RESUMO
The development of molecules embedding two distinct pharmacophores acting as muscarinic antagonists and ß2 agonists (MABAs) promises to be an excellent opportunity to reduce formulation issues and boost efficacy through cross-talk and allosteric interactions. Herein, we report the results of our drug discovery campaign aimed at improving the therapeutic index of a previous MABA series by exploiting the super soft-drug concept. The incorporation of a metabolic liability, stable at the site of administration but undergoing rapid systemic metabolism, to generate poorly active and quickly eliminated fragments was pursued. Our SAR studies yielded MABA 29, which demonstrated a balanced in vivo profile up to 24 h, high instability in plasma and the liver, as well as sustained exposure in the lung. In vitro safety and non-GLP toxicity studies supported the nomination of 29 (CHF-6366) as a clinical candidate, attesting to the successful development of a novel super-soft MABA compound.
Assuntos
Antagonistas Muscarínicos , Doença Pulmonar Obstrutiva Crônica , Administração por Inalação , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Broncodilatadores/uso terapêutico , Descoberta de Drogas , Humanos , Pulmão , Antagonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/uso terapêutico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológicoRESUMO
Although it is of great importance for healthcare professionals to ensure that patients' needs and concerns are valued and that they feel confident in the quality of the care they receive, there have been few studies specifically addressing the opinions, experiences and needs of patients with bronchiectasis, and more importantly the emotional impact of the disease, diagnosis and treatment. Using enterprise grade social listening tools, a comprehensive search around bronchiectasis was performed in five languages, on different social media platforms between January 2018 and December 2019 to obtain the perspectives of patients and caregivers from nine countries on symptoms, treatments and burden of the disease. Over 27â000 mentions of bronchiectasis were identified on social media channels, 38.8% of which were posted by patients and caregivers. Approximately 1600 posts were found on bronchiectasis symptoms, out of which persistent cough, shortness of breath and mucus production (22%, 20% and 18%, respectively) were the most commonly discussed. The research revealed that existing diagnostic tests often delay diagnosis or provide inaccurate results, leading to multiple rounds of consults and substantial delays in treatment initiation and management of the disease. Misdiagnosis was common across different age groups, especially among patients without severe symptoms, and this was associated with an emotional burden of anger, confusion, frustration and anxiety. Analysis of social media presents a new approach to derive insights on patients' experiences and emotions with bronchiectasis and has the potential to complement more traditional approaches to drive more patient-focused drug development.
RESUMO
The targeting of both the muscarinic and ß-adrenergic pathways is a well validated therapeutic approach for the treatment of chronic obstructive pulmonary disease (COPD). In this communication we report our effort to incorporate two pharmacologies into a single chemical entity, whose characteristic must be suitable for a once daily inhaled administration. Contextually, we aimed at a locally acting therapy with limited systemic absorption to minimize side effects. Our lung-tailored design of bifunctional compounds that combine the muscarinic and ß-adrenergic pharmacologies by the elaboration of the muscarinic inhibitor 7, successfully led to the potent, pharmacologically balanced muscarinic antagonist and ß2 agonist (MABA) 13.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Broncodilatadores/farmacologia , Descoberta de Drogas , Antagonistas Muscarínicos/farmacologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Administração por Inalação , Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Broncodilatadores/administração & dosagem , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Antagonistas Muscarínicos/administração & dosagem , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptor Muscarínico M3/antagonistas & inibidores , Receptor Muscarínico M3/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Relação Estrutura-AtividadeRESUMO
Neutrophil elastase (NE) is a crucial marker of neutrophilic inflammation. We aimed to compare different techniques to detect active NE in sputum samples of 50 Bronchiectasis (BE) and 50 Cystic Fibrosis (CF) patients. Three methods including a ProteaseTag® Active NE Immunoassay (ELISA) and two enzymatic digestion assays (chromogenic -CS- and fluorogenic -FS- substrate) were compared. Results of active NE were also correlated with clinical data. The three methods provided statistically different values for NE activity in the same sputum samples in both cohorts. In the BE cohort, the highest correlations between NE activity and Bronchiectasis Severity Index (rhoâ¯=â¯0.40, Pâ¯<â¯0.0001), sputum purulence (AUCâ¯=â¯0.79), and chronic infections due to any pathogen (AUCâ¯=â¯0.76) and P. aeruginosa (AUCâ¯=â¯0.80) were found when NE was measured through the activity-based immunoassay. In the CF cohort, the highest correlations between NE activity and sputum quantity (rhoâ¯=â¯0.71) and FEV1% (rhoâ¯=â¯0.42, Pâ¯=â¯0.03) were observed when the FS method was used, while similar correlations with chronic P. aeruginosa infection were identified with the FS and ELISA methods. NE activity in sputum correlates with clinical variables in both diseases. However, different methods to evaluate active NE in sputum lead to significantly different results, also in terms of correlation with clinical data.
