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WO2011066567A1 - Methods of treating diabetes - Google Patents

Methods of treating diabetes Download PDF

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Publication number
WO2011066567A1
WO2011066567A1 PCT/US2010/058413 US2010058413W WO2011066567A1 WO 2011066567 A1 WO2011066567 A1 WO 2011066567A1 US 2010058413 W US2010058413 W US 2010058413W WO 2011066567 A1 WO2011066567 A1 WO 2011066567A1
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Prior art keywords
inhibitor
seh
cox
mice
eet
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PCT/US2010/058413
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French (fr)
Inventor
Bruce D. Hammock
Ayala Luria
Fawaz George Haj
Ahmed Bettaieb
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The Regents Of The University Of California
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Publication of WO2011066567A1 publication Critical patent/WO2011066567A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention provides methods of reducing, preventing, inhibiting symptoms and/or the progression of diabetes, e.g., hyperglycemia, by coadministration of an agent that increases EETs in combination with an agent that inhibits cyclo-oxygenase and/or the 5 -lipoxygenase pathway (this includes inhibitors of 5-Lox, FLAP (fatty acid activating protein) inhibitors, and agonists of leukotriene receptors).
  • the agents can be co-administered in doses that are therapeutic, subtherapeutic or non-therapeutic for the individual agents.
  • nephropathy a process known as nephropathy.
  • the end stage of nephropathy is kidney failure, or end stage renal disease.
  • Nephropathy and kidney failure can result even when diabetes is controlled with drugs and exercise.
  • NIDDK National Institute of Diabetes and Digestive and Kidney Diseases
  • diabetes is the most common cause of kidney failure and is responsible for about 40% of the 100,000 cases of kidney failure that develop annually in the U.S.. Given the $20 billion annual cost of treating kidney failure in the U.S. alone, reducing nephropathy and kidney failure could significantly reduce the costs of treating this complication of diabetes.
  • Obesity a chronic inflammatory condition
  • Obese individuals exhibit a higher risk of chronic diseases including cardiovascular disease and type 2 diabetes.
  • the later is a complex, polygenic disease wherein a number of tissues are rendered insulin resistant (Biddinger and Kahn (2006) Annu Rev Physiol 68, 123-158). Insulin action is mediated by a complex network of signaling events (Lizcano and Alessi (2002) Curr Biol 12, R236-238) that modulate glucose homeostasis and hence regulate energy balance.
  • a fundamental mechanism for the maintenance of glucose homeostasis is the rapid action of insulin to stimulate glucose uptake and metabolism in peripheral tissues. This cascade initiates by binding of insulin to its cell surface receptor, followed by receptor
  • PI3K insulin receptor substrates
  • the mechanism underlying insulin resistance in type 2 diabetes contains many signaling players (Shulman (2000) J Clin Invest 106, 171-176 and Kobayashi (2005) Current drug targets 6, 525-529) resulting from pancreatic ⁇ -cell insufficiency with impairment of glucose-stimulated insulin secretion (Zhang, et al. (2001) Cell 105, 745-755) and destructed insulin receptor signaling (Taniguchi, et al. (2006) Nature reviews 7, 85-96).
  • Soluble epoxide hydrolase a therapeutic target for several disease models of hypertension and inflammation is also suggested to play a role in insulin resistance.
  • Polymorphism of the sEH gene is associated with insulin resistance in type 2 diabetic patients (Ohtoshi, et al. (2005) Biochem Biophys Res Commun 331, 347-350).
  • sEH is an enzyme that adds water to epoxides, forming their corresponding 1 ,2-diols (Newman, et al. (2005) Prog Lipid Res 44, 1-51). While, the enzyme possesses two functional domains i.e.
  • EETs Eicosatrienoic acids
  • EETs which are derived from arachidonic acid by epoxygenation through cytochrome P450 monooxygenases (CYP 2C), mediate endothelium-dependent vasodilation, promote angiogenesis, and have anti-inflammatory properties (Larsen, et al. (2007) Trends Pharmacol Sci 28, 32-38). Increased levels of sEH results in rapid metabolism of EET (regioisomers 5,6-, 8,9-, 11,12- and 14,15-EET) to
  • EETs dihydroxy eicosatrienoic acids
  • EETs might have beneficial effects on lipid metabolism and insulin sensitivity.
  • sEH protein and message levels are upregulated in the epididymal fat pad from mice that received a high fat diet (HFD) (De Taeye, et al. Obesity (2010) 18(3):489-98).
  • HFD high fat diet
  • CYP 2C expression is decreased and sEH expression is increased in obese Zucker rats, a commonly used animal model of obesity and insulin resistance (Luo, et al., J Pharmacol Exp Ther. (2010) 334(2):430-8).
  • sEH inhibition is a well established approach in cardiovascular, renal and inflammatory diseases in murine models, but its contribution in type 2 diabetes mellitus remains to be established. This study was designed to investigate the consequences of sEH-gene deletion on systemic insulin sensitivity and glucose homeostasis, and to support its mechanism of action using a pharmacological approach.
  • Epoxide hydrolases (“EH,” EC 3.3.2.3) are a family of enzymes which hydrolyze a variety of exogenous and endogenous epoxides to their corresponding diols. Epoxide hydrolases have been found in tissues of all mammalian species tested. The highest levels of the enzyme were found in liver and kidney cells (see Wixtrom and Hammock, Pharmacology and Toxicology (Zakim, D. and Vessey, D. A., ed.) 1 :1-93, Wiley, New York, 1985).
  • EH leukotriene epoxide hydrolase
  • cholesterol epoxide hydrolase cholesterol epoxide hydrolase
  • mEH microsomal EH
  • sEH soluble EH
  • the leukotriene EH acts on leukotriene A 4
  • the cholesterol EH hydrate compounds related to the 5,6-epoxide of cholesterol (Nashed, N. T., et al, Arch. Biochem. Biophysics., 241 : 149-162, 1985; Finley, B. and B. D. Hammock, Biochem. Pharmacol, 37:3169-3175,1988).
  • microsomal epoxide hydrolase metabolizes monosubstituted, 1,1- disubstituted, cz ' s- 1,2-disubstituted epoxides and epoxides on cyclic systems epoxides to their corresponding diols. Because of its broad substrate specificity, this enzyme is thought to play a significant role in ameliorating epoxide toxicity. Reactions of detoxification typically decrease the hydrophobicity of a compound, resulting in a more polar and thereby excretable substance.
  • Soluble EH is only very distantly related to mEH and hydrates a wide range of epoxides not on cyclic systems.
  • sEH is believed to play a role in the formation or degradation of endogenous chemical mediators.
  • cytochrome P450 epoxygenase catalyzes NADPH-dependent enatioselective epoxidation of arachidonic acid to four optically active czs-epoxyeicosantrienoic acids (“EETs”) (Karara, A., et al., J. Biol.
  • Soluble epoxide hydrolase has been shown in vivo to convert these compounds with regio- and enantiofacial specificity to the corresponding vzc-dihydroxyeicosatrienoic acids ("DHETs").
  • DHETs vzc-dihydroxyeicosatrienoic acids
  • Both liver and lung cytosolic fraction hydrolyzed 14,15-EET, 8,9-EET and 11,12-EET, in that order of preference.
  • the 5,6-EET is hydrolyzed more slowly.
  • Purified sEH selected 8S,9R- and 14R,15S-EET over their enantiomers as substrates.
  • EETs and their corresponding DHETs exhibit a wide range of biological activities as do their corresponding ⁇ -3 lipid homo logs. Some of these activities include involvements in luteinizing hormone -releasing hormone, stimulation of luteinizing hormone release, inhibition of Na + /K + ATPase, vasodilation of coronary artery, mobilization of Ca 2+ and inhibition of platelet aggregation. Soluble epoxide hydrolase is believed to play a role in these biological activities by contributing to the regulation of the steady state levels of EETs and DHETs as well as other biologically active epoxides and diols.
  • the present invention provides methods of maintaining and promoting glucose tolerance, and methods of reducing or inhibiting hyperglycemia, for example, postprandial hyperglycemia, by co-administering an agent that promotes increased levels of EETs in combination with an agent that inhibits cyclo-oxygenase or 5- lipoxygenase pathways.
  • the methods help retard the progression of diabetes in an individual in need thereof.
  • the invention provides methods of maintaining stable glucose levels in an individual in need thereof.
  • the methods comprise co-administering to the individual
  • Glucose levels can be measured in the blood, plasma or serum, as appropriate.
  • the invention provides methods of maintaining and promoting glucose tolerance, methods of maintaining stable glucose levels, and methods of reducing or inhibiting hyperglycemia, for example, postprandial hyperglycemia, in an individual in need thereof by administering an agent that promotes increased levels of EETs, e.g., (i) an effective amount of a first enzyme inhibitor that inhibits sEH, (ii) an epoxygenated fatty acid, (iii) both an inhibitor of sEH and an epoxygenated fatty acid, or (iv) a mimic of an epoxygenated fatty acid which is stable to epoxide hydrolase.
  • the individual is hyperglycemic. In some embodiments, the individual is hyperglycemic.
  • the individual has type 1 diabetes. In some embodiments, the individual has type 2 diabetes. In some embodiments, the individual is prediabetic.
  • the individual is (a) a person with diabetes mellitus whose blood pressure is 130/80 or less, (b) a person with metabolic syndrome whose blood pressure is less than 130/85, (c) a person with a triglyceride level over 215 mg/dL, or (d) a person with a cholesterol level over 200 mg/dL.
  • the invention provides methods for maintaining stable glucose levels, improving insulin sensitivity and accelerating glucose clearance in a prediabetic subject, the method comprising administering to the subject an effective amount of an inhibitor that inhibits sEH, (ii) an epoxygenated fatty acid, (iii) both an inhibitor of sEH and an epoxygenated fatty acid, or (iv) a mimic of an epoxygenated fatty acid which is stable to epoxide hydrolase.
  • the methods further comprise co-administering an effective amount of a second enzyme inhibitor that inhibits one or more enzymes selected from the group consisting of cyclo- oxygenase ("COX") -1, COX-2, and 5 -lipoxygenase ("5 LOX” or other enzymes and mediators in the LOX 5 pathway).
  • a second enzyme inhibitor that inhibits one or more enzymes selected from the group consisting of cyclo- oxygenase (“COX”) -1, COX-2, and 5 -lipoxygenase (“5 LOX” or other enzymes and mediators in the LOX 5 pathway).
  • one or both of the first and second enzyme inhibitors are administered at subtherapeutic doses. In some embodiments, one or both of the first and second enzyme inhibitors are administered at non-therapeutic doses. In some embodiments, one or both of the first and second enzyme inhibitors are administered at therapeutic doses.
  • one or both of the first and second enzyme inhibitors are administered in a sustained release or a controlled release formulation. In some embodiments, one or both of the first and second enzyme inhibitors are administered concurrently with or within 1 hour of a meal.
  • the epoxygenated fatty acid is an EET.
  • the EET is selected from the group consisting of 14,15-EET, 8,9-EET, 11,12-EET and 5,6-EET.
  • the epoxygenated fatty acid is an epoxide of docosahexaenoic acid ("DHA") or eicosapentaenoic acid (“EPA”), or epoxides of both DHA and of EPA.
  • the first enzyme inhibitor has a primary
  • pharmacophore selected from the group consisting of a urea, a carbamate and an amide.
  • the second enzyme inhibitor is an inhibitor of COX- 2. In some embodiments, the second enzyme inhibitor is a selective inhibitor of COX-2. In some embodiments, the second enzyme inhibitor is selected from the group consisting of celecoxib, valdecoxib, lumiracoxib, etoricoxib, and rofecoxib.
  • the second enzyme inhibitor is an inhibitor of COX- 1.
  • the second enzyme inhibitor is selected from the group consisting of aspirin, acetaminophen, diclofenac potassium, diclofenac sodium, disalsalate.
  • the second enzyme inhibitor is an inhibitor of 5- LOX. In some embodiments, the second enzyme inhibitor is a FLAP inhibitor or a leukotriene antagonist.
  • prediabetes and “prediabetic” interchangeably refer to a condition that involves impaired glucose tolerance (IGT) or impaired fasting glucose (IFG).
  • IGT is defined by a 2-h oral glucose tolerance test plasma glucose concentration >140 mg/dL (7.8 mmol/L) but ⁇ 200 mg/dL (11.1 mmol/L)
  • IFG is defined by a fasting plasma glucose concentration >100 mg/dL (5.6 mmol/L), but ⁇ 126 mg/dL (7.0 mmol/L). See, e.g., Pour and Dagogo-Jack, Clin Chem. (2010) Nov 9., PMID 21062906.
  • EETs "cz ' s-Epoxyeicosatrienoic acids"
  • EETs are biomediators synthesized by cytochrome P450 epoxygenases.
  • derivatives of EETs such as amides and esters (both natural and synthetic)
  • EETs analogs EETs homologs
  • stable EET mimics EETs optical isomers
  • EETs optical isomers can all be used in the methods of the invention, both in pure form and as mixtures of these forms.
  • EETs refers to all of these forms unless otherwise required by context.
  • Epoxide hydrolases (" ⁇ ;” EC 3.3.2.3) are enzymes in the alpha beta hydrolase fold family that add water to 3-membered cyclic ethers termed epoxides. The addition of water to the epoxides results in the corresponding 1 ,2-diols
  • EH leukotriene epoxide hydrolase
  • cholesterol epoxide hydrolase microsomal EH
  • sEH soluble EH
  • the leukotriene EH acts on leukotriene A4, whereas the cholesterol EH hydrates compounds related to the 5,6-epoxide of cholesterol.
  • microsomal epoxide hydrolase metabolizes monosubstituted, 1,1-disubstituted, cis-1,2- disubstituted epoxides and epoxides on cyclic systems to their corresponding diols. Because of its broad substrate specificity, this enzyme is thought to play a significant role in ameliorating epoxide toxicity. Reactions of detoxification typically decrease the hydrophobicity of a compound, resulting in a more polar and thereby excretable substance.
  • sEH Soluble epoxide hydrolase
  • DHETs dihydroxyeicosatrienoic acids
  • NCBI Entrez Nucleotide accession number L05779 sets forth the nucleic acid sequence encoding the protein, as well as the 5' untranslated region and the 3' untranslated region. The evolution and nomenclature of the gene is discussed in Beetham et al, DNA Cell Biol. 14(1):61-71 (1995). Soluble epoxide hydrolase represents a single highly conserved gene product with over 90% homology between rodent and human (Arand et al., FEBS Lett., 338:251-256 (1994)). Soluble EH is only very distantly related to mEH and hydrates a wide range of epoxides not on cyclic systems.
  • sEH In contrast to the role played in the degradation of potential toxic epoxides by mEH, sEH is believed to play a role in the formation or degradation of endogenous chemical mediators. Unless otherwise specified, as used herein, the terms “soluble epoxide hydrolase” and “sEH” refer to human sEH.
  • the terms "sEH inhibitor” (also abbreviated as "sEHI") or “inhibitor of sEH” refer to an inhibitor of human sEH.
  • the inhibitor does not also inhibit the activity of microsomal epoxide hydrolase by more than 25% at concentrations at which the inhibitor inhibits sEH by at least 50%, and more preferably does not inhibit mEH by more than 10% at that concentration.
  • the term "sEH inhibitor” as used herein encompasses prodrugs which are metabolized to active inhibitors of sEH.
  • reference herein to a compound as an inhibitor of sEH includes reference to derivatives of that compound (such as an ester of that compound) that retain activity as an sEH inhibitor.
  • COX is an abbreviation for "cyclo-oxygenase.”
  • COX-1 and COX-2 are recognized as of clinical significance, with COX-1 considered to be constitutively expressed and COX- 2 considered to be inducible and more prevalent at sites of inflammation. See, e.g., Hawkey, Best Pract Res Clin Gastroenterol. 15(5):801-20 (2001).
  • COX-1 inhibitor denotes an agent that inhibits COX-1 more than it inhibits COX-2
  • COX-2 inhibitor denotes an agent that inhibits COX-2 more than it inhibits COX-1.
  • All current non-steroidal anti-inflammatory drugs (NSAIDs) inhibit both COX-1 and COX-2, but most tend to inhibit the two isoforms to different degrees. Since both enzymes tend to be inhibited together to some degree, one can consider an inhibitor of either enzyme to be "COX inhibitor".
  • LOX is an abbreviation for "lipoxygenase.”
  • LOX Several LOX enzymes have been identified.
  • Arachidonate 5 -lipoxygenase (“5-LOX”, EC 1.13.11.34) is involved in the production of pro-inflammatory mediators.
  • Arachidonate 12-lipoxygenase (“12-LOX”, EC 1.13.11.31) and arachidonate 15 -lipoxygenase (“15-LOX”, EC 1.13.11.33) form trihydroxytetraenes known as "lipoxins” (“lipoxygenase interaction products”) from arachidonic acid. Lipoxins act as local anti-inflammatory agents.
  • FLAP 5 -lipoxygenase activating protein
  • FLAP is a protein required before 5-LOX can become catalytically active. Inhibiting FLAP activity reduces or prevents 5-LOX activation, decreasing the biosynthesis of leukotrienes.
  • Cytochrome P450 (“CYP450”) metabolism produces cis- epoxydocosapentaenoic acids (“EpDPEs”) and cz ' s-epoxyeicosatetraenoic acids (“EpETEs”) from docosahexaenoic acid (“DHA”) and eicosapentaenoic acid (“EPA”), respectively.
  • EpDPEs cis- epoxydocosapentaenoic acids
  • EpETEs cz ' s-epoxyeicosatetraenoic acids
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • EDHFs endothelium-derived hyperpolarizing factors
  • EDHFs are mediators released from vascular endothelial cells in response to acetylcholine and bradykinin, and are distinct from the NOS- (nitric oxide) and COX-derived (prostacyclin) vasodilators.
  • NOS- nitric oxide
  • COX-derived vasodilators epoxides, such as EETs,which are prime candidates for the active mediator(s).
  • 14(15)-EpETE for example, is derived via epoxidation of the 14,15-double bond of EPA and is the ⁇ -3 homolog of 14(15)-EpETrE ("14(15)EET”) derived via epoxidation of the 14,15-double bond of arachidonic acid.
  • IC 50 refers to the concentration of an agent required to inhibit enzyme activity by 50%.
  • physiological conditions an extracellular milieu having conditions (e.g., temperature, pH, and osmolality) which allows for the sustenance or growth of a cell of interest.
  • Micro-R A refers to small, noncoding RNAs of 18-25 nt in length that negatively regulate their complementary mRNAs at the posttranscriptional level in many eukaryotic organisms. See, e.g., Kurihara and Watanabe, Proc Natl Acad Sci USA 101(34): 12753-12758 (2004). Micro-RNAs were first discovered in the roundworm C. elegans in the early 1990s and are now known in many species, including humans. As used herein, it refers to exogenously administered miRNA unless specifically noted or otherwise required by context.
  • co-administration refers to the presence of both active agents in the blood at the same time. Active agents that are co-administered can be delivered concurrently (i.e., at the same time) or sequentially.
  • patient refers to a mammal, for example, a human or a non-human mammal, including primates (e.g., macaque, pan troglodyte, pongo), a domesticated mammal (e.g., felines, canines), an agricultural mammal (e.g., bovine, ovine, porcine, equine) and a laboratory mammal or rodent (e.g., rattus, murine, lagomorpha, hamster).
  • primates e.g., macaque, pan troglodyte, pongo
  • domesticated mammal e.g., felines, canines
  • an agricultural mammal e.g., bovine, ovine, porcine, equine
  • rodent e.g., rattus, murine, lagomorpha, hamster
  • the terms “reduce,” “inhibit,” “relieve,” “alleviate” refer to the detectable decrease in symptoms of diabetes, as determined by a trained clinical observer.
  • a reduction in symptoms of diabetes, or prognosis of diabetic condition can be measured using any test known in the art, including without limitation, decreased blood or plasma glucose levels, increased blood or plasma insulin levels, increased C- peptide levels, increased beta cell function.
  • terapéuticaally effective amount refers to an amount of the compound being administered sufficient to prevent or decrease the development of one or more of the symptoms of the disease, condition or disorder being treated.
  • subtherapeutic amount or “non-therapeutic amount” refers to an amount of the individual compound being administered that is insufficient to prevent or decrease the development of one or more of the symptoms of the disease, condition or disorder being treated. Subtherapeutic or non-therapeutic doses of individual agents are inefficacious for their intended purpose.
  • analgesic amount refers to that amount of the compound being administered sufficient to prevent or decrease pain in a subject under treatment.
  • controlled release sustained release
  • extended release extended release
  • timed release any drug-containing formulation in which release of the drug is not immediate, i.e., with a “controlled release” formulation, oral administration does not result in immediate release of the drug into an absorption pool.
  • controlled release sustained release
  • extended release extended release
  • the "absorption pool" represents a solution of the drug administered at a particular absorption site, and k r , k a and k e are first-order rate constants for (1) release of the drug from the formulation, (2) absorption, and (3) elimination, respectively.
  • the rate constant for drug release k r is far greater than the absorption rate constant k a .
  • the opposite is true, i.e., k r «k a , such that the rate of release of drug from the dosage form is the rate-limiting step in the delivery of the drug to the target area.
  • sustained release and extended release are used in their conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, for example, 12 hours or more, and that preferably, although not necessarily, results in substantially steady-state blood levels of a drug over an extended time period.
  • delayed release refers to a pharmaceutical preparation that passes through the stomach intact and dissolves in the small intestine.
  • synergy or “synergistic” interchangeably refer to the combined effects of two active agents that are greater than their additive effects. Synergy can also be achieved by producing an efficacious effect with combined inefficacious doses of two active agents. The measure of synergy is independent of statistical significance.
  • the phrase “consisting essentially of” refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any excipients inactive for the intended purpose of the methods or compositions. In some embodiments, the phrase “consisting essentially of expressly excludes the inclusion of one or more additional active agents other than the listed active agents, e.g., (i) an inhibitor of sEHi and/or an EET alone or in combination with (ii) an inhibitor of cyclo-oxygenase and/or 5-lipoxygenase.
  • Figure 1A-D illustrate whole-body Ephx2 gene deletion and sEH inhibition.
  • FIG. 1 Immunoblots of sEH in different tissues from WT, Ephx2-null (KO) mice and sEHI-treated WT mice. Tubulin expression is shown as a control for loading. Note that compared with control mice, sEH protein expression was ablated in tissues of KO mice, whereas sEH expression was unaffected in sEHI-treated mice.
  • Figures 2A-B illustrate body weights of WT and Ephx2-null mice fed chow or high fat diet.
  • Values depict mean ⁇ SEM of n 4.
  • t-test * P ⁇ 0.05, WT-chow vs. WT- chow+sEHI.
  • FIGS 3A-C illustrate food intake and feeding efficiency. Weekly measurements of food intake in Ephx2-null and WT male mice treated with or without TUPS (sEHI, 10 mg/L via drinking water in 1% PEG 400) during all study either on chow (A) or HFD (B).
  • (C) Feeding efficiency in WT, sEHI -treated WT and Ephx2-null mice. Values depict mean ⁇ SEM of n 4. t-test, * P ⁇ 0.05 WT vs.
  • Figures 5A-E illustrate morphological appearance of various tissue sections from WT, Ephx2-null and sEHI-treated WT mice. Representative histological appearance of hematoxylin-Eosin-stained sections from epididymal fat (A), liver (B), kidney (C), adrenal gland (D) and pancreas (E) sections taken from mice either on chow or high fat diets. Tissue collected at the end of the study from WT, sEHI- treated WT and Ephx2-null (KO) mice, weight, fixed and sectioned as described herein, showing a clear effect of the diet on cells size and fat accumulation. Other tissues appear normal.
  • FIGS 6A-B illustrate reduction of sEH activity reduces diet-induced insulin resistance.
  • Insulin tolerance test (ITT) and glucose tolerance test (GTT) were performed on WT, sEHI-treated WT and KO mice fed either chow diet (A, C) or HFD (B, D) for two months.
  • E and F Second cohort study with mice that started the diet at the age of 12 weeks.
  • ITT and F GTT in WT and Ephx2-null mice fed HFD or chow for 22 weeks (diet starts when mice were twelve weeks old).
  • A, B and E insulin test performed by injecting insulin (lmU/g; i.p.) to mice that were fasted four hours prior to ITT.
  • FIGS 7A-D illustrate reduction of sEH activity prevents diet-induced insulin resistance and sEH-gene deletion improves glucose homeostasis.
  • Insulin tolerance test (ITT) and glucose tolerance test (GTT) were performed on WT, Ephx2- null (KO) mice and sEHI-treated WT male mice fed either chow diet (A, C) or HFD (B, D) for five months.
  • Figures 8A-C illustrate ⁇ -islet size and vascularization density in response to Ephx2-gene deletion and inhibition.
  • A Representative insulin staining in pancreas sections from WT, sEHI-treated WT and Ephx2-null mice at the end of study either on chow or high-fat diets. Pancreata were stained
  • FIG. 9A-F illustrate immunofluorescence detection of insulin and glucagon in pancreatic ⁇ -islets. Representative pancreatic section showing
  • FIGS 10 A- J illustrate enhanced insulin signaling in mice with Ephx2- gene deletion or sEH inhibition.
  • Insulin receptor signaling in liver tissues from male mice on HFD Mice were fasted overnight then injected i.p. with saline or insulin (10 mU/g) and sacrificed after 10 minutes. Ly sates were either extracted and
  • IR insulin receptor
  • IRS-1 IRS-1
  • Phosphorylated state was detected by using specific antibodies for pIR serine 1 162/1163 (A), anti tyrosyl phosphorylation (C) and phospho-IRS-1 Tyr608 (C).
  • Membranes were stripped and reprobed for total protein levels of IR (A) and IRS-1 (C) to control for loading.
  • Other proteins in the signaling pathway were used, anti Akt Ser473 phosphorylation (G), anti MAPK (Thr202/Tyr204) (I) and anti pan p85 (C).
  • FIGS 11A-M illustrate einsulin signaling in mice with Ephx2-gene deletion or sEH inhibition. Insulin receptor signaling in adipose tissues from male mice on HFD. Mice were fasted overnight then injected i.p. with saline or insulin (10 mU/g) and sacrificed after 10 minutes. Ly sates were either extracted and
  • IR insulin receptor
  • IRS-1 IRS-1
  • Phosphorylated state was detected by using specific antibodies for pIR serine 1162/1163 (A) , anti tyrosyl phosphorylation (C) and phospho-IRS-1 Tyr608 (C).
  • Membranes were stripped and reprobed for total protein levels of IR (A) and IRS-1 (C) to control for loading.
  • Other proteins in the signaling pathway were used, anti Akt Ser473 phosphorylation (G), anti MAPK (Thr202/Tyr204) (I), anti pan p85 (C) and inflammatory mediators such as, TNFa and MCP1 (K-M).
  • FIG 12 illustrates blood glucose in an insulin tolerance test (ITT) performed on C57BL/6 wild-type (WT) and corresponding sEH knock-out (KO) mice after receiving either the regular diet (RD; 24% of calories from fat) or the high fat diet (HFD; 42% of calories from fat) for 4 weeks.
  • RD regular diet
  • HFD high fat diet
  • Figure 13 illustrates plasma glucose in a glucose tolerance test (GTT) performed on WT mice and corresponding sEH KO mice after receiving either the RD or the HFD for 5 weeks.
  • GTT glucose tolerance test
  • Figure 14 illustrates blood glucose in an ITT performed on WT mice fed the RD or the HFD for 7 weeks. Two groups of mice were given the sEH inhibitor 1-(1- Methanesulfonyl-piperidin-4-yl)-3-(14-trifluoromethoxy-phenyl)-urea (1709) in their drinking water. Mice fed the sEH inhibitor do not have high blood glucose from the HFD.
  • Figure 15 illustrates blood glucose in an ITT performed on WT mice or KO mice fed the RD or the HFD for 7 weeks. sEH KO mice do not exhibit high blood glucose from the HFD.
  • Figure 16 illustrates blood glucose in a GTT performed on WT mice fed the RD or the HFD for 8 weeks. Two groups of mice were given the sEH inhibitor 1709 in their drinking water.
  • Figure 17 illustrates blood glucose in a GTT performed on WT mice and corresponding sEH KO mice after receiving either the RD or the HFD for 8 weeks.
  • Figure 18 illustrates blood glucose in an ITT performed on WT mice fed the regular diet (RSD) or the HFD for 5 months. Two groups of mice were given the sEH inhibitor 1709 in their drinking water. Mice fed the sEH inhibitor do not have high blood glucose from the HFD.
  • Figure 19 illustrates blood glucose in an ITT performed on WT mice and corresponding sEH KO mice after receiving either the RSD or the HFD for 5 months. sEH KO mice do not exhibit high blood glucose from the HFD.
  • Figure 20 illustrates blood glucose in an ITT performed on WT mice and corresponding sEH KO mice after receiving either the RSD or the HFD for 6 months. sEH KO mice do not exhibit high blood glucose from the HFD.
  • Figure 21 illustrates blood glucose in an ITT performed on WT mice and corresponding sEH KO mice after receiving either the RSD or the HFD for 6 months. sEH KO mice do not exhibit high blood glucose from the HFD.
  • Figure 22 illustrates plasma glucose in an GTT performed on WT mice and corresponding sEH KO mice after receiving either the RSD or the HFD for 6 months.
