5-Fluorouracil (5FU) is a pyrimidine analogue, indicated for the therapy of proliferative skin diseases such as actinic keratosis (AK), superficial basal cell carcinoma and psoriasis. It has also been used for the treatment of solid tumours like colorectal, breast and liver carcinomas for nearly 40 years. Although 5FU has always been administered parenterally and orally, metabolism is rapid and absorption is erratic. Several severe side-effects are also commonly associated with 5FU therapy, including myelosuppression, hand-foot syndrome and gastrointestinal effects. Seeing that 5FU is an important part of the treatment of several malignant and pre-malignant disorders, it would be advantageous to find a delivery route and delivery system that negate absorption and metabolic variation and decrease side-effects. The transdermal route provides a promising alternative to the above-mentioned conventional delivery routes, solving most of the problems associated with parenteral and oral administration. That being said, the formidable barrier situated in the skin is not easily breached. The stratum corneum, the outermost skin layer, is mostly lipophilic in nature, preventing hydrophilic molecules such as 5FU from entering. 5FU-containing creams and lotions are currently commercially available, but absorption is still very limited. The transdermal absorption from these formulations has been compared to that obtained with the use of new transdermal delivery vehicles, with the newer formulations proving to be promising. It was decided to entrap 5FU in a novel therapeutic system, in the form of the Pheroid™ system, to increase its transdermal penetration. Pheroid™ vesicles are stable spherical structures in a unique, emulsion-like formulation, and fall in the submicron range. The main components of the Pheroid™ system are the ethyl esters of the essential fatty acids linoleic acid and linolenic acid, as well as the cys-form of oleic acid, and water. The formulation is saturated with nitrous oxide (N20). Although Pheroid™ vesicles may resemble other lipid-based vehicles, such as liposomes and micro-emulsions, they are unique in the sense that they have inherent therapeutic qualities as well. The Pheroid™ formulation can be specifically manipulated to yield different types of vesicles, ensuring a fast transport rate, high entrapment efficiency, rapid delivery and stability of the delivery system for a specific drug. In this study, 5FU was entrapped in the Pheroid™ formulation. Transdermal permeation studies were then performed to evaluate the influence of this delivery system on the transdermal flux of 5FU. Vertical Franz diffusion cells were utilised to determine the transdermal penetration of 5FU. Only Caucasian female abdominal skin was used to minimise physiological variables. Diffusion studies were done over 12 hour periods, with the entire receptor phase being withdrawn at predetermined intervals. Samples were analysed using high performance liquid chromatography (HPLC), after which the cumulative concentration of active was plotted against time. The linear portion of this graph represents the flux of 5FU through the skin. It was found that there were differences in the results between formulations containing 5FU in a phosphate buffer solution (PBS)-based Pheroid™ and water-based Pheroid™, though the difference was not statistically significant. The 0.5 % 5FU in water-based Pheroid™ resulted in a significantly bigger yield than the control (1 % 5FU in water) as well as a significant difference to the 1 % 5FU in PBS-based Pheroid™ formulation. In general the water-based Pheroid™ formulations had greater average cumulative concentrations, yields and fluxes than the other formulations. The fluxes obtained with the water-based Pheroid™ formulations also correlated well with a previous study done by Kilian (2004). Thus it can be concluded that the Pheroid™ therapeutic delivery system enhances the transdermal penetration of 5FU. Water-based Pheroid™ formulations proved to be more effective than PBS-based Pheroid™ formulations. It can also be concluded that a 0.5 % 5FU in water-based Pheroid™ formulation can be used instead of a 1 % formulation, because there were no statistically significant differences between the two formulations. This would be advantageous - patient compliance can be enhanced because of a more tolerable formulation with fewer side effects, while