FORMULATION AND IN VITRO - IN VIVO EVALUATION OF TETANUS TOXOID-MANNITOL DRY POWDER INHALATION FOR PULMONARY DELIVERY
Yıl 2023,
, 611 - 624, 20.05.2023
Mary Manoranjani Addanki
,
Prakash Katakam
,
Shanta Kumari Adıkı
,
Jessi Dason Vardhanapu
,
Nagabhushana Vinay Kumar Ala
,
Surekha Itamreddy
,
Sudhakar Muvvala
,
Parthiban Chınnachamy
Öz
Objective: As the conventional vaccines were accompanied by the limitations of pain, cold chain storage and sterility issues, a mucosal vaccine which is administered through pulmonary route was fabricated. A dry powder inhalation of tetanus toxoid (TT) and mannitol was prepared and evaluated for stability and immunogenicity in comparison to the conventional TT vaccine.
Material and Method: TT and mannitol dry powder inhalation was prepared and evaluated for particle size analysis, scanning electron microscopy, FTIR, flow properties, TT content estimation, flocculation, and in vitro drug vaccine release studies. Immunological studies of the formulation were performed on BALB/c mice.
Result and Discussion: The powder blend of tetanus toxoid and mannitol remained stable under the process conditions and after storage. The result was confirmed through a flocculation test. The FTIR analysis indictated no interactions between the components. The homogenization process yielded a powder with a geometrical particle size diameter of 1312 ± 1310.9 nm which was found suitable for pulmonary administration. The zeta potential and polydispersity index {PDI} were found to be -22.6 ± 0.16 mV and 0.499 ± 0.015, respectively. The diffusion studies indicated immediate release of the TT with 82.4 ± 6.7% of drug released within 2 h following the diffusion mechanism and zero order kinetics and it was found that mannitol didn’t retard the release of tetanus toxoid. Additionally, the flow properties of the dry powder inhalation were reported to have good flow properties. More importantly, the immunological studies inferred the induction of high systemic and mucosal immunity over conventional vaccines.
Teşekkür
We are grateful to Dano Vaccines and Biologicals Pvt. Ltd., Hyderabad, India, for providing tetanus toxoid gift samples and availing their facilities, Rodenta Bioserve, Hyderabad, India for conducting preclinical studies and Malla Reddy College of Pharmacy, Hyderabad, India, for providing research facilities for this study.
Kaynakça
- 1. Tetanus. (2021). Retrieved June 15, 2021, from Mayo Clinic website: https://www.mayoclinic.org/diseasesconditions/tetanus/symptoms-causes/syc-20351625 Accessed date 03.07.2021.
- 2. Office of Infectious Disease, & HIV/AIDS Policy (OIDP). (2021). Vaccines by disease. Retrieved April 26, 2022, from nhs.gov website: https://www.vaccines.gov/diseases/tetanus Accessed date 03.07.2021.
- 3. Vaccine Safety Basics (2022). Retrieved July 3, 2022, from Vaccine-safety-training.org website: https://openwho.org/courses/vaccine-safety-basics Accessed date 03.07.2021.