  • Figure 23 illustrates plasma glucose in an ITT performed on WT mice fed the RSD or the HFD for 7 months. Two groups of mice were given the sEH inhibitor 1709 in their drinking water. Mice fed the sEH inhibitor do not have high blood glucose from the HFD.
  • Figure 24 illustrates blood glucose in an GTT performed on WT mice fed the RSD or the HFD for 7 months. Two groups of mice were given the sEH inhibitor 1709 in their drinking water.
  • Figure 25 illustrates blood glucose in an GTT performed on WT mice fed the RSD or the HFD for 27 weeks. Two groups of mice were given the sEH inhibitor 1709 in their drinking water.
  • Figure 26 illustrates glucose to insulin ratios in treated mice groups, as indicated.
  • FIG. 27A illustrates that mice treated with high fat diet for two months have significantly higher levels of plasma glucose.
  • FIG 27B mice fed regular chow for two months did not have higher levels of plasma glucose.
  • Glucose (lmg/g body weight) was administered in order to measure the glucose clearance over time (Glucose Tolerance Test).
  • WT animals that did not receive an inhibitor of sEH (sEHi) required more time to clear the glucose (as indicated by the higher curve and larger area under the curve).
  • WT animals administered sEH inhibitor 1709 in drinking water (10 mg/L) show a significant reduction in the plasma glucose levels, increased as a result of the high fat diet.
  • the sEH knockout mice exhibit rapid clearance of glucose from the plasma, regardless of administration of an sEHi. The data are consistent with the conclusion that administration of an sEHi sensitizes signaling through the insulin receptor and accelerates glucose clearance.
  • the present invention is based, in part, on the surprising discovery that combined administration of an agent that increases EETs and an agent that inhibits cyclo-oxygenase and/or 5 -lipoxygenase finds use in reducing, preventing and/or ameliorating symptoms associated with diabetes.
  • an agent that increases EETs and an agent that inhibits cyclo-oxygenase and/or 5 -lipoxygenase finds use in reducing, preventing and/or ameliorating symptoms associated with diabetes.
  • an agent that promotes increased levels of EETs By co-administrating the inhibitor of cyclo-oxygenase and/or 5 -lipoxygenase with an agent that promotes increased levels of EETs, a therapeutic, subtherapeutic or non-therapeutic dose of the COX or 5-LOX inhibitor can be administered, thereby maintaining efficacy, reducing undesirable side effects and increasing safety to the patient receiving treatment.
  • Visceral obesity has been defined as an important element of the metabolic syndrome and contributes to the development of insulin resistance and cardiovascular disease.
  • the present invention is based, in part, on the discovery that increasing endogenous levels of the anti-hypertensive and anti-inflammatory mediators epoxyeicosatrienoic acids (EETs) attenuates the development of these diseases.
  • EETs epoxyeicosatrienoic acids
  • the availability of EETs is limited primarily by the soluble epoxide hydrolase (sEH, EPHX2), which metabolizes EETs to their less active diols.
  • mice with targeted gene deletion of sEH Ephx2-null mice
  • Knockout and inhibition of sEH prevents insulin resistance developed in obese mice fed a 'Western Diet'.
  • insulin resistance develops.
  • Knockout of sEH activity resulted in a significant increase in insulin sensitivity.
  • pancreatic ⁇ -islets were larger when sEH was disrupted associated with an increase in vasculature.
  • epoxyeicosatrienoic acids and other epoxylipids decrease inflammation.
  • epoxyeicosatrienoic acids and other epoxylipids regulate vascular tone, in part by preventing the activation of nuclear factor ⁇ .
  • These molecules transcriptionally downregulate the induced cyclooxygenase-2 and lipoxygenase-5 pathways resulting in synergism with NSAIDs, aspirin and also other cascade modulators in reducing the levels of inflammatory eicosanoids.
  • epoxyeicosatrienoic acids or epoxyeicosanoids are associated with a downregulation of prostaglandin PGE 2 , because they transcriptionally downregulate induction of cyclooxygenase. Thus, they could be considered to reduce adipocyte dysfunction.
  • Prostaglandins Other Lipid Mediat 82, 42-9 (2007); and De Taeye, et al. Obesity (Silver Spring) (2009) doi: 10.1038/oby.2009.227.
  • the inhibitors of soluble epoxide hydrolases also synergize with NSAIDs.
  • Nanomolar concentrations of 11,12-epoxyeicosatrienoic acid, or overexpression of CYP2J2 decreases upregulation of cell adhesion molecules, vascular adhesion molecule- 1, intercellular adhesion molecule- 1 and E-selectin induced by tumor necrosis factor, IL-l and LPS in cultured endothelial cells. This finding further validates their anti-inflammatory effects. Node, supra. These biological effects can also occur in the pancreas and in adipose tissue.
  • Anti-inflammatory agents such as NSAIDs, salicylates and aspirin reduce the severity of metabolic dysfunction. Yuan, et al. Science 293, 1673-7 (2001); Renna, et al., Clin Exp Pharmacol Physiol 36, 162-8 (2009); and Van Kerckhoven, et al., Cardiovasc Res 46, 316-23 (2000).
  • Soluble epoxide hydrolase inhibitors synergize the anti-inflammatory actions of these compounds, which suggests that low doses could be used in combination to reduce the symptoms of metabolic syndrome without affecting innate immunity. Inceoglu, supra and Schmelzer, supra.
  • the present methods find use in ameliorating symptoms in patients with type 1 or type 2 diabetes.
  • the individual is pre-diabetic (i.e., does not yet have diabetes).
  • the individual has hyperglycemia.
  • the individual may or may not be obese.
  • the individual may or may not have
  • the individual may or may not have metabolic syndrome.
  • Diabetes mellitus (generally referred to herein as “diabetes”) is a
  • the World Health Organization has set forth a classification scheme for diabetes mellitus that includes type 1 diabetes mellitus, type 2 diabetes mellitus, gestational diabetes, and other specific types of diabetes mellitus. These terms have largely displaced the formerly used terms IDDM (insulin-dependent diabetes mellitus), NIDDM (non- insulin dependent diabetes mellitus), juvenile-onset diabetes mellitus and adult-onset diabetes mellitus.
  • Type 1 diabetes results from an autoimmune destruction of the insulin- secreting B-cells of the pancreas.
  • islet cell autoantibodies autoantibodies to insulin
  • autoantibodies to glutamic acid decarboxylase (GAD65) autoantibodies to the tyrosine phosphatases
  • GAD65 glutamic acid decarboxylase
  • IA-2 and IA-2B tyrosine phosphatases
  • Type 2 diabetes disease usually develops after 40 years of age. It is much more common than type 1 diabetes and comprises approximately 90% of all individuals with diabetes. Insulin concentrations are mostly increased but they can be normal or decreased. Obesity is common. Diet and exercise regimens leading to weight reduction can ameliorate hyperglycemia. Oral hypoglycaemic drugs are also used in an effort to lower blood sugar. Nevertheless, insulin is sometimes required to correct hyperglycemia, particularly as patients grow older or as their ⁇ -cells fail.
  • Insulin resistance is defined as a decreased biological response to normal concentrations of circulating insulin and represents the primary underlying pathological process.
  • the second is the dysfunction of pancreatic B-cells, represented by the inability to produce sufficient amounts of insulin to overcome insulin resistance in the peripheral tissues.
  • insulin production can be insufficient to compensate for the insulin resistance due to B-cell dysfunction.
  • the common result is a relative deficiency of insulin.
  • Data support the concept that insulin resistance is the primary defect, preceding the derangement of insulin secretion. As with type 1 diabetes, the basis of the insulin resistance and insulin secretion defects is believed to be a combination of environmental and genetic factors.
  • Type 1 and type 2 diabetes comprise the great majority of cases of diabetes.
  • gestational diabetes which is usually asymptomatic, and a heterogeneous collection of specific types of diabetes resulting from pathologies of the pancreas, pathologies of the endocrine system, infection, or exposure to chemicals or drugs which damage the beta cells of the pancreas.
  • the present invention can be used with regard to any form of diabetes to the extent that it is associated with progressive damage to the kidney or kidney function.
  • persons with diabetes caused by autoimmune processes such as in type 1 diabetes, will benefit from the administration of sEH inhibitor, with or without EETs, in preferred embodiments relating to diabetes, the invention relates to persons whose diabetes is not caused by an autoimmune process. Therefore, in some preferred embodiments, the person has type 2 diabetes; in some preferred embodiments, the individual has one of the various types of diabetes caused by non-autoimmune processes described earlier in this paragraph.
  • the chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels.
  • the long-term complications of diabetes include retinopathy with potential loss of vision; nephropathy leading to renal failure; peripheral neuropathy with risk of foot ulcers, amputation, and Charcot joints.
  • Glycation of tissue proteins and other macromolecules and excess production of polyol compounds from glucose are among the mechanisms thought to produce tissue damage from chronic hyperglycemia.
  • the nonenzymatic glycation process is one in which glucose is chemically bound to amino groups of proteins, but without the help of enzymes. It is a covalent reaction where, by means of N- glycoside bonding, sugar-protein complex is formed through a series of chemical reactions described by Maillard. In Maillard reactions, sugar-reacts with protein to form complexes and represent an early product of nonenzymatic glycation and an intermediary that is a precursor of later compounds.
  • AGE advanced glycation endproducts
  • the "earliest clinical evidence of nephropathy is the appearance of low but abnormal levels ( 30 mg/day or 20 ⁇ g/min) of albumin in the urine, referred to as microalbuminuria.”
  • microalbuminuria the "earliest clinical evidence of nephropathy is the appearance of low but abnormal levels ( 30 mg/day or 20 ⁇ g/min) of albumin in the urine, referred to as microalbuminuria.”
  • type 1 diabetes juvenile diabetes, characterized by an inability to produce sufficient insulin
  • the Statement states that 80% of persons with microalbuminuria will gradually progress to overt nephropathy, with hypertension developing along the way, unless specific interventions are introduced, although they may have hypertension that becomes manifest about the time they develop microalbuminuria.
  • the Statement further indicates that a higher proportion of persons with type 2 diabetes (adult-onset, characterized by a reduced ability to respond to insulin) have microalbuminuria at diagnosis, and that 20-40% will progress to overt nephropathy without specific intervention.
  • the Statement indicates that one third of type 2 patients have hypertension at diagnosis, thereby indicating that two thirds do not. This is particularly important since the number of people with type 2 diabetes is significantly larger than the number that develop type 1 diabetes.
  • an agent that increases intracellular cAMP is coadministered with an agent that increases EETs.
  • Agents that increase EETs include EETs and inhibitors of sEH. a. Inhibitors of sEH
  • Scores of sEH inhibitors are known, of a variety of chemical structures.
  • Derivatives in which the urea, carbamate or amide pharmacophore (as used herein, "pharmacophore” refers to the section of the structure of a ligand that binds to the sEH) is covalently bound to both an adamantane and to a 12 carbon chain dodecane are particularly useful as sEH inhibitors.
  • Derivatives that are metabolically stable are preferred, as they are expected to have greater activity in vivo.
  • urea transition state mimetics that form a preferred group of sEH inhibitors.
  • N, N'-dodecyl-cyclohexyl urea (DCU) is preferred as an inhibitor, while N-cyclohexyl-N'-dodecylurea (CDU) is particularly preferred.
  • Some compounds, such as dicyclohexylcarbodiimide (a lipophilic diimide), can decompose to an active urea inhibitor such as DCU. Any particular urea derivative or other compound can be easily tested for its ability to inhibit sEH by standard assays, such as those discussed herein.
  • the production and testing of urea and carbamate derivatives as sEH inhibitors is set forth in detail in, for example, Morisseau et al, Proc Natl Acad Sci (USA) 96:8849-8854 (1999).
  • N-Adamantyl-N'-dodecyl urea (“ADU”) is both metabolically stable and has particularly high activity on sEH. (Both the 1- and the 2- admamantyl ureas have been tested and have about the same high activity as an inhibitor of sEH.) Thus, isomers of adamantyl dodecyl urea are preferred inhibitors. It is further expected that N, N'-dodecyl-cyclohexyl urea (DCU), and other inhibitors of sEH, and particularly dodecanoic acid ester derivatives of urea, are suitable for use in the methods of the invention. Preferred inhibitors include:
  • Another preferred group of inhibitors are piperidines.
  • the following Table sets forth some exemplar piperidines and their ability to inhibit sEH activity, expressed as the amount needed to reduce the activity of the enzyme by 50% (expressed as "IC 50 ").
  • U.S. Patent No. 5,955,496 also sets forth a number of sEH inhibitors which can be used in the methods of the invention.
  • One category of these inhibitors comprises inhibitors that mimic the substrate for the enzyme.
  • the lipid alkoxides e.g., the 9-methoxide of stearic acid
  • lipid alkoxides In addition to the inhibitors discussed in the '496 patent, a dozen or more lipid alkoxides have been tested as sEH inhibitors, including the methyl, ethyl, and propyl alkoxides of oleic acid (also known as stearic acid alkoxides), linoleic acid, and arachidonic acid, and all have been found to act as inhibitors of sEH.
  • oleic acid also known as stearic acid alkoxides
  • linoleic acid also known as arachidonic acid
  • the '496 patent sets forth sEH inhibitors that provide alternate substrates for the enzyme that are turned over slowly.
  • phenyl glycidols e.g., S, S-4- nitrophenylglycidol
  • chalcone oxides include 4-phenylchalcone oxide and 4-fluourochalcone oxide. The phenyl glycidols and chalcone oxides are believed to form stable acyl enzymes.
  • Additional inhibitors of sEH suitable for use in the methods of the invention are set forth in U.S. Patent Nos. 6,150,415 (the '415 patent) and 6,531,506 (the '506 patent).
  • Two preferred classes of sEH inhibitors of the invention are compounds of Formulas 1 and 2, as described in the '415 and '506 patents. Means for preparing such compounds and assaying desired compounds for the ability to inhibit epoxide hydrolases are also described.
  • the '506 patent in particular, teaches scores of inhibitors of Formula 1 and some twenty sEH inhibitors of Formula 2, which were shown to inhibit human sEH at concentrations as low as 0.1 ⁇ .
  • Any particular sEH inhibitor can readily be tested to determine whether it will work in the methods of the invention by standard assays. Esters and salts of the various compounds discussed above or in the cited patents, for example, can be readily tested by these assays for their use in the methods of the invention.
  • chalcone oxides can serve as an alternate substrate for the enzyme. While chalcone oxides have half lives which depend in part on the particular structure, as a group the chalcone oxides tend to have relatively short half lives (a drug's half life is usually defined as the time for the concentration of the drug to drop to half its original value. See, e.g., Thomas, G., Medicinal Chemistry: an
  • chalcone oxides and other inhibitors which have a half life whose duration is shorter than the practitioner deems desirable, are preferably administered in a manner which provides the agent over a period of time.
  • the inhibitor can be provided in materials that release the inhibitor slowly.
  • Methods of administration that permit high local concentrations of an inhibitor over a period of time are known, and are not limited to use with inhibitors which have short half lives although, for inhibitors with a relatively short half life, they are a preferred method of administration.
  • active derivatives can be designed for practicing the invention.
  • dicyclohexyl thio urea can be oxidized to dicyclohexylcarbodiimide which, with enzyme or aqueous acid
  • the drugs and prodrugs can be chiral for greater specificity. These derivatives have been extensively used in medicinal and agricultural chemistry to alter the pharmacological properties of the compounds such as enhancing water solubility, improving formulation chemistry, altering tissue targeting, altering volume of distribution, and altering penetration. They also have been used to alter toxicology profiles.
  • Such active proinhibitor derivatives are within the scope of the present invention, and the just-cited references are incorporated herein by reference. Without being bound by theory, it is believed that suitable inhibitors of the invention mimic the enzyme transition state so that there is a stable interaction with the enzyme catalytic site. The inhibitors appear to form hydrogen bonds with the nucleophilic carboxylic acid and a polarizing tyrosine of the catalytic site.
  • the sEH inhibitor used in the methods taught herein is a "soft drug.”
  • Soft drugs are compounds of biological activity that are rapidly inactivated by enzymes as they move from a chosen target site. EETs and simple biodegradable derivatives administered to an area of interest may be considered to be soft drugs in that they are likely to be enzymatically degraded by sEH as they diffuse away from the site of interest following administration. Some sEHI, however, may diffuse or be transported following administration to regions where their activity in inhibiting sEH may not be desired. Thus, multiple soft drugs for treatment have been prepared.
  • sEHI carbamates, esters, carbonates and amides placed in the sEHI, approximately 7.5 angstroms from the carbonyl of the central pharmacophore.
  • sEHI highly active sEHI that yield biologically inactive metabolites by the action of esterase and/or amidase.
  • Groups such as amides and carbamates on the central pharmacophores can also be used to increase solubility for applications in which that is desirable in forming a soft drug.
  • easily metabolized ethers may contribute soft drug properties and also increase the solubility.
  • sEH inhibition can include the reduction of the amount of sEH.
  • sEH inhibitors can therefore encompass nucleic acids that inhibit expression of a gene encoding sEH. Many methods of reducing the expression of genes, such as reduction of transcription and siRNA, are known, and are discussed in more detail below.
  • the inhibitor inhibits sEH without also significantly inhibiting microsomal epoxide hydrolase ("mEH").
  • mEH microsomal epoxide hydrolase
  • the inhibitor inhibits sEH activity by at least 50% while not inhibiting mEH activity by more than 10%.
  • Preferred compounds have an IC50 (inhibition potency or, by definition, the concentration of inhibitor which reduces enzyme activity by 50%) of less than about 500 ⁇ .
  • Inhibitors with IC50S of less than 500 ⁇ are preferred, with IC 50 s of less than 100 ⁇ being more preferred and, in order of increasing preference, an IC50 of 50 ⁇ , 40 ⁇ , 30 ⁇ , 25 ⁇ , 20 ⁇ , 15 ⁇ , 10 ⁇ , 5 ⁇ , 3 ⁇ , 2 ⁇ , 1 ⁇ or even less being still more preferred.
  • Assays for determining sEH activity are known in the art and described elsewhere herein. b. EETs
  • EETs which are epoxides of arachidonic acid, are known to be effectors of blood pressure, regulators of inflammation, and modulators of vascular permeability. Hydrolysis of the epoxides by sEH diminishes this activity. Inhibition of sEH raises the level of EETs since the rate at which the EETs are hydro lyzed into
  • DHETs dihydroxyeicosatrienoic acids
  • EETs administered systemically would be hydrolyzed too quickly by endogenous sEH to be helpful.
  • EETs were administered by catheters inserted into mouse aortas. The EETs were infused continuously during the course of the experiment because of concerns over the short half life of the EETs. See, Liao and Zeldin, International Publication WO 01/10438 (hereafter "Liao and Zeldin"). It also was not known whether endogenous sEH could be inhibited sufficiently in body tissues to permit administration of exogenous EET to result in increased levels of EETs over those normally present. Further, it was thought that EETs, as epoxides, would be too labile to survive the storage and handling necessary for therapeutic use.
  • sEHI, EETs, or co-administration of sEHIs and of EETs can be used in the methods of the present invention.
  • one or more EETs are administered to the patient without also administering an sEHI.
  • one or more EETs are administered shortly before or concurrently with administration of an sEH inhibitor to slow hydrolysis of the EET or EETs.
  • one or more EETs are administered after administration of an sEH inhibitor, but before the level of the sEHI has diminished below a level effective to slow the hydrolysis of the EETs.
  • EETs useful in the methods of the present invention include 14,15-EET, 8,9- EET and 11,12-EET, and 5,6 EETs.
  • the EETs are administered as the methyl ester, which is more stable.
  • the EETs are regioisomers, such as 8S,9R- and 14R,15S-EET.
  • 8,9-EET, 11,12-EET, and 14R,15S- EET are commercially available from, for example, Sigma-Aldrich (catalog nos. E5516, E5641, and E5766, respectively, Sigma-Aldrich Corp., St. Louis, MO).
  • EETs, analogs, or derivatives that retain activity can be used in place of or in combination with unmodified EETs.
  • Liao and Zeldin, supra define EET analogs as compounds with structural substitutions or alterations in an EET, and include structural analogs in which one or more EET olefins are removed or replaced with acetylene or cyclopropane groups, analogs in which the epoxide moiety is replaced with oxitane or furan rings and heteroatom analogs. In other analogs, the epoxide moiety is replaced with ether, alkoxides, urea, amide, carbamate,
  • the analogs or derivatives are relatively stable as compared to an unmodified EET because they are more resistant than an unmodified EET to sEH and to chemical breakdown.
  • “Relatively stable” means the rate of hydrolysis by sEH is at least 25% less than the hydrolysis of the unmodified EET in a hydrolysis assay, and more preferably 50% or more lower than the rate of hydrolysis of an unmodified EET.
  • Liao and Zeldin show, for example, episulfide and sulfonamide EETs derivatives.
  • Amide and ester derivatives of EETs and that are relatively stable are preferred embodiments.
  • Whether or not a particular EET analog or derivative has the biological activity of the unmodified EET can be readily determined by using it in standard assays, such as radio-ligand competition assays to measure binding to the relevant receptor.
  • EETs refers to unmodified EETs, and EETs analogs and derivatives unless otherwise required by context.
  • the EET or EETs are embedded or otherwise placed in a material that releases the EET over time.
  • Materials suitable for promoting the slow release of compositions such as EETs are known in the art.
  • one or more sEH inhibitors may also be placed in the slow release material.
  • the EET or EETs can be administered orally. Since EETs are subject to degradation under acidic conditions, EETs intended for oral administration can be coated with a coating resistant to dissolving under acidic conditions, but which dissolve under the mildly basic conditions present in the intestines. Suitable coatings, commonly known as "enteric coatings" are widely used for products, such as aspirin, which cause gastric distress or which would undergo degradation upon exposure to gastric acid. By using coatings with an appropriate dissolution profile, the coated substance can be released in a chosen section of the intestinal tract.
  • a substance to be released in the colon is coated with a substance that dissolves at pH 6.5-7, while substances to be released in the duodenum can be coated with a coating that dissolves at pH values over 5.5.
  • Such coatings are commercially available from, for example, Rohm Specialty Acrylics (Rohm America LLC, Piscataway, NJ) under the trade name "Eudragit®". The choice of the particular enteric coating is not critical to the practice of the invention.
  • any of a number of standard assays for determining epoxide hydrolase activity can be used to determine inhibition of sEH.
  • suitable assays are described in Gill,, et al., Anal Biochem 131 :273-282 (1983); and Borhan, et al., Analytical Biochemistry 231 :188-200 (1995)).
  • Suitable in vitro assays are described in Zeldin et al., J Biol. Chem. 268:6402-6407 (1993).
  • Suitable in vivo assays are described in Zeldin et al, Arch Biochem Biophys 330:87-96 (1996).
  • the enzyme also can be detected based on the binding of specific ligands to the catalytic site which either immobilize the enzyme or label it with a probe such as dansyl, fluoracein, luciferase, green fluorescent protein or other reagent.
  • the enzyme can be assayed by its hydration of EETs, its hydrolysis of an epoxide to give a colored product as described by Dietze et al, 1994, supra, or its hydrolysis of a radioactive surrogate substrate (Borhan et al, 1995, supra).
  • the enzyme also can be detected based on the generation of fluorescent products following the hydrolysis of the epoxide.
  • the assays are normally carried out with a recombinant enzyme following affinity purification. They can be carried out in crude tissue homogenates, cell culture or even in vivo, as known in the art and described in the references cited above. d. Other Means of Inhibiting sEH Activity
  • RNA molecules complementary to at least a portion of the human sEH gene can be used to inhibit sEH gene expression.
  • Means for inhibiting gene expression using short RNA molecules are known. Among these are short interfering RNA (siRNA), small temporal RNAs (stRNAs), and micro-RNAs (miRNAs). Short interfering RNAs silence genes through a mRNA degradation pathway, while stRNAs and miRNAs are approximately 21 or 22 nt RNAs that are processed from endogenously encoded hairpin-structured precursors, and function to silence genes via translational repression.
  • RNA interference a form of post-transcriptional gene silencing ("PTGS"), describes effects that result from the introduction of double-stranded RNA into cells (reviewed in Fire, A. Trends Genet 15:358-363 (1999); Sharp, P. Genes Dev 13: 139- 141 (1999); Hunter, C. Curr Biol 9:R440-R442 (1999); Baulcombe. D. Curr Biol 9:R599-R601 (1999); Vaucheret et al. Plant J 16: 651-659 (1998)).
  • RNAi RNA interference interference
  • RNAi The active agent in RNAi is a long double-stranded (antiparallel duplex) RNA, with one of the strands corresponding or complementary to the RNA which is to be inhibited.
  • the inhibited RNA is the target RNA.
  • the long double stranded RNA is chopped into smaller duplexes of approximately 20 to 25 nucleotide pairs, after which the mechanism by which the smaller RNAs inhibit expression of the target is largely unknown at this time. While RNAi was shown initially to work well in lower eukaryotes, for mammalian cells, it was thought that RNAi might be suitable only for studies on the oocyte and the preimplantation embryo. [0124] In mammalian cells other than these, however, longer RNA duplexes provoked a response known as "sequence non-specific RNA interference," characterized by the non-specific inhibition of protein synthesis.
  • dsRNA of greater than about 30 base pairs binds and activates the protein PKR and 2',5'-oligonucleotide synthetase (2',5'-AS).
  • PKR protein PKR
  • 2',5'-oligonucleotide synthetase 2',5'-AS.
  • Activated PKR stalls translation by phosphorylation of the translation initiation factors eIF2a
  • activated 2',5'-AS causes mRNA degradation by 2',5'-oligonucleotide-activated ribonuclease L.
  • RNAi would work in human cells if the RNA strands were provided as pre-sized duplexes of about 19 nucleotide pairs, and RNAi worked particularly well with small unpaired 3' extensions on the end of each strand (Elbashir et al. Nature 411 : 494-498 (2001)).
  • siRNA were applied to cultured cells by transfection in oligofectamine micelles. These RNA duplexes were too short to elicit sequence-nonspecific responses like apoptosis, yet they efficiently initiated RNAi.
  • Many laboratories then tested the use of siRNA to knock out target genes in mammalian cells. The results demonstrated that siRNA works quite well in most instances.
  • siRNAs to the gene encoding sEH can be specifically designed using computer programs.
  • the cloning, sequence, and accession numbers of the human sEH sequence are set forth in Beetham et al., Arch. Biochem. Biophys. 305(1): 197-201 (1993).
  • An exemplary amino acid sequence of human sEH (GenBank Accession No. L05779; SEQ ID NO: 1) and an exemplary nucleotide sequence encoding that amino acid sequence (GenBank Accession No. AAA02756; SEQ ID NO:2) are set forth in U.S. Patent No.
  • Target CAGTGTTCATTGGCCATGACTGG (SEQ ID NO:3)
  • Sense-siRNA 5' - GUGUUCAUUGGCCAUGACUTT- 3'
  • Antisense-siRNA 5' - AGUC AUGGC C AAUG AAC ACTT- 3' (SEQ ID NO:5)
  • Target CAAGCAGTGTTCATTGGCCATGA (SEQ ID NO: 12)
  • Sense-siRNA 5' - AGCAGUGUUCAUUGGCCAUTT- 3'
  • Antisense-siRNA 5' - AUGGCCAAUGAACACUGCUTT- 3' (SEQ ID NO: 14)
  • Sense-siRNA 5' - GCACAUGGAGGACUGGAUUTT- 3' (SEQ ID NO: 16)
  • Antisense-siRNA 5' - AAUCCAGUCCUCCAUGUGCTT- 3' (SEQ ID NO: 17)
  • siRNA can be generated using kits which generate siRNA from the gene.
  • the "Dicer siRNA Generation” kit (catalog number T510001, Gene Therapy Systems, Inc., San Diego, CA) uses the recombinant human enzyme "dicer” in vitro to cleave long double stranded R A into 22 bp siR As.
  • the kit permits a high degree of success in generating siRNAs that will reduce expression of the target gene.
  • the SilencerTM siRNA Cocktail Kit (RNase III) (catalog no. 1625, Ambion, Inc., Austin, TX) generates a mixture of siRNAs from dsRNA using RNase III instead of dicer.
  • RNase III cleaves dsRNA into 12-30 bp dsRNA fragments with 2 to 3 nucleotide 3' overhangs, and 5'-phosphate and 3'-hydroxyl termini.
  • dsRNA is produced using T7 RNA polymerase, and reaction and purification components included in the kit. The dsRNA is then digested by RNase III to create a population of siRNAs.
  • the kit includes reagents to synthesize long dsRNAs by in vitro transcription and to digest those dsRNAs into siRNA-like molecules using RNase III. The manufacturer indicates that the user need only supply a DNA template with opposing T7 phage polymerase promoters or two separate templates with promoters on opposite ends of the region to be transcribed.
  • the siRNAs can also be expressed from vectors. Typically, such vectors are administered in conjunction with a second vector encoding the corresponding complementary strand. Once expressed, the two strands anneal to each other and form the functional double stranded siRNA.
  • One exemplar vector suitable for use in the invention is pSuper, available from OligoEngine, Inc. (Seattle, WA).
  • the vector contains two promoters, one positioned downstream of the first and in antiparallel orientation. The first promoter is transcribed in one direction, and the second in the direction antiparallel to the first, resulting in expression of the complementary strands.
  • the promoter is followed by a first segment encoding the first strand, and a second segment encoding the second strand.
