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 (p¼0.0012) and patients with COPD (p<0.0148). Nuclear expression of Ku86, but not of DNA-PKcs, phospho-DNA-PKcs, Ku70 or g-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.
Flurbiprofen, a nonsteroidal antiinflammatory drug (NSAID), has been recently described to selectively inhibit beta-amyloid(1)(-)(42) (Abeta42) secretion, the most toxic component of the senile plaques present in the brain of Alzheimer patients. The use of this NSAID in Alzheimer's disease (AD) is hampered by a significant gastrointestinal toxicity associated with cyclooxygenase (COX) inhibition. New flurbiprofen analogues were synthesized, with the aim of increasing Abeta42 inhibitory potency while removing anti-COX activity. In vitro ADME developability parameters were taken into account in order to identify optimized compounds at an early stage of the project. Appropriate substitution patterns at the alpha position of flurbiprofen allowed for the complete removal of anti-COX activity, while modifications at the terminal phenyl ring resulted in increased inhibitory potency on Abeta42 secretion. In rats, some of the compounds appeared to be well absorbed after oral administration and to penetrate into the central nervous system. Studies in a transgenic mice model of AD showed that selected compounds significantly decreased plasma Abeta42 concentrations. These new flurbiprofen analogues represent potential drug candidates to be developed for the treatment of AD.
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 (IC 50 5 0.026 6 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 IC 50 values) the release of tumor necrosis factor-a 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-g from CD4 1 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 (ED 50 5 0.205 mmol/kg) and leukocyte infiltration (ED 50 5 0.188 mmol/kg) with an efficacy comparable to a high dose of budesonide (1 mmol/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.
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.
Background and purpose:We evaluated the effects of 1-(3′,4′-dichloro-2-fluoro[1,1′-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074), a new g-secretase modulator, on brain b-amyloid pathology and spatial memory in transgenic mice expressing the Swedish and London mutations of human amyloid precursor protein (hAPP). Experimental approach: Sixty 6-month-old hAPP mice were treated for 6 months with CHF5074 or ibuprofen (375 ppm in the diet) or standard diet. Twenty-one wild-type mice received standard diet. Key results: Compared with transgenic controls, CHF5074 treatment significantly reduced the area occupied by plaques in cortex (P = 0.003) and hippocampus (P = 0.004). The number of plaques were also reduced by CHF5074 in both cortex (P = 0.022) and hippocampus (P = 0.005). Plaque-associated microglia in CHF5074-treated animals was lower than in transgenic controls in cortex (P = 0.008) and hippocampus (P = 0.002). Ibuprofen treatment significantly reduced microglia area in cortex and hippocampus but not b-amyloid burden. On the last day of the Morris water maze, transgenic controls performed significantly worse than the non-transgenic animals and the CHF5074-treated transgenic mice, on the swimming path to reach the hidden platform. Ibuprofen-treated animals did not perform significantly better than transgenic controls. Conclusions and implications: Chronic CHF5074 treatment reduced brain b-amyloid burden, associated microglia inflammation and attenuated spatial memory deficit in hAPP mice. This novel g-secretase modulator is a promising therapeutic agent for Alzheimer's disease.
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.
Background Non-invasive delivery of nebulized surfactant has been a neonatology long-pursued goal. Nevertheless, the clinical efficacy of nebulized surfactant remains inconclusive, in part, due to the great technical challenges of depositing nebulized drugs in the lungs of preterm infants. The aim of this study was to investigate the feasibility of delivering nebulized surfactant ( poractant alfa ) in vitro and in vivo with an adapted, neonate-tailored aerosol delivery strategy. Methods Particle size distribution of undiluted poractant alfa aerosols generated by a customized eFlow-Neos nebulizer system was determined by laser diffraction. The theoretical nebulized surfactant lung dose was estimated in vitro in a clinical setting replica including a neonatal continuous positive airway pressure (CPAP) circuit, a cast of the upper airways of a preterm neonate, and a breath simulator programmed with the tidal breathing pattern of an infant with mild respiratory distress syndrome (RDS). A dose-response study with nebulized surfactant covering the 100–600 mg/kg nominal dose-range was conducted in RDS-modelling, lung-lavaged spontaneously-breathing rabbits managed with nasal CPAP. The effects of nebulized poractant alfa on arterial gas exchange and lung mechanics were assessed. Exogenous alveolar disaturated-phosphatidylcholine (DSPC) in the lungs was measured as a proxy of surfactant deposition efficacy. Results Laser diffraction studies demonstrated suitable aerosol characteristics for inhalation (mass median diameter, MMD = 3 μm). The mean surfactant lung dose determined in vitro was 13.7% ± 4.0 of the 200 mg/kg nominal dose. Nebulized surfactant delivered to spontaneously-breathing rabbits during nasal CPAP significantly improved arterial oxygenation compared to animals receiving CPAP only. Particularly, the groups of animals treated with 200 mg/kg and 400 mg/kg of nebulized poractant alfa achieved an equivalent pulmonary response in terms of oxygenation and lung mechanics as the group of animals treated with instilled surfactant (200 mg/kg). Conclusions The customized eFlow-Neos vibrating-membrane nebulizer system efficiently generated respirable aerosols of undiluted poractant alfa . Nebulized surfactant delivered at doses of 200 mg/kg and 400 mg/kg elicited a pulmonary response equivalent to that observed after treatment with an intratracheal surfactant bolus of 200 mg/kg. This bench-characterized nebulized surfactant delivery strategy is now under evaluation in Phase II clinical trial (EUDRACT No.:2016–004547-36). Electronic supplementary material The online version of this article (10.1186/s12931-019-1096-9) contains supplementary material, which is available to authorized users.
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