Mouse hepatocytes in primary monolayer culture (4 hr) were exposed for 10 min at 37°C to anisosmotic medium of altered NaCl concentration. Hepatocytes maintained constant relative cell volume (experimental volume/control volume) as a function of external medium relative osmolality (control mOsm/experimental mOsm), ranging from 0.8 to 1.5. In contrast, the relative cell volume fit a predicted Boyle-Van't Hoff plot when the experiment was done at 4°C. Mouse liver slices were used for electrophysiologic studies, in which hepatocyte transmembrane potential (Vm) and intracellular K+ activity (aKi) were recorded continuously by open-tip and liquid ion-exchanger ion-sensitive glass microelectrodes, respectively. Liver slices were superfused with control and then with anisosmotic medium of altered NaCl concentration. Vm increased (hyperpolarized) with hypoosmotic medium and decreased (depolarized) with hyperosmotic medium, and ln [10(experimental Vm/control Vm)] was a linear function of relative osmolality (control mOsm/experimental mOsm) in the range 0.8-1.5. The aKi did not change when medium osmolality was decreased 40-70 mOsm from control of 280 mOsm. Similar hypoosmotic stress in the presence of either 60 mm K+ or 1 mm quinine HCl or at 27°C resulted in no change in Vm compared with a 20-mV increase in Vm without the added agents or at 37°C. We conclude that mouse hepatocytes maintain their volume and aKi in response to anisosmotic medium; however, Vm behaves as an osmometer under these conditions. Also, increases in Vm by hypoosmotic stress were abolished by conditions or agents that inhibit K+ conductance.

The repeated emergence of Plasmodium falciparum resistance to first-line antimalarial drugs necessitates understanding the underlying resistance mechanisms to detect and monitor resistance in the field and to inform drug discovery efforts. With the advent of the FRG NOD human liver-chimeric (huHep) mouse model for P. falciparum genetic crosses, interest has renewed in harnessing this forward genetics tool to study traits including drug resistance. The antimalarial quinine (QN) is of particular interest as it has retained efficacy over 400 years as parasite resistance has been slow to develop against the drug, likely due to a multifactorial mechanism of which only several genes have been partially implicated. Chloroquine (CQ) is a former first-line drug for P. falciparum (that is still in use for P. vivax), and it’s phasing out has been associated with the recent emergence of CQ-sensitive P. falciparum parasites. While the CQ resistance transporter (PfCRT) is known to be the primary driver of resistance, studies have provided evidence for secondary modulators of CQ, of which only the multidrug resistance protein 1 (PfMDR1) transporter has been identified. This thesis addresses the hypotheses that additional mediators are involved in the parasite resistance mechanism to QN and that genes other than pfmdr1 modulate parasite resistance to CQ. In chapter 3, we present the P. falciparum genetic cross that we conducted between the QN- and CQ-sensitive African NF54 and QN- and CQ-resistant Cambodian Cam3.II parasites in huHep mice, in collaboration with Dr. Photini Sinnis’s laboratory at Johns Hopkins University. By applying different selective conditions to cross progeny bulk pools prior to cloning these bulks, we were able to recover 120 unique recombinant progeny from this cross. We observed minimal overlap in the progeny genotypes obtained from CQ and QN pressure, suggesting distinct mechanisms for parasite resistance to these drugs. Bulk progeny selection and progeny clone-based QN linkage mapping approaches identified quantitative trait loci (QTLs) on chromosomes 7 and 12, as well as minor QTLs on other chromosomes, consistent with a multifactorial resistance mechanism. We applied the latter approach to investigate parasite response to CQ and its active metabolite monodesethyl-CQ (md-CQ) and identified a novel chromosome 12 QTL in addition to pfcrt. Interestingly, while the chromosome 12 QTLs overlapped, the chromosome 7 QTL for high-grade QN resistance did not contain pfcrt. In chapter 4, we used bioinformatic approaches, whole-genome sequence data from our cross and field isolates, and literature review to identify the drug/metabolite transporter 1 (DMT1) as the top candidate of the chromosome 7 QTL, and S-adenosylmethionine mitochondrial carrier protein (SAMC), hydroxyethylthiazole kinase (ThzK), and ATP-dependent zinc metalloprotease (FtsH1) as the top candidates for the chromosome 12 QTLs. By harnessing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing methodologies (SNP-editing, knockout, and tagging), we obtained evidence favoring DMT1 as a marker of QN resistance and localized this transporter to structures associated with vesicular trafficking, PVM, lipid bodies, and the lysosome-like digestive vacuole. We also harnessed SNP-editing and identified FtsH1 as a potential mediator of QN resistance and a modulator of CQ and md-CQ resistance. QN, mefloquine, and lumefantrine belong to the same aryl-amino alcohol class, and we found that QN is structurally more similar to mefloquine than lumefantrine. We also showed that QN can partially inhibit heme detoxification. While conducting the work outlined in chapters 3 and 4, we identified an unmet need for quickly identifying clonal recombinant progeny and validating parasite identity, which inspired the study presented in chapter 5. We developed a genotyping method that can assess drug resistance-conferring SNPs directly from P. falciparum culture or infected blood as well as a multiplexed microsatellite genotyping method with five broadly informative markers. Both methods were applied in chapter 3 to identify clonal recombinant progeny, and the SNP genotyping method was used in chapter 4 to validate gene editing and progeny identity. We also tested the resolution, sensitivity, time, and cost of each method as well as whole-genome sequencing and recommended the ideal application for each genotyping method. Our data demonstrate that DMT1 is a novel marker for QN resistance, and a new chromosome 12 locus associates with CQ response, of which ftsh1 is a potential candidate. In chapter 6, we discuss the potential mechanisms by which DMT1 is involved in QN resistance, the potential impact of our findings, and future experiments that can further characterize the QN and CQ resistance mechanisms and the functional role of these candidate genes.