manufacturing cost is lowered by using a lower concentration of active. It is recommended that some aspects of the study be investigated further to optimise the transdermal delivery of 5FU using the Pheroid™ therapeutic system. These aspects include optimising the composition of the Pheroid formulation, investigating the entrapment process of 5FU within Pheroid™ spheres, the influence of PBS and water as basis of the Pheroid™ formulation and the amount of 5FU remaining in the epidermis after the 12 hour period of the diffusion study. Keywords: 5-Fluorouracil, Franz diffusion cell, Heat separated epidermis, Skin penetration, Transdermal, Drug delivery system, Pheroid™ / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

This thesis describes the use of functional genomics screens in yeast to study anticancer drug action and intracellular transport. The yeast Saccharomyces cerevisiae provides a particularly useful model system for global drug screens, due to the availability of knockout mutants for all yeast genes. A complete collection of yeast deletion mutants was screened for sensitivity to monensin, a drug that affects intracellular transport. A total of 63 deletion mutants were recovered, and most of them were in genes involved in transport beyond the Golgi. Surprisingly, none of the V-ATPase subunits were identified. Further analysis showed that a V-ATPase mutant interacts synthetically with many of the monensin-sensitive mutants. This suggests that monensin may act by interfering with the maintenance of an acidic pH in the late secretory pathway. The second part of the thesis concerns identification of the underlying causes for susceptibility and resistance to the anticancer drug 5-fluorouracil (5-FU). In a functional genomics screen for 5-FU sensitivity, 138 mutants were identified. Mutants affecting tRNA modifications were particularly sensitive to 5-FU. The cytotoxic effect of 5-FU is strongly enhanced in these mutants at higher temperature, which suggests that tRNAs are destabilized in the presence of 5-FU. Consistent with this, higher temperatures also potentiate the effect of 5-FU on wild type yeast cells. In a plasmid screen, five genes were found to confer resistance to 5-FU when overexpressed. Two of these genes, CPA1 and CPA2 encode the two subunits of the arginine-specific carbamoyl-phosphate synthase. The three other genes, HMS1, YAE1 and YJL055W are partially dependent on CPA1 and CPA2 for their effects on 5-FU resistance. The specific incorporation of [14C]5-FU into tRNA is diminished in all overexpressor strains, which suggest that they may affect the pyrimidine biosynthetic pathway.

The applications of dense gas technology (DGT) in modern particle engineering have shown promising results in producing submicron particles with uniform particle morphology. In this study, two configurations of dense gas antisolvent processes were employed for the micronization, encapsulation and co-precipitation of pharmaceutical compounds. The encapsulation of superparamagnetic iron oxide nanoparticles (SPIONs) by a pH-responsive polymer (Eudragit?? S100) was successfully performed using the supercritical antisolvent (SAS) process. Nanocomposites of less than 200nm in diameter with encapsulated SPIONs content as high as 16 wt% were achieved. Magnetic characterization of the product was also performed and the data were fitted by the Langevin equation. The superparamagnetic properties of the composites were preserved and the effective magnetic size was about 10 nm. The magnetically and pH-responsive nanocomposites can be potentially utilized as magnetic resonance imaging contrast agents and drug carriers. Screening experiments of 8 active pharmaceutical ingredients and 5 pharmaceutical excipients were performed using the recently patented atomized rapid injection solvent extraction (ARISE) process. Candidates with promising product morphology and recovery were selected for co-precipitation studies. The co-precipitation of the anti-cancer drug 5-fluorouracil (5FU) and poly l-lactic acid (PLLA) was conducted to develop a controlled release system. Experiments were designed based on a two-level, three-factor factorial design, in order to investigate the effects of processing parameters on product characteristics. Submicron PLLA-5FU composites (diameter<0.8 ??m) with a drug loading of 7.4 wt% were produced.

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.

