Inhalation of the prodrug PI3K inhibitor CL27c improves lung function in asthma and fibrosis

PI3K activation plays a central role in the development of pulmonary inflammation and tissue remodeling. PI3K inhibitors may thus offer an improved therapeutic opportunity to treat non-resolving lung inflammation but their action is limited by unwanted on-target systemic toxicity. Here we present CL27c, a prodrug pan-PI3K inhibitor designed for local therapy, and investigate whether inhaled CL27c is effective in asthma and pulmonary fibrosis. Mice inhaling CL27c show reduced insulin-evoked Akt phosphorylation in lungs, but no change in other tissues and no increase in blood glycaemia, in line with a local action. In murine models of acute or glucocorticoid-resistant neutrophilic asthma, inhaled CL27c reduces inflammation and improves lung function. Finally, inhaled CL27c administered in a therapeutic setting protects from bleomycin-induced lung fibrosis, ultimately leading to significantly improved survival. Therefore, local delivery of a pan-PI3K inhibitor prodrug reduces systemic on-target side effects but effectively treats asthma and irreversible pulmonary fibrosis.


Airways Inflammation
Score /4 Absent -(lack of inflammatory cells around airways) 0 Mild -(some airways have small number of cells) 1 Moderate -(some airways have significant number of cells) 2 Marked -(majority of airways have some inflammation) 3 Severe -(majority of airways are significant inflammed) 4 Score 2)

Mild -(some vessels have small number of cells) 1
Moderate -(some vessels have significant inflammation) 2 Marked -(majority of vessels have some inflammation) 3 Severe -(majority of vessels are significantly inflammed) 4
The reaction mixture was incubated at 25oC according to the assay specific incubation time. For

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Caliper based assay, the reaction was terminated by addition of EDTA buffer. Substrate and product were separated and quantified electrophoretically using the microfluidic-based and the assay plate was read on Caliper LabChip 3000 Drug Discovery System from Caliper Life Sciences (Walthan, MA, USA). ADP Glo was another method of detection used for some kinase assays where data was obtained by quantifying the intensity of luminescence. For ADP Glo based assay, the reaction was terminated by applying the Promega ADP Glo Kinase Assay kit and the assay plate was read on a LJL Biosystems Analyst HT.

Measurement of AKT phosphorylation and cell survival of Human Lung Fibroblasts
Control and IPF HLF (described above) were seeded in a 96-well tissue culture plates at 1 x 10 5 cells/well (Falcon, Germany) and starved in serum-free medium for 2 hours. Protein extraction was performed 10 minutes after serum stimulation. AKT phosphorylation was measured by western blot, as described in the "Western blot analysis" in the Methods section.

Measurement of Mucus Production
Lungs were removed and fixed in Milloning buffer solution (pH 7.4) with 4% paraformaldehyde.
For analysis of inflammation score around the bronchial region, the lung sections were stained with hematoxylin and eosin (H&E). Mucus production was analyzed from tissue sections stained with Harris hematoxylin stain and a combination of Periodic acid-Schiff (PAS) stain.
Photomicrographs of airways obtained at 400 × magnification were analyzed using the software Image-Pro Plus (Image-Pro Plus, 4.1; Media Cybernetics, Houston, TX, USA). Nine to twelve bronchial areas per lung were outlined and quantified. Results were expressed as PAS positive area (pixels). Peribronchial fibrosis was analyzed from tissue sections stained with hematoxylin and eosin stain and a combination of Masson's trichrome stain. Photomicrographs of airways 31 obtained at 200 × magnification were analyzed using the software Image-J (NIH, USA). Results were expressed as extracellular matrix deposition area (pixels).

Microsomial stability
All tests for the microsomial stability of CL27c were conducted by the Istituto di Ricerche Biomediche (RBM) SpA -Merck Serono, Ivrea, Italy.
Akt phosphorylation was measured by western blot as described in the "Western blot analysis" in the Methods section.

Preparation and analysis of Treg Cells.
Cells were obtained from Lymph nodes and spleen from naive C57Bl/6J and purified with CD4 T cell isolation Kit (Miltenyi Biotec #130-104-454) and with CD25 biotin Pharmacokinetic studies.