Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-21T15:25:14Z No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 3736513 bytes, checksum: 7a35a693236ba9e982e630b172b6f3cf (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:10:52Z (GMT) No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 3736513 bytes, checksum: 7a35a693236ba9e982e630b172b6f3cf (MD5) / Made available in DSpace on 2017-08-07T19:10:52Z (GMT). No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 3736513 bytes, checksum: 7a35a693236ba9e982e630b172b6f3cf (MD5) Previous issue date: 2013-02-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A quimioterapia atual para as leishmanioses está longe do ideal devido a uma série de problemas como alto custo e elevada toxicidade. Assim, existe uma necessidade imediata de obtenção de novos fármacos para o tratamento da doença. Neste contexto, derivados de quinolina têm demonstrado atividade leishmanicida promissora. Neste trabalho, foi avaliada a atividade leishmanicida de duas séries distintas de quinolinas: seis compostos derivados de 4-aminoquinolinas (4-AMQ) e seus híbridos com esteroide e nove derivados da amodiaquina (AQ). O efeito dos compostos foi testado em promastigotas e amastigotas de Leishmania sp e em macrófagos peritoneais de camundongos. Os resultados mostraram que a conjugação de 4-AMQ ao esteroide resultou em aumento significativo da atividade principalmente para formas promastigotas e amastigotas de L. major. O composto 6 foi o mais ativo em amastigota de L. major (CI50 de 1,9 µM), sendo três vezes mais efetivo do que a miltefosina. Demonstrou-se neste trabalho que a conjugação de dois grupos de grande aplicação biológica, quinolina e esteroide, pode ser uma estratégia interessante para o desenvolvimento racional de novos fármacos. Em relação aos derivados da AQ, observou-se que a grande maioria dos compostos foram ativos em promastigotas e amastigotas de Leishmania sp. Dentre os nove compostos avaliados, seis foram mais ativos em amastigotas de L. braziliensis do que o protótipo AQ e miltefosina. O composto 8 (CI50 de 0,4 µM) foi cerca de quatorze vezes mais ativo em amastigota do que a miltefosina. A maioria dos derivados de 4-AMQ e AQ foram mais seletivos e específicos para amastigotas. A atividade em potencial dos compostos avaliados pode ser considerada um importante avanço no estudo desta classe de derivados, visando-se o desenvolvimento de novos fármacos leishmanicidas mais eficazes, seletivos e não tóxicos para o hospedeiro humano. / Current chemotherapy for leishmaniasis is far from ideal due to a number of problems such as high cost and high toxicity. Thus, there is an immediate need to obtain new drugs for the treatment the disease. In this context, quinoline derivatives have shown promising antileishmanial activity. In this study, we evaluated the activity of two distinct series of quinolines, six 4-aminoquinolines (4-AMQ) derivatives and their hybrids with steroid and nine amodiaquine (AQ) derivatives in different Leishmania species. Effect of the compounds was assayed against Leishmania promastigotes and amastigotes and mouse peritoneal macrophages. Results showed that the combination of 4-AMQ to the steroid resulted in a significant increase in activity mainly for L. major promastigotes and amastigotes forms. Compound 6 was the most active against L. major amastigotes with IC50 of 1.9 µM, being three times more effective than miltefosine. It was demonstrated in this work that the combination of two groups with large biological application as quinoline derivatives and steroids may be an interesting strategy for the rational development of new drugs. Regarding to the AQ derivatives, it was observed that all compounds were most active against Leishmania promastigote and amastigote forms. Among the nine compounds evaluated, six were more active against L. braziliensis amastigotes than the prototype AQ and miltefosine. Compound 8 with IC50 of 0.4 µM was about fourteen times more active than miltefosine. In general, the majority of 4-AMQ and AQ derivatives are more selective and specific for amastigotes of Leishmania sp, forms clinically relevant. Potential activity of the compounds evaluated can be considered an important advance in the study of this class of derivatives in order to develop new leishmanicidal drugs more effective, selective and nontoxic to the human host.

