Malaria is an infectious disease caused by various forms of the Plasmodium parasite. It is responsible for thousands of deaths yearly with 90 % of those deaths being in sub-Saharan Africa, thus making it a disease of global importance. The global burden of malaria is worsened by resistance to current treatment, a lack in funding and limited research outputs. More alternative ways of treatment must be explored and may include the co-formulation of antimalarial drug substances as well as alternative ways of drug delivery. Antifolates are drugs which interfere with an organism’s folate metabolism by inhibiting dihydropteroate synthase (DHPS) or dihydrofolate reductase (DHFR). Dapsone is a synthetic sulfone which has a mechanism of action that is very similar to that of sulphonamides. The mechanism of action is characterised by the inhibition of folic acid synthesis through the inhibition of dihydropteroate synthase (DHPS). Another antifolate drug, proguanil, is the prodrug of cycloguanil. Its mechanism involves the inhibition of dihydrofolate reductase (DHFR), thus inhibiting the malaria parasite to metabolise folates and therefore stunting its growth. Unfortunately, dapsone has a serious side-effect in people with a deficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD) causing oxidative stress on the red blood cells leading to the rupturing of these cells. The main objective of this study was to formulate and characterise TMC-TPP microparticles loaded with the effective but toxic drug combination of dapsone and proguanil and to determine if these drug-containing microparticles had in vivo efficacy against malaria. N-trimethyl chitosan chloride (TMC), a partially quaternised chitosan derivative, shows good water solubility across a wide pH range thus having mucoadhesive properties and excellent absorption enhancing effects even at neutral pH. A faster, more efficient microwave irradiation method was developed as an alternative to the conventional synthesising method of TMC. TMC with the same degree of quaternisation (DQ), ± 60 %, was obtained in a quarter of the reaction time (30 min) by using the newly developed method. The TMC synthesised with the microwave irradiation method also exhibited less degradation of the polymer structure, thus limiting the chance for the formation of any unwanted by-products (Omethylation, N,N-dimethylation and N-monomethylation). The formation of complexes by ionotropic gelation between TMC and oppositely charged macromolecules, such as tripolyphosphate (TPP), has been utilised to prepare microparticles which are a suitable drug delivery system for the dapsone-proguanil combination. Both these drugs were successfully entrapped. These particles were characterised and the in vivo efficacy against the malaria parasites was determined. The microparticles with both the drugs, separately and in combination, displayed similar or better in vivo efficacy when compared to the drugs without the TMC microparticles. An in vitro dissolution study was also performed by subjecting the dapsone and proguanil TMC formulations to 0.1N HCl dissolution medium. Samples were withdrawn after predetermined time points and the drug concentration was determined with HPLC. It was found that the TMC microparticles resulted in a sustained release profile since only 73.00 ± 1.70 % (dapsone) and 55.00 ± 1.90 % (proguanil) was released after 150 minutes. The in vivo bioavailability of the dapsone and proguanil TMC formulations was evaluated in mice by collecting blood samples at predetermined time points and analysing the samples with a sensitive and accurate LC-MS/MS method. The in vivo bioavailability of the dapsone TMC formulation relative to the normal dapsone formulation was found to be 244 % and 123 % for the proguanil TMC formulation relative to the normal proguanil formulation. These TMC-TPP microparticles formulations showed better in vivo efficacy and bioavailability when compared to the normal formulation. Together with the sustained release, these formulations may be a promising cheaper and more effective treatment against malaria. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Malaria is an infectious disease caused by various forms of the Plasmodium parasite. It is responsible for thousands of deaths yearly with 90 % of those deaths being in sub-Saharan Africa, thus making it a disease of global importance. The global burden of malaria is worsened by resistance to current treatment, a lack in funding and limited research outputs. More alternative ways of treatment must be explored and may include the co-formulation of antimalarial drug substances as well as alternative ways of drug delivery. Antifolates are drugs which interfere with an organism’s folate metabolism by inhibiting dihydropteroate synthase (DHPS) or dihydrofolate reductase (DHFR). Dapsone is a synthetic sulfone which has a mechanism of action that is very similar to