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

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Figure 1.3: A schematic diagram showing clinical and potential protein delivery routes Abbreviation: absorption enhancers (AE) enzyme inhibitors (EI) and electroporation (EP), Adapted from Agu et al., 2001. 1.3 Improving protein absorption The use of permeation enhancers (PE), enzyme inhibitors (EI) and the exploitation of bioadhesive delivery systems have been investigated to overcome protein delivery challenges. 1.3.1 Permeation enhancers Permeation enhancers have been extensively studied to overcome the barriers to buccal protein and peptide delivery. Penetration enhancers act on the epithelium and increase the permeability of the membrane by either the paracellular or the transcellular pathway. Other mechanisms proposed for the improvement of mucosal peptide absorption by penetration enhancers include reduction of mucus layer elasticity and/or viscosity and increasing the thermodynamic activity of protein drugs which may be affected by the
composition of the delivery system with subsequent influence on micellization and solubility and by degree of drug and enhancer ion-pair formation (Veuillez et al., 2001). Examples of this class of compounds include surfactants, fatty acids, bile salts, and chelators such as ethylene diamine tetra acetate (EDTA). Many gel-forming polymers such as polycarbophil, chitosan and thiolated chitosans also act as permeation enhancers by binding to the extracellular Ca 2+ . Fatty acids, bile salts and surfactants mostly enhance permeability by increasing the fluidity of the lipid bilayer of the cell membranes. Local toxicity to the epithelium in the gastrointestinal tract is a major concern in using these systems in pharmaceutical products. Intracellular Ca 2+ plays an important role in regulating the permeability of the tight junction (Kan & Coleman, 1988). Thus, compounds such as EDTA that can bind the extracellular Ca 2+ , lowering the intracellular Ca 2+ concentration, can act as permeation enhancers by rendering the paracellular pathway more permeable (Khafagy et al., 2007). Some of these enhancers, however, can cause local damage to the mucosal membrane and even enter the systemic circulation, leading to systemic toxic effects. The potential damage to the epithelium and changes in the mucosal morphology, especially in long term multi-dosing regimen, as would be required in diabetes treatment, are major concerns in the clinical use of these agents. Another disadvantage of these compounds is their non-specificity in permeation enhancement. Increased permeability of the epithelium may also allow toxins and microbial pathogens in the buccal cavity to pass more easily into the systemic circulation, an event which negates the very function of the buccal mucosa (Sudhakar et al., 2006). Factors such as characteristics of permeant, composition of the delivery system and the previous treatment of tissue with or without enhancer, affect the degree of enhancement. Enhancers may therefore be selected based on safety, chemical and biological inertness, effectiveness and rapid but reversible effect (Nicolazzo et al., 2005). 1.3.2 Enzyme inhibitors The stability of protein and peptide drugs may be improved by the addition of enzyme inhibitors to formulations intended for absorption through the buccal mucosa (Khafagy et al., 2007). Concomitant administration of INS with aprotinin and soybean trypsin inhibitors has been shown to induce better hypoglycemic effect (Bernkop-Schnürch, 1998; Morishita, 1993). Polyacrylates such as Carbopol 934P and polycarbophil, which are typically used as bioadhesives, are also known to inhibit the activity of trypsin and chymotrypsin in a calciumdependent manner (Madsen & Peppas, 1999; Lueben et al., 1997). Bai et al., (1996) showed that in-situ absorption of INS was improved by carbomer polymers and also induced a
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 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: limited success. INS released in a
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
Page 71 and 72: (absorption in the range 260 to 320
Page 73 and 74: Figure 1.26: Representation of SEM.
Page 75 and 76: CHAPTER TWO FORMULATION DEVELOPMENT
Page 77 and 78: have recommended annealing time of
Page 79 and 80: Figure 2.1: Chemical structure of c
Page 81 and 82: 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,
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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
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6. Published abstract M1144 (2011).
Page 231:
8. Published abstract W1470 (2010).
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