Amir Smailagic

Altered lung function relates to inflammation in an acute LPS mouse model.

Preclinical in vivo models of lipopolysaccharide (LPS) -induced acute lung injury are commonly used to recapitulate pathophysiological features of chronic obstructive pulmonary disease and acute exacerbations. The LPS-induced lung inflammation is well described; however, whether the inflammatory response relates temporally to specific alterations in lung function has not been elucidated. We have investigated the effects of acute LPS inhalation in mice up to 96 h post LPS. Quantitation of inflammatory cells and inflammatory mediators in bronchoalveolar lavage fluid and non-invasive and invasive lung function measurements were performed at corresponding time points. The inhibitory effect of the glucocorticoid, budesonide, on LPS-induced lung inflammation and lung function was determined. LPS inhalation induced distinct histopathological changes, and infiltration of inflammatory cells to the lungs peaked at 48 h. At this time point, significantly increased inflammatory mediators and significantly altered lung capacity and mechanics parameters were observed. Budesonide given per os prevented the LPS-induced lung inflammation and lung dysfunction. These results demonstrate a temporal relationship between the peak of inflammatory cell influx and significant impairment of lung function, suggestive of a causative role of inflammation. These results allow better understanding of the functional consequences of lung inflammation in respiratory diseases.

The Discovery of Potent and Selective Non-Steroidal Glucocorticoid Receptor Modulators, Suitable for Inhalation.

We report the discovery of highly potent and selective non-steroidal glucocorticoid receptor modulators with PK properties suitable for inhalation. A high throughput screen of the AstraZeneca compound collection identified sulfonamide 3 as a potent non-steroidal glucocorticoid receptor ligand. Further optimization of this lead generated indazoles 30 and 48 that were progressed to characterization in in vivo models. X-ray crystallography was used to gain further insight into the binding mode of selected ligands.

(1)H and hyperpolarized (3)He MR imaging of mouse with LPS-induced inflammation

To evaluate the lipopolysaccharide (LPS) model of chronic obstructive pulmonary disease (COPD) in mouse with 1H and hyperpolarized (HP) 3He MR imaging.

Evaluation of excised lung gas volume measurements in animals with genetic or induced emphysema

Emphysema, a leading cause of respiratory disability and mortality in humans, is characterized by destruction of alveolar walls and enlargement of airspaces. Animal studies are critical in understanding the pathogenesis of emphysema. However, current measurements of airspace enlargement and emphysema in small laboratory animals are labor intensive and may not be sensitive enough for measuring alterations in lung function and structure at the early stages of emphysema. In this study, we have investigated the excised lung gas volume (ELGV) measurement as a potential index for determining airspace enlargement in pallid mice with developing emphysema, in tight-skin mice with developed emphysema, or in Wistar rats with emphysema induced by an intratracheal instillation of pancreatic elastase. Our results showed that values of both ELGV per lung and per gram lung tissue were significantly increased in all three emphysema models, compared to control. The ELGV values were correlated well with morphometric evaluation of emphysema. Variations in transpulmonary pressures caused by different termination procedures were critical factors influencing the ELGV values. The present study demonstrates that ELGV measurement is a simple and sensitive method to monitor the development of emphysema.

The discovery of a selective and potent A2a agonist with extended lung retention.

Although the anti‐inflammatory role of the A2a receptor is well established, controversy remains with regard to the therapeutic value for A2a agonists in treatment of inflammatory lung diseases, also as a result of unwanted A2a‐mediated cardiovascular effects. In this paper, we describe the discovery and characterization of a new, potent and selective A2a agonist (compound 2) with prolonged lung retention and limited systemic exposure following local administration. To support the lead optimization chemistry program with compound selection and profiling, multiple in vitro and in vivo assays were used, characterizing compound properties, pharmacodynamics (PD), and drug concentrations. Particularly, pharmacokinetic‐PD modeling was applied to quantify the effects on the cardiovascular system, and an investigative toxicology study in rats was performed to explore potential myocardial toxicities. Compound 2, in comparison to a reference A2a agonist, UK‐432,097, demonstrated higher solubility, lower lipophilicity, lower plasma protein binding, high rat lung retention (28% remaining after 24 h), and was efficacious in a lung inflammatory rat model following intratracheal dosing. Despite these properties, compound 2 did not provide a sufficient therapeutic index, that is, separation of local anti‐inflammatory efficacy in the lung from systemic side effects in the cardiovascular system. The plasma concentration that resulted in induction of hypotension (half maximal effective concentration; EC50 0.5 nmol/L) correlated to the in vitro A2a potency (rIC50 0.6 nmol/L). Histopathological lesions in the heart were observed at a dose level which is threefold above the efficacious dose level in the inflammatory rat lung model. In conclusion, compound 2 is a highly potent and selective A2a agonist with significant lung retention after intratracheal administration. Despite its local anti‐inflammatory efficacy in rat lung, small margins to the cardiovascular effects suggested limited therapeutic value of this compound for treatment of inflammatory lung disease by the inhaled route.

