Acute lymphoblastic leukemia, one of the most malignant cancer forms in children is commonly treated with the thiopurine 6-mercaptopurine (6-MP) in combination with a high dose of methotrexate (MTX). 6-Mercaptopurine is in the body metabolized by the enzyme thiopurine S-methyltransferase (TPMT). Polymorphic variants of TPMT express different catalytic activities, and for this reason the dosage of 6-MP needs to be individualized. In order to better optimize the treatment it is important to understand how mutations in TPMT affect its enzymatic activity. In this thesis we have investigated how the wild type and two variants of TPMT interact with different ligands using fluorescence and isothermal titration calorimetry. Experiments with MTX, ANS and furosemide resulted in a similar binding strength for the wild type and the variant TPMT*8, while the other variant TPMT*6 showed a slightly weaker binding. A binding affinity for polyglutamated MTX to TPMTwt was also determined which resulted in an almost twice as strong binding compared to MTX. Today’s methods to determine enzymatic activity are either based on radioactivity, time consuming or expensive. As an alternative the use of a spectrophotometric assay using 5-thio-2-nitrobenzoic acid (TNB) was investigated. The method showed positive results and could hopefully be adapted to plate readers in future experiments. Using 5.5’-dithiobis-(2-nitrobenzoic acid) (DTNB, also known as Ellman’s reagent) the amount of accessible thiol groups on the protein was estimated. This revealed a similar relationship between TPMTwt and TPMT*6, while the result for TPMT*8 was inconclusive.

Tez (Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, İç Hastalıkları Anabilim Dalı, 2008. / Kaynakça var.

Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine. / text

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Nöroloji Anabilim Dalı, 2006. / Bibliyografya var.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Fiziksel Tıp ve Rehabilitasyon Anabilim Dalı, 2003. / Bibliyografya var.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (doctoral)--Katholieke Universiteit te Nijmegen.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 205-218).

A área das Ciências Farmacêuticas busca constantemente por tratamentos mais eficientes, direcionados para alvos específicos, com diminuição da dose necessária e com a minimização dos efeitos adversos. Neste contexto, a área de Nanotecnologia Farmacêutica apresenta grande potencial de aplicabilidade, com resultados bastante promissores para o tratamento de diversas doenças. Os sistemas nanoestruturados têm sido avaliados para a incorporação de fármacos já utilizados em tratamentos administrados formas farmacêuticas convencionais que apresentam problemas farmacocinéticos ou farmacodinâmicos quando administrados. E, também, para a incorporação de novas moléculas com potencial para o tratamento de determinada doença. Neste trabalho de tese, nanocápsulas de núcleo lipídico versáteis contendo metotrexato na forma ácida e éster, bromelina, etanercept e infliximab foram desenvolvidas buscando contornar as limitações e aumentar a eficácia terapêutica desses fármacos. Inicialmente, as propriedades anti-inflamatórias de nanocápsulas de núcleo lipídico revestidas por micelas de polissorbato 80 contendo metotrexato encapsulado foram avaliadas em experimentos in vitro e in vivo, em células mononucleares obtidas a partir do líquido sinovial de pacientes com artrite reumatoide e em ratos Lewis com artrite induzida por adjuvante completo de Freund, respectivamente. As nanocápsulas de núcleo lipídico demonstraram serem altamente eficazes no controle da inflamação, sendo que os efeitos anti-inflamatórios in vivo foram alcançados em doses 75% menores que o metotrexato em solução. Na sequência, o tratamento in vitro da linhagem de células de carcinoma de mama humano, MCF-7, com nanocápsulas de núcleo lipídico multiparede funcionalizadas com bromelina demonstrou uma redução de 160 vezes na concentração necessária para obter o mesmo efeito quando comparada a uma solução de bromelina. A influência das pseudofases aniônicas e catiônicas no mecanismo de distribuição da indometacina, tacrolimus, aciclovir, metotrexato e éster etílico de metotrexato, foram avaliadas aplicando um algoritmo desenvolvido para nanocápsulas de núcleo lipídico. Verificou-se que somente a indometacina sofreu influência da presença de cargas, aumentando a afinidade pela fase dispersa das formulações. Formulações de nanocápsulas de núcleo lipídico multiparede contendo metotrexato na forma ácida e éster encapsulados e/ou funcionalizando a superfície das nanocápsulas foram desenvolvidas e testadas in vitro em linhagens de células tumorais (MCF-7) e em linhagens de células sadias (HaCaT). Essas formulações demonstraram atividade antiproliferativa maior