Transcript INSERM U-618 (National Institute for Health and Medical Research)

Intratracheal aerosolisation in Sprague Dawley rats, a new method for assessment of pulmonary toxicity of drug.
Protéases et
M. de Monte1, S. Le Guellec2, J. Montharu1, Y. Rabemampianina4, J. Guillemain3, B. Kittel4, F. Gauthier1, P. Diot1
Vectorisation
Pulmonaires
1- INSERM U-618 (National Institute for Health and Medical Research), Aerosol team, IFR135, Université François Rabelais, Tours, F-37000 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
Introduction
INSERM U-618
 BAL fluids analyses
Biochemical profiles of BAL fluids obtained from
AIR-CONTROL (■), COLLOIDS (■), LPS (■) and TEST (■) groups.
 Biochemical profiles of BAL fluid obtained from COLLOIDS and AIR-CONTROL groups were in compliance with
Local respiratory tolerance is a critical issue for inhaled drugs (1/2). It is usually assessed by In vivo
healthy lungs: No cell recruitment or TNF were detected and lower level of protein, MIP-2 and LDH (except for
studies using the intranasal route, which limit lung deposition, or inhalation chambers which require long
two animals in COLLOIDS group) were measured in supernatant.
exposition times and induce high fur deposition.
700.0
3500.0
600.0
3000.0
500.0
2500.0
400.0
2000.0
300.0
1500.0
200.0
1000.0
100.0
500.0
0.0
The aim of this study was to develop a new model for rapid evaluation of acute toxicity of aerosolized
 The profiles from animals aerosolized with LPS or with drug TEST differed:
drugs in rats. The model was designed with imaging and quantification of lung deposition and tested
LPS induced a statistical strong recruitment of PMNs (p=0.0001 vs. COLLOIDS group; p=0.003 vs. TEST group) and a
with a well-known irritant molecule (TEST group) and an inflammatory molecule, LPS.
significant increase of cytokines levels (MIP-2: p=0.0009 and TNF: p=0.0001 vs. COLLOIDS group).
gas-anesthetized rats (Aerrane® 4%, 3 minutes) and required only 30s per rat. AIR-CONTROL group were constituted by no aerosolized rats.
2.0
1.5
100.0
1.0
50.0
0.5
0.0
0.0
PROT µg/m l
TNF pg/m L
of this drug leads to a significant increase of proteins (p=0.0001 vs. COLLOIDS group) and cellular cytotoxicity in
4.0
80.0
3.5
70.0
BAL fluid supernatant.
3.0
60.0
2.5
50.0
 Results suggested lower inflammation, but higher toxicity.
2.0
40.0
1.5
30.0
1.0
20.0
0.5
10.0
<<
 Four groups of animals were constituted : LPS, TEST, COLLOIDS and AIR-CONTROL.
group, rats were aerosolized with the radioactive tracer alone, to test potential effects of technetium. Aerosolizations were performed on
2.5
200.0
150.0
Drug TEST produced a lower recruitment of PMNs with a high significant flow of RBC into the lungs. Aerosolization
 Rats belonging to LPS and TEST groups were aerosolized (microsprayer®) with 200µl of solutions mixed with 99mTc-colloids. In COLLOIDS
RBC /µL
3.0
250.0
 Results indicated acute inflammation.
Material and Methods
0.0
PNN /µL
0.0
 Histological findings
0.0
MIP-2 pg/m L
LDH %
 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
 Immediately after aerosolization, imaging of lung deposition
LPS group
TEST group
COLLOIDS group
AIR-CONTROL group
higher incidence in the LPS group.
was performed with a Biospace® gamma imager.
A
J0: Aerosolizations (200µL)
LPS (50µg)
+ 99mTc-colloids
 After 24h, bronchoalveolar lavage (BAL) was performed, in
Drug test (105µg)
+ 99mTc-colloids
Alveolar inflammation was present in LPS treated animals, characterized by a mixture of inflammatory cells present to some
99mTc-Colloids
extend in airways and vessels (A).
each group, for biochemical analyses and, lung and trachea were
removed for histological analyses.
Imaging of lung deposition (Biospace®)
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.
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.
B
 Histological analyses (Lungs and trachea)
C
D
E
 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®)
Results
Alveolar epithelial was degenerated and centroacinar lesions were accompanied by the presence of foamy macrophages (E), indicating initial alveolar
damage.
 Aerosol deposition
Similar to the change observed in the trachea, epithelial exfoliation, degeneration and regeneration were noted in the bronchi and bronchioles.
median % [q1;q3]
Trachea
Right lung
Left lung
Colloids
7.66% [6.27; 7.84]
52.53%
[41.16; 54.57]
35.21%
[27.32; 40.48]
LPS
7.70% [5.50; 8.08]
49.42%
[25.76; 52.72]
36.13%
[32.65; 52.78]
TEST
8.10% [6.75; 12.86]
45.27%
[41.84; 52.26]
36.93%
[34.83; 41.22]
Conclusion
LPS inflammatory processes and drug test irritant action on the lung were well discriminated on the basis on correlated
 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).
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.