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.

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.

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.

Activity of Antibiotics against Staphylococcus aureus in an In Vitro Model of Biofilms in the Context of Cystic Fibrosis: Influence of the Culture Medium.

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.

pH homeostasis in yeast; the phosphate perspective.

Recent research further clarified the molecular mechanisms that link nutrient signaling and pH homeostasis with the regulation of growth and survival of the budding yeast Saccharomyces cerevisiae. The central nutrient signaling kinases PKA, TORC1, and Sch9 are intimately associated to pH homeostasis, presumably allowing them to concert far-reaching phenotypical repercussions of nutritional cues. To exemplify such repercussions, we briefly describe consequences for phosphate uptake and signaling and outline interactions between phosphate homeostasis and the players involved in intra- and extracellular pH control. Inorganic phosphate uptake, its subcellular distribution, and its conversion into polyphosphates are dependent on the proton gradients created over different membranes. Conversely, polyphosphate metabolism appears to contribute in determining the intracellular pH. Additionally, inositol pyrophosphates are emerging as potent determinants of growth potential, in this way providing feedback from phosphate metabolism onto the central nutrient signaling kinases. All these data point towards the importance of phosphate metabolism in the reciprocal regulation of nutrient signaling and pH homeostasis.