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Peptides and Proteins: Pulmonary Absorption
a
Salomé-Juliette Koussoroplis , Rita Vanbever
a
a
Louvain Drug Research Institute, Pharmaceutics and Drug Delivery , Université catholique
de Louvain , Brussels , Belgium
To cite this entry: Salomé-Juliette Koussoroplis , Rita Vanbever . Peptides and Proteins: Pulmonary Absorption. In
Encyclopedia of Pharmaceutical Science and Technology, Fourth Edition. Taylor and Francis: New York, Published online: ;
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Salomé-Juliette Koussoroplis
Rita Vanbever
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Université catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug Delivery,
Brussels, Belgium
INTRODUCTION
Inhalation of drugs is very efficacious for the treatment of
lung diseases and a continuously increasing number of
inhaled drugs are becoming available on the market. Inhalation of drugs allows a targeted therapy with high drug
concentrations in the tissue of interest, low systemic drug
exposure, and thereby reduced systemic side effects. In
addition, it allows a rapid onset of therapeutic action and it
is a convenient mode of drug delivery.
Inhalation may also be an optimal route for the systemic administration of drugs because pulmonary drug
absorption is high and rapid and first-pass hepatic metabolism is avoided. Peptides and proteins are more efficiently absorbed from the lungs than from any other
noninvasive route of drug administration. For instance,
insulin can absorb from the lungs with a bioavailability of
30% relative to subcutaneous injection, while it reaches
at most 1% following oral, sublingual, nasal, or transdermal administration without chemical enhancer (1,2).
These absorption features originate from the large absorptive surface area of the alveoli, the very thin diffusion
path to the bloodstream as well as the local high blood
flow. Yet, administration of drugs to the healthy lungs can
raise toxicity issues in the long term, and it would be wise
to consider it for the treatment of short course diseases.
MAP Pharmaceuticals currently seeks FDA approval for
Levadex®, a dihydroergotamine inhalation aerosol for the
treatment of migraine, in line with this idea (3).
This chapter provides information about the advances in
pulmonary delivery of peptides and proteins used in both
local and systemic therapy. Inhaled peptides and proteins
that are currently undergoing various phases of clinical trials are presented. The different biological pathways that
these molecules can follow after deposition in the lung are
described, including pulmonary absorption to the bloodstream. Furthermore, the available in vitro and in vivo
models for the assessment of pulmonary absorption of peptides and proteins are outlined. The impact of smoking and
various pulmonary disease conditions on the pulmonary
fate of inhaled peptides and proteins is shown. Finally, a
discussion about stability issues that arise during their formulation, storage, and aerosolization is given.
LUNG PHYSIOLOGY
The respiratory system resembles an inverted tree where the
trachea divides into two main bronchi. Each bronchus further subdivides into progressively smaller bronchioles until
it reaches the smallest airspaces called alveoli. The lung
consists of two functional zones: the conducting zone
(16 first generations) and the respiratory zone (7 last generations). In the conducting region, the air is filtered,
warmed, and humidified, whereas in the respiratory region,
gas exchange between airspaces and blood capillaries
occurs. The airway bifurcations become smaller in diameter
and length but higher in number and larger in total crosssectional area. Consequently, the alveoli provide a total surface area that reaches 100 m2, which is substantially larger
compared to the 0.25 m2 surface area of the airways (4).
Two different epithelia line the conducting and respiratory zones (Fig. 1) (5,6). A pseudostratified columnar
epithelium lines the proximal conducting airways and is
composed of ciliated columnar cells, goblet or mucussecreting cells, and basal or progenitor cells (7). It is progressively replaced by a simple cuboidal cell layer in the
more distal airways and by a very thin epithelial lining in
the alveoli. Squamous type I pneumocytes cover 95% of
the alveolar surface, owing to their large apical surface and
thinness (0.05 µm). Cuboidal type II pneumocytes are
located in the corners of the alveoli. They produce the lung
surfactant and are progenitor for type I cells.
Mucociliary clearance is one of the most important
defense mechanisms to eliminate dust and microorganisms in the lungs (8). The mucus is produced by goblet
cells and sub-mucosa glands and protects the underlying
mucosa from dehydration. It covers the entire airway surface and its thickness ranges from 5 µm to 55 µm. It consists of an upper gel phase made of 95% water, 2% mucin
(a highly glycosylated and entangled protein) as well as
salts, proteins, and lipids (9). A periciliary liquid layer
underlies the mucus gel and its low viscosity allows effective cilia beating. The mucus is transported by the coordinated beating of the cilia and by expiratory airflow toward
the oropharynx. Mucus, cells, and debris coming from the
nasal cavities and from the lungs meet in the pharynx, are
mixed with saliva, and are swallowed. Mucus velocity
slows down when descending the respiratory tree, with
3 orders of magnitude faster mucus velocity at generation
0 as compared to generation 16. This counterbalances the
high number of peripheral airways and thereby the accumulating amounts of mucus to be cleared by the central
airways and trachea (10).
Pulmonary surfactant is responsible for biophysical stabilizing activities and innate defense mechanisms. It lines
the alveolar epithelial surfaces and overflows into the conductive
Encyclopedia of Pharmaceutical Science and Technology, Fourth Edition DOI: 10.1081/E-EPT4-120050324
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Peptides and Proteins: Pulmonary Absorption
Peptides and Proteins: Pulmonary Absorption
Encyclopedia of Pharmaceutical Science and Technology, Fourth Edition, Volume IV
Peptides and Proteins: Pulmonary Absorption
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2608
Human terminal
bronchioles
0.5–1 mm diameter
Human bronchi
3–5 mm diameter
Human alveoli
8 µm
58 µm
0.07 µm fluid
3 µm
10 µm
Ciliated
cell
Goblet Basal
cell
cell
Brush
cell
0.1–
0.2 µm
Type I cell
Basement
membrane
Figure 1 Comparison of human lung epithelia at different sites within the respiratory tract in terms of relative cell height and surface
liquid thickness. Source: From Ref. 5.
airways so that the surfactant film is continuous between
alveoli and central airways (11). Pulmonary surfactant is
composed of 80% phospholipids, 5–10% neutral lipids
(mainly cholesterol), 5–6% specific surfactant proteins,
and 3–4% nonspecific proteins (12). The phospholipids
are mainly responsible for forming the surface-active film
at the respiratory air–liquid interface. Half of surfactant
phospholipids by mass are composed of disaturated species, mainly dipalmitoylphosphatidylcholine. Specific
surfactant proteins include SP-A, SP-B, SP-C, and SP-D.
SP-A and SP-D are hydrophilic, whereas SP-B and SP-C
are hydrophobic. SP-A is able to bind multiple ligands,
including ligands on the surface of pathogens. SP-A
recognition by specific receptors on alveolar macrophages
stimulates phagocytosis. SP-B is strictly required for the
biogenesis of pulmonary surfactant. Both SP-B and SP-C
promote rapid transfer of phospholipids into air–liquid
interfaces.
Luminal airway and alveolar macrophages are at the
forefront of lung defense, and their primary role is to participate in innate immune responses, that is, chemotaxis,
phagocytosis, and microbial killing (13). They also downregulate adaptive immune responses and protect the lungs
from T-cell-mediated inflammation (14). Alveolar macrophages are tightly applied on the surface of respiratory epithelia. They are immersed in the lung lining fluid beneath
the surfactant film. Although they occupy only 1% of the
alveolar surface, they are capable of cleaning particles
from the entire alveolar surface due to amoeboid movements (13). In contrast to surface macrophages, interstitial
macrophages are primarily involved in adaptive immunity
by interfacing with lymphocytes through antigen presentation and production of cytokines (13).
The lung presents a lower level of metabolism than the
gastrointestinal tract and liver. Yet, various peptidases are
distributed on the surface of different cell types in the
lung, including bronchial and alveolar epithelial cells,
submucosal glands, smooth muscles, endothelial cells,
and connective tissue. Proteases are largely present in
lysosomes (15). Proteases that degrade the extracellular
matrix are secreted by different structural cells, or membrane bound (16). Proteases play an essential role in cell
and tissue growth, differentiation, repair, remodeling, cell
migration, and peptide-mediated inflammation (17). Proteases can also be released in the airspaces by activated
macrophages and neutrophils in case of inflammation in
the respiratory tract (18).