L’amertume fait partie intégrante de notre alimentation. Elle est par exemple fortement représentée dans certaines boissons (ex: café) ou dans certains légumes tels les crucifères. Néanmoins, la perception de l’amertume varie entre les individus et certains aliments considérés comme bénéfiques pour la santé peuvent être rejetés en raison de leur goût amer. Des facteurs génétiques (ex : polymorphisme génétique des récepteurs du goût amer) ou environnementaux (ex : âge, prise de médicaments) expliquent en partie les variations interindividuelles dans la perception de l’amertume. Cependant, d’autres facteurs péri-récepteurs pourraient intervenir, notamment la composition salivaire. Afin d’investiguer dans un premier temps le lien existant entre le protéome salivaire propre à un individu et sa sensibilité à l’amertume, le seuil de détection du goût amer de la caféine a été mesuré sur 29 hommes sains. Leur salive au repos a été étudiée par électrophorèse mono- et bidimensionnelle. L’analyse par électrophorèse bidimensionnelle de la salive au repos des 6 sujets les plus sensibles et 6 les sujets les moins sensibles à la caféine a permis la détection de 255 spots, dont 26 étaient significativement différents entre hyper- et hyposensibles. L’identification de ces 26 spots a révélé la surexpression de fragments d’alpha amylase, de fragments d’albumine sérique, et de sous-unités alpha de l’immunoglobuline A ainsi que la sous-expression de cystatine SN chez les hypersensibles. Ce dernier résultat a été confirmé par Western Blot. Ceci a permis de formuler une hypothèse sur le rôle de la protéolyse en bouche sur la sensibilité à l’amertume. Dans un deuxième temps et afin d’étudier l’effet des molécules amères sur la composition salivaire, une étude in vitro a été menée sur la lignée cellulaire de glandes salivaires humaines HSG différenciées en acini ou non. Après une mise au point des conditions de différenciation (culture dite en 3D), la cystatine SN a été détectée dans les cellules HSG par Western blot après traitement des cellules à la caféine, à la quinine, et à l’urée. Après traitement à la caféine à 5, 50 ou 100µM, une quantification par ELISA a mis en évidence que la cystatine SN était toujours plus abondante dans les cellules HSG différenciées que dans les cellules non-différenciées. Spécifiquement dans les cellules différenciées, l’exposition à la caféine induisait une sur-expression de cystatine SN, la teneur maximale en cystatine SN étant observée avec la caféine à 50 µM. La présence de cystatine SN a également été détectée dans les milieux de culture / Bitterness is present in every day beverages (e.g. coffee) and foods (e.g. vegetables such as cruciferous plants). However, bitterness is perceived differently among individuals and some foods considered as healthy may be rejected due to their bitter taste. Several genetic (eg. genetic polymorphism of bitter taste receptors) or environmental (eg. age, medications) factors partly explain the interindividual variability in bitterness perception. However, other peri-receptor factors may intervene, in particular salivary composition. First, in order to investigate the link between salivary proteome and sensitivity to bitterness, the detection threshold to the bitter taste of caffeine was measured in 29 male healthy subjects. Their resting saliva was studied by one- and two-dimensional electrophoresis. Two-dimensional electrophoresis revealed that 26 out of 255 spots were significantly different between the 6 hypersensitive and 6 hyposensitive subjects to the bitter taste of caffeine. Identification of the 26 spots revealed an overexpression of amylase-, serum albumin-, and immunoglobulin A fragments, and an underexpression of cystatin SN in hypersensitive subjects. The latter finding was confirmed by Western blotting. These results have led to formulate an hypothesis on the role of in-mouth proteolysis in bitterness perception. Second, in order to study the effect of bitter molecules on salivary composition, an in vitro study was performed on undifferentiated and differentiated human salivary cell line HSG. After setting the experimental conditions for HSG cell differentiation (culture in 3D conditions), cystatin SN was detected in HSG cells by Western blot after treatment with caffeine, quinine, and urea. After cell exposure with caffeine at 5, 50 and 100 µM, quantification by ELISA demonstrated that cystatin SN was always more abundant in differentiated vs undifferentiated HSG cells. Specifically in differentiated cells, caffeine exposure resulted in over-expression of cystatin SN, 50µM inducing the highest effect. Cystatin SN was also detected in culture media of the HSG cells

