Epoxy Fatty Acids Are Promising Targets for Treatment of Pain, Cardiovascular Disease and Other Indications Characterized by Mitochondrial Dysfunction, Endoplasmic Stress and Inflammation

Abstract

Bioactive lipid mediators resulting from the metabolism of polyunsaturated fatty acids (PUFA) are controlled by many pathways that regulate the levels of these mediators and maintain homeostasis to prevent disease. PUFA metabolism is driven primarily through three pathways. Two pathways, the cyclooxygenase (COX) and lipoxygenase (LO) enzymatic pathways, form metabolites that are mostly inflammatory, while the third route of metabolism results from the oxidation by the cytochrome P450 enzymes to form hydroxylated PUFA and epoxide metabolites. These epoxygenated fatty acids (EpFA) demonstrate largely anti-inflammatory and beneficial properties, in contrast to the other metabolites formed from the degradation of PUFA. Dysregulation of these systems often leads to chronic disease. Pharmaceutical targets of disease focus on preventing the formation of inflammatory metabolites from the COX and LO pathways, while maintaining the EpFA and increasing their concentration in the body is seen as beneficial to treating and preventing disease. The soluble epoxide hydrolase (sEH) is the major route of metabolism of EpFA. Inhibiting its activity increases concentrations of beneficial EpFA, and often disease states correlate to mutations in the sEH enzyme that increase its activity and decrease the concentrations of EpFA in the body. Recent approaches to increasing EpFA include synthetic mimics that replicate biological activity of EpFA while preventing their metabolism, while other approaches focus on developing small molecule inhibitors to the sEH. Increasing EpFA concentrations in the body has demonstrated multiple beneficial effects in treating many diseases, including inflammatory and painful conditions, cardiovascular disease, neurological and disease of the central nervous system. Demonstration of efficacy in so many disease states can be explained by the fundamental mechanism that EpFA have of maintaining healthy microvasculature and preventing mitochondrial and endoplasmic reticulum stress. While there are no FDA approved methods that target the sEH or other enzymes responsible for metabolizing EpFA, current clinical efforts to test for efficacy by increasing EpFA that include inhibiting the sEH or administration of EpFA mimics that block metabolism are in progress.

Keywords: Epoxide; Lipid metabolism; Oxylipin; PUFA; sEH.

Fig. 5.1

Metabolic fate of polyunsaturated fatty acids Free fatty acids are primarily metabolized through β-oxidation, Cyclooxygenase (COX), Lipoxygenase (LOX or LO), and CytochromeP450 (CYP450). A simplistic overview of the biological action of the resulting metabolites is shown in parenthesis. COX 1 and COX 2 metabolize PUFA to PGH2 which is the precursor to other inflammatory prostanoids and thromboxanes that regulate the immune system (PGD₂), increase pain and inflammation (PGE₂) and control platelet aggregation (PHI₂ and TXA₂). [108] 5-LO metabolizes PUFA to 5-HpETE or 15-LO to 15-HpETE, both are precursors to inflammatory leukotrienes and cytotoxic leukotoxins. These metabolites are important in exacerbating asthma by acting as powerful bronchoconstrictors, and they can also sustain inflammatory reactions through chemotaxis of inflammatory mediators. LO metabolism of omega-3 fatty acids result in pro-resolving lipid mediators called Resolvins, Protectins and Maresins. [139] CYP450 metabolizes PUFA to anti-i nflammatory epoxy fatty acids (EpFAs), n-terminal hydroxylated n-HETE, ω−1 oxidation, or allylic hydroxylations. 20-HETE regulates blood pressure by acting as a potent vasoconstrictor in kidneys and preventing sodium reabsorption in nephrons [140, 141]. The biological significance of mid-chain hydoxylations is less understood; however, biological significance has been observed with 12-HETE in corneal inflammation and neovascularization [142, 143]. Formation of EpFA, particularly if induced, is primarily accomplished by CYP2C and CYP2J subfamily; however, other CYP enzymes can generate EETs [144]. The EpFA act as homeostatic regulators to other metabolites in this pathway by stabilizing mitochondria, reducing ROS, decreasing ER-stress [20] and inflammation [15], regulating the vascular endothelium [88] and increasing bronchodilation [145]

Fig. 5.2

Formation of EpFA Polyunsaturated fatty acids (PUFAs) differ in both structure and function based on number of carbons and location of double bonds. They are incorporated as glycerides in fat cells, cellular membranes or circulating micelles and are liberated to free fatty acids by different phospholipases (PL) that act upon different areas of the glyceride or phospholipid (A). EpFA are formed through the oxygenation of FFA by CYP450. The metabolism of Arachidonic Acid (AA, 20:4 n-6) by cytochrome P450 yields EpFAs, epoxyeicosatrienoic acids (EET), which are further degraded by the soluble epoxide hydrolase into dihydroxyeicosatrienoic acids (DHET). The epoxide and diol on the 11,12 position is shown, but similar regioisomers are possible on all the double bonds in the PUFA. The n-6 fatty acid linoleic acid, LA, has been attributed to largely inflammatory epoxides, EPOMES; however, recent studies show that they are only toxic in the presence of sEH, suggesting that the diols of LA, DIHOMES, are responsible for this inflammatory action [137]. The 18:3 omega-3 fatty acid, linolenic acid (ALA), does not seem to have this same inflammatory action. The omega-6 fatty Adrenic Acid, AdA, is named for its abundance in the adrenal gland. Less is known about this PUFA and its metabolites, although the AdA EpFA, dihomoEETs are thought to regulate blood flow to the adrenal gland [138]. n-3 fatty acids alpha eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) form EpFA epoxyeicosatetraenoic acids (EEQs) and epoxydocosapentaenoic acids (EDPs) respectively. Omega-3 EpFA are largely beneficial with anti-inflammatory and pain resolving properties in vitro and in vivo. Little biological activity has been associated with the diols of Ada (dihomoDHET) or the diols of EPA and DHA, dihydroxyeicosatetraenoic acids (DHEQs) and dihydroxydocosapentaenoic acids (DiHDPAs) respectively. (see [86] for review)

Fig. 5.3

Mechanism of epoxide to diol metabolism by the soluble epoxide hydrolase (sEH) Two acidic tyrosines are oriented by pi-stacking to bind to and polarize the epoxide moiety. sEH inhibitors functionally mimic transient intermediates and the transition state of the enzyme (middle panel). The NH groups of the urea, amide, carbamate or other electronegative groups possibly encourage a salt bridge formation, for example between a polarized urea and the catalytic aspartic acid, likely stabilized by hydrogen bonds with the tyrosine. [146]

Fig. 5.4

Possible ways of increasing natural epoxy fatty acid chemical mediators to prevent and treat disease Increasing EpFA concentrations in the body have been associated with treating many diseases. The figure above demonstrates potential pathways of increasing their concentrations through increasing their biosynthesis or through preventing their metabolism. EpFA can be increased by increased PUFA consumption, increasing release from lipid membranes, or increased formation through CYP450 activity. Inhibiting the metabolic enzymes that convert EpFA to more polar compounds that are rapidly eliminated from the body, or supplementing the diet with EpFA mimics that prevent degradation by β oxidation are other ways in increasing the concentrations of the beneficial fatty acids