Assuntos
Bronquiectasia/enzimologia , Fibrose Cística/enzimologia , Elastase de Leucócito/metabolismo , Escarro/enzimologia , Adulto , Idoso , Bronquiectasia/fisiopatologia , Estudos de Coortes , Fibrose Cística/fisiopatologia , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Índice de Gravidade de DoençaRESUMO
Inhaled corticosteroids (ICSs) represent the first line therapy for the treatment of asthma and are also extensively utilized in chronic obstructive pulmonary disease. Our goal was to develop a new ICS with a basic group, which can allow solid state feature modulation, achieving at the same time high local anti-inflammatory effect and low systemic exposure. Through a rational drug design approach, a new series of pyrrolidine derivatives of budesonide was identified. Within the series, several compounds showed nanomolar binding affinity ( Ki) with GR that mostly correlated with the effect in inducing GR nuclear translocation in CHO cells and anti-inflammatory effects in macrophagic cell lines. Binding and functional cell-based assays allowed identifying compound 17 as a potent ICS agonist with a PK profile showing an adequate lung retention and low systemic exposure in vivo. Finally, compound 17 proved to be more potent than budesonide in a rat model of acute pulmonary inflammation.
Assuntos
Corticosteroides/química , Corticosteroides/farmacologia , Budesonida/química , Budesonida/farmacologia , Desenho de Fármacos , Pneumonia/tratamento farmacológico , Administração por Inalação , Corticosteroides/farmacocinética , Corticosteroides/uso terapêutico , Animais , Budesonida/farmacocinética , Budesonida/uso terapêutico , Células CHO , Cricetulus , Humanos , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica , Células RAW 264.7 , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Distribuição TecidualRESUMO
Phosphodiesterase 4 (PDE4) is a key cAMP-metabolizing enzyme involved in the pathogenesis of inflammatory disease, and its pharmacological inhibition has been shown to exert therapeutic efficacy in chronic obstructive pulmonary disease (COPD). Herein, we describe a drug discovery program aiming at the identification of novel classes of potent PDE4 inhibitors suitable for pulmonary administration. Starting from a previous series of benzoic acid esters, we explored the chemical space in the solvent-exposed region of the enzyme catalytic binding pocket. Extensive structural modifications led to the discovery of a number of heterocycloalkyl esters as potent in vitro PDE4 inhibitors. (S*,S**)-18e and (S*,S**)-22e, in particular, exhibited optimal in vitro ADME and pharmacokinetics properties and dose-dependently counteracted acute lung eosinophilia in an experimental animal model. The optimal biological profile as well as the excellent solid-state properties suggest that both compounds have the potential to be effective topical agents for treating respiratory inflammatory diseases.