  • the second strand is complementary to the palindrome of the first strand.
  • a section of RNA serving as a linker (sometimes called a "spacer") to permit the second strand to bend around and anneal to the first strand, in a configuration known as a "hairpin.”
  • RNAs hairpin RNAs
  • an siRNA expression cassette is employed, using a Polymerase III promoter such as human U6, mouse U6, or human HI .
  • the coding sequence is typically a 19-nucleotide sense siRNA sequence linked to its reverse complementary antisense siRNA sequence by a short spacer.
  • Nine-nucleotide spacers are typical, although other spacers can be designed.
  • the Ambion website indicates that its scientists have had success with the spacer TTCAAGAGA (SEQ ID NO: 18).
  • 5-6 T's are often added to the 3' end of the oligonucleotide to serve as a termination site for Polymerase III. See also, Yu et al., Mol Ther 7(2):228-36 (2003); Matsukura et al, Nucleic Acids Res 31(15):e77 (2003).
  • the siRNA targets identified above can be targeted by hairpin siRNA as follows.
  • sense and antisense strand can be put in a row with a loop forming sequence in between and suitable sequences for an adequate expression vector to both ends of the sequence.
  • the following are non- limiting examples of hairpin sequences that can be cloned into the pSuper vector:
  • Antisense strand 5'-AGCTAAAAAGTGTTCATTGGCCATGACTTCTCTT GAAAGTCATGGCCAATGAACACGGG -3' (SEQ ID NO:21)
  • Antisense strand 5'- AGCTAAAAAAAGGCTATGGAGAGTCATCTCTCTTGAA GATGACTCTCCATAGCCTTGGG -3' (SEQ ID NO:24)
  • Antisense strand 5'-
  • Target CAAGCAGTGTTCATTGGCCATGA (SEQ ID NO:28)
  • Sense strand 5'-GATCCCCAGCAGTGTTCATTGGCCATTTCAAGAGAATG GCCAATGAACACTGCTTTTTT -3' (SEQ ID NO:29)
  • Antisense strand 5'-
  • Antisense strand 5'-
  • nucleic acid molecule can be a DNA probe, a riboprobe, a peptide nucleic acid probe, a phosphorothioate probe, or a 2'-0 methyl probe.
  • the antisense sequence is substantially complementary to the target sequence.
  • the antisense sequence is exactly complementary to the target sequence.
  • the antisense polynucleotides may also include, however, nucleotide substitutions, additions, deletions, transitions, transpositions, or modifications, or other nucleic acid sequences or non-nucleic acid moieties so long as specific binding to the relevant target sequence corresponding to the sEH gene is retained as a functional property of the polynucleotide.
  • the antisense molecules form a triple helix- containing, or "triplex" nucleic acid.
  • Triple helix formation results in inhibition of gene expression by, for example, preventing transcription of the target gene (see, e.g., Cheng et al, 1988, J. Biol. Chem. 263:15110; Ferrin and Camerini-Otero, 1991, Science 354: 1494; Ramdas et al, 1989, J. Biol. Chem. 264: 17395; Strobel et al, 1991, Science 254: 1639; and Rigas et al, 1986, Proc. Natl. Acad. Sci. U.S.A. 83:9591)
  • Antisense molecules can be designed by methods known in the art. For example, Integrated DNA Technologies (Coralville, IA) makes available a program found on the worldwide web "biotools.idtdna.com/antisense/AntiSense.aspx", which will provide appropriate antisense sequences for nucleic acid sequences up to 10,000 nucleotides in length. Using this program with the sEH gene provides the following exemplar sequences:
  • ribozymes can be designed to cleave the mRNA at a desired position. (See, e.g., Cech, 1995, Biotechnology 13:323; and Edgington, 1992, Biotechnology 10:256 and Hu et al, PCT Publication WO 94/03596).
  • antisense nucleic acids can be made using any suitable method for producing a nucleic acid, such as the chemical synthesis and recombinant methods disclosed herein and known to one of skill in the art.
  • antisense RNA molecules of the invention may be prepared by de novo chemical synthesis or by cloning.
  • an antisense RNA can be made by inserting (ligating) a sEH gene sequence in reverse orientation operably linked to a promoter in a vector (e.g., plasmid).
  • the strand of the inserted sequence corresponding to the noncoding strand will be transcribed and act as an antisense oligonucleotide of the invention.
  • the oligonucleotides can be made using nonstandard bases (e.g., other than adenine, cytidine, guanine, thymine, and uridine) or nonstandard backbone structures to provides desirable properties (e.g., increased nuclease-resistance, tighter-binding, stability or a desired Tm).
  • nonstandard bases e.g., other than adenine, cytidine, guanine, thymine, and uridine
  • desirable properties e.g., increased nuclease-resistance, tighter-binding, stability or a desired Tm.
  • Techniques for rendering oligonucleotides nuclease-resistant include those described in PCT
  • oligonucleotides having a peptide-nucleic acid (PNA) backbone (Nielsen et al., 1991, Science 254: 1497) or incorporating 2'-0-methyl ribonucleotides, phosphorothioate nucleotides, methyl phosphonate nucleotides, phosphotriester nucleotides, phosphorothioate nucleotides, phosphoramidates.
  • PNA peptide-nucleic acid
  • Proteins have been described that have the ability to translocate desired nucleic acids across a cell membrane.
  • such proteins have amphiphilic or hydrophobic subsequences that have the ability to act as membrane-translocating carriers.
  • homeodomain proteins have the ability to translocate across cell membranes.
  • the shortest internalizable peptide of a homeodomain protein, Antennapedia was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996).
  • a linker can be used to link the oligonucleotides and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker or any other suitable chemical linker.
  • siRNAs can be introduced into mammals without eliciting an immune response by encapsulating them in
  • the nucleic acid is introduced directly into superficial layers of the skin or into muscle cells by a jet of compressed gas or the like.
  • Methods for administering naked polynucleotides are well known and are taught, for example, in U.S. Patent No. 5,830,877 and International Publication Nos. WO 99/52483 and 94/21797.
  • Devices for accelerating particles into body tissues using compressed gases are described in, for example, U.S. Patent Nos. 6,592,545, 6,475,181, and 6,328,714.
  • the nucleic acid may be lyophilized and may be complexed, for example, with polysaccharides to form a particle of appropriate size and mass for acceleration into tissue.
  • the nucleic acid can be placed on a gold bead or other particle which provides suitable mass or other characteristics.
  • a gold bead or other particle which provides suitable mass or other characteristics.
  • the nucleic acid can also be introduced into the body in a virus modified to serve as a vehicle without causing pathogenicity.
  • the virus can be, for example, adenovirus, fowlpox virus or vaccinia virus.
  • miPvNAs and siRNAs differ in several ways: miRNA derive from points in the genome different from previously recognized genes, while siRNAs derive from mRNA, viruses or transposons, miRNA derives from hairpin structures, while siRNA derives from longer duplexed RNA, miRNA is conserved among related organisms, while siRNA usually is not, and miRNA silences loci other than that from which it derives, while siRNA silences the loci from which it arises.
  • miRNAs tend not to exhibit perfect complementarity to the mRNA whose expression they inhibit. See, McManus et al, supra. See also, Cheng et al, Nucleic Acids Res.
  • an epoxygenated fatty acid is co-administered with an agent that increases intracellular cAMP.
  • exemplary epoxygenated fatty acids include epoxides of linoleic acid, eicosapentaenoic acid (“EPA”) and
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • Cytochrome P450 (“CYP450”) metabolism produces cis- epoxydocosapentaenoic acids (“EpDPEs”) and cz ' s-epoxyeicosatetraenoic acids (“EpETEs”) from docosahexaenoic acid (“DHA”) and eicosapentaenoic acid (“EPA”), respectively.
  • EpDPEs cis- epoxydocosapentaenoic acids
  • EpETEs cz ' s-epoxyeicosatetraenoic acids
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • EDHFs endothelium-derived hyperpolarizing factors
  • EDHFs are mediators released from vascular endothelial cells in response to acetylcholine and bradykinin, and are distinct from the NOS- (nitric oxide) and COX-derived (prostacyclin) vasodilators.
  • NOS- nitric oxide
  • COX-derived vasodilators epoxides, such as EETs,which are prime candidates for the active mediator(s).
  • 14(15)-EpETE for example, is derived via epoxidation of the 14,15-double bond of EPA and is the ⁇ -3 homolog of 14(15)-EpETrE ("14(15)EET”) derived via epoxidation of the 14,15-double bond of arachidonic acid.
  • EETs which are epoxides of the fatty acid arachidonic acid.
  • Our studies of the effects of EETs has led us to realization that the anti-inflammatory effect of EPA and DHA are likely due to increasing the levels of the epoxides of these two fatty acids.
  • increasing the levels of epoxides of EPA, of DHA, or of both, will act to reduce pain and
  • the epoxides of EPA and DHA are substrates for sEH.
  • the epoxides of EPA and DHA are produced in the body at low levels by the action of cytochrome P450s. Endogenous levels of these epoxides can be maintained or increased by the administration of sEHI.
  • the endogeous production of these epoxides is low and usually occurs in relatively special circumstances, such as the resolution of inflammation.
  • Our expectation is that administering these epoxides from exogenous sources will aid in the resolution of inflammation and in reducing pain, as well as with symptoms of diabetes and metabolic syndromes. It is further beneficial with pain or inflammation to inhibit sEH with sEHI to reduce hydrolysis of these epoxides, thereby maintaining them at relatively high levels.
  • EPA has five unsaturated bonds, and thus five positions at which epoxides can be formed, while DHA has six.
  • the epoxides of EPA are typically abbreviated and referred to generically as "EpETEs", while the epoxides of DHA are typically abbreviated and referred to generically as "EpDPEs”.
  • EpETEs the epoxides of EPA
  • EpDPEs epoxides of DHA
  • the specific regioisomers of the epoxides of each fatty acid are set forth in the following Table:
  • NSAIDs non-steroidal anti-inflammatory drugs
  • COX-2 is considered the enzyme associated with an inflammatory response
  • enzyme selectivity is generally measured in terms of specificity for COX-2.
  • cells of a target organ that express COX-1 or COX-2 are exposed to increasing levels of NSAIDs. If the cell does not normally produce COX-2, COX-2 is induced by a stimulant, usually bacterial lipopolysaccharide (LPS).
  • a stimulant usually bacterial lipopolysaccharide (LPS).
  • the relative activity of NSAIDs on COX-1 and COX-2 is expressed by the ratio of IC 50 S for each enzyme: COX-2 (ICso)/COX-l (IC 50 ).
  • various NSAIDs have been reported to have ratios of COX-2 (IC 5 o)/COX-l (IC 50 ) ranging from 0.33 to 122. See, Englehart et al., J Inflammatory Res 44:422-33 (1995).
  • Aspirin has an IC 50 ratio of 0.32, indicating that it inhibits COX-1 more than COX-2, while indomethacin is considered a COX-2 inhibitor since its COX-2 (IC 5 o)/COX-l (IC 50 ) ratio is 33. Even selective COX-2 inhibitors retain some COX-1 inhibition at therapeutic levels obtained in vivo. Cryer and Feldman, Am J Med. 104(5):413-21 (1998).
  • NSAIDs that find use in the methods and compositions of the invention include the traditional NSAIDs diclofenac potassium, diclofenac sodium, diclofenac sodium with misoprostol, diflunisal, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, ketoprofen,
  • LOX lipoxygenase
  • LTs leukotrienes
  • the primary inflammatory enzyme is 5 -lipoxygenase (“5-LOX”).
  • the 5-LOX cascade results in the formation of LTB4 and the cysteinyl LTs LTC4, LTD4, and LTE4.
  • LTB4 is a potent stimulator of leukocyte activation.
  • Cysteinyl LTs may participate in the damage of gastric mucosa by inducing mucosal microvascular injury and gastric vessel vasoconstriction, promoting breakdown of the mucosal barrier and stimulating the secretion of gastric acid, as well as the production of interleukin 1 ("IL1”) and proinflammatory cytokines.”
  • IL1 interleukin 1
  • IL1 interleukin 1
  • Additional lipoxygenases, 12-LOX and 15 -LOX exist that contribute to the formation of anti-inflammatory compounds known as lipoxins, or LXs.
  • lipoxins or LXs.
  • 5-LOX can also be inhibited by inhibiting the 5- lipoxygenase activating protein ("FLAP") by MK-886.
  • FLAP 5- lipoxygenase activating protein
  • This inhibitor induces apoptosis in some cell types and is best used in in vitro studies.
  • Other inhibitors are described in, e.g., U.S. Patent Application No. 20040198768 c. Joint COX/LOX inhibitors
  • Tepoxalin has also been shown to block the COX enzymes and LOX in humans and to be well tolerated.
  • a second inhibitor of COX and 5-LOX, licofelone (Merkle GmbH, Germany) is in Phase III clinical trials as a treatment for osteoarthritis and has shown gastric tolerability superior to naproxen. See, Bias et al., Am J Gastroenterol 99(4):611 (2004). See also, Martel- Pelletier 2003, supra; Tries et al., Inflamm Res 51 : 135-43 (2002).
  • a number of other dual COX/LOX inhibitors, and especially COX-2/5-LOX inhibitors, have been developed, as exemplified by U.S. Patent Nos.
  • compositions of the invention a COX-1, COX-2, or LOX inhibitor is combined with a sEHI.
  • the compositions further comprise one or more EETs or an epoxide of EPA, of DHA, or one or more epoxides of both.
  • the composition is of an epoxide or EPA, of DHA, or epoxides of both, and an sEHI.
  • the compositions of the invention can be prepared and administered in a wide variety of oral, parenteral and topical dosage forms.
  • compositions for use in the methods of the present invention can be administered orally, by injection, that is, intravenously, intramuscularly, intracutaneous ly, subcutaneously, intraduodenally, or intraperitoneally.
  • the compositions can also be administered by inhalation, for example, intranasally. Additionally, the compositions can be administered transdermally.
  • the methods of the invention permit administration of compositions comprising a pharmaceutically acceptable carrier or excipient, an inhibitor of COX-1, of COX-2, or of both, or an inhibitor of a LOX, a selected sEHI inhibitor or a pharmaceutically acceptable salt of the inhibitor and, optionally, one or more EETs or epoxides of EPA or of DHA, or of both.
  • the methods of the invention comprise administration of an sEHI and one or more epoxides of EPA or of DHA, or of both.
  • the pharmaceutically acceptable carriers can be either solid or liquid.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the carrier is a finely divided solid which is in a mixture with the finely divided active component.
  • the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain from 5% or 10% to 70% of the active compound.
  • Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • preparation is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
  • carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
  • cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
  • a low melting wax such as a mixture of fatty acid glycerides or cocoa butter
  • the active component is dispersed homogeneously therein, as by stirring.
  • the molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
  • Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.
  • liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
  • Transdermal administration can be performed using suitable carriers. If desired, apparatuses designed to facilitate transdermal delivery can be employed. Suitable carriers and apparatuses are well known in the art, as exemplified by U.S. Patent Nos. 6,635,274, 6,623,457, 6,562,004, and 6,274,166.
  • Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired.
  • Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active components in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
  • liquid forms include solutions, suspensions, and emulsions.
  • These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • the pharmaceutical preparation is preferably in unit dosage form.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects and animals, each unit containing a predetermined quantity of active material calculated to produce the desired pharmaceutical effect in association with the required pharmaceutical diluent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of this invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular effect to be achieved and (b) the limitations inherent in the art of compounding such an active material for use in humans and animals, as disclosed in detail in this specification, these being features of the present invention.
  • a therapeutically effective amount of one or more of the following: an sEH inhibitor, an EET, an EpDPE, or an EpETE, is employed to act as an analgesic alone or in combination with inhibitors of COX -1 or of -2, or both, or of a LOX enzyme.
  • the dosage of the specific compounds depends on many factors that are well known to those skilled in the art. They include for example, the route of administration and the potency of the particular compound.
  • An exemplary dose is from about 0.001 ⁇ g/kg to about 100 mg/kg body weight of the mammal.
  • EETs, EpDPEs, or EpETEs are unstable, and can be converted to the corresponding diols, in acidic conditions, such as those in the stomach.
  • EETs, EpDPEs, or EpETEs can be administered intravenously or by injection.
  • EETs, EpDPEs, or EpETEs intended for oral administration can be encapsulated in a coating that protects the compounds during passage through the stomach.
  • the EETs, EpDPEs, or EpETEs can be provided with a so-called "enteric" coating, such as those used for some brands of aspirin, or embedded in a formulation.
  • enteric coatings and formulations are well known in the art.
  • the compositions of the invention are embedded in a slow-release formulation to facilitate administration of the agents over time.
  • the sEHIs and, optionally, the EETs, EpDPEs, or EpETEs do not need to be combined with the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or COX/LOX inhibitor. They can instead be administered separately. If the sEHIs are administered separately (with or without EETs, EpDPEs, or
  • EpETEs they should be administered shortly before or concurrently with
  • COX/LOX inhibitor If the sEHI is administered after administration of the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or COX/LOX inhibitor, it should be administered as soon as possible after administration of the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or COX/LOX inhibitor to maximize the synergy with the other inhibitor. Administration of the sEHI will still be beneficial even if it follows the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or COX/LOX inhibitor by some time, however, so long as amounts of the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or COX/LOX inhibitor sufficient to inhibit the respective enzyme are still present.
  • sEHIs have half lives defined by the rate at which they are metabolized by or excreted from the body, and that the sEHIs will have a period following administration during which they will be present in amounts sufficient to be effective. If EETs, EpDPEs, or EpETEs are administered after the sEHI is administered, therefore, it is desirable that the EETs, EpDPEs, or EpETEs be administered during the period during which the sEHI will be present in amounts to be effective in delaying hydrolysis of the EETs, EpDPEs, or EpETEs. Typically, the EETs, EpDPEs, or EpETEs will be administered within 48 hours of administering an sEH inhibitor.
  • the EETs, EpDPEs, or EpETEs are administered within 24 hours of the sEHI, and even more preferably within 12 hours.
  • the EETs, EpDPEs, or EpETEs are administered within 10, 8, 6, 4, 2, hours, 1 hour, or one half hour after administration of the inhibitor.
  • the EETs, EpDPEs, or EpETEs are administered concurrently with the sEHI.
  • mice homozygotes or wild type mice were paired for subsequent breeding. All mice were maintained on a 12-hour light-dark cycle in a temperature-controlled facility, with free access to water and food. Genotyping was performed by PCR, using DNA extracted from tail as described earlier (Luria, et al. (2007) Journal of Biological Chemistry 282, 2891-2898). Studies were conducted using male WT and Ephx2-null mice at eight and 12 weeks of age. Mice were placed on standard chow diet (percent kcal from: protein 28.5 %, fat 13.5%, carbohydrates 58.0%; Rodent diet #5001, Test Diet, Richmond, IN) or a high fat diet (percent kcal from: protein 15.2%,
  • mice were further separated into two groups, in which one group was given a selective sEH inhibitor (TUPS, l-(l-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)- urea lOmg/L (laboratory number sEHI 1709) (Jones, et al. (2006) Bioorg Med Chem Lett 16, 5212-5216, Chiamvimonvat, et al. (2007) J Cardiovasc Pharmacol 50, 225- 237).
  • TUPS selective sEH inhibitor
  • Drug efficacy was already evaluated in a previous study, showing elevated sEH-substrate to product ratio during two weeks treatment using the same inhibitor and same route of administration (Enayetallah, et al. (2008) J Biol Chem 283, 36592- 36598). Animal handling, experimentation, and euthanasia were conducted in accordance with federal rules and guideline of IACUC at the University of California, Davis.
  • TUPS selective sEH inhibitor
  • TUPS was given via drinking water ad lib (10 mg/liter in 1% polyethylene glycol 400). Water consumption was monitored daily, and the concentration of TUPS in water monitored. A second set of WT animals was treated with 1% polyethylene glycol 400 (vehicle). At the end of the study, blood was collected by cardiac puncture from the right ventricle in EDTA-rinsed syringes following lethal injection of sodium pentobarbital (100 mg/kg body weight intraperitoneally). TUPS was extracted from blood, and selected organs (liver, epididymal fat and pancreas) and the concentration was analyzed by liquid
  • Glucose was measured in blood collected from the tail using a glucometer (Easy Check; Home Aide Diagnostics, Inc). Serum insulin was determined by enzyme-linked immunosorbent assay, using murine insulin as a standard (Crystal Chem Inc., IL). Free fatty acid (FFA) and triglyceride (TG) concentrations were measured by an enzymatic colorimetric method (Wako, Neuss, Germany). Serum leptin was assayed by enzyme-linked immunosorbent assay, using rat leptin standard (Crystal Chem, Inc). Fed glucose measurements were taken between 7-9 am and where indicated, from mice fasted for 12 hours. For insulin tolerance tests (ITTs), mice were fasted for 4 hrs and regular human insulin
  • mice were fasted over night, injected (i.p.) with a solution of 20% D-glucose (2 mg/g body weight), and blood glucose was measured before and 15, 30, 60, and 120 min following injection.
  • GTTs glucose tolerance tests
  • Immunoblots were performed with the anti- phosphotyrosine monoclonal antibodies 4G10 (Upstate Biotechnology), or antibodies against 3 ⁇ 4 ⁇ , IRS1, pAkt473, Akt, pErk (Cell Signaling) and Erk (Santa Cruz).
  • Proteins were visualized using enhanced chemiluminescence (ECL, Amersham Biosciences) and pixel intensities of immuno-reactive bands were quantified using FluorChem 9900 (Alpha Innotech, CA).
  • Pancreatic ⁇ -islet size was analyzed using Image J software (on the internet at rsb.info.nih.gov/ij). Endothelial cells were stained as previously described (Kostromina, et al., Endocrinology 2010 151(5):2050- 9). Briefly, anti platelet endothelial cell adhesion molecule- 1 (PEC AM 1)-CD31- conjugated Alexa Flour 488 antibodies (BD Pharmingen, Franklin Lakes, NJ) were used at 1 :200 dilution. After immunolabeling, sections were washed and mounted (Kostromina, et al., Endocrinology 2010 151(5):2050-9). Digital images were acquired using a fluorescence microscope (Zeiss, Jana, Germany).
  • Ephx2-null mice were healthy, fertile and morphologically indistinguishable from control littermates.
  • a PCR product of 290 bp indicates the Ephx2 sequence which distinguished WT from KO mice (band of 560 bp represents the sequence of neomycin gene) (Fig. 1 A).
  • Immunoblot analysis was used to determine the efficiency of sEH deletion in different tissues including those that are insulin-responsive (Fig. IB).
  • sEH protein was expressed in all examined tissue extracts of WT mice, and was absent in Ephx2-null mice indicating efficient deletion of the gene (Fig. IB).
  • a faint band corresponding to the size of sEH was detected in brain homogenates of KO mice, which could indicate trace expression of sEH.
  • sEH-null mice lack both the epoxide hydrolase and phosphatase domains.
  • a selective epoxide hydrolase inhibitor was used. Pharmacological inhibition was achieved by treating animals with l-(l-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy- phenyl)-urea (TUPS).
  • TUPS l-(l-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy- phenyl)-urea
  • This compound is an effective and potent inhibitor of sEH (IC50 of 5 and 3 nM for murine and human sEH, respectively) (Enayetallah, et al. (2008) J Biol Chem 283, 36592-36598 and Jones, et al.
  • TUPS was formulated in water using 1% polyethylene glycol 400 to increase solubility, and the efficacy of the compound in mice was previously reported (Enayetallah, supra).
  • concentration of TUPS in blood and tissue extracts was determined at the end of the study.
  • TUPS concentrations in blood ranged between 800-1000 ng/ml (2- 3 ⁇ TUPS) (Fig. 1C). These levels are significantly higher than the IC50 concentration determined with in vitro experiments using the recombinant murine sEH enzyme (Liu, et al. (2009) Br J Pharmacol 156, 284-296, Hwang, et al. (2007) J Med Chem 50, 3825-3840).
  • liver, epididymal fat and pancreas extracts revealed elevated levels of TUPS, with liver homogenates exhibiting highest concentrations (45 ⁇ g/g tissue or 0.12 ⁇ /g tissue) compared with epididymal fat and pancreas (3 and 10 ⁇ g/ g tissues, or 0.01 and 0.026 ⁇ / g tissue, respectively; Fig. ID).
  • inhibition of the epoxide hydrolase domain so far has been shown to have little, if any, affect its phosphatase activity.
  • mice were placed on chow or HFD, and body weights and food intake were measured weekly. As expected, mice on a HFD gained significantly more weight than their counterparts on chow diet (Fig. 2). However, mice treated with sEHI gained significantly more weight than their counterparts on chow diet (Fig. 2A). Increased weight appears to be caused, at least in part, by increased food intake in these mice (Fig. 3A), and is associated with elevated levels of the hormone leptin (Table 2).
  • pancreas sections Fig. 5 E
  • Larger pancreatic ⁇ -islets were observed in HFD treated Ephx2-null mice, and to a lesser extent in sEHI-treated mice (Fig. 5 E). Diet induced morphological changes were not seen in kidney and adrenal gland sections (Fig. 5 C and D), suggesting no adverse effect by TUPS treatment or disruption of the gene (Fig. 5 C and D).
  • mice sEHI-treated and KO mice exhibited lower levels of serum insulin levels on HFD compared with WT mice (Table 2). On chow diet, only KO mice showed lower levels of serum insulin (Table 2 fed conditions). When the ratio of insulin to glucose was calculated, KO mice either on chow or a HFD exhibited lower insulin to glucose ratios compared with WT counterparts. This is in line with improved insulin sensitivity. When mice were fasted, insulin to glucose ratios tended to be lower but did not reach statistical significance. As expected, the differences in these parameters are blunted when mice were fasting for 16 hours.
  • mice on HFD exhibited higher plasma leptin levels compared with those on chow diet.
  • significantly higher levels of leptin are detected in the sEHI -treated WT mice (Table 2). Since high secretion of leptin from the brain increases appetite, leptin is suggested to be a cause of the weight difference in sEHI-treated WT mice on chow diet.
  • mice were subjected to insulin tolerance tests (ITTs) at two (Fig. 6 A-D) and five months (Fig. 7) after the experiment started.
  • ITTs insulin tolerance tests
  • KO mice exhibited significantly greater reduction in blood glucose following insulin injection compared with controls (Fig. 7A). This effect was more pronounced in mice fed a HFD, with KO and sEHI-treated WT mice exhibiting improved insulin sensitivity compared with controls (Fig. 7B).
  • KO mice exhibited improved ability to clear glucose from the peripheral circulation during an intraperitoneal (i.p.) glucose tolerance test (GTT) both on chow and HFD (Fig. 7C and D). Additional ITT and GTT were performed on another independent cohort of mice on chow and HFD and comparable results were observed (Fig. 6 E and F).
  • mice with sEH inhibition or deletion have improved insulin sensitivity and enhanced glucose tolerance.
  • pancreas was stained for insulin and glucagon, and the size of the islets was determined.
  • KO and sEHI-treated WT mice on a HFD exhibited increase in islet size compared with WT controls (Fig. 8A and B).
  • Glucagon staining in the outer rim of the pancreatic ⁇ -islets shows no differences among all groups (Fig. 9). Quantitative data are shown in Fig. 8B, supporting the larger increase in islet size when sEH is inhibited either pharmacologically or
  • CD31 -labeled area in endocrine pancreas appeared to be enhanced particularly in the Ehpx2-null mice on HFD, and marginally enhanced in sEHI-treated WT mice (Fig.
  • VEGF is essential in the regulation of capillary network
  • mice were injected with insulin or saline (control), and the activation status of components in the insulin signaling pathway was examined in both liver (Fig. 10) and epididymal adipose (Fig. 11) extracts after HFD.
  • insulin increased IR tyrosyl phosphorylation (Yl 162/Y 1163) in both KO mice and those treated with TUPS (Fig. 10 A-B and 11 A-B).
  • the IR was basally
  • phosphorylation of IRS- 1 in KO and sEHI-treated WT mice upon insulin stimulation was higher than the WT controls, but not significantly different than 0' time point (Fig. 10C-D ). While, in adipose tissue this was more pronounced after insulin stimulation (Fig. 11 C-D). In line with that, phosphorylated-tyrosine residue 608 was higher basally and after insulin stimulation in both liver and adipose tissue of KO and EHI-treated WT mice (Fig. 10 and Fig. 11 C,E). Binding interaction between P85 and IRS-1 was greatly enhanced after insulin injection in KO liver extracts and in both KO and sEHI-treated WT adipose tissue extracts (Fig. 10 and 11 C,F).
  • Ephx2 gene deletion is sufficient to attenuate insulin resistance development in a murine -model of type 2 diabetes induced by obesity.
  • Loss of sEH activity results in enhanced insulin-sensitizing actions, marked increase in insulin receptor signaling that stabilizes serum glucose levels.
  • These observations were also supported with pharmacological inhibition of epoxide hydrolase activity, suggesting a therapeutic role for its substrates EETs.
  • These mediators are potent endogenous compounds with beneficial vascular actions (Larsen, et al. (2007) Trends Pharmacol Sci 28, 32-38), anti-inflammatory effects (Schmelzer, et al.
  • sEH activity limits EETs availability and hence inhibition of the enzyme has been a therapeutic approach for a variety of disease conditions. Therefore, sEH inhibition finds use in preventing progression of type 2 diabetes induced by high fat diet.
