Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references. Also available in print.

Το φυσικό προϊόν αρτεμισινίνη και τα παράγωγά της αποτελούν σήμερα φάρμακα επιλογής για την αντιμετώπιση της ελονοσίας ενώ πολλά απ’αυτά παρουσιάζουν και ιδιαίτερα σημαντική αντικαρκινική δράση. Στο πλαίσιο της παρούσας διπλωματικής εργασίας έγινε χημική τροποίηση του μορίου της αρτεμισίνης, προκειμένου να συντεθούν διμερή συζεύγματά της με πολυαμίνες (πουτρεσκίνη, σπερμιδίνη, σπερμίνη). Αρχικά, η αρτεμισινίνη τροποποιήθηκε κατάλληλα έτσι ώστε να φέρει συνδέτη με δεσμό C-O (10-oξo) ή C-C (10-καρβο) στη θέση-10 και N-C με αλλαγή του ετεροατόμου στην θέση-11, προκειμένου στη συνέχεια να προσδεθεί στις πολυαμίνες μέσω δεσμού ουρεθάνης. Για το λόγο αυτό, συντέθηκαν ενεργοποιημένα ανάλογα της αρτεμισινίνης, τα οποία μετά από αντίδραση με κατάλληλα προστατευμένες πολυαμίνες, οδηγούν στο σχηματισμό διμερών πολυαμινικών συζευγμάτων της αρτεμισινίνης. Επιπλέον, για προκαταρκτικές βιολογικές μελέτες, συντέθηκε ένα ασύμμετρο πολυαμινικό σύζευγμα της αρτεμισινίνης με το αντικαρκινικό φάρμακο χλωραμβουκίλη, χρησιμοποιώντας το 10-oξo ενεργοποιημένο ανάλογο και την πουτρεσκίνη. / The natural product Artemisinin and its derivatives are currently the drugs of choice for the treatment of malaria, which some of them showing important anticancer activity. In the context of the present dissertation, three chemical modifications of the molecule of artemisinin were accomplished towards the synthesis of several Artemisinin dimer conjugates with polyamines (putrescine, spermidine, spermine). Initially, artemisinin was modified at position 10 and 11, in order to synthesize analogues bearing suitable linkers, through C-O (10-oxo), C-C (10-carbo) and C-N (11-aza, by replacement of O with N in the ring A of Artemisinin) bonds, able to form carbamate bonds with amino groups of polyamines. For this purpose, the corresponding activated intermediates were synthesized, which upon reaction with suitably protected polyamines afforded the Artemisinin symmetric conjugates. In addition, for the sake of preliminary biological evaluation a new asymmetric conjugate consisted of an 10-oxo Artemisinin and a chlorambucil moiety, using putrescine as a polyamine-type linker, was synthesized.

Το φυσικό προϊόν αρτεμισινίνη και τα παράγωγά της αποτελούν σήμερα φάρμακα επιλογής για την αντιμετώπιση της ελονοσίας ενώ πολλά απ’αυτά παρουσιάζουν και ιδιαίτερα σημαντική αντικαρκινική δράση. Στο πλαίσιο της παρούσας διπλωματικής εργασίας έγινε χημική τροποποίηση του μορίου της αρτεμισινίνης, προκειμένου να συντεθούν διμερή συζεύγματά της με πολυαμίνες (πουτρεσκίνη, σπερμιδίνη, σπερμίνη). Αρχικά, η αρτεμισινίνη τροποποιήθηκε κατάλληλα έτσι ώστε, να φέρει συνδέτη με δεσμό C-O (10-oxo) στη θέση-10, προκειμένου στη συνέχεια να προσδεθεί στις πολυαμίνες μέσω δεσμού ουρεθάνης. Για το λόγο αυτό, συντέθηκε το ενεργοποιημένο ανάλογο(10-oxo , το οποίο μετά από αντίδραση με τις κατάλληλα προστατευμένες πολυαμίνες, έδωσαν τα διμερή συζεύγματα της αρτεμισινίνης 66-68. Επιπλέον, για προκαταρκτικές μελέτες βιολογικής δράσης, συντέθηκε ένα ασύμμετρο σύζευγμα της αρτεμισινίνης με το αντικαρκινικό χλωραμβουκίλη (69), χρησιμοποιώντας το 10-oxo ενεργοποιημένο ανάλογο και την πουτρεσκίνη ως πολυαμίνη. Όλα τα παραπάνω συζεύγματα, βρίσκονται στο στάδιο της αποτίμησης της βιολογικής τους δράσης, από συνεργαζόμενο εργαστήριο έναντι καρκινικών σειρών HL60. / The natural product Artemisinin and its derivatives are currently the drugs of choice for the treatment of malaria, with a large number of them showing important anticancer activity as well. In the context of the present dissertation, one chemical modification of artemisinin was accomplished towards the synthesis of several Artemisinin dimer conjugates with polyamines (putrescine, spermidine, spermine). Initially, artemisinin was modified at the 10- position, in order to synthesize analogue bearing suitable linkers, through C-O (10-oxo) bond, able to form urethane bonds with amino groups of polyamines. For this purpose, the activated intermediate(10-oxo) was synthesized, which upon reaction with suitably protected polyamines afforded the Artemisinin symmetric conjugates 66-68. In addition, for the sake of preliminary biological evaluation a new asymmetric conjugate (69) consisted of an 10-oxo Artemisinin and a chlorambucil moiety, using putrescine as a polyamine-type linker, was synthesized. All above conjugates are now under biological evaluation as anticancer agents, against HL60 cancer cells, by a collaborating research group. Further studies are underway, in order to evaluate the biological activity of the aforementioned analogues as antimalarials.

