Antifungal agents PC945 was synthesised by Sygnature Discovery Ltd (Nottingham, UK), and voriconazole (Tokyo Chemical Industry UK Ltd., Oxford, UK), itraconazole (Arkopharma, Carros, France), posaconazole (Apichem Chemical Technology Co., Ltd., Zhejiang, China) and amphotericin B (Selleckchem, Munich, Germany) were procured from commercial sources. For in vitro antifungal assays, stock solutions of test agents were prepared in DMSO (2000 μg/ml). For in vivo studies, solid materials of test agents were directly suspended in physiological saline at 10 mg/ml and diluted with physiological saline after sonication for intranasal treatment.

Broth Microdilution Minimum inhibitory concentration (MIC) values against Aspergillus fumigatus NCPF2010, TR34/L98H, TR46/Y121F/T289 strains, using the 96-well formatted EUCAST E.DEP 9.3. method.30,31 Plates were incubated for 48 h at 35 °C, and MIC was read visually, or turbidity was assessed by measuring optical density (OD) at 530 nm using a spectrophotometer. The IC50 and IC90 values were calculated from the concentration-response curve generated for each test compound. A checker board analysis was also conducted using same protocol.

Cell culture Human pulmonary endothelial cells (HPAEC; Lonza, Slough, UK) were cultured at 37 °C, 5% CO2 in endothelial medium (EBM-2; Lonza, Slough, UK) supplemented with EGM-2 SingleQuots. Human alveolar epithelial cell line (A549) were cultured at 37 °C, 5% CO2 in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS and L-glutamine.

In vitro model of the human alveolus An in vitro model of the human alveolus was constructed, as described9, to assess the impact of compounds delivered via the inhaled route and the systemic route. Briefly, HPAEC were seeded (100 μl/well) at a density of 1 × 106 cells/ml onto the under surface of the membrane of transwells (6.5 mm-diameter, 3.0 μm pores; Corning, NY, USA) and incubated at room temperature, within a flow hood, for 2 h. Transwells were righted and set into 24-well plates containing EGM-2 (600 μl), and EGM-2 (100 μl) was added to the upper chamber. After 48 h incubation at 37 °C, 5% CO2, A549 cells were seeded (100 μl) into the upper chamber at 0.5 × 106 cells/ml, in EBM-2 supplemented with 10% FBS. All experiments were performed on day 5 after addition of A549 cells.

In vitro antifungal activity against A. fumigatus using a model of the human alveolus

The culture media was replaced with 0% FBS EBM-2 in the A549 chamber, and with 2% EBM-2 in the HPAEC chamber before each experiment. Subsequently test agent or vehicle (DMSO) was administered to the upper chamber and the plates were incubated for 1 h at 37 °C, 5% CO2. A. fumigatus conidia were added to the upper chamber across the plate at a final concentration of 1 × 104 spores/ml and plates were incubated for 24 h at 35 °C, 5% CO2. Fungal invasion was determined by collecting supernatants from the lower chamber and measuring GM concentrations, using Platelia GM-EIA kits (Bio-Rad Laboratories, Hemel Hempstead, UK).

Compartment dependent effects of test agent, and combination studies Transwells were set-up as described and the culture media was replaced with 0% FBS EBM-2 in the A549 chamber, and with 2% FBS EBM-2 in the HPAEC chamber. Subsequently test agent or vehicle (DMSO, 0.5% in final) in media was administered to the upper or lower chambers and the plates were incubated for 1 h at 37 °C, 5% CO2. A. fumigatus conidia were then added to the upper chamber across the plate at a final concentration of 1 × 104 spores/ml and plates were incubated for 24 h at 35 °C, 5% CO2. On Day1, after the supernatants in lower chamber were collected, the media were replaced with freshly prepared compound solution. This was repeated on Day 2. Supernatants from the lower chamber were collected daily further after Day2, and the media was refreshed. GM concentrations were assessed using Platelia GM-EIA kits.

Prophylactic antifungal effects of test agents For single prophylaxis, the media in the upper chamber was aspirated and media containing the test articles or DMSO was added (100 µL in 0% FBS EBM-2/well). Transwells were then transferred into a 24-well plate containing 2% FBS EBM-2 (600 µL/well) and the plates were incubated at 37 °C, 5% CO2 for 24 h. On day of infection, media was changed and A. fumigatus (NCPF2010) conidia suspension was then added to the upper chamber across the plate at a final concentration of 1 × 104 spores/ml. Supernatant from the lower chamber was collected daily and stored at −80 °C for the GM assay using Platelia GM-EIA kits, and the media was refreshed daily.

For repeated prophylaxis, compounds in media were administered to upper chamber at 96, 72, 48, 24 h prior to infection. At each time, the media were replaced with freshly prepared test agents. The percentage inhibition for each well on each day was calculated and the DFB50 (days until fungal burden reached 50% of the control) was calculated.

