Malaria is a disease affecting millions of people in 109 malarious countries and territories, causing approximately one million deaths annually. In 2004 one of the parasites causing human malaria, Plasmodium falciparum, was among the leading global causes of death from a single infectious agent, especially in Africa (WHO, 2008:23). Treatment of this disease with single active pharmaceutical ingredients has led to the emergence of resistant P. falciparum parasites, resulting in the most severe form of this illness. Alarmingly, the poor quality of commercially available antimalarial products, especially in Africa, has increasingly been reported as a major cause of resistance to antimalarials. In Pakistan it was found that a P. falciparum epidemic that initially was attributed to drug resistance, was actually caused by substandard sulfadoxine/pyrimethamine products, causing a 50 times higher incidence of malaria in these areas than elsewhere (Leslie et al., 2009:1758). Other results indicated that up to 10% of sulfadoxine/pyrimethamine tablets, sampled in six African countries, failed the assay test, whilst up to 40% failed the USP dissolution test. Furthermore, the World Health Organization (WHO) reported that 20 - 90% of products failed quality requirements during 1999 and 2000 in seven African countries (WHO, 2003:263). Cases like these have raised the awareness of the vast number of inferior products that are being distributed. The subsequent need for establishing mechanisms to proactively detect substandard medicines, specifically antimalarials, easily and effectively had indirectly led to the origin of this study, long before it was formally undertaken. Testing monographs for pharmaceutical products are developed to formalise, or standardise, the regulation of pharmaceutical dosage forms. Problems have, however, been reported with regards to the inadequacy of existing antimalarial monographs in assuring quality medicines, fit for their intended use. The WHO had requested the Research Institute for Industrial Pharmacy, incorporating the Centre for Quality Assurance of Medicines (RIIP®/CENQAM®), both operating at the Potchefstroom Campus of the North–West University, to develop monographs for three immediate–release antimalaria dosage forms, namely amodiaquine tablets, sulfadoxine/pyrimethamine fixed–dose combination tablets and mefloquine tablets. The undertaking of these projects, to develop specifications for the quality control of these pharmaceutical products, formed the object of this research study. Data had been accumulated since 2000, as a result of continuous requests by the WHO to help solve problems that had been experienced with analytical test methods, especially from manufacturers. These requests either led to the refinement of existing methods, or to the development of new ones. The success with which these outcomes were implemented worldwide, finally led to the decision to publish these research findings under the umbrella of this project. The proud product is a comprehensive package of tests for three commercial antimalarial products, the outcomes of which are hoped to contribute towards the combat against resistance formation to these important disease fighters. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Malaria is a disease affecting millions of people in 109 malarious countries and territories, causing approximately one million deaths annually. In 2004 one of the parasites causing human malaria, Plasmodium falciparum, was among the leading global causes of death from a single infectious agent, especially in Africa (WHO, 2008:23). Treatment of this disease with single active pharmaceutical ingredients has led to the emergence of resistant P. falciparum parasites, resulting in the most severe form of this illness. Alarmingly, the poor quality of commercially available antimalarial products, especially in Africa, has increasingly been reported as a major cause of resistance to antimalarials. In Pakistan it was found that a P. falciparum epidemic that initially was attributed to drug resistance, was actually caused by substandard sulfadoxine/pyrimethamine products, causing a 50 times higher incidence of malaria in these areas than elsewhere (Leslie et al., 2009:1758). Other results indicated that up to 10% of sulfadoxine/pyrimethamine tablets, sampled in six African countries, failed the assay test, whilst up to 40% failed the USP dissolution test. Furthermore, the World Health Organization (WHO) reported that 20 - 90% of products failed quality requirements during 1999 and 2000 in seven African countries (WHO, 2003:263). Cases