- 4. Woodrow, K.A., Bennett, K.M., Lo, D.D. (2012). Mucosal vaccine design and delivery. Annual Review of Biomedical Engineering, 14(1), 17-46. [CrossRef]
- 5. Ogra, P.L., Faden, H., Welliver, R.C. (2001). Vaccination strategies for mucosal immune responses. Clinical Microbiology Reviews, 14(2), 430-445. [CrossRef]
- 6. Otczyk, D.C., Cripps, A.W. (2010). Mucosal immunization: A realistic alternative. Human Vaccines, 6(12), 978-1006. [CrossRef]
- 7. Cesta, M.F. (2006). Normal structure, function, and histology of mucosa-associated lymphoid tissue. Toxicologic Pathology, 34(5), 599-608. [CrossRef]
- 8. Medina, E., Guzmán, C.A. (2000). Modulation of immune responses following antigen administration by mucosal route. FEMS Immunology and Medical Microbiology, 27(4), 305-311. [CrossRef]
- 9. Neutra, M.R. (2006). Kozlowski. Mucosal Vaccines: The Promise and the Challenge. Nature Reviews, Immunology, 150-158. [CrossRef]
- 10. Arora, D.K., Goyal, A.R., Paliwal, S., Khurana, B.P., Vyas, S. (2010). Oral mucosal immunization: Recent advancement and future prospects. Current Immunology Reviews, 6(3), 234-259. [CrossRef]
- 11. Lu, D., Hickey, A.J. (2007). Pulmonary vaccine delivery. Expert Review of Vaccines, 6(2), 213-226. [CrossRef]
- 12. Buffa, V., Klein, K., Fischetti, L., Shattock, R.J. (2012). Evaluation of TLR agonists as potential mucosal adjuvants for HIV gp140 and tetanus toxoid in mice. PLoS One.7 (12), e50529. [CrossRef]
- 13. Tafaghodi, M., Rastegar, S. (2010). Preparation and in vivo study of dry powder microspheres for nasal immunization. Journal of Drug Targeting, 18(3), 235-242. [CrossRef]
- 14. Lamm, M.E. (1997). Interactions of antigens and antibodies at mucosal surfaces. Annual Reviews in Microbiology, 51. [CrossRef]
- 15. Srivastava, A., Gowda, D.V., Madhunapantula, S.V., Shinde, C.G., Iyer, M. (2015). Mucosal vaccines: a paradigm shift in the development of mucosal adjuvants and delivery vehicles. APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica, 123(4), 275-288. [CrossRef]
- 16. Koussoroplis, S.J., Vanbever, R. (2015). Peptides and Proteins: Pulmonary Absorption. Encyclopedia of Pharmaceutical Science and Technology, Fourth Edition, Volume IV. [CrossRef]
- 17. Sato, S., Kiyono, H. (2012). The mucosal immune system of the respiratory tract. Current Opinion in Virology, 2(3), 225-232. [CrossRef]
- 18. Kanig, J.L. (1963). Pharmaceutical aerosols. Journal of Pharmaceutical Sciences, 52(6), 513-535. [CrossRef]
- 19. Bosquillon, C., Lombry, C., Preat, V., Vanbever, R. (2001). Comparison of particle sizingtechniques in the case of inhalation dry powders. Journal of Pharmaceutical Sciences, 90(12), 2032-2041. [CrossRef]
- 20. Chaurasiya, B., Zhao, Y.Y. (2020). Dry powder for pulmonary delivery: A comprehensive review. Pharmaceutics, 13(1), 31. [CrossRef]
- 21. Bilati, U., Allémann, E., Doelker, E. (2003). Sonication parameters for the preparation of biodegradable nanocapsules of controlled size by the double emulsion method. Pharmaceutical Development and Technology, 8(1), 1-9. [CrossRef]
- 22. Ibrahim, M., Verma, R., Garcia-Contreras, L. (2015). Inhalation drug delivery devices: technology update. Medical Devices (Auckland, N.Z.), 8, 131-139. [CrossRef]
- 23. Melani, A.S., Bonavia, M., Cilenti, V., Cinti, C., Lodi, M., Martucci, P., Neri, M. (2011). Inhaler mishandling remains common in real life and is associated with reduced disease control. Respiratory Medicine, 105(6), 930-938. [CrossRef]
- 24. Hodder, R., Price, D. (2009). Patient preferences for inhaler devices in chronic obstructivepulmonary disease: experience with Respimat Soft Mist inhaler. International Journal of Chronic Obstructive Pulmonary Disease, 4, 381-390. [CrossRef]
- 25. Shetty, N., Cipolla, D., Park, H., Zhou, Q. T. (2020). Physical stability of dry powder inhaler formulations. Expert Opinion on Drug Delivery, 17(1), 77-96. [CrossRef]
- 26. Calvör, A., Müller, B.W. (1998). Production of microparticles by high-pressurehomogenization. Pharmaceutical Development and Technology, 3(3), 297-305. [CrossRef]
