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Defense Mechanisms of the Respiratory System and Aerosol Production Systems

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Medicinal Chemistry, 2014, 10, 000-000 1

Defense Mechanisms of the Respiratory System and Aerosol Production

Systems

Paul Zarogoulidis1,2*, Kaid Darwiche2, Lonny Yarmus3, Dionysios Spyratos1, Nevena Secen4, Wolfgang
Hohenforst-Schmidt5, Nikolaos Katsikogiannis6, Haidong Huang8, Andreas Gschwendtner7 and Konstantinos
Zarogoulidis1

1
Pulmonary Department, “G. Papanikolaou” General Hospital, Aristotle University of Thessaloniki. Thessaloniki,
Greece; 2University Pulmonary Department-Interventional Unit, University of Duisburg-Essen, Essen, Germany;
3
Division of Pulmonary and Critical Care, Johns Hopkins University, Baltimore, USA; 4Institute for Pulmonary
Diseases of Vojvodina, Clinic for Pulmonary Oncology, Faculty of Medicine, University of Novi Sad, Serbia; 4II Medi-
cal Clinic, Hospital of Coburg, University of Wuerzburg, Coburg, Germany; 6Surgery Department (NHS), University
General Hospital of Alexandroupolis, Alexandroupolis, Greece; 7Pathology Department, Clinic of Amberg, Amberg,
Germany; 8Department of Respiratory Diseases, Changhai Hospital/First Affiliated Hospital of the Second Military
Medical University, Shanghai, China

Abstract: Aerosolized therapies have been used in everyday clinical practice for decades. Experimentation with different
delivery systems have led to the creation of aerosolized insulin, antibiotics, gene therapy and chemotherapy. Several of
these therapies are already clinically available while others are being investigated in active clinical trials. The main factors
affecting the efficiency and safety of the aerosolized therapies are the production of the aerosol, distribution/deposition of
the aerosol throughout the lung parenchyma, respiratory defense mechanisms and tissue/pharmaceutical molecule interac-
tions. Current methods of aerosol production and distribution will be presented along with an overview of the respiratory
defense mechanisms. In addition, methods of aerosol evaluation in conjunction with a future perspective of the potential
development of aerosol therapies will be presented.

Keywords: Respiratory system, defense mechanisms, aerosol, dry powder, inhalers.

INTRODUCTION been previously demonstrated that main particles are depos-

The progressive anatomic branching and tortuosity of the
airways in combination with particle size and velocity result
in a greater probability of deposition by impaction. The
larger the particle, smaller airway and higher velocity, the
more efficient the deposition of particles throughout the air-
way [1]. The humid environment of the lung (99.5%) is a
major factor that results in particle size changes. A hygro-
scopic aerosol that is delivered from low temperature and
humidity, to high temperature and humidity is expected to
grow while being inhaled through the airways [2]. The
growth depends on the initial diameter of the particle. A fine
particle of <1μm is expected to increase by five-fold, while a
>2μm fine particle by two-to-three-fold [3]. Aqueous drop-
lets expand in the warm and humid environment of the air-
ways from 1-2μm to about 4.5 μm by the time they reach the
carina. Aerosolized particles less than 1μm in size diffuse
into the alveolar spaces due to random Brownian motion,
however their very small size is not an independent factor
that independently improves aerosol administration. It has
*Address correspondence to this author at the Pulmonary Department, “G.
Papanikolaou” General Hospital, Aristotle University of Thessaloniki, Thes-
saloniki, Greece; Tel: 004915779211742; Fax: 00302130992433;
E-mails: pzarog@hotmail.com and ppneumon@yahoo.com
ited by sedimentation in the lungs when they present in a
size of 1-5 μm due to improved laminar airflow [4].
As a result of these factors, the increase of the particle
size will directly affect the amount of the drug distribution
and deposition within the lung. The main factors affecting
the augmentation of the initial droplet size are the tonicity
and droplet size. After inhalation, the aerosol will then be
absorbed into the systemic circulation, degraded via the drug
metabolism or cleared via the airway clearing mechanism.
Particles deposited in the larger airways will be removed
through mucociliary clearance and to a lesser extent will be
absorbed by the airway epithelium into the blood and lym-
phatic circulation [5]. The alveolar macrophages have the
ability to engulf liposomes and microsphere carriers and
transport them to the lymphatic circulation [6]. The lung
defense mechanism is made up of the ciliated epithelium,
airway epithelial goblet cells and sub-mucosal glands. The
ciliated epithelium extends from the trachea to the terminal
bronchioles, moves the secretions to the upper airway, phar-
ynx and ultimately to the gastrointestinal tract. Airway
epithelial goblet cells and sub-mucosal glands consist of a
two-layer mucus blanket which traps insoluble particles and
with the help of the beating cilia are moved upwards and
cleared from the airway. Lipophilic molecules pass through