like these have raised the awareness of the vast number of inferior products that are being distributed. The subsequent need for establishing mechanisms to proactively detect substandard medicines, specifically antimalarials, easily and effectively had indirectly led to the origin of this study, long before it was formally undertaken. Testing monographs for pharmaceutical products are developed to formalise, or standardise, the regulation of pharmaceutical dosage forms. Problems have, however, been reported with regards to the inadequacy of existing antimalarial monographs in assuring quality medicines, fit for their intended use. The WHO had requested the Research Institute for Industrial Pharmacy, incorporating the Centre for Quality Assurance of Medicines (RIIP®/CENQAM®), both operating at the Potchefstroom Campus of the North–West University, to develop monographs for three immediate–release antimalaria dosage forms, namely amodiaquine tablets, sulfadoxine/pyrimethamine fixed–dose combination tablets and mefloquine tablets. The undertaking of these projects, to develop specifications for the quality control of these pharmaceutical products, formed the object of this research study. Data had been accumulated since 2000, as a result of continuous requests by the WHO to help solve problems that had been experienced with analytical test methods, especially from manufacturers. These requests either led to the refinement of existing methods, or to the development of new ones. The success with which these outcomes were implemented worldwide, finally led to the decision to publish these research findings under the umbrella of this project. The proud product is a comprehensive package of tests for three commercial antimalarial products, the outcomes of which are hoped to contribute towards the combat against resistance formation to these important disease fighters. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Resistance against anti-malarial drugs remains one of the greatest obstacles to the effective control of malaria. The current first-line treatment regimen for uncomplicated P.falciparum malaria is based on artemisinin combination therapies (ACTs). However, reports of an increase in tolerance of the malaria parasite to artemisinins used in ACTs have alarmed the malaria community. The spread of artemisinin-resistant parasites would impact negatively on malaria control. Chloroquine and amodiaquine are 4-aminoquinolines. Chloroquine and amodiaquine were evaluated in a primate model by comparing the bioavailability of these compounds in a reference formulation and also in a Pheroid® formulation. In vivo pharmacokinetic studies were conducted for chloroquine, and in vitro and in vivo drug metabolism and pharmacokinetic (DMPK) studies were conducted for amodiaquine. Pheroid® technology forms the basis of a colloidal drug delivery system, and it is the potential application of this technology in combination with the 4-aminoquinolines that was the focus of this thesis. Pheroid® is a registered trademark but for ease of reading will be referred to as pheroid(s) or pro-pheroid(s) throughout the rest of the thesis. The non-human primate model used for evaluation of the pharmacokinetic parameters was the vervet monkey (Chlorocebus aethiops). Chloroquine was administered orally at 20 mg/kg. A sensitive and selective LC-MS/MS method was developed to analyze the concentration of chloroquine in both whole blood and plasma samples. The Cmax obtained for whole blood was 1039 ± 251.04 ng/mL for the unformulated reference sample of chloroquine and 1753.6 ± 382.8 ng/mL for the pheroid formulation. The AUC0-inf was 37365 ± 6383 ng.h/mL (reference) and 52047 ± 11210 ng.h/mL (pheroid). The results indicate that the use of pheroid technology enhances the absorption of chloroquine. The effect of pheroid technology on the bioavailability of amodiaquine and N-desethylamodiaquine was determined in two groups of vervet monkeys, with the reference group receiving capsules containing the hydrochloride salt of amodiaquine and the test group receiving capsules containing a pro-pheroid formulation of amodiaquine. Amodiaquine was administered at 60 mg/kg. Blood concentrations of amodiaquine and N-desethylamodiaquine samples were monitored over 13 time points from 0.5 to 168 hours. Amodiaquine and pro-pheroid formulated amodiaquine were incubated in vitro with human and monkey liver (HLM and MLM) and intestinal (HIM and MIM) microsomes and recombinant cytochrome P450 enzymes. The in vitro metabolism studies confirm the rapid metabolism of amodiaquine to the main metabolite