Pharmaceutical Effects of Inhibiting the Soluble Epoxide Hydrolase in Canine Osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease that causes pain and bone deterioration driven by an increase in prostaglandins (PGs) and inflammatory cytokines. Current treatments focus on inhibiting prostaglandin production, a pro-inflammatory lipid metabolite, with NSAID drugs; however, other lipid signaling targets could provide safer and more effective treatment strategies. Epoxides of polyunsaturated fatty acids (PUFAs) are anti-inflammatory lipid mediators that are rapidly metabolized by the soluble epoxide hydrolase (sEH) into corresponding vicinal diols. Interestingly, diol levels are increased in the synovial fluid of humans with OA, warranting further research on the biological role of this lipid pathway in the progression of OA. sEH inhibitors (sEHI) stabilize these biologically active, anti-inflammatory lipid epoxides, resulting in analgesia in both neuropathic, and inflammatory pain conditions. Most experimental studies testing the analgesic effects of sEH inhibitors have used experimental rodent models, which do not completely represent the complex etiology of painful diseases. Here, we tested the efficacy of sEHI in aged dogs with natural arthritis to provide a better representation of the clinical manifestations of pain. Two sEHI were administered orally, once daily for 5 days to dogs with naturally occurring arthritis to assess efficacy and pharmacokinetics. Blinded technicians recorded the behavior of the arthritic dogs based on pre-determined criteria to assess pain and function. After 5 days, EC1728 significantly reduced pain at a dose of 5 mg/kg compared to vehicle controls. Pharmacokinetic evaluation showed concentrations exceeding the enzyme potency in both plasma and synovial fluid. In vitro data showed that epoxyeicosatrienoic acid (EETs), epoxide metabolites of arachidonic acid, decreased inflammatory cytokines, IL-6 and TNF-α, and reduced cytotoxicity in canine chondrocytes challenged with IL1β to simulate an arthritic environment. These results provide the first example of altering lipid epoxides as a therapeutic target for OA potentially acting by protecting chondrocytes from inflammatory induced cytotoxicity. Considering the challenges and high variability of naturally occurring disease in aged dogs, these data provide initial proof of concept justification that inhibiting the sEH is a non-NSAID, non-opioid, disease altering strategy for treating OA, and warrants further investigation

In vitro and in vivo Metabolism of a Potent Inhibitor of Soluble Epoxide Hydrolase, 1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (TPPU) is a potent soluble epoxide hydrolase (sEH) inhibitor that is used extensively in research for modulating inflammation and protecting against hypertension, neuropathic pain, and neurodegeneration. Despite its wide use in various animal disease models, the metabolism of TPPU has not been well-studied. A broader understanding of its metabolism is critical for determining contributions of metabolites to the overall safety and effectiveness of TPPU. Herein, we describe the identification of TPPU metabolites using LC-MS/MS strategies. Four metabolites of TPPU (M1–M4) were identified from rat urine by a sensitive and specific LC-MS/MS method with double precursor ion scans. Their structures were further supported by LC-MS/MS comparison with synthesized standards. Metabolites M1 and M2 were formed from hydroxylation on a propionyl group of TPPU; M3 was formed by amide hydrolysis of the 1-propionylpiperdinyl group on TPPU; and M4 was formed by further oxidation of the hydroxylated metabolite M2. Interestingly, the predicted α-keto amide metabolite and 4-(trifluoromethoxy)aniline (metabolite from urea cleavage) were not detected by the LC-MRM-MS method. This indicates that if formed, the two potential metabolites represent <0.01% of TPPU metabolism. Species differences in the formation of these four identified metabolites was assessed using liver S9 fractions from dog, monkey, rat, mouse, and human. M1, M2, and M3 were generated in liver S9 fractions from all species, and higher amounts of M3 were generated in monkey S9 fractions compared to other species. In addition, rat and human S9 metabolism showed the highest species similarity based on the quantities of each metabolite. The presence of all four metabolites were confirmed in vivo in rats over 72-h post single oral dose of TPPU. Urine and feces were major routes for TPPU excretion. M1, M4 and parent drug were detected as major substances, and M2 and M3 were minor substances. In blood, M1 accounted for ~9.6% of the total TPPU-related exposure, while metabolites M2, M3, and M4 accounted for <0.4%. All four metabolites were potent inhibitors of human sEH but were less potent than the parent TPPU. In conclusion, TPPU is metabolized via oxidation and amide hydrolysis without apparent breakdown of the urea. The aniline metabolites were not observed either in vitro or in vivo. Our findings increase the confidence in the ability to translate preclinical PK of TPPU in rats to humans and facilitates the potential clinical development of TPPU and other sEH inhibitors