1573-4064/14 $58.00+.00 © 2014 Bentham Science Publishers

2 Medicinal Chemistry, 2014, Vol. 10, No. ??
the airway epithelium with the method of passive transporta-
tion. Hydrophylic molecules cross the barrier with extracel-
lular pathways or endocytosis and exocytosis [7]. Alveolar
macrophages are the predominant lung defense using phago-
cytic cells against any inhaled particles. The insoluble drugs
are either cleared by the lymphatic circulation or the muco-
ciliaryescalator [8]. It has been reported that this process can
take weeks to months, however; in some inhaled solutions,
the lymphatic drainage occurred within minutes [9-11].
Soluble drugs are absorbed in the systemic circulation.
The pulmonary epithelium is more resistant to soluble parti-
cles than other organ epithelium linings [12]. Although the
rate of protein absorption from the alveolar is size dependent
[12], this is not true for all protein solutions, such as growth
hormone. This observation suggests that a receptor-mediated
endocytosis correlation exists based on local airway trans-
porters [8, 13]. In a recent study, epidermal growth factor
(EGF) was accumulated in a nanoparticle molecule with cis-
platin and was delivered as an aerosol with cancer cell lines
demonstrating higher efficiency than the nanoparticles with-
out the EGF addition [14].
Additional pathways targeted as active transport have
been investigated such as the fibroblast growth factor recep-
tor (FGFR)[15]. It has been previously observed that endo-
bronchial tumors do not have functional beating cilia; so on
the surface of an endobronchial tumor there is no other
mechanism of clearance other than the mucus produced from
the respiratory system and cough [16, 17]. In order to gain
the proper function of airway clearance, the structures of the
airway epithelial cells, ciliary structure-activity, depth-
chemical composition and mucus rheology must be intact
and fully functional [5]. However, there is a debate regarding
the efficacy and safety of administering inhaled drugs to
patients with underlying airway disease such as cystic fibro-
sis (CF), asthma, bronchiectasis, immotile cilia syndrome
and Chronic Obstructive Pulmonary Disease (COPD) (Table
1). The structures or mucus consistency can bealtered as a
consequence of these diseases and it would be expected that
the absorption of a delivered drug by inhalation would be
more inefficient [18].
Table 1. Modulation of Beating Cilia Function
Downregulation
Young syndrome
Kartagener syndrome
Mycoplasma
Upregulation
HaemophilusInfluenzae
Pseudomonas Aeroginosa
Interferon –
Tumor Necrosis Facror-

Interleukin-1
Zarogoulidis et al.
DEFENSE MECHANISMS
Upper Respiratory System
In the nasalcavity allmicroparticles from >10μm are en-
trapped by beating cilia as well as a large percentage of par-
ticles >5μm. Soluble and irritative aerosol gasses are also
absorbed. The particles entrapped are either exhaled with
cough or ingested with mucus into the gastrointestinal tract.
Lower Respiratory System
Airways
The airways are lined with epithelial cells and beating
cilia. Particles >2μm are trapped within the mucus and with
the help of cough are exhaled [19]. In order to work prop-
erly, the cough defense mechanism has to coordinate with
the movement of the beating cilia (pulse movement fre-
quency 12-14/sec). Mucus consists of an upperlayer, which
is thick, and the lower which is more soluble. Several condi-
tions have been identified where the beating cilia do not
work properly such as Young and Kartagener’s syndrome.
Other factors affecting proper ciliary function aremicrobes
(mycoplasma), oxidative factors and platelet derived growth
factor. The epithelial cells also produce proteins that bind to
iron (Fe+), anti-oxidative agents and anti-protease (Table 1).
Lactoferin has the ability to bind with iron and therefore the
microbes that need the iron for their proliferation are con-
trolled. Lysozyme destroys the wall of the microbes and fun-
gus. In addition, it has the protective role of inhibiting the
oxidative factors produced from inflammation. The airway
damage can be induced from the microbes as well as from
several factors that are produced during the phagocytosis.
Several antiproteases such as;
1-antithrypsin,
2-
antichymotripsine,
2-macrospharin and elaphin protect the
airways from the destructive effects of the produced oxida-
tive factors (Fig. 1).
Respiratory System Immunity
There are two main driving immune pathways within the
respiratory system, the endogenous and the specific [20, 21].
The basic difference between the two systems is the method
by which they identify microbes or foreign molecules. The
endogenous system identifies the harmful from the harmless
substances with the help of hydrocarbs. The purification of
the microbes from the alveoli is performed with the help of
cellular and chemical factors that include natural killer cells,
alveolar macrophages, alveolar surfactant and the supple-
ment.
Natural Killer Cells
The alveolar macrophages interact with microbes and in-
terleukin-12 (IL-12) is produced. Tumor necrosis factor-

(TNF-
) interacts with IL-12 and produces interferon-
(IFN-) from the natural killer cells (NK). The early produc-
tion of IFN- enhances the antimicrobial function of the al-
veolar macrophages [22].
Alveolar Macrophages
Macrophage populations are found in the large airways
(trachea) in small numbers and in larger numbers in the
smaller airways (alveoli) [23, 24]. Macrophages have the
Defense Mechanisms of the Respiratory System Medicinal Chemistry, 2014, Vol. 10, No. ?? 3