Assuntos
Inibidores da Fosfodiesterase 4/química , Inibidores da Fosfodiesterase 4/farmacologia , Relação Estrutura-Atividade , Administração por Inalação , Animais , Sítios de Ligação , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Estabilidade de Medicamentos , Humanos , Masculino , Inibidores da Fosfodiesterase 4/administração & dosagem , Eosinofilia Pulmonar/tratamento farmacológico , Pirrolidinas/química , Ratos Endogâmicos BN , Doenças Respiratórias/tratamento farmacológico , Tiazóis/químicaRESUMO
BACKGROUND: Asthma is a multifactorial disease for which a variety of mouse models have been developed. A major drawback of these models is represented by the transient nature of the airway pathology peaking 24-72 h after challenge and resolving in 1-2 weeks. We characterized the temporal evolution of pulmonary inflammation and tissue remodeling in a recently described mouse model of chronic asthma (8 week treatment with 3 allergens: Dust mite, Ragweed, and Aspergillus; DRA). METHODS: We studied the DRA model taking advantage of fluorescence molecular tomography (FMT) imaging using near-infrared probes to non-invasively evaluate lung inflammation and airway remodeling. At 4, 6, 8 or 11 weeks, cathepsin- and metalloproteinase-dependent fluorescence was evaluated in vivo. A subgroup of animals, after 4 weeks of DRA, was treated with Budesonide (100 µg/kg intranasally) daily for 4 weeks. RESULTS: Cathepsin-dependent fluorescence in DRA-sensitized mice resulted significantly increased at 6 and 8 weeks, and was markedly inhibited by budesonide. This fluorescent signal well correlated with ex vivo analysis such as bronchoalveolar lavage eosinophils and pulmonary inflammatory cell infiltration. Metalloproteinase-dependent fluorescence was significantly increased at 8 and 11 weeks, nicely correlated with collagen deposition, as evaluated histologically by Masson's Trichrome staining, and airway epithelium hypertrophy, and was only partly inhibited by budesonide. CONCLUSIONS: FMT proved suitable for longitudinal studies to evaluate asthma progression, showing that cathepsin activity could be used to monitor inflammatory cell infiltration while metalloproteinase activity parallels airway remodeling, allowing the determination of steroid treatment efficacy in a chronic asthma model in mice.
Assuntos
Remodelação das Vias Aéreas , Asma/patologia , Modelos Animais de Doenças , Inflamação/patologia , Animais , Asma/complicações , Líquido da Lavagem Broncoalveolar , Doença Crônica , Feminino , Fluorescência , Inflamação/complicações , Camundongos , Camundongos Endogâmicos BALB CRESUMO
We evaluated the autocrine activities of cysteinyl leukotrienes (cysteinyl-LTs) in HUVEC and studied the signaling and the pharmacological profile of the CysLT2 receptor (CysLT2R) expressed by ECs, finally assessing the role of the CysLT2R in permeability alterations in a model of isolated brain. Cysteinyl-LTs and their precursor LTA4 contracted HUVEC and increased permeability to macromolecules, increasing the formation of stress fibers through the phosphorylation of myosin light-chain (MLC) following Rho and PKC activation. Accordingly, in an organ model of cerebral vasculature with an intact intima, neutrophils challenge leaded to significant formation of cysteinyl-LTs and edema. Pretreatment with a selective CysLT2R antagonist prevented cytoskeleton rearrangement and HUVEC contraction, along with edema formation in the brain preparation, while leaving the synthesis of cysteinyl-LTs unaffected. We also demonstrate here that the CysLT1R antagonist zafirlukast, pranlukast, pobilukast and iralukast also possess CysLT2R antagonistic activity, which could help in reconsidering previous data on the role of cysteinyl-LTs in the cardiovascular system. The results obtained are further supporting a potential role for CysLT2R in cardiovascular disease.
