Malaria is currently a huge treat worldwide, as far as infections are concerned, and is responsible for thousands of deaths per annum. The dilemma associated with the development of anti–malarial drug resistance over the past few decades should be addressed as a matter of urgency. Novel drug delivery systems should be developed in order to employ new and existing anti–malarial drugs in the treatment and management of malaria. The aim of these delivery systems should include an improvement in the efficacy, specificity, acceptability and therapeutic index of anti–malarial drugs. Previous studies have suggested that liposomes have the ability to encapsulate, protect and to promote the sustained release of anti–malarial drugs. Two liposome formulations, namely liposomes and chloroquine entrapped in liposomes, were formulated during this thesis and evaluated by conducting a stability study and an in vitro study with the main focus on cell viability. The stability study consisted of a series of stability tests regarding the stability of nine liposome and nine chloroquine entrapped in liposome formulations over a period of twelve weeks. The in vitro study included three assays such as a reactive oxygen species assay, a lipid peroxidation assay and a hemolysis assay. The aims of these studies included the manufacturing of liposomes, the incorporation of chloroquine into liposomes, the determination of the stability of the formulations as well as the evaluation of the possible in vitro toxicity of liposomes. Results obtained from these studies revealed that liposomes remained more stable over the stability study period in comparison to chloroquine entrapped in liposomes. The entrapment of chloroquine within liposomes was possible, although the initial entrapment efficiency (%) of 14.55 % was much too low. The production of reactive oxygen species occurred to a small extent in the red blood cells and the infected red blood cells. Equal amounts of reactive oxygen species (%) was observed within both the red blood cells and the infected red blood cells with a maximum value of 23.27 % in the presence of the chloroquine entrapped in liposomes at varying concentrations. Red blood cells experienced the highest degree of lipid peroxidation (%) in the presence of chloroquine, at varying concentrations, entrapped in liposomes. The maximum amount of lipid peroxidation (%) was 79.61 %. No significant degree of hemolysis (%) was observed in the red blood cells neither in the presence of the liposomes nor in the presence of the chloroquine entrapped in liposomes at varying concentrations. It can be concluded that liposomes are a more stable formulation and have less toxic effects on red blood cells and infected red blood cells in comparison to the chloroquine entrapped in liposome formulations. Future studies should investigate the possibility of a more stable and less toxic chloroquine entrapped in liposome formulation. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Malaria is a curable disease that claims nearly one million lives each year. Problems with the treatment of malaria arise as resistance spreads and new treatment options are becoming less effective. The need for new treatments are of the utmost importance. Liposomes combined with antimalarials are a new avenue for research as liposomes can increase the efficacy of drugs against pathogens, as well as decreasing toxicity. Amodiaquine is a drug with known toxicity issues, but has proven to be effective and is, therefore, a prime candidate to be incorporated into the liposomal drug delivery system. The aim of this study was to prepare, characterize and evaluate the toxicity of the liposomes with incorporated amodiaquine. The solubility of amodiaquine was determined and liposomes formulated with, and without, amodiaquine entrapped. Accelerated stability studies (at 5 'C, 25 'C with relative humidity of 60% and 40 'C with a relative humidity of 40%) were conducted during which the size, pH, morphology and the entrapment efficacy was determined. The toxicity was determined in vitro by analysing the levels of reactive oxidative species and lipid peroxidation caused by the formulations to erythrocytes infected with P. falciparum as well as uninfected erythrocytes with flow cytometry. The solubility study of amodiaquine in different pH buffers showed that amodiaquine was more soluble at lower pH values. Solubility in solution with pH 4.5 was 36.3359 ± 0.7904mg/ml when compared to the solubility at pH 6.8, which was 15.6052 ± 1.1126 mg/ml. A buffer with a pH of 6 was used to ensure adequate solubility and acceptable compatibility with cells. Liposomes with incorporated amodiaquine were formulated with entrapment efficacies starting at 29.038 ± 