RESUMO
Arginase is a potential target for asthma treatment. However, there are currently no arginase inhibitors available for clinical use. Here, a novel class of arginase inhibitors was synthesized, and their efficacy was pharmacologically evaluated. The reference compound 2(S)-amino-6-boronohexanoic acid (ABH) and >200 novel arginase inhibitors were tested for their ability to inhibit recombinant human arginase 1 and 2 in vitro. The most promising compounds were separated as enantiomers. Enantiomer pairs SHK242 and SHK243, and SHK277 and SHK278 were tested for functional efficacy by measuring their effect on allergen-induced airway narrowing in lung slices of ovalbumin-sensitized guinea pigs ex vivo. A guinea pig model of acute allergic asthma was used to examine the effect of the most efficacious enantiopure arginase inhibitors on allergen-induced airway hyper-responsiveness (AHR), early and late asthmatic reactions (EAR and LAR), and airway inflammation in vivo. The novel compounds were efficacious in inhibiting arginase 1 and 2 in vitro. The enantiopure SHK242 and SHK277 fully inhibited arginase activity, with IC50 values of 3.4 and 10.5 µM for arginase 1 and 2.9 and 4.0 µM for arginase 2, respectively. Treatment of slices with ABH or novel compounds resulted in decreased ovalbumin-induced airway narrowing compared with control, explained by increased local nitric oxide production in the airway. In vivo, ABH, SHK242, and SHK277 protected against allergen-induced EAR and LAR but not against AHR or lung inflammation. We have identified promising novel arginase inhibitors for the potential treatment of allergic asthma that were able to protect against allergen-induced early and late asthmatic reactions. SIGNIFICANCE STATEMENT: Arginase is a potential drug target for asthma treatment, but currently there are no arginase inhibitors available for clinical use. We have identified promising novel arginase inhibitors for the potential treatment of allergic asthma that were able to protect against allergen-induced early and late asthmatic reactions. Our new inhibitors show protective effects in reducing airway narrowing in response to allergens and reductions in the early and late asthmatic response.
Assuntos
Alérgenos/efeitos adversos , Arginase/antagonistas & inibidores , Asma/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/uso terapêutico , Cobaias , MasculinoRESUMO
Airway inflammation and remodeling are major features of chronic obstructive pulmonary disease (COPD), whereas pulmonary hypertension is a common comorbidity associated with a poor disease prognosis. Recent studies in animal models have indicated that increased arginase activity contributes to features of asthma, including allergen-induced airway eosinophilia and mucus hypersecretion. Although cigarette smoke and lipopolysaccharide (LPS), major risk factors for COPD, may increase arginase expression, the role of arginase in COPD is unknown. This study aimed to investigate the role of arginase in pulmonary inflammation and remodeling using an animal model of COPD. Guinea pigs were instilled intranasally with LPS or saline twice weekly for 12 weeks and pretreated by inhalation of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or vehicle. Repeated LPS exposure increased lung arginase activity, resulting in increased l-ornithine/l-arginine and l-ornithine/l-citrulline ratios. Both ratios were reversed by ABH. ABH inhibited the LPS-induced increases in pulmonary IL-8, neutrophils, and goblet cells as well as airway fibrosis. Remarkably, LPS-induced right ventricular hypertrophy, indicative of pulmonary hypertension, was prevented by ABH. Strong correlations were found between arginase activity and inflammation, airway remodeling, and right ventricular hypertrophy. Increased arginase activity contributes to pulmonary inflammation, airway remodeling, and right ventricular hypertrophy in a guinea pig model of COPD, indicating therapeutic potential for arginase inhibitors in this disease.
