Galectin-3 inhibitor GB0139 protects against acute lung injury by inhibiting neutrophil recruitment and activation

Rationale: Galectin-3 (Gal-3) drives fibrosis during chronic lung injury, however, its role in acute lung injury (ALI) remains unknown. Effective pharmacological therapies available for ALI are limited; identifying novel concepts in treatment is essential. GB0139 is a Gal-3 inhibitor currently under clinical investigation for the treatment of idiopathic pulmonary fibrosis. We investigate the role of Gal-3 in ALI and evaluate whether its inhibition with GB0139 offers a protective role. The effect of GB0139 on ALI was explored in vivo and in vitro. Methods: The pharmacokinetic profile of intra-tracheal (i.t.) GB0139 was investigated in C57BL/6 mice to support the daily dosing regimen. GB0139 (1–30 µg) was then assessed following acute i.t. lipopolysaccharide (LPS) and bleomycin administration. Histology, broncho-alveolar lavage fluid (BALf) analysis, and flow cytometric analysis of lung digests and BALf were performed. The impact of GB0139 on cell activation and apoptosis was determined in vitro using neutrophils and THP-1, A549 and Jurkat E6 cell lines. Results: GB0139 decreased inflammation severity via a reduction in neutrophil and macrophage recruitment and neutrophil activation. GB0139 reduced LPS-mediated increases in interleukin (IL)-6, tumor necrosis factor alpha (TNFα) and macrophage inflammatory protein-1-alpha. In vitro, GB0139 inhibited Gal-3-induced neutrophil activation, monocyte IL-8 secretion, T cell apoptosis and the upregulation of pro-inflammatory genes encoding for IL-8, TNFα, IL-6 in alveolar epithelial cells in response to mechanical stretch. Conclusion: These data indicate that Gal-3 adopts a pro-inflammatory role following the early stages of lung injury and supports the development of GB0139, as a potential treatment approach in ALI.

Intra-tracheal pharmacokinetics in the mouse: Pharmacokinetics (PK) by the intra-tracheal (i.t.) route was investigated in female C57Bl/6J mice (n=81, body weight 19-20 g) housed in standard holding cages and maintained in a controlled environment with free access to food and water. GB0139 was dosed via the i.t. route (50 µL/mouse) at 0.5 mg/kg and 1 mg/kg (equivalent to 10 and 20 µg total lung dose). Bronchoalveolar lavage fluid (BALF) was collected 2, 4, 8, 12, 24 and 48 hours post-dosing by aspirating 0.5 mL PBS slowly into the trachea using a flexible butterfly catheter via a 3-way tap before the fluid was slowly withdrawn. This was repeated 3 times and BALF kept on ice prior to centrifugation (2,000 x g for 5 mins at 4 °C) and the cell pellet and BALF separated. The cell pellet was snap frozen and stored at -70 °C until day of analysis. Blood samples were taken at 15 mins, 30 mins, 1, 2, 4, 8, 12, 24 and 48 hours post-dosing via the facial vein into K2-EDTA tubes and put on ice prior to centrifugation (2,000 x g for 5 mins at 4 °C) with plasma recovered, snap frozen and stored at -70 °C until day of analysis.
Plasma analysis: Plasma samples from PK studies were thawed and mixed with acetonitrile (1:8) containing internal standard and centrifuged at 1500 x g for 10 minutes. Mouse bronchoalveolar lavage (BAL) cells were thawed and reconstituted in water (1 x 10 6 cells/mL) then mixed with acetonitrile (1:8) containing internal standard and centrifuged at 1500 x g for 10 minutes. The concentration of GB0139 in blood and BAL cells was then determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). to the insertion of a blunted metal 23-gauge intra-tracheal needle. LPS/Bleomycin ± GB0139 (in 50 μL 0.9% NaCl), was inserted into the needle via a pipette, and delivered into the lungs with a 2 x 100 μL bolus of air (using a 1 mL syringe). As a control, mice were given 50 μL PBS (i.t.). The needle was then removed, and the mouse kept upright to ensure entry into the lungs.

Induction of Acute
To reverse anaesthesia, Atipamezole (1 mg/kg, Antisedan, Pfizer) was injected sub-cutaneously 20 minutes after induction of anaesthesia, and animals left to recover at 29 °C overnight prior to retrieval. Bronchoalveolar Lavage: BALF was obtained by exposing the trachea and inserting a plasticcoated 25-gauge needle which was secured in place with elasticated thread. 3 boluses of 0.8 mL PBS were then instilled and retrieved prior to being stored on ice. The first bolus was kept separate from the subsequent two. Lavages were weighed to establish total volume, before being centrifuged at 350 x g for 5 minutes. Supernatants were removed and stored at -80 ⁰C prior to cytokine/protein analysis. Cell pellets were then combined in 1 mL PBS prior to performing a differential cell count on cytocentrifuged preparations stained with Quick-Diff kit (102164, Reagena, West Sussex, UK).
Histology and Immunohistochemistry Preparation: Following exsanguination and retrieval of BALF, lungs were perfused with 10 mL PBS through the right ventricle. Lungs were quickly dissected free and, after tying off the left bronchiole, the right lung was inflated with formalin (HT501128, Sigma-Aldrich) via the trachea and stored for 24 hours before being transferred to 70% ethanol. Lungs were then paraffin-wax embedded prior to being sectioned and stained with haematoxylin and eosin or Masson's trichrome. Total inflammation score and fibrosis score was assessed according to published protocols (Murao et al., 2003;Hübner et al., 2008).
Flow Cytometric Analysis of Lung Digests: Following exsanguination and retrieval of BALF, lungs were perfused with 10 mL PBS through the right ventricle. After tying off the left bronchiole, the left lung was dissected free and placed in a DNase (DNase 1, 1 mg/mL, DN-25, Sigma-Aldrich Company Ltd) and Collagenase (Collagenase D, 10 mg/mL, 11088866001, Roche) mix before being disrupted with scissors and incubated at 37 ⁰C for 1 hour. Cells were further released from tissue by vigorous pipetting using a 1 mL syringe and centrifuged at 300 x g for 15 minutes. The pellet was resuspended in 3 mL cold ACK buffer (Ammonium-Chloride-Potassium, A10492-01, Invitrogen, Carlsbad, California, USA) for 5 minutes on ice to lyse red cells before adding 2 mL PBS and centrifuged at 300 x g for 5 minutes. Following another wash, cells were resuspended in PBS and strained using a 40 µm cell strainer (352340, BD Biosciences). Fc block TM was added 1:100 to the lung suspension for 10 minutes at 4 ⁰C prior to another wash in PBS at 300 x g for 5 minutes. Cells were resuspended in antibody mix in PBS and stained on ice for 30 minutes before the addition of lysis fixation solution (349202, BD Europe). Samples were centrifuged at 350 x g for 5 minutes and resuspended in PBS prior to analysis. The cytometer was set to collect 10,000 events (LSR Fortessa, BD Biosciences).
Data analysis was performed using FlowJo software, version 7.2.4 (Tree Star Inc., USA).

Supplementary Figure 1
Neutrophil numbers and activation parameters were quantified via flow cytometry following tissue digest. d) Alveolar macrophage characterization. Alveolar macrophages (identified as CD11c hi , CD11b low Siglec F hi ) and phenotype (M1/M2) were quantified via flow cytometry following tissue digest. e) Interstitial macrophage characterization.