The aim of this study was to describe and characterize the pathophysiological changes occurring during the early inflammatory phase (first 3 days) in the rat bleomycin model of lung injury preceding the development of fibrosis. Further, we wanted to understand the kinetics and factors contributing to bleomycin‐induced acute lung injury (ALI) and provide a robust, reliable and reproducible framework of features of ALI readouts to assess effects of therapeutics on bleomycin‐induced ALI in rats. We induced ALI in rats with intratracheal (i.t.) installation of bleomycin. The animals were sacrificed on predetermined time points, that is, Day 0, 1, 2, and 3 post the bleomycin challenge. We analyzed bronchoalveolar lavage fluid (BALF) and lung tissue to establish and assess relevant experimental features of ALI. We demonstrated that bleomycin induced key features of experimental ALI including a profound increase in neutrophils in BALF (50–60%), pulmonary edema, and lung pathology on Day 3 after challenge. Furthermore, we showed that TGF‐β1, IL‐1β, TNF‐α, IL‐6, CINC‐1, TIMP‐1, and WISP‐1 were induced by studying their kinetic profile during the first 3 days after bleomycin injury consistent with their known role ALI. We also confirmed that detectable fibrogenesis occurs at the earliest on Day 3 after injury based on collagen content, along with changes in the TGF‐β/Smad signaling pathway and increased expression of Galectin‐3, Vimentin, and Fibronectin in lung homogenate. Our report presents robust features and contributing mediators/factors to the pathology of bleomycin‐induced ALI in rats on Day 3. The kinetic data provide insights on the progression of ALI and a detailed understanding of early events before actual fibrosis development. This set of experimental endpoints is very appropriate and invaluable for efficacy testing of potential novel therapeutic treatments (single or combined) in ALI and understanding their mechanism of action.

The long-sought-after “magic bullet” in systemic therapy remains unrealized for disease targets existing inside most tissues, theoretically because vascular endothelium impedes passive tissue entry and full target engagement. We engineered the first “dual precision” bispecific antibody with one arm pair to precisely bind to lung endothelium and drive active delivery and the other to precisely block TGF-β effector function inside lung tissue. Targeting caveolae for transendothelial pumping proved essential for delivering most of the injected intravenous dose precisely into lungs within one hour and for enhancing therapeutic potency by >1000-fold in a rat pneumonitis model. Ultra-low doses (μg/kg) inhibited inflammatory cell infiltration, edema, lung tissue damage, disease biomarker expression and TGF-β signaling. The prodigious benefit of active vs passive transvascular delivery of a precision therapeutic unveils a new promising drug design, delivery and therapy paradigm ripe for expansion and clinical testing.

Background The bleomycin mouse model is the most prevalent animal model used to study idiopathic pulmonary fibrosis (IPF), ranging from basic pathophysiological mechanisms to therapeutic modalities. High mortality rates, inconsistencies in disease induction, and great severity of the disease phenotype have limited its utility and clinical relevance. In this study, we identify conditions in the mouse bleomycin model that maintain the desired IPF phenotype found in patients while avoiding the usual, associated rapid mouse mortality. Methods In two independent studies, we analyzed the effects of incremental intratracheal (IT) high (1-3 U/kg) and low (0.05-0.5 U/kg) bleomycin doses on pulmonary fibrosis in mice. We observed dose effects on weight loss, morbidity and mortality, inflammation, lung collagen content, presence of various biomarkers in bronchoalveolar lavage fluid (BALf), and histology 14 days post-treatment, when the animals were in the active phase of fibrosis. Results We characterize the fibrotic effects of various bleomycin doses in mice on pulmonary inflammation, myofibroblast activation, vascular leakiness, angiogenesis, and extracellular tissue remodeling. Higher bleomycin doses induced acute disease with severe and saturated responses, extreme and progressive weight loss, and high morbidity and mortality rates. In contrast, lower doses of bleomycin induced a milder and chronic response, featuring robust, active-phase fibrosis with mild weight loss and no mortality. Conclusion Here we demonstrate that the bleomycin mouse model can be used to reproduce symptoms of IPF patients in an animal by using low doses of bleomycin. Low concentrations of bleomycin induce chronic and progressive fibrosis with no mortality.

The long-sought-after 'magic bullet' in systemic therapy remains unrealized for disease targets existing inside most tissues, theoretically because vascular endothelium impedes passive tissue entry and full target engagement. We engineered the first 'dual precision' bispecific antibody with one arm pair to precisely bind to lung endothelium and drive active delivery and the other to precisely block TGF-β effector function inside lung tissue. Targeting caveolae for transendothelial pumping proved essential for delivering most of the injected intravenous dose precisely into lungs within one hour and for enhancing therapeutic potency by >1000-fold in a rat pneumonitis model. Ultra-low doses (μg/kg) inhibited inflammatory cell infiltration, edema, lung tissue damage, disease biomarker expression and TGF-β signaling. The prodigious benefit of active vs passive transvascular delivery of a precision therapeutic unveils a new promising drug design, delivery and therapy paradigm ripe for expansion and clinical testing.