Content removed due to copyright restrictions: E I-Sherbiny, I.M., Lins, R.l , Abdel-Bary, E.M., Harding, D.R.K. Preparation, characterization, swelling and in vitro drug release behaviour of poly[Nacryloylglycine- chitosan] interpolymeric pH and thermally-responsive hydrogels. Eur. Polym. J. 41 (2005) 2584. E I-Sherbiny, I.M., Abdel-Bary, E.M., Harding, D. R.K. Preparation and swelling study of a pH-dependent interpolymeric hydrogel based on chitosan for controlled drug release. Inl. J. Polym. Mater. 55 (2006) 789. El-Sherbiny, I.M., Abdel-Bary, E.M., Harding, D. R. K. Swelling characteristics and in-vitro drug release study with pH and thermo-sensitive hydro gels based on modified chitosan. J. Appl. Polym. Sci. 102 (2006) 977. Abdelaal, M.Y., Abdel-Razik, E . A , Abdel-Bary, E.M., El-Sherbiny, I.M. Study on chitosan-poly(vinyl alcohol) interpolymeric pH-responsive hydrogel films for controlled drug delivery. J. Appl. Polym. Sci. (2006) in press. El-Sherbiny, I. M., Abdel-Bary, E.M., Harding, D. R. K. In-vitro investigation of new biodegradable pH-responsive hydrogel beads for oral delivery of protein drugs in the small intestine. New Zealand Institute of Chemistry Conference (NZIC), (2006) Rotorua, New Zealand, 2-6 December. / Controlled drug release enhances the safety, efficacy and reliability of drug therapy. Regulation of the drug release rate results in a reduction in the frequency of drug administration and should encourage patients to comply with dosing instructions. Hydrogels are crosslinked, three-dimensional hydrophilic polymers, which swell without dissolving when brought into contact with water or other biological fluids. The number of polymers suitable for the controlled release of viable therapeutics is quite limited because of inherent toxicity or lack of certain properties such as biodegradability. In this thesis, chitosan was chosen as the base polymer for the development of new hydrogels that can be tailored for use in the site-specific delivery of drugs to the gastrointestinal tract. Chitosan is a non-toxic and biodegradable polymer obtained through the alkaline deacetylation of natural chitin. The interesting characteristics of chitosan make it an ideal candidate for use in controlled drug release formulations. However, chitosan exhibits some shortcomings such as hydrophobicity and a high pH-dependency for its physical properties. Hence, it is very difficult to control drug release with chitosan itself because of the various pH values of the internal organs of the human body. This may negatively affect the human body because of drug under- or over-release. In a structured programme, some new chitosan-based hydrogels have been prepared for controlled drug release investigations by applying three main approaches to overcome the shortcomings of chitosan. The first approach was the incorporation of chitosan into interpenetrating polymer network hydrogels with either a hydrophilic polymer or with hydrophilic monomers treated to bring about in situ copolymerization in the presence of chitosan and a suitable crosslinking agent. The second approach was the chemical modification of chitosan by grafting of a suitable vinyl macromer such as poly(ethylene glycol)-diacrylate, then crosslinking this modified chitosan. The equilibrium swelling studies were carried out for the hydrogels prepared using these two approaches at 37 °C at pH 2.1 (simulated gastric fluid, SGF) and at pH 7.4 (simulated intestinal fluid, SIF). The swelling results showed a pH-responsive nature of these hydrogels. They attained higher swelling values in SGF than in SIF. 5-Fluorouracil (5-FU), an anti cancer drug, was entrapped as a model drug in all the hydrogels prepared using these two approaches. The in-vitro drug release studies were carried out at 37 °C in SGF and SIF. From the preliminary investigations of the prepared hydrogels, they may be customized and used to expand the utilization of these systems in drug delivery applications. In the third approach, chitosan was modified in such a fashion that the hydrogels produced were also pH-responsive but attained limited swelling in SGF and higher swelling in SIF. Hence, the resulting hydrogels could be tailored for utilization for intestine-targeted delivery of peptide and protein drugs with a potential protection of the drugs from the harsh acidity of the stomach. In this third approach the ionotropic gelation was used for the preparation of the hydrogels based on the modified chitosan with another natural polymer (sodium alginate) in the presence of a divalent ion. Bovine serum albumin (BSA) was entrapped as a model protein drug and the in-vitro drug release profiles were established at 37 °C in SGF and SIF. The results showed promising release profiles of BSA. However, this hydrogel study requires more effort to limit the swelling and consequently the loss of drug in the SGF, to act as an excellent candidate for intestine-specific delivery of peptide and protein drugs.

