressure in spontaneously hypertensive rats (SHR) and limited kidney damage secondary to high perfusion pressure. To investigate the mechanisms involved in PEA effect, we found that PEA reduced cytochrome P450 (CYP) hydroxylase CYP4A, epoxygenase CYP2C23 and soluble epoxide hydrolase enzyme expression in the kidney, accompanied by a reduction of 20-hydroxyeicosatetraenoic acid excretion in the urine. Moreover, it markedly reduced kidney oxidative and nitrosative stress accompanied by decreased expression of renal NAD(P)H oxidase and inducible nitric oxide synthase and increased expression of Cu/Zn superoxide dismutase, in the kidney of SHR. Moreover, angiotensin II receptor (AT) evaluation revealed a decrease in AT1 receptor expression and a restoration of AT2 receptor level in the kidney from PEA-treated SHR. Consistently, angiotensin converting enzyme expression was reduced, implying a decrease in angiotensin II synthesis. These results indicate that PEA treatment lowers blood pressure and can protect against hypertensive renal injury by increasing the antioxidant defense and anti-inflammatory response and modulating renin-angiotensin system.

Design, synthesis and anti-tuberculosis activity of 1-adamantyl-3-heteroaryl ureas with improved in vitro pharmacokinetic properties

Out of the prominent global ailments, tuberculosis (TB) is still one of the leading causes of death worldwide due to infectious disease. Development of new drugs that shorten the current tuberculosis treatment time and have activity against drug resistant strains is of utmost importance. Towards these goals we have focused our efforts on developing novel anti-TB compounds with the general structure of 1-adamantyl-3-phenyl urea. This series is active against Mycobacteria and previous lead compounds were found to inhibit the membrane transporter MmpL3, the protein responsible for mycolic acid transport across the plasma membrane. However, these compounds suffered from poor in vitro pharmacokinetic (PK) profiles and they have a similar structure/SAR to inhibitors of human soluble epoxide hydrolase (sEH) enzymes. Therefore, in this study the further optimization of this compound class was driven by three factors: (1) to increase selectivity for anti-TB activity over human sEH activity, (2) to optimize PK profiles including solubility and (3) to maintain target inhibition. A new series of 1-adamantyl-3-heteroaryl ureas was designed and synthesized replacing the phenyl substituent of the original series with pyridines, pyrimidines, triazines, oxazoles, isoxazoles, oxadiazoles and pyrazoles. This study produced lead isoxazole, oxadiazole and pyrazole substituted adamantyl ureas with improved in vitro PK profiles, increased selectivity and good anti-TB potencies with sub μg/mL minimum inhibitory concentrations.

Comparative efficacy of 3 soluble epoxide hydrolase inhibitors in rat neuropathic and inflammatory pain models

Epoxy-fatty acids have been recognized as important cell signaling molecules with multiple biological effects including anti-nociception. The main degradation pathway of these signaling molecules is via the soluble epoxide hydrolase (sEH) enzyme. Inhibitors of sEH extend the anti-nociceptive effects of fatty acid epoxides. In this study two models of pain with different etiology, streptozocin induced type I diabetic neuropathic pain and lipopolysaccharide induced inflammatory pain were employed to test sEH inhibitors. A dose range of three sEH inhibitors with the same central pharmacophore but varying substituent moieties was used to investigate maximal anti-allodynic effects in these two models of pain. Inhibiting the sEH enzyme in these models successfully blocked pain related behavior in both models. The sEH inhibitors were more potent and more efficacious than celecoxib in reducing both diabetic neuropathic pain and lipopolysaccharide induced inflammatory pain. Because of their ability to block diabetic neuropathic pain sEH inhibition is a promising new approach to treat chronic pain conditions.

The putative α/β-hydrolases of Dietzia cinnamea P4 strain as potential enzymes for biocatalytic applications

The draft genome of the soil actinomycete Dietzia cinnamea P4 reveals a versatile group of α/β-hydrolase fold enzymes. Phylogenetic and comparative sequence analyses were used to classify the α/β-hydrolases of strain P4 into six different groups: (i) lipases, (ii) esterases, (iii) epoxide hydrolases, (iv) haloacid dehalogenases, (v) C-C breaking enzymes and (vi) serine peptidases. The high number of lipases/esterases (41) and epoxide hydrolase enzymes (14) present in the relatively small (3.6 Mb) P4 genome is unusual; it is likely to be linked to the survival of strain P4 in its natural environment. Strain P4 is thus equipped with a large number of genes which would appear to confer survivability in harsh hot tropical soil. As such, this highly resilient soil bacterial strain provides an interesting genome for enzyme mining for applications in the field of biotransformations of polymeric compounds.

