Respirable Bacteriophages for the Treatment of Bacterial Lung Infections

Respirable Bacteriophages for the Treatmentof Bacterial Lung Infections

Susan Hoe, PhD,1 Diana D. Semler, MSc,2 Amanda D. Goudie, MSc,2 Karlene H. Lynch, PhD,2

Sadaf Matinkhoo, BSc,1 Warren H. Finlay, PhD,1 Jonathan J. Dennis, PhD,2 and Reinhard Vehring, Dr-Ing1

Abstract

This review article discusses the development of respiratory therapeutics containing bacteriophages indicatedfor lung infections, specifically those that have become increasingly difficult to treat because of antibiotic re-sistance. Recent achievements and remaining problems are presented for each step necessary to develop abacteriophage-containing dosage form for respiratory drug delivery, including selection of appropriate bacte-riophages for therapy, processing and purification of phage preparations, formulation into a stable, solid dosageform, and delivery device selection. Safety and efficacy studies in animals and human subjects are also reviewed.

Key words: bacteriophages, respiratory drug delivery, bacterial lung infections, antibiotic resistance, phagetherapy

Introduction

One of the most significant achievements of modernmedicine is the control of infections. Clinical procedures

such as surgery, transplantation, and chemotherapy are notfeasible without effective control of bacterial infections. An-tibiotics were discovered and put into clinical practice in thefirst half of the 20th century and contributed to a significantincrease in life expectancy.(1) Antibiotics are substances thatcan kill or inhibit the growth of bacteria by interfering withbacterial functions or growth processes.

Antibiotics can be categorized based on their specificity.Broad-spectrum antibiotics can be used against a wide rangeof bacteria, but even narrow-spectrum antibiotics typicallytarget a whole class of bacteria, such as Gram-negative orGram-positive. Due to the relatively nonspecific mechanismsof action of some broad-spectrum antibiotics, bacterialstrains are often not identified before therapy is initiated.However, the nonspecific activity also results in some of thecommonly observed antibiotic side effects, e.g., diarrhea,associated with the disruption of beneficial human micro-flora. Arguably the greatest weakness of antibiotics is thattheir effectiveness is diminished by the emergence of bacte-rial resistance. Within two decades after the discovery ofpenicillin, it was recognized that bacterial resistance wouldimpact the efficacy of antibiotics. Sir Alexander Fleming,

among others, warned early—and to no avail—against im-proper use of penicillin, e.g., using insufficient doses or ter-minating treatment prematurely. The emergence of resistantbacterial strains has been accelerated by a variety of factors,such as the overuse and misuse of antibiotics in clinicalpractice and in patients’ hands, and indiscriminate applica-tion of antibiotics in animal husbandry and fish farming. Ithas typically taken between 3 and 30 years following theintroduction of a new antibiotic to observe the onset ofresistance.(2)

Bacterial resistance has become a significant problem inthe treatment of infections of the respiratory system. Re-fractory or chronic infections are common in patients withcompromised lungs, such as cystic fibrosis (CF) patients.Their lungs are colonized by a variety of bacteria, often ini-tially by Staphylococcus aureus or Haemophilus influenzae andlater typically by bacteria of the Burkholderia cepacia complex(Bcc) or by Pseudomonas aeruginosa.(3) Many of these bacteriahave high intrinsic antimicrobial resistance or have acquiredmutations that result in resistance to a broad range of anti-biotics.(4,5) Similarly, highly resistant bacterial strains ofKlebsiella pneumoniae, Streptococcus pneumoniae,(6) and En-terobacter spp. are encountered in hospital emergency careunits and are a leading cause of mortality due to nosocomialpneumonia. Some of these bacterial species have now be-come resistant to all carbapenem antibiotics(7) via the transfer

1Department of Mechanical Engineering, University of Alberta, Edmonton, Alberta, T6G 2G8, Canada.2Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada.

JOURNAL OF AEROSOL MEDICINE AND PULMONARY DRUG DELIVERYVolume 26, Number 0, 2013ª Mary Ann Liebert, Inc.Pp. 1–19DOI: 10.1089/jamp.2012.1001

1

of genes that encode a highly effective b-lactamase en-zyme.(8) In addition, it is likely that at least 5% of the morethan 400,000 multidrug-resistant tuberculosis cases per yearworldwide are caused by extensively drug-resistant Myco-bacterium tuberculosis,(9) which is practically untreatable indeveloping countries.(10) Some of this high-level antibioticresistance is attributable to the fact that several pulmonarypathogens are capable of adopting a biofilm lifestyle,(11) inwhich the bacteria are encased in a self-synthesized polymericmatrix that serves to protect them from antibiotic activity andimmune responses. The exact mechanisms producing biofilmresistance remain undiscovered, but may be through a com-bination of bacterial adaptations, the net effect being thatbiofilm bacteria are nearly impossible to clear even usingcombinations of conventional chemical antibiotics.

More than 20 classes of antibiotics were introduced forclinical use between 1940 and 1962. However, only two havebeen added after this time, and very few are currently indevelopment. Given the recent increase in the use and

overuse of antibiotics, it is not surprising that now manybacterial species have developed resistance against morethan one antibiotic. A recent study by Coates et al. estimatesthat 20 new classes of antibiotics would have to be dis-covered and developed in the next five decades to controlbacterial infections at the current level.(12) Because it appearsunlikely that this goal can be accomplished, even if strongereconomic incentives for the development of new antibioticswere put in place, a search for alternatives to antibioticsseems warranted.

One alternative, phage therapy, has received renewed at-tention.(13,14) Bacteriophages, or phages, are viruses that in-fect and kill bacteria.(15) Figure 1 gives an overview of someselected milestones in the discovery of bacteriophages andtheir application to the treatment of respiratory infections.Phages were discovered independently by Frederick Twortand Felix d’Herelle at the beginning of the 20th century.D’Herelle soon thereafter initiated experimental treatment ofbacterial infections with phage ‘‘cocktails’’ (preparations

FIG. 1. A historical over-view of respiratory phagetherapy.

2 HOE ET AL.

consisting of a collection of different phages active againstmultiple bacterial strains or species), despite the fact thatlittle was known about phage particles at the time.(15,16) Earlyphage therapy was quite successful, leading to the devel-opment of many commercial phage preparations in the 1920sand 1930s by companies such as Eli Lilly & Company andwidespread use of phage therapy in Europe and the UnitedStates. However, early clinical trials also suffered frompoorly characterized phage preparations and a lack of un-derstanding of phage biology. As a result, the efficacy ofphage treatment was called into question, and phage therapyin the West was largely abandoned with the advent of che-mical antibiotics.(17) Nevertheless, phage research intensifiedin the 1940s as phages were recognized by Max Delbruck,among others, as powerful tools for the exploration of ge-netics. The American Phage Group, coordinated by Del-bruck, contributed largely to the scientific understanding ofphages that emerged in the 1940s and 1950s. In 1969, theNobel Prize in Physiology or Medicine was awarded to MaxDelbruck, Alfred D. Hershey, and Salvador E. Luria, ‘‘fortheir discoveries concerning the replication mechanism andthe genetic structure of viruses.’’(18) Phage research becamean exact science with rigorous quantitative methods. Some ofthe earlier failures of phage therapy could now be explained.It was realized that phages typically have a narrow bacterialhost range, and that this host range specificity depends on anexplicit match between the bacteriophage and bacterial sur-faces or surface structures.

The first reports of successful inhalation therapy usingphages emerged around 1960.(19,20) Table 1 outlines selectedstudies of inhalation phage therapy in humans. Delacostesuccessfully treated patients with refractory cough using anebulized phage cocktail.(19) Hoeflmayr nebulized a phagecocktail concomitantly with antimicrobial agents.(20) En-dotoxins were intentionally left in the preparation to stimulatean immune response in the patients. The treatment was suc-cessful in 90% of patients with chronic bronchitis. In EasternEurope, in particular in Russia, Poland, and Georgia, phagetherapy has been continuously applied and further developedsince the 1920s.(21) Phage therapy research centers include theHirszfeld Institute of Immunology and Experimental Ther-apy(22) in Wroc1aw, Poland, and the Eliava Institute(23) inTbilisi, Georgia. In Georgia, phage therapy is an accepted andwidely used mode of treatment. Commercial phage products,including phage cocktails that are available to the publicwithout prescription, are manufactured and approved inGeorgia and Russia.(21) The prospects for respirable phages asan alternative to antibiotics appear promising.