  • sEH is ubiquitously expressed in many tissues, as well as in the pancreas, muscle and adipose. Particularly, sEH is locally expressed in pancreatic ⁇ -islet cells (Luo, et al., J Pharmacol Exp Ther. (2010) 334(2):430-8). sEH also regulates adipogenesis and its expression levels are up regulated in response to high fat diet (De Taeye, et al.
  • sEH glucose homeostasis and insulin sensitivity in obese mice
  • Ephx2-null mice which have no expression of either the epoxide hydrolase or phosphatase domain.
  • Pharmacological inhibition of sEH was also employed, e.g., by using selective sEH inhibitor, TUPS.
  • This drug is a potent inhibitor of the epoxide hydrolase in vitro, and has no apparent effect on the phosphatase activity of the protein.
  • TUPS was used to confirm that the effects observed with Ephx2-null mice in the diabetes model were largely due to the absence of the epoxide hydrolase activity. Since this drug has high bioavailability and a long half life (Liu, et al.
  • Adipocytes can contribute to the maintenance of whole body glucose homeostasis either by the release of insulin-sensitizing adipose-derived hormones (adipokines) or through the sequestering of excess fatty acids and triglycerides that induce insulin resistance (Tontonoz and Spiegelman (2008) Ann Rev Biochem 77, 289-312; Rader (2007) Am J Med 120, S 12-18).
  • adipokines insulin-sensitizing adipose-derived hormones
  • We study reveals that sEH deficiency and inhibition improves insulin sensitivity on HFD.
  • Our biochemical data support the notion that improved whole-body glucose homeostasis and insulin sensitivity in sEH null and sEHI -treated mice is the direct result of increased insulin signaling.
  • pancreatic ⁇ -islets can respond in vivo to a sustained glucose stimulus by increasing their mass through hyperplasia or hypertrophy.
  • endothelial cells can affect the ability of pancreatic ⁇ -islets to grow in size when demands for insulin increase (Lammert, et al. (2003) Curr Biol 13, 1070-1074; Nikolova, et al. (2006) Developmental Cell 10, 397-405).
  • a previous study with sEH-null mice in a type 1 diabetes model suggests an increase insulin secretion via reduced apoptotic cell death in ⁇ -islets (Luo, et al, J Pharmacol Exp Ther. (2010) 334(2):430-8), and the link between CYP2C-derived EETs, VEGF and endothelial proliferation (Webler, et al. (2008) Am. J. Ajszo/ 295, C1292-1301; and Potente, et a/. (2002) J Biol Chem 277 ' , 15671-15676) can also contribute to the large islet size as seen in this study.

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Abstract

The present invention provides methods of reducing, preventing, inhibiting symptoms of diabetes, e.g., hyperglycemia, by administration of an agent that increases EETs, alone or in combination with an agent that inhibits cyclo-oxygenase and/or 5-lipoxygenase pathways. The agents can be co-administered in doses that are therapeutic, subtherapeutic or non therapeutic for the individual agents.

Description

METHODS OF TREATING DIABETES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S.
Provisional Application No. 61/265,250, filed on November 30, 2009, the entire disclosure of which is hereby incorporated herein by reference for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0002] This invention was made with government support under Grant Numbers R37 ES02710 and R01 ES013933 awarded by the National Institute on
Environmental Health Sciences, Grant Number P42 ES04699 awarded by the National Institute on Environmental Health Sciences Superfund Basic Research Program, and Grant UL1 RR024146 awarded by the National Center for Medical Rehabilitation Research (NCMRR). The government has certain rights in the invention.
FIELD OF THE INVENTION
[0003] The present invention provides methods of reducing, preventing, inhibiting symptoms and/or the progression of diabetes, e.g., hyperglycemia, by coadministration of an agent that increases EETs in combination with an agent that inhibits cyclo-oxygenase and/or the 5 -lipoxygenase pathway (this includes inhibitors of 5-Lox, FLAP (fatty acid activating protein) inhibitors, and agonists of leukotriene receptors). The agents can be co-administered in doses that are therapeutic, subtherapeutic or non-therapeutic for the individual agents.
BACKGROUND OF THE INVENTION
[0004] In 2003, the International Diabetes Federation estimated that there were 194 million people worldwide with diabetes. Of these, some 16 million were estimated to be in the United States. Many diabetes sufferers undergo a slow deterioration of the kidneys, a process known as nephropathy. The end stage of nephropathy is kidney failure, or end stage renal disease. Nephropathy and kidney failure can result even when diabetes is controlled with drugs and exercise. According to the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health, diabetes is the most common cause of kidney failure and is responsible for about 40% of the 100,000 cases of kidney failure that develop annually in the U.S.. Given the $20 billion annual cost of treating kidney failure in the U.S. alone, reducing nephropathy and kidney failure could significantly reduce the costs of treating this complication of diabetes.
[0005] Obesity, a chronic inflammatory condition, is an increasingly important public health issue reaching epidemic proportions (Smyth and Heron, (2006) Nat Med 12, 75-80). Obese individuals exhibit a higher risk of chronic diseases including cardiovascular disease and type 2 diabetes. The later is a complex, polygenic disease wherein a number of tissues are rendered insulin resistant (Biddinger and Kahn (2006) Annu Rev Physiol 68, 123-158). Insulin action is mediated by a complex network of signaling events (Lizcano and Alessi (2002) Curr Biol 12, R236-238) that modulate glucose homeostasis and hence regulate energy balance. A fundamental mechanism for the maintenance of glucose homeostasis is the rapid action of insulin to stimulate glucose uptake and metabolism in peripheral tissues. This cascade initiates by binding of insulin to its cell surface receptor, followed by receptor
autophosphorylation, and activation of receptor tyrosine kinases, which result in tyrosine phosphorylation of insulin receptor substrates (IRSs). Phosphatidylinositol 3-kinase (PI3K) activity is stimulated by its binding to IRS proteins, leading to activation of downstream targets such as protein kinase B/AKT (Luo and Cantley (2005) Cell Cycle 4, 1309-1312 and Avruch (1998) Mol Cell Biochem 182, 31-48). This will stimulate the translocation of insulin-mediated glucose transporter from intracellular vesicles to the plasma membrane and will initiate its uptake to peripheral tissues (Lizcano and Alessi, supra). The mechanism underlying insulin resistance in type 2 diabetes contains many signaling players (Shulman (2000) J Clin Invest 106, 171-176 and Kobayashi (2005) Current drug targets 6, 525-529) resulting from pancreatic β-cell insufficiency with impairment of glucose-stimulated insulin secretion (Zhang, et al. (2001) Cell 105, 745-755) and destructed insulin receptor signaling (Taniguchi, et al. (2006) Nature reviews 7, 85-96).
[0006] Soluble epoxide hydrolase (sEH), a therapeutic target for several disease models of hypertension and inflammation is also suggested to play a role in insulin resistance. Polymorphism of the sEH gene is associated with insulin resistance in type 2 diabetic patients (Ohtoshi, et al. (2005) Biochem Biophys Res Commun 331, 347-350). sEH is an enzyme that adds water to epoxides, forming their corresponding 1 ,2-diols (Newman, et al. (2005) Prog Lipid Res 44, 1-51). While, the enzyme possesses two functional domains i.e. a C-terminal epoxide hydrolase and an N- terminal lipid phosphatase, little is known about the identity and role of the phosphatase substrate(s) (Newman, et al. (2003) Proc Natl Acad Sci USA 100, 1558-1563 and Cronin, et al. (2003) Proc Natl Acad Sci USA 100, 1552-1557). Eicosatrienoic acids (EETs) are among the best substrates for the epoxide hydrolase domain. EETs, which are derived from arachidonic acid by epoxygenation through cytochrome P450 monooxygenases (CYP 2C), mediate endothelium-dependent vasodilation, promote angiogenesis, and have anti-inflammatory properties (Larsen, et al. (2007) Trends Pharmacol Sci 28, 32-38). Increased levels of sEH results in rapid metabolism of EET (regioisomers 5,6-, 8,9-, 11,12- and 14,15-EET) to
dihydroxy eicosatrienoic acids (DHETs). EETs are generally more bioactive products, their vicinal diols are less bioactive and rapidly cleared from the body.
[0007] In addition to their salutary effects in the vasculature, EETs might have beneficial effects on lipid metabolism and insulin sensitivity. sEH protein and message levels are upregulated in the epididymal fat pad from mice that received a high fat diet (HFD) (De Taeye, et al. Obesity (2010) 18(3):489-98). In Addition, CYP 2C expression is decreased and sEH expression is increased in obese Zucker rats, a commonly used animal model of obesity and insulin resistance (Luo, et al., J Pharmacol Exp Ther. (2010) 334(2):430-8). In type 1 diabetes model, genetic and pharmacological inhibition of sEH results in increased insulin secretion and attenuation of hyperglycemia (Luo, et al., supra). sEH inhibition is a well established approach in cardiovascular, renal and inflammatory diseases in murine models, but its contribution in type 2 diabetes mellitus remains to be established. This study was designed to investigate the consequences of sEH-gene deletion on systemic insulin sensitivity and glucose homeostasis, and to support its mechanism of action using a pharmacological approach.
[0008] Epoxide hydrolases ("EH," EC 3.3.2.3) are a family of enzymes which hydrolyze a variety of exogenous and endogenous epoxides to their corresponding diols. Epoxide hydrolases have been found in tissues of all mammalian species tested. The highest levels of the enzyme were found in liver and kidney cells (see Wixtrom and Hammock, Pharmacology and Toxicology (Zakim, D. and Vessey, D. A., ed.) 1 :1-93, Wiley, New York, 1985).
[0009] Four principal EH's are known: leukotriene epoxide hydrolase, cholesterol epoxide hydrolase, microsomal EH ("mEH"), and soluble EH ("sEH," previously called cytosolic EH). The leukotriene EH acts on leukotriene A4, whereas the cholesterol EH hydrates compounds related to the 5,6-epoxide of cholesterol (Nashed, N. T., et al, Arch. Biochem. Biophysics., 241 : 149-162, 1985; Finley, B. and B. D. Hammock, Biochem. Pharmacol, 37:3169-3175,1988).
[0010] The microsomal epoxide hydrolase metabolizes monosubstituted, 1,1- disubstituted, cz's- 1,2-disubstituted epoxides and epoxides on cyclic systems epoxides to their corresponding diols. Because of its broad substrate specificity, this enzyme is thought to play a significant role in ameliorating epoxide toxicity. Reactions of detoxification typically decrease the hydrophobicity of a compound, resulting in a more polar and thereby excretable substance.
[0011] Soluble EH is only very distantly related to mEH and hydrates a wide range of epoxides not on cyclic systems. In contrast to the role played in the degradation of potential toxic epoxides by mEH, sEH is believed to play a role in the formation or degradation of endogenous chemical mediators. For instance, cytochrome P450 epoxygenase catalyzes NADPH-dependent enatioselective epoxidation of arachidonic acid to four optically active czs-epoxyeicosantrienoic acids ("EETs") (Karara, A., et al., J. Biol. Chem., 264: 19822-19877, (1989)). Soluble epoxide hydrolase has been shown in vivo to convert these compounds with regio- and enantiofacial specificity to the corresponding vzc-dihydroxyeicosatrienoic acids ("DHETs"). Both liver and lung cytosolic fraction hydrolyzed 14,15-EET, 8,9-EET and 11,12-EET, in that order of preference. The 5,6-EET is hydrolyzed more slowly. Purified sEH selected 8S,9R- and 14R,15S-EET over their enantiomers as substrates. Studies have revealed that EETs and their corresponding DHETs exhibit a wide range of biological activities as do their corresponding ω-3 lipid homo logs. Some of these activities include involvements in luteinizing hormone -releasing hormone, stimulation of luteinizing hormone release, inhibition of Na+/K+ ATPase, vasodilation of coronary artery, mobilization of Ca2+ and inhibition of platelet aggregation. Soluble epoxide hydrolase is believed to play a role in these biological activities by contributing to the regulation of the steady state levels of EETs and DHETs as well as other biologically active epoxides and diols.
BRIEF SUMMARY OF THE INVENTION
[0012] The present invention provides methods of maintaining and promoting glucose tolerance, and methods of reducing or inhibiting hyperglycemia, for example, postprandial hyperglycemia, by co-administering an agent that promotes increased levels of EETs in combination with an agent that inhibits cyclo-oxygenase or 5- lipoxygenase pathways. The methods help retard the progression of diabetes in an individual in need thereof.
[0013] Accordingly, in one aspect, the invention provides methods of maintaining stable glucose levels in an individual in need thereof. In some embodiments, the methods comprise co-administering to the individual
a) (i) an effective amount of a first enzyme inhibitor that inhibits sEH, (ii) an epoxygenated fatty acid, (iii) both an inhibitor of sEH and an epoxygenated fatty acid, or (iv) a mimic of an epoxygenated fatty acid which is stable to epoxide hydrolase, and
b) an effective amount of a second enzyme inhibitor that inhibits one or more enzymes selected from the group consisting of cyclo-oxygenase ("COX") -1, COX-2, and 5 -lipoxygenase ("5 LOX" or other enzymes and mediators in the LOX 5 pathway). Glucose levels can be measured in the blood, plasma or serum, as appropriate.
[0014] In a related aspect, the invention provides methods of maintaining and promoting glucose tolerance, methods of maintaining stable glucose levels, and methods of reducing or inhibiting hyperglycemia, for example, postprandial hyperglycemia, in an individual in need thereof by administering an agent that promotes increased levels of EETs, e.g., (i) an effective amount of a first enzyme inhibitor that inhibits sEH, (ii) an epoxygenated fatty acid, (iii) both an inhibitor of sEH and an epoxygenated fatty acid, or (iv) a mimic of an epoxygenated fatty acid which is stable to epoxide hydrolase. [0015] In some embodiments, the individual is hyperglycemic. In some
embodiments, the individual has type 1 diabetes. In some embodiments, the individual has type 2 diabetes. In some embodiments, the individual is prediabetic.
[0016] In some embodiments, the individual is (a) a person with diabetes mellitus whose blood pressure is 130/80 or less, (b) a person with metabolic syndrome whose blood pressure is less than 130/85, (c) a person with a triglyceride level over 215 mg/dL, or (d) a person with a cholesterol level over 200 mg/dL.
[0017] In a further aspect, the invention provides methods for maintaining stable glucose levels, improving insulin sensitivity and accelerating glucose clearance in a prediabetic subject, the method comprising administering to the subject an effective amount of an inhibitor that inhibits sEH, (ii) an epoxygenated fatty acid, (iii) both an inhibitor of sEH and an epoxygenated fatty acid, or (iv) a mimic of an epoxygenated fatty acid which is stable to epoxide hydrolase. In some embodiments, the methods further comprise co-administering an effective amount of a second enzyme inhibitor that inhibits one or more enzymes selected from the group consisting of cyclo- oxygenase ("COX") -1, COX-2, and 5 -lipoxygenase ("5 LOX" or other enzymes and mediators in the LOX 5 pathway).
[0018] In some embodiments, one or both of the first and second enzyme inhibitors are administered at subtherapeutic doses. In some embodiments, one or both of the first and second enzyme inhibitors are administered at non-therapeutic doses. In some embodiments, one or both of the first and second enzyme inhibitors are administered at therapeutic doses.
[0019] In some embodiments, one or both of the first and second enzyme inhibitors are administered in a sustained release or a controlled release formulation. In some embodiments, one or both of the first and second enzyme inhibitors are administered concurrently with or within 1 hour of a meal.
[0020] In some embodiments, the epoxygenated fatty acid is an EET. In some embodiments, the EET is selected from the group consisting of 14,15-EET, 8,9-EET, 11,12-EET and 5,6-EET. In some embodiments, the epoxygenated fatty acid is an epoxide of docosahexaenoic acid ("DHA") or eicosapentaenoic acid ("EPA"), or epoxides of both DHA and of EPA. [0021] In some embodiments, the first enzyme inhibitor has a primary
pharmacophore selected from the group consisting of a urea, a carbamate and an amide.
[0022] In some embodiments, the second enzyme inhibitor is an inhibitor of COX- 2. In some embodiments, the second enzyme inhibitor is a selective inhibitor of COX-2. In some embodiments, the second enzyme inhibitor is selected from the group consisting of celecoxib, valdecoxib, lumiracoxib, etoricoxib, and rofecoxib.
[0023] In some embodiments, the second enzyme inhibitor is an inhibitor of COX- 1. In some embodiments, the second enzyme inhibitor is selected from the group consisting of aspirin, acetaminophen, diclofenac potassium, diclofenac sodium, disalsalate. diclofenac sodium with misoprostol, diflunisal, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate sodium, mefenamic acid, meloxicam, nabumetone, naproxen sodium, piroxicam, tolmetin sodium, magnesium salicylate, choline salicylate, salicylic acid esters, salsalate, and sodium salicylate.
[0024] In some embodiments, the second enzyme inhibitor is an inhibitor of 5- LOX. In some embodiments, the second enzyme inhibitor is a FLAP inhibitor or a leukotriene antagonist.
DEFINITIONS
[0025] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. Terms not defined herein have their ordinary meaning as understood by a person of skill in the art.
[0026] The terms "prediabetes" and "prediabetic" interchangeably refer to a condition that involves impaired glucose tolerance (IGT) or impaired fasting glucose (IFG). IGT is defined by a 2-h oral glucose tolerance test plasma glucose concentration >140 mg/dL (7.8 mmol/L) but <200 mg/dL (11.1 mmol/L), and IFG is defined by a fasting plasma glucose concentration >100 mg/dL (5.6 mmol/L), but <126 mg/dL (7.0 mmol/L). See, e.g., Pour and Dagogo-Jack, Clin Chem. (2010) Nov 9., PMID 21062906.
[0027] "cz's-Epoxyeicosatrienoic acids" ("EETs") are biomediators synthesized by cytochrome P450 epoxygenases. As discussed further in a separate section below, while the use of unmodified EETs is the most preferred, derivatives of EETs, such as amides and esters (both natural and synthetic), EETs analogs, EETs homologs, stable EET mimics and EETs optical isomers can all be used in the methods of the invention, both in pure form and as mixtures of these forms. For convenience of reference, the term "EETs" as used herein refers to all of these forms unless otherwise required by context.
[0028] "Epoxide hydrolases" ("ΕΗ;" EC 3.3.2.3) are enzymes in the alpha beta hydrolase fold family that add water to 3-membered cyclic ethers termed epoxides. The addition of water to the epoxides results in the corresponding 1 ,2-diols
(Hammock, B. D. et al., in Comprehensive Toxicology: Biotransformation (Elsevier, New York), pp. 283-305 (1997); Oesch, F. Xenobiotica 3:305-340 (1972)). Four principal EH's are known: leukotriene epoxide hydrolase, cholesterol epoxide hydrolase, microsomal EH ("mEH"), and soluble EH ("sEH," previously called cytosolic EH). The leukotriene EH acts on leukotriene A4, whereas the cholesterol EH hydrates compounds related to the 5,6-epoxide of cholesterol. The microsomal epoxide hydrolase metabolizes monosubstituted, 1,1-disubstituted, cis-1,2- disubstituted epoxides and epoxides on cyclic systems to their corresponding diols. Because of its broad substrate specificity, this enzyme is thought to play a significant role in ameliorating epoxide toxicity. Reactions of detoxification typically decrease the hydrophobicity of a compound, resulting in a more polar and thereby excretable substance.
[0029] "Soluble epoxide hydrolase" ("sEH") is an epoxide hydrolase which in many cell types converts EETs to dihydroxy derivatives called dihydroxyeicosatrienoic acids ("DHETs"). The cloning and sequence of the murine sEH is set forth in Grant et al., J. Biol. Chem. 268(23): 17628-17633 (1993). The cloning, sequence, and accession numbers of the human sEH sequence are set forth in Beetham et al., Arch. Biochem. Biophys. 305(1): 197-201 (1993). NCBI Entrez Nucleotide accession number L05779 sets forth the nucleic acid sequence encoding the protein, as well as the 5' untranslated region and the 3' untranslated region. The evolution and nomenclature of the gene is discussed in Beetham et al, DNA Cell Biol. 14(1):61-71 (1995). Soluble epoxide hydrolase represents a single highly conserved gene product with over 90% homology between rodent and human (Arand et al., FEBS Lett., 338:251-256 (1994)). Soluble EH is only very distantly related to mEH and hydrates a wide range of epoxides not on cyclic systems. In contrast to the role played in the degradation of potential toxic epoxides by mEH, sEH is believed to play a role in the formation or degradation of endogenous chemical mediators. Unless otherwise specified, as used herein, the terms "soluble epoxide hydrolase" and "sEH" refer to human sEH.
[0030] Unless otherwise specified, as used herein, the terms "sEH inhibitor" (also abbreviated as "sEHI") or "inhibitor of sEH" refer to an inhibitor of human sEH. Preferably, the inhibitor does not also inhibit the activity of microsomal epoxide hydrolase by more than 25% at concentrations at which the inhibitor inhibits sEH by at least 50%, and more preferably does not inhibit mEH by more than 10% at that concentration. For convenience of reference, unless otherwise required by context, the term "sEH inhibitor" as used herein encompasses prodrugs which are metabolized to active inhibitors of sEH. Further for convenience of reference, and except as otherwise required by context, reference herein to a compound as an inhibitor of sEH includes reference to derivatives of that compound (such as an ester of that compound) that retain activity as an sEH inhibitor.
[0031] "COX" is an abbreviation for "cyclo-oxygenase." Several COX enzymes have been identified. Two isozymes, COX-1 and COX-2, are recognized as of clinical significance, with COX-1 considered to be constitutively expressed and COX- 2 considered to be inducible and more prevalent at sites of inflammation. See, e.g., Hawkey, Best Pract Res Clin Gastroenterol. 15(5):801-20 (2001).
[0032] As used herein, a "COX-1 inhibitor" denotes an agent that inhibits COX-1 more than it inhibits COX-2, while a "COX-2 inhibitor" denotes an agent that inhibits COX-2 more than it inhibits COX-1. All current non-steroidal anti-inflammatory drugs (NSAIDs) inhibit both COX-1 and COX-2, but most tend to inhibit the two isoforms to different degrees. Since both enzymes tend to be inhibited together to some degree, one can consider an inhibitor of either enzyme to be "COX inhibitor".
[0033] "LOX" is an abbreviation for "lipoxygenase." Several LOX enzymes have been identified. Arachidonate 5 -lipoxygenase ("5-LOX", EC 1.13.11.34) is involved in the production of pro-inflammatory mediators. Arachidonate 12-lipoxygenase ("12-LOX", EC 1.13.11.31) and arachidonate 15 -lipoxygenase ("15-LOX", EC 1.13.11.33) form trihydroxytetraenes known as "lipoxins" ("lipoxygenase interaction products") from arachidonic acid. Lipoxins act as local anti-inflammatory agents.
[0034] "5 -lipoxygenase activating protein," or "FLAP," is a protein required before 5-LOX can become catalytically active. Inhibiting FLAP activity reduces or prevents 5-LOX activation, decreasing the biosynthesis of leukotrienes.
[0035] Cytochrome P450 ("CYP450") metabolism produces cis- epoxydocosapentaenoic acids ("EpDPEs") and cz's-epoxyeicosatetraenoic acids ("EpETEs") from docosahexaenoic acid ("DHA") and eicosapentaenoic acid ("EPA"), respectively. These epoxides are known endothelium-derived hyperpolarizing factors ("EDHFs"). These EDHFs, and others yet unidentified, are mediators released from vascular endothelial cells in response to acetylcholine and bradykinin, and are distinct from the NOS- (nitric oxide) and COX-derived (prostacyclin) vasodilators. Overall cytochrome P450 (CYP450) metabolism of polyunsaturated fatty acids produces epoxides, such as EETs,which are prime candidates for the active mediator(s).
14(15)-EpETE, for example, is derived via epoxidation of the 14,15-double bond of EPA and is the ω-3 homolog of 14(15)-EpETrE ("14(15)EET") derived via epoxidation of the 14,15-double bond of arachidonic acid.
[0036] "IC50" refers to the concentration of an agent required to inhibit enzyme activity by 50%.
[0037] By "physiological conditions" is meant an extracellular milieu having conditions (e.g., temperature, pH, and osmolality) which allows for the sustenance or growth of a cell of interest.
[0038] "Micro-R A" ("miR A") refers to small, noncoding RNAs of 18-25 nt in length that negatively regulate their complementary mRNAs at the posttranscriptional level in many eukaryotic organisms. See, e.g., Kurihara and Watanabe, Proc Natl Acad Sci USA 101(34): 12753-12758 (2004). Micro-RNAs were first discovered in the roundworm C. elegans in the early 1990s and are now known in many species, including humans. As used herein, it refers to exogenously administered miRNA unless specifically noted or otherwise required by context.
[0039] The term "co-administration" refers to the presence of both active agents in the blood at the same time. Active agents that are co-administered can be delivered concurrently (i.e., at the same time) or sequentially.
[0040] The terms "patient," "subject" or "individual" interchangeably refers to a mammal, for example, a human or a non-human mammal, including primates (e.g., macaque, pan troglodyte, pongo), a domesticated mammal (e.g., felines, canines), an agricultural mammal (e.g., bovine, ovine, porcine, equine) and a laboratory mammal or rodent (e.g., rattus, murine, lagomorpha, hamster).
[0041] The terms "reduce," "inhibit," "relieve," "alleviate" refer to the detectable decrease in symptoms of diabetes, as determined by a trained clinical observer. A reduction in symptoms of diabetes, or prognosis of diabetic condition, can be measured using any test known in the art, including without limitation, decreased blood or plasma glucose levels, increased blood or plasma insulin levels, increased C- peptide levels, increased beta cell function.
[0042] The term "therapeutically effective amount" refers to an amount of the compound being administered sufficient to prevent or decrease the development of one or more of the symptoms of the disease, condition or disorder being treated.
[0043] The term "subtherapeutic amount" or "non-therapeutic amount" refers to an amount of the individual compound being administered that is insufficient to prevent or decrease the development of one or more of the symptoms of the disease, condition or disorder being treated. Subtherapeutic or non-therapeutic doses of individual agents are inefficacious for their intended purpose.
[0044] The term "analgesic amount" refers to that amount of the compound being administered sufficient to prevent or decrease pain in a subject under treatment.
[0045] The terms "controlled release," "sustained release," "extended release," and "timed release" are intended to refer interchangeably to any drug-containing formulation in which release of the drug is not immediate, i.e., with a "controlled release" formulation, oral administration does not result in immediate release of the drug into an absorption pool. The terms are used interchangeably with
"nonimmediate release" as defined in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippencott Williams & Wilkins (2006). As discussed therein, immediate and nonimmediate release can be defined kinetically by reference to the following equation:
Dosage Absorption -a
· Target ■ Form drug Pool absorption Area elimination release
[0046] The "absorption pool" represents a solution of the drug administered at a particular absorption site, and kr, ka and ke are first-order rate constants for (1) release of the drug from the formulation, (2) absorption, and (3) elimination, respectively. For immediate release dosage forms, the rate constant for drug release kr is far greater than the absorption rate constant ka. For controlled release formulations, the opposite is true, i.e., kr «ka, such that the rate of release of drug from the dosage form is the rate-limiting step in the delivery of the drug to the target area.
[0047] The terms "sustained release" and "extended release" are used in their conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, for example, 12 hours or more, and that preferably, although not necessarily, results in substantially steady-state blood levels of a drug over an extended time period.
[0048] As used herein, the term "delayed release" refers to a pharmaceutical preparation that passes through the stomach intact and dissolves in the small intestine.
[0049] As used herein, "synergy" or "synergistic" interchangeably refer to the combined effects of two active agents that are greater than their additive effects. Synergy can also be achieved by producing an efficacious effect with combined inefficacious doses of two active agents. The measure of synergy is independent of statistical significance.
[0050] As used herein, the phrase "consisting essentially of refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any excipients inactive for the intended purpose of the methods or compositions. In some embodiments, the phrase "consisting essentially of expressly excludes the inclusion of one or more additional active agents other than the listed active agents, e.g., (i) an inhibitor of sEHi and/or an EET alone or in combination with (ii) an inhibitor of cyclo-oxygenase and/or 5-lipoxygenase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] Figure 1A-D illustrate whole-body Ephx2 gene deletion and sEH inhibition.
(A) Representative genotypic analysis using genomic DNA from murine tails. PCR product of sEH sequence recognizes a 290-bp gene product in WT animals, while a gene product of the neomycin resistant gene (560-bp) is shown in Ephx2-null mice.
(B) Immunoblots of sEH in different tissues from WT, Ephx2-null (KO) mice and sEHI-treated WT mice. Tubulin expression is shown as a control for loading. Note that compared with control mice, sEH protein expression was ablated in tissues of KO mice, whereas sEH expression was unaffected in sEHI-treated mice. (C) Plasma and (D) tissue concentrations of l-(l-methylsulfonyl-piperidin-4-yl)-3-(4- trifluoromethoxy-phenyl)-urea ("TUPS") from WT mice that are untreated (control) and treated with TUPS (n= 4). Values depict mean ± SEM of n=4. * P < 0.05, *** P < 0.001 t-test of WT untreated vs. WT treated with TUPS.
