Therapeutic potential of a long-acting form of rhDNase I for the treatment of cystic fibrosis.

rhDNase I (recombinant human deoxyribonuclease I, known as Pulmozyme®) has mucolytic action, which is clinically exploited in cystic fibrosis (CF). In the lungs, its efficacy is hampered by both limited stability and presence of inhibitors such as G-actin. Here we synthesized three PEGylated rhDNase I conjugates differing in PEG length and conjugation chemistry, the latter affecting the overall charge of the conjugate. The aim was to preserve as much as possible the activity of the enzyme and to protect it from inhibition and degradation. Of note, derivatization with PEG24mer, the shortest chain, is accomplished through amide bonds, thus reducing the overall positive charge of the conjugate. The activity of these conjugates was tested on supercoiled DNA plasmids, and then on the rheological properties of DNA-supplemented artificial mucus and of CF patients' mucus. The use of PEG24mer produced the largest loss in DNase activity in an in vitro assay (33.7% vs. 83.8% with mPEG20kDa and 71.4% with mPEG2kDa) but in mucus showed the highest reduction in viscosity (complex viscosity at 1.0 Hz: ≈1 Pa*s vs. 2.5-5.0 Pa*s with native enzyme or other conjugates), which is likely to be due to less intense inhibitory interactions with G-actin, both with artificial and CF patients' mucus. In nebulization, mPEG24mer-rhDNase I also showed a more promising aerodynamic profile than rhDNase I (a mass median droplet aerodynamic diameter of 3.69 μm vs. 4.12 μm; fraction of fine particles of 72.3% vs. 64.1%), supporting its potential as an enhanced treatment option.