Assuntos
Comunicação Autócrina , Cisteína/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Leucotrienos/metabolismo , Receptores de Leucotrienos/metabolismo , Transdução de Sinais , Animais , Comunicação Autócrina/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Leucotrieno A4/farmacologia , Leucotrieno C4/farmacologia , Cadeias Leves de Miosina/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Permeabilidade/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide] is a novel phosphodiesterase 4 (PDE4) inhibitor designed for use in pulmonary diseases by inhaled administration. Intratracheal administration of CHF6001 to ovalbumin-sensitized Brown-Norway rats suppressed the antigen-induced decline of lung functions (ED50 = 0.1 µmol/kg) and antigen-induced eosinophilia (ED50 = 0.03 µmol/kg) when administered (0.09 µmol/kg) up to 24 hours before antigen challenge, in agreement with CHF6001-sustained lung concentrations up to 72 hours after intratracheal treatment (mean residence time 26 hours). Intranasal, once daily administration of CHF6001 inhibited neutrophil infiltration observed after 11 days of tobacco smoke exposure in mice, both upon prophylactic (0.15-0.45 µmol/kg per day) or interventional (0.045-0.45 µmol/kg per day) treatment. CHF6001 was ineffective in reversing ketamine/xylazine-induced anesthesia (a surrogate of emesis in rat) up to 5 µmol/kg administered intratracheally, a dose 50- to 150-fold higher than anti-inflammatory ED50 observed in rats. When given topically to ferrets, no emesis and nausea were evident up to 10 to 20 µmol/kg, respectively, whereas the PDE4 inhibitor GSK-256066 (6-[3-(dimethylcarbamoyl)phenyl]sulfonyl-4-(3-methoxyanilino)-8-methylquinoline-3-carboxamide) induced nausea at 1 µmol/kg intratracheally. A 14-day inhalation toxicology study in rats showed a no-observed-adverse-effect level dose of 4.4 µmol/kg per day for CHF6001, lower than the 0.015 µmol/kg per day for GSK-256066. CHF6001 was found effective and extremely well tolerated upon topical administration in relevant animal models, and may represent a step forward in PDE4 inhibition for the treatment of asthma and chronic obstructive respiratory disease.
Assuntos
Anti-Inflamatórios/administração & dosagem , Inibidores da Fosfodiesterase 4/administração & dosagem , Sulfonamidas/administração & dosagem , para-Aminobenzoatos/administração & dosagem , Administração por Inalação , Administração Tópica , Animais , Anti-Inflamatórios/química , Avaliação Pré-Clínica de Medicamentos/métodos , Furões , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Fosfodiesterase 4/química , Ratos , Ratos Endogâmicos BN , Ratos Wistar , Sulfonamidas/química , para-Aminobenzoatos/químicaRESUMO
This study examined the pharmacologic characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, respectively, in inhibiting PDE4 enzymatic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A-D with equal potency, showed an elevated ratio of high-affinity rolipram binding site versus low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity versus PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4(+) T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 µmol/kg) and leukocyte infiltration (ED50 = 0.188 µmol/kg) with an efficacy comparable to a high dose of budesonide (1 µmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions associated with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.
Assuntos
Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Inibidores da Fosfodiesterase 4/administração & dosagem , Inibidores da Fosfodiesterase 4/metabolismo , Administração por Inalação , Administração Tópica , Animais , Furões , Masculino , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos BN , Ratos Sprague-DawleyRESUMO
We studied in vivo the potential involvement of nuclear factor-κB (NF-κB) pathway in the molecular mechanism of the anti-inflammatory and immunomodulatory activity of azithromycin in the lung. Mice transiently transfected with the luciferase gene under the control of a NF-κB responsive element were used to assess in vivo NF-κB activation by bioluminescence imaging. Bioluminescence as well as inflammatory cells and concentrations of proinflammatory cytokines in bronchoalveolar lavage fluids, were monitored in an acute model of pulmonary inflammation resulting from intratracheal instillation of lipopolysaccharide. Lipopolysaccharide (LPS) instillation induced a marked increase in lung bioluminescence in mice transiently transfected with the luciferase gene under the control of an NF-κB responsive element, with significant luciferase expression in resident cells such as endothelial and epithelial cells, as assessed by duoplex immunofluorescence staining. Activation of NF-κB and inflammatory cell lung infiltration linearly correlated when different doses of bortezomib were used to inhibit NF-κB activation. Pretreatment with azithromycin significantly decreased lung bioluminescence and airways cell infiltration induced by LPS, also reducing proinflammatory cytokines concentrations in bronchoalveolar lavages and inhibiting NF-κB nuclear translocation. The results obtained using a novel approach to monitor NF-κB activation, provided, for the first time, in vivo evidence that azithromycin treatment results in pulmonary anti-inflammatory activity associated with the inhibition of NF-κB activation in the lung.