Chronic Doxorubicin Cardiotoxicity Modulates Cardiac Cytochrome P450-Mediated Arachidonic Acid Metabolism in Rats

Doxorubicin [(DOX) Adriamycin] is an effective anticancer agent whose major limiting side effect is cardiotoxicity. This cardiotoxicity is predicted only by the cumulative dose of DOX where the clinical situation involves chronic drug administration. Therefore, we investigate the effect of chronic DOX cardiotoxicity on expression of the cardiac cytochrome P450 (P450) enzymes and arachidonic acid (AA) metabolism in male Sprague-Dawley (SD) rats. The chronic toxicity was induced by multiple intraperitoneal injections for a cumulative dose of 15 mg/kg divided into six injections within 2 weeks. After 14 days of the last injection, the heart, liver, and kidney were harvested, and the expression of different genes was determined by real-time polymerase chain reaction. In addition, microsomal protein from the heart was prepared and incubated with AA. Thereafter, different AA metabolites were analyzed by liquid chromatography-electrospray ionization-mass spectrometry. The chronic DOX cardiotoxicity significantly induced gene expression of hypertrophic markers, apoptotic markers, CYP2E1, CYP4A3, CYP4F1, CYP4F5, and soluble epoxide hydrolase (sEH) enzyme, which was accompanied by an increase in the activity of P450 ω-hydroxylases and sEH. In addition, both the sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid, and the ω-hydroxylase inhibitor, N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016), significantly prevented the DOX-mediated induction of the hypertrophic markers in the cardiac-derived H9c2 cells, which further confirms the role of these enzymes in DOX cardiotoxicity. Furthermore, gene expression of P450 and sEH was altered in an organ-specific manner. As a result, the chronic DOX administration leads to an imbalance between P450-mediated cardiotoxic and cardioprotective pathways. Therefore, P450 ω-hydroxylases and sEH might be considered as novel targets to prevent and/or treat DOX cardiotoxicity.

Phenytoin Hypersensitivity Syndrome Associated with Glutathione S-Transferase, N-Acetyltransferase, Cytochrome P450 Polymorphisms

Observation: The anticonvulsant hypersensitivity syndrome is rare complication that occurs with the use of antiepileptic medications. All of the aromatic anticonvulsant drugs are metabolised by the cytochrome P-450 enzyme to a common arene oxide metabolite that is normally detoxified by epoxide hydrolase. A genetically determined inability to detoxify the toxic metabolites due to a defect in the enzyme epoxide hydrolase has been observed in patients with this hypersensitivity syndrome. We present a patient with severe phenytoin-induced hypersensitivity syndrome associated with glutathione S-transferase, N-acetyltransferase, and cytochrome P450 polymorphisms.

Novel soluble epoxide hydrolase inhibitor protects mitochondrial function following stress

Epoxyeicosatrienoic acids (EETs) are active metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase enzyme (sEH) to dihydroxyeicosatrienoic acid. EETs are known to render cardioprotection against ischemia reperfusion (IR) injury by maintaining mitochondrial function. We investigated the effect of a novel sEH inhibitor (sEHi) in limiting IR injury. Mouse hearts were perfused in Langendorff mode for 40 min and subjected to 20 min of global no-flow ischemia followed by 40 min of reperfusion. Hearts were perfused with 0.0, 0.1, 1.0 and 10.0 µmol·L(-1) of the sEHi N-(2-chloro-4-methanesulfonyl-benzyl)-6-(2,2,2-trifluoro-ethoxy)-nicotinamide (BI00611953). Inhibition of sEH by BI00611953 significantly improved postischemic left-ventricular-developed pressure and reduced infarct size following IR compared with control hearts, and similar to hearts perfused with 11,12-EETs (1 µmol·L(-1)) and sEH(-/-) mice. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 µmol·L(-1)), or the plasma membrane K(ATP) channels (pmK(ATP)) inhibitor (glibenclamide, 10 µmol·L(-1)) abolished the improved recovery by BI00611953 (1 µmol·L(-1)). Mechanistic studies in H9c2 cells demonstrated that BI0611953 decreased ROS generation, caspase-3 activity, proteasome activity, increased HIF-1∝ DNA binding, and delayed the loss of mitochondrial membrane potential (ΔΨ(m)) caused by anoxia-reoxygenation. Together, our data demonstrate that the novel sEHi BI00611953, a nicotinamide-based compound, provides significant cardioprotection against ischemia reperfusion injury.

