Clinical trials of phage therapy for lung infections.
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.
- pulmonary delivery
- multidrug-resistant bacteria
- respiratory infection
- dry powder inhaler
- phage formulation
- inhaled phage therapy
Lung infection is a leading cause of morbidity and mortality worldwide . Currently, antibiotics remain the mainstay treatment options for bacterial lung infections . 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, 4, 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 . Overall, phage therapy for respiratory infections have not been extensively studied and only a handful of human studies reported [6, 7, 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 . 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
Aslam et al. reported the early clinical experience of phage therapy in lung transplant recipients in the USA . Three patients with life-threatening MDR infections caused by
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
|ClinicalTrails. gov Identifier||Phase||Target condition/Disease||Phage||Design||Trail status|
|NCT01818206||NA||Cystic Fibrosis||A cocktail of 10 bacteriophages||Single Group Assignment||Completed in 2012|
|NCT04596319||1b/2a||Chronic ||AP-PA02 cocktail||Parallel Assignment (Randomized, double-blind, placebo-controlled)||Recruiting as of the preparation of this book chapter|
|NCT04636554||NA||Covid-19 patients with bacterial co-infections||Phages against ||Expanded Access (Intermediate-size Population, Treatment IND/Protocol)||Recruiting as of the preparation of this book chapter|
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
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, 20, 21], LC Sprint jet nebulizer , AeroEclipse  and atomizer  have been used to deliver therapeutic phages. Leung et al. showed the air-jet nebulization had negligible impacts on the stability of the
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, 24, 25]. Golshahi et al. showed both the LCstar (air-jet) and eFlow (mesh) nebulizers were suitable for the delivery of phages active against
Ultrasonic nebulizers use a piezoelectric transducer to generate ultrasonic wave in the liquid drug formulation and aerosolize it at the solution surface. Upon the 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 . 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
In vivoefficacy of inhaled phage therapy achieved with nebulization
Carrigy et al. recently demonstrated the prophylactic function of nebulized D29 phage for protection against
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 . Cao et al. explored the phage antibacterial effect of hemorrhagic pneumonia in a mink model . 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.
4. Dry powder inhalers
4.1 Powder formulation
Although nebulization has been the method of choice for phage delivery in treating lung infections in clinical settings, dry powder formulations are preferred to liquid formulations in terms of storage, transportation and administration . Compared to nebulizers, dry powder inhalers (DPIs) are easier to handle without the need of a power source, fewer cleaning requirements and quick delivery . Current research on pharmaceutical development of inhaled phage dry powder mainly focuses on formulation optimization for sufficient powder dispersibility to deliver phage to the lung and storage stability. The choice of excipients plays a key role among all the techniques to produce phage dry powder. Zhang et al. published a comprehensive review to discuss how the choice of excipients affecting the stability of phage in the solid-state . Overall, sucrose, lactose and trehalose are the most popular disaccharides in phage powder formulations. Freeze drying (FD), spray drying (SD) and spray freeze drying (SFD) have been used to generate inhalable phage dry powders with these excipients.
FD is a commonly employed technique to stabilize drugs in solid state . Puapermpoonsiri et al. used FD to generate dry powder of phage-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres designed for pulmonary delivery . Although phages were successfully incorporated into the PLGA microparticles, the poor shelf-life of the encapsulated phage which completely deactivated within 7 days either stored at 4 °C or 22 °C was discouraging. In their follow-up study, they investigated the feasibility of using a high concentration of sucrose (0.5 M) or PEG6000 (5%) to stabilize the FD phage cake . Although rapid phage reduction was still noted over the first 7–14 days, phage remained relatively stable in the powder formulations thereafter. Since then, a number of studies have studied the impacts of various excipients on the production loss and storage stability of FD phages [40, 41, 42, 43]. Among all excipients examined, sucrose and trehalose were identified as the most promising stabilizers to preserve phage viability upon the dehydration in the drying process and upon storage. The residual moisture content was found to play an important role in maintaining phage stability. Similar to other protein therapeutics, a 3–6% moisture content of the powder cake was found to be optimal for phage preservation [39, 41]. Although the mechanisms of phage stabilization in dry powder by these sugars are still unclear. Two most acceptable hypotheses for the stabilization of proteins in the solid state by sugars are water replacement and vitrification, which may also be applicable to phages because they are mostly composed of proteins.
