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the end of July 2017. His research is in pulmonary drug delivery. In
particular, he specialises in the engineering, physicochemical
characterisation, and electrostatics of pharmaceutical aerosol
formulations. He has collaborated with academic and industrial
researchers, both locally and internationally, on formulation-focused as
well as interdisciplinary projects.
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(1)
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
J. Lam, P. C. L. Kwok (eds.), RespiratoryDeliveryofBiologics,NucleicAcids,and
Vaccines, AAPS Introductions in the Pharmaceutical Sciences 8
https://doi.org/10.1007/978-3-031-47567-2_1
DesignStrategiesofDryPowdersfor
PulmonaryDeliveryofPharmaceutical
Peptides
Hideyuki Sato
1
Laboratory of Biopharmacy, School of Pharmaceutical Sciences,
University of Shizuoka, Shizuoka, Japan
HideyukiSato
Email:h.sato@u-shizuoka-ken.ac.jp
Abstract
Pharmaceutical peptides have gradually become more attractive
therapeutic molecules than small-molecule drugs, since
pharmaceutical peptides are more selective and effective, and have
fewer side effects, compared to small-molecule drugs. Generally, owing
to their poor oral absorbability and gastrointestinal stability, peptides
are mainly administered via intravenous and intramuscular routes,
which adversely affects patient compliance. Pulmonary characteristics,
such as large surface area, abundant capillary network, thin membrane
with adequate permeability for macromolecules, reduced enzymatic
degradation, and lack of irst-pass metabolism, facilitate the use of
inhalable formulations to achieve local and systemic actions. Precise
control of powder properties and appropriate design of respirable
particles to stabilize target peptides are necessary for their eficient
pulmonary delivery. This chapter discusses the strategies for
formulation and eficient delivery of peptide-loaded dry powders.
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Keywords Dry powders – Particle design – Peptides – Pulmonary
delivery
1 Introd uction
Recent advances in formulation development, such as drug engineering
and recombinant DNA technology, facilitate the eficient formulation
and delivery of medium and high molecular weight compounds such as
peptides, nucleic acids, antibodies, and proteins, which have speciic
and potent pharmacological actions owing to their high target
selectivity. In 2019, eight biologic drugs were among the top 10 global
sales [10]. Recently, middle molecular weight drugs with a molecular
weight of about 500–6000 Da, peptides and nucleic acids, have
attracted signiicant interest as they are relatively easy to design,
synthesize, and control, compared with proteins and antibodies.
However, similar to other biologics, the oral bioavailability of peptides
is poor, owing to their molecular size, degradation by digestive
enzymes in the gastrointestinal (GI) tract, low permeability through
the epithelial barriers in the GI tract, and irst-pass metabolism. Thus,
most peptide drugs are administered via the parenteral routes, like
subcutaneous, intramuscular, or intravenous injections. Parenteral
administration has several limitations, such as pain, risk of injury at
the injection site, dificulties with self-administration, cold chain
storage, production of needles, syringes, and other waste materials
that are dificult to dispose of. To overcome these drawbacks, safe,
effective, and non-invasive routes and drug delivery technologies are
being developed.