Blood supply to the lungs is divided among the pulmonary and systemic circulations (19). The pulmonary circulation consists of the pulmonary artery that leaves the right
heart, branches into a dense pulmonary capillary bed that
surrounds the alveoli, and finally coalesces into the pulmonary vein that drains into the left heart. One hundred percent of the cardiac output flows through the pulmonary
circulation. Its principal functions are gas exchange with
air in the alveoli and nutrients supply to terminal respiratory units. The lungs receive a second blood supply through
the systemic circulation, commonly referred to as the bronchial circulation. The bronchial circulation originates from
the aorta and provides oxygenated blood and nutrients to
all structures of the tracheobronchial tree. Lymphatic vessels exist in close proximity of major blood vessels and
airways (20).
DEVELOPMENT STATUS OF INHALED
PEPTIDES AND PROTEINS
Although the efforts performed in the field of pulmonary
delivery of peptides and proteins have been tremendous,
there are still a very limited number of inhaled macromolecules available on the market (Table 1). Yet, there are a
growing number of inhaled peptides and proteins undergoing various phases of clinical trials, those developed for
local therapy being the most promising.
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Table 1 Status of inhaled peptides and proteins in development
Peptide/Protein
Trade name
Indication
Lead company
Status
Natural surfactant
containing SP-B and
SP-C (bovine)
Survanta®
Neonatal RDS
Abbott Laboratories
On the market since 1991
Natural surfactant
containing SP-B and
SP-C (porcine)
Curosurf®
Neonatal RDS
Chiesi Farmaceutici S.P.A.
On the market since 1992
Recombinant human
deoxyribonuclease I
Pulmozyme®
Cystic fibrosis
Roche
On the market since 1994
Natural surfactant
containing SP-B and
SP-C (bovine)
Infasurf®
Neonatal RDS
ONY Inc.
On the market since 1999
Insulin
AFREZZA®
Type I and II diabetes mellitus
MannKind corporation
Under review by the FDA
Glucagon-like peptide
_
Type II diabetes
MannKind corporation
In initial clinical trials
Glycan-binding decoy
protein
_
COPD, chronic lung
inflammations
ProtAffin
Phase I clinical trials
in early 2012
Nanobody: antibodyderived therapeutic
protein
_
Respiratory tract infections
caused by human
respiratory syncytial virus
Ablynx
Phase I clinical trials
Abbreviations: RDS, Respiratory distress syndrome; COPD, chronic obstructive pulmonary disease.
Three protein-based products used for the treatment of
neonatal respiratory distress syndrome are available on the
market since the 90’s. These intratracheally administered
drugs are basically composed of natural surfactant containing polar lipids and surfactant proteins, mainly SP-B. Infasurf® and Survanta® come from bovine source, whereas
Curosurf® comes from porcine source.
Recombinant human deoxyribonuclease I (Pulmozyme®) is used for the treatment of cystic fibrosis and is
available on the market since 1994. Cystic fibrosis is a
genetic disease where thick secretions are retained in the
airways, leading to reduced pulmonary function and exacerbation of infection. Mucus thickness is partly due to the
release of high quantities of DNA by degenerating leukocytes. Recombinant human deoxyribonuclease I cleaves
DNA in airway secretions and reduces their viscoelasticity.
The glycoprotein is administered by inhalation of an aerosol mist produced by a pneumatic nebulizer (21).
The first inhaled insulin product for the treatment of
patients with type 1 and type 2 diabetes mellitus was
approved under the name Exubera® in January 2006. However, less than 2 years later, the drug was withdrawn from
the market due to disappointing sales. Prescriptions
amounted to less than 1% of the insulin market because the
dry powder inhaler failed to gain acceptance of patients
and physicians. Exubera® did not present improved efficacy
as compared to short-acting subcutaneous insulins (Fig. 2).
The time to maximum serum insulin concentrations was
similar following inhalation using Exubera® and following
injection of rapid-acting insulin analogs (22). Insulin bioavailability using Exubera® was approximately 10% relative to subcutaneous regular human insulin. Exubera®
marginally decreased patients’ breathing ability, and regulators
required patients to take lung function tests before and
during treatment, which increased cost and inconvenience (23).
The large size of the Exubera® inhaler was also an issue.
Another inhaled insulin product, AFREZZA™, is currently
under review by the Food and Drug Administration (FDA)
for use in patients with diabetes (24). AFREZZA™ is an
ultra-rapid acting insulin comprising Technosphere® insulin powder in unit-dose cartridges for administration with
the inhaler. The Technosphere® powder formulation is prepared by precipitating insulin from solution onto preformed
diketopiperazine particles, which readily dissolve once in
the lung environment. AFREZZA™ appears to overcome
several limitations of Exubera®. Technosphere® insulin is
both rapidly absorbed and eliminated, and its pharmacokinetic profile mimics more closely normal physiologic insulin release than injection of regular human insulin and of
rapid-acting analogs. Insulin bioavailability using
AFREZZA™ reaches 30% relative to subcutaneous regular human insulin. The inhaler is small and discrete.
AFREZZA™ has demonstrated a favorable safety and tolerability profile in clinical studies. However, a small reduction in pulmonary function also appeared in patients who
received Technosphere® insulin (1,25).
The development of inhaled insulin began in 1990 and
led to the investigation of the pulmonary administration
of many other systemically acting therapeutic peptides
and proteins. Preclinical studies have been numerous but
only a few small-scale clinical trials have been conducted. These included studies on LHRH analogs (27),
salmon calcitonin (28), human growth hormone (hGH)
(29), and an erythropoietin-Fc fusion protein (30). Yet,
following withdrawal of Exubera® from the market, these
clinical trials have not been pursued. Only one pharmaceutical company (MannKind corporation) currently
pursues the development of inhaled peptides for a
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Inhaled insulin
SC regular insulin
SC insulin lispro
10
GIR (mg.kg–1.min–1)
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8
6
4
2
0
0
100
200
300
Time (min)
400
500
600
Figure 2 Pharmacodynamic response (glucose infusion rates) over 10 hr after insulin inhalation (Exubera®) compared to injected regular insulin and injected lispro. Source: From Ref. 26.
systemic action, these include insulin (AFFREZA™)
and glucagon-like peptide 1 (GLP-1). Pulmonary administration of GLP-1 adsorbed on Technosphere microparticles is undergoing initial clinical investigation (31).
The pulsatile administration of GLP-1 through the lungs
is beneficial as the gastrointestinal intolerance observed
after subcutaneous injection is avoided. This drug may
be used alone or in combination with prandial insulin in
patients with type 2 diabetes.
While FDA approval of new molecular entities has
steadily decreased over the last 15 years (from 53 approvals in 1996 to 15 in 2010), FDA approval of biomolecules
has remained constant with an average of four to five
approvals per year (32). Therefore, the proportion of biomolecules delivered by inhalation is also expected to grow.
A decoy form of IL-8 can potentially treat chronic obstructive pulmonary disease (COPD) following pulmonary
administration (33). COPD is characterized by a neutrophilic
inflammation of the airways where IL-8, a major chemokine, plays a central role. This decoy protein is an engineered version of human IL-8, with higher affinity for
glycosaminoglycans present on endothelium cell surfaces
and in which the neutrophil-binding domain has been
removed. Therefore, it acts as an anti-IL-8 product. This
protein is currently in phase I study which started in early
2012. A single-variable domain of a camelid immunoglobulin, called a nanobody®, has been used for the binding of
the respiratory syncytial virus fusion protein and thereby
for virus neutralization (34). This candidate drug entered
Phase I clinical trials in December 2011. Nanobodies®
appear suitable for inhalation as they are very stable with a
low propensity to aggregate. It is also noteworthy that they
can be manufactured at relatively low cost in microbial
systems.
FATE OF PEPTIDES AND PROTEINS
IN THE LUNGS
The fate of drugs following inhalation depends on their site
of deposition within the lungs. Aerosol particles deposited
in the tracheobronchial tree come into contact with the
mucus, and the peptide or protein transported within the
particle dissolves within the mucus. The macromolecule
can then be cleared by the mucociliary escalator into the
gastrointestinal tract or can diffuse in the mucus and cross
the airway epithelium. On the other hand, particles deposited in the alveolar region initially come into contact with
the thin layer of lining fluid coating the alveolar epithelium. The dissolved peptide or protein can then be subjected to clearance by alveolar macrophages or can be
transported across the alveolar epithelium into the bloodstream. The lungs exhibit a decreased proteolytic activity
compared to the gut, enzymes are present though. Peptides
and proteins may be enzymatically degraded either extracellularly (by membrane-associated proteases and peptidases) and/or intracellularly (within macrophages and
epithelial cells) (35).