Fig. (1). The airways have more than 50 different cell types. The beating cilia move the mucus with microbes and foreign material from the
lower respiratory system to the mouth cavity and with the additional help of cough the phlegm is exhaled. Otherwise the phlegm is swal-
lowed. The macrophages of the airways phagocytose the microbes and foreign materials. The additional immunologic response towards these
two stimulation factors depends from the dendritic cells which are loculated among the airway epithelial cells. Lymphocytes also exist among
the epithelial airway cells and they are equipped with T receptors.

ability to move through the different layers of the airway
structures and lung parenchyma. The microbial function of
the macrophages depends on signal recognition, immigration
to the site of the signal) phagocytosis (size of the harmful
factor) and secreted mediators. The macrophages recognize
hydrocarbs [25], with the help of receptors. These receptors
include the Mannose receptor, liposacharide receptor, com-
plement receptor 1 and 2 and scavenger receptors type 1, 2
with collagenous structure. The microbe is initially engulfed
by the pseudopods of the macrophage and then inserted in-
side the phagosomeand destroyed. The liposacharides that
exist in the membrane of the microbes are the most powerful
signals that attract the macrophages. Interferons
and  are
produced from the liposacharides of the microbes and en-
4 Medicinal Chemistry, 2014, Vol. 10, No. ?? Zarogoulidis et al.

Fig. (2). The function of the macrophages depends from the signal. The signal is usually a microbe or a foreign material (molecule). The
macrophages upon their stimulation phagocytose the microbes or the foreign material and produce mediators.

hance the phagocytic capability of the macrophages. Addi- Surfactant

tionally, the growth macrophage-cell stimulating factor
(GM-CSF) is a powerful signal that also enhances the
phagocytic function of the macrophages. The macrophages
are less effective than monocytessince the alveolar macro-
phages have minimum myeloperoxidase (MPO). Further-
more, defensins a peptide whose phagocytic function de-
pends on the macrophages are responsible for the effective
destruction of both Gram negative and positive microbes,
viruses and fungus (Figs. 2 and 3).
Surfactant consists of the protein A (SP-A) and D (SP-D)
and is a member of the collectin family. SP-A and SP-D do
not have the ability to destroy microbes; however, they are
responsible for the enhancement of the macrophage phago-
cytosis.
Supplement
Complement is stimulated by the hydrocarbs on the outer
membrane of the microbes or the collectins. The production
Defense Mechanisms of the Respiratory System Medicinal Chemistry, 2014, Vol. 10, No. ?? 5

Fig. (3). The macrophage produce mediators when they phagocytose microbes and foreign materials, such as; IL-12; Interleukin-12, TNF-;
Tumor ecrosis Factor-, GM-CSF; Granulocyte Macrophage-Colony Stimulating Factor. Many cytokines stimulate the macrophages. TNF-
 and L-12, stimulate the production of interferon- from Natural Killer cells.