Pulmonary delivery can offer rapid absorption of drugs and higher
systemic exposure because of the extensive vascularization in the lung,
high tissue permeability of epithelial cells, relatively low activity of
metabolic enzymes, and large pulmonary surface area [57], leading to
several advantages over conventional, non-invasive administration
routes in the treatment of systemic diseases. The pulmonary
administration system can deliver a larger amount of drug directly to a
local disease site with minimal systemic exposure. There have been
many reports on achieving eficacious drug delivery to the disease site
for the treatment of asthma, chronic obstructive pulmonary disease,
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and respiratory infections [28]. Therefore, pulmonary delivery is a
viable option for effective and safe delivery of therapeutic peptides for
topical and systemic actions, depending on the target disease. Thus,
much attention has been paid to develop and investigate pulmonary
delivery systems for therapeutic peptides. For the systemic actions,
pulmonary delivery of metabolic hormones, including insulin,
calcitonin, growth hormones, somatostatin, thyroid-stimulating
hormone, and follicle-stimulating hormone, to humans and
experimental animals has been reported [1]. For the treatment of
respiratory diseases like asthma, chronic obstructive pulmonary
diseases, and cystic ibrosis and lung infections, applications of
vasoactive intestinal peptide analogues, neuropeptide Y, cyclosporine
A, and colistin have been investigated [7, 16, 79]. However, dificulty in
particle design and formulation strategies limits the practical
applications of pulmonary delivery of therapeutic peptides. Generally,
for orally inhaled particles, aerodynamic particle size between 1 and
5 μm ensures eficient delivery of inhaled particles [13]. Particles with
aerodynamic diameters larger than 10 μm are deposited in the
extrathoracic sites, such as the mouth, pharynx, and larynx, and
particle sizes between 5–10 μm are deposited in the tracheobronchial
tree. Particles that are too small cannot adhere to the surface of the
respiratory tract because they are removed from the respiratory tract
by exhaled airlow. Compared with conventional small-molecule drugs,
therapeutic peptides tend to be at risk of degradation and deactivation,
owing to their poor stability against chemical and physical stress
during the production process and storage [28]. In addition, even after
reaching the respiratory site, some physiological factors cause their
degradation/denaturalization and elimination from the target site
[30]. Thus, along with the precise control of particle size, the selection
of appropriate excipients and formulation design are key
considerations for the successful pulmonary delivery of therapeutic
peptides.
There are three commonly used systems for inhalation medication:
nebulizers, metered dose inhalers, and dry powder inhalers (DPIs).
Among these, DPIs are the most commonly used inhalation devices in
adults patients due to a lot of practical advantages including ease of
use, portability, and relatively high pulmonary delivery eficiency [70].
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Additionally, considering the chemical stability of peptides,
powderization would be one of the options to stabilize the target
molecule, since the presence of water could accelerate the degradation
of peptides. Thus, this chapter mainly focuses on the physicochemical
and biological challenges of therapeutic peptides and particle
design/formulation strategies for DPIs to achieve eficient peptide
delivery by oral inhalation.
2 FactorsAffectingPeptideStabilityin
ManufacturingProcess
During the manufacturing process of inhalable formulations, many
factors cause the degradation, aggregation, and/or deactivation of
target peptides due to preparation conditions and chemical and
physical stresses such as thermal stress, oxidative stress, shear stress,
and pressure. Deamidation, covalent aggregation, oxidation, and
Maillard reaction are the main degradation pathways [77]. Additionally,
in some cases, aggregation is irreversible and reduces the physical
stability of the peptide, not only leading to a loss of activity but also
other critical problems such as toxicity and immunogenicity [54].
Thus, to prevent the risk of loss of quality and toxicity of the
formulation, appropriate conditions and excipients should be selected.
This section briely summarizes general information on the potential
factors inluencing peptide stability (Table 1).
Table1 Factors affecting peptide stability in manufacturing process
Factor Comment
pH Solution pH can inluence the charge state of dissolved peptide
depending on the isoelectric point (pI), changing the dispersion
state by electric repulsion.
Physical stress
(heat, pressure,
and shear stress)
Physical stresses in manufacturing process can alter molecular
interactions including electrostatic force, hydrophobic
interactions, hydrogen bonding, van der Waals forces, and local
peptide interactions, possibly changing the folding state and
causing aggregations.
Oxidative stress Amino acids with reactive side chain in a peptide (histidine,
methionine, cysteine, tyrosine, and tryptophan) can be
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Factor Comment
potentially damaged by reaction with any of a number of
reactive oxygen species during formulation process and storage.
Excipient Buffering agents and salts have potential to inluence on peptide
stability due to changing pH and ionic strength of peptide
solution. Some excipients like surfactants and antioxidants can
stabilize peptide.