While the molecular defect that cause sickle cell disease has well been established, the cause of vaso-occlusive crisis remains elusive and largely debated upon. Majority of studies have linked the painful episodes to polymerization of sickle hemoglobin following its deoxygenation. The variability of the disease symptoms among patients, compounds efforts for a holistic therapy. Hydroxyurea, a stimulator of Hb F induction and a widely used treatment, has ameliorated the complication of SCD but it is only effective in 50% of the patients. Expression of Hb F lowers the content of Hb S in blood and hence reduces oxidative stress caused by Hb S denaturation. Sickle cell disease severity depends on several factors. Most importantly, the ability of red cell to sickle dominates all other determinants. While deoxygenation of sickle hemoglobin may be inevitable, the duration with which the red cell remains in the deoxygenated state can be manipulated. Deoxygenation is a transient process that when compared to the time taken to develop the long filaments of deoxyhemoglobin to causes severe sickling, the red cell would have been cycled back to the lungs and re-oxygenated to restore the healthy conditions of the cell. In fact, if sickle cells would flow as fast as healthy erythrocytes, the detrimental impacts of sickling such as vaso-occlusive crisis, would not be a concern for this disease. Unfortunately, the unstable sickle hemoglobin undergoes denaturation through auto-oxidation, which imposes oxidative stress to the cells. The oxidative stress inhibits erythrocytes tyrosine phosphatases, a course which subsequently impair their constitutive action against the tyrosine kinases. In the end, a net tyrosine phosphorylation state in the red cell membrane proteins, most notably the transmembrane protein band 3, succeeds. Band 3 tyrosine phosphorylation abrogates the protein’s interaction with ankyrin and spectrin-actin cytoskeleton, hence the cytoskeleton loses its major anchorage to the membrane thus engendering membrane destabilization. A destabilized erythrocyte sheds membrane fragments in form of microvesicles/microparticles and discharges free hemoglobin into the extra cellular matrix. In consequence, the microparticles power initiation of coagulation cascade through activation of thrombin, while free Hb inflicts inflammation, scavenges nitric oxide which is necessary for vasodilation and induces further oxidative stress within the microvasculature, and activates expression of adhesion receptors on the endothelium. Taken together, these events culminate in entrapment of red cells (not naming leucocytes and platelets) in the microvasculature, blockade of blood vessels and further damage of erythrocytes through prolonged deoxygenated state thus terminating in tissue injury, strokes, and organ damage, amid vaso-occlusive episodes which always require hospitalization and extensive medical care for survival. Band 3 tyrosine phosphorylation and membrane weakening is not unique just to SCD, but also a druggable target for malaria. Malaria, a disease that is touted as the evolutionary cause of sickle cell disease, surprisingly thrives through the same mechanism. Briefly, malaria parasite consumes hemoglobin for its DNA synthesis, and in the process generate reactive oxygen species from denatured hemoglobin that feeds into the oxidative stress which triggers band 3 tyrosine phosphorylation. In this case however, a destabilized membrane offers perfect conditions for merozoites’ (malaria daughter parasites) egress/exit out of the cell to begin infecting other red cells. Ultimately, the ensuing anemia and organ dysfunction leads to patient’s death. Treatment of diseased cells with imatinib and other Syk inhibitors effectively reversed membrane weakening. A stabilized membrane not only survives longer in circulation to alleviate SCD symptoms but also traps and starves malaria parasite leading to termination of the parasitic infection. With band 3 tyrosine phosphorylation at center stage, this dissertation explores the above events in an effort to unveil a novel therapy for sickle cell and malaria diseases. First, the therapeutic strategy regarding SCD is discussed in detail beginning with non-transfused patients and ending in additional mechanistic study on inactivation of the principal erythrocyte’s protein tyrosine phosphatase 1 B, PTP1B. The dissertation then provides an initial proof of concept on efficacy of imatinib in treatment of malaria as a monotherapy and its efficacy when used in a triple combination therapy with the standard of care treatment. Finally, I outline an alternative possible mechanism of action of quinine against malaria.

