ABSTRACT
Title
Efficacy of NCX 226, a compound donating nitric oxide while blocking endothelin receptors, on bleomycin-induced lung fibrosis in mice and monocrotaline-induced pulmonary hypertension in rats
Authors
A. Pini1, D. Bani1, E. Masini2, S. Viappiani3, V. Girod4, S. Robelet4, M. Bolla3
Departments of: 1Anatomy Histology & Forensic Medicine; 2Preclinical & Clinical Pharmacology, University of Florence, Florence, Italy. 3NicOx Research, Bresso, Italy. 4Syncrosome, Marseille, France
Departments of: 1Anatomy Histology & Forensic Medicine; 2Preclinical & Clinical Pharmacology, University of Florence, Florence, Italy. 3NicOx Research, Bresso, Italy. 4Syncrosome, Marseille, France
Abstract
Endothelin receptor blockers act by inhibiting the vasocontracting, pro-inflammatory and pro-fibrotic effects of endothelin (ET). NCX 226 is a novel nitric oxide (NO)-donating compound based on the structure of bosentan thus possessing ET receptor antagonism while releasing NO. Pre-clinical data suggest that NO may exert important anti-inflammatory and anti-fibrotic actions and improve microcirculation.We tested the activity of NCX 226 in bleomycin-induced lung fibrosis in the mouse and in monocrotaline-induced pulmonary hypertension in the rat, in comparison with bosentan.
Lung fibrosis model: Bleomycin was instilled intra-tracheally in C57BL/6 mice then treated orally for 14 days with NCX 226 or bosentan (equimolar at 10 and 30 mg/kg). Airway resistance was measured at the end of the treatment period. At sacrifice, lung specimens were collected for histological (collagen and microvascular density, arterial dilation) and biochemical analyses. Parameters of lung inflammation and fibrosis, such as accumulation of leukocytes (myeloperoxidase, MPO), oxidative stress (thiobarbituric acid reactive substances, TBARS; 8-hydroxy-2’-deoxyguanosine, 8-OHdG), and tissue levels of the pro-fibrotic cytokine TGF-β and collagen-derived hydroxyproline, were measured. Pulmonary hypertension model: subcutaneous monocrotaline (60 mg/kg) was administered to Sprague Dawley rats then treated orally (once a day) for 21 days with vehicle, test compounds (NCX 226 and bosentan in equimolar doses to 100 and 300 mg/kg) or 40 mg/kg tadalafil (PDE5 inhibitor) as positive control. At the end of treatment period, 2 hours post last dose, mean arterial pressure, right ventricular pressure (RVP) and cardiac mass ratio (weight ratio of right ventricle/left ventricle+septum, RV/LV+S) were assessed.
Lung fibrosis results: Both test drugs prevented bleomycin-induced airway stiffness, with NCX 226 being better. High dose NCX 226 was significantly more effective than equimolar bosentan in reducing lung tissue levels of myeloperoxidase (10.9, 4.8*# and 6.9* mU/mg prot), TGF-β (357.5, 134*# and 200.6* pg/mg prot) and thiobarbituric-acid reactive substances (10, 4.4*# and 6.6* nmol/mg prot); values for vehicle, 37 mg/kg NCX 226 and 30 mg/kg bosentan, respectively (p<0.01 vs vehicle* or equimolar bosentan #). Both compounds decreased 8-OHdG, lung sclerosis and inflammation-induced vasculogenesis, with a trend for NCX 226 as being more potent than bosentan, although the differences do not reach statistical significance. Hydroxyproline measurements showed inconsistent variability between groups.
Pulmonary hypertension results: At day 21 post monocrotaline, none of the test compounds affected the mean arterial pressure of the rats, while pulmonary hypertension has developed (vehicle group RVP: 54.3±8.6 mmHg). NCX 226 (both doses), bosentan (high dose only) and tadalafil treatment significantly reduced RVP (39.4±8.0, 39.0±4.5, 41.0±10.3 and 40.5±6.4 mmHg, respectively). Only tadalafil and high dose NCX 226 had a significant reduction in the cardiac weight ratio (RV/LV+S: 0.53±0.11, 0.40±0.05 and 0.40±0.08 for vehicle, tadalafil and NCX 226, respectively).
