Potential of Inhaled Bacteriophage Therapy for Bacterial Lung Infection

*Wei Yan, Subhankar Mukhopadhyay, Kenneth Kin Wah To and Sharon Shui Yee Leung*

### **Abstract**

Phage therapy as a promising alternative antimicrobial to treat multidrug resistant (MDR) bacteria related lung infections, has drawn significant attention in clinical trials and bench-scale study in the recent decade, and the therapeutic effect of local delivery of phage has been demonstrated by several clinical reports. This book chapter discusses the current clinical development of inhaled phage therapy followed by the advancement of phage formulation designs for respiratory delivery of phage using various inhalation devices and their *in vivo* efficacy. The development of combination therapy of phage and antibiotics to combat MDR bacteria associated lung infections is also covered to reflect the current clinical practice. Lastly, we also share our insights on the challenges of advancing inhaled phage therapy and potential directions for future research.

**Keywords:** pulmonary delivery, multidrug-resistant bacteria, respiratory infection, dry powder inhaler, nebulization, phage formulation, inhaled phage therapy

### **1. Introduction**

Lung infection is a leading cause of morbidity and mortality worldwide [1]. Currently, antibiotics remain the mainstay treatment options for bacterial lung infections [2]. With the rapid emergence of multidrug-resistant (MDR) bacteria, last-line antibiotics such as colistin and carbapenem have been increasingly used for life-threatening infections. However, nosocomial outbreaks caused by pan-drug resistant (PDR) 'superbugs' have also been increasingly reported worldwide, creating significant therapeutic challenges for the treatment of lung infections [3–5].

Bacteriophage (phage) therapy has been proposed as a promising alternative to antibiotics in combating bacterial infections, including those caused by the MDR pathogens. A comprehensive review from Abedon summarized earlier clinical studies of phage application, with most reported cases from Eastern Europe as these countries more practical experience [6]. Overall, phage therapy for respiratory infections have not been extensively studied and only a handful of human studies reported [6–8].

Although recent failure of the "Phagoburn" trial against burn wound infections is discouraging, a lesson we learnt is the importance of the stability of phage preparations and the efficient delivery of sufficient amount of viable phage to the site of infections [9]. Pulmonary delivery of phage would hold the greatest promise in achieving optimal concentration of phage in the lung for effective treatment. In this book chapter, we first introduce the clinical progress of inhaled phage therapy and highlight recent advancement made in the delivery of phage preparations using various inhalation devices. As most experimental phage therapeutic investigations were conducted with concomitant antibiotic treatment, we also discuss the development of phage-antibiotic combinations to treat lung infections. Lastly, we summarize the challenges that must be overcome in order to translate inhaled phage therapy to clinical applications.

## **2. Clinical development of inhaled phage therapy**

In the past decade, a few success stories in experimental inhaled phage therapy were reported. Hoyle et al. reported a successful inhaled phage therapy to manage chronic lung infection caused by MDR *Achromobacter xylosoxidans* [10]. The 17-year-old female patient was unsuccessfully treated with many rounds of antibiotics before she was given a phage cocktail treatment containing two *Achromobacter* phages in the Eliava Phage Therapy Center. The phage cocktail was given by nebulization once daily and orally twice daily for 20 days. The treatment was repeated 4 times at 1, 3, 6 and 12 months after the initial treatment. The patient's subjective conditions were significantly improved and her lung function-FEV1 increased from 1.83 L to 3.33 L together with intermittent antibiotic regimen. Successful phage treatment was also reported for a 12-year-old lung-transplanted cystic fibrosis (CF) patient suffered from persistent lung infection caused by PDR *A. xylosoxidans* [11]. After two rounds of inhaled phage therapy, the patient's respiratory condition slowly improved and the bacterial load was significantly reduced. Similar favorable therapeutic efficacy was also reported in another clinical case [12], where a fiveyear-old cystic fibrosis patient suffering from severe lung infections was treated with a commercially available phage preparation (pyophage) by nebulization.

Aslam et al. reported the early clinical experience of phage therapy in lung transplant recipients in the USA [13]. Three patients with life-threatening MDR infections caused by *Pseudomonas aeruginosa* (n = 2) and *Burkholderia dolosa* (n = 1) received phage cocktails via both intravenous injection and nebulization with concurrent antibiotic treatments for variable duration. Two patients responded clinically with the phage treatments and were discharged from hospitals, while the third patient infected by *B. dolosa* was dead due to infection relapsed. Nonetheless, no phage therapy-related adverse events were identified. While these experimental use of inhaled phage therapy as an adjunct treatment has demonstrated the clinical benefits in treating lung infections caused by MDR superbugs, well-designed clinical trials are needed to convincingly evaluate its clinical efficacy.

