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ispartof: AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE vol:207 issue:8 pages:E71-E72 ispartof: location:United States status: published

Conjugation to polyethylene glycol (PEG) is a widely used approach to improve the therapeutic value of proteins essentially by prolonging their body residence time. PEGylation may however induce changes in the structure and/or the stability of proteins and thus on their function(s). The effects of PEGylation on the thermodynamic stability can either be positive (stabilization), negative (destabilization), or neutral (no effect). Moreover, various factors such as the PEG length and PEGylation site can influence the consequences of PEGylation on the structure and stability of proteins. In this study, the effects of PEGylation on the structure, stability, and polymerization of alpha1-antitrypsin (AAT) were investigated, using PEGs with different lengths, different structures (linear or 2-armed) and different linking chemistries (via amine or thiol) at two distinct positions of the sequence. The results show that whatever the size, position, and structure of PEG chains, PEGylation (a) does not induce significant changes in AAT structure (either at the secondary or tertiary level); (b) does not alter the stability of the native protein upon both chemical- and heat-induced denaturation; and (c) does not prevent AAT to fully refold and recover its activity following chemical denaturation. However, the propensity of AAT to aggregate upon heat treatment was significantly decreased by PEGylation, although PEGylation did not prevent the irreversible inactivation of the enzyme. Moreover, conjugation to PEG, especially 2-armed 40 kDa PEG, greatly improved the proteolytic resistance of AAT. PEGylation of AAT could be a promising strategy to prolong its half-life after infusion in AAT-deficient patients and thereby decrease the frequency of infusions.

Vaccination via the pulmonary route could be an attractive alternative to parenteral administration. Research towards the best site of antigen deposition within the lungs to induce optimal immune responses has conflicting results which might be dependent on the type of vaccine and/or its physical state. Therefore, in this study, we explored whether deep lung deposition is crucial for two different vaccines, i.e., influenza and hepatitis B vaccine. In view of this, influenza subunit vaccine and hepatitis B surface antigen were labeled with a fluorescent dye and then spray-dried. Imaging data showed that after pulmonary administration to mice the powders were deposited in the trachea/central airways when a commercially available insufflator was used while deep lung deposition was achieved when an in-house built aerosol generator was used. Immunogenicity studies revealed that comparable immune responses were induced upon trachea/central airways or deep lung targeting of dry influenza vaccine formulations. However, for hepatitis B vaccine, no immune responses were induced by trachea/central airways deposition whereas they were considerable after deep lung deposition. Thus, we conclude that deep lung targeting is not a critical parameter for the efficacy of pulmonary administered influenza vaccine whereas for hepatitis B vaccine it is., Graphical abstract The influence of pulmonary deposition on immune response was investigated for influenza and hepatitis B (Hep-B) vaccine candidates. Powder vaccines were targeted to different regions of the respiratory tract by Penn-insufflator (commercial) and in-house aerosol generator. Site of deposition was of minor relevance for influenza but of major importance for Hep-B vaccine.Image 1

Background Li-ion batteries (LIB) are increasingly used worldwide. They are made of low solubility micrometric particles, implying a potential for inhalation toxicity in occupational settings and possibly for consumers. LiCoO2 (LCO), one of the most used cathode material, induces inflammatory and fibrotic lung responses in mice. LCO also stabilizes hypoxia-inducible factor (HIF) -1α, a factor implicated in inflammation, fibrosis and carcinogenicity. Here, we investigated the role of cobalt, nickel and HIF-1α as determinants of toxicity, and evaluated their predictive value for the lung toxicity of LIB particles in in vitro assays. Results By testing a set of 5 selected LIB particles (LCO, LiNiMnCoO2, LiNiCoAlO2) with different cobalt and nickel contents, we found a positive correlation between their in vivo lung inflammatory activity, and (i) Co and Ni particle content and their bioaccessibility and (ii) the stabilization of HIF-1α in the lung. Inhibition of HIF-1α with chetomin or PX-478 blunted the lung inflammatory response to LCO in mice. In IL-1β deficient mice, HIF-1α was the upstream signal of the inflammatory lung response to LCO. In vitro, the level of HIF-1α stabilization induced by LIB particles in BEAS-2B cells correlated with the intensity of lung inflammation induced by the same particles in vivo. Conclusions We conclude that HIF-1α, stabilized in lung cells by released Co and Ni ions, is a mechanism-based biomarker of lung inflammatory responses induced by LIB particles containing Co/Ni. Documenting the Co/Ni content of LIB particles, their bioaccessibility and their capacity to stabilize HIF-1α in vitro can be used to predict the lung inflammatory potential of LIB particles.

