A Comparison of Drug Transport in Pulmonary Absorption Models: Isolated Perfused rat Lungs, Respiratory Epithelial Cell Lines and Primary Cell Culture
A Comparison of Drug Transport in Pulmonary Absorption Models: Isolated Perfused rat Lungs, Respiratory Epithelial Cell Lines and Primary Cell Culture
Cynthia Bosquillon 0 1 2 3
Michaela Madlova 0 1 2 3
Nilesh Patel 0 1 2 3
Nicola Clear 0 1 2 3
Ben Forbes 0 1 2 3
0 School of Pharmacy, University of Reading , Whiteknights, Reading RG6 6AP , UK
1 Faculty of Pharmacy, Charles University in Prague , Hradec Kralove , Czech Republic
2 King′s College London, Pharmaceutical Science Division , Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH , UK
3 Pfizer R&D , Sandwich, Kent CT13 9NJ , UK
Purpose To evaluate the ability of human airway epithelial cell layers and a simple rat isolated perfused lung (IPL) model to predict pulmonary drug absorption in rats in vivo. Method The permeability of seven compounds selected to possess a range of lipophilicity was measured in two airway cell lines (Calu-3 and 16HBE14o-), in normal human bronchial epithelial (NHBE) cells and using a simple isolated perfused lungs (IPL) technique. Data from the cell layers and ex vivo lungs were compared to published absorption rates from rat lungs measured in vivo. Results A strong relationship was observed between the logarithm of the in vivo absorption half-life and the absorption half-life in the IPL (r = 0.97; excluding formoterol). Good log-linear relationships were also found between the apparent first-order absorption rate in vivo and cell layer permeability with correlation coefficients of 0.92, 0.93, 0.91 in Calu-3, 16HBE14o- and NHBE cells, respectively. Conclusion The simple IPL technique provided a good prediction of drug absorption from the lungs, making it a useful method for empirical screening of drug absorption in the lungs. Permeability measurements were similar in all the respiratory epithelial cell models evaluated, with Calu-3 having the advantage for routine permeability screening purposes of being readily availability, robust and easy to culture.
16HBE14o-; biopharmaceutics; calu-3; inhalation; isolated perfused lungs (IPL); NHBE; permeability; pulmonary
School of Pharmacy, University of Nottingham, Boots Science
Building, University Park, Nottingham NG7 2RD, UK
The rate and extent of absorption of inhaled drugs are
determined by the relative rates of the different clearance
mechanisms that operate in the lungs [
]. Clearance by absorptive
transfer from the lung lumen is predominately controlled by
the epithelial permeability of free (unbound) drug. Although
in vitro epithelial cell culture [
] and ex vivo lung methods [
are available to screen the permeability of drug candidates for
development as orally inhaled products, there is no standard
experimental method for measuring drug permeability or
predicting lung absorption [
]. As drug permeability in the
lungs has been proposed recently to be a key factor in a
biopharmaceutical classification system being developed for
inhaled compounds (iBCS; [
]), the validation of screening
techniques for predicting absorptive clearance from the lungs is of
The use of human epithelial cell lines as models for drug
transport in the lungs is limited to airway cell lines because
established and newer alveolar epithelial cell lines, (A549 [
and TTI [
] cell lines, respectively) have proved unsuitable as
models for screening drug permeability as they do not form
cell layers with barrier properties representative of the lung
]. The potential for the human airway
epithelial cell lines Calu-3 and 16HBE14o- to be cultured as drug
absorption models was recognized in the late 1990’s [
and they have become the pre-eminent human respiratory
epithelial cell lines for measuring drug permeability.
Methods have been optimized for culturing 16HBE14o- cells
] and Calu-3 cells [
] such that they exhibit
epithelial barrier-like properties, and the permeability of a wide
variety of compounds has been measured in these cell layers in
different laboratories [
]. Furthermore, the drug permeability
in Calu-3 [
] and 16HBE14o- cells [
] has been correlated
with absorption from the lungs in vivo and ex vivo, respectively.
Although these models have been evaluated individually, to
date the permeability of solutes in the two cell lines has not
been compared directly or matched to permeability in
primary normal human bronchial epithelial cells.
