Development of novel formulations for mucosal delivery of protein ...

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ABSTRACT Stable mucoadhesive lyophilised chitosan and thiolated chitosan xerogels have been developed for potential delivery of proteins via the buccal mucosa. Membrane dialysis to eliminate sodium acetate (NaAc) and annealing during lyophilisation cycle (developed by differential scanning calorimetry; DSC) were critical for obtaining optimised porous xerogels. Based on characteristic performance and structural integrity, xerogels containing 10 % (per polymer weight) each of plasticizer (glycerol) and cryoprotectant (D-mannitol) were loaded with bovine serum albumin (BSA) and insulin (INS) as model drugs for further development. The optimised xerogels were loaded with enzyme inhibitor (EI) and permeation enhancer (PE) to enhance permeability of the buccal mucosa and backed with impervious ethylcellulose (EC) laminate to ensure unidirectional release. Characterisation of xerogels using 1 HNMR and ATR-FT-IR spectroscopy confirmed the functional groups in chitosan and TG-chitosan. The amount of thiol groups on TG-chitosan was quantified by Ellman’s reaction with polymer molecular weight monitoring by gel permeation chromatography (GPC). The stability of the secondary structures of BSA and INS were by ATR-FT-IR and circular dichroism (CD) while xerogel crystallinity was examined by XRPD. SEM micrographs showed highly porous xerogels due to annealing which allowed for high drug loading and hydration capacities of the dialysed and annealed xerogels which also exhibited optimal moisture content for maintaining protein stability. Disulphide bond formation in thiolated xerogels however limited the hydration capacity. Storage at 25 °C/ 60 % RH for six months led to protein instability while storage at 5 °C maintained protein stability. 2% mucin concentration was found optimum for mucoadhesion studies of the chitosan based xerogels. EI (glutathione; GSH and aprotinin; APR) effect on mucoadhesion was concentration dependent and inhibitor specific. In vitro BSA release profiles from annealed xerogels were similar and significantly higher than nonannealed xerogels but were not affected by thiolation. Crystalline NaAc in the undialysed xerogel led to a three-fold reduction in BSA release. Significant reductions in BSA and INS release were also observed with the addition of EIs to TG-chitosan-BSA and TG-chitosan- INS xerogel respectively. The drug dissolution data from xerogels fitted best with model dependent First order, Hixson-Crowell and Korsmeyer-Peppas equations. Permeation studies’ using EpiOral TM showed 12- to 14-fold increase in BSA permeation but was reduced by GSH for both BSA and INS loaded xerogels. APR containing xerogel enhanced INS permeation through sheep buccal membrane and demonstrated a good linear correlation with EpiOral TM . These results demonstrate the potential application of lyophilised chitosan and thiolated chitosan xerogels for buccal mucosa delivery of proteins with improved mucoadhesion, penetration enhancing and enzyme inhibition characteristics.
CONTENTS DECLARATION .................................................................................................................... II ACKNOWLEDGEMENTS .................................................................................................. III ABBREVIATIONS ...............................................................................................................IV ABSTRACT ........................................................................................................................... V CONTENTS ..........................................................................................................................VI TABLES .............................................................................................................................. XII FIGURES ........................................................................................................................... XIV CHAPTER ONE ....................................................................................................................... 1 INTRODUCTION AND LITERATURE REVIEW ............................................................. 1 1.1 OVERVIEW ......................................................................................................................... 1 1.2 PROTEIN STRUCTURE AND AGGREGATION .......................................................................... 4 1.2.1 Use of proteins as therapeutic agents ....................................................................... 5 1.2.2 Model protein drugs .................................................................................................. 6 1.2.2.1 Bovine serum albumin (BSA) ............................................................................ 6 1.2.2.2 Insulin (INS)....................................................................................................... 8 1.2.3 Protein delivery and challenges .............................................................................. 11 1.3 IMPROVING PROTEIN ABSORPTION ................................................................................... 12 1.3.1 Permeation enhancers ............................................................................................. 12 1.3.2 Enzyme inhibitors .................................................................................................... 13 1.3.3 Mucoadhesive delivery systems ............................................................................... 15 1.3.3.1 Theories of mucoadhesion ................................................................................ 15 1.3.3.2 Factors affecting mucoadhesion ....................................................................... 17 1.3.3.3 Mucin ................................................................................................................ 18 1.4 MUCOSAL DELIVERY ROUTES .......................................................................................... 20 1.4.1 The oral route .......................................................................................................... 20 1.4.2 The nasal route..................................................................................................... 21 1.4.3 The pulmonary route ............................................................................................... 23 1.4.4 The ocular route ...................................................................................................... 23 1.4.5 Vaginal and rectal routes ........................................................................................ 23 1.4.6 Buccal route ............................................................................................................. 24 1.5 BUCCAL DELIVERY OF PROTEINS ...................................................................................... 25 1.5.1 Pros and cons of protein delivery via the buccal mucosa ....................................... 26 1.5.2 Mechanism of protein transport across buccal mucosa. ......................................... 27
Page 1 and 2: Greenwich Academic Literature Archi
Page 3 and 4: DECLARATION “I certify that this
Page 5: ABBREVIATIONS ATR-FT-IR (attenuated
Page 9 and 10: CHAPTER TWO .......................
Page 11 and 12: CHAPTER FOUR ......................