RESUMO
Polymorphonuclear cells diapedesis has an important contribution to the induced Mannhemia haemolytica (M. haemolytica) infection lung inflammation and IL-8 is the primary polymorphonuclear chemoattractant. Using a bovine IL-8/luciferase transiently transgenized mouse model, the orchestration among M. haemolytica, IL-8 promoter activation and neutrophilia was followed in real time by in vivo image analysis.
Assuntos
Modelos Animais de Doenças , Interleucina-8/metabolismo , Mannheimia haemolytica/imunologia , Neutrófilos/imunologia , Infecções por Pasteurella/imunologia , Pneumonia Bacteriana/imunologia , Animais , Bovinos , Feminino , Luciferases/metabolismo , Substâncias Luminescentes/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Neutrófilos/microbiologiaRESUMO
The first steps in the selection process of a new anti-inflammatory drug for the inhaled treatment of asthma and chronic obstructive pulmonary disease are herein described. A series of novel ester derivatives of 1-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3,5-dichloropyridin-4-yl) ethanol have been synthesized and evaluated for inhibitory activity toward cAMP-specific phosphodiesterase-4 (PDE4). In particular, esters of variously substituted benzoic acids were extensively explored, and structural modification of the alcoholic and benzoic moieties were performed to maximize the inhibitory potency. Several compounds with high activity in cell-free and cell-based assays were obtained. Through the evaluation of opportune in vitro ADME properties, a potential candidate suitable for inhaled administration in respiratory diseases was identified and tested in an in vivo model of pulmonary inflammation, proving its efficacy.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Asma/tratamento farmacológico , Benzoatos/síntese química , Pneumopatias Obstrutivas/tratamento farmacológico , Inibidores da Fosfodiesterase 4/síntese química , Sulfonamidas/síntese química , para-Aminobenzoatos/síntese química , Administração por Inalação , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Benzoatos/química , Benzoatos/farmacologia , Linhagem Celular , Doença Crônica , Cristalografia por Raios X , Eosinofilia/tratamento farmacológico , Eosinofilia/imunologia , Eosinofilia/patologia , Ésteres , Cobaias , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Simulação de Acoplamento Molecular , Ovalbumina , Inibidores da Fosfodiesterase 4/química , Inibidores da Fosfodiesterase 4/farmacologia , Conformação Proteica , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , para-Aminobenzoatos/química , para-Aminobenzoatos/farmacologiaRESUMO
The role of the receptor for advanced glycation end products (RAGE) in promoting the inflammatory response through activation of NF-κB pathway is well established. Recent findings indicate that RAGE may also have a regulative function in apoptosis, as well as in cellular proliferation, differentiation, and adhesion. Unlike other organs, lung tissue in adulthood and during organ development shows relatively high levels of RAGE expression. Thus a role for the receptor in lung organogenesis and homeostasis may be proposed. To evaluate the role of RAGE in lung development and adult lung homeostasis, we generated hemizygous and homozygous transgenic mice overexpressing human RAGE, and analyzed their lungs from the fourth postnatal day to adulthood. Moderate RAGE hyperexpression during lung development influenced secondary septation, resulting in an impairment of alveolar morphogenesis and leading to significant changes in morphometric parameters such as airspace number and the size of alveolar ducts. An increase in alveolar cell apoptosis and a decrease in cell proliferation were demonstrated by the terminal deoxy-nucleotidyltransferase-mediated dUTP nick end labeling reaction, active caspase-3, and Ki-67 immunohistochemistry. Alterations in elastin organization and deposition and in TGF-ß expression were observed. In homozygous mice, the hyperexpression of RAGE resulted in histological changes resembling those changes characterizing human bronchopulmonary dysplasia (BPD). RAGE hyperexpression in the adult lung is associated with an increase of the alveolar destructive index and persistent inflammatory status leading to "destructive" emphysema. These results suggest an important role for RAGE in both alveolar development and lung homeostasis, and open new doors to working hypotheses on the pathogenesis of BPD and chronic obstructive pulmonary disease.