2.599% and increasing to 51.914 ± 1.683%. The accelerated stability studies showed the median sizes and span values remained constant for both liposome and amodiaquine incorporated liposomes at 5 'C. The higher temperatures, i.e. 25 'C and 40 'C, displayed increases in the median size, and decreases in the span for both formulations. The conclusion can, therefore, be made that both liposome and amodiaquine incorporated liposomes are stable at lower temperatures. The entrapment efficacy increased from initial values to nearly 100% during the course of the stability study. This was attributed to amodiaquine precipitating from the solution. The pH values of the liposomes and amodiaquine incorporated liposomes remained constant for each formulation; though the amodiaquine incorporated liposomes had a lower starting pH, the formulations are both thought to be stable in terms of the pH. Toxicity studies revealed low levels of reactive oxygen species as well as low levels of lipid peroxidation for both liposome and amodiaquine incorporated liposomes, on both erythrocyte and Plasmodium infected erythrocytes. From the toxicity studies it can be concluded that liposomes and amodiaquine incorporated liposomes are not toxic to erythrocytes and infected erythrocytes. It was concluded that liposomes incorporating amodiaquine could possibly be used as a treatment option for malaria. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Malaria is currently a huge treat worldwide, as far as infections are concerned, and is responsible for thousands of deaths per annum. The dilemma associated with the development of anti–malarial drug resistance over the past few decades should be addressed as a matter of urgency. Novel drug delivery systems should be developed in order to employ new and existing anti–malarial drugs in the treatment and management of malaria. The aim of these delivery systems should include an improvement in the efficacy, specificity, acceptability and therapeutic index of anti–malarial drugs. Previous studies have suggested that liposomes have the ability to encapsulate, protect and to promote the sustained release of anti–malarial drugs. Two liposome formulations, namely liposomes and chloroquine entrapped in liposomes, were formulated during this thesis and evaluated by conducting a stability study and an in vitro study with the main focus on cell viability. The stability study consisted of a series of stability tests regarding the stability of nine liposome and nine chloroquine entrapped in liposome formulations over a period of twelve weeks. The in vitro study included three assays such as a reactive oxygen species assay, a lipid peroxidation assay and a hemolysis assay. The aims of these studies included the manufacturing of liposomes, the incorporation of chloroquine into liposomes, the determination of the stability of the formulations as well as the evaluation of the possible in vitro toxicity of liposomes. Results obtained from these studies revealed that liposomes remained more stable over the stability study period in comparison to chloroquine entrapped in liposomes. The entrapment of chloroquine within liposomes was possible, although the initial entrapment efficiency (%) of 14.55 % was much too low. The production of reactive oxygen species occurred to a small extent in the red blood cells and the infected red blood cells. Equal amounts of reactive oxygen species (%) was observed within both the red blood cells and the infected red blood cells with a maximum value of 23.27 % in the presence of the chloroquine entrapped in liposomes at varying concentrations. Red blood cells experienced the highest degree of lipid peroxidation (%) in the presence of chloroquine, at varying concentrations, entrapped in liposomes. The maximum amount of lipid peroxidation (%) was 79.61 %. No significant degree of hemolysis (%) was observed in the red blood cells neither in the presence of the liposomes nor in the presence of the chloroquine entrapped in liposomes at varying concentrations. It can be concluded that liposomes are a more stable formulation and have less toxic effects on red blood cells and infected red blood cells in comparison to the chloroquine entrapped in liposome formulations. Future studies should investigate the possibility of a more stable and less toxic chloroquine entrapped in liposome formulation. