Ubiquitous exposure to various exogenous factors imparts both acute and chronic adverse effects on human health. Common molecular modes of action include induction of inflammation and activation of the mitochondrial dysfunction-reactive oxygen species-endoplasmic reticulum (ER) stress axis. The kidney is especially susceptible to noxious impacts due to several structural and functional characteristics. Preservation of inflammation-mediated and organelle-linked health impacts is further hampered by a lack of safe and efficacious reno-protective agents and unreliable biomarkers.

The cytochrome P450 (CYP) branch of polyunsaturated fatty acid (PUFA) metabolism oxygenates PUFAs to a class of signaling molecules known as epoxy fatty acids (EpFAs). These are primarily inflammation resolving and mitochondria-ER stabilizing lipid mediators. Since numerous points within the P450 pathway can be capitalized upon and/or manipulated, CYP-mediated EpFA biosynthesis and metabolism offer novel therapeutic targets to ameliorate and monitor disease.

Thus, this dissertation describes new and complementing research methodologies that were explored to elucidate the pharmacological and diagnostic potential of this PUFA metabolizing route. More precisely, Chapter 1 reveals the prevalence and therapeutic relevance of an overlooked group of EpFAs. Chapter 2 furthers the quest for naturally occurring, dietarily-attained enzyme inhibitors that block the primary route of EpFA metabolism and exert desired medicinal effects. Chapter 3 illuminates a biochemically active functional group that can be employed as a lead for the design of new and improved EpFA-like therapies. Chapter 4 delivers a modern and clinically pertinent analytical method for quantitation of the downstream metabolites of EpFAs derived from a pervasive dietary fat to exploit their exceptional biomarker functions.

Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are the most widely pharmacologically utilized group of EpFAs. However, the presence and therapeutic activity of their 22 carbon- homologues, adrenic acid-derived epoxydocosatrienoic acids (EDTs), is unstudied, despite the abundance of its parent PUFA in the kidney and vasculature. EDTs were found to occur naturally in vivo and exert anti-ER stress effects in kidney cells and relief from inflammatory pain in an animal model, thus illuminating another medicinally relevant group of EpFAs. Inhibition of the soluble epoxide hydrolase (sEH) enzyme curbs the major route of EpFA degradation, facilitating their bioactivities. sEH inhibitors (sEHI) derived from natural sources, such as botanicals, offer unique applicatory benefits over synthetic drugs. Accordingly, macamides derived from maca (Lepidium meyenii) were found to be the most promising natural sEHI group to date for a combination of inhibitory potency towards sEH and abundance in plant root. The single most relevant macamide was orally available and alleviated inflammatory pain pre-clinically. Design of structural analogues of EpFAs provides biochemically robust therapeutics that mimic EpFA activity. Accordingly, replacement of the labile epoxide group with more stable alkyl ether groups yielded a new class of EpFA analogues. These alkoxy-based analogues of EETs attenuated nephrotoxicity of a widely used chemotherapeutic (i.e., cisplatin) in vitro through downregulation of mitochondrial oxidative stress and termination of downstream apoptotic signaling. Downstream sEH-generated dihydroxy fatty acids, most notably the predominant linoleic acid-derived dihydroxyoctadecenoic acids (DiHOMEs), have been closely implicated in numerous pro-inflammatory pathologies and hence provide a promising biomarker for assessment of health outcomes. Reliable analytical methods are necessary for their detection and enzyme-linked immunoassays (ELISA) possess distinct benefits as complements to classically used liquid chromatography-tandem mass spectrometry (LC-MS/MS). Therefore, utilizing polyHRP technology, a highly sensitive ELISA for DiHOMEs was developed, validated for use in human plasma, the most clinically relevant matrix, and authenticated with LC-MS/MS.

In conclusion, the multitude of strategies discussed here highlight the medicinal potential and malleability of the CYP route of PUFA metabolism and advance the pursuit of potential biochemical means to mitigate and track the deleterious health effects of environmental stressors.

