nÃo hà / A mucosite oral (MO) à um efeito colateral freqÃente e dose limitante da terapia do cÃncer, caracterizada por intensa reaÃÃo inflamatÃria na mucosa e formaÃÃo de Ãlceras na cavidade orofarÃngea. O objetivo deste trabalho foi avaliar o efeito da atorvastatina (ATV), droga utilizada para reduzir os nÃveis sÃricos de colesterol e que tem atividade antiinflamatÃria, na mucosite oral induzida por 5-fluorouracil (5-FU) em hamsters Golden Sirian. A mucosite oral foi induzida pela administraÃÃo intraperitoneal (i.p.) de 5-FU no 1 e 2 dias do experimento (60 e 40 mg/kg, respectivamente), com subseqÃentes escoriaÃÃes na mucosa jugal, no quarto dia. Os animais foram tratados intraperitonealmente (i.p.) com ATV 1, 5 ou 10 mg/kg ou salina ou salina/etanol 5% vol/vol 30 minutos antes de cada injeÃÃo do 5-FU e diariamente por 5 ou 10 dias. Os animais foram sacrificados no 5 ou 10 dia e amostras de mucosa jugal e dos principais ÃrgÃos vitais foram coletados para anÃlise histopatolÃgica. A determinaÃÃo dos nÃveis de TNF-α, IL-1β, nitrito, grupos sulfidrilas nÃo proteicos (NP-SH), ensaio da mieloperoxidase (MPO), imunohistoquÃmica para TNF-α, Ãxido nÃtrico sintase induzida (iNOS), NF-KB-p50, NF-kB-p50 NLS (sequÃncia de localizaÃÃo nuclear) e a expressÃo do NF-kB-p50 por Western Blot foram outros parÃmetros avaliados em amostras coletadas da mucosa jugal dos animais. O sangue foi coletado para leucograma, anÃlise dos parÃmetros bioquÃmicos e anÃlise da bacteremia. Atorvastatina nas doses de 1 e 5 mg/kg reduziu o dano na mucosa e a inflamaÃÃo, bem como os nÃveis de citocinas, nitrito e a atividade da MPO no 5 e 10 dia da MO. Ademais, atorvastatina 1 e 5 mg/Kg diminuiu a marcaÃÃo imunohistoquÃmica para TNF-α, NOSi, NF-kB-p50, NF-kB-p50 NLS, bem como a expressÃo do NF-kB-p50 na mucosa jugal dos hamsters submetidos a MO, no 5 dia. ATV 1 mg/Kg aumentou os nÃveis de NP-SH na mucosa jugal dos animais, quando comparado com os grupos 5-FU no 10 dia da MO. A associaÃÃo de ATV 5 mg/Kg e 5-FU diminuiu a taxa de sobrevida, amplificou a leucopenia dos animais, aumentou os nÃveis sÃricos de transaminases e causou lesÃo hepÃtica. NÃs tambÃm detectamos a presenÃa de bacilos Gram negativos no sangue de 100% dos animais tratados com ATV 5mg/Kg + 5-FU. Esses resultados sugerem que a atorvastatina previne o dano na mucosa oral e a inflamaÃÃo associada com MO induzida por 5-FU, entretanto a combinaÃÃo de altas doses de ATV com 5-FU induz hepatotoxicidade, amplificaÃÃo da leucopenia e bacteremia, que merecem atenÃÃo e futuros estudos em humanos. / Oral mucositis (OM) is a frequent side effect and dose-limiting of cancer therapy, characterized by intense inflammatory mucosal reaction and formation of ulcers in oropharyngeal cavity. The aim of this study was to evaluate the effect of atorvastatin (ATV) â cholesterol lowering drug with anti-inflammatory activity - in oral mucositis induced by 5-fluorouracil (5-FU) in Golden Sirian hamsters. Oral mucositis was induced by intraperitoneal (i.p.) administration of 5-FU in the first and second days of experiment (60 and 40 mg/kg i.p., respectively), with subsequent excoriations of the cheek pouch mucosa on the fourth day. The animals were treated by intraperitoneal route (i.p.) with ATV 1, 5 or 10 mg/kg or saline or saline and 5% vol/vol ethanol 30 min before 5-FU injection and daily for 5 or 10 days. The animals were sacrificed on the 5th or 10th day and samples of cheek pouches and major vital organs were removed for histopathological analysis. The determination of TNF-α, IL-1β, nitrite, non-protein sulfhydryl group (NP-SH) levels, myeloperoxidase (MPO) assay, immunohistochemistry for TNF-α, induced nitric oxide synthase (iNOS), NF-kB-p50, NF-kB-p50 NLS and the expression of NF-kB-p50 by Western Blot were other parameters evaluated in the samples collected from the oral mucosa of animals. Blood was collected for a leukogram, analysis of biochemical parameters and analysis of bacteremia. Atorvastatin at doses