Quantification of Lung Fibrosis in IPF-Like Mouse Model and Pharmacological Response to Treatment by Micro-Computed Tomography

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive degenerative lung disease leading to respiratory failure and death. Although anti-fibrotic drugs are now available for treating IPF, their clinical efficacy is limited and lung transplantation remains the only modality to prolong survival of IPF patients. Despite its limitations, the bleomycin (BLM) animal model remains the best characterized experimental tool for studying disease pathogenesis and assessing efficacy of novel potential drugs. In the present study, the effects of oropharyngeal (OA) and intratracheal (IT) administration of BLM were compared in C57BL/6 mice. The development of lung fibrosis was followed in vivo for 28 days after BLM administration by micro-computed tomography and ex vivo by histological analyses (bronchoalveolar lavage, histology in the left lung to stage fibrosis severity and hydroxyproline determination in the right lung). In a separate study, the antifibrotic effect of Nintedanib was investigated after oral administration (60 mg/kg for two weeks) in the OA BLM model. Lung fibrosis severity and duration after BLM OA and IT administration was comparable. However, a more homogeneous distribution of fibrotic lesions among lung lobes was apparent after OA administration. Quantification of fibrosis by micro-CT based on % of poorly aerated tissue revealed that this readout correlated significantly with the standard histological methods in the OA model. These findings were further confirmed in a second study in the OA model, evaluating Nintedanib anti-fibrotic effects. Indeed, compared to the BLM group, Nintedanib inhibited significantly the increase in % of poorly aerated areas (26%) and reduced ex vivo histological lesions and hydroxyproline levels by 49 and 41%, respectively. This study indicated that micro-computed tomography is a valuable in vivo technology for lung fibrosis quantification, which will be very helpful in the future to better evaluate new anti-fibrotic drug candidates

Physiological, biochemical, and biophysical characterization of the lung-lavaged spontaneously-breathing rabbit as a model for respiratory distress syndrome

Nasal continuous positive airway pressure (nCPAP) is a widely accepted technique of non-invasive respiratory support in spontaneously-breathing premature infants with respiratory distress syndrome (RDS). Surfactant administration techniques compatible with nCPAP ventilation strategy are actively investigated. Our aim is to set up and validate a respiratory distress animal model that can be managed on nCPAP suitable for surfactant administration techniques studies. Surfactant depletion was induced by bronchoalveolar lavages (BALs) on 18 adult rabbits. Full depletion was assessed by surfactant component analysis on the BALs samples. Animals were randomized into two groups: Control group (nCPAP only) and InSurE group, consisting of a bolus of surfactant (Poractant alfa, 200 mg/kg) followed by nCPAP. Arterial blood gases were monitored until animal sacrifice, 3 hours post treatment. Lung mechanics were evaluated just before and after BALs, at the time of treatment, and at the end of the procedure. Surfactant phospholipids and protein analysis as well as surface tension measurements on sequential BALs confirmed the efficacy of the surfactant depletion procedure. The InSurE group showed a significant improvement of blood oxygenation and lung mechanics. On the contrary, no signs of recovery were appreciated in animals treated with just nCPAP. The surfactant-depleted adult rabbit RDS model proved to be a valuable and efficient preclinical tool for mimicking the clinical scenario of preterm infants affected by mild/moderate RDS who spontaneously breathe and do not require mechanical ventilation. This population is of particular interest as potential target for the non-invasive administration of surfactant

CHF6297: a novel potent and selective p38 MAPK inhibitor with robust anti-inflammatory activity and suitable for inhaled pulmonary administration as dry powder

Inhibition of p38 mitogen-activated protein kinase (MAPKs) is a potential therapeutic approach for the treatment of acute and chronic pulmonary inflammatory conditions. Here, we report the in vitro and in vivo characterization of the anti-inflammatory effects of CHF6297, a novel potent and selective p38α inhibitor designed for inhalation delivery as a dry powder formulation. CHF6297 has been proven to inhibit p38α enzymatic activity with sub-nanomolar potency (IC50 = 0.14 ± 0.06 nM), with >1,000-fold selectivity against p38γ and p38δ. In human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharides (LPS), as well as in human bronchial epithelial cells (BEAS2B) stimulated with TNF-α or cigarette smoke extract (CSE), CHF6297 inhibited interleukin (IL)-8 release with low nanomolar potency. CHF6297 administered to rats by using a nose-only inhalation device as a micronized dry powder formulation blended with lactose dose-dependently inhibited the LPS-induced neutrophil influx in the bronchoalveolar lavage fluid (BALF). CHF6297 administered intratracheally to rats dose-dependently counteracted the IL-1β (0.3 mg/kg)-induced neutrophil influx (ED50 = 0.22 mg/kg) and increase in IL-6 levels (ED50 = 0.82 mg/kg) in the BALF. In mice exposed to tobacco smoke (TS), CHF6297, administered intranasally (i.n.) for 4 days at 0.03 or 0.3 mg/kg, dose-dependently inhibited the corticosteroid-resistant TS-induced neutrophil influx in the BALF. In a murine house dust mite (HDM) model of asthma exacerbated by influenza virus A (IAV) (H3N3), CHF6297 (0.1 mg/kg, i.n.) significantly decreased airway neutrophilia compared to vehicle-treated IAV/HDM-challenged mice. When CHF6297, at a dose ineffective per se (0.03 mg/kg), was added to budesonide, it augmented the anti-inflammatory effects of the steroid. Overall, CHF6297 effectively counteracted lung inflammation in experimental models where corticosteroids exhibit limited anti-inflammatory activity, suggesting a potential for the treatment of acute exacerbations associated with chronic obstructive pulmonary disease (COPD) and asthma, acute lung injury (ALI), and viral-induced hyperinflammation

The PDE4 Inhibitor Tanimilast Restrains the Tissue-Damaging Properties of Human Neutrophils

: Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001-is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide-a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, degranulation, extracellular DNA traps casting, and cytokine secretion. In contrast, it promoted neutrophil survival by decreasing both spontaneous apoptosis and cell death in the presence of pro-survival factors. The present work suggests that tanimilast can alleviate the severe tissue damage caused by massive recruitment and activation of neutrophils in inflammatory diseases such as COPD