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Analysis of Secondary Metabolites from Aspergillus fumigatus and Penicillium nalgiovense : Antimicrobial Compounds from Filamentous Fungi Isolated from Extreme EnvironmentsSvahn, Stefan January 2015 (has links)
This thesis describes the cultivation and extraction of filamentous fungi isolated from extreme environments in the search for new antibiotic compounds. Filamentous fungi are a rich source of medicines including antibiotics, and it is believed that many currently unknown fungal species and bioactive fungal metabolites remain to be discovered. Aspergillus fumigatus and Penicillium nalgiovense strains were isolated from an antibiotic-contaminated riverbed near Hyderabad, India, and soil taken from a penguin’s nest on Paulete Island, Antarctica, respectively. It was anticipated that the extreme conditions within these environments would exert unusual selective pressures on their filamentous fungi, possibly causing the secretion of new bioactive compounds. The cultivation, extraction and analysis of metabolites from the A. fumigatus strain resulted in the isolation of the antimicrobial substance gliotoxin. Subsequent investigations revealed that this strain’s secretion of gliotoxin was increased by as much as 65 % when it was cultivated in the presence of pathogen-associated molecular patterns. These results indicate the existence of a fungal receptor/signaling system for detecting nearby bacteria. The scope for using gliotoxin and the related metabolite bis(methyl)gliotoxin as biomarker metabolites for diagnosing the lethal pulmonary condition invasive aspergillosis was also investigated. Bronchoalveolar lavage fluid from 42 patients with and without possible invasive aspergillosis was extracted and analyzed. The results obtained suggest that gliotoxin and bis(methyl)gliotoxin are not suitable markers for diagnosing invasive aspergillosis. Studies on the P. nalgiovense strain from Antarctica resulted in the isolation of the antifungal agent amphotericin B. The secretion of this compound increased when P. nalgiovense was cultured on a potato-dextrose agar enriched with coconut flakes rather than liquid RPMI 1640 medium. This was the first time amphotericin B was isolated from any organism other than the bacterium Streptomyces nodosus. The results presented in this thesis will be useful in the continuing search for novel bioactive compounds, the diagnosis of fungal infections, and as a source of insight into the interactions between microorganisms. Moreover, they show that even extensively studied fungal genera such as Aspergillus and Penicillium are not completely understood and may produce unexpected or previously unknown bioactive metabolites under appropriate conditions.
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Cellular differentiation and antibiotic production by Streptomyces nodosus immobilised in alginate capsulesPereira, Marie Antoinette Tanya. January 2007 (has links)
Thesis (PhD) -- University of Western Sydney, 2007. / A thesis submitted to the University of Western Sydney, College of Health and Science, School of Natural Sciences, as a requirement for the degree of Doctor of Philosophy. Includes bibliography.
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Microemulsões biocompatíveis de anfotericina B para administração oral : estudo estrutural, liberação in vitro e farmacocinética pré-clínica /Pestana, Kelly Chrystina. January 2009 (has links)
Resumo: Anfotericina B (AmB) é o fármaco de escolha para o tratamento das infecções fúngicas invasivas, importante causa de morbidade e mortalidade em pacientes imunodeprimidos. A toxicidade da AmB na forma convencional tem estimulado o desenvolvimento de novas formulações para a administração do fármaco. Neste trabalho foram estudadas microemulsões óleo/água, contendo fosfatidilcolina de soja/tween 20 como agentes tensoativos, CaptexTM 200 como fase oleosa e tampão fosfato 50mM pH 7,2 como fase aquosa, com o objetivo de reduzir a toxicidade e aumentar a absorção oral da AmB. Os sistemas obtidos com diferentes proporções dos componentes foram descritos através de um diagrama de fase pseudo-ternário. As microestruturas foram caracterizadas por espalhamento dinâmico de luz (DLS), reologia, microscopia de luz polarizada e espalhamento de