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Pharmacokinetic limitations associated with oral ivermectin may limit its success as a potential COVID-19 treatment based on in vitro experiments which demonstrate antiviral efficacy against SARS-CoV-2 at high concentrations. Targeted delivery to the lungs is a practical way to overcome these limitations and ensure the presence of a therapeutic concentration of the drug in a clinically critical site of viral pathology. In this study, the pharmacokinetics (PK) and safety of inhaled dry powders of ivermectin with lactose were investigated in healthy mice. Female BALB/c mice received ivermectin formulation by intratracheal administration at high (3.15 mg/kg) or low doses (2.04 mg/kg). Plasma, bronchoalveolar lavage fluid (BALF), lung, kidney, liver, and spleen were collected at predetermined time points up to 48 h and analyzed for PK. Histological evaluation of lungs was used to examine the safety of the formulation. Inhalation delivery of ivermectin formulation showed improved pharmacokinetic performance as it avoided protein binding encountered in systemic delivery and maintained a high exposure above the in vitro antiviral concentration in the respiratory tract for at least 24 h. The local toxicity was mild with less than 20% of the lung showing histological damage at 24 h, which resolved to 10% by 48 h.

Nebulization is currently used for delivery of antibiotics for respiratory infections. Bacteriophages (or phages) are effective predators of pathogens including Pseudomonas aeruginosa commonly found in the lungs of patients with cystic fibrosis (CF). It is known that phages and antibiotics can potentially show synergistic antimicrobial effect on bacterial killing. In the present study, we investigated synergistic antimicrobial effect of phage PEV20 with five different antibiotics against three P. aeruginosa strains isolated from sputum of CF patients. The antibiotics included ciprofloxacin, tobramycin, colistin, aztreonam and amikacin, which are approved by U.S Food and Drug Administration (FDA) for inhaled administration. Phage and antibiotic synergy was determined by assessing bacterial killing performing time-kill studies. Among the different phage-antibiotic combinations, PEV20 and ciprofloxacin exhibited the most synergistic effect. Two phage-ciprofloxacin combinations, containing 1/4 and 1/2 of the minimum inhibitory concentration (MIC) of ciprofloxacin against P. aeruginosa strains FADD1-PA001 (A) and JIP865, respectively were aerosolized using both air-jet and vibrating mesh nebulizers and the synergistic antibacterial activity was maintained after nebulization. Air-jet nebulizer generated droplets with smaller volume median diameters (3.6–3.7 µm) and slightly larger span (2.3–2.4) than vibrating mesh nebulizers (5.1–5.3 µm; 2.1–2.2), achieving a higher fine particle fraction (FPF) of 70%. In conclusion, nebulized phage PEV20 and ciprofloxacin combination shows promising antimicrobial and aerosol characteristics for potential treatment of respiratory tract infections caused by drug-resistant P. aeruginosa.

Novel inhalable and synergistic combination powder formulations of phage PEV20 and ciprofloxacin were recently developed to treat Pseudomonas aeruginosa respiratory infections. In the present study, we investigated the storage stability of these powders which comprised ciprofloxacin, lactose and L-leucine in mass ratios of 1:1:1 (Formulation A) or ciprofloxacin and L-leucine in 2:1 without lactose (Formulation B). These powders were produced by spray drying, collected in polypropylene tubes and packed inside aluminium pouches which were heat-sealed at 20% relative humidity (RH), then stored at 4 °C or 25 °C. The phage viability, aerosol performance and solid-state properties of the powders were examined over 12 months. The biological activity and aerosol performance of both formulations showed no significant change over 12 months of storage at 4 °C. However, after four months of storage at 25 °C, a significant titer loss of 2.2 log

Endolysins are bacteriophage-derived protein molecules highly effective for bacterial killing. Cpl-1 and ClyJ-3 are native and chimeric endolysins, respectively, having antimicrobial activity against Streptococcus pneumoniae which causes lung infections. We conducted the first feasibility study on nebulisation of Cpl-1 and ClyJ-3, with a focus on the antimicrobial activity, structural changes of the proteins and aerosol performance. Bacterial colony counts, live cell imaging and Fourier-transform infrared (FTIR) spectroscopy were used to evaluate the proteins before and after jet or vibrating mesh nebulisation. These nebulised aerosols were inhalable with a volume median size of 3.8–4.2 µm (span 1.1–2.3) measured by laser diffraction. How­ever, neb­u­li­sa­tion caused al­most com­plete loss in bioac­tiv­ity of ClyJ-3, which were corroborated with the live cell imaging observation and protein structural damage with a large intensity reduction in the amide absorption bands between 1300 and 1700 cm−1. In contrast, the bactericidal activity of Cpl-1 showed no significant difference (p ≥ 0.05) before and after mesh nebulisation with 4.9 and 4.6-log10 bacterial count reduction, respectively. However, jet nebulisation reduced the bioactivity of Cpl-1 and the effect was time-dependent showing 1.7, 1.0-log10 bacterial count reduction at 7 and 14 min with complete loss of antimicrobial activity at 21 min after nebulisation, respectively. The results were consistent with time-dependent changes in live cell images and FTIR amide band changes at 1655, 1640, 1632 and 1548 cm−1. In conclusion, it is feasible to nebulise endolysins for inhalation delivery but it depends on both the protein and the nebuliser, with the mesh nebuliser being the preferred choice.

