RESUMO
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.
Assuntos
Eosinófilos , Eosinófilos/metabolismo , OxirreduçãoRESUMO
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.
Assuntos
Eosinófilos , Receptores Eicosanoides , Animais , Anti-Inflamatórios/farmacologia , Fatores Quimiotáticos/farmacologia , Haplorrinos/metabolismoRESUMO
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.
Assuntos
Asma , Pneumonia , Alérgenos , Animais , Asma/tratamento farmacológico , Eosinófilos , Pneumonia/tratamento farmacológico , Pneumonia/prevenção & controle , Primatas , Receptores EicosanoidesRESUMO
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.
Assuntos
Alérgenos , Antialérgicos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Dermatite/prevenção & controle , Eosinofilia/prevenção & controle , Eosinófilos/efeitos dos fármacos , Receptores Eicosanoides/antagonistas & inibidores , Pele/efeitos dos fármacos , Animais , Antialérgicos/síntese química , Antialérgicos/farmacocinética , Antígenos de Helmintos/imunologia , Ácidos Araquidônicos , Ascaris suum/imunologia , Células Cultivadas , Dermatite/imunologia , Dermatite/metabolismo , Modelos Animais de Doenças , Eosinofilia/imunologia , Eosinofilia/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Proteínas de Insetos/imunologia , Macaca fascicularis , Macaca mulatta , Masculino , Projetos Piloto , Pyroglyphidae/imunologia , Receptores Eicosanoides/metabolismo , Transdução de Sinais , Pele/imunologia , Pele/metabolismoRESUMO
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.
Assuntos
Antialérgicos/farmacocinética , Anti-Inflamatórios/farmacocinética , Indóis/farmacocinética , Neutrófilos/efeitos dos fármacos , Receptores Eicosanoides/antagonistas & inibidores , Ativação Metabólica , Administração Oral , Animais , Antialérgicos/sangue , Antialérgicos/síntese química , Anti-Inflamatórios/sangue , Anti-Inflamatórios/síntese química , Cálcio/metabolismo , Feminino , Meia-Vida , Humanos , Hidroxilação , Indóis/sangue , Indóis/síntese química , Macaca fascicularis , Neutrófilos/metabolismo , Receptores Eicosanoides/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Ácidos Araquidônicos/antagonistas & inibidores , Fatores Quimiotáticos/antagonistas & inibidores , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Ácidos Pentanoicos/farmacologia , Receptores Eicosanoides/antagonistas & inibidores , Animais , Cálcio/metabolismo , Feminino , Humanos , Concentração Inibidora 50 , Macaca fascicularis , Ácidos Pentanoicos/química , Ácidos Pentanoicos/metabolismo , Ácidos Pentanoicos/farmacocinética , Estereoisomerismo , Distribuição TecidualRESUMO
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 24â¯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.
Assuntos
Ácidos Araquidônicos/farmacologia , Fatores Quimiotáticos/farmacologia , Eosinófilos/efeitos dos fármacos , Indóis/farmacocinética , Receptores Eicosanoides/antagonistas & inibidores , Administração Oral , Animais , Quimiotaxia/efeitos dos fármacos , Eosinófilos/metabolismo , Feminino , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Haplorrinos , Masculino , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Oxirredução/efeitos dos fármacos , RatosRESUMO
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.
Assuntos
Antiasmáticos/farmacocinética , Anti-Inflamatórios não Esteroides/farmacocinética , Ácidos Araquidônicos/antagonistas & inibidores , Fatores Quimiotáticos/antagonistas & inibidores , Indóis/farmacocinética , Cetoácidos/farmacocinética , Receptores Eicosanoides/antagonistas & inibidores , Administração Oral , Alquilação , Animais , Antiasmáticos/administração & dosagem , Antiasmáticos/sangue , Antiasmáticos/farmacologia , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/sangue , Anti-Inflamatórios não Esteroides/farmacologia , Ácidos Araquidônicos/metabolismo , Fatores Quimiotáticos/metabolismo , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Eosinófilos/metabolismo , Feminino , Glucuronídeos/sangue , Glucuronídeos/química , Glucuronídeos/farmacologia , Humanos , Hidroxilação , Inativação Metabólica , Indóis/administração & dosagem , Indóis/sangue , Indóis/química , Indóis/farmacologia , Cetoácidos/administração & dosagem , Cetoácidos/sangue , Cetoácidos/química , Cetoácidos/farmacologia , Macaca fascicularis , Estrutura Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores Eicosanoides/agonistas , Receptores Eicosanoides/metabolismo , EstereoisomerismoRESUMO
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.
Assuntos
Ácidos Araquidônicos/farmacologia , Asma/metabolismo , Eosinófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Actinas/genética , Actinas/metabolismo , Alérgenos/imunologia , Animais , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Ácidos Araquidônicos/biossíntese , Asma/induzido quimicamente , Asma/genética , Asma/imunologia , Benzenoacetamidas/farmacologia , Benzotiazóis/farmacologia , Líquido da Lavagem Broncoalveolar/citologia , Gatos , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Cynodon/química , Cynodon/imunologia , Modelos Animais de Doenças , Eosinófilos/metabolismo , Eosinófilos/patologia , Feminino , Expressão Gênica , Humanos , Leucotrieno B4/farmacologia , Masculino , Neutrófilos/metabolismo , Neutrófilos/patologia , Polimerização , Cultura Primária de Células , Prostaglandina D2/farmacologia , Receptores Eicosanoides/antagonistas & inibidores , Receptores Eicosanoides/genética , Receptores Eicosanoides/metabolismoRESUMO
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.