- 27. Kaialy, W., Nokhodchi, A. (2013). Treating mannitol in a saturated solution of mannitol: a novel approach to modify mannitol crystals for improved drug delivery to the lungs. International Journal of Pharmaceutics, 448(1), 58-70. [CrossRef]
- 28. Kaialy, W., Nokhodchi, A. (2013). Freeze-dried mannitol for superior pulmonary drug delivery via dry powder inhaler. Pharmaceutical Research, 30(2), 458-477. [CrossRef]
- 29. Kaialy, W., Larhrib, H., Ticehurst, M.D., Nokhodchi, A. (2012). Influence of batch cooling crystallization on mannitol physical properties and drug disposition from dry-powder inhalers. Crystal Growth & Design, 12, 3006-3017. [CrossRef]
- 30. Kaialy, W., Martin, G.P., Ticehurst, M.D., Momin, M.N., Nokhodchi, A. (2010). The enhancedaerosol performance of salbutamol from dry powders containing engineered mannitol asexcipient. International Journal of Pharmaceutics, 392(1-2), 178-188. [CrossRef]
- 31. Nokhodchi, A., Larhrib, H. (2013). Overcoming the undesirable properties of dry-powder inhalers with novel engineered mannitol particles. Therapeutic Delivery, 4(8), 879-882. [CrossRef]
- 32. Lazaridou, M., Christodoulou, E., Nerantzaki, M., Kostoglou, M., Lambropoulou, D., Katsarou, A., Pantopoulos, K., Bikiaris, D. (2020). Formulation and in-vitro characterization of chitosan-nanoparticlesloaded with the iron Chelator deferoxaminemesylate (DFO). Pharmaceutics, 12(3), 238. [CrossRef]
- 33. Mahmoud, A.A., El-Feky, G.S., Kamel, R., Awad, G.E.A. (2011). Chitosan/sulfobutylether-β-cyclodextrin nanoparticles as a potential approach for ocular drug delivery. International Journal of Pharmaceutics, 413(1-2), 229-236. [CrossRef]
- 34. Lau (deceased), E. (2001). Preformulation studies. Separation Science and Technology, 3 173-233. [CrossRef]
- 35. Lee, S.H., Zhang, Z., Feng, S.S. (2007). Nanoparticles of poly (lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymers for protein drug delivery. Biomaterials, 28(11), 2041-2050. [CrossRef]
- 36. Arthanari, S., Renukadevi P., Mani, K.R. (2011). Preparation and evaluation of sucrose stabilizedtetanus toxoid encapsulated into chitosan microspheres. Genomic Medicine, Biomarkers and Health Sciences, 3(3-4), 91-97. [CrossRef]
- 37. Lyng, J. (1990). Quantitative estimation of diphtheria and tetanus toxoids. 4. Toxoids as international reference materials defining Lf-units for diphtheria and tetanus toxoids. Biologicals: Journal of the International Association of Biological Standardization, 18(1), 11-17. [CrossRef]
- 38. Fulthorpe, A.J. (1958). Estimation of tetanus toxoid by different methods, including haemagglutination inhibition. Immunology, 1(4), 365-372. [CrossRef]
- 39. International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use ich harmonised tripartite guideline stability testing of new drug substances and products. Ich.org. 2003 [cited 2022 Dec 27]. Available from: https://database.ich.org/sites/default/files/Q1A%28R2%29%20Guideline.pdf
- 40. Chaurasiya, B., Zhou, M., Tu, J., Sun, C. (2018). Design and validation of a simple device for insufflation of dry powders in a mice model. European Journal of Pharmaceutical Sciences, 123, 495-501. [CrossRef]
- 41. Pirouzmand, H., Khameneh, B., Tafaghodi, M. (2017). Immunoadjuvant potential of cross-linked dextran microspheres mixed with chitosan nanospheres encapsulated with tetanustoxoid. Pharmaceutical Biology, 55(1), 212-217. [CrossRef]
- 42. Labiris, N.R., Dolovich, M.B., (2003). Pulmonary drug delivery. Part I: Physiological factors affecting therapeutic effectiveness of aerosolized medications: Physiological factors affecting the effectiveness of inhaled drugs. British Journal of Clinical Pharmacology, 56(6), 588-599. [CrossRef]
- 43. Peng, T., Zhang, X., Huang, Y., Zhao, Z., Liao, Q., Xu, J., Huang, Z., Zhang, J., Wu, CY., Pan., X, Wu, C. (2017). Nanoporous mannitol carrier prepared by non-organic solvent spray drying technique to enhance the aerosolization performance for dry powder inhalation. Scientific Reports, 7(1), 46517. [CrossRef]
- 44. Indian Pharmacopoeia. Vol II. Delhi: Controller of Publications; 1996. p. A.p. 33.