Isolated perfused rat lungs (IPL) provide an ex vivo intact
organ model with many applications for evaluating
pulmonary biopharmaceutics [
], including estimation of drug
absorption. The ability of an IPL model to predict drug
absorption from the lungs has been reported by Tronde and
]. However, most IPL methods use bespoke
apparatus to preserve and monitor the mechanical
functioning of the lungs ex vivo, e.g. negative pressure ventilation,
monitoring of perfusion pressure and airway compliance together
with custom spray or aerosol delivery systems to distribute
drugs as evenly and as deeply as possible throughout the lung.
This complexity has been a barrier to widespread adoption of
the IPL as a biopharmaceutical screening technique . We
investigated whether drug permeability in the lungs can be
measured using a much simpler IPL in which drug solution
is instilled into statically inflated lungs from which drug
transfer into vascular perfusate is measured.
This study was designed to provide a systematic
comparison of solute permeability in different human airway epithelial
cell culture systems and a simple rat IPL, avoiding
interlaboratory variation. Model compounds were selected
carefully to possess a range of log P and reported pulmonary
absorption data in rats; these included two drugs licensed as
inhaled medicines (Table I). Novel features of the study
included, (i) measuring solute permeability in 16HBE14o- and
Calu-3 models compared to NHBE cells to evaluate whether
Table I Panel of Test Compounds for Permeability Evaluation
Ka in vivo (min−1)
In vivo Ka data are from (1) ref. [
], (2) ref. [
] and (3) ref. [
the measurements in the cell line monocultures were
comparable to permeability in the more physiologically-based
primary cell layers which feature both ciliated and goblet cells, (ii)
measuring the absorptive transport of the same drugs in a
deliberately simple IPL system to investigate whether this
technically less demanding model could determine drug
permeability similarly to more complex perfused rat lungs [
and the cell models. Finally, the relationship between the
experimental data and reported absorption of the same
compounds from rat lungs in vivo was evaluated.
MATERIALS AND METHODS
Chemicals and Reagents
Test compounds; [3H]-formoterol, [3H]-terbutaline,
[3H]-metoprolol were purchased from Vitrax (Placentia, USA),
[3H]-propranolol from Amersham (Amersham, UK), [3H]-imipramine
from Perkin-Elmer (Bucks, UK) and [14C]-dextran 10 K from
Sigma-Aldrich (Poole, UK). Paracellular markers; [3H]-mannitol
and [14C]-mannitol were obtained from Sigma-Aldrich and
Amersham (Amersham, UK), respectively. Ready Protein+®
scintillation cocktail was purchased from Beckman Coulter
(High Wycombe, UK). Cell culture supports were obtained from
Corning Costar (Corning, UK). All cell culture reagents and all
other chemicals were obtained from Sigma-Aldrich (Poole, UK).
Simple Isolated Perfused rat Lung Method
Eight-week old male Wistar rats were obtained from Harlan UK
Ltd. (Oxon, Oxfordshire). They were fed with a SDS RM1(E)
maintenance diet (Special Diets Services Ltd., Essex). They were
maintained at 20–21°C and 45–60% humidity with a 12 h light/
dark cycle. All procedures performed on these animals were in
accordance with regulations and established guidelines and were
reviewed and approved by an Institutional Animal Care and Use
Committee or through an ethical review process.
Rats were sacrificed with a lethal injection of pentobarbital
(130 mg/kg body weight). As soon as they were unconscious,
rats were secured in a supine position on a board inclined at
approximately 45°. A midline incision was made from the neck
to the abdomen using a scalpel blade and the rat was
exsanguinated by severing the main abdominal vessels. The trachea was
exposed and carefully pierced through one wall with a 21 G
needle. A 3 cm long cannula made of a polyethylene tubing
(PolyE 240, Harvard Apparatus Ltd., Edenbridge, UK)
mounted on a blunt 21 G needle was introduced into the
trachea. This was securely tied with two suture threads (Silk black
braid USP size 4.0, Harvard apparatus Ltd) and a 25 mm
Dieffenbach’s bulldog artery clip (Scientific Laboratory
Supplies Ltd., Nottingham, UK). The diaphragm was cut
open, 0.5 mL of air was administered to the lungs to partly
re-inflate them and the rib cage was laterally incised with
scissors taking care not to damage the lung tissue.