Thesis submitted in fulfilment of the requirements for the degree Magister Technologiae: Chemistry in the Faculty of Applied Science at the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY 2012 / Chirality is very important to the pharmaceutical industry as enantiomers have the same macroproperties except for their optical and pharmacological activity. Industrial research has thus focused to find the most effective resolution technique. However, our aim was to obtain more information regarding the discrimination process. In this project the structures of the hydrates of di-quininium L-malate, (2QUIN+)(L-MA2-)•2H2O and the di-quininium D-malate, (2QUIN+)(D-MA2-)•2H2O have been investigated. (-)-Quinine (QUIN) did not show selectivity between the D and L malic acid and the structure of (2QUIN+)(DL-MA2-)•2H2O was obtained. Effect of solvents was demonstrated in the study and the structure of (QUIN+)(D-MA-)•H2O) was reported. The relationship between C-O bonds of the carboxylate and carboxylic moieties and ÄpKa was explored in salt and co-crystal formation. Kinetics of absorption was conducted for the reaction of (+)-deoxycholic acid (DCA) with n-propylamine and DCA with racemic sec-butylamine. The rate constants of the reactions were determined. Kinetics of desolvation was performed on the powder samples of mixtures of DCA and sec-butylamine and DCA with di-n-butylamine. Non-isothermal methods were used where a series of TG analyses was carried out at different heating rates (2, 4, 10, 32 K min-1). The structures of DCA with n-propylamine and di-n-butylamine were elucidated. The selectivity of DCA was investigated. The host compound was found to be able to successfully resolve racemic sec-butylamine (2-BUAM) and 2-amino-3-methylbutane (MeBUAM). The structures of DCA with enantiomers of these guests are reported in the study. The structures of R-BUAM and S-BUAM were solved in different space groups while R-MeBUAM and S-MeBUAM crystallized in the same space group.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-10-10T14:13:58Z No. of bitstreams: 1 ARYELE PINTO IZAGUIRRY SET 2019.pdf: 6182820 bytes, checksum: 5b47d16cee80dd95195380917e9ac189 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-10-10T14:14:11Z (GMT) No. of bitstreams: 1 ARYELE PINTO IZAGUIRRY SET 2019.pdf: 6182820 bytes, checksum: 5b47d16cee80dd95195380917e9ac189 (MD5) / Made available in DSpace on 2017-10-10T14:14:11Z (GMT). No. of bitstreams: 1 ARYELE PINTO IZAGUIRRY SET 2019.pdf: 6182820 bytes, checksum: 5b47d16cee80dd95195380917e9ac189 (MD5) Previous issue date: 2017-08-05 / A quinina é um alcaloide extraído da casca da árvore chinchona, a qual apresenta ação antimalárica. A malária é a doença parasitária mais prevalente no mundo, causada pelo protozoário do gênero Plasmodium. Atualmente existem diversas terapias medicamentosas para o tratamento da malária, entretanto devido aos elevados efeitos colaterais e a resistência dos parasitos às terapias, a utilização de ferramentas como os nanosistemas tem sido uma alternativa a estas limitações. A utilização de nanocápsulas contendo quinina já apresentou efeito satisfatório frente a parasitemia induzida por P. berghei em roedores. Entretanto os efeitos toxicológicos desta formulação sobre o sistema reprodutivo e desenvolvimento fetal são desconhecidos. Desta forma, este trabalho teve como objetivo avaliar os efeitos da quinina livre e nanoencapsulada sobre o sistema reprodutivo, bem como sobre o desenvolvimento pré-natal. Para o desenvolvimento do estudo, o trabalho foi dividido em 2 experimentos. No experimento I, ratos da raça Wistar adultos machos e fêmeas foram divididos em 4 grupos: Controle, Nanocápsula branca (B-NC), Quinina Livre (25mg/Kg) e Quinina Nanoencapsulada (Q-NC) (25mg/kg). Após 7 dias de tratamento, verificou-se dano testicular e ovariano induzido pela quinina, evidenciados por aumento nos níveis de espécies reativas e malondialdeído, diminuição da atividade da enzima esteroidogênica 17 β-hidroxiesteróide desidrogenase, bem como alterações na capacidade antioxidante total. Em fêmeas verificou-se diminuição da viabilidade folicular e em machos diminuição da integridade de membrana dos espermatozoides, bem como alterações histológicas moderadas nos testículos após exposição à quinina. Os machos tratados com Q-NC, apresentaram níveis de espécies reativas diminuídos, capacidade antioxidante total aos níveis de controle 6 bem como espermatozoides com 100% de integridade de membrana. Entretanto os outros parâmetros apresentaram as mesmas alterações dos animais tratados com quinina livre. Após