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Spray-dried Respirable Powders Containing Bacteriophages for the Treatment of Pulmonary Infections
Spray-Dried Respirable Powders Containing Bacteriophages for the Treatment of Pulmonary Infections SADAF MATINKHOO,1 KARLENE H. LYNCH,2 JONATHAN J. DENNIS,2 WARREN H. FINLAY,1 REINHARD VEHRING1 1
Department of Mechanical Engineering, University of Alberta, Edmonton, Alberta T6G 2G8, Canada
2
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
Received 9 April 2011; revised 14 June 2011; accepted 11 July 2011 Published online 23 August 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22715 ABSTRACT: Myoviridae bacteriophages were processed into a dry powder inhalable dosage form using a low-temperature spray-drying process. The phages were incorporated into microparticles consisting of trehalose, leucine, and optionally a third excipient (either a surfactant or casein sodium salt). The particles were designed to have high dispersibility and a respirable particle size, and to preserve the phages during processing. Bacteriophages KS4- M, KS14, and cocktails of phages KZ/D3 and KZ/D3/KS4-M were spray-dried with a processing loss ranging from 0.4 to 0.8 log pfu. The aerosol performance of the resulting dry powders as delivR dry powder inhaler (DPI) exceeded the performance of commercially ered from an Aerolizer available DPIs; the emitted mass and the in vitro total lung mass of the lead formulation were 82.7% and 69.7% of filled capsule mass, respectively. The total lung mass had a mass median aerodynamic diameter of 2.5–2.8 :m. The total in vitro lung doses of the phages, delivered from a single actuation of the inhaler, ranged from 107 to 108 pfu, levels that are expected to be efficacious in vivo. Spray drying of bacteriophages into a respirable dry powder was found to be feasible. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:5197–5205, 2011 Keywords: aerosols; formulation; pulmonary drug delivery; spray drying; biomaterials; processing; microparticles
INTRODUCTION Bacterial infections have been treated successfully with antibiotics for decades, but many bacteria have now evolved to the point that many of the currently available antimicrobials are ineffective, thus affecting the treatment of pulmonary infections. The compromised lungs of cystic fibrosis patients are at an elevated risk of infection1 by bacteria of the Burkholderia cepacia complex2 and by Pseudomonas aeruginosa. Several strains of these bacteria have emerged that demonstrate significant resistance against a broad range of antibiotics.3–5 Bacterial pneumonia is also becoming more difficult to treat, with few antibiotics available that are effective against multidrugresistant strains, such as those of Streptococcus pneumoniae.6 Similarly, a genetic mutation first identified in Klebsiella pneumoniae strain NDM-1 enables the bacterium to produce an enzyme, known as Correspondence to: Reinhard Vehring (Telephone: +780-4925180; Fax: +780-492-2200; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 100, 5197–5205 (2011) © 2011 Wiley-Liss, Inc. and the American Pharmacists Association
New Delhi metallo-$-lactamase, that inactivates carbapenem antibiotics.7 This gene can be transferred between different bacteria and permits complete resistance against all aminoglycoside, quinolone, and $lactam antibiotics.8 Another bacterium of particular concern is multidrug-resistant Mycobacterium tuberculosis, with about 400,000 cases per year worldwide.9 It is likely that in more than 5% of these cases, extensively drug resistant tuberculosis is present that is practically untreatable in resource-limited settings and is associated with high mortality.10 Given the concerns about antibiotic resistance, an alternative or supplemental treatment for bacterial infections, bacteriophage therapy, has become the focus of renewed attention.11–14 Bacteriophages are viruses that infect and kill bacteria.15 Because bacteriophages and bacteria have coevolved, it is unlikely that bacteria can develop a general resistance against bacteriophages.16 Unlike chemical antibiotics, which are unchanging, bacteriophages can mutate and adapt to changes in their bacterial hosts. Because a bacteriophage species typically has a narrow host range, bacteriophage therapy may avoid
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systemic side effects that are common in aggressive antibiotic treatment. This specificity also has a drawback in that the causative agent of an infection must be identified before an appropriate phage for its treatment can be selected. Alternatively, administration of phage cocktails,17 consisting of phages active against multiple bacterial strains or species, may be effective. Late-stage clinical trials in accordance with current regulatory requirements in North America or Europe have not yet been completed, but safety risks associated with phage administration appear low based on extensive experimental phage therapy experience in Eastern Europe and the former Soviet Union. Successful bacteriophage treatment for respiratory tract infections in cystic fibrosis patients has been reported,13 but questions remain about the best route of delivery. Bacterial infections in the lung may reside in macrophages or may exist in colonies that are isolated from the vasculature, for example, in mucus layers or walled off by biofilms. Hence, the feasibility of respiratory bacteriophage delivery via the airways has been investigated in an attempt to provide an effective alternative delivery route. First reports of inhaled bacteriophages in mouse models are promising; a P. aeruginosa infection could be cured by a single nasal administration of bacteriophages.18 Golshahi et al.19 have demonstrated that nebulization can be used to deliver bacteriophages to the lungs in quantities that are expected to be sufficient for phage therapy. Development of a stabilized dry dosage form for phage inhalation is critical for the global distribution and therapeutic use of phages.20 This strategy is promising because many bacteriophages can be stabilized in the solid state for years.21 Freezedrying with subsequent micronization has been successfully applied to bacteriophages for respiratory delivery.22 An acceptable titer loss was found on processing and the in vitro performance of the resulting powder demonstrated efficient delivery by a dry powder inhaler (DPI). The work presented in this paper investigates the feasibility of spray drying to manufacture an inhalable bacteriophage powder in a single, scalable processing step. Spray drying is an established technique for the manufacturing of respiratory therapeutics.23,24 It also provides the opportunity to design microparticles with an internal structure that improves dispersibility and provides protection against processing stresses.25 Because bacteriophages are known to be susceptible to thermal stress,26 traditional methods of preserving phages include storage at −80◦ C or low-temperature drying processes such as freeze-drying or vacuum drying.21,27 Evidence of bacteriophage resistance to spray drying has been found by the dairy industry, where these viruses can interfere with cheese making by killing the bacteria that start the fermentation proJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
Figure 1. (Left panel) Electron micrograph of bacteriophage KS4-M and (right panel) morphology comparison of the phages used in the spray-drying study. The drawings for KS4-M and KS14 phages are based on electron micrographs of phages with contracted tail sheaths (dark grey). On the basis of their genome sequence, these phages are also expected to have tail fibers that are not shown in the cartoon because they were not clearly visible in the electron micrographs.
cess. In a study investigating the inactivation of bacteriophages in milk following pasteurization or spray drying,28 14 of 16 bacteriophages showed a titer loss of more than 1 log after being subjected to a standard spray-drying process. Decreasing the outlet temperature of the dryer from 96◦ C to 85◦ C reduced the bacteriophage inactivation efficiency of the drying process, indicating that low temperature spray drying may be a promising technique for the processing of inhalable bacteriophage powders. To the best of our knowledge, spray drying for the preservation of bacteriophages has not been reported previously.
