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Production of Inhalation Phage Powders Using Spray Freeze Drying and Spray Drying Techniques for Treatment of Respiratory Infections

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Abstract

Purpose

The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders.

Method

A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1 = 60:20:20 and F2 = 40:40:20). The phage titer loss after the SFD and SD processes and in vitro aerosol performance of the produced powders were assessed.

Results

A significant titer loss (~2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss. In vitro aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1 = 13.1 ± 1.7 × 104 pfu and SD-F2 = 11.0 ± 1.4 × 104 pfu) than from their counterpart SFD formulations (SFD-F1 = 8.3 ± 1.8 × 104 pfu and SFD-F2 = 2.1 ± 0.3 × 104 pfu).

Conclusion

Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2.

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Abbreviations

CF:

Cystic fibrosis

cfu:

Colony formation unit

DSC:

Differential scanning calorimetry

DVS:

Dynamic vapor sorption

FPF:

Fine particle fraction

HPLC:

High performance liquid chromatography

HPMC:

Hydroxypropyl methylcellulose

MDR:

Multidrug-resistant

MSLI:

Multi-stage liquid impinger

NB:

Nutrient broth

pfu:

Plaque formation unit

RH:

Relative humidity

SD:

Spray drying

SEM:

Scanning electron microscope

SFD:

Spray freeze drying

SMB:

Salt-magnesium buffer

Tg :

Glass transition temperature

TGA:

Thermogravimetric analysis

XRD:

X-ray diffraction

References

  1. Williams BG, Gouws E, Boschi-Pinto C, Bryce J, Dye C. Estimates of world-wide distribution of child deaths from acute respiratory infections. Lancet Infect Dis. 2002;2(1):25–32.

    Article  PubMed  Google Scholar 

  2. In: Foundation TAL, editor. The Australian Lung Foundation’s case statement: respiratory infectious disease burden in Australia. 2007.

  3. Govan JRW, Deretic V. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev. 1996;60(3):539–74.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Boucher HW, Talbot GH, Bradley JS, Edwards Jr JE, Gilbert D, Rice LB, et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis. 2009;48(1):1–12.

    Article  PubMed  Google Scholar 

  5. Li J, Nation RL, Turnidge JD, Milne RW, Coulthard K, Rayner CR, et al. Colistin: the re-emerging antibiotic for multidrug-resistant Gram-negative bacterial infections. Lancet Infect Dis. 2006;6(9):589–601.

    Article  CAS  PubMed  Google Scholar 

  6. Chmiel JF, Aksamit TR, Chotirmall SH, Dasenbrook EC, Elborn JS, LiPuma JJ, et al. Antibiotic management of lung infections in cystic fibrosis. I. The microbiome, methicillin-resistant Staphylococcus aureus, gram-negative bacteria, and multiple infections. Ann Am Thorac Soc. 2014;11(7):1120–9.

    Article  CAS  PubMed  Google Scholar 

  7. Hoiby N. Recent advances in the treatment of Pseudomonas aeruginosa infections in cystic fibrosis. Bmc Med. 2011;9:32.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Nordmann P, Naas T, Fortineau N, Poirel L. Superbugs in the coming new decade; multidrug resistance and prospects for treatment of Staphylococcus aureus, Enterococcus spp. and Pseudomonas aeruginosa in 2010. Curr Opin Microbiol. 2007;10(5):436–40.

    Article  CAS  PubMed  Google Scholar 

  9. Hoe S, Semler DD, Goudie AD, Lynch KH, Matinkhoo S, Finlay WH, et al. Respirable bacteriophages for the treatment of bacterial lung infections. J Aerosol Med Pulm Drug Deliv. 2013;26(6):317–35.

    Article  CAS  PubMed  Google Scholar 

  10. Sulakvelidze A, Alavidze Z, Morris JG. Bacteriophage therapy. Antimicrob Agents Chemother. 2001;45(3):649–59.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kutter E, De Vos D, Gvasalia G, Alavidze Z, Gogokhia L, Kuhl S, et al. Phage therapy in clinical practice: treatment of human infections. Curr Pharm Biotechnol. 2010;11(1):69–86.