Assuntos
Envelhecimento , Pulmão/crescimento & desenvolvimento , Receptores Imunológicos/fisiologia , Animais , Sequência de Bases , Caspase 3/metabolismo , Primers do DNA , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Antígeno Ki-67/metabolismo , Pulmão/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Receptor para Produtos Finais de Glicação Avançada , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1. METHODOLOGY/PRINCIPAL FINDINGS: By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells and fibroblasts, acrolein and CS extract evoked IL-8 release, a response selectively reduced by TRPA1 antagonists. Capsaicin, agonist of the transient receptor potential vanilloid 1 (TRPV1), a channel co-expressed with TRPA1 by airway sensory nerves, and acrolein or CS (TRPA1 agonists), or the neuropeptide substance P (SP), which is released from sensory nerve terminals by capsaicin, acrolein or CS), produced neurogenic inflammation in mouse airways. However, only acrolein and CS, but not capsaicin or SP, released the keratinocyte chemoattractant (CXCL-1/KC, IL-8 analogue) in bronchoalveolar lavage (BAL) fluid of wild-type mice. This effect of TRPA1 agonists was attenuated by TRPA1 antagonism or in TRPA1-deficient mice, but not by pharmacological ablation of sensory nerves. CONCLUSIONS: Our results demonstrate that, although either TRPV1 or TRPA1 activation causes airway neurogenic inflammation, solely TRPA1 activation orchestrates an additional inflammatory response which is not neurogenic. This finding suggests that non-neuronal TRPA1 in the airways is functional and potentially capable of contributing to inflammatory airway diseases.
Assuntos
Canais de Cálcio/biossíntese , Canais de Cálcio/fisiologia , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/fisiologia , Sistema Respiratório/patologia , Canais de Potencial de Receptor Transitório/biossíntese , Canais de Potencial de Receptor Transitório/fisiologia , Animais , Líquido da Lavagem Broncoalveolar , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica/métodos , Inflamação , Interleucina-8/biossíntese , Interleucina-8/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Liso/metabolismo , Fumar , Canal de Cátion TRPA1 , Canais de Cátion TRPV/biossínteseRESUMO
The purpose of this study was to characterize enzyme, receptor, and signaling involved in the synthesis and the activity of cysteinyl leukotrienes (cys-LTs) in human umbilical vein endothelial cells (HUVECs). We used primary cultures of HUVECs and evaluated the formation of cys-LTs by RP-HPLC. Suicide inactivation and subcellular localization of the enzyme responsible for the conversion of leukotriene (LT) A(4) into LTC(4) were studied by repeated incubations with LTA(4) and immunogold electron microscopy. The CysLT(2) receptor in HUVECs was characterized by equilibrium binding studies, Western blot analysis, and immunohistochemistry. Concentration-response curves in HUVECs and in transfected COS-7 cells were used to characterize a novel specific CysLT(2) receptor antagonist (pA(2) of 8.33 and 6.79 against CysLT(2) and CysLT(1) receptors, respectively). The results obtained provide evidence that the mGST-II synthesizing LTC(4) in HUVECs is pharmacologically distinguishable from the LTC(4)-synthase (IC(50) of MK886 <5 µM for LTC(4)-synthase and >30 µM for mGST-II), is not suicide-inactivated and is strategically located on endothelial transport vesicles. The CysLT(2) receptor is responsible for the increase in intracellular Ca(2+) following exposure of HUVECs to cys-LTs and is coupled to a pertussis toxin-insensitive G(q) protein. The synthesis of cys-LTs from LTA(4) by endothelial cells is directly associated with the activation of the CysLT(2) receptor (EC(50) 0.64 µM) in a typical autocrine fashion.