NCX 226 and bosentan counteract lung inflammation and airway stiffness in an in vivo mouse model of bleomycin-induced lung fibrosis. NCX 226 shows improved efficacy over bosentan in reducing key inflammatory parameters, such as leukocyte infiltration, TGF-β secretion and oxidative stress. NCX 226 showed improved efficacy over bosentan on RVP and cardiac mass ratio in a pulmonary hypertension model. The data confirm a potential role for the modulation of NO pathway in the control of these pathologies.
Lung fibrosis model: Bleomycin was instilled intra-tracheally in C57BL/6 mice then treated orally for 14 days with NCX 226 or bosentan (equimolar at 10 and 30 mg/kg). Airway resistance was measured at the end of the treatment period. At sacrifice, lung specimens were collected for histological (collagen and microvascular density, arterial dilation) and biochemical analyses. Parameters of lung inflammation and fibrosis, such as accumulation of leukocytes (myeloperoxidase, MPO), oxidative stress (thiobarbituric acid reactive substances, TBARS; 8-hydroxy-2’-deoxyguanosine, 8-OHdG), and tissue levels of the pro-fibrotic cytokine TGF-β and collagen-derived hydroxyproline, were measured. Pulmonary hypertension model: subcutaneous monocrotaline (60 mg/kg) was administered to Sprague Dawley rats then treated orally (once a day) for 21 days with vehicle, test compounds (NCX 226 and bosentan in equimolar doses to 100 and 300 mg/kg) or 40 mg/kg tadalafil (PDE5 inhibitor) as positive control. At the end of treatment period, 2 hours post last dose, mean arterial pressure, right ventricular pressure (RVP) and cardiac mass ratio (weight ratio of right ventricle/left ventricle+septum, RV/LV+S) were assessed.
Lung fibrosis results: Both test drugs prevented bleomycin-induced airway stiffness, with NCX 226 being better. High dose NCX 226 was significantly more effective than equimolar bosentan in reducing lung tissue levels of myeloperoxidase (10.9, 4.8*# and 6.9* mU/mg prot), TGF-β (357.5, 134*# and 200.6* pg/mg prot) and thiobarbituric-acid reactive substances (10, 4.4*# and 6.6* nmol/mg prot); values for vehicle, 37 mg/kg NCX 226 and 30 mg/kg bosentan, respectively (p<0.01 vs vehicle* or equimolar bosentan #). Both compounds decreased 8-OHdG, lung sclerosis and inflammation-induced vasculogenesis, with a trend for NCX 226 as being more potent than bosentan, although the differences do not reach statistical significance. Hydroxyproline measurements showed inconsistent variability between groups.
Pulmonary hypertension results: At day 21 post monocrotaline, none of the test compounds affected the mean arterial pressure of the rats, while pulmonary hypertension has developed (vehicle group RVP: 54.3±8.6 mmHg). NCX 226 (both doses), bosentan (high dose only) and tadalafil treatment significantly reduced RVP (39.4±8.0, 39.0±4.5, 41.0±10.3 and 40.5±6.4 mmHg, respectively). Only tadalafil and high dose NCX 226 had a significant reduction in the cardiac weight ratio (RV/LV+S: 0.53±0.11, 0.40±0.05 and 0.40±0.08 for vehicle, tadalafil and NCX 226, respectively).
NCX 226 and bosentan counteract lung inflammation and airway stiffness in an in vivo mouse model of bleomycin-induced lung fibrosis. NCX 226 shows improved efficacy over bosentan in reducing key inflammatory parameters, such as leukocyte infiltration, TGF-β secretion and oxidative stress. NCX 226 showed improved efficacy over bosentan on RVP and cardiac mass ratio in a pulmonary hypertension model. The data confirm a potential role for the modulation of NO pathway in the control of these pathologies.