[0052] Figures 2A-B illustrate body weights of WT and Ephx2-null mice fed chow or high fat diet. Body weight curves of age-matched wild type (WT), KO and WT mice treated with a selective sEH inhibitor (TUPS, 10 mg/L via drinking water) fed regular chow diet (CHOW) (A) or a high fat diet (HFD) (B) for 20 weeks post weaning. Values depict mean ± SEM of n=4. t-test, * P < 0.05, WT-chow vs. WT- chow+sEHI.
[0053] Figures 3A-C illustrate food intake and feeding efficiency. Weekly measurements of food intake in Ephx2-null and WT male mice treated with or without TUPS (sEHI, 10 mg/L via drinking water in 1% PEG 400) during all study either on chow (A) or HFD (B). (C) Feeding efficiency in WT, sEHI -treated WT and Ephx2-null mice. Values depict mean ± SEM of n=4. t-test, * P < 0.05 WT vs.
WT+sEHI; # P < 0.05 WT vs. KO; & P < 0.05 WT+sEHI vs. KO. [0054] Figure 4 illustrates organ mass analysis. Organ mass in WT treated with or without TUPS and Ephx2-null mice fed either chow or HF diets. Data (AVG±SEM) presented as organ to body weight ratio (n=4). BAT, brown adipose tissue; SUBQ, subcutaneous fat, Retro, retroperitoneal fat. t-test, * P < 0.05, WT vs. WT+sEHI; #P < 0.05, WT vs. KO; & P < 0.05 WT+sEHI vs. KO on either diet. Significant differences between diets (t-test), ΛΡ < 0.05, WT HFD vs. chow; n P < 0.05, WT+sEHI HFD vs. chow; § P < 0.05, KO HFD vs. chow.
[0055] Figures 5A-E illustrate morphological appearance of various tissue sections from WT, Ephx2-null and sEHI-treated WT mice. Representative histological appearance of hematoxylin-Eosin-stained sections from epididymal fat (A), liver (B), kidney (C), adrenal gland (D) and pancreas (E) sections taken from mice either on chow or high fat diets. Tissue collected at the end of the study from WT, sEHI- treated WT and Ephx2-null (KO) mice, weight, fixed and sectioned as described herein, showing a clear effect of the diet on cells size and fat accumulation. Other tissues appear normal. Note a marked increase in cytoplasmic vacuolization of hepatocytes, which is thought to be fatty degeneration along with nuclei displaced to the periphery were observed microscopically in mice fed a HFD compared with mice fed chow diet. The epididymal fat pads were visibly larger in mice fed a HFD, and adipocytes were also enlarged. While, histological analysis of kidney and adrenal gland shows no apparent differences among genotypes and diets. While, clear differences in pancreatic β-islet size is observed in sEHI -treated WT and Ephx2-null mice.
[0056] Figures 6A-B illustrate reduction of sEH activity reduces diet-induced insulin resistance. Insulin tolerance test (ITT) and glucose tolerance test (GTT) were performed on WT, sEHI-treated WT and KO mice fed either chow diet (A, C) or HFD (B, D) for two months. E and F, Second cohort study with mice that started the diet at the age of 12 weeks. (E) ITT and (F) GTT in WT and Ephx2-null mice fed HFD or chow for 22 weeks (diet starts when mice were twelve weeks old). A, B and E, insulin test performed by injecting insulin (lmU/g; i.p.) to mice that were fasted four hours prior to ITT. At indicated times, blood samples (n=3) were taken from each animal and glucose was measured via Glucometer. C, D and F, glucose test performed by injecting glucose (2mg/g of body mass; i.p.) to mice that were fasted 16 hours prior to GTT. At indicated times, blood samples (n=3) were taken from each animal and glucose was measured via Glucometer. Note lower blood glucose levels in both sEHI- treated WT and KO mice fed HFD at zero time. Values are mean ± SEM of n=4. * P < 0.05, WT vs. WT plus sEH inhibitor (sEHI); #P < 0.05, ##P < 0.01, WT vs. KO on either diets.
[0057] Figures 7A-D illustrate reduction of sEH activity prevents diet-induced insulin resistance and sEH-gene deletion improves glucose homeostasis. Insulin tolerance test (ITT) and glucose tolerance test (GTT) were performed on WT, Ephx2- null (KO) mice and sEHI-treated WT male mice fed either chow diet (A, C) or HFD (B, D) for five months. A and B, insulin test performed by injecting insulin (ImU/g) intraperitoneally to mice that were fasted four hours prior to ITT. At indicated times, blood samples (n=3) were taken from each animal and glucose was measured via Glucometer. C and D, glucose test performed by injecting glucose (2mg/g of body mass) intraperitoneally to mice that were fasted 16 hours prior to GTT. At indicated times, blood samples (n=3) were taken from each animal and glucose was measured via Glucometer. Note lower blood glucose levels in both WT with TUPS and KO mice fed HFD at zero time. Values are mean ± SEM of n=4. *P < 0.05, **P < 0.01 WT vs. WT+sEHI; #P < 0.05, ##P < 0.01, WT vs. KO; & P < 0.05, && P < 0.01 WT+sEHI vs. KO on either diets.
[0058] Figures 8A-C illustrate β-islet size and vascularization density in response to Ephx2-gene deletion and inhibition. (A) Representative insulin staining in pancreas sections from WT, sEHI-treated WT and Ephx2-null mice at the end of study either on chow or high-fat diets. Pancreata were stained
immunohistochemically for insulin (Red). Bar represents 200 μιη.
(B) Measurements of β-islet size as measured by the size of the stained area under the same magnification. * indicates significant difference (t-test) between WT vs. WT +sEHI or WT vs. KO. (C) Endothelial cell marker (CD31) staining in pancreatic sections from WT, sEHI-treated WT and Ephx2-null mice on chow or HFD. CD31 immunolabeled with specific antibodies (green) represents vascular density in the islets (e.g. inside white boundary). Vascular density was significantly enhanced in the islets of sEH-null mice and sEHI-treated WT mice on HFD. Nuclei were stained by DAPI (blue). [0059] Figures 9A-F illustrate immunofluorescence detection of insulin and glucagon in pancreatic β-islets. Representative pancreatic section showing
immunofluorescence staining of β-islets from WT, sEHI-treated WT and Ephx2-null mice at the end of study fed either chow or high-fat diets. Pancreata were stained using antibodies for insulin (Red), glucagon (green) and overlay (merge). Nuclei were stained by DAPI (blue). Bar represents 200 μιη.
[0060] Figures 10 A- J illustrate enhanced insulin signaling in mice with Ephx2- gene deletion or sEH inhibition. Insulin receptor signaling in liver tissues from male mice on HFD. Mice were fasted overnight then injected i.p. with saline or insulin (10 mU/g) and sacrificed after 10 minutes. Ly sates were either extracted and
immunob lotted or immunoprecipitated first with IR (A) or IRS-1 (B) antibodies and immunoblotted. Phosphorylated state was detected by using specific antibodies for pIR serine 1 162/1163 (A), anti tyrosyl phosphorylation (C) and phospho-IRS-1 Tyr608 (C). Membranes were stripped and reprobed for total protein levels of IR (A) and IRS-1 (C) to control for loading. Other proteins in the signaling pathway were used, anti Akt Ser473 phosphorylation (G), anti MAPK (Thr202/Tyr204) (I) and anti pan p85 (C). Immunoblots were quantified and bar graphs represent pooled, normalized data (arbitrary units) for KO (n= 4) and WT (n= 4) mice (B, D-F, H and J). Statistical analysis was performed using one-way AN OVA followed by post hoc Tukey's honestly significant difference (HSD) test. * indicates significant difference (P < 0.05) to WT mice injected with saline (0'), # indicates significant difference to WT mice injected with insulin (10 min), and φ indicates significant differences between WT and WT-sEHI at 0' time. **; ## or φ φ P < 0.01; *** ; ### or φφφ P < 0.001.
[0061] Figures 11A-M illustrate einsulin signaling in mice with Ephx2-gene deletion or sEH inhibition. Insulin receptor signaling in adipose tissues from male mice on HFD. Mice were fasted overnight then injected i.p. with saline or insulin (10 mU/g) and sacrificed after 10 minutes. Ly sates were either extracted and
immunoblotted or immunoprecipitated first with IR (A) or IRS-1 (B) antibodies and immunoblotted. Phosphorylated state was detected by using specific antibodies for pIR serine 1162/1163 (A) , anti tyrosyl phosphorylation (C) and phospho-IRS-1 Tyr608 (C). Membranes were stripped and reprobed for total protein levels of IR (A) and IRS-1 (C) to control for loading. Other proteins in the signaling pathway were used, anti Akt Ser473 phosphorylation (G), anti MAPK (Thr202/Tyr204) (I), anti pan p85 (C) and inflammatory mediators such as, TNFa and MCP1 (K-M).
Immunoblots were quantified and bar graphs represent pooled, normalized data (arbitrary units) for KO (n= 4) and WT (n= 4) mice (B, D-F, H, J and L-M).
Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's honestly significant difference (HSD) test. * indicates significant difference (P < 0.05) to WT mice injected with saline (0'), # indicates significant difference to WT mice injected with insulin (10 min), and φ indicates significant differences between WT and WT-sEHI at 0' time. **; ## or φ φ P < 0.01; *** ; ### or φφφ P < 0.001.
[0062] Figure 12 illustrates blood glucose in an insulin tolerance test (ITT) performed on C57BL/6 wild-type (WT) and corresponding sEH knock-out (KO) mice after receiving either the regular diet (RD; 24% of calories from fat) or the high fat diet (HFD; 42% of calories from fat) for 4 weeks. sEH KO mice do not exhibit high blood glucose from the HFD. In all studies, 6 or more mice were in each group.
[0063] Figure 13 illustrates plasma glucose in a glucose tolerance test (GTT) performed on WT mice and corresponding sEH KO mice after receiving either the RD or the HFD for 5 weeks.
[0064] Figure 14 illustrates blood glucose in an ITT performed on WT mice fed the RD or the HFD for 7 weeks. Two groups of mice were given the sEH inhibitor 1-(1- Methanesulfonyl-piperidin-4-yl)-3-(14-trifluoromethoxy-phenyl)-urea (1709) in their drinking water. Mice fed the sEH inhibitor do not have high blood glucose from the HFD.
[0065] Figure 15 illustrates blood glucose in an ITT performed on WT mice or KO mice fed the RD or the HFD for 7 weeks. sEH KO mice do not exhibit high blood glucose from the HFD.
[0066] Figure 16 illustrates blood glucose in a GTT performed on WT mice fed the RD or the HFD for 8 weeks. Two groups of mice were given the sEH inhibitor 1709 in their drinking water.
[0067] Figure 17 illustrates blood glucose in a GTT performed on WT mice and corresponding sEH KO mice after receiving either the RD or the HFD for 8 weeks. [0068] Figure 18 illustrates blood glucose in an ITT performed on WT mice fed the regular diet (RSD) or the HFD for 5 months. Two groups of mice were given the sEH inhibitor 1709 in their drinking water. Mice fed the sEH inhibitor do not have high blood glucose from the HFD.
[0069] Figure 19 illustrates blood glucose in an ITT performed on WT mice and corresponding sEH KO mice after receiving either the RSD or the HFD for 5 months. sEH KO mice do not exhibit high blood glucose from the HFD.
[0070] Figure 20 illustrates blood glucose in an ITT performed on WT mice and corresponding sEH KO mice after receiving either the RSD or the HFD for 6 months. sEH KO mice do not exhibit high blood glucose from the HFD.
[0071] Figure 21 illustrates blood glucose in an ITT performed on WT mice and corresponding sEH KO mice after receiving either the RSD or the HFD for 6 months. sEH KO mice do not exhibit high blood glucose from the HFD.
[0072] Figure 22 illustrates plasma glucose in an GTT performed on WT mice and corresponding sEH KO mice after receiving either the RSD or the HFD for 6 months.
[0073] Figure 23 illustrates plasma glucose in an ITT performed on WT mice fed the RSD or the HFD for 7 months. Two groups of mice were given the sEH inhibitor 1709 in their drinking water. Mice fed the sEH inhibitor do not have high blood glucose from the HFD.
[0074] Figure 24 illustrates blood glucose in an GTT performed on WT mice fed the RSD or the HFD for 7 months. Two groups of mice were given the sEH inhibitor 1709 in their drinking water.
[0075] Figure 25 illustrates blood glucose in an GTT performed on WT mice fed the RSD or the HFD for 27 weeks. Two groups of mice were given the sEH inhibitor 1709 in their drinking water.
[0076] Figure 26 illustrates glucose to insulin ratios in treated mice groups, as indicated.
[0077] Figure 27A illustrates that mice treated with high fat diet for two months have significantly higher levels of plasma glucose. In Figure 27B, mice fed regular chow for two months did not have higher levels of plasma glucose. Glucose (lmg/g body weight) was administered in order to measure the glucose clearance over time (Glucose Tolerance Test). WT animals that did not receive an inhibitor of sEH (sEHi) required more time to clear the glucose (as indicated by the higher curve and larger area under the curve). WT animals administered sEH inhibitor 1709 in drinking water (10 mg/L) show a significant reduction in the plasma glucose levels, increased as a result of the high fat diet. The sEH knockout mice exhibit rapid clearance of glucose from the plasma, regardless of administration of an sEHi. The data are consistent with the conclusion that administration of an sEHi sensitizes signaling through the insulin receptor and accelerates glucose clearance.
DETAILED DESCRIPTION
1. Introduction
[0078] The present invention is based, in part, on the surprising discovery that combined administration of an agent that increases EETs and an agent that inhibits cyclo-oxygenase and/or 5 -lipoxygenase finds use in reducing, preventing and/or ameliorating symptoms associated with diabetes. By co-administrating the inhibitor of cyclo-oxygenase and/or 5 -lipoxygenase with an agent that promotes increased levels of EETs, a therapeutic, subtherapeutic or non-therapeutic dose of the COX or 5-LOX inhibitor can be administered, thereby maintaining efficacy, reducing undesirable side effects and increasing safety to the patient receiving treatment.
[0079] Visceral obesity has been defined as an important element of the metabolic syndrome and contributes to the development of insulin resistance and cardiovascular disease. The present invention is based, in part, on the discovery that increasing endogenous levels of the anti-hypertensive and anti-inflammatory mediators epoxyeicosatrienoic acids (EETs) attenuates the development of these diseases. The availability of EETs is limited primarily by the soluble epoxide hydrolase (sEH, EPHX2), which metabolizes EETs to their less active diols. To address the physiological role of EETs in regulating glucose homeostasis and insulin signaling, mice with targeted gene deletion of sEH (Ephx2-null mice) were used, followed by a subsequent study using a selective sEH inhibitor. Knockout and inhibition of sEH prevents insulin resistance developed in obese mice fed a 'Western Diet'. When wild type mice are fed a high fat diet, insulin resistance develops. Knockout of sEH activity resulted in a significant increase in insulin sensitivity. These findings are characterized by enhancement of total tyrosyl phosphorylation of the insulin receptor and insulin receptor substrate 1, and its down stream cascade. In addition, pancreatic β-islets were larger when sEH was disrupted associated with an increase in vasculature. These observations were supported by pharmacological inhibition of sEH, suggesting that the enhancement of islets size and hence improved insulin signaling and sensitivity is due to reduction of epoxide hydrolase leading to increased levels of EETs.
[0080] Unlike the cyclooxygenase and lipoxygenase metabolites,
epoxyeicosatrienoic acids and other epoxylipids decrease inflammation. In addition, epoxyeicosatrienoic acids and other epoxylipids regulate vascular tone, in part by preventing the activation of nuclear factor κΒ. Campbell, W.B. Trends Pharmacol Sci 21, 125-7 (2000); and Node, et al. Science 285, 1276-9 (1999). These molecules transcriptionally downregulate the induced cyclooxygenase-2 and lipoxygenase-5 pathways resulting in synergism with NSAIDs, aspirin and also other cascade modulators in reducing the levels of inflammatory eicosanoids. Schmelzer, et al. Proc Natl Acad Sci USA (2006) 103,13646-13651. Increased levels of
epoxyeicosatrienoic acids or epoxyeicosanoids are associated with a downregulation of prostaglandin PGE2, because they transcriptionally downregulate induction of cyclooxygenase. Thus, they could be considered to reduce adipocyte dysfunction. Inceoglu, et al. Prostaglandins Other Lipid Mediat 82, 42-9 (2007); and De Taeye, et al. Obesity (Silver Spring) (2009) doi: 10.1038/oby.2009.227.
[0081] The inhibitors of soluble epoxide hydrolases also synergize with NSAIDs. Schmelzer, supra. Nanomolar concentrations of 11,12-epoxyeicosatrienoic acid, or overexpression of CYP2J2 decreases upregulation of cell adhesion molecules, vascular adhesion molecule- 1, intercellular adhesion molecule- 1 and E-selectin induced by tumor necrosis factor, IL-l and LPS in cultured endothelial cells. This finding further validates their anti-inflammatory effects. Node, supra. These biological effects can also occur in the pancreas and in adipose tissue. Interestingly in humans, single nucleotide polymorphisms in the gene that encodes soluble epoxide hydrolase are associated with an increased risk of cardiovascular disease,( Lee, et al. Hum Mol Genet (2006) 15,1640-1649) insulin resistance (Ohtoshi, et al. Biochem Biophys Res Commun (2005) 331,347-350) and hypertension in patients with diabetes mellitus (Burdon, et al. Diab Vase Dis Res (2008) 5,128-134). [0082] A recent study has reported expression and regulation of soluble epoxide hydrolases in adipose tissue from mice fed either a normal or high-fat diet. De Taeye, et al. Obesity (Silver Spring) (2009) doi: 10.1038/oby.2009.227. Although soluble epoxide hydrolase m NA and protein levels in adipose tissue did not differ between normal and fat-fed animals, total adipose soluble epoxide hydrolases activity was increased in obese mice. De Taeye, supra. Given the involvement of soluble epoxide hydrolase in inflammation, this study suggests that mimicking or increasing epoxyeicosatrienoic acid concentrations is important in controlling obesity and the symptoms of the metabolic syndrome (e.g., hyperglycemia and diabetes).
[0083] Anti-inflammatory agents such as NSAIDs, salicylates and aspirin reduce the severity of metabolic dysfunction. Yuan, et al. Science 293, 1673-7 (2001); Renna, et al., Clin Exp Pharmacol Physiol 36, 162-8 (2009); and Van Kerckhoven, et al., Cardiovasc Res 46, 316-23 (2000). However, the very high doses necessary can lead to a variety of adverse effects ranging from gastrointestinal problems to tinnitus. Soluble epoxide hydrolase inhibitors synergize the anti-inflammatory actions of these compounds, which suggests that low doses could be used in combination to reduce the symptoms of metabolic syndrome without affecting innate immunity. Inceoglu, supra and Schmelzer, supra.
2. Conditions Subject to Treatment
[0084] The present methods find use in ameliorating symptoms in patients with type 1 or type 2 diabetes. In some embodiments, the individual is pre-diabetic (i.e., does not yet have diabetes). In some embodiments, the individual has hyperglycemia. The individual may or may not be obese. The individual may or may not have
hypertension. The individual may or may not have metabolic syndrome.
[0085] Diabetes mellitus (generally referred to herein as "diabetes") is a
heterogeneous group of metabolic disorders, connected by raised plasma glucose concentration and disturbance of glucose metabolism. It is a chronic condition characterized by the presence of fasting hyperglycemia and the development of widespread premature atherosclerosis. The hyperglycemia in diabetes mellitus generally results from defects in insulin secretion, insulin action, or both. The World Health Organization (WHO) has set forth a classification scheme for diabetes mellitus that includes type 1 diabetes mellitus, type 2 diabetes mellitus, gestational diabetes, and other specific types of diabetes mellitus. These terms have largely displaced the formerly used terms IDDM (insulin-dependent diabetes mellitus), NIDDM (non- insulin dependent diabetes mellitus), juvenile-onset diabetes mellitus and adult-onset diabetes mellitus.
[0086] Type 1 diabetes results from an autoimmune destruction of the insulin- secreting B-cells of the pancreas. There are several markers of this autoimmune destruction, detectable in body fluids and tissues, including islet cell autoantibodies, autoantibodies to insulin, autoantibodies to glutamic acid decarboxylase (GAD65), and autoantibodies to the tyrosine phosphatases IA-2 and IA-2B. While genetic factors are strongly implicated, the concordance rate in twin studies is under 50% and supports a role for environmental factors, which are said to include viral infections. The autoimmune process typically begins many years before clinical detection and presentation. The rate of B-cell destruction is quite variable, being rapid in some individuals (mainly infants and children) and usually slow in adults.
[0087] Type 2 diabetes disease usually develops after 40 years of age. It is much more common than type 1 diabetes and comprises approximately 90% of all individuals with diabetes. Insulin concentrations are mostly increased but they can be normal or decreased. Obesity is common. Diet and exercise regimens leading to weight reduction can ameliorate hyperglycemia. Oral hypoglycaemic drugs are also used in an effort to lower blood sugar. Nevertheless, insulin is sometimes required to correct hyperglycemia, particularly as patients grow older or as their β-cells fail.
[0088] Two groups of disorders may be said to typify type 2 diabetes mellitus. The first one is a decreased ability of insulin to act on peripheral tissues, usually referred to as "insulin resistance." Insulin resistance is defined as a decreased biological response to normal concentrations of circulating insulin and represents the primary underlying pathological process. The second is the dysfunction of pancreatic B-cells, represented by the inability to produce sufficient amounts of insulin to overcome insulin resistance in the peripheral tissues. Eventually, insulin production can be insufficient to compensate for the insulin resistance due to B-cell dysfunction. The common result is a relative deficiency of insulin. Data support the concept that insulin resistance is the primary defect, preceding the derangement of insulin secretion. As with type 1 diabetes, the basis of the insulin resistance and insulin secretion defects is believed to be a combination of environmental and genetic factors.
[0089] Type 1 and type 2 diabetes comprise the great majority of cases of diabetes. In addition to these, there is gestational diabetes, which is usually asymptomatic, and a heterogeneous collection of specific types of diabetes resulting from pathologies of the pancreas, pathologies of the endocrine system, infection, or exposure to chemicals or drugs which damage the beta cells of the pancreas. The present invention can be used with regard to any form of diabetes to the extent that it is associated with progressive damage to the kidney or kidney function. While persons with diabetes caused by autoimmune processes, such as in type 1 diabetes, will benefit from the administration of sEH inhibitor, with or without EETs, in preferred embodiments relating to diabetes, the invention relates to persons whose diabetes is not caused by an autoimmune process. Therefore, in some preferred embodiments, the person has type 2 diabetes; in some preferred embodiments, the individual has one of the various types of diabetes caused by non-autoimmune processes described earlier in this paragraph.
[0090] The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels. The long-term complications of diabetes include retinopathy with potential loss of vision; nephropathy leading to renal failure; peripheral neuropathy with risk of foot ulcers, amputation, and Charcot joints.
[0091] Glycation of tissue proteins and other macromolecules and excess production of polyol compounds from glucose are among the mechanisms thought to produce tissue damage from chronic hyperglycemia. The nonenzymatic glycation process is one in which glucose is chemically bound to amino groups of proteins, but without the help of enzymes. It is a covalent reaction where, by means of N- glycoside bonding, sugar-protein complex is formed through a series of chemical reactions described by Maillard. In Maillard reactions, sugar-reacts with protein to form complexes and represent an early product of nonenzymatic glycation and an intermediary that is a precursor of later compounds. Numerous intermediary products are then formed, followed by complex product polymerization reactions resulting in heterogeneous structures called advanced glycation endproducts (AGE). It has also been reported that AGEs progressively accumulate on the tissues and organs that develop chronic complications of diabetes mellitus like retinopathy, nephropathy, neuropathy and progressive atherosclerosis. Immunohistochemical methods have demonstrated the presence of different AGE compounds in glomeruli and tubuli cells in both experimental and human diabetic nephropathy. Glycation in diabetes and AGEs are discussed in, for example, U.S. Application Nos. 20030203973 and 20030092744 and U.S. Patent Nos. 6,624,178 and 5,518,720.
[0092] In 2002, the American Diabetes Association published a position statement entitled "Diabetic Nephropathy," at Diabetes Care 25:S85-S89 (2002) (the
"Statement"). According to the Statement, the "earliest clinical evidence of nephropathy is the appearance of low but abnormal levels ( 30 mg/day or 20 μg/min) of albumin in the urine, referred to as microalbuminuria." In persons with type 1 diabetes (juvenile diabetes, characterized by an inability to produce sufficient insulin), the Statement states that 80% of persons with microalbuminuria will gradually progress to overt nephropathy, with hypertension developing along the way, unless specific interventions are introduced, although they may have hypertension that becomes manifest about the time they develop microalbuminuria. The Statement further indicates that a higher proportion of persons with type 2 diabetes (adult-onset, characterized by a reduced ability to respond to insulin) have microalbuminuria at diagnosis, and that 20-40% will progress to overt nephropathy without specific intervention. The Statement indicates that one third of type 2 patients have hypertension at diagnosis, thereby indicating that two thirds do not. This is particularly important since the number of people with type 2 diabetes is significantly larger than the number that develop type 1 diabetes.
3. Agents that Increase EETs
[0093] In some embodiments, an agent that increases intracellular cAMP is coadministered with an agent that increases EETs. Agents that increase EETs include EETs and inhibitors of sEH. a. Inhibitors of sEH
[0094] Scores of sEH inhibitors are known, of a variety of chemical structures. Derivatives in which the urea, carbamate or amide pharmacophore (as used herein, "pharmacophore" refers to the section of the structure of a ligand that binds to the sEH) is covalently bound to both an adamantane and to a 12 carbon chain dodecane are particularly useful as sEH inhibitors. Derivatives that are metabolically stable are preferred, as they are expected to have greater activity in vivo. Selective and competitive inhibition of sEH in vitro by a variety of urea, carbamate, and amide derivatives is taught, for example, by Morisseau et al, Proc. Natl. Acad. Sci. U. S. A, 96:8849-8854 (1999), which provides substantial guidance on designing urea derivatives that inhibit the enzyme.
[0095] Derivatives of urea are transition state mimetics that form a preferred group of sEH inhibitors. Within this group, N, N'-dodecyl-cyclohexyl urea (DCU), is preferred as an inhibitor, while N-cyclohexyl-N'-dodecylurea (CDU) is particularly preferred. Some compounds, such as dicyclohexylcarbodiimide (a lipophilic diimide), can decompose to an active urea inhibitor such as DCU. Any particular urea derivative or other compound can be easily tested for its ability to inhibit sEH by standard assays, such as those discussed herein. The production and testing of urea and carbamate derivatives as sEH inhibitors is set forth in detail in, for example, Morisseau et al, Proc Natl Acad Sci (USA) 96:8849-8854 (1999).
[0096] N-Adamantyl-N'-dodecyl urea ("ADU") is both metabolically stable and has particularly high activity on sEH. (Both the 1- and the 2- admamantyl ureas have been tested and have about the same high activity as an inhibitor of sEH.) Thus, isomers of adamantyl dodecyl urea are preferred inhibitors. It is further expected that N, N'-dodecyl-cyclohexyl urea (DCU), and other inhibitors of sEH, and particularly dodecanoic acid ester derivatives of urea, are suitable for use in the methods of the invention. Preferred inhibitors include:
12-(3-Adamantan-l-yl-ureido)dodecanoic acid (AUDA),
Figure imgf000026_0001
12-(3-Adamantan-l-yl-ureido)dodecanoic acid butyl ester (AUDA-BE),
Figure imgf000027_0001
Adamantan- 1 -yl-3 - {5 - [2-(2-ethoxyethoxy)ethoxy]pentyl} urea (compound 950, also referred to herein as "AEPU"), and
Figure imgf000027_0002
[0097] Another preferred group of inhibitors are piperidines. The following Table sets forth some exemplar piperidines and their ability to inhibit sEH activity, expressed as the amount needed to reduce the activity of the enzyme by 50% (expressed as "IC50").
Table 1. IC50 values for selected alkylpiperidine-based sEH inhibitors
Figure imgf000027_0003
a As determined via a kinetic fluorescent assay.
[0098] A number of other sEH inhibitors which can be used in the methods and compositions of the invention are set forth in co-owned applications
PCT/US2008/072199, PCT/US2007/006412, PCT/US2005/038282,
PCT/US2005/08765, PCT/US2004/010298 and U.S. Published Patent Application Publication 2005/0026844, each of which is hereby incorporated herein by reference in its entirety for all purposes. [0099] U.S. Patent No. 5,955,496 (the '496 patent) also sets forth a number of sEH inhibitors which can be used in the methods of the invention. One category of these inhibitors comprises inhibitors that mimic the substrate for the enzyme. The lipid alkoxides (e.g., the 9-methoxide of stearic acid) are an exemplar of this group of inhibitors. In addition to the inhibitors discussed in the '496 patent, a dozen or more lipid alkoxides have been tested as sEH inhibitors, including the methyl, ethyl, and propyl alkoxides of oleic acid (also known as stearic acid alkoxides), linoleic acid, and arachidonic acid, and all have been found to act as inhibitors of sEH.
[0100] In another group of embodiments, the '496 patent sets forth sEH inhibitors that provide alternate substrates for the enzyme that are turned over slowly.
Exemplars of this category of inhibitors are phenyl glycidols (e.g., S, S-4- nitrophenylglycidol), and chalcone oxides. The '496 patent notes that suitable chalcone oxides include 4-phenylchalcone oxide and 4-fluourochalcone oxide. The phenyl glycidols and chalcone oxides are believed to form stable acyl enzymes.
