ARTICLE IN PRESS

EXPERIMENTAL
ANDTOXICOLOGIC

PATHOLOGY
Experimental and Toxicologic Pathology 57 (2006) S2, 4953
www.elsevier.de/etp

Novel strategies of aerosolic pharmacotherapy

David A. Groneberga,b,, Hanna Paulb, Tobias Weltea
a
Departmnt of Respiratory Medicine, Hannover Medical School, Carl-Neuberg-Strae 1, 30625 Hannover, Germany
b
Allergy-Centre-Charite, Charite School of Medicine, Free University and Humboldt University, Augustenburger Platz 1,
13353 Berlin, Germany

Received 21 October 2005; accepted 16 February 2006

Abstract
The pulmonary administration of drugs plays a crucial role in the management of various respiratory and systemic
diseases. While the cellular properties of airway epithelial cells offer a great potential to deliver drugs into the lungs or
the circulation, only little is known about the exact transport pathways.
Recently, the high-afnity proton-coupled drug and peptide transporter PEPT2 was identied in the human
respiratory tract. The expression of transporter mRNA and protein was localized to the airway epithelium and alveolar
type II pneumocytes. In addition, transport studies revealed transporter-mediated uptake of substrates into epithelial
cells indicating that the transporter is the molecular basis for the transport of peptides and peptidomimetic drugs in
pulmonary epithelial cells. Since genotype analysis revealed no signicant differences amongst different transporter
genotypes concerning expression and function, the transporter displays an interesting novel target for pulmonary
delivery of drugs.
r 2006 Elsevier GmbH. All rights reserved.

Keywords: Aerosol; Airways; Asthma; COPD; Drug; Inhalation; Transport

Introduction (Beringer, 1999), bacterial pneumonia (Wood and

The aerosolic administration of drugs plays an
important role in the treatment of pulmonary and
systemic diseases and the unique cellular properties of
airway epithelium offer a great potential to deliver new
compounds (Groneberg et al., 2004a). Using aerosolized
drugs diseases such as bronchial asthma (Rubin and
Fink, 2001), chronic obstructive pulmonary disease
(COPD) (Newhouse and Corkery, 2001), cystic brosis
Corresponding author. Allergy-Centre-Charite, Charite School of
Medicine, Free University and Humboldt-University, Augustenburger
Platz 1/OR-1, 13353 Berlin, Germany. Tel.: +49 30 450559 055;
fax: +49 30 450559 951.
E-mail address: david.groneberg@charite.de (D.A. Groneberg).
Boucher, 2000; Flume and Klepser, 2002) or viral
respiratory infections (ORiordan and Faris, 1999;
Groneberg et al., 2003d, 2005b, c) may be treated
(Table 1). Also, non-respiratory diseases may be treated
via the pulmonary route (Table 2).
As the relative contributions from the large airways to
the alveolar space are important factors concerning the
local and systemic availability of target compounds, the
sites and mechanism of uptake and transport need to be
known.
Among the different cells of the respiratory tract, the
ciliated epithelial cells of the larger and smaller airways
and the type I and type II pneumocytes are the key
players in pulmonary transport of drugs (Groneberg et al.,
2003c). They each have diverse cellular characteristics and

0940-2993/$ - see front matter r 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.etp.2006.02.007
 ARTICLE IN PRESS
50 D.A. Groneberg et al. / Experimental and Toxicologic Pathology 57 (2006) S2, 4953