In general, FD powder is not respirable, and a separate milling step is required to reduce the particle size to <5 μm, suitable for pulmonary delivery. However, the high-energy milling may cause additional phage loss due to the generation of heat and mechanical stresses. Golshahi et al. prepared FD formulations of KS4-M and ΦKZ phages with 60% lactose and 40% lactoferrin suitable for pulmonary delivery without milling . The size of the phage powder was within the inhalable range (< 5 μm) and acceptable aerosol performance with a fine particle dose of >106 pfu using an Aerolizer was achieved. The production loss was 1–2 log which was not desirable, but the FD phage powders were stable with negligible titer reduction within 3 months storing either at 4 °C or 22 °C.
SD is a well-established single-step technique employed for the production of many inhaled pharmaceutical products . Matinkhoo et al. were among the first to study the feasibility of using SD to produce inhalable phage powders comprising trehalose and leucine with or without a third excipients (a surfactant or casein sodium salt) . In these formulations, trehalose was used to protect phage against dehydration; leucine forming a crystalline shell at the particle surface was used to enhance the dispersibility of powders; and a surfactant was employed to reduce aggregation of phage during the drying process. Due to the thermal sensitivity of phage, a low drying temperature was used to produce SD powders with acceptable production loss (0.4–0.8 log) and phage lung dose (7–8 log pfu). Trehalose-alone formulation was employed by Vandenheuvel et al., but the production loss was found to be phage dependent . On the other hand, trehalose-leucine and lactose-leucine systems could stabilize a panel of
SFD is a relatively new drying technique to produce inhalable dry powders. The produced powders are superior to those prepared by traditional FD in terms of structure, quality, and the retention of volatiles and bioactive compounds . The suitability of SFD porous mannitol carriers for pulmonary delivery of drug nanoparticles and biologics have been demonstrated [58, 59, 60]. Leung et al. produced SFD phage powder and compared their differences of powder properties with the SD phage powders (Figure 1d). With the use of a high frequency of ultrasonic nozzle in the SFD process, a significant titer reduction (>2 log) was noted in the spraying process, making the overall production loss inferior compared with the SD process . Nonetheless, the larger porous carrier provided a larger extent of protection of the embedded phage during aerosolization with a higher recovery of viable phage compared with the SD counterparts. The conventional SFD process is a two-step manufacturing process, which hinders scaling up. Ly et al. used an atmospheric spray freeze-drying (ASFD) technique, which is a single step process, to prepare D29 phage powder . An acceptable titer loss (~0.6 log) was noted due to the use of a twin-fluid nozzle and improved mass and heat transfer rates.
In vivoefficacy of inhalable phage dry powder
Pulmonary delivery of dry powder to small animals is challenging as they cannot inhale powder actively. Intratracheal delivery using a dry powder insufflator, either the commercially available Penn-Century models or custom-made insufflators , are commonly employed to introduce powders directly into the lungs of the experimental animals. Chang et al. explored the
5. Other inhalation devices
5.1 Metered dose inhaler
Pressurized metered-dose inhalers (pMDIs) are the most popular inhalers for the treatment of asthma and chronic obstructive pulmonary diseases. To date, only one study has attempted this type of device to aerosolize phage . The phage cocktail suspension containing FKZ/D3 and KS4-M phages, was formulated in a reverse emulsion with Tyloxapol surfactant using hydrofluoroalkane 134a as the propellant. A limited loss of phage activity (0.5–0.9 log) upon the actuation was observed, but the long term storage stability of the phages was not assessed. Further studies to examine the interactions between phage and liquefied propellant gas , and maximum loading capacity of phage/puff are required to move this inhaler choice forward.
5.2 Soft mist inhaler
Soft mist inhaler (SMI) is a relatively new generation, propellant-free inhaler that delivers drugs to the lung more efficiently than pMDIs because of the lower spray velocity and longer duration time . Carrigy et al. compared the delivery efficiency of phage among vibrating mesh nebulizer, jet nebulizer and SMI . SMI was showed to deliver phage D29 at high titers quickly (~5 × 108 pfu/actuation) with an acceptable titer reduction (0.6 log pfu/ml) and a higher lung delivery (3.2 × 106 pfu/actuation of inhalable active phage). This compact and light weight device may act as an attractive option for self-administration of phage aerosols.
