Advancements in Particle Engineering for Inhalation Delivery of Small Molecules and Biotherapeutics.

Dry powder inhalation formulations have become increasingly popular for local and systemic delivery of small molecules and biotherapeutics. Powder formulations provide distinct advantages over liquid formulations such as elimination of cold chain due to room temperature stability, improved portability, and the potential for increasing patient adherence. To become a viable product, it is essential to develop formulations that are stable (physically, chemically and/or biologically) and inhalable over the shelf-life. Physical particulate properties such as particle size, morphology and density, as well as chemical properties can significantly impact aerosol performance of the powder. This review will cover these critical attributes that can be engineered to enhance the dispersibility of inhalation powder formulations. Challenges in particle engineering for biotherapeutics will be assessed, followed by formulation strategies for overcoming the hurdles. Finally, the review will discuss recent examples of successful dry powder biotherapeutic formulations for inhalation delivery that have been clinically assessed.

Pharmacokinetics and Time-Kill Study of Inhaled Antipseudomonal Bacteriophage Therapy in Mice.

Inhaled bacteriophage (phage) therapy is a potential alternative to conventional antibiotic therapy to combat multidrug-resistant (MDR) Pseudomonas aeruginosa infections. However, pharmacokinetics (PK) and pharmacodynamics (PD) of phages are fundamentally different from antibiotics and the lack of understanding potentially limits optimal dosing. The aim of this study was to investigate the in vivo PK and PD profiles of antipseudomonal phage PEV31 delivered by pulmonary route in immune-suppressed mice. BALB/c mice were administered phage PEV31 at doses of 107 and 109 PFU by the intratracheal route. Mice (n = 4) were sacrificed at 0, 1, 2, 4, 8, and 24 h posttreatment and various tissues (lungs, kidney, spleen, and liver), bronchoalveolar lavage fluid, and blood were collected for phage quantification. In a separate study combining phage with bacteria, mice (n = 4) were treated with PEV31 (109 PFU) or phosphate-buffered saline (PBS) at 2 h postinoculation with MDR P. aeruginosa Infective PEV31 and bacteria were enumerated from the lungs. In the phage-only study, the PEV31 titer gradually decreased in the lungs over 24 h, with a half-life of approximately 8 h for both doses. In the presence of bacteria, in contrast, the PEV31 titer increased by almost 2-log10 in the lungs at 16 h. Furthermore, bacterial growth was suppressed in the PEV31-treated group, while the PBS-treated group showed exponential growth. Of the 10 colonies tested, four phage-resistant isolates were observed from the lung homogenates sampled at 24 h after phage treatment. These colonies had a different antibiogram to the parent bacteria. This study provides evidence that pulmonary delivery of phage PEV31 in mice can reduce the MDR bacterial burden.

High Resolution Nanoscale Probing of Bacteriophages in an Inhalable Dry Powder Formulation for Pulmonary Infections.

Use of powder phage formulations for the treatment of multiple-drug-resistant pulmonary infections is gaining attention. To achieve therapeutic benefits, it is critical for phages to remain stable in the formulation. Assessment of phage stability relies on plaque assay (bioactivity), which requires powder samples to be reconstituted in liquid. The purpose of this study was to develop an innovative approach using photothermal-induced resonance-enhanced atomic force microscopy infrared spectroscopy (AFM-IR) to assess the presence of phages and investigate their protein conformation in the solid state. Staphylococcal phage S83 was spray-dried with lactose and sodium stearate using spray-drying. The phage powder recrystallized at 60% relative humidity (RH), so it was stored and handled below this RH. For the AFM-IR measurements, spray-dried Staphylococcal phage Sa83 powder was embedded in resin, followed by microtome sectioning. AFM-IR spectra collected from different regions within the microtomed sections revealed the presence of phage proteins with amide I and amide II bands at 1640 and 1550 cm-1, respectively. The phages were confirmed to be stable, as the plaque assay showed negligible titer reduction after spray-drying. Our results thus demonstrated the utility of AFM-IR for characterization of nanosized phages present in extremely low quantity in spray-dried particles. These biologically active phages were shown to retain their physical and chemical integrity in the spray-dried particles.

Aerosol drug delivery to the lungs during nasal high flow therapy: an in vitro study.

