In vitro and in vivo characterization of poractant alfa supplemented with budesonide for safe and effective intratracheal administration.

BackgroundThe intratracheal (IT) administration of budesonide using surfactant as a vehicle has been shown to reduce the incidence of bronchopulmonary dysplasia (BPD) in preterm infants. The objective of this study was to characterize the in vitro characteristics and in vivo safety and efficacy of the extemporaneous combination of budesonide and poractant alfa.MethodsThe stability, minimum surface tension, and viscosity of the preparation were evaluated by means of high-performance liquid chromatography (HPLC), Wilhelmy balance, and Rheometer, respectively. The safety and efficacy of the IT administration of the mixture were tested in two respiratory distress syndrome (RDS) animal models: twenty-seventh day gestational age premature rabbits and surfactant-depleted adult rabbits.ResultsA pre-formulation trial identified a suitable procedure to ensure the homogeneity and stability of the formulation. Wilhelmy Balance tests clarified that budesonide supplementation has no detrimental effect on poractant alfa surface tension activity. The addition of budesonide to poractant alfa did not affect the physiological response to surfactant treatment in both RDS animal models, and was associated to a significant reduction of lung inflammation in surfactant-depleted rabbits.ConclusionOur in vitro and in vivo analysis suggests that the IT administration of a characterized extemporaneous combination of poractant alfa and budesonide is a safe and efficacious procedure in the context of RDS.

CHF6001 II: a novel phosphodiesterase 4 inhibitor, suitable for topical pulmonary administration--in vivo preclinical pharmacology profile defines a potent anti-inflammatory compound with a wide therapeutic window.

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.

Enlightened Mannhemia haemolytica lung inflammation in bovinized mice.

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.

Persistency of Mesenchymal Stromal/Stem Cells in Lungs.

Mesenchymal stromal/stem cells (MSCs) are a fibroblast-like cell population with high regenerative potential that can be isolated from many different tissues. Several data suggest MSCs as a therapeutic tool capable of migrating to a site of injury and guide tissue regeneration mainly through their secretome. Pulmonary first-pass effect occurs during intravenous administration of MSCs, where 50 to 80% of the cells tend to localize in the lungs. This phenomenon has been exploited to study MSC potential therapeutic effects in several preclinical models of lung diseases. Data demonstrated that, regardless of the lung disease severity and the delivery route, MSCs were not able to survive longer than 24 h in the respiratory tract but still surprisingly determined a therapeutic effect. In this work, two different mouse bone marrow-derived mesenchymal stromal/stem cell (mBM-MSC) lines, stably transduced with a third-generation lentiviral vector expressing luciferase and green fluorescent protein reporter genes tracking MSCs in vivo biodistribution and persistency, have been generated. Cells within the engrafted lung were in vivo traced using the high-throughput bioluminescence imaging (BLI) technique, with no invasiveness on animal, minimizing biological variations and costs. In vivo BLI analysis allowed the detection and monitoring of the mBM-MSC clones up to 28 days after implantation independently from the delivery route. This longer persistency than previously observed (24 h) could have a strong impact in terms of pharmacokinetics and pharmacodynamics of MSCs as a therapeutic tool.

In-vivo lung fibrosis staging in a bleomycin-mouse model: a new micro-CT guided densitometric approach.

Although increasing used in the preclinical testing of new anti-fibrotic drugs, a thorough validation of micro-computed tomography (CT) in pulmonary fibrosis models has not been performed. Moreover, no attempts have been made so far to define density thresholds to discriminate between aeration levels in lung parenchyma. In the present study, a histogram-based analysis was performed in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis by micro-CT, evaluating longitudinal density changes from 7 to 21 days after BLM challenge, a period representing the progression of fibrosis. Two discriminative densitometric indices (i.e. 40th and 70th percentiles) were extracted from Hounsfield Unit density distributions and selected for lung fibrosis staging. The strong correlation with histological findings (rSpearman = 0.76, p < 0.01) confirmed that variations in 70th percentile could reflect a pathological lung condition and estimate the effect of antifibrotic treatments. This index was therefore used to define lung aeration levels in mice distinguishing in hyper-inflated, normo-, hypo- and non-aerated pulmonary compartments. A retrospective analysis performed on a large cohort of mice confirmed the correlation between the proposed preclinical density thresholds and the histological outcomes (rSpearman = 0.6, p < 0.01), strengthening their suitability for tracking disease progression and evaluating antifibrotic drug candidates.

Heterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice.

Idiopathic pulmonary fibrosis is a very common interstitial lung disease derived from chronic inflammatory insults, characterized by massive scar tissue deposition that causes the progressive loss of lung function and subsequent death for respiratory failure. Bleomycin is used as the standard agent to induce experimental pulmonary fibrosis in animal models for the study of its pathogenesis. However, to visualize the establishment of lung fibrosis after treatment, the animal sacrifice is necessary. Thus, the aim of this study was to avoid this limitation by using an innovative approach based on a double bleomycin treatment protocol, along with the in vivo images analysis of bleomycin-treated mice. A reporter gene construct, containing the luciferase open reading frame under the matrix metalloproteinase-1 promoter control region, was tested on double bleomycin-treated mice to investigate, in real time, the correlation between bleomycin treatment, inflammation, tissue remodeling and fibrosis. Bioluminescence emitted by the lungs of bleomycin-treated mice, corroborated by fluorescent molecular tomography, successfully allowed real time monitoring of fibrosis establishment. The reporter gene technology experienced in this work could represent an advanced functional approach for real time non-invasive assessment of disease evolution during therapy, in a reliable and translational living animal model.

In vivo image analysis of BoHV-4-based vector in mice.

Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVlucΔTK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVlucΔTK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVlucΔTK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVlucΔTK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVlucΔTK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver.

In vivo imaging of transiently transgenized mice with a bovine interleukin 8 (CXCL8) promoter/luciferase reporter construct.

One of the most remarkable properties of interleukin 8 (CXCL8/IL-8), a chemokine with known additional functions also in angiogenesis and tissue remodeling, is the variation of its expression levels. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by several folds in response to proinflammatory cytokines, bacterial or viral products, and cellular stress. Although mouse cells do not bear a clear homologous IL-8 gene, the murine transcriptional apparatus may well be capable of activating or repressing a heterologous IL-8 gene promoter driving a reporter gene. In order to induce a transient transgenic expression, mice were systemically injected with a bovine IL-8 promoter-luciferase construct. Subsequently mice were monitored for luciferase expression in the lung by in vivo bioluminescent image analysis over an extended period of time (up to 60 days). We demonstrate that the bovine IL-8 promoter-luciferase construct is transiently and robustly activated 3-5 hours after LPS and TNF-α instillation into the lung, peaking at 35 days after construct delivery. Bovine IL-8 promoter-luciferase activation correlates with white blood cell and neutrophil infiltration into the lung. This study demonstrates that a small experimental rodent model can be utilized for non-invasively monitoring, through a reporter gene system, the activation of an IL-8 promoter region derived from a larger size animal (bovine). This proof of principle study has the potential to be utilized also for studying primate IL-8 promoter regions.