Soluble epoxide hydrolase as a therapeutic target for pain, inflammatory and neurodegenerative diseases

Abstract

Eicosanoids are biologically active lipid signaling molecules derived from polyunsaturated fatty acids. Many of the actions of eicosanoid metabolites formed by cyclooxygenase and lipoxygenase enzymes have been characterized, however, the epoxy-fatty acids (EpFAs) formed by cytochrome P450 enzymes are newly described by comparison. The EpFA metabolites modulate a diverse set of physiologic functions that include inflammation and nociception among others. Regulation of EpFAs occurs primarily via release, biosynthesis and enzymatic transformation by the soluble epoxide hydrolase (sEH). Targeting sEH with small molecule inhibitors has enabled observation of the biological activity of the EpFAs in vivo in animal models, greatly contributing to the overall understanding of their role in the inflammatory response. Their role in modulating inflammation has been demonstrated in disease models including cardiovascular pathology and inflammatory pain, but extends to neuroinflammation and neuroinflammatory disease. Moreover, while EpFAs demonstrate activity against inflammatory pain, interestingly, this action extends to blocking chronic neuropathic pain as well. This review outlines the role of modulating sEH and the biological action of EpFAs in models of pain and inflammatory diseases.

Keywords: Alzheimer's disease; Depression; Epoxy-fatty acids (EpFAs); Inflammatory pain; Neuropathic pain; Soluble epoxide hydrolase (sEH).

Figure 1. Long chain polyunsaturated acid metabolism through the CYP450 pathway

Arachidonic acid (ARA) and other long chain polyunsaturated fatty acids (LC-PUFA) are metabolized by cytochrome P450 enzymes (CYP450) into the epoxy-fatty acids (EpFA). For simplicity, the metabolism of omega-6 ARA is depicted here as an example of LC-PUFA metabolism. A class of EpFA, the epoxyeicosatrienoic acids (EETs), are formed from ARA. Four individual regioisomers can be formed by the epoxidation of any one of the four double bonds with the 14,15 EET depicted. In addition to the epoxides from LC-PUFA, any fatty acids with an olefinic bond may form epoxidized metabolites. The soluble epoxide hydrolase (sEH) adds water to the oxirane ring to yield the diol, in the case of ARA metabolites are termed dihydroxyeicosatrienoic acids (DHETs). This process is the same for omega-3 LC-PUFA including DHA and EPA which form potent biologically active classes of EpFA.

Figure 2. EpFA block inflammation through several mechanisms

Regioisomers of the EET class of EpFA inhibit VCAM-1, E-selectin and ICAM-1 expression in endothelial cells which blocks the adherence and infiltration of activated monocytes. EETs also reduce inflammation by blocking the nuclear translocation of NFκB, and sEHI administration which elevates all EpFA, blocks the increase of phospho-ΙκΒα levels which activate NFκB and thus inhibit NFκB signaling. This results in the downregulation of several enzymes including calcium-insensitive nitric oxide synthase (iNOS), lipoxygenase-5 (LOX-5), and cyclooxygenase-2 (COX-2, pictured) that are upregulated in inflammation. The downregulation of COX-2 also limits the production of prostaglandin E₂ (PGE₂) which is a potent inflammatory agent. EETs also decrease TNFα secretion from monocytic cells and inflammatory cytokines in several tissues. Activation of signal transducer and activator of transcription 3 (STAT3) and other nuclear receptors such as peroxisome proliferator activated receptor (PPAR) alpha and gamma are additional anti-inflammatory mechanisms that have been described for EpFA in blocking downstream inflammation.

Figure 3. sEH inhibition and EpFA blocks natural arthritic pain

Male and female beagle dogs (ages 8–14 years) with naturally occurring osteoarthritis were administered the sEHI t-TUCB at 5 mg/kg orally in a capsule or placebo control for five days (n=7 dogs/group). The results are presented as an average of 5 days of testing pre-treatment (pooled baseline) compared to an average of 5 days of testing under treatment (placebo or t-TUCB). The sEHI significantly lowered pain scores compared to both pooled baseline and placebo control (p ≤ 0.001).

Figure 4. sEH inhibition and EpFA blocks chronic neuropathic pain

The use of surgical models of neuropathic pain allow the testing of the chronic nature of the developed pain state. In the chronic constriction injury (CCI) model a 3.0 mg/kg oral gavage dose of the sEHI TPPU was effective in male and female rats against neuropathy assessed with a von Frey assay. TPPU treatment was highly significant compared to PEG300 vehicle control in males (**p<0.001) and was also significant in females compared to vehicle control in females (*p<0.010).

Figure 5. sEH inhibition and EpFA block Endoplasmic Reticulum Stress (ER Stress)

A variety of biological signals can influence the ER Stress pathway such as unfolded and miss folded proteins, high glucose as in diabetes, or reactive oxygen species (ROS) which can be contributed by mitochondrial dysfunction and other sources. EETs reduce the effects of ROS on the ER Stress pathway and stabilize mitochondria (not shown). The three key protein sensors of the ER Stress are inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6 (ATF6) and PKR-like endoplasmic reticulum-resident kinase (PERK). Downstream of IRE1α X-box binding protein-1 when spliced (XBP1s) is activated, similarly ATF6 is cleaved to release the active NH2-terminal domain ATF6(N) and both enter the nucleus as transcription factors. Phosphorylated PERK results in the phosphorylation of eukaryotic initiation factor 2 (eIF2α), ATF4 activation and transcription of C/EBP homologous protein (CHOP) a major participant in genes involved in apoptosis. Apoptotic responses occur when ER Stress is excessive, prolonged, or insufficiently neutralized and is initiated through downstream pathways such as ER-associated protein degradation (ERAD) and CHOP. The enzymatic flow of a single EET regioisomer is depicted on the right. ARA released by phospholipase A2 from the phospholipid bilayer is acted on by CYP450 epoxygenase to form 14,15 EET and would be metabolized by the sEH into a less active 14,15 DHET. Inhibiting sEH maintains the EpFA which block phosphorylation of PERK, elF2α, and IRE1α and significantly decrease XBP1s and ATF6(N) expression. In addition to this action, sEHI also normalize phospho-p38 and phospho-JNK, kinase mediators of neuropathic pain. However, in healthy rats, sEH inhibition does not lead to changes in ER Stress pathways.