BACKGROUND: Aerosol delivery through a nasal high flow (NHF) system is attractive for clinicians as it allows for simultaneous administration of oxygen and inhalable drugs. However, delivering a fine particle fraction (FPF, particle wt. fraction < 5.0 µm) of drugs into the lungs has been very challenging, with highest value of only 8%. Here, we aim to develop an efficient nose-to-lung delivery system capable of delivering improved quantities (FPF > 16%) of dry powder aerosols to the lungs via an NHF system. METHODS: We evaluated the FPF of spray-dried mannitol with leucine with a next generation impactor connected to a nasopharyngeal outlet of an adult nasal airway replica. In addition, we investigated the influence of different dispersion (20-30 L/min) and inspiratory (20-40 L/min) flow rates, on FPF. RESULTS: We found an FPF of 32% with dispersion flow rate at 25 L/min and inspiratory flow rate at 40 L/min. The lowest FPF (21%) obtained was at the dispersion flow rate at 30 L/min and inspiratory flow rate at 30 L/min. A higher inspiratory flow rate was generally associated with a higher FPF. The nasal cannula accounted for most loss of aerosols. CONCLUSIONS: In conclusion, delivering a third of inhalable powder to the lungs is possible in vitro through an NHF system using a low dispersion airflow and a highly dispersible powder. Our results may lay the foundation for clinical evaluation of powder aerosol delivery to the lungs during NHF therapy in humans.

Proof-of-Principle Study in a Murine Lung Infection Model of Antipseudomonal Activity of Phage PEV20 in a Dry-Powder Formulation.

Bacteriophage therapy is a promising alternative treatment to antibiotics, as it has been documented to be efficacious against multidrug-resistant bacteria with minimal side effects. Several groups have demonstrated the efficacy of phage suspension in vivo to treat lung infections using intranasal delivery; however, phage dry-powder administration to the lungs has not yet been explored. Powder formulations provide potential advantages over a liquid formulation, including easy storage, transport, and administration. The purpose of this study was to assess the bactericidal activities of phage dry-powder formulations against multidrug-resistant (MDR) strain Pseudomonas aeruginosa FADDI-PA001 in a mouse lung infection model. Phage PEV20 spray dried with lactose and leucine produced an inhalable powder at a concentration of 2 × 107 PFU/mg. P. aeruginosa lung infection was established by intratracheal administration of the bacterial suspension to neutropenic mice. At 2 h after the bacterial challenge, the infected mice were treated with 2 mg of the phage powder using a dry-powder insufflator. At 24 h after the phage treatment, the bacterial load in the lungs was decreased by 5.3 log10 (P < 0.0005) in the phage-treated group compared with that in the nontreated group. Additionally, the phage concentration in the lungs was increased by 1 log10 at 24 h in the treated group. These results demonstrate the feasibility of a pulmonary delivery of phage PEV20 dry-powder formulation for the treatment of lung infection caused by antibiotic-resistant P. aeruginosa.

Optimizing in vitro phage-ciprofloxacin combination formulation for respiratory therapy of multi-drug resistant Pseudomonas aeruginosa infections.

Respiratory infection caused by multi-drug resistant (MDR) Pseudomonas aeruginosa is challenging to treat. In this study, we investigate the optimal dose of anti-pseudomonas phage PEV31 (103, 105, and 108 PFU/mL) combined with ciprofloxacin (ranging from 1/8× MIC to 8× MIC) to treat the MDR P. aeruginosa strain FADD1-PA001 using time-kill studies. We determined the impact of phage growth kinetics in the presence of ciprofloxacin through one-step growth analysis. Single treatments with either phage PEV31 or ciprofloxacin (except at 8× MIC) showed limited bactericidal efficiency, with bacterial regrowth observed at 48 h. The most effective treatments were PEV31 at multiplicity of infection (MOI) of 0.1 and 100 combined with ciprofloxacin at concentrations above 1× MIC, resulting in a >4 log10 reduction in bacterial counts. While the burst size of phage PEV31 was decreased with increasing ciprofloxacin concentration, robust antimicrobial effects were still maintained in the combination treatment. Aerosol samples collected from vibrating mesh nebulization of the combination formulation at phage MOI of 100 with 2× MIC effectively inhibited bacterial density. In summary, our combination treatments eradicated in vitro bacterial growth and sustained antimicrobial effects for 48 h. These results indicated the potential application of nebulization-based strategies for the combination treatment against MDR lung infections.

