Magnetic Resonance Imaging of Protease-Mediated Lung Tissue Inflammation and Injury.

Pulmonary inflammation usually involves strong neutrophil recruitment with a marked release of proteases such as neutrophil elastase (NE). Noninvasive in vivo assessment of unregulated elastase activity in the lungs would provide a valuable diagnostic tool. Here, it is proposed to use Overhauser-enhanced magnetic resonance imaging (OMRI) in mice where inflammation was induced by the instillation of lipopolysaccharide (LPS). OMRI contrast in the lungs was generated by a dedicated NE free radical substrate. The free radical decayed more rapidly in LPS-treated mouse lungs than in control mice, indicating the occurrence of increased proteolysis under inflammation. Preclinical detection of abnormal proteolysis opens the way for new diagnosis modality and antiprotease testing in vivo.

An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide.

Pulmonary inflammatory diseases are a major burden worldwide. They have in common an influx of neutrophils. Neutrophils secrete unchecked proteases at inflammation sites consequently leading to a protease/inhibitor imbalance. Among these proteases, neutrophil elastase is responsible for the degradation of the lung structure via elastin fragmentation. Therefore, monitoring the protease/inhibitor status in lungs non-invasively would be an important diagnostic tool. Herein we present the synthesis of a MeO-Suc-(Ala)2-Pro-Val-nitroxide, a line-shifting elastase activity probe suitable for Electron Paramagnetic Resonance spectroscopy (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI). It is a fast and sensitive neutrophil elastase substrate with Km = 15 ±â€¯2.9 µM, kcat/Km = 930,000 s-1 M-1 and Km = 25 ±â€¯5.4 µM, kcat/Km = 640,000 s-1 M-1 for the R and S isomers, respectively. These properties are suitable to detect accurately concentrations of neutrophil elastase as low as 1 nM. The substrate was assessed with broncho-alveolar lavages samples derived from a mouse model of Pseudomonas pneumonia. Using EPR spectroscopy we observed a clear-cut difference between wild type animals and animals deficient in neutrophil elastase or deprived of neutrophil Elastase, Cathepsin G and Proteinase 3 or non-infected animals. These results provide new preclinical ex vivo and in vivo diagnostic methods. They can lead to clinical methods to promote in time lung protection.