Dissertações/Teses

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2019
Teses
1
  • FRANCISCO ALEXANDRINO JÚNIOR
  • Desenvolvimento de Sistemas Terapêuticos para Leishmaniose Cutânea

  • Orientador : ERYVALDO SOCRATES TABOSA DO EGITO
  • MEMBROS DA BANCA :
  • ERYVALDO SOCRATES TABOSA DO EGITO
  • HELVECIO VINICIUS ANTUNES ROCHA
  • ADRIANA RAFFIN POHLMANN
  • ELIANA MARTINS LIMA
  • FREDERIC JEAN GEORGES FREZARD
  • Data: 26/09/2019

  • Mostrar Resumo
  • Esta tese é parte dos esforços do grupo para desenvolver alternativas terapêuticas contendo anfotericina B (AmB) para o tratamento da leishmaniose. Uma vez que a instabilidade da AmB, quando presente em meio aquoso, é um mecanismo complexo, dependente de fatores como o estado de agregação, grau de ionização (pKa) e pH do meio, o primeiro capítulo objetivou determinar os valores de pKa através do gráfico de Bjerrum, auxiliando na compreensão dos mecanismos envolvidos na instabilidade da AmB em soluções hidroalcoólicas, fornecendo subsídio para a elaboração de um modelo matemático com adequada capacidade preditiva e explanatória. O segundo capítulo objetivou avaliar os perfis cinéticos de liberação da AmB contida em sistemas poliméricos com distinta geometria (fibras e filmes) e composição (poli(álcool vinílico) (PVA) e poli(ácido lático) (PLA)), e correlacionar tais perfis com os parâmetros termodinâmicos. Os dados demonstraram que a liberação se apresentou como um processo endotérmico e não-espontâneo, com as fibras e filmes ajustando-se ao modelo de Peppas–Sahlin and Higuchi respectivamente. Dos sistemas avaliados os hidrogéis de PVA foram os que demonstraram melhor controle na liberação e foram escolhidos para compor o terceiro capítulo, onde suas propriedades como potencial sistema terapêutico foram avaliadas in vitro. Os resultados demonstraram que o sistema foi capaz de controlar a permeação ao vapor d’agua em níveis compatíveis com a pele em seu estado fisiológico, e simultaneamente apresentou eficiente atividade antifúngica e leishmanicida, sem apresentar citotoxicidade potencial para células VERO. No capítulo quatro são apresentados os resultados obtidos no desenvolvimento e avaliação de microemulsões (ME) termorresponsivas contendo AmB. Foi possível observar que ME desenvolvida possibilitou a retenção dérmica da AmB, e que a instabilidade causada por termodesestabilização da foi atenuada ao incorporar o poloxamer 407 ao sistema. Assim, os resultados aqui obtidos demonstram que os hidrogéis de PVA e as ME termorresponsivas são potenciais sistemas a serem usados no tratamento tópico ambulatorial da leishmaniose tegumentar americana.


  • Mostrar Abstract
  • This thesis is part of the group's efforts to develop therapeutic alternatives containing amphotericin B (AmB) for the treatment of leishmaniasis. Since the instability of AmB, when present in an aqueous medium, is a complex mechanism, dependent on factors such as the state of aggregation, degree of ionization (pKa) and pH of the medium, the first chapter aimed to determine the pKa values of AmB in distinct hydroalcoholic solutions through Bjerrum plot. Such an approach provided support to elaborate a mathematical model with suitable predictive and explanatory capacity. Therefore, allowing a better understanding of the mechanisms involved in the aqueous instability of AmB. The second chapter aimed to evaluate the kinetic profiles of AmB released from polymeric systems with different geometry (fibers and films) and composition (polyvinyl alcohol (PVA) and poly(lactic acid) (PLA)) and to correlate such profiles with thermodynamic parameters. The results suggest that the drug release was an endothermic and non-spontaneous process, with kinetic profiles of fibers and films consistent to the Peppas-Sahlin and Higuchi model, respectively. Since the PVA hydrogels demonstrated a better control in the AmB release, its properties as a potential therapeutic system were evaluated in vitro in the third chapter. Wherein the results revealed that the system was able to control the water vapor permeation to levels compatible with the skin in its physiological state. Additionally, it displayed effective antifungal and antileishmanial activity, without potential cytotoxicity for VERO cells. The fourth chapter reports the results obtained in the development and evaluation of AmB-loaded thermo-responsive microemulsions (ME). In which the ME enabled the AmB skin retention, and the instability phenomena of ME caused by thermodestabilization were hindered by adding poloxamer 407 into the system. Thus, the results obtained herein demonstrate that PVA hydrogels and thermo-responsive ME are potential systems to be used in the topical outpatient treatment of cutaneous leishmaniasis.