The role of metals in biological processes has been discussed with particular emphasis on the importance of chelation. Furst has suggested that many anticancer drugs may owe their activity to their chelating properties. A number of new amidoximes and hydroxylamine have been made from the appropriate imidoyl chloride and a hydroxylaqine. The spectral and chelating properties of these compounds are discussed. All the compounds have been submitted to the anticancer screening programme of the National Cancer Institute. The results available so far are presented and indicate that the compounds are toxic but inactive against experimental tumours. Sodium sulphide reduces a-phenyl-o-nitro-cinnamonitrile (106) to 2-amino-3-phenylquinoline-1-oxide (107) in good yield. Extension of the reduction to other o-nitrocinnamonitriles gives very poor results. The spectral properties, configurations and conformations of the o-nitrocinnamonitriles are discussed. The 12-aminoquinoline-1-oxide group is shown to form solid metal complexes. That from 2-amino-3-phenylquinoline-1-oxide (107) with nickel has been prepared. Antitumour screening results are presented for several 2-aminoquinoline derivatives. None of these compounds appears to be active.

The role of metals in biological processes has been discussed with particular emphasis on the importance of chelation. Furst has suggested that many anticancer drugs may owe their activity to their chelating properties. A number of new amidoximes and hydroxylamine have been made from the appropriate imidoyl chloride and a hydroxylaqine. The spectral and chelating properties of these compounds are discussed. All the compounds have been submitted to the anticancer screening programme of the National Cancer Institute. The results available so far are presented and indicate that the compounds are toxic but inactive against experimental tumours. Sodium sulphide reduces a-phenyl-o-nitro-cinnamonitrile (106) to 2-amino-3-phenylquinoline-1-oxide (107) in good yield. Extension of the reduction to other o-nitrocinnamonitriles gives very poor results. The spectral properties, configurations and conformations of the o-nitrocinnamonitriles are discussed. The 12-aminoquinoline-1-oxide group is shown to form solid metal complexes. That from 2-amino-3-phenylquinoline-1-oxide (107) with nickel has been prepared. Antitumour screening results are presented for several 2-aminoquinoline derivatives. None of these compounds appears to be active.

The role of metals in biological processes has been discussed with particular emphasis on the importance of chelation. Furst has suggested that many anticancer drugs may owe their activity to their chelating properties. A number of new amidoximes and hydroxylamine have been made from the appropriate imidoyl chloride and a hydroxylaqine. The spectral and chelating properties of these compounds are discussed. All the compounds have been submitted to the anticancer screening programme of the National Cancer Institute. The results available so far are presented and indicate that the compounds are toxic but inactive against experimental tumours. Sodium sulphide reduces a-phenyl-o-nitro-cinnamonitrile (106) to 2-amino-3-phenylquinoline-1-oxide (107) in good yield. Extension of the reduction to other o-nitrocinnamonitriles gives very poor results. The spectral properties, configurations and conformations of the o-nitrocinnamonitriles are discussed. The 12-aminoquinoline-1-oxide group is shown to form solid metal complexes. That from 2-amino-3-phenylquinoline-1-oxide (107) with nickel has been prepared. Antitumour screening results are presented for several 2-aminoquinoline derivatives. None of these compounds appears to be active.

