[Show abstract][Hide abstract] ABSTRACT: Inhalation aerosols offer a targeted therapy for respiratory diseases. However, the therapeutic efficacy of inhaled biopharmaceuticals is limited by the rapid clearance of macromolecules in the lungs. The aim of this research was to study the effects of the PEGylation of antibody fragments on their local residence time after administration to the respiratory tract. We demonstrate that the conjugation of a two-armed 40-kDa polyethylene glycol (PEG) chain to anti-interleukin-17A (IL-17A) F(ab')2 and anti-IL-13 Fab' greatly prolonged the presence of these fragments within the lungs of mice. The content of PEGylated antibody fragments within the lungs plateaued up to 4hours post-delivery, whereas clearance of unconjugated proteins started immediately after administration. Forty-eight hours post-delivery, F(ab')2 and Fab' content in the lungs had decreased to 10 and 14 % of the dose initially deposited, respectively. However, this value was 40 % for both PEG40-F(ab')2 and PEG40-Fab'. The prolonged pulmonary residency of the anti-IL-17A PEG40-F(ab')2 translated into an improved efficacy in reducing lung inflammation in a murine model of house dust mite-induced lung inflammation. We demonstrate that PEGylated proteins were principally retained within the lung lumen rather than the nasal cavities or lung parenchyma. In addition, we report that PEG increased pulmonary retention of antibody fragments through mucoadhesion and escape from alveolar macrophages rather than increased hydrodynamic size or improved enzymatic stability. The PEGylation of proteins might find broad application in the local delivery of therapeutic proteins to diseased airways.
Journal of controlled release : official journal of the Controlled Release Society. 05/2014;
[Show abstract][Hide abstract] ABSTRACT: In several inflammatory conditions such as rheumatoid arthritis or sepsis, the regulatory mechanisms of inflammation are inefficient and the excessive inflammatory response leads to damage to the host. Sirtuins are class III histone deacetylases that modulate the activity of several transcription factors that are implicated in immune responses. In this study, we evaluated the impact of sirtuin inhibition on the activation of lipopolysaccharide (LPS)-stimulated J774 macrophages by assessing the production of inflammatory cytokines. The pharmacologic inhibition of sirtuins decreased the production of tumour necrosis factor-alpha (TNF-α) interleukin 6 (IL-6) and Rantes. The reduction of cytokine production was associated with decreased nuclear factor kappa B (NF-κB) activity and inhibitor kappa B alpha (IκBα) phosphorylation while no impact was observed on the phosphorylation status of p38 mitogen-activated kinase (p38 MAPK). This work shows that sirtuin pharmacologic inhibitors are a promising tool for the treatment of inflammatory conditions.
Biochemical and Biophysical Research Communications 03/2012; 420(4):857-61. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This review presents the different pathways that nanomedicines can follow after deposition in the lung. These include their interactions with the air–liquid interface, their diffusion in and clearance with mucus, their uptake by lung-surface macrophages, their transport across respiratory epithelia and protein metabolism. These processes mostly occur simultaneously in the lung and their respective rates determine the dominant pathways followed by the particular nanomedicine. Accordingly, the fate of nanomedicines in the lung is highly dependent on the physico-chemical as well as biological properties of the compound considered. IgG are endocytosed by alveolar macrophages and transported across respiratory epithelia by receptor-mediated endocytosis while insulin is not taken up by alveolar macrophages and rapidly crosses the epithelium towards the systemic circulation via paracellular diffusion. Inhaled proteins are usually cleared from the lung within 24h. Nanoparticles largely escape uptake by lung-surface macrophages and can remain in the lung for weeks, without significant translocation across respiratory epithelia.
Current Opinion in Colloid & Interface Science 06/2011; 16(3):246-254. · 6.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neutrophils constitute the first line of host defense against invading microorganisms. Yet their removal from the inflammatory environment is fundamental for injury restraint and resolution of inflammation. Nicotinamide, a component of vitamin B(3), is known to modulate cell survival. In this study, we assessed the influence of nicotinamide on neutrophil apoptosis, both in vitro and in vivo in a mouse model of endotoxin-induced lung inflammation. In vitro, nicotinamide promoted apoptosis of human blood neutrophils in a dose-dependent manner in the presence of the apoptosis inhibitors granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor. The highest concentration of nicotinamide completely neutralized the pro-survival effect of granulocyte (macrophage) colony-stimulating factor. Nicotinamide proapoptotic effect was associated with enhanced caspase-3 activity. In addition, nicotinamide slightly reduced neutrophil chemotaxis in vitro. In vivo, pulmonary nicotinamide delivery decreased the levels of cellular and biochemical inflammation markers and increased the percentage of apoptotic neutrophils in bronchoalveolar lavages. Our findings suggest that nicotinamide is an apoptotic stimulus for neutrophils, thereby contributing to the resolution of neutrophilic inflammation in the lungs.
