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Caracterização bioquímica e atividade citotóxica in vitro e antitumoral in vivo de proteínas do látex de Calotropis procera / Biochemical characterization and cytotoxicity in vitro and antitumor activity in vivo of protein from the of latex Calotropis proceraOliveira, Jefferson Soares de January 2011 (has links)
OLIVEIRA, Jefferson Soares de. Caracterização bioquímica e atividade citotóxica in vitro e antitumoral in vivo de proteínas do látex de Calotropis procera. 2011. 149 f. : Tese (Doutorado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2011. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-20T14:23:08Z
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Previous issue date: 2011 / Latex of Calotropis procera was described as a source of pharmacologically active proteins such as anti-inflammatory and analgesic activities. This study evaluated the cytotoxic activity in vitro of proteins (LP) recovered from the latex of the medicinal plant C. procera against human cancer cells and the in vivo growth inhibition of Sarcoma 180. LP exhibited significant cytotoxicity for cell lines with IC50 values ranging from 0.11 to 1.36 µg/ml for tested cell lines (HL-60, SF295, HCT-8 and MDA-MB-435). There were no visible effects on the viability or morphology of healthy mononuclear cells exposed to PL (10 µg / ml) for 72 h, showing that PL was selective for malignant cells. Fractionation of PL by ion exchange chromatography (pH 5.0) gave rise to three new protein fractions (PI, PII and PIII) and almost all cytotoxicity present in PL was retained in fraction PI. The cytotoxic effects of PL and PI were diminished when pre-treated with pronase or 2-mercaptoethanol, reinforcing the protein nature of active molecules. PI was absent on cysteine protease activity, indicating that this enzyme abundantly found in PL is not involved in cytotoxicity. Mechanistic studies of LP cytotoxicity using HL-60 cells revealed that PL induces apoptosis probably due to changes in DNA topology since PL interfered in the activity of topoisomerase I. The cytotoxic activity present in PI seems to be performed by the synergic action of different proteins. This hypothesis is suggested since PI subjected to gel filtration chromatography produced distinct protein peaks that shared cytotoxic activity, although with lower extent than PI. Studies on growth inhibition of Sarcoma 180 showed that animals treated with PL by oral (10 or 20 mg/kg) or intraperitoneal (2 or 5 mg/kg) rout reduced tumor growth significantly (up 51.83%, po) and increased life span of transplanted animals for up to four days. The inhibitory activity of tumor growth was lost when the LP was subjected to proteolysis, acidic treatment or collected in iodoacetamide. On the other hand, LP maintained its in vivo activity after heat treatment, suggesting that thermo stable proteins are involved in the suppression of tumor growth. Biochemical parameters such as the enzymatic activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the content of urea in serum were not affected in animals treated with LP. Treatment of animals with LP induced increasing of leukocyte numbers and protected from leukopenia induced by 5-FU administration. In addition, no significant changes in the histopathology of liver of animals treated with oral LP were seen. In vivo antitumor activity was retained in the PII and this activity was observed even when animals received a single dose of PII. It seems that the in vivo action of latex proteins is related to an immunestimulant event and not to the cytotoxic action of protein on cells Sarcoma 180. PII-3, recovered after PII fractionation on ion exchange column at pH 6.0, retained the tumor growth inhibition activity found in PII. PII-3 was shown to possess cysteine proteinase and papain inhibitor activities; however is not completely clear weather this molecules are involved in the antitumor activity. This study confirms the pharmacological potential of latex proteins from C. procera to control the development of tumor cells. / O látex de Calotropis procera foi descrito como uma fonte de proteínas farmacologicamente ativas como atividade antiinflamatória e analgésica. O presente trabalho avaliou a atividade citotóxica in vitro das proteínas (PL) recuperadas do látex da planta medicinal C. procera contra células de câncer humano e a inibição do crescimento do Sarcoma 180 transplantado em camundongos. PL apresentou significante citotoxicidade para as linhagens celulares com valores de IC50 variando entre 0,11 a 1,36 µg/ml para as linhagens celulares testadas (HL-60, SF295, HCT-8 e MDA-MB-435). Não foram observados efeitos visíveis sobre a viabilidade ou a morfologia de células mononucleares saudáveis expostas a PL (10 µg / ml) por 72 h, mostrando que PL apresentou seletividade para células tumorais. O fracionamento de PL por cromatografia de troca iônica (pH 5,0) deu origem a três novas frações (PI, PII e PIII) e quase toda citotoxicidade presente em PL ficou retida na fração PI. Os efeitos citotóxicos de PL e PI foram diminuídos quando previamente tratados com pronase, ou 2-mercaptoetanol, sugerindo uma natureza protéica de moléculas ativas. PI não apresentou atividade de proteinase cisteínica, indicando que esta enzima, encontrada em abundância em PL, não está envolvida na citotoxicidade. Estudos do mecanismo da ação citotóxica de PL utilizando células HL-60 revelou que PL induz apoptose