Nebulization of PEGylated recombinant human deoxyribonuclease I using vibrating membrane nebulizers: A technical feasibility study.

Recombinant human deoxyribonuclease I (rhDNase, Pulmozyme®) is the most frequently used mucolytic agent for the symptomatic treatment of cystic fibrosis (CF) lung disease. Conjugation of rhDNase to polyethylene glycol (PEG) has been shown to greatly prolong its residence time in the lungs and improve its therapeutic efficacy in mice. To present an added value over current rhDNase treatment, PEGylated rhDNase needs to be efficiently and less frequently administrated by aerosolization and possibly at higher concentrations than existing rhDNase. In this study, the effects of PEGylation on the thermodynamic stability of rhDNase was investigated using linear 20 kDa, linear 30 kDa and 2-armed 40 kDa PEGs. The suitability of PEG30-rhDNase to electrohydrodynamic atomization (electrospraying) as well as the feasibility of using two vibrating mesh nebulizers, the optimized eFlow® Technology nebulizer (eFlow) and Innospire Go, at varying protein concentrations were investigated. PEGylation was shown to destabilize rhDNase upon chemical-induced denaturation and ethanol exposure. Yet, PEG30-rhDNase was stable enough to withstand aerosolization stresses using the eFlow and Innospire Go nebulizers even at higher concentrations (5 mg of protein per ml) than conventional rhDNase formulation (1 mg/ml). High aerosol output (up to 1.5 ml per min) and excellent aerosol characteristics (up to 83% fine particle fraction) were achieved while preserving protein integrity and enzymatic activity. This work demonstrates the technical feasibility of PEG-rhDNase nebulization with advanced vibrating membrane nebulizers, encouraging further pharmaceutical and clinical developments of a long-acting PEGylated alternative to rhDNase for treating patients with CF.

Production and characterization of mono-PEGylated alpha-1 antitrypsin for augmentation therapy.

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.

PEGylation of recombinant human deoxyribonuclease I decreases its transport across lung epithelial cells and uptake by macrophages.

Conjugation to high molecular weight (MW ≥ 20 kDa) polyethylene glycol (PEG) was previously shown to largely prolong the lung residence time of recombinant human deoxyribonuclease I (rhDNase) and improve its therapeutic efficacy following pulmonary delivery in mice. In this paper, we investigated the mechanisms promoting the extended lung retention of PEG-rhDNase conjugates using cell culture models and lung biological media. Uptake by alveolar macrophages was also assessed in vivo. Transport experiments showed that PEGylation reduced the uptake and transport of rhDNase across monolayers of Calu-3 cells cultured at an air-liquid interface. PEGylation also decreased the uptake of rhDNase by macrophages in vitro whatever the PEG size as well as in vivo 4 h following intratracheal instillation in mice. However, the reverse was observed in vivo at 24 h due to the higher availability of PEGylated rhDNase in lung airways at 24 h compared with rhDNase, which is cleared faster. The uptake of rhDNase by macrophages was dependent on energy, time, and concentration and occurred at rates indicative of adsorptive endocytosis. The diffusion of PEGylated rhDNase in porcine tracheal mucus and cystic fibrosis sputa was slower compared with that of rhDNase. Nevertheless, no significant binding of PEGylated rhDNase to both media was observed. In conclusion, decreased transport across lung epithelial cells and uptake by macrophages appear to contribute to the longer retention of PEGylated rhDNase in the lungs.

Biodistribution and elimination pathways of PEGylated recombinant human deoxyribonuclease I after pulmonary delivery in mice.

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.

The influence of organic modification on the cytotoxicity of clay particles to keratinocytes, hepatocytes and macrophages; an investigation towards the safe use of polymer-clay nanocomposite packaging.

Organically modified clays can be used as nanofillers in polymer-clay nanocomposites to create bio-based packaging with improved strength and barrier properties. The impact of organic modification on the physico-chemical properties and toxicity of clays has yet to be fully investigated but is essential to ensure their safe use. Two organoclays, named N116_HDTA and N116_TMSA, were prepared using a commercially available sodium bentonite clay and the organic modifiers hexadecyl trimethyl ammonium bromide (HDTA) and octadecyl trimethyl ammonium chloride (TMSA). An in vitro hazard assessment was performed using HaCaT skin cells, C3A liver cells, and J774.1 macrophage-like cells. Organic modification with HDTA and TMSA increased the hazard potential of the organoclays in all cell models, as evidenced by the higher levels of cytotoxicity measured. N116_TMSA caused the greatest loss in viability with IC50 values of 3.2, 3.6 and 6.1 µg/cm2 calculated using J774.1, HaCaT and C3A cell lines, respectively. Cytotoxic effects were dictated by the amount of free or displaced organic modifier present in the exposure suspensions. The parent bentonite clay also caused distinct cytotoxic effects in J774.1 macrophage-like cells with associated TNF-α release. Such information on the hazard profile of organoclays, can feed into risk assessments for these materials.