This article was downloaded by: [T&F Internal Users], [Kelly Daugherty] On: 30 July 2013, At: 06:25 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Encyclopedia of Pharmaceutical Science and Technology, Fourth Edition Publication details, including instructions for authors and subscription information: http://staging.www.tandfonline.com/doi/book/10.1081/E-EPT4 Peptides and Proteins: Pulmonary Absorption Salomé-Juliette Koussoroplis a , 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: ; 2607-2618. To link to this chapter: http://dx.doi.org/10.1081/E-EPT4-120050324 PLEASE SCROLL DOWN FOR CHAPTER Full terms and conditions of use: http://staging.www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
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This article was downloaded by: [T&F Internal Users], [Kelly Daugherty]On: 30 July 2013, At: 06:25Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK
Encyclopedia of Pharmaceutical Science andTechnology, Fourth EditionPublication details, including instructions for authors and subscription information:http://staging.www.tandfonline.com/doi/book/10.1081/E-EPT4
Peptides and Proteins: Pulmonary AbsorptionSalomé-Juliette Koussoroplis a , Rita Vanbever aa Louvain Drug Research Institute, Pharmaceutics and Drug Delivery , Université catholiquede Louvain , Brussels , Belgium
To cite this entry: Salomé-Juliette Koussoroplis , Rita Vanbever . Peptides and Proteins: Pulmonary Absorption. InEncyclopedia of Pharmaceutical Science and Technology, Fourth Edition. Taylor and Francis: New York, Published online: ;2607-2618.
To link to this chapter: http://dx.doi.org/10.1081/E-EPT4-120050324
PLEASE SCROLL DOWN FOR CHAPTER
Full terms and conditions of use: http://staging.www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses shouldbe independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly inconnection with or arising out of the use of this material.
Salomé-Juliette KoussoroplisRita VanbeverUniversité catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug Delivery, Brussels, Belgium
INTRODUCTIONInhalation of drugs is very effi cacious for the treatment of lung diseases and a continuously increasing number of inhaled drugs are becoming available on the market. Inha-lation 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 sys-temic administration of drugs because pulmonary drug absorption is high and rapid and fi rst-pass hepatic metab-olism is avoided. Peptides and proteins are more effi -ciently 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 transder-mal administration without chemical enhancer (1,2). These absorption features originate from the large absorp-tive surface area of the alveoli, the very thin diffusion path to the bloodstream as well as the local high blood fl ow. 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 tri-als are presented. The different biological pathways that these molecules can follow after deposition in the lung are described, including pulmonary absorption to the blood-stream. Furthermore, the available in vitro and in vivo models for the assessment of pulmonary absorption of pep-tides 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 for-mulation, storage, and aerosolization is given.
LUNG PHYSIOLOGYThe respiratory system resembles an inverted tree where the trachea divides into two main bronchi. Each bronchus fur-ther subdivides into progressively smaller bronchioles until
it reaches the smallest airspaces called alveoli. The lung consists of two functional zones: the conducting zone (16 fi rst generations) and the respiratory zone (7 last gen-erations). In the conducting region, the air is fi ltered, warmed, and humidifi ed, 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 cross-sectional area. Consequently, the alveoli provide a total sur-face 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 resp-iratory zones (Fig. 1) (5,6). A pseudostratifi ed columnar epithelium lines the proximal conducting airways and is composed of ciliated columnar cells, goblet or mucus-secreting cells, and basal or progenitor cells (7). It is pro-gressively 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 microorgan-isms 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 sur-face and its thickness ranges from 5 µm to 55 µm. It con-sists 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 effec-tive cilia beating. The mucus is transported by the coordi-nated beating of the cilia and by expiratory airfl ow 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 accu-mulating amounts of mucus to be cleared by the central airways and trachea (10).