[Show abstract][Hide abstract] ABSTRACT: Although there is a modest body of literature on the absorption of inhaled pharmaceuticals by normal lungs and some limited information from diseased lungs, there is still a surprising lack of mechanistic knowledge about the details of the processes involved. Where are molecules absorbed, what mechanisms are involved, how well are different lung regions penetrated, what are the determinants of metabolism and dissolution, and how best can one retard the clearance of molecules deposited in the lung or induce intracellular uptake by lung cells? Some general principles are evident: (1) small hydrophobic molecules are absorbed very fast (within tens of seconds) usually with little metabolism; (2) small hydrophilic molecules are absorbed fast (within tens of minutes), again with minimal metabolism; (3) very low water solubility of the drug can retard absorption; (4) peptides are rapidly absorbed but are significantly metabolized unless chemically protected against peptidases; (5) larger proteins are more slowly absorbed with variable bioavailabilities; and 6) insulin seems to be best absorbed distally in the lungs while certain antibodies appear to be preferentially absorbed in the upper airways. For local lung disease applications, and some systemic applications as well, many small molecules are absorbed much too fast for convenient and effective therapies. For systemic delivery of peptides and proteins, absorption may sometimes be too fast. Bioavailabilities are often too low for cost-effective and reliable treatments. A better understanding of the determinants of pulmonary drug dissolution, absorption, metabolism, and how to target specific regions and/or cells in the lung will enable safer and more effective inhaled medicines in the future.
Journal of Aerosol Medicine and Pulmonary Drug Delivery 12/2010; 23 Suppl 2:S71-87. · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lung comprises an interesting route of administration not only for topical drugs but also for systemically acting drugs. Over the last years, several models have been developed in order to study the efficacy and safety of pulmonary drug delivery.
This review describes relevant drug delivery models for preclinical evaluation of inhaled drug products.
Epithelial cell culture models, the isolated perfused lung and in vivo models are reviewed. The suitability and limitations of each method are discussed. This review is mostly based on publications from the last 10 years.
Cell cultures are ideal models to compare transport rates of molecules and to study their mechanisms of transepithelial transport. Yet the most complete assessment of pulmonary drug delivery including delivery efficacy and safety remains provided by studies performed in vivo in animal models.
Expert Opinion on Drug Delivery 11/2009; 6(11):1231-45. · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sialoadhesin (Sn) is a macrophage-restricted receptor that was first characterised on mouse resident bone marrow macrophages as a receptor that mediates the binding, without ingestion, of sheep erythrocytes. Sn is highly conserved in mammals but its expression on tissue macrophages is heterogeneous. In the mouse, high levels of erythrocytes binding are shown on macrophages from lymphoid tissues but a low erythrocytes binding activity is detectable on macrophages isolated from the broncho-alveolar space. Yet, Sn expression has been demonstrated on human, rat and pig alveolar macrophages (AM) using methods of molecular biology. Therefore, the present study aimed to investigate the expression of Sn on mouse AM in order to confirm the presence of the protein on this population of murine macrophages. Using cytometrical analyses, we showed that Sn was expressed on mouse AM surface. Following desialylation, AM largely bound erythrocytes and this binding was inhibited by 3D6, an anti-mouse Sn monoclonal antibody, in a dose-dependent manner. This indicates that Sn is expressed on mouse AM but that the sialic acid binding activity mediated by this molecule is naturally masked by endogenous sialic acid within the glycocalyx on the cell surface.
[Show abstract][Hide abstract] ABSTRACT: N-butyldeoxynojyrimicin (NB-DNJ, miglustat [Zavesca]) an approved drug for treating Gaucher disease, was reported to be able to correct the defective trafficking of the F508del-CFTR protein.
To evaluate the efficacy of in vivo airway delivery of miglustat for restoring ion transport in cystic fibrosis (CF).
We used nasal transepithelial potential difference (PD) as a measure of sodium and chloride transport. The effect of nasal instillation of a single dose of miglustat was investigated in F508del, cftr knockout and normal homozygous mice. The galactose iminosugar analog N-butyldeoxygalactonojirimycin (NB-DGJ) was used as a placebo.
