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Protein Formulations for Pulmonary Delivery

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Protein Formulations for Pulmonary Delivery PROTEIN FORMULATIONS FOR PULMONARY DELIVERY BILDAD KIMANI NYAMBURA A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 2007 DEPARTMENT OF PHARMACEUTICS, THE SCHOOL OF PHARMACY, UNIVERSITY OF LONDON, 29/39 BRUNSWICK SQUAgE^L^DON WCIN lAX, UK. ^ 4^p) ' ProQuest Number: 10104298 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10104298 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Biotechnology advances have led to the discovery of new biopharmaceuticals. This has subsequently increased the search for new safe and effective delivery systems that can be used to administer protein therapeutics via inhalation. The purpose of this study was to design, develop and characterise novel delivery systems that can be used to administer protein drugs via the pulmonary route for systemic action. Lysozyme and insulin were selected as model protein drugs for this study. Initially, large porous microparticles (LPMs) containing protein (40% w/w) were produced by spray-drying emulsions. Protein stability was determined using a turbidimetric method and High Performance Liquid Chromatography (HPLC). Aerosolisation properties were assessed using a laser diffraction technique and multistage liquid impinger (MSLI). Approximately 99% retained activity of lysozyme was achieved while HPLC studies showed that insulin was not degraded after formulation processing. Aerosolisation studies showed that the LPMs were suitable for alveolar deposition, with approximately 44 % w/w was deposited as fine particle fraction (FPF<i. 7^m). To increase the protein content of the formulation, nanoparticles containing protein (80% w/w) were produced by nanoprecipitation or émulsification processes followed by freeze-drying. The nanoparticles were purified by washing off excess surfactant and were suspended in HFA 134a using ethanol plus surfactant or essential oils (cineole and citral) to form pMDI formulations. Turbidimetric studies showed that approximately 98% retained activity of lysozyme was achieved while HPLC, size exclusion chromatography, circular dichroism and fluorescence spectroscopy studies showed that the native structure of insulin was retained after formulation processing. Aerosolisation studies showed that the nanoparticles were^ suitable for alveolar deposition, with approximately 45% w/w was deposited as FPF<i.7pm. In conclusion, the formulations developed in this study have the potential to deliver protein therapeutics via inhalation for systemic action. Declaration This thesis describes research conducted in The School of Pharmacy, University of London between October, 2002 and October, 2006 under the supervision of Professor Ian W. Keliaway and Professor Kevin M.G. Taylor. I certify that the research described is original and that any parts of the work that have been conducted by collaboration are clearly indicated. I also certify that I have written all the text herein and have clearly indicated by suitable citation any part of this thesis that has already appeared in publication. — I a Signature Date Acknowledgements I would like to express my sincere gratitude to my academic supervisors Professors Ian W. Keliaway and Kevin M.G. Taylor for their advice, guidance and encouragement throughout the research project. I am also grateful for the university technical staff, in particular Dave for all the SEM work, and Keith (retired), Lionel (retired) and John for their technical support with equipment on a number of occasions. I would also thank Dr Alfred Drake and Dr Tam Bui of Kings College, Molecular Biophysics Group, for all of the circular dichroism work. Thanks also to Lipoid GMBH (Germany) for their kind donation of lecithin that was used in this research project. Thanks to all postgraduates and alumni from the Department of Pharmaceutics Rob, Kristin, Claire, Mei, Simon and Amina who were very helpful and made my time at the School of Pharmacy thoroughly enjoyable. Finally, I would like to sincerely thank my family and friends for their unwavering support, motivation and encouragement throughout the project. CONTENTS Title page ...........................................................................................................................1 Abstract .............................................................................................................................2 Declaration ....................................................................................................................... 3 Acknowledgement ...........................................................................................................4 List of figures ................................................................................................................. 10 List of tables ................................................................................................................... 13 Abbreviations ................................................................................................................. 15 Chapter 1: Introduction............................................................................................. 18 1.1 General introduction ............................................................................................... 19 1.2 Fundamentals of protein structure ........................................................................ 19 1.2.1 Amino acids ................................................................................................... 20 1.2.2 The primary structures of proteins ............................................................... 21 1.2.3 The secondary structures of proteins ........................................................... 22 1.2.3.1 The helices .........................................................................................22 1.2.3.2 The p - sheet ..................................................................................... 23 1.2.3.3 The turns ............................................................................................23 1.2.4 The tertiary structure of proteins ...................................................................24 1.2.5 The quaternary structure of proteins ............................................................ 25 1.3 Stabilisation of proteins ..........................................................................................25 1.3.1 Factors affecting protein stability .................................................................25 1.3.2 Formulation to stabilise protein ....................................................................29 1.4 Protein therapeutics ................................................................................................. 35 1.5 Delivery routes of proteins/peptides ......................................................................37 1. 6 Respiratory tract ...................................................................................................... 38 1.6 .1 The anatomy of the respiratory tract ............................................................ 38 1.6.2 The cellular profile of the respiratory tract ..................................................39 1.6.3 Drug targeting in the respiratory tract .......................................................... 40 1.6.4 Particle dynamics in the respiratory tract .....................................................41 1.6.5 The absorption of drugs in the respiratory tract .......................................... 42 1.6 . 6 The barriers to pulmonary drug delivery .....................................................43 1.7 Design and engineering of respirable particles .....................................................44 5 1.8 Production of respirable particles ..........................................................................47 1.8.1 Spray-drying .................................................................................................. 47 1.8.1.1 Atomisation ........................................................................................48 1.8.1.2 Drying ................................................................................................ 50 1.8.1.3 Powder separation ............................................................................. 52 1.8.2 Freeze-drying ................................................................................................. 53 1.8.2.1 Freezing stage ................................................................................... 54 1.8.2.2 Primary drying .................................................................................. 55 1.8.2.3 Secondary drying .............................................................................. 56 1.9 Delivery systems of respirable drug particles .......................................................56 1.9.1 Dry powder inhalers .......................................................................................57 1.9.2 Pressurised metered dose inhalers ...............................................................
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