    Article  CAS  PubMed  Google Scholar 

  12. Kutateladze M, Adamia R. Phage therapy experience at the Eliava Institute. Med Mal Infect. 2008;38(8):426–30.

    Article  CAS  PubMed  Google Scholar 

  13. Debarbieux L, Leduc D, Maura D, Morello E, Criscuolo A, Grossi O, et al. Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections. J Infect Dis. 2010;201(7):1096–104.

    Article  CAS  PubMed  Google Scholar 

  14. Essoh C, Blouin Y, Loukou G, Cablanmian A, Lathro S, Kutter E, et al. The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages. Plos One. 2013;8(4).

  15. Larche J, Pouillot F, Essoh C, Libisch B, Straut M, Lee JC, et al. Rapid identification of international multidrug-resistant Pseudomonas aeruginosa clones by multiple-locus variable number of tandem repeats analysis and investigation of their susceptibility to lytic bacteriophages. Antimicrob Agents Chemother. 2012;56(12):6175–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Semler DD, Goudie AD, Finlay WH, Dennis JJ. Aerosol phage therapy efficacy in Burkholderia cepacia Complex respiratory infections. Antimicrob Agents Chemother. 2014;58(7):4005–13.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Morello E, Saussereau E, Maura D, Huerre M, Touqui L, Debarbieux L. Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards treatment and prevention. Plos One. 2011;6(2), e16963.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Alemayehu D, Casey PG, McAuliffe O, Guinane CM, Martin JG, Shanahan F, et al. Bacteriophages phi MR299-2 and phi NH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells. Mbio. 2012;3(2), e00029.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wright A, Hawkins CH, Anggard EE, Harper DR. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a preliminary report of efficacy, in Clin Otolaryngol. 2009;349–357.

  20. Khawaldeh A, Morales S, Dillon B, Alavidze Z, Ginn AN, Thomas L, et al. Bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection. J Med Microbiol. 2011;60(11):1697–700.

    Article  CAS  PubMed  Google Scholar 

  21. Kutter E, Borysowski J, Miedzybrodzki R, Gorski A, Weber-Dabrowska B, Kutateladze M, et al. Clinical phage therapy. In: Borysowski J, Miedzybrodzki R, Gorski A, editors. Therapy: current research and applications. Caister Academic Press; 2014. p. 257–88.

  22. Keen EC, Adhya SL. Phage therapy: current research and applications. Clin Infect Dis Off Publ Infect Dis Soc Am. 2015;61(1):141–2.

    Article  Google Scholar 

  23. Kutateladze M, Adamia R. Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends Biotechnol. 2010;28(12):591–5.

    Article  CAS  PubMed  Google Scholar 

  24. Semler DD, Lyunch KH, Dennis JJ. The promise of bacteriophage therapy for Burkholderia cepacia complex respiratory infections. Frontiers in cellular and infection microbiology, 2012. 1. UNSP 27.

  25. Ackermann HW. 5500 phages examined in the electron microscope. Arch Virol. 2007;152(2):227–43.

    Article  CAS  PubMed  Google Scholar 

  26. Carmody LA, Gill JJ, Summer EJ, Sajjan US, Gonzalez CF, Young RF, et al. Efficacy of bacteriophage therapy in a model of Burkholderia cenocepacia pulmonary infection. J Infect Dis. 2010;201(2):264–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Golshahi L, Seed KD, Dennis JJ, Finlay WH. Toward modern inhalational bacteriophage therapy: nebulization of bacteriophages of Burkholderia cepacia Complex. J Aerosol Med Pulm Drug Deliv. 2008;21(4):351–9.

    Article  CAS  PubMed  Google Scholar 

  28. Sahota JS, Smith CM, Radhakrishnan P, Winstanley C, Goderdzishvili M, Chanishvili N, et al. Bacteriophage delivery by nebulization and efficacy against phenotypically diverse Pseudomonas aeruginosa from cystic fibrosis patients. J Aerosol Med Pulm Drug Deliv. 2015;28(5):353–60.

    Article  CAS  PubMed  Google Scholar 

  29. Golshahi L, Lynch KH, Dennis JJ, Finlay WH. In vitro lung delivery of bacteriophages KS4-M and Theta KZ using dry powder inhalers for treatment of Burkholderia cepacia complex and Pseudomonas aeruginosa infections in cystic fibrosis. J Appl Microbiol. 2011;110(1):106–17.