2.1 pH
Generally, the pH can inluence the aggregation and chemical
degradation of peptides. Electrostatic interactions are known to play a
major role in stabilizing the dispersion state. More speciically, a higher
net charge can contribute to the suppression of the aggregation
potential of peptides owing to the electronic repulsion between
peptide molecules [60]. Additionally, the ionic strength and nature of
the cations and anions in solution, and the presence of polyelectrolytes
also have the potential to affect the rate and extent of aggregation. To
estimate the effect of solution conditions on aggregation, the
isoelectric point of peptides, where net charges are essentially zero, is
useful information [38], suggesting the importance of selecting
suitable excipients for stabilizing target peptides in the formulation
process and subsequent products. When the solution pH is
signiicantly different from the isoelectric point, the protein becomes
highly charged, resulting in electric repulsions.
2.2 Temperature
During the pharmaceutical process, active ingredients experience
physical stress, such as thermal stress, pressure, and shear stress.
Temperature affects the structural stability of peptides via molecular
interactions, conformational stability (secondary structure), solubility,
and chemical degradation. Electrostatic forces, hydrophobic
interactions, hydrogen bonding, van der Waals forces, and local peptide
interactions are responsible for the free energy of the folding state.
Temperature plays a crucial role in reaction kinetics because rate
constants increase exponentially with temperature, i.e., the rate of
aggregation increases at high temperatures because of the rise in
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global molecular mobility (kinetic energy) [6]. Therefore, an increase
in temperature accompanies an increase in the probability and number
of collisions with suficient energy to overcome the activation energies
for the reaction, possibly leading to the acceleration of aggregation
formation. Consequently, they may compromise their therapeutic
eficacy and cause potential safety concerns. The lyophilization/freezedrying technique is a very common method to increase both chemical
and physical stability [18] and reduce the thermal stress for
solidiication. However, in some cases, these processes result in
conformational changes in peptides, leading to increased aggregation
after reconstitution in water, depending on their physicochemical
characteristics. Considering these points, the thermal stress during
drying and evaporation conditions should be carefully selected to
minimize the risk of aggregation, and even using freeze-drying, the
possible changes in conformational changes and redispersibility of the
peptide should be evaluated by spectroscopic analysis such as circular
dichroism spectroscopy.
2.3 OxidativeStress
Oxidation of peptides is one of the major problems associated with
chemical and physical instability. It is well known that some amino acid
chains can be oxidatively modiied during peptide puriication,
formulation, and storage [47, 71]. These peptides included aromatic
amino acids (Tyr, Trp, and His) and sulphur-containing side chains
(Met and Cys). Thus, there have been many reports on the application
of antioxidants, such as ascorbic acid, for stabilization [35].
Methionine, catalase, sodium thiosulfate, and chelating agents such as
ethylenediaminetetraacetic acid (EDTA) and diethylene triamine
pentaacetic acid (DTPA) have also been used as stabilizers against
oxidative stress [40, 80]. Replacement of these sensitive amino acids
with other chemical moieties may increase the chemical stability of
peptide. In this case, the conformational changes and afinity to the
target site for pharmacological action should be carefully evaluated
after modiication of the amino acid sequence and/or chemical
moieties of target peptide.
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2.4 Excipients
Different excipients are used in pharmaceutical development of
peptide formulations, including salts, polyols, sugars, surfactants,
osmolytes, chelators, antioxidants, speciic ligands, and carbohydrates
[77]. As mentioned above, pH conditions can strongly affect the
stability and potential of aggregation. Therefore, buffering agents such
as acetate, citrate, histidine, phosphate, and Tris can alter stability.
Salts also have a complicated inluence on peptide stability by altering
both conformational and colloidal stability [35]. Depending on the
difference in surface charge, the overall effect of a salt on physical
stability is a balance of different and multiple mechanisms by which
the salt interacts with water and biomolecules (Hofmeister effects and
Debye–Hu ckel effects). Using a surfactant, the exposure of the
hydrophobic part of the peptide to water is minimized owing to its
amphiphilic properties, resulting in the stabilization of the peptide.
Pharmaceutical excipients have both positive and negative effects on
the stability of peptides, suggesting the necessity of careful selection
depending on the physicochemical properties of the target peptides.
