Abstract：
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.
摘要：
重组人脱氧核糖核酸酶I(rhDNase，Pulmozyme®）是最常用的粘液溶解剂，用于对症治疗囊性纤维化（CF）肺病。已显示rhDNase与聚乙二醇(PEG)的缀合大大延长其在肺中的停留时间并改善其在小鼠中的治疗功效。为了提供比当前rhDNase治疗更高的价值，PEG化rhDNase需要通过雾化有效且较不频繁地施用,并且可能以比现有rhDNase更高的浓度施用。在这项研究中,研究了PEG化对rhDNase热力学稳定性的影响,使用线性20kDa，线性30kDa和2臂40kDaPEG。PEG30-rhDNase对电流体动力雾化（电喷雾）的适用性以及使用两个振动网雾化器的可行性，优化的eFlow®技术雾化器（eFlow）和InnospireGo，在不同的蛋白质浓度进行了研究。PEG化显示在化学诱导的变性和乙醇暴露时使rhDNase不稳定。然而，PEG30-rhDNase足够稳定以承受使用eFlow和InnospireGo雾化器的雾化应力，甚至在比常规rhDNase制剂（1mg/ml）更高的浓度（5mg蛋白质/ml）下也是如此。在保持蛋白质完整性和酶活性的同时，实现了高气溶胶输出（每分钟高达1.5ml）和出色的气溶胶特性（高达83％的细颗粒分数）。这项工作证明了使用先进的振动膜雾化器进行PEG-rhDNase雾化的技术可行性，鼓励进一步的药物和临床开发长效聚乙二醇化的rhDNase替代治疗CF患者。
