A series of acute (96h) toxicity tests were conducted on six species of crayfish inthe family Cambaridae. Toxicity values fell into a sensitive group comprising Orconectesplacidus and Procambarus acutus (LC 50= 0.368mg Cd/L - 0.487mg Cd/L; LC10= 0.048mg Cd/L - 0.092 mg Cd/L) and tolerant group comprising O. juvenilis, O. virilis, P.alleni, and P. clarkii (LC 50= 2.44 mg Cd/L - 3.30 mg Cd/L; LC10= 0.386 mg Cd/L -0.947 mg Cd/L). For juvenile crayfish, the LC50 and LC10 values were as follows: O.juvenilis, 0.060 and 0.014 mg Cd/L; O. placidus, 0.037 and 0.002 mg Cd/L; P. clarkii,0.624 and 0.283 mg Cd/L. Cd exposure decreased molting success highlighting theimportance of this sensitive process. Behavioral responses were assessed in O. placidus,O. virilis, P. acutus, P. alleni, and P. clarkii. The tail-flip predator avoidance behaviorwas significantly reduced by cadmium exposure. In most species tested, the claw raisedefensive behavior was significantly increased by Cd exposure. Between species, as bodymass increased, the tail-flip response frequency decreased, and the claw-raise responseincreased in frequency. P. clarkii was also analyzed for the effect of Cd exposure onheart rate and response to two stimuli. The data indicate that heart rate may be a usefulphysiological marker of Cd toxicity. The major organ systems were dissected fromsurvivors of four adult crayfish toxicity tests (O. juvenilis, O. placidus, P. acutus, P.clarkii) and analyzed for metal content. Cadmium tissue content correlated with Cdexposure. Cadmium accumulated more in the hepatopancreas, gills and green glands ofsensitive species than in tolerant species. Zn showed negative correlations with Cdexposure in the hepatopancreas. Cu increased in green glands, gills, and hemolymph and,in some cases, decreased in the hepatopancreas. Ca, Fe, Mg, and Zn also showedsignificant trends. Zn accumulated in the exposure water over 24h. These data indicatethat Cd may displace Cu and/or Zn in the hepatopancreas and the displaced metal thenmay move into other tissues, especially the gills and green glands, possibly to beexcreted.

Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-09-06T11:19:09Z No. of bitstreams: 2 Tese - Danillo Fabrini Maciel Costa Veloso - 2018.pdf: 15996172 bytes, checksum: 30a5841de71ac68f55a18e1b917ed259 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-09-10T10:40:59Z (GMT) No. of bitstreams: 2 Tese - Danillo Fabrini Maciel Costa Veloso - 2018.pdf: 15996172 bytes, checksum: 30a5841de71ac68f55a18e1b917ed259 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-09-10T10:40:59Z (GMT). No. of bitstreams: 2 Tese - Danillo Fabrini Maciel Costa Veloso - 2018.pdf: 15996172 bytes, checksum: 30a5841de71ac68f55a18e1b917ed259 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-07-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Voriconazole, a second-generation triazole with a large spectrum of action is one of the most recommended systemic antifungal agents as the first line therapy against several clinically important fungal pathogens, among them Candida albicans. This antifungal has moderate water solubility and exhibits a nonlinear pharmacokinetic profile due to metabolic clearance saturation. By entrapping voriconazole into liposomes it is possible to circumvent its physico-chemical limitations, avoid the high toxicity of the antimicrobial, caused by sulfobutyl ether-betacyclodextrin, vehicle used to increase its solubility, present in its commercially available formulation: VFEND®. Pharmacokinetics and biodistribution of voriconazole modified by encapsulation in liposomes allowed its antifungal activity to be potentiated, leading to increased specificity and tissue penetration, protection the drug from biological degradation and reduced metabolism. Liposomes entrapping voriconazole (LVCZ) showed a particle size of 95.3 ± 1.27 nm, PdI of 0.09 ± 0.01, zeta potential near to neutrality, as well as a high efficiency of encapsulation of the antifungal and vesicles presenting spherical morphology uni and/or multilamellar. In vitro and in vivo evaluations of the performance of the liposomal formulation containing voriconazole were all performed in a comparative fashion with the commercially available formulation, VFEND®. In the in vitro assays using different species of fungal isolates of Candida and Aspergillus sp. the liposomal formulation was equivalent or superior to VFEND®. In a non-clinical pharmacokinetic assay in Balb/c mice, using a dose of 10 mg/kg, the main pharmacokinetic parameters were obtained: Cmax (μg/mL) = 1.23 ± 0.28 and 0.61 ± 0.15; AUC0-24 (μg/ml*h) = 4.86 ± 1.01 and 1.96 ± 0.30; Cl (mL/h) = 52.75 ± 8.88 and 100.91 ± 20.14; Vd (mL) = 230.18 ± 53.61 and 314.18 ± 106.24 for LVCZ and VFEND®, respectively. In all calculated/observed