Assuntos
Comunicação Autócrina/fisiologia , Células Endoteliais/metabolismo , Leucotrieno C4/biossíntese , Receptores de Leucotrienos/metabolismo , Animais , Transporte Biológico/fisiologia , Plaquetas/metabolismo , Células COS , Sinalização do Cálcio/fisiologia , Chlorocebus aethiops , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Leucotrieno A4/metabolismo , Receptores de Leucotrienos/genéticaRESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by oxidative stress and increased risk of lung carcinoma. Oxidative stress causes DNA damage which can be repaired by DNA-dependent protein kinase complex. OBJECTIVES: To investigate DNA damage/repair balance and DNA-dependent protein kinase complex in COPD lung and in an animal model of smoking-induced lung damage and to evaluate the effects of oxidative stress on Ku expression and function in human bronchial epithelial cells. METHODS: Protein expression was quantified using immunohistochemistry and/or western blotting. DNA damage/repair was measured using colorimetric assays. RESULTS: 8-OH-dG, a marker of oxidant-induced DNA damage, was statistically significantly increased in the peripheral lung of smokers (with and without COPD) compared with non-smokers, while the number of apurinic/apyrimidinic (AP) sites (DNA damage and repair) was increased in smokers compared with non-smokers (p = 0.0012) and patients with COPD (p < 0.0148). Nuclear expression of Ku86, but not of DNA-PKcs, phospho-DNA-PKcs, Ku70 or γ-H2AFX, was reduced in bronchiolar epithelial cells from patients with COPD compared with normal smokers and non-smokers (p < 0.039). Loss of Ku86 expression was also observed in a smoking mouse model (p < 0.012) and prevented by antioxidants. Oxidants reduced (p < 0.0112) Ku86 expression in human bronchial epithelial cells and Ku86 knock down modified AP sites in response to oxidative stress. CONCLUSIONS: Ineffective DNA repair rather than strand breakage per se accounts for the reduced AP sites observed in COPD and this is correlated with a selective decrease of the expression of Ku86 in the bronchiolar epithelium. DNA damage/repair imbalance may contribute to increased risk of lung carcinoma in COPD.
Assuntos
Dano ao DNA , Neoplasias Pulmonares/genética , Doença Pulmonar Obstrutiva Crônica/genética , Adulto , Idoso , Animais , Antígenos Nucleares/metabolismo , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Células Cultivadas , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Autoantígeno Ku , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estresse Oxidativo/genética , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumar/efeitos adversosRESUMO
Acetaminophen [N-acetyl-p-aminophenol (APAP)] is the most common antipyretic/analgesic medicine worldwide. If APAP is overdosed, its metabolite, N-acetyl-p-benzo-quinoneimine (NAPQI), causes liver damage. However, epidemiological evidence has associated previous use of therapeutic APAP doses with the risk of chronic obstructive pulmonary disease (COPD) and asthma. The transient receptor potential ankyrin-1 (TRPA1) channel is expressed by peptidergic primary sensory neurons. Because NAPQI, like other TRPA1 activators, is an electrophilic molecule, we hypothesized that APAP, via NAPQI, stimulates TRPA1, thus causing airway neurogenic inflammation. NAPQI selectively excites human recombinant and native (neuroblastoma cells) TRPA1. TRPA1 activation by NAPQI releases proinflammatory neuropeptides (substance P and calcitonin gene-related peptide) from sensory nerve terminals in rodent airways, thereby causing neurogenic edema and neutrophilia. Single or repeated administration of therapeutic (15-60 mg/kg) APAP doses to mice produces detectable levels of NAPQI in the lung, and increases neutrophil numbers, myeloperoxidase activity, and cytokine and chemokine levels in the airways or skin. Inflammatory responses evoked by NAPQI and APAP are abated by TRPA1 antagonism or are absent in TRPA1-deficient mice. This novel pathway, distinguished from the tissue-damaging effect of NAPQI, may contribute to the risk of COPD and asthma associated with therapeutic APAP use.