In this article, we provide an overview of the individualsteps that need to be taken to develop a bacteriophage dos-age form for administration to the respiratory system. Thefirst sections review the selection of adequate phages, theiramplification, and purification. Then recent advances in theformulation of phage preparations into stable, respirabledosage forms are discussed and delivery device options arereviewed. Finally, the efficacy and safety of phage therapy asestablished by recent clinical trials are summarized.

Bacteriophage Selection

The first steps in the development of a phage therapeuticare the isolation and selection of safe and efficacious

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BACTERIOPHAGES FOR BACTERIAL LUNG INFECTIONS 3

bacteriophages. Phages are found in vast quantities andabundant variety in all environments that harbor bacteria.Phages specific to a certain bacterial strain usually can beisolated from samples containing the host bacteria, suchas soil or sewage. Phages are obligate parasites of bacteria.They have been shown to coevolve with their bacterialhosts.(24) Consequently, phages specific to a mutated bacte-rium have been observed to emerge soon after mutation ofthe bacterial host.(25)

An effective phage must: (1) successfully bind to the sur-face of the bacterium; (2) transfer its genetic material into thehost bacterium; (3) synthesize progeny virions; and (4) lysethe bacterial cell.

This process of infection is referred to as the ‘‘lytic lifecycle’’ as shown in Figure 2. One lytic cycle is completed inabout 15–60 min and produces typically 100–200 new pha-ges.(21,26–32) In phage therapy, the concentration of phagesin vivo will theoretically grow rapidly if the bacterial host ispresent in sufficient quantity. When used therapeutically, thehigh interaction specificity between a phage and the bacterialcell prevents the viral particles from destroying human cells.However, this also limits the usefulness of certain phagesdue to their extreme selectivity. In selecting a phage, or set ofphages for clinical use, the most important criterion is bac-terial host specificity. Phage host ranges are limited due tofactors such as the inability to bind the host or to transferDNA, the cleavage of phage DNA by host proteins, or bac-terial abortive infection systems.(33) The narrow host range ofphages is beneficial, because it avoids the adverse effects onthe normal flora observed following broad-spectrum antibi-otic treatment.(25) A disadvantage of this specificity, how-ever, is that a panel of phages must be screened on theinfecting bacterium prior to administration in order to assesswhich phages are the most effective in lysing the bacterialcells.(16)

To mitigate this issue, phages with expanded bacterialhost ranges can be isolated or developed using classical ormolecular techniques.(34,35) In addition, multiple phages maybe used together in a cocktail formulation, and this is espe-cially useful for polymicrobial infections where multiplebacterial pathogens are present (as might be found in the CFlung).(34) This can be achieved by selecting phages that infect

the most common bacterial species or strains associated witha particular infection, or by first determining which bacterialspecies or strains are involved in the infection and selectingphages for each. A CF patient, for example, might be treatedwith a cocktail of phages capable of infecting a number ofdifferent P. aeruginosa, Burkholderia cenocepacia, and S. aureusstrains, because these are common pathogens found in thelungs of CF patients.(36) Phage cocktails can also be used toreduce the chance of bacterial resistance occurring, becausephages specific for two different bacterial receptors wouldrequire that a single target cell undergo simultaneous mu-tation of two different receptors, a highly improbable event.

Virulent versus temperate phages

Two other important criteria for phage selection are anobligately lytic lifestyle and the absence of genes that encodevirulence factors. Phages can be classified as either obligatelylytic (also referred to as virulent) or temperate. Obligatelylytic phages follow the lytic life cycle, causing lysis of thebacterial host and release of progeny virions at the end ofeach infection cycle.(37) In contrast, temperate phages mayeither lyse the bacterial host and release virions or, alterna-tively, the phage DNA may be incorporated into the cell aspart of the bacterial chromosome or as a plasmid, remainingdormant as a ‘‘prophage’’ through successive bacterial celldivisions until triggered to reenter the lytic life cycle.(37)

During lysogeny, the host cell is not killed by the phage andbecomes resistant to infection by all other related phages.(38)

The integrated prophage genome may occasionally containsequences that encode virulence proteins such as adhesins ortoxins.(39) In the case of Vibrio cholerae (in which virulencefactors, cholera toxin, and the toxin-coregulated pilus arefound to be prophage-encoded), these genes can have dra-matic effects on the pathogenicity of the bacterial host.(40,41)

Temperate phages may also transfer bacterial virulencegenes via specialized transduction, a process in which hostDNA is packaged into progeny virions along with phageDNA following induction.(42)

Genome sequencing and analysis can be used to assessboth the lifestyle (obligately lytic or temperate) and the genecontent of a phage prior to clinical use. Temperate phageshave characteristic sequences that lytic phages lack, includ-ing a repressor gene, operator sequences to which the re-pressor protein binds, and (in most cases) an integrasegene.(38) It should be noted that, in rare cases, phages withthese sequences have been identified that do not form stablelysogens (lysogenized bacteria).(43,44) Therefore, assessmentof phage lifestyle should include both genomics analysis andscreening for lysogens or turbid plaques (characteristic oftemperate phages).(42) Virulence genes can also be identifiedthrough bioinformatics analysis and may show similarity toproteins that mediate cell adherence, immune evasion, toxinproduction, or other processes.(39)

The concerns discussed above usually lead to a recom-mendation to select lytic phages for therapeutic applications.However, several cases have been identified in which phageswith temperate lifestyles or virulence genes have been shownto be effective in phage therapy applications. For instance,most phages of Clostridium difficile and the Bcc appear to betemperate.(45,46) Out of necessity, phages used (and shown tobe effective) in infection models of these bacterial species areFIG. 2. Life cycle of lytic bacteriophages.

4 HOE ET AL.

either temperate or putatively temperate.(47–50) Temperatephages have also been shown to be effective against S. au-reus, even in a case where the phage carried putative leu-kocidin and enterotoxin A genes.(51–53) Instead of discardingphages encoding proteins involved in lysogeny or virulence,genetic modification may be an option to improve safetyprior to clinical use. Lytic mutants of temperate phages havebeen shown to be effective against Escherichia coli in a mousemodel,(54) S. aureus in human volunteers and a mouse woundmodel,(55) and B. cenocepacia in an invertebrate model.(48)

These phages may be spontaneous mutants (with a charac-teristic clear plaque morphology) or generated by random ortargeted mutagenesis of the operator or repressor se-quences.(42,48,55) Although the use of obligately lytic phagesprevents lysogenic conversion,(56) virulence genes can beremoved from the temperate phage genome such that theydo not introduce problematic genetic material upon recom-bination with the bacterial chromosome during infection.

It has been shown that some phages can penetrate biofilmmatrices, or possess surface-exposed enzymes that are ca-pable of degrading the polymeric matrix components ofbacterial biofilms.(57,58) Such polysaccharide depolymerasesor glycanases are capable of allowing some phages access tobacterial host cells deeper within biofilms, thereby enhancinglysis and degradation of the bacterial biofilm.(59,60) The genesencoding such phage biofilm-degrading enzymes can betransferred to and expressed by other phages in order toenhance phage penetration of biofilms.

