Concise Syntheses of Microsomal Metabolites of a Potent OXE (Oxoeicosanoid) Receptor Antagonist.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most potent eosinophil chemoattractant among lipid mediators, and its actions are mediated by the selective oxoeicosanoid (OXE) receptor. Our group previously developed a highly potent indole-based OXE antagonist, S-C025, with an IC50 value of 120 pM. S-C025 was converted to a number of metabolites in the presence of monkey liver microsomes. Complete chemical syntheses of authentic standards enabled us to identify that the four major metabolites were derived by the oxidation at its benzylic and N-methyl carbon atoms. Herein we report concise syntheses of the four major metabolites of S-C025.

Metabolism of anti-inflammatory OXE (oxoeicosanoid) receptor antagonists by nonhuman primates.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the only product of the proinflammatory 5-lipoxygenase pathway with potent chemoattractant effects for human eosinophils, suggesting an important role in eosinophilic diseases such as asthma. 5-Oxo-ETE, acting through its selective OXE receptor, induces dermal eosinophilia in both humans and monkeys. To block its effects, we designed selective indole-based OXE antagonists containing hexyl (S-230) or phenylhexyl (S-C025 and S-Y048) side chains, which inhibit allergen-induced dermal and pulmonary inflammation in monkeys, suggesting that they may be useful therapeutic agents in humans. In this study we identified two metabolic pathways for the phenylhexyl-containing antagonists in liver microsomes: benzylic and N-methyl hydroxylation, resulting in ω-hydroxy, ω-oxo, and NH-containing products with reduced potencies that were identified by mass spectrometry and comparison with synthetic standards. Products of both pathways were also identified in monkey plasma following oral administration of S-C025 and S-Y025, but were less abundant than the α-hydroxy metabolites that we previously identified. Interestingly, the α-hydroxy compounds were not detected in microsomal incubations, suggesting a different origin. The relative rates of metabolism of these antagonists were S-230 >> S-C025 > S-Y048, which may help to explain the differences in their plasma half-lives (S-230 < S-C025 < S-Y048). In conclusion, S-C025 and S-Y048 are metabolized by liver microsomes by benzylic and N-methyl hydroxylation but not by α-hydroxylation, whereas all three pathways exist in vivo. Addition of a phenyl group to the hexyl side chain of these antagonists dramatically reduced their rates of metabolism, which would explain their prolonged in vivo half-lives.

Targeting the OXE receptor with a selective antagonist inhibits allergen-induced pulmonary inflammation in non-human primates.

BACKGROUND AND PURPOSE: The 5-lipoxygenase product, 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid), is a potent chemoattractant for eosinophils and neutrophils. However, little is known about its pathophysiological role because of the lack of a rodent ortholog of the oxoeicosanoid (OXE) receptor. The present study aimed to determine whether the selective OXE receptor antagonist S-Y048 can inhibit allergen-induced pulmonary inflammation in a monkey model of asthma. EXPERIMENTAL APPROACH: Monkeys sensitized to house dust mite antigen (HDM) were treated with either vehicle or S-Y048 prior to challenge with aerosolized HDM, and bronchoalveolar (BAL) fluid was collected 24 h later. After 6 weeks, animals that had initially been treated with vehicle received S-Y048 and vice versa for animals initially treated with S-Y048. Eosinophils and neutrophils in BAL and lung tissue samples were evaluated, as well as mucus-containing cells in bronchi. KEY RESULTS: HDM significantly increased the numbers of eosinophils, neutrophils, and macrophages in BAL fluid 24 h after challenge. These responses were all significantly inhibited by S-Y048, which also reduced the numbers of eosinophils and neutrophils in lung tissue 24 h after challenge with HDM. S-Y048 also significantly reduced the numbers of bronchial epithelial cells staining for mucin and MUC5AC after antigen challenge. CONCLUSION AND IMPLICATIONS: This study provides the first evidence that 5-oxo-ETE may play an important role in inducing allergen-induced pulmonary inflammation and could also be involved in regulating MUC5AC in goblet cells. OXE receptor antagonists such as S-Y048 may useful therapeutic agents in asthma and other eosinophilic as well as neutrophilic diseases.

