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

Endocrine resistance is a significant clinical problem in the treatment of estrogen (E2) receptor positive breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation respectively. While ER positive breast cancers typically express a high ratio of ERα to ERβ, the acquisition of antiestrogen resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers ERβ has been shown to function in opposition to the ERα in the presence of E2. Here we demonstrate that exposure to two different ERβ agonists results in decreased cell viability, and produced a marked reduction in G2/M phase in antiestrogen resistant breast cancer cell line in conjunction with altered cyclin D1, and cyclin E expression relative to E2. ERβ agonists also strongly downregulated Bcl-2 expression and recruited both ERs to the Bcl-2 and pS2 E2-response elements resulting in a reduction in mRNA transcripts from both of these genes. Bcl-2 reduction correlated with increased lipidation of LC3-I to LC3-II, indicative of increased autophagic flux. Although ERβ agonist treatment alone did not induce apoptosis, remarkably, the coaddition of ERβ agonist and the autophagy inhibitor, chloroquine, resulted in robust cell death. Lastly, in vivo studies demonstrate that preferential-ERβ agonists are not estrogenic in the uterus or mammary gland. Together, these observations suggest that combined therapies including an ERβ agonist and an autophagy inhibitor may provide the basis for a safe, novel approach to the treatment of antiestrogen-resistant breast cancers.

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

O funcionamento pleno e harmônico de uma célula está intimamente associado à sua capacidade de manter a integridade genômica. Diversos agentes químicos e físicos exógenos, bem como produtos do próprio metabolismo celular, podem interagir com o DNA, causando danos a esta molécula. Em respota a esses eventos, um intrincado mecanismo de resposta a danos ao DNA é ativado, podendo culminar tanto na correção das lesões, como na ativação de programas de morte celular, como a apoptose, sempre com o intuito de preservar a homeostase tecidual. Falhas neste mecanismo estão associadas a um aumento nas taxas de mutação, que apesar de constituírem a base da diversidade genética e evolução das espécies, está intimimamente associado à tumorigênese e ao envelhecimento. Neste trabalho, dividido em duas partes, utilizamos células de glioma humano como modelo de estudo para quimioterapia adjuvante, bem como também utilizamos neurônios humanos obtidos à partir de células-tronco pluripotente-induzidas como modelo de estudo para a neurodegeneração característica da síndrome de Cockayne, uma doença genética na qual os pacientes apresentam deficiências em mecanismos de reparo de DNA, bem como envelhecimento precoce. Na primeira etapa, avaliamos a resposta de células de glioma a cloroquina, um promissor adjuvante no tratamento desta enfermidade, e notamos que a resistência das células a esta droga estava intimamente relacionada ao seu potencial de membrana mitocondrial, o qual podia ser desfeito por meio da inibição da quinase ATR. Apesar da função canônica desta proteína se dar através da regência da resposta a danos ao DNA, notamos que a sua participação como agente promotor de resistência à cloroquina se dava independentemente deste mecanismo. Também notamos que a combinação da cloroquina com a inibição de ATR via silenciamento gênico exercia um potente efeito tóxico sobre as células tumorais tratadas com o quimioterápico Temozolomida. Já na etapa final desta tese, através do emprego da reprogramação celular, obtivemos, pela primeira vez, neurônios humanos de pacientes portadores da síndrome de Cockayne a partir de fibroblastos de pele. Com este modelo de estudo, foi possível observar que esses neurônios apresentavam uma reduzida densidade de puncta sináptica, bem como uma aparente deficiência na sincronia de suas atividades. Por fim, por meio do sequenciamento do RNA destes neurônios, identificamos uma desregulação na expressão de diversas vias relacionadas ao funcionamento e comunicação neural. As implicações para o uso da cloroquina como adjuvante no tratamento de gliomas, bem como as vantagens do uso de neurônios humanos de Cockayne em detrimento aos modelos atualmente disponíveis, também são discutidos. / Genome integrity is constantly threatened by chemical and physical exogenous agents, as well as products of cells own metabolism, and capability of cells to overcome these challenges is essential to achieve homeostasis. In response to DNA lesions, cells activate a dynamic and intricate DNA damage response that ultimately results either in lesion resolution, or in cell death through apoptosis. Regardless the fate chosen, tissue homeostasis is the ultimate goal. Flaws in this mechanism are associated to an increase in mutation rates. Although it constitutes the basis of genetic diversity and evolution, it is also strictly associated to tumorigenesis and aging. In this thesis, separated in two chapters, we used human glioma cells as a model to study adjuvant chemotherapy, and induced pluripotent stem cells-derived human neurons as a model to study neurodegeneration in Cockayne syndrome, a genetic disease in which patients display defects in DNA repair mechanisms, and also premature aging. In the first chapter, we investigated the response of cancer cells to chloroquine, a promising adjuvant drug in glioma therapy, and we noticed that cellsresistance to this drug was strictly associated to its mitochondrial membrane potential values, which could be dismantled through ATR inhibition. Interestingly, we noticed that the ability of ATR to promote resistance of glioma cells to chloroquine was independent of its canonical role in the DNA damage response. We also noticed that combined treatment of chloroquine to ATR inhibition through gene silencing exerted a powerful toxic effect on glioma cells treated with the chemotherapeutic Temozolomide. In the second chapter of this thesis, we employed cell reprogramming technique to obtain, for the first time, human neurons from Cockayen Syndrome patients from skin fibroblasts. With this model, we were able to identify a reduced density of synaptic puncta, as well as reduced synchrony in the activity of the patients neurons. Through RNA sequencing, we noticed several pathways related to synapses and neuronal function deregulated in Cockayne Sydrome patients neurons. Implications for the use of chloroquine as an adjuvant drug in glioma therapy, as well as the advantage of using iduced pluripotent stem cells-derived Cockayne syndrome human neurons (instead of currently available models) to study this disease, are also discussed.