[0101] Additional inhibitors of sEH suitable for use in the methods of the invention are set forth in U.S. Patent Nos. 6,150,415 (the '415 patent) and 6,531,506 (the '506 patent). Two preferred classes of sEH inhibitors of the invention are compounds of Formulas 1 and 2, as described in the '415 and '506 patents. Means for preparing such compounds and assaying desired compounds for the ability to inhibit epoxide hydrolases are also described. The '506 patent, in particular, teaches scores of inhibitors of Formula 1 and some twenty sEH inhibitors of Formula 2, which were shown to inhibit human sEH at concentrations as low as 0.1 μΜ. Any particular sEH inhibitor can readily be tested to determine whether it will work in the methods of the invention by standard assays. Esters and salts of the various compounds discussed above or in the cited patents, for example, can be readily tested by these assays for their use in the methods of the invention.
[0102] As noted above, chalcone oxides can serve as an alternate substrate for the enzyme. While chalcone oxides have half lives which depend in part on the particular structure, as a group the chalcone oxides tend to have relatively short half lives (a drug's half life is usually defined as the time for the concentration of the drug to drop to half its original value. See, e.g., Thomas, G., Medicinal Chemistry: an
introduction, John Wiley & Sons Ltd. (West Sussex, England, 2000)). Since the various uses of the invention contemplate inhibition of sEH over differing periods of time which can be measured in days, weeks, or months, chalcone oxides, and other inhibitors which have a half life whose duration is shorter than the practitioner deems desirable, are preferably administered in a manner which provides the agent over a period of time. For example, the inhibitor can be provided in materials that release the inhibitor slowly. Methods of administration that permit high local concentrations of an inhibitor over a period of time are known, and are not limited to use with inhibitors which have short half lives although, for inhibitors with a relatively short half life, they are a preferred method of administration.
[0103] In addition to the compounds in Formula 1 of the '506 patent, which interact with the enzyme in a reversible fashion based on the inhibitor mimicking an enzyme- substrate transition state or reaction intermediate, one can have compounds that are irreversible inhibitors of the enzyme. The active structures such as those in the Tables or Formula 1 of the '506 patent can direct the inhibitor to the enzyme where a reactive functionality in the enzyme catalytic site can form a covalent bond with the inhibitor. One group of molecules which could interact like this would have a leaving group such as a halogen or tosylate which could be attacked in an SN2 manner with a lysine or histidine. Alternatively, the reactive functionality could be an epoxide or Michael acceptor such as an α/β-unsaturated ester, aldehyde, ketone, ester, or nitrile.
[0104] Further, in addition to the Formula 1 compounds, active derivatives can be designed for practicing the invention. For example, dicyclohexyl thio urea can be oxidized to dicyclohexylcarbodiimide which, with enzyme or aqueous acid
(physiological saline), will form an active dicyclohexylurea. Alternatively, the acidic protons on carbamates or ureas can be replaced with a variety of substituents which, upon oxidation, hydrolysis or attack by a nucleophile such as glutathione, will yield the corresponding parent structure. These materials are known as prodrugs or protoxins (Gilman et al., The Pharmacological Basis of Therapeutics, 7th Edition, MacMillan Publishing Company, New York, p. 16 (1985)) Esters, for example, are common prodrugs which are released to give the corresponding alcohols and acids enzymatically (Yoshigae et al, Chirality, 9:661-666 (1997)). The drugs and prodrugs can be chiral for greater specificity. These derivatives have been extensively used in medicinal and agricultural chemistry to alter the pharmacological properties of the compounds such as enhancing water solubility, improving formulation chemistry, altering tissue targeting, altering volume of distribution, and altering penetration. They also have been used to alter toxicology profiles.
[0105] There are many prodrugs possible, but replacement of one or both of the two active hydrogens in the ureas described here or the single active hydrogen present in carbamates is particularly attractive. Such derivatives have been extensively described by Fukuto and associates. These derivatives have been extensively described and are commonly used in agricultural and medicinal chemistry to alter the pharmacological properties of the compounds. (Black et al., Journal of Agricultural and Food Chemistry, 21(5):747-751 (1973); Fahmy et al, Journal of Agricultural and Food Chemistry, 26(3):550-556 (1978); Jojima et al, Journal of Agricultural and Food Chemistry, 31(3):613-620 (1983); and Fahmy et al, Journal of Agricultural and Food Chemistry, 29(3):567-572 (1981).)
[0106] Such active proinhibitor derivatives are within the scope of the present invention, and the just-cited references are incorporated herein by reference. Without being bound by theory, it is believed that suitable inhibitors of the invention mimic the enzyme transition state so that there is a stable interaction with the enzyme catalytic site. The inhibitors appear to form hydrogen bonds with the nucleophilic carboxylic acid and a polarizing tyrosine of the catalytic site.
[0107] In some embodiments, the sEH inhibitor used in the methods taught herein is a "soft drug." Soft drugs are compounds of biological activity that are rapidly inactivated by enzymes as they move from a chosen target site. EETs and simple biodegradable derivatives administered to an area of interest may be considered to be soft drugs in that they are likely to be enzymatically degraded by sEH as they diffuse away from the site of interest following administration. Some sEHI, however, may diffuse or be transported following administration to regions where their activity in inhibiting sEH may not be desired. Thus, multiple soft drugs for treatment have been prepared. These include but are not limited to carbamates, esters, carbonates and amides placed in the sEHI, approximately 7.5 angstroms from the carbonyl of the central pharmacophore. These are highly active sEHI that yield biologically inactive metabolites by the action of esterase and/or amidase. Groups such as amides and carbamates on the central pharmacophores can also be used to increase solubility for applications in which that is desirable in forming a soft drug. Similarly, easily metabolized ethers may contribute soft drug properties and also increase the solubility.
[0108] In some embodiments, sEH inhibition can include the reduction of the amount of sEH. As used herein, therefore, sEH inhibitors can therefore encompass nucleic acids that inhibit expression of a gene encoding sEH. Many methods of reducing the expression of genes, such as reduction of transcription and siRNA, are known, and are discussed in more detail below.
[0109] Preferably, the inhibitor inhibits sEH without also significantly inhibiting microsomal epoxide hydrolase ("mEH"). Preferably, at concentrations of 500 μΜ, the inhibitor inhibits sEH activity by at least 50% while not inhibiting mEH activity by more than 10%. Preferred compounds have an IC50 (inhibition potency or, by definition, the concentration of inhibitor which reduces enzyme activity by 50%) of less than about 500 μΜ. Inhibitors with IC50S of less than 500 μΜ are preferred, with IC50s of less than 100 μΜ being more preferred and, in order of increasing preference, an IC50 of 50 μΜ, 40 μΜ, 30 μΜ, 25 μΜ, 20 μΜ, 15 μΜ, 10 μΜ, 5 μΜ, 3 μΜ, 2 μΜ, 1 μΜ or even less being still more preferred. Assays for determining sEH activity are known in the art and described elsewhere herein. b. EETs
[0110] EETs, which are epoxides of arachidonic acid, are known to be effectors of blood pressure, regulators of inflammation, and modulators of vascular permeability. Hydrolysis of the epoxides by sEH diminishes this activity. Inhibition of sEH raises the level of EETs since the rate at which the EETs are hydro lyzed into
dihydroxyeicosatrienoic acids ("DHETs") is reduced.
[0111] It has long been believed that EETs administered systemically would be hydrolyzed too quickly by endogenous sEH to be helpful. For example, in one prior report of EETs administration, EETs were administered by catheters inserted into mouse aortas. The EETs were infused continuously during the course of the experiment because of concerns over the short half life of the EETs. See, Liao and Zeldin, International Publication WO 01/10438 (hereafter "Liao and Zeldin"). It also was not known whether endogenous sEH could be inhibited sufficiently in body tissues to permit administration of exogenous EET to result in increased levels of EETs over those normally present. Further, it was thought that EETs, as epoxides, would be too labile to survive the storage and handling necessary for therapeutic use.
[0112] Studies from the laboratory of the present inventors, however, showed that systemic administration of EETs in conjunction with inhibitors of sEH had better results than did administration of sEH inhibitors alone. EETs were not administered by themselves in these studies since it was anticipated they would be degraded too quickly to have a useful effect. Additional studies from the laboratory of the present inventors have since shown, however, that administration of EETs by themselves has had therapeutic effect. Without wishing to be bound by theory, it is surmised that the exogenous EET overwhelms endogenous sEH, and allows EETs levels to be increased for a sufficient period of time to have therapeutic effect. Thus, EETs can be administered without also administering an sEHI to provide a therapeutic effect. Moreover, EETs, if not exposed to acidic conditions or to sEH are stable and can withstand reasonable storage, handling and administration.
[0113] In short, sEHI, EETs, or co-administration of sEHIs and of EETs, can be used in the methods of the present invention. In some embodiments, one or more EETs are administered to the patient without also administering an sEHI. In some embodiments, one or more EETs are administered shortly before or concurrently with administration of an sEH inhibitor to slow hydrolysis of the EET or EETs. In some embodiments, one or more EETs are administered after administration of an sEH inhibitor, but before the level of the sEHI has diminished below a level effective to slow the hydrolysis of the EETs.
[0114] EETs useful in the methods of the present invention include 14,15-EET, 8,9- EET and 11,12-EET, and 5,6 EETs. Preferably, the EETs are administered as the methyl ester, which is more stable. Persons of skill will recognize that the EETs are regioisomers, such as 8S,9R- and 14R,15S-EET. 8,9-EET, 11,12-EET, and 14R,15S- EET, are commercially available from, for example, Sigma-Aldrich (catalog nos. E5516, E5641, and E5766, respectively, Sigma-Aldrich Corp., St. Louis, MO).
[0115] If desired, EETs, analogs, or derivatives that retain activity can be used in place of or in combination with unmodified EETs. Liao and Zeldin, supra, define EET analogs as compounds with structural substitutions or alterations in an EET, and include structural analogs in which one or more EET olefins are removed or replaced with acetylene or cyclopropane groups, analogs in which the epoxide moiety is replaced with oxitane or furan rings and heteroatom analogs. In other analogs, the epoxide moiety is replaced with ether, alkoxides, urea, amide, carbamate,
difluorocycloprane, or carbonyl, while in others, the carboxylic acid moiety is stabilized by blocking beta oxidation or is replaced with a commonly used mimic, such as a nitrogen heterocycle, a sulfonamide, or another polar functionality. In preferred forms, the analogs or derivatives are relatively stable as compared to an unmodified EET because they are more resistant than an unmodified EET to sEH and to chemical breakdown. "Relatively stable" means the rate of hydrolysis by sEH is at least 25% less than the hydrolysis of the unmodified EET in a hydrolysis assay, and more preferably 50% or more lower than the rate of hydrolysis of an unmodified EET. Liao and Zeldin show, for example, episulfide and sulfonamide EETs derivatives. Amide and ester derivatives of EETs and that are relatively stable are preferred embodiments. Whether or not a particular EET analog or derivative has the biological activity of the unmodified EET can be readily determined by using it in standard assays, such as radio-ligand competition assays to measure binding to the relevant receptor. As mentioned in the Definition section, above, for convenience of reference, the term "EETs" as used herein refers to unmodified EETs, and EETs analogs and derivatives unless otherwise required by context.
[0116] In some embodiments, the EET or EETs are embedded or otherwise placed in a material that releases the EET over time. Materials suitable for promoting the slow release of compositions such as EETs are known in the art. Optionally, one or more sEH inhibitors may also be placed in the slow release material.
[0117] Conveniently, the EET or EETs can be administered orally. Since EETs are subject to degradation under acidic conditions, EETs intended for oral administration can be coated with a coating resistant to dissolving under acidic conditions, but which dissolve under the mildly basic conditions present in the intestines. Suitable coatings, commonly known as "enteric coatings" are widely used for products, such as aspirin, which cause gastric distress or which would undergo degradation upon exposure to gastric acid. By using coatings with an appropriate dissolution profile, the coated substance can be released in a chosen section of the intestinal tract. For example, a substance to be released in the colon is coated with a substance that dissolves at pH 6.5-7, while substances to be released in the duodenum can be coated with a coating that dissolves at pH values over 5.5. Such coatings are commercially available from, for example, Rohm Specialty Acrylics (Rohm America LLC, Piscataway, NJ) under the trade name "Eudragit®". The choice of the particular enteric coating is not critical to the practice of the invention. c. Assays for Epoxide Hydrolase Activity
[0118] Any of a number of standard assays for determining epoxide hydrolase activity can be used to determine inhibition of sEH. For example, suitable assays are described in Gill,, et al., Anal Biochem 131 :273-282 (1983); and Borhan, et al., Analytical Biochemistry 231 :188-200 (1995)). Suitable in vitro assays are described in Zeldin et al., J Biol. Chem. 268:6402-6407 (1993). Suitable in vivo assays are described in Zeldin et al, Arch Biochem Biophys 330:87-96 (1996). Assays for epoxide hydrolase using both putative natural substrates and surrogate substrates have been reviewed (see, Hammock, et al. In: Methods in Enzymology, Volume III, Steroids and Isoprenoids, Part B, (Law, J.H. and H.C. Rilling, eds. 1985), Academic Press, Orlando, Florida, pp. 303-311 and Wixtrom et al. , In: Biochemical
Pharmacology and Toxicology, Vol. 1 : Methodological Aspects of Drug
Metabolizing Enzymes, (Zakim, D. and D.A. Vessey, eds. 1985), John Wiley & Sons, Inc., New York, pp. 1-93. Several spectral based assays exist based on the reactivity or tendency of the resulting diol product to hydrogen bond {see, e.g., Wixtrom, supra, and Hammock. Anal. Biochem. 174:291-299 (1985) and Dietze, et al. Anal. Biochem. 216: 176-187 (1994)).
[0119] The enzyme also can be detected based on the binding of specific ligands to the catalytic site which either immobilize the enzyme or label it with a probe such as dansyl, fluoracein, luciferase, green fluorescent protein or other reagent. The enzyme can be assayed by its hydration of EETs, its hydrolysis of an epoxide to give a colored product as described by Dietze et al, 1994, supra, or its hydrolysis of a radioactive surrogate substrate (Borhan et al, 1995, supra). The enzyme also can be detected based on the generation of fluorescent products following the hydrolysis of the epoxide. Numerous methods of epoxide hydrolase detection have been described (see, e.g., Wixtrom, supra). [0120] The assays are normally carried out with a recombinant enzyme following affinity purification. They can be carried out in crude tissue homogenates, cell culture or even in vivo, as known in the art and described in the references cited above. d. Other Means of Inhibiting sEH Activity
[0121] Other means of inhibiting sEH activity or gene expression can also be used in the methods of the invention. For example, a nucleic acid molecule
complementary to at least a portion of the human sEH gene can be used to inhibit sEH gene expression. Means for inhibiting gene expression using short RNA molecules, for example, are known. Among these are short interfering RNA (siRNA), small temporal RNAs (stRNAs), and micro-RNAs (miRNAs). Short interfering RNAs silence genes through a mRNA degradation pathway, while stRNAs and miRNAs are approximately 21 or 22 nt RNAs that are processed from endogenously encoded hairpin-structured precursors, and function to silence genes via translational repression. See, e.g., McManus et al, RNA, 8(6):842-50 (2002); Morris et al, Science, 305(5688): 1289-92 (2004); He and Hannon, Nat Rev Genet. 5(7):522-31 (2004).
[0122] "RNA interference," a form of post-transcriptional gene silencing ("PTGS"), describes effects that result from the introduction of double-stranded RNA into cells (reviewed in Fire, A. Trends Genet 15:358-363 (1999); Sharp, P. Genes Dev 13: 139- 141 (1999); Hunter, C. Curr Biol 9:R440-R442 (1999); Baulcombe. D. Curr Biol 9:R599-R601 (1999); Vaucheret et al. Plant J 16: 651-659 (1998)). RNA
interference, commonly referred to as RNAi, offers a way of specifically inactivating a cloned gene, and is a powerful tool for investigating gene function.
[0123] The active agent in RNAi is a long double-stranded (antiparallel duplex) RNA, with one of the strands corresponding or complementary to the RNA which is to be inhibited. The inhibited RNA is the target RNA. The long double stranded RNA is chopped into smaller duplexes of approximately 20 to 25 nucleotide pairs, after which the mechanism by which the smaller RNAs inhibit expression of the target is largely unknown at this time. While RNAi was shown initially to work well in lower eukaryotes, for mammalian cells, it was thought that RNAi might be suitable only for studies on the oocyte and the preimplantation embryo. [0124] In mammalian cells other than these, however, longer RNA duplexes provoked a response known as "sequence non-specific RNA interference," characterized by the non-specific inhibition of protein synthesis.
[0125] Further studies showed this effect to be induced by dsRNA of greater than about 30 base pairs, apparently due to an interferon response. It is thought that dsRNA of greater than about 30 base pairs binds and activates the protein PKR and 2',5'-oligonucleotide synthetase (2',5'-AS). Activated PKR stalls translation by phosphorylation of the translation initiation factors eIF2a, and activated 2',5'-AS causes mRNA degradation by 2',5'-oligonucleotide-activated ribonuclease L. These responses are intrinsically sequence-nonspecific to the inducing dsRNA; they also frequently result in apoptosis, or cell death. Thus, most somatic mammalian cells undergo apoptosis when exposed to the concentrations of dsRNA that induce RNAi in lower eukaryotic cells.
[0126] More recently, it was shown that RNAi would work in human cells if the RNA strands were provided as pre-sized duplexes of about 19 nucleotide pairs, and RNAi worked particularly well with small unpaired 3' extensions on the end of each strand (Elbashir et al. Nature 411 : 494-498 (2001)). In this report, siRNA were applied to cultured cells by transfection in oligofectamine micelles. These RNA duplexes were too short to elicit sequence-nonspecific responses like apoptosis, yet they efficiently initiated RNAi. Many laboratories then tested the use of siRNA to knock out target genes in mammalian cells. The results demonstrated that siRNA works quite well in most instances.
[0127] For purposes of reducing the activity of sEH, siRNAs to the gene encoding sEH can be specifically designed using computer programs. The cloning, sequence, and accession numbers of the human sEH sequence are set forth in Beetham et al., Arch. Biochem. Biophys. 305(1): 197-201 (1993). An exemplary amino acid sequence of human sEH (GenBank Accession No. L05779; SEQ ID NO: 1) and an exemplary nucleotide sequence encoding that amino acid sequence (GenBank Accession No. AAA02756; SEQ ID NO:2) are set forth in U.S. Patent No.
5,445,956. The nucleic acid sequence of human sEH is also published as GenBank Accession No. NM 001979.4; the amino acid sequence of human sEH is also published as GenBank Accession No. NP 001970.2. [0128] A program, siDESIGN from Dharmacon, Inc. (Lafayette, CO), permits predicting siRNAs for any nucleic acid sequence, and is available on the World Wide Web at dharmacon.com. Programs for designing siRNAs are also available from others, including Genscript (available on the Web at genscript.com/ssl-bin/app/rnai) and, to academic and non-profit researchers, from the Whitehead Institute for Biomedical Research found on the worldwide web at
"jura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/."
[0129] For example, using the program available from the Whitehead Institute, the following sEH target sequences and siRNA sequences can be generated:
1) Target: CAGTGTTCATTGGCCATGACTGG (SEQ ID NO:3) Sense-siRNA: 5' - GUGUUCAUUGGCCAUGACUTT- 3' (SEQ ID NO:4) Antisense-siRNA: 5' - AGUC AUGGC C AAUG AAC ACTT- 3' (SEQ ID NO:5)
2) Target: GAAAGGCTATGGAGAGTCATCTG (SEQ ID NO:6) Sense-siRNA: 5' - AAGGCUAUGGAGAGUCAUCTT - 3' (SEQ ID NO:7) Antisense-siRNA: 5'- GAUGACUCUCCAUAGCCUUTT - 3' (SEQ ID NO:8)
3) Target AAAGGCTATGGAGAGTCATCTGC (SEQ ID NO:9) Sense-siRNA: 5' - AGGCUAUGGAGAGUCAUCUTT- 3' (SEQ ID NO: 10) Antisense-siRNA: 5' - AGAUGACUCUCCAUAGCCUTT- 3' (SEQ ID NO: 11)
4) Target: CAAGCAGTGTTCATTGGCCATGA (SEQ ID NO: 12) Sense-siRNA: 5' - AGCAGUGUUCAUUGGCCAUTT- 3' (SEQ ID NO: 13) Antisense-siRNA: 5' - AUGGCCAAUGAACACUGCUTT- 3' (SEQ ID NO: 14)
5) Target: CAGCACATGGAGGACTGGATTCC (SEQ ID NO: 15)
Sense-siRNA: 5' - GCACAUGGAGGACUGGAUUTT- 3' (SEQ ID NO: 16)
Antisense-siRNA: 5' - AAUCCAGUCCUCCAUGUGCTT- 3' (SEQ ID NO: 17)
[0130] Alternatively, siRNA can be generated using kits which generate siRNA from the gene. For example, the "Dicer siRNA Generation" kit (catalog number T510001, Gene Therapy Systems, Inc., San Diego, CA) uses the recombinant human enzyme "dicer" in vitro to cleave long double stranded R A into 22 bp siR As. By having a mixture of siRNAs, the kit permits a high degree of success in generating siRNAs that will reduce expression of the target gene. Similarly, the Silencer™ siRNA Cocktail Kit (RNase III) (catalog no. 1625, Ambion, Inc., Austin, TX) generates a mixture of siRNAs from dsRNA using RNase III instead of dicer. Like dicer, RNase III cleaves dsRNA into 12-30 bp dsRNA fragments with 2 to 3 nucleotide 3' overhangs, and 5'-phosphate and 3'-hydroxyl termini. According to the manufacturer, dsRNA is produced using T7 RNA polymerase, and reaction and purification components included in the kit. The dsRNA is then digested by RNase III to create a population of siRNAs. The kit includes reagents to synthesize long dsRNAs by in vitro transcription and to digest those dsRNAs into siRNA-like molecules using RNase III. The manufacturer indicates that the user need only supply a DNA template with opposing T7 phage polymerase promoters or two separate templates with promoters on opposite ends of the region to be transcribed.
[0131] The siRNAs can also be expressed from vectors. Typically, such vectors are administered in conjunction with a second vector encoding the corresponding complementary strand. Once expressed, the two strands anneal to each other and form the functional double stranded siRNA. One exemplar vector suitable for use in the invention is pSuper, available from OligoEngine, Inc. (Seattle, WA). In some embodiments, the vector contains two promoters, one positioned downstream of the first and in antiparallel orientation. The first promoter is transcribed in one direction, and the second in the direction antiparallel to the first, resulting in expression of the complementary strands. In yet another set of embodiments, the promoter is followed by a first segment encoding the first strand, and a second segment encoding the second strand. The second strand is complementary to the palindrome of the first strand. Between the first and the second strands is a section of RNA serving as a linker (sometimes called a "spacer") to permit the second strand to bend around and anneal to the first strand, in a configuration known as a "hairpin."
[0132] The formation of hairpin RNAs, including use of linker sections, is well known in the art. Typically, an siRNA expression cassette is employed, using a Polymerase III promoter such as human U6, mouse U6, or human HI . The coding sequence is typically a 19-nucleotide sense siRNA sequence linked to its reverse complementary antisense siRNA sequence by a short spacer. Nine-nucleotide spacers are typical, although other spacers can be designed. For example, the Ambion website indicates that its scientists have had success with the spacer TTCAAGAGA (SEQ ID NO: 18). Further, 5-6 T's are often added to the 3' end of the oligonucleotide to serve as a termination site for Polymerase III. See also, Yu et al., Mol Ther 7(2):228-36 (2003); Matsukura et al, Nucleic Acids Res 31(15):e77 (2003).
[0133] As an example, the siRNA targets identified above can be targeted by hairpin siRNA as follows. To attack the same targets by short hairpin RNAs, produced by a vector (permanent RNAi effect), sense and antisense strand can be put in a row with a loop forming sequence in between and suitable sequences for an adequate expression vector to both ends of the sequence. The following are non- limiting examples of hairpin sequences that can be cloned into the pSuper vector:
1) Target: CAGTGTTCATTGGCCATGACTGG (SEQ ID NO: 19)
Sense strand: 5'-GATCCCCGTGTTCATTGGCCATGACTTTCAA
GAGAAGTCATGGCCAATGAACACTTTTT-3' (SEQ ID NO:20)
Antisense strand: 5'-AGCTAAAAAGTGTTCATTGGCCATGACTTCTCTT GAAAGTCATGGCCAATGAACACGGG -3' (SEQ ID NO:21)
2) Target: GAAAGGCTATGGAGAGTCATCTG (SEQ ID NO:22)
Sense strand: 5'-GATCCCCAAGGCTATGGAGAGTCATCTTCAAGAGAGA TGACTCTCCATAGCCTTTTTTT -3' (SEQ ID NO:23)
Antisense strand: 5'- AGCTAAAAAAAGGCTATGGAGAGTCATCTCTCTTGAA GATGACTCTCCATAGCCTTGGG -3' (SEQ ID NO:24)
3) Target: AAAGGCTATGGAGAGTCATCTGC (SEQ ID NO:25)
Sense strand: 5'-GATCCCCAGGCTATGGAGAGTCATCTTTCAAGAGAAG ATGACTCTCCATAGCCTTTTTT -3' (SEQ ID NO:26)
Antisense strand: 5'-
AGCTAAAAAAGGCTATGGAGAGTCATCATCTCTTGAAAGATGACTCTCCA TAGCCTGGG -3' (SEQ ID NO:27)
4) Target: CAAGCAGTGTTCATTGGCCATGA (SEQ ID NO:28) Sense strand: 5'-GATCCCCAGCAGTGTTCATTGGCCATTTCAAGAGAATG GCCAATGAACACTGCTTTTTT -3' (SEQ ID NO:29)
Antisense strand: 5'-
AGCTAAAAAAGCAGTGTTCATTGGCCATTCTCTTGAAATG GCCAATGAACACTGCTGGG -3' (SEQ ID NO:30)
5) Target: CAGCACATGGAGGACTGGATTCC (SEQ ID N0:31)
Sense strand 5'-GATCCCCGCACATGGAGGACTGGATTTTCAAGAGAAATC CAGTCCTCCATGTGCTTTTT -3' (SEQ ID NO:32)
Antisense strand: 5'-
AGCTAAAAAGCACATGGAGGACTGGATTTCTCTTGAAAA TCCAGTCCTCCATGTGCGGG -3' (SEQ ID NO:33)
[0134] In addition to siR As, other means are known in the art for inhibiting the expression of antisense molecules, ribozymes, and the like are well known to those of skill in the art. The nucleic acid molecule can be a DNA probe, a riboprobe, a peptide nucleic acid probe, a phosphorothioate probe, or a 2'-0 methyl probe.
[0135] Generally, to assure specific hybridization, the antisense sequence is substantially complementary to the target sequence. In certain embodiments, the antisense sequence is exactly complementary to the target sequence. The antisense polynucleotides may also include, however, nucleotide substitutions, additions, deletions, transitions, transpositions, or modifications, or other nucleic acid sequences or non-nucleic acid moieties so long as specific binding to the relevant target sequence corresponding to the sEH gene is retained as a functional property of the polynucleotide. In one embodiment, the antisense molecules form a triple helix- containing, or "triplex" nucleic acid. Triple helix formation results in inhibition of gene expression by, for example, preventing transcription of the target gene (see, e.g., Cheng et al, 1988, J. Biol. Chem. 263:15110; Ferrin and Camerini-Otero, 1991, Science 354: 1494; Ramdas et al, 1989, J. Biol. Chem. 264: 17395; Strobel et al, 1991, Science 254: 1639; and Rigas et al, 1986, Proc. Natl. Acad. Sci. U.S.A. 83:9591)
[0136] Antisense molecules can be designed by methods known in the art. For example, Integrated DNA Technologies (Coralville, IA) makes available a program found on the worldwide web "biotools.idtdna.com/antisense/AntiSense.aspx", which will provide appropriate antisense sequences for nucleic acid sequences up to 10,000 nucleotides in length. Using this program with the sEH gene provides the following exemplar sequences:
1) UGUCCAGUGCCCACAGUCCU (SEQ ID NO:34)
2) UUCCCACCUGACACGACUCU (SEQ ID NO:35)
3) GUUCAGCCUCAGCCACUCCU (SEQ ID NO:36)
4) AGUCCUCCCGCUUCACAGA (SEQ ID NO:37)
5) GCCCACUUCCAGUUCCUUUCC (SEQ ID NO:38)
[0137] In another embodiment, ribozymes can be designed to cleave the mRNA at a desired position. (See, e.g., Cech, 1995, Biotechnology 13:323; and Edgington, 1992, Biotechnology 10:256 and Hu et al, PCT Publication WO 94/03596).
[0138] The antisense nucleic acids (DNA, RNA, modified, analogues, and the like) can be made using any suitable method for producing a nucleic acid, such as the chemical synthesis and recombinant methods disclosed herein and known to one of skill in the art. In one embodiment, for example, antisense RNA molecules of the invention may be prepared by de novo chemical synthesis or by cloning. For example, an antisense RNA can be made by inserting (ligating) a sEH gene sequence in reverse orientation operably linked to a promoter in a vector (e.g., plasmid).
Provided that the promoter and, preferably termination and polyadenylation signals, are properly positioned, the strand of the inserted sequence corresponding to the noncoding strand will be transcribed and act as an antisense oligonucleotide of the invention.