MATERIALS AND METHODS Materials
Bacteriophages Different bacteriophages of the Myoviridae family were chosen for the present spray-drying study. Bacteriophage KS4-M29 is a liquid-clearing variant of bacteriophage KS44 that has been demonstrated to be active against a clinically relevant strain of the Burkholderia cepacia complex in a wax moth larvae model. KS4-M has an icosahedral head and contractile tail (Fig. 1) and can be assigned to the order Caudovirales and family Myoviridae based on this morphology. Phage KS4-M has been used successfully in both nebulization and lyophilization studies22,19 and is easy to obtain in high titers. Phage KS14 is a myovirus of similar size, active against the Burkholderia cepacia complex.29 Its genome has been sequenced and characterized and its morphology has been analyzed previously.30 The DOI 10.1002/jps
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drawing in Figure 1 shows its size in comparison to the other phages used in this study. From its depiction in Figure 1, which was derived from a previously published cryo-electron microscopy study,31 it is clear that the third Myoviridae phage, KZ, is much larger than the other two phages considered here. It is active against P. aeruginosa, is well described in the literature,32 has been used successfully in a lyophilization study,22 and is easy to obtain in high titers. Broth cultures of bacterial host strains (Burkholderia cenocepacia C6433 for KS14, B. cenocepacia K562 for KS4- M, and P. aeruginosa PAO for KZ) were grown in half-strength Luria–Bertani ( 21 LB) broth and incubated aerobically with shaking at 30◦ C overnight. Soft agar overlays on 12 LB solid medium (containing 100 :L phage stock, 100 :L broth culture, and 3 mL 12 LB medium containing 0.7% agar) were incubated aerobically at 30◦ C overnight. Plates exhibiting confluent lysis were overlaid with sterile water and incubated on a platform rocker 4–8 h at 4◦ C. The supernatant containing the phages was pelleted by centrifugation at 10,000 rcf ≤10 min to remove debris, filter sterilized using a 0.45 :m filter, passed through a Pierce Detoxi-Gel Endotoxin Removing Column (Thermo Fisher Scientific, Waltham, Massachusetts), and stored at 4◦ C. A verification of the phage preparations found that the KZ preparation consisted of a cocktail of KZ and P. aeruginosa phage D3,33,34 denoted KZ/D3. Both phages were assayed together, that is, the reported titers correspond to the sum of the titers of the individual phages in the cocktail.
Excipients The main component of the spray-dried particles was ","-trehalose (CAS number 6138-23-4). It was added with the intent to minimize the processing loss upon spray drying. The potential of trehalose to protect biologic material against desiccation and thermal stress has been well documented35–37 and due to its low toxicology risk, its use as an excipient for pulmonary drug delivery has been advanced.38 Dairy phages in milk show significantly higher survival under thermal stress when compared with phages suspended in water.39 The protective effect is not due to the fat component of milk because it is also seen in reconstituted nonfat dry skim milk.40 As casein is the main protein in cow milk, it was also considered as an excipient that may have the potential to provide protection against processing loss. Casein sodium salt from bovine milk (Sigma–Aldrich, St. Louis, MO) was used. This product is a mixture of four similar phosphoproteins with molecular weights between 19 and 24 kDa. Surfactants Tyloxapol [4-(1,1,3,3-tetramethylbutyl phenol polymer with formaldehyde and oxirane, DOI 10.1002/jps
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Figure 2. Schematic of the spray-drying apparatus.
CAS number 25301-02- 4] and Pluronic F68 (polyoxyethylene–polyoxypropylene block copolymer, CAS number 9003-11- 6) were added to some formulations to aid in dispersion of bacteriophages in the feed solution.41 All formulations contained L-leucine to achieve efficient drug delivery from DPIs by improving the dispersibility of the powders. It has been shown that leucine is compatible with spray drying of peptides42 and biological material41 and has the potential to reduce cohesive forces between particles.43,44 Selection of the excipient ratios was guided by the results of a recently published study45 on trehalose–leucine microparticles designed for respiratory drug delivery of biological actives. Experimental Methods
Spray Drying ¨ Spray drying was performed using a Buchi Nano ¨ Spray Dryer B-90 (Buchi Labortechnik AG, Flawil 1, Switzerland). The manufacturing setup is shown in Figure 2. The spray dryer used a vibrating mesh atomizer with 111 orifices each having a diameter of 4 :m corresponding to an atomized droplet diameter of 7 :m.46,47 The feedstock was circulated through the spray head at atmospheric pressure. A cellulose acetate and cellulose nitrate membrane filter with a pore width of 0.45 :m was used in line for filtration of the feedstock. Process conditions were chosen to provide a low outlet temperature of 40◦ C–45◦ C and an outlet relative humidity of less than 7%. The lowoutlet temperature was chosen because significant JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
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inactivation of phages at high-outlet temperatures has been shown previously.28 The corresponding inlet conditions were as follows: drying air flow rate, 100 L/min; feed flow rate, 0.33 mL/min; inlet temperature, 75◦ C. The collector of the system charges the dry particles via a high voltage corona discharge and separates them electrostatically from the air flow onto a ring electrode. The spray dryer was housed in a dry enclosure with glove ports to allow collection of powder from the system without exposure to moisture.
Aerosol Performance Testing Aerosol performance of the bacteriophage powders was tested in a commercially available DPI, the R , (Novartis Pharmaceuticals, Dorval, QueAerolizer bec, Canada), using an inspiratory flow rate of 60 L/ min for 4 s. A powder dose of 25–28 mg was filled by hand into hard gelatin capsules (#3, Capsule Connection, Prescott, Arizona) for a single actuation in the R device. Aerolizer The experimental setup used to measure the emitted powder mass, mouth–throat mass, total lung mass, and total lung dose has been described in detail elsewhere.22 Briefly, the Alberta Idealized Throat (Copley Scientific, Nottingham, UK) was used as an in vitro measure to assess deposition in the mouth and throat of adult patients. This throat geometry48 reflects extrathoracic deposition more accurately than the United States Pharmacopeia/European Pharmacopoeia induction ports frequently used for impactor measurements.49 The powder mass collected in the silicone-coated Alberta Idealized Throat was determined by aqueous extraction of the deposited powder from the throat and quantification of trehalose in the extract using an anthrone assay.50 To determine the respirable fraction of the aerosol, that is, the mass of powder that can be inspired into the lung, the total lung mass was measured using a low-resistance filter that was attached directly to the outlet of the Alberta Idealized Throat. The mass of powder on the filter was determined by extracting the filter with water and assaying for trehalose as referenced above. This provided the powder mass because the mass fraction of trehalose was accurately known for each formulation. The infective dose of bacteriophages that can be inhaled into the lung, that is, the total lung dose, was determined by performing a bacteriophage plaque assay on the filter extract. The aerodynamic particle size distribution of the total lung fraction was determined by attaching an Anderson cascade impactor (Graseby Andersen, Smyrna, Georgia) to the outlet of the Alberta Idealized Throat. The impactor was operated at 60 L/min with a reduced number of stages, 0, 1, 2, 3, 4, and 5, with effective stage cutoff aerodynamic diameters of 5. 6, 4. 3, 3. 4, 2. 0, 1. 1, and 0.51 :m,51 respectively. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
The powder mass on the silicone-coated stages was determined gravimetrically.
Plaque Assay Powders containing bacteriophages were dissolved in an aqueous suspension medium [50 mM Tris–HCl (pH 7. 5), 100 mM NaCl, 10 mM MgSO4 , 0.01% gelatin solution] to a concentration of 50 mg/mL. To perform phage counts, serial dilutions of the powder solution were made in suspension medium. The dilutions were plated in triplicate in soft agar overlays with the corresponding host strain, and plaques were counted following overnight incubation at 30◦ C. For KS4-M/KZ spray-dried cocktail powders, duplicate counts were performed using K56-2 and PAO separately as hosts. For total lung dose determination, filters were overlaid with 4–5 mL suspension medium and incubated 5 minutes at room temperature. The solution was then serially diluted, plated, and counted as above. Each batch was tested in duplicate.
Electron Microscopy For transmission electron microscopy, 10 :L of hightiter KS4-M lysate (filter sterilized with a 0.22 :m filter) was incubated for 5 minutes at room temperature on a carbon-coated copper grid. Residual lysate was removed and the grid was stained for 2 minutes at room temperature with 2% phosphotungstate. Micrographs were taken at 140,000-fold magnification using a Philips/FEI (Morgagni) transmission electron microscope with charge-coupled device camera (Department of Biological Sciences, Advanced Microscopy Facility, University of Alberta). For scanning electron microscopy, particle images were taken using a Hitachi SEM S-2500 (Hitachi Ltd., Tokyo, Japan). Samples were prepared on aluminum pin stubs using double-sided carbon tape and sputter coated with gold.