In addition to removing lysogeny and virulence genes,or adding genes encoding enzymes capable of enhancingbiofilm degradation, other phage components can be genet-ically manipulated prior to therapeutic use.(61) Such modi-fications include bacterial host range expansion(35,62) andthe introduction of antibacterial toxin genes.(63–65) En-gineered phages have been shown to be active against sev-eral species in mouse models, including E. coli, S. aureus, andP. aeruginosa.(53,64–66)

Potential host effects

Phages are considered to be biological entities and, whenused as therapeutic agents, have the potential to interact withor stimulate the host’s immune system. However, phages asantibacterial agents are not unique in this respect. Severalapproved protein-based pharmaceuticals also have the abil-ity to stimulate an immune response, and chemical antibi-otics that achieve their effects through bacterial lysis willcause the release of bacterial cell wall components, includingendotoxin. Current dogma suggests that phage particles inan animal or human host are cleared by the reticuloendo-thelial system within a few days.(54,67,68) However, the im-munogenicity of phage particles is likely highly variable,partially depending on where the phages are initially ad-ministered (bloodstream versus body surface). For example,Carmody et al. were able to provide a small therapeutic effectto a pulmonary bacterial infection via intraperitoneal phagedelivery, suggesting that some phages can easily penetratedifferent body cavities without activating an anti-phage im-mune response.(47) Within the lung, phages showed ampli-fication and, at the same time, markers of pathogen-inducedinflammation were reduced. Similarly, Merril et al. were ableto evolve long-circulating phages that were presumably less

immunogenic than parental types, suggesting that somephages may be relatively invisible to an immune re-sponse.(54) Although little rigorous information exists aboutimmune stimulation by phages in vivo, the paucity of suchdata suggests that phage lysis of bacteria in an animal orhuman host is of little consequence and, at least, does notworsen the host’s condition. Several recently completedphage therapy clinical trials suggest that the presence ofphages in different body cavities does not enhance the dis-ease state due to immune activation.(16,69) In support of thisview, several phage-related products have now passed someregulatory standards and have been classified by the U.S.Food and Drug Administration as GRAS (Generally Re-garded As Safe), have been registered with the U.S. En-vironmental Protection Agency, or have been approved foruse by the U.S. Department of Agriculture.(70,71)

Summary

By using conventional screening techniques together withgenetic modification, one can select phages for clinical usethat are bacterial host-specific, obligately lytic, free of viru-lence genes, and highly active in vivo. There is little existingdata on the human or animal immune response to phages,but also little evidence of phages having a negative effect ondisease state.

Bacteriophage Preparation

As discussed, there are already many phages availablethat are capable of infecting a wide range of antibiotic-resistant bacteria, and more are continually being isolatedand characterized. The procedures for growing phages arewell described elsewhere; a summary of these preparationsteps is provided in Figure 3.

Phages are obtained from environmental or biologicalsamples, such as soil, river water, sewage effluent, and fecalmatter. One or a panel of bacterial strains is used to propa-gate any desired phages in the sample before filter sterili-zation to remove particulate and cellular matter. Samples areoften mixed with a culture medium along with the bacterialhost, and the entire preparation is incubated to allow phagesto bind to potential hosts. If host-specific phages are presentin the sample, the bacterial cells will be lysed to releaseprogeny virions. The lysate is then centrifuged or passedthrough a membrane filter, or both, to elute the phages in asterilized sample. This sample is then plated against bacterialstrains to isolate the single desired phage.(16,72–74)

Once phages have been isolated, they can then be grownto the desired concentration. This is known as phage am-plification. As with enrichment, the phage is cultured withthe host bacterial strain or strains in a suitable medium andincubated. This can be done in liquid culture or on agarplates, followed by an overlay of sterile water or suspensionmedium to suspend the phages in solution. Separation of thephage from cellular debris is often carried out by centrifu-gation and membrane filtration (Fig. 3).

Early phage preparations were often just filtered lysatesthat still contained bacterial endo- and exotoxins at the pointof human delivery.(75) There are no reports of serious sideeffects due to endotoxins in the early literature covering ex-perimental bacteriophage therapy. Nevertheless, current ex-pectations regarding the safety of biological preparations


require removal of bacterial endotoxin from phage prepara-tions before proceeding with any other modifications (i.e.,spray-drying, freeze-drying, nebulizing, etc.). Endotoxin is apart of the cell wall of Gram-negative bacteria known aslipopolysaccharide. The lipid component is associated withtoxicity, whereas the polysaccharide component is respon-sible for eliciting an immune response.(76)

Lipopolysaccharide is typically present in initial phagepreparations once phages have lysed bacterial host cells.There are a number of commercially available products thatremove endotoxin from phage samples. For large-scalepreparations, ion-exchange or gel filtration chromatographymay be used.(76–78) Another method of removing endotoxininvolves a series of phage dialysis and purification stepsusing an organic solvent such as octyl or butyl alcohol.(79)

Because laboratory-scale methods of bacteriophagegrowth and purification tend to be labor-intensive and time-consuming, commercial production of phage preparationsrequires optimization of manufacturing steps during scale-up. The production process for live attenuated virus vaccineshas been developed to commercial stage and is now a rou-tine operation.(80) Many of the processing steps used in virusvaccine production are similar to the ones needed for bac-teriophage manufacturing.(72) Specific improvements forphage manufacturing processes can be found in the patentliterature: Bujanover proposed the use of a semisolid culturemedium instead of liquid medium for incubation with thehost bacterial strain.(81) This is followed by a repeating pro-cess of dilution, centrifugation, and extraction of supernatantto achieve a bacteriophage titer of at least 1010 plaque-forming units (PFU)/mL in the final extract. Lenherr andBartsch outlined a fully automated purification process.(73)

A prepurification step involves passing the unprocessedpostamplification phage and bacteria preparation through arotating membrane prefilter with a low-pressure pump. Thefiltrate is subjected to subsequent filtration steps with pro-

gressively finer pore size. The phages are then washed off thefinal filter and collected.

Summary

Bacteriophage preparation, from raw environmentalsamples to purified endotoxin-free phage product, can beaccomplished using established biotechnology processes.Similar production processes for viral vaccines have beenscaled up and are routinely used in vaccine manufacturing.

Formulation

After the selection of an appropriate phage and prepara-tion of phage stock, the next step in the development of arespirable dosage form is to incorporate the phage into astable, inhalable formulation.

The reported shelf life of phage preparations varies, andalthough many phages can be stored for years without anysignificant loss, not all phages are suited to long-term storagewithout further stabilization measures. Clark (1962) testedthe effect of 2 years of different storage conditions on thepreservation of 14 different phages.(82) He found that mostphages survived better when stored at 4�C. At this temper-ature, 10 phages survived best when freeze-dried, four whensuspended in liquid, and three when stored in 50% glycerol.More recently, it has been reported that lactococcal phagesare stored best at - 30�C in a broth supplemented with cal-cium, but are less stable when freeze-dried.(83) However, Bccand P. aeruginosa phages KS4-M and FKZ, respectively, canbe stored without significant titer loss after lyophilizing in alactose/lactoferrin matrix.(84)

Storage conditions for phage preparations will, therefore,depend on the individual phage, or phages, that they con-tain. A variety of commercial phage preparations in Georgiaare marketed as liquid suspensions with a shelf life of 2 yearswhen stored at 2–15�C(85) and are also available in a

FIG. 3. Processing steps inthe manufacture of bacterio-phage preparations.

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lyophilized form with 1.5 years of shelf life at unspecifiedstorage conditions.(86) Given that phages are protein struc-tures, they are potentially susceptible to factors that areknown to cause protein denaturation, including temperature,pH, interfacial adsorption, and ionic strength. Table 2 pro-vides a summary of the studies discussed in this section.