celular provavelmente devido a alterações na topologia de DNA, já que PL interferiu na atividade de topoisomerase I. A atividade citotóxica presente em PI parece ser desempenhada pela ação combinada de diferentes proteínas uma vez que PI submetida à cromatografia de filtração em gel gerou picos protéicos distintos que compartilharam atividade citotóxica, embora com menor potência que PI. Estudo de inibição do crescimento do Sarcoma 180 revelou que animais tratados com PL por via oral (10 or 20 mg/kg) ou intraperitoneal (2 or 5 mg/kg) reduziram de modo significativo o crescimento do tumor (em até 51,83%; v.o.) e prolongou o tempo de sobrevivência dos animais transplantados por até quatro dias. A atividade inibitória do crescimento do tumor foi perdida quando a fração PL foi submetida à proteólise, tratamento ácido ou com iodoacetamida. No entanto, PL conservou a sua atividade in vivo após o tratamento térmico, sugerindo que proteínas termoestáveis estão envolvidas na supressão do crescimento tumoral. Os parâmetros bioquímicos, como a atividade enzimática da aspartato aminotransferase (AST) e alanina aminotransferase (ALT) e o teor de uréia no soro, não foram afetados nos animais tratados com PL. PL induziu aumento no número de leucócitos de animais tratados e ainda eliminou completamente a leucopenia induzida pela administração do 5-FU. Em adição, não foram observadas mudanças na histopatologia do fígado de animais tratados com PL por via oral. Atividade antitumoral in vivo ficou retida no PII e esta atividade foi observada mesmo quando animais transplantados receberam uma única dose de PII sugerindo que a ação in vivo de proteínas do látex está relacionada a um evento imunoestimunate de proteínas e não à ação citotóxica sobre as células do Sarcoma 180. PII-3, obtido após fracionamento de PII em coluna de troca iônica em pH 6,0 reteve a atividade de inibição do crescimento tumoral de PII. Esta fração possui atividade de proteinase cisteínica e atividade de inibidor de papaína, porém não é completamente claro o envolvimento dessas moléculas na atividade in vivo. Este estudo confirma o potencial farmacológico das proteínas do látex de C. procera para controlar o desenvolvimento de células tumorais.
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Adsorção de 5 fluorouracil e 5 clorouracil sobre HOPG : um estudo da importância do estado de protonação via microscopia de varredura por tunelamentoPassos, Renata Almeida 30 June 2011 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work explored the adsorption of organic molecules to achieve change on surfaces. In this context it was proposed, the adsorption of organic molecules of halogenated derivatives of uracil, 5 Fluorouracil and 5 Chlorouracil, in monocrystalline substrate of HOPG (highly oriented pyrolytic graphite), sizzling was the technique used for construction of self-assembled monolayers (SAM). The atomic resolution of the STM (Scanning Tunneling Microscope) images of HOPG substrate prove its hexagonal structure and that their interatomic distance is 0.246 nm. While in the molecular resolution images of 5 Fluorouracil and of 5 Chlorouracil adsorbed on the HOPG substrate, is presented evidence that after in the adsorption some changes occur in the intermolecular distances in the structures and the positioning of these molecular films formed on the substrate because the molecular films behaved in a horizontal position and were stabilized laterally by hydrogen bonds when they are protonated, but when we performed the same procedure of adsorption for these organic molecules, only varying the pH of the environment, the change caused the deprotonation of the molecules, thus allowing one to simulate electrochemical environment and being able to extract information about the pKa of adsorbates using UV-Vis spectroscopy, and was also observed that these deprotonated molecular films behaved in a vertical position on the substrate. / Neste trabalho foi explorada a adsorção de moléculas orgânicas para realizar modificação em superfícies. Neste contexto foi proposta a adsorção das moléculas orgânicas de derivados halogenados do uracil, o 5 Fluorouracil e o 5 Clorouracil no, substrato monocristalino do HOPG (Grafite Pirolítico Altamente Orientado do inglês, Highly Oriented Pyrolitic Graphite), sendo usada a técnica de sizzling para construção de monocamadas automontadas (SAM Self-Assembled Monolayers). As imagens de STM (Scanning Tunneling Microscopy) de resolução atômica do substrato de HOPG comprovam sua estrutura hexagonal e sua distância interatômica que é de 0,246 nm. Já nas imagens de resolução molecular do 5 Fluorouracil e do 5 Clorouracil adsorvido no substrato do HOPG, são apresentadas evidências de que após a adsorção ocorrem mudanças nas distâncias intermoleculares, nas estruturas e no posicionamento desses filmes moleculares formados no substrato, pois os filmes moleculares se comportaram em um posicionamento horizontal e são estabilizados lateralmente por ligações de hidrogênio quando estão protonadas, mas quando foi realizado o mesmo procedimento de adsorção para essas moléculas orgânicas, só que variando o pH do meio, esta variação provocou a deprotonação das moléculas, assim permitindo simular um ambiente eletroquímico e sendo possível extrair informação sobre o pKa dos adsorbatos usando a espectroscopia UV-Vis, e foi observado também que estes filmes moleculares deprotonados se comportaram em um posicionamento vertical no substrato.