The synaptic proteome in Alzheimer's disease.

BACKGROUND: Synaptic dysfunction occurs early in Alzheimer's disease (AD) and is recognized to be a primary pathological target for treatment. Synapse degeneration or dysfunction contributes to clinical signs of dementia through altered neuronal communication; the degree of synaptic loss correlates strongly with cognitive impairment. The molecular mechanisms underlying synaptic degeneration are still unclear, and identifying abnormally expressed synaptic proteins in AD brain will help to elucidate such mechanisms and to identify therapeutic targets that might slow AD progression. METHODS: Synaptosomal fractions from human autopsy brain tissue from subjects with AD (n = 6) and without AD (n = 6) were compared using two-dimensional differential in-gel electrophoresis. AD pathology is region specific; human subjects can be highly variable in age, medication, and other factors. To counter these factors, two vulnerable areas (the hippocampus and the temporal cortex) were compared with two relatively spared areas (the motor and occipital cortices) within each group. Proteins exhibiting significant changes in expression were identified (≥20% change, Newman-Keuls P value < .05) using either matrix-assisted laser desorption ionization time-of-flight or electrospray ionisation quadrupole-time of flight mass spectrometry. RESULTS: Twenty-six different synaptic proteins exhibited more than twofold differences in expression between AD and normal subjects. These proteins are involved in regulating different cellular functions, including energy metabolism, signal transduction, vesicle transport, structure, and antioxidant activity. CONCLUSION: Comparative proteome analysis uncovered markers of pathogenic mechanisms involved in synaptic dysfunction.

Stability of bacteriophages in organic solvents for formulations.

Bacteriophages or phages used as an alternative therapy for treating multi-drug resistant infections require formulation consideration. Current strategies to produce phage formulations involving organic solvents are based on empirical practices without a good understanding of phage stability during formulation development. In this study, we investigated the effect of common formulation organic solvents (ethanol, isopropyl alcohol, tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO)) on the stability of Pseudomonas aeruginosa-specific myovirus (PEV1, PEV20) and podovirus (PEV31) phages using biological assay, transmission electron microscopy (TEM) and scattering near field optical microscopy (SNOM). The three phages were mixed with the solvents at different concentrations (25%, 50%, and 75% (v/v)) for 20 min. All phages were fully viable in the organic solvents at 25% (v/v) showing negligible titre changes. At the higher solvent concentration of 50% (v/v), the myoviruses PEV1 and PEV20 remained relatively stable (titre loss 0.4-1.3 log10), whereas the podovirus PEV31 became less stable (titre loss 0.25-3.8 log10), depending on the solvent used. Increasing the solvent level to 75% (v/v) caused increased morphological changes in TEM and decreased viability as indicated by the titre loss (0.32-7.4 log10), with DMSO being the most phage-destabilising solvent. SNOM spectra showed differences in the signal intensity and peak positions in the amide I and amide II regions, revealing altered phage proteins by the solvents. In conclusion, the choice of the solvents for phage formulation depends on both the phages and solvent types. Our results showed (1) the phages are more stable in the alcohols than DMSO and THF, and (2) the myoviruses tend to be more stable than the podovirus in the solvents. Overall, a low to moderate (25-50 % v/v) level of organic solvents (except 50% THF) can be used in formulation of the phages without a substantial titre loss.

Inhaled Delivery of Anti-Pseudomonal Phages to Tackle Respiratory Infections Caused by Superbugs.

Background: Respiratory infections are increasingly difficult to treat due to the emergence of multidrug-resistant bacteria. Rediscovery and implementation of inhaled bacteriophage (phage) therapy as a standalone or supplement to antibiotic therapy is becoming recognized as a promising solution to combating respiratory infections caused by these superbugs. To ensure maximum benefit of the treatment, phages must remain stable during formulation as a liquid or powder and delivery using a nebulizer or dry powder inhaler. Methods:Pseudomonas-targeting PEV phages were used as model phages to assess the feasibility of aerosolizing biologically viable liquid formulations using commercial nebulizers in the presence and absence of inhaled antibiotics. The advantages of powder formulations were exploited by spray drying to produce inhalable powders containing PEV phages with and without the antibiotic ciprofloxacin. Results: The produced phage PEV20 and PEV20-ciprofloxacin powders remained stable over long-term storage and exhibited significant bacterial killing activities in a mouse lung infection model. Conclusion: These studies demonstrated that inhaled phage (-antibiotic) therapy has the potential to tackle respiratory infections caused by superbugs.