Genetic Polymorphism of Microsomal Epoxide Hydrolase Enzyme Gene in Preeclamptic Females

IntroductionMicrosomal epoxide hydrolase enzyme is involved in xenobiotics detoxification. It catalyzes the phase I hydrolysis of epoxides and plays a role in the detoxification processes and in the metabolism of endogenous and exogenous compounds. Preeclampsia, which is one of the most serious complications of pregnancy, may be due to an imbalance between these compounds, such as lipid peroxides and oxygen-free radicals and detoxifying and scavenging substances. Two variants of human epoxide hydrolase enzyme with different enzyme activity have been described; exon 3 polymorphism is associated with lower enzyme activity whereas exon 4 polymorphism is associated with higher activity. The authors tried to investigate the association between these genetic polymorphisms and preeclampsia. MethodThirty preeclamptic females together with 30 normal pregnant females as controls were included in the study. Genotyping for exons 3 and 4 of microsomal epoxide hydrolase enzyme was done by polymerase chain reaction–restriction fragment length polymorphism. ResultsThere was no statistical significant difference in the distribution of exon 3 genotype between cases and controls (P= 0.4); on the other hand, a highly statistical significant difference was found between cases and controls as regard exon 4 genotype (P= 0.002). ConclusionThere may be an association between epoxide hydrolase enzyme polymorphism and the risk of preeclampsia.

Highlights of This Issue

Although mammographic density is a strong marker of breast cancer risk, it is unclear whether tumors arise specifically within dense breast tissue. Pinto Pereira and colleagues studied 231 British women diagnosed with breast cancer and assessed whether tumor location was related to localized mammographic density 5 years prior to diagnosis. Tumor locations, identified on digitized diagnostic films, were aligned with serial images obtained from the same woman before diagnosis. The authors report that prediagnostic mammographic density was higher in areas that subsequently contained tumors. This work supports a model in which tumors predominantly arise within radiodense breast tissue and suggests that localized mammographic density may be a predictor of subsequent tumor location.To uncover new biomarkers for bladder cancer, Eissa and colleagues retrospectively analyzed the methylation status of the RARβ2 and APC genes in urine samples from bladder cancer patients. The authors report that the methylation of these two genes was higher in cancer patients compared with healthy controls. Methylated RARβ2 and APC were found in all bladder cancer grades and stages. These studies suggest that methylated RARβ2 and APC might represent valuable urinary molecular markers for the early detection of bladder cancer.Previous studies, using serotype-derived blood typing, have shown an association between ABO blood type and gastric cancer. In this study, Nakao and colleagues further explored this association using single-nucleotide polypeptide analysis to evaluate ABO blood genotype and the risks of gastric cancer, atrophic gastritis and Helicobacter pylori infection. They report a significant association between ABO genotype and gastric cancer risk. In addition, the study revealed that ABO genotype may also influence atrophic gastritis prevalence and H. pylori infection.The microsomal epoxide hydrolase enzyme (mEH), encoded by EPHX1, has an established role in the detoxification of smoking-induced oxidative substances. Lee and colleagues explored the association between two EPHX1 polymorphisms and cancer risk in a large Danish population. In these studies, 47,000 individuals were genotyped and divided into groups with predicted fast, intermediate, and slow mEH phenotypes. The authors then evaluated the risks of several cancer diagnoses and found that individuals with predicted intermediate and slow mEH activities were at higher risk of tobacco-related cancers, compared with subjects with fast mEH phenotypes. This work represents one of the largest studies testing the hypothesis that genetically lowered mEH activities are associated with cancer risk.

The structure–activity relationship of urea derivatives as anti-tuberculosis agents

The treatment of tuberculosis is becoming more difficult due to the ever increasing prevalence of drug resistance. Thus, it is imperative that novel anti-tuberculosis agents, with unique mechanisms of action, be discovered and developed. The direct anti-tubercular testing of a small compound library led to discovery of adamantyl urea hit compound 1. In this study, the hit was followed up through the synthesis of an optimization library. This library was generated by systematically replacing each section of the molecule with a similar moiety until a clear structure–activity relationship was obtained with respect to anti-tubercular activity. The best compounds in this series contained a 1-adamantyl-3-phenyl urea core and had potent activity against Mycobacterium tuberculosis plus an acceptable therapeutic index. It was noted that the compounds identified and the pharmacophore developed is consistent with inhibitors of epoxide hydrolase family of enzymes. Consequently, the compounds were tested for inhibition of representative epoxide hydrolases: M. tuberculosis EphB and EphE; and human soluble epoxide hydrolase. Many of the optimized inhibitors showed both potent EphB and EphE inhibition suggesting the antitubercular activity is through inhibition of multiple epoxide hydrolase enzymes. The inhibitors also showed potent inhibition of humans soluble epoxide hydrolase, but limited cytotoxicity suggesting that future studies must be towards increasing the selectivity of epoxide hydrolase inhibition towards the M. tuberculosis enzymes.