Submitted by Raquel Bester Liszbinski (raquelb.biomed@gmail.com) on 2018-06-12T15:27:51Z No. of bitstreams: 1 Dissertação Raquel Bester Liszbinski 04.2018 .pdf: 2684297 bytes, checksum: 39e14fcba58d472dfe21ab816c211c29 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-06-13T16:59:06Z (GMT) No. of bitstreams: 1 liszbinski_rb_me_bot.pdf: 2684297 bytes, checksum: 39e14fcba58d472dfe21ab816c211c29 (MD5) / Made available in DSpace on 2018-06-13T16:59:06Z (GMT). No. of bitstreams: 1 liszbinski_rb_me_bot.pdf: 2684297 bytes, checksum: 39e14fcba58d472dfe21ab816c211c29 (MD5) Previous issue date: 2018-04-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O câncer colorretal (CCR) é o terceiro tipo mais prevalente de câncer no mundo, com frequentes complicações metastáticas muitas vezes associadas ao desenvolvimento de resistência adquirida aos fármacos. Uma das estratégias terapêuticas utilizadas no tratamento de pacientes que não respondem bem à quimioterapia convencional é a imunoterapia baseada na aplicação de anticorpos monoclonais cetuximab ou panitumumab, ambos dirigidos ao receptor do fator de crescimento epidérmico (EGFR). Em alguns casos observa-se associação da resistência aos anticorpos com a superexpressão de HER2, outro membro da família do EGFR. Assim, esta dissertação foi elaborada para testar a hipótese de que o uso conjunto de anticorpos anti-EGFR e anti-HER será capaz de reduzir o escape das células tumorais e que a associação com fármacos antitumorais tornará mais efetiva sua eliminação. Com esse propósito, nosso objetivo foi utilizar nanopartículas ouro (AuNP) para o transporte de 5-fluorouracil (5FU), endereçando esses nanocarreadores para as células de câncer colorretal recobrindo-os com anticorpos contra os receptores para fator de crescimento epidérmico EGFR (ErbB-1) e HER2 (ErbB-2). Os nanocarreadores foram testados in vitro quanto a sua interação por células de câncer colorretal HCT-116 e Caco-2 por citometria de fluxo, sua capacidade de induzir apoptose/necrose e seu efeito sobre a atividade proliferativa das células testadas. Nossos dados indicam que as AuNP produzidas e funcionalizadas com as moléculas de interesse possuem estrutura esférica, no entanto, algumas formulações apresentaram aglomeração devido a incorporação das moléculas orgânicas. Os ensaios biológicos mostraram que a AuNP 5FU EGFR teve efeito significativo na atividade citotóxica pela via apoptótica recente quando as células foram expostas à maior concentração testada, independentemente do tempo de exposição. Consequentemente, a proliferação celular foi cessada neste mesmo tratamentos e ambos os fenômenos foram observados de forma mais evidente na linhagem Caco-2, inclusive demonstrando efeito superior ao quimioterápico na forma livre. / Colorectal cancer (CRC) is the third most prevalent type of cancer in worldwide and is frequently associated with metastatic complications. Therapeutic strategies to treat those patients who do not respond to conventional chemotherapy is based on the application of monoclonal antibodies cetuximab or panitumumab, both directed targeting the epidermal growth factor receptor (EGFR). However, in some cases resistance is associated with an overexpression of HER2, another receptor belonging to the EGFR family. Therefore, this dissertation was designed to test the hypothesis that the combination of anti-EGFR and anti-HER antibodies will be able to reduce the escape of tumor cells, and that their association with antitumor drugs could improve the elimination of tumor cells. Then, our goal was to use gold nanoparticles (AuNP) for transporting 5-fluorouracil (5FU), addressing them to colorectal cancer cells by coating them with antibodies against the receptors of epidermal growth factor EGFR (ErbB-1) and HER2 (ErbB-2). Nanocarriers were tested in vitro by flow cytometry for their interaction and internalization by the colorectal cancer cell line HCT-116 and Caco-2, their ability to induce apoptosis/necrosis and stop cell proliferation. Our results show that AuNP produced and functionalized with the molecules of interest have spherical structure, however, some formulations showed agglomeration due to the incorporation of organic molecules. Biological assays showed that AuNP 5FU EGFR had a significant effect on cytotoxic activity by the recent apoptotic pathway when cells were exposed to the highest concentration tested independently of the time of exposure. As a result, cell proliferation was stopped at this same treatment and both phenomena were observed most clearly in the Caco-2 cell line, including demonstrating superior effect to chemotherapy in free form. / FAPESP: 2015/26729-2.