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Estudo do papel do Ãxido nÃtrico nas mucosites oral e intestinal induzidas por 5-fluorouracil e metotrexato e efeito da glutamina e alanil-glutamina na mucosite oral induzida por 5-fluorouracil / Study of the paper of nitric oxide in the induced mucosites verbal and intestinal for 5-fluorouracil and metotrexato and effect of the glutamina and alanil-glutamina in the induced verbal mucosite for 5-fluorouracilRenata Ferreira de Carvalho LeitÃo 20 April 2007 (has links)
Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A mucosite induzida por quimioterÃpicos à um efeito colateral importante e limitante da terapia do cÃncer, cuja fisiopatologia nÃo à completamente compreendida. O presente estudo visa investigar o papel do Ãxido nÃtrico (NO) na patogÃnese das mucosites oral e intestinal induzidas por 5-fluorouracil (5-FU) e metotrexato (MTX) e os efeitos da glutamina (GLU) e alanil-glutamina (AL-GLU) na mucosite oral induzida por 5-FU. A mucosite oral foi induzida por duas administraÃÃes intraperitoneais (i.p.) de 5-FU nos 1 e 2 dias (60 e 40 mg/kg respectivamente), em hamsters. Os animais foram tratados subcutaneamente (s.c.) com os inibidores da Ãxido nÃtrico sintase (NOS), N-(3-(Aminomethyl)benzyl)acetamidina (1400W; 1 mg/kg), aminoguanidina (AG; 5 or 10 mg/kg), Nφ-Nitro-L-Arginina Methyl Ester (L-NAME; 5, 10 or 20 mg/kg) ou salina (0,4 ml), uma hora antes do 5-FU e, diariamente, atà o sacrifÃcio, no 10 dia. Em outro ciclo de experimentos, os animais receberam salina, suspensÃo de GLU ou de AL-GLU (100 mM) uma hora antes do 5-FU e, diariamente, atà o sacrifÃcio, nos 10 e 14 dias. A mucosite intestinal foi induzida pela administraÃÃo de MTX (2,5 mg/kg; s.c.) nos primeiros trÃs dias de experimentos, em ratos Wistar. Os animais foram tratados com AG (10 mg/Kg; i.p.), ou L-NAME (20 mg/Kg; i.p.), uma hora antes do MTX e, diariamente, atà o sacrifÃcio, no 5 dia. Na investigaÃÃo do papel do NO na mucosite oral induzida por 5-FU, os seguintes parÃmetros foram avaliados: anÃlises micro e macroscÃpica, atividade de mieloperoxidade (MPO) e da NOS, nÃveis teciduais de nitrito, imunohistoquÃmica para NOSi e detecÃÃo de morte celular. O efeito do 5-FU na produÃÃo salivar tambÃm foi avaliado. No estudo dos efeitos da GLU e AL-GLU, anÃlises micro e macroscÃpicas, atividade de MPO, detecÃÃo de morte celular, estoques teciduais de glutationa e concentraÃÃo sÃrica de glutamina foram os parÃmetros avaliados. No estudo do papel do NO na mucosite intestinal, foram realizados anÃlise histopatolÃgica, medida da altura de vilos nos trÃs segmentos do intestino delgado, atividade de MPO, detecÃÃo de apoptose, assim como, western blot e imunohistoquÃmica para NOSi. 1400W e AG, contrariamente ao L-NAME, reduziram os parÃmetros macro e microscÃpicos da mucosite oral e a infiltraÃÃo de cÃlulas inflamatÃrias, detectada na histopatologia e na atividade de MPO. Foram observados ainda, no 10 dia, maior atividade da NOS e marcaÃÃo imunohistoquÃmica para NOSi. 1400W reverteu a diminuiÃÃo da secreÃÃo salivar induzida por 5-FU. A mucosite oral induzida por 5-FU resultou na diminuiÃÃo dos nÃveis sÃricos de glutamina, bem como dos estoques teciduais de glutationa, no 10 dia, efeitos que foram revertidos pela administraÃÃo de GLU e AL-GLU. Apesar de nÃo ter prevenido a mucosite oral no 10 dia, o tratamento com GLU ou AL-GLU reduziu os parÃmetros macro e microscÃpicos da mucosite oral, e a atividade de MPO, no 14 dia. Na mucosite intestinal, AG e L-NAME preveniram o encurtamento de vilos e reduziram a necrose de criptas, assim como o infiltrado inflamatÃrio, efeitos induzidos pelo MTX, sendo esse Ãltimo constatado pela anÃlise histopatolÃgica e atividade de MPO. Foi detectado maior marcaÃÃo imunohistoquÃmica para NOSi no jejuno de ratos submetidos à mucosite intestinal. Esses resultados sugerem o papel relevante do NO na fisiopatologia das mucosites oral e intestinal. O presente estudo demonstrou ainda que a GLU e AL-GLU aceleraram a recuperaÃÃo da mucosa dos animais submetidos a mucosite oral por 5-FU, aumentando os nÃveis teciduais de glutationa, reduzindo a inflamaÃÃo e promovendo reepitelizaÃÃo / Mucositis induced by antineoplastic drugs is an important, dose-limiting and costly side effect of cancer therapy, which pathophysiology is not completely understood. The aim of the present study was to investigate the role of nitric oxide (NO) on the pathogenesis of oral and intestinal mucositis induced by 5-fluorouracil (5-FU) and methotrexate (MTX) and the effect of glutamine (GLU) and alanyl-glutamine (AL-GLU) on 5-FU-induced experimental mucositis. Oral mucosistis was induced by two intraperitoneal (i.p) administrations of 5-FU on the 1st and 2nd days (60 and 40 mg/kg, respectively) in hamsters. Animals were treated subcutaneously (s.c.) with the nitric oxide synthase (NOS) inhibitors N-(3-(Aminomethyl)benzyl)acetamidine (1400W; 1 mg/kg), aminoguanidine (AG; 5 or 10 