Inhibition of Soluble Epoxide Hydrolase Confers Cardioprotection and Prevents Cardiac Cytochrome P450 Induction by Benzo(a)pyrene

We recently demonstrated that benzo(a)pyrene (BaP) causes cardiac hypertrophy by altering arachidonic acid metabolism through the induction of the expression of CYP ω-hydroxylases and soluble epoxide hydrolase (sEH) enzymes. The inhibition of CYP ω-hydroxylase enzymes partially reversed the BaP-induced cardiac hypertrophy. Therefore, it is important to examine whether the inhibition of sEH also confers cardioprotection. For this purpose, male Sprague-Dawley rats were injected intraperitoneally daily with either the sEH inhibitor 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS; 0.65 mg/kg), BaP (20 mg/kg), or the combination of BaP (20 mg/kg) and TUPS (0.65 mg/kg) for 7 days. Thereafter, the heart, liver, and kidney were harvested, and the heart to body weight ratio was measured. The expression of the hypertrophic markers, sEH, heme oxygenase-1, and CYP450 enzymes was determined. Our results demonstrate that BaP alone significantly induced the expression of sEH and CYP ω-hydroxylases in the heart, liver, and kidney tissues. Treatment with TUPS significantly reversed the BaP-mediated induction of the hypertrophic markers, completely prevented the increase in the heart to body weight ratio, and reduced the BaP-induced CYP1A1, CYP1B1, CYP4F4, and CYP4F5 genes in the heart. The current study demonstrates the cardioprotective effect of sEH inhibitor, TUPS, against BaP-induced cardiac hypertrophy and further confirms the role of sEH and CYP450 enzymes in the development of cardiac hypertrophy.

Cytochrome P450 eicosanoids and cerebral vascular function

The eicosanoids 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs), which are generated from the metabolism of arachidonic acid by cytochrome P450 (CYP) enzymes, possess a wide array of biological actions, including the regulation of blood flow to organs. 20-HETE and EETs are generated in various cell types in the brain and cerebral blood vessels, and contribute significantly to cerebral blood flow autoregulation and the coupling of regional brain blood flow to neuronal activity (neurovascular coupling). Investigations are beginning to unravel the molecular and cellular mechanisms by which these CYP eicosanoids regulate cerebral vascular function and the changes that occur in pathological states. Intriguingly, 20-HETE and the soluble epoxide hydrolase (sEH) enzyme that regulates EET levels have been explored as molecular therapeutic targets for cerebral vascular diseases. Inhibition of 20-HETE, or increasing EET levels by inhibiting the sEH enzyme, decreases cerebral damage following stroke. The improved outcome following cerebral ischaemia is a consequence of improving cerebral vascular structure or function and protecting neurons from cell death. Thus, the CYP eicosanoids are key regulators of cerebral vascular function and novel therapeutic targets for cardiovascular diseases and neurological disorders.

Cytochrome P450-Derived Epoxyeicosatrienoic Acids and Pulmonary Hypertension: Central Role of Transient Receptor Potential C6 Channels

Hypoxia induces the constriction of pulmonary resistance arteries, which results in the redistribution of blood from poor to better ventilated areas, thus optimizing its oxygenation. Many different oxygen-sensing mechanisms have been proposed to regulate this process, including cytochrome P450 enzymes. These enzymes, which convert substrates such as arachidonic acid into bioactive epoxides (the epoxyeicosatrienoic acids [EETs]), are highly expressed in the lung as is the soluble epoxide hydrolase which metabolizes the epoxides to their less active diols. The EETs play a well-documented role as endothelium-derived vasodilators in the systemic vasculature, but in the pulmonary circulation, they are generated in vascular smooth muscle cells and potentiate vasoconstriction. Preventing the breakdown of 11,12-EET by the inhibition or genetic deletion of the soluble epoxide hydrolase strongly augments the response to hypoxia. Mechanistically, 11,12-EET potentiates the contractile response by recruiting transient receptor potential C6 channels to caveolae. Indeed, neither 11,12-EET nor hypoxia is able to elicit pulmonary vasoconstriction in TRPC6 knockout mice. The cytochrome and soluble epoxide hydrolase enzymes are also implicated in the vascular remodeling associated with chronic hypoxia and pulmonary hypertension. Thus, targeting this pathway may be in an attractive new therapeutic approach to treat this incapacitating disease.