The aqueous solubility of an active pharmaceutical ingredient (API) is an important property that requires evaluation during early development and prior to formulation of the final product. With general, experimental, solubility testing of different APIs, the question always arises as to whether particle size had been determined beforehand or not. All available literature suggests that particle size, for pharmaceutical powders, does not significantly affect equilibrium solubility. The dissolution rate will differ according to different particle sizes, but the overall results should be identical after equilibrium is established. This study was therefore planned to investigate as to whether different particle size fractions of the same API, dissolving at different rates, would all reach solubility equilibrium within 24 hours. Also, APIs from different solubility classes were investigated, because poorly soluble substances would most likely require a longer period of time to equilibrate. The time period of 24 hours was selected, because many published solubility studies report using that interval and is it the standard for our research group also. Available APIs were selected to determine the influence (if any) of particle size on their equilibrium solubilities and the time required for attaining that status. For the purpose of this investigation, five APIs were selected from compounds at our disposal in-house, ranging from freely soluble to poorly soluble in the order: chloroquine phosphate > pyrazinamide > mefloquine hydrochloride > closantel sodium > roxithromycin. Solubility studies were successfully completed on four of the five APIs selected. For closantel sodium, pyrazinamide and roxithromycin it was demonstrated that the 24 hour test period was sufficient for the attainment of equilibrium solubility, regardless of the particle size fractions tested. Surprisingly, the only API in this study for which 24 hours was an insufficient test period was mefloquine HCl, which was not the least soluble compound tested. Further testing would be required to clarify this anomaly. What was evident from the outcomes of this investigation was that although the ubiquitous 24 hour solubility test may work well in many cases, its suitability should be reviewed on a case-by-case basis and not just for the most poorly soluble compounds. Researchers testing solubility at temperatures lower than 37°C should be especially cautious of using a standardised test period, because equilibrium solubility would take longer to achieve with less energy available to the system. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

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 currently one of the most imperative parasitic diseases in developing countries. Artesunate has a short half-life, low aqueous solubility and resultant poor and erratic absorption upon oral administration, which translate to low bioavailability. Mefloquine is eliminated slowly with a terminal elimination half-life of approximately 20 days and has neuropsychiatric side effects. Novel drug delivery systems have been utilised to optimise chemotherapy with currently available antimalarial drugs. Pheroid™ technology is a patented drug delivery system which has the ability to capture, transport and deliver pharmaceutical compounds. Pheroid™ technology may play a key role in ensuring effective delivery and enhanced bioavailability of novel antimalarial drugs. The aim of this study was to evaluate the possible efficacy and bioavailability enhancement of the selected antimalarial drugs, artesunate and mefloquine, in combination with Pheroid™ vesicles. The in vitro efficacy of artesunate and mefloquine co-formulated in the oil phase of Pheroid™ vesicles and entrapped in Pheroid™ vesicles 24 hours after manufacturing were investigated against a 3D7 chloroquine-sensitive strain of Plasmodium falciparum. Parasitemia (%) was quantified with flow cytometry after incubation periods of 48 and 72 hours. Drug sensitivity was expressed as 50% inhibitory concentration (IC50) values. An in vivo bioavailability study with artesunate and mefloquine was also conducted in combination with Pheroid™ vesicles, using a mouse model. A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to analyse the drug levels. C57 BL6 mice were used during this study. The selected antimalarial drugs were administered at a dose of 20 mg/kg with an oral gavage tube. Blood samples were collected by means of tail bleeding. The in vitro drug sensitivity assays revealed that artesunate, co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period, decreased the IC50 concentration significantly by 90%. Extending the incubation period to 72 hours decreased the IC50 concentration of artesunate, also co-formulated in the oil phase of Pheroid ™ vesicles significantly by 72%. No statistically significant differences between the reference and Pheroid™ vesicle groups were achieved when