Le paludisme est l’une des maladies infectieuses la plus fréquente au monde avec 40% de la population mondiale exposée. En dépit des stratégies actuelles de lutte notamment la prise en charge rapide des cas, l’utilisation de matériaux imprégnés et la pulvérisation intra domiciliaire d’insecticide, le paludisme reste une des premières causes de morbidité et de mortalité notamment en Afrique subsaharienne. Cette partie du monde totalise à elle seule plus de 90% des cas de décès par paludisme dont 88% chez les enfants de moins de moins de 5 ans. En absence de vaccin utilisable en santé publique, il y a donc un besoin urgent de trouver une stratégie efficiente et simple de contrôle du paludisme. Le traitement préventif intermittent (TPI) définie comme l’administration d’un antipaludique à dose curative à des intervalles de temps prédéfinis réduit l’incidence du paludisme et apparaît aujourd’hui comme une des stratégies les plus prometteuses. Cette stratégie couplée au Programme Elargi de Vaccination (PEV) chez les enfants de moins de 1 an réduit l’incidence du paludisme de 30%. Des résultats plus importants sont obtenus chez les enfants de 0 à 5 ans voire de 0 à 10 ans lorsque la stratégie est appliquée en ciblant la saison de transmission. Nos travaux de recherche au Mali ont porté sur :- l’impact de la mise en œuvre du TPI couplé à la vaccination du PEV (TPin) sur i) la résistance P. falciparum à la Sulfadoxine pyrimethamine (SP), ii) la couverture des vaccins du PEV, iii) le taux de mortalité des enfants âgés de 4 à 18 mois.- l’efficacité du TPI chez les enfants ciblant la saison de transmission (TPIe) dans un contexte de faible et de forte couverture en des Moustiquaires Imprégnés d’Insecticides (MII). Nos résultats ont montré qu’après une année de mise en œuvre à l’échelle du district sanitaire, le TPIn a entrainé une augmentation de la couverture des vaccins du PEV. Cette couverture était de 53% en zone de non-intervention contre 69.5% en zone d’intervention (p<0.01). Il y a eu une réduction de la mortalité globale de 27% (RR= 0,73, IC95% : 0,55-0,97, p=0,029) chez les enfants âgés de 4 à 18 mois. Les fréquences des marqueurs moléculaires de la résistance de P. falciparum à SP en début et en fin la mise en œuvre et entre la zone d’intervention et la zone de non –intervention après une année de mise en œuvre étaient similaires. Deux doses de SP données en TPI à 8 semaines d’ intervalle durant la saison de transmission réduit le taux d’incidence du paludisme pendant la saison de transmission de 69,4% chez les enfants de moins de 5 ans et de 63,4% chez les enfants de 5-10 ans dans un contexte de très faible utilisation de MII (<5%). Dans une autre étude que nous avons menée, le TPI avec SP + Amodiaquine (AQ) donné en 3 occasions à un mois d’ intervalle pendant la saison de transmission a réduit le taux d’ incidence du paludisme clinique non compliqué de 82% (IC à 95%: 78%– 85%; P<0.001) et les formes graves de paludisme de 87% (IC à 95% 42% – 99%, P=0.001) chez les enfants âgés de 3 à 59 mois en dépit un taux d’utilisation des MII de plus de 99%. Nous n’avons pas documenté d’événement indésirable grave lié à l’utilisation de la SP ou de la SP + AQ en TPI durant ces deux études. Nos résultats étayent la recommandation du TPI, ciblant la saison de transmission ou couplée au PEV, pour la lutte antipaludique chez les enfants. / Malaria is one of the most common infectious diseases in the world and 40% of the world population is exposed to malaria. Despite the current control strategies such as rapid diagnosis and treatment of disease cases, use of insecticide impregnated materials and indoor residuals spraying with insecticides, malaria remained a main cause of morbidity and mortality particularly in sub Saharan Africa. More than 90% of the deaths due to malaria occurred in this region and 88% of these deaths occurred in children aged less than 5 years of age. In absence of vaccine that can be used in public health, there is an urgent need for a simple and efficient control strategy. Malaria intermittent preventive treatment (IPT) defined as the administration of curative dose of anti-malarial drug at predefined time intervals, appears as one of the most promising strategies. Given through the Expanded Program of Immunization (EPI), the strategy reduced the incidence