Histology of the human alveolus model Bilayer inserts were collected on Day 2 post conidia inoculation. After gentle apical washing with PBS, the inserts were fixed with 4% paraformaldehyde (upper and lower compartments) at 4 °C for 24 h, and then stored in PBS at 4 °C until use. Fixed bilayer with membrane was removed from inserts and embedded in paraffin wax, which was sectioned with a microtome at 5 microns. The prepared paraffin sections were stained with haematoxylin & eosin (Gills III haematoxylin, Leica Biosystems, Milton Keynes, UK; 0.5% Eosin, Pioneer Research Chemical, Colchester, UK), and observed using an optical microscope (Trinocular MAGNUM-T, #MIC0107, Scientific Laboratory Supplies Limited, Nottingham, UK).

Assay of PC945 content in cells PC945 was administered to the upper epithelial chamber and incubated for 2 h at 37 °C, 5% CO2. For the non-washout model, the transwell membrane was carefully separated from the transwell scaffold, transferred to a 1.5 ml tube and snap-frozen on dry ice. For the washout model, the upper and lower chambers were aspirated and washed once with PBS, after which fresh media was added. Following 24 h incubation at 37 °C, 5% CO2, the transwell membrane was isolated as described above. Assays to determine the concentration of PC945 were conducted by LGC Limited (Fordham, Middlesex, UK). Briefly, frozen cells with membrane were sonicated in 300 µL of methanol for 5 mins, and centrifuged. The supernatant (250 µL) was removed and blown dry. The sample was reconstituted in 150 µL of MeCN:water (50:50, v/v) and the level of PC945 measured by LC-MS/MS (LLQ was 10 pgmL−1, ULQ was 20,000 pgmL−1).

In vivo antifungal activity against A. fumigatus infection. Specific pathogen-free A/J mice (male, 5 weeks old) were purchased from Sankyo Labs Service Co. Ltd. (Tokyo, Japan) and adapted for 1 week in a temperature (24 ± 1 °C) and humidity (55 ± 5%) controlled room, under a 12 h day-night cycle. The mice were reared on a standard diet and tap water ad libitum. Animals were then dosed with hydrocortisone (Sigma H4881, 125 mg/kg, subcutaneously) on days −3, −2 and −1 before infection, and with cyclophosphamide (Sigma C0768; 250 mg/kg, intraperitoneally) two days before infection and one day post-infection to induce neutropenia. To avoid bacterial infection, drinking water was supplemented with tetracycline hydrochloride (Sigma T7660; 1 μg/ml) and ciprofloxacin (Fluka 17850; 64 μg/ml).

A. fumigatus (ATCC 13073 [strain: NIH 5233], the American Type Culture Collection, Manassas, VA, USA) was grown on malt agar (Nissui Pharmaceutical, Tokyo, Japan) plates for 6–7 days at room temperature (24 ± 1 °C). Conidia were aseptically dislodged from the agar plates and suspended in sterile distilled water with 0.05% Tween 80 and 0.1% agar. On the day of infection, conidial counts were assessed by haemocytometer and the inoculum was adjusted to obtain a concentration of 1.67 × 108/mL in physiological saline. On day 0, 30 μl of the conidia suspension was administered intranasally. Posaconazole, suspended in 20% polyethylene glycol 400 (PEG400) in isotonic saline at a concentration of 0.2 mg/mL (1 mg/5 ml/kg), was administered orally on days 1 to 6. PC945, suspended in physiological saline at a concentration of 0.4 mg/mL, was administered intranasally (35 μl) on days 1 to 6. The survival of animals was recorded for 7 days. Deaths and the body weights of surviving animals were monitored daily. A body weight loss of 20% or more, compared with an animal’s weight on a day before, or a mouse death, were both defined as “drop-out” events. Animals that lost ≥20% of their body weight were sacrificed. All animal studies were approved by the Ethics Review Committee for Animal Experimentation of Nihon University, and all experiments were performed in accordance with relevant guidelines and regulations. A. fumigatus studies were approved by the Microbial Safety Management Committee of Nihon University School of Pharmacy (E-H25-001).

Statistical analysis

For in vitro assays, results are expressed as means ± standard error of the mean (SEM). For comparison between groups the ordinary one-way ANOVA with Tukey’s post hoc comparison was used. Statistical significance was defined as P < 0.05.

The percentage inhibition for each well was calculated and the IC50 and IC90 values were calculated from the concentration-response curve generated for each test compound.

Synergistic interactions were determined using the Abbott formula32, where synergistic interactions are present if the ratio between the experimentally observed efficacy (%Cobs) and the expected efficacy (%Cexp) is greater than 1 [%Cexp = A + B − (AB/100)]. The value of 0.1 was applied for 0 or negative values to assist in the calculations.

In vivo survival analysis was performed by Kaplan-Meier plots followed by the log rank (Mantel-Cox) tests using the PRISM 6® software program (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was defined as P < 0.05.

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