After the thymus was removed, the heart was twisted slightly
to expose the pulmonary artery and then stretched down using a
Halstead’s artery clamp (Scientific Laboratory Supplies). An
incision was made and the pulmonary artery was cannulated using
a cannula similar to the tracheal one. This was secured with a
micro aneurysm clip (Harvard apparatus Ltd). Lungs were
perfused using a single pass constant flow rate of 8 mL/min. The
perfusate was a modified Krebs-Ringer solution (NaCl 118 mM,
KCl 4.7 mM, CaCl2 2.5 mM, MgSO4 1.2 mM, NaHCO3
24.9 mM, KH2PO4 1.2 mM, HEPES 10 mM, D-glucose
11 mM, 4.5% w/v BSA, heparin 35 kU/mL, pH = 7.4)
maintained at 37°C and saturated with 95% O2 and 5% CO2. The
pericardium was dissected free to allow free efflux of the perfusate
and lungs were inflated manually with 1.5 mL air using a 10 mL
syringe connected to the intratracheal cannula. As soon as the
tissue blanched, the lungs were removed carefully from the chest
cavity while maintaining the perfusion and a semi micro Rexaloy
clamp (Fisher Scientific, Loughborough, UK) was used to
suspend the lungs vertically above a funnel and beaker.
Absorptive Drug Transfer in the Isolated Perfused Lung
The drugs investigated, 200 nM [3H]-formoterol, 130 nM
[3H]terbutaline, 130 nM [3H]-metoprolol, 275 nM
[3H]-propranolol, 100 nM [3H]-imipramine and 65 μM [14C]-dextran 10 K
solutions, were made up in Hank’s balanced salt solution (HBSS)
at concentrations determined according to their specific activity.
A paracellular marker; [3H]-mannitol 12.5 nM or
[14C]-mannitol 65 μM to allow dual counting, was added to the test
compound solutions as a control of the lung barrier properties.
After isolation, the lungs were allowed to stabilize for 1–2 min.
The syringe attached to the intratracheal cannula was then
disconnected and 100 μL of the test solutions were instilled into the
airways using a Hamilton microsyringe. Lungs were re-inflated
with 1.5 mL of air and sampling was performed by collecting the
effluent solution dripping from the left atrium at different time
intervals for 90 min. Lung viability was assessed by visual
inspection for any sign of oedema as well as by the profile of mannitol
airway to perfusate transfer. Samples were assayed by liquid
scintillation after addition of 5 mL of Ready Protein +® scintillant
using a 1209 Rackbeta dual scintillation counter.
The cumulative percentage of drug transferred from the
airways to the perfusate in 90 min was calculated as the fraction of
the administered dose recovered in the perfusate. The time
needed for 50% of the drug recovered after 90 min to pass into the
perfusate was defined as the absorption half-life (t1/2 abs) [
The apparent first-order absorption rate constant (KaIPL) was
calculated as follows:
KaIPL ¼ t1
Absorption data were collected using 4 or 5 IPL
Calu-3 cells were purchased from the American Type Culture
Collection (ATCC, Rockville, USA) and 16HBE14o- cells were
a gift from Dieter Gruenert (California Pacific Medical Center,
San Francisco, USA). Normal human bronchial epithelial
(NHBE) cells (Clonetics™, 1st passage) and bronchial epithelial
cell growth medium (BEGM) bullet kit were obtained from
Cambrex BioScience, Inc. (Walkersville, MD, USA).
Calu-3 cells (passages 26–31) were grown in Dulbecco’s
modified Eagle’s medium (DMEM) nutrient mixture F-12 Ham
supplemented with 10% foetal bovine serum, 100 UI/mL penicillin,
100 μg/mL streptomycin, 20 mM L-glutamine and 1% v/v
nonessential amino acids. For solute permeability experiments, cells
were seeded onto 24-well polyester Clear Transwell® cell culture
inserts (0.4 μm pore size, 0.33 cm2 surface area, Costar Corning)
at a density of 100,000 cells/cm2. After 24 h in culture, the
medium was removed from the apical compartment to allow
cells to grow at an air-interface as described previously [
Cell layers were used after 10–14 d in culture.