Objetivos: Avaliar fatores de risco e diferentes métodos diagnósticos para retinopatia nos usuários crônicos do difosfato de cloroquina, portadores de lúpus eritematoso sistêmico. Métodos: Foram avaliados 72 olhos de 36 pacientes consecutivos, seguidos no Serviço de Reumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, entre julho de 2007 e abril de 2008. Dados demográficos e clínicos foram pesquisados para estudar os fatores de alto risco (dose diária acima de 3,0 mg/Kg, dose cumulativa, alterações renais, alterações hepáticas, idade acima de 60 anos) e compará-los com os seguintes métodos diagnósticos: acuidade visual, testes de Amsler com grade branca e vermelha, biomicroscopia da córnea, biomicroscopia do fundo, retinografia, angiofluoresceinografia da retina, campo visual dos 10 graus centrais com mira branca, testes de visão de cores, Panel D15 e HRR. Resultados: Dos 36 pacientes, 34 (94,4 %) eram mulheres. A média de idade foi 39,9±9,8 anos, com tempo de doença igual a 13,9±6,6 anos e tempo de uso da cloroquina igual a 11,9±5,1 anos. Além do uso crônico, os pacientes apresentaram altas doses diárias (4,4±0,4 mg, segundo peso ideal e 5,4±0,6 mg, segundo peso magro) e cumulativas (1092,2±476,6 g). Não foi observada relação entre os fatores de alto risco estudados e maior prevalência de retinopatia. A prevalência de retinopatia, confirmada por alterações bilaterais, centrais ou paracentrais e reprodutíveis nos exames de campo visual, foi de 38.9 %. Outros exames indicados para seguimento, como acuidade visual, biomicroscopia da córnea, biomicroscopia do fundo, teste de Amsler, Panel D15 e angiofluoresceinografia do fundo, não foram capazes de diagnosticar a maioria das alterações confirmadas pelo campo visual. O teste de visão de cores HRR apresentou alta sensibilidade e boa especificidade. A intensidade dos achados no HRR mostrou relação com a intensidade dos achados no campo visual. Conclusão: Foi observada alta prevalência de retinopatia por cloroquina entre os pacientes usuários crônicos da cloroquina. A avaliação desses pacientes deve considerar a realização do exame de campo visual e testes de visão de cores mais específicos para as maculopatias adquiridas, mesmo quando não há suspeita clínica. / Purpose: To evaluate risk factors and screnning methods for retinopathy in systemic lupus erythematosus patients after chronic use of chloroquine. Methods: Seventy-two eyes of 36 consecutive patients, followed in the Division of Rheumatology of Hospital das Clínicas, School of Medicine, São Paulo University, were analised from July 2007 to April 2008. Demographic and clinical data were evaluated in order to study risk factors and compare different ophthalmological methods as following: visual acuity, Amsler grid, corneal biomicroscopy, fundus examination, retinography, fluorescein retinography, visual field, color vision tests. Results: From 36 patients, 34 (94,4 %) were female. The mean age was 39,9± 9,8 years and disease duration was 13,9±6,6 years. Besides chronic use of chloroquine, patients also showed high daily (4,4±0,4 mg) and cumulative (1092,2 ±476,6 g) doses. These high risk factors were not related to higher prevalence of retinopathy. Visual field showed 38.9 % retinopathy prevalence. Other ophthalmological methods failed in detecting most cases. Color vision test HRR was highly sensitive but not so specific in detecting retinopathy. The intensity of alterations in HRR were related to the intensity of visual field alterations. Conclusion: High prevalence of retinopathy in chronic users of chloroquine patients was detected by visual field test, but other screening methods failed in detecting alterations. Ophthalmological assessment of these patients should include visual field and color vision tests specific for acquired maculopathies, even in the absence of clinical alterations.

