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

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1.6 BUCCAL ADHESIVE POLYMERS ......................................................................................... 27 1.6.1 Chitosan ................................................................................................................... 29 1.6.2 Pharmaceutical and medicinal uses of chitosan ..................................................... 30 1.6.3 Chitosan derivatives ................................................................................................ 31 1.6.3.1 Thiolated chitosans ........................................................................................... 31 1.6.4 Functional properties of thiolated chitosan ............................................................ 32 1.6.4.1 Mucoadhesion ................................................................................................... 32 1.6.4.2 Permeation enhancement .................................................................................. 33 1.6.6.3 Thiolated chitosans as matrices for controlled drug release ............................. 35 1.7 FORMULATION TECHNIQUES ............................................................................................ 35 1.7.1 Lyophilisation .......................................................................................................... 35 1.7.1.1 Lyophilisation principles .................................................................................. 35 1.7.1.2 Critical lyophilisation parameters ..................................................................... 36 1.7.1.3 Lyophilisation cycle ......................................................................................... 37 1.7.1.4 Freezing ............................................................................................................ 37 1.7.1.5 Primary drying .................................................................................................. 38 1.7.1.6 Secondary drying .............................................................................................. 38 1.7.2 The Freeze-drier machine ....................................................................................... 39 1.7.3 Gel formation ........................................................................................................... 40 1.7.4.1 Polymeric films for drug delivery ......................................................................... 40 1.7.4.2 Lyophilised xerogels (wafers) as drug delivery systems ...................................... 41 1.8 ANALYTICAL TECHNIQUES ............................................................................................... 42 1.8.1 Nuclear magnetic resonance (NMR) spectroscopy ................................................. 42 1.8.2 Gel permeation chromatography (GPC)................................................................. 43 1.8.3 Fourier transform infrared (FT-IR) spectroscopy................................................... 44 1.8.3.1 Attenuated total reflection (ATR-FT-IR) spectroscopy ................................... 44 1.8.4 X-ray powder diffractometry (XRPD) ..................................................................... 46 1.8.4.1 X-ray generation and properties ....................................................................... 46 1.8.4.2 Powder diffraction ............................................................................................ 47 1.8.5 Differential scanning calorimetry (DSC) ................................................................ 48 1.8.5.1 Interpreting DSC thermogram in lyophilisation ............................................... 49 1.8.6 Circular dichroism (CD) spectroscopy ................................................................... 50 1.8.7 Thermogravimetry (TG) ........................................................................................ 51 1.8.8 Scanning electron microscope (SEM) ..................................................................... 52 1.9 AIMS AND OBJECTIVES ..................................................................................................... 54
CHAPTER TWO .................................................................................................................... 55 FORMULATION DEVELOPMENT AND OPTIMISATION .......................................... 55 2.1 INTRODUCTION ................................................................................................................ 55 2.1.1 Annealing ................................................................................................................. 56 2.1.2 Effect of cryoprotectants .......................................................................................... 57 2.1.3 Effect of plasticisers ................................................................................................ 59 2.1.4 Permeation enhancers ............................................................................................. 59 2.1.4.1 Brij L23 (Brij 35) .............................................................................................. 60 2.1.5 Enzyme inhibitors (EI)............................................................................................. 60 β.1.5.1 Glutathione (GSH, -L-Glutamyl-L-cysteinyl-glycine) ................................... 61 2.1.5.2 Aprotinin (APR) ............................................................................................... 62 2.2 MATERIALS AND METHODS .............................................................................................. 64 2.2.1 Materials .................................................................................................................. 64 2.2.2 Preliminary investigations: solution preparation and membrane dialysis ............. 65 2.2.3 Synthesis of chitosan-4-thioglycolic acid (TG-chitosan) ........................................ 66 2.2.4 Preparation and permeability studies of ethyl cellulose (EC) backing membrane . 67 2.2.5 Annealing studies ..................................................................................................... 68 2.2.6 Lyophilisation .......................................................................................................... 68 2.3 RESULTS AND DISCUSSION ............................................................................................... 69 2.3.1 Preliminary investigations: ..................................................................................... 69 2.3.1.1 Gel preparation ................................................................................................. 69 2.3.1.2 Membrane dialysis ............................................................................................ 72 2.3.2 Synthesis of chitosan-4-thioglycolic acid (TG-chitosan) ........................................ 72 2.3.3 Preparation of ethylcellulose (EC) backing membrane .......................................... 73 2.3.4 Annealing studies ..................................................................................................... 74 2.3.5 Lyophilisation .......................................................................................................... 75 2.4 CONCLUSIONS .................................................................................................................. 76 CHAPTER THREE ................................................................................................................ 78 ANALYTICAL, PHYSICO-CHEMICAL CHARACTERISATION OF XEROGELS AND STABILITY EVALUATION OF MODEL PROTEIN DRUGS (BSA, INSULIN) IN OPTIMISED TG-CHITOSAN XEROGELS ................................................................. 78 3.1 INTRODUCTION ................................................................................................................ 78 3.2 MATERIALS AND METHODS .............................................................................................. 79
Page 1 and 2: Greenwich Academic Literature Archi
Page 3 and 4: DECLARATION “I certify that this
Page 5 and 6: ABBREVIATIONS ATR-FT-IR (attenuated
Page 7: CONTENTS DECLARATION ..............
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
Page 59 and 60:
the product matrix, the vacuum of t
Page 61 and 62:
an oven at an appropriate temperatu
Page 63 and 64:
Several analytical procedures have
Page 65 and 66:
transmitting crystal with a high re
Page 67 and 68:
1.8.4.2 Powder diffraction Powder X
Page 69 and 70:
1.8.5.1 Interpreting DSC thermogram
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(absorption in the range 260 to 320
Page 73 and 74:
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
Page 195 and 196:
drug delivery systems via the bucca
Page 197 and 198:
33. Bunte, H., Drooge, D.J., Ottjes
Page 199 and 200:
56. Engles, L. 2005. Review and app
Page 201 and 202:
(chitooligosaccharide derivatives)
Page 203 and 204:
of single-point mutants of an amylo
Page 205 and 206:
121. Koschier, F., Kostrubsky, V.,
Page 207 and 208:
142. Liu, W., Wang, D.Q., Nail, S.
Page 209 and 210:
160. Modi, P., Mihic, M. And Lewin,
Page 211 and 212:
180. Pastore, A., Piemonte, F., Loc
Page 213 and 214:
204. Robinson, J., R. 1990. Rationa
Page 215 and 216:
224. Silva, C.A., Nobre, T.M., Pavi
Page 217 and 218:
245. Tamburic, S., Graig, D.Q.M. 19
Page 219 and 220:
265. Wang W. 2000. Lyophilisation a
Page 221 and 222:
285. Zor, T., Selinger, Z. 1996. Li
Page 223 and 224:
APPENDIX B: Manuscripts in preparat
Page 225 and 226:
2. Accepted abstract for podium and
Page 227 and 228:
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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