16HBE14o- cells (passages 31–33) were cultured in Minimum
Essential Medium (MEM) supplemented with 10% foetal bovine
serum, 100 UI/mL penicillin, 100 μg/mL streptomycin, 20 mM
L-glutamine and 1% v/v non-essential amino acids. They were
seeded onto 24-well polyester Clear Transwell® cell culture
inserts (0.4 μm pore size, 0.33 cm2 surface area) at a density of 2.5 x
105 cells/cm2 and were grown as described previously [
] for 7
d before drug transport studies.
NHBE were cultured in a cell culture flask using the BEGM
bullet kit provided by the supplier until reaching 70–80%
confluence. They were then seeded onto 12-well polyester Clear
Transwell® cell culture inserts (0.4 μm pore size, 1.13 cm2 surface
area) at a density of 2.5 x 105 cells/cm2 in serum-free
BEGM:DMEM/F12 Ham 1:1 supplemented with
hydrocortisone (0.5 mg/mL), insulin (5 mg/mL), transferrin (10 mg/mL),
epinephrine (0.5 mg/mL), triiodothyronine (6.5 mg/mL),
gentamicin (50 mg/mL), amphotericin-B (50 mg/mL), retinoic acid
(0.1 ng/mL), and epidermal growth factor (0.5 ng/mL human
]. After 24 h, cells were cultured at an air
interface. Cell layers were used for transport studies after 14 d in
All cells were maintained in a 5% C02, 95% air atmosphere at
37°C and provided with fresh medium every 2–3 d (Calu-3 and
16HBE14o-) or every 1–2 d (NHBE). Development of confluent
cell layers with suitable tight junctions was monitored by
transepithelial electrical resistance (TER) measurement using
an epithelial VoltOhmMeter (World Precision Instruments,
Stevenage, UK) with silver chloride chopstick electrodes.
In Vitro Drug Permeability Measurements
Drugs were presented in HBSS for transport studies in Calu-3
and NHBE or in serum-free medium for transport studies in
16HBE14o-. Non-radiolabelled compounds were added to the
test solutions to reach a total drug concentration of 10 μM;
solutions were buffered at pH 7.4. 3H– or 14C–labelled
mannitol was added to the solution to produce a 10 μM
concentration of paracellular marker to serve as an internal standard for
cell layer integrity.
All solutions used in the transport experiment were
prewarmed to 37°C. In preparation for transport experiments,
cell layers were washed twice with HBSS (Calu-3, NHBE) or
serum-free medium (16HBE14o-). After 30 min equilibration,
the pre-experiment TER of each monolayer was measured.
The resistance of the cell-free culture support was subtracted
from the gross resistance to yield the TER of the epithelial cell
layers. Cell layers with TER > 200 Ω cm2 were used in
transport experiments and TER was monitored to remain within
10% of the initial value over the course of experiments. Drug
transport was measured in the absorptive apical (A) to
basolateral (B) direction. To initiate the transport
measurements, test solutions were added to the apical donor chamber
and cell culture supports transferred into a base plate
containing HBSS (Calu-3 and NHBE cells) or medium (16HBE14o-)
supplemented with 1% bovine serum albumin. Within 1 min,
10 μL of the test solution was removed from the donor
chambers to establish the initial donor concentration (Co). Cell
layers were placed in a 37°C incubator on an orbital shaker
rotating at 100 rpm. Every 30 min for 2 h, cell inserts were
carefully removed from the basolateral chambers and
transferred to a fresh base plate containing pre-warmed transport
medium. At each time point, 500 μL were sampled from the
receiver compartments. Between samples, the cell layers were
returned to the 37°C incubator. After 2 h, 10 μL of sample
was removed from the apical chamber to determine the final
donor concentration and the post experiment TER was
Samples were analysed by liquid scintillation counting,
after addition of 5 mL of Ready Protein+® scintillant
using a 1209 Rackbeta dual scintillation counter.