para as MCF-7 (com redução em mais de 50% na viabilidade celular) em comparação com as soluções de metotrexato e éster etílico de metotrexato e esta atividade foi maior para as formulações em que as moléculas foram funcionalizadas na superfície das nanopartículas. A captação das nanopartículas pelas células também foi maior para as formulações funcionalizadas com metotrexato ou éster etílico de metotrexato em comparação com a formulação em que o éster de metotrexato está encapsulado. As três formulações contendo metotrexato na forma ácida ou éster não demonstraram ação antiproliferativa em linhagens de células sadias (HaCaT). Devido à baixa expressão de receptores de folato nessas células, não houve aumento da captação celular em comparação à formulação sem fármaco. Por último, foram desenvolvidas satisfatoriamente formulações de nanocápsulas de núcleo lipídico multiparede funcionalizadas com os anticorpos monoclonais infliximab e etanercept, e contendo éster etílico de metotrexato encapsulado, demonstrando que são adequadas para futuros estudos visando o tratamento da artrite reumatoide. Esse conjunto de resultados demonstra que as nanocápsulas de núcleo lipídico com funcionalização de superfície versátil, sejam revestidas com polissorbato 80 ou multiparede funcionalizadas são um sistema bastante promissor para a administração de fármacos de modo a aumentar sua especificidade e eficácia. / The Pharmaceutical Sciences field is constantly searching for more effective treatments, aiming specific targets, with dose reduction and minimization of side effects. In this context, the Pharmaceutical Nanotechnology field presents great applicability potential, with highly promising results for the treatment of several diseases. Nanostructured systems have been evaluated for the encapsulation of drugs approved for use in conventional pharmaceutical dosage forms that, however, exhibit pharmacokinetic or pharmacodynamics problems when administered, and for the encapsulation of novel molecules with potential to treat a determined disease. In the present thesis, versatile lipid-core nanocapsules containing methotrexate in the acid and ester forms, bromelain, etanercept and infliximab were developed, seeking to circumvent the limitations and increase the therapeutic efficacy of these drugs. Initially, the anti-inflammatory properties of methotrexate-loaded lipid-core nanocapsules coated with polysorbate 80 micelles were evaluated in in vitro and in vivo experiments, using mononuclear cells obtained from the synovial fluid of rheumatoid arthritis patients and Lewis rats with Freund complete adjuvant-induced arthritis. Lipid-core nanocapsules demonstrated to be highly effective in the control of inflammation, and the in vivo anti-inflammatory effects were reached in a dose 75% lower than the methotrexate in solution. In the sequence, the in vitro treatment of a human breast cancer cell line, MCF-7, with bromelina-functionalized multiple-wall lipid-core nanocapsules demonstrated a 160-fold reduction of the concentration required to obtain the same effect when compared with a bromelain solution. The influence of the anionic and cationic pseudo-phases in the distribution mechanism of indomethacin, tacrolimus, acyclovir, methotrexate and methotrexate ethyl ester was evaluated through an algorithm developed for lipid-core nanocapsules. It was verified that only indomethacin underwent influence in the presence of charge, increasing the affinity by the disperse phase of the formulations. Multiple-wall lipid-core nanocapsules formulations containing methotrexate in the acid and ester forms encapsulated and/or functionalizing the surface of the nanoparticles were developed and tested in vitro in tumour MCF-7 cells and in a healthy cell line (HaCaT). These formulations demonstrated higher anti-proliferative activity for the MCF-7 cells (reduction of over 50 % in cellular viability) in comparison with the methotrexate and methotrexate ethyl ester solutions and this activity was higher for the formulations in which the molecules were functionalized in the surface of the nanoparticles. A higher cellular uptake was observed for the formulations functionalized with methotrexate or methotrexate ethyl ester in comparison with the formulations in which the methotrexate ester is encapsulated. The three formulations containing methotrexate in the acid or ester form did not demonstrate anti-proliferative activity in non-tumour cell lines (HaCaT). Since these cells have a small expression of folate receptors, the uptake was not increased in comparison with the formulation without drug. Lastly, formulations of methotrexate ethyl ester-loaded multiwall lipid core nanocapsules functionalized with monoclonal antibodies infliximab and etanercept were successfully developed demonstrating suitability for future studies aiming the treatment of rheumatoid arthritis. These groups of results demonstrate that versatile lipid core nanocapsules, either coated with polysorbate 80 or multiwalled functionalized are a very promising system for the administration of drugs aiming their specificity and efficacy.