Mechanisms of retention of PEGylated recombinant human deoxyribonuclease i (rhDNase) in the lungs

Conjugation of recombinant human deoxyribonuclease I (rhDNase) to large (≥ 20 kDa) polyethylene glycol (PEG) has been shown to prolong the lung residence time of the enzyme in mice. Here, we investigated the mechanisms of this extended retention of PEG-rhDNase conjugates. PEG-rhDNase conjugates were detected in lung airspaces of mice for a longer time, at least 7 days post intratracheal instillation in the case of PEG30-rhDNase compared to ≤ 24 h for rhDNase. PEG30-rhDNase was significantly less absorbed than rhDNase in vivo from the lungs and in vitro across monolayers of lung epithelial cells. The uptake of PEGylated rhDNase by macrophages was delayed in vitro and in vivo whatever the PEG size. Moreover, local degradation in the lungs was observed for both proteins; however, more extensively for rhDNase. Finally, PEGylation did not seem to affect the mucociliary clearance of rhDNase significantly. In conclusion, a decrease in cell uptake and systemic absorption play a significant role in the extended 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

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.</p

Mechanisms of retention of PEGylated recombinant human deoxyribonuclease I (rhDNase) in the lungs

Conjugation of recombinant human deoxyribonuclease I (rhDNase) to large (≥ 20 kDa) polyethylene glycol (PEG) has been shown to prolong the lung residence time of the enzyme in mice. Here, we investigated the mechanisms of this extended retention of PEG-rhDNase conjugates. PEG-rhDNase conjugates were detected in lung airspaces of mice for a longer time, at least 7 days post intratracheal instillation in the case of PEG30-rhDNase compared to ≤ 24 h for rhDNase. PEG30-rhDNase was significantly less absorbed than rhDNase in vivo from the lungs and in vitro across monolayers of lung epithelial cells. The uptake of PEGylated rhDNase by macrophages was delayed in vitro and in vivo whatever the PEG size. Moreover, local degradation in the lungs was observed for both proteins; however, more extensively for rhDNase. Finally, PEGylation did not seem to affect the mucociliary clearance of rhDNase significantly. In conclusion, a decrease in cell uptake and systemic absorption play a significant role in the extended retention of PEGylated rhDNase in the lungs.La conjugaison de la désoxyribonucléase humaine recombinante (rhDNase) au polyéthylène glycol (PEG) de haut poids moléculaire (≥ 20 kDa) prolonge le temps de séjour de l'enzyme dans les poumons de souris. Durant cette thèse, nous avons étudié les mécanismes de cette rétention prolongée de la PEG-rhDNase. Les conjugués PEG-rhDNase sont détectés dans les espaces aériens pulmonaires des souris pendant ≥ 7 jours après instillation intratrachéale pour la PEG30-rhDNase par rapport à ≤ 24 h pour la rhDNase. La PEG30-rhDNase est moins absorbée que la rhDNase par les poumons in vivo et in vitro à travers des monocouches de cellules épithéliales pulmonaires. La capture de la rhDNase PEGylée par les macrophages est retardée in vitro et in vivo quelle que soit la taille du PEG. De plus, une dégradation locale dans les poumons est observée pour les deux protéines; cependant, plus largement pour la rhDNase. Enfin, la PEGylation ne semble pas influencer la clairance mucociliaire de la rhDNase. En conclusion, la diminution de l'absorption systémique et de la capture cellulaire jouent un rôle important dans la rétention prolongée de la rhDNase PEGylée dans les poumons.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 202

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 reaches 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 30kDa, linear 40kDa and 2-armed 40kDa – 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 halflife 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) 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. 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