Primaquina e cloroquina são agentes antimaláricos amplamente utilizados profilática e terapeuticamente contra esta enfermidade. A interação destes fármacos com sistemas modelo podem fornecer informações úteis no entendimento dos mecanismos envolvidos em sistemas biológicos reais. Neste sentido, através das técnicas de calorimetria diferencial de varredura e ressonância paramagnética eletrônica, estudamos as interações entre os fármacos antimaláricos supracitados e modelos de membrana, no intuito de investigarmos as modificações provocadas por ambos na estrutura lipídica. Os resultados obtidos indicam que a associação da cloroquina com membranas de DMPC em pH fisiológico é limitada. Uma perturbação desta molécula na estrutura e dinâmica lipídica foi detectada apenas numa região próxima ao carbono sete das cadeias acila das fosfatidilcolinas. Os experimentos de DSC mostram que este fármaco tem efeito apenas na diminuição da cooperatividade da transição principal das membranas. Por outro lado, a redução da temperatura de transição de fase lipídica observada nos estudos calorimétricos demonstra que a primaquina promove uma desestabilização da fase gel. Os experimentos de RPE corroboram esse resultado, evidenciado pelo aumento da fluidez da membrana. Adicionalmente, o aumento do empacotamento provocado no centro da bicamada lipídica sugere penetração deste fármaco até esta região. Não foram observadas alterações da estrutura e dinâmica das cadeias lipídicas na fase fluida da membrana. Os resultados obtidos fornecem um melhor entendimento das interações fármacos-lipídios em um nível molecular, que podem ser aplicados no desenvolvimento de sistemas carreadores de ambos os fármacos. / Primaquine (PQ) and Chloroquine (CQ) are potent therapeutic agents used in the treatment of malaria. The investigation of drug-lipid interactions is pivotal for understanding their biological activity. Electron Spin Resonance (ESR) and Differential Scanning Calorimetry (DSC) were used to investigate the effects of drug binding on the lipid phase transition and acyl chain dynamics of model membranes made up of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) phospholipids. Labels located at different positions along the lipid chain were used to monitor different membrane regions. ESR results indicated that PQ is more effective in changing the membrane structure than CQ. PQ is effective in perturbing the whole chain of DMPC vesicles, whereas the effect of CQ is more pronounced near the polar headgroup region. Furthermore, PQ causes a slight increase of the lipid packing close to the membrane center, suggesting a deeper insertion of this molecule into DMPC bilayers. DSC thermograms revealed that PQ interacts with DMPC decreasing the main transition temperature (TM) by ca. 2ºC and completely abolishing its pre-transition. On the other hand, CQ effects are mainly noticed as a decrease in the cooperativity of the main transition. Because of its lipophilic character, PQ penetrates into the bilayer hydrocarbon region causing considerable disorganization. Electrostatic interaction between CQ and the phosphatidylcholine phosphate groups is probably related with its low membrane permeability. These results shed light on the molecular mechanism of druglipid interaction, which may be useful for the development of lipid drug delivery systems of antimalarial drugs.