raios X a baixo ângulo (SAXS). Foi desenvolvido e validado um método por CLAE para a determinação de AmB em plasma para aplicação em estudo do perfil farmacocinético do fármaco veiculado na ME em ratos. A nefrotoxicidade da AmB foi avaliada pela determinação da creatinina plasmática dos ratos após administração oral da nova formulação desenvolvida (50 mg/kg), e comparada à administração da formulação convencional na mesma dose. Foi observado que o tamanho das gotículas das microemulsões aumenta quando a AmB é incorporada ao sistema. As amostras apresentaram comportamento Newtoniano e, dependendo da composição do sistema, antitixotropia foi observada. Também foi observado que a viscosidade aumenta com o aumento da fase oleosa assim como a formação de estruturas lamelares ordenadas que são desfavorecidas com a adição do fármaco. A incorporação do fármaco depende das proporções de fase oleosa e tensoativo. As interações da AmB... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Amphotericin B (AmB) is the drug of choice for therapy of invasive fungal infections, an important cause of morbidity and mortality among immunodeficient patients. The high toxicity of AmB in its conventional formulation have induced the development of innovative formulations for the drug administration. In this work, oil-in-water microemulsions containing soya phosphatidylcholine/Tween® 20 (1:1) as surfactant, captexTM 200 as oil phase, and phosphate buffer 50mM, pH 7.2 as aqueous phase were studied in order to reduce AmB toxicity and increase its oral absorption. Systems obtained with different proportions of the components were described by pseudoternary phase diagram. The microstructures of the system were characterized by dynamic light scattering (DLS), rheological behavior, polarized light microscopy and small angle X-ray scattering (SAXS). Was developed and validated a fast and selective HPLC method to determine AmB for application to study of pharmacokinetic profile of drug in rats. The nephrotoxicity of AmB was assessed by determining of rat plasma creatinine after oral administration of the novel formulation (50 mg/kg) and comparing with it a conventional formulation in the same dose. It was observed that the oil droplets size increase when AmB is incorporated into the system. The samples presented Newtonian behavior depending on the system composition. An anti-thixotropic behavior was found, as well, the viscosity increases withthe oil phase. The data showed the formation of ordered structures with lamellar arrangements in the drug unloaded systems and that order decrease with the drug incorporation. The AmB incorporation into the system was dependent on both the oil phase and surfactant. The interactions of AmB with the systems can control both the drug solubility and release... (Complete abstract click electronic access below) / Orientador: Anselmo Gomes de Oliveira / Coorientador: Rosangela Gonçalves Peccinini / Banca: Maria Palmira Daflon Gremião / Banca: Marco Vinícius Chaud / Banca: Newton Andreo Filho / Banca: Raul Cesar Evangelista / Doutor
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Tratamento da pitiose em membros de equinos por meio de perfusão regional intravenosa com anfotericina BDória, Renata Gebara Sampaio [UNESP] 31 March 2009 (has links) (PDF)
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doria_rgs_dr_jabo.pdf: 805921 bytes, checksum: 7baf913cbb38741225210e93ed796dd6 (MD5) / Avaliaram-se os efeitos da perfusão regional intravenosa com anfotericina B, um antimicótico, em membros de equinos acometidos por pitiose, combinada com a excisão cirúrgica. Foram utilizados 16 equinos, jovens e adultos, da raça pantaneira, com pitiose em membro torácico ou pélvico, distalmente às articulações do cotovelo ou joelho, distribuídos em dois grupos experimentais. Um constituído de 12 equinos tratados, após excisão cirúrgica e termocauterização, por perfusão regional intravenosa do membro com anfotericina B (Ganf) e outro constituído de quatro equinos não tratados (controle, Gc). As feridas foram fotografadas e avaliadas antes do início do tratamento (D0) e após sete (D7), catorze (D14), vinte e um (D21), vinte e oito (D28), trinta e cinco (D35) e sessenta (D60) dias e foi realizado exame do aparelho locomotor. No Ganf, 92% dos animais apresentaram cicatrização