Deposition in the Respiratory Tract
Deposition is the process that determines the fraction of the
inhaled particles that will be caught in the respiratory tract
and will not be exhaled. The aerodynamic diameter, daer, of
an inhaled particle has a major impact on its site of deposition within the lungs. The daer can be conceptualized as the
diameter of a spherical particle with a density of 1 g/cm3
(ρ0), such as a water droplet, which has the same velocity
as the particle of interest in still air. It is defined by the
equation:
d aer = d
r
r0 c
(1)
where d is the geometric diameter of the particle, ρ is the
particle density, and χ is the particle dynamic shape factor
denoting deviation of shape from sphericity (36).
Pulmonary deposition of particles occurs mainly by
three mechanisms: inertial impaction, gravitational settling,
and Brownian diffusion. It depends on aerodynamic particle size, on inhalation flow, and on lung anatomy. Large
particles (daer > 5 µm) deposit in upper airways (mouth,
trachea, and main bronchi) by inertial impaction where
the airstream velocity is maximum. Inertia refers to the
inability of inhaled particles to follow the changes in
direction and speed of the inspired airflow within the
respiratory tract. Therefore, particles retain their original
direction, “crashing” on the airway wall. Smaller particles (daer = 1 to 5 µm) usually pass through the larger airways and reach the deep lung (lower airways and
respiratory bronchioles), where they deposit by gravitational settling. In this region, the airstream velocity markedly decreases due to the dramatic increase in total airway
cross-sectional area. Therefore, the particles “fall” on the
airway wall because of gravity. Very small particles
(daer < 1 µm) remain suspended in the air and up to 80%
of the inhaled bolus can be exhaled due to low inertia and
low sedimentation.
The effectiveness of inertial impaction and sedimentation varies with the breathing pattern and with the anatomy
of the respiratory tract. Slow inhalation is generally preferred to minimize inertial impaction in upper airways and
to increase penetration into the lungs. A breath hold gives
time to particles that have penetrated deep into the lungs to
sediment on airway surfaces. Variations in airways anatomy between individuals, that is, airway dimensions and
branching angles, lead to variations in aerosol deposition
between subjects. In patients with asthma, COPD and cystic fibrosis, there is a systematic variability in airway anatomy, because the pulmonary airways may be narrowed by
a combination of bronchospasm, inflammation, and mucus
hypersecretion. Airway narrowing increases the likelihood
of deposition by impaction, as well as creates turbulent airflow in regions of the lungs where airflow would otherwise
be laminar. Aerosol deposition in central airways may
therefore occur more readily in patients than in healthy
subjects, and peripheral airway deposition may consequently be lower (37).
Drug delivery inhalers can be divided into three different
categories: nebulizers, metered-dose inhalers, and dry
powder inhalers. Therapeutic peptides and proteins have
been delivered to the lungs using nebulizers and dry powder inhalers. The drug must reach the target receptors in an
adequate amount to effectively treat the disease in focus.
Medical inhalers generate particles with daer in the micronsize range for both local and systemic treatment. Particles
with a daer between 3 µm and 10 µm are used for deposition
in the tracheobronchial tree to treat the airways; whereas
particles with a daer between 1 and 3 µm are used for deposition in the alveolar region for systemic drug absorption.
Conventional inhalers typically deliver 10% of their
nominal doses to the lungs and lung deposition generally
increases with peak inspiratory flow rate (38). New technology inhalers have largely improved these features. For
instance, the AIR dry powder pulmonary system reaches a
lung deposition of the nominal doses of 50%, and lung
deposition does not depend on peak inspiratory flow rate
(38). An AERx prototype high technology nebulizer was
capable of delivering 80% of the nominal dose to the lungs
(39). The MedTone dry powder inhaler, used to deliver
Technosphere insulin, reaches a lung deposition of 40% of
the initial cartridge load (40).
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Interaction with the Air–Liquid Interface
Following pulmonary deposition, macromolecules interact
with the air–liquid interface. The large surface area of the
lungs is favorable to adsorption of proteins at the air–liquid
interface, to protein unfolding and aggregation (41–43).
Proteins can also bind endogenous components in the lung
lining fluid and form agglomerates. Protein aggregates are
likely to be scavenged by alveolar macrophages and be
degraded. For instance, hGH has been shown to aggregate
in the lungs following intratracheal instillation in adult rats
(42). After deposition in the alveoli, hGH was concentrated
in a thin layer at the air-epithelial boundary, little hGH penetrated respiratory epithelia, and the protein was largely
taken up by alveolar macrophages (42,43). Aggregates of
hGH were visible in a gel filtration chromatogram carried
out on lung homogenates (42).
Soluble proteins may minimally perturb surface-active
lipid films, with minor reorganization at low concentrations of the protein (44). Interactions of serum and serum
proteins with pulmonary surfactant have been largely
investigated in vitro because serum leakage into the alveolar space has been assumed to be the primary cause of surfactant dysfunction in acute respiratory distress syndrome.
Air interface adsorbed films of bovine lipid extract surfactant could not attain equilibrium surface tension value
in vitro, in a tensiometer, when bovine serum albumin was
added to the surfactant. Albumin itself being surface active
adsorbed at the air–liquid interface and inhibited the surface adsorption of the lipid extract surfactant. Yet, these
effects were observed at high albumin to surfactant relative
concentrations (1:1 w/w), concentrations which are well
above the concentrations of therapeutic proteins attained
locally following pulmonary delivery.
Mucociliary Clearance
Mucociliary clearance clears proteins that stick to mucin
fibers or freely diffuse in the mucus but are unable to cross
the airway epithelium. Mucin forms a network with a mesh
spacing between 20 nm and 800 nm, which is much larger
than the hydrodynamic diameter of most globular proteins
(2–15 nm) (45). Yet, some proteins can make low-affinity
bonds with the mucin and their diffusion can be hindered in
mucus. Adhesion can involve electrostatic interactions
with the carboxyl or sulfate groups on the mucin, lowaffinity bonds between hydrophobic domains and hydrogen bonds.
Olmsted et al. studied the diffusion of macromolecules
in human cervical mucus. Nearly every soluble globular
protein investigated diffused in mucus as fast as it diffused
in PBS (Dmucus/DPBS = 1) (45–47). However, polyvalent
antibodies were retarded in mucus due to low-affinity
bonds of the Fc domains with mucin fibers (45). This was
concluded after a comparative study of the diffusion rate in
mucus of full-length IgM and IgM after removal of Fab
regions, that is, a pentameric ring of Fcs joined by the IgM
j-chain. Both proteins were identically slowed in mucus.
The binding between antibodies and mucin must have very
low affinity because the diffusion of IgG, with only one Fc
region, was not slowed significantly in mucus but antibodies
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Nonwalk
HPV
5000
1
0.1
slgAagg
0.01
HSV
Serum hCG (mIU/ml)
4000
lgM Fc5µ lgM
Dmuc / Dpbs
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2612
3000
2000
1000
0.001
1
10
Radius (nm)
100
Figure 3 Normalized diffusion coefficients of proteins and
viruses in mucus. The proteins tested were lysozyme, myoglobin, pepsin, lactoferrin, IgG, IgA, IgM, IgM Fc, and ferritin.
The viruses tested were human papilloma virus (HPV) and herpes
simplex virus (HSV). Source: From Ref. 45.
with multiple Fcs as IgM and small aggregates of IgA were
significantly slowed (Fig. 3). This suggests that antibodies
accumulating on the surface of a pathogen may be able to
form a sufficient number of low-affinity bonds to trap the
pathogen in the mucus.
Lay et al. have monitored the retention and clearance of
radiolabeled human serum albumin and radiolabeled sulfur
colloid (220 nm insoluble particles) following localized
deposition in a bronchus in dogs (48). Both compounds
were cleared by mucociliary clearance but albumin was
cleared more slowly than sulfur colloid. This indicates that
a low-permeating water-soluble material as albumin
remains in contact with the airway epithelium to a greater
extent than does a solid insoluble particle. Albumin likely
diffused to a greater extent than sulfur colloid into the periciliary sol layer, which is transported less efficiently than
the mucus gel layer during mucociliary clearance.