mg/kg), Nφ-Nitro-L-Arginine Methyl Ester (L-NAME; 5, 10 or 20 mg/kg) or saline (0.4 ml), one hour before the injections of 5-FU and daily until sacrifice, on the 10th day. In another set of experiments, animals received saline, GLU or AL-GLU suspension (100 mM) one hour before the injections of 5-FU and daily until sacrifice, on the 10th or 14th day. Intestinal mucositis was induced by three administrations of MTX (2.5 mg/kg; s.c.) on the first three days of the experiment, in Wistar rats. Animals were treated i.p. with AG (10 mg/Kg), or L-NAME (20 mg/Kg), one hour before the injections of MTX and daily until sacrifice, on the 5th day. In the investigation of the role of NO on 5-FU induced oral mucositis, the following parameters were evaluated: microscopic and macroscopic analysis, myeloperoxidade (MPO) and NOS activities, nitrite level, immunohistochemistry for NOSi, salivary secretion, and cell death. In order to study the effect of GLU and AL-GLU, microscopic and macroscopic analysis, MPO activity, cell death, glutathione stores and the serum concentration of glutamine, were evaluated. In the MTX-induced intestinal mucositis, histopathological analysis was evaluated and the villus height in all three small intestine segments was measured. MPO activity, cell death, as well as, western blot and immunohistochemistry to evaluated the expression of the NOSi, were also conducted. 1400W or AG, but not L-NAME, reduced macroscopic and histological parameters of oral mucositis, and reduced the inflammatory cell infiltration as detected on histopathology and by MPO activity. Increased NOS activity and immunostaining for NOSi were detected. 5-FU induced a decrease in salivary secrection, observed on 4th day, and this effect was prevented by 1400W. The 5-FU-induced oral mucositis significantly decreased the serum GLU level as well as the cheek pouch glutathione stores, observed on day 10. GLU or AL-GLU reversed the 5-FU effects, restoring serum GLU levels and cheek pouch glutathione stores, observed on day 10, but did not prevent oral mucositis at this time. However, GLU and AL-GLU reduced macroscopic and histological parameters of oral mucositis, and reduced the MPO activity on day 14. In the MTX-induced mucositis, AG and L-NAME significantly prevented villous blunting, lamina propria cell death, and reduced crypt necrosis induced by MTX, decreasing neutrophil infiltration as detected by histopathology and by MPO activity. These data were associated with the detection of iNOS expression by Western blot and by immunohistochemistry in the jejunum tissue. These results suggest an important role of NO in the pathogenesis of oral and intestinal mucositis induced by 5-FU and MTX. The present study also demonstrated that GLU or AL-GLU hastens mucosal recovery increasing mucosal tissue glutathione stores, reducing inflammatory parameters and speeding reepithelization
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5-Fluorouracil-Spiegelbestimmung unter neoadjuvanter Radiochemotherapie und adjuvanter Chemotherapie beim lokal fortgeschrittenen Rektumkarzinom / Effects of a body surface area based 5-fluoruracil dosing under the neoadjuvant radiochemotherapy and adjuvant chemotherapy in locally advanced rectal cancerQuack, Henriette 19 May 2015 (has links)
No description available.
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Estudo prospectivo da adição de metformina a 5-fluorouracil em pacientes com adenocarcinoma colorretal metastático refratário / Phase II Trial of Metformin Combined With 5-Fluorouracil in Patients With Refractory Metastatic Colorectal CancerMiranda, Vanessa da Costa 23 November 2016 (has links)
INTRODUÇÃO: Estudos observacionais e pré-clinicos sugerem o efeito antitumoral da metformina em tumores sólidos, incluindo o câncer colorretal. Entretanto, os efeitos da metformina no câncer colorretal ainda não foram testados em estudos prospectivos. PACIENTES E MÉTODOS: Este foi um estudo de fase II, unicêntrico, braço único de pacientes com câncer colorretal metastático em progressão e previamente tratados com 5-FU, irinotecano, oxaliplatina e anti-EGFR, se RAS selvagem. Os pacientes receberam metformina 850 mg VO duas vezes por dia e 5-FU 425 mg/m2 e leucovorin 50 mg IV semanal até progressão de doença, toxicidade inaceitável ou retirada de consentimento. O desfecho primário foi controle de doença em 8 semanas. RESULTADOS: Dos 50 pacientes incluídos, 11 (22%) alcançaram o desfecho primário. Para toda coorte, a SLP foi de 1,8 meses e a SG mediana de 7,9 meses. Quando avaliamos somente os 11 pacientes que alcançaram controle de doença na semana 8, a SLP foi de 5,6 meses e a SG foi de 16,2 meses. Houve tendência a maior sobrevida entre os obesos (12,4 vs 5,8 meses de acordo com IMC maior ou menor que 30) e aqueles que tiveram maior intervalo livre de 5FU antes de entrar no estudo. O tratamento foi bem tolerado e os principais efeitos colaterais de qualquer grau foram diarreia, náusea, vômito