The effect of excipients on the stability and aerosol performance of salmon calcitonin dry powder inhalers prepared via the spray freeze drying process

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Spray freeze drying was developed to produce dry powders suitable for applications such as inhalation delivery. In the current study, the spray freeze drying technique was employed to produce inhalable salmon calcitonin microparticles. Effects of the carrier type, concentration of hydroxyl propyl-β-cyclodextrin and the presence of Tween 80 on the chemical and structural stability, as well as on the aerosol performance of the particles were investigated. The results indicated that hydroxyl propyl-β-cyclodextrin had the most important effect on the chemical stability of the powder and strongly increased its stability by increasing its concentration in the formulation. Chemically stable formulations (over 90 % recovery) were selected for further examinations. Fluorescence spectroscopy and circular dichroism suggested that the formulations were structurally stable. Aerosol performance showed that the Tween-free powders produced higher fine particle fraction values than the formulations containing Tween (53.7 vs. 41.92 % for trehalose content and 52.85 vs. 43.06 % for maltose content).

1. J. P. Meyer, J. T. Pelton, J. Hoflack and V. Saudek, Solution structure of salmon calcitonin, Biopolymers 31 (1991) 233–241; DOI: 10.1002/bip.360310210.

2. C. L. Stevenson, Advances in peptide pharmaceuticals, Cur. Pharm. Biotechnol. 10 (2009) 122–137; DOI: 10.2174/138920109787048634.

3. L. Jorgensen, E. H. Moeller, M. Van de Weert, H. M. Nielsen and S. Frokjaer, Preparing and evaluating delivery systems for proteins, Eur. J. Pharm. Sci. 29 (2006) 174–182; DOI: 10.1016/j.eps.2006.05.008.

4. Z. Antosova, M. Mackova, V. Kral and T. Macek, Therapeutic application of peptides and proteins: parenteral forever? Trends Biotech. 27 (2009) 628–635; DOI: 10.1016/j.tibtech.2009.07.009.

5. A. Makhlof, M. Werle, Y, Tozuka and H. Takeuchi, Nanoparticles of glycol chitosan and its thiolated derivative significantly improved the pulmonary delivery of calcitonin, Int. J. Pharm. 397 (2010) 92–95; DOI: 10.1016/j.ijpharm.2010.07.00.

6. H. Yamamoto, Y. Kuno, S. Sugimoto, H. Takeuchi and Y. Kawashima, Surface-modified PLGA nanosphere with chitosan improved pulmonary delivery of calcitonin by mucoadhesion and opening of the intercellular tight junctions, J. Control. Release 102 (2005) 373–381; DOI: 10.1016/j.conrel.2004010.010.

7. J. Varshosaz, M. Minaiyan and M. Forghanian, Prolonged hypocalcemic effect by pulmonary delivery of calcitonin loaded poly (methyl vinyl ether maleic acid) bioadhesive nanoparticles, Biomed. Res. Int. 2014, 1–13; DOI: 10.1155/2014/932615.

8. S. A. Shoyele and S. Cawthorne, Particle engineering techniques for inhaled biopharmaceuticals, Adv. Drug. Deliv. Rev. 58 (2006) 1009–1029; DOI: 10.1016/j.addr.2006.07.010.

9. Y. F. Maa, P. A. Nguyen, T. Sweeney, S. J. Shire and C. C. Hsu, Protein inhalation powders: spray drying vs. spray freeze drying, Pharm. Res. 16 (1999) 249–254; DOI: 10.1023/A:1018828425184.

10. D. A. Edwards, J. Hanes, G. Caponetti, J. Hrkach, A. Ben-Jebria, M. L. Eskew, J. Mintzes, D. Deaver, N. Lotan and R. Langer, Large porous particles for pulmonary drug delivery, Science 276 (1997) 1868–1872; DOI: 10.1126/science.276.5320.1868.

11. Z. Yu, K. P. Johnston and R. O. Williams III, Spray freezing into liquid versus spray-freeze drying: Influence of atomization on protein aggregation and biological activity, Eur. J. Pharm. Sci. 27 (2006) 9–18; DOI: 10.1016/j.ejps.2005.08.010.

12. F. Depreter, G. Pilcer and K. Amighi, Inhaled proteins: Challenges and perspectives, Int. J. Pharm. 447 (2013) 251–280; DOI: 10.1016/j.ijpharm.2013.02.031.