Alveolar Macrophages
Alveolar macrophages are a primary barrier to the transport
of large proteins from the airway lumen into the bloodstream (41). Lombry et al. showed that depletion of alveolar
macrophages by liposome-encapsulated dichloromethylene
diphosphonate (Cl2MDP) caused severalfold enhancement
in systemic absorption of immunoglobulin G (150 kDa) and
human chorionic gonadotropin (39.5 kDa) following intratracheal instillation in rats (Fig. 4). Large proteins are
slowly transported across the alveolo-capillary barrier and
can remain within the airspaces for several hours. This
gives time to alveolar macrophages to engulf them by pinocytosis or “cell drinking,” the uptake of fluids and soluble
compounds. In contrast to large proteins, no increase in pulmonary absorption of the peptide insulin (5.8 kDa) and of
the small protein hGH (22 kDa) was associated with the
depletion of alveolar macrophages (49). Insulin and growth
hormone remained in the airspaces for less than 1 hr in rats,
indicating that these compounds crossed the alveolar epithelium
0
0
6
12
18
24
30
Time (h)
36
42
48
Figure 4 Pharmacokinetics of pulmonary human chorionic
gonadotropin in alveolar macrophages-depleted and control rats.
Rats received a human chorionic gonadotropin (hCG) dose of
100 µg by intratracheal instillation 1 day after Cl2MDP (○, n = 4)
or PBS (□, n = 6) liposome administration or no treatment (▲,
n = 6). The bioavailability of hCG increased from 4.4 ± 1.1%
(untreated rats) to 17.6 ± 3.7% (PBS liposomes) to 59.7 ± 9.2%
(Cl2MDP liposomes) compared with an intravenous dose of 10 µg
(Δ, n = 4). Values are averages ± SE. Source: From Ref. 41.
quickly, presumably preventing major uptake and degradation by alveolar macrophages.
It should be noted that a large molecular weight does not
systematically involve a long residence time within the airspaces and consequently an uptake by alveolar macrophages. Other mechanisms can affect the rate of transport
of proteins through the epithelium relative to the rate of
alveolar macrophages uptake. For instance, there is evidence that for certain endogenous molecules that normally
occur in lung lining fluids, for example, albumin, immunoglobulins, and transferrin, there are specific receptor-mediated transport mechanisms on the alveolar epithelial cell
that enable these proteins to be absorbed at higher rates
than expected (50). In addition, the rate of endocytosis by
alveolar macrophages can be affected by the physicochemical properties of the proteins, with hydrophobicity and a
global cationic charge increasing adsorptive endocytosis of
proteins (49).
Peptidase and Protease Activity
Although the lungs are a far less hostile metabolic environment than the gastrointestinal tract, proteolytic enzymes
are still present (51). Enzymatic degradation of inhaled
proteins and peptides may occur prior or along their transport across the lung epithelium. Baginski et al. analyzed
the mRNA expression of proteolytic enzymes in cell lines
and primary cells of the human respiratory epithelium (52).
They focused their investigation on secreted and membrane-bound peptidases. Many enzymes were shown to be
expressed in human respiratory epithelial cells, but at different
Encyclopedia of Pharmaceutical Science and Technology, Fourth Edition, Volume IV
Table 2 Bioavailability and Tmax of peptides and proteins following pulmonary delivery to humans using high technology inhalers
Peptide/Protein
MW (Da)
Bioavailabilitya (%)
Tmax (hr)
Reference
LHRH analogs
1,200
18
1.6
(63)
Salmon calcitonin
3,400
11–18b
0.3–0.7
(28)
GLP-1
3,200
NAc
0.1
(31)
PTH(1–34)
4,120
48
0.2
(64)
Insulin
6,000
10
0.7–1.6
(65) (Exubera®)
6,000
30
0.2
(1,24) (AFREZZA™)
3.5–7.6
1–4
(29)
20
(30)
Insulin
Human growth hormone
Erythropoietin-Fc fusion protein
22,000
112,000
c
NA
a
Relative to subcutaneous injection and dose loaded in the inhaler.
Relative to subcutaneous injection and dose deposited in the lungs.
c
Data not available.
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b
levels according to the cell type. All respiratory cells
expressed a smaller number of peptidases than Caco-2
cells, an intestinal epithelial cell line.
Small peptides are prone to degradation by peptidases
located at the apical surface of the airway and alveolar
epithelium (53). Somatostatin and glucagon are very
poorly transported to the bloodstream following pulmonary administration due to severe local peptidase degradation (54). Protection of the amino acid terminus of
peptides may inhibit peptidase attack though, leading to
increased bioavailabilities (54). Pang et al. (55) showed
that lung ectopeptidases were responsible for the metabolism of inhaled insulin and not insulin-degrading enzyme.
Bacitracin, an ectopeptidase inhibitor, decreased the nonabsorptive loss of insulin in the isolated perfused rat lung
while inhibitors of insulin-degrading enzyme did not.
Baginski et al. (56) studied the impact of epithelial proteases on the pulmonary fate of salmon calcitonin using
monolayers of human respiratory epithelial cells. The
peptide remained unaltered over 2 hr when incubated in
cell supernatant or in cell monolayers. When incubated in
cell homogenates, salmon calcitonin was degraded to
varying extents. When incubated with neutrophil elastase,
trypsin, or chymotrypsin, salmon calcitonin was rapidly
degraded. These proteases could be involved in the enzymatic breakdown of calcitonin following pulmonary
delivery as Western blot analysis showed their expression
in cell lines.
Large proteins are not as good substrates as small peptides for proteolytic enzymes. Their large size and globular
structure may prevent enzymes to fit them into their catalytic sites (57). Nonspecific endocytosis transport of proteins across lung epithelia may lead to their partial
degradation in the lysosomal compartment. One representative example is given by horseradish peroxidase, a nonspecific fluid-phase endocytosis marker (58). About half of the
FITC-labeled species present in either the apical or basolateral receiver fluid of rat alveolar epithelial cell monolayers
was intact horseradish peroxidase, suggesting that part of
the internalized protein underwent cellular metabolism.
Conjugation of polyethylene glycol (PEG) to proteins
and peptides may increase proteolytic stability after pulmonary administration by shielding them from proteolytic
enzymes (59). For instance, the site-specific substitution of
salmon calcitonin with a PEG of 5 kDa led to a thousandfold increase in proteolytic resistance in rat lung homogenate (60). Lee et al. (61) have demonstrated the beneficial
effect of PEGylation on GLP-1. The PEG conjugates were
found to have 10- to 20-fold more resistance to rat lung
enzymes, as compared to the unmodified version.
Transport Across the Airway
and Alveolar Epithelium
Transport of macromolecules occurs across both the airway
pseudostratified columnar epithelium as well as across the
thin alveolar epithelium. Yet, macromolecules are absorbed
into the bloodstream in larger amounts when they are delivered to the deep lung than when they are delivered to central
aiways (39,62). This likely originates from higher absorption rate in alveoli because of the large surface area of the
alveolar epithelium as well as of the short diffusion path
between the alveolar epithelium and the capillary endothelium. Fast absorption from the alveoli reduces the time of
exposure to degradation processes occurring in the airspace
and respiratory tissue, thereby increasing the drug fraction
absorbed systemically. Bioavailabilities of proteins following pulmonary delivery range from 48% to 3.5% (Table 2),
indicating that their transport across the alveolar epithelium
toward the systemic circulation, in many cases, does not
represent the most significant pathway in their fate.
Transport of peptides and proteins across respiratory
epithelia may take place through paracellular or transcellular routes, and the main mechanism of transepithelial
transport depends on the macromolecule molecular weight.
Protein-specific transcytosis and peptide-specific proteolysis can enhance and reduce transport, respectively. Up to a
molecular weight of approximately 40 kDa, peptides and
proteins with no specific receptor on epithelial cells are
transported by paracellular diffusion. Above this molecular
size, nonspecific pinocytosis occurs. For instance, growth
hormone (22 kDa) diffuses between alveolar epithelial
cells, whereas horseradish peroxidase (40 kDa) traverses
across the epithelium by nonspecific pinocytosis (66,67).
Matsukawa et al. determined the permeability coefficient across the alveolar epithelium for dextrans of different
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molecular weights (68). Dextran transport rates decreased
gradually up to 40 kDa and then plateaued at
70 kDa and 150 kDa. Lowering experimental temperature
from 37°C to 4°C led to 50% decrease in transport rate for
dextrans up to 40 kDa, consistent with paracellular diffusion. In contrast, transport rates of 70 kDa and 150 kDa
dextrans decreased by 90% when lowering experimental
temperature, indicating a pinocytic transport pathway.