that of sulphonamides. The mechanism of action is characterised by the inhibition of folic acid synthesis through the inhibition of dihydropteroate synthase (DHPS). Another antifolate drug, proguanil, is the prodrug of cycloguanil. Its mechanism involves the inhibition of dihydrofolate reductase (DHFR), thus inhibiting the malaria parasite to metabolise folates and therefore stunting its growth. Unfortunately, dapsone has a serious side-effect in people with a deficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD) causing oxidative stress on the red blood cells leading to the rupturing of these cells. The main objective of this study was to formulate and characterise TMC-TPP microparticles loaded with the effective but toxic drug combination of dapsone and proguanil and to determine if these drug-containing microparticles had in vivo efficacy against malaria. N-trimethyl chitosan chloride (TMC), a partially quaternised chitosan derivative, shows good water solubility across a wide pH range thus having mucoadhesive properties and excellent absorption enhancing effects even at neutral pH. A faster, more efficient microwave irradiation method was developed as an alternative to the conventional synthesising method of TMC. TMC with the same degree of quaternisation (DQ), ± 60 %, was obtained in a quarter of the reaction time (30 min) by using the newly developed method. The TMC synthesised with the microwave irradiation method also exhibited less degradation of the polymer structure, thus limiting the chance for the formation of any unwanted by-products (Omethylation, N,N-dimethylation and N-monomethylation). The formation of complexes by ionotropic gelation between TMC and oppositely charged macromolecules, such as tripolyphosphate (TPP), has been utilised to prepare microparticles which are a suitable drug delivery system for the dapsone-proguanil combination. Both these drugs were successfully entrapped. These particles were characterised and the in vivo efficacy against the malaria parasites was determined. The microparticles with both the drugs, separately and in combination, displayed similar or better in vivo efficacy when compared to the drugs without the TMC microparticles. An in vitro dissolution study was also performed by subjecting the dapsone and proguanil TMC formulations to 0.1N HCl dissolution medium. Samples were withdrawn after predetermined time points and the drug concentration was determined with HPLC. It was found that the TMC microparticles resulted in a sustained release profile since only 73.00 ± 1.70 % (dapsone) and 55.00 ± 1.90 % (proguanil) was released after 150 minutes. The in vivo bioavailability of the dapsone and proguanil TMC formulations was evaluated in mice by collecting blood samples at predetermined time points and analysing the samples with a sensitive and accurate LC-MS/MS method. The in vivo bioavailability of the dapsone TMC formulation relative to the normal dapsone formulation was found to be 244 % and 123 % for the proguanil TMC formulation relative to the normal proguanil formulation. These TMC-TPP microparticles formulations showed better in vivo efficacy and bioavailability when compared to the normal formulation. Together with the sustained release, these formulations may be a promising cheaper and more effective treatment against malaria. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Over the past couple of years there has been rapid progress in the development and design of safe and effective delivery systems for the administration of protein and peptide drugs. The effective delivery of these type of drugs are not always as simple as one may think, due to various inherent characteristics of these compounds. Due to the hydrophilic nature and molecular size of peptide and protein drugs, such as recombinant human growth hormone, they are poorly absorbed across mucosal epithelia, both transcellularly and paracellularly. This problem can be overcome by the inclusion of absorption enhancers in peptide and protein drug formulations but this is not necessarily the best method to follow. This investigation focussed specifically on the evaluation of the ability of the PheroidTM carrier system to transport recombinant human growth hormone across mucosal epithelia especially when administered via the nasal cavity. The PheroidTM delivery system is a patented system consisting of a unique submicron emulsion type formulation. The PheroidTM delivery system, based on PheroidTM technology, will for ease of reading be called Pheroid(s) only throughout the rest of this dissertation. The Pheroid carrier system is a unique microcolloidal drug delivery system. A Pheroid is a stable structure within a novel therapeutic system which can be manipulated in terms of morphology, structure, size and function. Pheroids consist mainly of plant and essential fatty acids and can entrap, transport and deliver pharmacologically active compounds and other useful substances to the desired site of action. The specific objectives of this study can be summarised as follows: a literature study on Pheroid technology; a literature study on chitosan and N-trimethyl chitosan chloride; a literature study on recombinant human growth hormone (somatropin); a literature study on nasal drug administration; formulation of a suitable Pheroid carrier; entrapment of somatropin in the Pheroid carrier, and in vivo evaluation of nasal absorption of somatropin in Sprague-Dawley rats. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Approximately 350 million people worldwide suffer from diabetes mellitus (DM) and this number increases yearly. Since the discovery and clinical application of insulin in 1921, subcutaneous injections have been the standard treatment for DM. Because insulin is hydrophilic and has a high molecular weight and low bioavailability, this molecule is poorly absorbed if administered orally. The aim of this study is to evaluate nasal delivery systems for insulin, using Sprague Dawley rats as the nasal absorption model. Pheroid technology and N-trimethyl chitosan chloride (TMC) with different dosages of insulin (4, 8 and 12 IU/kg bodyweight insulin) was administered in the left nostril of the rat by using a micropipette. Pheroid technology is a patented (North-West University) carrier system consisting of a unique oil/water emulsion that actively transports drug actives through various physiological barriers. These formulations were administered nasally to rats in a volume of 100 p/kg bodyweight in different types of Pheroids (vesicles, with a size of 1.7 1 - 1.94 pm and microsponges, with a size of 5.7 1 - 8.25 pm). The systemic absorption of insulin was monitored by measuring arterial blood glucose levels over a period of 3 hours. The TMC formulation with 4 IU/kg insulin produced clinically relevant levels of insulin in the blood and as a result also the maximal hypoglycaemic effect. TMC is a quaternary derivative of chitosan and is able to enhance the absorption of various peptide drugs by opening tight junctions between epithelial cells. Pheroid formulations were also effective in lowering blood glucose levels but only at higher doses (8 and 12 IU/kg) of insulin. This study indicated that Pheroid rnicrosponges had a faster onset of action and a slightly better absorption of insulin when compared to Pheroid vesicles, but many more studies are needed in this field. Although the results of this study with absorption enhancers are encouraging, nasal insulin bioavailability is still very low, and the Pheroid formulations and long-term safety of nasal insulin therapy have yet to be investigated. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Advances in biotechnology and recombinant technologies have lead to the production of several classes of new drugs such as peptide and protein drugs. These compounds are mostly indicated for chronic use but their inherent characteristics such as size, polarity and stability prevent them from incorporation in novel dosage forms. The bioavailability of nearly all peptide drugs is very low due to poor absorption from the administration site. Several challenges confront the pharmaceutical scientist in developing effective and innovative dosage forms for these classes of drugs. A lot of attention has been given to the nasal route of drug administration for delivery of peptide drugs. The availability of several promising classes of absorption enhancers and new drug delivery technologies has also prompt scientists to develop new delivery systems for nasal administration of peptide drugs. It has been shown in recent years that N-trimethyl chitosan chloride (TMC), a quaternary derivative of chitosan, is effective in enhancing the absorption of several peptide drugs, both in the peroral route and in the nasal route of drug administration. Early indications are that new drug delivery technologies such as Pheroid technology will also be able to enhance peptide drug absorption in the nasal route. The aim of this study was to evaluate and compare the absorption enhancing abilities of TMC and Pheroid technology in the nasal delivery of calcitonin, a peptide hormone with low bioavailability. Pheroid vesicles and Pheroid microsponges were prepared and characterized for their morphology and size distribution. Calcitonin was entrapped into these vesicles and microsponges and TMC and TMO solutions (0.5 % w/v), containing calcitonin, was also prepared. These formulations were administered nasally to rats in a volume of 100 μl/kg body-weight to obtain a final concentration of 10 IU/kg body-weight of calcitonin. Plasma calcitonin and calcium levels were determined over a period of 3 hours. The results of this study clearly indicated that both Pheroid formulations and the TMC formulation increase the nasal absorption of calcitonin with a resulting decrease in plasma calcium levels, indicating an increased absorption of calcitonin. The highest increase in calcitonin absorption was obtained with the TMC formulation and this was explained by the difference in the mechanism of action in enhancing peptide absorption between TMC and Pheroid technology. It was concluded that Pheroid technology is also a potent system to enhance peptide drug delivery and that the exact mechanism of action should be investigated further. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