of 1 and 5 mg/kg reduced mucosal damage and inflammation, as well as the levels of cytokines, nitrite, and myeloperoxidase activity on the 5th and 10th day of OM. Moreover, ATV 1 and 5 mg/kg decreased the immunohistochemical staining for TNF-α, iNOS, NF-kB-p50, NF-kB-p50 NLS and expression of NF-kB-p50 of the cheek pouch mucosa on the 5th day of OM. ATV at 1 mg/kg increased cheek pouch NP-SH when compared to 5-FU groups on the 10th day of OM. The association of ATV 5 mg/kg and 5-FU decreased the survival rate, amplified the leukopenia of animals, increased transaminase serum levels and caused liver lesions. We also detected the presence of Gram negative bacillus in the blood of 100% of the animals treated with ATV 5 mg/kg + 5-FU. These results suggest that atorvastatin prevent mucosal damage and inflammation associated with 5-FU-induced OM, but the association of a higher dose of ATV with 5-FU induced hepatotoxicity, amplified leucopenia and bacteremia, which deserves attention and further research in humans.

Orientador: Edgard Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T18:26:23Z (GMT). No. of bitstreams: 1 Moreira_FernandadosSantos_M.pdf: 1478167 bytes, checksum: 57f6f26580823a79292af5a274beaa2d (MD5) Previous issue date: 2014 / Resumo: A ácido graxo sintase (FASN) é a principal enzima envolvida na lipogênese neoplásica e apontada como uma oncoproteína metabólica por favorecer o crescimento e sobrevivência das células tumorais, nas quais sua expressão é em geral elevada. Vários são os compostos capazes de inibir a atividade de FASN, dentre eles o orlistat (Xenical®), que possui efeitos antineoplásicos em cânceres de mama e próstata e nos melanomas. O carcinoma espinocelular (CEC) representa aproximadamente 90% de todas as neoplasias malignas que acometem a cavidade oral, sendo diagnosticado em estágios avançados em grande parte dos casos. Nestes pacientes, geralmente com metástases, é realizada uma abordagem sistêmica com agentes quimioterápicos como a cisplatina, o 5-fluorouracil (5-FU) e o paclitaxel, de maneira isolada ou combinada. Este trabalho tem como objetivo investigar in vitro se cisplatina, 5-FU ou paclitaxel potencializam o efeito antitumoral do orlistat no CEC oral. Para isto, células de CEC de língua altamente metastáticas (SCC-9 LN1) foram tratadas com estas drogas isoladamente ou combinadas com orlistat e avaliadas com relação às taxas de apoptose e necrose, progressão do ciclo celular e secreção de VEGFA e VEGFA165b. As maiores taxas de apoptose foram encontradas com o uso da combinação de orlistat com paclitaxel, após 48 horas de tratamento. Com relação ao ciclo celular, houve acúmulo de células na fase S com a combinação de orlistat com cisplatina e na fase G2 com a combinação de orlistat com paclitaxel. O tratamento das células SCC-9 LN1 com os agentes quimioterápicos reduziu a secreção dos fatores VEGFA e VEGFA165b, em comparação ao tratamento com orlistat isolado ou em combinação. Em conjunto, estes resultados mostram a existência de sinergismo na combinação de orlistat com paclitaxel com evidente potencialização do efeito pró-apoptótico nas células derivadas de CEC oral metastático / Abstract: Fatty acid synthase (FASN) is the main enzyme involved in the neoplastic lipogenesis. The expression of FASN is elevated in many tumor cells, suggesting a role as a metabolic oncoprotein, with the ability to promote growth and survival of tumor cells. There are several compounds that inhibit FASN activity, including orlistat. Orlistat has evident antineoplastic effects in breast and prostate cancers, as well as melanomas. Oral squamous cell carcinoma (OSCC), corresponds to approximately 90% of all cancers that affect the oral cavity, and is diagnosed in advanced stages in most cases. Distant metastases of OSCC are systemically treated with chemotherapeutic agents, such as cisplatin, 5-fluorouracil (5-FU) and paclitaxel. This study aims to investigate the in vitro antitumor effect of orlistat combined