Equivalent pore analysis based on permeability coefficients of hydrophilic solutes yielded a pore radius of 6 nm
for diffusional paracellular pathways, suggesting that proteins with a radius >6 nm (~50 kDa) are excluded from
paracellular transport (67).
Bur et al. assessed the transport rates of a series of serum
and therapeutic proteins across primary human alveolar
epithelial cell monolayers in vitro (66). Several proteins,
including GLP-1, albumin, transferrin, and immunoglobulin G, were actively transported across the monolayer with
higher transport in the apical to basolateral direction than
in the reverse. Parathyroid hormone, insulin, and growth
hormone did not show transport directionality. Although
receptor-mediated transcytosis of insulin was demonstrated
(69), the active process is totally saturated at therapeutic
insulin concentrations and paracellular diffusion is the relevant mechanism for insulin transport (55,66). The transalveolar transport of immunoglobulin G has also been
analyzed in alveolar epithelial cell monolayers and shown
to involve FcRn-mediated transcytosis (70). The expression of FcRn was localized in nonhuman primate lung
using immunohistochemistry and shown to be higher in
epithelial cells in airways than in alveoli (71). Several therapeutic proteins were fused to the Fc-domain of an IgG1,
and Fc-fusion proteins were well absorbed into the bloodstream following delivery to upper and central airways in
monkeys, through Fc-Rn-mediated transport (72).
In contrast to the oral, nasal, and transdermal routes of
administration, the bioavailability of a drug delivered to the
lung does not systematically decrease with an increase in
molecular weight (Table 2). However, similarly to other
noninvasive routes of drug administration, the larger the
molecular size, the slower the absorption rate and the later
the time to peak plasma concentration (Tmax; Table 2). The
rate of diffusion between epithelial cells decreases with
increasing molecular size (68). In contrast, the total amount
of a drug absorbed from the lung depends on its biological
stability during its residence within the pulmonary tissue.
Large proteins cross the alveolar epithelium slowly and can
remain within the alveolar space for several hours. If they
undergo limited degradation within the alveoli during this
time, their systemic absorption can be high. However, the
comparison of, for instance, LHRH analogs and insulin indicates that the correlation between molecular weight and Tmax
is not perfect and that other parameters are involved in the
pharmacokinetic profile in vivo as the elimination half-life.
IN VITRO AND IN VIVO MODELS FOR
DETERMINATION OF PULMONARY
ABSORPTION
Several models are available for the assessment of pulmonary absorption of peptides and proteins (6). These include
in vitro cell cultures models, the ex vivo-isolated perfused
lung model, and in vivo animal models.
In vitro cell culture models are interesting because they
provide information on peptide and protein transport rates
and mechanisms across respiratory epithelia and because
they bring up few ethical questions. Both continuous and
primary cell cultures can be used. Primary cells present
cells characteristics and state of differentiation more similar to the in vivo situation than cell lines. In both cellular
models, it is important that epithelial cells form a tight
monolayer to represent the natural epithelial barrier. The
Calu-3 cell line derives from bronchial epithelial cells of a
human adenocarcinoma and is the most commonly used
respiratory cell line. It can be used in both liquid-covered
and air-interface conditions. Air-interface cultures are
more representative of the in vivo situation where drug
deposition and dissolution occur in a small volume of cell
lining fluid. Calu-3 cells grown in an air-interface also
shows greater similarity to airway’s epithelial morphology
than liquid-covered culture (73). Most primary cell cultures consist of alveolar epithelial cells. Type II pneumocytes are isolated from normal lung tissue of humans, rats,
or pigs and undergo differentiation into type I-like cells in
culture. After 1 week in culture, the cells form a tight
monolayer consisting mainly of type I-like cells and some
interspersed type II cells (74).
In the ex vivo-isolated perfused lung model, the lung is
isolated from rats, guinea pigs, or rabbits, and is suspended,
together with the heart, in a humidified jacketed chamber
maintained at 37°C (75). The lung is then perfused through
the pulmonary artery and the perfusion solution collected
from the pulmonary vein. Drugs can be delivered by the
intratracheal route or by injection in the perfusate solution
to simulate a systemic administration. As compared to
in vitro cell culture models, the isolated perfused lung is a
more complete model as structural integrity and interactions between the different cells in the lung are maintained
and the impact of particle size and site of deposition within
the lung can be assessed. As compared to in vivo, the isolated perfused lung allows studies on drug absorption from
the lung without the influence of the other organs. However, the model does not include absorption from the airways as the tracheobronchial circulation is severed during
surgery, and it demands significant surgical skills.
The most complete assessment of pulmonary absorption
is provided in vivo using animal models (6). Small rodents
are common models for initial studies on pulmonary drug
delivery because they can be used in large numbers. Mice
have been widely used for assessing pulmonary delivery of
locally acting drugs. Pharmacokinetic studies following
pulmonary delivery of systemically acting drugs have
often been performed in rats, as blood samples at all sampling times can be collected in one rat. Guinea pigs have
been widely used as an animal model of allergic asthma
and infectious diseases because the airway anatomy and
the response to inflammatory stimuli are comparable to the
human case. Confirmatory testing can be conducted in the
rabbit, the dog, the sheep, or the monkey. The dog is a good
model for assessing systemic drug delivery by the pulmonary route as well as toxicity. Monkeys have very similar
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anatomy and physiology as humans, but their use is
restricted to advanced research. Drugs can be delivered to
the animal lung by passive inhalation of an aerosol or
directly in the trachea as a liquid or powder aerosol or by
instillation of a liquid bolus.
SAFETY ASPECTS
There are some limitations and safety concerns to be taken
into consideration when designing and delivering peptide
and protein drugs to the lungs. These include local side
effects, immunogenicity, and the need of a safe drug carrier.
Pulmonary administration of peptides and proteins is
generally well tolerated in the short term (29,76,77). However, few cases allow the determination of its safety in the
long term. Side effects attributed to inhaled recombinant
human deoxyribonuclease I in clinical trials and post-marketing are rare (1 < 1,000) and, in most cases, side effects
are mild and transient. Several Phase III trials have investigated the safety of inhaled insulin (25,78,79). All studies
indicated that inhaled insulin was well tolerated, and the
most common respiratory event reported was a mild transient cough occurring within minutes of inhalation. There
was no difference in hypoglycemic events between subcutaneous and inhaled insulin. Lung function declined over
the years following both injection and inhalation, consistent with aging. Yet, inhalation of insulin induced a small
decrement in forced expiratory volume in 1 sec and carbon
monoxide diffusing capacity but this decrement was nonprogressive and reversible (79).
Following delivery of proteins, the immune defense system may recognize the native or the denaturated protein as
an antigen and trigger an immune response. The antibodies
generated against the delivered protein can bind and neutralize it and cause the loss of protein bioactivity (80).
Increased insulin antibody levels have been noted following
inhalation of insulin as compared to its subcutaneous
administration (79). These increased antibody levels might
be related to the higher insulin doses given to the lungs (due
to the reduced bioavailability) as compared to injection.
Yet, these insulin-specific antibody levels were not correlated with any clinical signs. In any case, care must be given
to only deliver the native protein to the lungs to reduce
immunogenicity and the risk of decreased biological activity over treatment time.
Following pulmonary delivery, rapid drug absorption
occurs, which may be a limitation for local treatment and
may lead to multiple daily dosing. Various efforts have
been made for sustaining the release of peptide and protein
drugs within the lungs using carrier-based or polymer-conjugation strategies (51). Special attention should be given
to the selection of the carrier or polymer for the sustained
release: the carrier or polymer needs to be biocompatible,
in this regard, chitosan is not adequate for pulmonary
delivery as it opens tight junctions (81); large molecular
mass polymers should be avoided as accumulation in the
lung may occur; and high drug loading in the carrier should
be achievable as masses delivered to the lungs are limited
(82). Engineered or modified peptides and proteins should
be considered as new chemical entities with novel biochemical
2615
properties. They may exhibit different potential risks from
their unmodified version (83).
IMPACT OF SMOKING AND PULMONARY
DISEASE CONDITIONS
Smoking increases insulin absorption from the lungs
(84,85). Smokers also appear less sensitive to insulin
glucodynamic effects than nonsmokers following both
subcutaneous injection and inhalation (84). Smoking abstinence attenuates the enhancement in pulmonary insulin
absorption due to smoking, but rechallenge with a single
cigarette restores it. Therefore, it is not recommended that
smokers and those at risk of recidivism use inhaled insulin.