tratamento com Q-NC, as fêmeas apresentaram níveis de espécies reativas, capacidade antioxidante total, atividade da enzima 17 β-hidroxiesteróide desidrogenase e viabilidade folicular aos níveis do grupo controle. No experimento II avaliamos a toxicidade pré-natal, onde ratas prenhes foram tratadas do 7º ao 13º dia de gestação com quinina livre e Q-NC (25mg/kg) e eutanasiadas no 20º dia da prenhes e os fetos foram removidos para as análises morfológicas e bioquímicas. As fêmeas grávidas não apresentavam sinais visíveis de toxicidade. Não houve alteração no consumo de alimentos e água das fêmeas, bem como não houve diferença no ganho de peso. Os fetos que receberam tratamento perinatal com Q-NC não apresentaram anormalidades externas ou esqueléticas significativas e não houveram alterações nas medidas biométricas. Em ambos os grupos, foi verificado a presença de quinina no tecido cerebral e hepático dos fetos, demonstrando que a quinina atravessa a barreira transplacentária, entretanto, não foi verificada diferença significativa entre os grupos. Desta forma, pode-se verificar que nanocápsulas são bons carreadores para quinina, diminuindo os efeitos nocivos induzidos pela quinina em tecido ovariano e testicular, bem como não induziu danos maternos e fetais nos parâmetros avaliados, demonstrando ser uma terapia promissora para o tratamento da malária, inclusive em gestantes. / Quinine is an alkaloid extracted from the bark of the chinchona tree, which has antimalarial activity. Malaria is the most prevalent parasitic disease in the world, caused by the protozoan of the Plasmodium genus. Currently, there are several drug therapies for the malaria treatment. However, due to the high side effects and resistance of the parasites to the therapies, the use of tools such as nanosystems has been an alternative to these limitations. The use of quinine-loaded nanocapsules demonstrated a satisfactory effect against parasitemia induced by P. berghei in rodents, however the toxicological effects of this formulation on the reproductive system and foetal development are unknown. Thus, this work evaluated the effects of free and nanoencapsulated quinine on reproductive system, as well as prenatal development. For the development of the study, the work was divided into 2 experiments. Experiment I: Male and female adult Wistar rats were divided into 4 groups: Control, Blank Nanocapsules (B-NC), Free Quinine (25mg / kg) and Quinine-loaded Nanocapsules (Q-NC) (25mg / kg). After 7 days of treatment, testicular and ovarian damage quinine-induced was evidenced by increased levels of reactive species and malondialdehyde, decreased steroidogenic 17β-hydroxysteroid dehydrogenase enzyme activity, and changes in total antioxidant capacity. In females there was a decrease in follicular viability and in males decrease of spermatozoa membrane integrity, as well as moderate histological changes in the testes after exposure to quinine. Males treated with Q-NC showed decreased reactive species levels, total antioxidant capacity at control levels as well as spermatozoa with 100% of membrane integrity. However, the other parameters showed the same alterations as those treated with free quinine. After treatment with Q-NC, the females showed levels 8 of reactive species, total antioxidant capacity, 17 β-hydroxysteroid dehydrogenase enzyme activity and follicular viability at the levels of the control group. In experiment II we evaluated the prenatal toxicity, where pregnant rats were treated from the 7th to the 13th day of pregnancy with free quinine and Q-NC (25mg / kg) and euthanized on the 20th day of pregnancy and the fetuses were removed for morphological analysis and biochemical. Pregnant females showed no visible signs of toxicity. There was no change in the food and water consumption of the females, as well was there was no difference in the weight gain. Fetuses receiving perinatal Q-NC treatment did not present significant external or skeletal abnormalities and there were no changes in biometric measurements. In both groups, the presence of quinine in the cerebral and hepatic tissues of the fetuses was verified, demonstrating that quinine crosses the transplacental barrier, however, there was no significant difference between the groups. Thus, we verified that nanocapsules are good carriers for quinine, reducing the harmful effects induced by quinine in ovarian and testicular tissue, as well as did not induce maternal and fetal damages in the evaluated parameters, demonstrating to be a promising therapy for the malaria treatment, including in pregnancy.