Conjugation of recombinant human deoxyribonuclease I (rhDNase) to polyethylene glycol (PEG) of 20 to 40 kDa was previously shown to prolong the residence time of rhDNase in the lungs of mice after pulmonary delivery while preserving its full enzymatic activity. This work aimed to study the fate of native and PEGylated rhDNase in the lungs and to elucidate their biodistribution and elimination pathways after intratracheal instillation in mice. In vivo fluorescence imaging revealed that PEG30 kDa-conjugated rhDNase (PEG30-rhDNase) was retained in mouse lungs for a significantly longer period of time than native rhDNase (12 days vs 5 days). Confocal microscopy confirmed the presence of PEGylated rhDNase in lung airspaces for at least 7 days. In contrast, the unconjugated rhDNase was cleared from the lung lumina within 24 h and was only found in lung parenchyma and alveolar macrophages thereafter. Systemic absorption of intact rhDNase and PEG30-rhDNase was observed. However, this was significantly lower for the latter. Catabolism, primarily in the lungs and secondarily systemically followed by renal excretion of byproducts were the predominant elimination pathways for both native and PEGylated rhDNase. Catabolism was nevertheless more extensive for the native protein. On the other hand, mucociliary clearance appeared to play a less prominent role in the clearance of those proteins after pulmonary delivery. The prolonged presence of PEGylated rhDNase in lung airspaces appears ideal for its mucolytic action in patients with cystic fibrosis.

Alpha-1 antitrypsin (AAT) is an endogenous inhibitor of serine proteases which, in physiological conditions, neutralizes the excess of neutrophil elastase and other serine proteases in tissues and especially the lungs. Weekly intravenous infusion of plasma-purified human AAT is used to treat AAT deficiency-associated lung disease. However, only 2 % of the AAT dose reach the lungs after intravenous infusion. Inhalation of AAT might offer an alternative route of administration. Yet, the rapid clearance of AAT from the respiratory tract results in high and frequent dosing by inhalation and limited efficacy. In the present study, we produced and characterized in vitro a PEGylated version of AAT which could offer a prolonged body residence time and thereby be useful for augmentation therapy by the intravenous and inhalation routes. Two PEGylation reactions - N-terminal and thiol PEGylation - and three polyethylene glycol (PEG) chains - linear 30 kDa, linear 40 kDa and 2-armed 40 kDa - were used. The yields of mono-PEGylated AAT following purification by anion exchange chromatography were 40-50 % for N-terminal PEGylation and 60-70% for thiol PEGylation. The PEG-AAT conjugates preserved the ability to form a protease-inhibitor complex with neutrophil elastase and proteinase 3 as well as the full inhibitory capacity to neutralize neutrophil elastase activity. These results open up interesting prospects for PEGylated AAT to achieve a prolonged half-life and an improved therapeutic efficacy in vivo.

Highly viscous mucus and its impaired clearance characterize the lungs of patients with cystic fibrosis (CF). Pulmonary secretions of patients with CF display increased concentrations of high molecular weight components such as DNA and actin. Recombinant human deoxyribonuclease I (rhDNase) delivered by inhalation cleaves DNA filaments contained in respiratory secretions and thins them. However, rapid clearance of rhDNase from the lungs implies a daily administration and thereby a high therapy burden and a reduced patient compliance. A PEGylated version of rhDNase could sustain the presence of the protein within the lungs and reduce its administration frequency. Here, we evaluated the enzymatic activity of rhDNase conjugated to a two-arm 40 kDa polyethylene glycol (PEG40) in CF sputa. Rheology data indicated that both rhDNase and PEG40-rhDNase presented similar mucolytic activity in CF sputa, independently of the purulence of the sputum samples as well as of their DNA, actin and ions contents. The macroscopic appearance of the samples correlated with the DNA content of the sputa: the more purulent the sample, the higher the DNA concentration. Finally, quantification of the enzymes in CF sputa following rheology measurement suggests that PEGylation largely increases the stability of rhDNase in CF respiratory secretions, since 24-fold more PEG40-rhDNase than rhDNase was recovered from the samples. The present results are considered positive and provide support to the continuation of the research on a long acting version of rhDNase to treat CF lung disease. ispartof: CLINICAL SCIENCE vol:132 issue:13 pages:1439-1452 ispartof: location:England status: published