N-desethylamodiaquine in monkeys. Although the pharmacokinetic parameters varied greatly, parameters for both the parent compound and main metabolite were lower in the test formulation compared to the reference formulation. For HLM, MLM and CYP2C8, the pro-pheroid test formulation showed significantly longer amodiaquine clearance and slower formation of N-desethylamodiaquine. However, the effect was reversed in MIM. Pheroid technology impacts differently on the bioavailability of the various pharmaceutical classes of anti-malarials. Pheroid technology did not enhance the bioavailability of amodiaquine or N-desethylamodiaquine. This is contrary to the observed effects of pheroid technology on the pharmacokinetics of other drugs such as artemisone and chloroquine where it increases the area under the curve and prolongs the drug half-life. / Thesis (PhD (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

Resistance against anti-malarial drugs remains one of the greatest obstacles to the effective control of malaria. The current first-line treatment regimen for uncomplicated P.falciparum malaria is based on artemisinin combination therapies (ACTs). However, reports of an increase in tolerance of the malaria parasite to artemisinins used in ACTs have alarmed the malaria community. The spread of artemisinin-resistant parasites would impact negatively on malaria control. Chloroquine and amodiaquine are 4-aminoquinolines. Chloroquine and amodiaquine were evaluated in a primate model by comparing the bioavailability of these compounds in a reference formulation and also in a Pheroid® formulation. In vivo pharmacokinetic studies were conducted for chloroquine, and in vitro and in vivo drug metabolism and pharmacokinetic (DMPK) studies were conducted for amodiaquine. Pheroid® technology forms the basis of a colloidal drug delivery system, and it is the potential application of this technology in combination with the 4-aminoquinolines that was the focus of this thesis. Pheroid® is a registered trademark but for ease of reading will be referred to as pheroid(s) or pro-pheroid(s) throughout the rest of the thesis. The non-human primate model used for evaluation of the pharmacokinetic parameters was the vervet monkey (Chlorocebus aethiops). Chloroquine was administered orally at 20 mg/kg. A sensitive and selective LC-MS/MS method was developed to analyze the concentration of chloroquine in both whole blood and plasma samples. The Cmax obtained for whole blood was 1039 ± 251.04 ng/mL for the unformulated reference sample of chloroquine and 1753.6 ± 382.8 ng/mL for the pheroid formulation. The AUC0-inf was 37365 ± 6383 ng.h/mL (reference) and 52047 ± 11210 ng.h/mL (pheroid). The results indicate that the use of pheroid technology enhances the absorption of chloroquine. The effect of pheroid technology on the bioavailability of amodiaquine and N-desethylamodiaquine was determined in two groups of vervet monkeys, with the reference group receiving capsules containing the hydrochloride salt of amodiaquine and the test group receiving capsules containing a pro-pheroid formulation of amodiaquine. Amodiaquine was administered at 60 mg/kg. Blood concentrations of amodiaquine and N-desethylamodiaquine samples were monitored over 13 time points from 0.5 to 168 hours. Amodiaquine and pro-pheroid formulated amodiaquine were incubated in vitro with human and monkey liver (HLM and MLM) and intestinal (HIM and MIM) microsomes and recombinant cytochrome P450 enzymes. The in vitro metabolism studies confirm the rapid metabolism of amodiaquine to the main metabolite N-desethylamodiaquine in monkeys. Although the pharmacokinetic parameters varied greatly, parameters for both the parent compound and main metabolite were lower in the test formulation compared to the reference formulation. For HLM, MLM and CYP2C8, the pro-pheroid test formulation showed significantly longer amodiaquine clearance and slower formation of N-desethylamodiaquine. However, the effect was reversed in MIM. Pheroid technology impacts differently on the bioavailability of the various pharmaceutical classes of anti-malarials. Pheroid technology did not enhance the bioavailability of amodiaquine or N-desethylamodiaquine. This is contrary to the observed effects of pheroid technology on the pharmacokinetics of other drugs such as artemisone and chloroquine where it increases the area under the curve and prolongs the drug half-life. / Thesis (PhD (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

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 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.