Malaria is a parasitic disease claiming one million lives worldwide annually. Unfortunately, malaria-endemic countries in need of good quality medicines are also overwhelmed with counterfeit or substandard medicine. This results in treatment inefficacy, resistance towards treatment and death. Counterfeit or substandard quinine sulfate tablets are known to have infiltrated the market, however at this point in time, treatment efficacy of quinine sulfate has fortunately not yet been significantly impaired by resistance, but immediate action is required to prevent it from becoming obsolete. Validated analytical methods with justified specifications are effective in controlling the quality of medicines and to minimise the effect of poor quality medicines. Pharmacopoeia specifies analytical quality control procedures and accompanying specifications to standardise acceptable levels of product quality. Understandably, different monographs of different pharmacopoeias are developed by different independent laboratories and therefore their respective test procedures/specifications for the same FPP may differ from each other. Institutions such as the Pharmacopoeial Dicussion Group (PDG) aim to harmonise pharmacopoeia in order to synchronise final outcomes. This study evaluated the relevancy of differences in analytical procedures, results and specifications for quinine sulfate tablets set by the United States Pharmacopoeia (USP), British Pharmacopoeia (BP) and International Pharmacopoeia (Ph.Int.) in an aim to ensure that these different methods all provide with similar final outcomes and that they be effective in successfully evaluating the quality of quinine sulfate tablets. Four quinine sulfate tablet products were obtained from different manufacturers and were subjected to the tests of all three pharmacopoeia – BP, USP and Ph.Int. The results from identification, assay and related substance testing concluded that the outcomes were the same between the pharmacopoeia despite their differences in techniques/procedures/specifications. The assay, identification and related substances methods and specifications set by each respective monograph were deemed appropriate to evaluate the quality of quinine sulfate tablets. Even with differences in methodology, quantitative techniques and specifications, the USP and BP dissolution methods for quinine sulfate tablets shared the same final outcome at the first stage of dissolution, whereas none of the products achieved a compliant outcome using the Ph.Int. dissolution method. Possible reasons for the poor dissolution (when using the Ph.Int. method) were identified and investigated. Investigation into the solubility of quinine sulfate found the Ph.Int. dissolution method conditions to be too stringent, as the solubility of quinine sulfate in phosphate buffer pH 6.8 (dissolution medium specified by the Ph.Int.) was found to be much less than in acidic media (as proposed by the BP and USP dissolution methods). Several adapted dissolution methods (called developmental studies) were investigated to serve as potential alternatives for the Ph.Int. dissolution method. The developmental studies investigated an alternative dissolution medium, agitation rates (50 rpm, 75 rpm, 100 rpm) and medium volumes (500 ml, 750 ml, 900 ml and 1000 ml). Developmental study 6 was proposed as an alternative dissolution method. Developmental study 6 stipulates the use of the same medium as the original Ph.Int. method, as it was deemed the medium of choice for its discriminatory ability. To address the impaired solubility of quinine sulfate in phosphate buffer, the medium volume and agitation were increased (in reference to the original method) to 900 ml and 100 rpm respectively. The same analytical quantitation technique (UV-Vis spectroscopy) is proposed for Developmental study 6. The newly proposed method provided with final outcomes comparable to that of the USP and BP, however having more discriminatory power than the USP and BP. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Malaria is a parasitic disease claiming one million lives worldwide annually. Unfortunately, malaria-endemic countries in need of good quality medicines are also overwhelmed with counterfeit or substandard medicine. This results in treatment inefficacy, resistance towards treatment and death. Counterfeit or substandard quinine sulfate tablets are known to have infiltrated the market, however at this point in time, treatment efficacy of quinine sulfate has fortunately not yet been significantly impaired by resistance, but immediate action is required to prevent it from becoming obsolete. Validated analytical methods with justified specifications are effective in controlling the quality of medicines and to minimise the effect of poor quality medicines. Pharmacopoeia specifies analytical quality control procedures and accompanying specifications to standardise acceptable levels of product quality. Understandably, different monographs of different pharmacopoeias are developed by different independent laboratories and therefore their respective test procedures/specifications for the same FPP may differ from each other. Institutions such as the