In F508del mice, sodium conductance (evaluated by basal hyperpolarization) and chloride conductance (evaluated by perfusing the nasal mucosa with chloride-free solution in the presence of amiloride and forskolin) were normalized 1 hour after an intranasal dose of 50 picomoles of miglustat. Chloride conductance in the presence of 200 microM 4-4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of alternative chloride channels, was much higher after miglustat than after placebo. In cftr knockout mice, a normalizing effect was observed on sodium but not on chloride conductance.
Our results provide clear evidence that nasal delivery of miglustat, at picomolar doses, normalizes sodium and Cftr-dependent chloride transport in F508del transgenic mice; they highlight the potential of topical miglustat as a therapy for CF.
American Journal of Respiratory and Critical Care Medicine 04/2009; 179(11):1022-8. · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Poly(ethylene glycol) (PEG) 5 kDa and 20 kDa have been previously conjugated to two anti-sialoadhesin (Sn) monoclonal antibodies (mAbs), SER-4 and 3D6, and shown to dramatically increase their inhibitory potency in solid-phase red blood cell binding assays. In the present study, we evaluated the effect of anti-Sn SER-4 and 3D6 mAbs PEGylation on their inhibition of cell adhesion in mouse peritoneal macrophages. We also examined whether Sn-mediated PEGylation could affect plasma membrane functions of macrophages as to prevent accessibility, binding, and endocytosis of macromolecules and particles. Conjugation of PEG to plasma membrane is known to cause immune tolerance by impairing protein-protein and cell-cell interactions. PEGylation of SER-4 and 3D6 mAbs increased by 4-fold their inhibition of Sn-mediated erythrocyte binding to macrophages. PEGylated SER-4 and 3D6 mAbs did not impair macrophage membrane integrity, cell metabolism, nor pinocytosis of macromolecules and phagocytosis of latex particles. Thus, PEGylation of antibodies directed to cell surface receptors could be potentially exploited in a therapeutic setting to increase inhibitory potency of antibodies without impairing vital functions of cells.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the influence of formulation excipients on physical characteristics of inhalation dry powders prepared by spray-drying. The excipients used were a series of amino acids (glycine, alanine, leucine, isoleucine), trehalose and dipalmitoylphosphatidylcholine (DPPC). The particle diameter and the powder density were assessed by laser diffraction and tap density measurements, respectively. The aerosol behaviour of the powders was studied in a Multi-Stage Liquid Impinger. The nature and the relative proportion of the excipients affected the aerosol performance of the powders, mainly by altering powder tap density and degree of particle aggregation. The alanine/trehalose/DPPC (30/10/60 w/w/w) formulation showed optimal aerodynamic behaviour with a mass median aerodynamic diameter of 4.7 μm, an emitted dose of 94% and a fine particle fraction of 54% at an airflow rate of 100 L/min using a Spinhaler inhaler device. The powder had a tap density of 0.10 g/cm3. The particles were spherical with a granular surface and had a 4 μm volume median diameter. In conclusion, optimization of the aerosolization properties of inhalation dry powders could be achieved by appropriately selecting the composition of the particles.
European Journal of Pharmaceutics and Biopharmaceutics 11/2008; · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PEGylation of antibodies is known to increase their half-life in systemic circulation, but nothing is known regarding whether PEGylation can improve the inhibitory potency of antibodies against target receptors. In this paper, we have examined this question using antibodies directed to Sialoadhesin (Sn), a macrophage-restricted adhesion molecule that mediates sialic acid dependent binding to different cells. Anti-Sn monoclonal antibodies (mAbs), SER-4 and 3D6, were conjugated to PEG 5 kDa or and PEG 20 kDa, resulting in the incorporation of up to 3 molecules of PEG per mAb molecule. Following purification of PEGylated mAbs by anion exchange chromatography, it was shown that PEGylation had little or no effect on antigen binding activity but led to a dramatic increase in inhibitory potency that was proportional to both the size of the PEG and the degree of derivatization. Thus, PEGylation of antibodies directed to cell surface receptors could be a powerful approach to improve the therapeutic efficacy of antibodies, not only by increasing their half-life in vivo, but also by increasing their inhibitory potency for blocking receptor-ligand interactions.