Temperature

It is well known that the application of high temperaturesleads to thermal denaturation of proteins, a fact that can beused for sterilization or pasteurization purposes. Phagesshow typical behavior in response to short-term temperatureexposure: S. aureus-specific phages FH5 and FA72 werefound to maintain initial titer ( > 4 · 105 PFU/mL) after sus-pension in ultra-high-temperature–processed, whole-fat milkwhen stored at 4�C for 8 hr, showed approximately 20–25%titer loss at 37�C, and showed complete loss at 72�C.(87)

Caldeira and Peabody demonstrated thermal stability of E.coli-specific MS2 phages in storage media, from 20 to 60�C,after 2-min exposure to temperature.(88) Briers et al. identifiedthe peptidoglycan hydrolase gp181 as an important part ofphage FKZ infectivity against P. aeruginosa, and found thatits activity gradually declined from 100% to 0% when ex-posed to temperatures of 25–90�C for 60 min.(89) The study ofphage survival is well established in the dairy industry,where the presence of phages can inactivate the bacterialcultures required for fermentation. For example, the Lacto-coccus lactis phage P008 experienced 6 log10 titer loss whenheld at 72�C for 30 min(90); the heat-resistant variant phageP680 experienced a 2 to 3 log10 titer loss when held at 90�Cfor 10 min.(91) Short-term temperature exposure is not aconcern in the manufacturing of phage preparations, becausehigh-temperature exposure is unnecessary or avoidable in allprocessing steps. However, temperature excursions duringtransport or administration may need to be considered.

pH

The pH is an important formulation parameter that iscommonly controlled in feedstock preparation for furtherprocessing steps such as freeze-drying. In general, phages arewell adapted to pH ranges typically encountered in livingsystems. Four P. aeruginosa-specific phages were found toachieve 100% survival at pH 7 and 80–100% survival at pH 9,but declined in phage activity when pH was decreased to1.5.(92) However, formulation pH may affect the charge car-ried on protein surfaces, resulting in an alteration to elec-trostatic and hydrophobic interactions, and a change inprotein conformation.(93) For MS2 bacteriophages, pH con-ditions close to or below the isoelectric point were shownto favor phage aggregation, associated with a greater than1 log10 loss in PFU count.(94)

Ionic strength

The ionic strength of a formulation may affect the os-motic pressure on bacteriophages in a suspension; if thedifference in osmotic pressure inside and outside the phageis too great, the phages may be destroyed by osmoticshock.(95) This may be of particular concern if the ionicstrength changes rapidly, for example, during removal ofwater in drying processes. The peptidoglycan hydrolase

gp181 used in phage FKZ infectivity maintained optimalactivity at a pH of 6.2 and ionic strength of 140 mM; vari-ation in pH and ionic strength in either direction (pH 5–8,20–320 mM) only produced a significant decline in enzy-matic activity.(89) Golshahi et al. observed a 74% drop inKS4-M bacteriophage titer when prepared in a hypotonicsuspension (105 mOsm), compared against an isotonic (282–290 mOsm) suspension.(96)

Interfacial adsorption

When presented with an interface, e.g., during filtration orin the process of dry particle formation, proteins may unfoldto assume the conformation of lowest energy; its hydro-phobic regions are exposed to the hydrophobic phase (air ororganic solvent) and its hydrophilic regions (charged andpolar groups) to the hydrophilic phase (aqueous or polarsolvent). This has ramifications for the design of a stablebacteriophage formulation, where the phages may need to beprotected from the liquid–air interface. Pharmaceutical for-mulations often use nonionic surfactants to prevent interfa-cial adsorption of proteins.(97)

In addition to thermal and chemical stresses, phages maybe susceptible to damage by mechanical stresses inducedduring processing. In particular, shear stress from high-speed mixing, filtration, and centrifugation is a potentialsource of denaturation.(98) Short-term and long-term stresson bacteriophages during processing and storage apply tophages regardless of their mode of delivery, and standardtechniques for stabilization such as cryoprotection and de-siccoprotection may be applied. For respirable dosage forms,additional criteria must be met. The final dosage form mustbe suitable for efficient inhalation and delivery to the ap-propriate disease target.

Summary

Formulation of stable phage dosage forms appears feasi-ble, given that over-the-counter phage preparations are al-ready available. Formulation variables to consider includepH, temperature, osmotic pressure, ionic strength, and me-chanical stress. However, formulation of biological activesinto a dosage form suitable for respirable delivery remainsan active area of research.

Phage Delivery

The efficacy of phage delivery to an infected respiratorytract is reliant upon a number of factors common to respi-rable dosage forms. Two significant factors are their aero-dynamic behavior under inhalational airflow and theintended target for delivery.

Aerodynamic particle characteristics

Aerodynamic particle diameter is often used in in vitroaerosol studies to estimate regional deposition in the respi-ratory tract. The actual deposition pattern of particles in theairways is obviously also affected by device and patientfactors, such as the breathing profile and individual airwaygeometry. As depicted in Figure 4, for typical breathingpatterns and normal airways, it is generally accepted thatparticles with an aerodynamic diameter between 1 and 5 lmare respirable, whereas most particles larger than 5 lm are

8 HOE ET AL.

expected to deposit in the extrathoracic airways, and parti-cles smaller than 1 lm (but larger than 100 nm) are liable tobe lost in exhalation.(99) From aerodynamic particle diametermeasurements, the parameters of fine particle dose (mass ofdelivered particles smaller than 5 lm), mass median aero-dynamic diameter (MMAD; diameter at which 50% of thedelivered mass is smaller than this value), and geometricstandard distribution can be used along with delivered doseto predict the amount of aerosol delivered into the lung andthe delivery efficiency.(100)

Delivery target

While in vitro studies of aerosol particle size distributionoften attempt to correlate fine particle dose to lung deposi-tion, the architecture and airways surface liquid composi-tion of an acute or chronically diseased lung weakensthis in vitro–in vivo correlation further. If we were to take CFas an example, features of the disease, such as bronchiec-tasis, peribronchial thickening, mucus plugging, and air-trapping,(101) would be expected to alter the aerodynamicprofile of the pulmonary system, as shown in Figure 4, notonly due to morphological changes, but also as a result ofchanges in inhalation profile.

The results of chest radiography studies in CF patientsshow a more complicated range of disease presentation,making it difficult to mathematically model the diseasedrespiratory system.(100) Kaza et al. identified diffuse bilateraldisease from chest radiographs in 62% of the adult patientsstudied (n = 68), compared with 28% with predominant up-per lobe disease and 7% with lower lobe disease.(102) Incomparison, a small study of 17 children younger than 4years of age found six patients with the greatest disease inthe right upper lobe and another six in the right lower lobe,but the left upper lobe was also qualitatively categorized ashaving the least amount of disease in six subjects.(103)

Another study of 60 children (aged 6–10 years of age) foundbronchiectasis and mucous plugging to be distributed simi-larly between the left and right, and upper and lower,lobes.(104)

Nevertheless, numerical models have been devised to assistin vitro prediction of aerosol deposition in CF patients. In oneexample, deposition of coarse particles ( > 5 lm MMAD) by im-paction was predicted to be two to three times higher in the largeapical and basal airways (generations 1 through 12) of CF pa-tients than in those of healthy subjects.(105) However, to keep themodel workable, assumptions were made in their simulation:disease was assumed to progress from the small to large airways,sedimentation and impaction were the only deposition mecha-nisms included, and the disease was more severe at the apex.

Based on other aerosol formulations in CF, and due touncertainties in predicting deposition patterns in diseasedlungs and considerable patient variability (both intersubjectand temporal intrasubject variability), it seems advisable todesign bacteriophage dosage forms with an MMAD smallerthan approximately 5 lm for lung infections, and an MMADlarger than approximately 5 lm for infections of the extra-thoracic airways.

At the time of this publication, three formulation tech-niques have been used to manufacture a respirable phagedosage form: suspension of phages in a liquid medium,freeze-drying, and spray-drying. These formulations havethen been loaded into inhalation devices, intended for de-livery into the human respiratory tract. This section willoutline the phage formulations that have been successfullyproduced and the outcomes of the in vitro aerosol testingperformed for these formulations (see Table 3 for a summaryof the studies discussed in this section).