das feridas de pitiose e no Gc 100% apresentaram recrudescência da afecção. No Ganf, 58% exigiram administração única de anfotericina B pela técnica de perfusão regional do membro e 42% exigiram uma readministração, após 14 dias. No Ganf, 33% apresentaram ulceração no local da administração da anfotericina B e 42% apresentaram aumento de volume do membro e dor à palpação na região perfundida pela anfotericina B. No Ganf, 100% dos animais apresentavam claudicação severa no D0 e 92% não apresentavam claudicação no D21. Conclui-se que a administração de anfotericina B, por perfusão regional intravenosa, promove a cura da pitiose, com mínimas reações adversas, sendo uma alternativa terapêutica para o tratamento de pitiose em membros de equinos. / The effects of intravenous regional perfusion with amphotericin B, an antifungal drug, in equine limbs with pythiosis associated with surgical excision were evaluated. 16 horses, young and adults, pantaneira breed, with pythiosis in thoracic or pelvic limbs, distally to the elbow or knee joints, were allocated into two experimental groups. One constituted by 12 horses treated, after surgical excision and thermocauterization, by intravenous regional limb perfusion with amphotericin B (Ganf) and other constituted by four not treated horses (control, Gc). The wounds were photographed and evaluated before the beginning of the treatment (D0) and after seven (D7), fourteen (D14), twenty-one (D21), twenty-eight (D28), thirty-five (D35) and sixty (D60) days and examination of the locomotor system was performed. 92% of the animals with pythiosis presented wound healing in Ganf and, in Gc, 100% presented recrudescence of the disease. In the Ganf, 58% of the animals needed single administration of amphotericin B by regional limb perfusion and 42% of the animals needed one more administration after 14 days. In the Ganf, 33% of the animals presented ulceration in the amphotericin B injection site and 42% of the animals presented limb edema and pain during palpation of the amphotericin B perfused region. In Ganf, 100% of the animals presented severe lameness at D0 and 92% of the animals presented no lameness at D21. It can be concluded that the administration of amphotericin B by intravenous regional perfusion promotes pythiosis remission with minimal side effects, being a therapeutic alternative to the pythiosis treatment in equine limbs.
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Microemulsões biocompatíveis de anfotericina B para administração oral: estudo estrutural, liberação in vitro e farmacocinética pré-clínicaPestana, Kelly Chrystina [UNESP] 20 August 2009 (has links) (PDF)
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pestana_kc_dr_arafcf.pdf: 1397611 bytes, checksum: 139f2c10e325976c56a9928f8e54e882 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Anfotericina B (AmB) é o fármaco de escolha para o tratamento das infecções fúngicas invasivas, importante causa de morbidade e mortalidade em pacientes imunodeprimidos. A toxicidade da AmB na forma convencional tem estimulado o desenvolvimento de novas formulações para a administração do fármaco. Neste trabalho foram estudadas microemulsões óleo/água, contendo fosfatidilcolina de soja/tween 20 como agentes tensoativos, CaptexTM 200 como fase oleosa e tampão fosfato 50mM pH 7,2 como fase aquosa, com o objetivo de reduzir a toxicidade e aumentar a absorção oral da AmB. Os sistemas obtidos com diferentes proporções dos componentes foram descritos através de um diagrama de fase pseudo-ternário. As microestruturas foram caracterizadas por espalhamento dinâmico de luz (DLS), reologia, microscopia de luz polarizada e espalhamento de raios X a baixo ângulo (SAXS). Foi desenvolvido e validado um método por CLAE para a determinação de AmB em plasma para aplicação em estudo do perfil farmacocinético do fármaco veiculado na ME em ratos. A nefrotoxicidade da AmB foi avaliada pela determinação da creatinina plasmática dos ratos após administração oral da nova formulação desenvolvida (50 mg/kg), e comparada à administração da formulação convencional na mesma dose. Foi observado que o tamanho das gotículas das microemulsões aumenta quando a AmB é incorporada ao sistema. As amostras apresentaram comportamento Newtoniano e, dependendo da composição do sistema, antitixotropia