Assuntos
Remodelação das Vias Aéreas , Arginase/metabolismo , Doença Pulmonar Obstrutiva Crônica/enzimologia , Animais , Arginase/antagonistas & inibidores , Fibrose , Cobaias , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/imunologia , Hipertensão Pulmonar/patologia , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/imunologia , Hipertrofia Ventricular Direita/patologia , Interleucina-8/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/irrigação sanguínea , Pulmão/enzimologia , Pulmão/patologia , Mucina-5AC/metabolismo , Neutrófilos/patologia , Pneumonia/enzimologia , Pneumonia/imunologia , Pneumonia/patologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologiaRESUMO
Airway remodelling, characterised by increased airway smooth muscle (ASM) mass, subepithelial fibrosis, goblet cell hyperplasia and mucus gland hypertrophy, is a feature of chronic asthma. Increased arginase activity could contribute to these features via increased formation of polyamines and l-proline downstream of the arginase product l-ornithine, and via reduced nitric oxide synthesis. Using the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH), we studied the role of arginase in airway remodelling using a guinea pig model of chronic asthma. Ovalbumin-sensitised guinea pigs were treated with ABH or PBS via inhalation before each of 12 weekly allergen or saline challenges, and indices of arginase activity, and airway remodelling, inflammation and responsiveness were studied 24 h after the final challenge. Pulmonary arginase activity of repeatedly allergen-challenged guinea pigs was increased. Allergen challenge also increased ASM mass and maximal contraction of denuded tracheal rings, which were prevented by ABH. ABH also attenuated allergen-induced pulmonary hydroxyproline (fibrosis) and putrescine, mucus gland hypertrophy, goblet cell hyperplasia, airway eosinophilia and interleukin-13, whereas an increased l-ornithine/l-citrulline ratio in the lung was normalised. Moreover, allergen-induced hyperresponsiveness of perfused tracheae was fully abrogated by ABH. These findings demonstrate that arginase is prominently involved in allergen-induced airway remodelling, inflammation and hyperresponsiveness in chronic asthma.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Arginase/fisiologia , Asma/enzimologia , Asma/fisiopatologia , Remodelação das Vias Aéreas/efeitos dos fármacos , Alérgenos/efeitos adversos , Aminocaproatos/uso terapêutico , Animais , Antiasmáticos/uso terapêutico , Arginase/antagonistas & inibidores , Compostos de Boro/uso terapêutico , Hiper-Reatividade Brônquica/tratamento farmacológico , Doença Crônica , Citrulina/análise , Eosinofilia/tratamento farmacológico , Glândulas Exócrinas/efeitos dos fármacos , Células Caliciformes/efeitos dos fármacos , Cobaias , Interleucina-13/análise , Pulmão/química , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Ornitina/análise , Ovalbumina/efeitos adversos , Fibrose Pulmonar/tratamento farmacológico , Traqueia/efeitos dos fármacos , Traqueia/fisiopatologiaRESUMO
Airway remodelling and emphysema are major structural abnormalities in chronic obstructive pulmonary disease (COPD). In addition, pulmonary vascular remodelling may occur and contribute to pulmonary hypertension, a comorbidity of COPD. Increased cholinergic activity in COPD contributes to airflow limitation and, possibly, to inflammation and airway remodelling. This study aimed to investigate the role of acetylcholine in pulmonary inflammation and remodelling using an animal model of COPD. To this aim, guinea pigs were instilled intranasally with lipopolysaccharide (LPS) twice weekly for 12 weeks and were treated, by inhalation, with the long-acting muscarinic receptor antagonist tiotropium. Repeated LPS exposure induced airway and parenchymal neutrophilia, and increased goblet cell numbers, lung hydroxyproline content, airway wall collagen and airspace size. Furthermore, LPS increased the number of muscularised microvessels in the adventitia of cartilaginous airways. Tiotropium abrogated the LPS-induced increase in neutrophils, goblet cells, collagen deposition and muscularised microvessels, but had no effect on emphysema. In conclusion, tiotropium inhibits remodelling of the airways as well as pulmonary inflammation in a guinea pig model of COPD, suggesting that endogenous acetylcholine plays a major role in the pathogenesis of this disease.
Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Antagonistas Colinérgicos/farmacologia , Pneumonia/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Derivados da Escopolamina/farmacologia , Acetilcolina/fisiologia , Remodelação das Vias Aéreas/imunologia , Animais , Animais não Endogâmicos , Modelos Animais de Doenças , Enfisema/tratamento farmacológico , Enfisema/imunologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/imunologia , Células Caliciformes/metabolismo , Cobaias , Lipopolissacarídeos/toxicidade , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Masculino , Mucina-5AC/metabolismo , Antagonistas Muscarínicos/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Pneumonia/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/imunologia , Brometo de TiotrópioRESUMO
Peroxynitrite has been shown to be crucially involved in airway hyperresponsiveness (AHR) after the late asthmatic reaction (LAR). Peroxynitrite production may result from simultaneous synthesis of nitric oxide (NO) and superoxide by inducible NO-synthase (iNOS) at low L-arginine concentrations. L-arginine availability to iNOS is regulated by its cellular uptake, which can be inhibited by eosinophil-derived polycations and by arginase, which competes with iNOS for the common substrate. Using a guinea pig model of allergic asthma, we investigated whether aberrant L-arginine homeostasis could underlie peroxynitrite-mediated AHR after the LAR. After the LAR, arginase activity in the airways and eosinophil peroxidase release from bronchoalveolar lavage cells were increased. These changes were associated with a 2.0-fold AHR to methacholine as measured in isolated perfused tracheal preparations. AHR was reduced by exogenous L-arginine administration. Moreover, both the arginase inhibitor N(omega)-hydroxy-nor-L-arginine (nor-NOHA) and the polycation antagonist heparin normalised airway responsiveness. These effects were reversed by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME), indicating that both agents reduced AHR by restoring bronchodilating NO production. In conclusion, in allergen-challenged guinea pigs, the AHR after the LAR is caused by arginase- and polycation-induced attenuation of L-arginine availability to iNOS, which may switch the enzyme to simultaneous production of superoxide and NO, and, consequently, peroxynitrite.