with cisplatin, 5-FU and/or paclitaxel in OSSC cells. Highly metastatic tongues squamous cell carcinoma cells (SCC-9-LN1) were treated with these drugs separately or combined with orlistat, in concentrations close to their respective IC50. Next, the cultures were evaluated regarding the rates of cell death (apoptosis and necrosis), cell cycle progression and the secretion VEGFA and VEGFA165b. The highest rate of apoptosis was observed with the combination of orlistat and paclitaxel, after 48 hours of treatment. Cell cycle analysis assay demonstrate as an accumulation of cells SCC-9 LN1 in the S phase, after incubation with the combination of orlistat with cisplatin, and in the G2 phase with orlistat plus paclitaxel. The 5-FU alone, promoted accumulation of cells in the G1/S. Additionally, secretion of VEGFA and was VEGFA165b was inhibited in SCC-9-LN1cells by the combined treatments. These results demonstrate the synergism existence of the mixture orlistat and paclitaxel with potentiation of their pro-apoptotic effects in SCC-9 LN1 cells / Mestrado / Estomatologia / Mestra em Estomatopatologia

Orientador: Francisco Benedito Teixeira Pessine / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T02:12:00Z (GMT). No. of bitstreams: 1 Calderini_Adriana_D.pdf: 8667238 bytes, checksum: 93c066670f54801635af79d770afae4c (MD5) Previous issue date: 2011 / Resumo: 5-Fluorouracil (5-FU) e um dos fármacos mais úteis no tratamento de tumores sólidos em adultos, especificamente carcinomas do trato gastrointestinal (estômago, cólon e reto) e da mama. Em resumo, seu maior efeito bioquímico e a inibição da síntese do DNA, pois a concentração que inibe sua síntese pode ainda permitir a sintese do RNA. Causa severos efeitos adversos como mielo supressão, mucosite, dermatite, diarréia e toxicidade cardíaca. Em razão disto, têm sido realizadas várias tentativas para encapsular 5-FU a fim de reduzir os efeitos adversos que ele provoca. O objetivo deste projeto foi encapsular este anti-neoplásico utilizando nano-esferas de ácido poli(lático-co-glicólico), PLGA, como sistema carreador, acoplando à sua superfície quitosana e folato de quitosana para melhor endereçamento aos locais de ação, bioadesividade e menor toxicidade. A encapsulação de 5-FU em nano-esferas de PLGA foi aperfeiçoada através de planejamento experimental e por análise quimiométrica. Muitos fatores foram estudados: métodos de preparação, temperatura, quantidade inicial de 5-FU e pH. Na caracterização destes sistemas foram utilizadas diversas técnicas: espalhamento dinâmico de luz, determinação do potencial Zeta, calorimetria diferencial de varredura, análise termogravimétrica, difratometria de raios-X e microscopia eletrônica de varredura. Além disso, foram realizados ensaios de perfil de liberação, de estabilidade coloidal e estudo do comportamento desses sistemas em relação às células in vitro. O planejamento experimental permitiu obter nanoparticulas com capacidade de carregamento em torno de 11% e eficiência de encapsulação de 32%. O acoplamento de quitosana e folato de quitosana permitiu retardar a liberação do fármaco em solução. Para armazenagem destas partículas, observou-se que elas foram menos degradadas quando estão liofilizadas e mantidas a 4 °C. A melhor concentração de sacarose para liofilizá-las, sem que ocorra aumento de tamanho e polidispersão, foi 250 mmol/L. Os testes in vitro comprovaram a eficácia destas formulações / Abstract: 5-fluorouracil (5-fluoro-1H-pyrimidine-2,4-dione) is one of the most used drug to treat solid tumors in adults, specifically gastrointestinal (stomach and colorectal) and breast carcinomas. In summary, the major biochemical effect of 5-FU is inhibition of DNA synthesis, since concentrations which inhibit this synthesis may still permit RNA synthesis. It causes severe adverse effects as myelosuppression, mucositis, dermatitis, diarrhea and cardiac toxicity. Its encapsulation in nanoparticles can reduce these adverse effects, prolong