2017
Teses
1
  • ANDREZA ROCHELLE DO VALE MORAIS
  • Freeze-drying to increase stability of Amphotericin B-loaded microemulsion for leishmaniasis treatment

  • Orientador : ERYVALDO SOCRATES TABOSA DO EGITO
  • MEMBROS DA BANCA :
  • ERYVALDO SOCRATES TABOSA DO EGITO
  • FRÉDERIC JEAN GEORGES FREZARD
  • GILLIAN BARRATT
  • HATEM FESSI
  • PHILIPPE LEGRAND
  • PHILIPPE LOISEAU
  • Data: 20/10/2017

  • Mostrar Resumo
  • A leishmaniose visceral é uma doença tropical negligenciada que pode ser fatal se não tratada. A Anfotericina B (AmB) é eficiente no tratamento desta doença, porém o seu elevado custo ou sua alta toxicidade torna necessário o desenvolvimento de novos sistemas terapêuticos para solucionar tais inconvenientes. Nesse contexto, pode-se utilizar o aquecimento da formulação micelar de AmB aquecida (A-AmB), e o desenvolvimento de uma microemulsão (ME) contendo AmB (MEAmB). Adicionalmente, é desejada a remoção da água desses sistemas microemulsionados a fim de diminuir instabilidades relacionadas à contaminação microbiológica e a hidrólise. Desta forma, o objetivo deste trabalho foi desenvolver e avaliar a atividade e toxicidade in vitro e in vivo da A-AmB e da MEAmB contra Leishamania donovani (LV9), além de otimizar um sistema microemulsionado liofilizado contendo AmB. Caracterizações da reologia, do tamanho e da morfologia da gotícula mostraram que a MEAmB apresentou tamanhos médios de 35 nm, um comportamento Newtoniano e uma morfologia esférica. A caracterização da A-AmB mostrou a formação de superagregados, que, são menos tóxicos que os outros estados de agregação. Análises in vitro, tanto para a forma amastigota axênica como para a intramacrofágica mostraram que as atividades da A-AmB e da MEAmB foram semelhantes ao do Ambisome®. Além disso, foi observado um alto índice de seletividade da A-AmB e da MEAmB comparada a formulação não aquecida. Adicionalmente, essas duas formulações, quando comparadas ao Ambisome®, mostraram elevada atividade contra cepas AmB resistentes. Essas formulações foram testadas in vivo a fim de avaliar sua atividade e toxicidade. Os resultados não apresentaram diferenças significativas entre as atividades das amostras contendo AmB, e, com relação a toxicidade, não mostraram diferenças expressivas capazes de causar uma disfunção renal ou hepática. Portanto, tanto a A-AmB como a MEAmB podem ser usados como alternativa no tratamento contra LV9, apresentando a vantagem sobre o Ambisome® devido aos menores custos de suas produções. Por conseguinte, a fim de liofilizar o sistema microemulsionado, foi realizado um planejamento experimental completo, no qual o observou-se que MEs com menores tamanhos de gotículas foram obtidos quando utilizado maltose como crioprotetor na concentração de 5 %, com congelamento a – 80 ºC e por um período de liofilização de 24 h. Além disso, foi observado que a ME contendo AmB não teve mudanças significativas quanto ao conteúdo do fármaco, quando comparado o produto antes e após o processo de liofilização. Desta forma, a ME, em sua forma liofilizada, pode manter a estabilidade do sistema perante os danos que poderiam ser causados pela quantidade de água.