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Methotrexate (MTX) is an antimetabolite drug analogue of folic acid with wide clinical application, used in high doses for the treatment of cancer and in low doses for the treatment of autoimmune diseases such as rheumatoid arthritis and psoriasis. Although effective, the MTX has several side effects. The oxidative stress seems to be involved with the toxicity caused by the MTX in various organs. Despite the effect on the imbalance of oxidative metabolism, the influence of polymorphisms of antioxidant enzymes on MTX toxicity is not well studied. In this context, the present study aimed to examine whether the Ala16Val polymorphism of the antioxidant enzyme superoxide dismutase manganese dependent (SOD2), which affects the efficiency of the detox enzyme, could have an effect on the cytotoxic response to MTX. For this, an in vitro study using peripheral blood mononuclear cells (PBMC) obtained from healthy donors harboring different genotypes of polymorphism Ala16Val-SOD (= genotypes AA, VV and AV) was performed. Once obtained, PBMCs were treated with MTX at concentrations of 10 and 100 μM for 24 and 72 hours and analyzed for the effect on viability, modulation of the oxidative metabolism-inflammatory and apoptotic. PBMC-AA which have a naturally SOD2 30 to 40% more efficient than the PBMC- VV, showed more resistance to treatment with MTX compared to PBMC-AV/VV assessed. As production levels of EROS and lipid peroxidation significantly increased in cells exposed to MTX, regardless of genotype, however, increased levels of protein carbonylation were observed only in PBMC-AV/VV. The PBMC-AA demonstrated decreased activity of SOD2 and the levels of glutathione peroxidase with PBMC-AA were higher. The levels of caspase-8 and -3 were increased in PBMC exposed to MTX, but the modulation of these genes, as well as Bax and Bcl-2 genes involved in apoptotic route, was genotype dependent. The MTX was able to raise the levels of inflammatory cytokines and decrease the level of anti-inflammatory cytokine IL-10, regardless of genotype. The results suggest that Ala16Val-SOD2 polymorphism is capable of modulating the cytotoxic response of PBMC to the MTX. / O Metotrexato (MTX) é um fármaco antimetabólito análogo do ácido fólico, com vasta aplicação clinica, utilizado em doses elevadas no tratamento de neoplasias e em baixas doses para o tratamento de doenças autoimunes, como a artrite reumatoide e a psoríase. Apesar de efetivo, o MTX possui diversos efeitos colaterais. Assim, o estresse oxidativo parece estar envolvido com a toxicidade causada pelo MTX a diversos órgãos. Apesar do efeito no desequilíbrio do metabolismo oxidativo, a influência de polimorfismos de enzimas antioxidantes sobre a toxicidade ao MTX ainda não é bem estudada. Nesse contexto, o presente estudo teve como objetivo analisar se o polimorfismo Ala16Val da enzima antioxidante superóxido dismutase dependente de manganês (SOD2), que afeta a eficiência detoxificadora da enzima, poderia ter efeito sobre a resposta citotóxica ao MTX. Para tanto, foi realizado um estudo in vitro utilizando células mononucleares do sangue periférico (CMSP), obtidas de doadores saudáveis portadores de diferentes genótipos do polimorfismo Ala16Val-SOD (genótipos = AA, VV e AV). Uma vez obtidas, as CMSPs foram tratadas com MTX nas concentrações de 10 e 100 μM por 24 e 72 horas, sendo posteriormente analisado o efeito sobre a viabilidade, modulação do metabolismo oxidativo-inflamatório e apoptótico. As CMSP-AA, que naturalmente apresentam uma SOD2 30 a 40% mais eficiente do que as CMSP-VV, apresentaram maior resistência ao tratamento com MTX em relação às CMSP-AV/VV. Quanto aos níveis de produção de EROS (espécies reativas de oxigênio) e lipoperoxidação, houve aumento significativo nas células expostas ao MTX independente do genótipo, entretanto, o aumento dos níveis de carbonilação de proteínas foi observado apenas em CMSP-AV/VV. Nas CMSP-AA houve diminuição da atividade da SOD2. Já quanto aos níveis de Glutationa Peroxidase, as CMSP-AA apresentaram uma elevação mais intensa. Os níveis das caspases 3 e 8 foram aumentados nas CMSP expostas ao MTX, mas a modulação desses genes, assim como do Bax e Bcl-2 (genes envolvidos rota apoptótica), foi genótipo-dependente. O MTX foi capaz de elevar os níveis das citocinas inflamatórias e diminuir o nível da citocina antiinflamatória IL-10, independente do genótipo. Os resultados sugerem que o polimorfismo Ala16Val-SOD2 é capaz de modular a resposta citotóxica de CMSP ao MTX.