Pharmacopoeial Dicussion Group (PDG) aim to harmonise pharmacopoeia in order to synchronise final outcomes. This study evaluated the relevancy of differences in analytical procedures, results and specifications for quinine sulfate tablets set by the United States Pharmacopoeia (USP), British Pharmacopoeia (BP) and International Pharmacopoeia (Ph.Int.) in an aim to ensure that these different methods all provide with similar final outcomes and that they be effective in successfully evaluating the quality of quinine sulfate tablets. Four quinine sulfate tablet products were obtained from different manufacturers and were subjected to the tests of all three pharmacopoeia – BP, USP and Ph.Int. The results from identification, assay and related substance testing concluded that the outcomes were the same between the pharmacopoeia despite their differences in techniques/procedures/specifications. The assay, identification and related substances methods and specifications set by each respective monograph were deemed appropriate to evaluate the quality of quinine sulfate tablets. Even with differences in methodology, quantitative techniques and specifications, the USP and BP dissolution methods for quinine sulfate tablets shared the same final outcome at the first stage of dissolution, whereas none of the products achieved a compliant outcome using the Ph.Int. dissolution method. Possible reasons for the poor dissolution (when using the Ph.Int. method) were identified and investigated. Investigation into the solubility of quinine sulfate found the Ph.Int. dissolution method conditions to be too stringent, as the solubility of quinine sulfate in phosphate buffer pH 6.8 (dissolution medium specified by the Ph.Int.) was found to be much less than in acidic media (as proposed by the BP and USP dissolution methods). Several adapted dissolution methods (called developmental studies) were investigated to serve as potential alternatives for the Ph.Int. dissolution method. The developmental studies investigated an alternative dissolution medium, agitation rates (50 rpm, 75 rpm, 100 rpm) and medium volumes (500 ml, 750 ml, 900 ml and 1000 ml). Developmental study 6 was proposed as an alternative dissolution method. Developmental study 6 stipulates the use of the same medium as the original Ph.Int. method, as it was deemed the medium of choice for its discriminatory ability. To address the impaired solubility of quinine sulfate in phosphate buffer, the medium volume and agitation were increased (in reference to the original method) to 900 ml and 100 rpm respectively. The same analytical quantitation technique (UV-Vis spectroscopy) is proposed for Developmental study 6. The newly proposed method provided with final outcomes comparable to that of the USP and BP, however having more discriminatory power than the USP and BP. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Thesis (Ph. D.)--University of California, San Diego, 2009. / Title from first page of PDF file (viewed July 9, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 369-389).

A malária, causada por protozoários intracelulares do gênero Plasmodium, é uma das doenças tropicais mais devastadoras existentes. Mais de 3 bilhões de pessoas vivem em regiões endêmicas para a malária. Cinco espécies de Plasmodium (falciparum, vivax, ovale, malariae e knowlesi) causam doenças em humanos e a infecção com P. falciparum, o mais letal desses parasitas, resulta em mais de 1 milhão de mortes anualmente. O desenvolvimento de resistência aos fármacos antimaláricos tradicionais, leva ao uso de combinações de fármacos como a quinina (QN) e a doxiciclina (DOX). Nesse contexto, os objetivos deste trabalho foram desenvolver e caracterizar formulação de nanopartículas lipídicas sólidas (NLS) contendo a associação de QN/DOX, avaliar sua eficácia em um modelo in vivo de malária berghei, determinar a sua farmacocinética e o coeficiente de partição nos eritrócitos dos fármacos livres e nanoencapsulados. A formulação de NLS contendo QN/DOX (2,0/0,2 mg/mL) foi preparada pela técnica de homogeneização a alta pressão, utilizando polissorbato 80 e Lipoid® como emulsionantes e palmitato de cetila como matriz lipídica. No estudo preliminar de estabilidade, a formulação de NLS contendo QN/DOX apresentou tamanho de partícula adequado (152,8 ± 5,26 nm), índice de polidispersão (0,173 ± 0,006), potencial zeta (-38,6 ± 1,82 mV), alto conteúdo dos fármacos (95,9% ± 0,70/ 94,1% ± 2,41) e adequada eficiência de encapsulação (94,2% ± 1,14/83,0% ± 2,52), após 21 dias de armazenamento em temperatura ambiente. Para a análise do teor um método rápido e específico de cromatografia líquida-acoplada a espectrometria de massa (LC-MS/MS) foi desenvolvido e validado para a determinação simultânea de QN e DOX nas formulações. O método por LC-MS/MS utilizou coluna Waters Sun Fire C18 (50 mm x 3,0 mm de diâmetro) e a fase móvel foi composta de acetonitrila:ácido fórmico 0,1% (75:25, v/v), no fluxo de 0,45 mL/min (split 1:3). O volume de injeção foi de 10 μL. Ratos Wistar infectados por P. berghei foram utilizados para avaliar a eficácia da formulação de NLS contendo QN/DOX utilizando diferentes regimes de dose. As doses efetivas da