foi observada. Também foi observado que a viscosidade aumenta com o aumento da fase oleosa assim como a formação de estruturas lamelares ordenadas que são desfavorecidas com a adição do fármaco. A incorporação do fármaco depende das proporções de fase oleosa e tensoativo. As interações da AmB... / Amphotericin B (AmB) is the drug of choice for therapy of invasive fungal infections, an important cause of morbidity and mortality among immunodeficient patients. The high toxicity of AmB in its conventional formulation have induced the development of innovative formulations for the drug administration. In this work, oil-in-water microemulsions containing soya phosphatidylcholine/Tween® 20 (1:1) as surfactant, captexTM 200 as oil phase, and phosphate buffer 50mM, pH 7.2 as aqueous phase were studied in order to reduce AmB toxicity and increase its oral absorption. Systems obtained with different proportions of the components were described by pseudoternary phase diagram. The microstructures of the system were characterized by dynamic light scattering (DLS), rheological behavior, polarized light microscopy and small angle X-ray scattering (SAXS). Was developed and validated a fast and selective HPLC method to determine AmB for application to study of pharmacokinetic profile of drug in rats. The nephrotoxicity of AmB was assessed by determining of rat plasma creatinine after oral administration of the novel formulation (50 mg/kg) and comparing with it a conventional formulation in the same dose. It was observed that the oil droplets size increase when AmB is incorporated into the system. The samples presented Newtonian behavior depending on the system composition. An anti-thixotropic behavior was found, as well, the viscosity increases withthe oil phase. The data showed the formation of ordered structures with lamellar arrangements in the drug unloaded systems and that order decrease with the drug incorporation. The AmB incorporation into the system was dependent on both the oil phase and surfactant. The interactions of AmB with the systems can control both the drug solubility and release... (Complete abstract click electronic access below)
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Intérêt de la lyophilisation pour améliorer la stabilité des microémulsions charges en Amphotéricine B destinées au traitement de la leishmanioseMorais, Andreza Rochelle do Vale 20 October 2017 (has links)
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Previous issue date: 2017-10-20 / 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. / 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.
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Tratamento da pitiose em membros de equinos por meio de perfusão regional intravenosa com anfotericina B /Dória, Renata Gebara Sampaio. January 2009 (has links)
Orientador: Carlos Augusto Araújo Valadão / Banca: Rita de Cássia Campbell / Banca: José Wanderley Cattelan / Banca: Antônio Carlos Alessi / Banca: Juliana Regina Peiró / Resumo: Avaliaram-se os efeitos da perfusão regional intravenosa com anfotericina B, um antimicótico, em membros de equinos acometidos por pitiose, combinada com a excisão cirúrgica. Foram utilizados 16 equinos, jovens e adultos, da raça pantaneira, com pitiose em membro torácico ou pélvico, distalmente às articulações do cotovelo ou joelho, distribuídos em dois grupos experimentais. Um constituído de 12 equinos tratados, após excisão cirúrgica e termocauterização, por perfusão regional intravenosa do membro com anfotericina B (Ganf) e outro constituído de quatro equinos não tratados (controle, Gc). As feridas foram fotografadas e avaliadas antes do início do tratamento (D0) e após sete (D7), catorze (D14), vinte e um (D21), vinte e oito (D28), trinta e cinco (D35) e sessenta (D60) dias e foi realizado exame do aparelho locomotor. No Ganf, 92% dos animais apresentaram cicatrização das feridas de pitiose e no Gc 100% apresentaram recrudescência da afecção. No Ganf, 58% exigiram administração única de anfotericina B pela técnica de perfusão regional do membro e 42% exigiram uma readministração, após 14 dias. No Ganf, 33% apresentaram ulceração no local da administração da anfotericina B e 42% apresentaram aumento de volume do membro e dor à palpação na região perfundida pela anfotericina B. No Ganf, 100% dos animais apresentavam claudicação severa no D0 e 92% não apresentavam claudicação no D21. Conclui-se que a administração de anfotericina B, por perfusão regional intravenosa, promove a cura da pitiose, com mínimas reações adversas, sendo uma alternativa terapêutica para o tratamento de pitiose em membros de equinos. / Abstract: The effects of intravenous regional perfusion with amphotericin B, an antifungal drug, in equine limbs with pythiosis associated with surgical excision were evaluated. 