Pulmonary administration of anti-cytokine antibodies offers a targeted therapy in asthma. However, the rapid elimination of proteins from the lungs limits the efficacy of inhaled medications. PEGylation has been shown to increase the residence time of anti-interleukin (IL)-17A and anti-IL-13 antibody fragments in the lungs and to improve their therapeutic efficacy. Yet, little is known about the factors that affect the residence time of PEGylated antibody fragments in the lungs following pulmonary delivery. In this study, we showed that the molecular weight of polyethylene glycol (PEG), 20kDa or 40kDa, had a moderate effect on the residence time of an anti-IL-17A Fab' fragment in the lungs of mice. By contrast, the site of delivery of the anti-IL-17A and anti-IL-13 Fab' fragments within the lungs had a major impact on their residence time, with the deeper the delivery, the more prolonged the residence time. The nature of the Fab' fragment had an influence on its residence time as well and the anti-IL-17A Fab' benefited more from PEGylation than the anti-IL-13 Fab' did. Acute lung inflammation slightly shortened the residence time of the anti-IL-17A and anti-IL-13 Fab' fragments in the lungs but PEGylation was able to prolong their presence in both the healthy and inflamed lungs. Antibody fragments were predominately located within the airway lumen rather than the lung parenchyma. Transport experiments on monolayers of Calu-3 cells and studies of fluorescence recovery after photobleaching in respiratory mucus showed that mechanisms involved in the prolonged presence of PEGylated Fab' in the airway lumen might include binding to the mucus, reduced uptake by respiratory cells and reduced transport across lung epithelia. Finally, using I125-labeled anti-IL-17A Fab', we showed that the protein fragment hardly penetrated into the lungs following subcutaneous injection, as opposed to pulmonary delivery.

More and more therapeutic proteins are developed for an administration by inhalation to treat respiratory diseases. PEGylation is an interesting approach for sustaining the residence time of these biopharmaceuticals in the lungs and thereby decrease the frequency of administration and the daily burden of inhalation therapies. Several PEGylated proteins have been delivered to the lungs in rodents and shown to be retained in the respiratory tract for longer periods than unconjugated counterparts. Mechanisms involved in their pulmonary retention might include increased molecular size, mucoadhesion, enhanced proteolytic resistance and escape from the uptake by alveolar macrophages. Pulmonary delivery of PEGylated peptides and proteins is also interesting as a non-invasive route of administration of long-acting biopharmaceuticals to the bloodstream. However, PEGylation decreases the systemic absorption of the compounds, especially when the PEG size is large. This review presents the recent work carried out on the pulmonary delivery of PEGylated biopharmaceuticals, the factors affecting residence time in the lungs and systemic absorption as well as the safety of the approach in preclinical studies.

The PEGylation of antibody fragments has been shown to greatly prolong their residence time in the lungs in mice. The purpose of this research was to confirm the effect of PEGylation in higher animal species, that is, the rat and the rabbit. An anti-IL-17A Fab' antibody fragment was conjugated to a two-armed 40kDa polyethylene glycol (PEG) via site-selective thiol PEGylation. PEGylation did not significantly alter the binding activity of the Fab' fragment but it largely enhanced its inhibitory potency. PEGylation increased the residence time of the Fab' in the lungs of mice, rats and rabbits. Following intratracheal administration, the unconjugated Fab' was cleared from the lungs within 24h while large quantities of the PEGylated Fab' remained present up to 48h. No significant differences in clearance were noted between the three animal species although there was a tendency of longer residence time in higher species. PEGylation represents a promising approach to sustain the presence of antibody fragments in the lungs and to enhance their therapeutic efficacy in respiratory diseases.

The WST-1 assay is the most common test to assess the in vitro cytotoxicity of chemicals. Tetrazolium-based assays can, however, be affected by the interference of tested chemicals, including carbon nanotubes or Mg particles. Here, we report a new interference of Mn materials with the WST-1 assay. Endothelial cells exposed to Mn particles (Mn alone or Fe-Mn alloy from 50 to 1600 μg/ml) were severely damaged according to the WST-1 assay, but not the ATP content assay. Subsequent experiments revealed that Mn particles interfere with the reduction of the tetrazolium salt to formazan. Therefore, the WST-1 assay is not suitable to evaluate the in vitro cytotoxicity of Mn-containing materials, and luminescence-based assays such as CellTiter-Glo® appear more appropriate.