- 45. Manivannan, R., Dhanaraj, S.A., Rao, Y.U.B., Balasubramaniam, A., Gowrishankar, N.L., Jawahar, N., (2008). In vivo evaluation of single dose tetanus toxoid vaccine formulation with chitosan microspheres. Indian Journal of Pharmaceutical Sciences, 70(1), 11-15. [CrossRef]
- 46. Jung, T., Kamm, W., Breitenbach, A., Hungerer, K.D., Hundt, E., Kissel, T. (2001). Tetanus toxoid loaded nanoparticles from sulfobutylatedpoly(vinyl alcohol)-graft-poly(lactide-co-glycolide): Evaluation of antibody response after oral and nasal application in mice. Pharmaceutical Research, 18(3), 352-360. [CrossRef]
PULMONER VERİLİŞE YÖNELİK TETANOZ TOKSOİD-MANNİTOL KURU TOZ İNHALASYON FORMÜLASYONU VE İN VİTRO - İN VİVO DEĞERLENDİRİLMESİ
Yıl 2023,
, 611 - 624, 20.05.2023
Mary Manoranjani Addanki
,
Prakash Katakam
,
Shanta Kumari Adıkı
,
Jessi Dason Vardhanapu
,
Nagabhushana Vinay Kumar Ala
,
Surekha Itamreddy
,
Sudhakar Muvvala
,
Parthiban Chınnachamy
Öz
Amaç: Konvansiyonel aşılara ağrı sınırlamaları, soğuk zincir depolama ve sterilite sorunları eşlik ettiğinden, pulmoner yolla uygulanan mukozal bir aşı üretimiştir. Tetanoz toksoidi (TT) ve mannitolün kuru toz inhalasyonu hazırlanmış ve konvansiyonel tetanoz toksoid aşısına kıyasla stabilite ve immünojenisite açısından değerlendirilmiştir.
Gereç ve Yöntem: TT ve mannitol kuru toz inhalasyonu hazırlandı ve partikül boyutu analizi, FTIR, akış özellikleri, kapsülleme etkinliği, flokülasyon ve in vitro ilaç aşısı salım çalışmaları açısından değerlendirildi. Formülasyonun immünolojik çalışmaları, BALB/c fareleri üzerinde gerçekleştirildi.
Sonuç ve Tartışma: TT ve mannitolün toz karışımı, işlem koşulları altında ve depolama sonrasında stabil kalmıştır. Sonuç, bir flokülasyon testi ile doğrulanmıştır. FTIR analizine göre hiçbir etkileşim tespit edilmemiştir. Homojenizasyon işlemi ile, 1312 ± 1310.9 nm'lik geometrik partikül boyutuna sahip, pulmoner uygulama için uygun bulunan bir toz elde edilmiştir. Zeta potansiyeli ve polidispersite indeksi (PDI) sırasıyla -22.6 ± 0.16 mV ve 0.499 ± 0.015 olarak bulunmuştur. Difüzyon çalışmaları, difüzyon mekanizması ve sıfır derece kinetiği takiben 2 saat içinde açığa çıkan etkin maddenin %82.4 ±% 6.7'si ile TT'nin derhal salındığını göstermiş ve mannitolün tetanoz toksoidinin salınımını geciktirmediği bulunmuştur. Ek olarak, kuru toz inhalasyonunun akış özelliklerinin iyi akış özelliklerine sahip olduğu rapor edilmiştir. Daha da önemlisi, immünolojik çalışmalar, geleneksel aşılara göre yüksek sistemik ve mukozal bağışıklığın indüklendiğini ortaya çıkarmıştır.