[0139] It will be appreciated that the oligonucleotides can be made using nonstandard bases (e.g., other than adenine, cytidine, guanine, thymine, and uridine) or nonstandard backbone structures to provides desirable properties (e.g., increased nuclease-resistance, tighter-binding, stability or a desired Tm). Techniques for rendering oligonucleotides nuclease-resistant include those described in PCT
Publication WO 94/12633. A wide variety of useful modified oligonucleotides may be produced, including oligonucleotides having a peptide-nucleic acid (PNA) backbone (Nielsen et al., 1991, Science 254: 1497) or incorporating 2'-0-methyl ribonucleotides, phosphorothioate nucleotides, methyl phosphonate nucleotides, phosphotriester nucleotides, phosphorothioate nucleotides, phosphoramidates.
[0140] Proteins have been described that have the ability to translocate desired nucleic acids across a cell membrane. Typically, such proteins have amphiphilic or hydrophobic subsequences that have the ability to act as membrane-translocating carriers. For example, homeodomain proteins have the ability to translocate across cell membranes. The shortest internalizable peptide of a homeodomain protein, Antennapedia, was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996). Another subsequence, the h (hydrophobic) domain of signal peptides, was found to have similar cell membrane translocation characteristics (see, e.g., Lin et al., J. Biol. Chem. 270: 14255-14258 (1995)). Such subsequences can be used to translocate oligonucleotides across a cell membrane. Oligonucleotides can be conveniently derivatized with such sequences. For example, a linker can be used to link the oligonucleotides and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker or any other suitable chemical linker.
[0141] More recently, it has been discovered that siRNAs can be introduced into mammals without eliciting an immune response by encapsulating them in
nanoparticles of cyclodextrin. Information on this method can be found on the worldwide web at "nature.com/news/2005/050418/full/050418-6.html."
[0142] In another method, the nucleic acid is introduced directly into superficial layers of the skin or into muscle cells by a jet of compressed gas or the like. Methods for administering naked polynucleotides are well known and are taught, for example, in U.S. Patent No. 5,830,877 and International Publication Nos. WO 99/52483 and 94/21797. Devices for accelerating particles into body tissues using compressed gases are described in, for example, U.S. Patent Nos. 6,592,545, 6,475,181, and 6,328,714. The nucleic acid may be lyophilized and may be complexed, for example, with polysaccharides to form a particle of appropriate size and mass for acceleration into tissue. Conveniently, the nucleic acid can be placed on a gold bead or other particle which provides suitable mass or other characteristics. Use of gold beads to carry nucleic acids into body tissues is taught in, for example, U.S. Patent Nos. 4,945,050 and 6,194,389.
[0143] The nucleic acid can also be introduced into the body in a virus modified to serve as a vehicle without causing pathogenicity. The virus can be, for example, adenovirus, fowlpox virus or vaccinia virus.
[0144] miPvNAs and siRNAs differ in several ways: miRNA derive from points in the genome different from previously recognized genes, while siRNAs derive from mRNA, viruses or transposons, miRNA derives from hairpin structures, while siRNA derives from longer duplexed RNA, miRNA is conserved among related organisms, while siRNA usually is not, and miRNA silences loci other than that from which it derives, while siRNA silences the loci from which it arises. Interestingly, miRNAs tend not to exhibit perfect complementarity to the mRNA whose expression they inhibit. See, McManus et al, supra. See also, Cheng et al, Nucleic Acids Res. 33(4): 1290-7 (2005); Robins and Padgett, Proc Natl Acad Sci U S A. 102(11):4006-9 (2005); Brennecke et al, PLoS Biol. 3(3):e85 (2005). Methods of designing miRNAs are known. See, e.g., Zeng et al, Methods Enzymol. 392:371-80 (2005); Krol et al, J Biol Chem. 279(40):42230-9 (2004); Ying and Lin, Biochem Biophys Res Commun. 326(3):515-20 (2005).
4. Epoxygenated Fatty Acids
[0145] In some embodiments, an epoxygenated fatty acid is co-administered with an agent that increases intracellular cAMP. Exemplary epoxygenated fatty acids include epoxides of linoleic acid, eicosapentaenoic acid ("EPA") and
docosahexaenoic acid ("DHA").
[0146] The fatty acids eicosapentaenoic acid ("EPA") and docosahexaenoic acid ("DHA") have recently become recognized as having beneficial effects, and fish oil tablets, which are a good source of these fatty acids, are widely sold as supplements. In 2003, it was reported that these fatty acids reduced pain and inflammation. Sethi, S. et al, Blood 100: 1340-1346 (2002). The paper did not identify the mechanism of action, nor the agents responsible for this relief.
[0147] Cytochrome P450 ("CYP450") metabolism produces cis- epoxydocosapentaenoic acids ("EpDPEs") and cz's-epoxyeicosatetraenoic acids ("EpETEs") from docosahexaenoic acid ("DHA") and eicosapentaenoic acid ("EPA"), respectively. These epoxides are known endothelium-derived hyperpolarizing factors ("EDHFs"). These EDHFs, and others yet unidentified, are mediators released from vascular endothelial cells in response to acetylcholine and bradykinin, and are distinct from the NOS- (nitric oxide) and COX-derived (prostacyclin) vasodilators. Overall cytochrome P450 (CYP450) metabolism of polyunsaturated fatty acids produces epoxides, such as EETs,which are prime candidates for the active mediator(s).
14(15)-EpETE, for example, is derived via epoxidation of the 14,15-double bond of EPA and is the ω-3 homolog of 14(15)-EpETrE ("14(15)EET") derived via epoxidation of the 14,15-double bond of arachidonic acid.
[0148] As mentioned, it is beneficial to elevate the levels of EETs, which are epoxides of the fatty acid arachidonic acid. Our studies of the effects of EETs has led us to realization that the anti-inflammatory effect of EPA and DHA are likely due to increasing the levels of the epoxides of these two fatty acids. Thus, increasing the levels of epoxides of EPA, of DHA, or of both, will act to reduce pain and
inflammation, and symptoms associated with diabetes and metabolic syndromes, in mammals in need thereof. This beneficial effect of the epoxides of these fatty acids has not been previously recognized. Moreover, these epoxides have not previously been administered as agents, in part because, as noted above, epoxides have generally been considered too labile to be administered.
[0149] Like EETs, the epoxides of EPA and DHA are substrates for sEH. The epoxides of EPA and DHA are produced in the body at low levels by the action of cytochrome P450s. Endogenous levels of these epoxides can be maintained or increased by the administration of sEHI. However, the endogeous production of these epoxides is low and usually occurs in relatively special circumstances, such as the resolution of inflammation. Our expectation is that administering these epoxides from exogenous sources will aid in the resolution of inflammation and in reducing pain, as well as with symptoms of diabetes and metabolic syndromes. It is further beneficial with pain or inflammation to inhibit sEH with sEHI to reduce hydrolysis of these epoxides, thereby maintaining them at relatively high levels.
[0150] EPA has five unsaturated bonds, and thus five positions at which epoxides can be formed, while DHA has six. The epoxides of EPA are typically abbreviated and referred to generically as "EpETEs", while the epoxides of DHA are typically abbreviated and referred to generically as "EpDPEs". The specific regioisomers of the epoxides of each fatty acid are set forth in the following Table:
TABLE A
Regioisomers of Eicosapentaenoic acid ("EPA") epoxides:
1. Formal name: (±)5(6)- epoxy- 8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid,
Synonym 5(6)- epoxy Eicosatetraenoic acid
Abbreviation 5(6)- EpETE
2. Formal name: (±)8(9)- epoxy- 5Z, 11Z, 14Z, 17Z -eicosatetraenoic acid,
Synonym 8(9)- epoxy Eicosatetraenoic acid
Abbreviation 8(9)- EpETE
3. Formal name: (±)11(12)- epoxy- 5Z, 8Z, 14Z, 17Z -eicosatetraenoic acid,
Synonym 11(12)- epoxy Eicosatetraenoic acid
Abbreviation 11(12)- EpETE
4. Formal name: (±)14(15)- epoxy- 5Z, 8Z, 11Z, 17Z-eicosatetraenoic acid,
Synonym 14(15)- epoxy Eicosatetraenoic acid
Abbreviation 14(15)- EpETE
5. Formal name: (±)17(18)- epoxy- 5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid,
Synonym 17(18)- epoxy Eicosatetraenoic acid
Abbreviation 17(18)- EpETE
Regioisomers of Docosahexaenoic acid ("DHA") epoxides:
1. Formal name: (±) 4(5)- epoxy- 7Z, 10Z, 13Z, 16Z, 19Z -docosapentaenoic acid,
Synonym 4(5)- epoxy Docosapentaenoic acid
Abbreviation 4(5)- EpDPE
2. Formal name: (±) 7(8)- epoxy- 4Z, 10Z, 13Z, 16Z, 19Z -docosapentaenoic acid,
Synonym 7(8)- epoxy Docosapentaenoic acid
Abbreviation 7(8)- EpDPE
3. Formal name: (±)10(11)- epoxy- 4Z, 7Z, 13Z, 16Z, 19Z -docosapentaenoic acid,
Synonym 10(11)- epoxy Docosapentaenoic acid
Abbreviation 10(11)- EpDPE
4. Formal name: (±)13(14)- epoxy- 4Z, 7Z, 10Z, 16Z, 19Z -docosapentaenoic acid,
Synonym 13(14)- epoxy Docosapentaenoic acid
Abbreviation 13(14)- EpDPE
5. Formal name: (±) 16(17)- epoxy- 4Z, 7Z, 10Z, 13Z, 19Z -docosapentaenoic acid, Synonym 16(17)- epoxy Docosapentaenoic acid
Abbreviation 16(17)- EpDPE
6. Formal name: (±) 19(20)- epoxy- 4Z, 7Z, 10Z, 13Z, 16Z -docosapentaenoic acid, Synonym 19(20)- epoxy Docosapentaenoic acid
Abbreviation 19(20)- EpDPE
[0151] Any of these epoxides, or combinations of any of these, can be administered in the compositions and methods of the invention.
5. Agents that Inhibit COX and/or 5-LOX a. Inhibitors of COX-1 and/or COX-2
[0152] Current non-steroidal anti-inflammatory drugs (NSAIDs) inhibit both isoforms, but most tend to inhibit the two isoforms to different degrees. Since COX-2 is considered the enzyme associated with an inflammatory response, enzyme selectivity is generally measured in terms of specificity for COX-2. Typically, cells of a target organ that express COX-1 or COX-2 are exposed to increasing levels of NSAIDs. If the cell does not normally produce COX-2, COX-2 is induced by a stimulant, usually bacterial lipopolysaccharide (LPS).
[0153] The relative activity of NSAIDs on COX-1 and COX-2 is expressed by the ratio of IC50S for each enzyme: COX-2 (ICso)/COX-l (IC50). The smaller the ratio, the more specific the NSAID is for COX-2. For example, various NSAIDs have been reported to have ratios of COX-2 (IC5o)/COX-l (IC50) ranging from 0.33 to 122. See, Englehart et al., J Inflammatory Res 44:422-33 (1995). Aspirin has an IC50 ratio of 0.32, indicating that it inhibits COX-1 more than COX-2, while indomethacin is considered a COX-2 inhibitor since its COX-2 (IC5o)/COX-l (IC50) ratio is 33. Even selective COX-2 inhibitors retain some COX-1 inhibition at therapeutic levels obtained in vivo. Cryer and Feldman, Am J Med. 104(5):413-21 (1998).
[0154] Commercially available NSAIDs that find use in the methods and compositions of the invention include the traditional NSAIDs diclofenac potassium, diclofenac sodium, diclofenac sodium with misoprostol, diflunisal, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, ketoprofen,
meclofenamate sodium, mefenamic acid, meloxicam, nabumetone, naproxen sodium, piroxicam, tolmetin sodium, the selective COX-2 inhibitors celecoxib, rofecoxib, and valdecoxib, the acetylated salicylates, such as aspirin, and the non-acetylated salicylates, such as magnesium salicylate, choline salicylate, salsalate, salicylic acid esters and sodium salicylate. b. Inhibitors of 5-LOX
[0155] Metabolism of arachidonic acid through the lipoxygenase ("LOX") pathway lead to the formation of leukotrienes ("LTs") that are implicated in a range of pathologies. The primary inflammatory enzyme is 5 -lipoxygenase ("5-LOX"). The 5-LOX cascade results in the formation of LTB4 and the cysteinyl LTs LTC4, LTD4, and LTE4. LTB4 is a potent stimulator of leukocyte activation. Cysteinyl LTs "may participate in the damage of gastric mucosa by inducing mucosal microvascular injury and gastric vessel vasoconstriction, promoting breakdown of the mucosal barrier and stimulating the secretion of gastric acid, as well as the production of interleukin 1 ("IL1") and proinflammatory cytokines." Martel-Pelletier et al., Ann. Rheumatic Dis 62:501-509 (2003) ("Martel-Pelletier 2003"). Additional lipoxygenases, 12-LOX and 15 -LOX, exist that contribute to the formation of anti-inflammatory compounds known as lipoxins, or LXs. Thus, for purposes of reducing inflammation, it is desirable to inhibit 5-LOX without also inhibiting 12-LOX and 15-LOX.
[0156] Because of its role in inflammation, a number of inhibitors of 5-LOX have been developed. See, e.g., Julemont et al, Expert Opinion on Therapeutic Patents, 13(1): 1-13 (2003) (review of patents directed to 5-LOX inhibitors for 1999-2002). One orally effective inhibitor is REV 5901 [alpha-pentyl-3-(2-quinolinylmethoxy)- benzene-methanol] (see, Van Inwegen et al., Pharmacol Exp Therapeutics
241(1): 117-124 (1987)). 5-LOX can also be inhibited by inhibiting the 5- lipoxygenase activating protein ("FLAP") by MK-886. (see, Smirnov et al., Br J Pharmacol 124:572-578 (1998)). This inhibitor, however, induces apoptosis in some cell types and is best used in in vitro studies. Other inhibitors are described in, e.g., U.S. Patent Application No. 20040198768 c. Joint COX/LOX inhibitors
[0157] Because of the inflammatory effects of prostaglandins and leukotrienes, and because blocking the COX pathway has been thought to shuttle arachidonic acid into the LOX pathway, it has been suggested that dual inhibition of both COX-2 and 5- LOX would maximize the inhibition of inflammation. See, e.g., Martel-Pelletier 2003, supra. Several compounds have been developed to block both COX-2 and 5- LOX. One, tepoxalin, blocks COX-1, COX-2, and 5-LOX, and is commercially available as a veterinary pharmaceutical for dogs, under the name Zubrin® (Schering Plough Animal Health Corp., Union, NJ). Tepoxalin has also been shown to block the COX enzymes and LOX in humans and to be well tolerated. A second inhibitor of COX and 5-LOX, licofelone (Merkle GmbH, Germany), is in Phase III clinical trials as a treatment for osteoarthritis and has shown gastric tolerability superior to naproxen. See, Bias et al., Am J Gastroenterol 99(4):611 (2004). See also, Martel- Pelletier 2003, supra; Tries et al., Inflamm Res 51 : 135-43 (2002). A number of other dual COX/LOX inhibitors, and especially COX-2/5-LOX inhibitors, have been developed, as exemplified by U.S. Patent Nos. 6,753,344 (thiophene substituted hydroxamic acid derivatives), 6,696,477 (heterocyclo substituted hydroxamic acid derivatives), 6,677,364 (substituted sulfonylphenylheterocycles), and U.S. Patent Application Nos. 20040248943 (pyrazole substituted hydroxamic acid derivatives), 20040147565 (substituted sulfonylphenylheterocycles), 20030180402 (flavans isolated from the genus A cacia), and 20030176708 (thiophene substituted hydroxamic acid derivatives).
6. Therapeutic Administration
[0158] In the compositions of the invention, a COX-1, COX-2, or LOX inhibitor is combined with a sEHI. Optionally, the compositions further comprise one or more EETs or an epoxide of EPA, of DHA, or one or more epoxides of both. In some embodiments, the composition is of an epoxide or EPA, of DHA, or epoxides of both, and an sEHI. The compositions of the invention can be prepared and administered in a wide variety of oral, parenteral and topical dosage forms. In preferred forms, compositions for use in the methods of the present invention can be administered orally, by injection, that is, intravenously, intramuscularly, intracutaneous ly, subcutaneously, intraduodenally, or intraperitoneally. The compositions can also be administered by inhalation, for example, intranasally. Additionally, the compositions can be administered transdermally. Accordingly, in some embodiments, the methods of the invention permit administration of compositions comprising a pharmaceutically acceptable carrier or excipient, an inhibitor of COX-1, of COX-2, or of both, or an inhibitor of a LOX, a selected sEHI inhibitor or a pharmaceutically acceptable salt of the inhibitor and, optionally, one or more EETs or epoxides of EPA or of DHA, or of both. In some embodiments, the methods of the invention comprise administration of an sEHI and one or more epoxides of EPA or of DHA, or of both.
[0159] For preparing the pharmaceutical compositions, the pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
[0160] In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from 5% or 10% to 70% of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
[0161] For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
[0162] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution. Transdermal administration can be performed using suitable carriers. If desired, apparatuses designed to facilitate transdermal delivery can be employed. Suitable carriers and apparatuses are well known in the art, as exemplified by U.S. Patent Nos. 6,635,274, 6,623,457, 6,562,004, and 6,274,166. [0163] Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active components in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium
carboxymethylcellulose, and other well-known suspending agents.
[0164] Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
[0165] The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
[0166] The term "unit dosage form", as used in the specification, refers to physically discrete units suitable as unitary dosages for human subjects and animals, each unit containing a predetermined quantity of active material calculated to produce the desired pharmaceutical effect in association with the required pharmaceutical diluent, carrier or vehicle. The specifications for the novel unit dosage forms of this invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular effect to be achieved and (b) the limitations inherent in the art of compounding such an active material for use in humans and animals, as disclosed in detail in this specification, these being features of the present invention.
[0167] A therapeutically effective amount of one or more of the following: an sEH inhibitor, an EET, an EpDPE, or an EpETE, is employed to act as an analgesic alone or in combination with inhibitors of COX -1 or of -2, or both, or of a LOX enzyme. The dosage of the specific compounds depends on many factors that are well known to those skilled in the art. They include for example, the route of administration and the potency of the particular compound. An exemplary dose is from about 0.001 μg/kg to about 100 mg/kg body weight of the mammal.
[0168] EETs, EpDPEs, or EpETEs are unstable, and can be converted to the corresponding diols, in acidic conditions, such as those in the stomach. To avoid this, EETs, EpDPEs, or EpETEs can be administered intravenously or by injection. EETs, EpDPEs, or EpETEs intended for oral administration can be encapsulated in a coating that protects the compounds during passage through the stomach. For example, the EETs, EpDPEs, or EpETEs can be provided with a so-called "enteric" coating, such as those used for some brands of aspirin, or embedded in a formulation. Such enteric coatings and formulations are well known in the art. In some formulations, the compositions of the invention are embedded in a slow-release formulation to facilitate administration of the agents over time.
[0169] It will be appreciated that the sEHIs and, optionally, the EETs, EpDPEs, or EpETEs, do not need to be combined with the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or COX/LOX inhibitor. They can instead be administered separately. If the sEHIs are administered separately (with or without EETs, EpDPEs, or
EpETEs), they should be administered shortly before or concurrently with
administration of the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or
COX/LOX inhibitor. If the sEHI is administered after administration of the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or COX/LOX inhibitor, it should be administered as soon as possible after administration of the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or COX/LOX inhibitor to maximize the synergy with the other inhibitor. Administration of the sEHI will still be beneficial even if it follows the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or COX/LOX inhibitor by some time, however, so long as amounts of the COX-1 inhibitor, COX-2 inhibitor, LOX inhibitor, or COX/LOX inhibitor sufficient to inhibit the respective enzyme are still present.
[0170] It is understood that, like all drugs, sEHIs have half lives defined by the rate at which they are metabolized by or excreted from the body, and that the sEHIs will have a period following administration during which they will be present in amounts sufficient to be effective. If EETs, EpDPEs, or EpETEs are administered after the sEHI is administered, therefore, it is desirable that the EETs, EpDPEs, or EpETEs be administered during the period during which the sEHI will be present in amounts to be effective in delaying hydrolysis of the EETs, EpDPEs, or EpETEs. Typically, the EETs, EpDPEs, or EpETEs will be administered within 48 hours of administering an sEH inhibitor. Preferably, the EETs, EpDPEs, or EpETEs are administered within 24 hours of the sEHI, and even more preferably within 12 hours. In increasing order of desirability, the EETs, EpDPEs, or EpETEs are administered within 10, 8, 6, 4, 2, hours, 1 hour, or one half hour after administration of the inhibitor. Most preferably, the EETs, EpDPEs, or EpETEs are administered concurrently with the sEHI.
EXAMPLES
[0171] The following examples are offered to illustrate, but not to limit the claimed invention.
EXAMPLE 1 - MATERIALS AND METHODS
[0172] Mouse studies. Mice with targeted disruption in exon 1 of the Ephx2 gene were originally obtained from the NIH (Sinai, et al. (2000) J Biol Chem 275, 40504- 40510). These mice were back-crossed onto a C57BL6 (Jackson Laboratories, Bar Harbor, ME) genetic background an additional 10 generations prior to use in this study (Luria, et al. (2007) Journal of Biological Chemistry 282, 2891-2898, Luria, et al. (2009) Am J Physiol Endocrinol Metab 297, E37 '5-383). Ephx2-null
homozygotes or wild type (WT) mice were paired for subsequent breeding. All mice were maintained on a 12-hour light-dark cycle in a temperature-controlled facility, with free access to water and food. Genotyping was performed by PCR, using DNA extracted from tail as described earlier (Luria, et al. (2007) Journal of Biological Chemistry 282, 2891-2898). Studies were conducted using male WT and Ephx2-null mice at eight and 12 weeks of age. Mice were placed on standard chow diet (percent kcal from: protein 28.5 %, fat 13.5%, carbohydrates 58.0%; Rodent diet #5001, Test Diet, Richmond, IN) or a high fat diet (percent kcal from: protein 15.2%,
carbohydrate 42.7%, fat 42.0%; TD 88137, Harlan Teklad, Madison, WI). WT mice were further separated into two groups, in which one group was given a selective sEH inhibitor (TUPS, l-(l-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)- urea lOmg/L (laboratory number sEHI 1709) (Jones, et al. (2006) Bioorg Med Chem Lett 16, 5212-5216, Chiamvimonvat, et al. (2007) J Cardiovasc Pharmacol 50, 225- 237). Drug efficacy was already evaluated in a previous study, showing elevated sEH-substrate to product ratio during two weeks treatment using the same inhibitor and same route of administration (Enayetallah, et al. (2008) J Biol Chem 283, 36592- 36598). Animal handling, experimentation, and euthanasia were conducted in accordance with federal rules and guideline of IACUC at the University of California, Davis.
[0173] Drug Concentrations. Inhibition of sEH was achieved by using the selective sEH inhibitor (TUPS). TUPS was given via drinking water ad lib (10 mg/liter in 1% polyethylene glycol 400). Water consumption was monitored daily, and the concentration of TUPS in water monitored. A second set of WT animals was treated with 1% polyethylene glycol 400 (vehicle). At the end of the study, blood was collected by cardiac puncture from the right ventricle in EDTA-rinsed syringes following lethal injection of sodium pentobarbital (100 mg/kg body weight intraperitoneally). TUPS was extracted from blood, and selected organs (liver, epididymal fat and pancreas) and the concentration was analyzed by liquid
chromatography-tandem mass spectrometry (Hwang, et al. (2007) J Med Chem 50, 3825-3840).
[0174] Body weights, food intake and organ weights. Animal body weight along with food intake was monitored weekly. At the end of the feeding period, various fat pads (epididymal, subcutaneous, retroperitoneum, visceral, and brown adipose tissues), liver, pancreas, brain, muscle and kidney were harvested and weighed; one part of the tissues was snap-frozen in liquid nitrogen and stored at -80 °C for further analysis while another part was fixed in 10% formalin or Z fix for H&E staining (Kushner, et al. (2004) Diabetes 53, 61-66).
[0175] Metabolic measurements. Glucose was measured in blood collected from the tail using a glucometer (Easy Check; Home Aide Diagnostics, Inc). Serum insulin was determined by enzyme-linked immunosorbent assay, using murine insulin as a standard (Crystal Chem Inc., IL). Free fatty acid (FFA) and triglyceride (TG) concentrations were measured by an enzymatic colorimetric method (Wako, Neuss, Germany). Serum leptin was assayed by enzyme-linked immunosorbent assay, using rat leptin standard (Crystal Chem, Inc). Fed glucose measurements were taken between 7-9 am and where indicated, from mice fasted for 12 hours. For insulin tolerance tests (ITTs), mice were fasted for 4 hrs and regular human insulin
(HumulinR; Eli Lilly Corp., Indianapolis, IN) was administered intraperitoneally at a dose of 0.75-1.0 mU/g. Blood glucose values were measured before and 15, 30, 45, 60, 90, and 120 min post injection. For glucose tolerance tests (GTTs), mice were fasted over night, injected (i.p.) with a solution of 20% D-glucose (2 mg/g body weight), and blood glucose was measured before and 15, 30, 60, and 120 min following injection.
[0176] Biochemical analyses. For insulin signaling experiments, animals were fasted overnight, injected (i.p) with insulin (10 mU/g body weight), and sacrificed 10 minutes after injection. Tissues were dissected and immediately frozen in liquid nitrogen. Tissues homogenates were lysed using radio-immunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% TritonX-100, 1% sodium deoxycholate, 5 mM EDTA, 1 mM NaF, 1 mM sodium ortho vanadate and protease inhibitors). Lysates were clarified by
centrifugation at 13,000 rpm for 10 min and protein concentrations were determined using bicinchoninic acid protein assay kit (Pierce Chemical, IL). Proteins (500-1000 μg) were immunoprecipitated with anti-IR (Santa Cruz) and anti-IRSl antibodies (Millipore, MA). Immune complexes were collected on protein G-Sepharose beads (GE Healthcare) and washed with lysis buffer. Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. Immunoblots were performed with the anti- phosphotyrosine monoclonal antibodies 4G10 (Upstate Biotechnology), or antibodies against ¾β, IRS1, pAkt473, Akt, pErk (Cell Signaling) and Erk (Santa Cruz).
Proteins were visualized using enhanced chemiluminescence (ECL, Amersham Biosciences) and pixel intensities of immuno-reactive bands were quantified using FluorChem 9900 (Alpha Innotech, CA).
[0177] Immunohistochemistry. Pancreas tissue was collected from each animal and was fixed in Z-fix (Fisher Scientific) and imbedded in paraffin. Sections (1 μιη) were prepared from each sample and stained for insulin and glucagon using guinea pig anti insulin and rabbit anti glucagon, respectively (Zymed Laboratories). Detection of the first antibody was performed using a fluorescein-conjugated secondary antibodies (Jackson Immunoresearch). Nuclei were detected by 4',6'-diamino-2-phenyl inodole (DAPI; Vector Laboratories, Burlingame, CA). Pancreatic β-islet size was analyzed using Image J software (on the internet at rsb.info.nih.gov/ij). Endothelial cells were stained as previously described (Kostromina, et al., Endocrinology 2010 151(5):2050- 9). Briefly, anti platelet endothelial cell adhesion molecule- 1 (PEC AM 1)-CD31- conjugated Alexa Flour 488 antibodies (BD Pharmingen, Franklin Lakes, NJ) were used at 1 :200 dilution. After immunolabeling, sections were washed and mounted (Kostromina, et al., Endocrinology 2010 151(5):2050-9). Digital images were acquired using a fluorescence microscope (Zeiss, Jana, Germany).
[0178] Statistical analyses. Results are presented as means ± standard error of the means (SEM). Statistical analyses were performed using the JMP program (SAS Institute). Comparisons between groups were made by unpaired two-tailed Student's t test. ITTs and GTTs were analyzed by repeated measures analysis of variance (ANOVA) and area under the curve. Post hoc analysis was performed using Tukey- Kramer honestly significant difference test.
EXAMPLE 2 - RESULTS
[0179] Whole-body soluble epoxide hydrolase deletion and inhibition in mice. To investigate the role of sEH in regulating glucose homeostasis, the physiological effects of its removal was assessed. Disruption of sEH was achieved through the use of whole-body knockout (KO, Ephx2-null) mice (Sinai, et al. (2000) J Biol Chem 275, 40504-40510 and Luria, et al. (2007) Journal of Biological Chemistry 282, 2891-2898) and pharmacological inhibition using a selective sEH inhibitor (Liu, et al. (2009) Br J Pharmacol 156, 284-296). Ephx2-null mice were healthy, fertile and morphologically indistinguishable from control littermates. A PCR product of 290 bp indicates the Ephx2 sequence which distinguished WT from KO mice (band of 560 bp represents the sequence of neomycin gene) (Fig. 1 A). Immunoblot analysis was used to determine the efficiency of sEH deletion in different tissues including those that are insulin-responsive (Fig. IB). sEH protein was expressed in all examined tissue extracts of WT mice, and was absent in Ephx2-null mice indicating efficient deletion of the gene (Fig. IB). A faint band corresponding to the size of sEH was detected in brain homogenates of KO mice, which could indicate trace expression of sEH.