of malaria by 30%. More drastic reductions were obtained in children aged 0-5 years and even 0-10 years when the malaria transmission season was targeted for the administration of the strategy. Our research work in Mali has assessed the following:- The impact of implementation of IPT administrated through EPI (IPTi) on: i) the resistance of P. falciparum to Sulfadoxine pyrimethamine (SP); ii) EPI vaccine coverage, and iii) mortality of children of 4-18 months of age. - The efficacy of IPT in children targeting the malaria transmission season (IPTe) in a context of low and high coverage of insecticide impregnated nets (ITN).We have found that the implementation of IPTi at the district level has resulted in an augmentation of the EPI vaccine coverage. The EPI vaccine coverage was 53% in the non-intervention zone compared to 69.5% in the intervention zone (p<0,01). There was a reduction in all cause mortality of 27% (RR= 0.73, 95% CI : 0.55-0.97, p=0.029) in children aged 4-18 months. The frequencies of molecular markers of the resistance of P. falciparum to SP were similar at the beginning and the end of the one year implementation period and between the intervention and non-intervention zones.Two doses of SP given at 8 weeks interval during the transmission season, reduced the incidence of malaria episodes during the transmission season by 69.4% in children aged less than 5 years and by 63.4% in children aged 5-10 years in a context of very low ITN use (<5%). In another study that we have conducted, IPT with SP + Amodiaquine (AQ) given at three occasions at one month interval during the transmission season reduced the incidence rate of clinical malaria by 82% (95% CI: 78%– 85%; P<0.001), and the incidence of severe and complicated malaria by 87% (95% IC 42% – 99%, P=0.001) in children aged 3 to 59 months of age despite an ITN use of greater than 99%.There was no serious adverse event related to the use of SP or SP+AQ in IPT during the two studies. Our results support the recommendation of IPT targeting the transmission season and IPT given through the EPI for malaria control in children.

This research had as main objective to develop an electroanalytical procedure for the determination of amodiaquine in human milk, using a carbon paste electrode modified with hemin, hemoglobin functional group responsible for transporting oxygen. The amodiaquine (a derivative 4-aminoquinoquinolínico) is a drug used to treat malaria with anti-inflammatory and antipyretic properties. The characterization and preparation of carbon paste electrode modified with hemin was investigated. The electrochemical behavior of modified electrode was explored using differential pulse voltammetry. The best voltammetric response was obtained for the composition of carbon paste 10% hemin solution in Britton-Robson buffer pH 7.0. To study the electrochemical behavior of amodiaquine was used the technique of cyclic voltammetry, which presented in a neutral medium oxidation process at 0.054 V vs. Ag / AgCl at a rate of 20 mV s-1. The voltammetric techniques used to develop the methodology where the differential pulse voltammetry and square wave voltammetry. In this study the optimized conditions were: pH 7, scan rate = 40 mV s-1, amplitude = 200 mV and frequency = 150 Hz, where the parameters indicated the square wave voltammetry as the best technique for determining the voltammetric amodiaquine has a minor detection limit. The limits of detection and quantification were obtained in the determination of 8.7 x 10-7 mol L-1 and 2.9 x 10-6 mol L-1, respectively. After optimization of all relevant parameters, the analytical method developed was successfully applied in samples of breast milk. The concentration of amodiaquine obtained was 1.7 x 10-5 mol L-1. The method was efficient and reproducible in the determination of amodiaquine present in breast milk, there is no need any pretreatment of the sample. / Este trabalho teve como objetivo desenvolver uma metodologia eletroanalítica para a determinação de amodiaquina em leite materno, utilizando um biossensor a base de hemina, uma porfirina presente na hemoglobina e responável pelo transporte de oxigênio. A amodiaquina, por sua vez, é um derivado 4-aminoquinoquinolínico amplamente utilizada no tratamento da malária, com propriedades antiinflamatória e