Smoking is believed to affect major alveolar clearance
mechanisms in the lungs, such as absorption, alveolar macrophages, and metabolism of peptides and proteins
(86–88). The mechanisms involved in the increased permeability of the lung epithelial barrier are believed to be
related to changes in the integrity of tight junctions and
cytoskeletal proteins (89,90). Petecchia et al. (89) have
studied the effect of exposure to cigarette smoke on tight
junction’s integrity using two human bronchial epithelial
cells, BEA-2B and 16HBE14o-. The exposure of the two
cell lines to cigarette smoke resulted in concentration- and
time-dependent tight junction’s disassembly and DNA
fragmentation. Olivera et al. (90) have investigated the
effects of cigarette smoke on Calu-3 airway epithelial cells.
Cigarette smoke exposure led to increased polymerized
actin, redistribution of the tight junction proteins from the
normal apical circumferential band to a more basal location as well as to decreased association between two tight
junction proteins, thereby increasing permeability to small
solutes and macromolecules. Yet, the increased permeability induced by cigarette smoke appears reversible and the
lung epithelium is able to recover within a few days (86).
Chronic lung diseases have been shown to affect pulmonary absorption of proteins as well. Asthma is associated
with a 30% to 40% lower absorption of inhaled insulin and
with lesser glucose-lowering effects (91). However, prior
administration of a bronchodilator can reverse airway
obstruction and restore pulmonary insulin absorption (91).
Pulmonary insulin absorption was also reduced by 35% in
subjects with chronic bronchitis and by 20% in subjects
with emphysema, relative to healthy subjects (92).
STABILITY ISSUES
A key issue in pulmonary delivery of peptides and proteins
is the preservation of the structural and biological integrity
of the therapeutic during formulation, storage, and aerosolization. Many proteins are structurally unstable and susceptible to physical and chemical degradation following
exposure to various stresses as elevated temperature,
extreme pH, shear strain, and surface adsorption. The dried
state provides a more stable environment to the protein
than the solution as shear-induced denaturation and hydrolysis and deamidation reactions are reduced (93).
Spray-drying is a fairly common process for preparing
inhalation dry powders of proteins (28,38). However, the
generation of small droplets during drying provides a vast
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increase in air–liquid interface, which may cause unfolding
of the protein followed by aggregation. Therefore, addition
of excipients to the formulation is needed to prevent physical degradation. Maa et al. have optimized the spray-drying
conditions of recombinant hGH and have shown that adding the surfactant polysorbate 20 and divalent zinc ions to
the liquid feed reduced the formation of hGH aggregates
during spray-drying (94). Other technologies as supercritical fluid processes are also developed for the preparation of
fine powders. Similarly to spray-drying, appropriate stabilizing excipients are required to retain protein stability during processing (95).
Nebulization of protein solutions in aerosols involves
the formation of a large air–liquid interface and requires
appropriate additives to stabilize the protein as well. Niven
et al. studied the stability of lactate dehydrogenase and
recombinant granulocyte colony-stimulating factor to airjet nebulization (96). Pneumatic nebulization of lactate
dehydrogenase resulted in a time-dependent loss of enzymatic activity, and the extent of inactivation was dependent upon applied air pressure and upon the volume of
fluid in the nebulizer reservoir. Nebulization of recombinant granulocyte colony-stimulating factor resulted in
aggregation and chemical degradation, by-products
accounting for 40% of the protein after 10 min. Polyethylene glycol 1000 added at 1% w/v markedly reduced the
deleterious effects of nebulization on both proteins. Thermal denaturation can be an additional degradation mechanism during ultrasonic nebulization because the protein
solution warms during operation. Steckel et al. showed
that aviscumine, a 57 kDa protein, lost 50% activity after
20 min of nebulization. Ultrasonic nebulization was more
deleterious to the protein than air-jet nebulization (97).
Yet, about 70% of the aviscumine activity could be
retained by the addition of a surfactant and buffer salts. In
contrast to conventional nebulizers that involve multiple
recirculation of the solutions, single-pass systems that
form aerosol by extrusion of the solution through a fine
nozzle do not appear to cause protein denaturation (77).
lungs have been tested in animal models, more work should
be done to bring the most promising strategies to clinical
development.
Understanding the fate of peptides and proteins in the
lungs is important because fate and therapeutic action are
closely linked. Future investigations should confirm the
formation of protein aggregates in the lung lining fluid.
The metabolism of therapeutic proteins in the lung tissue
has been little studied and would deserve further investigation. Finally, studying the fate of drug carriers following
delivery to the lungs could also give useful information for
their optimal design.
ARTICLES OF FURTHER INTEREST
Drug Delivery: Pulmonary Delivery, p. 1164
Dry Powder Aerosols: Emerging Technologies, p. 1295
Inhalation: Dry Powder, p. 1954
Inhalation: Liquids, p. 1967
Metered Dose Inhalers, p. 2107
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
CONCLUSIONS
Pulmonary delivery offers great potential for local as well
as systemic delivery of peptides and proteins. Applications
of local therapies are expected to expand because inhalation of drugs readily allows drug targeting to the diseased
airways. Inhalation may also be an optimal route for the
systemic administration of peptides and proteins particularly in the case of short-course diseases. This route of
macromolecules administration has not been fully exploited
yet, but the extensive development of high technology
inhalers driven by inhaled insulin has paved the way
toward novel applications.
The rapid pulmonary absorption of peptides and proteins
may be a limitation for local therapies as it may imply multiple
daily dosing. Attaining sustained drug release in the lungs
is challenging because the lungs present efficient clearance
mechanisms to maintain lung homeostasis and to protect
the lungs from foreign substances. Although various
approaches to obtain sustained release of proteins in the
10.
11.
12.
13.
14.
15.
16.
Pfützner A, Mann AE, Steiner SS. Technosphere™/insulin-a
new approach for effective delivery of human insulin via the
pulmonary route. Diabetes Technol Ther 2002; 4: 589–94.
Illum L. Nasal drug delivery: new developments and strategies.
Drug Discov Today 2002; 7: 1184–9.
MAP Pharmaceuticals, Inc. LEVADEX® (dihydroergotamine
mesylate, USP) inhalation aerosol [Internet]. [Available from:
http://www.mappharma.com/product-portfolio/levadex] [Cited
2012 Feb 14].
Mercer RR, Russell ML, Roggli VL, Crapo J. Cell number and
distribution in human and rat airways. Am J Respir Cell Mol
Biol 1994; 10: 613.
Patton JS. Mechanisms of macromolecule absorption by the
lungs. Adv Drug Deliv Rev 1996; 19: 3–36.
Fernandes CA, Vanbever R. Preclinical models for pulmonary
drug delivery. Expert Opin Drug Deliv 2009; 6: 1231–45.
Parkes WR. Morphology of the respiratory tract. Occupational
Lung Disorders, 3rd edn. Hobber Arnold Publishers, 1994:
1–17.
Van der Schans CP. Bronchial mucus transport. Respir Care
2007; 52: 1150.
Bansil R, Turner BS. Mucin structure, aggregation, physiological functions and biomedical applications. Curr Opin Colloid
Interface Sci 2006; 11: 164–70.
Asgharian B, Hofmann W, Bergmann R. Particle deposition in
a multiple-path model of the human lung. Aerosol Sci Technol
2001; 34: 332–9.
Bernhard W, Haslam PL, Floros J. From birds to humans: new
concepts on airways relative to alveolar surfactant. Am J Respir
Cell Mol Biol 2004; 30: 6–11.
Pérez-Gil J. Structure of pulmonary surfactant membranes and
films: the role of proteins and lipid-protein interactions. Biochim Biophys Acta Biomembr 2008; 1778: 1676–95.
Geiser M. Update on macrophage clearance of inhaled microand nanoparticles. J Aerosol Med Pulm Drug Deliv 2010; 23:
207–17.
Holt PG, Strickland DH, Wikström ME, Jahnsen FL. Regulation of immunological homeostasis in the respiratory tract. Nat
Rev Immunol 2008; 8: 142–52.
Bühling F, Waldburg N, Reisenauer A, et al. Lysosomal cysteine proteases in the lung: role in protein processing and immunoregulation. Eur Respir J 2004; 23: 620–8.