However, previous studies using this KO strain indicate no sEH protein expression in the brain (Iliff, et al. (2007) Experimental Physiology 92, 653-658; Marowsky, et al. (2009) Neuroscience 163, 646-661). Liver and adipose tissue exhibit high and comparable expression of sEH. As expected, treatment of WT mice with sEH inhibitor (sEHI) did not alter sEH expression (Fig. IB).
[0180] sEH-null mice lack both the epoxide hydrolase and phosphatase domains. To ensure that effects are attributed by the epoxide hydrolase domain, a selective epoxide hydrolase inhibitor was used. Pharmacological inhibition was achieved by treating animals with l-(l-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy- phenyl)-urea (TUPS). This compound is an effective and potent inhibitor of sEH (IC50 of 5 and 3 nM for murine and human sEH, respectively) (Enayetallah, et al. (2008) J Biol Chem 283, 36592-36598 and Jones, et al. (2006) Bioorg Med Chem Lett 16, 5212-5216). TUPS was formulated in water using 1% polyethylene glycol 400 to increase solubility, and the efficacy of the compound in mice was previously reported (Enayetallah, supra). The concentration of TUPS in blood and tissue extracts was determined at the end of the study. TUPS concentrations in blood ranged between 800-1000 ng/ml (2- 3μΜ TUPS) (Fig. 1C). These levels are significantly higher than the IC50 concentration determined with in vitro experiments using the recombinant murine sEH enzyme (Liu, et al. (2009) Br J Pharmacol 156, 284-296, Hwang, et al. (2007) J Med Chem 50, 3825-3840). Additionally, liver, epididymal fat and pancreas extracts revealed elevated levels of TUPS, with liver homogenates exhibiting highest concentrations (45 μg/g tissue or 0.12 μιηοΐ/g tissue) compared with epididymal fat and pancreas (3 and 10 μg/ g tissues, or 0.01 and 0.026 μιηοΐ/ g tissue, respectively; Fig. ID). Of note, inhibition of the epoxide hydrolase domain so far has been shown to have little, if any, affect its phosphatase activity. Taken together, these findings indicate that KO mice exhibit efficient gene-deletion and indicate that the sEH inhibitor is efficiently delivered.
[0181] Effects of sEH gene deletion and inhibition on body mass and adiposity. To study the role of sEH in body mass regulation, WT, KO and sEHI -treated WT mice were placed on chow or HFD, and body weights and food intake were measured weekly. As expected, mice on a HFD gained significantly more weight than their counterparts on chow diet (Fig. 2). However, mice treated with sEHI gained significantly more weight than their counterparts on chow diet (Fig. 2A). Increased weight appears to be caused, at least in part, by increased food intake in these mice (Fig. 3A), and is associated with elevated levels of the hormone leptin (Table 2). No differences in food intake were observed between genotypes in mice fed a HFD (Fig. 3B), suggesting that no contribution to body weight or fat deposition is caused by inhibition of sEH under high fat-fed conditions. On chow diet, sEHI-treated mice show tendency to eat more than their littermates (Fig. 3A). Taken together, these data suggest that differences in insulin sensitivity and glucose homeostasis in these mice are primary and not due to body weight alterations. Consistent with food intake and as expected from diet enriched with fat, feeding efficiency was higher in mice when fed HFD (Fig. 3 C).
Table 2
Metabolic parameters of WT, sEHI-treated and
Figure imgf000057_0001
mice
CHOW HFD
WT KO WT+sEHI WT KO WT+SE: iicose (mg/dL)
Fed 194±2.6* 180±8.8* 184±9.3 218±7.7 b 224±8.5 c 170±14.1 Fasted 1 15±12.5** 101±5.3* 138±21 168±4.5 a 137±9.2 167±3.1 l Insulin (mg/ml)
Fed 0.69±0.1* 0.5±0.1 * 1.2±0.28 c 4.5±1.6 2.8±0.7 1.7±0.i Fasted 0.54±0.2 0.46±0.1 1.2±0.4 0.8±0.1 0.75±0.2 0.7±0.C in/Glucose Ratio
Fed 0.35±0.04* 0.31±0.04*c 0.67±0.1 b,c 2.2±0.8b 1.25±0.4 0.96±0. Fasted 0.45±0.1 0.46±0.01 0.89±0.3 0.5±0.06 0.55±0.1 0.43±0.i eptin (ng/ml)
Fed 8.8±0.4* 13.3±2.6 16.5±1.7 b 21.7±1.8 21.3±2.9 20.6±1 , Fasted 3.2±0.3* 3.9±1.1* 12.7+0.6 b* 18.2±1.6 19.5±2.1 19.7±1. lonectin ^g/ml)
Fed 6.3±0.8 5.0±0.4 5.8±0.8 5.6±0.35 5.3±0.6 5.5±0.C Fasted 6.7±0.4 6.6±0.1 * 7.0±0.8 4.6±1.3 4.8±0.1 4.3±1.! ycerides (mg/ml)
Fed 0.48±0.1 0.8±0.06 a* 0.64+0.03* 0.4±0.04 0.4±0.05 0.3±0.C Fasted 0.29±0.01 0.3±0.04* 0.27±0.03* 0.4±0.03 0.43±0.02 0.4±0.C
Serum was collected from fed and fasted male WT, KO and WT+sEHI mice (five months on chow or HFD), and the indicated metabolic parameters were measured. Values are expressed as the mean ± SEM of measurements obtained four animals per genotype. * Significant difference (*P<0.05) between HFD and Chow; a, indicates significant difference between KO and WT mice, b, significant difference between WT and WT+sEHI, and c, significant difference between KO and WT+sEHI treated group.
[0182] Diet-induced obesity is reflected in adiposity (Fig. 4). Several fat depots such as brown adipose tissue, subcutaneous fat pad, epididymal, visceral and
retroperitoneum fat pads were dissected and weighed. Consistent with increased body mass, adipose tissue weight was significantly increased in all animals fed a HFD (Fig.
4). Surprisingly, despite the body weight difference between sEHI-treated WT mice to KO and WT mice fed chow diet, no differences in fat depot weights were observed (Fig. 4). H&E staining was further assessed to see any pathological differences between groups and diets and to rule out any potential toxic effects due to the treatment (Fig. 5). Analysis of adipose tissue, liver, kidney, adrenal glands and pancreas sections showed no adverse effects due to sEH inhibition (Fig. 5).
However, significant morphological changes due to the diet were detected in epididymal fat and liver sections (Fig. 5 A and B). Diet-induced hypertrophy in adipocytes and fatty liver were clearly seen in all animal groups on HFD (Fig. 5 A and B). Although diet-induced obesity is known to cause leukocyte infiltration (Kubota, et al. (1999) Molecular Cell 4, 597-609; Lumeng, et al. (2007) Diabetes 56, 16-23; and Xu, et al. (2003) J Clin Invest 112, 1821-1830), no significant changes in leukocytes were seen in HFD-treated liver and fat tissues (Fig. 5 A and B).
Interestingly, a difference was noticed in pancreas sections (Fig. 5 E). Larger pancreatic β-islets were observed in HFD treated Ephx2-null mice, and to a lesser extent in sEHI-treated mice (Fig. 5 E). Diet induced morphological changes were not seen in kidney and adrenal gland sections (Fig. 5 C and D), suggesting no adverse effect by TUPS treatment or disruption of the gene (Fig. 5 C and D).
[0183] Improved insulin sensitivity and glucose tolerance in mice with Ephx2 deletion and sEH inhibition. Several metabolic parameters were assayed in Ephx2- null, WT and sEHI-treated WT mice on chow and a HFD (Table 2). These parameters were analyzed in both fasted and fed conditions. Despite their similar body weights and adiposity, fasted glucose levels in Ephx2-null mice on a HFD were lower than those of WT and sEHI -treated mice (Table 1), indicating improved insulin sensitivity. When animals were normally fed, glucose levels on HFD and chow were comparable (Table 2). Consistent with this observation, sEHI-treated and KO mice exhibited lower levels of serum insulin levels on HFD compared with WT mice (Table 2). On chow diet, only KO mice showed lower levels of serum insulin (Table 2 fed conditions). When the ratio of insulin to glucose was calculated, KO mice either on chow or a HFD exhibited lower insulin to glucose ratios compared with WT counterparts. This is in line with improved insulin sensitivity. When mice were fasted, insulin to glucose ratios tended to be lower but did not reach statistical significance. As expected, the differences in these parameters are blunted when mice were fasting for 16 hours. sEHI -treated WT mice exhibited lower glucose levels, serum insulin levels and insulin to glucose ratios only when they were not fasting and on HFD (Table 2). It is suggested that the increased weight in this group (Fig. 1 A) masks any differences in these parameters. In addition to that, several other parameters of whole-body homeostasis were measured. As expected, mice on HFD exhibited higher plasma leptin levels compared with those on chow diet. In line with body weight, significantly higher levels of leptin are detected in the sEHI -treated WT mice (Table 2). Since high secretion of leptin from the brain increases appetite, leptin is suggested to be a cause of the weight difference in sEHI-treated WT mice on chow diet. Although Ephx2-null and sEHI-treated WT mice show insulin sensitivity, no significant alterations in adiponectin, an insulin-sensitizing adipokine (Kadowaki and Yamauchi (2005) Endocrine Reviews 26, 439-451) level were observed. Finally, no significant differences were seen in triglyceride levels in mice fed HFD. However, when mice were on chow diet, inhibition of sEH tends to increase serum levels of triglycerides (Table 2, fed conditions).
[0184] To directly assess insulin sensitivity in vivo, mice were subjected to insulin tolerance tests (ITTs) at two (Fig. 6 A-D) and five months (Fig. 7) after the experiment started. On chow diet, KO mice exhibited significantly greater reduction in blood glucose following insulin injection compared with controls (Fig. 7A). This effect was more pronounced in mice fed a HFD, with KO and sEHI-treated WT mice exhibiting improved insulin sensitivity compared with controls (Fig. 7B). In addition to their improved insulin sensitivity, KO mice exhibited improved ability to clear glucose from the peripheral circulation during an intraperitoneal (i.p.) glucose tolerance test (GTT) both on chow and HFD (Fig. 7C and D). Additional ITT and GTT were performed on another independent cohort of mice on chow and HFD and comparable results were observed (Fig. 6 E and F).
[0185] To test whether the difference observed in GTT was caused by alterations in insulin concentration over time, serum insulin concentrations were measured at 0, 15 and 30 minutes during GTTs. No significant differences in insulin levels were observed between KO, sEHI-treated WT and WT control mice on chow diet (Table 3). However, on HFD, increased insulin levels were measured in KO mice at 30 minutes (Table 3). Collectively, these data indicate that mice with sEH inhibition or deletion have improved insulin sensitivity and enhanced glucose tolerance. Table 3
Glucose-stimulated insulin secretion during glucose tolerance test
iicose (mg/dL) CHOW HFD
Time (min) \\ I KO WT+sEHI WT KO WT+sE]
0 115±13 101±3 a 139±15 c 176±9 140±8 a 167±3
15 541 ±4 480±29 376±39 c,b 547±39 450±17 a 553±24
30 637±14 427±37 a 598±33 c 634±16 507±13 a 639±11 sulin (mg/ml) CHOW HFD
Time (min) \\ I KO WT+sEHI WT KO WT+sE]
0 0.8±0.05 0.6±0.04 0.8±0.2 0.7±0.06 1.3±0.3 a 0.8+0.
15 5.0±0.05 5.8±0.4 5.2±0.5 6.4±0.6 8.0±1.0 6.4+0.:
30 5.0±0.3 6.3±0.3 6.6±0.6 b 6.5±0.8 4.8±0.1 a 6.3±0.4
Blood was collected from overnight fasted WT, sEHI-treated WT (TUPS, lOmg/L via drinking water, ad libitum) or Ephx2-null male mice fed a high fat diet (HFD) or regular standard chow diet (test was taken after 24 weeks on diet). The indicated values of glucose and Insulin were measured before and after intraperitoneal injection of glucose (2mg/g of body mass). The values are expressed as the means SEM (n=4). a, b, c, P<0.05, using each pair comparison T-TEST; a, WT vs. KO; b, WT vs.
WT+sEHI; c, KO vs. WT+sEHI.
[0186] Effect of sEH deletion and inhibition on β-islet size and vascularization.
To better understand the mechanism underlying enhanced glucose-stimulated insulin secretion in KO mice on HFD, pancreas was stained for insulin and glucagon, and the size of the islets was determined. Notably, KO and sEHI-treated WT mice on a HFD exhibited increase in islet size compared with WT controls (Fig. 8A and B).
Glucagon staining in the outer rim of the pancreatic β-islets shows no differences among all groups (Fig. 9). Quantitative data are shown in Fig. 8B, supporting the larger increase in islet size when sEH is inhibited either pharmacologically or
genetically. In order to explain the pancreatic size increase in KO and sEHI-treated
WT mice, pancreas sections for endothelial cells were stained, using CD31
immunostaining as a marker. As seen in Fig. 8C, vascular density, as indicated by
CD31 -labeled area in endocrine pancreas appeared to be enhanced particularly in the Ehpx2-null mice on HFD, and marginally enhanced in sEHI-treated WT mice (Fig.
8C). Moreover, it is also very clear that KO-HFD mice, and to a lesser extent the sEHI -treated mice, have more nuclei stained in both the endocrine and exocrine areas (Fig. 8C), might be a result of either an increase in proliferation or decrease in
apoptosis. Because VEGF is essential in the regulation of capillary network
formation, these data suggest a link between elevated EETs, VEGF and
vascularization. Previous studies show a strong link between EETs and VEGF
secretion (Webler, et al. (2008) Am. J. Physiol 295, C1292-1301). Furthermore, it is well known that high expression levels of VEGF observed in endocrine cells are responsible for strong islet vascularization and hence improved physiological function of islets (Cleaver and Melton (2003) Nat Med 9, 661-668; Lammert, et al. (2001) Science 294, 564-567; and Lammert, et al. (2003) Curr Biol 13, 1070-1074).
Measurements of plasma VEGF and assessment of it in pancreatic homogenates are needed to establish the link between VEGF, EETs and pancreatic vasculature.
[0187] Increased insulin signaling in liver and adipose of mice with sEH gene deletion or inhibition. To investigate the molecular basis for enhanced insulin sensitivity in Ephx2-null and sEHI-treated mice, mice were injected with insulin or saline (control), and the activation status of components in the insulin signaling pathway was examined in both liver (Fig. 10) and epididymal adipose (Fig. 11) extracts after HFD. In the liver and adipose homogenates, insulin increased IR tyrosyl phosphorylation (Yl 162/Y 1163) in both KO mice and those treated with TUPS (Fig. 10 A-B and 11 A-B). Interestingly, the IR was basally
hyperphosphorylated in liver extracts of KO mice (Fig. 10A-B, at time 0'), presumably represents higher IR sensitivity state. In liver, total tyrosyl
phosphorylation of IRS- 1 in KO and sEHI-treated WT mice upon insulin stimulation was higher than the WT controls, but not significantly different than 0' time point (Fig. 10C-D ). While, in adipose tissue this was more pronounced after insulin stimulation (Fig. 11 C-D). In line with that, phosphorylated-tyrosine residue 608 was higher basally and after insulin stimulation in both liver and adipose tissue of KO and EHI-treated WT mice (Fig. 10 and Fig. 11 C,E). Binding interaction between P85 and IRS-1 was greatly enhanced after insulin injection in KO liver extracts and in both KO and sEHI-treated WT adipose tissue extracts (Fig. 10 and 11 C,F). Interestingly, binding between these proteins was more pronounced in basal state in sEHI-treated WT liver and KO adipose tissue (Fig. 10 F and Fig. 11 F, respectively). Consistent with the increased IRS-1 tyrosyl phosphorylation and PI3K binding, insulin stimulated downstream pathway, Akt phosphorylation (Ser473) and MAPK were also enhanced in KO mice and mice treated with TUPS in both tissue homogenates (Fig. 10 G-J and Fig. 11 G-J). Our data show no differences in TNF and MCP1 expression levels between WT and KO mice from liver and adipose extracts (Fig. 11 K-M). Taken together, these data suggest that the increased insulin sensitivity in KO and sEHI -treated mice result from enhanced activation of PI3K/Akt and MAPK signaling pathway downstream of the IR in liver and adipose tissue.
EXAMPLE 3 - DISCUSSION
[0188] The present study clearly shows that Ephx2 gene deletion is sufficient to attenuate insulin resistance development in a murine -model of type 2 diabetes induced by obesity. Loss of sEH activity results in enhanced insulin-sensitizing actions, marked increase in insulin receptor signaling that stabilizes serum glucose levels. These observations were also supported with pharmacological inhibition of epoxide hydrolase activity, suggesting a therapeutic role for its substrates EETs. These mediators are potent endogenous compounds with beneficial vascular actions (Larsen, et al. (2007) Trends Pharmacol Sci 28, 32-38), anti-inflammatory effects (Schmelzer, et al. (2005) Proc Natl Acad Sci USA 102, 9772-9777) and anti-hypertensive modulators (Imig, et al. (2002) Hypertension 39, 690-694). sEH activity limits EETs availability and hence inhibition of the enzyme has been a therapeutic approach for a variety of disease conditions. Therefore, sEH inhibition finds use in preventing progression of type 2 diabetes induced by high fat diet.
[0189] With regard to insulin resistance and metabolic syndrome, reduced sEH activity and the resulting increased in EETs have a beneficial effect on insulin sensitivity in a diabetes type 1 model (Luo, et al, J Pharmacol Exp Ther. (2010) 334(2):430-8). It has been reported that cytochrome P450 enzyme (CYP 2J,C) expression is decreased and sEH expression is increased in mesenteric arteries of obese Zucker rats (Zhao, et al. (2005) Am J Physiol Regul Integr Comp Physiol 288, R188-196). On the other hand, streptozotocin-induced diabetic mice have lower sEH expression in liver and kidney, as a result of increase in reactive oxygen species generation (Oguro, et al. (2009) Drug Metabolism and Pharmacokinetics 24, 438- 445). Although this observation suggests a link between bioavailability of EETs and development of metabolic syndrome, the physiological roles of sEH in diabetes are still unknown. The level of expression and the distribution pattern of sEH and CYP450 epoxygenases in any particular tissue may alter the availability of EETs. sEH is ubiquitously expressed in many tissues, as well as in the pancreas, muscle and adipose. Particularly, sEH is locally expressed in pancreatic β-islet cells (Luo, et al., J Pharmacol Exp Ther. (2010) 334(2):430-8). sEH also regulates adipogenesis and its expression levels are up regulated in response to high fat diet (De Taeye, et al.
Obesity (2010) 18(3):489-98). These observations suggest a direct link between sEH and glucose homeostasis.
[0190] In order to further understand the physiological roles of sEH in glucose homeostasis and insulin sensitivity in obese mice, genetically disrupted Ephx2-null mice (KO) were used, which have no expression of either the epoxide hydrolase or phosphatase domain. Pharmacological inhibition of sEH was also employed, e.g., by using selective sEH inhibitor, TUPS. This drug is a potent inhibitor of the epoxide hydrolase in vitro, and has no apparent effect on the phosphatase activity of the protein. Thus, TUPS was used to confirm that the effects observed with Ephx2-null mice in the diabetes model were largely due to the absence of the epoxide hydrolase activity. Since this drug has high bioavailability and a long half life (Liu, et al.
(2009) Br J Pharmacol 156, 284-296), the high doses used resulted in high blood and tissue levels of TUPS generating a good chemical knockout. Efficacy was measured in previous studies, showing that plasma levels of EETs are elevated in the presence of this drug or naturally enhanced in Ephx2-null mice (Enayetallah, et al. (2008) J Biol Chem 283, 36592-36598). In spite of the high doses of TUPS, no adverse effects were seen in the pathological analysis of these organs. Evidence provided shows that both Ephx2 knockout and sEHI-treated mice store similar amounts of excess of fat compared with WT mice when provided a hypercaloric diet. As expected from the diet, insulin-responses organs were dramatically affected by the high levels of fats.
[0191] Despite the absence of any differences in body weight and/or adiposity when mice fed a HFD, disruption of Ephx2 exhibit marked improvements in whole-body glucose homeostasis and insulin sensitivity, as evidenced by a considerably enhanced ability to clear glucose from peripheral circulation during GTT and a significantly greater decrease in blood glucose during ITT. Thus, the effect of sEH on insulin sensitivity appears not due to impairment of adipocyte function, but rather is a direct effect on the production and response to insulin. Improved glucose homeostasis was only seen with the null mice, suggesting that elimination of the phosphatase or some other genetic alteration in the Ephx2-null mice could be involved.
[0192] Adipocytes can contribute to the maintenance of whole body glucose homeostasis either by the release of insulin-sensitizing adipose-derived hormones (adipokines) or through the sequestering of excess fatty acids and triglycerides that induce insulin resistance (Tontonoz and Spiegelman (2008) Ann Rev Biochem 77, 289-312; Rader (2007) Am J Med 120, S 12-18). Our study reveals that sEH deficiency and inhibition improves insulin sensitivity on HFD. Our biochemical data support the notion that improved whole-body glucose homeostasis and insulin sensitivity in sEH null and sEHI -treated mice is the direct result of increased insulin signaling. This mechanism is accomplished through highly complex pathways involving many different molecules, cell types, and organs. Obesity-induced insulin resistance results in insufficient circulating levels of serum insulin and decreased responsiveness of insulin receptors and its downstream signaling in 'insulin-responses tissues' (Biddinger and Kahn (2006) Annu Rev Physiol 68, 123-158; Uysal, et al. (1997) Nature 389, 610-614; and Farese (2001) Exp Biol Med 226, 283-295). The marked improvement in glucose homeostasis and systemic insulin sensitivity is evidently due to increased insulin signaling and enhanced activation of downstream signaling. Our data provide insights into the regulation of insulin signaling modulate by sEH. Mice lacking sEH exhibit enhanced basal and insulin-initiated IR tyrosyl phosphorylation, followed by an increase in its substrate, IRS-1 tyrosyl
phosphorylation. The higher basal phosphorylation of amino acid tyrosine (Y608) in both KO and sEHI-treated WT mice, probably represents maximal phosphorylation of this site. Hence, further enhancement of phosphorylation upon insulin treatment is not observed. Consistent with this, PI3K association with IRS-1 is elevated and MAPK and AKT pathways were initiated. As previously described, these pathways lead into glucose transporter (GLUT4) translocation to the plasma membrane and glucose uptake into the cells (Schmitz-Peiffer and Whitehead (2003) IUBMB Life 55, 367-374). The signaling cascade observed in KO mice was supported using TUPS. Hence, our findings demonstrate that high endogenous levels of EETs lead to a significant enhancement in insulin response.
[0193] Large number of studies showing that sEH inhibition and the resulting increase in EETs exert beneficial vascular and anti-inflammatory effects (33, 42). Although diet-induced obesity is defined as a sub-inflammatory disease (Kubota, et al. (1999) Molecular Cell 4, 597-609; Lumeng, et al. (2007) Diabetes 56, 16-23; Xu, et al. (2003) J Clin Invest 112, 1821-1830), no anti-inflammatory response was observed. In a different model of insulin resistance due to heme oxygenase 2 gene disruption, the addition of EETs with sEHI caused a marked improvement in all metabolic parameters including inflammatory mediators, TNFa and MCP-1 (Sodhi, et al. (2009) J Pharmacol Exp Ther 331, 906-916). Another striking observation of this study is the increase in the size of pancreatic β-islets and the associated vasculature in both Ephx2-null and sEHI-treated WT mice. Pancreatic β-islet compensatory response to insulin resistance is a recognized feature in obesity and type 2 diabetes. However, the signal and proteins that mediate this important adaptive response are poorly understood (Flier, et al. (2001) Proc Natl Acad Sci USA 98, 7475-7480). Pancreatic β-islets can respond in vivo to a sustained glucose stimulus by increasing their mass through hyperplasia or hypertrophy. Furthermore, endothelial cells can affect the ability of pancreatic β-islets to grow in size when demands for insulin increase (Lammert, et al. (2003) Curr Biol 13, 1070-1074; Nikolova, et al. (2006) Developmental Cell 10, 397-405). A previous study with sEH-null mice in a type 1 diabetes model suggests an increase insulin secretion via reduced apoptotic cell death in β-islets (Luo, et al, J Pharmacol Exp Ther. (2010) 334(2):430-8), and the link between CYP2C-derived EETs, VEGF and endothelial proliferation (Webler, et al. (2008) Am. J. Ajszo/ 295, C1292-1301; and Potente, et a/. (2002) J Biol Chem 277 ', 15671-15676) can also contribute to the large islet size as seen in this study.
[0194] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, practice the present invention to its fullest extent.
[0195] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:
1. A method of maintaining stable glucose levels in an individual in need thereof, the method comprising co-administering to the individual
a) (i) a first enzyme inhibitor that inhibits soluble epoxide hydrolase ("sEH"), (ii) an epoxygenated fatty acid, or (iii) both an inhibitor of sEH and an epoxygenated fatty acid, and
b) a second enzyme inhibitor that inhibits one or more enzymes selected from the group consisting of cyclo-oxygenase ("COX") -1, COX-2, and 5- lipoxygenase ("5-LOX").
2. The method of claim 1, wherein the individual is hyperglycemic.
3 . The method of claim 1, wherein the individual is prediabetic.
4. The method of claim 1, wherein the individual has type 1 diabetes.
5. The method of claim 1, wherein the individual has type 2 diabetes.
6. The method of claim 1 , wherein one or both of the first and second enzyme inhibitors are administered at subtherapeutic doses.
7. The method of claim 1 , wherein one or both of the first and second enzyme inhibitors are administered in a sustained release formulation.
8. The method of claim 1, wherein the epoxygenated fatty acid is an EET.
9. The method of claim 8, wherein the EET is selected from the group consisting of 14,15-EET, 8,9-EET, 11,12-EET and 5,6-EET.
10. The method of claim 1 , wherein the epoxygenated fatty acid is an epoxide of docosahexaenoic acid ("DHA") or eicosapentaenoic acid ("EPA"), or epoxides of both DHA and of EPA.
11. The method of claim 1 , wherein the first enzyme inhibitor has a primary pharmacophore selected from the group consisting of a urea, a carbamate and an amide.
12. The method of claim 1 , wherein the second enzyme inhibitor inhibits COX-2.
13. The method of claim 12, wherein the second enzyme inhibitor is selected from the group consisting of celecoxib, valdecoxib, lumiracoxib, etoricoxib, and rofecoxib.
14. The method of claim 1 , wherein the second enzyme inhibitor inhibits COX-1.
15. The method of claim 14, wherein the second enzyme inhibitor is selected from the group consisting of aspirin, acetaminophen, diclofenac potassium, diclofenac sodium, diclofenac sodium with misoprostol, diflunisal, disalsalate, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate sodium, mefenamic acid, meloxicam, nabumetone, naproxen sodium, piroxicam, tolmetin sodium, magnesium salicylate, choline salicylate, salsalate, salicylic acid esters and sodium salicylate.
16. The method of claim 1 , wherein the second enzyme inhibitor inhibits 5-LOX.
17. The method of claim 16, wherein the second enzyme inhibitor is a FLAP inhibitor or a leukotriene antagonist.
18. A method of improving insulin receptor sensitivity and/or glucose clearance in a prediabetic subject, the method comprising administering to the subject an effective amount of an agent selected from the group consisting of (i) an inhibitor of sEH, (ii) an epoxygenated fatty acid, or (iii) both an inhibitor of sEH and an epoxygenated fatty acid.
19. The method of claim 18, wherein the epoxygenated fatty acid is an EET.
20. The method of claim 19, wherein the EET is selected from the group consisting of 14,15-EET, 8,9-EET, 11,12-EET and 5,6-EET.
21. The method of claim 19, wherein the epoxygenated fatty acid is an epoxide of docosahexaenoic acid ("DHA") or eicosapentaenoic acid ("EPA"), or epoxides of both DHA and of EPA.
22. The method of claim 18, wherein the inhibitor of sEH has a primary pharmacophore selected from the group consisting of a urea, a carbamate and an amide.
23. The method of claim 18, further comprising co-administering to the subject an inhibitor of one or more enzymes selected from the group consisting of cyclo-oxygenase ("COX") -1, COX-2, and 5 -lipoxygenase ("5-LOX").
24. The method of claim 23, wherein an inhibitor of COX-2 is co-administered.
25. The method of claim 24, wherein the inhibitor of COX-2 is selected from the group consisting of celecoxib, valdecoxib, lumiracoxib, etoricoxib, and rofecoxib.
26. The method of claim 23, wherein an inhibitor of COX-1 is co-administered.
27. The method of claim 26, wherein the inhibitor of COX-1 is selected from the group consisting of aspirin, acetaminophen, diclofenac potassium, diclofenac sodium, diclofenac sodium with misoprostol, diflunisal, disalsalate, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate sodium, mefenamic acid, meloxicam, nabumetone, naproxen sodium, piroxicam, tolmetin sodium, magnesium salicylate, choline salicylate, salsalate, salicylic acid esters and sodium salicylate.
28. The method of claim 23, wherein an inhibitor of 5-LOX is coadministered.
29. The method of claim 28, wherein the inhibitor of 5-LOX is a FLAP inhibitor or a leukotriene antagonist.
30. The method of claim 23, wherein one or both of the inhibitor of sEH and the inhibitor of COX-1, COX-2 or 5-LOX are administered at subtherapeutic doses.
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