antipirética. Sua transferência para o leite materno é moderada, porém mesmo em baixas dosagens pode ser considerada prejudicial ao lactante. A caracterização e a preparo do biossensor foi a primeira etapa deste trabalho. O comportamento eletroquímico do biossensor foi explorado pelo emprego da técnica voltametria de pulso diferencial. As melhores respostas voltamétricas foram obtidas com a composição da pasta de carbono contendo 10% de hemina em solução-tampão Britton-Robson de pH 7,0. Para o estudo do comportamento eletródico da amodiaquina foi utilizada a técnica de voltametria cíclica, que apresentou em meio neutro um processo de oxidação em 0,054 V vs. Ag/AgCl na velocidade de 20 mV s-1 em pH 7,0. As técnicas voltamétricas utilizadas para o desenvolvimento da metodologia foram a voltametria de pulso diferencial e a voltametria de onda quadrada. Nesse estudo as condições otimizadas encontradas foram: pH = 7; velocidade de varredura = 40 mV s-1; amplitude = 200 mV e freqüência = 150 Hz, onde os parâmetros indicaram a voltametria de onda quadrada como a melhor técnica para a determinação voltamétrica da amodiaquina por apresentar melhor desempenho. Os limites de detecção e quantificação obtidos na determinação da amodiaquina por voltametria de onda quadrada foram de 8,7 x 10-7 mol L-1 e 2,9 x 10-6 mol L-1, respectivamente. Após a otimização de todos os parâmetros relevantes, o método analítico desenvolvido foi aplicado com sucesso em amostras de leite materno. A concentração de amodiaquina analisada foi de 1,7 x 10 -5 mol L-1. O método desenvolvido mostrou-se eficiente e reprodutível na determinação de amodiaquina presente no leite materno, não sendo necessário qualquer tratamento prévio da amostra.

<p>This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper.</p><p>Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. </p><p>Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil).</p><p>Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE.</p><p>Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.</p>

This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper. Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil). Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE. Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.

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.

Introduction: Emergence of resistance to artemisinin-based combination therapy (ACT) is a major threat to combat Plasmodium falciparum malaria. Regular therapeutic studies to monitor treatment efficacy is essential, and genotyping of molecular makers is useful for mapping development and spread of resistance. Aims: The study aims are to assess efficacy of artesunate-amodiquine (ASAQ) and prevalence of molecular markers of drug resistance in Zanzibar in 2017. Methods: Treatment efficacy of the clinical trial conducted in 2017 was compared with efficacies in 2002 and 2005. A total of 142 samples were genotyped for single nucleotide polymorphisms (SNPs) in the P. falciparum chloroquine resistance transporter gene (pfcrt) gene, the P. falciparum multi drug resistance 1 (pfmdr1) gene, and in the P. falciparum Kelch 13 (PfK13) propeller region. Prevalence of SNPs were assessed during the period 2002-2017. Results: Cure rate was 100% in 2017, compared to 94% and 96%, in 2002-2003 and 2005, respectively. Day 3 fever clearance rate were also high 93% (2002-3), 99% (2005) and 98% (2017) in all studies. Prevalence of pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y and pfmdr1 (86Y, 184Y and 1246Y) YYY haplotypes were significantly decreased between 2002-3 and 2017 (p &lt; 0.001). No SNP in the PfK13 gene related to artemisinin resistance was identified. Conclusion: Efficacy of ASAQ remains high after fourteen years as first-line treatment, despite the wide-scale use of ASAQ, and there is no evidence of selection of resistance markers in Zanzibar. Continuous monitoring of drug efficacy and resistance markers is recommended. / <p>This master thesis is a collaboration project between Institutionen för kvinnors och barns hälsa, Department of Women's and Children's Health, Uppsala Universtiy and Anders Björkman group, Department of Microbiology, Tumor and Cell Biology (MTC), C1, Karolinska Institutet. Laboratory examinations were mainly conducted at MTC house, Karolinska Institutet.</p>