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.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Oral mucositis (OM) induced by antineoplasic drugs is an important, dose-limiting, and costly side effect of cancer therapy. Calotropis procera is a plant plant constitutively produces abundant latex that is reported to possess anti-inflammatory, bacteriolytic, insecticidal, analgesic properties. The present work aimed to describe the effect of laticifer proteins of Calotropis procera (LP) in the expression of pro-inflammatory cytokines and inducible enzymes, such as, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the model of OM in Hamsters. OM was induced by two intraperitoneal (i.p.) administrations of 5-Fluorouracil (5-FU) on the 1st and 2nd days (60 and 40 mg/kg, respectively) in hamsters (n=5). LP (0,25; 1; 5 E 25 mg / kg) was injected i.p. 24h before and 24h after mechanical trauma of the cheek pouches. Control group received only saline. On the 10th day, the animals were sacrificed and tissues from the cheek pouches were harvested. Macroscopical and histopathological (inflammatory cell infiltration, edema, hemorrhage and the formation of ulcerations and abscess) analysis as well as immunohistochemistry for TNF-&#61537;, IL-1&#946;, iNOS and COX-2 was performed in the cheek pouch tissue. Kruskal Wallis/Dunn was used as statistical tests. P<0.05 was accepted. Ethics Committee 036/10. The LP significantly inhibited macroscopical and histopathological parameters when compared to control group with maximum effect in macroscopic scores reaching 75% and 66% of maximum effect at the histopathological evaluation. The MPO activity was also significantly inhibited by LP in 91% at the same dose (p<0,001) and also inhibited the lost weight of 5- FU induced-oral mucositis. The cheek pouches of hamsters submitted to OM showed marked immunostaining for TNF-&#61537;, IL-1&#946;, iNOS and COX-2 on inflamed conjunctive (Cj) and epithelial (Ep) tissue compared with the cheek pouches of the normal control group. LP caused considerable reduction in the immunostaining for TNF-&#61537; (62%,Cj; 70%,Ep), IL-1&#946; (87%,Cj; 80%,Ep), iNOS (82%Cj; 52%Ep) and COX-2 (70%,Cj; 100%,Ep) in the check pouches tissue when compared with the group of animals subjected to experimental mucositis that received saline instead of LP. These findings show anti-inflammatory effects of LP in 5-FU-induced OM. The protective effect could be supported by the reduction of the expression of pro-inflammatory cytokines, such as TNF-&#61537; and IL-1&#946; and the enzymes iNOS and COX-2. The protective mechanism appears to involve inhibition of the expression of iNOS, COX-2, TNF-&#61537;, and IL- 1&#946;. / Mucosite oral (MO) induzida por drogas antineoplÃsicas à um importante fator limitante da dose e efeitos colaterais da terapia do cÃncer. Calotropis procera à uma planta que produz lÃtex constitutivamente abundante que à relatado possuir propriedades antiinflamatÃrias, bactericidas, inseticidas, analgÃsicas. O presente trabalho teve como objetivo descrever o efeito das proteÃnas do lÃtex da Calotropis procera (LP) na expressÃo de citocinas prÃ-inflamatÃrias (TNF-&#945; e IL-1&#61538;) e enzimas induzÃveis, como, ciclooxigenase-2 (COX-2) e Ãxido nÃtrico sintase induzÃvel (NOSi) no modelo de MO em hamsters. A mucosite oral foi induzida por duas administraÃÃes intraperitoneal (i.p) de 5-fluorouracil (5-FU) no 1  e 2 dias nas doses de 60 e 40 mg/kg, respectivamente nos animais (n = 5). As LP (0,25; 1; 5 E 25 mg/kg) foi injetado via i.p. 24h antes e 24h apÃs o trauma mecÃnico da mucosa jugal. O grupo controle recebeu apenas soluÃÃo salina. No 10 dia, os animais foram sacrificados e os tecidos da mucosa jugal foram colhidos. Foram realizadas no tecido mucosa jugal as anÃlises macroscÃpicas e histopatolÃgicas (infiltraÃÃo de cÃlulas inflamatÃrias, edema, hemorragia e à formaÃÃo de ulceraÃÃes e abscessos), bem