of C3b and C5a is initiated and finally the complex C5b-C9
is constructed and attached on the surface of the microbes
and destroys their membrane.
Inflammatory Reactions
The airway macrophages are an efficient defense
mechanism when the microbial load is in a range where their
population can control it. However, there are situations
where the macrophage population may not be capable of
efficiently destroying the microbe load due to the high toxic-
ity and population. Several mediators are produced due to
the toxic agents of the microbes and polymorphonuclear
cells are attracted to assist in the phagocytosis. Cytokines
and Chemokines attract lymphocytes and polymorphonu-
clear cells at the site of the infection. There are four cytoki-
nes CXC, CC, C and CXXC [26]. The CXC family consists
of interleukin-8 (IL-8) and the macrophage inflammatory
protein-2 (MIP-2). The CC family consists of monocyte
6 Medicinal Chemistry, 2014, Vol. 10, No. ??
chemo attractant proteins -1, 2, 3 (MCP 1, 2, 3), RANTES
and MIP-1
and MIP-1. The CXXC consists of fractalkine
and C family from lemphotacin [27, 28]. The CXC family is
responsible for the production of IL-8, TNF-
and MIP-2.
These factors are closely related to favorable outcomes in the
control of respiratory infections. Lower levels of these fac-
tors indicate a prolonged infection [27].
Specific Immune Reactions
There are two systems responsible for the immune re-
sponse towards microbes antibodies and cellular immunity;
both of which are controlled by B and T lymphocytes. The
receptors from B-lymphocytes recognize proteins or hydro-
carbons. The T lymphocytes recognize peptides. After the
infections have been controlled, clones from the antigen
which induces the infection circulate creating lymphocytes
which are specific to the circulating antigen. Initially these
lymphocytes are observed in small populations, however;
they have the ability to act fast upon the recolonization of the
known antigen giving the immune system the time to pro-
duce more specific lymphocytes. The T lymphocytes are also
stimulated by cytokines such as IL-2. The dendritic cells
which can be found between alveolar barriers and interstitial
tissue act as a scouter for the T lymphocytes. The alveolar
macrophages down-regulate the function of the T lympho-
cytes. Although a powerful action against the microbes and
harmful molecules is necessary, there are many cytokines
and chemokines produced throughout this process that are
harmful for the lung parenchyma. Therefore if GM-CSF and
TNF-
are overproduced due to large microbe load, the al-
veolar macrophages do not inhibit any more action of T
lymphocytes. T lymphocyte population will be attracted to
the site of the infection [29-31].
T-Helper-1 cells are responsible for the production of IL-
2, IFN-, TNF-
and GM-CSF, while TH-2 is responsible
for IL-4, IL-5 IL-6 and IL-10 [32]. The modification of the
TH cells to TH1 requires IL-12 and TNF-
, while TH-2 need
sIL-4. Mast cells and eosinophils produce IL-4 due to spe-
cific antigens. TH-2 cells population is down-regulated by
IFN- which is produced by TH-1. The IL-10 and IL-4
which are produced by TH-2, down-regulate the population
of TH-1.
Infectious Factors and Immune Response
An investigation regarding specific microbes and viruses
has been performed in animal models. It has been observed
that mice with a lack of CD4+ T cells had infection spread-
ing outside the lung parenchyma when infected with Crypto-
coccus neoformans. Moreover, these mice had a low number
of CD8+ cells and disrupted airway clearance. The CD8+
cells either phagocytose the microbe or destroy the infected
cells of the respiratory system. Furthermore, they augment
the phagocytic function of monocytes by producing cytoki-
nes. Other infectious organisms such as Candida Albicans,
Blastomyces Dermatitis and Histoplasma Capsulatum; al-
though they are phagocytosed, they continue to be repro-
duced within the macrophages, therefore the produced cyto-
kines form CD8+ cells are necessary to augment their
phagocytic ability [33].
Tuberculosis has also been investigated in a mouse
model. The CD4+ cells and CD8+ cells assist in the phago-
Zarogoulidis et al.
cytosis of the microbe by producing cytokines such as; IL-2,
IL-4, TNF-
and IFN-. TNF-
and IFN- have a synergistic
effect towards the antimicrobial capacity of macrophages for
tuberculosis. TNF-
has also a synergistic effect with GM-
CSF towards the control of tuberculosis. In addition, these
cytokines act as a signal by which the monocytes and lym-
phocytes are attracted to the microbes and create granuloma
in order to contain the infection. Tuberculosis microbes con-
tinue to hibernate within the phagosome after ingestion.
Their destruction depends on whether the phagosome will be
connected with a lysosome [34]. At each point Nitric Oxide
assists in the destruction of tuberculosis bacillus within the
macrophages.
Viral infections of the respiratory system are controlled
with CD8+ cells which exist between the epithelial cells
which recognizes the viral gene that acts as an antigen.
Immune Responses in the Lower Airways
When an antigen reaches the lower respiratory airways
and bypasses all the non-immune/specific defense mecha-
nisms, lymphnodes are activated and produce antibodies.
The antibody forming cells (AFC) are produced in the lym-
phnodes and pass through the systematic and lymphatic cir-
culation to reach the lung parenchyma [35]. The AFC cells
can also move through vessel walls and diffuse into the
blood circulation. The diffusion is increased with inflamma-
tion; therefore more AFCs are attracted locally to the site of
infection/inflammation. The antigens produce locally mem-
ory cells with the additional help of the dendritic cells and
therefore locally there is the ability for the antibodies to be
produced faster. The antibodies are produced with a much
smaller population of antigens. Moreover, the B line memory
cells have the ability to produce antibodies within the inter-
stitial tissue. When the B cells are activated, they can pro-
duce over 1000 plasmatocytes and more than 30.000 immu-
noglobulins [36] (Fig. 4).
Immunoglobulins of the Respiratory System
Immunoglobulins usually can be found in the respiratory
tract [37]. Bronchoalveolar lavage (BAL) samples reveal the
presence of IgG, IgM and IgA. The IgA are diminished from
the upper to the lower respiratory tracts, while the IgG are
inversely increased. IgA and IgG are included in the defense
mechanisms of the respiratory tract. IgA assists as a defense
mechanism against viruses and prevents microbes from at-
taching to the respiratory wall. IgG assists in preventing mi-
crobes from attaching to the bronchial walls and the macro-
phages to phagocytose the microbes.
Significant Properties Required for Efficient Inhaled
Solution
The physical properties that influence the efficiency and
safety of inhaled solutions can be summarized as follows:
a) Viscosity: A liquid's viscosity depends on the size and
shape of its particles and the attractions between the par-
ticles, Zero viscosity is observed only at very low tem-
peratures in superfluids.
b) Ionic Strength is responsible for the dissociation or the
solubility of different salts within a solution
Defense Mechanisms of the Respiratory System Medicinal Chemistry, 2014, Vol. 10, No. ?? 7

Fig. (4). The proliferation and differentiation of B-Cells is taking part in the hilar lymph nodes. Dendritic cells inside the lymph nodes assist
in the maturation of the B-cells in order to produce antibodies. B-cells migrate to bone marrow and lung parenchyma where they will convert
to plasmatocytes which produce antibodies. The antibodies assists in the phagocytic process of the microbes and enhance in the recognition
of the microbes from the antigens that are produced during the phagocytic process.