To date, there have been three phage therapy clinical studies registered with the ClinicalTrials.gov to evaluate the safety and efficacy of phage therapy against lung infections (**Table 1**). "MUCOPHAGES" (NCT01818206) assessed the effect of a cocktail of 10 phages on *P. aeruginosa* from sputum samples isolated from CF patients. Although the trial was completed in 2012 according to the clinical trial registry, no information about the outcome of this trial was published. In 2020, two other trials were launched. One trial (NCT04636554) is attempting to apply personalized phage treatment in Covid-19 patients with bacterial co-infections microbial for pneumonia or bacteremia/septicemia. Another trial launched by Armata Pharmaceuticals is a Phase 1b/2a, double-blind, randomized, placebocontrolled trial (NCT04596319) aiming to study the safety, tolerability, and preliminary efficacy of inhaled AP-PA02 in subjects with CF and chronic pulmonary


*Potential of Inhaled Bacteriophage Therapy for Bacterial Lung Infection DOI: http://dx.doi.org/10.5772/intechopen.96660*

#### **Table 1.**

*Clinical trials of phage therapy for lung infections.*

*P. aeruginosa* infection. This is the first randomized trial on inhaled phage therapy and the AP-PA02 cocktail is an advanced version of AP-PA01 which was used in the successful experimental study documented in Aslam et al. [13]. The findings from this trial are expected to set a landmark for the development of inhaled phage therapy.

### **3. Nebulization**

### **3.1 Liquid formulation**

Majority of the phage studies for lung delivery focus on liquid formulations as minimal formulation development is required to prepare phage cocktails with sufficient stability for a short storage period. The long term storage stability of phage in liquid formulations was often reported. Cooper et al. demonstrated a phage cocktail of 3 *Pseudomonas* phages (GL-1, GL-12.5 and LP-M10) suspended in phosphate buffered saline (PBS) was stable at both 4 °C and room temperature with no statistically significant titer loss (≤ 0.5 log) for 6 months [14]. As most commonly used phage stabilizers, including PBS, salt-magnesium buffer (SMB) and Tris-H buffer are not yet approved for inhalation. Dilution of phage suspension with 0.9% sodium chloride (NaCl) is usually needed for pulmonary administration [10]. Carrigy et al. showed minimal impacts on the phage stability with the NaCl dilution process, suggesting the suitability of this approach [15].

To date, nebulization has been the exclusive choice for pulmonary delivery of phage suspension in human studies due to its high delivery efficiency and capability of delivering a large volume of liquid phage formulation (> 1 mL) to patients including those cannot administer the dose voluntarily. Several types of commercial nebulizers are available to aerosolize phage into fine droplets using different aerosol generation mechanisms, including air-jet nebulization, vibrating mesh nebulization, ultrasonic nebulization, and colliding liquid jets [16, 17]. The suitability of these nebulizers in delivering phage to lungs has been previously evaluated in terms of deactivation of phage upon the nebulization process.

Jet nebulizers use compressed air to atomize the liquid phage suspension into primary droplets and their subsequent impaction onto the baffle would further breakdown into smaller droplets suitable for inhalation. LC-star nebulizer [16, 18], Collison 6-jet [19–21], LC Sprint jet nebulizer [22], AeroEclipse [23] and atomizer [24] have been used to deliver therapeutic phages. Leung et al. showed the air-jet nebulization had negligible impacts on the stability of the *Podovidae* PEV2 phage, while significant titer loss was found in *Myoviridae* PEV40 phage (~1 log loss) and *Siphoviridae* D29 phage (~3 log loss) [22]. Based on the cryo-transmission electron microscopy analysis, they found the nebulization-induced titer loss was correlated with morphological damage to phages. They further suggested that the length of phage tail may be an important consideration when delivering phages via jet nebulization, particularly for phage cocktails containing phages of different morphologies. The influence of the final formulation composition for nebulization of D29 phage was evaluated by Liu et al. using a Collison 6-jet nebulizer [19, 21]. They reported that deionized water was the optimal spray liquid for D29 aerosol generation and they postulated that the high ion strength and salt concentrations in the PBS and 0.9% NaCl were detrimental to the phage upon jet nebulization. These results were in accord with Carrigy et al. and Leung et al. nebulizing buffered D29 using other jet nebulizers [15, 22]. Liu et al. also studied the impact of relative humidity (RH) on the stability of nebulized D29 and found a low environmental humidity condition was more favorable for D29 nebulization [19]. Later, Verreault et al. reported that the stability of nebulized phage aerosols at different temperatures and humidity is phage-dependent with some being more robust and some being more vulnerable [21]. Overall, these studies highlighted the importance of controlling the temperature and RH for phage nebulization.