The purpose of this research project was to develop and characterise matrix type microspheres prepared from Eudragit® L100, containing insulin as model peptide drug as well as an absorption enhancer, N-trimethyl chitosan chloride (TMC), to improve intestinal absorption via the paracellular route. Insulin loaded microspheres were prepared using a single water in oil emulsification/evaporation method in accordance with a fractional factorial design (23) and subsequently characterised in terms of morphology as well as internal structure. Also, insulin and TMC loading were determined using a high pressure liquid chromatography analysis (HPLC) and colorimetric assay, respectively. Scanning electron microscopic characterisation revealed that most microsphere formulations showed a spherical shape and smooth surface with a sponge-like internal structure as well as relatively good homogeneity in terms of size distribution. Insulin loading ranged from 27.9 ± 14.25 – 52.4 ± 2.72% between the different formulations. TMC loading was lower than for insulin and ranged from 29.1 ± 3.3 - 37.7 ± 2.3% between the different formulations. The pronounced difference in insulin and TMC loading between the microsphere formulations is probably the result of the multitude parameters involved as well as the complex physicochemical processes which govern emulsification/solvent evaporation. Based on the microsphere characterisation results, two formulations were selected (i.e. B and F) for further characterisation (i.e. particle size distribution, dissolution behaviour, and enteric nature) and for in vitro evaluation of insulin transport across excised Fischer (FSR) rat intestinal tissue using a Sweetana-Grass diffusion chamber. Particle size analysis by means of laser light diffraction of the two selected microsphere formulations revealed that the mean particle size (based on volume) ranged from 135.7 ± 41.05 to 157.3 ± 31.74 m. Dissolution results for microsphere Formulations B and F revealed that both insulin and TMC were released from the microsphere formulations in an alkaline environment (pH 7.4). The mean dissolution time (MDT) for insulin ranged from 34.5 ± 4.01 to 42.6 ± 9.06 min, while the MDT for TMC ranged from 1.2 ± 1.73 to 6.8 ± 6.42 min. Statistical analysis revealed no significant differences in the MDT of either insulin or TMC (p-value > 0.05) between the two formulations, although the difference between insulin and TMC of each formulation was significant (p-value < 0.05). Microsphere formulations B and F released 36.92 and 48.21% of their total drug content over a period of 1 h in 0.1 M HCl. Microsphere Formulation B showed 8.3 ± 0.52% and formulation F 8.9 ± 2.26% transport of the initial insulin dose after a period of 120 min across excised rat intestinal tissue. The increase in insulin transport by the microsphere formulations compared to that of the control group (i.e. insulin alone) correlated well with the decrease in transepithelial electrical resistance (TEER) caused by the microsphere formulations. The transport of insulin from Formulations B and F represented transport enhancement ratios of 10.67 and 9.68, respectively. Insulin loaded EudragitL100 microspheres containing TMC were successfully prepared by emulsification/solvent evaporation that demonstrated promising potential to serve as oral drug delivery systems for insulin. The microspheres exhibited improved insulin permeability across intestinal epithelial tissue; however, its enteric properties should be improved and clinical effectiveness need to be confirmed by future in vivo studies. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

The purpose of this research project was to develop and characterise matrix type microspheres prepared from Eudragit® L100, containing insulin as model peptide drug as well as an absorption enhancer, N-trimethyl chitosan chloride (TMC), to improve intestinal absorption via the paracellular route. Insulin loaded microspheres were prepared using a single water in oil emulsification/evaporation method in accordance with a fractional factorial design (23) and subsequently characterised in terms of morphology as well as internal structure. Also, insulin and TMC loading were determined using a high pressure liquid chromatography analysis (HPLC) and colorimetric assay, respectively. Scanning electron microscopic characterisation revealed that most microsphere formulations