Suspension formulations

So far, the production of liquid suspension for directphage delivery into the respiratory tract has been for nebu-lization. To the best of our knowledge, no work has beenpublished reporting suspensions of bacteriophage in pro-pellants for metered dose inhaler delivery. Animal studiesand experimental clinical work have adopted nasal andpleural instillation and insufflation of phage suspensions as amethod of delivery.(47,106,107)

Commercial nebulizers for inhalational drug deliveryemploy different types of aerosol dispersion,(108,109) includ-ing jet (air-blast) nebulization, vibrating mesh plate nebuli-zation, ultrasonic nebulization, as well as colliding jets. Withjet nebulization, compressed air is passed through a gap in athin tube, leading to a Venturi effect that creates a pressuregradient that draws the nebulizer solution or suspension upa feed tube. Droplets are generated via a viscosity-inducedinstability,(100) whereby nonlinear growth of surface wavesand subsequent drop breakup and filtering by impaction onbaffles occur. Vibrating mesh nebulization may be carriedout by two methods: (1) a piezoelectric crystal vibrates athigh frequency, transferring the vibration to a transducerhorn that pushes the liquid through a mesh plate (‘‘passive’’);or (2) a piezoelectric element directly vibrates a mesh plate,causing micrometer-range vertical displacement of the meshthat generates droplets from the liquid drawn through(‘‘active’’).(110,111) Ultrasonic nebulization does away with themesh plate altogether, and involves the oscillation of a pie-zoelectric crystal transducer, which transfers the oscillationenergy to the liquid. Droplets are formed by the breakup ofsurface capillaries or the collapse of cavitation bubbleswithin the liquid.(109,111)

FIG. 4. Common delivery device options [metered doseinhaler (MDI), dry powder inhaler (DPI), or nebuliser], anddelivery targets in healthy and diseased lungs.


Nebulization was the mode of delivery in the early reportson phage inhalation reviewed in the introduction.(19,20)

However, few device and formulation details are given, andinformation on possible titer loss during nebulization cannotbe derived from these publications. Phages have been neb-ulized to test the efficacy of biological respirator filters,alongside aerosolized bacteria, fungi, and viruses (e.g.,Balazy et al.,(112) Eninger et al.(113)). However, the focus ofthese studies is typically on particles in the most penetratingsize range (a few hundred nanometers in diameter) andnormally involves evaporated nebulized aerosols usuallyproduced by the standard Collison nebulizer, which iscommonly used in laboratory studies in occupational hy-giene. However, such submicrometer particle sizes are notoptimal for respiratory delivery due to their low efficiency ofdeposition. In addition, the Collison nebulizer does notmatch current commercial nebulizer designs in its ability todeliver therapeutic aerosols to the human respiratory tract.For example, Liu et al. used the six-jet Collison nebulizer toaerosolize D29 phages (for M. tuberculosis).(114) With an initialphage titer of 108 PFU/mL, the phage suspension wasaerosolized at 10 L/min, into a chamber for 10 min, beforethe aerosol was sampled at 12.5 L/min for 5 min in a liquidimpinger. Regardless of the suspension medium (PBS orsaline), the sampling medium (PBS, saline, or deionizedwater), or relative humidity in the chamber (25, 55, or 85%),there was a 2 to 3 log10 titer loss upon collection of theaerosolized phage, supporting the conclusion that the Colli-son nebulizer may not be the best choice for phage lungdelivery.(114)

A study by Golshahi et al. reported on nebulizer suspen-sions designed with the intent to deliver phages to the re-spiratory tract.(96) The phage selected for the study was KS4-M, which is selective for one strain of the highly antibiotic-resistant Bcc. KS4-M was prepared to an initial phage titer of2.15 – 1.63 · 108 PFU/mL. Two commercial nebulizers wereused in this study: the Pari LC Star jet nebulizer, and the ParieFlow vibrating mesh nebulizer.(96) The eFlow system is ofparticular interest, given that it is has been suggested that itsactive vibration nebulization subjects the liquid to less shearstress,(115) a feature that could potentially reduce phage titerloss from nebulization. Each nebulizer was loaded with2.5 mL of the KS4-M suspension and aerosolized under asimulated inhalation profile generated by a breathing ma-chine (tidal volume = 800 mL, respiratory rate = 14 breaths/min, duty cycle = 0.5). No significant difference in total neb-ulizer output was found between the eFlow and LC Starsystems, with both producing about 108 PFU. This equates toless than 1 log10 titer loss, which is an ideal outcome for arespiratory delivery method; although the vibration meshnebulizer did not appear to give an advantage in phagesurvival, both nebulizers demonstrate a clear advantage overthe Collison nebulizer used by Eninger et al.(113) There werealso no significant differences between the two nebulizers forthe KS4-M droplet size distributions, with a mass meangeometric diameter of 5–6 lm.

The distribution of these nebulized droplets in the lungwas predicted by the incorporation of particle size distribu-tion and breathing pattern data into a numerical respiratorytract deposition model. Alveolar deposition was approxi-mately 3 · 107 PFU for both systems. These distribution datawere then used in a follow-on model of airway surface liquid

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10 HOE ET AL.

in the lungs to estimate KS4-M concentration within thedifferent lung generations, dependent on mucus productionand velocity. For both the LC Star and eFlow nebulizers,concentration decreased with conducting airway generation,from 1.9–2.2 · 107 PFU/mL (generation 1) to 6.0–7 · 106

PFU/mL (generation 13). It should be noted that althoughthese deposition predictions are promising, the numericalmodels are based on healthy patient lung architecture.

Dry powder formulations

In designing an inhalable dry powder formulation, animportant factor for consideration is the ability of the deliv-ery device to entrain and deaggregate the powder, whichrequires a dispersible powder. Most dry powder inhalers(DPIs) are of the passive type, requiring the patient’s inha-lation effort to generate a sufficient air flow rate to dispersethe dry powder into the respiratory tract.

Although milling is commonly used to produce powdersfor inhaled aerosol formulations, destructive forces in millsmay result in reduced survival of phage formula-tions.(84,116,117) Instead, lyophilization and spray-drying havebeen used to produce dry powder formulations intended forinhalation therapy. To the best of our knowledge, only twopublished articles have extended bacteriophage dry powderformulation studies as far as in vitro aerosol testing: oneusing a lyophilized dosage form and the other a spray-driedpowder.

Lyophilization (freeze-drying). Lyophilization has longbeen used for the preservation of commercial protein for-mulations, including vaccines, antihemophilic factors, hu-man growth hormone, and interferons.(118) Lyophilizationhas also been used to preserve phages; however, phagesexhibit different stabilities (short- and long-term) dependingon the formulation contents and method of lyophiliza-tion,(119–121) and there are few data specifically dealing withlyophilization of phages infecting problematic respiratorybacterial species such as P. aeruginosa and the BCC.

Traditional lyophilization involves three stages: freezing,primary drying, and secondary drying. During the freezingstage, the solvent crystallizes amongst solid amorphous orcrystalline solute. Solid solvent sublimation occurs duringprimary drying, and moisture desorption during secondarydrying, leaving a solid porous ‘‘cake’’ of the solute.(122) Ex-cipients such as polyethylene glycol (PEG), trehalose, man-nitol, and sucrose are commonly added as cryoprotectants,protein stabilizers, and bulking agents, to give the cakepharmaceutically acceptable physical properties.(122,123) Re-lated to traditional lyophilization, spray freeze-drying,whereby primary freezing occurs upon spraying into coldliquid, has been proposed.(124) In addition, atmosphericspray freeze-drying, where primary freezing occurs uponspraying into cold gas in order to ease commercial scale-upissues, has been used to prepare aerosol powders of bacte-ria(125) with 90% survival rates in the prepared aerosolpowder.

Using traditional lyophilization, Golshahi et al. carriedout a study on freeze-dried phage formulations designedfor respiratory delivery.(84) The phages KS4-M (for a Bccstrain) and FKZ (for P. aeruginosa) were each formulatedwith lactose and lactoferrin. Lactose served as a carrier and

bulking agent.(84) Lactoferrin, a protein abundant in airwaysurface liquid, has been shown to inhibit P. aeruginosa biofilmformation.(126,127) In addition, lactose and lactoferrin wereselected as cryoprotectants and stabilizers for the phages. A60:40 % w/w ratio of lactose to lactoferrin was selected onthe basis of its optimal aerodynamic particle size distributionand protection in preliminary studies. Viability tests postlyophilization demonstrated a 2 log10 titer loss for KS4-Mand a 1 log10 loss for FKZ. The authors also reported nofurther phage loss after a milling procedure in a mixer mill(without the use of beads) to deagglomerate the lyophilizedpowders.