e mielotoxicidade. CONCLUSÃO: Neste estudo de fase II, a metformina combinada ao 5FU apresentou atividade modesta na população geral de pacientes com câncer colorretal metastático refratário. Em análise de subgrupo, obesos e maior intervalo livre de 5FU foram associados a controle de doença prolongado. Estudos prospectivos randomizados com metformina em câncer colorretal devem ser realizados / BACKGROUND: Observational and pre-clinical studies have suggested that metformin has antitumor effects in solid tumors, including colorectal cancer. However the effects of metformin in colorectal cancer have not been tested in clinical trials. PATIENTS AND METHODS: This was a single center, single-arm phase II clinical trial where histologically confirmed colorectal cancer patients with measurable and progressing metastatic disease previously treated with 5-FU, irinotecan, oxaliplatin and an anti-EGFR, if the tumor was RAS wild type, were enrolled to receive metformin 850 mg orally bid continuously plus 5-FU 425 mg/m2 and leucovorin 50 mg IV weekly until disease progression, unacceptable toxicity or consent withdrawn. The primary endpoint was disease control rate at 8 weeks. RESULTS: Among 50 patients included, 11 (22%) met the primary endpoint. The median progression free survival was 1.8 months and the median overall survival was 7.9 months. Analyzing only those 11 patients who achieved disease control rate at 8 weeks, their median progression-free survival was 5.6 months and their median overall survival was 16.2 months. There was a trend for prolonged median survival for obese patients (12.4 vs 5.8 months) and those longer off 5FU. The treatment was well tolerated and the main side effects were diarrhea, nausea, vomiting and myelotoxicity. CONCLUSION: Metformin and 5FU showed an overall modest but intriguing activity in refractory colorectal cancer patients in this phase II study. Some patients presented long-term disease control. Further trials are needed to confirm these results, particularly in obese patients with colorectal cancer
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Investigation of mechanisms of drug resistance in colorectal cancer : a proteomic and pharmacological study using newly developed drug-resistant human cell line subclonesDuran, M. Ortega January 2017 (has links)
Despite therapeutic advances, colorectal cancer still has a 45% mortality rate, and one of the most crucial problems is the development of acquired resistance to treatment with anticancer drugs. Thus the aims of this project are to develop drug-resistant colon cancer cell lines in order to identify mechanisms of resistance for the most commonly drugs used in colorectal cancer: 5-fluorouracil, oxaliplatin, and irinotecan. Following evaluation of drug sensitivity to these agents in an initial panel of eight colorectal cancer cell lines, 3 lines (DLD-1, KM-12 and HT-29) were selected for the development of 5-FU (3 lines), oxaliplatin (2) and irinotecan (1) resistant sublines by continuous drug exposure, with resistance confirmed using the MTT assay. Consistently resistant sublines were subject to a „stable isotope labelling with amino acids in cell culture‟ (SILAC) approach and a MudPIT proteomics strategy, employing 2D LC and Orbitrap Fusion mass spectrometric analysis, to identify novel predictive biomarkers for resistance. An average of 3622 proteins was quantified for each resistant and parent cell line pair, with on average 60-70 proteins up-regulated and 60-70 down-regulated in the drug resistant sublines. The validity of this approach was further confirmed using immunodetection techniques. These studies have provided candidate proteins which can be assessed for their value as predictive biomarkers, or as therapeutic targets for the modulation of acquired drug resistance in colorectal cancer.
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Desenvolvimento de um sistema terapêutico micro-/nanoestruturado contendo 5-fluorouracil para administração pulmonarZatta, Kelly Cristine January 2016 (has links)
A inexistência de um agente terapêutico único satisfatório para o tratamento do melanoma metastático e a potencialidade do quimioterápico 5FU (5-fluorouracil) motivou esta pesquisa, a qual teve por objetivo o desenvolvimento tecnológico de sistemas carreadores micro-/ e nanoestruturados contendo 5FU a fim de aumentar sua eficácia terapêutica e reduzir a toxicidade por meio da administração pulmonar. Duas formulações pulverulentas foram desenvolvidas com polímeros naturais, sulfato de condroitina e hidroxipropil-metil-celulose, denominadas 5FU-MS e 5FU-NS, utilizando as técnicas de aspersão e atomização vibracional piezoelétrica, respectivamente. Ambas as formulações foram avaliadas quanto às características físicas e químicas, perfil toxicológico in vivo (C. elegans e em ratos Wistar), e penetração e biodisponibilidade no tecido pulmonar pela quantificação da fração livre de fármaco por