Transcript Diapositive 1 - PSTANIMALERIES

Pulmonary delivery of dry powders to rats: the limits of an intra-tracheal
administration model
J.
1:
2
Montharu , A.
1
Guillon ,
V.
2
Schubnel ,
G.
2
Roseau ,
L.
3
Vecellio ,
J.
4
Guillemain ,
P.
1
Diot ,
F.
1
Gauthier ,
M. de
1
Monte
Unité INSERM U618 « Protéases et Vectorisation Pulmonaires », 2: PPF Animaleries, Université François Rabelais, Faculté de Médecine, Tours. 3: AERODRUG, Faculté de Médecine, Tours. 4: SESAME, Tours.
Introduction
Rodent models to deliver locally-acting or systemic therapies to the lung were based on methods using inhalation chambers and nose-only
apparatus, resulting in limited lung deposition. Intubation of rats with endotracheal probes generating liquid aerosols allows administering precise
and controlled large quantities of drug directly into the lungs. However, preliminary data related to intratracheal dry powder administration
procedures have demonstrated some artefacts which were related to precise quantification of administered powder and to lung tolerability.
The aim of the study was to evaluate the reproducibility and the lung safety of a method based on the intratracheal administration of a
dry powder in a rat model.
Method
The Microsprayer® is a delivery tube connected to a
holding chamber (aerosol generator). Number and
volume of air puffs necessary to empty the chamber
from the loaded powders were determined in vitro. In
each case, air puffs were administered by a plastic
syringe or by to the AP-1 Air Pump (PennCentury)
connected to the chamber (Fig. 2).
Twelve groups of 6 female Sprague Dawley rats were
subjected to single administrations of air alone (2, 2X2
or 5X2 mL), lactose (10 mg/rat) or magnesium stearate
(2 or 10 mg/rat), using a DP-4 Microsprayer®
(PennCentury, U.S.A.) - (Fig. 1)
Administration procedure
 Rats were anesthetized
with Isoflurane at a 4%
vaporizer output
concentration,
Aerosol generator or holding chamber 
contained loaded powders
 and placed in a supine
position, suspended by their
incisors, on a work stand
allowing a 45° angle.
 Tongue was pulled out of
the mouth, in order to get a
clear view of the trachea.
Air Pump-1 (0-5mL capacity)
 air puffs pushed powders into the
delivery tube
 An otoscope was used to
keep the mouth open and to
immobilize the tongue at its
position outside the mouth.
Fig.1: Intratracheal administration of an aérosol to a rat, using a Microsprayer®
Endotracheal metal 2’’ delivery tube
 tip located close to the carina in
order to obtain optimal lung deposition
 Microsprayer® was
inserted into the trachea,
through larynx, and air or
powders were administered to
the lungs.
Fig.2: Dry Powder-4 (DP-4) Microsprayer® connected to Air Pump-1 (AP-1)
Results
Fig. 3: Percentages/ranges of delivered powder according to compound/dose
 Actual volume of powder administered (by weighting device before and after dosing)
100
6 rats / groupe
90
7 - 100
80
The actual volume of powder given to each rat varied statistically according to compounds and/or doses
administered (Fig. 3).
2mg MS - Syringe (2mL air)
2mg MS - Air pump (2mL air)
70
14 - 85
10mg MS - Syringe (2X2mL air)
60
10mg MS - Air pump (2X2mL air)
11 - 80
50
10mg L - Syringe (5X2mL air)
10mg L - Air pump (5X2mL air)
40
 The total amount of loaded powder could rarely be administered whatever compound or doses.
30
2 - 71
8 - 58
20
2 – 97
10
0
 Clinical response related to administration
Lactose
1
Magnesium stearate
Short duration respiratory arrests (~ 4-5s) were observed during 13/72 administration procedures (12/13 with magnesium stearate, 1/13 with
lactose). Only one death occurred. This was not observed during administration of air alone.
 About 1/3 powder dosing procedures resulted in respiratory failures.
Fig. 4: Percentages of rats according to number/gravity of lung lesions after treatment - 6rats / group
 Lung gross pathology
100%
90%
80%
 83% and 56% of the lungs were impaired in animals respectively
administered a powder or air alone.
 Presence/absence of lung impairment varied statistically according to
air and compound/dose administered i.e. the number of rats with lung
impairment increased with insufflations of a higher air volume and/or
with presence of powder (Fig. 4).
70%
60%
50%
40%
30%
20%
10%
No lung impairment
Lung with slight impairment
0%
2mL
seringue
2mL air
pump
2X2mL 2X2mL air 5X2mL 5X2mL air
seringue
pump
seringue
pump
Stéarate
2mgseringue
Stéarate
2mg-Air
Pump
Administration of air alone
Stéarate
10mgseringue
Stéarate
10mg-Air
Pump
Lactose
10mgseringue
Lactose
10mg-Air
Pump
Lungs with severe impairment
Generalized lung trauma
Administration of powders
 Gravity of lesions varied in the same manner i.e. lung lesions were
more severe in rats receiving powders than in rats administered air alone,
but generalized trauma, could also be induced by delivering air alone
(Fig. 4, 5).
 Air alone or powder spraying using air as a vehicle could induce
lung lesions due to high pressure and/or high volume traumatism
Lung with “slight impairment” i.e. one
lobe with edema, hemorrhage and atelectasia
Lung with “severe impairment” i.e. > 1
lobe with edema, hemorrhage or atelectasia
Lungs with “generalized trauma” i.e. all lobes with edema, hemorrhage or
atelectasia
Fig. 5: Lung gross pathology, each lung being classified in one category according to the severity of external pulmonary impairment
 The severity of lung lesion was statistically linked to respiratory arrest (9/13 respiratory failures were observed in rats demonstrating
lungs impairment with “generalized trauma”).