Magister Pharmaceuticae - MPharm / The declining efficacy of antimalarial drugs against resistant Plasmodium falciparum strains in several endemic regions has amplified the world’s burden of neglected diseases. This has highlighted the need for alternate strategies for chemotherapy and chemoprophylaxis. Since malaria is prevalent primarily in third world countries, it is critical for novel therapies to be affordable. Previous research has found that some antihistamines possess inherent antimalarial activity and cause a marked reversal of chloroquine resistance in vitro and in vivo. Promising results have been demonstrated when chlorpheniramine was combined with chloroquine to reverse chloroquine resistance in two African studies (Sowunmi et al, 1997; Abok., 1997).Recently, astemizole and its principle human metabolite desmethylastemizole were identified as potent inhibitors of Plasmodium falciparum at sub-micromolar concentrations in both chloroquine sensitive and chloroquine resistant parasites, showing efficacy in vitro and in two mouse models. The promising results observed with these studies warrant a more comprehensive understanding of how antihistamines interact with the malaria parasite. Additionally, analysing the different structural and mechanistic characteristics of antihistamines may lead to the design and development of effective and affordable antimalarial agents or chloroquine resistance modulators.This thesis describes the antimalarial activity of mainly off-patent (generic) antihistamines by comparing the efficacy of a total of 24 antihistamines, representing histamine1, histamine2, and histamine3 receptor antagonists, against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. Cyproheptadine, ketotifen, loratadine, desloratadine, 3-(1HImidazol-4-yl) propyldi (p-fluorophenyl) methyl ether hydrochloride and ciproxifan display IC50 values less than 4μg/ml. There was no significant difference in the sensitivity to antihistamines among the chloroquine sensitive and resistant parasites tested. A tricyclic nucleus appears to be an important structural scaffold for antihistamines which exhibit low IC50 values. Synergistic studies indicate that enhancement of the antimalarial effect of chloroquine on P.falciparum was observed with the ethanolamines against the chloroquine sensitive parasites.Cyproheptadine, ketotifen and desloratadine exerted a marked synergistic action with chloroquine against chloroquine sensitive and resistant parasites. Chlorpheniramine exhibited synergism with chloroquine against resistant parasites only.Microscopic studies illustrate the effect of antihistamines on parasite morphology when compared to control. Using immunofluorescence microscopy, it was seen that ketotifen decreases haemoglobin localization while cyproheptadine increases haemoglobin localization in the parasite’s food vacuole. Western blots have confirmed these results, in addition to indicating that chlorpheniramine decreases the haemoglobin content in the parasite. The results confirm that certain antihistamines do indeed cause a reduction in the growth of malaria parasites. Furthermore, the histamine1 and histamine3 receptor antagonists are most active while histamine2 receptor antagonists have no antimalarial activity. Microscopic studies suggest that antihistamines do not exert their antimalarial effect via a single mechanism of action.I wish to express my sincere appreciation to the following people and institutions whose supervision and assistance made the presentation of this thesis possible:My supervisor, Prof. Henry Leng. Thank for always believing in me. Your encouragement, kindness and calm temperament has given me the strength to complete this thesis even when times were tough. Your wisdom and understanding will always be remembered.My co-supervisor, Prof. Pete Smith. I sincerely thank you for allowing me the opportunity to work in your laboratory and for welcoming me into the department. Your kindness and welcoming attitude will forever be appreciated. Thank you for always being patient and understanding.Dr. Uschi Wiehart. Thank you for all the help in the laboratory and always being there for me. I truly value and appreciate your contribution to this thesis. Your friendship has added so much positive energy to my life. Thank you for your wisdom, inspirational advice and unfaltering encouragement Sumaya and Ntokosi, your help, advice and company in tissue culture, are truly appreciated.The UCT, Pharmacology students. Thank for all your assistance.My dearest Pharmaceutical Chemistry colleagues, Jaques Joubert, for your friendship and support and for always listening and Prof. Peter Eagles, your kindness, support and wise advice has given me strength when I needed it most. To my other School of Pharmacy colleagues. Prof. Sarel Malan and team, for your support and motivation.To my family for all your support and wisdom and to my baby brothers; Omar and Uzair for all the joy that you bring to my life.And finally to my dearest husband, Zaheer for all your love and support throughout my studies and for taking me to UCT to culture parasites every weekend

Endocrine resistance is a significant clinical problem in the treatment of estrogen (E2) receptor positive breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation respectively. While ER positive breast cancers typically express a high ratio of ERα to ERβ, the acquisition of antiestrogen resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers ERβ has been shown to function in opposition to the ERα in the presence of E2. Here we demonstrate that exposure to two different ERβ agonists results in decreased cell viability, and produced a marked reduction in G2/M phase in antiestrogen resistant breast cancer cell line in conjunction with altered cyclin D1, and cyclin E expression relative to E2. ERβ agonists also strongly downregulated Bcl-2 expression and recruited both ERs to the Bcl-2 and pS2 E2-response elements resulting in a reduction in mRNA transcripts from both of these genes. Bcl-2 reduction correlated with increased lipidation of LC3-I to LC3-II, indicative of increased autophagic flux. Although ERβ agonist treatment alone did not induce apoptosis, remarkably, the coaddition of ERβ agonist and the autophagy inhibitor, chloroquine, resulted in robust cell death. Lastly, in vivo studies demonstrate that preferential-ERβ agonists are not estrogenic in the uterus or mammary gland. Together, these observations suggest that combined therapies including an ERβ agonist and an autophagy inhibitor may provide the basis for a safe, novel approach to the treatment of antiestrogen-resistant breast cancers.