Drug Inhalation is one of the most effective administration routes; in fact, first pass metabolism is bypassed, rapid action onset is enabled and drug doses can be kept relatively low compared to other administration routes. The most recent 3D in vitro models allow to mimic some of the pulmonary tissue functionalities. These models reproduce the air-liquid interface, with beating cilia and mucus production, since different types of cells are present. Therefore, these models could be possibly applied to multi-disciplinary investigations following liquid, dry powder or aerosol treatment. In this thesis, an integrated strategy is proposed, with the aim to increase the rate of success in the drug candidate selection phase. Ideally, safety data should be integrated with early pharmacokinetics (PK) and efficacy indications in order to increase chances to select the candidate with the highest safety margins. With this ambitious objective in mind, a human-based 3D model were evaluated as model for the toxicity assessment and drug permeability evaluation. In particular, a commercially available 3D respiratory model SmallAir™ has been qualified for: a) sensibility and specificity in the evaluation of lung toxicity potential of new compounds; b) permeability to test drug transport through tissues for formulation screening purposes; c) quantitative cytokine secretion on cell supernatant; d) cilia beating and muco-ciliary clearance evaluation by image analysis. These tests performed on a human tissue could provide more reliable results also because all tests were performed in the same model and this could be helpful in data integration. This approach could allow to fill gaps in drug discovery for human-relevant screening of new chemical entities (NCEs), best formulation selection, including physiochemical equivalence evaluation generic drug development. In vitro and in silico data can be helpful in predicting PK and toxicity profiles prior to preclinical and clinical studies. This allows to respect the 3Rs principle of replacement, reduction and refinement of in vivo studies. The proposed integrated testing strategy (ITS) has the potential to reduce the attrition in drug development, to optimize the inhaled formulation, to screen compounds for candidate selection and to reduce in vivo studies.For the toxicity tests, well-known respiratory toxic compound were tested both in the SmallAir™ model and in the A549 cell line model. On the basis of results, the SmallAir™ model seemed to be less sensitive than A549, probably due to the 3D structure physiological features. Cilia beating and mucus production can indeed protect the cells from the toxic effect miming the in vivo response. For the permeability study, well-known inhalation compounds with very different permeability values were evaluated both in SmallAir™ model and in the standard Caco2 cell model. For low and high permeable compounds results obtained were comparable in the two test considered systems. The most evident difference was observed with medium permeable compounds, suggesting that the SmallAir™ model should express different efflux pumps on their surface form the standard Caco2 cell model. The SmallAir™ model was also evaluated as in vitro model for the inflammatory mediators assessment. The treatment with TGF-β allowed to confirm the activation of the signalling via Smad2 while, inconclusive results were obtained with regards to cytokines and ROS release following Bleomycin treatment. The SmallAir™ model was finally evaluated as in vitro model for the assessment of the Muco-ciliary Clearance (MCC). Results obtained in this project, showed that the SmallAir™ can be a promising model to assess the MCC in vitro after treatment with compound acting on ATP release and Cystic fibrosis transmembrane conductance Inhibitor-172 (CFTR172inh). More test considering different compound, study design and end points has to be conducted, in order to identify a human relevant in vitro lung model to be applied in many fields of analysis.

Characterization of an in vitro 3D Human Small Airway Epithelia model for the application of integrated strategies in inhaled drug development

Illaria Masotto
2021-01-01

Abstract

Drug Inhalation is one of the most effective administration routes; in fact, first pass metabolism is bypassed, rapid action onset is enabled and drug doses can be kept relatively low compared to other administration routes. The most recent 3D in vitro models allow to mimic some of the pulmonary tissue functionalities. These models reproduce the air-liquid interface, with beating cilia and mucus production, since different types of cells are present. Therefore, these models could be possibly applied to multi-disciplinary investigations following liquid, dry powder or aerosol treatment. In this thesis, an integrated strategy is proposed, with the aim to increase the rate of success in the drug candidate selection phase. Ideally, safety data should be integrated with early pharmacokinetics (PK) and efficacy indications in order to increase chances to select the candidate with the highest safety margins. With this ambitious objective in mind, a human-based 3D model were evaluated as model for the toxicity assessment and drug permeability evaluation. In particular, a commercially available 3D respiratory model SmallAir™ has been qualified for: a) sensibility and specificity in the evaluation of lung toxicity potential of new compounds; b) permeability to test drug transport through tissues for formulation screening purposes; c) quantitative cytokine secretion on cell supernatant; d) cilia beating and muco-ciliary clearance evaluation by image analysis. These tests performed on a human tissue could provide more reliable results also because all tests were performed in the same model and this could be helpful in data integration. This approach could allow to fill gaps in drug discovery for human-relevant screening of new chemical entities (NCEs), best formulation selection, including physiochemical equivalence evaluation generic drug development. In vitro and in silico data can be helpful in predicting PK and toxicity profiles prior to preclinical and clinical studies. This allows to respect the 3Rs principle of replacement, reduction and refinement of in vivo studies. The proposed integrated testing strategy (ITS) has the potential to reduce the attrition in drug development, to optimize the inhaled formulation, to screen compounds for candidate selection and to reduce in vivo studies.For the toxicity tests, well-known respiratory toxic compound were tested both in the SmallAir™ model and in the A549 cell line model. On the basis of results, the SmallAir™ model seemed to be less sensitive than A549, probably due to the 3D structure physiological features. Cilia beating and mucus production can indeed protect the cells from the toxic effect miming the in vivo response. For the permeability study, well-known inhalation compounds with very different permeability values were evaluated both in SmallAir™ model and in the standard Caco2 cell model. For low and high permeable compounds results obtained were comparable in the two test considered systems. The most evident difference was observed with medium permeable compounds, suggesting that the SmallAir™ model should express different efflux pumps on their surface form the standard Caco2 cell model. The SmallAir™ model was also evaluated as in vitro model for the inflammatory mediators assessment. The treatment with TGF-β allowed to confirm the activation of the signalling via Smad2 while, inconclusive results were obtained with regards to cytokines and ROS release following Bleomycin treatment. The SmallAir™ model was finally evaluated as in vitro model for the assessment of the Muco-ciliary Clearance (MCC). Results obtained in this project, showed that the SmallAir™ can be a promising model to assess the MCC in vitro after treatment with compound acting on ATP release and Cystic fibrosis transmembrane conductance Inhibitor-172 (CFTR172inh). More test considering different compound, study design and end points has to be conducted, in order to identify a human relevant in vitro lung model to be applied in many fields of analysis.
lung, 3D cell culture, toxicity, permeability, muco-ciliary clearance, drug development
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/1043759
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