Table 1. Examples of aerosolized compounds for the topical The two major cell types found in the alveolar
treatment of pulmonary diseases (except common drugs used epithelium are type I and type II pneumocytes. Type I
for obstructive diseases such as asthma and COPD) cells have a very thin cell body with long membranous
extensions and occupy an area of about 95% of the
Class Drugs Diseases
alveolar surface (Hirai and Ogawa, 1986). The type II
Vaccines Viral/ Infectious diseases pneumocytes have a more cuboidal morphology and
bacterial cover about 5% of the total alveolar surface (Sorokin,
Antibacterial Aminoglycosides 1970; Mason and Crystal, 1998). Although type II
Cystic brosis pneumocytes express transport proteins (Groneberg
Penicillins Bronchiectasis et al., 2001d, 2002c), their main functions are the
Pentamidine AIDS generation of surfactant proteins and the differentiation
Antiviral Ribavirin RSV infections
into type I cells after epithelial barrier injuries.
Surfactant ARDS/IRDS
The pulmonary blood-gas barrier is composed of the
Immunosuppressive
drugs alveolar epithelium and the capillary endothelium with
Steroids Lung brosis an intervening extracellular matrix consisting of the two
Prostaglandins Pulmonary basement membranes of the two cell layers (Low, 1952).
hypertension The type I cells display most likely the rate-limiting step
Protease Trypsin Alveolar proteinosis regarding transport of drugs into the pulmonary
circulation since it was reported that these cells have a
103 times lower permeability for substances such as
sucrose in comparison to the endothelial cells (Wan-
Table 2. Aerosolized drugs for systemic diseases
gensteen et al., 1969). There are two main reasons for
Diseases Drugs the lower permeability: (1) difference in the pore size
between alveolar cells (0.61 nm) and endothelial cells
Diabetes Insulin (45.8 nm) (Taylor and Gaar, 1970); (2) the tight
Anticoagulation Heparin
junction depth is with 0.26170.023 mm in type I cells
Osteoporosis Calcitonin
signicantly higher than the tight junction depths of
Headache Ergotamine
capillary endothelial cells (0.16670.011 mm) (Inoue
et al., 1976).
offer unique uptake possibilities. Next to the importance
of knowledge about the transport properties of each of
these cells, the structure of aerosolized drugs, the
characteristics of delivery systems and the deposition Administration of drugs and deposition
and pulmonary clearance display crucial factors in
pulmonary drug delivery mechanisms. There are two main modes of pulmonary drug
Based on the growing knowledge on cell biology and administration: nasal or oral inhalation. The nasal
pathophysiology due to modern methods of molecular inhalation is limited by anatomical features such as a
biology such as gene depletion or overexpression narrower airway lumen. Therefore, the route of oral
(Kerzel et al., 2003; Springer et al., 2004c), the future inhalation of compounds is generally preferred and
characterization of pulmonary drug transport pathways previous studies demonstrated a better oral inhalative
can lead to new strategies in aerosol drug therapy. administration of 5 mm diameter particles with a
concentration loss of only 20% in comparison to 85%
by nasal administration (Lippmann and Albert, 1969;
Cellular and morphological aspects of drug Lippmann et al., 1980). The three principal mechanisms
transport leading to pulmonary deposition are inertial impaction,
sedimentation and diffusion. The inertial impaction
The transepithelial transport along the respiratory takes place during the passage through the oropharynx
epithelium from the upper airways to the lower and large conducting airways if the administered particle
respiratory tract is characterized by large quantitative possesses a certain mass and velocity. The process can
differences. In the upper airways the transport is limited be inuenced by hyperventilation and does not occur at
by a smaller surface area and a lower regional blood a particle size below 3 mm diameter. Particles with a
ow. The upper airways also possess a high ltering lower diameter are subject to sedimentation by gravita-
capacity and can remove between 70% and 90% of tional force occurring in smaller airways. This mechan-
pressurized particles while the smaller airways and the ism is inuenced by breath-holding. From a range below
alveolar space account for more than 95% of the lungs 0.51 mm, particles are deposited by diffusion (Ariya-
total surface area (Weibel, 1979). nanda et al., 1996).
 ARTICLE IN PRESS
D.A. Groneberg et al. / Experimental and Toxicologic Pathology 57 (2006) S2, 4953 51