  • Mostrar Abstract
  • Visceral leishmaniasis is a neglected tropical disease that can be fatal if left untreated. Amphotericin B (AmB) is effective in the treatment of this disease, but the conventional formulation, Fungizone® has dose-limiting toxicity while the less toxic lipid-based forms such as Ambisome® are expensive. Therefore, the need for new therapeutic systems remains. In this respect, the heating of the Fungizone® formulation (H-AmB), and the development of a microemulsion (ME) containing AmB (MEAmB) are possible solutions. In addition, it is desirable to remove water from microemulsion systems in order to reduce instability due to microbiological contamination and hydrolysis. Therefore, the objective of this work was to develop and to evaluate the activity and toxicity in vitro and in vivo of H-AmB and MEAmB against Leishmania donovani (strain LV9) and, furthermore, to optimize a lyophilized microemulsion system containing AmB. Rheological, size and morphology studies showed that MEAmB presented average droplet sizes of 35 nm, a Newtonian behavior and spherical morphology. Spectroscopic characterization of H-AmB showed the formation of superaggregates, which are less toxic than the other states of aggregation. In-vitro evaluation on both the axenic and intramacrophagic amastigote forms showed that H-AmB and MEAmB showed similar activity to Ambisome®. A high selectivity index of H-AmB and MEAmB was observed compared with unheated Fungizone®. Furthermore, both new formulations showed high activity against AmB-resistant strains compared with Ambisome®. In-vivo experiments designed to evaluate their activity and toxicity did not reveal significant differences in activity between the new and reference formulations. There were no significant differences either in indicators of renal and hepatic toxicity. Therefore, both H-AmB and MEAmB can be used as an alternative for the treatment of LV9, presenting an advantage over AmBisome® in their lower costs of production. Therefore, a complete experimental design was performed in order to optimize the lyophilisation of the microemulsion system. It was observed that microemulsions with smaller droplet sizes were obtained using maltose as a cryoprotectant at a concentration of 5%, with freezing at -80 ° C, and a lyophilization process period of 24 h. Furthermore, it was observed that ME containing AmB showed no significant changes in drug content before and after the lyophilization process. Therefore, in its lyophilized form, the ME can remain stable and avoid degradation due to the presence of water.

2
  • ÉVERTON DO NASCIMENTO ALENCAR
  • Chemical stability of Amphotericin B in lipid-based media

  • Orientador : ERYVALDO SOCRATES TABOSA DO EGITO
  • MEMBROS DA BANCA :
  • ELQUIO ELEAMEN OLIVEIRA
  • ERYVALDO SOCRATES TABOSA DO EGITO
  • GILSON ANDRADE RAMALDES
  • LEE E. KIRSCH
  • LOURENA MAFRA VERISSIMO
  • Nereide Stela Santos Magalhães
  • Data: 14/12/2017

  • Mostrar Resumo
  • This thesis is part of research efforts with the intent of applying chemical stability and drug degradation kinetics knowledge to lipid-based systems, as amphotericin B (AmB), usually requires a nanotechnological drug delivery system to overcome its biopharmaceutical issues. The aim of this thesis was to determine the instability pathway and degradation kinetics of AmB in oil solutions, since stability of drug in delivery systems are chemically complex to be investigated due to their multi-phased aspect. The first section of this thesis was designed to provide a background on the current efforts regarding microemulsions containing Amphotericin B on the context of the research developed by our research group, for this purpose, literature reviews were published and showed that microemulsions containing AmB are systems capable of incorporating therapeutic concentrations of AmB. Those systems show effective anti-leishmania and antifungal activities. The use of lipidic formulations decrease the toxicity of AmB. In addition, freeze-drying has been one of the methods successfully used to increase stability of emulsioned systems, since the removal of water decreases degradation of lipids and drugs associated to hydrolysis mechanisms. The second section of this thesis was driven on the hypothesis that AmB´s toxicity might be associated to drug degradation instead of what is reported by the literature, which is mainly related to the drug´s aggregation state. The experimental research herein was conducted in order to investigate the chemical
    instability pathways and kinetics of AmB in oils, aiming to generate knowledge that can be useful on a larger context involving nanotechnological drug delivery systems currently studied by our research group, mainly emulsions and microemulsions. For this purpose, reaction mixtures containing AmB were done using different solvents and co-solutes. Samples were stored under different conditions of temperature and light exposure. AmB aggregation state in reaction mixtures was investigated by UV-Vis spectroscopy. The degradation pathway of AmB in oil was investigated. Use of hydrogen donator antioxidants decreased the degradation of AmB under thermal stress. AmB degradation under dark conditions was not linearly temperature dependent. Use of radical initiator increased the degradation of AmB in methanol greatly. Based on the obtained results, adsorption and aggregation did not appear to play a role in AmB degradation in oil. Under dark conditions, the most likely pathway for AmB degradation in oil was autoxidation. Whereas under light exposure, the most likely degradation pathway was light catalyzed oxidation. Empirical degradation schemes were drawn and differential equations were developed to explain AmB loss process. A kinetic model was successfully used to describe AmB loss in different solvents under dark environment. The model proved that AmB undergoes a complex degradation pathway, once a simple autocatalytic model could not describe its loss. In fact, a reversible loss probably related to hydrolysis might be involved, as demonstrated by the scheme and the kinetic model. A second model described AmB loss under light exposure successfully. AmB loss in this condition showed to be pseudo-first order. Due to complex degradation pathway, the kinetics of the different processes leading to AmB degradation
    could not be distinguished. The information generated by this research will help to predict AmB instability in
    microemulsions once data of AmB in aqueous phase and surfactants are associated. Additionally, further efforts on trying to generate and isolate the degradants here suggested can help on the investigation of toxicity related to degradation products.