Sensitivity of Disease Parameters to Flexible Budesonide/Formoterol Treatment in an Allergic Rat Model

RATIONALE Clinical studies show that flexible dosing (maintenance and symptom-driven dose adjustments) of budesonide and formoterol (BUD/FORM) improves control of asthma exacerbations as compared to fixed maintenance dosing protocols (maintenance therapy) even when the latter utilize higher BUD/FORM doses. This suggests that dose-response relationships for certain pathobiologic mechanisms in asthma shift over time. Here, we have conducted animal studies to address this issue. OBJECTIVES (1) To test in an animal asthma-like model whether it is possible to achieve the same or greater pharmacological control over bronchoconstriction and airway/lung inflammation, and with less total drug used, by flexible BUD/FORM dosing (upward adjustment of doses) in association with allergen challenges. (2) To determine whether the benefit requires adjustment of both drug components. METHODS Rats sensitized on days 0 and 7 were challenged intratracheally with ovalbumin on days 14 and 21. On days 13-21, rats were treated intratracheally with fixed maintenance or flexible BUD/FORM combinations. On day 22, rats were challenged with methacholine and lungs were harvested for analysis. RESULTS A flexible BUD/FORM dosing regimen (using 3.3 times less total drug than the fixed maintenance high dose regimen), delivered the same or greater reductions of excised lung gas volume (a measure of gas trapped in lung by bronchoconstriction) and lung weight (a measure of inflammatory oedema). When either BUD or FORM alone was increased on days of challenge, the benefit of the flexible dose upward adjustment was lost. CONCLUSIONS Flexible dosing of the BUD/FORM combination improves the pharmacological inhibition of allergen-induced bronchoconstriction and an inflammatory oedema in an allergic asthma-like rat model.

¹H and hyperpolarized ³He magnetic resonance imaging clearly detect the preventative effect of a glucocorticoid on endotoxin-induced pulmonary inflammation in vivo.

Introduction: Proton (1H) magnetic resonance imaging (MRI) can be utilized to quantify pulmonary edema in endotoxin-induced pulmonary inflammation and hyperpolarized (HP) 3He MRI can assess pulmonary ventilation. Neither of the methods has been applied to assess the impact of a drug on endotoxin-induced pulmonary inflammation in vivo. The aim of the current study was to evaluate the capability of 1H and HP 3He MRI to assess the effects of a glucocorticoid on endotoxin-induced pulmonary inflammation in vivo. Materials and Methods: Mice were exposed to an aerosol of either saline or endotoxin (5 mg/ml) for 10 min. Half of the endotoxin-exposed mice were pretreated with a glucocorticoid (budesonide 3 mg/kg; 2 times/day) and the other half with vehicle p.o. The first budesonide treatment was administered 1 h prior to the aerosol inhalation. Forty-eight hours after the aerosol exposure, the mice were anaesthetized for subsequent imaging. Hyperpolarized 3He was administered and axial MR images of the lungs obtained. Matching 1H MR images were then acquired. The mice were sacrificed and broncho-alveolar lavage (BAL) samples were harvested to determine total and cell differential counts. Results: The lesion volume on both 1H and 3He MRI, were markedly increased by endotoxin exposure (P<0.001). Budesonide strongly reduced lesion volume (P<0.001). The BAL cell count correlated strongly with both 3He (P<0.001; r = 0.96) and 1H lesion volumes (P<0.001; r = 0.97). Conclusions: Hyperpolarized 3He MRI and 1H MRI clearly visualized the preventative effect of budesonide on the impact of endotoxin on pulmonary ventilation and edema, respectively. The fact that ventilation defects on 3He MRI corresponded to findings from conventional 1H MRI, as well as to counts of BAL inflammatory cells suggests that these imaging techniques constitute promising tools for non-invasive monitoring of pulmonary inflammation in vivo.

Variable flip angle 3D ultrashort echo time (UTE) T1 mapping of mouse lung: A repeatability assessment: VFA 3D-UTE T1 Mapping of Mouse Lung

Lung T1 is a potential translational biomarker of lung disease. The precision and repeatability of variable flip angle (VFA) T1 mapping using modern 3D ultrashort echo time (UTE) imaging of the whole lung needs to be established before it can be used to assess response to disease and therapy.