Targeting the OXE receptor as a potential novel therapy for asthma.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is an arachidonic acid metabolite formed by oxidation of the 5-lipoxygenase (5-LO) product 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5S-HETE) by the NADP+-dependent enzyme 5-hydroxyeicosanoid dehydrogenase. It is the only 5-LO product with appreciable chemoattractant activity for human eosinophils. Its actions are mediated by the selective OXE receptor, which is highly expressed on eosinophils, basophils, neutrophils and monocytes. Orthologs of the OXER1 gene, which encodes this receptor, are found in many species except for rodents. Intradermal injection of 5-oxo-ETE into humans and monkeys elicits eosinophil infiltration into the skin, raising the possibility that it may play a pathophysiological role in eosinophilic diseases. To investigate this and possibly identify a novel therapy we sought to prepare synthetic antagonists that could selectively block the OXE receptor. We synthesized a series of indole-based compounds bearing substituents that mimic the regions of 5-oxo-ETE that are required for biological activity, which we modified to reduce metabolism. The most potent of these OXE receptor antagonists is S-Y048, which is a potent inhibitor of 5-oxo-ETE-induced calcium mobilization (IC50, 20 pM) and has a long half-life following oral administration. S-Y048 inhibited allergen-induced eosinophil infiltration into the skin of rhesus monkeys that had been experimentally sensitized to house dust mite and inhibited pulmonary inflammation resulting from challenge with aerosolized allergen. These data provide the first evidence for a pathophysiological role for 5-oxo-ETE in mammals and suggest that potent and selective OXE receptor antagonists such as S-Y048 may be useful therapeutic agents in asthma and other eosinophilic diseases.

Inhibition of allergen-induced dermal eosinophilia by an oxoeicosanoid receptor antagonist in non-human primates.

BACKGROUND AND PURPOSE: 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), acting via the OXE receptor, is unique among 5-lipoxygenase products in its ability to directly induce human eosinophil migration, suggesting its involvement in eosinophilic diseases. To address this hypothesis, we synthesized selective indole-based OXE receptor antagonists. Because rodents lack an OXE receptor orthologue, we sought to determine whether these antagonists could attenuate allergen-induced skin eosinophilia in sensitized monkeys. EXPERIMENTAL APPROACH: In a pilot study, cynomolgus monkeys with environmentally acquired sensitivity to Ascaris suum were treated orally with the "first-generation" OXE antagonist 230 prior to intradermal injection of 5-oxo-ETE or Ascaris extract. Eosinophils were evaluated in punch biopsy samples taken 6 or 24 hr later. We subsequently treated captive-bred rhesus monkeys sensitized to house dust mite (HDM) allergen with a more recently developed OXE antagonist, S-Y048, and evaluated its effects on dermal eosinophilia induced by either 5-oxo-ETE or HDM. KEY RESULTS: In a pilot experiment, both 5-oxo-ETE and Ascaris extract induced dermal eosinophilia in cynomolgus monkeys, which appeared to be reduced by 230. Subsequently, we found that the related OXE antagonist S-Y048 is a highly potent inhibitor of 5-oxo-ETE-induced activation of rhesus monkey eosinophils in vitro and has a half-life in plasma of about 6 hr after oral administration. S-Y048 significantly inhibited eosinophil infiltration into the skin in response to both intradermally administered 5-oxo-ETE and HDM. CONCLUSIONS AND IMPLICATIONS: 5-Oxo-ETE may play an important role in allergen-induced eosinophilia. Blocking its effects with S-Y048 may provide a novel therapeutic approach for eosinophilic diseases.

Novel highly potent OXE receptor antagonists with prolonged plasma lifetimes that are converted to active metabolites in vivo in monkeys.