RESULTS AND DISCUSSION Powder Properties and Manufacturability Table 1 shows the composition of the four formulations that were chosen for more extensive analysis. All formulations mainly consisted of trehalose (T) with 19% of leucine (L) added. Two formulations contained 2% of a surfactant, tyloxapol (X), or Pluronic (P). The last formulation contained casein sodium salt (C) instead. Several other excipient ratios, for example, trehalose and leucine in a 1:1 ratio, were tested for titer loss on spray drying but were abandoned because they were inferior to the ones listed in Table 1 with respect to titer. All formulations were spray-dried with a feed concentration, cF , that was selected to produce a mass median aerodynamic diameter, da , of the primary DOI 10.1002/jps
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Table 1.
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Formulation, Manufacturing Parameters, and Aerosol Performance of Four Lead Formulations
Formulation Code Mass fraction, trehalose Mass fraction, leucine Mass fraction, surfactant Mass fraction, casein sodium salt Feed concentration in mg/mL Manufacturing yield in % Emitted mass in % of capsule mass Mouth–throat fraction in % of emitted mass
LT
LTX
LTP
LTC
0.77 0.19 0 0 27 62.5 ± 5.3 (n = 6) NA NA
0.76 0.19 0.02 0 27.5 82.6 ± 5.6 (n = 6) 82.9 ± 4.7 (n = 2) 28.7± 2.8 (n = 2)
0.76 0.19 0.02 0 27.5 78.1 ± 4.1 (n = 6) 91.7 ± 9.5 (n = 6) 29.3 ± 2.8 (n = 6)
0.76 0.19 0 0.02 27.5 59.2 ± 3.8 (n = 18) 82.7 ± 4.5 (n = 8) 21.8 ± 3.1 (n = 8)
L, T, X, P, and C denote leucine, trehalose, tyloxapol, pluronic, and casein sodium salt, respectively. Error margins refer to a single standard deviation. n denotes the number of replicates.
particles of 1.4–1.9 :m according to the following equation25 : da =
6
ρP ρ ref
3
cF d0 ρ ref
The particle density, ρP , was estimated to be in the range of 0.1–0.5 kg/L and the reference density, ρref , is 1 kg/ L. d0 denotes the initial diameter of the atomized droplets. Figure 3 shows representative electron micrographs of the four formulations described in Table 1. The formulations were used for the spray drying of different phages. The species of phage incorporated in the formulation did not have a discernible influence on the morphology of the resulting particles and for this reason only four electron micrographs for the different formulations are shown. All powders had a well-dispersed appearance. No fused particles were observed. All formulations showed evidence of internal voids in the particles, in particular in the larger particles. The particles had a spherical shape and the leucine–trehalose (LT) and leucine–trehalose–casein (LTC) formulations had clearly increased rugosity, which is known to improve dispersibility.52 The morphology of the particles for all four formulations appears suitable for respiratory delivery applications. The manufacturing yield of the surfactantcontaining formulations was significantly higher than that of the other two formulations (Table 1). The type of phage in the formulation did not have a statis-
tically significant influence on yield so the data for the different phages were pooled. However, all four formulations had an acceptable to high yield, given the small batch sizes of 200–500 mg. Manufacturing yields are expected to increase for the production of larger batches.
Aerosol Performance R Table 1 shows the emitted dose from the Aerolizer DPI. The emitted dose was tested for formulations that contained surfactant or casein. No powder residue was visible in the capsules after administration. The powder mass left in the device was derived by subtracting the assayed mouth–throat mass and total lung mass from the capsule mass. In all 16 tests, the emitted dose was higher than 76%, reaching 100% in several cases. The 60 L/min flow rate for the emitted dose tests was chosen conservatively. With a comfortable inspiratory pressure drop of 4 kPa across the R , a flow rate of approximately 90 L/min will Aerolizer be established.53 Most patients exceed the test flow rate by a large margin,54 which is expected to lead to nearly complete delivery of the powders described in Table 1. In comparison, most commercially available DPI emits less than two thirds of the packaged powder mass from the device.55 For a subset of tests, the powder deposited in the Alberta Idealized Throat was assayed to determine the mouth–throat fraction, reported in Table 1. In all tests, less than a third of the emitted mass was deposited in the mouth–throat. The difference in the
Figure 3. Scanning electron micrographs of formulations listed in Table 1. From left to right: leucine–trehalose–pluronic (LTP), leucine–trehalose–tyloxapol (LTX), leucine–trehalose (LT), leucine–trehalose–casein (LTC). DOI 10.1002/jps
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Figure 4. Total lung mass in percentage of capsule mass for bacteriophage powders containing KS4-M or KZ/D3 or a cocktail of phages in different formulations. The formulation codes are explained in Table 1. Error bars represent the standard deviation.
mouth–throat fraction of the surfactant-containing formulations relative to the casein-containing formulation was statistically significant. The latter had an exceptionally low extrathoracic deposition. The mouth–throat fraction measured for the spray-dried powders demonstrates a large improvement relative to freeze-dried bacteriophage preparations where the mouth–throat fraction was 58%.22 The total lung mass, that is, the mass of powder delivered past the mouth–throat relative to the powder fill mass in the capsule, is shown in Figure 4. In all cases, the total lung mass exceeded 50%. The most consistent delivery across all phages including the phage cocktails was accomplished with the leucine–trehalose–casein formulation, which was chosen as lead formulation. In comparison, the total lung dose of 12 commercially available DPIs was reported in a range from 5.5% to 40.5% with a mean of 23%.55 The total lung mass of the lead formulation shows a more than twofold improvement relative to recent freeze-dried formulations.22 These comparisons demonstrate that the particle design approach45,56 used in this study has accomplished the target of a highly dispersible powder, suitable for efficient powder delivery to the lung. The aerosol size distribution of the total lung mass for two lead formulations is shown in Figure 5. The distribution was found to be consistent for all tests and showed only minor differences for the two phages tested. The mass median aerodynamic diameter was less than 3 :m in all cases, which is considered suitable for delivery to all areas of the lung.57 The increased deposition on stage 5, with a stage cutoff of 0.51 :m, is likely caused by particle bounce in the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
Figure 5. Aerosol size distribution and mass median aerodynamic diameter (MMAD) of the total lung mass for two selected bacteriophage formulations in a leucine–trehalose–casein (LTC) formulation. The error bars represent the standard deviation of duplicate tests on two independent batches, that is, n = 4. A single capsule was actuated for each test.
impactor, which is difficult to eliminate for highly dispersible powders, even with silicone-coated plates.
Bacteriophage Titer The results in Figure 6 show the loss in titer after spray drying. All Myoviridae phages used in this study were successfully processed and reconstituted with a titer loss of less than 1 log pfu/mL. A titer loss of less than 1 log during the process of development is acceptable in terms of manufacturability of drugs. Phage cocktail KZ/D3 proved to be most robust with a titer loss of less than 0.5 log pfu/mL, irrespective of the formulation in which it was processed. Phage KS4-M was more sensitive to processing stress as seen previously in lyophilization experiments.22 Although the titer of freeze-dried KS4-M preparations
Figure 6. Titer loss of the phages both separately and in a cocktail using different formulations during the process. The error bars represent the standard deviations. DOI 10.1002/jps
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doses in the above-mentioned animal and human trials by orders of magnitude. The titer of the powder ranged from 1.3 × 106 pfu/mg for phage KZ in the phage cocktail to 1.7 × 107 pfu/mg for phage KS4- M. Assuming a total lung mass of greater than 70%, this indicates that a dose of 106 pfu could be administered from an inhaler using a powder mass of 100 :g to 1.1 mg per actuation.
