Heparan Sulfate Mimetics as a new Therapy for Pulmonary Lesions – MAT-PL

Development and validation of the human precision cut lung slices (hPCLS), an ex vivo model to test to test the safety and efficacy of new antifibrotic agent

Proteomic analysis on hPCLS was developed and validated

Method and synthesis of new antifibrotic agent labelled with fluorochrome validated and tested in vitro in lung cells.

For cell culture, HUVEC (P2 to P9) and MLE-15 (P21 to P29) were purchased from ATCC and from a collaboration with Partner H&P, respectively.

For pro-fibrotic cell model, bleomycin agent as genotoxic molecule from Sigma-Aldrich was used at the concentration of 10 to 20 ug/mL for 72h in HUVEC and at the concentration of 100 ug/mL in MLE-15.

For Low GAG expression model, β-D-Xyloside was used at the concentration of 2mM for 72h in HUVEC. The reduction of total GAG was confirmed using an anti-HS antibody by flow cytometry.

For internalization assay analysis of OTR4132-Alexa647, we used the fluorescent microscopy (Zeiss and Zoe).

For analysis of GAGs:

Detection of different heparan sulfate species in lung tissue by histological methods using anti-HS antibodies.

Detection and quantification of heparan sulfate by biochemical methods (biochemical DMMB).

Detection and quantification of heparan sulfates by HPLC with MS detection.

Methods of detection of heparan sulfate species in lung tissue by histological and biochemical methods is validated. Quantification of GAGs, HS and CS, by biochemical methods is validated from frozen lung tissue is validated. GAGs seems to be implicated in profibrotic processes induced by bleomycin in vitro. OTR4132 is rapidly internalized and distributed on different cell compartments in a time-dependent manner in endothelial cells.

Our data confort the use of OTR4132 in pulmonary diseases and allow financing request(s) to continue building the dossier for a clinical trial for pulmonary fibrosis

Enhancement of lung tissue regeneration and functional recovery following an acute pulmonary insult by regenerative therapy based on the use of heparan sulfate mimetics (HSM) is a promising approach for the treatment of diffuse alveolar damage (DAD), observed in acute respiratory distress syndrome (ARDS), acute exacerbations of fibrotic interstitial lung diseases or severe COVID-19 pneumonitis. The beneficial effects of HSM would be mainly related to the regeneration of the injured tissue by restructuring the destroyed matrix, protecting the cellular communicating peptides (growth factors, cytokines, chemokines), and by limiting the fibrosis usually observed during the repair process. Using of innovative approaches and models developed by 4 partners including a private one, the MAT-PL project will explore the ability of a heparan sulphate mimetic agent (OTR4132) to reduce inflammation, dysregulated angiogenesis and fibrosis, and improves the outcome of acute pulmonary injuries characteristic of DAD, thus opening a new approach for the prevention and the treatment of lung fibrosis.