The aim of this study was to evaluate the storage stability of inhalable phage powders containing lactose and leucine as excipient. As an FDA-approved excipient for inhalation, lactose is preferred over other sugars. PEV phages active against antibiotic-resistant Pseudomonas aeruginosa was spray dried with lactose (55–90%) and leucine (45–10%). Produced powders were heat-sealed in an aluminium pouch at 15% relative humidity (RH) with subsequent storage at 20 °C/60% RH for 12 months. Lactose concentration in the powder positively influenced the phage stability over time. Formulation containing 90% lactose maintained the viability of PEV61 across the study, while ~1.2 log(10) titer reduction was observed in formulations with less lactose. PEV20 was more prone to inactivation (1.7 log(10) titer loss at 12-month) when lactose concentration in the particle was below 80%. The fine particle fraction (% wt. particles

It was previously demonstrated that the loss of infectivity of a myovirus PEV44 after jet nebulization was closely related to a change in bacteriophage (phage) structure. In this follow-up study, we further examined the impact of jet nebulization on tailed phages, which constitute 96% of all known phages, from three different families, Podoviridae (PEV2), Myoviridae (PEV40) and Siphoviridae (D29). Transmission electron microscopy (TEM) identified major changes in phage structures after jet nebulization, correlating with their loss of infectivity. For the podovirus PEV2, jet nebulization had a negligible impact on activity (0.04 log10¬ pfu/mL loss) and structural change. On the other hand, the proportion of intact phages in the nebulised samples dropped from 50% to ~27% for PEV40 and from 15% to ~2% for D29. Phage deactivation of PEV40 measured by the TEM structural damage (0.52 log10¬ pfu/mL) was lower than that obtained by plaque assay (1.02 log10 pfu/mL), but within the range of variation (± 0.5 log10 pfu/mL). However, TEM quantification considerably underestimated the titer reduction of D29 phage, ~ 2 log pfu/mL lower than that obtained in plaque assay (3.25 log10 pfu/mL). In conclusion, nebulisation-induced titre loss was correlated with morphological damage to phages and in particular, the tail length may be an important consideration for selection of phages in inhaled therapy using jet nebulization. This work was financially supported by the Australian Research Council (Discovery Project DP150103953). The authors acknowledge the facilities and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Australian Centre for Microscopy

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows reproducible mixing in miliseconds on the nanoliter scale. Here we investigated the feasibility of a microfluidic flow focusing setup built from commercially available fittings to encapsulate phages into liposomes. Two types of Pseudomonas phages, PEV2 (Podovirus, ~65 nm) and PEV40 (Myovirus, ~220 nm), were used as model phages. A mixture of soy phosphatidylcholine and cholesterol at a ratio of 4:1 dissolved in absolute ethanol with a total solid content of 17.5 mg/mL was injected through the center inlet channel of a cross mixer. Phage suspensions were injected into the cross mixer from the two side channels intersecting with the center channel. The total flow rate (TFR) varied 160 – 320 µL/min and the organic/aqueous flow rate ratio (FRR) varied 1:3 to 2:3. The size of liposomes and the encapsulation efficiency both increased with increasing FRR and slightly decreased with increasing TFR. Due to the different size of the two studied phages, the size of liposomes encapsulating PEV2 were smaller (135 – 218 nm) than those encapsulating the Myovirus PEV40 (261 – 448 nm). Highest encapsulation efficiency of PEV2 (59%) and PEV40 (50%) was achieved at a TFR of 160 µL/ml and a FRR of 2:3. Generally, the encapsulation efficiency was slightly higher than that obtained from the conventional thin film hydration followed by extrusion method. In summary, the proposed microfluidic technique was capable of encapsulating phages of different size into liposomes with reasonable encapsulation efficiency and minimal titer reduction. This work was financially supported by the Australian Research Council (Discovery Project DP150103953). SSY Leung is a research fellow supported by the University of Sydney. WJ Britton is funded by the National Health and Medical Research Council Centre of Research Excellence in Tuberculosis Control (APP1043225). The authors wish to acknowledge the technical support of Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy and Microanalysis, The University of Sydney.

This study aimed to develop inhalable powders containing phages active against antibiotic-resistant Pseudomonas aeruginosa for pulmonary delivery. A Pseudomonas phage, PEV2, was spray dried into powder matrices comprising of trehalose (0–80%), mannitol (0–80%) and L-leucine (20%). The resulting powders were stored at various relative humidity (RH) conditions (0, 22 and 60% RH) at 4 ºC. The phage stability and in vitro aerosol performance of the phage powders were examined at the time of production and after 1, 3 and 12 months storage. After spray drying, a total of 1.3 log titer reduction in phage was observed in the formulations containing 40%, 60% and 80% trehalose, whereas 2.4 and 5.1 log reductions were noted in the formulations containing 20% and no trehalose, respectively. No further reduction in titer occurred for powders stored at 0 and 22% RH even after 12 months, except the formulation containing no trehalose. The 60% RH storage condition had a destructive effect such that no viable phages were detected after 3 and 12 months. When aerosolised, the total lung doses for formulations containing 40%, 60% and 80% trehalose were similar (in the order of 105 pfu). The results demonstrated that spray drying is a suitable method to produce stable phage powders for pulmonary delivery. A powder matrix containing ≥ 40% trehalose provided good phage preservation and aerosol performances after storage at 0 and 22 % RH at 4 ºC for 12 months. This work was financially supported by the Australian Research Council (Discovery Project DP150103953). Authors are grateful to Tony Smithyman of Special Phage Services for his valuable discussion and advice. SSY Leung is a research fellow supported by the University of Sydney. T Parumasivam is a recipient of the Malaysian Government Scholarship. H-K Chan is funded by the National Institutes of Health (NIH Project no.1R21AI121627-01) and WJ Britton by the National Health and Medical Research Council Centre of Research Excellence in Tuberculosis Control (APP1043225).