[Show abstract][Hide abstract] ABSTRACT: The present study reports animal immuno-toxicological data of pulmonary vaccination against inactivated seasonal influenza. Its aims were (i) to monitor the temporal kinetics of lung inflammation in normal mice over a period of 2 weeks following pulmonary vaccination in order to assess the risk of chronic lung inflammation, (ii) to evaluate the impact of pulmonary vaccination on the asthmatic phenotype in an established allergen-sensitized murine model of asthma. Both sets of experiments were performed using high doses of split influenza virus vaccine. In the first part of this study, we showed that pulmonary influenza vaccination induced a slight local inflammatory response which was limited in duration since it was no longer observed at 2 weeks post-vaccination. At this time point, it has previously been shown that the immunogenic efficacy was maintained. In the second part, we demonstrated that pulmonary influenza vaccination did not significantly exacerbate the cardinal features of asthma, i.e., allergen-specific IgE formation, the development of airway hyperreactivity (AHR) and eosinophilic airway inflammation. Our data therefore suggest that the overall immuno-toxicological profile of pulmonary vaccination against seasonal influenza was acceptable, even in an animal model of pulmonary hypersensitivity.
[Show abstract][Hide abstract] ABSTRACT: Pulmonary vaccination is a promising immunization route. However, there still remains a crucial need to characterize the different parameters affecting the efficacy of inhaled vaccination. This study aimed at assessing the impact of antigen distribution within the respiratory tract on the immune response to a monovalent A/Panama/2007/99 H3N2 influenza split virus vaccine administered to BALB/c mice. Varying the administration technique allowed the targeting of the vaccine to different sites of the mouse respiratory tract, i.e. the nasal cavity, the upper or central airways, or the deep lung. This targeting was verified by using ovalbumin as a tracer compound. The immune responses generated following influenza vaccine administration to the different respiratory tract sites were compared to each other and to those elicited by intramuscular and peroral intragastric immunization. Delivery of the vaccine to the different respiratory regions generated systemic, local and cellular virus-specific immune responses, which increased with the depth of vaccine deposition, culminating in deep-lung vaccination. The latter induced virus-specific serum immunoglobulin G and neutralizing antibody titres as elevated as intramuscular vaccination, whereas the production of mucosal secretory immunoglobulin A was significantly superior in deep-lung-vaccinated animals. The analysis of cytokines secreted by mononuclear cells during an in vitro recall response indicated that deep-lung vaccination induced a local shift of the cellular immune response towards a T helper type 1 phenotype as compared to intramuscular vaccination. In conclusion, antigen distribution within the respiratory tract has a major effect on the immune response, with the deep lung as the best target for inhaled influenza vaccination.
[Show abstract][Hide abstract] ABSTRACT: Infection with influenza virus has been associated with seemingly opposing effects on the development of asthma. However, there are no data about the effects of mucosal vaccination with inactivated influenza on the inception of allergic asthma.
To assess the immunological effects of inhaled inactivated influenza vaccine, using two different types of flu vaccines, on the inception of allergic sensitization and allergen-mediated airway disease in a mouse model.
BALB/c mice were intranasally or intratracheally vaccinated with whole or split influenza virus vaccine (days -1 or -1, 27) before systemic sensitization with ovalbumin (OVA) (days 1, 14) and repeated airway allergen challenges (days 28-30). Allergen sensitization (IgE serum levels), airway inflammation (differential cells in bronchoalveolar lavage fluid) and airway hyper-reactivity (AHR) (in vivo lung function) were analysed.
The intranasal instillation of whole influenza vaccine before allergen sensitization significantly reduced the serum levels of total and OVA-specific IgE as well as allergen-induced AHR. Prevention was due to an allergen-specific shift from a predominant T helper (Th)2- towards a Th1-immune response. Application of split influenza vaccine did not show the same preventive effect.
Intranasal administration of inactivated whole influenza vaccine reduced subsequent allergen sensitization and prevented allergen-induced AHR. Our results show that the composition of the influenza vaccine has a major influence on subsequent development of allergen-induced sensitization and AHR, and suggest that mucosal inactivated whole influenza vaccination may represent a step towards the development of a preventive strategy for atopic asthma.
[Show abstract][Hide abstract] ABSTRACT: Our laboratory recently demonstrated that alveolar macrophages (AM) comprised a major barrier to the transport of two large proteins, IgG and human chorionic gonadotropin, from the airways into the bloodstream, while they had no impact on the peptide insulin. This study was aimed at assessing the role of AM in the alveolar clearance of human growth hormone (hGH), a smaller therapeutic protein being investigated as an inhalation aerosol in clinical trials. Using intratra- cheal instillation of liposome-encapsulated clodronate to deplete AM of rat lungs, we studied the pulmonary absorption of hGH in AM-depleted versus normal animals. The systemic absorption of hGH, following pulmonary administration, did not show significant alterations following AM depletion; absolute bioavailabilities reached 11.4%, 10.7% and 9.0% in clodronate liposome-, PBS liposome- and PBS-treated rats. Absorption from the lungs was rapid in all tested conditions, indicating that hGH crossed the alveolar epithelium quickly, presumably preventing major up- take and degradation by AM. AM uptake of proteins appears to be a significant local elimination mechanism for proteins above a certain size which implies a residence time within the alveolar spaces long enough for significant AM endocytosis.