parameters, the liposomal formulation presented superior performance, using the same dose as the commercial formulation. Increases in antifungal concentrations found in blood, liver and kidneys and lower amounts of the inactive metabolite formed when using the liposomal formulation can be attributed to the ability of liposomes to alter the pharmacokinetics and biodistribution of voriconazole in the body mainly because of their capacity to protect the drug from accelerated metabolism. In vivo efficacy evaluation of voriconazole was also performed in a systemic candidiasis model in immunosuppressed animals, as well as parameters such as weight loss, fungal burden in the liver and kidneys and histological alterations caused by infection and treatment. As a consequence of voriconazole pharmacokinetics and biodistribution modified by the encapsulation in liposomes, the antifungal activity of drug was potentiated, leading to greater specificity and tissue penetration. In conclusion, in order to provide appropriate dose regimens for the treatment of systemic fungal infections, avoiding obstacles such as toxicity and resistance mechanisms we developed na alternative therapeutic platform, able to lead to safe and effective treatment. / O voriconazol, um triazólico de segunda geração de amplo espectro de ação, é um dos agentes antifúngicos sistêmicos mais recomendados como terapia de primeira linha contra vários patógenos fúngicos clinicamente importantes, dentre eles Candida albicans. Este antifúngico apresenta algumas desvantagens do ponto de vista tecno-farmacêutico: baixa solubilidade em água e perfil farmacocinético não-linear devido à saturação de depuração metabólica. Alem disso, sua formulação comercialmente disponível (VFEND®), utiliza um agente complexante, a sulfobutil éter-beta-ciclodextrina, para aumentar a solubilidade do voriconazol em água, porém esse agente apresenta consideráveis efeitos de toxicidade. Diante dessas limitações, encapsular o antifúngico em uma formulação lipossomal, que utiliza componentes biodegradáveis e biocompatíveis, os fosfolipídeos, é uma alternativa para aumentar a solubilidade do voriconazol em água, possibilitando o uso intravenoso. Além de contornar suas limitações físico-químicas, o uso de lipossomas permite eliminar o uso do veiculo tóxico presente na formulação comercial, além de possibilitar melhoras na farmacocinética e na distribuição tecidual do voriconazol. Os lipossomas contendo voriconazol (LVCZ) apresentaram tamanho de partícula de 95,3 ± 1,27 nm, índice de polidispersão de 0,09 ± 0,01, potencial zeta próximo da neutralidade, bem como uma elevada eficiência de encapsulação do antifúngico (aproximadamente 80%) e vesículas de morfologia esférica uni e/ou multilamelares. As avaliações in vitro e in vivo da performance da formulação lipossomal contendo voriconazol foram realizadas de forma comparativa com o VFEND®. Nos ensaios in vitro utilizando diferentes espécies de isolados fúngicos de Candida e Aspergillus sp. a formulação lipossomal se mostrou equivalente ou superior ao VFEND®. No estudo não-clínico de farmacocinética em camundongos Balb/c com dose de 10 mg/kg, foram obtidos os principais parâmetros farmacocinéticos: Cmax (μg/mL) = 1,23 ± 0,28 e 0,61 ± 0,15; AUC0-24 (μg/mL*h) = 4,86 ± 1,01 e 1,96 ± 0,30; Cl (mL/h) = 52,75 ± 8,88 e 100,91± 20,14; Vd (mL) = 230,18 ± 53,61 e 314,18 ± 106,24 para LVCZ e VFEND®, respectivamente. Em todos os parâmetros calculados/observados, a formulação lipossomal apresentou melhor desempenho, utilizando a mesma dose que a formulação comercial. Aumentos nas concentrações do antifúngico encontradas no sangue, fígado e nos rins e menores quantidades do metabólito inativo (voriconazol-N-óxido) formado, quando se utilizou a formulação lipossomal podem ser atribuídos à capacidade dos lipossomas de alterar a farmacocinética e biodistribuição do voriconazol no organismo, principalmente devido à sua capacidade de proteger o fármaco do metabolismo acelerado. A avaliação da eficácia in vivo do voriconazol também foi realizada em modelo de candidíase sistêmica em animais imunossuprimidos, assim como parâmetros como a perda de peso, a carga fúngica no fígado e rins e alterações histológicas provocadas pela infecção e pelo tratamento. Como consequência da farmacocinética e da biodistribuição de voriconazol modificadas devido à encapsulação do voriconazol em lipossomas, a atividade antifúngica do fármaco foi potencializada, levando a maior especificidade e penetração tecidual. Em conclusão, a fim de fornecer regimes de dose adequados para o tratamento de infecções fúngicas sistêmicas, evitando obstáculos, como mecanismos de resistência e alta toxicidade, desenvolvemos uma plataforma terapêutica alternativa, capaz de prover a um tratamento seguro e eficaz.