In Vivo Target Engagement Assessment of Nintedanib in a Double-Hit Bleomycin Lung Fibrosis Rat Model

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, <i>p</i> < 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.