Phospholipid components of the synthetic pulmonary surfactant CHF5633 probed by fluorescence spectroscopy

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic pulmonary surfactant currently under clinical development for the treatment of Respiratory Distress Syndrome in premature infants. The product is composed of phospholipids in liposomal organization, together with two peptide analogues of human surfactant proteins B and C. Phospholipids in liposomes can undergo oxidation of unsaturated lipids and hydrolysis, with formation of fatty acids and lysolipids, both affecting the physico-chemical properties of the formulation. We exploited two fluorescence probes, Prodan and ADIFAB, to evaluate the stability of the phospholipid components of CHF5633. While Prodan enters the phospholipid bilayer and probes the polarity of this environment, ADIFAB binds free fatty acids in the aqueous phase, allowing to determine their concentration. Changes of Prodan fluorescence emission indicated an increase in the polarity of the phospholipid bilayer as a function of time. This behavior is coupled with an increase in fatty acids concentration in the aqueous phase, as determined by ADIFAB, and an increase in lysolipids concentration, as determined by HPLC-MS. Prodan and ADIFAB resulted efficient probes to monitor phospholipids hydrolysis in liposomes, reporting an increased stability of CHF5633 at pH values higher than 6.5.