c) Particle size (1-3μm) for alveolar deposition
d) Optimal ventilator parameters respiratory rate and tidal
volume
e) Site residence time to allow for efficient absorption from
local transporters and connection with antibodies
f) Site of deposition in the airways(different site, different
transporters and gene expression)
g) Osmolarity which is per volume of solvent and is an
important factor related tot he safety of the alveoli as a
low osmolarity might destroy the alveolar structure. If
the osmolarityis not within normal values, bronchocon-
striction can occur which reduced the efficenxy of drug
absorption [38, 39].
H) Shape (>20μm in one axis prevents the phagocytosis
from the macrophages and induces cough)
8 Medicinal Chemistry, 2014, Vol. 10, No. ??
I) Particle charge is important for solution stability-and
efficient local delivery
J) pH(< 6.5 not applicable for the respiratory system). A
pH <3.5 is considered toxic for the epithelium in con-
trast with the gastrointestinal tract.
Deposition Mechanisms
There are 3 main mechanisms of deposition: inertial im-
paction, gravitational sedimentation and brownian diffusion.
The first mechanism affects particles >5μm. Particles >5μm
tend to keep their trajectory and when sudden gas flow
change occurs they deviate from their initial course and im-
pact on airway walls. The gravitational sedimentation affects
particles with size between 1-8μm and is the dominant
mechanism in small airways and alveoli region. It also de-
pends on the time the particle resides in the airways and al-
veoli in relation to the particle size as the higher the particle
size, the more is affected. In addition, particles tend to ex-
pand while moving from the upper airways to the lower air-
way as they come in contact with the humidity of the respira-
tory airways. Again this mechanism increases its influence
while the particle is moving from the upper to the lower air-
ways. The Brownian diffusion occurs with <0.5μm and is the
main mechanism of deposition in the alveoli region. The
smaller the particles, the more random trafficking is ob-
served from the multiple collisions between the molecules
[40].
The larger the particles are, the more obvious the gravita-
tional sedimentation and the inertial impaction act in the
same way. For particles about 1μm, the three factors will
affect deposition together in the same level. The deposition
mechanism of inertial effect depend on both particle diame-
ter and mass density as well as the inflow velocity of the gas
(breath intensity) [41].
Methods of Enhancing the Aerosol Inhalation
The main method for enhancing the aerosol deposition in
an animal model was found to be the addition of 5% CO2
[42-48]. The addition of CO2 while nebulizing, has the abil-
ity to lower the respiratory rateand increase vital capacity
(deep breaths) [49]. Another option would be the nebuliza-
tion of the drug formulation in Heliox which is a low density
gas mixture that has the ability to reduce flow resistance and
allow more aerosol to be deposited in the alveoli region [50].
The intensity of breath not only improves the inhalation of
aerosols, but also improves deposition in the respiratory
tract.
Deposition in Lung Disease
There are several inhaled therapies used in everyday
clinical practice including antibiotic administration and in-
haled insulin [51-53]. Although these therapies are used, it
was unclear if a patient with an underlying respiratory dis-
ease absorbs the same amount of the administered compoun-
das a healthy person. It is well known and previously dis-
cussed that several respiratory diseases alter the lung paren-
chyma [18]. Moreover, several manifestations of an exacer-
bation such as broncho constriction (airway narrowing) and
local inflammation alter the aerosol distribution and absorp-
tion. There are several alterations that occur to cause turbu-
lent flow including bifurcation angles and increased mucus
Zarogoulidis et al.
secretion which divert the aerosol to non-obstructed areas of
the lung and prevent the administered drug from being ab-
sorbed.
Inflammation itself can increase the clearance of the de-
livered formulations through the macrophages. This becomes
an issue when an antibiotic treatment is administered to con-
trol a local infection [54-56]. However, other aerosol thera-
pies, such as inhaled insulin showed an administration depo-
sition that was efficient and safe, but nevertheless a more
frequent check of the blood glucose was advised [52]. It was
observed that the combinationof bronchodilation therapy and
inhaled corticosteroids assisted in the increase of forced ex-
piratory volume in 1 second (FEV1) and reduction of local
airway inflammation making the absorption of the inhaled
compound more efficient, especially in the obstructive dis-
ease pattern [56-58].
Bronchial-Lymphatic Circulation
The lung is the most well vascularized organ which is
most pronounced in the alveolar region and respiratory bron-
chioles [59]. The bronchial circulation distributes aerosol-
ized drugs to regions outside the lung parenchyma. It was
found in an animal model that induced broncho constriction
increased the blood flow and the same was observed in hu-
man patients by 1%-30% [59]. Inhaled drug administration
in previous studies revealed that the administered com-
pounds had high concentrations observed in the regional
mesothoracic lymph nodes [10, 11].
AEROSOL DELIVERY SYSTEMS
The development of an efficient inhalant agent for loco-
regional therapy depends, not only on the molecular formu-
lation, but also on a well-designed inhalation delivery sys-
tem.
Nebulizers and Enhancement Method
Nebulizers have been used for several respiratory dis-
eases. As described above, the most important physical
properties regarding the molecule formulation for a drug to
be nebulized are viscosity, pH, ionic strength, osmolarity,
and surface tension [60]. There are several types of nebuliz-
ers currently used in the market that each has its own advan-
tages and disadvantages. The two basic types are the jet
nebulizers and the ultrasonic nebulizers. The jet nebulizer is
based on the Bernoulli principle and it uses gas (air or oxy-
gen) to produce aerosol droplets. The ultrasonic nebulizers
use a piezoelectric crystal vibrating at a high frequency (1-3
MHz) and generate small aerosol droplets. The higher the
frequency, the smaller the produced droplets [60]. These
nebulizers can aerosolize most drug solutions and provide
large drug doses with no necessary patient-apparatus coordi-
nation and no education regarding their usage. Although they
are inexpensive the compressors used to supply the gas are
not. In addition, they are time consuming and drug consum-
ing. In many cases more than 50% of the nebulized drug is
lost and only approximately 10% of the drug placed in the
drug reservoir is deposited to the lungs [61].
Recent technological advantages in the field have over-
come technical obstacles and enhanced the drug deposition.
The aerogens aerosol generator can determine the aerosol
particle size and flow rate through the flow hole, making it
Defense Mechanisms of the Respiratory System Medicinal Chemistry, 2014, Vol. 10, No. ?? 9