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Malaria is a curable disease that claims nearly one million lives each year. Problems with the treatment of malaria arise as resistance spreads and new treatment options are becoming less effective. The need for new treatments are of the utmost importance. Liposomes combined with antimalarials are a new avenue for research as liposomes can increase the efficacy of drugs against pathogens, as well as decreasing toxicity. Amodiaquine is a drug with known toxicity issues, but has proven to be effective and is, therefore, a prime candidate to be incorporated into the liposomal drug delivery system. The aim of this study was to prepare, characterize and evaluate the toxicity of the liposomes with incorporated amodiaquine. The solubility of amodiaquine was determined and liposomes formulated with, and without, amodiaquine entrapped. Accelerated stability studies (at 5 'C, 25 'C with relative humidity of 60% and 40 'C with a relative humidity of 40%) were conducted during which the size, pH, morphology and the entrapment efficacy was determined. The toxicity was determined in vitro by analysing the levels of reactive oxidative species and lipid peroxidation caused by the formulations to erythrocytes infected with P. falciparum as well as uninfected erythrocytes with flow cytometry. The solubility study of amodiaquine in different pH buffers showed that amodiaquine was more soluble at lower pH values. Solubility in solution with pH 4.5 was 36.3359 ± 0.7904mg/ml when compared to the solubility at pH 6.8, which was 15.6052 ± 1.1126 mg/ml. A buffer with a pH of 6 was used to ensure adequate solubility and acceptable compatibility with cells. Liposomes with incorporated amodiaquine were formulated with entrapment efficacies starting at 29.038 ± 2.599% and increasing to 51.914 ± 1.683%. The accelerated stability studies showed the median sizes and span values remained constant for both liposome and amodiaquine incorporated liposomes at 5 'C. The higher temperatures, i.e. 25 'C and 40 'C, displayed increases in the median size, and decreases in the span for both formulations. The conclusion can, therefore, be made that both liposome and amodiaquine incorporated liposomes are stable at lower temperatures. The entrapment efficacy increased from initial values to nearly 100% during the course of the stability study. This was attributed to amodiaquine precipitating from the solution. The pH values of the liposomes and amodiaquine incorporated liposomes remained constant for each formulation; though the amodiaquine incorporated liposomes had a lower starting pH, the formulations are both thought to be stable in terms of the pH. Toxicity studies revealed low levels of reactive oxygen species as well as low levels of lipid peroxidation for both liposome and amodiaquine incorporated liposomes, on both erythrocyte and Plasmodium infected erythrocytes. From the toxicity studies it can be concluded that liposomes and amodiaquine incorporated liposomes are not toxic to erythrocytes and infected erythrocytes. It was concluded that liposomes incorporating amodiaquine could possibly be used as a treatment option for malaria. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

The human body has evolved certain defence mechanisms to cope with the high occurrence of free radicals. These radicals are obtained endogenously from the mitochondria, peroxisomes, the cytochrome P450 (CYP 450) system and neutrophils, or exogenously from the environment. Lack of antioxidants and/or increased production of free radicals will result in oxidative stress, which has been implicated in certain human diseases such as hypertension, inflammation, ageing, autoimmunity, atherosclerosis, Parkinson?s disease, cancer and diabetes. Although the initial aim was to standardise a single assay to quantify both 8–OHdG and DHN–MA, this could not be achieved in this study due to the vast difference in the chemical properties of these two metabolites. Following the decision to use two separate assays for the quantification of the mentioned biomarkers, the 8–OHdG assay was standardised and validated. The intrabatch variation of the assay was 4.18% and the interbatch variation was 17.37%. Unfortunately, the DHN–MA assay could not be standardised within the time frame of this study due to experimental difficulties. Therefore, only urinary 8–OHdG and serum ROS levels were quantified. Urinary 8–OHdG levels were measured in 409 participants (209 Caucasians, 101 males and 108 females and 200 Africans, 100 males and 100 females) from the SABPA study. After removal of outliers from the data matrix, the effect of gender and ethnicity was investigated on the measured urinary 8–OHdG levels. No significant difference in the urinary 8–OHdG levels between Caucasian males (n=87) and females (n=96) were observed (p = 0.68). A similar observation was made for the African males (n=86) and females (n=84), where no significant difference in 8–OHdG levels was detected (p = 0.053). Thus, from the results obtained in this study, it seems that urinary 8–OHdG levels are not