L'artémisinine (ART) est un composé antipaludique majeur très actif sur les souches multi-résistantes de Plasmodium falciparum. Cependant, son application clinique est limitée par sa faible solubilité en milieu aqueux et sa faible biodisponibilité par voie orale. La mise en œuvre de cyclodextrines (CDs) bioestérifiées et de dérivés amphiphiles polyoxyéthylénés permet de viser deux objectifs : i) d'une part d'améliorer la concentration aqueuse d'ART à travers son association aux vecteurs colloïdaux de CDs modifiées et ii) d'autre part d'améliorer sa biodisponibilité et son efficacité thérapeutique à travers la décoration de la surface des vecteurs. La décoration surfacique est sensée augmenter le temps de circulation sanguine des nanovecteurs de manière à maintenir des doses plasmatiques efficaces d'ART sur une longue période, suite à une administration intraveineuse. Des suspensions colloïdales stables suffisamment chargées d'ART ont été mises au point permettant de palier le problème d'insolubilité en milieu aqueux de la molécule active et donc d'envisager son administration par voie parentérale. Les essais de lyodisponibilité indiquent que l'ART est libérée des nanosystèmes pendant une période de 4 jours pour les nanoréservoirs et de 11 jours pour les nanosphères. En plus des caractérisations physico-chimiques et pharmacotechniques, les potentialités des nanoparticules décorées en surface ont été évaluées et comparées au cours de tests biologiques. In vitro, l'ART mise en forme a montré une bonne efficacité sur des souches plasmodiales choloroquino-sensibles (3D7) et chloroquino-résistantes (K1) avec des CI50 très faibles de l'ordre de 3 à 6 ng/mL. Le concept de co-nano-assemblage de dérivés amphiphiles de γ-CD-C10 bioestérifiée et de polyéthylène glycol (PEG) dans les conditions de nanoprécipitation semble être une approche intéressante pour conférer des propriétés de furtivité aux nano-systèmes de γ-CD-C10. En effet, les études in vitro mettent en évidence une diminution significativement de la phagocytose par les cellules macrophagiques et/ou de l'adsorption des protéines du complément sérique à la surface des nanoparticules de γ-CD-C10 décorées par le polysorbate 80, le stéarate de PEG1500, et le DMPE-mPEG2000. In vivo, nous observons une augmentation du temps de circulation sanguine des nanoréservoirs γ-CD-C10/polysorbate 80 et des nanosphères γ-CD-C10/DMPE-mPEG2000. Enfin, les études de pharmacocinétique réalisées chez le rat montrent que les paramètres pharmacocinétiques de l'ART sont améliorés lorsqu'elle est associée aux deux systèmes nanoparticulaires précédents. En effet, des valeurs de clairance plasmatique très faibles et de temps de demi-vie plasmatique longs (3 et 5 heures, respectivement) de l'ART ont été enregistrées avec ces deux formulations. Ces formes vectorisées d'ART mises au point ouvrent de perspectives intéressantes pour la prise en charge thérapeutiques des crises de paludisme sévère. / Artemisinin (ART), an endoperoxide sesquiterpene lactone antimalarial drug isolated from a Chinese medicinal herb (Artemisia annua), has a fast action against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, making it very effective in the treatment of multidrug-resistant malaria. However, its poor aqueous solubility, short half-life, and high first-pass metabolism limit its use in therapeutics. The purpose of the present study was to investigate the potential of self-assembled bio-transesterified CD-based nanocarriers as ART delivery systems for an intravenous route. The objective of our study was twofold: (i) to improve the ART dosage through its association with CD esters-based nanocarriers, (ii) to ensure a sufficient blood circulation time of the nanocarriers through their surface decoration, which is a prerequisite to reach infected erythrocytes after systemic administration. Stable colloidal suspensions with good ART association rate were developed to solve the problem of insolubility in aqueous medium of the active molecule and therefore consider its parenteral administration. The γ-CD-C 10 based nanospheres showed a significant sustained release profile of ART extended up to 4 days for nanosphères and 11 days for nanoreservoirs. In addition to the physicochemical characterizations, the potential of nanoparticles decorated on the surface were evaluated and compared in biological tests. The results from this study indicate that ART-loaded nanosystems, mainly PEGylated ones exhibit satisfactory in vitro activity against Chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of P. falciparum with very low IC50 of the order of 3 to 6 ng / mL. The concept of co-assembly of nano-amphiphilic derivatives of γ-CD-C10 bioestérifiée and polyethylene glycol (PEG) under conditions of nanoprecipitation seems to be an interesting approach to impart stealth nano-systems-γ-CD C10. Indeed, in vitro studies show a significant decrease in phagocytosis by macrophages and/or adsorption of serum complement proteins on the surface of nanoparticles of γ-CD-C10 decorated by polysorbate 80, PEG1500 stearate, and mPEG2000-DMPE. In vivo, we observed an increase in blood circulation time of nanoreservoirs γ-CD-C10/polysorbate 80 and nanospheres γ-CD-C10/DMPE-mPEG2000. Finally, pharmacokinetic studies in rats show that the pharmacokinetics of ART are enhanced when combined with previous two nanoparticle systems. Indeed, values of plasma clearance and very low time of long plasma half-life (3 to 5 hours, respectively) of ART were recorded with both formulations. These colloidal forms of ART developed open up interesting prospects for the therapeutic treatment of severe malaria.

Submitted by THALITA CRISTINA MARQUES CERVEZAN null (tha_cerv@hotmail.com) on 2017-07-28T02:30:55Z No. of bitstreams: 1 Tese_final_Cervezan2017.pdf: 3225815 bytes, checksum: 72cc707821ac3d0f6e9ccdefd2c22aa4 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-07-31T19:54:42Z (GMT) No. of bitstreams: 1 cervezan_tcm_dr_bot.pdf: 3225815 bytes, checksum: 72cc707821ac3d0f6e9ccdefd2c22aa4 (MD5) / Made available in DSpace on 2017-07-31T19:54:42Z (GMT). No. of bitstreams: 1 cervezan_tcm_dr_bot.pdf: 3225815 bytes, checksum: 72cc707821ac3d0f6e9ccdefd2c22aa4 (MD5) Previous issue date: 2017-05-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A malária é considerada uma doença infecciosa grave que atinge milhares de pessoas, principalmente em países tropicais e subtropicais. O controle dessa doença pode ser feito através de alguns tipos de medicamentos semissintéticos e bioativos naturais, entre eles os derivados da espécie Artemisia annua L.. Os principais derivados bioativos de A. annua L. são a artemisinina, diidroartemisinina, deoxiartemisinina, ácido artemisínico e arteanuina B. Com o aumento do valor dos princípios ativos naturais, estudos relacionados à pós-colheita e secagem de material vegetal tornam-se importantes para melhor conservação de suas propriedades fitoterápicas. O presente estudo teve como objetivos avaliar o efeito de diferentes tipos de processamento pós-colheita sobre a qualidade do fármaco e seus derivados. Folhas de A. annua L. foram submetidas a quatro tratamentos de desidratação sendo eles: campo em pleno Sol, campo na sombra, estufa de circulação de ar a temperatura de 28°C +/-2 e câmara de crescimento 15°C +/-2, por 29 dias. Foram realizadas as análises de teor de umidade, flavonoides, rendimento e quantificação de óleo essencial, avaliação do teor de artemisinina, diidroartemisinina, deoxiartemisinina, ácido artemisínico e arteanuina B. Cada tratamento apresentou influência distinta sobre o acúmulo de bioativos estudados. Entretanto, quando a biomassa foi processada em estufa com temperatura controlada, houve maior estabilidade para a maioria dos princípios ativos avaliados, com exceção da artemisinina. / Malaria is considered a serious infectious disease that affects thousands of people, occurring in tropical and subtropical countries. Disease control can be done through some types of semi-synthetic and bioactive natural drugs, including them the derivatives of the species Artemisia annua L.. Some major bioactive derivatives of A. annua L. are artemisinin, dihydroartemisinin, deoxiartemisinin, artemisinic acid and arteanuin B. With the increase in the value of the natural active principles, studies related to post-harvesting and drying of plant material become important for better conservation of its phytotherapic properties. The present study aims to evaluate the effect of different types of post-harvest processing on the quality of the drug and its derivatives. The leaves of A. annua L. were submitted to 4 post-harvest treatments: Leaves of A. annua L. were submitted to 4 post-harvest treatments: field in full sun, field shaded, air circulation oven dry at 28 °C +/-2 and grown chamber 15 °C +/-2 for 29 days. The analysis of moisture content, flavonoids, quantification of essential oil yield, evaluation of the artemisinin, dihydroartemisinin, deoxiartemisinin, artemisinic acid and arteanuin B contents were carried out. Were analyzed treatment had a different influence on the bioactives under study, however, the oven dry demonstrated to have greater stability in most of the active principles with the exception of artemisinin.

