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

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6.1 INTRODUCTION .............................................................................................................. 155 6.2 MATERIALS AND METHODS ........................................................................................... 157 6.2.1 Materials ................................................................................................................ 157 6.2.2 EpiOral permeation studies ............................................................................... 157 6.2.3 Tissue integrity (MTT assay) ................................................................................. 159 6.2.4 In vitro buccal permeation studies (sheep buccal tissue) ...................................... 159 6.2.5 Permeation correlation between EpiOral TM and sheep buccal tissues. ................ 160 6.3 RESULTS AND DISCUSSION ............................................................................................. 160 6.3.1 EpiOral TM permeation studies ............................................................................... 160 6.3.2 Tissue integrity (MTT assay) ................................................................................. 164 6.3.3 In vitro buccal permeation studies (sheep buccal tissue) ...................................... 165 6.3.4 Permeation correlation between EpiOral TM and sheep buccal tissues. ................ 166 6.3 CONCLUSIONS ................................................................................................................ 167 CHAPTER SEVEN .............................................................................................................. 169 SUMMARY CONCLUSIONS AND FUTURE WORK ................................................... 169 7.1 SUMMARY CONCLUSIONS ............................................................................................... 169 7.2 KEY RESEARCH FINDINGS .............................................................................................. 172 7.3 FUTURE WORK ............................................................................................................... 172 CHAPTER EIGHT .............................................................................................................. 174 REFERENCES ..................................................................................................................... 174 CHAPTER NINE .................................................................................................................. 202 APPENDIX ........................................................................................................................... 202 APPENDIX A: LIST OF PUBLICATIONS .............................................................................. 202 APPENDIX B: MANUSCRIPTS IN PREPARATION ................................................................. 203 APPENDIX C: ABSTRACTS AND POSTERS FROM CONFERENCE PROCEEDINGS .................... 204
TABLES Table 1.1: Relationship of isomeric forms of bovine serum albumin (Forster, 1977)............... 7 Table 1.2: Transmucosal routes of insulin delivery systems. Adapted from Khafagy et al., (2007). ...................................................................................................................................... 10 Table 1.3 Marketed/under development protein oral delivery technology by companies. Adapted from Park et al., 2011. ................................................................................................ 22 Table 2.1: Details of materials used in the formulation development and optimisation ......... 64 Table 2.2: Details of consumables used in the formulation development and optimisation ... 65 Table 2.3: Matrix chitosan formulations prepared in preliminary stages ................................ 65 Table 2.4: Optimised drug loaded chitosan/TG-chitosan formulations with permeation enhancer and various amounts of different enzyme inhibitors (cf Table 2.3) .......................... 66 Table 2.5: Lyophilisation cycles with and without an annealing process ............................... 69 Table 2.6: Optimised chitosan/TG-chitosan formulations loaded with INS. .......................... 69 Table 3.1: Materials ................................................................................................................. 79 Table 3.2: 1 H parameters for Jeol ECA, 500 MHz FT NMR .................................................. 80 Table 3.3: Xerogels and materials characterised by ATR-FT-IR ............................................ 81 Table 3.4: ICH conditions for drug products intended for storage in a refrigerator ............... 84 Table 3.5: 1 H-NMR signal assignments for chitosan and TG-chitosan .................................. 86 Table 3.6: Mean residue ellipticity ratios at 209 and 222 nm for INS loaded Xerogels ....... 100 Table 3.7: Percentage drug loading capacity and moisture content of xerogels ................... 100 Table 4.1: Details of materials used in hydration and mucoadhesion studies. ...................... 116 Table 4.2: Texture analyser settings for determining the peak adhesive force (PAF), total work of adhesion (TWA) and cohesiveness of formulations. ................................................ 118 Table 4.3 Hydration capacity of xerogels .............................................................................. 121 Table 4.4 Effect of reducing agent 10 mM DTT on hydration capacity of thiolated chitosan xerogels ................................................................................................................................... 122 Table 5.1: Diffusion exponent and drug diffusion mechanism for cylindrical shaped matrices ................................................................................................................................................ 134 Table 5.2: Materials for drug release studies ......................................................................... 135 Table 5.3 Various plots with corresponding kinetic /mechanism model ............................... 137 Table 5.4: Release parameters obtained from fitting experimental drug dissolution (release) data to different kinetic equations for chitosan and TG-chitosan xerogels loaded with BSA. ................................................................................................................................................ 148
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 and 8: CONTENTS DECLARATION ..............
Page 9 and 10: CHAPTER TWO .......................
Page 11: CHAPTER FOUR ......................
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
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transmitting crystal with a high re
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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
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Figure 1.26: Representation of SEM.
Page 75 and 76:
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 )
Page 185 and 186:
Average percentage viability was pl
Page 187 and 188:
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
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(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
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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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