Engineering the right formulation for enhanced drug delivery.

Dry powder inhalers (DPIs) can be used with a wide range of drugs such as small molecules and biologics and offer several advantages for inhaled therapy. Early DPI products were intended to treat asthma and lung chronic inflammatory disease by administering low-dose, high-potency drugs blended with lactose carrier particles. The use of lactose blends is still the most common approach to aid powder flowability and dose metering in DPI products. However, this conventional approach may not meet the high demand for formulation physical stability, aerosolisation performance, and bioavailability. To overcome these issues, innovative techniques coupled with modification of the traditional methods have been explored to engineer particles for enhanced drug delivery. Different particle engineering techniques have been utilised depending on the types of the active pharmaceutical ingredient (e.g., small molecules, peptides, proteins, cells) and the inhaled dose. This review discusses the challenges of formulating DPI formulations of low-dose and high-dose small molecule drugs, and biologics, followed by recent and emerging particle engineering strategies utilised in developing the right inhalable powder formulations for enhanced drug delivery.

Phage-Antibiotic Therapy as a Promising Strategy to Combat Multidrug-Resistant Infections and to Enhance Antimicrobial Efficiency.

Infections caused by multidrug-resistant (MDR) bacteria have highlighted the importance of the development of new antimicrobial agents. While bacteriophages (phages) are widely studied as alternative agents to antibiotics, combined treatments using phages and antibiotics have exhibited Phage-Antibiotic Synergy (PAS), in which antibiotics promote phage replication and extraordinary antimicrobial efficacy with reduced development of bacterial resistance. This review paper on the current progress of phage-antibiotic therapy includes aspects of the mechanisms of PAS and the therapeutic performance of PAS in combating multidrug-resistant bacterial infections. The choice of phages and antibiotics, the administration time and sequence, and the concentrations of the two agents impact the bacterial inhibitory effects to different extents.

Novel antimicrobial agents for combating antibiotic-resistant bacteria.

Antibiotic therapy has become increasingly ineffective against bacterial infections due to the rise of resistance. In particular, ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have caused life-threatening infections in humans and represent a major global health threat due to a high degree of antibiotic resistance. To respond to this urgent call, novel strategies are urgently needed, such as bacteriophages (or phages), phage-encoded enzymes, immunomodulators and monoclonal antibodies. This review critically analyses these promising antimicrobial therapies for the treatment of multidrug-resistant bacterial infections. Recent advances in these novel therapeutic strategies are discussed, focusing on preclinical and clinical investigations, as well as combinatorial approaches. In this 'Bad Bugs, No Drugs' era, novel therapeutic strategies can play a key role in treating deadly infections and help extend the lifetime of antibiotics.

Topical liquid formulation of bacteriophages for metered-dose spray delivery.

Bacteriophage (phage) therapy is a promising treatment strategy to combat antibiotic-resistant bacteria. Clinical reports from a century ago, as well as recent reports have revealed safety and efficacy of phage therapy for bacterial wound infections. However, the conventional liquid phage formulation and delivery platforms reported lack of dose control as it easily runs off from the infection site and it is impossible to determine total volume transfer. The aim of this study was to formulate phage liquids for topical delivery using a metered-dose spray. Two types of anti-Pseudomonas phages, PEV1 (myovirus) and PEV31 (podovirus) were formulated in 35% ethanol in water containing non-ionic polymers. The formulations were evaluated for physical properties, ease of spray, dripping upon spraying, drying time, in vitro release profiles, antibacterial activity, and storage stability. The optimized phage-polymer spray formulations were easily sprayable with minimal dripping and fast drying time. Phages were rapidly released from the formulation and inhibited the growth of Pseudomonas aeruginosa. Both PEV1 and PEV31 remained biologically stable in the optimized formulations during storage at 4 °C for eight weeks. This study showed the topical spray formulations containing non-ionic polymers in ethanol/water could be a promising and innovative therapeutic system for delivering phages.

Advances in the development of antimicrobial peptides and proteins for inhaled therapy.