CONCLUSION
Figure 7. In vitro total lung dose and powder titer of LTC (leucine–trehalose–casein) formulations for different phages alone and in a phage cocktail. The diamond represents the median (horizontal line) and one standard deviation (vertices). The whiskers describe the minimum and maximum value and the closed symbol represents the mean. Each test had six replicates.
decreased more than 2 logs upon processing, the low temperature spray-drying process used here led to a loss of about 1 log or less for the formulations without casein sodium salt and less than 0.5 log for the caseincontaining formulation. The processing loss of phage KS14 was similar to that of KS4-M in this formulation. The LTC formulation was also suitable for the processing of a KS4-M/KZ/D3 phage cocktail. The processing loss for the components in the cocktail was not statistically different from the loss measured in the powders of the individual components. The batch sizes produced in this study were too small for formal storage stability tests. A titer retest in triplicate after 3 months of refrigerated storage for KZ/D3 in formulations LT and LTC showed less than 0.15 log titer loss in all cases. Figure 7 shows a statistical evaluation of the in vitro total lung dose of bacteriophages delivered R DPI. Also listed is the to the lung with the Aerolizer titer of the powder that was filled into the capsule of the DPI to allow for total lung dose estimates with other delivery devices. In a study in mice infected with lethal doses of P. aeruginosa, a single intranasal dose of bacteriophages was found to be efficacious at 3 × 106 pfu, curative at 3 × 107 pfu and provided complete prophylactic protection at 3 × 108 pfu.18 A controlled human clinical trial on patients with chronic ear infections caused by antibiotic-resistant P. aeruginosa showed efficacy after topical administration of a single dose of a phage cocktail containing 6 × 105 pfu.58 The total lung doses shown in Figure 7 exceed the efficacious DOI 10.1002/jps
Bacteriophages can be processed into a respirable dry dosage form by low temperature spray drying. Different phage species, even from the same family, have different susceptibility to processing loss. Development of a bacteriophage therapeutic for pulmonary delivery appears promising; acceptable process loss was achieved with excipients that pose a low pulmonary toxicity risk, the manufacturing process had an adequate yield given the small batch sizes, and the resulting powders had outstanding aerosol performance when delivered from a simple DPI. Powder titers were sufficiently high to be compatible with a wide range of inhalation devices. The in vitro lung doses of all tested bacteriophages and a bacteriophage cocktail were much higher than levels shown to be efficacious in animal models.
ACKNOWLEDGMENTS Jonathan J. Dennis. and Warren H. Finlay gratefully acknowledge funding from Canadian Institutes of Health Research and Cystic Fibrosis (CF), Canada. Sadaf Matinkhoo and Reinhard Vehring gratefully acknowledge funding from Natural Sciences and Engineering Research Council of Canada (NSERC). Karlene H. Lynch thanks the Killam Trusts, NSERC, Alberta Heritage Foundation for Medical Research, and CF Canada for financial support.
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6. Reinert R. 2009. The antimicrobial resistance profile of Streptococcus pneumoniae. Clinical Microbiol Infect 15:7– 11. 7. Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, Walsh TR. 2009. Characterization of a New Metallo-{beta}lactamase gene, blaNDM- 1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 53:5046–5054. 8. Karthikeyan K, Thirunarayan M, Krishnan P. 2010. Coexistence of blaOXA-23 with blaNDM-1 and armA in clinical isolates of Acinetobacter baumannii from India. J Antimicrob Chemother 65:2253–2254. 9. WHO. 2010. Multidrug and extensively drug-resistant TB (M/ XDR-TB): 2010 global report on surveillance and response. ISBN 978 924 159 9191. 10. Jassal M, Bishai WR. 2009. Extensively drug-resistant tuberculosis. Lancet Infect Dis 9:19–30. 11. Abedon ST, Thomas-Abedon C. 2010. Phage therapy pharmacology. Curr Pharm Biotechnol 11:28–47. 12. Broxmeyer L. 2004. Bacteriophages: Antibacterials with a future?. Med Hypotheses 62:889–893. 13. Kutateladze M, Adamia R. 2010. Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends Biotechnol 28:591–595. 14. Monk A, Rees C, Barrow P, Hagens S, Harper D. 2010. Under the microscope: Bacteriophage applications: Where are we now?. Lett Appl Microbiol 51:363–369. 15. Parisien A, Allain B, Zhang J, Mandeville R, Lan C. 2008. Novel alternatives to antibiotics: Bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides. J Appl Microbiol 104:1–13. 16. Morgan AD, Bonsall MB, Buckling A. 2010. Impact of bacterial mutation rate on coevolutionary dynamics between bacteria and phages. Evolution 64:2980–2987. 17. Merabishvili M, Pirnay JP, Verbeken, G, Chanishvili N, Tediashvili M, Lashkhi N, Glonti T, Krylov V, Mast J, Van Parys L. 2009. Quality-controlled small-scale production of a welldefined bacteriophage cocktail for use in human clinical trials. PLoS ONE 4:1–10. 18. Morello E, Saussereau E, Maura D, Huerre M, Touqui L, Debarbieux L, Aziz R. 2011. Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: First steps towards treatment and prevention. PLoS ONE 6:e16963. 19. Golshahi L, Seed K, Dennis J, Finlay W. 2008. Toward modern inhalational bacteriophage therapy: Nebulization of bacteriophages of Burkholderia cepacia complex. J Aerosol Med Pulm Drug Deliv 21:351–360. 20. Sou T, Meeusen EN, de Veer M, Morton DAV, Kaminskas LM, McIntosh MP. 2011. New developments in dry powder pulmonary vaccine delivery. Trends Biotechnol 29:191– 198. 21. Ackermann HW, Tremblay D, Moineau S. 2004. Long-term bacteriophage preservation. World Fed Cult Collections Newsl 38:35–40. 22. Golshahi L, Lynch K, Dennis J, Finlay W. 2010. In vitro lung delivery of bacteriophages KS4 M and KZ using dry powder inhalers for treatment of Burkholderia cepacia complex and Pseudomonas aeruginosa infections in cystic fibrosis. J Appl Microbiol 110:106–117. 23. Broadhead J, Edmond Rouan SK, Rhodes CT. 1992. The spray drying of pharmaceuticals. Drug Dev Ind Pharm 18:1169–1206. 24. Weers JG, Tarara TE, Clark AR. 2007. Design of fine particles for pulmonary drug delivery. Expert Opin Drug Deliv 4:297–313. 25. Vehring R. 2008. Pharmaceutical particle engineering via spray drying. Pharm Res 25:999–1022. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
26. Muller-Merbach M, Rauscher T, Hinrichs J. 2005. Inactivation of bacteriophages by thermal and high-pressure treatment. Int Dairy J 15:777–784. 27. Skurnik M, Pajunen M, Kiljunen S. 2007. Biotechnological challenges of phage therapy. Biotechnol Lett 29:995– 1003. 28. Chopin MC. 1980. Resistance of 17 mesophilic lactic Streptococcus bacteriophages to pasteurization and spray-drying. J Dairy Res 47:131–139. 29. Seed KD, Dennis JJ. 2009. Experimental bacteriophage therapy increases survival of Galleria mellonella larvae infected with clinically relevant strains of the Burkholderia cepacia complex. Antimicrob Agents Chemother 53:2005–2008. 30. Lynch K, Stothard P, Dennis J. 2010. Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex. BMC Genomics 11:599. 31. Fokine A, Battisti A, Bowman V, Efimov A, Kurochkina L, Chipman P, Mesyanzhinov V, Rossmann M. 2007. Cryo-EM study of the Pseudomonas bacteriophage [phi] KZ. Structure 15:1099–1104. 32. Mesyanzhinov V, Robben J, Grymonprez B, Kostyuchenko V, Bourkaltseva M, Sykilinda N, Krylov V, Volckaert G. 2002. The genome of bacteriophage [phi] KZ of Pseudomonas aeruginosa. J Mol Biol 317:1–19. 33. Holloway B, Cooper G. 1962. Lysogenic conversion in Pseudomonas aeruginosa. J Bacteriol 84:1321–1324. 34. Holloway B, Egan J, Monk M. 1960. Lysogeny in Pseudomonas aeruginosa. Aust J Exp Biol Med Sci 38:321–329. 35. Ameri M, Maa Y-F. 2006. Spray drying of biopharmaceuticals: Stability and process considerations. Drying Technol 24:763–768. 