There is currently an urgent need for safe and effective treatment options for chronic diseases, especially pain, given that current medicines are accompanied by severe side effects. Bioactive lipid mediators resulting from the metabolism of polyunsaturated fatty acids (PUFA) are controlled by many pathways that maintain homeostasis and prevent disease, and regulation of this pathway presents a novel and safe approach for treating many diseases, including pain. Epoxygenated fatty acids (EpFA), formed through metabolism by cytochrome P450 enzymes, are regulatory lipids that demonstrate largely anti-inflammatory properties that reduce endoplasmic reticulum stress to help resolve disease; however, their utility is limited by rapid degradation by the soluble epoxide hydrolase (sEH) into corresponding vicinal diols. Inhibiting its activity increases concentrations of beneficial EpFA, and often disease states correlate to increased sEH activity that results in decreased concentrations of EpFA in the body. Research described in this dissertation detail the pharmacological properties of EC1728, a novel soluble epoxide hydrolase inhibitor (sEHI), as an option for pain control in companion animals. The exposure and distribution are described in target animal species and demonstrate that there are differences in exposures between species that need to be considered for drug development. An efficacy study demonstrating pain relief after treatment with EC1728 in dogs with naturally occurring arthritis is also presented. Fundamentally, inhibition of the sEH is thought to offer therapeutic advantages by increasing EpFA; however, the last chapter explores lipidomics profiles in patients with severe COVID-19 disease to advance the idea that metabolism products of EpFA, specifically the diols resulting from linoleic acid metabolism, are also responsible for driving severe inflammatory disease. These studies demonstrate that sEH inhibition is beneficial by both increasing EpFA and decreasing inflammatory diol metabolites. In summary, pharmacological properties of EC1728 demonstrate suitable properties for developing as a pain-relieving therapy in companion animals, and also validation of sEHI as a therapeutic target by demonstrating inhibition in natural disease in addition to laboratory models. The methods described here of characterizing detailed drug exposure, characterizing efficacy in companion animals with natural disease, as well as identification of biomarkers in disease settings represents a novel approach to drug discovery that may help advance selection of safe and effective treatments for multimodal diseases.  

Paraquat is one of the most common and acutely toxic herbicides used in developing nations, with epidemiological studies linking chronic paraquat exposure to Parkinson’s disease. Numerous studies in mice have demonstrated a chronic paraquat-mediated reduction in dopaminergic neural markers in the midbrain. However, given the scarcity of research surrounding the effect of chronic paraquat on striatal neurotoxicity, there is a need for further investigation into the effect of paraquat on the induction of endoplasmic reticulum stress and inflammation in mouse striatum. This study attempts to observe changes in both inflammatory and endoplasmic reticulum stress markers in mouse striatum following chronic paraquat administration. Furthermore, previous studies have shown that inhibition of soluble epoxide hydrolase mitigates MPTP-mediated loss of dopaminergic neurons and endoplasmic reticulum stress in mouse striatum. Thus, this study seeks to determine if inhibition of soluble epoxide hydrolase mitigates paraquat-induced neurotoxicity, as well as whether epoxyeicosatrienoic acids, endogenous substrates of the soluble epoxide hydrolase, reduce TLR4-mediated inflammation in primary astrocytes and microglia. Our results show that while the pro-inflammatory effect of chronic paraquat injection on mouse striata is relatively small, there are significant inductions of inflammatory and cellular stress markers such as COX2 and CHOP in vivo and inflammatory markers in vitro that can be mitigated through a prophylactic administration of a soluble epoxide hydrolase inhibitor.

This article focuses on the role of omega-3 fatty acids as precursors for lipid signaling molecules known as oxylipins. Although omega-3 fatty acids are beneficial in autoimmune disorders, inflammatory diseases and heart disease, they are generally underrepresented in the American diet. A literature review confirms that the consumption of omega-3 fatty acids — whether in food sources such as walnuts, flax seeds and fatty fish (including salmon and sardines), or in supplements — is associated with decreased morbidity and mortality. This growing body of evidence, including the results of a recent study of patients with kidney disease, highlights the need to measure omega-3 fatty acids and their oxylipin products as markers of metabolic health and biomarkers of disease. In addition, there is substantial evidence of the need to increase the omega-3 fatty acid content of American diets to optimize metabolic health.

Background

Methods

Results

Conclusion