16 horses, young and adults, pantaneira breed, with pythiosis in thoracic or pelvic limbs, distally to the elbow or knee joints, were allocated into two experimental groups. One constituted by 12 horses treated, after surgical excision and thermocauterization, by intravenous regional limb perfusion with amphotericin B (Ganf) and other constituted by four not treated horses (control, Gc). The wounds were photographed and evaluated before the beginning of the treatment (D0) and after seven (D7), fourteen (D14), twenty-one (D21), twenty-eight (D28), thirty-five (D35) and sixty (D60) days and examination of the locomotor system was performed. 92% of the animals with pythiosis presented wound healing in Ganf and, in Gc, 100% presented recrudescence of the disease. In the Ganf, 58% of the animals needed single administration of amphotericin B by regional limb perfusion and 42% of the animals needed one more administration after 14 days. In the Ganf, 33% of the animals presented ulceration in the amphotericin B injection site and 42% of the animals presented limb edema and pain during palpation of the amphotericin B perfused region. In Ganf, 100% of the animals presented severe lameness at D0 and 92% of the animals presented no lameness at D21. It can be concluded that the administration of amphotericin B by intravenous regional perfusion promotes pythiosis remission with minimal side effects, being a therapeutic alternative to the pythiosis treatment in equine limbs. / Doutor
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Avaliação da atividade mutagênica e carcinogênica da anfotericina B em células somáticas de Drosophila melanogasterSaturnino, Rosiane Soares 20 November 2012 (has links)
Conselho Nacional de Desenvolvimento Científico e Tecnológico / CHAPTER II: Amphotericin B (AmB) is an antifungal extracted from Streptomyces nodosus. Its fungicidal activity depends primarily on its binding to the sterol group, present in the membrane of fungi. Due to the toxicity of this drug this study aimed to evaluate their mutagenic, carcinogenic and recombinogenic activity. To do so, we used Somatic Mutation and Recombination Test (SMART) and the test for the detection of Epithelial Tumors (wts), both in Drosophila melanogaster. In the assessment using SMART, larvae from 72 h, descendants from the standard (ST) cross and the high bioactivation (HB) cross were treated with different concentrations of Amphotericin B (0.01; 0.02 e 0.04 mg/mL). The results revealed that the AmB is a promutagen exhibiting increase in the number of spots on individuals from high bioactivation (HB) cross with a high level of the cytochrome P450. The results also indicate that recombinogenicity is the main genotoxic event induced by AmB. Homologous recombination can function as a determinant at different stages of carcinogenesis. For verification, larvae from the wts test, descendants from the wts/TM3 virgin female and mwh/mwh male cross, were treated with the same three AmB concentrations separately and in association with mitomicin C (0.1 mM). The results did not, however, provide evidence that AmB has carcinogenic potential. / CAPÍTULO II: A Anfotericina B (Anf B) é um antifúngico extraído de Streptomyces nodosus, que possui atividade antibiótica efetiva contra diversas espécies de fungos patogênicos. Devido à toxicidade desta droga, o presente trabalho foi desenvolvido com o objetivo de avaliar sua atividade mutagênica, recombinogênica e carcinogênica. Para tanto, foram utilizados os testes para Detecção de Mutação e Recombinação Somática (SMART) e o Teste para Detecção de Tumores Epiteliais (wts), ambos realizados em Drosophila melanogaster. No SMART foram utilizadas larvas de 4 horas provenientes do cruzamento padrão (ST) e de alta capacidade de bioativação metabólica (HB). As larvas provenientes