The water solubility of transition metals constrain their use in the bioinorganic chemistry of organisms. Iron(III) is one of the most sought-after metals by organisms; however, it has poor solubility in water at physiological pH. Prior to the great oxidation event (GOE) 2.4 billion years ago, iron was more bioavailable in the 2+ oxidation state. This higher water solubility sustained a greater supply of iron to organisms that required it. Since the GOE and the oxidation of Fe(II) to Fe(III), organisms have been evolving methods to sequester iron from the environment. One method to obtain iron is through the use of low molecular weight molecules called siderophores which solubilize Fe(III) from their environment through chelation. The strongest siderophore to date is enterobactin (ent) which forms a Fe-ent complex with an association constant of 1049. While iron is utilized by most organisms, titanium is also a bioactive metal. Titanium is second to iron in abundance of transition metals in the Earth’s crust and is becoming increasingly relevant in medicine, technology, and commercial goods. Titanium(IV) (the most common oxidation state of titanium) also has a larger charge to size ratio than Fe(III) and may outcompete iron for binding. The already high abundance and increasing usage of titanium in the developed world leads to possible undocumented interactions between titanium and siderophores. In an attempt to address the natural selection of the chemical elements during bioinorganic evolution, the solution interactions of Ti(III), Ti(IV), Fe(II), and Zn(II) with sulfide in a solution mimicking the sulfidic early ocean were explored. Anaerobic metal solutions were added to solutions with ocean-mimicking salts, acetic acid buffer, and increasing concentrations of sodium sulfide. After equilibration, the soluble metal ion concentrations were measured using ICP-OES. Catechol ligands are important bioinorganic chelators. The synthesis and crystallization of the disodium, dilithium, and diammonium salts of the model catechol ligand 4,5-dihydroxy-1,3-benzenedisulfonate (tiron) are described. The dilithium and diammonium salts were synthesized from the disodium tiron salt by cation metathesis. The crystals were obtained by solvent evaporation in either alcohols or water. Crystal structures were acquired, solved, and analyzed revealing inter- and intramolecular interactions in the dication tiron crystal structures. The synthesis of the siderophore enterobactin (ent) was improved upon from published methods to increase purity. Following the synthesis of ent, spectrophotometric techniques were used to analyze Ti-ent complex stability. Spectrophotometric titrations were carried out from p[H] 2-12 revealed that the fully catecholate bound [Ti(ent)]2- complex was the only species in the p[H] range 2-9. Above a p[H] value of 9 the trilactone backbone apparently hydrolyzed. This result indicates that protons cannot outcompete Ti(IV) binding at low p[H] and hydroxide cannot outcompete ent for binding to Ti(IV) at high p[H]. Using various Ti(IV)-ligand complexes, the log b110 for Ti-ent was probed at pH 6. The complex [Ti(DFOB)]+ (log b110 = 38.1) with 1,000 fold excess desferrioxamine B (DFOB) was unable to compete with ent and every addition of ent lead to the formation of [Ti(ent)]2- yielding a lower limit log b110 of 52.9. The catecholate ligand tiron was used in a 4,000:1 ratio of tiron:Ti(IV) forming [Ti(tiron)3]8- (log b130 = 57.6) to attempt to compete with ent. This yielded a lower limit log b110 for [Ti(ent)]2- of 61.2. Kinetic studies of titanium sequestration from titanium containing species was also investigated. Using Ti(IV) complexes such as [Ti(DFOB)]+, [Ti(citrate)3]8-, and the titanium complex of human serum transferrin (Ti2HsTf) as the Ti(IV) source, the rate of titanium sequestration by ent was probed. Taken together, these findings indicate that ent is a strong binder of Ti(IV); however, it binds relatively slowly. Strong binding ligands are able to coordinate and dissolve metal ions from the surface of metal oxides. Titanium mostly exists in the solid