Malaria endemic areas not only pose a public health threat, but affects 3.3 billion people worldwide. In 2011, estimated malaria related deaths amounted to 660 000 out of 219 million reported cases, with 81% of these and 91% of malaria related mortality occurred in the African region. Those most affected were pregnant women, children under the age of five and immunocompromised individuals. Malaria is the fifth deadliest disease worldwide and accounts for the second highest death rate in Africa, following HIV/Aids. To combat this parasitic infection of antiquity, the ideal malaria pharmacotherapy would be a cost effective and easily obtainable monotherapy. The malaria parasite, however, has an intrinsic ability to develop drug resistance through various mechanisms. Widespread resistance towards antimalarial drugs has rendered traditionally used drugs therapeutically ineffective, hence accentuating the efficacy of the artemisinins as first line treatment option for uncomplicated Plasmodium falciparum (P. falciparum). A devastating reality of the challenging battle against malaria is the confirmed prolonged parasitic clearance times of the artemisinins, despite adequate drug exposure, which emphasises the urgent need for identifying and developing new, effective and safe therapies. During this study, 9-aminoacridines and artemisinin-acridine hybrids were successfully synthesised through nucleophillic substitution and their chemical structures confirmed by means of nuclear magnetic resonance spectroscopy (NMR), high resolution mass spectroscopy (HRMS) and infrared spectroscopy (IR). The hybrid compounds were synthesised through microwave assisted radiation, by covalently linking the artemisinin- and amino-functionalised acridine pharmacophores by means of a liable aminoethyl ether chain. The target compounds were screened in vitro for antimalarial activity against both the chloroquine sensitive (NF54) and chloroquine resistant (Dd2) strains of P. falciparum. Their cytotoxicities were assessed against various mammalian cells of different origins, viz. the Chinese hamster ovarian cells (CHO) from animal origin, and from human origin, hepatocellular- (HepG2), neuroblastoma- (SH-SY5Y) and cervical cancer (HeLa) cells. The synthesised hybrids exhibited antimalarial activity against both Plasmodium strains. Compound 7, featuring an ethylenediamine moiety in the linker, was the most active hybrid, with 50% inhibitory concentration (IC50) values of 2.6 nM and 35.3 nM against the NF54 and Dd2 strains, respectively. It had gametocytocidal activity against the NF54 strain, comparable to dihydroartemisinin (DHA) and artesunate (AS) and it is significantly more potent than chloroquine (CQ), whilst possessing a resistance index value of 14, indicative of a significant loss of activity against the CQ resistant strain. Contrary, the promising hybrid 10, containing a 2-methylpiperazine linker, had gametocytocidal activity, comparable to CQ and was found to be six-fold more potent than CQ against the Dd2 strain, with a resistance index (RI) value of 2, whilst it further showed highly selective action towards the parasitic cells. Compound 10 was also found to possess anticancer activity against the HeLa cell line, comparable to DHA and AS, but fivefold higher than that of CQ, with the same levels of hepatotoxicity and neurotoxicity. The artemisinin-acridine hybrids displayed superior antimalarial activity, compared to the derived 9-aminoacridines against both the Plasmodium strains. They, however, did not have the ability to overcome resistance, reduce the toxicity of acridine, nor induce synergistic activity. The hybrids, indeed displayed promising anticancer activity against HeLa cells. It is anticipated that these compounds may stand as drug candidates for further investigation in the search for new anti-cervical cancer drugs, rather than as antimalarials. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Malaria endemic areas not only pose a public health threat, but affects 3.3 billion people worldwide. In 2011, estimated malaria related deaths amounted to 660 000 out of 219 million reported cases, with 81% of these and 91% of malaria related mortality occurred in the African region. Those most affected were pregnant women, children under the age of five and immunocompromised individuals. Malaria is the fifth deadliest disease worldwide and accounts for the second highest death rate in Africa, following HIV/Aids. To combat this parasitic infection of antiquity, the ideal malaria pharmacotherapy would be a cost effective and easily obtainable monotherapy. The malaria parasite, however, has an intrinsic ability to develop drug resistance through various mechanisms. Widespread resistance towards antimalarial drugs has rendered traditionally