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

Pulmonary toxicity of drug assessment: a subacute study using intratracheal aerosolisation in Sprague Dawley rats.
Protéases et
M. de Monte1, S. Le Guellec2, J. Montharu1, Y. Rabemampianina4, J. Guillemain3, B. Kittel4, P. Diot1 and F. Gauthier1
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
Local respiratory tolerance is a critical issue for inhaled drugs (2/2). In the previous study, a model of
intratracheal aerosolization in SD rats, designed with imaging and quantification of lung deposition, allowed us
to assess the acute toxicity of a TEST drug.
 BAL fluids profiles of COLL and SALB group were characterized by a few number of PNNs and/or RBC. Drug test produced a lower dose-dependent
recruitment of PNNs along with a dose-dependent flow of RBC into BAL fluids, suggesting lower inflammation, and high toxicity.
 A repeat-dosing effect was observed on protein, MIP-2 and LDH, which was particularly perceptible in COLL group. After 4-day dosing, 3 parameters levels
were significantly higher than levels obtained in the previous study, after a single dosing (MW: p=0.0002).
We test this model to evaluate toxicity of the same drug according to a subacute repeated dose protocol.
Three doses TEST groups were compared with salbutamol and vehicle administrated control groups.
 Also observed with TEST molecule, this latter effect was confused with that induced by the
molecule itself (in particular with MIP-2). However, its aerosolization leads to an dose-dependant
Material and Methods
significant increase of proteins in BAL fluid when the 50µgx4-dose was used (vs. COLLOIDS and vs.
Biochemical profiles of BAL
fluids obtained from
COLLOIDS (■), SALBUTAMOL
(■), TEST-2.5 (■), TEST-25 (■)
and TEST-50 (■) groups.
TEST-25, MW: p=0.0001).
100.0
450.0
90.0
400.0
80.0
350.0
70.0
300.0
60.0
250.0
50.0
200.0
40.0
150.0
30.0
20.0
100.0
10.0
50.0
0.0
0.0
PNN/µl
LDH levels were significantly increased only after repeated doses of 2.5 and 50µg UK (MW: p=0.009).
RBC/µl
450.0
100.0
8000.0
400.0
90.0
7000.0
 Five groups of animals were constituted : COLLOIDS (COLL), SALBUTAMOL (SALB, 50µg/rat), TEST-2.5, TEST-25 and TEST-50 (2.5µg, 25µg or
350.0
80.0
50µg/rat).
200.0
250.0
150.0
 Rats belonging to the three TEST groups and SALB group were aerosolized (microsprayer®) with 150µl of solutions mixed with 99mTccolloids. In COLL group, rats were aerosolized with the radioactive tracer alone. Aerosolizations were performed on gas-anesthetized rats
(Aerrane® 4%, 3 minutes) and required only 30s per rat. All animals were aerosolized once a day during 4 days.
 Epithelial flattening in larynx and trachea (with exfoliation and
gamma imager, every day before and after each aerosolization.
 Biochemical analyses (on BAL fluid): Cells count (PMNs, macrophages and red blood
emphasizing the hypothesis that this damage was caused by the
cells RBC) and protein assay.
procedure of administration (repeated microsprayer® insertions).
group, for biochemical analyses and, lung and trachea were
deposition were determined for right and left lung and for trachea (ROIs).
removed for histological analyses.
 Statistical analyses: A multivariate repeated measure analysis (MANOVA) was
performed to analyse aerosol deposition data (SYSTAT®). Kruskall and Wallis test and
Wilcoxon-Mann-Whitney test were used to analyze biochemical and aerosol deposition
data (StatXact-3®).
50.0
4000.0
40.0
3000.0
100.0
20.0
50.0
10.0
2000.0
1000.0
0.0
0.0
MIP-2 pg/m l
LDH %
 Histological findings
degeneration) was seen in all animals whatever their group (A),
 Lung deposition (TESTs, Salbutamol and Colloids groups): Percents of aerosol
5000.0
PROT µg/m l
Analyses
 At day 5, bronchoalveolar lavage (BAL) was performed, in each
60.0
30.0
0.0
 Imaging of lung deposition was performed with a Biospace®
 Histological analyses (Lungs and trachea)
6000.0
70.0
300.0
A
 Light inflammation due to “repeat-dosing effect” was observed in all
animals' lungs.
 Lungs damages observed for TEST-groups were similar to changes observed for animals which were subjected, in the previous study, to an acute dose
(such as alveolar oedema, haemorrhage and moderate inflammation).
However, the four days of consecutive administration of this drug were well tolerated: lesions were more diffuse than after an acute administration of a high
dose, and were also characterized by “centroacinar inflammation (B), granulocytic infiltrations (C) and hyperplasia of bronchi (D)”.
Results
B
C
D
 Aerosol deposition
median % [q1;q3]
Trachea
Left lung
Right lung
Colloids
9.35% [5.79, 21.47]
42.77% [34.23, 49.77]
38.30% [29.95, 48.84]
Salbutamol
12.51% [8.00, 22.50]
40.70% [31.44, 47.55]
37.38%
TEST-2.5µg
8.04% [6.13, 11.06]
46.51% [37.99, 54.23]*
39.25% [29.14, 48.59]
TEST-25µg
TEST-50µg
8.71% [5.36, 21.09]
8.21% [4.68, 12.22]
46.59% [40.07, 53.97]*
47.41% [34.21, 51.21]
[31.71, 46.91]
36.87% [31.24, 42.46]
40.59% [33.40, 51.66]
 Repetition of aerosolization for 4 consecutive days had no effect on repartition of aerosol between the different pulmonary areas (MANOVA: p>0.05 for repeated
measures in trachea, left and right lungs).
 Treatment-related findings were in dose-response relation from 25µg/rat/day. NOEL at 2.5µg/rat/day.
 Histological findings were well correlated with biochemical data.
Conclusion
 Repartition of aerosol in the different pulmonary areas was generally homogenous between all groups (KW>0.05). However, there was a significant asymmetry
between right and left lung in TEST-2.5µg and TEST-25µg groups (MW<0.05; *).
 The amount of compound actually deposited into trachea and lungs after 4 aerosolizations was very close to the total quantity of compound loaded in the
microsprayer. Indeed, 80 to 89% of loaded dose was deposited in the respiratory tract (salbutamol: 161.87/200µg; TEST-2.5µg: 8.60/10µg; TEST-25µg: 80.27/100µg and
TEST-50µg: 177.58/200µg).
Results indicated that intratracheal aerosolization is a suitable highly reproducible method to test respiratory tolerance of
drugs. Repeat-dosing effect and dose response treatment-related effects were well discriminated on the basis on a good
correlation obtained between biochemical and histological parameters, demonstrating the validity of implemented method.