Apparent permeability coefficient (Papp) were calculated
using the following equation:
dQ .dt .ðACoÞ
Where dQ/dt is the transport rate; A is the surface area of
the cell culture support, and Co the initial drug concentration
in the donor chamber. Transport data were obtained from 6
cell layers from 2 different passages in Calu-3 and
16HBE14oand from 3 cell layers from passage 2 in NHBE.
Differences in IPL drug permeability data were compared
u s i n g K r u s k a l - W a l l i s n o n - p a r a m e t r i c A N O V A .
Relationships between IPL transport parameters, in vivo
absorption data in rats and in vitro solute permeability in airway
cell layers were analyzed using the Spearman’s correlation
coefficient. The statistical analysis was performed using
SPSS 14.0 for Windows software (SPSS Inc., Chicago,
Absorptive Drug Transfer in Isolated Perfused Lungs
Following instillation of test solution using a micro-syringe,
absorptive solute transfer into the perfusate was measured as
cumulative % transferred over 90 min (Fig. 1). Transfer of the
high molecular compound, dextran 10 K, was linear with only
9.5 ± 1.9% transported at 90 min (Table II), demonstrating
that the epithelial barrier of the lung was maintained over the
duration of the experiment in the simple IPL model. The
mean proportion of the low molecular weight compounds
transferred ranged between 46–65% (Table II). As the
cumulative drug transferred to perfusate either reached a
plateau (metoprolol and propranolol) or was approaching
a plateau (other small molecules) (Fig. 1), this appeared to
Fig. 1 Cumulative percentage of initial dose transferred to the perfusate vs
time profiles (data not fitted) after intratracheal instillation to isolated
perfused rat lungs. Dextran = dextran 10 K. Data are presented as mean ± SD
(n = 4 or 5).
represent the proportion of dose available for transfer
under the experimental conditions. It is interesting that four
of the small compounds were not fully absorbed after
90 min; this is most likely related to retarded transport
kinetics specific to the properties of the individual
compounds, i.e. tissue binding of small basic compounds,
although redistribution after instillation to regions of the
lungs from which absorption in the IPL can occur may play
The more lipophilic compounds imipramine,
metoprolol and propranolol were transferred into the
perfusate faster than the hydrophilic compounds as evident
from the absorptive profiles (Fig. 1) and t1/2 (Table II).
The data were analysed using the approach of Tronde
et al. [
] for simplicity and to enable comparison with
the published data. This approach includes an
assumption of first order kinetics, although the full cumulative
absorptive transfer profile is not utilized for compounds
which do not reach a plateau in 90 min, thus the KaIPL
parameter may be misleading. However, differences in
absorptive flux of the test compounds were clearly
apparent and the calculated parameters provided a ranking
reflective of transfer profiles in the first 5–10 min, where
the greatest differences in solute transfer to the perfusate
were observed. Absorptive drug transfer in the IPL was
compared with reported pulmonary absorption data
from rat lungs [
]. A strong relationship was
observed between the logarithm of the in vivo absorption
half-life (log T50%) and the absorptive half-time (t½ abs)
in the IPL (r = 0.97, p < 0.01, Fig. 2a) when formoterol,
which appeared as an outlier, was excluded from the
Drug Permeability in Airway Epithelial Cell Layers
The absorptive permeability of each compound was also
measured in the airway epithelial cell drug transport
models. The cell lines produced cell layers with TER
and Papp of mannitol within the normal range for these
m o d e l s [
] : C a l u - 3 : T E R = 2 8 0 ± 1 0 Ω c m 2 ,
Papp = 0.48 ± 0.06 x 10−6 cm s−1; 16HBE14o-:
TER = 240 ± 20, Papp = 3.7 ± 0.5 x 10−6 cm s−1. The
primary cell layers produced similar resistances and
permeability to mannitol: NHBE: TER = 330 ± 110 Ω cm2,
Papp = 1.6 ± 0.5 x 10−6, cm s−1. The recovery (mass
balance) of compounds was >70% (except for imipramine
in NHBE for which recovery was 56.5 ± 2.9%; losses
likely due to binding to plasticware and drug in the
cellular compartment) and the cumulative drug transported
vs time profiles were linear in all instances (R2 > 0.98). In
each of the cell culture models, the permeability of the
hydrophilic molecules mannitol and dextran 10 K, which
permeate cell layers exclusively via the tight junctions and
serve as paracellular markers, was lower than that of the
more lipophilic therapeutic molecules. The permeability
of the cell layers to the paracellular markers ranked
Calu3 > NHBE >16HBE14o- (Table III). The rank order of
permeability for the compounds investigated was identical
in Calu-3 and 16HBE14o- layers, but varied for the more
lipophilic compounds in NHBE cell layers (Table III). Log
Fig. 2 Relationship between pulmonary absorption in vivo in rats and
absorption / permeability in (a) isolated perfused rat lungs - half-time of solute
absorbed in 90 min in the IPL (t½ abs IPL). Formoterol (square on the plot)
has been excluded from the correlation. (b) human airway epithelial cell layers
- the apparent permeability coefficient (logarithm of Papp; cm/s) in cell culture
absorption models based on the Calu-3 and 16HBE14o- cell lines and normal
human bronchial epithelial cells (NHBE).