Staphylococcus aureus is a highly prevalent pathogen in the respiratory tract of young patients with cystic fibrosis (CF) and causes biofilm-related infections. Here, we set up an in vitro model of a biofilm grown in Trypticase soy broth supplemented with glucose and NaCl (TGN) or in artificial sputum medium (ASM) and used it to evaluate on a pharmacodynamic basis the activity of antibiotics used in CF patients and active on staphylococci (meropenem, vancomycin, azithromycin, linezolid, rifampin, ciprofloxacin, tobramycin). Rheological studies showed that ASM was more elastic than viscous, as was also observed for sputa from CF patients, with elastic and viscous moduli being, respectively, similar to and slightly lower than those of CF sputa. Biofilms formed by methicillin-sensitive S. aureus strain ATCC 25923 and methicillin-resistant S. aureus strain ATCC 33591 reached maturity after 24 h, with biomass (measured by crystal violet staining) and metabolic activity (assessed by following resazurin metabolization) being lower in ASM than in TGN and viability (assessed by bacterial counts) being similar in both media. Full concentration-response curves of antibiotics obtained after 24 h of incubation of biofilms showed that all antibiotics were drastically less potent and less efficient in ASM than in TGN toward viability, metabolic activity, and biomass. Tobramycin selected for small-colony variants, specifically in biofilms grown in ASM; the auxotrophism of these variants could not be established. These data highlight the major influence exerted by the culture medium on S. aureus responsiveness to antibiotics in biofilms. The use of ASM may help to determine effective drug concentrations or to evaluate new therapeutic options against biofilms in CF patients.

Graphical abstract, PEGylation is a promising approach to increase the residence time of antibody fragments in the lungs and sustain their therapeutic effects. However, concerns arise as to the potential pulmonary toxicity of antibody fragments conjugated to high molecular weight (HMW) polyethylene glycol (PEG), notably after repeated administrations, and the possibility of PEG accumulation in the lungs. The purpose of this proof-of-concept study is to give insights about the safety of lung administration of a Fab’ anti-IL17A antibody fragment conjugated to two-armed 40 kDa PEG (PEG40). The presence of the PEG40 moiety inside alveolar macrophages remained stable for at least 24 h after intratracheal administration of PEG40-Fab’ to mice. PEG40 was then progressively cleared from alveolar macrophages. Incubation of PEG40 alone with macrophages in vitro did not significantly harm macrophages and did not affect phagocytosis or the production of inflammatory markers. After acute or chronic administration of PEG40-Fab’ to mice, no signs of significant pulmonary toxicity or inflammatory cell accumulation were observed. A vacuolization of alveolar macrophages not associated with any inflammation was noticed when PEG40, PEG40-Fab’, or unPEGylated Fab’ were administered. To conclude this preliminary proof of concept study, acute or repeated pulmonary administrations of PEGylated Fab’ appear safe in rodents.

The purpose of this study was to assess whether cationic nanoliposomes could address tumor vaccines to dendritic cells in the lungs in vivo. Nanoliposomes were prepared using a cationic lipid, dimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol) or dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the most abundant phospholipid in lung surfactant. The liposomes presented a size below 175 nm and they effectively entrapped tumor antigens, an oligodeoxynucletotide containing CpG motifs (CpG) and the fluorescent dye calcein used as a tracer. Although the liposomes could permanently entrap a large fraction of the actives, they could not sustain their release in vitro. Liposomes made of DOTAP were safe to respiratory cells in vitro, while liposomes composed of DC-cholesterol were cytotoxic. DOTAP nanoliposomes were mainly taken up by alveolar macrophages following delivery to the lungs in mice. Few dendritic cells took up the liposomes, and interstitial macrophages did not take up liposomal calcein more than they took up soluble calcein. Stimulation of the innate immune system using liposomal CpG strongly enhanced uptake of calcein liposomes by all phagocytes in the lungs. Although a small percentage of dendritic cells took up the nanoliposomes, alveolar macrophages represented a major barrier to dendritic cell access in the lungs.