oxide form in the Earth’s crust, thus solubilization of these oxides may be important for Ti(IV) incorporation into an organism. To determine the effects of ent in solution at a TiO2 interface, 50 mM ent was added to a series of suspensions of TiO2 at pH 5 ± 0.5 with different loadings for 176 h. The [Ti]max for all anatase trials averaged to be approximately 20 – 25 mM indicating 40 – 50% ent efficacy for TiO2 dissolution. This value was compared to previously reported TiO2 dissolution by 2 mM DFOB at pH 2 as well as dissolution of 50 mM and 150 mM DFOB and tiron, respectively, at pH 5 ± 0.5. Surface interactions between ent and anatase were probed using ATR-IR. One extension of this work related to the avid Fe(III) ligand deferasirox (DSX). DSX is an orally administered chelator for Fe(III) overload. A Ti(IV)-DSX complex may be useful as an anticancer drug. The solution chemistry of DSX with Ti(IV) was characterized utilizing spectrophotometric and mass spectrometric techniques. A series of titrations with Ti(IV) and DSX from pH 6.7-11.2 revealed two distinct species. Additions of [Ti(HDSX)(DSX)]- to bovine serum albumin (BSA) indicated that the [Ti(HDSX)(DSX)]- complex binds to BSA. This transfer is possibly an important step in the efficacy of Ti(IV) anticancer drugs and may relate to the possible mechanism for Ti(IV) delivery into cancerous cells. / Chemistry

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T18:53:57Z : No. of bitstreams: 1 belinijunior_e_me_sjrp.pdf: 995631 bytes, checksum: d866ab976032b462435b7ca7899faf6d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A causa principal da doença falciforme (DF) é uma mutação pontual no gene β globina que codifica uma hemoglobina (Hb) com características diferentes da Hb normal, denominada de Hb S. Sua fisiopatologia multifacetada envolve múltiplas alterações nos eritrócitos falcêmicos, episódios vaso-oclusivos, hemólise, ativação de mediadores inflamatórios, disfunção das células endoteliais e estresse oxidativo. Com isso, há uma diminuição do fluxo sanguíneo e obstrução da microcirculação acarretando aos portadores, anemia, crises de dor e insuficiência de múltiplos órgãos. Visto que o excesso de espécies reativas de oxigênio é citotóxico modificando vários mecanismos celulares e, consequentemente, ocasionando danos aos órgãos, pretendeu-se avaliar a capacidade antioxidante e o estresse oxidativo em DF, correlacionando com os genótipos da DF, haplótipos do cluster β globina e o uso de medicação específica. Em um estudo longitudinal, foram avaliadas, para o Tempo 1 (T1) de análise, 69 amostras de sangue e, para o Tempo 2 (T2), 55 amostras de DF do Hemocentro de São José do Rio Preto (SJRP) e do Hemocentro da Santa Casa de São Paulo (SP). As amostras foram submetidas a testes eletroforéticos, cromatográficos e moleculares para a identificação dos genótipos da DF e haplótipos do cluster , bem como dosagens bioquímicas para determinação das espécies reativas ao ácido tiobarbitúrico (TBARS) e da capacidade antioxidante em equivalência ao Trolox (TEAC). Por meio da caracterização genotípica da DF, encontramos um maior número de Hb SS, tanto em SP quanto em SJRP. Houve maior frequência da interação Hb S/Beta talassemia em SJRP (10,9%) com as mutações características de países mediterrâneos como a CD39 e IVS-I-110. O haplótipo Bantu foi mais frequente nos dois grupos (60,6%); o haplótipo II esteve associado à mutação... / The main cause of sickle cell disease (SCD) is a single β-globin gene mutation that encodes a hemoglobin (Hb) with different characteristics from normal Hb, called Hb S. This disease is characterized by presenting a multifaceted pathophysiology which involves multiple changes in sickle erythrocytes, vaso-occlusive episodes, hemolysis, inflammatory mediators activation, oxidative stress and endothelical cells dysfunction. Thereby, there is a decrease in blood flow and obstruction of the microcirculation leading the patients to anemia, pain crises and failure of multiple organs. Since the excess of reactive oxygen species is cytotoxic, modifying various cellular mechanisms, and thus, causing