Kaynakça
- 1. Tetanus. (2021). Retrieved June 15, 2021, from Mayo Clinic website: https://www.mayoclinic.org/diseasesconditions/tetanus/symptoms-causes/syc-20351625 Accessed date 03.07.2021.
- 2. Office of Infectious Disease, & HIV/AIDS Policy (OIDP). (2021). Vaccines by disease. Retrieved April 26, 2022, from nhs.gov website: https://www.vaccines.gov/diseases/tetanus Accessed date 03.07.2021.
- 3. Vaccine Safety Basics (2022). Retrieved July 3, 2022, from Vaccine-safety-training.org website: https://openwho.org/courses/vaccine-safety-basics Accessed date 03.07.2021.
- 4. Woodrow, K.A., Bennett, K.M., Lo, D.D. (2012). Mucosal vaccine design and delivery. Annual Review of Biomedical Engineering, 14(1), 17-46. [CrossRef]
- 5. Ogra, P.L., Faden, H., Welliver, R.C. (2001). Vaccination strategies for mucosal immune responses. Clinical Microbiology Reviews, 14(2), 430-445. [CrossRef]
- 6. Otczyk, D.C., Cripps, A.W. (2010). Mucosal immunization: A realistic alternative. Human Vaccines, 6(12), 978-1006. [CrossRef]
- 7. Cesta, M.F. (2006). Normal structure, function, and histology of mucosa-associated lymphoid tissue. Toxicologic Pathology, 34(5), 599-608. [CrossRef]
- 8. Medina, E., Guzmán, C.A. (2000). Modulation of immune responses following antigen administration by mucosal route. FEMS Immunology and Medical Microbiology, 27(4), 305-311. [CrossRef]
- 9. Neutra, M.R. (2006). Kozlowski. Mucosal Vaccines: The Promise and the Challenge. Nature Reviews, Immunology, 150-158. [CrossRef]
- 10. Arora, D.K., Goyal, A.R., Paliwal, S., Khurana, B.P., Vyas, S. (2010). Oral mucosal immunization: Recent advancement and future prospects. Current Immunology Reviews, 6(3), 234-259. [CrossRef]
- 11. Lu, D., Hickey, A.J. (2007). Pulmonary vaccine delivery. Expert Review of Vaccines, 6(2), 213-226. [CrossRef]
- 12. Buffa, V., Klein, K., Fischetti, L., Shattock, R.J. (2012). Evaluation of TLR agonists as potential mucosal adjuvants for HIV gp140 and tetanus toxoid in mice. PLoS One.7 (12), e50529. [CrossRef]
- 13. Tafaghodi, M., Rastegar, S. (2010). Preparation and in vivo study of dry powder microspheres for nasal immunization. Journal of Drug Targeting, 18(3), 235-242. [CrossRef]
- 14. Lamm, M.E. (1997). Interactions of antigens and antibodies at mucosal surfaces. Annual Reviews in Microbiology, 51. [CrossRef]
- 15. Srivastava, A., Gowda, D.V., Madhunapantula, S.V., Shinde, C.G., Iyer, M. (2015). Mucosal vaccines: a paradigm shift in the development of mucosal adjuvants and delivery vehicles. APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica, 123(4), 275-288. [CrossRef]
- 16. Koussoroplis, S.J., Vanbever, R. (2015). Peptides and Proteins: Pulmonary Absorption. Encyclopedia of Pharmaceutical Science and Technology, Fourth Edition, Volume IV. [CrossRef]
- 17. Sato, S., Kiyono, H. (2012). The mucosal immune system of the respiratory tract. Current Opinion in Virology, 2(3), 225-232. [CrossRef]
- 18. Kanig, J.L. (1963). Pharmaceutical aerosols. Journal of Pharmaceutical Sciences, 52(6), 513-535. [CrossRef]