13. N. R. Maddux, S. B. Joshi, D. B. Volkin, J. P. Ralston and C. R. Middaugh, Multidimensional methods for the formulation of biopharmaceuticals and vaccines, J. Pharm. Sci. 100 (2011) 4171–4197; DOI: 10.1002/jps.22618.

14. T. Serno, R. Geidobler and G. Winter, Protein stabilization by cyclodextrins in the liquid and dried state, Adv. Drug Deliv. Rev. 63 (2011) 1086–1106; DOI: 1016/j/addr.2011.08.003.

15. J. Iwai, N. Ogawa, H. Nagase, T. Endo, T. Loftsson and H. Ueda, Effects of various cyclodextrins on the stability of freeze dried lactate dehydrogenase, J. Pharm. Sci. 96 (2007) 3140–3143; DOI: 10.1002/jps.20847.

16. K. Izutsu, S. Yoshioka and T. Terao, Stabilization of [beta]-galactosidase by amphiphilic additives during freeze-drying, Int. J. Pharm. 90 (1993) 187–194; DOI: 10.1016/0378-5173(93)90190-Q.

17. H. R. Costantino, L. Firouzabadian, C. Wu, K. G. Carrasquillo, K. Griebenow, S. E. Zale and M. A. Tracy, Protein spray freeze drying. 2. Effect of formulation variables on particle size and stability, J. Pharm. Sci. 91 (2002) 388–395; DOI: 10.1002/jps.10059.

18. W. C. Johnson, Protein secondary structure and circular dichroism: a practical guide, Proteins 7 (1990) 205–214; DOI: 10.1002/prot.340070302.

19. P. Manavalan and W. C. Johnson, Sensitivity of circular dichroism to protein tertiary structure class, Nature 305 (1983) 831–832; DOI: 10.1038/305831a0.

20. H. R. Costantino, H. Culley, L. Chen, D. Morris, M. Houston, S. Roth, M. J. Phoenix, C. Foerder, J. S. Philo, T. Arakawa, L. Eideuschink, N. H. Andersen, G. Brandt and S. C. Quav, Development of calcitonin salmon nasal spray: similarity of peptide formulated in chlorobutanol compared to benzalkonium chloride as preservative, J. Pharm. Sci. 98 (2009) 3691–3706; DOI: 10.1002/jps.21690.

21. M. A. Capelle, R. Gurny and T. Arvinte, High throughput screening of protein formulation stability: practical considerations, Eur. J. Pharm. Biopharm. 65 (2007) 131–148; DOI: 10.1016/j.ejpb.2006.09.009.

22. J. T. Vivian and P. R. Callis, Mechanisms of tryptophan fluorescence shifts in proteins, Biophys. J. 80 (2001) 2093–2109; DOI: 10.1016150006-349(01)76183-8.

23. D. J. van Drooge, W. LJ. Hinrichs, B. H. J. Dickhoff, M. N. A. Elli, M. R. Visser, G. S. Zijlstra, W. Henddeik and H. W. Frijlink, Spray freeze drying to produce a stable Delta-9-tetrahydrocannabinol containing inulin-based solid dispersion powder suitable for inhalation, Eur. J. Pharm. Sci. 26 (2005) 231–240; DOI: 10.1016/j.ejps.2005.06.007.

24. S. A. Shoyele, N. Sivadas and S. A. Cryan, The effects of excipients and particle engineering on the biophysical stability and aerosol performance of parathyroid hormone (1-34) prepared as a dry powder for inhalation, AAPS Pharmscitech. 12 (2011) 304–311; DOI: 10.1208/s12249-011-9585-2.

25. D. A. Edwards, G. Caponetti, J. S. Hrkach, N. Lotan, J. Hanes and A. A. Ben-Jebria, Aerodynamically light particles for pulmonary drug delivery, U.S. Pat. 5874064. 23 Feb. 1999.

26. Y. Kato, T. Matsuda, N. Kato and R. Nakamura, Maillard reaction of disaccharides with protein: suppressive effect of nonreducing and pyranoside groups on browning and protein polymerization, J. Agri. Food Chem. 37 (1989) 1077–1081; DOI: 10.1021/jf00088a057.

27. W. Wang, Y. J. Wang and D. Q. Wang, Dual effects of Tween 80 on protein stability, Int. J. Pharm. 347 (2008) 31–38; DOI: 10161j.ijpharm.2007.06.042.

28. N. B. Bam, J. L. Cleland and T. W. Randolph, Molten globule intermediate of recombinant human growth hormone: stabilization with surfactants, Biotechnol. Prog. 12 (1996) 801–809; DOI: 10.1021/bp960068b.

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