damage to organs, the aim of this work was to assess the antioxidant capacity and oxidative stress in SCD, correlating with SCD genotypes, cluster β-globin haplotypes and specific medication. In a longitudinal study were evaluated for the T1: 69 samples of peripheral blood, and for the T2: 55 samples of the SCD patients from Blood Center of the São José do Rio Preto (SJRP) and the Blood Center of the Santa Casa Medical School of São Paulo (SP). We performed electrophoretics, chromatographic and molecular assays for SCD genotypes and cluster β-globin identification, such as biochemical measurements for the determination of thiobarbituric acid reactive species (TBARS) and Trolox equivalence antioxidant capacity (TEAC). Through SCD genetic characterization, we found a greater number of Hb SS in both SP and in SJRP. There was a higher frequency of interaction Hb S/Beta-thalassemia in SJRP (10.9%) with typical mutation of Mediterranean countries such as CD39 and IVS-I -110. The Bantu haplotype was more frequent in both groups (60.6%); the haplotype II was associated with CD39 mutation and the haplotype I with the IVS-I-110. We found a patient with Cameroon haplotype, rare in the Brasilian... (Complete abstract click electronic access below)

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul / The oomycete Pythium insidiosum, classified in the kingdom Straminipila, is the agent of pythiosis, a chronic disease that can be hard to treat and life-threatening. The predisposing factors in mammals are unknown, but the iron overload is suspected to act directly or indirectly in the development of the infection. This aim of this study was: to evaluate the iron metabolism in rabbits with experimental pythiosis; to verify the antifungal activity and the mechanism of action in vitro of the iron chelator deferasirox against P. insidiosum isolates and to compared the treatment and the mechanism of action of the drug with immunotherapy in vivo; to verify the antifungal activity of micafungin, alone and in combination with deferasirox, in vitro and in vivo; to develop an experimental model of pythiosis using the fruit-fly Drosophila melanogaster with iron overload. The drug susceptibility tests included 17 P. insidiosum clinical isolates, and were performed according to the CLSI M38-A2 guidelines. The mechanism of action of deferasirox was evaluated by the XTT and DiBAC assays. For the in vivo tests, five P. insidiosum infected rabbits were included per group, as follows: infected treated with placebo, treated with immunotherapy, treated with deferasirox, treated with immunotherapy and deferasirox, treated with micafungin and treated with micafungin and deferasirox. Five uninfected rabbits were used as controls. Hematological and biochemical analyses were performed at days 0 25, 50 and 75 post-infection. The quantification of iron in the hepatocytes was performed after 50 days of treatment (day 75). The mechanism of action of deferasirox was evaluated by the quantification of the catabolic enzyme adenosine deaminase and by the histology of the subcutaneous lesions. The results show that P. insidiosum is poorly susceptible to deferasirox in vitro, with minimum inhibition concentrations (MICs) ranging from 12.5 to 50 μg/ml and minimum fungicidal concentrations between 50 and 100 μg/ml. No activity against P. insidiosum was observed for micafungin in vitro (MICs > 128 μg/ml) and in rabbits with pythiosis. Notwithstanding, synergism was observed in 88% of the isolates when micafungin was combined with deferasirox, and the lesions were decreased in comparison to the other groups (P = 0.06). In general, the rabbits showed microcytic hypochromic anemia with depleted iron stores, which was less prominent in the groups treated with immunotherapy or deferasirox. Despite the iron chelator has failed to thwart the infection, deferasirox showed toxicity against P. insidiosum hyphae in vitro and an immunomodulatory action was observed in the lesions, in a similar pattern to that seen in immunotherapy-treated rabbits. However, the use of deferasirox favored the dissemination of the