Assuntos
Acetaminofen/efeitos adversos , Acetaminofen/metabolismo , Analgésicos não Narcóticos/efeitos adversos , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/metabolismo , Acroleína/análogos & derivados , Acroleína/farmacologia , Analgésicos não Narcóticos/metabolismo , Animais , Asma/induzido quimicamente , Benzoquinonas , Lavagem Broncoalveolar , Cálcio/metabolismo , Linhagem Celular , Cromatografia Líquida , Cobaias , Humanos , Iminas , Imuno-Histoquímica , Masculino , Camundongos , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Espectrometria de Massas por Ionização por Electrospray , Canal de Cátion TRPA1 , Espectrometria de Massas em Tandem , Canais de Potencial de Receptor Transitório/genéticaRESUMO
PURPOSE: To study the in vivo effect of intratracheal administration of lipopolysaccharide (LPS) in mice by magnetic resonance imaging (MRI) and to investigate the correlation with ex vivo histological evaluation of lung inflammation and oedema. MATERIALS AND METHODS: LPS (or phosphate buffered saline) was administered intratracheally to thirty male Balb/C mice at a concentration of 0.3 mg/ml in a total volume of 100 microl. Animals were divided into fifteen LPS-treated and fifteen control mice. MR images were acquired 24 h after challenge in freely breathing animals with standard ECG-gated Gradient-Echo (GRE) sequences and, in a limited number of animals, with ECG-gated Ultrashort-echo time (UTE) sequences. After MRI, animals were sacrificed, and lungs were fixed and processed for histological analysis of the total volume of healthy lung tissue. RESULTS: GRE images revealed the presence of high intensity signal in lungs of LPS-treated mice that was attributable to oedema caused by alveolar inflammation. In histological slices, regions of alterations in the normal alveolar microstructure were observed that could account for MRI findings. A good correlation was observed between the volumes of lesioned tissue measured by MRI and by histology. The volume of the lesion detected by GRE sequences was lower than the volume detected by UTE sequences. CONCLUSIONS: The effect of intratracheal administration of LPS in mice was investigated by MRI and histology. A good correlation was observed between GRE-MRI and histological findings. MR images obtained with UTE sequences appear to be more sensitive to the presence of lesions than those obtained by standard GRE acquisitions.
Assuntos
Lipopolissacarídeos , Pulmão/patologia , Imageamento por Ressonância Magnética/métodos , Pneumonia/induzido quimicamente , Pneumonia/diagnóstico , Animais , Biópsia/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Traqueia/efeitos dos fármacos , Traqueia/patologiaRESUMO
We studied the urinary excretion of the isoprostane 8-iso-prostaglandin F(2alpha) as an index of in vivo oxidant stress, and the production of leukotriene (LT) B(4) (LTB(4)) by neutrophils in subjects with chronic obstructive pulmonary disease (COPD) and normal subjects. Overnight urinary excretion of the isoprostane was significantly higher in patients with COPD than in control subjects, and LTB(4) production by challenge of neutrophils obtained from patients with COPD was also significantly higher than that observed in control neutrophils. Treatment with a standardized polyphenol extract caused a significant decrease in isoprostane excretion, accompanied by a statistically significant increase of Pa(O(2)). Furthermore, changes in FEV(1) significantly correlated with the changes in isoprostane urinary excretion observed from enrollment to the end of treatment. The results of this study suggest that enhanced oxidative stress in subjects with COPD is paralleled by the increased ability of neutrophils to synthesize the chemotactic factor LTB(4), and may ultimately contribute to the infiltration/activation of neutrophils into the airways of subjects with COPD. Antioxidant treatment in subjects with COPD is effective in reducing oxidant stress as shown by the decrease of urinary isoprostane, a reduction that correlates with the severity of the disease, as indicated by changes in Pa(O(2)) and FEV(1).