artesunate was entrapped 24 hours after manufacturing of Pheroid™ vesicles. Mefloquine co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period decreased the IC50 concentration by 36%. Extending the incubation period to 72 hours increased the efficacy of the Pheroid™ vesicles and the IC50 concentration was significantly decreased by 51%. In contrast with the results obtained with artesunate, entrapment of mefloquine in Pheroid™ vesicles 24 hours after manufacturing decreased the IC50 concentration significantly by 66%. The LC-MS/MS method was found to be sensitive, selective and accurate for the determination of artesunate and its active metabolite, dihydroartemisinin (DHA) in mouse plasma and mefloquine in mouse whole blood. Most of the artesunate plasma concentrations were below the limit of quantification in the reference group and relatively high outliers were observed in some of the samples. The mean artesunate levels of the Pheroid™ vesicle group were lower compared to the reference group, but the variation within the Pheroid™ vesicle group lessened significantly. The mean DHA concentrations of the Pheroid™ vesicle group were significantly higher. DHA obtained a higher peak plasma drug concentration with the Pheroid™ vesicle group (173.0 ng/ml) in relation to the reference group (105.0 ng/ml) and at a much faster time (10 minutes in Pheroid™ vesicles in contrast to 30 minutes of the reference group). Pharmacokinetic models could not be constructed due to blood sampling per animal limitation. The incorporation of mefloquine in Pheroid™ vesicles did not seem to have improved results in relation to the reference group. No statistical significant differences were observed in the pharmacokinetic parameters between the two groups. The relative bioavailability (%) of the Pheroid™ vesicle incorporated mefloquine was 7% less bioavailable than the reference group. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Stability plays an important role in the development of a new drug product. High Performance Liquid Chromatography (HPLC) is considered a stability indicating method of analysis. It is widely used in the pharmaceutical industry for the quantification of small organic molecules during stability testing. Previous stability studies conducted on Pheroid™-based drug products, experienced problems with the generation of reliable data by means of HPLC analysis. With these studies it was concluded that the inconclusive results could either be attributed to the stability of the delivery system itself and the compatibility of the active pharmaceutical ingredients (API's) with the delivery system, or to the usage of unsuitable HPLC methods. The aims of this study were to: i. determine if the Pheroid™ delivery system changes significantly over time at accelerated storage conditions and how these changes influence the HPLC analysis, ii. determine the effect of the anti-oxidant tert-butylhydroquinone (TBHQ) on the stability and HPLC analysis of the Pheroid™ delivery system, and iii. to suggest a suitable approach for the analysis of Pheroid™-based drug products. Pheroid™ microsponges, containing no API's, were prepared and stored for a period of three months at 5°C, 25°C+60%RH, 30°C+65%RH and 40°C+75%RH. Two of the four Pheroid™ formulations contained an extra anti-oxidant, namely TBHQ. Monthly HPLC analyses were done using existing methods for mefloquine and artesunate. In addition to HPLC analysis, particle size analysis and Confocal Laser Scanning Microscopy (CLSM) were undertaken to support the HPLC results and provide information concerning the overall stability of the Pheroid™ delivery system. After the completion of the above analyses, experiments were carried out to determine whether adjustments to some of the key chromatographic parameters could improve the separation of Pheroid™-based samples. The parameters that were subjected to change included the organic solvent, isocratic versus gradient separation, pH and detection wavelength. Two pro-Pheroid vesicles formulations were prepared and stored for a three month period at 40°C+75%RH only. No API was added to the one formulation while the other contained 2 mg/ml of mefloquine hydrochloride. Results obtained indicated that the Pheroid™ formulations changed after exposure to elevated temperature and humidity. The number of detectable peaks increased, longer run times became necessary and solubility in the sample solvent (methanol) decreased. Solubility of the Pheroid™ formulations in methanol was preserved to some extent by the presence of TBHQ. Physical signs of instability like discolouration and creaming were noted for TBHQ-containing formulations. TBHQ also seemed to have influenced the particle sizes, particle size distributions and structure of the Pheroid™ microsponges. With adjustments made to the HPLC method it was found that: i. the sample solvent is incompatible with the HPLC system, ii. very hydrophobic compounds are