Antimicrobial peptides and proteins (APPs) are becoming increasingly important in targeting multidrug-resistant (MDR) bacteria. APPs is a rapidly emerging area with novel molecules being produced and further optimised to enhance antimicrobial efficacy, while overcoming issues associated with biologics such as potential toxicity and low bioavailability resulting from short half-life. Inhalation delivery of these agents can be an effective treatment of respiratory infections owing to the high local drug concentration in the lungs with lower exposure to systemic circulation hence reducing systemic toxicity. This review describes the recent studies on inhaled APPs, including in vitro and in vivo antimicrobial activities, toxicity assessments, and formulation strategies whenever available. The review also includes studies on combination of APPs with other antimicrobial agents to achieve enhanced synergistic antimicrobial effect. Since different APPs have different biological and chemical stabilities, a targeted formulation strategy should be considered for developing stable and inhalable antimicrobial peptides and proteins. These strategies include the use of sodium chloride to reduce electrostatic interaction between APP and extracellular DNA in sputum, the use of D-enantiomers or dendrimers to minimise protease-mediated degradation and or the use of prodrugs to reduce toxicity. Although great effort has been put towards optimising the biological functions of APPs, studies assessing biological stability in inhalable aerosols are scarce, particularly for novel molecules. As such, formulation and manufacture of inhalable liquid and powder formulations of APPs are underexplored, yet they are crucial areas of research for clinical translation.

A dual action of D-amino acids on anti-biofilm activity and moisture-protection of inhalable ciprofloxacin powders.

Although inhalation powder aerosols of antibiotics have been used to treat respiratory infections caused by Pseudomonas aeruginosa, biofilms are difficult to clear. Ciprofloxacin and D-amino acids (D-Met, D-Trp and D-Phe) were shown to facilitate P. aeruginosa biofilm removal. Spray dried powders for inhalation tend to be amorphous, hence unstable to moisture which causes deterioration in the aerosol performance. Hydrophobic L-amino acids such as leucine can impart moisture protection. In this study, we hypothesized that co-spray dried formulations of ciprofloxacin and hydrophobic D-amino acids will offer the combined benefits of both anti-biofilm and moisture protection properties. Of the three D-amino acids tested, D-Met and D-Trp (at 5 mM) but not D-Phe reduced clinical isolate P. aeruginosa biofilm loads and the extent of biofilm clearance was further enhanced in the presence of ciprofloxacin. Subsequently, ciprofloxacin was spray dried alone or in combination with 30% (w/w) D-Met or D-Trp. The biological and physicochemical properties of the powders were assessed, including the minimum inhibitory concentration, anti-biofilm activity, particle size distribution and morphology, solid-state properties, water sorption, and aerosol performance. The spray dried combination powders were physically stable and inhalable with fine particle fraction (<5 µm) values of 50-57% when aerosolized. The powders exhibited enhanced anti-biofilm activity compared with ciprofloxacin alone. The presence of D-amino acids provided moisture protection, with the recrystallization event shifting from 50% RH to 80% RH in powders containing D-Trp. In conclusion, the use of D-amino acids (D-Met or D-Trp) is an attractive formulation strategy which offers dual benefits of anti-biofilm effect and moisture protection.

The effects of different doses of inhaled bacteriophage therapy for Pseudomonas aeruginosa pulmonary infections in mice.

OBJECTIVES: Inhaled phage therapy has been revisited as a potential treatment option for respiratory infections caused by multidrug-resistant Pseudomonas aeruginosa; however, there is a distinct gap in understanding the dose-response effect. The aim of this study was to investigate the dose-response effect of Pseudomonas-targeting phage PEV31 delivered by the pulmonary route in a mouse lung infection model. METHODS: Neutropenic BALB/c mice were infected with multidrug-resistant P. aeruginosa (2 × 104 colony-forming units) through the intratracheal route and then treated with PEV31 at three different doses of 7.5 × 104 (Group A), 5 × 106 (Group B), and 5 × 108 (Group C) plaque-forming units, or phosphate-buffered saline at 2 hours postinoculation. Mice (n = 5-7) were euthanized at 2 hours and 24 hours postinfection, and lungs, kidneys, spleen, liver, bronchoalveolar lavage fluid, and blood were collected for bacteria and phage enumeration. RESULTS: At 24 hours postinfection, all phage-treated groups exhibited a significant reduction in pulmonary bacterial load by 1.3-1.9 log10, independent of the delivered phage dose. The extent of phage replication was negatively correlated with the dose administered, with log10 titre increases of 6.2, 2.7, and 9 for Groups A, B, and C, respectively. Phage-resistant bacterial subpopulations in the lung homogenate samples harvested at 24 hours postinfection increased with the treatment dose (i.e. 30%, 74%, and 91% in respective Groups A-C). However, the mutants showed increased susceptibility to ciprofloxacin, impaired twitching motility, and reduced blue-green pigment production. The expression of the inflammatory cytokines (IL-1ß and IL-6, and TNF-α) was suppressed with increasing PEV31 treatment dose. DISCUSSION: This study provides the dose-response effect of inhaled phage therapy that may guide dose selection for treating P. aeruginosa respiratory infections in humans.