Stamenkovic I. Extracellular matrix remodelling: the role of
matrix metalloproteinases. J Pathol 2003; 200: 448–64.
17.
18.
19.
20.
21.
Downloaded by [T&F Internal Users], [Kelly Daugherty] at 06:25 30 July 2013
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
van der Velden V, Hulsmann A. Peptidases: structure, function
and modulation of peptide-mediated effects in the human lung.
Clin Exp Allergy 1999; 29: 445–56.
Buhling F, Groneberg D, Welte T. Proteases and their role in
chronic inflammatory lung diseases. Curr Drug Targets 2006; 7:
751–9.
Altiere R, Thompson D. Pulmonary physiology and pharmacology of the airways. In: Hickey A, ed. Inhalation Aerosols: Physical and Biological Basis for Therapy. New York: Marcel
Dekker, 1996: 96–9.
El-Chemaly S, Pacheco-Rodriguez G, Ikeda Y, Malide D, Moss
J. Lymphatics in idiopathic pulmonary fibrosis: new insights
into an old disease. Lymphat Res Biol 2009; 7: 197–203.
Shak S, Capon DJ, Hellmiss R, Marsters SA, Baker CL.
Recombinant human DNase I reduces the viscosity of cystic
fibrosis sputum. Proc Natl Acad Sci USA 1990; 87: 9188.
Patton JS, Bukar JG, Eldon MA. Clinical pharmacokinetics and
pharmacodynamics of inhaled insulin. Clin Pharmacokinet
2004; 43: 781–801.
Kling J. Inhaled insulin’s last gasp? Nat Biotechnol 2008; 26:
479–80.
Neumiller JJ, Campbell RK. Technosphere insulin: an inhaled
prandial insulin product. BioDrugs 2010; 24: 165–72.
Raskin P, Heller S, Honka M, et al. Pulmonary function over
2 years in diabetic patients treated with prandial inhaled Technosphere Insulin or usual antidiabetes treatment: a randomized
trial. Diabetes Obes Metab 2012; 14: 163–73.
Rave K, Bott S, Heinemann L, et al. Time-action profile of
inhaled insulin in comparison with subcutaneously injected
insulin lispro and regular human insulin. Diabetes Care 2005;
28: 1077–82.
Adjei A, Sundberg D, Miller J, Chun A. Bioavailability of leuprolide acetate following nasal and inhalation delivery to rats
and healthy humans. Pharm Res 1992; 9: 244–9.
Clark A, Kuo M, Newman S, et al. A comparison of the pulmonary bioavailability of powder and liquid aerosol formulations
of salmon calcitonin. Pharm Res 2008; 25: 1583–90.
Walvoord EC, de la Peña A, Park S, et al. Inhaled growth hormone (GH) compared with subcutaneous GH in children with
GH deficiency: pharmacokinetics, pharmacodynamics, and
safety. J Clin Endocrinol Metab 2009; 94: 2052.
Dumont JA, Bitonti AJ, Clark D, et al. Delivery of an erythropoietin-Fc fusion protein by inhalation in humans through an immunoglobulin transport pathway. J Aerosol Med 2005; 18: 294–303.
Marino M, Costello D, Baughman R, et al. Pharmacokinetics
and pharmacodynamics of inhaled GLP-1 (MKC253): proofof-concept studies in healthy normal volunteers and in
patients with type 2 diabetes. Clin Pharmacol Ther 2010; 88:
243–50.
Mullard A. 2010 FDA drug approvals. Nat Rev Drug Discov
2011; 10: 82–5.
Adage T, Rek A, Kungl AJ. Pharmacological profile of a novel
decoy CXCL8-based biologic therapeutic in murine models of
lung inflammation. Am J Respir Crit Care Med 2011;
183:A4071.
Schepens B, Ibañez LI, De Baets S, et al. Nanobodies® specific for
respiratory syncytial virus fusion protein protect against infection
by inhibition of fusion. J Infect Dis 2011; 204: 1692–701.
Todoroff J, Vanbever R. Fate of nanomedicines in the lungs.
Curr Opin Colloid Interface Sci 2011; 16: 246–54.
Hinds WC. Aerosol Technology: Properties, Behaviour, and
Measurement of Airborne Particles, 2nd edn. John Wiley &
Sons, Inc, 1999.
Newman S, Anderson P, Byron R, Dalby R, Peart J. Aerosol
properties and deposition principles. Respiratory Drug Delivery: Essential Theory and Practice. 2009: 29–52.
DeLong M, Wright J, Dawson M, et al. Dose delivery characteristics of the AIR® pulmonary delivery system over a range of
inspiratory flow rates. J Aerosol Med 2005; 18: 452–9.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
2617
Sangwan S, Agosti JM, Bauer LA, et al. Aerosolized protein
delivery in asthma: gamma camera analysis of regional deposition and perfusion. J Aerosol Med 2001; 14: 185–95.
Cassidy JP, Amin N, Marino M, et al. Insulin lung deposition
and clearance following Technosphere® insulin inhalation powder administration. Pharm Res 2011; 28: 2157–64.
Lombry C, Edwards DA, Préat V, Vanbever R. Alveolar macrophages are a primary barrier to pulmonary absorption of macromolecules. Am J Physiol Lung Cell Mol Physiol 2004; 286: L1002–8.
Patton J, McCabe J, Hansen SE, Daugherty A. Absorption of
human growth hormone from the rat lung. Biotechnol Ther
1989; 1: 213.
Bosquillon C, Préat V, Vanbever R. Pulmonary delivery of
growth hormone using dry powders and visualization of its
local fate in rats. J Control Release 2004; 96: 233–44.
Nag K, Vidyashankar S, Devraj R, Garcia MF, Panda AK.
Physicochemical studies on the interaction of serum albumin
with pulmonary surfactant extract in films and bulk bilayer
phase. J Colloid Interface Sci 2010; 352: 456–64.
Olmsted SS, Padgett JL, Yudin AI, et al. Diffusion of macromolecules and virus-like particles in human cervical mucus.
Biophys J 2001; 81: 1930–7.
Lai SK, Wang YY, Hanes J. Mucus-penetrating nanoparticles
for drug and gene delivery to mucosal tissues. Adv Drug Deliv
Rev 2009; 61: 158–71.
Cone RA. Barrier properties of mucus. Adv Drug Deliv Rev
2009; 61: 75–85.
Lay JC, Stang MR, Fisher PE, Yankaskas JR, Bennett WD. Airway retention of materials of different solubility following
local intrabronchial deposition in dogs. J Aerosol Med 2003;
16: 153–66.
Ducreux J, Vanbever R. Crucial biopharmaceutical issues facing macromolecular candidates for inhalation: the role of macrophages in pulmonary protein clearance. Respir Drug Deliv
Eur 2007; 2007: 31–41.
Patton JS, Fishburn CS, Weers JG. The lungs as a portal of
entry for systemic drug delivery. Proc Am Thorac Soc 2004; 1:
338–44.
Cryan SA. Carrier-based strategies for targeting protein and
peptide drugs to the lungs. AAPS J 2005; 7: 20–41.
Baginski L, Tachon G, Falson F, et al. Reverse Transcription
Polymerase Chain Reaction (RT-PCR) analysis of proteolytic
enzymes in cultures of human respiratory epithelial cells.
J Aerosol Med Pulm Drug Deliv 2011; 24: 89–101.
Hastings RH, Folkesson HG, Matthay MA. Mechanisms of
alveolar protein clearance in the intact lung. Am J Physiol Lung
Cell Mol Physiol 2004; 286: L679–89.
Adjei AL, Gupta PK. Inhalation Delivery of Therapeutic Peptides and Proteins. Informa HealthCare, 1997.
Pang Y, Sakagami M, Byron PR. The pharmacokinetics of pulmonary insulin in the in vitro isolated perfused rat lung: implications of metabolism and regional deposition. Eur J Pharm Sci
2005; 25: 369–78.
Baginski L, Tewes F, Buckley ST, et al. Investigations into the
fate of inhaled salmon calcitonin at the respiratory epithelial
barrier. Pharm Res 2012; 29: 332–41.
Patton J, Nagarajan S, Clark A. Pulmonary absorption and metabolism of peptides and proteins. Respir Drug Deliv 1998; 7: 17–24.
Matsukawa Y, Yamahara H, Lee VHL, Crandall ED, Kim KJ.
Horseradish peroxidase transport across rat alveolar epithelial
cell monolayers. Pharm Res 1996; 13: 1331–5.