36. Sastry GM, Agmon N. 1997. Trehalose prevents myoglobin collapse and preserves its internal mobility. Biochem 36:7097–7108. 37. Satpathy GR, T¨or¨ok Z, Bali R, Dwyre DM, Little E, Walker NJ, Tablin F, Crowe JH, Tsvetkova, NM. 2004. Loading red blood cells with trehalose: A step towards biostabilization. Cryobiology 49:123–136. 38. Tarara TE, Stevenson CL, Thompson S, Vehring R, Sadrzadeh N. 2007. Methionine-containing protein or peptide compositions and methods of making and using. International Patent Application WO 2007/095288 A2. ¨ 39. Muller-Merbach M, Neve H, Hinrichs J. 2005. Kinetics of the thermal inactivation of the Lactococcus lactis bacteriophage P008. J Dairy Res 72:281–286. 40. Quiberoni A, Guglielmotti DM, Reinheimer JA. 2003. Inactivation of Lactobacillus delbrueckii bacteriophages by heat and biocides. Int J Food Microbiol 84:51–62. 41. Wong Y-L, Sampson S, Germishuizen WA, Goonesekera S, Caponetti G, Sadoff J, Bloom BR, Edwards D. 2007. Drying a tuberculosis vaccine without freezing. Proc Natl Acad Sci U S A 104:2591–2595. 42. Garcia-Contreras L, Fiegel J, Telko MJ, Elbert K, Hawi A, Thomas M, VerBerkmoes J, Germishuizen WA, Fourie PB, Hickey AJ, Edwards D. 2007. Inhaled large porous particles of capreomycin for treatment of tuberculosis in a guinea pig model. Antimicrob Agents Chemother 51:2830–2836. 43. Learoyd T, Burrows J, French E, Seville P. 2009. Sustained delivery by leucine-modified chitosan spray-dried respirable powders. Int J Pharm 372:97–104. 44. Raula J, Thielmann F, Naderi M, Lehto V, Kauppinen E. 2010. Investigations on particle surface characteristics vs. dispersion behaviour of l-leucine coated carrier-free inhalable powders. Int J Pharm 385:79–85. 45. Feng A, Boraey M, Gwin M, Finlay P, Kuehl P, Vehring R. 2011. Mechanistic models facilitate efficient development of leucine containing microparticles for pulmonary drug delivery. Int J Pharm 409:156–163. DOI 10.1002/jps
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46. Li X, Anton N, Arpagaus C, Belleteix F, Vandamme T. 2010. Nanoparticles by spray drying using innovative new technol¨ ogy: The Buchi nano spray dryer b-90. J Controlled Release 147:304–310. 47. Schmid K, Arpagaus C, Friess W. 2011. Evaluation of the nano spray dryer B-90 for pharmaceutical applications. Pharm Dev Technol 16:287–294. 48. Stapleton K, Guentsch E, Hoskinson M, Finlay W. 2000. On the suitability of k-turbulence modeling for aerosol deposition in the mouth and throat: A comparison with experiment. J Aerosol Sci 31:739–749. 49. Grgic B, Finlay W, Burnell P, Heenan A. 2004. In vitro intersubject and intrasubject deposition measurements in realistic mouth–throat geometries. J Aerosol Sci 35:1025–1040. 50. Khanmohammadi M, Moeini M, Bagheri Garamarudi A, Sotudehnia A, Zarrabi M. 2009. Simultaneous determination of sucrose and trehalose in olive leaves by spectrophotometry utilizing partial least squares method. Acta Physiologiae Plantarum 31:865–869. 51. Nichols S, Brown D, Smurthwaite M. 1998. New concept for the variable flow rate Andersen cascade impactor and calibration data. J Aerosol Med 11:133–138.
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52. Chew NYK, Tang P, Chan H-K, Raper JA. 2005. How much particle surface corrugation is sufficient to improve aerosol performance of powders?. Pharm Res 22:148–152. 53. Assi K, Chrystyn H. 2000. The device resistance of recently introduced dry-powder inhalers. J Pharm Pharmacol 52:58– 58. ¨ 54. Bronsky EA, Grossman J, Henis MJ, Gallo PP, Yegen U,
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Cioppa GD, Kottakis J, Mehra S. 2004. Inspiratory flow rates and volumes with the Aerolizer dry powder inhaler in asthmatic children and adults. Curr Med Res Opin 20:131–137. Newman S, Busse W. 2002. Evolution of dry powder inhaler design, formulation, and performance. Respir Med 96:293–304. Ivey J, Vehring R. 2010. The use of modeling in spray drying of emulsions and suspensions accelerates formulation and process development. Comput Chem Eng 34:1030–1035. Finlay WH. 2001. The mechanics of inhaled pharmaceutical aerosols. San Diego: Academic Press. ¨ Wright A, Hawkins C, Anggård E, Harper D. 2009. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic resistant Pseudomonas aeruginosa: A preliminary report of efficacy. Clin Otolaryngol 34:349–357.
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Department of Mechanical Engineering, University of Alberta, Edmonton, Alberta T6G 2G8, Canada
2
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
Received 9 April 2011; revised 14 June 2011; accepted 11 July 2011 Published online 23 August 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22715 ABSTRACT: Myoviridae bacteriophages were processed into a dry powder inhalable dosage form using a low-temperature spray-drying process. The phages were incorporated into microparticles consisting of trehalose, leucine, and optionally a third excipient (either a surfactant or casein sodium salt). The particles were designed to have high dispersibility and a respirable particle size, and to preserve the phages during processing. Bacteriophages KS4- M, KS14, and cocktails of phages KZ/D3 and KZ/D3/KS4-M were spray-dried with a processing loss ranging from 0.4 to 0.8 log pfu. The aerosol performance of the resulting dry powders as delivR dry powder inhaler (DPI) exceeded the performance of commercially ered from an Aerolizer available DPIs; the emitted mass and the in vitro total lung mass of the lead formulation were 82.7% and 69.7% of filled capsule mass, respectively. The total lung mass had a mass median aerodynamic diameter of 2.5–2.8 :m. The total in vitro lung doses of the phages, delivered from a single actuation of the inhaler, ranged from 107 to 108 pfu, levels that are expected to be efficacious in vivo. Spray drying of bacteriophages into a respirable dry powder was found to be feasible. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:5197–5205, 2011 Keywords: aerosols; formulation; pulmonary drug delivery; spray drying; biomaterials; processing; microparticles
INTRODUCTION Bacterial infections have been treated successfully with antibiotics for decades, but many bacteria have now evolved to the point that many of the currently available antimicrobials are ineffective, thus affecting the treatment of pulmonary infections. The compromised lungs of cystic fibrosis patients are at an elevated risk of infection1 by bacteria of the Burkholderia cepacia complex2 and by Pseudomonas aeruginosa. Several strains of these bacteria have emerged that demonstrate significant resistance against a broad range of antibiotics.3–5 Bacterial pneumonia is also becoming more difficult to treat, with few antibiotics available that are effective against multidrugresistant strains, such as those of Streptococcus pneumoniae.6 Similarly, a genetic mutation first identified in Klebsiella pneumoniae strain NDM-1 enables the bacterium to produce an enzyme, known as Correspondence to: Reinhard Vehring (Telephone: +780-4925180; Fax: +780-492-2200; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 100, 5197–5205 (2011) © 2011 Wiley-Liss, Inc. and the American Pharmacists Association
New Delhi metallo-$-lactamase, that inactivates carbapenem antibiotics.7 This gene can be transferred between different bacteria and permits complete resistance against all aminoglycoside, quinolone, and $lactam antibiotics.8 Another bacterium of particular concern is multidrug-resistant Mycobacterium tuberculosis, with about 400,000 cases per year worldwide.9 It is likely that in more than 5% of these cases, extensively drug resistant tuberculosis is present that is practically untreatable in resource-limited settings and is associated with high mortality.10 Given the concerns about antibiotic resistance, an alternative or supplemental treatment for bacterial infections, bacteriophage therapy, has become the focus of renewed attention.11–14 Bacteriophages are viruses that infect and kill bacteria.15 Because bacteriophages and bacteria have coevolved, it is unlikely that bacteria can develop a general resistance against bacteriophages.16 Unlike chemical antibiotics, which are unchanging, bacteriophages can mutate and adapt to changes in their bacterial hosts. Because a bacteriophage species typically has a narrow host range, bacteriophage therapy may avoid