Pulmonary delivery offers an attractive route of administration for chemotherapeutic agents, with the advantages of high drug concentrations locally and low side effects systemically. However, fast clearance mechanisms result in short residence time of small molecule drugs in the lungs. Moreover, the local toxicity induced by antineoplastic drugs is considered a major obstacle for the clinical application of inhaled chemotherapy. In this study, we explored the utility of 6 kDa and 20 kDa polyethylene glycol-paclitaxel (PEG-PTX) conjugates to retain paclitaxel within the lungs, achieve its sustained release locally, and thereby, improve its efficacy and reduce its pulmonary toxicity. The conjugates increased the maximum tolerated dose of paclitaxel by up to 100-fold following intratracheal instillation in healthy mice. PEG-PTX conjugates induced lung inflammation. However, the inflammation was lower than that induced by an equivalent dose of the free drug and it was reversible. Conjugation of paclitaxel to both PEG sizes significantly enhanced its anti-tumor efficacy following intratracheal instillation of a single dose in a Lewis lung carcinoma model in mice. PEG-PTX 20k showed equivalent efficacy as PEG-PTX 6k delivered at a 2.5-fold higher dose, suggesting that the molecular weight of the conjugate plays a role in anti-cancer activity. PEG-PTX 20k conjugate presented a prolonged residency and a sustained paclitaxel release within the lungs. This study showed that PEGylation of paclitaxel offers a potential delivery system for inhalation with improved anti-cancer efficacy, prolonged exposure of lung-resident tumors to the antineoplastic drug and reduced local toxicity.

International audience; Rechargeable Li-ion batteries (LIB) are increasingly produced and used worldwide. LIB electrodes are made of micrometric and low solubility particles, consisting of toxicologically relevant elements. The health hazard of these materials is not known. Here, we investigated the respiratory hazard of three leading LIB components (LiFePO4 or LFP, Li4Ti5O12 or LTO, and LiCoO2 or LCO) and their mechanisms of action. Particles were characterized physico-chemically and elemental bioaccessibility was documented. Lung inflammation and fibrotic responses, as well as particle persistence and ion bioavailability, were assessed in mice after aspiration of LIB particles (0.5 or 2 mg); crystalline silica (2 mg) was used as reference. Acute inflammatory lung responses were recorded with the 3 LIB particles and silica, LCO being the most potent. Inflammation persisted 2 m after LFP, LCO and silica, in association with fibrosis in LCO and silica lungs. LIB particles persisted in the lungs after 2 m. Endogenous iron co-localized with cobalt in LCO lungs, indicating the formation of ferruginous bodies. Fe and Co ions were detected in the broncho-alveolar lavage fluids of LFP and LCO lungs, respectively. Hypoxia-inducible factor (HIF) -1α, a marker of fibrosis and of the biological activity of Co ions, was upregulated in LCO and silica lungs. This study identified, for the first time, the respiratory hazard of LIB particles. LCO was at least as potent as crystalline silica to induce lung inflammation and fibrosis. Iron and cobalt, but not lithium, ions appear to contribute to LFP and LCO toxicity, respectively.

Administration of influenza vaccines via the respiratory tract has potential benefits over conventional parenteral administration, inducing immunity directly at the site of influenza exposure as well as being needle free. In this study, we investigated the suitability of Advax™, a stable particulate polymorph of inulin, also referred to as delta inulin, as a mucosal adjuvant for whole inactivated influenza vaccine (WIV) administered either as a liquid or dry powder formulation. Spray freeze-drying produced Advax-adjuvanted WIV powder particles in a size range (1–5 μm) suitable for inhalation. The physical and biological characteristics of both WIV and Advax remained unaltered both by admixing WIV with Advax and by spray freeze drying. Upon intranasal or pulmonary immunization, both liquid and dry powder formulations containing Advax induced significantly higher systemic, mucosal and cellular immune responses than non-adjuvanted WIV formulations. Furthermore, pulmonary immunization with Advax-adjuvanted WIV led to robust memory B cell responses along with an increase of lung localization factors i.e. CXCR3, CD69, and CD103. A less pronounced but still positive effect of Advax was seen on memory T cell responses. In contrast to animals immunized with WIV alone, all animals pulmonary immunized with a single dose of Advax-adjuvanted WIV were fully protected with no visible clinical symptoms against a lethal dose of influenza virus. These data confirm that Advax is a potent mucosal adjuvant that boosts vaccine-induced humoral and cellular immune responses both in the lung and systemically with major positive effects on B-cell memory and complete protection against live virus. Hence, respiratory tract immunization, particularly via the lungs, with Advax-adjuvanted WIV formulation as a liquid or dry powder is a promising alternative to parenteral influenza vaccination., Graphical abstract Unlabelled Image, Highlights • Effective Advax-adjuvanted powder influenza vaccine formulation can be prepared. • Advax enhances B and T cell immune responses to influenza vaccine. • Complete protection with Advax-adjuvanted influenza vaccine by pulmonary route. • Advax elicits robust B and T cell memory responses upon pulmonary administration. • Advax is a potent mucosal adjuvant.