Page 13 and 14: TABLES Table 1.1: Relationship of i
Page 15 and 16: FIGURES Figure 1.1: Molecular model
Page 17 and 18: [Accessed; 24/05/2012]. ...........
Page 19 and 20: Figure 4.2: In-vitro mucoadhesion m
Page 21 and 22: CHAPTER ONE INTRODUCTION AND LITERA
Page 23 and 24: freezing rate at the start of a lyo
Page 25 and 26: aggregates may lead to an immunogen
Page 27 and 28: Figure 1.1: Molecular model of seru
Page 29 and 30: One of the greatest milestones in m
Page 31 and 32: limited success. INS released in a
Page 33 and 34: composition of the delivery system
Page 35 and 36: ioavailability of proteins using pe
Page 37 and 38: 1.3.3.2 Factors affecting mucoadhes
Page 39 and 40: Human salivary glands secrete 1000-
Page 41 and 42: 1.4.2 The nasal route Nasal protein
Page 43 and 44: 1.4.3 The pulmonary route The lungs
Page 45 and 46: and enzymes from foods and beverage
Page 47 and 48: (Veuillez et al., 2001; Nagai, 1986
Page 49 and 50: 1.6.1 Chitosan Chitosan together wi
Page 51 and 52: 1.6.3 Chitosan derivatives The exce
Page 53 and 54: with increasing amounts of immobili
Page 55 and 56: 1.6.6.3 Thiolated chitosans as matr
Page 57 and 58:
stability studies among others can
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the product matrix, the vacuum of t
Page 61 and 62:
an oven at an appropriate temperatu
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Several analytical procedures have
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transmitting crystal with a high re
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1.8.4.2 Powder diffraction Powder X
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1.8.5.1 Interpreting DSC thermogram
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(absorption in the range 260 to 320
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Figure 1.26: Representation of SEM.
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CHAPTER TWO FORMULATION DEVELOPMENT
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have recommended annealing time of
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Figure 2.1: Chemical structure of c
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model proteins for buccal delivery.
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microemulsion formulation containin
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Table 2.2: Details of consumables u
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chitosan was stirred continuously f
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Table 2.5: Lyophilisation cycles wi
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a b c d Figure 2.6: Photographs of
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simple one step coupling reaction i
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2.3.5 Lyophilisation The primary dr
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The foremost implication of these f
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3.2 Materials and methods 3.2.1 Mat
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were analysed with Agilent Chemstat
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previously tarred 70 µl aluminium
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3.2.13.2 Assay of BSA and INS conte
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A H1 (GluNAc) H3 -H6 NAc H2 (GluNH
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V o V e Intensity ElutionTime (min)
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c c r er % Transmittance 110 90 70
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during the xerogel preparation proc
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and without APR respectively. The r
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3.3.7 Physical stability of BSA and
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15 Circular dichroism (mdeg) 10 5 0
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Since no product melt-back was obse
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0.8 Absorbance ratio 595/450 0.7 0.
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A B Dialysed chitosan-BSA 100 µm 1
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High magnification SEM micrograph o
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BSA content (%) 100.0 80.0 60.0 40.
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conditions leads to protein hydroly
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3.4 Conclusions Analytical characte
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CHAPTER FOUR HYDRATION CAPACITY AND
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4.2.2.1 Effect of formulation proce
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substrate in order to select the op
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Table 4.3 Hydration capacity of xer
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4.3.2.1 Factors affecting mucoadhes
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impacted on the mucoadhesive proper
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and TG-chitosan xerogels. The chito
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PAF (N) TWA (mJ) Cohesiveness (mm)
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Annealed TG-chitosan xerogel simila
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that each model makes assumptions t
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independent difference and similari
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elease profiles plotted. Blank cont
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the second phase of the release pro
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100 Cummulative BSA Release (%) 80
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controlled drug release has been de
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Cummulative INS release (%) 100 80
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that of non-annealed dialysed chito
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Table 5.5: Release parameters obtai
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The effect of formulation processes
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Table 5.9: Model independent differ
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CHAPTER SIX PERMEATION STUDIES USIN
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6.2 Materials and Methods 6.2.1 Mat
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Permeation studies were conducted f
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fold increase in transport of BSA f
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Cumulative INS permeated (g/cm 2 )
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Average percentage viability was pl
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5.5 A TG-chitosan-INS permeation (E
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CHAPTER SEVEN SUMMARY CONCLUSIONS A
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was observed for dialysed and annea
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2. The long-term stability evaluati
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drug delivery systems via the bucca
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33. Bunte, H., Drooge, D.J., Ottjes
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56. Engles, L. 2005. Review and app
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(chitooligosaccharide derivatives)
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of single-point mutants of an amylo
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121. Koschier, F., Kostrubsky, V.,
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142. Liu, W., Wang, D.Q., Nail, S.
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160. Modi, P., Mihic, M. And Lewin,
Page 211 and 212:
180. Pastore, A., Piemonte, F., Loc
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204. Robinson, J., R. 1990. Rationa
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224. Silva, C.A., Nobre, T.M., Pavi
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245. Tamburic, S., Graig, D.Q.M. 19
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265. Wang W. 2000. Lyophilisation a
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285. Zor, T., Selinger, Z. 1996. Li
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APPENDIX B: Manuscripts in preparat
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2. Accepted abstract for podium and
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4. Abstract # 43 awarded podium and
Page 229 and 230:
6. Published abstract M1144 (2011).
Page 231:
8. Published abstract W1470 (2010).
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