Downstream purification and analysis of a model biopharmaceutical protein (recombinant human myelin basic protein) is described. The recombinant protein was expressed in the milk of transgenic cows and was found exclusively associated with the casein micellar phase. Binding of milk calcium to the active sites of a cation exchanger resin was used beneficially in this study in order to gently disrupt the casein micelles and liberate the recombinant protein. This approach was found superior to the conventional micelle disruption procedures with respect to product recovery, resin fouling due to milk components and column hydrodynamic properties. Further purification was carried out using Ni2+ affinity chromatography and resulted in purity more than 90% and a total recovery of 78%. A capillary electrophoresis total protein assay employing large volume sample stacking and a microsphere-based, sandwich-type immunoassay were developed and validated. Both methods were successfully integrated with the downstream purification protocol in order to evaluate various quality attributes of the recombinant protein. A onestep capillary isoelectric focusing protocol was developed in order to monitor the recombinant protein in milk samples. The results showed extra protein bands in the transgenic milk that had isoelectric points significantly lower than the theoretically calculated one which indicated that the protein had been modified during expression. The association between the recombinant protein and bovine milk caseins was explored at the molecular level using the surface plasmon resonance technique. Results showed a calciummediated interaction between the recombinant protein and the phosphorylated caseins. This selective interaction was not noted between the human myelin basic protein and milk caseins which indicated mammary gland-related posttranslational modifications, most likely phosphorylation. The co-expression of the recombinant protein and caseins in the mammary gland, along with the ability of the recombinant protein to form calcium bridges with caseins explained its association with the casein micellar phase in the transgenic milk. Despite this and owing to the low expression levels of the recombinant protein in milk, light scattering investigations using diffusing wave spectroscopy showed no significant differences between the transgenic and the non-transgenic milk samples with respect to the average micelle size and the micelle surface charges.

Milk is provided to new borns as their first food source and it contains essential nutrients, vitamins and other beneficial components, such as enzymes and antibodies that are required for rapid growth and development of the new born and for sustained growth over time. Milk contains two main types of proteins; casein proteins and whey proteins. Although casein proteins account for up to 80% of the proteins found in bovine milk, it is the whey protein that has become of high interest because of its bioactive content. Whey, a very watery mixture of lactose, proteins, minerals and trace amounts of fat, is formed from milk when the milk is coagulated and/or the casein proteins are removed from the milk. Bovine whey protein, including both the acidic and basic fractions (low and high isoelectric point, respectively), has previously been studied in vitro (cell based) and in vivo (using rats) for its impact on bone to determine if it can help improve bone mineral density and help reduce the risk of developing bone diseases, such as osteoporosis. Bone is constantly undergoing a remodelling process of being dissolved and reformed and the two main cell types responsible for this bone remodelling process are mature osteoclasts, which dissolve (resorb) bone, and osteoblasts, which reform the bone. Prior work has shown that acidic protein fractions derived from different sources of whey protein concentrate (WPC) have both in vivo and in vitro activity on bone, particularly anti-resorptive properties. However, the component(s) which confer activity have not yet been identified. In this thesis, work was undertaken to better understand the analytical composition of three types of WPC (cheese, mineral acid and lactic acid) and their associated acidic protein fractions and relate this to bone activity in the hope of identifying where the activity lies. Bone activity was assessed using in vitro screening with osteoblast cells (MC3T3-E1) and osteoclast cells (RAW 264.7). Comparison of the cell-based bone activity of the parent WPCs and corresponding acidic fractions indicated that the acidic fractions derived from both mineral acid and lactic WPC were superior in their ability to inhibit osteoclast development. Although compositional data was complex and definitive correlations with both bone bioactivities could not be made, it appeared that elements common to both the acidic fractions were a higher proportion of GLYCAM-1 and bone sialoprotein-1 (osteopontin). Further studies to more closely investigate the bone bioactivity of the acidic fractions are warranted.