Administration of antibiotics by inhalation can greatly improve drug targeting to the site of respiratory infections. In addition, dry powder inhalers are particularly convenient for the patients. The purposes of this study were to demonstrate the interest of pulmonary temocillin delivery to reach high temocillin concentrations locally in the lungs as well as to prepare a spray-dried temocillin powder for inhalation using a minimal amount of generally recognized as safe excipients. Intratracheal instillation of a temocillin solution allowed to reach higher and more sustained drug concentrations in the lungs than intravenous injection in mice, although a 10-fold lower temocillin dose was delivered intratracheally than systemically. A spray-dried powder of pure temocillin presented a fine particle fraction of 9% of the dose loaded in the inhaler. However, the incorporation of 0.5% to 20% of dipalmitoylphosphatidylcholine (DPPC) in the powder increased the fine particle fraction 4- to 5-fold. X-ray photoelectron spectroscopy and X-ray diffraction revealed that DPPC concentrated at the particle surface with its aliphatic chains laterally packed. The minimal amount of DPPC needed to improve the aerosol performance of temocillin supports the use of this excipient in the formulation of cohesive antibiotic powders for inhalation.

Rechargeable Li-ion batteries (LIB) are increasingly produced and used worldwide. LIB electrodes are made of micrometric and low solubility particles, consisting of toxicologically relevant elements. The health hazard of these materials is not known. Here, we investigated the respiratory hazard of three leading LIB components (LiFePO

Recombinant human deoxyribonuclease I (rhDNase) is the mucolytic agent most widely used for the treatment of respiratory disease in cystic fibrosis. However, rhDNase is rapidly cleared from the lungs which implies a high dosing frequency and limited patient adherence. The aim of this study was to produce a long-acting PEGylated derivative of rhDNase presenting a preserved enzymatic activity. Site-specific PEGylation on the N-terminal (N-ter) leucine residue of rhDNase was achieved by reductive alkylation at acidic pH using linear 20kDa, linear 30kDa or two-arm 40kDa polyethylene glycol (PEG) propionaldehydes. Yields of mono-PEGylated products ranged between 45% and 61%. Conjugation to PEG fully preserved the secondary structure and the in vitro enzymatic activity of the native protein. These properties offer interesting perspectives for in vivo inhalation studies of the PEGylated enzyme.

Drug delivery to the lungs by inhalation offers a targeted drug therapy for respiratory diseases. However, the therapeutic efficacy of inhaled drugs is limited by their rapid clearance in the lungs. Carriers providing sustained drug release in the lungs can improve therapeutic outcomes of inhaled medicines because they can retain the drug load within the lungs and progressively release the drug locally at therapeutic levels. This review presents the different formulation strategies developed to control drug release in the lungs including microparticles and the wide array of nanomedicines. Large and porous microparticles offer excellent aerodynamic properties. Their large geometric size reduces their uptake by alveolar macrophages, making them a suitable carrier for sustained drug release in the lungs. Similarly, nanocarriers present significant potential for prolonged drug release in the lungs because they largely escape uptake by lung-surface macrophages and can remain in the pulmonary tissue for weeks. They can be embedded in large and porous microparticles in order to facilitate their delivery to the lungs. Conjugation of drugs to polymers as polyethylene glycol can be particularly beneficial to sustain the release of proteins in the lungs as it allows high protein loading. Drug conjugates can be readily delivered to respiratory airways by any current nebulizer device. Nonetheless, liposomes represent the formulation most advanced in clinical development. Liposomes can be prepared with lipids endogenous to the lungs and are particularly safe. Their composition can be adjusted to modulate drug release and they can encapsulate both hydrophilic and lipophilic compounds with high drug loading.

The aim of this study was to maximize the yield of the production of mono-PEGylated anti-interleukin-17A (anti-IL-17A) antibody fragments using large (≥ 20 kDa) polyethylene glycol (PEG) chains. Particular attention was paid to selectively yield mono-PEGylated species to maintain the maximum possible functionality and to simplify the purification. Neutralization of IL-17A by antibody constructs might find application for the treatment of bronchial hyperreactivity. Amino-directed and sulfhydryl-directed PEGylation of the native antibody fragments were compared. The former was selected as it produced the most interesting construct in terms of yield and preservation of biological activity. In particular, the F(ab')2-PEG conjugate with one 40 kDa branched PEG prepared in this study was produced at a 42% yield. The conjugate presented only a slight decrease in its binding activity and in its in vitro inhibitory potency offering interesting perspectives for in vivo studies.