Table 3. Particle sizes and generating devices some of these agents may be applied as aerosolic drugs,
the localization and function of the oligopeptide
Particle size Particle size Generating device transporter PEPT2 in the respiratory tract were
(mm) uniformity
assessed.
135 Heterogenous Metered-dose inhaler It was shown that the high-afnity transporter PEPT2
1.26.9 Heterogenous Jet nebulizer is expressed in the respiratory tract. Northern
3.710.5 Heterogenous Ultrasonic nebulizer blotting and RT-PCR technologies showed that PEPT2
1.330 Monodisperse Spinning disc but not the intestinal transporter PEPT1 is expr-
0.550 Monodisperse Vibrating orice essed in the mammalian respiratory tract (Groneberg
1.1 Monodisperse Condensation et al., 1999). To identify the cellular sources of
0.14 Heterogenous/ Solid particle PEPT2 non-isotopic mRNA in situ hybridization
monodisperse
studies were carried out. They revealed PEPT2 mRNA
Flow- Heterogenous Dry powder
expression in the rat respiratory epithelium and
related
type II pneumocytes (Groneberg et al., 2001d).
After the development of peptide transporters anti-
bodies (Ogihara et al., 1996; Doring et al., 1998),
Delivery devices immunohistochemistry was performed in rat and murine
airways and a colocalization of PEPT2 protein and
Although there are numerous devices that can be used mRNA in the respiratory epithelium and type II cells
for particle generation (Newman, 1991; Thompson, was found.
1998) (Table 3), the most common systems are Further experiments were performed that led to the
nebulizers, metered dose inhalers (MDIs), and dry visualization of cell types that exhibit a transport
powder inhalers (PDIs). The currently used standard function. Using the uorophore-conjugated dipeptide
inhalation devices produce aerosols which are hetero- substrate D-Ala-Lys-AMCA the incubation of murine
disperse in size. While monodisperse particle-sized lungs with the reporter molecule revealed an uptake and
aerosols are superior concerning targeted delivery to intracellular accumulation of the labelled PEPT2 sub-
the lower airways, the production of these monodisperse strate along the respiratory tract.
particles is limited by complex and expensive generation A specic uptake of the reporter substrate into type II
processes (Newman et al., 2000). cells as well as into bronchial and tracheal epithelial cells
was found and conrmed the morphological data of
immunohistochemistry and in situ hybridization
(Groneberg et al., 2001d) which was found to be similar
Pulmonary drug transporter in murine, rat and human airways (Groneberg et al.,
2000, 2001d).
The cDNAs encoding two families of proton-coupled Patients with cystic brosis and recurrent pulmonary
oligopeptide transporters have been cloned from epithe- infections represent a main indication for an aerosolic
lial cells of intestine (PEPT1) and kidney cortex treatment with antibiotics. Since the transporter may be
(PEPT2) (Daniel and Rubio-Aliaga, 2003). While used as a drug delivery system the expression in human
PEPT1 is expressed in the intestine (Groneberg cystic brosis airway tissues was examined and un-
et al., 2001b) and to smaller extent in kidney (Daniel changed expresssion levels were found (Groneberg et al.,
and Rubio-Aliaga, 2003) but not lung (Groneberg et al., 2002c). Uptake assays were carried out to analyse the
2001d), PEPT2 is expressed in the kidney (Groneberg activity of the transporter to carry drugs after topical
et al., 2002a; Rubio-Aliaga et al., 2003), nervous administration. It was shown that the transporter
system (Groneberg et al., 2001c, 2004b) and in a variety exhibited a regular function (Groneberg et al., 2002c)
of peripheral tissues including lung (Groneberg et al., but the total accumulation of labelled substrate was
2002b, c). Both isoforms possess 12 membrane signicantly reduced due to the large quantities of
spanning domains and share an identity of approxi- airway obstructing mucus (Groneberg et al., 2001a).
mately 47% at the protein level. The carrier proteins This mucus was later shown to consist of mucus-
mediate electrogenic uphill peptide transport by forming mucin proteins from both epithelial goblet cells
coupling substrate translocation to the movement of and mucus gland cells (Groneberg et al., 2002d, 2003a;
H+/H3O+ with the transmembrane electrochemical Chung et al., 2004).
proton gradient as the driving force. In addition Future studies need to assess the expression and
to di-/tripeptides, both isoforms transport several function of the transporter system in human tissues and
peptidomimetics such as aminocephalosporins, amino- animal models of diseases such as bronchial asthma,
penicillins, bestatin, d-ALA and selected ACE inhibitors COPD, pneumonia and pulmonary neoplasms to dene
as substrate (Daniel and Rubio-Aliaga, 2003). Since its potential therapeutic role.
 ARTICLE IN PRESS
52 D.A. Groneberg et al. / Experimental and Toxicologic Pathology 57 (2006) S2, 4953
Conclusion
Pulmonary drug administration plays a major role in
the treatment of both respiratory and systemic diseases
and is attractive for future drug development. The
effects of aerosol-based drugs depend on multiple
factors including the nature of the compounds, cellular
aspects of transport and characteristics of delivery
systems and aerosol administration. Thus, multiple
ways to optimize drug delivery exist.
Concerning the cellular drug transport via drug
transporters, future studies addressing the unique
molecular, biochemical, and physiological characteris-
tics of various respiratory regions have to be carried out
applying modern techniques of molecular biology
(Groneberg et al., 2003b; Springer et al., 2004b),
morphology (Dinh et al., 2004; Heppt et al., 2004;
Fischer et al., 2005; Groneberg et al., 2005a; Springer
et al., 2005) and physiology (Quarcoo et al., 2004;
Springer et al., 2004a; Trevisani et al., 2004).
Acknowledgements
This study was supported by the German Academic
Exchange Service (DAAD, D/00/10559), and the Ger-
man Research Community DFG (GR 2014/2-1).
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