linear relationships between drug permeability in the cell
layers in vitro and the apparent first order absorption rate
constant in vivo were observed (r = 0.92, 0.93 and 0.91 in
Calu-3, 16HBE14o- and NHBE cells, respectively; Fig.
2b, p < 0.05). Even stronger log-linear relationships were
obtained between solute permeability measured in the
different in vitro models, i.e. r = 0.97 for Calu-3 and
0.96 for 16HBE14o- vs NHBE (Fig. 3a) and r = 0.98 for
Calu-3 vs 16HBE14o- (Fig. 3b).
In Vitro – Ex Vivo Correlations
Linear relationships were obtained between the logarithm
of the Papp in cell layers and the absorption half-life in the
IPL when formoterol, an outlier in the IPL-in vivo
correlation, was excluded from the analysis. These relationships
were stronger for the cell lines compared to the primary
cell model (r = 0.92 for Calu-3 cells; r = 0.93 for
16HBE14o- cells; r = 0.89 for NHBE cells; Fig. 4,
p < 0.01). This was similar to previous evaluations
comparing solute permeability in 16HBE14o- cell layers [
and Calu-3 cell layers [
] with drug transfer/absorption
from the lungs.
A recent AAPS/FDA/USP workshop considered a
systematic framework to classify pulmonary drugs to
provide a tool for formulators and discovery chemists
working in the pulmonary drug delivery field [
permeability or the rate of absorption was identified as an
important predictor of local residence time and,
therefore, duration of effect for locally-acting drugs. When
drugs are administered to the lungs for the purpose of
systemic delivery, drug permeability is a critical
log Papp NHBE
Log Papp Calu-3
Fig. 3 Comparison of permeability measured using in vitro methods. (a) The
relationship between apparent permeability (logarithm Papp; cm/s) in the
Calu3 and 16HBE14o- cell lines compared to normal human bronchial epithelial
cells (NHBE). (b) Correlation between the apparent permeability (Papp) in
Calu-3 and 16HBE140- cell layers.
determinant of bioavailability. If pulmonary drug
permeability and the rate of absorption are to be utilized in an
iBCS, there is requirement for simple and reliable
methods to screen inhaled drug candidates for this
property at an early stage of their development.
The IPL preparation has been explored as an ex vivo model
for screening the pulmonary absorption of drugs by
] and GlaxoSmithKline [
]. There are a
number of methodological variations in how the IPL
t1/2 abs in IPL (min)
Fig. 4 Comparison of permeability measured using in vitro and ex vivo
methods. The relationship between absorption half-life in the IPL (t½ abs IPL)
and permeability in airway epithelial cell layers (logarithm Papp; cm/s).