    Article  CAS  PubMed  Google Scholar 

  30. Matinkhoo S, Lynch KH, Dennis JJ, Finlay WH, Vehring R. Spray-dried respirable powders containing bacteriophages for the treatment of pulmonary infections. J Pharm Sci. 2011;100(12):5197–205.

    Article  CAS  PubMed  Google Scholar 

  31. Vandenheuvel D, Singh A, Vandersteegen K, Klumpp J, Lavigne R, Van den Mooter G. Feasibility of spray drying bacteriophages into respirable powders to combat pulmonary bacterial infections. Eur J Pharm Biopharm. 2013;84(3):578–82.

    Article  CAS  PubMed  Google Scholar 

  32. Vandenheuvel D, Meeus J, Lavigne R, Van den Mooter G. Instability of bacteriophages in spray-dried trehalose powders is caused by crystallization of the matrix. Int J Pharm. 2014;472(1–2):202–5.

    Article  CAS  PubMed  Google Scholar 

  33. Merabishvili M, Vervaet C, Pirnay J-P, De Vos D, Verbeken G, Mast J, et al. Stability of Staphylococcus aureus phage ISP after freeze-drying (Lyophilization). Plos One. 2013;8(7).

  34. Ackermann HW, Tremblay D, Moineau S. Long-term bacteriophage preservation. World Fed Cult Collect Newsl. 2004;38:35–40.

    Google Scholar 

  35. Jepson CD, March JB. Bacteriophage lambda is a highly stable DNA vaccine delivery vehicle. Vaccine. 2004;22(19):2413–9.

    Article  CAS  PubMed  Google Scholar 

  36. D’Addio SM, Chan JGY, Kwok PCL, Benson BR, Prud’homme RK, Chan H-K. Aerosol delivery of nanoparticles in uniform mannitol carriers formulated by ultrasonic spray freeze drying. Pharm Res. 2013;30(11):2891–901.

    Article  PubMed  Google Scholar 

  37. Ceyssens P-J, Brabban A, Rogge L, Lewis MS, Pickard D, Goulding D, et al. Molecular and physiological analysis of three Pseudomonas aeruginosa phages belonging to the “N4-like viruses”. Virology. 2010;405(1):26–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Carlson K. Appendix. Working with bacteriophages: common techniques and methodological approaches. In: Kutter E, and Sulakvelidze A, editors. Bacteriophages: biology and applications. Boca Raton: CRC Press; 2005. p. 437–94.

  39. Chopin MC. Resistance of 17 mesophilic lactic streptococcus bacteriophages to pasteurization and spray-drying. J Dairy Res. 1980;47(1):131–9.

    Article  CAS  PubMed  Google Scholar 

  40. Dixit N, Maloney KM, Kalonia DS. Protein-silicone oil interactions: comparative effect of nonionic surfactants on the interfacial behavior of a fusion protein. Pharm Res. 2013;30(7):1848–59.

    Article  CAS  PubMed  Google Scholar 

  41. Wang W. Lyophilization and development of solid protein pharmaceuticals. Int J Pharm. 2000;203(1–2):1–60.

    Article  CAS  PubMed  Google Scholar 

  42. Leuenberger H. Process of drying a particulate material and apparatus for implementing the process. 1987. US patent 4,608,764.

  43. Wang ZL, Finlay WH, Peppler MS, Sweeney LG. Powder formation by atmospheric spray-freeze-drying. Powder Technol. 2006;170(1):45–52.

    Article  CAS  Google Scholar 

  44. Sonner C, Maa YF, Lee G. Spray-freeze-drying for protein powder preparation: particle characterization and a case study with trypsinogen stability. J Pharm Sci. 2002;91(10):2122–39.

    Article  CAS  PubMed  Google Scholar 

  45. Feng AL, Boraey MA, Gwin MA, Finlay PR, Kuehl PJ, Vehring R. Mechanistic models facilitate efficient development of leucine containing microparticles for pulmonary drug delivery. Int J Pharm. 2011;409(1–2):156–63.

    Article  CAS  PubMed  Google Scholar 

  46. Surana R, Pyne A, Suryanarayanan R. Effect of preparation method on physical properties of amorphous trehalose. Pharm Res. 2004;21(7):1167–76.

    Article  CAS  PubMed  Google Scholar 

  47. Burnett DJ, Thielmann F, Booth J. Determining the critical relative humidity for moisture-induced phase transitions. Int J Pharm. 2004;287(1–2):123–33.