The current Bacille Calmette-Guérin vaccine provides variable protection against tuberculosis and new vaccination approaches are urgently needed. Pulmonary vaccination could be the best way to induce a protective immunity against Mycobacterium tuberculosis as it targets its natural site of infection. The aim of this study was to investigate the potential of Poloxamer 407 (P407) combined with a CpG oligonucleotide (CpG) to enhance immune responses to M. tuberculosis antigen 85A (Ag85A) following pulmonary delivery in BALB/c mice. An additional goal of this study was to localize the optimal delivery site of Ag85A within the lungs for generating the most intense immunity. We also investigated the capacity of P407 to prolong the residence time of the antigen within the lungs and we studied the safety of the adjuvants following pulmonary delivery. Targeting the antigen to the deep lungs produced more intense specific immune responses than targeting it to the upper airways. P407 and CpG further increased humoral immune responses and splenocyte proliferation in vitro. CpG strongly increased the Th-1 immune response with high IgG2a/IgG1 ratio, high IFN-γ and TNF-α productions by spleen mononuclear cells in vitro. P407 tended to induce a Th-2 response, as indicated by the slight decrease in IgG2a/IgG1 ratio and the slight increase in IL-5 levels. The combination of P407 and CpG produced the highest Th-1 and Th-17 responses by generating IFN-γ, TNF-α, IL-2, and IL-17A cytokines. Targeting the antigen to the deep lungs and the presence of P407 increased the residence time of the antigen within the lungs. This might explain the enhancement of immune responses induced by these factors. CpG did not induce inflammation in the lungs while P407 produced a reversible alteration of the alveolo-capillary barrier. Adding CpG to P407 did not further increase this alteration of the alveolo-capillary barrier. In conclusion, delivery of Ag85A formulated in a combination of P407 and CpG to the deep lungs induced strong immune responses, with a polyfunctional T cells phenotype.

Monoclonal antibody (mAb) therapeutics have tremendous potential to benefit patients with lung diseases, for which there remains substantial unmet medical need. To capture the current state of mAb research and development in the area of respiratory diseases, the Research Center of Respiratory Diseases (CEPR-INSERM U1100), the Laboratory of Excellence “MAbImprove,” the GDR 3260 “Antibodies and therapeutic targeting,” and the Grant Research program ARD2020 “Biotherapeutics” invited speakers from industry, academic and government organizations to present their recent research results at the Therapeutic Monoclonal Antibodies for Respiratory Diseases: Current challenges and perspectives congress held March 31 – April 1, 2016 in Tours, France.

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.

Pulmonary drug delivery is considered an attractive route of drug administration for lung cancer chemotherapy. However, fast clearance mechanisms result in short residence time of small molecule drugs in the lung. Therefore, achieving a sustained presence of chemotherapeutics in the lung is very challenging. In this study, we synthesized two different polyethylene glycol-paclitaxel ester conjugates with molecular weights of 6 and 20 kDa in order to achieve sustained release of paclitaxel in the lung. One structure was synthesized with azide linker using “click” chemistry and the other structure was synthesized with a succinic spacer. The physicochemical and biological properties of the conjugates were characterized in vitro. Conjugation to polyethylene glycol improved the solubility of paclitaxel by up to four orders of magnitude. The conjugates showed good stability in phosphate buffer saline pH 6.9 (half-life ≥72 h) and in bronchoalveolar lavage (half-life of 3 to 9 h) at both molecular weights, but hydrolyzed quickly in mouse serum (half-life of 1 to 3 h). The conjugates showed cytotoxicity to B16-F10 melanoma cells and LL/2 Lewis lung cancer cells but less than free paclitaxel or Taxol, the commercial paclitaxel formulation. These properties imply that the conjugates have the potential to retain paclitaxel in the lung for a prolonged duration and to sustain its release locally for a better efficacy.

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 24 h. Nanoparticles largely escape uptake by lung-surface macrophages and can remain in the lung for weeks, without significant translocation across respiratory epithelia. Major recent advances The fate of therapeutic proteins within the lungs has been partly revealed over the past 15 years. Yet, many recent studies have investigated the pulmonary fate of nanoparticles and have highlighted their persistence within the lung, their low uptake by lung-surface macrophages and their limited translocation across respiratory epithelia towards the systemic circulation.