Conclusion
This model for powder administration suffers from artefacts which may limit its relevance. These restrictions are of particular importance,
especially in the frame of regulatory toxicity studies .
Protéases et
Vectorisation
Pulmonaires
INSERM U-618
Pulmonary delivery of dry powders to macaques: set up of an intra-tracheal
administration model
A.
1:
1
Guillon ,
J.
2
Montharu ,
V.
2
Schubnel ,
G.
2
Roseau ,
3
Vecellio ,
L.
J.
4
Guillemain ,
P.
1
Diot ,
F.
1
Gauthier ,
M. de
1
Monte
Unité INSERM U618 « Protéases et Vectorisation Pulmonaires », 2: PPF Animaleries, Université François Rabelais, Faculté de Médecine, Tours. 3: AERODRUG, Faculté de Médecine, Tours. 4: SESAME, Tours.
Introduction
The inhaled route to deliver both locally-acting and systemic therapies is developing. Rodent models are widely used to evaluate the pulmonary safety
of dry power administration. However, regulatory toxicity studies required several animal species. Monkeys, that more closely replicate human
disease, require specialized management. Repeated aerosol drug administration to anaesthetized monkey is probably not free of consequences, even
with inactive drug aerosol (lactose).
Thus, the aim of the study was the subacute tolerance of repeated lactose powder administration by intra tracheal route, in monkeys.
Method
Two male cynomolgus monkeys (5.3 and 7.5 kg) were administered lactose 7 times in 15 days (2.5mg/kg every 48 hours), using a Microsprayer®
(Pencentury, USA). Clinical, laboratory investigations (haematology, biochemistry), and histopathology of the lung and trachea were performed.
The Microsprayer® is a delivery tube connected to a holding
chamber (aerosol generator). Air puffs were administered by
a plastic syringe connected to the chamber.
Administration procedure:
 Monkeys were sedated with an intramuscular injection of ketamine.
 Anaesthesia was induced with isoflurane at a 5% vaporizer output
concentration in oxygen via a facial mask.
 Tracheal intubation was performed under direct laryngoscopy.
 Microsprayer® (Fig. 1) was inserted into the trachea probe, and
lactose powder were administered to the lungs.
Fig.1: Dry Powder-4 (DP-4) Microsprayer®
Plastic seringe 
air puffs pushed powders into the delivery
tube
Aerosol generator or holding chamber
 contained loaded powders
Anaesthesia
 Sedation
Endotracheal metal 11.8’’ delivery tube 
tip located close to the carina in order to
obtain optimal lung deposition
Monotoring procedure:
Animals kept spontaneous ventilation during anaesthesia.
Monitoring included pulse-oximetry and heart rate recording.
 Monitoring
Blood sampling
 Administration
 Intubation
Blood sampling:
Toxicokinetic studies were performed on the first and the last
days (7 blood sample of 1.0 mL in 24h). ml
Results
Clinical evaluation  No clinical sign related to administration of lactose was recorded, no body weight loss was observed.
Laboratory investigations  Hyperleucocytosis, hepatic cytolysis and rhabdomyolysis were recorded at the end of the study period
(Table 1 - median values). Hepatic cytolysis could be explained by the repeated fasting periods. Rhabdomyolysis was probably due to prolonged
anaesthesia and rise in the compartment pressure.
Table 1
Before
treatment:
-10 days
After
treatment:
Day 14
UREA CREAT GLU
mmol/L µmol/L mmol/L
5.15
89
3.45* 81.5*
GGT
IU/L
ALAT ASAT
IU/L IU/L
4.15 116.5 33.5
3.7
44
ALP
IU/L
CK
IU/L
566
603
101.5 65* 106* 481
CA++ PHOS
Na+
K+
Clmmol/L mmol/L mmol/L mmol/L mmol/L
2.5
1.63
ALB
g/L
145
3.9
106
41.5
3536* 2.61 0.91* 146
4.2
106
42.5
PROT BIL
g/L µmol/L
81
4.25
WBC
g/L
N
%
7.45
38
HGB
g/dL
HCT
%
14.15 42.05
PLT
g/L
PT
s
FIB
g/L
APTT
s
348
11.45
2.21
22.95
10.9
1.99
21.35
79.5 4.05 12.7* 66.5* 13.55 40.85 336.5
CREAT: creatinin; GLU: glucose, GGT: Gamma glutamyltransferase; ALAT: Alanine aminitransferase; ASAT: Aspartate aminotransferase; ALP: Alkaline phosphatase; CK: Creatinine kinase; PHOS: Phosphorus, ALB: Albumin; PROT: Protein; BIL: Bilirubin; WBC: White blood cells; N:
Neutrophil, HGB: Heamoglobin; HCT: Haematocrit; PLT: Platelet; PT: Prothrombin time; FIB: Fibrinogen; APTT: Activate dprothrombin time; *: « Modified » values (no statistical test due to low number of animals)
Lung and trachea histopathology  No relevant histological effects were induced by powdered lactose administration
Conclusion
Repeated aerosol of lactose did not have local consequences after macroscopic and histological examination. This result suggests that this
model is suitable to evaluate sub acute local toxicity after aerosol drug administration. Lactose aerosol administration can be used as a
negative control. However, experimental procedures need to be improved in order to limit consequences of repeated anaesthesia.
Protéases et
Vectorisation
Pulmonaires
INSERM U-618