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systemic side effects that are common in aggressive antibiotic treatment. This specificity also has a drawback in that the causative agent of an infection must be identified before an appropriate phage for its treatment can be selected. Alternatively, administration of phage cocktails,17 consisting of phages active against multiple bacterial strains or species, may be effective. Late-stage clinical trials in accordance with current regulatory requirements in North America or Europe have not yet been completed, but safety risks associated with phage administration appear low based on extensive experimental phage therapy experience in Eastern Europe and the former Soviet Union. Successful bacteriophage treatment for respiratory tract infections in cystic fibrosis patients has been reported,13 but questions remain about the best route of delivery. Bacterial infections in the lung may reside in macrophages or may exist in colonies that are isolated from the vasculature, for example, in mucus layers or walled off by biofilms. Hence, the feasibility of respiratory bacteriophage delivery via the airways has been investigated in an attempt to provide an effective alternative delivery route. First reports of inhaled bacteriophages in mouse models are promising; a P. aeruginosa infection could be cured by a single nasal administration of bacteriophages.18 Golshahi et al.19 have demonstrated that nebulization can be used to deliver bacteriophages to the lungs in quantities that are expected to be sufficient for phage therapy. Development of a stabilized dry dosage form for phage inhalation is critical for the global distribution and therapeutic use of phages.20 This strategy is promising because many bacteriophages can be stabilized in the solid state for years.21 Freezedrying with subsequent micronization has been successfully applied to bacteriophages for respiratory delivery.22 An acceptable titer loss was found on processing and the in vitro performance of the resulting powder demonstrated efficient delivery by a dry powder inhaler (DPI). The work presented in this paper investigates the feasibility of spray drying to manufacture an inhalable bacteriophage powder in a single, scalable processing step. Spray drying is an established technique for the manufacturing of respiratory therapeutics.23,24 It also provides the opportunity to design microparticles with an internal structure that improves dispersibility and provides protection against processing stresses.25 Because bacteriophages are known to be susceptible to thermal stress,26 traditional methods of preserving phages include storage at −80◦ C or low-temperature drying processes such as freeze-drying or vacuum drying.21,27 Evidence of bacteriophage resistance to spray drying has been found by the dairy industry, where these viruses can interfere with cheese making by killing the bacteria that start the fermentation proJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
Figure 1. (Left panel) Electron micrograph of bacteriophage KS4-M and (right panel) morphology comparison of the phages used in the spray-drying study. The drawings for KS4-M and KS14 phages are based on electron micrographs of phages with contracted tail sheaths (dark grey). On the basis of their genome sequence, these phages are also expected to have tail fibers that are not shown in the cartoon because they were not clearly visible in the electron micrographs.
cess. In a study investigating the inactivation of bacteriophages in milk following pasteurization or spray drying,28 14 of 16 bacteriophages showed a titer loss of more than 1 log after being subjected to a standard spray-drying process. Decreasing the outlet temperature of the dryer from 96◦ C to 85◦ C reduced the bacteriophage inactivation efficiency of the drying process, indicating that low temperature spray drying may be a promising technique for the processing of inhalable bacteriophage powders. To the best of our knowledge, spray drying for the preservation of bacteriophages has not been reported previously.
MATERIALS AND METHODS Materials
Bacteriophages Different bacteriophages of the Myoviridae family were chosen for the present spray-drying study. Bacteriophage KS4-M29 is a liquid-clearing variant of bacteriophage KS44 that has been demonstrated to be active against a clinically relevant strain of the Burkholderia cepacia complex in a wax moth larvae model. KS4-M has an icosahedral head and contractile tail (Fig. 1) and can be assigned to the order Caudovirales and family Myoviridae based on this morphology. Phage KS4-M has been used successfully in both nebulization and lyophilization studies22,19 and is easy to obtain in high titers. Phage KS14 is a myovirus of similar size, active against the Burkholderia cepacia complex.29 Its genome has been sequenced and characterized and its morphology has been analyzed previously.30 The DOI 10.1002/jps
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drawing in Figure 1 shows its size in comparison to the other phages used in this study. From its depiction in Figure 1, which was derived from a previously published cryo-electron microscopy study,31 it is clear that the third Myoviridae phage, KZ, is much larger than the other two phages considered here. It is active against P. aeruginosa, is well described in the literature,32 has been used successfully in a lyophilization study,22 and is easy to obtain in high titers. Broth cultures of bacterial host strains (Burkholderia cenocepacia C6433 for KS14, B. cenocepacia K562 for KS4- M, and P. aeruginosa PAO for KZ) were grown in half-strength Luria–Bertani ( 21 LB) broth and incubated aerobically with shaking at 30◦ C overnight. Soft agar overlays on 12 LB solid medium (containing 100 :L phage stock, 100 :L broth culture, and 3 mL 12 LB medium containing 0.7% agar) were incubated aerobically at 30◦ C overnight. Plates exhibiting confluent lysis were overlaid with sterile water and incubated on a platform rocker 4–8 h at 4◦ C. The supernatant containing the phages was pelleted by centrifugation at 10,000 rcf ≤10 min to remove debris, filter sterilized using a 0.45 :m filter, passed through a Pierce Detoxi-Gel Endotoxin Removing Column (Thermo Fisher Scientific, Waltham, Massachusetts), and stored at 4◦ C. A verification of the phage preparations found that the KZ preparation consisted of a cocktail of KZ and P. aeruginosa phage D3,33,34 denoted KZ/D3. Both phages were assayed together, that is, the reported titers correspond to the sum of the titers of the individual phages in the cocktail.
Excipients The main component of the spray-dried particles was ","-trehalose (CAS number 6138-23-4). It was added with the intent to minimize the processing loss upon spray drying. The potential of trehalose to protect biologic material against desiccation and thermal stress has been well documented35–37 and due to its low toxicology risk, its use as an excipient for pulmonary drug delivery has been advanced.38 Dairy phages in milk show significantly higher survival under thermal stress when compared with phages suspended in water.39 The protective effect is not due to the fat component of milk because it is also seen in reconstituted nonfat dry skim milk.40 As casein is the main protein in cow milk, it was also considered as an excipient that may have the potential to provide protection against processing loss. Casein sodium salt from bovine milk (Sigma–Aldrich, St. Louis, MO) was used. This product is a mixture of four similar phosphoproteins with molecular weights between 19 and 24 kDa. Surfactants Tyloxapol [4-(1,1,3,3-tetramethylbutyl phenol polymer with formaldehyde and oxirane, DOI 10.1002/jps
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Figure 2. Schematic of the spray-drying apparatus.