its release and the drug can be placed directly on its site of action. The goal of this work was to encapsulate this anti-neoplasic drug using poly (lactic-co-glycolic acid) PLGA nanospheres as carrier system, attaching on their surface chitosan and folate-chitosan to attain an enhanced targeting, bioadesivity and less toxicity. The encapsulation of 5-FU in PLGA nanospheres was improved through an experimental design and chemometry. Many factors were studied: methods of preparation, temperature, initial amount of 5-FU and pH. In the characterization, many techniques were employed: dynamic light scattering, determination of Zeta potential, differential scanning calorimetry, thermo gravimetric analysis, X-ray difractometry and scanning electron microscopy. Besides, we also analyzed the release profile, colloidal stability and the behavior of these systems in relation to in vitro cancer cells. The experimental design allowed obtaining nanoparticles with drug loading around 11% and encapsulation efficiency of 32%. The attachment of chitosan and folate-chitosan also allowed prolonging the drug release in solution. To store these formulations, we observed that lyophilized particles kept at 4 °C were less degraded. The best sucrose concentration to freeze-drying these particles with no size and polidispersity change was 250 mmol/L. The in vitro tests proved the efficacy of these formulations / Doutorado / Físico-Química / Doutor em Ciências

On the basis of clinical studies, some researchers have advocated that the neurohormone and antioxidant melatonin, shown to possess intrinsic anticancer properties, be used as co-therapy in cancer patients being treated with the antineoplastic agent 5-fluorouracil, as increased patient survival times and enhanced quality of life have been observed. The focus of this research was thus to investigate the mechanisms of this seemingly beneficial drug interaction between 5-fluorouracil and melatonin. Metabolism studies were undertaken, in which it was established that there is no hepatic metabolic drug interaction between these agents by cytochrome P450, and that neither agent alters the activity of this enzyme system. Co-therapy with melatonin is thus unlikely to alter plasma levels of 5-fluorouracil by this mechanism. Novel mechanisms by which 5-fluorouracil is toxic were elucidated, such as the induction of lipid peroxidation, due to the formation of reactive oxygen species; decreases in brain serotonin, dopamine and norepinephrine levels, possibly leading to depression; hippocampal shrinkage and morphological alterations and lysis of hippocampal cells, which may underlie cognitive impairment; and a reduction in the nociceptive threshold when administered acutely. All these deleterious effects are attenuated by the co-administration of melatonin, suggesting that the agent exhibits antidepressive and analgesic properties, in addition to its known antioxidative and free radical-scavenging abilities. This suggests that melatonin cotherapy can significantly decrease 5-fluorouracil-induced toxicity, but this may also exert a protective effect on cancer cells and thus compromise the anticancer efficacy of 5-fluorouracil. It was, furthermore, found that stimulation of indoleamine 2,3-dioxygenase activity, mediated by increases in superoxide anion and interferon-γ levels, may underlie resistance to 5-fluorouracil therapy. Melatonin was shown to increase superoxide anion levels in vivo, and this is believed to be by conversion to the metabolite and known oxidant 6- hydroxymelatonin. This highlights that the possible deleterious effects of melatonin metabolites should be studied further. Serum corticosterone levels and cytokine profiles are unaltered by both 5-FU and melatonin, suggesting that these agents may be used by HIV infected individuals without promoting the progression to AIDS. It can thus be concluded that melatonin co-therapy is potentially useful in countering 5-fluorouracil toxicity.