microdiálise pulmonar. A análise físico-química revelou a obtenção de partículas micrométricas para 5FU-MS e submicrométricas para 5FU-NS, com diâmetros médios de partícula de 2,546 ± 0,07 m e 0,652 ± 0,03 m, e fração respirável (FR%) de 55,12 ± 2,98 e 76,84 ± 0,07, respectivamente. Ambas demonstraram características e propriedades adequadas para administração pulmonar, com capacidade de deposição nas porções média e profunda. A toxicidade das formulações avaliada em C. elegans considerou o percentual de morte, desenvolvimento, DL50 e produção de ROS para os nematodos sob tratamento agudo e crônico. Os resultados evidenciaram redução significativa da toxicidade proporcionada pela redução da taxa de morte e maior desenvolvimento dos grupos tratados com as formulações 5FU-MS e 5FU-NS em comparação ao fármaco livre, sugerindo perfis de segurança satisfatórios para administração. Além disso, 5FU-MS revelou-se um agente pró-oxidante, representando um diferencial promissor deste sistema, podendo alcançar maior sensibilização das células tumorais com menores doses. A toxicidade pulmonar aguda foi avaliada pela análise de LDH e proteínas totais no fluido de lavagem bronco-alveolar (BALF) após a administração combinada das formulações 5FU-MS e 5FU-NS para administração como um sistema terapêutico único (5FU-MS/NS), e análise de dano tecidual pulmonar em ratos. Os resultados da análise bioquímica e histológica indicaram o baixo potencial de indução de lesão tecidual a partir da administração pulmonar combinada das formulações, em relação ao fármaco livre. A análise do perfil farmacocinético por microdiálise pulmonar evidenciou o êxito no desenvolvimento dos sistemas carreadores, tornando possível duplicar o t1/2 do 5FU e aumentar significativamente a biodisponibilidade no tecido pulmonar. Os resultados obtidos indicam a eficiência das formulações 5FU-MS e 5FU-NS em alcançar os benefícios terapêuticos do fármaco 5FU com menores doses e maiores intervalos de administração. Este trabalho de tese apresenta uma abordagem promissora na terapia de neoplasias com recorrência de metástase pulmonar. / The absence of a single therapeutic agent suitable for the treatment of metastatic melanoma and the potential of 5FU chemotherapy (5-fluorouracil) motivated this study, which aimed the development of carrier systems based on micro-/ and nanostructures containing 5FU to increase the therapeutic efficacy and reduce toxicity of this drug by pulmonary administration. Two different formulations of dry powders were developed with natural polymers, chondroitin sulfate and hydroxypropyl-methyl-cellulose, denomined 5FU-MS and 5FU-NS, using the spray-drying and vibrational piezoelectric atomization techniques, respectively. Both formulations were evaluated in terms of physico-chemical characteristics, in vivo toxicological behaviors (C. elegans and in Wistar rats), bioavailability and penetration in the lung tissue by quantifying of drug free fraction by lung microdialysis. The physicochemical analysis showed that were obtained as micrometric (5FU-MS) and submicron particles (5FU-NS), with average diameters of particle 2.546 ± 0.07 m and 0.652 ± 0.03 m, and respirable fraction (FR%) of 55.12 ± 2.98 and 76.84 ± 0.07, respectively. Both showed suitable characteristics and properties for pulmonary delivery, with deposition capacity in the middle and deep lung portions. The toxicity of the formulations evaluated in C. elegans considered the death rate, body development, DL50 and production of ROS to nematodes under acute and chronic treatment. The results showed significant reduction of toxicity, reducing the death rate and greater development of the groups treated with 5FU-MS and 5FU-NS formulations compared to the free drug, suggesting satisfactory safety profile for administration. In addition, 5FU-MS proved to be a pro-oxidant agent, representing a promising differential of this system which can achieve greater sensitization of tumoral cells with lower doses. Acute pulmonary toxicity was evaluated by analyzing LDH, and total protein in the bronchoalveolar lavage fluid (BALF) after combined administration of 5FU-MS formulations and 5FU-NS for administration as a single therapeutic system (5FU-MS/NS) and analysis of lung tissue damage in rats. The results of biochemical and histological analysis indicated the low potential to induce tissue damage from the pulmonary administration of combined formulations, compared to free drug. Analysis of the pharmacokinetic profile for pulmonary microdialysis showed the successful development of carrier systems, making it possible to double the t1/2 of 5FU and significantly increase bioavailability in lung tissue. The results indicate the effectiveness of the formulations 5FU-MS and 5FU-NS in achieving the therapeutic benefits of the drug 5FU at lower doses and higher dosing intervals. This thesis work presents a promising approach to cancer therapy with lung metastasis recurrence.