disease to the lung by unrevealed mechanisms. D. melanogaster flies were resistant to P. insidiosum infection, independently of the iron overload. Conversely, toll-deficient D. melanogaster flies were susceptible to the infection, highlighting the importance of these receptors in pythiosis. In conclusion, rabbits with pythiosis showed iron deficiency anemia and, despite the chelating effect of deferasirox, the administration of the drug should be evaluated with care because of the dissemination of the disease observed in some of the treated animals. / O oomiceto Pythium insidiosum, classificado no Reino Straminipila, é o agente etiológico da pitiose, uma doença crônica, de difícil tratamento e que pode levar à morte. Os fatores predisponentes da doença em mamíferos não são conhecidos, mas suspeita-se que a sobrecarga de ferro exerça um papel direto ou indireto no desenvolvimento da infecção. Este estudo teve por objetivos: avaliar o metabolismo do ferro em coelhos com pitiose experimental; verificar a atividade antifúngica e o mecanismo de ação in vitro do quelante de ferro deferasirox frente a isolados de P. insidiosum e comparar o tratamento e o mecanismo de ação do fármaco com a imunoterapia in vivo; verificar a atividade antifúngica da micafungina, sozinha e em combinação com deferasirox, in vitro e in vivo; desenvolver um modelo experimental de pitiose utilizando-se a mosca-das-frutas Drosophila melanogaster com sobrecarga de ferro. Os testes de susceptibilidade aos fármacos foram realizados com 17 isolados clínicos de P. insidiosum, utilizando-se o protocolo de microdiluição M38-A2 do CLSI. O mecanismo de ação in vitro do deferasirox foi avaliado através da técnica de XTT e DiBAC. Para os testes in vivo, foram utilizados cinco coelhos infectados por P. insdiosum por grupo, conforme o delineamento a seguir: infectados tratados com placebo, tratados com imunoterápico, tratados com deferasirox, tratados com imunoterápico e deferasirox, tratados com micafungina e tratados com micafungina e deferasirox. Cinco coelhos não infectados foram utilizados como controle negativo. Realizaram-se avaliações hematológicas e bioquímicas, incluindo os níveis séricos de ferro, transferrina e ferritina nos dias 0, 25, 50 e 75 pós-infecção. A quantificação do ferro depositado nos hepatócitos foi realizada após 50 dias de tratamento (dia 75). O mecanismo de ação do deferasirox in vivo foi avaliado através da mensuração da atividade da enzima catabólica adenosina deaminase e da histologia das lesões subcutâneas. Os resultados mostram que P. insidiosum demonstrou ser pouco susceptível ao deferasirox in vitro, com concentrações inibitórias mínimas (CIMs) que variaram de 12,5 a 50 μg/ml e concentrações fungicidas mínimas entre 50 e 100 μg/ml. A micafungina não apresentou atividade antifúngica contra P. insidiosum in vitro (CIMs > 128 μg/ml) e nos coelhos com pitiose. Contudo, sinergismo foi observado em 88% dos isolados quando a micafungina foi associada ao deferasirox, e as lesões dos coelhos estavam diminuídas em relação aos demais grupos (P = 0,06). No geral, os coelhos infectados apresentaram anemia microcítica hipocrômica com depleção das reservas corporais de ferro, que foi menos acentuada nos grupos tratados com imunoterápico ou quelante de ferro. Apesar do deferasirox não ter sido capaz de conter a infecção, o mesmo teve ação tóxica sobre as hifas de P. insidiosum in vitro e teve ação imunomodulatória sobre as lesões, em um padrão similar ao observado nos coelhos tratados com imunoterapia. Contudo, o uso do deferasirox favoreceu a disseminação pulmonar da doença por mecanismos não compreendidos. Moscas D. melanogaster foram resistentes à infecção por P. insidiosum, independente da sobrecarga de ferro. Entretanto, D. melanogaster deficientes de receptores do tipo Toll foram susceptíveis à infecção, evidenciando a importância desses receptores na pitiose. Em conclusão, coelhos com pitiose apresentaram anemia por deficiência de ferro e, apesar do efeito quelante do deferasirox, a utilização do mesmo deve ser avaliada com cuidado devido à disseminação da doença em alguns animais.