Submitted by Adriana Martinez (amartinez@unoeste.br) on 2017-08-15T19:12:52Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Bianca.pdf: 748434 bytes, checksum: 25858a86b136011052140d31f626de30 (MD5) / Made available in DSpace on 2017-08-15T19:12:52Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Bianca.pdf: 748434 bytes, checksum: 25858a86b136011052140d31f626de30 (MD5) Previous issue date: 2016-11-29 / The purpose of this study was to evaluate the effect of supplementation with L-arginine, in different doses, on body weight, hemogram, production of IgA, IgG and IgM immunoglobulins and in the small bowel inflammatory process of Wistar rats subjected to chemotherapy. A total of 32 Rattus novergicus Wistar rats were randomly distributed in 4 groups (8 rats / group) with similar body weight: control group (CG), the rats were given ration and water; G5-FU was given ration, water and a dose of 5-FU; Garg295 + 5-FU and Garg458 + 5-FU were given, respectively, 295 mg and 458 mg of L-arginine, which was added to the water and a dose of 5-FU. 5-FU was applied after the adaptation period (day 7) in a single dose of 200 mg of 5-FU / kg body weight, intraperitoneally. The rats were weighed individually, always in the morning and without previous fasting, to determine the weight gain before and after the application of 5-FU. Four days after application of 5-FU, the rats were anesthetized with thionembutal, blood and small intestine samples were collected. Supplementation with 295 mg of L-arginine was shown to be beneficial in reducing body weight loss during treatment with the 5-FU chemotherapeutic. The concentration of platelets was lower (P <0.05) in the G5-FU and GArg458 + 5-FU groups, but similar between the GC and GArg295 + 5-FU groups. The total leukocyte concentration showed a significant reduction in the G5-FU, GArg295 + 5-FU and GArg458 + 5-FU groups when compared to the GC group, however, there was a significant reduction of neutrophils only in the G5-FU group. There was an increase (P <0.05) in fibrinogen concentration in the groups treated with L-arginine (GArg295 + 5-FU and GArg458 + 5-FU). In the GArg295 + 5-FU and GArg458 + 5FU groups, the serum IgA concentration increased significantly in relation to the G5-FU group. Rats supplemented with L-arginine had a minor inflammatory process in the small intestine. It was concluded that supplementation with 295 mg of L-arginine / day attenuated the immediate body weight loss of Wistar rats subjected to 5-FU chemotherapy and that daily supplementation with both 295 mg and 458 mg of L-arginine may ease some side effects of 5-FU such as thrombocytopenia, neutropenia and immunomodular production of IgA, in addition to reducing intestinal inflammatory processes. / Este estudo teve o objetivo deste estudo foi avaliar o efeito da suplementação com L-arginina, em diferentes doses, no peso corporal, no hemograma, na produção das imunoglobulinas IgA, IgG e IgM e no processo inflamatório do intestino delgado de ratos Wistar submetidos à quimioterapia. Foram utilizados 32 Rattus novergicus da linhagem Wistar, distribuídos aleatoriamente em 4 grupos (8 ratos/grupo) com peso corporal semelhante: grupo controle (GC), os ratos receberam ração e água; G5-FU receberam ração, água e uma dose de 5-FU; Garg295+5-FU