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Efeitos da formulação mucoadesiva com extrato de Curcuma longa L. em animais portadores de mucosite intestinal induzida por 5-fluorouracilSantos Filho, Edvande Xavier dos 20 March 2014 (has links)
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Previous issue date: 2014-03-20 / Introduction: Intestinal mucositis is a frequent limiting factor in antineoplastic therapy. There is no truly effective treatment targeted to cure this side effect. Curcuma longa L. has been reported to have antioxidant, antitumor and anti-inflammatory properties. Objective: This study aimed to evaluate the effects of a mucoadhesive formulation with Curcuma longa L. extract (MCL) in mice bearing intestinal mucositis induced by 5-FU. Methods: Swiss male adult mice (35-40g) were randomly allocated into three experimental groups of 5 animals each: control (mucoadhesive formulation 0.6mL/animal, via gavage, from day 1-6); exposed to 5-fluorouracil (5-FU 200mg/kg, i.p., in days 4-6); treated prophylactically and throughout mucositis with FMCL and exposed to 5-FU (MCL 15mg/kg, via gavage, from day 1-6, plus 5-FU 200mg/kg, i.p., in days 4-6). In the 7th day, animals were anesthetized by xylazine-ketamine followed by cervical dislocation. Duodenal samples, 10 cm after pyloric sphincter were removed to perform the essays. The parameters evaluated were: body weight assessment, morphometrics and histo-pathological analysis, apoptosis (p53/Bax, Bcl-2), cell proliferation (Ki-67), myelo-peroxidase (MPO) and malondialdehyde (MDA). Results: 5-FU induced intestinal mucositis characterized by villus shortening, crypt deepening, intense inflammatory infiltration, vacuolization and mucosal edema. Besides, 5-FU induced severe mice body mass reduction, apoptosis in cells of villus and crypts (p<0.001), increase in MPO activity and MDA formation (p<0.05), when compared to the control group. Treatment with MCL attenuated body mass loss, protected intestinal mucosa from villus shortening and crypt deepening, decrease MPO activity and MDA formation (p<0.05). In this group of animals was also observed high expression of the cell proliferation marker Ki-67 in epithelial cells lining of villus and crypts. Conclusion: Our data confirm the therapeutic potential of MCL for the treatment of intestinal mucositis in mice. Further studies are needed to assess this formulation potential for human use. / Introdução: A mucosite intestinal é um dos efeitos adversos limitantes das terapias antineoplásicas. Não existe tratamento realmente efetivo direcionado a cura deste efeito colateral grave. A Curcuma longa L. tem sido proposta como candidata ao tratamento de várias doenças por possuir propriedades antioxidante, antitumoral e anti-inflamatória. Objetivo: Este estudo objetivou avaliar os efeitos da formulação mucoadesiva com extrato de Curcuma longa L. (FMCL) em animais portadores de mucosite intestinal induzida por 5-FU. Métodos: Camundongos Swiss adultos, machos (35-40g) foram aleatoriamente alocados em três grupos experimentais de 5 animais cada: [1] controle (formulação mucoadesiva 0.6mL/animal, via gavagem, do dia 1-6); [2] exposto ao 5-fluorouracil (5-FU 200mg/kg, i.p., dos dias 4-6), [3] tratado profilaticamente e ao curso da mucosite com FMCL e exposto ao 5-FU (FMCL 15mg/kg, via gavagem dos dias 1-6, mais 5-FU 200mg/kg, i.p., dos dias 4-6). No sétimo dia, os animais foram anestesiados por xilazina-ketamina seguido por deslocamento cervical. Amostras de duodeno, 10 cm após o esfíncter pilórico, foram coletadas para realização dos ensaios. Os parâmetros avaliados foram: avaliação de massa corporal, análise morfométrica e histopatológica, apoptose (p53/Bax; Bcl-2), proliferação celular (Ki-67), mieloperoxidase (MPO) e malondialdeído (MDA) Resultados: A administração de 5-FU induziu mucosite intestinal caracterizada por encurtamento das vilosidades, aprofundamento das criptas, intenso infiltrado infla-matório, vacuolização e edema na mucosa. Além disso, o 5-FU induziu grave redução de massa corporal nos camundongos e apoptose nas células das vilosida-des e criptas, quando comparado ao grupo controle (p<0,001). Foi observado ainda aumento na atividade de MPO e formação de MDA, quando comparado ao grupo controle (p<0,05). Por outro lado, o tratamento com a FMCL atenuou a perda de massa corporal dos animais com mucosite. Ademais, protegeu a mucosa intestinal da redução no tamanho das vilosidades e criptas induzidas pelo 5-FU. Neste grupo de animais foi observado ainda aumento na expressão do marcador de proliferação celular Ki-67 nas células epiteliais de revestimento das vilosidades e criptas intestinais. O tratamento também diminuiu significantemente a atividade de MPO e formação de MDA (p<0,05). Conclusão: Os dados confirmam o potencial terapêutico da FMCL no tratamento da mucosite intestinal em camundongos. Estudos adicionais são necessários para que esta formulação possa se tornar uma alternativa segura para uso em seres humanos.
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Fabrication and characterization of 5-Fu loaded poly(lactide-Co-Glycolide) millirods: assessment of their suitability for local tumor treatmentLeelakanok, Nattawut 01 August 2017 (has links)
The synthetic chemotherapeutic agent, 5-FU, has been used for the treatment of a variety cancers, with colorectal cancer being among the most susceptible. Administration of 5-FU by continuous intravenous infusion has proven to yield greater antitumor efficacy and lower hematotoxicity compared to administration of 5-FU by intravenous bolus injections. Nevertheless, systemic application of 5-FU is often limited by its narrow therapeutic threshold, and therefore in certain situations, such as tumor resection, it may be more appropriate to provide local rather than systemic delivery of 5-FU. It was therefore proposed that 5-FU loaded PLGA millirods may be capable of providing sustained release of 5-FU at a local level which may have equivalent or greater antitumor activity and less cytotoxicity than the systemic or local delivery of soluble 5-FU.