Pulmonary surfactant is responsible for biophysical sta-bilizing activities and innate defense mechanisms. It lines the alveolar epithelial surfaces and overfl ows into the conductive
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airways so that the surfactant fi lm is continuous between alveoli and central airways (11). Pulmonary surfactant is composed of 80% phospholipids, 5–10% neutral lipids (mainly cholesterol), 5–6% specifi c surfactant proteins, and 3–4% nonspecifi c proteins (12). The phospholipids are mainly responsible for forming the surface-active fi lm at the respiratory air–liquid interface. Half of surfactant phospholipids by mass are composed of disaturated spe-cies, mainly dipalmitoylphosphatidylcholine. Specifi c 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 specifi c 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 par-ticipate in innate immune responses, that is, chemotaxis, phagocytosis, and microbial killing (13). They also down-regulate adaptive immune responses and protect the lungs from T-cell-mediated infl ammation (14). Alveolar macro-phages are tightly applied on the surface of respiratory epi-thelia. They are immersed in the lung lining fl uid beneath the surfactant fi lm. Although they occupy only 1% of the alveolar surface, they are capable of cleaning particles from the entire alveolar surface due to amoeboid move-ments (13). In contrast to surface macrophages, interstitial macrophages are primarily involved in adaptive immunity by interfacing with lymphocytes through antigen presenta-tion 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 mem-brane bound (16). Proteases play an essential role in cell and tissue growth, differentiation, repair, remodeling, cell migration, and peptide-mediated infl ammation (17). Pro-teases can also be released in the airspaces by activated macrophages and neutrophils in case of infl ammation in the respiratory tract (18).
Blood supply to the lungs is divided among the pulmo-nary and systemic circulations (19). The pulmonary circu-lation consists of the pulmonary artery that leaves the right heart, branches into a dense pulmonary capillary bed that surrounds the alveoli, and fi nally coalesces into the pulmo-nary vein that drains into the left heart. One hundred per-cent of the cardiac output fl ows through the pulmonary circulation. Its principal functions are gas exchange with air in the alveoli and nutrients supply to terminal respira-tory units. The lungs receive a second blood supply through the systemic circulation, commonly referred to as the bron-chial circulation. The bronchial circulation originates from the aorta and provides oxygenated blood and nutrients to all structures of the tracheobronchial tree. Lymphatic ves-sels exist in close proximity of major blood vessels and airways (20).
DEVELOPMENT STATUS OF INHALED PEPTIDES AND PROTEINSAlthough the efforts performed in the fi eld of pulmonary delivery of peptides and proteins have been tremendous, there are still a very limited number of inhaled macromol-ecules available on the market (Table 1). Yet, there are a growing number of inhaled peptides and proteins undergo-ing various phases of clinical trials, those developed for local therapy being the most promising.
Human bronchi3–5 mm diameter
Human terminalbronchioles
0.5–1 mm diameter Human alveoli
3 µm
58 µm
8 µm
0.07 µm fluid0.1–
0.2 µm
Type I cell
10 µm
Brushcell
Basalcell
Basementmembrane
Ciliatedcell
Gobletcell
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.
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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 contain-ing polar lipids and surfactant proteins, mainly SP-B. Infa-surf® and Survanta® come from bovine source, whereas Curosurf® comes from porcine source.
Recombinant human deoxyribonuclease I (Pulmo-zyme®) is used for the treatment of cystic fi brosis and is available on the market since 1994. Cystic fi brosis is a genetic disease where thick secretions are retained in the airways, leading to reduced pulmonary function and exac-erbation of infection. Mucus thickness is partly due to the release of high quantities of DNA by degenerating leuko-cytes. Recombinant human deoxyribonuclease I cleaves DNA in airway secretions and reduces their viscoelasticity. The glycoprotein is administered by inhalation of an aero-sol mist produced by a pneumatic nebulizer (21).