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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) EFETIVIDADE DA ELIPTICINA E ARTEMISININA-atual.pdf: 1595775 bytes, checksum: b76659782dbf4751e7f12e9f356c42fe (MD5) Previous issue date: 2018-02-27 / Parasite resistance to commercially available antimalarial drugs is a reality, which makes it possible to search for new drugs and to develop researches with plants that have pharmacological properties. Numerous discussions on rapidlyreleasing antimalarial drugs have been gaining strength and adherence, as it has advantages related to its efficacy, specificity and patient tolerance in therapy. In this way, nanotechnology offers important tools for the development of new drugs and nanoparticles can contribute to a more specific therapy of selective action in the distribution and absorption of drugs. According to the Amazonian biodiversity, the active principle, ellipticina involved in research as antimalarial was isolated from Aspidosperma vargasii, whose popular name is Amarelão or Carapanaúba branca. Ellipticin exhibits antiplasmodial activity that has been demonstrated in in vitro test because it inhibited the growth of heme crystal that occurs in the digestive vacuole of Plasmodium ssp. and is associated with parasite growth. Another active principle, which is extracted from Artemisia annua L. is artemisinin. It is considered essential in the treatment of malaria, by its potency and its rapid action. Interventions of artemisinin-based combination therapy have reduced morbidity and mortality associated with malaria in several parts of the world. The combinations of artemeter-lumifantrine, artesunate-mefloquine and artesunateamodiaquine, are related to artoisinin's gametocytocidal properties that inhibit the transmission of parasites to probably reduce the development of antimalarial resistance. Recent studies show that artesunate may be a good alternative for the treatment of some types of cancer by the antiangiogenic mechanism. Its low toxicity represents a promising chemotherapeutic. It is important to note that artesunate is not carried by P-glycoproteins, so it is not involved in multidrug resistance. In the fight against these pathologies, several inconveniences are pointed out for the patient as some adverse effects and dosing schedule. Thus, it considers that the evaluation of new drug release systems can improve the therapeutic efficacy of future nanoformulations for antimalarial and antitumor drugs. In this sense, this work aims to develop, characterize and evaluate nanoparticles in their physico-chemical and cytotoxic aspect by means of bioassays against Plasmodium falciparum and antitumor cells. The substances used in the synthesis of the nanospheres were sodium tetrachloropalladium and ellipticine hydrochloride. The substances involved in the synthesis of nanofibers were artemisinin hydrochloride, polyvinylpyrrolidone and polybutylene. / A resistência parasitária aos fármacos antimaláricos comercialmente disponível é uma realidade, o que possibilita a busca por novas drogas e o desenvolvimento de pesquisas com plantas que possuem propriedades farmacológicas. As inúmeras discussões sobre as drogas antimaláricas de liberação rápida, vem ganhando força e adesão, pois apresenta vantagens relacionadas a sua eficácia, especificidade e tolerância por parte do paciente na terapia. Desta forma, a nanotecnologia oferece ferramentas importantes para o desenvolvimento de novos fármacos e as nanopartículas podem contribuir para uma terapia mais específica de ação seletiva na distribuição e absorção das drogas. De acordo com a biodiversidade Amazônica, o príncipio ativo, elipticina envolvida em pesquisas como antimalárico foi isolado da Aspidosperma vargasii, cujo nome popular é Amarelão ou Carapanaúba branca. A elipticina apresenta atividade antiplasmodial que foi demonstrado em teste in vitro, pois inibiu o crescimento do cristal de heme que ocorre no vacúolo digestivo do Plasmodium ssp. e está associado ao crescimento do parasito. Outro princípio ativo, que é extraído da Artemisia annua L. é a artemisinina. É considerada fundamental no tratamento da malária, por sua potência e sua rápida ação. As intervenções da terapia combinada baseada em artemisinina, diminuíram a morbidade e a mortalidade associadas a malária em várias partes do mundo. As combinações de artemeter-lumifantrine, artesunato-mefloquina e artesunato-amodiaquina, está relacionado a propriedades gametocitócidas da artemisinina que inibe a transmissão de parasitas para provavelmente reduzir o desenvolvimento de resistência antipalúdica. Recentes estudos mostram que o artesunato pode ser uma boa alternativa para o tratamento de alguns tipos de câncer, pelo mecanismo antiangiogênico. A sua baixa toxicidade representa um quimioterápico promissor. É importante salientar que o artesunato não é transportado por P-glicoproteínas, portanto não está envolvido em resistência a multidrogas. Na