como a imunohistoquÃmica para TNF-&#945;, IL-1&#946;, NOSi e COX-2. Foram utilizados Kruskal Wallis / Dunn como testes estatÃsticos, onde P <0,05 foi aceito. O estudo foi submetido ao Comità de Ãtica sob o protocolo 036/10. Observou-se que a LP inibiu significativamente parÃmetros macroscÃpicos e histopatolÃgicos, quando comparado ao grupo controle, com efeito mÃximo nos escores macroscÃpicos atingindo 75% e 66% do efeito mÃximo na avaliaÃÃo histopatolÃgica. A atividade de Mieloperoxidase (MPO) tambÃm foi significativamente inibida por LP em 91% com a mesma dose (p <0,001) quando comparado ao grupo controle e tambÃm inibiu a perda de peso em animais submetidos a mucosite oral e tratados com LP. A mucosa jugal dos animais submetidos a MO mostrou imunomarcaÃÃo para TNF-&#945;, IL-1&#946;, NOSi e COX-2 na conjuntiva inflamada (Cj) e tecido epitelial (Ep) em comparaÃÃo com o tecido jugal do grupo normal. LP causou reduÃÃo considerÃvel na imunomarcaÃÃo para TNF-&#945; (62%, Cj, 70%, Ep), IL-1&#946; (87%, Cj, 80%, Ep), NOSi (82% Cj; Ep 52%) e COX -2 (70%, Cj, 100%, Ep) no tecido jugal quando comparado com o grupo de animais submetidos à mucosite experimental que receberam salina, em vez de LP. Esses achados demonstram efeitos anti-inflamatÃrios de LP em MO induzida por 5-FU. O efeito protetor poderia ser suportado pela reduÃÃo da expressÃo das citocinas prÃ-inflamatÃrias, como TNF-&#945; e IL-1&#946; e na expressÃo de enzimas COX-2 e NOSi. O mecanismo de proteÃÃo parece envolver a expressÃo inibitÃria da NOSi, COX-2, TNF-&#945; e IL-1&#946;.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19597号 / 医博第4104号 / 新制||医||1014(附属図書館) / 32633 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 野田 亮, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Colorectal cancer is the third most common cancer in the United States and there is currently a lot of research going into new antitumor agents to kill the cancer. One method for replicating the tumor response to a drug in vivo is by creating an in vitro drug testing model to replicate the in vivo condition. This research project was conducted to determine the efficacy of testing tumor cultures in a microfluidic device as a way to provide accurate drug responses in vitro instead of using in vivo subjects in clinical trials. A total of four experiments were conducted with each experiment increasing the complexity of the culture model. The first experiment was a 2-dimensional tumor culture that was seeded in a well plate to study how 5-fluorouracil treatments affected the tumor cell viability. The second experiment was a 2-dimensional tumor culture that was seeded on top of a fibrin extracellular matrix (ECM) gel to determine how the tumor cells would respond to the 5-luorouracil treatments while growing on the fibrin. The third experiment was to create a 3-dimensional tumor culture that was seeded inside the fibrin ECM gel. This experiment was conducted to determine if tumor cells cultured within the fibrin gel could receive nutrients from the medium diffusing through the gel. Once the tumors responded as expected in the fibrin gel, the gel could be injected into a microfluidic device for the fourth experiment. The fourth experiment was a proof of concept to determine if the tumor cells could survive in the microfluidic device and be properly treated with 5-fluorouracil. The experiment with the cells seeded in the well plates showed that an increase in 5-fluorouracil concentration caused a significant decrease in cell viability. Both fibrin gel experiments showed that the average tumor size, total tumor area, and tumor count decreased as the 5-fluorouracil concentration increased. The tumor cells were successfully able to be cultured in the microfluidic device and the average tumor size decreased significantly when the culture was exposed to the 5-fluorouracil treatment.

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.