- 19. Bosquillon, C., Lombry, C., Preat, V., Vanbever, R. (2001). Comparison of particle sizingtechniques in the case of inhalation dry powders. Journal of Pharmaceutical Sciences, 90(12), 2032-2041. [CrossRef]
- 20. Chaurasiya, B., Zhao, Y.Y. (2020). Dry powder for pulmonary delivery: A comprehensive review. Pharmaceutics, 13(1), 31. [CrossRef]
- 21. Bilati, U., Allémann, E., Doelker, E. (2003). Sonication parameters for the preparation of biodegradable nanocapsules of controlled size by the double emulsion method. Pharmaceutical Development and Technology, 8(1), 1-9. [CrossRef]
- 22. Ibrahim, M., Verma, R., Garcia-Contreras, L. (2015). Inhalation drug delivery devices: technology update. Medical Devices (Auckland, N.Z.), 8, 131-139. [CrossRef]
- 23. Melani, A.S., Bonavia, M., Cilenti, V., Cinti, C., Lodi, M., Martucci, P., Neri, M. (2011). Inhaler mishandling remains common in real life and is associated with reduced disease control. Respiratory Medicine, 105(6), 930-938. [CrossRef]
- 24. Hodder, R., Price, D. (2009). Patient preferences for inhaler devices in chronic obstructivepulmonary disease: experience with Respimat Soft Mist inhaler. International Journal of Chronic Obstructive Pulmonary Disease, 4, 381-390. [CrossRef]
- 25. Shetty, N., Cipolla, D., Park, H., Zhou, Q. T. (2020). Physical stability of dry powder inhaler formulations. Expert Opinion on Drug Delivery, 17(1), 77-96. [CrossRef]
- 26. Calvör, A., Müller, B.W. (1998). Production of microparticles by high-pressurehomogenization. Pharmaceutical Development and Technology, 3(3), 297-305. [CrossRef]
- 27. Kaialy, W., Nokhodchi, A. (2013). Treating mannitol in a saturated solution of mannitol: a novel approach to modify mannitol crystals for improved drug delivery to the lungs. International Journal of Pharmaceutics, 448(1), 58-70. [CrossRef]
- 28. Kaialy, W., Nokhodchi, A. (2013). Freeze-dried mannitol for superior pulmonary drug delivery via dry powder inhaler. Pharmaceutical Research, 30(2), 458-477. [CrossRef]
- 29. Kaialy, W., Larhrib, H., Ticehurst, M.D., Nokhodchi, A. (2012). Influence of batch cooling crystallization on mannitol physical properties and drug disposition from dry-powder inhalers. Crystal Growth & Design, 12, 3006-3017. [CrossRef]
- 30. Kaialy, W., Martin, G.P., Ticehurst, M.D., Momin, M.N., Nokhodchi, A. (2010). The enhancedaerosol performance of salbutamol from dry powders containing engineered mannitol asexcipient. International Journal of Pharmaceutics, 392(1-2), 178-188. [CrossRef]
- 31. Nokhodchi, A., Larhrib, H. (2013). Overcoming the undesirable properties of dry-powder inhalers with novel engineered mannitol particles. Therapeutic Delivery, 4(8), 879-882. [CrossRef]
- 32. Lazaridou, M., Christodoulou, E., Nerantzaki, M., Kostoglou, M., Lambropoulou, D., Katsarou, A., Pantopoulos, K., Bikiaris, D. (2020). Formulation and in-vitro characterization of chitosan-nanoparticlesloaded with the iron Chelator deferoxaminemesylate (DFO). Pharmaceutics, 12(3), 238. [CrossRef]
- 33. Mahmoud, A.A., El-Feky, G.S., Kamel, R., Awad, G.E.A. (2011). Chitosan/sulfobutylether-β-cyclodextrin nanoparticles as a potential approach for ocular drug delivery. International Journal of Pharmaceutics, 413(1-2), 229-236. [CrossRef]