The fi rst 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. How-ever, 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 effi cacy 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 bio-availability using Exubera® was approximately 10% rela-tive 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® insu-lin powder in unit-dose cartridges for administration with the inhaler. The Technosphere® powder formulation is pre-pared 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 pharmacoki-netic profi le mimics more closely normal physiologic insu-lin release than injection of regular human insulin and of rapid-acting analogs. Insulin bioavailability using AFREZZA™ reaches 30% relative to subcutaneous regu-lar human insulin. The inhaler is small and discrete. AFREZZA™ has demonstrated a favorable safety and tol-erability profi le in clinical studies. However, a small reduc-tion 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 con-ducted. 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 pharma-ceutical company (MannKind corporation) currently pursues the development of inhaled peptides for a
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 fi brosis 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 FDAGlucagon-like peptide _ Type II diabetes MannKind corporation In initial clinical trialsGlycan-binding decoy
protein_ COPD, chronic lung
infl ammationsProtAffi n Phase I clinical trials
in early 2012Nanobody: antibody-
derived therapeutic protein
_ Respiratory tract infections caused by human respiratory syncytial virus
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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 depos-ited in the alveolar region initially come into contact with the thin layer of lining fl uid coating the alveolar epithe-lium. The dissolved peptide or protein can then be sub-jected to clearance by alveolar macrophages or can be transported across the alveolar epithelium into the blood-stream. The lungs exhibit a decreased proteolytic activity compared to the gut, enzymes are present though. Peptides and proteins may be enzymatically degraded either extra-cellularly (by membrane-associated proteases and pepti-dases) and/or intracellularly (within macrophages and epithelial cells) (35).
Deposition in the Respiratory TractDeposition 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 deposi-tion 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 defi ned by the equation:
0aerd d r
r 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 par-ticle size, on inhalation fl ow, and on lung anatomy. Large particles (daer > 5 µm) deposit in upper airways (mouth,
systemic action, these include insulin (AFFREZA™) and glucagon-like peptide 1 (GLP-1). Pulmonary admin-istration of GLP-1 adsorbed on Technosphere micropar-ticles is undergoing initial clinical investigation (31). The pulsatile administration of GLP-1 through the lungs is benefi cial 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 approv-als in 1996 to 15 in 2010), FDA approval of biomolecules has remained constant with an average of four to fi ve approvals per year (32). Therefore, the proportion of bio-molecules delivered by inhalation is also expected to grow. A decoy form of IL-8 can potentially treat chronic obstruc-tive pulmonary disease (COPD) following pulmonary administration (33). COPD is characterized by a neutrophilic infl ammation of the airways where IL-8, a major chemo-kine, plays a central role. This decoy protein is an engi-neered version of human IL-8, with higher affi nity 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 immunoglob-ulin, 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 LUNGSThe 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
Figure 2 Pharmacodynamic response (glucose infusion rates) over 10 hr after insulin inhalation (Exubera®) compared to injected regu-lar insulin and injected lispro. Source: From Ref. 26.
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nInteraction with the Air–Liquid InterfaceFollowing 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 fl uid 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 pen-etrated respiratory epithelia, and the protein was largely taken up by alveolar macrophages (42,43). Aggregates of hGH were visible in a gel fi ltration chromatogram carried out on lung homogenates (42).
Soluble proteins may minimally perturb surface-active lipid fi lms, with minor reorganization at low concentra-tions of the protein (44). Interactions of serum and serum proteins with pulmonary surfactant have been largely investigated in vitro because serum leakage into the alveo-lar space has been assumed to be the primary cause of sur-factant dysfunction in acute respiratory distress syndrome. Air interface adsorbed fi lms of bovine lipid extract surfac-tant 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 sur-face 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 ClearanceMucociliary clearance clears proteins that stick to mucin fi bers 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-affi nity 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, low-affi nity bonds between hydrophobic domains and hydro-gen 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-affi nity bonds of the Fc domains with mucin fi bers (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 affi nity because the diffusion of IgG, with only one Fc region, was not slowed signifi cantly in mucus but antibodies
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 airfl ow within the respiratory tract. Therefore, particles retain their original direction, “crashing” on the airway wall. Smaller parti-cles (daer = 1 to 5 µm) usually pass through the larger air-ways and reach the deep lung (lower airways and respiratory bronchioles), where they deposit by gravita-tional settling. In this region, the airstream velocity mark-edly 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 sedimenta-tion varies with the breathing pattern and with the anatomy of the respiratory tract. Slow inhalation is generally pre-ferred 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 anat-omy between individuals, that is, airway dimensions and branching angles, lead to variations in aerosol deposition between subjects. In patients with asthma, COPD and cys-tic fi brosis, there is a systematic variability in airway anat-omy, because the pulmonary airways may be narrowed by a combination of bronchospasm, infl ammation, and mucus hypersecretion. Airway narrowing increases the likelihood of deposition by impaction, as well as creates turbulent air-fl ow in regions of the lungs where airfl ow 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 conse-quently 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 pow-der 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 micron-size 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 depo-sition 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 fl ow rate (38). New tech-nology 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 fl ow 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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quickly, presumably preventing major uptake and degrada-tion by alveolar macrophages.