The lyophilized powders were also subjected to in vitroaerosol testing; 10 mg of powder was dispersed froman Aerolizer DPI into the Alberta idealized throat and filterat a vacuum air flow rate of 60 L/min, where the mass ofpowder recovered from the filter was representative of re-spirable dose. The results showed an acceptable titer dropfrom capsule dose to respirable dose, with the maximumloss observed being 1.2 log10 for KS4-M, and 0.84 log10 forFKZ. Respirable doses of 3.4 – 2.5 · 106 for KS4-M and1.9 – 0.6 · 107 for FKZ were achieved.

The phage powders were stored at 21 – 2% relative hu-midity and temperatures of 4�C and 21�C for 3 months aspart of a short stability study. There was no evidence of titerloss for either KS4-M or FKZ, indicating that phage activitywas preserved. Long-term stability results were not reported.

Alfadhel et al. did perform a 12-month phage stabilitystudy on a formulation designed for nasal delivery.(128) In-cluded in the formulation besides an S. aureus-specific phagewas hydroxypropylmethylcellulose (HPMC), to alter vis-cosity and increase residence time in the nasal cavity, andmannitol, as a lyoprotectant and bulking agent. Active phagetiter was decreased by 1 log10 after lyophilization. Duringstorage of the freeze-dried powder at 4�C, there was a further1 log10 loss after 1 month, another 1 log10 loss after 2 months,and a total of 3 log10 loss from the beginning of refrigerationto the end of the 12-month period.(128) Ultimately, the ac-ceptability of this magnitude of titer loss depends on itsvariability, the necessary delivered dosage, and whether aformulation with sufficiently high initial titer can be practi-cably prepared to offset the loss.

Furthermore, the addition of mannitol did not appear toaffect phage survival, with the formulations containingHPMC (1 or 2% w/v) as the only excipient performingsimilarly to those with both HPMC and mannitol. A man-nitol-only phage formulation was found to exhibit 6 log10

titer loss just after lyophilization. These results pointed to-ward HPMC acting to maintain protein conformation, andthe authors suggested the stability of amorphous HPMC tobe crucial (the glass transition temperature for lyophilizedHPMC was approximately 187�C).(128) Although the formu-lations tested in this study were intended for nasal delivery,they demonstrate that high-molecular-weight polymers mayhave a future role in maintaining phage activity during ly-ophilization and storage.

Spray-drying. Spray-drying is an established techniquefor the manufacture of pharmaceutical products.(129) Asshown in Figure 5, a liquid feedstock is atomized, typicallyusing atomization gas in a twin-fluid nozzle, and mixed withdrying gas in a drying chamber. The atomized droplets


rapidly evaporate, and dry particles with a respirable parti-cle diameter are produced, separated from the gas stream,e.g., in a cyclone, and collected.

When drying biological material, the design of the spray-drying process(130,131) must be conducted in a way thatminimizes stresses, including shear, temperature, and des-iccation stress.(132) Low-temperature spray-drying processesfor heat-sensitive biological pharmaceutical ingredients havebeen developed.(133–135)

Walbeck submitted a patent application detailing the useof a rotary atomization spray-dryer system for the produc-tion of E. coli O157:H7-specific phages in dry powder.(136)

The process used an inlet temperature of 200–220�C and anoutlet temperature of 90–130�C. The authors predicted thepowders to contain 2–4 · 109 PFU/mL; the actual phage titerof the spray-dried powders was 1.1–1.8 · 102 PFU/mL, a 7log10 titer loss.(136) Given that existing literature has estab-lished that some phages are thermally labile at temperaturesbelow 100�C (as described above), it is unsurprising thatsuch high outlet temperatures would result in significant ti-ter losses.

Matinkhoo et al. presented a study on spray-dried bac-teriophage powder formulated for inhalation from aDPI.(137) The authors selected the Bcc phage KS4-M andthe P. aeruginosa phage cocktail FKZ/D3 for study. Eachphage or phage cocktail was formulated with trehalose forprotection against thermal stress and desiccation, andleucine as a dispersibility enhancer in the ratio 76:19, withother excipients in the remaining 5%. Given the consid-erations to be made about phage inactivation from thermalstress, a relatively low spray-dryer outlet temperature of40–45�C was selected. A vibrating mesh atomizer with4 lm mesh orifice diameter was used to produce 7 lmdroplets.(137)

The authors reported less than 1 log titer loss after spray-drying for all tested phages, regardless of formulation com-position.(137) The FKZ/D3 cocktail was particularly resilient,with less than 0.5 log10 titer loss in all cases. These results notonly indicate that inactivation by thermal stress was largelyprevented, but also that the other factors for inactivation, likeshear stress or desiccation stress, were not significant influ-ences for the chosen formulations. Also reported was a lessthan 0.15 log10 titer loss for FKZ/D3 spray-dried powderafter 3 months of refrigerated storage. However, as with thefreeze-drying studies, long-term stability results were notreported, but would be necessary to advance the phageformulations into clinical trials.(137)

Matinkhoo et al. also subjected the spray-dried powders toin vitro aerosol testing, using an Aerolizer DPI and the same

methodology as described for the lyophilized powderproduced by Golshahi et al.,(84) but with 25–28 mg of powderloaded into the capsule.(137) The respirable dose was *70%of loaded powder mass, regardless of phage type. Phagetiters for the respirable dose were *107 PFU for FKZ/D3,and *108 PFU for KS4-M. Separate particle size measure-ments with the Andersen Cascade Impactor showed theKS4-M and FKZ/D3 in leucine-trehalose-casein formulationsto have an MMAD of < 3 lm, suggesting that the powder ishighly dispersible and capable of reaching the smaller air-ways.(137) An efficacious dose for local phage treatment oflung infections has not been established, but the titers re-ported in this study are two to three orders of magnitudehigher than the minimum titer in commercial phage prepa-rations for P. aeruginosa infections distributed in Georgia.(85)

Summary

The feasibility of efficiently delivering phages to the lunghas been demonstrated using different processing and deviceoptions. More work in developing a stable respirable dosageform is needed, and long-term stability of such dosage formsstill needs to be evaluated.

Reports on Safety and Efficacy in Animals

The study of aerosol phage therapy in animals has been arelatively recent addition to the body of research beingconducted in phage therapy, and further studies need to beconducted. The aerosol phage therapy animal studiesreviewed here (Carmody et al.,(47) Debarbieux et al.,(138)

Morello et al.,(106) Alemayehu et al.(59)) all follow a similarprotocol. Mice are infected and treated via an intranasal in-stillation of the liquid. Intranasal inhalation requires theliquid containing either the bacteria or phage to be drippedonto the nares of the mouse, and from there the droplets ofthe liquid are inhaled into the lungs.

Although intranasal instillation is a much easier deliveryroute to use in animal studies because there are no additionalaerosol generators or delivery systems required, it should benoted that it does not accurately mimic aerosol drug delivery inclinical environments, and as such it is difficult to extrapolatethe results of these studies to a prediction of aerosol therapyefficacy. Indeed, a previous study has shown that intranasalinstillation of bacteria does not mimic aerosol particle deposi-tion within the lungs of mice: Halperin et al. demonstrated thatmice given Bordetella pertussis respiratory infections via twomethods, either intranasal inhalation or a whole-body expo-sure chamber, had very different bacterial lung counts.(139) Themice receiving intranasal instillation showed a far higher de-gree of variability in bacterial load in comparison with thosereceiving aerosolized bacteria (a 1,000-fold variability as com-pared with a five-fold variability, respectively). The mice re-ceiving intranasal instillation also showed a far greatervariability in bacterial distribution within the lungs (right lung,43–84%; left lung, 16–57%) in comparison with those receivingaerosols via the whole-body exposure chamber (right lung, 60–68%; left lung, 32–40%).(139) This may also translate into highervariability when treating mice in phage therapy studies, and itwould be of interest to see additional studies performed using awhole-body exposure chamber or nose-only inhalation cham-ber, in order to observe the effect of aerosol phage deliverymethod on treatment efficacy.