used drugs therapeutically ineffective, hence accentuating the efficacy of the artemisinins as first line treatment option for uncomplicated Plasmodium falciparum (P. falciparum). A devastating reality of the challenging battle against malaria is the confirmed prolonged parasitic clearance times of the artemisinins, despite adequate drug exposure, which emphasises the urgent need for identifying and developing new, effective and safe therapies. During this study, 9-aminoacridines and artemisinin-acridine hybrids were successfully synthesised through nucleophillic substitution and their chemical structures confirmed by means of nuclear magnetic resonance spectroscopy (NMR), high resolution mass spectroscopy (HRMS) and infrared spectroscopy (IR). The hybrid compounds were synthesised through microwave assisted radiation, by covalently linking the artemisinin- and amino-functionalised acridine pharmacophores by means of a liable aminoethyl ether chain. The target compounds were screened in vitro for antimalarial activity against both the chloroquine sensitive (NF54) and chloroquine resistant (Dd2) strains of P. falciparum. Their cytotoxicities were assessed against various mammalian cells of different origins, viz. the Chinese hamster ovarian cells (CHO) from animal origin, and from human origin, hepatocellular- (HepG2), neuroblastoma- (SH-SY5Y) and cervical cancer (HeLa) cells. The synthesised hybrids exhibited antimalarial activity against both Plasmodium strains. Compound 7, featuring an ethylenediamine moiety in the linker, was the most active hybrid, with 50% inhibitory concentration (IC50) values of 2.6 nM and 35.3 nM against the NF54 and Dd2 strains, respectively. It had gametocytocidal activity against the NF54 strain, comparable to dihydroartemisinin (DHA) and artesunate (AS) and it is significantly more potent than chloroquine (CQ), whilst possessing a resistance index value of 14, indicative of a significant loss of activity against the CQ resistant strain. Contrary, the promising hybrid 10, containing a 2-methylpiperazine linker, had gametocytocidal activity, comparable to CQ and was found to be six-fold more potent than CQ against the Dd2 strain, with a resistance index (RI) value of 2, whilst it further showed highly selective action towards the parasitic cells. Compound 10 was also found to possess anticancer activity against the HeLa cell line, comparable to DHA and AS, but fivefold higher than that of CQ, with the same levels of hepatotoxicity and neurotoxicity. The artemisinin-acridine hybrids displayed superior antimalarial activity, compared to the derived 9-aminoacridines against both the Plasmodium strains. They, however, did not have the ability to overcome resistance, reduce the toxicity of acridine, nor induce synergistic activity. The hybrids, indeed displayed promising anticancer activity against HeLa cells. It is anticipated that these compounds may stand as drug candidates for further investigation in the search for new anti-cervical cancer drugs, rather than as antimalarials. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Malaria affects about forty percent of the world’s population. Annually more than 1.5 million fatalities due to malaria occur and parasite resistance to existing antimalarial drugs such as mefloquine has already reached disturbingly high levels in South-East Asia and on the African continent. Consequently, there is a dire need for new drugs or formulations in the prophylaxis and treatment of malaria. Artesunate, an artemisinin derivative, represents a new category of antimalarials that is effective against drug-resistant Plasmodium falciparum strains and is of significance in the current antimalarial campaign. As formulating an ACT double fixed-dose combination is technically difficult, it is essential that fixed-dose combinations are shown to have satisfactory ingredient compatibility, stability, and dissolution rates similar to the separate oral dosage forms. Since the general deployment of a combination of artesunate and mefloquine in 1994, the cure rate increased again to almost 100% from 1998 onwards, and there has been a sustained decline in the incidence of Plasmodium falciparum malaria in the experimental studies (Nosten et al., 2000:297; WHO, 2010:17). However, the successful formulation of a solid oral dosage form and fixed dosage combination of artesunate and mefloquine remains both a market opportunity and a challenge. Artesunate and mefloquine both exhibited poor flow properties. Furthermore, different elimination half-lives, treatment dosages as well as solubility properties of artesunate and mefloquine required different formulation approaches. To substantiate the FDA’s pharmaceutical quality by design concept, the double fixed-dose combination of artesunate