3 BiologicalBarriers
Active ingredients can act in the mucus layer or on pathogens for
topical pharmacological actions in the respiratory tract, whereas for
systemic delivery, target drugs have to permeate the bronchial mucus
layer and alveolar epithelial cells to enter systemic circulation (Fig. 1).
The thickness of the mucus layer is between 5 and 55 μm depending on
the depth of the site in the respiratory tract [29] and varies depending
on the physiological conditions [27], resulting in the dificulty of
suficient delivery of target peptides in obstructive diseases, which
causes increases in mucus production and viscosity of mucus. Thus,
controlling diffusiveness within the mucus layer by mucoadhesive and
mucopenetrating properties of the formulation could contribute to the
enhanced and/or sustained absorbability from the lung (described in
detail in Sect. 5.1 Adjusting mucodiffusiveness). Although pulmonary
mucus is not a signiicant barrier for macromolecules with sizes less
than 10 nm (−500 kDa), aggregates of peptides and nano/microparticle
formulations (>10 nm) could be trapped by the mucus layer owing to
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the mesh-like structure of the mucus layer. They are transported from
the deep lung to the pharynx by mucociliary clearance and are removed
via phagocytosis by macrophages.
Fig.1 Biological barriers for pulmonary delivery of peptide drugs
For systemic action of the target peptides, they must penetrate the
epithelial layer. For permeation via passive diffusion, there is a suitable
range of lipophilicity (log P value: 2–9) and a cutoff value for molecular
size (above 1000 Da) [4, 48]. Thus, they can permeate the cellular
membrane via transcytosis (which may be receptor-mediated or
carrier-mediated), paracellular routes, and large transitory pores in the
epithelial layer. Proton-coupled peptide transporters (PEPT) are
generally known as the main transporter for the permeation of di-and
tripeptides; however, the contribution of PEPT to the absorption of
inhaled peptides in the pulmonary tract is not signiicant. If target
peptides can be captured by endocytosis and pinocytosis during the
absorption process, they can be found in lysosomes, possibly leading to
enzymatic degradation by hydrolases in the lysosomes. Therefore,
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appropriate strategies are required to avoid inactivation by enzymatic
degradation.
In the respiratory tract, the activities of metabolic enzymes are
lower than those in the gastrointestinal tract and liver tissues, and
molecules absorbed via pulmonary delivery can avoid irst-pass
metabolism, suggesting great advantages for peptide delivery via
inhalation systems. However, degradation of peptides and proteins
also occurs in the respiratory tract, and the extent of degradation
depends on the molecular size. Relatively small peptides with
molecular sizes of less than 3 kDa are more sensitive than larger
proteins (6–500 kDa) [73]. In bronchoalveolar lavage luid,
angiotensin-converting enzyme, cathepsin D, cathepsin H, and
dipeptidyl peptidase IV are abundant enzymes involved in the
degradation of peptides and proteins.
Proteins and peptides deposited on ciliated epithelium are not
signiicantly absorbed owing to mucociliary transportation up the
airways and movement into the gastrointestinal tract, resulting in the
degradation and denaturation of the molecules. The clearance
mechanism in the alveolar region includes phagocytosis by
macrophages, paracellular diffusion through tight junctions, vesicular
endocytosis or pinocytosis, and receptor-mediated transcytosis. For
soluble proteins, clearance via phagocytosis by alveolar macrophages
does not seem important as a clearance system because macrophages
preferentially capture relatively insoluble particles.
Some respiratory diseases, including asthma, chronic obstructive
pulmonary diseases, lung ibrosis, and pulmonary hypertension, cause
physiological changes such as bronchial constriction, emphysema, and
increase of mucus thickness and viscosity. It has also been reported
that the activities of several proteases are altered due to the migration
and accumulation of inlammatory cells at the inlammatory site and
the release of protease from inlammatory cells [22]. Thus, peptide
degradation increases in most lung diseases. An increase in the
thickness of the mucus layer in the diseased state is also a nonenzymatic barrier for the delivery of peptides to target cells. In patients
with cystic ibrosis, a combination of increased mucus production and
viscosity can be observed, indicating the dificulty of pulmonary drug
delivery compared with the healthy state [27]. Additionally, tissue
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