Chemotherapy induced peripheral neuropathy (CIPN) is a particularly pernicious form of neuropathy and the associated pain is the primary dose-limiting factor of life-prolonging chemotherapy treatment. The prevalence of CIPN is high and can last long after treatment has been stopped. Currently, late in the COVID-19 pandemic, there are still increased psychological pressures on cancer patients as well as additional challenges in providing analgesia for them. These include the risks of nonsteroidal anti-inflammatory drug (NSAID) analgesics potentially masking early infection symptoms and the immunosuppression of steroidal and opiate based approaches. Even without these concerns, CIPN is often inadequately treated with few therapies that offer significant pain relief. The experiments we report use soluble epoxide hydrolase inhibitors (sEHI) which relieved this intractable pain in preclinical models. Doses of EC5026, an IND candidate intended to treat neuropathic pain, elicited dose dependent analgesic responses in multiple models including platinum-based, taxane, and vinca alkaloid-based CIPN pain in Sprague Dawley rats. At the same time as a class, the sEHI are known to result in fewer debilitating side effects of other analgesics, likely due to their novel mechanism of action. Overall, the observed dose-dependent analgesia in both male and female rats across multiple models of chemotherapy induced neuropathic pain holds promise as a useful tool when translated to the clinic.

Effectively treating chronic pain remains a therapeutic challenge in the clinic. Recent evidence has shown the inhibition of the soluble epoxide hydrolase (sEH) to be an effective strategy to limit chronic pain in preclinical models, horses and companion animals. Determining the safety of sEH inhibition in addition to this demonstrated efficacy is a critical step to the further development of sEH inhibitors (sEHI) as analgesics. Here we describe a comparison of the sEHI TPPU with other first in class analgesics for human chronic pain. We assess the development of tolerance to the analgesia mediated by TPPU with extended use. We also assess for CNS effects by measuring changes in motor control and functioning. The sEHI are multimodal analgesics that have demonstrated potent efficacy against chronic pain. They have previously been tested and show no reward potential using operant methods. The results of the current experiments show that they lack motor function effects and also lack the development of tolerance with extended dosing.

The soluble epoxide hydrolase (sEH) enzyme is a major regulator of bioactive lipids. The enzyme is highly expressed in liver and kidney and modulates levels of endogenous epoxy-fatty acids, which have pleiotropic biological effects including limiting inflammation, neuroinflammation, and hypertension. It has been hypothesized that inhibiting sEH has beneficial effects on limiting obesity and metabolic disease as well. There is a body of literature published on these effects, but typically only male subjects have been included. Here, we investigate the role of sEH in both male and female mice and use a global sEH knockout mouse model to compare the effects of diet and diet-induced obesity. The results demonstrate that sEH activity in the liver is modulated by high-fat diets more in male than in female mice. In addition, we characterized the sEH activity in high fat content tissues and demonstrated the influence of diet on levels of bioactive epoxy-fatty acids. The sEH KO animals had generally increased epoxy-fatty acids compared to wild-type mice but gained less body weight on higher-fat diets. Generally, proinflammatory prostaglandins and triglycerides were also lower in livers of sEH KO mice fed HFD. Thus, sEH activity, prostaglandins, and triglycerides increase in male mice on high-fat diet but are all limited by sEH ablation. Additionally, these changes also occur in female mice though at a different magnitude and are also improved by knockout of the sEH enzyme.

Chronic pain is a complicated condition which causes substantial physical, emotional, and financial impacts on individuals and society. However, due to high cost, lack of efficacy and safety problems, current treatments are insufficient. There is a clear unmet medical need for safe, nonaddictive and effective therapies in the management of pain. Epoxy-fatty acids (EpFAs), which are natural signaling molecules, play key roles in mediation of both inflammatory and neuropathic pain sensation. However, their molecular mechanisms of action remain largely unknown. Soluble epoxide hydrolase (sEH) rapidly converts EpFAs into less bioactive fatty acid diols in vivo; therefore, inhibition of sEH is an emerging therapeutic target to enhance the beneficial effect of natural EpFAs. In this review, we will discuss sEH inhibition as an analgesic strategy for pain management and the underlying molecular mechanisms.

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.