Veronese FM, Pasut G. PEGylation, successful approach to
drug delivery. Drug Discov Today 2005; 10: 1451–8.
Youn YS, Kwon MJ, Na DH, et al. Improved intrapulmonary
delivery of site-specific PEGylated salmon calcitonin: optimization by PEG size selection. J Control Release 2008; 125: 68–75.
Lee KC, Chae SY, Kim TH, et al. Intrapulmonary potential of
polyethylene glycol-modified glucagon-like peptide-1s as a
type 2 anti-diabetic agent. Regul Pept 2009; 152: 101–7.
Peptides and Proteins: Pulmonary Absorption
Encyclopedia of Pharmaceutical Science and Technology, Fourth Edition, Volume IV
Peptides and Proteins: Pulmonary Absorption
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62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
Encyclopedia of Pharmaceutical Science and Technology, Fourth Edition, Volume IV
Codrons V, Vanderbist F, Ucakar B, Préat V, Vanbever R.
Impact of formulation and methods of pulmonary delivery on
absorption of parathyroid hormone (1–34) from rat lungs.
J Pharm Sci 2004; 93: 1241–52.
Adjei A, Garren J. Pulmonary delivery of peptide drugs: effect
of particle size on bioavailability of leuprolide acetate in
healthy male volunteers. Pharm Res 1990; 7: 565–9.
Pfutzner A, Flacke F, Pohl R, et al. Pilot Study with Technosphere/PTH (1-34)-A new approach for effective pulmonary
delivery of parathyroid hormone (1-34). Horm Metabol Res
2003; 35: 319–23.
De Galan B, Simsek S, Tack C, Heine R. Efficacy and safety of
inhaled insulin in the treatment of diabetes mellitus. Neth
J Med 2006; 64: 319–25.
Bur M, Huwer H, Lehr CM, et al. Assessment of transport rates
of proteins and peptides across primary human alveolar epithelial cell monolayers. Eur J Pharm Sci 2006; 28: 196–203.
Kim KJ, Malik AB. Protein transport across the lung epithelial
barrier. Am J Physiol Lung Cell Mol Physiol 2003; 284:
L247–59.
Matsukawa Y, Lee VHL, Crandall ED, Kim KJ. Size dependent
dextran transport across rat alveolar epithelial cell monolayers.
J Pharm Sci 1997; 86: 305–9.
Yamahara H, Lehr CM, Lee VHL, Kim KJ. Fate of insulin during transit across rat alveolar epithelial cell monolayers. Eur J
Pharm Biopharm 1994; 40: 294–8.
Kim KJ, Fandy TE, Lee VHL, et al. Net absorption of IgG via
FcRn-mediated transcytosis across rat alveolar epithelial cell
monolayers. Am J Physiol Lung Cell Mol Physiol 2004; 287:
L616–22.
Bitonti AJ, Dumont JA, Low SC, et al. Pulmonary delivery of
an erythropoietin Fc fusion protein in non-human primates
through an immunoglobulin transport pathway. Proc Natl Acad
Sci USA 2004; 101: 9763.
Bitonti AJ, Dumont JA. Pulmonary administration of therapeutic proteins using an immunoglobulin transport pathway. Adv
Drug Deliv Rev 2006; 58: 1106–18.
Grainger CI, Greenwell LL, Lockley DJ, Martin GP, Forbes B.
Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier. Pharm Res 2006;
23: 1482–90.
Kim KJ, Borok Z, Crandall ED. A useful in vitro model for
transport studies of alveolar epithelial barrier. Pharm Res 2001;
18: 253–5.
Tronde A, Krondahl E, von Euler-Chelpin H, et al. High airway-to-blood transport of an opioid tetrapeptide in the isolated
rat lung after aerosol delivery. Peptides 2002; 23: 469–78.
Nelson HS, Busse WW, Sanger M, et al. Short-term safety of
somatropin inhalation powder in adults with mild to moderate
asthma. OceanSide Publications, Inc, 2009: 325–32.
Diaz KT, Skaria S, Harris K, et al. Delivery and safety of
inhaled interferon-γ in idiopathic pulmonary fibrosis. J Aerosol
Med Pulm Drug Deliv 2012; 25: 1–9.
Garg SK, Mathieu C, Rais N, et al. Two-year efficacy and
safety of AIR inhaled insulin in patients with type 1 diabetes: an
open-label randomized controlled trial. Diabetes Technol Ther
2009; 11: 5–16.
Rosenstock J, Cefalu WT, Hollander PA, et al. Safety and efficacy of inhaled human insulin (Exubera) during discontinuation and readministration of therapy in adults with type 2
diabetes: a 3-year randomized controlled trial. Diabetes Technol Ther 2009; 11: 697–705.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
Malucchi S, Gilli F, Caldano M, et al. One-year evaluation of
factors affecting the biological activity of interferon beta in
multiple sclerosis patients. J Neurol 2011; 258: 895–903.
Kotzé AR, Lueβen HL, de Leeuw BJ, et al. N-trimethyl chitosan chloride as a potential absorption enhancer across mucosal
surfaces: in vitro evaluation in intestinal epithelial cells (Caco-2).
Pharm Res 1997; 14: 1197–202.
Patton JS, Bukar J, Nagarajan S. Inhaled insulin. Adv Drug
Deliv Rev 1999; 35: 235–47.
Rogueda PGA, Traini D. The nanoscale in pulmonary delivery.
Part 1: deposition, fate, toxicology and effects. Expert Opin
Drug Deliv 2007; 4: 595–606.
Wise S, Chien J, Yeo K, Richardson C. Smoking enhances
absorption of insulin but reduces glucodynamic effects in individuals using the Lilly-Dura inhaled insulin system. Diabet
Med 2006; 23: 510–15.
Pan AX, De La Peña A, Yeo KP, et al. Effects of smoking cessation, acute re-exposure and nicotine replacement in smokers
on AIR® inhaled insulin pharmacokinetics and glucodynamics.
Br J Clin Pharmacol 2008; 65: 480–7.
Mason G, Uszler J, Effros R, Reid E. Rapidly reversible alterations of pulmonary epithelial permeability induced by smoking.
Chest 1983; 83: 6–11.
Monick MM, Powers LS, Walters K, et al. Identification of an
autophagy defect in smokers’ alveolar macrophages. J Immunol 2010; 185: 5425.
Olsson B, Bondesson E, Borgström L, et al. Pulmonary drug
metabolism, clearance, and absorption. Control Pulm Drug
Deliv 2011; 21–50.
Petecchia L, Sabatini F, Varesio L, et al. Bronchial airway epithelial cell damage following exposure to cigarette smoke
includes disassembly of tight junction components mediated by
the extracellular signal-regulated kinase 1/2 pathway. Chest
2009; 135: 1502–12.
Olivera D, Knall C, Boggs S, Seagrave JC. Cytoskeletal modulation and tyrosine phosphorylation of tight junction proteins
are associated with mainstream cigarette smoke-induced permeability of airway epithelium. Exp Toxicol Pathol 2010; 62:
133–43.
Mudaliar S, Henry RR. Inhaled insulin in patients with asthma
and chronic obstructive pulmonary disease. Diabetes Technol
Ther 2007; 9: 83–92.
Rave K, de la Peña A, Tibaldi FS, et al. AIR inhaled insulin in
subjects with chronic obstructive pulmonary disease. Diabetes
Care 2007; 30: 1777–82.
Ohtake S, Kita Y, Arakawa T. Interactions of formulation excipients with proteins in solution and in the dried state. Adv Drug
Deliv Rev 2011; 63: 1053–73.
Maa YF, Nguyen PAT, Hsu SW. Spray-drying of air–liquid
interface sensitive recombinant human growth hormone.
J Pharm Sci 1998; 87: 152–9.
Cape SP, Villa JA, Huang ETS, et al. Preparation of active
proteins, vaccines and pharmaceuticals as fine powders using
supercritical or near-critical fluids. Pharm Res 2008; 25:
1967–90.
Niven RW, Ip AY, Mittelman SD, et al. Protein nebulization: I.
Stability of lactate dehydrogenase and recombinant granulocyte-colony stimulating factor to air-jet nebulization. Int
J Pharm 1994; 109: 17–26.
Steckel H, Eskandar F, Witthohn K. Effect of excipients on the
stability and aerosol performance of nebulized aviscumine.
J Aerosol Med 2003; 16: 417–32.