Intratracheal aerosolisation in Sprague Dawley rats, a new method for assessment of pulmonary toxicity of drug. M. de Monte 1, S. Le Guellec 2, J. Montharu 1, Y. Rabemampianina 4, J. Guillemain 3, B. Kittel 4, F. Gauthier 1, P. Diot 1 1- INSERM U-618 (National Institute for Health and Medical Research), Aerosol team, IFR135, Université François Rabelais, Tours, F France 2- ATOMISOR - DTF, La Diffusion Technique Française, F-42003, Saint Etienne, France 3- ADREMI, Faculté de Pharmacie, F-37000, Tours, France 4- PFIZER Global Research and Development, ZI Pocé-sur-Cisse, BP159, F-37041, Amboise, France Protéases et Vectorisation Pulmonaires INSERM U-618 Introduction Local respiratory tolerance is a critical issue for inhaled drugs (1/2). It is usually assessed by In vivo studies using the intranasal route, which limit lung deposition, or inhalation chambers which require long exposition times and induce high fur deposition. The aim of this study was to develop a new model for rapid evaluation of acute toxicity of aerosolized drugs in rats. The model was designed with imaging and quantification of lung deposition and tested with a well-known irritant molecule (TEST group) and an inflammatory molecule, LPS. COLLOIDS group LPS (50µg) + 99mTc-colloids J0: Aerosolizations (200µL) Drug test (105µg) + 99mTc-colloids 99mTc-Colloids Imaging of lung deposition (Biospace ® ) J0+24h : Sacrifice and analyses  Biochemical analyses (on BAL fluid): Cells count (PMNs, Macrophages and red blood cells RBC); Protein, cellular cytotoxicity (LDH) and cytokine (TNF-  ) assays.  Histological analyses (Lungs and trachea)  Lung deposition (LPS, TEST and Colloids groups): Percents of aerosol deposition were determined for right and left lung and for trachea (ROIs).  Statistical analyses: Kruskall and Wallis test, and Wilcoxon-Mann-Whitney test (StatXact-3 ® ) TEST groupLPS groupAIR-CONTROL group  Four groups of animals were constituted : LPS, TEST, COLLOIDS and AIR-CONTROL.  Rats belonging to LPS and TEST groups were aerosolized (microsprayer®) with 200µl of solutions mixed with 99mTc-colloids. In COLLOIDS group, rats were aerosolized with the radioactive tracer alone, to test potential effects of technetium. Aerosolizations were performed on gas-anesthetized rats (Aerrane® 4%, 3 minutes) and required only 30s per rat. AIR-CONTROL group were constituted by no aerosolized rats.  Immediately after aerosolization, imaging of lung deposition was performed with a Biospace® gamma imager.  After 24h, bronchoalveolar lavage (BAL) was performed, in each group, for biochemical analyses and, lung and trachea were removed for histological analyses. Material and Methods  80 to 89% of the administered dose (loaded dose) is deposited in the respiratory tract: deposition was < 10% in the trachea, 45 to 53% in the right lung and 35 to 37% in left lung.  Repartition of aerosol deposition in lungs and trachea was homogeneous and not different in COLLOIDS, LPS, and TEST groups (KW >0.05). Results  Aerosol deposition  BAL fluids analyses  Histological findings  Biochemical profiles of BAL fluid obtained from COLLOIDS and AIR-CONTROL groups were in compliance with healthy lungs: No cell recruitment or TNF were detected and lower level of protein, MIP-2 and LDH (except for two animals in COLLOIDS group) were measured in supernatant. Biochemical profiles of BAL fluids obtained from AIR-CONTROL (■), COLLOIDS (■), LPS (■) and TEST (■) groups.  In the larynx and trachea of most of rats, epithelial flattening was observed (due to microsprayer® insertions). Moreover, in TEST group, several animals had treatment related lesions mostly of slight degree (necrosis/exfoliation, degenerative/regenerative epithelium changes).  In lungs, perivascular infiltration of cells was seen in all aerosolized animals, which was very slight in COLLOIDS group, moderate in TEST group and in a higher incidence in the LPS group. Alveolar epithelial was degenerated and centroacinar lesions were accompanied by the presence of foamy macrophages (E), indicating initial alveolar damage. Similar to the change observed in the trachea, epithelial exfoliation, degeneration and regeneration were noted in the bronchi and bronchioles. Alveolar inflammation was present in LPS treated animals, characterized by a mixture of inflammatory cells present to some extend in airways and vessels (A). A BCD Drug TEST treatment induced alveolar oedema (B), haemorrhage (C) and moderate inflammation (D), most often bronchoalveolar and sometimes extensive and diffuse comprising a whole lobe. E median % [q1;q3] TracheaRight lungLeft lung Colloids7.66% [6.27; 7.84] 52.53% [41.16; 54.57] 35.21% [27.32; 40.48] LPS7.70% [5.50; 8.08] 49.42% [25.76; 52.72] 36.13% [32.65; 52.78] TEST8.10% [6.75; 12.86] 45.27% [41.84; 52.26] 36.93% [34.83; 41.22]  The profiles from animals aerosolized with LPS or with drug TEST differed: LPS induced a statistical strong recruitment of PMNs (p= vs. COLLOIDS group; p=0.003 vs. TEST group) and a significant increase of cytokines levels (MIP-2: p= and TNF: p= vs. COLLOIDS group).  Results indicated acute inflammation. Drug TEST produced a lower recruitment of PMNs with a high significant flow of RBC into the lungs. Aerosolization of this drug leads to a significant increase of proteins (p= vs. COLLOIDS group) and cellular cytotoxicity in BAL fluid supernatant.  Results suggested lower inflammation, but higher toxicity. LPS inflammatory processes and drug test irritant action on the lung were well discriminated on the basis on correlated histological and biochemical data. The method was highly reproducible, leading to homogeneous aerosol distribution in the respiratory tract and high amount of drug deposition into the lungs (80 to 89% of the delivered dose). This study validated intratracheal aerosolisation as a new model for rapid acute pulmonary toxicity assessment of molecules. Conclusion