BACKGROUND AND PURPOSE: The 5-lipoxygenase product 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE), acting through the OXE receptor, is a potent eosinophil chemoattractant that may be an important proinflammatory mediator in eosinophilic diseases such as asthma. We previously identified a series of indole-based OXE receptor antagonists that rapidly appear in the blood following oral administration but have limited lifetimes. The objective of this study was to increase the potency and plasma half-lives of these compounds and thereby identify the optimal candidate for future preclinical studies in monkeys, as rodents do not have an OXE receptor orthologue. EXPERIMENTAL APPROACH: We synthesized a series of substituted phenylalkyl indoles and compared their antagonist potencies, pharmacokinetics, and metabolism to those of our earlier compounds. The potencies of some of their metabolites were also investigated. KEY RESULTS: Among the compounds tested, the S-enantiomer of the m-chlorophenyl compound (S-Y048) was the most potent, with an pIC50 of about 10.8 for inhibition of 5-oxo-ETE-induced calcium mobilization in human neutrophils. When administered orally to cynomolgus monkeys, S-Y048 rapidly appeared in the blood and had a half-life in plasma of over 7 hr, considerably longer than any of the other OXE analogues tested. A major hydroxylated metabolite, with a potency close to that of its precursor, was identified in plasma. CONCLUSION AND IMPLICATIONS: Because of its highly potent antagonist activity and its long lifetime in vivo, S-Y048 may be a useful anti-inflammatory agent for the treatment of eosinophilic diseases such as asthma, allergic rhinitis, and atopic dermatitis.

Novel Highly Potent and Metabolically Resistant Oxoeicosanoid (OXE) Receptor Antagonists That Block the Actions of the Granulocyte Chemoattractant 5-Oxo-6,8,11,14-Eicosatetraenoic Acid (5-oxo-ETE).

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent lipid mediator that induces tissue eosinophilia via the selective OXE receptor (OXE-R), which is an attractive therapeutic target in eosinophilic diseases. We previously identified indole OXE-R antagonists that block 5-oxo-ETE-induced primate eosinophil activation. Although these compounds possess good oral absorption, their plasma levels decline rapidly due to extensive oxidation of their hexyl side chain. We have now succeeded in dramatically increasing antagonist potency and resistance to metabolism by replacing the hexyl group with phenylpentyl or phenylhexyl side chains. Compared with our previous lead compound S-230, our most potent antagonist, S-C025, has an IC50 (120 pM) over 80 times lower and a substantially longer plasma half-life. A single major metabolite, which retains antagonist activity (IC50, 690 pM) and has a prolonged lifetime in plasma was observed. These new highly potent OXE-R antagonists may provide a novel strategy for the treatment of eosinophilic disorders like asthma.

Metabolism and pharmacokinetics of a potent N-acylindole antagonist of the OXE receptor for the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) in rats and monkeys.

We previously identified the indole 264 as a potent in vitro antagonist of the human OXE receptor that mediates the actions of the powerful eosinophil chemoattractant 5-oxo-ETE. No antagonists of this receptor are currently commercially available or are being tested in clinical studies. The lack of a rodent ortholog of the OXE receptor has hampered progress in this area because of the unavailability of commonly used mouse or rat animal models. In the present study, we examined the feasibility of using the cynomolgus monkey as an animal model to investigate the efficacy of orally administered 264 in future in vivo studies. We first confirmed that 264 is active in monkeys by showing that it is a potent inhibitor of 5-oxo-ETE-induced actin polymerization and chemotaxis in granulocytes. The major microsomal metabolites of 264 were identified by cochromatography with authentic chemically synthesized standards and LC-MS/MS as its ω2-hydroxy and ω2-oxo derivatives, formed by ω2-oxidation of its hexyl side chain. Small amounts of ω1-oxidation products were also identified. None of these metabolites have substantial antagonist potency. High levels of 264 appeared rapidly in the blood following oral administration to both rats and monkeys, and declined to low levels by 24 h. As with microsomes, its major plasma metabolites in monkeys were ω2-oxidation products. We conclude that the monkey is a suitable animal model to investigate potential therapeutic effects of 264. This, or a related compound with diminished susceptibility to ω2-oxidation, could be a useful therapeutic agent in eosinophilic disorders such as asthma.