Milk is provided to new borns as their first food source and it contains essential nutrients, vitamins and other beneficial components, such as enzymes and antibodies that are required for rapid growth and development of the new born and for sustained growth over time. Milk contains two main types of proteins; casein proteins and whey proteins. Although casein proteins account for up to 80% of the proteins found in bovine milk, it is the whey protein that has become of high interest because of its bioactive content. Whey, a very watery mixture of lactose, proteins, minerals and trace amounts of fat, is formed from milk when the milk is coagulated and/or the casein proteins are removed from the milk. Bovine whey protein, including both the acidic and basic fractions (low and high isoelectric point, respectively), has previously been studied in vitro (cell based) and in vivo (using rats) for its impact on bone to determine if it can help improve bone mineral density and help reduce the risk of developing bone diseases, such as osteoporosis. Bone is constantly undergoing a remodelling process of being dissolved and reformed and the two main cell types responsible for this bone remodelling process are mature osteoclasts, which dissolve (resorb) bone, and osteoblasts, which reform the bone. Prior work has shown that acidic protein fractions derived from different sources of whey protein concentrate (WPC) have both in vivo and in vitro activity on bone, particularly anti-resorptive properties. However, the component(s) which confer activity have not yet been identified. In this thesis, work was undertaken to better understand the analytical composition of three types of WPC (cheese, mineral acid and lactic acid) and their associated acidic protein fractions and relate this to bone activity in the hope of identifying where the activity lies. Bone activity was assessed using in vitro screening with osteoblast cells (MC3T3-E1) and osteoclast cells (RAW 264.7). Comparison of the cell-based bone activity of the parent WPCs and corresponding acidic fractions indicated that the acidic fractions derived from both mineral acid and lactic WPC were superior in their ability to inhibit osteoclast development. Although compositional data was complex and definitive correlations with both bone bioactivities could not be made, it appeared that elements common to both the acidic fractions were a higher proportion of GLYCAM-1 and bone sialoprotein-1 (osteopontin). Further studies to more closely investigate the bone bioactivity of the acidic fractions are warranted.

Milk is provided to new borns as their first food source and it contains essential nutrients, vitamins and other beneficial components, such as enzymes and antibodies that are required for rapid growth and development of the new born and for sustained growth over time. Milk contains two main types of proteins; casein proteins and whey proteins. Although casein proteins account for up to 80% of the proteins found in bovine milk, it is the whey protein that has become of high interest because of its bioactive content. Whey, a very watery mixture of lactose, proteins, minerals and trace amounts of fat, is formed from milk when the milk is coagulated and/or the casein proteins are removed from the milk. Bovine whey protein, including both the acidic and basic fractions (low and high isoelectric point, respectively), has previously been studied in vitro (cell based) and in vivo (using rats) for its impact on bone to determine if it can help improve bone mineral density and help reduce the risk of developing bone diseases, such as osteoporosis. Bone is constantly undergoing a remodelling process of being dissolved and reformed and the two main cell types responsible for this bone remodelling process are mature osteoclasts, which dissolve (resorb) bone, and osteoblasts, which reform the bone. Prior work has shown that acidic protein fractions derived from different sources of whey protein concentrate (WPC) have both in vivo and in vitro activity on bone, particularly anti-resorptive properties. However, the component(s) which confer activity have not yet been identified. In this thesis, work was undertaken to better understand the analytical composition of three types of WPC (cheese, mineral acid and lactic acid) and their associated acidic protein fractions and relate this to bone activity in the hope of identifying where the activity lies. Bone activity was assessed using in vitro screening with osteoblast cells (MC3T3-E1) and osteoclast cells (RAW 264.7). Comparison of the cell-based bone activity of the parent WPCs and corresponding acidic fractions indicated that the acidic fractions derived from both mineral acid and lactic WPC were superior in their ability to inhibit osteoclast development. Although compositional data was complex and definitive correlations with both bone bioactivities could not be made, it appeared that elements common to both the acidic fractions were a higher proportion of GLYCAM-1 and bone sialoprotein-1 (osteopontin). Further studies to more closely investigate the bone bioactivity of the acidic fractions are warranted.