  • Mostrar Abstract
  • This thesis is part of research efforts with the intent of applying chemical stability and drug degradation kinetics knowledge to lipid-based systems, as amphotericin B (AmB), usually requires a nanotechnological drug delivery system to overcome its biopharmaceutical issues. The aim of this thesis was to determine the instability pathway and degradation kinetics of AmB in oil solutions, since stability of drug in delivery systems are chemically complex to be investigated due to their multi-phased aspect. The first section of this thesis was designed to provide a background on the current efforts regarding microemulsions containing Amphotericin B on the context of the research developed by our research group, for this purpose, literature reviews were published and showed that microemulsions containing AmB are systems capable of incorporating therapeutic concentrations of AmB. Those systems show effective anti-leishmania and antifungal activities. The use of lipidic formulations decrease the toxicity of AmB. In addition, freeze-drying has been one of the methods successfully used to increase stability of emulsioned systems, since the removal of water decreases degradation of lipids and drugs associated to hydrolysis mechanisms. The second section of this thesis was driven on the hypothesis that AmB´s toxicity might be associated to drug degradation instead of what is reported by the literature, which is mainly related to the drug´s aggregation state. The experimental research herein was conducted in order to investigate the chemical
    instability pathways and kinetics of AmB in oils, aiming to generate knowledge that can be useful on a larger context involving nanotechnological drug delivery systems currently studied by our research group, mainly emulsions and microemulsions. For this purpose, reaction mixtures containing AmB were done using different solvents and co-solutes. Samples were stored under different conditions of temperature and light exposure. AmB aggregation state in reaction mixtures was investigated by UV-Vis spectroscopy. The degradation pathway of AmB in oil was investigated. Use of hydrogen donator antioxidants decreased the degradation of AmB under thermal stress. AmB degradation under dark conditions was not linearly temperature dependent. Use of radical initiator increased the degradation of AmB in methanol greatly. Based on the obtained results, adsorption and aggregation did not appear to play a role in AmB degradation in oil. Under dark conditions, the most likely pathway for AmB degradation in oil was autoxidation. Whereas under light exposure, the most likely degradation pathway was light catalyzed oxidation. Empirical degradation schemes were drawn and differential equations were developed to explain AmB loss process. A kinetic model was successfully used to describe AmB loss in different solvents under dark environment. The model proved that AmB undergoes a complex degradation pathway, once a simple autocatalytic model could not describe its loss. In fact, a reversible loss probably related to hydrolysis might be involved, as demonstrated by the scheme and the kinetic model. A second model described AmB loss under light exposure successfully. AmB loss in this condition showed to be pseudo-first order. Due to complex degradation pathway, the kinetics of the different processes leading to AmB degradation
    could not be distinguished. The information generated by this research will help to predict AmB instability in
    microemulsions once data of AmB in aqueous phase and surfactants are associated. Additionally, further efforts on trying to generate and isolate the degradants here suggested can help on the investigation of toxicity related to degradation products.

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