Structure-activity relationship study of ß-oxidation resistant indole-based 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) receptor antagonists.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is formed from 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by the 5-lipoxygenase (5-LO) pathway under conditions associated with oxidative stress. 5-Oxo-ETE is an important pro-inflammatory mediator, which stimulates the migration of eosinophils via a selective G-protein coupled receptor, known as the OXE receptor (OXE-R). Previously, we designed and synthesized structural mimics of 5-oxo-ETE such as 1 using an indole scaffold. In the present work, we added various substituents at C-3 of this moiety to block potential ß-oxidation of the 5-oxo-valerate side chain, and investigated the structure-activity relationships of the resulting novel ß-oxidation-resistant antagonists. Cyclopropyl and cyclobutyl substituents were well tolerated in this position, but were less potent as the highly active 3S-methyl compound. It seems likely that 3-alkyl substituents can affect the conformation of the 5-oxovalerate side chain containing the critical keto and carboxyl groups, thereby affecting interaction with the OXE-receptor.

In vivo α-hydroxylation of a 2-alkylindole antagonist of the OXE receptor for the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid in monkeys.

We have developed a selective indole antagonist (230) targeting the OXE receptor for the potent eosinophil chemoattractant 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid), that may be useful for the treatment of eosinophilic diseases such as asthma. In previous studies we identified ω2-oxidation of the hexyl side chain of racemic 230 as a major metabolic route in monkeys, but also obtained evidence for another pathway that appeared to involve hydroxylation of the hexyl side chain close to the indole. The present study was designed to investigate the metabolism of the active S-enantiomer of 230 (S230) and to identify the novel hydroxy metabolite and its chirality. Following oral administration, S230 rapidly appeared in the blood along with metabolites formed by a novel and highly stereospecific α-hydroxylation pathway, resulting in the formation of αS-hydroxy-S230. The chirality of α-hydroxy-S230 was determined by the total synthesis of the relevant diastereomers. Of the four possible diastereomers of α-hydroxy-230 only αS-hydroxy-S230 has significant OXE receptor antagonist activity and only this diastereomer was found in significant amounts in blood following oral administration of S230. Other novel metabolites of S230 identified in plasma by LC-MS/MS were αS,ω2-dihydroxy-S230 and glucuronides of S230 and ω2-hydroxy-S230. Thus the alkyl side chain of S230, which is essential for its antagonist activity, is also the major target of the metabolic enzymes that terminate its antagonist activity. Modification of this side chain might result in the development of related antagonists with improved metabolic stability and efficacy.

Design and synthesis of affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase.

Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5-oxo-ETE.

Pharmacokinetics and Metabolism of Selective Oxoeicosanoid (OXE) Receptor Antagonists and Their Effects on 5-Oxo-6,8,11,14-eicosatetraenoic Acid (5-Oxo-ETE)-Induced Granulocyte Activation in Monkeys.

The potent eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a 5-lipoxygenase product that acts via the selective OXE receptor, which is present in many species, but not rodents. We previously reported that the indole 230 is a potent human OXE receptor antagonist. The objective of the present study was to determine whether the monkey would be a suitable animal model to investigate its pharmaceutical potential. We found that monkey leukocytes synthesize and respond to 5-oxo-ETE and that 230 is a potent antagonist of the OXE receptor in monkey eosinophils. Pharmacokinetic studies revealed that 230 appears rapidly in the blood following oral administration. Using chemically synthesized standards, we identified the major microsomal and plasma metabolites of 230 as products of ω2-hydroxylation of the alkyl side chain. These studies demonstrate that the monkey is a promising animal model to investigate the drug potential of OXE receptor antagonists.

Biosynthesis and actions of 5-oxoeicosatetraenoic acid (5-oxo-ETE) on feline granulocytes.