Made available in DSpace on 2015-07-22T20:49:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-22. Added 1 bitstream(s) on 2015-08-11T03:26:02Z : No. of bitstreams: 1 Publico-12515.pdf: 978359 bytes, checksum: 3be0798a82267f20204fe25244cbed7d (MD5) / Objetivo: Avaliar a evidência de eficácia e segurança do deferasirox para o tratamento da sobrecarga de ferro relacionada à betatalassemia, doença falciforme e síndrome mielodisplásica, em comparação a outros quelantes de ferro disponíveis no Brasil (desferroxamina e deferiprona). Método: Para síntese da evidência foi realizada busca por relatórios de agências de avaliação de tecnologias em saúde e por revisões sistemáticas no Medline (março 2009). Adicionalmente, foi feita busca por ensaios clínicos para atualização da informação disponível. Das referências identificadas, foi selecionado o trabalho de McLeod (2009), por ser uma revisão sistemática e estudo de avaliação econômica elaborado por uma agência de avaliação de tecnologias em saúde de alta confiabilidade. Considerando o contexto econômico local e a disponibilidade dos medicamentos e insumos laboratoriais, a evidência foi adaptada e avaliada criticamente. Resultados: A evidência encontrada é baseada em três ensaios clínicos, de baixa qualidade metodológica, que avaliaram o uso do deferasirox em relação à desferroxamina. Não foram localizados estudos comparativos entre o deferasirox e a deferiprona. Nos ensaios clínicos identificados, a concentração hepática de ferro (exame não disponível no Brasil) foi considerada como desfecho primário, apesar de na prática clínica a quantidade de ferro ser monitorada pela concentração de ferritina sérica. As mudanças na ferritina sérica revelam-se mais favoráveis em pacientes com betatalassemia e doença falciforme (maior prevalência no Brasil) que receberam desferroxamina, do que aqueles que receberam deferasirox. As informações econômicas levantadas indicam que o deferasirox, em comparação a desferroxamina pode ser uma opção custo-efetiva. Conclusões: Como o deferasirox foi introduzido recentemente no mercado brasileiro e é considerado alternativa de tratamento no sistema público de saúde, informações de farmacovigilância precisam ser monitoradas, principalmente no que se refere ao risco de insuficiência renal, citopenias (agranulocitose e trombocitopenia), além de distúrbios gastrointestinais, hepáticos, renais e sanguíneos. / Objectives: to evaluate the evidence on efficacy and safety regarding deferasirox for treatment of iron overload relating to beta-thalassemia, sickle cell disease and myelodysplastic syndrome, in comparison with other iron chelators available in Brazil (deferoxamine and deferiprone). Methods: to produce a synthesis of the evidence, a search was conducted using reports from HTA agencies and systematic reviews in Medline (March 2009). Additionally, a search for clinical trials was conducted to update the information available. Among the references identified, the study by McLeod (2009) was selected because this was a systematic review and economic evaluation produced by a highly trustworthy HTA agency. Considering the local economic context and the availability of medications and laboratory supplies, the evidence was adapted and critically evaluated. Results: the evidence found was based on three clinical trials of low methodological quality that evaluated the use of deferasirox in relation to deferoxamine. No comparative studies between deferasirox and deferiprone were found. In the clinical trials identified, hepatic iron concentration (a test unavailable in Brazil) was taken to be the primary outcome, although in clinical practice, the iron levels were monitored by means of the serum ferritin concentration. The changes in serum ferritin concentration were more favorable among patients with beta thalassemia and sickle cell disease (highest prevalence in Brazil) who received deferoxamine than among those who received deferasirox. Previous economic evaluation suggests that deferasirox may be a cost-effective strategy. Conclusions: since deferasirox was only recently introduced onto the Brazilian market and is considered to be an alternative for treatment within the public healthcare system, drug surveillance information is required, particularly regarding the risks of kidney failure, cytopenia (agranulocytosis and thrombocytopenia) and gastrointestinal, hepatic, renal and blood disorders. / TEDE / BV UNIFESP: Teses e dissertações