formulação, i.v. (75/7,5 mg/kg/dia) e oral (105/10,5 mg/kg/dia), representam uma redução de quase 30% em comparação com os fármacos livres utilizados em associação. A farmacocinética foi avaliada após a administração dos fármacos livres ou nanoencapsulados pela vias i.v. (10/1 mg/ kg) e oral (25/2,5 mg/kg) em ratos Wistar infectados. Para a quantificação das amostras de plasma dos ratos, método por LC-MS/MS foi desenvolvido e validado. A QN, a DOX e a cimetidina (padrão interno, PI) foram extraídos do plasma através de precipitação de proteínas e a fase móvel consistiu de metanol/ácido fórmico 0,1% (70:30, v/v), no fluxo de 0,5 mL / min (split 1:3). A detecção foi realizada através da ionização por electrospray positivo, no modo de monitoramento de reações múltiplas, onde foram monitoradas as transições 325,0>307,0, 445,0>428,1 e 252,8>159,0, para QN, DOX e PI, respectivamente. A análise foi realizada em 2,0 min e o método foi linear na faixa de concentração plasmática entre 5-5000 ng/mL. Nenhuma alteração significativa dos parâmetros farmacocinéticos foi observada para ambos os fármacos e vias de administração, após a nanoencapsulação. O coeficiente de partição da QN nos eritrócitos infectados por P. berghei aumentou (5,53 ± 0,28) quando a formulação de NLS contendo QN/DOX foi usada em comparação com os fármacos livres em associação (3,81± 0,23). Nenhuma alteração significativa na penetração intraeritrocitária da DOX foi observada com a nanoencapsulação. Os resultados demonstram que a nanoencapsulação da QN/DOX em NLS diminui a dose efetiva para o tratamento da malária, sendo uma alternativa interessante a ser investigada para o tratamento da malária falciparum resistente. / Malaria is one of the most devastating tropical diseases caused by intracellular protozoan parasites of the genus Plasmodium. More than 3 billion people live in malarial endemic regions. Five species of Plasmodium (falciparum, vivax, ovale, malariae and knowlesi) cause disease in humans and infection with P. falciparum, the most deadly of these parasites, results in more than 1 million deaths annually. The development of resistance to traditional antimalarial drugs leads to the use of drug combinations such as quinine (QN)/doxycycline (DOX). In this context, the aims of this work were to develop and characterize solid lipid nanoparticles (SLN) loaded with QN/ DOX, to evaluate their efficacy in an in vivo model of berghei malaria, and to determine their pharmacokinetics and erythrocyte partition coefficient compared to the non-encapsulated (free) drug association. The SLN were prepared by high pressure homogenization technique using polysorbate 80 and Lipoid® as emulsifiers and cetyl palmitate as lipid matrix. In the preliminary stability study, QN/DOX-loaded SLN (2.0/0.2 mg/mL) presented adequate particle size (152.8 ± 5.26 nm), polydispersion index (0.173 ± 0.006), zeta potential (-38.6 ± 1.82 mV), high drug content (95.9% ± 0.70/94.1% ± 2.41) and appropriate encapsulation efficiency (94.2% ± 1.14/83.0% ± 2.52) after 21 days of storage at room temperature. For the assay analysis, a fast and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of QN and DOX. The LC-MS/MS method was carried out on a Sun Fire Waters C18 column (50 mm x 3.0 mm I.D.) and the mobile phase consisted of acetonitrile:0.1% formic acid (75:25, v/v), run at a flow rate of 0.45 mL/min (split 1:3). The injection volume was 10 μL. Plasmodium berghei infected Wistar rats were used to evaluate the efficacy of QN/DOX-loaded SLN using different dosing regimens. The effective QN/DOX-loaded SLN i.v. (75/7.5 mg/kg/day) and oral (105/10.5 mg/kg/day) doses represent an almost 30% reduction compared to the free drugs in association. Plasma pharmacokinetics was evaluated after administration of free or nanoencapsulated QN/DOX by i.v. (10/1 mg/kg) and oral (25/2.5 mg/kg) routes to infected Wistar rats. For the quantification of the rat plasma samples, a fast, sensitive and specific LC-MS-MS method was developed and validated for the determination of QN and DOX. QN, DOX and cimetidine (internal standard, IS) were extracted from the plasma by protein precipitation and the mobile phase consisted of methanol/formic acid 0.1% (70:30, v/v), run at a flow rate of 0.5 mL/min (split 1:3). Detection was carried out by positive Electrospray Ionization in multiple reaction monitoring mode, monitoring the transitions 325.0>307.0, 445.0>428.1 and 252.8>159.0, for QN, DOX and IS, respectively. The analysis was carried out in 2.0 min and the method was linear in the plasma concentration range of 5-5000 ng/mL. No significant alteration of pharmacokinetic parameters was observed for both drugs and routes of dosing after nanoencapsulation. QN partition coefficient into P. berghei infected erythrocyte was increased (5.53 ± 0.28) when the QN/DOX-loaded SLN was used in comparison with the free drugs in association (3.81 ± 0.23). No significant alteration on DOX erythrocyte partition coefficient was observed. In summary, the results showed that QN/DOX nanoencapsulation into SLN allows the reduction of the effective antimalarial dose being an interesting alternative to be investigated for the treatment of falciparum resistant malaria.