- 34. Lau (deceased), E. (2001). Preformulation studies. Separation Science and Technology, 3 173-233. [CrossRef]
- 35. Lee, S.H., Zhang, Z., Feng, S.S. (2007). Nanoparticles of poly (lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymers for protein drug delivery. Biomaterials, 28(11), 2041-2050. [CrossRef]
- 36. Arthanari, S., Renukadevi P., Mani, K.R. (2011). Preparation and evaluation of sucrose stabilizedtetanus toxoid encapsulated into chitosan microspheres. Genomic Medicine, Biomarkers and Health Sciences, 3(3-4), 91-97. [CrossRef]
- 37. Lyng, J. (1990). Quantitative estimation of diphtheria and tetanus toxoids. 4. Toxoids as international reference materials defining Lf-units for diphtheria and tetanus toxoids. Biologicals: Journal of the International Association of Biological Standardization, 18(1), 11-17. [CrossRef]
- 38. Fulthorpe, A.J. (1958). Estimation of tetanus toxoid by different methods, including haemagglutination inhibition. Immunology, 1(4), 365-372. [CrossRef]
- 39. International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use ich harmonised tripartite guideline stability testing of new drug substances and products. Ich.org. 2003 [cited 2022 Dec 27]. Available from: https://database.ich.org/sites/default/files/Q1A%28R2%29%20Guideline.pdf
- 40. Chaurasiya, B., Zhou, M., Tu, J., Sun, C. (2018). Design and validation of a simple device for insufflation of dry powders in a mice model. European Journal of Pharmaceutical Sciences, 123, 495-501. [CrossRef]
- 41. Pirouzmand, H., Khameneh, B., Tafaghodi, M. (2017). Immunoadjuvant potential of cross-linked dextran microspheres mixed with chitosan nanospheres encapsulated with tetanustoxoid. Pharmaceutical Biology, 55(1), 212-217. [CrossRef]
- 42. Labiris, N.R., Dolovich, M.B., (2003). Pulmonary drug delivery. Part I: Physiological factors affecting therapeutic effectiveness of aerosolized medications: Physiological factors affecting the effectiveness of inhaled drugs. British Journal of Clinical Pharmacology, 56(6), 588-599. [CrossRef]
- 43. Peng, T., Zhang, X., Huang, Y., Zhao, Z., Liao, Q., Xu, J., Huang, Z., Zhang, J., Wu, CY., Pan., X, Wu, C. (2017). Nanoporous mannitol carrier prepared by non-organic solvent spray drying technique to enhance the aerosolization performance for dry powder inhalation. Scientific Reports, 7(1), 46517. [CrossRef]
- 44. Indian Pharmacopoeia. Vol II. Delhi: Controller of Publications; 1996. p. A.p. 33.
- 45. Manivannan, R., Dhanaraj, S.A., Rao, Y.U.B., Balasubramaniam, A., Gowrishankar, N.L., Jawahar, N., (2008). In vivo evaluation of single dose tetanus toxoid vaccine formulation with chitosan microspheres. Indian Journal of Pharmaceutical Sciences, 70(1), 11-15. [CrossRef]
- 46. Jung, T., Kamm, W., Breitenbach, A., Hungerer, K.D., Hundt, E., Kissel, T. (2001). Tetanus toxoid loaded nanoparticles from sulfobutylatedpoly(vinyl alcohol)-graft-poly(lactide-co-glycolide): Evaluation of antibody response after oral and nasal application in mice. Pharmaceutical Research, 18(3), 352-360. [CrossRef]