present in the Pheroid™-based samples, iii. acetontrile and methanol are unsuitable for both gradient and isocratic separation of Pheroid™-based samples, iv. more Pheroid™ components absorb at shorter wavelengths, and v. small changes in the pH values usually implemented do not influence the retention and selectivity of the Pheroid™ components. The Pheroid™ delivery system proved to be too complex and reversed hydrophobic for phase HPLC analysis. Preparation of the sample by only diluting the Pheroid™ formulations with pure methanol was not optimal. These samples introduced compounds to the column of which some caused interferences with the analyte peak while others were difficult to elute from the column. To continue using HPLC for the analysis of Pheroid™-based drug products, it is therefore recommended that attention should be given to the development of a more appropriate sample preparation procedure, like solid phase extraction or liquid-liquid extraction, one that will eliminate the effects of the Pheroid™ components. Physical instabilities noticed with the addition of TBHQ, suggest that there should also be attended to the compatibility and stability of each of the components in the Pheroid™ delivery system during formulation development. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

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 currently one of the most imperative parasitic diseases in developing countries. Artesunate has a short half-life, low aqueous solubility and resultant poor and erratic absorption upon oral administration, which translate to low bioavailability. Mefloquine is eliminated slowly with a terminal elimination half-life of approximately 20 days and has neuropsychiatric side effects. Novel drug delivery systems have been utilised to optimise chemotherapy with currently available antimalarial drugs. Pheroid™ technology is a patented drug delivery system which has the ability to capture, transport and deliver pharmaceutical compounds. Pheroid™ technology may play a key role in ensuring effective delivery and enhanced bioavailability of novel antimalarial drugs. The aim of this study was to evaluate the possible efficacy and bioavailability enhancement of the selected antimalarial drugs, artesunate and mefloquine, in combination with Pheroid™ vesicles. The in vitro efficacy of artesunate and mefloquine co-formulated in the oil phase of Pheroid™ vesicles and entrapped in Pheroid™ vesicles 24 hours after manufacturing were investigated against a 3D7 chloroquine-sensitive strain of Plasmodium falciparum. Parasitemia (%) was quantified with flow cytometry after incubation periods of 48 and 72 hours. Drug sensitivity was expressed as 50% inhibitory concentration (IC50) values. An in vivo bioavailability study with artesunate and mefloquine was also conducted in combination with Pheroid™ vesicles, using a mouse model. A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to analyse the drug levels. C57 BL6 mice were used during this study. The selected antimalarial drugs were administered at a dose of 20 mg/kg with an oral gavage tube. Blood samples were collected by means of tail bleeding. The in vitro drug sensitivity assays revealed that artesunate, co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period, decreased the IC50 concentration significantly by 90%. Extending the incubation period to 72 hours decreased the IC50 concentration of artesunate, also co-formulated in the oil phase of Pheroid ™ vesicles significantly by 72%. No statistically significant differences between the reference and Pheroid™ vesicle groups were achieved when artesunate was entrapped 24 hours after manufacturing of Pheroid™ vesicles. Mefloquine co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period decreased the IC50 concentration by 36%. Extending the incubation period to 72 hours increased the efficacy of the Pheroid™ vesicles and the IC50 concentration was significantly decreased by 51%. In contrast with the results obtained with artesunate, entrapment of mefloquine in Pheroid™ vesicles 24 hours after manufacturing decreased the IC50 concentration significantly by 66%. The LC-MS/MS method was found to be sensitive, selective and accurate for the determination of artesunate and its active metabolite, dihydroartemisinin (DHA) in mouse plasma and mefloquine in mouse whole blood. Most of the artesunate plasma concentrations were below the limit of quantification in the reference group and relatively high outliers were observed in some of the samples. The mean artesunate levels of the Pheroid™ vesicle group were lower compared to the reference group, but the variation within the Pheroid™ vesicle group lessened significantly. The mean DHA concentrations of the Pheroid™ vesicle group were significantly higher. DHA obtained a higher peak plasma drug concentration with the Pheroid™ vesicle group (173.0 ng/ml) in relation to the reference group (105.0 ng/ml) and at a much faster time (10 minutes in Pheroid™ vesicles in contrast to 30 minutes of the reference group). Pharmacokinetic models could not be constructed due to blood sampling per animal limitation. The incorporation of mefloquine in Pheroid™ vesicles did not seem to have improved results in relation to the reference group. No statistical significant differences were observed in the pharmacokinetic parameters between the two groups. The relative bioavailability (%) of the Pheroid™ vesicle incorporated mefloquine was 7% less bioavailable than the reference group. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Stability plays an important role in the development of a new drug product. High Performance Liquid Chromatography (HPLC) is considered a stability indicating method of analysis. It is widely used in the pharmaceutical industry for the quantification of small organic molecules during stability testing. Previous stability studies conducted on Pheroid™-based drug products, experienced problems with the generation of reliable data by means of HPLC analysis. With these studies it was concluded that the inconclusive results could either be attributed to the stability of the delivery system itself and the compatibility of the active pharmaceutical ingredients (API's) with the delivery system, or to the usage of unsuitable HPLC methods. The aims of this study were to: i. determine if the Pheroid™ delivery system changes significantly over time at accelerated storage conditions and how these changes influence the HPLC analysis, ii. determine the effect of the anti-oxidant tert-butylhydroquinone (TBHQ) on the stability and HPLC analysis of the Pheroid™ delivery system, and iii. to suggest a suitable approach for the analysis of Pheroid™-based drug products. Pheroid™ microsponges, containing no API's, were prepared and stored for a period of three months at 5°C, 25°C+60%RH, 30°C+65%RH and 40°C+75%RH. Two of the four Pheroid™ formulations contained an extra anti-oxidant, namely TBHQ. Monthly HPLC analyses were done using existing methods for mefloquine and artesunate. In addition to HPLC analysis, particle size analysis and Confocal Laser Scanning Microscopy (CLSM) were undertaken to support the HPLC results and provide information concerning the overall stability of the Pheroid™ delivery system. After the completion of the above analyses, experiments were carried out to determine whether adjustments to some of the key chromatographic parameters could improve the separation of Pheroid™-based samples. The parameters that were subjected to change included the organic solvent, isocratic versus gradient separation, pH and detection wavelength. Two pro-Pheroid vesicles formulations were prepared and stored for a three month period at 40°C+75%RH only. No API was added to the one formulation while the other contained 2 mg/ml of mefloquine hydrochloride. Results obtained indicated that the Pheroid™ formulations changed after exposure to elevated temperature and humidity. The number of detectable peaks increased, longer run times became necessary and solubility in the sample solvent (methanol) decreased. Solubility of the Pheroid™ formulations in methanol was preserved to some extent by the presence of TBHQ. Physical signs of instability like discolouration and creaming were noted for TBHQ-containing formulations. TBHQ also seemed to have influenced the particle sizes, particle size distributions and structure of the Pheroid™ microsponges. With adjustments made to the HPLC method it was found that: i. the sample solvent is incompatible with the HPLC system, ii. very hydrophobic compounds are present in the Pheroid™-based samples, iii. acetontrile and methanol are unsuitable for both gradient and isocratic separation of Pheroid™-based samples, iv. more Pheroid™ components absorb at shorter wavelengths, and v. small changes in the pH values usually implemented do not influence the retention and selectivity of the Pheroid™ components. The Pheroid™ delivery system proved to be too complex and reversed hydrophobic for phase HPLC analysis. Preparation of the sample by only diluting the Pheroid™ formulations with pure methanol was not optimal. These samples introduced compounds to the column of which some caused interferences with the analyte peak while others were difficult to elute from the column. To continue using HPLC for the analysis of Pheroid™-based drug products, it is therefore recommended that attention should be given to the development of a more appropriate sample preparation procedure, like solid phase extraction or liquid-liquid extraction, one that will eliminate the effects of the Pheroid™ components. Physical instabilities noticed with the addition of TBHQ, suggest that there should also be attended to the compatibility and stability of each of the components in the Pheroid™ delivery system during formulation development. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 135-171).