de ambos os cruzamentos foram tratadas com diferentes concentrações de Anfotericina B (0,01; 0,02 e 0,04 mg/mL). Os resultados revelaram que a Anf B causou aumento significativo no número de manchas nos indivíduos provenientes do cruzamento HB, o que sugere que esse fármaco atua como um prómutageno, manifestando tal efeito após a biotransformação pelo sistema citocromo P450. Os resultados indicam, também, que a recombinogenicidade é o principal evento induzido pela Anf B. A recombinação homóloga pode ter função determinante nos diferentes estágios da carcinogênese. Para esta verificação, larvas provenientes do teste wts, descendentes do cruzamento de fêmeas virgens wts/TM3 com machos mwh/mwh, foram tratadas com as mesmas concentrações de Anf B isoladas e em associação com a mitomicina C (0,1 mM). Os resultados não revelaram atividade carcinogênica para a Anf B. / Mestre em Genética e Bioquímica
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Développement et caractérisation physico-chimique d'une nouvelle forme galénique à base d'Amphotéricine B adaptée à la voie orale et pulmonaire / Development and physicochemical characterization of a new galenic form based on Amphotericin B adapted to the oral and pulmonary routeMehenni, Lyes 07 March 2018 (has links)
Les infections fongiques peuvent s’exprimer sous différentes formes (leishmaniose cutanée ouviscérale, aspergillose pulmonaire à caractère allergique ou invasive) et connaissent unerecrudescence dans les pays développés en affectant principalement les personnesimmunodéprimées comme les cancéreux, les malades ayant subis des greffes d’organes ou lespatients atteints du VIH. Ces infections sont une cause de mortalité chez 30% des sujetsatteints. L’approche médicamenteuse conventionnelle consiste en l’administration par voieintraveineuse (IV) d’antifongiques. L'Amphotéricine B (AmB), un antibiotiquede la familledes polyènes, reste à ce jour l'un des agents les plus efficaces dans le traitement des infectionsfongiques systémiques mais entraîne aussi des effets indésirables comme une néphrotoxicitéaigüe après injection. Pour améliorer l’index thérapeutique de cette molécule active, nousavons développé différents outils galéniques adaptés à d’autres voies d’administration. Dansun premier temps, nous avons mis au point des liposomes à base de céramides végétales,adaptés pour une administration orale et dans le but de traiter la leishmaniose viscérale. Cesliposomes ont été testés dans un modèle in vitro Estomac/Duodénum pour mimer lesconditions physiologiques et ont montré une bonne stabilité dans un tel milieu avec un tauxd’encapsulation satisfaisant. Des liposomes formulés à partir de Dimyristoylphosphatidylcholine(DMPC)/Dimyristoylphosphatidylglycérol (DMPG) et encapsulantl’AmB ont été étudiés à l’aide de la résonnance magnétique nucléaire (RMN31P et 1H) et parrésonnance paramagnétique électronique (RPE). Ces liposomes, dont la composition pourraitêtre adaptée pour une administration par voie pulmonaire, ont été utiliséscomme modèlemembranaire pour une étude des interactions du principe actif dans la matrice liposomale.Enfin, nous décrivons le développement et la caractérisation physico-chimique d’une nouvellegénération de dispersions solides amorphes sphériques formulées à base de polymères decyclodextrines très hydrophiles et d’amphotéricine B.Les formulations ont été obtenues sousforme de poudre sèche par spray-drying, une méthode de séchage par atomisation. Cessystèmes ont été caractérisés par diverses techniques : spectroscopie infrarouge à transforméede Fourier (FT-IR), spectroscopie Raman, granulométrie laser, microscopie électronique àtransmission et microscopie électronique à balayage. La distribution aérodynamique de cesdispersions solides amorphes a été évaluée à l’aide d’un impacteur à cascades afin de testerleur intérêt pour des applications potentielles par voie pulmonaire. / Résumé en anglais non disponible
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Développement de nanocomplexes antileishmaniens lipidiques administrables par voie orale / Development of Lipid Nanocomplexes for Oral Administration of Anti-leishmanial DrugsPham, Thi Thu Hanh 11 July 2013 (has links)