Formoterol has been excluded from the correlation.
technique is configured [
], and a factor limiting the wider
adoption of the IPL as a drug absorption model has been the
perceived requirement for sophisticated systems for delivering
drugs and maintaining the organ preparation. In contrast, the
approach taken in this study was to evaluate the minimum
requirements, i.e. the simplest system, that will permit the
lungs to be used ex vivo to obtain absorptive drug transport
data. A low-cost, simple IPL model requiring no specialist
equipment in which the lung viability was maintained for
more than 90 min was developed. This is within the 20–
120 min duration over which airspace-to-perfusate drug
transfer has been reported [
]. Drug administration by
instillation was adopted for simplicity; and is favoured by some
investigators  although fine sprays [
] and aerosol
] have been used, but introduce complexity.
In this study, the profiles of drug transfer from the airspaces to
the perfusate (Fig. 1) were comparable to those reported after
administration of drug by nebulizer catheter to a more
complex physiologically-controlled IPL system [
], and a similar
correlation with pulmonary drug absorption in vivo was
obtained. A number of different systems are available for delivering
aerosols to the IPL, e.g. the PreciseInhale® system from
Inhalation Sciences, which is an important aspect if aerosol
formulation-driven absorption kinetics are to be studied.
Although instillation may not penetrate the airways as fully
as aerosol administration, this method of delivery gives precise
control over dosimetry and allows discrimination between the
absorptive transport of drugs on the basis of their
physicochemical characteristics [
The β2-adrenoreceptor agonist formoterol appeared as a
poorly transported outlier in the IPL/in vivo correlation; if
formoterol is excluded from the analysis, the correlation is
r = 0.97. By contrast, in a study by Tronde et al., formoterol
fitted well with the IPL/in vivo linear relationship obtained
], although both studies suffered from low numbers of
compounds for in vitro-in vivo correlation. The airway
absorption of inhaled β2-adrenergic agonists is complex.
Although formoterol has relatively low lipid solubility due
to a net positive charge, lung tissue retention is observed
due to high levels of tissue binding [
]. Formoterol charge
is highly pH dependent over the range pH 6–8 (speciation
vs pH plot [
]) which can influence both passive
permeability and interaction with the pH-dependent cation
transporters that transport formoterol in the airway [
]. It is
possible that small pH changes in the lung lining fluid may
have occurred, pH was not measured and it would be
interesting to study in detail the effect of pH changes with
regard to formoterol transport in the IPL.
IPL has become sufficiently well established and valued for
drug permeability screening that it has been used to generate
quantitative structure activity relationship (QSAR) [
Quality control and validation of the IPL system is important
if the model is to be useful as a screen for pulmonary drug
absorption, and used to evaluate transport mechanisms,
including the effects of drug transporters [
]. Thus, it will be
important to establish benchmarks, controls and acceptance
criteria for the technique, which may need to be specific for
different applications. The data presented herein provides a
proof-of-principle that the simple IPL provides useful drug
transport data, but is limited by the modest number of
compounds evaluated. To establish definitively that the technique
is predictive of pulmonary drug absorption would require a
larger range of compounds (for which in vivo data is available),
separated into a probe set to establish a predictive model and
a test set with which to test it.
The benefit of the simplified IPL described in this study is
that it avoids the need for specialized equipment and requires
only the skills of an in vivo pharmacologist to isolate and
maintain the lungs ex vivo. The technique is not in itself sparing of
the use of animals in research and it is more costly and has
lower capacity than cell culture. However, it is a method
under which lung processes, such as absorption or metabolism,
can be isolated and studied in a system which preserves
threedimensional organ architecture under carefully controlled
conditions, enabling studies to obtain answers with fewer
replicates by avoiding interference from systemic influences. In
addition to measuring the intrinsic permeability of drugs, the
IPL technique is being used to evaluate the effectiveness of a
variety of absorption-modifying drug delivery strategies on
absorptive clearance from the lungs, including nanoparticles
], sequence-specific phage display-derived peptide
conjugated dendrimers [
], drug-ester polymer conjugates [
] and polymer microparticles [
Drug permeability in respiratory epithelial cell lines is
wellcorrelated with the pulmonary absorption rate constant in rats
], which makes the cell lines useful for rank-ordering and
screening drugs with respect to their intrinsic lung absorption
rates . We compared directly the permeability of seven
molecules in Calu-3, 16HBE14o- and NHBE cell layers, deriving
log-linear relationships between their permeability in Calu-3,
16HBE14o- or NHBE cell layers and the absorption rate
constant determined after pulmonary delivery to rats (Fig. 2b).