CAS number 25301-02- 4] and Pluronic F68 (polyoxyethylene–polyoxypropylene block copolymer, CAS number 9003-11- 6) were added to some formulations to aid in dispersion of bacteriophages in the feed solution.41 All formulations contained L-leucine to achieve efficient drug delivery from DPIs by improving the dispersibility of the powders. It has been shown that leucine is compatible with spray drying of peptides42 and biological material41 and has the potential to reduce cohesive forces between particles.43,44 Selection of the excipient ratios was guided by the results of a recently published study45 on trehalose–leucine microparticles designed for respiratory drug delivery of biological actives. Experimental Methods
Spray Drying ¨ Spray drying was performed using a Buchi Nano ¨ Spray Dryer B-90 (Buchi Labortechnik AG, Flawil 1, Switzerland). The manufacturing setup is shown in Figure 2. The spray dryer used a vibrating mesh atomizer with 111 orifices each having a diameter of 4 :m corresponding to an atomized droplet diameter of 7 :m.46,47 The feedstock was circulated through the spray head at atmospheric pressure. A cellulose acetate and cellulose nitrate membrane filter with a pore width of 0.45 :m was used in line for filtration of the feedstock. Process conditions were chosen to provide a low outlet temperature of 40◦ C–45◦ C and an outlet relative humidity of less than 7%. The lowoutlet temperature was chosen because significant JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
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inactivation of phages at high-outlet temperatures has been shown previously.28 The corresponding inlet conditions were as follows: drying air flow rate, 100 L/min; feed flow rate, 0.33 mL/min; inlet temperature, 75◦ C. The collector of the system charges the dry particles via a high voltage corona discharge and separates them electrostatically from the air flow onto a ring electrode. The spray dryer was housed in a dry enclosure with glove ports to allow collection of powder from the system without exposure to moisture.
Aerosol Performance Testing Aerosol performance of the bacteriophage powders was tested in a commercially available DPI, the R , (Novartis Pharmaceuticals, Dorval, QueAerolizer bec, Canada), using an inspiratory flow rate of 60 L/ min for 4 s. A powder dose of 25–28 mg was filled by hand into hard gelatin capsules (#3, Capsule Connection, Prescott, Arizona) for a single actuation in the R device. Aerolizer The experimental setup used to measure the emitted powder mass, mouth–throat mass, total lung mass, and total lung dose has been described in detail elsewhere.22 Briefly, the Alberta Idealized Throat (Copley Scientific, Nottingham, UK) was used as an in vitro measure to assess deposition in the mouth and throat of adult patients. This throat geometry48 reflects extrathoracic deposition more accurately than the United States Pharmacopeia/European Pharmacopoeia induction ports frequently used for impactor measurements.49 The powder mass collected in the silicone-coated Alberta Idealized Throat was determined by aqueous extraction of the deposited powder from the throat and quantification of trehalose in the extract using an anthrone assay.50 To determine the respirable fraction of the aerosol, that is, the mass of powder that can be inspired into the lung, the total lung mass was measured using a low-resistance filter that was attached directly to the outlet of the Alberta Idealized Throat. The mass of powder on the filter was determined by extracting the filter with water and assaying for trehalose as referenced above. This provided the powder mass because the mass fraction of trehalose was accurately known for each formulation. The infective dose of bacteriophages that can be inhaled into the lung, that is, the total lung dose, was determined by performing a bacteriophage plaque assay on the filter extract. The aerodynamic particle size distribution of the total lung fraction was determined by attaching an Anderson cascade impactor (Graseby Andersen, Smyrna, Georgia) to the outlet of the Alberta Idealized Throat. The impactor was operated at 60 L/min with a reduced number of stages, 0, 1, 2, 3, 4, and 5, with effective stage cutoff aerodynamic diameters of 5. 6, 4. 3, 3. 4, 2. 0, 1. 1, and 0.51 :m,51 respectively. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
The powder mass on the silicone-coated stages was determined gravimetrically.
Plaque Assay Powders containing bacteriophages were dissolved in an aqueous suspension medium [50 mM Tris–HCl (pH 7. 5), 100 mM NaCl, 10 mM MgSO4 , 0.01% gelatin solution] to a concentration of 50 mg/mL. To perform phage counts, serial dilutions of the powder solution were made in suspension medium. The dilutions were plated in triplicate in soft agar overlays with the corresponding host strain, and plaques were counted following overnight incubation at 30◦ C. For KS4-M/KZ spray-dried cocktail powders, duplicate counts were performed using K56-2 and PAO separately as hosts. For total lung dose determination, filters were overlaid with 4–5 mL suspension medium and incubated 5 minutes at room temperature. The solution was then serially diluted, plated, and counted as above. Each batch was tested in duplicate.
Electron Microscopy For transmission electron microscopy, 10 :L of hightiter KS4-M lysate (filter sterilized with a 0.22 :m filter) was incubated for 5 minutes at room temperature on a carbon-coated copper grid. Residual lysate was removed and the grid was stained for 2 minutes at room temperature with 2% phosphotungstate. Micrographs were taken at 140,000-fold magnification using a Philips/FEI (Morgagni) transmission electron microscope with charge-coupled device camera (Department of Biological Sciences, Advanced Microscopy Facility, University of Alberta). For scanning electron microscopy, particle images were taken using a Hitachi SEM S-2500 (Hitachi Ltd., Tokyo, Japan). Samples were prepared on aluminum pin stubs using double-sided carbon tape and sputter coated with gold.
RESULTS AND DISCUSSION Powder Properties and Manufacturability Table 1 shows the composition of the four formulations that were chosen for more extensive analysis. All formulations mainly consisted of trehalose (T) with 19% of leucine (L) added. Two formulations contained 2% of a surfactant, tyloxapol (X), or Pluronic (P). The last formulation contained casein sodium salt (C) instead. Several other excipient ratios, for example, trehalose and leucine in a 1:1 ratio, were tested for titer loss on spray drying but were abandoned because they were inferior to the ones listed in Table 1 with respect to titer. All formulations were spray-dried with a feed concentration, cF , that was selected to produce a mass median aerodynamic diameter, da , of the primary DOI 10.1002/jps
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Table 1.
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Formulation Code Mass fraction, trehalose Mass fraction, leucine Mass fraction, surfactant Mass fraction, casein sodium salt Feed concentration in mg/mL Manufacturing yield in % Emitted mass in % of capsule mass Mouth–throat fraction in % of emitted mass
LT
LTX
LTP
LTC
0.77 0.19 0 0 27 62.5 ± 5.3 (n = 6) NA NA
0.76 0.19 0.02 0 27.5 82.6 ± 5.6 (n = 6) 82.9 ± 4.7 (n = 2) 28.7± 2.8 (n = 2)
0.76 0.19 0.02 0 27.5 78.1 ± 4.1 (n = 6) 91.7 ± 9.5 (n = 6) 29.3 ± 2.8 (n = 6)
0.76 0.19 0 0.02 27.5 59.2 ± 3.8 (n = 18) 82.7 ± 4.5 (n = 8) 21.8 ± 3.1 (n = 8)
L, T, X, P, and C denote leucine, trehalose, tyloxapol, pluronic, and casein sodium salt, respectively. Error margins refer to a single standard deviation. n denotes the number of replicates.
particles of 1.4–1.9 :m according to the following equation25 : da =
6
ρP ρ ref
3
cF d0 ρ ref
The particle density, ρP , was estimated to be in the range of 0.1–0.5 kg/L and the reference density, ρref , is 1 kg/ L. d0 denotes the initial diameter of the atomized droplets. Figure 3 shows representative electron micrographs of the four formulations described in Table 1. The formulations were used for the spray drying of different phages. The species of phage incorporated in the formulation did not have a discernible influence on the morphology of the resulting particles and for this reason only four electron micrographs for the different formulations are shown. All powders had a well-dispersed appearance. No fused particles were observed. All formulations showed evidence of internal voids in the particles, in particular in the larger particles. The particles had a spherical shape and the leucine–trehalose (LT) and leucine–trehalose–casein (LTC) formulations had clearly increased rugosity, which is known to improve dispersibility.52 The morphology of the particles for all four formulations appears suitable for respiratory delivery applications. The manufacturing yield of the surfactantcontaining formulations was significantly higher than that of the other two formulations (Table 1). The type of phage in the formulation did not have a statis-
tically significant influence on yield so the data for the different phages were pooled. However, all four formulations had an acceptable to high yield, given the small batch sizes of 200–500 mg. Manufacturing yields are expected to increase for the production of larger batches.