FIG. 5. Schematic of a spray-dryer for respirable powders.

12 HOE ET AL.

Debarbieux et al. demonstrated the efficacy of aerosolphage therapy via intranasal instillation for treating acuterespiratory infections using a bioluminescent strain of P.aeruginosa PAK.(138) Provided that the phage titer was highenough and treatment was given soon enough, the phagetreatment proved effective. Untreated mice all died within48 hr of infection, whereas those treated 2 hr after infectionwith a multiplicity of infection (MOI) of 10 all survived to theend of the trial (12 days). Twenty-four hours after treatment,the bacterial load in treated mice was already 6 log10 lowerthan in untreated mice; 48 hr after treatment, the inflamma-tion levels in the phage-treated mice (as indicated by tumornecrosis factor-a and interleukin-6) had decreased to baselinelevels, whereas levels remained high in untreated mice.(138)

Morello et al. tested the efficacy of phage therapy via in-tranasal instillation in treating a P. aeruginosa strain isolatedfrom a CF patient.(106) Mice were treated 2 hr after infectionwith a high enough phage titer to provide a 95% survivalrate (MOI 100). These mice survived to the end of the 16-daytrial as opposed to untreated mice, which had 0% survival by2 days post infection. In addition to survival and an ap-proximately 3 log10 reduction in bacterial lung titer 20 hrafter infection, treated mice also had significantly reducedinflammation and significantly reduced lung damage (asdemonstrated through histology). Interestingly, immuno-histochemistry performed 20 hr after infection revealed thatuntreated mice had bacteria distributed through multipleareas of the lung, including the macrophages, extracellularspace, and alveoli, whereas treated mice were found to havefew bacteria remaining in the lungs, and these were mainlylocalized in lung macrophages.(106) This demonstrates thatthe immune system works in conjunction with the action ofphages to remove bacteria from the lungs.

Debarbieux et al. and Morello et al. both demonstrated thatphages could protect mice from P. aeruginosa infection. Allmice receiving phage either 1 day (Debarbieux et al.) or 4days (Morello et al.) prior to infection were protected fromdeath.(106,138) Although this effect may not be as clinicallysignificant as infection treatment, it demonstrates the efficacyof phage usage to prevent infection.

Additionally, Alemayehu et al. demonstrated that a phagecocktail of FNH-4 and FMR299-2 delivered 2 hr after infec-tion via intranasal instillation was able to clear P. aeruginosarespiratory infections in mice.(59) Two separate biolumines-cent strains of P. aeruginosa (MR299 and NH57388A) weredelivered intranasally, and the bioluminescence within thelungs was measured at 2-hr intervals up to 8 hr after infec-tion. The luminescence within the lungs (which is propor-tional to bacterial concentration) showed a significantdecrease in phage-treated mice, whereas untreated miceshowed a three-fold increase in luminescence intensity.(59)

Carmody et al. studied phage treatment via intranasalinstillation in a respiratory infection of B. cenocepaciaAU0728.(47) Phages were administered 24 hr after infectionvia either an intranasal or intraperitoneal route. The bacterialload was determined within the lungs of all mouse groups48 hr after treatment. In contrast to the three studiesreviewed above (Debarbieux et al.,(138) Morello et al.,(106)

Alemayehu et al.,(59)), phage delivered intranasally did notproduce a significant reduction in bacterial load within thelungs (approximately 1 log10 reduction), whereas phagedelivered intraperitoneally did significantly reduce the

bacterial load (approximately a 2 log10 reduction), eventhough a higher phage titer was isolated from mice treatedintranasally. The authors suggest that these results indicatethat phage therapy in lung infections is more effective whenadministered systemically rather than topically.(47) There areseveral reasons why these results may have been obtained.With respect to the reference described above (Halperinet al.(139)), intranasal phage application does not give phageaerosol deposition in the lungs. As we have observed the lossof aerosolized phages out of the lung into the peritoneum(unpublished data), it is not inconceivable that phages cancross from the peritoneum into the lungs. In this case, itappears that peritoneal injection of high titers of phages wasmore effective than the ineffective intranasal deposition ofphages on the mouse nares.

In either case, the 1 to 2 log10 drop in bacterial numbersobserved by Carmody et al.(47) may not be an overly effectivetreatment if the bacterial load is high, and the load is notreduced far enough for the human immune system to dealwith the remainder of the infection. One could argue that, ineither condition, phage therapy in this study was not effi-cacious. It is also interesting that more than one phage wasnot tested, as we have observed notable differences in phageactivity in vivo, as compared with their in vitro activity. Fi-nally, due to the controversial nature of these findings, it isunfortunate that these experiments were not repeated withother bacterial species (such as P. aeruginosa) in order todetermine if intraperitoneal phage administration wouldhave a similar effect with a proven and published phagetherapy candidate.(23,138)

Summary

Animal studies of phage therapy have been limited tointranasal delivery. Although these studies have demon-strated varying levels of efficacy in reducing bacterial loadand increasing survival, work is still needed to investigatethe safety and efficacy of phage aerosol delivery into thelungs.

Reports on Safety and Efficacy in Humans

Although there have been studies investigating aerosolphage formulations (see Suspension formulations and Drypowder formulations), there are relatively few reports ofaerosol phage therapy in humans. However, case studieshave shown promising results (see Table 1). In Eastern Eur-ope, it is often applied to infections with antibiotic-resistantstrains, and typically 80–90% of cases are cured. This in-cludes phage treatment for respiratory tract infections.(23)

Slopek et al. reported treatment success between 82 and 92%for different patient groups with respiratory infections trea-ted with oral or locally administered phages.(107) Successfultreatment of a pediatric CF patient was also reported recentlyby Kvachadze et al.(140) In this case, phage cocktails wereadministered to the lung via a nebulizer. However, no con-trolled clinical trials have been performed to evaluate aerosolphage delivery to patients. Evidence for the clinical safety ofphage therapy in humans may be found in studies on oraland topical phage treatment. Four recent trials will be re-viewed (Bruttin and Brussow,(141) Merabishvili et al.,(16)

Rhoads et al.,(142) Wright et al.(69)). One of these trials (Wrightet al.) also addresses phage therapy efficacy.


The first of the recent phage administration human safetytrials in the English literature was performed by Bruttin andBrussow (2005).(141) Fifteen volunteers were given either aplacebo, a low-phage titer preparation (103 PFU/mL), or ahigh-phage titer preparation (105 PFU/mL) orally on 2 con-secutive days followed by 5 days without phage adminis-tration. This was repeated for the following 2 weeks witheach patient receiving a different phage preparation or pla-cebo each week. Throughout the study, five adverse effectswere reported; however, there was no difference in thenumber of adverse effects in the placebo, low-phage titer,and high-phage titer groups, and the effects were deemed tobe unrelated to the study. Phages were never recovered fromstool samples prior to phage administration or when theplacebo was administered. However, phages were recoveredfrom stool samples in a dose-dependent manner after phageadministration, demonstrating that the phages were able tosurvive transit through the gastrointestinal system. Interest-ingly, the presence of phage T4 did not correlate with a de-crease in natural E. coli counts in the stool samples,suggesting that the phage did not affect the natural micro-flora of the volunteers, albeit as measured by numbers ofonly one bacterial species. Throughout the study, there wasno change in serum alanine aminotransferase and aspartateaminotransferase levels, which are liver enzymes that wouldhave indicated an increase in liver toxicity. There was also nodetected immune response to the phages, demonstrating thesafety of phage administration.(141)