Vibrating mesh nebulizers produce aerosol droplets by extruding the liquid formulation through a membrane with calibrated holes based on the converse piezoelectric effects. Several studies compared the aerosol delivery of phage between jet and mesh nebulizers [15, 16, 23–25]. Golshahi et al. showed both the LCstar (air-jet) and eFlow (mesh) nebulizers were suitable for the delivery of phages active against *Burkholderia cepacia* Complex by imaging the lung deposition and mathematical model prediction [16]. In some studies, mesh nebulizers were found to be more detrimental to phage than air-jet nebulizers [23, 24], but reasons for the poorer delivery of mesh nebulizer were unclear. In contrast, better phage recovery was noted after nebulizing using a mesh nebulizer compared with the jet nebulization in some other studies [15, 25]. Visual evidence on the correlation between the titer reduction and morphological change of a *Myoviridae* PEV44 phage after nebulization was provided by Leung et al., showing more "intact" phage was detected in the mesh-nebulized phage samples under TEM image. The more destructive effect of jet nebulization is likely caused by stresses associated with the droplet production and re-nebulization processes. Based on the collected experimental data and a mathematical model, Carrigy et al. estimated phage were re-nebulized an average of 96 times before exiting the mouthpiece of the jet nebulizer [15]. A review from Prichard et al. revealed that 86% of the disclosed nebulizer technology have chosen vibration-mesh nebulizers as the delivery devices, particularly for stress-sensitive drugs [26]. The mixed findings of phage nebulization in the literature can be attributed to many factors, such as phage types, formulation composition, experimental conditions (like temperature, humidity and sample collection methods) and different models of the same nebulizer type. Therefore, the survival of individual phages within a cocktail should be tested with different delivery devices for the optimization of phage cocktail – inhalation device combinations.

Ultrasonic nebulizers use a piezoelectric transducer to generate ultrasonic wave in the liquid drug formulation and aerosolize it at the solution surface. Upon the

*Potential of Inhaled Bacteriophage Therapy for Bacterial Lung Infection DOI: http://dx.doi.org/10.5772/intechopen.96660*

nebulization process, a portion of the ultrasonic energy converts to heat, which could be detrimental to heat-sensitive biologics, like phages. Only one study reported the use of an ultrasonic nebulizer to deliver phage to treat lung infections in a mink model, but little data on the nebulization process was available [27]. More recently, Marqus et al. assessed the capability of a novel low cost and portable hybrid surface and bulk acoustic wave (HYDRA) nebulizer to deliver a *Myoviridae* phage K and lysostaphin to target *Staphylococcus aureus* [28]. Negligible titer reduction was noted (0.1 log loss), possibly due to the relatively low powers and high frequencies (approximately 10 MHz) of the nebulizer. Furthermore, the size of the aerosols generated by HYDRA is smaller (DV50 1.85 μm), well within the respirable range, demonstrating its suitability for pulmonary delivery of phages.

### **3.2** *In vivo* **efficacy of inhaled phage therapy achieved with nebulization**

The *in vivo* efficacy of phage liquid formulation has been studied in rodent and mink models. Semler et al. established *B. cenocepacia* respiratory infection model in mice and then treated with liquid phage formulation delivered by a LC-star jet nebulizer or intraperitoneal injection (IP) [18]. After a 2-day treatment, the lung bacterial load was only reduced by ~0.5 log in mice received phage via IP injection, but a 2-log bacterial reduction was observed in mice treated with inhaled phage. This finding is in contradiction with a previous study showing that phage delivered by the IP route was more efficacious than intranasal instillation in treating a *B. cenocepacia* respiratory infection in mice [29]. Semler et al. accounted the discrepancy to the efficiency of phage delivery to lungs that nebulization is a more effective way in delivering phage particles to the lung than intranasal instillation. Also, the capability of IP injected phage reaching lung is significantly affected by the clearance rate of phage in blood which is phage-dependent. The *in vivo* delivery efficiency of D29 phage using a Collison 6-jet nebulizer and IP route was compared by Liu et al. [20]. Approximately 10% of D29 phage could reach to the lung of mice after nebulization and complete phage elimination was noted in 72 h, whereas only 0.1% of the phage could reach the lung by IP injection and no phage was detected after 12 h. The importance of phage dose on the pharmacokinetics/pharmacodynamics (PK/PD) of inhaled phage therapy was recently confirmed by Chow et al. using *Pseudomonas* phage PEV31 [30].

Carrigy et al. recently demonstrated the prophylactic function of nebulized D29 phage for protection against *Mycobacterium tuberculosis* infection in a mouse model [31]. Phage was delivered with a vibrating mesh nebulizer and a dose of 6.6 log phage reached the lung and remained there for 90 min post-delivery, suggesting that phage was not rapidly cleared in the mouse lung. Low doses of *M. tuberculosis* (5–100 CFU) were given to mice 30 min post phage administration. This phage pretreatment was able to significantly reduce the bacterial burden in mouse lungs at 24 h and 3 weeks post infection. The prophylactic effect of phage was also demonstrated in a rat model against methicillin-resistant *S. aureus* infection [32]. Phage was given by a vibrating mesh nebulizer 4 h before the bacterial challenge, higher survival rate (60–70% improvement) with a 2 log bacterial reduction in the rat lungs were observed. Both studies demonstrated prophylactic treatment with sufficient dose of nebulized phage may provide protection to immunocompromised individuals and health care professionals who are at risk of exposure to "superbugs".

There is accumulating evidence that bacterial clearance by phage therapy requires the synergy between phage and host immune system. Therefore, the translation of preclinical data collected from rodent to humans should be treated with care due to the significant difference in their immune systems [33]. Cao et al. explored the phage antibacterial effect of hemorrhagic pneumonia in a mink model [27]. Effective treatment

outcomes were achieved at multiplicity of infection (MOI) of 10 with an 80% survival rate at 12 days after phage administrated by means of ultrasonic nebulization.