6. Combination of phage therapy and antibiotic to treat lung infections
6.1 Mechanisms of phage-antibiotic synergy
With the emergence of phage-resistant bacteria , the combination therapy of antibiotics and phages has drawn increasing attention. Synergistic effect of antibiotic and phage against
Interestingly, the sequence of phage and antibiotic administration was found to be critical in the overall antibacterial effect from the combination treatment. Chaudhry et al. showed the efficiency of removing
6.2 Novel tools for selection of optimum phage-antibiotic combination
Since the exact mechanisms responsible for PAS are still unclear and the choice of the combinations is mostly empirical, it is not surprising that mixed results were reported in the literature [72, 82]. Also, the concentration of antibiotics used in previous studies was limited to one or two levels, which is not enough to predict the efficacious concentration when applied in clinical treatment. To solve these problems, Liu et al. developed a high-throughput platform called synogram by combining an optically based real-time microtiter plate readout with a matrix-like heat map to quickly assess the effects of various phage and antibiotic concentrations on bacterial growth . They concluded that PAS is highly dependent on the antibacterial mechanism of action for antibiotic and phage pairs and their stoichiometry.
To guide the choice of phage-antibiotic combination, Rodriguez-Gonzalez et al.  developed an
6.3 Formulations of phage-antibiotic combination to treat lung infections
7. Challenges for pulmonary delivery of phage and future perspective
Phage therapy is evolving as a promising alternative or an adjuvant to antibiotics for the battle against MDR bacteria. Although a few randomized, double-blind and placebo-controlled clinical trials have been conducted to assess tolerance and/or efficacy of phage therapy in the past few years, none of the completed trials have yielded data supporting the promising observations noted in the experimental phage therapy conducted in animals and humans. Górski et al. highlighted the importance of the quality and titer of the phage preparations and their delivery efficiency to the target sites to ensure a sufficient high phage to bacteria concentration in the vicinity of infected tissues . For lung infection, directly delivering phage preparation to the airways enhance the incidence of phage getting access to its host bacteria, avoiding the rapid clearance in systemic circulation. Advancements have been made in the past decade to improve the formulations for pulmonary delivery of phage. Here we highlight some hurdles remained to be tackled to bring inhaled phage therapy to clinical settings beyond compassionate use and a few prospective research directions for the commercial application of aerosol formulations.
As a sufficient amount of phage at the site of infection is the prerequisite for successful therapy, nebulizers and DPI are better choice for pulmonary delivery of phage compared with pMDI and SMI due to their capacity of high dose delivery. The detrimental effect of the various type of nebulizers to phage was found to be phage-specific, likely attributing to the tail morphology of phage  and compositions of the phage formulations . Systematic studies to confirm their impacts on phage nebulization will provide important information in developing new phage cocktail formulations. Although liquid formulations are commonly used for phage therapy, solid phage formulations are more desirable for long-term storage and transportation. While stable phage powder formulations have been successfully achieved with storage at ambient temperature, they are usually required to be handled and stored at low humidity conditions (RH < 20%) [48, 49, 50]. These would be easily achievable in a manufacturing setting and with pharmaceutical packaging designs. As excessive environmental moisture could also be relevant in patients’ homes or in healthcare settings, the impacts of humidity on powdered phage administration should be evaluated to ensure the phage product could be used successfully in different geographic regions over the world. In preparing phage-powder formulation, trehalose, lactose, and leucine are commonly employed to stabilize phage. However, these excipients have not been approved for inhalation except lactose was approved as a carrier which is not expected to be delivered to the lower respiratory tract. Further
The role of the immune system on phage therapy is largely unexplored in animal studies and human trials [33, 88]. Depending on administration route, phage type and phage dose, and duration of phage therapy can lead to the generation of neutralizing antibodies . Together with increasing evidences showing the interactions between phage and mammalian cells [95, 96, 97], it would be worthwhile to explore the interaction between phage formulations with lung leukocytes and epithelial cells lining the alveolar surface and the conducting airways.
Current phage formulation research is largely empirical based. To speed up the research progress for phage therapy,
In the past decade, highly acceptable formulations have been achieved with minimal phage loss and desirable stability for pulmonary delivery using both nebulizers and dry powder inhalers. The synergistic effect of the phage-antibiotic combination provides an efficient way to prevent the emergence of bacterial resistance and reduce the toxicity of antibiotic use. However, systematic PKPD profile of phage after administration by inhalation, and the modern tools to accurately predict the result of combination therapy are still pending. With the advent of phage research, the sound manufacturing and regulatory guidelines towards successful clinical trials to bring phage therapy to clinical settings will be beneficial to the patients suffering from bacterial infections.
The authors gratefully acknowledge the provision of graduate studentship from CUHK to W. Yan and S. Mukhopadhyay is supported by the HKPFS. The funding support from University Grants Committee Hong Kong (ref. 24300619) for our phage research is greatly acknowledged.