and mefloquine required strict preliminary formulation considerations regarding compatibility between excipients and between the APIs. Materials and process methods were only considered if theoretically and experimentally proved safe. Infrared absorption spectroscopy (IR) and X-ray powder diffraction (XRPD) data proved compatibility between ingredients and stability during the complete manufacturing process by a peak by peak correlation. Scanning Electron Micrographs (SEM) provided explanations for the inferior flow properties exhibited by the investigated APIs. Particle size analysis and SEM micrographs confirmed that the larger, rounder and more consistently sized particles of the granulated APIs contributed to improved flow under the specified testing conditions. A compressible mixture containing 615 mg of the APIs in accordance with the WHO recommendation of 25 mg/kg of mefloquine taken in two or three divided dosages, and 4 mg/kg/day for 3 days of artesunate for uncomplicated falciparum malaria was developed. Mini-tablets of artesunate and mefloquine were compressed separately and successfully with the required therapeutic dosages and complied with pharmacopoeial standards. Preformulation studies eventually led to a formula for a double fixed-dose combination and with the specific aim of delaying the release of artesunate due to its short half-life. A factorial design revealed the predominant factors contributing to the successful wet granulation of artesunate and mefloquine. A fractional factorial design identified the optimum factors and factor levels. The application of the granulation fluid (20% w/w) proved to be sufficient by a spraying method for both artesunate and mefloquine. A compatible acrylic polymer and coating agent for artesunate, Eudragit® L100 was employed to delay the release of approximately half of the artesunate dose from the double fixed-dose combination tablet until a pH of 6.8. A compressible mixture was identified and formulated to contain 200 mg of artesunate and 415 mg of mefloquine per tablet. The physical properties of the tablets complied with BP standards. An HPLC method from available literature was adapted and validated for analytical procedures. Dissolution studies according to a USP method were conducted to verify and quantify the release of the APIs in the double fixed-dose combination. The initial dissolution rate (DRi) of artesunate and mefloquine in the acidic dissolution medium was rapid as required. The enteric coated fraction of the artesunate exhibited no release in an acidic environment after 2 hours, but rapid release in a medium with a pH of 6.8. The structure of the granulated particles of mefloquine may have contributed to its first order release profile in the dissolution mediums. A linear correlation was present between the rate of mefloquine release and the percentage of mefloquine dissolved (R2 = 0.9484). Additionally, a linear relationship was found between the logarithm of the percentage mefloquine remaining against time (R2 = 0.9908). First order drug release is the dominant release profile found in the pharmaceutical industry today and is coherent with the kinetics of release obtained for mefloquine. A concept pre-clinical phase, double fixed-dose combination solid oral dosage form for artesunate and mefloquine was developed. The double fixed-dose combination was designed in accordance with the WHO’s recommendation for an oral dosage regimen of artesunate and mefloquine for the treatment of uncomplicated falciparum malaria. The specifications of the double fixed-dose combination were developed in close accordance with the FDA’s quality by design concept and WHO recommendations. An HPLC analytical procedure was developed to verify the presence of artesunate and mefloquine. The dissolution profiles of artesunate and mefloquine were investigated during the dissolution studies. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Malaria affects about forty percent of the world’s population. Annually more than 1.5 million fatalities due to malaria occur and parasite resistance to existing antimalarial drugs such as mefloquine has already reached disturbingly high levels in South-East Asia and on the African continent. Consequently, there is a dire need for new drugs or formulations in the prophylaxis and treatment of malaria. Artesunate, an artemisinin derivative, represents a new category of antimalarials that is effective against drug-resistant Plasmodium falciparum strains and is of significance in the current antimalarial campaign. As formulating an ACT double fixed-dose combination is technically difficult, it is essential that fixed-dose combinations are shown to have satisfactory ingredient compatibility, stability, and dissolution rates similar to the separate