very useful when specific parameters have to be used in sev-
eral clinical applications. They are portable, use batteries, are
relatively quiet and the remaining solution (residual volume)
is minimal. Finally, they require a shorter time for nebuliza-
tion and the range of nebulization is from 0.3 to 0.6ml. The
aerogen`s aerosol generator efficiently aerosolizes proteins
and peptides [62, 63]. In addition, the Aeroneb® portable
nebulizer system was designed as a hand-held inhaler de-
signed for hospital and domiciliary use. It has a three- to five
fold higher efficiency of delivering drug particles when
compared to jet and ultrasonic nebulizers [63]. Therefore
lower nominal doses can be used when this equipment is
used. The Omron`s technology incorporates a piezoelectric
crystal that vibrates at high frequency when electric current
is applied. These are portable, run either with batteries or are
current powered and generate fine-particle mist due to a
mesh plate with more than 6000 tapered holes [64]. In addi-
tion, the remaining residual volume of the drug in the resid-
ual cup is negligible [65]. Finally, the Touch Spray technol-
ogy utilizes a perforate membrane, vibrating at high frequen-
cies against a body of fluid [66]. This technique can aerolize
a wide range of solutions and molecules as well as control
the particle size and flow rate. Their cost is slightly higher
than conventional jet-nebulizers, but lower than some ultra-
sonic and the patients can easily be trained to use them.
A very important section of the nebulization administra-
tion is the part where the aerosol is actually delivered to the
nose and mouth cavity. This transition is being accomplished
through a specialized facemask with an inspiration and expi-
ration valve. Older systems used facemasks which had two
holes in order for the aerosol that was not inhaled to leak into
the environment. The newly introduced inlets with the two
valves actually allow for delivery control with a breath-
actuated administration and higher drug delivery efficiency
[52]. In previous studies, it has been observed that by adding
5-7% of CO2 in inhaled aerosol, the efficacy of the inhaled
compound was increased. This was due to the increased
depth of each breath resulting in a 180% increase in tidal
volume while the respiratory frequency was reduced. How-
ever, if more than 7% of CO2 was added, the patients expe-
rienced dizziness, confusion and sleepiness [49, 67, 68].
When used correctly, this additional mode of inhalation can
enhance the drug peripheral deposition (Table 2).
Soft Mist Inhalers
Soft mist inhalers use the mechanical force of a spring to
produce a fine aerosol mist. The only one currently available
on the market is the respimat Spiriva®. It is cheap, easy to
use and it has an increased lung deposition to the periphery
[69-71]. The delivered dose is independent of the patients

Table 2. Aerosol Delivery Systems Advantages Disadvantages

No patient-apparatus coordination, inexpensive,

Jet-Nebulizers (gas-Brenoulli principal) 50% loss of drug, time consuming
aerosolize any compound, portable

No patient-apparatus coordination, inexpensive,

Ultrasound nebulizers (piezoelectric crystal) 50% loss of drug, time consuming
aerosolize any compound, portable

Portable, quiet, shorter time of aerosol production,

Aerogen`s aerosol generator ability to control the particle size and flow rate, expensive
efficiently aerolises proteins and peptides

hand-held inhaler, three- to five fold higher effi-

® ciency of delivering drug particles when compared
Aeroneb expensive
to jet and ultrasonic nebulizers, remaining residual
volume of the drug in the residual cup is negligible

remaining residual volume of the drug in the resid-

Omron`s technology, piezoelectric crystal ual cup is negligible, ability to control the particle expensive
size and flow rate

ability to control the particle size and flow rate,

TouchSpray technology expensive
aerosolize any compound

The delivered dose is in depended of the patients

Soft Mist Inhaler cheap, easy to use respiratory capacity and lower doses are needed in
comparison to Handihaler devices

The lung deposition varies between 12-40% and

20-25% of the produced cloud is retained within
outpatient inhalation device, single dose and multi- the device, hand-mouth discordination, 50-80%
Metered Dose Inhalers
ple dose inhalation devices, oropharyngeal deposition due to high velocity of
the produced particles, the technique of the patients
still imposed a major factor

need fast and large inhalation efforts (>60 l min-1),

Dry Powder Inhalers breath actuated
efficiency depends from the nature of the powder
10 Medicinal Chemistry, 2014, Vol. 10, No. ?? Zarogoulidis et al.