Orientador: Claudia Regina Bonini Domingos / Banca: Dorotéia Rossi Silva Souza / Banca: Ivan de Lucena Angulo / Resumo: A causa principal da doença falciforme (DF) é uma mutação pontual no gene β globina que codifica uma hemoglobina (Hb) com características diferentes da Hb normal, denominada de Hb S. Sua fisiopatologia multifacetada envolve múltiplas alterações nos eritrócitos falcêmicos, episódios vaso-oclusivos, hemólise, ativação de mediadores inflamatórios, disfunção das células endoteliais e estresse oxidativo. Com isso, há uma diminuição do fluxo sanguíneo e obstrução da microcirculação acarretando aos portadores, anemia, crises de dor e insuficiência de múltiplos órgãos. Visto que o excesso de espécies reativas de oxigênio é citotóxico modificando vários mecanismos celulares e, consequentemente, ocasionando danos aos órgãos, pretendeu-se avaliar a capacidade antioxidante e o estresse oxidativo em DF, correlacionando com os genótipos da DF, haplótipos do cluster β globina e o uso de medicação específica. Em um estudo longitudinal, foram avaliadas, para o Tempo 1 (T1) de análise, 69 amostras de sangue e, para o Tempo 2 (T2), 55 amostras de DF do Hemocentro de São José do Rio Preto (SJRP) e do Hemocentro da Santa Casa de São Paulo (SP). As amostras foram submetidas a testes eletroforéticos, cromatográficos e moleculares para a identificação dos genótipos da DF e haplótipos do cluster , bem como dosagens bioquímicas para determinação das espécies reativas ao ácido tiobarbitúrico (TBARS) e da capacidade antioxidante em equivalência ao Trolox (TEAC). Por meio da caracterização genotípica da DF, encontramos um maior número de Hb SS, tanto em SP quanto em SJRP. Houve maior frequência da interação Hb S/Beta talassemia em SJRP (10,9%) com as mutações características de países mediterrâneos como a CD39 e IVS-I-110. O haplótipo Bantu foi mais frequente nos dois grupos (60,6%); o haplótipo II esteve associado à mutação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The main cause of sickle cell disease (SCD) is a single β-globin gene mutation that encodes a hemoglobin (Hb) with different characteristics from normal Hb, called Hb S. This disease is characterized by presenting a multifaceted pathophysiology which involves multiple changes in sickle erythrocytes, vaso-occlusive episodes, hemolysis, inflammatory mediators activation, oxidative stress and endothelical cells dysfunction. Thereby, there is a decrease in blood flow and obstruction of the microcirculation leading the patients to anemia, pain crises and failure of multiple organs. Since the excess of reactive oxygen species is cytotoxic, modifying various cellular mechanisms, and thus, causing damage to organs, the aim of this work was to assess the antioxidant capacity and oxidative stress in SCD, correlating with SCD genotypes, cluster β-globin haplotypes and specific medication. In a longitudinal study were evaluated for the T1: 69 samples of peripheral blood, and for the T2: 55 samples of the SCD patients from Blood Center of the São José do Rio Preto (SJRP) and the Blood Center of the Santa Casa Medical School of São Paulo (SP). We performed electrophoretics, chromatographic and molecular assays for SCD genotypes and cluster β-globin identification, such as biochemical measurements for the determination of thiobarbituric acid reactive species (TBARS) and Trolox equivalence antioxidant capacity (TEAC). Through SCD genetic characterization, we found a greater number of Hb SS in both SP and in SJRP. There was a higher frequency of interaction Hb S/Beta-thalassemia in SJRP (10.9%) with typical mutation of Mediterranean countries such as CD39 and IVS-I -110. The Bantu haplotype was more frequent in both groups (60.6%); the haplotype II was associated with CD39 mutation and the haplotype I with the IVS-I-110. We found a patient with Cameroon haplotype, rare in the Brasilian... (Complete abstract click electronic access below) / Mestre