oral dosage forms. Since the general deployment of a combination of artesunate and mefloquine in 1994, the cure rate increased again to almost 100% from 1998 onwards, and there has been a sustained decline in the incidence of Plasmodium falciparum malaria in the experimental studies (Nosten et al., 2000:297; WHO, 2010:17). However, the successful formulation of a solid oral dosage form and fixed dosage combination of artesunate and mefloquine remains both a market opportunity and a challenge. Artesunate and mefloquine both exhibited poor flow properties. Furthermore, different elimination half-lives, treatment dosages as well as solubility properties of artesunate and mefloquine required different formulation approaches. To substantiate the FDA’s pharmaceutical quality by design concept, the double fixed-dose combination of artesunate and mefloquine required strict preliminary formulation considerations regarding compatibility between excipients and between the APIs. Materials and process methods were only considered if theoretically and experimentally proved safe. Infrared absorption spectroscopy (IR) and X-ray powder diffraction (XRPD) data proved compatibility between ingredients and stability during the complete manufacturing process by a peak by peak correlation. Scanning Electron Micrographs (SEM) provided explanations for the inferior flow properties exhibited by the investigated APIs. Particle size analysis and SEM micrographs confirmed that the larger, rounder and more consistently sized particles of the granulated APIs contributed to improved flow under the specified testing conditions. A compressible mixture containing 615 mg of the APIs in accordance with the WHO recommendation of 25 mg/kg of mefloquine taken in two or three divided dosages, and 4 mg/kg/day for 3 days of artesunate for uncomplicated falciparum malaria was developed. Mini-tablets of artesunate and mefloquine were compressed separately and successfully with the required therapeutic dosages and complied with pharmacopoeial standards. Preformulation studies eventually led to a formula for a double fixed-dose combination and with the specific aim of delaying the release of artesunate due to its short half-life. A factorial design revealed the predominant factors contributing to the successful wet granulation of artesunate and mefloquine. A fractional factorial design identified the optimum factors and factor levels. The application of the granulation fluid (20% w/w) proved to be sufficient by a spraying method for both artesunate and mefloquine. A compatible acrylic polymer and coating agent for artesunate, Eudragit® L100 was employed to delay the release of approximately half of the artesunate dose from the double fixed-dose combination tablet until a pH of 6.8. A compressible mixture was identified and formulated to contain 200 mg of artesunate and 415 mg of mefloquine per tablet. The physical properties of the tablets complied with BP standards. An HPLC method from available literature was adapted and validated for analytical procedures. Dissolution studies according to a USP method were conducted to verify and quantify the release of the APIs in the double fixed-dose combination. The initial dissolution rate (DRi) of artesunate and mefloquine in the acidic dissolution medium was rapid as required. The enteric coated fraction of the artesunate exhibited no release in an acidic environment after 2 hours, but rapid release in a medium with a pH of 6.8. The structure of the granulated particles of mefloquine may have contributed to its first order release profile in the dissolution mediums. A linear correlation was present between the rate of mefloquine release and the percentage of mefloquine dissolved (R2 = 0.9484). Additionally, a linear relationship was found between the logarithm of the percentage mefloquine remaining against time (R2 = 0.9908). First order drug release is the dominant release profile found in the pharmaceutical industry today and is coherent with the kinetics of release obtained for mefloquine. A concept pre-clinical phase, double fixed-dose combination solid oral dosage form for artesunate and mefloquine was developed. The double fixed-dose combination was designed in accordance with the WHO’s recommendation for an oral dosage regimen of artesunate and mefloquine for the treatment of uncomplicated falciparum malaria. The specifications of the double fixed-dose combination were developed in close accordance with the FDA’s quality by design concept and WHO recommendations. An HPLC analytical procedure was developed to verify the presence of artesunate and mefloquine. The dissolution profiles of artesunate and mefloquine were investigated during the dissolution studies. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2014