Milk is provided to new borns as their first food source and it contains essential nutrients, vitamins and other beneficial components, such as enzymes and antibodies that are required for rapid growth and development of the new born and for sustained growth over time. Milk contains two main types of proteins; casein proteins and whey proteins. Although casein proteins account for up to 80% of the proteins found in bovine milk, it is the whey protein that has become of high interest because of its bioactive content. Whey, a very watery mixture of lactose, proteins, minerals and trace amounts of fat, is formed from milk when the milk is coagulated and/or the casein proteins are removed from the milk. Bovine whey protein, including both the acidic and basic fractions (low and high isoelectric point, respectively), has previously been studied in vitro (cell based) and in vivo (using rats) for its impact on bone to determine if it can help improve bone mineral density and help reduce the risk of developing bone diseases, such as osteoporosis. Bone is constantly undergoing a remodelling process of being dissolved and reformed and the two main cell types responsible for this bone remodelling process are mature osteoclasts, which dissolve (resorb) bone, and osteoblasts, which reform the bone. Prior work has shown that acidic protein fractions derived from different sources of whey protein concentrate (WPC) have both in vivo and in vitro activity on bone, particularly anti-resorptive properties. However, the component(s) which confer activity have not yet been identified. In this thesis, work was undertaken to better understand the analytical composition of three types of WPC (cheese, mineral acid and lactic acid) and their associated acidic protein fractions and relate this to bone activity in the hope of identifying where the activity lies. Bone activity was assessed using in vitro screening with osteoblast cells (MC3T3-E1) and osteoclast cells (RAW 264.7). Comparison of the cell-based bone activity of the parent WPCs and corresponding acidic fractions indicated that the acidic fractions derived from both mineral acid and lactic WPC were superior in their ability to inhibit osteoclast development. Although compositional data was complex and definitive correlations with both bone bioactivities could not be made, it appeared that elements common to both the acidic fractions were a higher proportion of GLYCAM-1 and bone sialoprotein-1 (osteopontin). Further studies to more closely investigate the bone bioactivity of the acidic fractions are warranted.

Milk is provided to new borns as their first food source and it contains essential nutrients, vitamins and other beneficial components, such as enzymes and antibodies that are required for rapid growth and development of the new born and for sustained growth over time. Milk contains two main types of proteins; casein proteins and whey proteins. Although casein proteins account for up to 80% of the proteins found in bovine milk, it is the whey protein that has become of high interest because of its bioactive content. Whey, a very watery mixture of lactose, proteins, minerals and trace amounts of fat, is formed from milk when the milk is coagulated and/or the casein proteins are removed from the milk. Bovine whey protein, including both the acidic and basic fractions (low and high isoelectric point, respectively), has previously been studied in vitro (cell based) and in vivo (using rats) for its impact on bone to determine if it can help improve bone mineral density and help reduce the risk of developing bone diseases, such as osteoporosis. Bone is constantly undergoing a remodelling process of being dissolved and reformed and the two main cell types responsible for this bone remodelling process are mature osteoclasts, which dissolve (resorb) bone, and osteoblasts, which reform the bone. Prior work has shown that acidic protein fractions derived from different sources of whey protein concentrate (WPC) have both in vivo and in vitro activity on bone, particularly anti-resorptive properties. However, the component(s) which confer activity have not yet been identified. In this thesis, work was undertaken to better understand the analytical composition of three types of WPC (cheese, mineral acid and lactic acid) and their associated acidic protein fractions and relate this to bone activity in the hope of identifying where the activity lies. Bone activity was assessed using in vitro screening with osteoblast cells (MC3T3-E1) and osteoclast cells (RAW 264.7). Comparison of the cell-based bone activity of the parent WPCs and corresponding acidic fractions indicated that the acidic fractions derived from both mineral acid and lactic WPC were superior in their ability to inhibit osteoclast development. Although compositional data was complex and definitive correlations with both bone bioactivities could not be made, it appeared that elements common to both the acidic fractions were a higher proportion of GLYCAM-1 and bone sialoprotein-1 (osteopontin). Further studies to more closely investigate the bone bioactivity of the acidic fractions are warranted.