Soluble Epoxide Hydrolase Regulation of Lipid Mediators Limits Pain

The role of lipids in pain signaling is well established and built on decades of knowledge about the pain and inflammation produced by prostaglandin and leukotriene metabolites of cyclooxygenase and lipoxygenase metabolism, respectively. The analgesic properties of other lipid metabolites are more recently coming to light. Lipid metabolites have been observed to act directly at ion channels and G protein-coupled receptors on nociceptive neurons as well as act indirectly at cellular membranes. Cytochrome P450 metabolism of specifically long-chain fatty acids forms epoxide metabolites, the epoxy-fatty acids (EpFA). The biological role of these metabolites has been found to mediate analgesia in several types of pain pathology. EpFA act through a variety of direct and indirect mechanisms to limit pain and inflammation including nuclear receptor agonism, limiting endoplasmic reticulum stress and blocking mitochondrial dysfunction. Small molecule inhibitors of the soluble epoxide hydrolase can stabilize the EpFA in vivo, and this approach has demonstrated relief in preclinical modeled pain pathology. Moreover, the ability to block neuroinflammation extends the potential benefit of targeting soluble epoxide hydrolase to maintain EpFA for neuroprotection in neurodegenerative disease

Improved ELISA for linoleate-derived diols in human plasma utilizing a polyHRP-based secondary tracer

Dihydroxyoctadecenoic acids (DiHOMEs) are cytochrome P450 pathway-derived metabolites of linoleic acid, a highly abundant dietary fatty acid. They serve thermogenic functions at low concentrations but, at high concentrations, are involved in proinflammatory and deleterious outcomes in a wide range of pathologies. Hence, the development of a reliable analytical method is critical to elucidate their potential as biomarkers of health, and enzyme-linked immunoassay (ELISA)-based approaches offer unique benefits as alternatives to traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems. Accordingly, an earlier ELISA for DiHOMEs was dramatically improved employing new secondary tracers and geared towards use in human plasma, a universal matrix in biomedical applications, as well as urine. Three ELISA formats, two utilizing polyHRP-based secondary labels for signal amplification, were compared. The best format involved a biotinylated detection antibody and a polyHRP-conjugated streptavidin tracer. Assay detectability was enhanced 20-fold, relative to the original immunoassay, and performance assessments validated precision, selectivity, and robustness. Fast and easy extraction-clean up steps yielded high analytical recovery and permitted the assay to operate in moderate concentrations (up to 20%) of plasma, expanding its practical relevance. Finally, the ELISA was applied towards detection of DiHOMEs in clinical samples and authenticated with complementary LC-MS/MS analysis. Hence, the method provides a valuable analytical tool to investigate the diverse and extensive roles of DiHOMEs in regulatory biology

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

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

sEH-derived metabolites of linoleic acid drive pathologic inflammation while impairing key innate immune cell function in burn injury

Fatty acid composition in the Western diet has shifted from saturated to polyunsaturated fatty acids (PUFAs), and specifically to linoleic acid (LA, 18:2), which has gradually increased in the diet over the past 50 y to become the most abundant dietary fatty acid in human adipose tissue. PUFA-derived oxylipins regulate a variety of biological functions. The cytochrome P450 (CYP450)–formed epoxy fatty acid metabolites of LA (EpOMEs) are hydrolyzed by the soluble epoxide hydrolase enzyme (sEH) to dihydroxyoctadecenoic acids (DiHOMEs). DiHOMEs are considered cardioprotective at low concentrations but at higher levels have been implicated as vascular permeability and cytotoxic agents and are associated with acute respiratory distress syndrome in severe COVID-19 patients. High EpOME levels have also correlated with sepsis-related fatalities; however, those studies failed to monitor DiHOME levels. Considering the overlap of burn pathophysiology with these pathologies, the role of DiHOMEs in the immune response to burn injury was investigated. 12,13-DiHOME was found to facilitate the maturation and activation of stimulated neutrophils, while impeding monocyte and macrophage functionality and cytokine generation. In addition, DiHOME serum concentrations were significantly elevated in burn-injured mice and these increases were ablated by administration of 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a sEH inhibitor. TPPU also reduced necrosis of innate and adaptive immune cells in burned mice, in a dose-dependent manner. The findings suggest DiHOMEs are a key driver of immune cell dysfunction in severe burn injury through hyperinflammatory neutrophilic and impaired monocytic actions, and inhibition of sEH might be a promising therapeutic strategy to mitigate deleterious outcomes in burn patients