PLGA millirods loaded with 5-FU were successfully fabricated by a hot-melt extrusion technique and characterized for in vitro and in vivo release rates. It was demonstrated that percentage loading by weight of 5-FU could be adjusted to modify its release kinetics. It was also shown that millirods could be stably stored under a variety of conditions for at least 2 months.
An optimal millirod formulation (PLGA 50:50 loaded with 5-FU (50% w/w)) was tested for antitumor activity and general toxicity in vivo. At the dose of 120 mg/kg 5-FU, millirods (delivered peritumorally) were efficacious (with 100% survival rates) against solid thymomas in tumor-challenged mice (causing complete regression). Whilst the soluble form of 5-FU (delivered intraperitoneally (IP) at 120 mg/kg) was also highly efficacious (90% survival rates) against thymomas it was also more hematotoxic. In addition, the millirod form provided significantly greater antitumor activity against colorectal tumors in mice compared to the soluble form of 5-FU. In terms of in vivo toxicity, surprisingly, the type of formulation did not have a significant effect on mouse weight despite both IP and subcutaneous (SC) delivery causing death of some mice. Importantly, it was found that 5-FU loaded PLGA millirods were significantly less hematotoxic than soluble 5-FU delivered by either IP or SC injection at the equivalent dose. Thus, locally implanted 5-FU loaded PLGA millirods appeared to be less toxic and possessed overall greater antitumor potency than soluble 5-FU delivered by IP or SC injection.
This study further investigated whether the combination of 5-FU loaded PLGA millirods with eniluracil (in both thymoma and colorectal tumor models) or immune checkpoint inhibitors (in the colorectal tumor model) could enhance the antitumor efficacy of 5-FU millirods in mice challenged with colorectal tumors. It was found that the combination of 5-FU loaded PLGA millirods and eniluracil (millirod or solution forms) did not significantly enhance the antitumor efficacy of 5-FU millirods in either tumor models. It was also found that immune checkpoint inhibitors did not enhance the antitumor efficacy of 5-FU loaded PLGA millirods in the colorectal tumor model.
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Intraperitoneal 5-Fluorouracil treatment of cancer - clinical and experimental studiesÖman, Mikael January 2004 (has links)
<p>Background:Pancreas cancer is a most aggressive malignancy. More than 80% of patients diagnosed with pancreas cancer, exhibit such advanced disease, that curative surgery is impossible. Systemic chemotherapy prolongs survival to 5-9 months. High concentrations of chemotherapeutic agents in the abdominal cavity and in the lymphatics draining the area is achieved by intraperitoneal administration. Vasopressin decreases splanchnic blood flow, reducing the intraperitoneal uptake of drugs, thus raising the local and lymphatic dose intensity.</p><p>Aim: The aim of the study was to investigate the feasibility and tumour response of intraperitoneal 5-Fluorouracil (5-FU) treatment in non-resectable pancreas cancer, using vasopressin to improve the pharmacokinetic profile. Further, to study the effect of vasopressin on peritoneal blood flow, altered by intraperitoneal 5-FU or the presence of peritoneal carcinomatosis.</p><p>Methods: In the animal experiments, the 133Xe-clearance technique and as a comparison Laser doppler flow, were used to identify changes of peritoneal blood flow caused by vasopressin in unmanipulated animals and in animals with peritoneal carcinomatosis or animals given intraperitoneal 5-FU. In the clinical studies, 68 (39 women/29 men) patients, with a non-resectable ductal pancreas cancer and a Karnovsky Index ≥70 were included. Patients were treated with 750-1500 mg/m2 5-FU intraperitoneally through a Port-a-cath and Leucovorin 100 mg/m2 intravenously on two consecutive days every 21 days until progression. Seventeen patients, receiving 750 mg/m2 5-FU, were given concomitant vasopressin 0.1 IU/min during 180 minutes, alternatively day 1 or 2.</p><p>Results: In the animal experiments, vasopressin 0.07 IU/kg/min significantly reduced the 133Xe-clearance. Intraperitoneal 5-FU decreased the basal peritoneal blood flow and abrogated the vasopressin effect for 1-2 days. The presence of peritoneal carcinomatosis did not influence the basal peritoneal blood flow, nor the reduction of peritoneal blood flow caused by vasopressin. In the clinical studies, the treatment with intraperitoneal 5-FU was well tolerated, with no WHO Grade 3 or 4 toxicity with doses up to 1250 mg/m2. Thirty patients achieved at least stable disease at three months. The median survival time was 8.0 (range 0.8-54.1) months. There was a significant reduction of 5-FU Cmax on day 2, but no significant reduction of AUC, when vasopressin was given.</p><p>Conclusion: Peritoneal blood flow changes caused by vasopressin can be estimated with the 133Xe-clearance technique. Intraperitoneal 5-FU but not peritoneal carcinomatosis decreases the vasopressin induced 133Xe-clearance reduction, 1-2 days after administration. In patients with non-resectable pancreas cancer, intraperitoneal 5-FU up to 1250 mg/m2 for two days every third week can be given without WHO grade 3 and 4 toxicity. The treatment is well tolerated with few and minor side effects. Tumour responses were observed. Addition of vasopressin does not significantly enhance the pharmacokinetics of intraperitoneal 5-Flurorouracil, but adds toxicity.</p>
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