luta contra essas patologias, são apontados vários inconvenientes para o paciente como alguns efeitos adversos e horário de dosagens. Assim, considera que a avaliação de novos sistemas de liberação de medicamentos podem melhorar a eficácia terapêutica de futuras nanoformulações para fármacos antimaláricos e antitumorais. Neste sentido, este trabalho tem por objetivo desenvolver, caracterizar e avaliar as nanopartículas no seu aspecto físico-químico e citotóxico por meio de bioensaios contra Plasmodium falciparum e células antitumorais. As substâncias utilizadas na síntese das nanoesferas foram sódio tetracloropaládio e cloridrato de elipticina. As substâncias envolvidas na síntese das nanofibras foram o cloridrato de artemisinina, polyvinilpirrolidona e polibutileno.

Orientadores: Fabio Trindade Maranhão Costa, Stefanie Costa Pinto Lopes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T13:56:29Z (GMT). No. of bitstreams: 1 NaranjoPrado_IsabelCristina_M.pdf: 2482256 bytes, checksum: 0074002c6c9825a945839bdec23357f3 (MD5) Previous issue date: 2014 / Resumo: A malária é responsável por cerca de 300 milhões de casos de infecção e 1 milhão de mortes por ano. No Brasil, em 2012, foram registrados cerca de 1500 casos sendo a malária vivax responsável por 85% destes. Ainda, é frequentemente reportada falha terapêutica aos antimaláricos convencionais (como cloroquina e mefloquina) em infecções por P. falciparum, principalmente, mas também por P. vivax. Sendo assim, terapias combinadas com artemisinina e seus derivados (ACT) são atualmente recomendadas. No entanto, resistência aos derivados da artemisinina já é evidente e a busca por novos compostos com atividade antimalárica é urgente. A violaceína, pigmento violeta extraído de bactérias Gram negativas, demostrou apresentar elevada atividade antimalárica in vitro e in vivo em trabalho anterior de nosso grupo. Neste sentido, este projeto tem como objetivo aprofundar a investigação a respeito da atividade antimalárica da violaceína avaliando seu papel em isolados amazônicos de P. falciparum adaptados recentemente em cultura, e em P. vivax após imediata coleta de sangue de pacientes infectados. Além disso, investigamos o potencial da violaceína como terapia combinada junto ao artesunato no tratamento de parasitas murinos resistentes a este e a outros antimaláricos como a cloroquina. Inicialmente foram testadas dois diferentes tipos de violaceína in vitro contra P. falciparum 3D7: uma comercial extraída de Janthinobacterium lividum (vJl- IC50: 227 nM) e outra extraída de Chromobacterium violaceum (vCv- IC50: 390 nM). Apesar de não termos encontrado uma diferença na atividade antimalárica entre as duas violaceínas, a extraída de C. violaceum teve uma baixa toxicidade em eritrócitos (<400 nM) em células de hepatoma humano (<800 nM). Devido a esta baixa toxicidade, somente vCv foi avaliada quanto sua atividade antimalárica. Demonstramos que vCv apresentou IC50 similar ao encontrado para P. falciparum 3D7 (IC50 média= 419,8 nM) em 7 isolados de campo de P. falciparum. Ainda, em ensaios in vivo, utilizando cepas murinas de P. chabaudi, vCv conseguiu diminuir significativamente (P<0,05) a parasitemia no dia pico da infeção em cepas resistentes à cloroquina (30CQ) e a artesunato e mefloquina (ATNMF1). Adicionalmente, nos testes realizados em P. vivax a vCv parece evitar o amadurecimento parasitário nos quatro isolados testados. Coletivamente, podemos concluir que a vCv apresentou um efeito antimalárico em cepas de P. falciparum e pode ser especialmente útil quando usada em combinação com artesunato no tratamento de camundongos infectados com cepas resistentes. Finalmente, ensaios adicionais de amadurecimento com isolados de P. vivax necessitam ser conduzidos para a comprovação do efeito da vCv nesta espécie / Abstract: Malaria is responsible for about 300 million infections and one million deaths per year. In Brazil in 2012, about 1500 cases were reported and malaria vivax accounts for 85% of these. Still, it is frequently reported treatment failure with conventional antimalarials (as chloroquine and mefloquine) mainly in infections with P. falciparum but also by P. vivax parasite. Because of that combination therapies with artemisinin and its derivatives (ACT) are now currently recommended. In a previous work, our group demonstrated that violacein was able to inhibit the in vitro growth of laboratory strains of P. falciparum and also to strongly control the parasitemia of mice infected with P. chabaudi. This project aims to investigate further the antimalarial activity of violacein evaluating its activity in Amazonian isolates of P. falciparum recently adapted in culture, and in P. vivax isolates immediately after collecting blood from infected patients. Furthermore, we investigate the potential of violacein as combination therapy with artesunate in the treatment of murine strains which are resistant in different levels to artesunate, mefloquine and