The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most powerful human eosinophil chemoattractant among lipid mediators and could play a major pathophysiological role in eosinophilic diseases such as asthma. Its actions are mediated by the OXE receptor, orthologs of which are found in many species from humans to fish, but not rodents. The unavailability of rodent models to examine the pathophysiological roles of 5-oxo-ETE and the OXE receptor has substantially hampered progress in this area. As an alternative, we have explored the possibility that the cat could serve as an appropriate animal model to investigate the role of 5-oxo-ETE. We found that feline peripheral blood leukocytes synthesize 5-oxo-ETE and that physiologically relevant levels of 5-oxo-ETE are present in bronchoalveolar lavage fluid from cats with experimentally induced asthma. 5-Oxo-ETE (EC50, 0.7nM) is a much more potent activator of actin polymerization in feline eosinophils than various other eicosanoids, including leukotriene (LT) B4 and prostaglandin D2. 5-Oxo-ETE and LTB4 induce feline leukocyte migration to similar extents at low concentrations (1nM), but at higher concentrations the response to 5-oxo-ETE is much greater. Although high concentrations of selective human OXE receptor antagonists blocked 5-oxo-ETE-induced actin polymerization in feline granulocytes, their potencies were about 200 times lower than for human granulocytes. We conclude that feline leukocytes synthesize and respond to 5-oxo-ETE, which could potentially play an important role in feline asthma, a common condition in this species. The cat could serve as a useful animal model to investigate the pathophysiological role of 5-oxo-ETE.

Biosynthesis, biological effects, and receptors of hydroxyeicosatetraenoic acids (HETEs) and oxoeicosatetraenoic acids (oxo-ETEs) derived from arachidonic acid.

Arachidonic acid can be oxygenated by a variety of different enzymes, including lipoxygenases, cyclooxygenases, and cytochrome P450s, and can be converted to a complex mixture of oxygenated products as a result of lipid peroxidation. The initial products in these reactions are hydroperoxyeicosatetraenoic acids (HpETEs) and hydroxyeicosatetraenoic acids (HETEs). Oxoeicosatetraenoic acids (oxo-ETEs) can be formed by the actions of various dehydrogenases on HETEs or by dehydration of HpETEs. Although a large number of different HETEs and oxo-ETEs have been identified, this review will focus principally on 5-oxo-ETE, 5S-HETE, 12S-HETE, and 15S-HETE. Other related arachidonic acid metabolites will also be discussed in less detail. 5-Oxo-ETE is synthesized by oxidation of the 5-lipoxygenase product 5S-HETE by the selective enzyme, 5-hydroxyeicosanoid dehydrogenase. It actions are mediated by the selective OXE receptor, which is highly expressed on eosinophils, suggesting that it may be important in eosinophilic diseases such as asthma. 5-Oxo-ETE also appears to stimulate tumor cell proliferation and may also be involved in cancer. Highly selective and potent OXE receptor antagonists have recently become available and could help to clarify its pathophysiological role. The 12-lipoxygenase product 12S-HETE acts by the GPR31 receptor and promotes tumor cell proliferation and metastasis and could therefore be a promising target in cancer therapy. It may also be involved as a proinflammatory mediator in diabetes. In contrast, 15S-HETE may have a protective effect in cancer. In addition to GPCRs, higher concentration of HETEs and oxo-ETEs can activate peroxisome proliferator-activated receptors (PPARs) and could potentially regulate a variety of processes by this mechanism. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Two Potent OXE-R Antagonists: Assignment of Stereochemistry.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is formed by the oxidation of 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE), which is a major metabolite of enzymatic oxidation of arachidonic acid (AA). 5-Oxo-ETE is the most potent lipid chemoattractant for human eosinophils. Its actions are mediated by the selective OXE receptor, which is therefore an attractive target in eosinophilic diseases such as allergic rhinitis and asthma. Recently, we have reported two excellent OXE receptor antagonists that have IC50 values at low nanomolar concentrations. Each of these antagonists has a chiral center, and the isolation of the individual enantiomers by chiral high-performance liquid chromatography (HPLC) revealed that in each case one enantiomer is over 300 times more potent than the other. To unambiguously assign the stereochemistry of these enantiomers and to provide access to larger amounts of the active compounds for biological testing, we report here their total synthesis.

Base-dependent formation of cis and trans olefins and their application in the synthesis of 5-oxo-ETE receptor antagonists.