influenced by gender. However, 8–OHdG levels were dramatically influenced by ethnicity. Caucasian males (n=87) excreted 70% higher amounts of 8–OHdG compared to African males (n=86) (p < 0.001). Caucasian females (n=96) also excreted larger urinary 8–OHdG amounts (42%) compared to African females (n=84) (p < 0.001). Therefore, it seems that urinary 8–OHdG levels are dramatically influenced by ethnicity. Finally, urinary 8–OHdG levels were compared to serum ROS levels, but no significant correlation between the measured metabolites was observed (r = –0.045). Hence, urinary 8–OHdG and serum ROS levels are not related in these subjects. Even though the initial aim of this study was to standardise an analytical method to quantify both urinary 8–OHdG and DHN–MA, this could not be achieved due to time constraints. vi The human body has evolved certain defence mechanisms to cope with the high occurrence of free radicals. These radicals are obtained endogenously from the mitochondria, peroxisomes, the cytochrome P450 (CYP 450) system and neutrophils, or exogenously from the environment. Lack of antioxidants and/or increased production of free radicals will result in oxidative stress, which has been implicated in certain human diseases such as hypertension, inflammation, ageing, autoimmunity, atherosclerosis, Parkinson?s disease, cancer and diabetes. Although the initial aim was to standardise a single assay to quantify both 8–OHdG and DHN–MA, this could not be achieved in this study due to the vast difference in the chemical properties of these two metabolites. Following the decision to use two separate assays for the quantification of the mentioned biomarkers, the 8–OHdG assay was standardised and validated. The intrabatch variation of the assay was 4.18% and the interbatch variation was 17.37%. Unfortunately, the DHN–MA assay could not be standardised within the time frame of this study due to experimental difficulties. Therefore, only urinary 8–OHdG and serum ROS levels were quantified. Urinary 8–OHdG levels were measured in 409 participants (209 Caucasians, 101 males and 108 females and 200 Africans, 100 males and 100 females) from the SABPA study. After removal of outliers from the data matrix, the effect of gender and ethnicity was investigated on the measured urinary 8–OHdG levels. No significant difference in the urinary 8–OHdG levels between Caucasian males (n=87) and females (n=96) were observed (p = 0.68). A similar observation was made for the African males (n=86) and females (n=84), where no significant difference in 8–OHdG levels was detected (p = 0.053). Thus, from the results obtained in this study, it seems that urinary 8–OHdG levels are not influenced by gender. However, 8–OHdG levels were dramatically influenced by ethnicity. Caucasian males (n=87) excreted 70% higher amounts of 8–OHdG compared to African males (n=86) (p < 0.001). Caucasian females (n=96) also excreted larger urinary 8–OHdG amounts (42%) compared to African females (n=84) (p < 0.001). Therefore, it seems that urinary 8–OHdG levels are dramatically influenced by ethnicity. Finally, urinary 8–OHdG levels were compared to serum ROS levels, but no significant correlation between the measured metabolites was observed (r = –0.045). Hence, urinary 8–OHdG and serum ROS levels are not related in these subjects. Even though the initial aim of this study was to standardise an analytical method to quantify both urinary 8–OHdG and DHN–MA, this could not be achieved due to time constraints. However, an LC–MS/MS analytical assay was standardised and validated for the quantification of urinary 8–OHdG. The method proved reliable for the quantification of 8–OHdG from urine samples and can thus be used for further studies on oxidative DNA damage. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

The human body has evolved certain defence mechanisms to cope with the high occurrence of free radicals. These radicals are obtained endogenously from the mitochondria, peroxisomes, the cytochrome P450 (CYP 450) system and neutrophils, or exogenously from the environment. Lack of antioxidants and/or increased production of free radicals will result in oxidative stress, which has been implicated in certain human diseases such as hypertension, inflammation, ageing, autoimmunity, atherosclerosis, Parkinson?s disease, cancer and diabetes. Although the initial aim was to standardise a single assay to quantify both 8–OHdG and DHN–MA, this could not be achieved in this study due to the vast difference in the chemical properties of these two metabolites. Following the decision to use two separate assays for the quantification of the mentioned biomarkers, the 8–OHdG assay was standardised and validated. The intrabatch variation of