Development of Improved Double-Nanobody Sandwich ELISAs for Human Soluble Epoxide Hydrolase Detection in Peripheral Blood Mononuclear Cells of Diabetic Patients and the Prefrontal Cortex of Multiple Sclerosis Patients.

Nanobodies have been progressively replacing traditional antibodies in various immunological methods. However, the use of nanobodies as capture antibodies is greatly hampered by their poor performance after passive adsorption to polystyrene microplates, and this restricts the full use of double nanobodies in sandwich enzyme-linked immunosorbent assays (ELISAs). Herein, using the human soluble epoxide hydrolase (sEH) as a model analyte, we found that both the immobilization format and the blocking agent have a significant influence on the performance of capture nanobodies immobilized on polystyrene and the subsequent development of double-nanobody sandwich ELISAs. We first conducted epitope mapping for pairing nanobodies and then prepared a horseradish-peroxidase-labeled nanobody using a mild conjugation procedure as a detection antibody throughout the work. The resulting sandwich ELISA using a capture nanobody (A9, 1.25 μg/mL) after passive adsorption and bovine serum albumin (BSA) as a blocking agent generated a moderate sensitivity of 0.0164 OD·mL/ng and a limit of detection (LOD) of 0.74 ng/mL. However, the introduction of streptavidin as a linker to the capture nanobody at the same working concentration demonstrated a dramatic 16-fold increase in sensitivity (0.262 OD·mL/ng) and a 25-fold decrease in the LOD for sEH (0.03 ng/mL). The streptavidin-bridged double-nanobody ELISA was then successfully applied to tests for recovery, cross-reactivity, and real samples. Meanwhile, we accidentally found that blocking with skim milk could severely damage the performance of the capture nanobody by an order of magnitude compared with BSA. This work provides guidelines to retain the high effectiveness of the capture nanobody and thus to further develop the double-nanobody ELISA for various analytes

Development of Improved Double-Nanobody Sandwich ELISAs for Human Soluble Epoxide Hydrolase Detection in Peripheral Blood Mononuclear Cells of Diabetic Patients and the Prefrontal Cortex of Multiple Sclerosis Patients.

Nanobodies have been progressively replacing traditional antibodies in various immunological methods. However, the use of nanobodies as capture antibodies is greatly hampered by their poor performance after passive adsorption to polystyrene microplates, and this restricts the full use of double nanobodies in sandwich enzyme-linked immunosorbent assays (ELISAs). Herein, using the human soluble epoxide hydrolase (sEH) as a model analyte, we found that both the immobilization format and the blocking agent have a significant influence on the performance of capture nanobodies immobilized on polystyrene and the subsequent development of double-nanobody sandwich ELISAs. We first conducted epitope mapping for pairing nanobodies and then prepared a horseradish-peroxidase-labeled nanobody using a mild conjugation procedure as a detection antibody throughout the work. The resulting sandwich ELISA using a capture nanobody (A9, 1.25 μg/mL) after passive adsorption and bovine serum albumin (BSA) as a blocking agent generated a moderate sensitivity of 0.0164 OD·mL/ng and a limit of detection (LOD) of 0.74 ng/mL. However, the introduction of streptavidin as a linker to the capture nanobody at the same working concentration demonstrated a dramatic 16-fold increase in sensitivity (0.262 OD·mL/ng) and a 25-fold decrease in the LOD for sEH (0.03 ng/mL). The streptavidin-bridged double-nanobody ELISA was then successfully applied to tests for recovery, cross-reactivity, and real samples. Meanwhile, we accidentally found that blocking with skim milk could severely damage the performance of the capture nanobody by an order of magnitude compared with BSA. This work provides guidelines to retain the high effectiveness of the capture nanobody and thus to further develop the double-nanobody ELISA for various analytes