It should be noted that a large molecular weight does not systematically involve a long residence time within the air-spaces and consequently an uptake by alveolar macro-phages. 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 evi-dence that for certain endogenous molecules that normally occur in lung lining fl uids, for example, albumin, immuno-globulins, and transferrin, there are specifi c receptor-medi-ated 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 physicochem-ical properties of the proteins, with hydrophobicity and a global cationic charge increasing adsorptive endocytosis of proteins (49).
Peptidase and Protease ActivityAlthough the lungs are a far less hostile metabolic environ-ment than the gastrointestinal tract, proteolytic enzymes are still present (51). Enzymatic degradation of inhaled proteins and peptides may occur prior or along their trans-port 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 mem-brane-bound peptidases. Many enzymes were shown to be expressed in human respiratory epithelial cells, but at different
with multiple Fcs as IgM and small aggregates of IgA were signifi cantly slowed (Fig. 3). This suggests that antibodies accumulating on the surface of a pathogen may be able to form a suffi cient number of low-affi nity 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 peri-ciliary sol layer, which is transported less effi ciently than the mucus gel layer during mucociliary clearance.
Alveolar MacrophagesAlveolar macrophages are a primary barrier to the transport of large proteins from the airway lumen into the blood-stream (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 intra-tracheal 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 pino-cytosis or “cell drinking,” the uptake of fl uids and soluble compounds. In contrast to large proteins, no increase in pul-monary 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
Nonwalk
lgM Fc5µ lgM
slgAagg
HPV
HSV
1
0.1
0.01
0.0011 10 100
Radius (nm)
Dm
uc
/ D
pb
s
Figure 3 Normalized diffusion coeffi cients of proteins and viruses in mucus. The proteins tested were lysozyme, myoglo-bin, 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.
5000
4000
3000
2000
1000
00 6 12 18 24 30 36
Time (h)
Ser
um
hC
G (
mIU
/ml)
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.
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enzymes (59). For instance, the site-specifi c substitution of salmon calcitonin with a PEG of 5 kDa led to a thousand-fold increase in proteolytic resistance in rat lung homoge-nate (60). Lee et al. (61) have demonstrated the benefi cial 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 unmodifi ed version.
Transport Across the Airway and Alveolar EpitheliumTransport of macromolecules occurs across both the airway pseudostratifi ed columnar epithelium as well as across the thin alveolar epithelium. Yet, macromolecules are absorbed into the bloodstream in larger amounts when they are deliv-ered to the deep lung than when they are delivered to central aiways (39,62). This likely originates from higher absorp-tion 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 endothe-lium. 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 follow-ing 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 signifi cant pathway in their fate.
Transport of peptides and proteins across respiratory epithelia may take place through paracellular or transcel-lular routes, and the main mechanism of transepithelial transport depends on the macromolecule molecular weight. Protein-specifi c transcytosis and peptide-specifi c proteoly-sis can enhance and reduce transport, respectively. Up to a molecular weight of approximately 40 kDa, peptides and proteins with no specifi c receptor on epithelial cells are transported by paracellular diffusion. Above this molecular size, nonspecifi c pinocytosis occurs. For instance, growth hormone (22 kDa) diffuses between alveolar epithelial cells, whereas horseradish peroxidase (40 kDa) traverses across the epithelium by nonspecifi c pinocytosis (66,67).
