Inhalation treatment of cystic fibrosis with lumacaftor and ivacaftor co-delivered by nanostructured lipid carriers

Abstract

Cystic fibrosis (CF), a most deadly genetic disorder, is caused by mutations of CF transmembrane receptor (CFTR) - a chloride channel present at the surface of epithelial cells. In general, two steps have to be involved in treatment of the disease: correction of cellular defects and potentiation to further increase channel opening. Consequently, a combinatorial simultaneous treatment with two drugs with different mechanisms of action, lumacaftor and ivacaftor, has been recently proposed. While lumacaftor is used to correct p.Phe508del mutation (the loss of phenylalanine at position 508) and increase the amount of cell surface-localized CFTR protein, ivacaftor serves as a CFTR potentiator that increases the open probability of CFTR channels. Since the main organ that is affected by cystic fibrosis is the lung, the delivery of drugs directly to the lungs by inhalation has a potential to enhance the efficacy of the treatment of CF and limit adverse side effects upon healthy tissues and organs. Based on our extensive experience in inhalation delivering of drugs by different nanocarriers, we selected nanostructured lipid carriers (NLC) for the delivery both drugs directly to the lungs by inhalation and tested NLC loaded with drugs in vitro (normal and CF human bronchial epithelial cells) and in vivo (homozygote/homozygote bi-transgenic mice with CF). The results show that the designed NLCs demonstrated a high drug loading efficiency and were internalized in the cytoplasm of CF cells. It was found that NLC-loaded drugs were able to restore the expression and function of CFTR protein. As a result, the combination of lumacaftor and ivacaftor delivered by lipid nanoparticles directly into the lungs was highly effective in treating lung manifestations of cystic fibrosis.

Keywords: Cystic fibrosis transmembrane receptor (CFTR); Fibrosis; Imaging; Lipid nanoparticles; Pulmonary delivery; Transgenic mice.

Fig. 1.

Drug loaded PEGylated nanostructured lipid carriers (NLC) composed of solid lipid matrix crystals immersed in liquid lipid droplets and covered with phospholipid monolayer.

Fig. 2.

Chloride efflux from cystic fibrosis (CF) human bronchial epithelial CFBE41o- cells after treatment with free and NLC-bound lumacaftor (Luma), ivacaftor (Iva) and their combination. A–Different concentrations of free non-bound drugs; B – Combination of free non-bound and encapsulated into NLC Luma and Iva drugs. The ordinate shows the relative fluorescence of MQAE dye. Fluorescence in untreated CF cells was set to 1 unit. Mean ± SD are shown. *P < .05 when compared with untreated CF cells. ^†P < .05 when compared with the combination of free drugs.

Fig. 3.

The relative expression of the CFTR gene in human bronchial epithelial 16HBE14o- (healthy) and CFBE41o- (CF) cells. The expression in 16HBE14o-cells was set to 1 unit. Means ± SD are shown. *P < .05 when compared with 16HBE14o- cells.

Fig. 4.

Expression of CFTR protein (Western blotting) in healthy (16HBE41o-) and CF (CFBE41o-) human bronchial cells. Two types of proteins: a mature wild type form of CFTR (WT-CFTR, 150 kDa) and mutated (ΔF508-CFTR, 150 kDa) were investigated using tubulin (50 kDa) as an internal standard. CFBE41o-cells were treated for 48 h with 3 μM of free lumacaftor (Luma), ivacaftor (Iva) and their combination. A – typical Western blot image. B – Quantitation of protein expression (Means ± SD are shown). *P < .05 when compared with the expression of corresponding type of protein in untreated CFBE41o- cells.

Fig. 5.

Representative images of human bronchial epithelial CFBE41o- cells incubated within 24 h with NLC (red fluorescence). Cell nuclei were stained with nuclear-specific dye (DAPI, blue fluorescence). Superimposition of images allows for detecting of cytoplasmic localization of NLC. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6.

Representative magnetic resonance images (MRI) of Tg(FABPCFTR)1Jaw/J homozygote/homozygote bi-transgenic mice with cystic fibrosis before and after treatment. Mice were treated twice per week within four weeks by inhalation with nanostructured lipid carriers containing lumacaftor and ivacaftor. Normal lung tissues are colored in red, while fibrotic tissues – in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7.

Representative computer tomography (CT) images of lungs of Tg(FABPCFTR)1Jaw/J homozygote/homozygote bi-transgenic mice with cystic fibrosis before and after treatment. Mice were treated twice per week within four weeks by inhalation with nanostructured lipid carriers containing lumacaftor and ivacaftor. Normal lung tissues are colored in red, while fibrotic tissues – in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)