chloroquine. The antimalarial activity of violacein were initially investigated in vitro against P. falciparum 3D7 using two different types of violacein, one comercial, extracted from Janthinobacterium lividum (vJl-IC50: 227 nM), and another extracted from Chromobacterium violaceum (vCv-IC50: 390 nM) by our collaborators. In spite of no difference in the antimalarial activity between the two violaceins, the one extracted from C. violaceum had the lowest toxicity in erythrocytes (<400 nM) and in human hepatoma cells (<800 nM). Because of this, the antimalarial activity only of vCv was evaluated against 7 field isolates of P. falciparum showing a similar IC50 to that found for P. falciparum 3D7 (IC50= 419.8 nM). The antimalarial activity was also evaluated in murine strains of P. chabaudi showing a significant (P < 0.05) parasitemia decrease in the peak day in the two clones of P. chabaudi tested, one resistant to chloroquine (30CQ) and another resistant to artesunate and mefloquine (ATNMF1). Additionally, in P. vivax vCv was capable to reduce the parasite maturation in the four isolates tested. Therefore we can conclude that the vCv has an antimalarial effect on field isolates of P. falciparum and can be especially useful when used in combination with artesunate in the treatment of mice infected with resistant strains. Moreover, more assays should be conducted using blood infected with P. vivax to corroborate its effect in inhibiting the maturation of trophozoites / Mestrado / Imunologia / Mestra em Genética e Biologia Molecular

Resistance to artemisinin combination therapies (ACTs) has emerged in southeast Asia threatening the most widely used treatment against antimalarial-resistant Plasmodium falciparum worldwide. Artemisinin resistance has been associated with a reduced rate of parasite clearance following treatment with an ACT and is attributed to increased survival of ring-stage parasites. Single nucleotide polymorphisms (SNPs) in kelch gene (K13) has been associated with delayed in vivo clearance half-life of artemisinin-resistant P. falciparum and is the only known molecular marker of resistance. The absence of reliable in vitro phenotypes for artemisinin resistance has limited our understanding of the resistance mechanism(s) and fitness costs, therefore we have culture adapted and cloned patient isolates from Thailand and Cambodia that had clinical resistant phenotypes. Stable reduced susceptibility to artemisinin derivatives and mefloquine was observed using a modified [3H]hypoxanthine drug susceptibility assay. In addition we devised an in vitro phenotype assay of artemisinin resistance, known as the delayed clearance assay (DCA), that was positively correlated with the in vivo delayed clearance and presence of K13 SNPs of artemisinin resistance. Remarkably we discovered for the first time altered patterns of intraerythrocytic development in artemisinin-resistant P. falciparum. In the absence of drug pressure most artemisinin-resistant clones have a prolonged ring phenotype with temporally compressed trophozoite stage, yet with the normal overall asexual life cycle period. Parasites remain in ring-stage up to 14 hours longer than wild-type, whereas time spent in trophozoite-stage is dramatically reduced. One parasite, PL08-09 (5C), exhibited an accelerated 36-hour life cycle in the absence of drug pressure and progressed through asexual development in equal time spent at each intraerythrocytic stage. These data support our hypothesis that parasite resistance to artemisinin results from reduced exposure to drug at the most susceptible stage of development (trophozoite). Interestingly, the most prevalent K13 mutation C580Y is associated with both cell cycle phenotypes. Another cell cycle phenotype, ring-stage dormancy, has been associated with artemisinin resistance in vitro reducing the dormant period of artemisinin-resistant parasites following dihydroartemisinin exposure allowing resistant parasite cultures recrudesce before wild-type. However, sensitive parasites have the ability to enter ring-stage dormancy causing recrudescence in vitro. It is possible that multiple cell cycle phenotypes enhance the survival and fitness of the resistant parasite population as a whole in the face of antimalarial exposure. We have demonstrated that there is a fitness cost associated with artemisinin resistance and remains an important component in the spread of genetic mutations associated with artemisinin resistance. Resistant parasites outcompeted sensitive in vitro only when exposed to dihydroartemisinin. Two mutations associated with artemisinin resistance, including a mutation in K13, were lost in artemisinin resistant parasite by the end of the study. Conversely, parasite cultures maintained artemisinin resistance phenotypes in vitro only if exposed to artemisinin drug pressure every 21-42 days. The mechanism of artemisinin resistance remains elusive and how the parasites alter their intraerythrocytic