Ce travail porte sur la mise au point des nanocochléates intégrant à la fois l’amphotéricine B (AmB) et la miltéfosine (hexadécylphosphocholine, HePC) dotés de propriétés adéquates pour une administration par voie orale pour le traitement de la leishmaniose viscérale. Notre premier axe de recherche a été l’étude des interactions de l’AmB et de l’HePC avec les monocouches de lipides de DOPS (dioleoyl phosphatidylsérine) et de Cho (cholestérol) dans le développement de nanocochléates chargés avec ces deux principes actifs. Les résultats expérimentaux nous ont permis de déterminer le rapport drogue/lipides optimal pour la formulation des nanocochléates : 9DOPS/1Cho/0,5AmB/0,5HePC et de confirmer que dans la formulation de nanocochléates, l’AmB et l’HePC sont incorporés de façon stable et se situent au sein des bicouches lipidiques entre des lipides, plutôt que dans la phase aqueuse entre les bicouches.Notre second axe de recherche a porté sur la formulation et la caractérisation des nanocochléates chargés en AmB et en HePC. Ces nanocochléates ont été formulés à partir des liposomes unilamellaires encapsulant ces deux principes actifs. Les paramètres expérimentaux majeurs (taille de liposomes, proportion DOPS/1Cho) ont été optimisés. Les propriétés physico-chimiques des nanocochléates, telles que la taille, la charge de surface, la morphologie, le rendement d'encapsulation, l’organisation des principes actifs et la stabilité pendant le stockage au cours de temps ont été étudiées. En vue d'une formulation orale, la libération des principes actifs in vitro ainsi que la stabilité des nanocochléates in vitro dans les milieux gastro-intestinaux ont également été étudiés d’après la Pharmacopée des Etats-Unis. Enfin, une étude in vivo préliminaire chez le rat portant sur la pharmacocinétique plasmatique d'AmB après administration orale de nanocochléates chargés en AmB avec ou sans HePC en comparaison avec l’Ambisome® et la Fungizone® a été entamée. Les résultats préliminaires ont démontré une absorption significative par voie orale de l’AmB.Ce travail a permis de développer des nanocochléates avec des propriétés appropriées pour une formulation contenant à la fois l’AmB et l’HePC administrable par voie orale. Néanmoins, des optimisations restent à réaliser avant d’envisager une application clinique. / The aim of this work was to formulate nanocochleates containing both Amphotericin B (AmB) and miltefosine (hexadecylphosphocholine, HePC) with properties suitable for administration by the oral route for the treatment of visceral leishmaniasis. The first part of this work was a fundamental study of the interactions between AmB and HePC and monolayers of dioleylphosphatidylserine (DOPS) with or without cholesterol (Cho), to provide a basis for the formulation of nanocochleates containing the two active molecules. The results allowed us to define the optimal proportions for the formulation of nanocochleates: 9DOPS/1Cho/0.5AmB/0.5HePC and to predict that AmB and HePC would be incorporated stably into the formulation and would be located between the lipids in the bilayers rather than in the aqueous phase between the bilayers. The second part of the work was the formulation and characterization of nanococheates containing both AmB and HePC. These were derived from unilamellar liposomes containing the two active molecules. The crucial experimental parameters (size of the liposomes, ratio of DOPS to Cho) were optimized. The physico-chemical properties of the nanocochleates, such as the size, surface charge, morphology, encapsulation yield, the organization of the active molecules and the stability during long-term storage were studied. Since the formulation was destined for the oral route, in-vitro drug release and the stability of the nanocochleates in simulated gastro-intestinal media were studied according to the recommendations of the US Pharmacopeia. Finally, a preliminary in-vivo study of the plasma pharmacokinetics of AmB after oral gavage to rats of nanocochleates contained AmB with or without HePC, in comparison with AmBisome and Fungizone, was carried out. A significant oral absorption of AmB was observed. This work has led to the formulation of nanocochleates containing AmB and HePC with appropriate properties for oral administration. However, further optimization is necessary before such particles will be suitable for clinical use.
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