Pharmacokinetics and safety of inhaled ivermectin in mice as a potential COVID-19 treatment.

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.

Inhalation delivery technology for genome-editing of respiratory diseases.

The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has significant therapeutic potentials for lung congenital diseases such as cystic fibrosis, as well as other pulmonary disorders like lung cancer and obstructive diseases. Local administration of CRISPR/Cas9 therapeutics through inhalation can achieve high drug concentration and minimise systemic exposure. While the field is advancing with better understanding on the biological functions achieved by CRISPR/Cas9 systems, the lack of progress in inhalation formulation and delivery of the molecule may impede their clinical translation efficiently. This forward-looking review discussed the current status of formulations and delivery for inhalation of relevant biologics such as genes (plasmids and mRNAs) and proteins, emphasising on their design strategies and preparation methods. By adapting and optimising formulation strategies used for genes and proteins, we envisage that development of inhalable CRISPR/Cas9 liquid or powder formulations for inhalation administration can potentially be fast-tracked in near future.

Phage cocktail powder for Pseudomonas aeruginosa respiratory infections.

Phage cocktail broadens the host range compared with a single phage and minimizes the development of phage-resistant bacteria thereby promoting the long-term usefulness of inhaled phage therapy. In this study, we produced a phage cocktail powder by spray drying three Pseudomonas phages PEV2 (podovirus), PEV1 and PEV20 (both myovirus) with lactose (80 wt%) and leucine (20 wt%) as excipients. Our results showed that the phages remained viable in the spray dried powder, with little to mild titer reduction (ranging from 0.11 to 1.3 logs) against each of their specific bacterial strains. The powder contained spherical particles with a small volume median diameter of 1.9 µm (span 1.5), a moisture content of 3.5 ± 0.2 wt%., and was largely amorphous with some crystalline peaks, which were assigned to the excipient leucine, as shown in the X-ray diffraction pattern. When the powder was dispersed using the low- and high-resistance Osmohalers, the fine particle fraction (FPF, wt. % of particles < 5 µm in the aerosols relative to the loaded dose) values were 45.37 ± 0.27% and 62.69 ± 2.1% at the flow rate of 100 and 60 L/min, respectively. In conclusion, the PEV phage cocktail powder produced was stable, inhalable and efficacious in vitro against various MDR P. aeruginosa strains that cause pulmonary infections. This formulation will broaden the bactericidal spectrum and reduce the emergence of resistance in bacteria compared with single-phage formulations reported previously.

Enteric-coated bacteriophage tablets for oral administration against gastrointestinal infections.

Intestinal Pseudomonas aeruginosa is highly problematic in immunocompromised patients such as those in intensive care units in hospitals. Phage therapy is an attractive alternative or supplementary therapy to antibiotics as it not only kills multidrug-resistant bacteria, but also minimises the disruption of gut microflora. Solid oral dosage forms (i.e., tablets) have the potential to effectively deliver viable phages to the gastrointestinal tract, but formulation studies have been scarce. In this study, Pseudomonas-targeting phage PEV20 was used as a model to produce tablets suitable for oral delivery by utilising industry-scale tablet compression and tablet coating machines. Phage tablets were produced by (i) spray drying of phages, (ii) direct compression of the phage powders into tablets, and then (iii) tablet coating. The resulting phage tablets had negligible phage titre reduction throughout the process and passed the British Pharmacopeia tests, including friability, weight variation, disintegration and dissolution of the tablets as well as weight gain and disintegration (in 0.1 M HCl and pH 7.4 phosphate buffer) of coated tablets. The developed formulation method can be utilised to produce tablets containing other phages and phage cocktails that are effective against enteric bacterial infections.