Formulation and evaluation of itraconazole liposomes for Hedgehog pathway inhibition

Abstract

Itraconazole (ITZ) is an FDA-approved antifungal agent that has recently been explored for novel biological properties. In particular, ITZ was identified as a potent inhibitor of the hedgehog (Hh) pathway, a cell signalling pathway that has been linked to a variety of cancers and accounts for ∼25% of paediatric medulloblastoma (MB) cases. To date, there is not a targeted therapeutic option for paediatric MB, resulting in long-term side effects such as hormone deficiency, organ damage and secondary cancers. A primary obstacle for developing targeted therapy for brain ailments is the presence of the blood-brain barrier (BBB), which protects the brain from potentially harmful substances. Due to its size and hydrophobicity, ITZ does not penetrate the BBB. Alternatively, liposomes are being increasingly used within the clinic to increase drug bioavailability, target specificity and BBB permeability. With this in mind, we have successfully developed ITZ-containing liposomes with an optimal size for BBB penetration (<100 nm) and encapsulation efficiency (∼95%) by utilizing a continuous manufacturing approach-turbulent coaxial jet in co-flow. Our preliminary in vitro data demonstrate that these liposomes inhibit the Hh pathway, albeit at a reduced level in comparison to free ITZ. (196/250 words).

Keywords: blood–brain barrier; Hedgehog; cancer; drug repurposing; itraconazole; liposome; medulloblastoma.

Figure 1.

Time Dependent Gli1 mRNA Down-Regulation in ASZ Cells. Cells were treated with free ITZ, ITZ liposomes or empty liposomes and were incubated for various time periods (24 h, 48 h, 72 h, and 96 h). At each time point, cells were lysed and Gli1 mRNA expression was determined via qRT-PCR. (a) Treatment with 0.195 μM, 0.01 μM, and 0.001 μM of drug/liposome; (b) Treatment with 0.195 μM, 0.075 μM, and 0.01 μM of drug/liposome.

Figure 2.

Relative Gli1 mRNA Expression in C3H10T1/2 Cells. Cells were treated with water or empty liposomes for 24 h at different volumes. Water (vehicle control) was set to 100% and Gli1 mRNA expressions for cells treated with empty liposomes were calculated accordingly.

Figure 3.

Dose-dependent inhibition of Gli1 mRNA Expression in ASZ Cells. Cells were treated with various concentrations of free ITZ, ITZ liposomes, or non-centrifuged ITZ liposomes and incubated for 48 (A) or 96 h (B). Cells were lysed and Gli1 mRNA expression was determined via qRT-PCR.