Antifibrotic efficacy of a nintedanib−peptide conjugate and diagnostic potential of a fluorescent companion probe targeting αVβ6 integrin in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic pathology currently treated with two antifibrotic drugs, nintedanib and pirfenidone; however, more effective and safer cell-specific therapeutic agents are needed to overcome their limited efficacy and tolerability. alpha v beta 6 integrin is a clinically validated fibrosis biomarker, and several alpha v beta 6-targeted small molecules and positron emission tomography (PET) tracers have recently proven their therapeutic and diagnostic potential in IPF. Surprisingly, alpha v beta 6-targeted and fibrosis-related drug conjugates are still lacking. Two molecular conjugates, namely the previously reported peptide-drug conjugate (PDC) 1 and the novel fluorescent probe 2, were developed here, where a alpha v beta 6-targeted cyclopeptide is covalently linked to either nintedanib or the near-infrared (NIR) ZW800-1 fluorescent tag via robust linkers. Chemical synthesis of the two compounds, molecular docking studies of 1 in complex with alpha v beta 6, mouse and human plasma stability measurement, binding affinity evaluation toward the isolated alpha v beta 6 receptor, and in vitro human IPF-derived fibroblast cell internalization and antifibrotic studies were performed. Then, in vivo and ex vivo assessments of the antifibrotic efficacy of 1 and the diagnostic potential of 2 were carried out in a bleomycin (BLM)-induced lung fibrosis mouse model. Conjugate 1 demonstrated superior antifibrotic efficacy as compared to the separated peptide and drug components, and probe 2 specifically accumulated in the fibrotic lesions of mice lungs. The molecular conjugates 1 and 2 represent a promising theranostic couple for lung fibrosis and alpha v beta 6-related pathologies and a useful proof-of-principle tool testifying how the simultaneous cell-targeted inhibition of multiple fibrosis-related receptors could be more impactful than the inhibition of one sole receptor.