development is unknown. Therefore. we transfected green fluorescent protein (GFP) or luciferase into artemisinin-resistant P. falciparum clones from Thailand and Cambodia to aid the study the cell cycle phenotypes associated with artemisinin resistance. Artemisinin resistance phenotypes were maintained in stably transfected clones. Increased growth of artemisinin-resistant clones was observed following exposure to ACT partner drug. Low concentrations of antimalarials synchronized the luciferase expression of artemisinin-resistant parasites having different cell cycle phenotypes in the absence of drug pressure. Ring-stage dormancy was observed with many antimalarial drugs and contributes to recrudescence observed by antimalarials other than artemisinin. Our results show evidence that current ACT treatments are selecting multidrug resistant parasites in the field that are better able to tolerate all antimalarials through regulatory cell cycle mechanisms. These cell cycle phenotypes associated with artemisinin resistance contribute to reducing the fitness cost associated with genetic mutations causing artemisinin resistance. This leads to the survival of the most fit population of parasites that survive combination drug treatments, thus demonstrating the importance of discovering novel drugs to target ACT-resistant P. falciparum.

Magister Public Health - MPH / In Zambia, there had been a large scaling up of new interventions to control malaria since 2003, which included the distribution of rapid diagnostic tests (RDTs), used to immediately determine if someone with symptoms suggestive of malaria actually has malaria; training of health workers in the use of the RDTs; and the prescription of artemisinin-based combination therapy (ACT) to which the malaria parasite is sensitive, rather than the old treatment regime of chloroquine to which the malaria parasite had become resistant. The use of RDTs to confirm the presence of malaria before treating for it with ACT became known as the „test and treat‟ policy. Previously, since the 1960s, in malaria endemic areas such as Zambia, children presenting with fever (the commonest symptom of malaria) without any obvious other cause for the fever, were assumed to have malaria and were hence treated for it with chloroquine. This was known as "presumptive treatment" of malaria. The combination of "presumptive treatment" and the use of a single medication led to the development of high levels of resistance to chloroquine, to the extent that it is now no longer an effective treatment for malaria. Years after the introduction of the "test and treat" policy, it was still unclear to what extent it was being implemented, as there was initial reluctance by health workers to test all children presenting with fever for malaria and if they did test they may not have followed the management guidelines of treating those who test positive with ACT and further investigating those who test negative for the cause of the fever. It seemed that staff had gotten used to the "presumptive treatment" approach to malaria over almost 4 decades and hence were quite reluctant to abandon it. The conflicting guidelines for malaria treatment in children between IMCI and "test and treat‟ has promoted a paradox between presumptive treatment for malaria and "test and treat" approach as IMCI teaches health workers to treat febrile children presumptively for malaria whereas the "test and treat" approach requires them to first make a definitive diagnosis before treating. Hence although the "test and treat" approach was instituted to overcome the problems with presumptive treatment approach it now had to contend with the competing and contradictory influence of the IMCI approach. This study therefore aimed to assess what proportion of children aged five years and younger who presented with fever were managed via the "test and treat" guidelines and which factors were associated with this, in Lusaka District, Zambia. Methodology: A cross sectional analytical study design was used based on a review of medical records. A sample size of 800 medical records of children presenting with fever was selected from 10 out of the 23 health care facilities in Lusaka, using a multistage stratified random sampling technique. Four hundred records were sampled from 2008 records (five years after commencement of the "test and treat" policy) and 400 from 2011 records (eight years after commencement of the "test and treat" policy). Trained data collectors used a data extraction tool to transcribe demographic and clinical data from the medical records in a standardized manner. Data Analysis: Univariate descriptive statistics analysis was performed using measures of central tendency and measures of dispersion to analyze numerical (continuous) variables such as age, weight and body temperature; and using frequencies for categorical variables such as gender, area of residence, RDTs/microscopy malaria tests conducted, received