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23080号 / 医博第4707号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 伊藤 貴浩, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Magister Pharmaceuticae - MPharm / The aim of this study was to investigate the mechanism of fenfluramine-induced pulmonary phospholipidosis, by comparing the profile and levels of induced phospholipids in the rat and the mode of phospholipase inactivation, both relative to that produced by chlorphentermine. Wistar and BD9 rats were injected with fenfluramine (FF) and chlorphentermine (CP) intra-peritoneally daily over a six week period to induce phospholipidosis. The lungs isolated from such treated and untreated animals, were grouped into unlavaged lungs and lungs to be lavaged and from the latter group the alveolar macrophages were isolated. Small sections of the unlavaged lungs were microscopically examined to verify the induction of phospholipidosis. Further the levels of phosphatidyl choline (PC), spingomyelin (SPM), phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidyl serine (PS) and phosphatidic acid (PA) were determined in both groups of lungs using a TLC method. To assess whether the drug-mediated inactivation of the phospholipases (PL) occurred via direct inhibition of the enzymes or via the drug-phospholipid complex, the hydrolysis of the above phospholipids by PL-A or PL-C were monitored using colorimetric methods. The feasibility of the phospholipid-drug complex-mediated mechanism was further explored, by assessing the effect the two drugs had on the phase transition temperature of the phospholipids. Electron microscopy revealed the presence of hypertrophied and elevated counts of alveolar macrophages in the treated-Wistar and -BD9 rats. In the FF- and CP treated Wistar and BD9 rats there were, compared to the saline-treated rats, a 200 % and 235 % increase in macrophage counts, respectively, for the FF-treated rats and a 700 % and 965 % increase in macrophage counts, respectively, for the CP treated rats. The levels of all the phospholipids in the unlavaged lungs of both rat strains were elevated, except that for PG, PS and PA. In both rat strains following the treatment with both drugs the PG levels were not elevated and the PS levels were not elevated following CP treatment. Following the treatment for both drugs, the PA levels were also not elevated in the BD9 rats. Relative to the levels found in the unlavaged lungs of the control rats, the increases ranged from a minimum of 9 to a maximum of 216 %. In general, Wistar rats appeared to be more susceptible to both FF and CP treatment. In both rat strains, lavaging of the lungs considerably reduced the levels of phospholipids remaining in the lung and the differences between the treated and untreated animals became less striking. The addition of FF or CP, whether directly to the enzyme, or in the form of the drug phospholipid complex, resulted in significant decreases in the PL-A-mediated or PL-C-mediated hydrolysis of virtualy all the test phospholipids. The average decrease ranged from 0.811 to 4.04 ,.,.FFAbbb ,.,.1-1sample min-I, for the PL-A activity and 0.023 to 0.827 ,.,.gIp'CC100 ,.,.1-1 sample min-I, for the PL-C activity. In the case of FF, the inhibition of PL-A activity could not be ascribed exclusively to either direct inhibition of the enzyme or reduced susceptibility of the phospholipid substrate-drug complex. The PL-C activity appeared to be inhibited to a greater extent via the phospholipid substrate-drug complex rather than by direct inhibition. On the other hand, CP induced a small, but significantly greater degree of inhibition of PL-A activity, more via direct inhibition, rather than by the phospholipid substrate-drug complex. The PL-C activity appeared to be inhibited to a greater extent via phospholipid substrate-drug complexation than by direct inhibition. From the above data, considered collectively, it was not possible to declare either of the two possible mechanisms as the more likely one for FF or CP-induced inhibition of the phospholipases. The feasibility of the indirect mode was further explored, by determining the phase transition temperatures for the phospholipid-drug complexes of each drug. The addition of each drug caused a depression of the phase transition temperature of all the phospholipids with a .1T'dd ranging from 0.52 to 15.73 °C. This appears to support the notion that both drugs bind to the phospholipids and the differences in the extent of the phase transition temperature depression of the individual phospholipids may indicate differences in the binding capacities of these drugs. The following major conclusions may be drawn from the results of this investigation. Fenfluramine induces a phospholipidosis syndrome in the lungs of Wistar and BD9 rats that are histologically similar to that induced by CP. It induces the elevation of essentially the same phospholipids as CP, primarily in the alveolar spaces and macrophages, and by implication, most likely via similar mechanisms. For both FF and CP, both direct inhibition and phospholipid-drug complex-mediated inhibition of phospholipases were found to be a viable mechanism for this syndrome. The mechanism for FF-induced pulmonary phospholipidosis thus appears to be similar to that of CP; small quantitative differences in essentially similar mechanisms, may explain the differences in the levels of induced phospholipidosis found in this study.