A phase I safety trial was performed by Rhoads et al. toensure that the application of WPP-201, a phage cocktailcontaining eight lytic phages targeting S. aureus, P. aerugi-nosa, and E. coli, would not pose any health risks to patientswith venous leg ulcers.(142) Thirty-nine patients completedthe 24-week trial, with every patient receiving the samecompression therapy and wound dressings, with the excep-tion that approximately half received WPP-201 applied to-pically, whereas the controls received only a sterile salineapplication. Phage application occurred weekly for 12 weeks,while dressings were changed three times weekly for thesame duration. Patients were monitored weekly for the first12 weeks of the study, and data collected included woundsize and severity, vital signs, and adverse events, which mayor may not have been related to the phage application (ad-verse events included a variety of infections, injuries, medicalprocedures, pain, and cardiovascular issues). Wound bacte-rial counts, blood chemistry, and blood cell counts wereperformed every other week as well. Two additional as-sessments were made on weeks 16 and 24. There were nostatistically significant differences between the test andcontrol groups in either the number of adverse events ex-perienced or the frequency with which wounds healed. Thisphase I clinical study did not set out to evaluate phage ef-ficacy, but instead appraised the safety of phage application.With this objective successfully completed, the researcherscan now proceed to phase II studies, which will evaluate theefficacy of WPP-201.(142)

Merabishvili et al. developed and began evaluating BFC-1,a phage cocktail consisting of three phages that targetedP. aeruginosa and S. aureus strains isolated from burn woundsof patients in the Burn Centre of the Queen Astrid MilitaryHospital in Belgium.(16) The cocktail was sprayed onto theburn wounds of eight patients using a micromister. None of

these patients was reported to experience any adverse eventsresulting from the phage application.(16)

Chronic otitis caused by P. aeruginosa can be problematicto treat, as P. aeruginosa is highly antibiotic-resistant. Wrightet al. conducted phase I/II trials to evaluate the treatment ofthis infection using Biophage-PA, a cocktail of six phagesspecific for P. aeruginosa.(169) The 24 volunteers in this study,each with a history of chronic otitis lasting 2 to 58 years, hadP. aeruginosa infections sensitive to Biophage-PA prior toparticipation. A single dose of either Biophage-PA or placebowas applied directly into the ear of each participant. Themedian bacterial counts significantly decreased in phage-treated volunteers on days 21 and 42 (swabs were taken ondays 0, 7, 21, and 42), whereas there was no significant de-crease in placebo-treated volunteers. This study demon-strated the advantage of administering a self-replicatingtreatment, as the mean phage count isolated on days 7, 21,and 42 was 1.27 · 108 PFU, whereas the initial phage countadministered was 6 · 105 PFU. The phage persisted at the siteof the infection for an average of 23.1 days, and the phagewas cleared in cases where the infection was successfullytreated. Each case was also scored on a visual analogue scale(VAS) by the patient and the physician. Patients scored theinfection on discomfort, itchiness, wetness, and odor,whereas the physician scored the infection on inflammation,ulceration/granulation/polyps, discharge quantity, dis-charge type, and odor. When the patient VAS scores fromdays 7, 21, and 42 were averaged, there was a significantdecrease from days 0 for discomfort, itchiness, and wetness,as well as overall patient VAS scores, whereas placebo-treated volunteers did not show any significant reductions.When the physician VAS scores from days 7, 21, and 42 wereaveraged, there was a significant decrease in inflammation,ulceration/granulation/polyps, discharge type, and odor, aswell as overall physician VAS scores, whereas the placebogroup did not show any significant reductions. As with theother clinical trials reviewed, none of the adverse events re-ported during the course of the trial was deemed to be re-lated to phage treatment. This study provided sufficientevidence of phage therapy efficacy to warrant the design of aphase III clinical trial.(69)

It is interesting to note that these studies delivered a rangeof phage titers to the infected sites and, in the phase I/II trial,only one phage dose was required for treatment.(69) Thishighlights a unique difference in the pharmacokinetics ofphage therapy in contrast with traditional chemical antibi-otics. Traditional antibiotics must be administered at regularintervals as they are metabolized and excreted. In some sit-uations, similar delivery schedules for phages are used. Thisis known as passive phage therapy, because the treatmentdoes not rely on phage replication within the host bacteri-um.(143) In some situations, a single treatment is sufficientbecause phages can replicate at the site of infection, allowingfor the continued release of new antibacterial progeny. Thisis known as active phage therapy.

The bacterial density at the site of infection may also affectthe treatment strategy. Active phage therapy is effective inareas of high bacterial density, whereas passive phage ther-apy may be effective in areas of reduced bacterial density.(30)

Interestingly, mathematical modeling has demonstrated thatthe timing of phage administration is also very impor-tant.(143) As expected, delayed administration of phages may

14 HOE ET AL.

be detrimental to treatment; however, treating an infectiontoo early (e.g., low bacterial numbers) may also be detri-mental. This, again, relates to the density of bacterial hostsavailable at the site of an infection. A very low densitybacterial load may be more treatable once it has replicated tothe point at which the density is high enough for effectiveactive phage therapy. The studies reviewed above (Bruttinand Brussow,(141) Merabishvili et al.,(16) Rhoads et al.,(142)

Wright et al.(69)) demonstrate, however, that regardless of thephage delivery strategy used, phages are safe for use inphage therapy applications.

Summary

Recent trials of oral and topical phage therapy have shownthe safety of phage therapy in humans. Experimental phagetherapy has been reported to be efficacious in older studiesand in a recent phase I/II trial; however, there have not beencontrolled clinical trials for aerosol phage therapy.

Conclusion

The current experience with phage treatment of bacterialinfections indicates that it is likely that safe and efficaciousphage therapeutics can be developed. Phages have the po-tential to become a much-needed tool in the fight againstextensively antibiotic-resistant bacteria, including thosecausing respiratory infections. However, many pharmaco-kinetic questions remain open, and the ideal route of deliveryfor lung infections has not yet been determined. Althoughthere is evidence to show the safety and efficacy of phagetherapy, there is a real need for in vitro and in vivo studiesspecifically investigating aerosol phage delivery to the lung.Local administration of inhalable phages is an attractiveprospect, as it may provide advantages, in particular forinfections that are walled off from the systemic circulatorysystem, such as in CF patients.

Initial studies probing the feasibility of inhalable phagedosage forms appear promising, and compatibility withnebulization and dry powder delivery has been shown. Mostchemistry, manufacturing, and controls questions, except forlong-term storage stability, either have been addressed suc-cessfully or can be answered using the extensive develop-ment history with viral vaccine manufacturing.

The future of phage therapy in the West will depend oneconomic considerations as phage products move into moreexpensive late-stage clinical trials. Whether it will becomeattractive to industry to advance these much needed thera-peutics to the commercial stage will be decided by patent-ability, market projections, potential financial incentives bygovernmental or nonprofit organizations, and by the pre-scribed regulatory pathway for these products. Currently,there are no specific regulatory guidelines for the develop-ment and approval of bacteriophage therapeutics in Europeand North America. Specific guidelines or clarifications re-garding current applicable regulations would be desirable toremove development risks for sponsors. The regulatoryframework should take into account that bacteriophagestrains will likely have to be updated rapidly and may haveto be adjusted to locally prevalent bacterial strains,(144)

similar to the modifications required in the case of attenuatedviral influenza vaccines. For this reason, a standard formu-lation and delivery system for bacteriophages that can be

proven to be generally safe and suitable for a wide variety ofinterchangeable phage strains appears highly desirable.

Author Disclosure Statement

The authors declare that no conflicts of interest exist.

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Received on August 2, 2012in final form, December 9, 2012

Reviewed by:Brian Gilbert

Anthony Hickey

Address correspondence to:Dr. Reinhard Vehring

Department of Mechanical EngineeringUniversity of Alberta

Edmonton, AlbertaT6G 2G8

Canada

E-mail: [email protected]


Respirable Bacteriophages for the Treatment of Bacterial Lung Infections

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