ACT if RDT positive, presence of an ACT treatment chart on the health centre wall and availability of a weighing scale. To determine the relationship between variables, bivariate analysis via the prevalence ratio was conducted. Results: Just over half (55%) of all children with fever were tested for malaria in 2008 and this gratifyingly increased to (73%) in 2011. Overall, the proportion of children correctly and appropriately treated with ACT, which means that those who tested positive for malaria were given ACT, was 85% in 2008 but regrettably dropped to 72% in 2011. Although "presumptive treatment" decreased from 24% in 2008 to 11% in 2011, the proportion of children with fever not tested for malaria, and although not treated for malaria, but left without a definitive diagnosis of their fever being made, remained high but dropping (22% in 2008 and 16% in 2011). Similarly the proportion of children who tested negative for malaria but then did not undergo any further investigation also unfortunately remained very high and rising (57% in 2008 and 89% in 2011). A combination of the above poor clinical management practises resulted in only 38% of children with fever in 2008 and unfortunately dropping to only 33% in 2011 being correctly managed (tested for malaria via RDT or microscopy and treated with ACT if positive, while further investigated for the cause of fever if negative). On preparedness of the health facility to implement the "test and treat" policy, it was noted that only 4 out of 10 health facilities were at least minimally prepared to do so, but paradoxically on bivariate analysis those minimally prepared were less likely (PR 0.62; 95% CI 0.41-0.94) to correctly manage the patients in 2011 than those who were unprepared. A similar paradox occurred for those correctly treated with ACT after testing positive, with facilities which were minimally prepared being less likely to do so (PR 0.28; 95% CI 0.14-0.58) in 2011 than those facilities which were unprepared to implement the "test and treat" policy. However these associations were inconsistent over time, as the associations were not present in 2008. Similarly all other factors such as staff category (doctor, nurse, clinical officer) and type of presenting symptoms besides fever (anorexia, lethargy, pallor) assessed, were not consistently associated with testing for malaria in both 2008 and 2011. The same applied for the other two main outcome variables of 'treated with ACT after test positive for malaria' and 'correctly managed child with fever', in that there were no factors that showed a consistent association with them in both 2008 and 2011. Conclusion: Testing of children with fever for malaria is at a low level but rose between 2008 and 2011. Paradoxically the proportion of those diagnosed with malaria who were correctly treated with ACT dropped between 2008 and 2011, as did the proportion of children with fever who were correctly managed. No factors assessed in this study were found to be consistently associated in both 2008 and 2011 with either testing for malaria, or treating confirmed malaria cases with ACT, or managing patients with fever correctly. Recommendations: In order for health workers to correctly implement the "test and treat" policy, which involves a series of complex steps, they ought to be formally trained to do so, mentored and constructively supervised. Additionally health facilities should be adequately equipped to enable health workers to fully implement the policy. Further studies to assess factors associated with the correct management of malaria via the "test and treat" policy are warranted.

Artemisinin-based combination therapies (ACTs) are the gold standard for the treatment of malaria, but the efficacy is threatened by the development of parasite resistance. Histone deacetylase inhibitors (HDACis) are an emerging new class of potential antiplasmodial drugs. In this work, we present the design, synthesis, and biological evaluation of a mini library of dihydroartemisinin– HDACi hybrid molecules. The screening of the hybrid molecules for their activity against selected human HDAC isoforms, asexual blood stage P. falciparum parasites, and a panel of leukemia cell lines delivered important structure–activity relationships. All synthesized compounds demonstrated potent activity against the 3D7 and Dd2 line of P. falciparum with IC50 values in the single-digit nanomolar range. Furthermore, the hybrid ()-7c displayed improved activity against artemisininresistant parasites compared to dihydroartemisinin. The screening of the compounds against five cell lines from different leukemia entities revealed that all hydroxamate-based hybrids (7a–e) and the ortho-aminoanilide 8 exceeded the antiproliferative activity of dihydroartemisinin in four out of five cell lines. Taken together, this series of hybrid molecules represents an excellent starting point toward the development of antimalarial and antileukemia drug leads.