Matsukawa et al. determined the permeability coeffi -cient across the alveolar epithelium for dextrans of different
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 pulmo-nary administration due to severe local peptidase degra-dation (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 metabo-lism of inhaled insulin and not insulin-degrading enzyme. Bacitracin, an ectopeptidase inhibitor, decreased the non-absorptive 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 prote-ases 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 enzy-matic 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 pep-tides for proteolytic enzymes. Their large size and globular structure may prevent enzymes to fi t them into their cata-lytic sites (57). Nonspecifi c endocytosis transport of pro-teins across lung epithelia may lead to their partial degradation in the lysosomal compartment. One representa-tive example is given by horseradish peroxidase, a nonspe-cifi c fl uid-phase endocytosis marker (58). About half of the FITC-labeled species present in either the apical or basolat-eral receiver fl uid 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 pul-monary administration by shielding them from proteolytic
Table 2 Bioavailability and Tmax
of peptides and proteins following pulmonary delivery to humans using high technology inhalers
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®)Insulin 6,000 30 0.2 (1,24) (AFREZZA™)Human growth hormone 22,000 3.5–7.6 1–4 (29)Erythropoietin-Fc fusion protein 112,000 NAc 20 (30)aRelative to subcutaneous injection and dose loaded in the inhaler.bRelative to subcutaneous injection and dose deposited in the lungs.cData not available.
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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 simi-lar 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 fl uid. 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 cul-tures consist of alveolar epithelial cells. Type II pneumo-cytes 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 humidifi ed 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 interac-tions 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 iso-lated perfused lung allows studies on drug absorption from the lung without the infl uence of the other organs. How-ever, the model does not include absorption from the air-ways as the tracheobronchial circulation is severed during surgery, and it demands signifi cant 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 sam-pling 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 infl ammatory stimuli are comparable to the human case. Confi rmatory 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 pulmo-nary route as well as toxicity. Monkeys have very similar
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 diffu-sion. 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 coeffi -cients of hydrophilic solutes yielded a pore radius of 6 nm for diffusional paracellular pathways, suggesting that pro-teins 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 immunoglobu-lin 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 rel-evant mechanism for insulin transport (55,66). The transal-veolar transport of immunoglobulin G has also been analyzed in alveolar epithelial cell monolayers and shown to involve FcRn-mediated transcytosis (70). The expres-sion 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 ther-apeutic proteins were fused to the Fc-domain of an IgG1, and Fc-fusion proteins were well absorbed into the blood-stream 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 indi-cates that the correlation between molecular weight and Tmax is not perfect and that other parameters are involved in the pharmacokinetic profi le in vivo as the elimination half-life.
IN VITRO AND IN VIVO MODELS FOR DETERMINATION OF PULMONARY ABSORPTIONSeveral models are available for the assessment of pulmo-nary absorption of peptides and proteins (6). These include
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nproperties. They may exhibit different potential risks from their unmodifi ed version (83).
IMPACT OF SMOKING AND PULMONARY DISEASE CONDITIONSSmoking 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 absti-nence 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 mac-rophages, and metabolism of peptides and proteins (86–88). The mechanisms involved in the increased per-meability 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 loca-tion as well as to decreased association between two tight junction proteins, thereby increasing permeability to small solutes and macromolecules. Yet, the increased permeabil-ity 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 pulmo-nary 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 ISSUESA 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 aerosol-ization. Many proteins are structurally unstable and sus-ceptible 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 hydro-lysis 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
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 ASPECTSThere 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). How-ever, 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-mar-keting are rare (1 < 1,000) and, in most cases, side effects are mild and transient. Several Phase III trials have investi-gated 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 tran-sient cough occurring within minutes of inhalation. There was no difference in hypoglycemic events between subcu-taneous and inhaled insulin. Lung function declined over the years following both injection and inhalation, consis-tent 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 non-progressive and reversible (79).
Following delivery of proteins, the immune defense sys-tem 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 neu-tralize 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-specifi c antibody levels were not corre-lated 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 activ-ity 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-con-jugation 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 modifi ed peptides and proteins should be considered as new chemical entities with novel biochemical
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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 confi rm the formation of protein aggregates in the lung lining fl uid. The metabolism of therapeutic proteins in the lung tissue has been little studied and would deserve further investiga-tion. Finally, studying the fate of drug carriers following delivery to the lungs could also give useful information for their optimal design.
ARTICLES OF FURTHER INTERESTDrug Delivery: Pulmonary Delivery, p. 1164Dry Powder Aerosols: Emerging Technologies, p. 1295Inhalation: Dry Powder, p. 1954Inhalation: Liquids, p. 1967Metered Dose Inhalers, p. 2107
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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 effi cient 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
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