Nintedanib is an anti-fibrotic medication endowed with a multi-kinase inhibitor profile and approved for the treatment of Idiopathic Pulmonary Fibrosis (IPF). Nintedanib is believed to inhibit mainly Vascular Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF), and Fibroblast Growth Factor (FGF) receptor kinases. The main objective was to identify potential tissue and/or circulating biomarkers to demonstrate Nintedanib’s target engagement and support its in vivo pharmacodynamic activity, consistent with its proposed mechanism(s) of action. In four independent experiments of bleomycin (BLM)-induced lung fibrosis model in rats, animals received Nintedanib (oral, 100 mg/kg/day) from day 7 post-BLM for 3 weeks. As expected, Nintedanib significantly reduced lung weight, the levels of lung fibrotic markers, and fibrotic areas. Moreover, Nintedanib-treated animals expressed lower levels of FGF2 in lung homogenates and higher plasma and lung levels of VEGF (≥3-fold, p < 0.05) compared to control animals. Lung proteomic analysis revealed the inhibition of receptor tyrosine kinases signaling in Nintedanib-treated animals. Circulating and lung levels of Nintedanib confirmed an optimal tissue distribution in the rat, consistent with the data reported for humans. Although VEGF ligand levels are elevated in the lungs of Nintedanib-treated animals, the VEGF signaling pathway remained functionally downregulated, strongly suggesting compensatory VEGF feedback delivery to its receptor blockade by Nintedanib. In summary, based on the present experimental findings in rats and supporting clinical preliminary evidence, increased VEGF levels can be reasonably considered an indicator of target engagement for Nintedanib and potentially for other VEGF modulators.

Introduction: Bleomycin (BLM)-induced lung fibrosis is currently the main pre-clinical rodent model in use for the screening of potential therapeutics in Idiopathic Pulmonary Fibrosis (IPF). Typically, histological lung fibrosis scoring is observer-dependent and consequently subject to variability, precluding comparative studies between different laboratories. Thus, a reliable and observer-independent quantification of lung fibrosis is needed. Aims and Objectives: We developed automated image Analysis Protocol Packages (APPs) by using a flexible software (Visiopharm) to quantify fibrotic lung lesions and collagen deposition in digital images of lung sections from mice and rats treated with a double intratracheal administration of BLM. Methods: The first APP was set-up to automatically quantify the fibrotic tissue, detecting accumulation of connective tissue, excess of collagen deposition and cellular hyperproliferation in Masson’s trichrome stained lung sections. The second APP was developed to specifically quantify collagen deposition on Mallory-Azan’s trichrome stained slices. Results: We confirmed a significant increase in BLM-induced lung fibrosis and collagen deposition in both mice and rats at day 14 and 21 post-BLM, respectively, with a significant correlation with the Ashcroft score. Moreover, the antifibrotic effect by oral Nintedanib (60 mg/kg/day in mice and 100 mg/kg/day in rats from day 7 post-BLM) was clearly established. Conclusions: Overall, these data describe a novel observer-independent measure of BLM-induced lung fibrosis in rodents, which could be beneficial for the evaluation of potential preclinical drugs in the lung fibrosis field.

Background: Idiopathic Pulmonary Fibrosis (IPF), is a progressive lung disease characterized by basal and subpleural fibrosis associated with honeycomb changes. In experimental settings, single intratracheal (IT) administration of bleomycin (BLM) to rodents is the most commonly used model. In order to more closely mimic human IPF, a novel experimental procedure with two IT administrations of BLM in rats is described. Aims and objectives: Histological and biomolecular findings following single and double IT administration of BLM in the rat were compared. Furthermore, the effect of Nintedanib was assessed. Methods: Single (4U/Kg, day 1) or double (2 X 2U/Kg, day 1 and day 4) administration of BLM was given IT to male SD rats. Nintedanib (100 mg/kg, oral) was administered once daily for three weeks starting from day 7. Morphometric quantification of different patterns of lung fibrosis and cluster gene expression on tissue homogenate (Real Time PCR) were performed. Results: A single BLM administration induced focal collagen deposition mostly surrounding the main bronchi. In contrast, two BLM administrations seemed to evoke a more diffuse pattern of interstitial collagen deposition widening lung parenchyma. Reduction of lung fibrosis by Nintedanib was more effective in the 2 X 2U/Kg group (~46%) compared to 4U/Kg group (~11%). Moreover, normalisation by Nintedanib of differentially modulated IPF-associated genes was more evident in 2 X 2U/Kg group. Conclusions: Our study provides a novel improved BLM rat model of IPF that is sensitive to the effect of Nintedanib and functional to assess efficacy in drug development.