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 B3, 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.

BACKGROUND: 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. OBJECTIVE: This review describes relevant drug delivery models for preclinical evaluation of inhaled drug products. METHODS: 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. RESULTS/CONCLUSION: 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.

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.

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.

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.

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.

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 (

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.

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.

The objectives of this work were (i) to validate electrical zone sensing and laser diffraction for the analysis of primary particle size in the case of inhalation dry powders and (ii) to study the influence of the aggregation state of the powder on the sizing techniques. Free-flowing dry powders were prepared by spray-drying with a combination of albumin, lactose, and dipalmitoylphosphatidylcholine. The replacement of lactose by mannitol, the removal of albumin, and the atomization at high relative humidity all increased powder cohesion. Automated measurements were compared with primary particle sizes collected by light and electron microscopy. The mass mode obtained by electrical zone sensing and the mass median diameter measured by laser diffraction following dispersion with compressed air at a pressure of 3 bar or following suspension in water and ultrasonic dispersion at a power of 60 W for 30 s each provided primary particle sizes close to microscopy measurements. However, these conditions only applied in the case of slightly to moderately aggregated powders. For strongly agglomerated powders, an exact measurement of the size was only collected by laser diffraction in the wet state combined with ultrasonic dispersion. Our study underlies how measurement of primary particle size highly depends on both powder material and proper particle dispersion.

The objective of this study was to determine the effects of formulation excipients and physical characteristics of inhalation particles on their in vitro aerosolization performance, and thereby to maximize their respirable fraction. Dry powders were produced by spray-drying using excipients that are FDA-approved for inhalation as lactose, materials that are endogenous to the lungs as albumin and dipalmitoylphosphatidylcholine (DPPC); and/or protein stabilizers as trehalose or mannitol. Dry powders suitable for deep lung deposition, i.e. with an aerodynamic diameter of individual particles

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.

Conventional slow-acting insulin preparations for subcutaneous injection, e.g., suspensions of the complex with protamine and/or zinc, were reformulated as dry powders for inhalation and the insoluble aerosol tested for providing sustained insulin plasma levels. Large porous particles made of lactose, albumin, and dipalmitoylphosphatidylcholine, and incorporating insulin, protamine, and/or zinc chloride were prepared using spray-drying. Integrity of insulin after spray-drying and insulin insolubilization in spraydried particles was verified in vitro. The pharmacokinetic profile of the formulation delivered by inhalation and subcutaneous injection was assessed in vivo in the rat. The formulation process of insulin as dry powders did not alter insulin integrity and did not impede, in most cases, insulin insolubilization by protamine and/or zinc. Large porous insulin particles presented 7 μm mass mean geometric particle diameters, 0.1 g/ cm3 bulk powder tap densities and theoretical aerodynamic diameters suitable for deep lung deposition (in the range of 2.2-2.5 μm). The dry powders exhibited 40% respirable fractions in the Andersen cascade impactor and 58-75% in the Aero-Breather. Insoluble inhaled insulin provided sustained insulin plasma levels for half a day, similar to injected insulin, and exhibited a bioavailability of 80.5% relative to subcutaneous injection of the same formulation.

A combined electrical (HV, "high voltage", pulsing) and chemical (topical sodium thiosulfate) intervention is hypothesized to create enlarged aqueous pathways that allow large quantities of macromolecules to be transported through human skin's stratum corneum (SC), the dominant barrier for transdermal drug delivery and biochemical analyte extraction. This expectation is based on the known structure and composition of the SC, and previous models and experiments for local transport regions (LTRs) due to transdermal HV pulsing. In vitro experiments demonstrated that transdermal macromolecule fluxes of 10(-9) to 10(-8) mol h(-1) cm(-2) (10 to 100 microg h(-1) cm(-2)) or greater are possible for lactalbumin and an antibody (IgG), which are potentially therapeutic values for peptides, proteins and nucleic acids. In the absence of sodium thiosulfate, only a small molecule (sulforhodamine) flux increased significantly, consistent with many previous studies. Significant macromolecule transdermal fluxes occurred only if a pathway enlarging molecule (sodium thiosulfate) was present. Our results also provide support for the mechanism hypothesis that HV pulses leading to transdermal voltages U(skin) > 50 V create straight-through aqueous pathways that penetrate multilamellar bilayer membranes, corneocyte envelopes and corneocyte interiors within the SC.