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.

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.

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.

The TORC1-Sch9 pathway as a crucial mediator of chronological lifespan in the yeast Saccharomyces cerevisiae.

The concept of ageing is one that has intrigued mankind since the beginning of time and is now more important than ever as the incidence of age-related disorders is increasing in our ageing population. Over the past decades, extensive research has been performed using various model organisms. As such, it has become apparent that many fundamental aspects of biological ageing are highly conserved across large evolutionary distances. In this review, we illustrate that the unicellular eukaryotic organism Saccharomyces cerevisiae has proven to be a valuable tool to gain fundamental insights into the molecular mechanisms of cellular ageing in multicellular eukaryotes. In addition, we outline the current knowledge on how downregulation of nutrient signaling through the target of rapamycin (TOR)-Sch9 pathway or reducing calorie intake attenuates many detrimental effects associated with ageing and leads to the extension of yeast chronological lifespan. Given that both TOR Complex 1 (TORC1) and Sch9 have mammalian orthologues that have been implicated in various age-related disorders, unraveling the connections of TORC1 and Sch9 with yeast ageing may provide additional clues on how their mammalian orthologues contribute to the mechanisms underpinning human ageing and health.

pH homeostasis links the nutrient sensing PKA/TORC1/Sch9 ménage-à-trois to stress tolerance and longevity.

The plasma membrane H+-ATPase Pma1 and the vacuolar V-ATPase act in close harmony to tightly control pH homeostasis, which is essential for a vast number of physiological processes. As these main two regulators of pH are responsive to the nutritional status of the cell, it seems evident that pH homeostasis acts in conjunction with nutrient-induced signalling pathways. Indeed, both PKA and the TORC1-Sch9 axis influence the proton pumping activity of the V-ATPase and possibly also of Pma1. In addition, it recently became clear that the proton acts as a second messenger to signal glucose availability via the V-ATPase to PKA and TORC1-Sch9. Given the prominent role of nutrient signalling in longevity, it is not surprising that pH homeostasis has been linked to ageing and longevity as well. A first indication is provided by acetic acid, whose uptake by the cell induces toxicity and affects longevity. Secondly, vacuolar acidity has been linked to autophagic processes, including mitophagy. In agreement with this, a decline in vacuolar acidity was shown to induce mitochondrial dysfunction and shorten lifespan. In addition, the asymmetric inheritance of Pma1 has been associated with replicative ageing and this again links to repercussions on vacuolar pH. Taken together, accumulating evidence indicates that pH homeostasis plays a prominent role in the determination of ageing and longevity, thereby providing new perspectives and avenues to explore the underlying molecular mechanisms.

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.

The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability.

The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase) to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc) homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.

Yeast buddies helping to unravel the complexity of neurodegenerative disorders.

Neurodegenerative disorders have a profound effect on the quality of life of patients and their environment. However, the development of adequate therapies requires accurate understanding of the underlying disease pathogenesis. On that account, yeast models can play an important role, as they enable the elucidation of the mechanisms leading to neurodegenerative disorders. Furthermore, by using so-called humanized yeast systems, the findings in yeast can be interpolated to humans. In this review, we will give an overview of the current body of knowledge on the use of yeast models with regard to Huntington's, Parkinson's and Alzheimer's disease. In addition to the results, obtained with the baker's yeast Saccharomyces cerevisiae, we also consider the existing literature on the less common but promising fission yeast Schizosaccharomyces pombe.

The protein kinase Sch9 is a key regulator of sphingolipid metabolism in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae protein kinase Sch9 is an in vitro and in vivo effector of sphingolipid signaling. This study examines the link between Sch9 and sphingolipid metabolism in S. cerevisiae in vivo based on the observation that the sch9Δ mutant displays altered sensitivity to different inhibitors of sphingolipid metabolism, namely myriocin and aureobasidin A. Sphingolipid profiling indicates that sch9Δ cells have increased levels of long-chain bases and long-chain base-1 phosphates, decreased levels of several species of (phyto)ceramides, and altered ratios of complex sphingolipids. We show that the target of rapamycin complex 1-Sch9 signaling pathway functions to repress the expression of the ceramidase genes YDC1 and YPC1, thereby revealing, for the first time in yeast, a nutrient-dependent transcriptional mechanism involved in the regulation of sphingolipid metabolism. In addition, we establish that Sch9 affects the activity of the inositol phosphosphingolipid phospholipase C, Isc1, which is required for ceramide production by hydrolysis of complex sphingolipids. Given that sphingolipid metabolites play a crucial role in the regulation of stress tolerance and longevity of yeast cells, our data provide a model in which Sch9 regulates the latter phenotypes by acting not only as an effector but also as a regulator of sphingolipid metabolism.

Molecular mechanisms linking the evolutionary conserved TORC1-Sch9 nutrient signalling branch to lifespan regulation in Saccharomyces cerevisiae.

The knowledge on the molecular aspects regulating ageing in eukaryotic organisms has benefitted greatly from studies using the budding yeast Saccharomyces cerevisiae. Indeed, many aspects involved in the control of lifespan appear to be well conserved among species. Of these, the lifespan-extending effects of calorie restriction (CR) and downregulation of nutrient signalling through the target of rapamycin (TOR) pathway are prime examples. Here, we present an overview on the molecular mechanisms by which these interventions mediate lifespan extension in yeast. Several models have been proposed in the literature, which should be seen as complementary, instead of contradictory. Results indicate that CR mediates a large amount of its effect by downregulating signalling through the TORC1-Sch9 branch. In addition, we note that Sch9 is more than solely a downstream effector of TORC1, and documented connections with sphingolipid metabolism may be particularly interesting for future research on ageing mechanisms. As Sch9 comprises the yeast orthologue of the mammalian PKB/Akt and S6K1 kinases, future studies in yeast may continue to serve as an attractive model to elucidate conserved mechanisms involved in ageing and age-related diseases in humans.