    Article  CAS  PubMed  Google Scholar 

  48. Kim AI, Akers MJ, Nail SL. The physical state of mannitol after freeze-drying: effects of mannitol concentration, freezing rate, and a noncrystallizing cosolute. J Pharm Sci. 1998;87(8):931–5.

    Article  CAS  PubMed  Google Scholar 

  49. Chen T, Bhowmick S, Sputtek A, Fowler A, Toner M. The glass transition temperature of mixtures of trehalose and hydroxyethyl starch. Cryobiology. 2002;44(3):301–6.

    Article  CAS  PubMed  Google Scholar 

  50. Burger A, Henck JO, Hetz S, Rollinger JM, Weissnicht AA, Stottner H. Energy/temperature diagram and compression behavior of the polymorphs of D-mannitol. J Pharm Sci. 2000;89(4):457–68.

    Article  CAS  PubMed  Google Scholar 

  51. Lahde A, Raula J, Malm J, Kauppinen EI, Karppinen M. Sublimation and vapour pressure estimation of L-leucine using thermogravimetric analysis. Thermochim Acta. 2009;482(1–2):17–20.

    Article  Google Scholar 

  52. Vehring R. Pharmaceutical particle engineering via spray drying. Pharm Res. 2008;25(5):999–1022.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Maury M, Murphy K, Kumar S, Mauerer A, Lee G. Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G. Eur J Pharm Biopharm. 2005;59(2):251–61.

    Article  CAS  PubMed  Google Scholar 

  54. Maa YF, Costantino HR, Nguyen PA, Hsu CC. The effect of operating and formulation variables on the morphology of spray-dried protein particles. Pharm Dev Technol. 1997;2(3):213–23.

    Article  CAS  PubMed  Google Scholar 

  55. Soothill JS. Treatment of experimental infections of mice with bacteriophages. J Med Microbiol. 1992;37(4):258–61.

    Article  CAS  PubMed  Google Scholar 

  56. Payne RJH, Jansen VAA. Pharmacokinetic principles of bacteriophage therapy. Clin Pharmacokinet. 2003;42(4):315–25.

    Article  CAS  PubMed  Google Scholar 

  57. Abedon ST. Phage therapy pharmacology: calculating phage dosing. Adv Appl Microbiol. 2011;77:1–40.

    Article  PubMed  Google Scholar 

  58. Curtright AJ, Abedon ST. Phage therapy: emergent property pharmacology. J Bioanal Biomed. 2011; S6, article 002.

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ACKNOWLEDGMENTS AND DISCLOSURES

This work was financially supported by the Australian Research Council (Discovery Project DP150103953). Authors are grateful to Tony Smithyman of Special Phage Services for his valuable discussion and advice. Sharon Leung is a research fellow supported by the University of Sydney. Thaigarajan Parumasivam is a recipient of the Malaysian Government Scholarship. H-KC is funded by the National Institutes of Health (NIH Project no.1R21AI121627-01).

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Authors and Affiliations

  1. Faculty of Pharmacy, University of Sydney, Sydney, NSW, 2006, Australia

    Sharon S. Y. Leung, Thaigarajan Parumasivam, Fiona G. Gao & Hak-Kim Chan

  2. Department of Mechanical Engineering, University of Alberta, Edmonton, Alberta, T6G 2G8, Canada

    Nicholas B. Carrigy, Reinhard Vehring & Warren H. Finlay

  3. AmpliPhi Biosciences AU, 7/27 Dale Street, Brookvale, Sydney, NSW, 2100, Australia

    Sandra Morales

  4. Tuberculosis Research Program, Centenary Institute and Sydney Medical School, University of Sydney, Sydney, NSW, 2006, Australia

    Warwick J. Britton

  5. The Evergreen State College, Olympia, Washington, 98502, USA

    Elizabeth Kutter

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  1. Sharon S. Y. Leung
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  2. Thaigarajan Parumasivam
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Correspondence to Hak-Kim Chan.

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Leung, S.S.Y., Parumasivam, T., Gao, F.G. et al. Production of Inhalation Phage Powders Using Spray Freeze Drying and Spray Drying Techniques for Treatment of Respiratory Infections. Pharm Res 33, 1486–1496 (2016). https://doi.org/10.1007/s11095-016-1892-6

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