Table 3. Highlights

Methodology

The parameters that have to be included when a new aerosol therapy is investigated are the method of the aerosol delivery, the pharmaceutical bioequiva-
lence to the human respiratory system and proper design of local tissue/pharmaceutical interaction.

Aerosol Delivery Systems

Several apparatus have to be investigated before selecting a method and experiments have to be performed in animals with a respiratory system similar to
human. Several pharmaceuticals might induce emphysema or interstitial disease in small animals such as BALBC mice, while larger animals such as pig or
sheeps, might not present such adverse effects.

Pharmaceutical Local Delivery

A conjugation with a sustain release molecule or a molecule protecting from the defense mechanisms of the respiratory system is also necessary. Passive or
Active transportation of the aerosolized pharmaceuticals has to be clarified before initiating the protocol

Aerosol Therapy

Enhancing the delivery method is warranted and close observation is necessary when the subject has underlying pulmonary disease

respiratory capacity and lower doses are needed in compari-
son to Handihaler devices [70] (Table 2).
Dry Powder Inhalers (DPIs)
Dry powder inhalers were mainly introduced to the mar-
ket to eliminate the use of chlorofluorocarbons (CFC) and
resolve the breath-inhalation co-ordination problem of
MDIs. There are two types in the market; the single dose and
the multiple dose inhalation devices. The lung deposition
varies between 12-40% and 20-25% of the produced cloud is
retained within the device [72, 73]. Factors that affect the
lung deposition are high humidity, large temperature
changes, and mode of the inspiratory flow rate that consists
of the tidal volume and respiratory rate. These factors affect
the de-aggregation of the fine drug particles, making the
produced fine particle cloud inefficient [60]. In contrast to
metered dose inhalers (MDIs), the dry powder inhalers
(DPIs) need fast and large inhalation efforts (>60 l min-1). It
has demonstrated that when increasing this parameter from
35-60 l min-1, the lung deposition is also increased [74]. The
de-aggregation of the DPI powder and dispersion deepens on
shear turbulent forces in order to break up the powder into
fine particles and hence provide improved drug lung deposi-
tion. Several efforts have been made to improve the deposi-
tion and minimize the inspiratory effort of the patients. This
was accomplished with the use of battery-driven propeller
and by using compressed air to increase the de-aggregation
and dispersion of the powder in a holding chamber. In addi-
tion, breath actuated DPIs have been introduced in the mar-
ket improving further on lung deposition and making them
more efficient for patients with low inspiratory flow rates.
DPIs that are currently on the market need an inspiratory
flow rate between 30 to 130 l ml-1 to be efficient due to the
nature of the powder and forces needed to de-aggregate and
disperse them (Table 2).
Metered Dose Inhalers (MDIs)
Metered dose inhalers are mainly used as an outpatient
inhalation device. They use propellants, such as chloro-
fluorocarbons (CFC) and more recently, hydrofluoroalkanes
(HFAs). Most of the old devices with CFC have now been
retired from the market due to environmental safety reasons
[75]. The main disadvantages of these inhalers are the hand-
mouth discoordination found in more than 51% [76], 50-
80% oropharyngeal deposition due to high velocity of the
produced particles [77] and only 10-20% of the emitted dose
is deposited to the lung parenchyma. Factors positively af-
fecting the lung deposition involve increased tidal volume
(TV), slow respiratory frequency, breath holding at the end
of the inspiration, and particle size (mass median aerody-
namic diameter MMAD optimal 2.5-3.5μm for reasons pre-
viously presented in the text). Fast inhalation (>60 l min-1)
and large particle size result in oropharyngeal deposition,
ingestion of the produced cloud and exhalation [57, 78].
Several actions have been made to improve the administra-
tion disadvantages. Different spacer tubes and valved hold-
ing chambers with or without mouthpiece extensions have
been developed. These additions eliminated the breath-
actuation coordination problem, produced a finer mass me-
dian aerodynamic diameter (MMAD) particle size cloud
(<25% than before) within the chamber and reduced the cold
freon effect [60]. Despite the inefficiencies surrounding drug
delivery, patients with airway obstruction using this modality
did have evidence of improved lung deposition [79]. As a
result, breath-actuated MDIs were introduced in the market
and provided improvement in the lung deposition (7.2%-
20.8%), however, it was noted that the technique had major
limitations in clinical efficacy [80]. The deep continuous
inhalation and slow respiratory rate continue to be the main
factors that must be perfectly controlled by the patient for
maximal lung deposition to be achieved (Table 2).
Bioequivalence and Evaluation Methods for Inhaled
Drug Formulations
There are a variety of different inhalation drug delivery
systems in existence. Each one has the capability to generate
drug aerosol with differences in particle size, respirable drug
dosage, lung deposition and distribution [60, 81, 82]. An
appropriate matching of the inhalation drug delivery system
and molecule formulation should be made in order to in-
crease the efficiency of this mode of therapy.
Defense Mechanisms of the Respiratory System Medicinal Chemistry, 2014, Vol. 10, No. ?? 11