Higher apparent permeability coefficients were obtained in
16HBE14o- and NHBE compared to Calu-3 cells, whereas
the same rank order was obtained in the cell lines,
16HBE14o- and Calu-3. In terms of molecular properties,
the molecules with log P > 0.1 possessed higher permeability
and were clustered with Papp values in the range 5.2–24.5 x
10−6 cm.s−1 in all the cell models. The Calu-3 cell layers were
more restrictive to the large molecule, dextran MW 10,000,
and hydrophilic small molecule, mannitol. Strong in vitro/in vivo
correlations have been reported previously with compounds
possessing a wider range of molecular weight, and therefore a
wider range of permeability, i.e. dextrans MW 4000, 10,000,
40,000 and 70,000 [
]; in contrast dextran 10,000 was the
only non-small molecule in the data sets reported herein.
When selecting in vitro models for pre-clinical screening,
there is generally a trade-off between practicalities (simplicity,
economy, reproducibility, capacity) and biorelevance (human
systems, mixed cell types, structural/morphological/dynamic
features). Although NHBE cells provide a more biorelevant
model, for routine use this advantage is outweighed by the
convenience, low cost and robustness of cell lines, especially
if no advantage of the primary cell model can be rationalized
or demonstrated. For the compounds used in this study, in vitro
permeability correlated with in vivo absorption for each of the
cell models, with no advantage apparent in regard of utilizing
one cell model over another. Although comparison of
compound permeability in the respiratory cell-based models
showed strong relationships with absorption from the IPL
(Fig. 4) and rat lungs in vivo (Fig. 2b), a similar relationship
has been reported between permeability in Caco-2 cells and
absorptive transfer in IPL [
]. For other aspects of lung
absorption/retention, e.g. drug transport mechanisms,
lungtargeting strategies and the efficacy of inhaled medicines,
more organ-specific models may be required with
requirements which should be carefully considered on a
case-bycase basis for each application [
]. If respiratory cell lines
are to be used to generate decision-making data, e.g. for
selecting compounds for development as orally inhaled
products, similar principles to those advocated for the use of
Caco2 in predicting intestinal drug permeability should be applied
to maximise data quality, i.e. standardized practices for
culture of cells, conduct of experiments, use of benchmarks and
data analysis [
The physicochemical properties of molecules that confer good
biopharmaceutical performance when inhaled are not fully
]. In vitro and ex vivo techniques provide
experimental models in which drug permeability in the lungs
can be derived empirically. Of the techniques available for
pre-clinical characterization of drug permeability, a hierarchy
ranging from in silico methods to in vivo studies transport has
been proposed previously [
]. Under such a model, cell
cultures should be used for initial screening of drug permeability
before proceeding to ex vivo and in vivo techniques for lead
candidate optimization. Interestingly, non-cellular PAMPA
methods have been developed for certain epithelia, e.g.
intestinal and blood brain barrier [
], but not to date for lung
permeability measurements. In this absence, cell lines provide
an opportunity to reduce animal testing and can be used to
determine intrinsic drug permeability in drug design and
] and generate essential inputs for mechanistic
modelling. In our study, drug permeability in a much simpler
IPL method than previously reported was indicative of in vivo
lung absorption and concorded with findings in the cell
culture models. All of the techniques were suitable for
empirical screening of drug absorption in the lungs, with Calu-3 out
of the cell models having the advantage for routine drug
permeability screening purposes of being commercially available
and more robust in forming tighter air-interfaced cell layers
compared to 16HBE14o- cells and more economic and
simpler to culture than NHBE.
ACKNOWLEDGMENTS AND DISCLOSURES
This work was partly funded by a grant from Pfizer R&D,
Sandwich, UK. Cynthia Bosquillon was a University of
London Maplethorpe fellow. Michaela Madlova was
supported by the GALENOS-Network and an EST Marie Curie
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License
permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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