[Show abstract][Hide abstract] ABSTRACT: Systemic drug delivery using inhalation aerosols presents requirements and challenges. To be well absorbed from the lung, a compound needs to be delivered to the alveolar region and recent high technology inhaler systems have allowed increased efficiency of drug administration to the deep lung. Yet, clearance mechanisms within the respiratory tissue operate effectively and considerably diminish bioavailabilities. Methods for enhancing drug absorption from the lung have been investigated. Viable and recent strategies to accelerate drug transport across respiratory epithelia or to decrease the rate of local degradation processes are reported.Section editors:Daan J.A. Crommelin – Department of Pharmaceutical Sciences, Utrecht University, Utrecht, The NetherlandsGerrit Borchard – Enzon Pharmaceuticals, Piscataway, NJ, USA
[Show abstract][Hide abstract] ABSTRACT: Powder aerosols made of albumin, dipalmitoylphosphatidylcholine (DPPC) and a protein stabilizer (lactose, trehalose or mannitol) were prepared by spray-drying and analyzed for aerodynamic behavior, surface composition and physical state. The powders exited a Spinhaler inhaler as particle aggregates, the size of which depending on composition, spray-drying parameters and airflow rate. However, due to low bulk powder tap density (<0.15 g/cm3), the aerodynamic size of a large fraction of aggregates remained respirable (<5 microm). Fine particle fractions ranged between 21% and 41% in an Andersen cascade impactor operated at 28.3 l/min, with mannitol and lactose providing the most cohesive and free-flowing powders, respectively. Particle surface analysis by X-ray photoelectron spectroscopy (XPS) revealed a surface enrichment with DPPC relative to albumin for powders prepared under certain spray-drying conditions. DPPC self-organized in a gel phase in the particle and no sugar or mannitol crystals were detected by X-ray diffraction. Water sorption isotherms showed that albumin protected lactose from moisture-induced crystallization. In conclusion, a proper combination of composition and spray-drying parameters allowed to obtain dry powders with elevated fine particle fractions (FPFs) and a physical environment favorable to protein stability.
Journal of Controlled Release 10/2004; 99(3):357-67. · 7.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this work was to optimize the absorption of parathyroid hormone 1-34 (PTH) from the lungs by determining factors favoring its transport from the air spaces into the bloodstream. We simultaneously conducted pharmacokinetic and regional lung deposition studies in vivo in the rat following intratracheal administration of PTH in solution or dry powder form. Dry powders of PTH or albumin were prepared by spray-drying using lactose and dipalmitoylphosphatidylcholine (DPPC). Deposition in the trachea, peripheral, and central lobe sections was assessed after tissue grinding using albumin as a marker. The method of intratracheal instillation had a significant impact on PTH absorption from the lungs, and the deeper the deposition within the respiratory tract, the higher the absorption. Inhalation of the PTH powder resulted in high systemic bioavailability despite deposition of the formulation principally in upper airways. We demonstrated that the increased absorption resulted from DPPC that had permeation enhancer properties even though it was abundantly present locally in pulmonary surfactant. Optimization of PTH absorption from the lungs could be attained by targeting the peripheral lungs as well as codelivering DPPC.
Journal of Pharmaceutical Sciences 06/2004; 93(5):1241-52. · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We demonstrate that a primary source of elimination of inhaled macromolecules after delivery to the lungs and before absorption into the systemic circulation owes to clearance by alveolar macrophages (AM). Depletion of AM by liposome-encapsulated dichloromethylene diphosphonate is shown to cause severalfold enhancement in systemic absorption of IgG and human chorionic gonadotropin after intratracheal instillation in rats. Lowering the doses of IgG delivered to the lungs alleviates local degradation and results in a dramatic increase in systemic absorption of the protein as well. Chemical and physical means of minimizing uptake of macromolecules by AM are proposed as novel methods for enhancing protein absorption from the lungs. Such strategies may have important ramifications on the development of inhalation as an attractive mode of administration of therapeutic proteins to the bloodstream.