Different nebulizer models produce various particles eign aerosol particles and destroy them through phagocyto-
sizes based on the residual volume, initial drug volume and sis. Therefore a stealthmolecule covering the outer layer of
flow rate of compressed gas [83-85]. There are no guidelines the drug can be used [94]. Future aerosol drug design should
on various drug formulations regarding the nebulization sys- take under consideration the defense/immunologic response
tem that clinicians should use in order to achieve the optimal of the human respiratory system. Local transporters and
anticipated drug efficiency. Several studies using different genes in every part of the respiratory system should be con-
nebulizer models showed that MMAD plays a crucial role in sidered upon development of a drug formulation [95, 96].
the efficiency of the bronchodilator treatment. Specifically, The respiratory system is a huge vessel (80-100m2) through
large particles (size >7.7μm) deposited only in the upper which the diffusion of several formulations is absorbed al-
respiratory tract make the bronchodilation treatment ineffi- most immediately. Antibiotics are already used in the mar-
cient since most of the 2-receptors are localized in the small ket, new products of inhaled insulin [97] are currently being
airways [86]. In other studies, where the same drug formula- evaluated and new treatment modalities such as inhaled che-
tion with different nebulizer models was evaluated, aerolized motherapy [98-101] and inhaled gene therapy [102-106] are
drug efficiency varied between 30% and <5% of the initial currently being explored.
dose [87, 88]. HFA formulations in the market with salbuta-
mol, salmeterol and fluticasone demonstrated the same effi- CONCLUSION
ciency as the respective CFC formulations [89, 90]. Never-
theless, differences between HFA inhalation devices were The defense mechanisms of the respiratory system should
observed when finer particles were produced. The efficiency always be considered in the design of an experimental aero-
was increased when finer particle aerosol was produced [91, sol therapy. The different mechanisms can either prevent the
92]. particles from being inhaled, or they can destroy the inhaled
particles. The activation of the immune system locally can
Four main methods of aerosol drug evaluation: activate a cascade of cytokine and chemokine production,
A) In vitro particle size distribution: The main disadvantage which in turn can induce severe inflammation. Therefore
is the ambient conditions under which the measurements further studies are needed on the effect of aerosolized thera-
are being done. It has been previously described that the pies on different cells lines in addition to the creation of an
humid environment of the lung expands the molecule efficient delivery system prior to clinical trials on human
(nanocomplexe) which decreases deposition. The lung subjects.
defense mechanisms influence, which exists in the lung
environment, hasnot been evaluated with this method CONFLICT OF INTEREST
[93]; The authors confirm that this article content has no con-
B) Radiolabeled aerosol drug deposition: This method flicts of interest.
should apply to patients with underlying respiratory dis-
ease. ACKNOWLEDGEMENTS
C) Pharmacokinetic studies: These studies are not directly Declared none.
correlated to the doses administered since a large
amount of the drug is deposited locally and lung disease ABBREVIATIONS
plays a crucial role since mucociliary secretion is in-
creased in several conditions (e.g COPD, infection). In EGF = Epidermal growth factor
addition, part of the drug is absorbed through the gastro- FGFR = Fibroblast growth factor receptor
intestinal tract. Therefore charcoal should be used to
block the gastrointestinal tract absorption in order to de- CF = Cystic fibrosis
fine the net drug inhaled formulation. COPD = Chronic Obstructive Pulmonary Disease
D) Comparative studies, evaluating efficiency and safety: IL-12 = Interleukin-12
Standard methodology for inhalation therapies hasnotyet
TNF-
= Tumor necrosis factor-

been applied by the various government agencies.
IFN- = Interferon-
The concepts mentioned above indicate that there is a
need for a standardized algorithm of bioequivalence for in- NK = Natural killer cells
haled therapies as we move from treating respiratory dis-
GM-CSF = Growth macrophage-cell stimulating factor
eases to treating systemic diseases through aerosolized respi-
ratory tract administration. MPO = Myeloperoxidase
IL-8 = Interleukin-8
DISCUSSION
MIP-2 = Macrophage inflammatory protein-2
Aerosol therapies should be designed to be easy to use
for a variety of patients (Table 2). The pharmaceuticals de- AFC = Antibody forming cells
livered should be designed to deliver a drug formulation lo- FEV1 = Forced expiratory volume in 1 second
cally based on the time necessary for the drug to act. The
aerosolized formulation should be relatively inert to the res- CFC = Chlorofluorocarbons
piratory system and not induced early or late immunologic DPI = Dry powder inhalers
responses. Macrophages have the ability to attack any for-
12 Medicinal Chemistry, 2014, Vol. 10, No. ??
MDI = Metered dose inhalers
TV = Tidal volume
MMAD = Mass median aerodynamic diameter
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Accepted: August 30, 2013