5-Oxo-ETE is the most potent eosinophil chemoattractant among lipid mediators. We have developed two 5-oxo-ETE receptor antagonists. In the course of the work, we have developed a procedure to selectively introduce a cis and trans double bond in an alkyl side chain. Reacting indolecarboxaldehydes with alkyl ylides using the Li base affords the trans olefins, whereas using the K base yields the cis olefins.

Inhibition of 5-oxo-6,8,11,14-eicosatetraenoic acid-induced activation of neutrophils and eosinophils by novel indole OXE receptor antagonists.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a 5-lipoxygenase product that is a potent granulocyte chemoattractant, which induces the infiltration of eosinophils into human skin when injected intradermally. It could therefore be an important proinflammatory mediator in eosinophilic diseases such as asthma and allergic rhinitis, and the OXE receptor, which mediates its actions, is therefore an attractive drug target. Using a structure-based approach in which substituents mimicking the essential polar (C1-C5) and hydrophobic (C15-C20) regions of 5-oxo-ETE were incorporated on an indole scaffold, we identified two potent selective OXE antagonists with IC50 values of about 30 nM. Neither compound displayed agonist activity and both inhibited 5-oxo-ETE-induced chemotaxis and actin polymerization and were relatively resistant to metabolism by rat liver homogenates. The active enantiomers of these racemic antagonists were even more potent, with IC50 values of <10 nM. These selective OXE antagonists could potentially be useful therapeutic agents in allergic diseases such as asthma.

The eosinophil chemoattractant 5-oxo-ETE and the OXE receptor.

5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is formed from the 5-lipoxygenase product 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH). The cofactor NADP(+) is a limiting factor in the synthesis of 5-oxo-ETE because of its low concentrations in unperturbed cells. Activation of the respiratory burst in phagocytic cells, oxidative stress, and cell death all dramatically elevate both intracellular NADP(+) levels and 5-oxo-ETE synthesis. 5-HEDH is widely expressed in inflammatory, structural, and tumor cells. Cells devoid of 5-lipoxygenase can synthesize 5-oxo-ETE by transcellular biosynthesis using inflammatory cell-derived 5-HETE. 5-Oxo-ETE is a chemoattractant for neutrophils, monocytes, and basophils and promotes the proliferation of tumor cells. However, its primary target appears to be the eosinophil, for which it is a highly potent chemoattractant. The actions of 5-oxo-ETE are mediated by the highly selective OXE receptor, which signals by activating various second messenger pathways through the release of the ßγ-dimer from Gi/o proteins to which it is coupled. Because of its potent effects on eosinophils, 5-oxo-ETE may be an important mediator in asthma, and, because of its proliferative effects, may also contribute to tumor progression. Selective OXE receptor antagonists, which are currently under development, could be useful therapeutic agents in asthma and other allergic diseases.

Quantification of lipid mediator metabolites in human urine from asthma patients by electrospray ionization mass spectrometry: controlling matrix effects.

Eicosanoids (e.g., prostaglandins and leukotrienes) are inflammatory signaling molecules that are metabolized and excreted in urine. The quantification of eicosanoid metabolites in human urine has been demonstrated to provide insight into the inflammatory and oxidative stress status of the individual. However, urine is a complex matrix that can exhibit profound matrix effects for quantification via liquid chromatography coupled to mass spectrometry (LC-MS/MS). This phenomenon can lead to impairment and biasing of results, because the sample background is dependent on the fluid intake and water-salt balance. Herein we describe an analytical methodology to address these limitations via the normalization of extracted urine volume by the ratio of absorbance at 300 nm to an optimized reference material. The platform is composed of 4 LC-MS/MS methods that collectively quantify 26 lipid mediators and their metabolites, with on-column limits of detection between 0.55 and 15 fmol. Prior to optimization, internal standards exhibited strong matrix effects with up to 50% loss of signal. Notably, the accuracy of exact deuterated structural analogues was found to vary based upon the number of incorporated deteurium. The platform was used to analyze urine from 16 atopic asthmatics under allergen provocation, showing increases in metabolites of prostaglandin D2, cysteinyl leukotrienes, and isoprostanes following the challenge. This method presents a functional and reproducible approach to addressing urine-specific matrix effects that can be readily formatted for quantifying large numbers of samples.