Aerosol Performance R Table 1 shows the emitted dose from the Aerolizer DPI. The emitted dose was tested for formulations that contained surfactant or casein. No powder residue was visible in the capsules after administration. The powder mass left in the device was derived by subtracting the assayed mouth–throat mass and total lung mass from the capsule mass. In all 16 tests, the emitted dose was higher than 76%, reaching 100% in several cases. The 60 L/min flow rate for the emitted dose tests was chosen conservatively. With a comfortable inspiratory pressure drop of 4 kPa across the R , a flow rate of approximately 90 L/min will Aerolizer be established.53 Most patients exceed the test flow rate by a large margin,54 which is expected to lead to nearly complete delivery of the powders described in Table 1. In comparison, most commercially available DPI emits less than two thirds of the packaged powder mass from the device.55 For a subset of tests, the powder deposited in the Alberta Idealized Throat was assayed to determine the mouth–throat fraction, reported in Table 1. In all tests, less than a third of the emitted mass was deposited in the mouth–throat. The difference in the
Figure 3. Scanning electron micrographs of formulations listed in Table 1. From left to right: leucine–trehalose–pluronic (LTP), leucine–trehalose–tyloxapol (LTX), leucine–trehalose (LT), leucine–trehalose–casein (LTC). DOI 10.1002/jps
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Figure 4. Total lung mass in percentage of capsule mass for bacteriophage powders containing KS4-M or KZ/D3 or a cocktail of phages in different formulations. The formulation codes are explained in Table 1. Error bars represent the standard deviation.
mouth–throat fraction of the surfactant-containing formulations relative to the casein-containing formulation was statistically significant. The latter had an exceptionally low extrathoracic deposition. The mouth–throat fraction measured for the spray-dried powders demonstrates a large improvement relative to freeze-dried bacteriophage preparations where the mouth–throat fraction was 58%.22 The total lung mass, that is, the mass of powder delivered past the mouth–throat relative to the powder fill mass in the capsule, is shown in Figure 4. In all cases, the total lung mass exceeded 50%. The most consistent delivery across all phages including the phage cocktails was accomplished with the leucine–trehalose–casein formulation, which was chosen as lead formulation. In comparison, the total lung dose of 12 commercially available DPIs was reported in a range from 5.5% to 40.5% with a mean of 23%.55 The total lung mass of the lead formulation shows a more than twofold improvement relative to recent freeze-dried formulations.22 These comparisons demonstrate that the particle design approach45,56 used in this study has accomplished the target of a highly dispersible powder, suitable for efficient powder delivery to the lung. The aerosol size distribution of the total lung mass for two lead formulations is shown in Figure 5. The distribution was found to be consistent for all tests and showed only minor differences for the two phages tested. The mass median aerodynamic diameter was less than 3 :m in all cases, which is considered suitable for delivery to all areas of the lung.57 The increased deposition on stage 5, with a stage cutoff of 0.51 :m, is likely caused by particle bounce in the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
Figure 5. Aerosol size distribution and mass median aerodynamic diameter (MMAD) of the total lung mass for two selected bacteriophage formulations in a leucine–trehalose–casein (LTC) formulation. The error bars represent the standard deviation of duplicate tests on two independent batches, that is, n = 4. A single capsule was actuated for each test.
impactor, which is difficult to eliminate for highly dispersible powders, even with silicone-coated plates.
Bacteriophage Titer The results in Figure 6 show the loss in titer after spray drying. All Myoviridae phages used in this study were successfully processed and reconstituted with a titer loss of less than 1 log pfu/mL. A titer loss of less than 1 log during the process of development is acceptable in terms of manufacturability of drugs. Phage cocktail KZ/D3 proved to be most robust with a titer loss of less than 0.5 log pfu/mL, irrespective of the formulation in which it was processed. Phage KS4-M was more sensitive to processing stress as seen previously in lyophilization experiments.22 Although the titer of freeze-dried KS4-M preparations
Figure 6. Titer loss of the phages both separately and in a cocktail using different formulations during the process. The error bars represent the standard deviations. DOI 10.1002/jps
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doses in the above-mentioned animal and human trials by orders of magnitude. The titer of the powder ranged from 1.3 × 106 pfu/mg for phage KZ in the phage cocktail to 1.7 × 107 pfu/mg for phage KS4- M. Assuming a total lung mass of greater than 70%, this indicates that a dose of 106 pfu could be administered from an inhaler using a powder mass of 100 :g to 1.1 mg per actuation.
CONCLUSION
Figure 7. In vitro total lung dose and powder titer of LTC (leucine–trehalose–casein) formulations for different phages alone and in a phage cocktail. The diamond represents the median (horizontal line) and one standard deviation (vertices). The whiskers describe the minimum and maximum value and the closed symbol represents the mean. Each test had six replicates.
decreased more than 2 logs upon processing, the low temperature spray-drying process used here led to a loss of about 1 log or less for the formulations without casein sodium salt and less than 0.5 log for the caseincontaining formulation. The processing loss of phage KS14 was similar to that of KS4-M in this formulation. The LTC formulation was also suitable for the processing of a KS4-M/KZ/D3 phage cocktail. The processing loss for the components in the cocktail was not statistically different from the loss measured in the powders of the individual components. The batch sizes produced in this study were too small for formal storage stability tests. A titer retest in triplicate after 3 months of refrigerated storage for KZ/D3 in formulations LT and LTC showed less than 0.15 log titer loss in all cases. Figure 7 shows a statistical evaluation of the in vitro total lung dose of bacteriophages delivered R DPI. Also listed is the to the lung with the Aerolizer titer of the powder that was filled into the capsule of the DPI to allow for total lung dose estimates with other delivery devices. In a study in mice infected with lethal doses of P. aeruginosa, a single intranasal dose of bacteriophages was found to be efficacious at 3 × 106 pfu, curative at 3 × 107 pfu and provided complete prophylactic protection at 3 × 108 pfu.18 A controlled human clinical trial on patients with chronic ear infections caused by antibiotic-resistant P. aeruginosa showed efficacy after topical administration of a single dose of a phage cocktail containing 6 × 105 pfu.58 The total lung doses shown in Figure 7 exceed the efficacious DOI 10.1002/jps
Bacteriophages can be processed into a respirable dry dosage form by low temperature spray drying. Different phage species, even from the same family, have different susceptibility to processing loss. Development of a bacteriophage therapeutic for pulmonary delivery appears promising; acceptable process loss was achieved with excipients that pose a low pulmonary toxicity risk, the manufacturing process had an adequate yield given the small batch sizes, and the resulting powders had outstanding aerosol performance when delivered from a simple DPI. Powder titers were sufficiently high to be compatible with a wide range of inhalation devices. The in vitro lung doses of all tested bacteriophages and a bacteriophage cocktail were much higher than levels shown to be efficacious in animal models.
ACKNOWLEDGMENTS Jonathan J. Dennis. and Warren H. Finlay gratefully acknowledge funding from Canadian Institutes of Health Research and Cystic Fibrosis (CF), Canada. Sadaf Matinkhoo and Reinhard Vehring gratefully acknowledge funding from Natural Sciences and Engineering Research Council of Canada (NSERC). Karlene H. Lynch thanks the Killam Trusts, NSERC, Alberta Heritage Foundation for Medical Research, and CF Canada for financial support.
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