the assay was 4.18% and the interbatch variation was 17.37%. Unfortunately, the DHN–MA assay could not be standardised within the time frame of this study due to experimental difficulties. Therefore, only urinary 8–OHdG and serum ROS levels were quantified. Urinary 8–OHdG levels were measured in 409 participants (209 Caucasians, 101 males and 108 females and 200 Africans, 100 males and 100 females) from the SABPA study. After removal of outliers from the data matrix, the effect of gender and ethnicity was investigated on the measured urinary 8–OHdG levels. No significant difference in the urinary 8–OHdG levels between Caucasian males (n=87) and females (n=96) were observed (p = 0.68). A similar observation was made for the African males (n=86) and females (n=84), where no significant difference in 8–OHdG levels was detected (p = 0.053). Thus, from the results obtained in this study, it seems that urinary 8–OHdG levels are not influenced by gender. However, 8–OHdG levels were dramatically influenced by ethnicity. Caucasian males (n=87) excreted 70% higher amounts of 8–OHdG compared to African males (n=86) (p < 0.001). Caucasian females (n=96) also excreted larger urinary 8–OHdG amounts (42%) compared to African females (n=84) (p < 0.001). Therefore, it seems that urinary 8–OHdG levels are dramatically influenced by ethnicity. Finally, urinary 8–OHdG levels were compared to serum ROS levels, but no significant correlation between the measured metabolites was observed (r = –0.045). Hence, urinary 8–OHdG and serum ROS levels are not related in these subjects. Even though the initial aim of this study was to standardise an analytical method to quantify both urinary 8–OHdG and DHN–MA, this could not be achieved due to time constraints. vi The human body has evolved certain defence mechanisms to cope with the high occurrence of free radicals. These radicals are obtained endogenously from the mitochondria, peroxisomes, the cytochrome P450 (CYP 450) system and neutrophils, or exogenously from the environment. Lack of antioxidants and/or increased production of free radicals will result in oxidative stress, which has been implicated in certain human diseases such as hypertension, inflammation, ageing, autoimmunity, atherosclerosis, Parkinson?s disease, cancer and diabetes. Although the initial aim was to standardise a single assay to quantify both 8–OHdG and DHN–MA, this could not be achieved in this study due to the vast difference in the chemical properties of these two metabolites. Following the decision to use two separate assays for the quantification of the mentioned biomarkers, the 8–OHdG assay was standardised and validated. The intrabatch variation of the assay was 4.18% and the interbatch variation was 17.37%. Unfortunately, the DHN–MA assay could not be standardised within the time frame of this study due to experimental difficulties. Therefore, only urinary 8–OHdG and serum ROS levels were quantified. Urinary 8–OHdG levels were measured in 409 participants (209 Caucasians, 101 males and 108 females and 200 Africans, 100 males and 100 females) from the SABPA study. After removal of outliers from the data matrix, the effect of gender and ethnicity was investigated on the measured urinary 8–OHdG levels. No significant difference in the urinary 8–OHdG levels between Caucasian males (n=87) and females (n=96) were observed (p = 0.68). A similar observation was made for the African males (n=86) and females (n=84), where no significant difference in 8–OHdG levels was detected (p = 0.053). Thus, from the results obtained in this study, it seems that urinary 8–OHdG levels are not influenced by gender. However, 8–OHdG levels were dramatically influenced by ethnicity. Caucasian males (n=87) excreted 70% higher amounts of 8–OHdG compared to African males (n=86) (p < 0.001). Caucasian females (n=96) also excreted larger urinary 8–OHdG amounts (42%) compared to African females (n=84) (p < 0.001). Therefore, it seems that urinary 8–OHdG levels are dramatically influenced by ethnicity. Finally, urinary 8–OHdG levels were compared to serum ROS levels, but no significant correlation between the measured metabolites was observed (r = –0.045). Hence, urinary 8–OHdG and serum ROS levels are not related in these subjects. Even though the initial aim of this study was to standardise an analytical method to quantify both urinary 8–OHdG and DHN–MA, this could not be achieved due to time constraints. However, an LC–MS/MS analytical assay was standardised and validated for the quantification of urinary 8–OHdG. The method proved reliable for the quantification of 8–OHdG from urine samples and can thus be used for further studies on oxidative DNA damage. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.