showed a spherical shape and smooth surface with a sponge-like internal structure as well as relatively good homogeneity in terms of size distribution. Insulin loading ranged from 27.9 ± 14.25 – 52.4 ± 2.72% between the different formulations. TMC loading was lower than for insulin and ranged from 29.1 ± 3.3 - 37.7 ± 2.3% between the different formulations. The pronounced difference in insulin and TMC loading between the microsphere formulations is probably the result of the multitude parameters involved as well as the complex physicochemical processes which govern emulsification/solvent evaporation. Based on the microsphere characterisation results, two formulations were selected (i.e. B and F) for further characterisation (i.e. particle size distribution, dissolution behaviour, and enteric nature) and for in vitro evaluation of insulin transport across excised Fischer (FSR) rat intestinal tissue using a Sweetana-Grass diffusion chamber. Particle size analysis by means of laser light diffraction of the two selected microsphere formulations revealed that the mean particle size (based on volume) ranged from 135.7 ± 41.05 to 157.3 ± 31.74 m. Dissolution results for microsphere Formulations B and F revealed that both insulin and TMC were released from the microsphere formulations in an alkaline environment (pH 7.4). The mean dissolution time (MDT) for insulin ranged from 34.5 ± 4.01 to 42.6 ± 9.06 min, while the MDT for TMC ranged from 1.2 ± 1.73 to 6.8 ± 6.42 min. Statistical analysis revealed no significant differences in the MDT of either insulin or TMC (p-value > 0.05) between the two formulations, although the difference between insulin and TMC of each formulation was significant (p-value < 0.05). Microsphere formulations B and F released 36.92 and 48.21% of their total drug content over a period of 1 h in 0.1 M HCl. Microsphere Formulation B showed 8.3 ± 0.52% and formulation F 8.9 ± 2.26% transport of the initial insulin dose after a period of 120 min across excised rat intestinal tissue. The increase in insulin transport by the microsphere formulations compared to that of the control group (i.e. insulin alone) correlated well with the decrease in transepithelial electrical resistance (TEER) caused by the microsphere formulations. The transport of insulin from Formulations B and F represented transport enhancement ratios of 10.67 and 9.68, respectively. Insulin loaded EudragitL100 microspheres containing TMC were successfully prepared by emulsification/solvent evaporation that demonstrated promising potential to serve as oral drug delivery systems for insulin. The microspheres exhibited improved insulin permeability across intestinal epithelial tissue; however, its enteric properties should be improved and clinical effectiveness need to be confirmed by future in vivo studies. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

Approximately 350 million people worldwide suffer from diabetes mellitus (DM) and this number increases yearly. Since the discovery and clinical application of insulin in 1921, subcutaneous injections have been the standard treatment for DM. Because insulin is hydrophilic and has a high molecular weight and low bioavailability, this molecule is poorly absorbed if administered orally. The aim of this study is to evaluate nasal delivery systems for insulin, using Sprague Dawley rats as the nasal absorption model. Pheroid technology and N-trimethyl chitosan chloride (TMC) with different dosages of insulin (4, 8 and 12 IU/kg bodyweight insulin) was administered in the left nostril of the rat by using a micropipette. Pheroid technology is a patented (North-West University) carrier system consisting of a unique oil/water emulsion that actively transports drug actives through various physiological barriers. These formulations were administered nasally to rats in a volume of 100 p/kg bodyweight in different types of Pheroids (vesicles, with a size of 1.7 1 - 1.94 pm and microsponges, with a size of 5.7 1 - 8.25 pm). The systemic absorption of insulin was monitored by measuring arterial blood glucose levels over a period of 3 hours. The TMC formulation with 4 IU/kg insulin produced clinically relevant levels of insulin in the blood and as a result also the maximal hypoglycaemic effect. TMC is a quaternary derivative of chitosan and is able to enhance the absorption of various peptide drugs by opening tight junctions between epithelial cells. Pheroid formulations were also effective in lowering blood glucose levels but only at higher doses (8 and 12 IU/kg) of insulin. This study indicated that Pheroid rnicrosponges had a faster onset of action and a slightly better absorption of insulin when compared to Pheroid vesicles, but many more studies are needed in this field. Although the results of this study with absorption enhancers are encouraging, nasal insulin bioavailability is still very low, and the Pheroid formulations and long-term safety of nasal insulin therapy have yet to be investigated. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Advances in biotechnology and recombinant technologies have lead to the production of several classes of new drugs such as peptide and protein drugs. These compounds are mostly indicated for chronic use but their inherent characteristics such as size, polarity and stability prevent them from incorporation in novel dosage forms. The bioavailability of nearly all peptide drugs is very low due to poor absorption from the administration site. Several challenges confront the pharmaceutical scientist in developing effective and innovative dosage forms for these classes of drugs. A lot of attention has been given to the nasal route of drug administration for delivery of peptide drugs. The availability of several promising classes of absorption enhancers and new drug delivery technologies has also prompt scientists to develop new delivery systems for nasal administration of peptide drugs. It has been shown in recent years that N-trimethyl chitosan chloride (TMC), a quaternary derivative of chitosan, is effective in enhancing the absorption of several peptide drugs, both in the peroral route and in the nasal route of drug administration. Early indications are that new drug delivery technologies such as Pheroid technology will also be able to enhance peptide drug absorption in the nasal route. The aim of this study was to evaluate and compare the absorption enhancing abilities of TMC and Pheroid technology in the nasal delivery of calcitonin, a peptide hormone with low bioavailability. Pheroid vesicles and Pheroid microsponges were prepared and characterized for their morphology and size distribution. Calcitonin was entrapped into these vesicles and microsponges and TMC and TMO solutions (0.5 % w/v), containing calcitonin, was also prepared. These formulations were administered nasally to rats in a volume of 100 μl/kg body-weight to obtain a final concentration of 10 IU/kg body-weight of calcitonin. Plasma calcitonin and calcium levels were determined over a period of 3 hours. The results of this study clearly indicated that both Pheroid formulations and the TMC formulation increase the nasal absorption of calcitonin with a resulting decrease in plasma calcium levels, indicating an increased absorption of calcitonin. The highest increase in calcitonin absorption was obtained with the TMC formulation and this was explained by the difference in the mechanism of action in enhancing peptide absorption between TMC and Pheroid technology. It was concluded that Pheroid technology is also a potent system to enhance peptide drug delivery and that the exact mechanism of action should be investigated further. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

Over the past couple of years there has been rapid progress in the development and design of safe and effective delivery systems for the administration of protein and peptide drugs. The effective delivery of these type of drugs are not always as simple as one may think, due to various inherent characteristics of these compounds. Due to the hydrophilic nature and molecular size of peptide and protein drugs, such as recombinant human growth hormone, they are poorly absorbed across mucosal epithelia, both transcellularly and paracellularly. This problem can be overcome by the inclusion of absorption enhancers in peptide and protein drug formulations but this is not necessarily the best method to follow. This investigation focussed specifically on the evaluation of the ability of the PheroidTM carrier system to transport recombinant human growth hormone across mucosal epithelia especially when administered via the nasal cavity. The PheroidTM delivery system is a patented system consisting of a unique submicron emulsion type formulation. The PheroidTM delivery system, based on PheroidTM technology, will for ease of reading be called Pheroid(s) only throughout the rest of this dissertation. The Pheroid carrier system is a unique microcolloidal drug delivery system. A Pheroid is a stable structure within a novel therapeutic system which can be manipulated in terms of morphology, structure, size and function. Pheroids consist mainly of plant and essential fatty acids and can entrap, transport and deliver pharmacologically active compounds and other useful substances to the desired site of action. The specific objectives of this study can be summarised as follows: a literature study on Pheroid technology; a literature study on chitosan and N-trimethyl chitosan chloride; a literature study on recombinant human growth hormone (somatropin); a literature study on nasal drug administration; formulation of a suitable Pheroid carrier; entrapment of somatropin in the Pheroid carrier, and in vivo evaluation of nasal absorption of somatropin in Sprague-Dawley rats. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.