Enzyme-linked immunosorbent assays (ELISA) for the detection of soluble epoxide hydrolase (sEH), a key enzyme in the metabolism of fatty acids and a biomarker, may increasingly represent an important diagnostic tool. However, there is a lack of ELISAs for mouse sEH quantification, thus resulting in a bottleneck in understanding the pathogenesis of many diseases related to sEH based on mouse models. In this work, nanobodies recognizing mouse sEH were obtained through rebiopanning against mouse sEH in the previous phage display library of human sEH. Later, we developed four ELISAs involving a combination of anti-mouse sEH polyclonal antibodies (pAbs) and nanobodies. It was found that the double antibodies worked as dual filters and had a huge impact on both the sensitivity and selectivity of sandwich immunoassays. The switch from anti-human sEH pAbs to anti-mouse sEH pAbs led to over a 100-fold increase in the sensitivity and a dramatic decrease of the limit of detection to a picogram per milliliter range in format B (pAb/biotin-VHH/streptavidin-poly-horseradish peroxidase). Moreover, we found that the four sandwich ELISAs might demonstrate excellent selectivities to mouse sEH, despite the antibodies alone showing significant cross-reactivity to the matrix, indicating the enhanced selectivity of double antibodies as dual filters. Eventually, for the first time, the ELISA (format B) was successfully used to measure the mouse sEH level in cancer cells with ultralow abundances. The ELISAs proposed here represent a sensitive tool for tracking sEH in various biological processes and also provide deep insights into developing sandwich immunoassays against various targets in terms of both the sensitivity and selectivity.

Adrenic acid (AdA, 22:4) is an ω-6 polyunsaturated fatty acid (PUFA), derived from arachidonic acid. Like other PUFAs, it is metabolized by cytochrome P450s to a group of epoxy fatty acids (EpFAs), epoxydocosatrienoic acids (EDTs). EpFAs are lipid mediators with various beneficial bioactivities, including exertion of analgesia and reduction of endoplasmic reticulum (ER) stress, that are degraded to dihydroxy fatty acids by the soluble epoxide hydrolase (sEH). However, the biological characteristics and activities of EDTs are relatively unexplored, and, alongside dihydroxydocosatrienoic acids (DHDTs), they had not been detected in vivo. Herein, EDT and DHDT regioisomers were synthesized, purified, and used as standards for analysis with a selective and quantitative high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. Biological verification in AdA-rich tissues suggests that basal metabolite levels are highest in the liver, with 16,17-EDT concentrations consistently being the greatest across the analyzed tissues. Enzyme hydrolysis assessment revealed that EDTs are sEH substrates, with greatest relative rate preference for the 13,14-EDT regioisomer. Pretreatment with an EDT methyl ester regioisomer mixture significantly reduced the onset of tunicamycin-stimulated ER stress in human embryonic kidney cells. Finally, administration of the regioisomeric mixture effectively alleviated carrageenan-induced inflammatory pain in rats. This study indicates that EDTs and DHDTs are naturally occurring lipids, and EDTs could be another therapeutically relevant group of EpFAs.

Fatty acid amide hydrolase (FAAH) is responsible for regulating concentrations of the endocannabinoid arachidonoyl ethanolamide. Multiple FAAH inhibitors have been developed for clinical trials and have failed to demonstrate efficacy at treating pain, despite promising preclinical data. One approach toward increasing the efficacy of FAAH inhibitors is to concurrently inhibit other targets responsible for regulating pain. Here, we designed dual inhibitors targeting the enzymes FAAH and soluble epoxide hydrolase (sEH), which are targets previously shown to synergize at reducing inflammatory and neuropathic pain. Exploration of the sEH/FAAH inhibitor structure-activity relationship started with PF-750, a FAAH inhibitor (IC₅₀ = 19 nM) that weakly inhibited sEH (IC₅₀ = 640 nM). Potency was optimized resulting in an inhibitor with improved potency on both targets (11, sEH IC₅₀ = 5 nM, FAAH IC₅₀ = 8 nM). This inhibitor demonstrated good target selectivity, pharmacokinetic properties (AUC = 1200 h nM, t _1/2 = 4.9 h in mice), and in vivo target engagement.

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.

Over the last two decades polypharmacology has emerged as a new paradigm in drug discovery, even though developing drugs with high potency and selectivity toward a single biological target is still a major strategy. Often, targeting only a single enzyme or receptor shows lack of efficacy. High levels of inhibitor of a single target also can lead to adverse side effects. A second target may offer additive or synergistic effects to affecting the first target thereby reducing on- and off-target side effects. Therefore, drugs that inhibit multiple targets may offer a great potential for increased efficacy and reduced the adverse effects. In this review we summarize recent findings of rationally designed multitarget compounds that are aimed to improve efficacy and safety profiles compared to those that target a single enzyme or receptor. We focus on dual inhibitors/modulators that target the soluble epoxide hydrolase (sEH) as a common part of their design to take advantage of the beneficial effects of sEH inhibition.