Background: Evaluation of fibrosis and its severity is the commonest parameter to screen potential new medicines in rodents treated with bleomycin (BLM). Results are mainly reported by histological analysis and indirect measurement of collagen through hydroxyproline (HYP) quantification. Objectives: In the present study we performed a comparative analysis of different methods to assess collagens induced by BLM in the mouse lung. Methods: BLM (0.02 U/kg) was administered by oropharyngeal aspiration to male C57BL/6 mice on day 0 and day 4. Oral Nintedanib (60 mg/kg/day) started from day 7. Three and four weeks after the first BLM administration, histopathological examination and collagen genes expression analysis were applied. Moreover, HYP and collagen isoforms (LC-MS) quantification in lung homogenates were assessed. Results: Ashcroft score and automated measure of lung fibrosis revealed significant fibrotic lesions in BLM-treated animals. Specific collagen genes appeared up-regulated in BLM group. LC-MS quantification of collagen isoforms correlated with HYP levels although, interestingly, showed a higher therapeutic window between control and BLM group (1.5 vs. 0.5 fold, respectively). Nintedanib significantly affected all the evaluated readouts. Conclusions: Our study provides a comprehensive analysis of changes in collagen genes and proteins following BLM treatment in mice and the effect of Nintedanib. Importantly, our data indicate for the first time LC-MS as a sensitive and reliable method for collagen quantification in lung homogenate. This approach might implement the most employed HYP assay to achieve reliable predictions of efficacy for therapeutics in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) remains a progressive and fatal disease despite major advances in antifibrotic therapy. Pirfenidone and nintedanib slow lung function decline, and the PDE4B inhibitor nerandomilast and treprostinil have recently shown promise as next-generation treatments. However, current therapies neither halt nor reverse disease progression, and tolerability issues often limit adherence. We compared the antifibrotic activity and plasma levels of nintedanib administered by either oral gavage or dietary supplementation in a rat model of pulmonary fibrosis. Male Sprague-Dawley rats received intratracheal bleomycin (1 U/kg) on days 0 and 4. From day 7 to day 28, animals were treated with nintedanib (100 mg/kg/day) by either oral gavage or medicated chow. Lung weight, fibrosis biomarkers (procollagen-I, metalloproteinase-7 or MMP7, WNT1-inducible signaling pathway protein or WISP-1), epithelial injury marker KL-6, target engagement biomarkers (Fibroblast Growth Factor 2 or FGF-2, Vascular Endothelial Growth Factor or VEGF), plasma drug levels, and histological fibrosis scores were evaluated. Both administration regimens significantly reduced procollagen-I, WISP-1 and KL-6, and histological fibrosis scores. Oral gavage produced approximately fourfold-higher peak plasma concentrations of nintedanib 30 min after dosing, whereas dietary administration resulted in lower, more stable plasma levels with reduced variability while resulting in comparable antifibrotic efficacy. In conclusion, nintedanib retained robust antifibrotic activity when administered via dietary supplementation despite lower, but continuous, active plasma concentrations. Collectively, these findings indicate that optimizing drug delivery and absorption kinetics may achieve sustained therapeutic efficacy while reducing unnecessary exposure to supratherapeutic concentrations, thereby potentially improving tolerability.

Tanimilast is an inhaled phosphodiesterase‐4 inhibitor in development for chronic obstructive pulmonary disease and asthma. We conducted two studies to evaluate tanimilast pharmacokinetics, one in subjects with mild, moderate, or severe hepatic impairment and matched healthy controls, and one in subjects with mild, moderate, or severe renal impairment and matched healthy controls. Both studies were single‐center, open‐label, and parallel group; all subjects inhaled a single 800 μg dose of tanimilast. The primary objective was to evaluate systemic exposure of tanimilast in subjects with hepatic or renal impairment, and in matched healthy subjects, in terms of maximum observed plasma concentration (C_max) and area under the plasma concentration–time curve from time 0 to the last quantifiable concentration (AUC_0–t). A total of 44 subjects were enrolled in each study (8 with mild, moderate or severe hepatic/renal impairment, and 20 controls), all of whom completed. In the hepatic impairment study, there were no statistically significant differences in tanimilast C_max and AUC_0–t between controls and subjects with mild or moderate impairment. For severe hepatic impairment, C_max did not differ significantly from controls, but AUC_0–t increased by 104% (point estimate of ratio [90% confidence interval], 204.03 [134.36; 309.81]). In the renal impairment study, there were no statistically significant differences in tanimilast C_max and AUC_0–t between controls and subjects with mild‐to‐severe impairment. Three subjects had adverse events in each study, all mild‐to‐moderate, and none were study treatment related. These data suggest that the pharmacokinetics of tanimilast are not impacted by mild‐to‐moderate hepatic impairment, or by mild‐to‐severe renal impairment.Trial Registration: ClinicalTrials.gov (hepatic impairment NCT05373953; renal impairment NCT05431426) and EudraCT (hepatic impairment 2021‐003729‐31; renal impairment 2021‐005567‐43)