e Garg458+5-FU que receberam ração, 295 mg e 458 mg de L-arginina, respectivamente, adicionada na água e uma dose de 5-FU. A 5-FU foi aplicada após o período de adaptação (dia 7) na dose única de 200 mg de 5-FU/Kg de peso corporal, por via intraperitoneal. Os ratos foram pesados individualmente, sempre no período da manhã e sem jejum prévio, para determinação do ganho de peso antes e depois da aplicação da 5-FU. Quatro dias após da aplicação da 5-FU, os ratos foram anestesiados com tionembutal e colheu-se amostras de sangue e do intestino delgado. A suplementação com 295 mg de L-arginina mostrou-se benéfica na redução de perda de peso corporal durante o tratamento com o quimioterápico 5-FU. A concentração de plaquetas foi menor (P < 0,05) nos grupos G5-FU e GArg458+5-FU, porém, semelhantes entre os grupos GC e GArg295+5-FU. A concentração de leucócitos totais apresentou redução significativa nos grupos G5-FU, GArg295+5-FU e GArg458+5-FU quando comparados ao grupo GC, entretanto, houve redução significativa de neutrófilos somente no grupo G5-FU. Ocorreu um aumento (P < 0,05) na concentração de fibrinogênio nos grupos tratados com L-arginina (GArg295+5-FU e GArg458+5-FU). Nos grupos GArg295+5-FU e GArg458+5FU, a concentração sérica de IgA aumentou significativamente em relação ao grupo G5-FU. Os ratos suplementados com L-arginina apresentaram menor processo inflamatório no intestino delgado. Conclui-se que a suplementação com 295 mg de L-arginina/dia atenuou a perda de peso corporal imediata dos ratos Wistar submetidos à quimioterapia com 5-FU e que a suplementação diária tanto com 295 mg como 458 mg de L-arginina pode amenizar alguns efeitos colaterais da 5-FU como a plaquetopenia, a neutropenia e imunomodular a produção de IgA, além de reduzir processos inflamatórios intestinais.

Nasopharyngeal carcinomas (NPC) is highly induced by Epstein-Barr virus (EBV). EBV infection encoded latent membrane protein 1 (LMP1) is expressed in latent stage II and III of EBV infection nasopharyngeal cells. LMP1 was reported to be associated with increased tumorigenesis, through the activation of nuclear factor-£eB (NF-£eB). In this study, LMP1 overexpressing NPC cell line (TW04) was used for assays of cell proliferation, apoptosis and migration under drug (Sodium Salicylate, Cisplatin and 5-Fluorouracil) treatment. Sodium Salicylate is one of non-steroidal anti-inflammatory (NSAID) drug, which inhibits the downstream of NF-£eB pathway, e.g., COX-1. Cisplatin and 5-Fluorouracil are traditional chemotherapy durgs used in many cancers. Our result shows that overexpression of LMP1 affects cell proliferation, apoptosis , invasion and migration ability. It indicates that LMP1-overexpression is an important marker for NPC therapy.

The present study explored the effect of three anti-tumor drugs (cisplatin, fluorouracil, and temozolomide) on viability and cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells. The effect of cisplatin related mitogen-activated protein kinases (MAPKs) phosphorylation was also examined. Cisplatin at concentration of 25-150 £gM decreased viability in a concentration-dependent manner, and so did fluorouracil (50-1000 £gM) and temozolomide (50-600 £gM). The three anti-tumor drugs all failed to induce a [Ca2+]i increase; thus it seemed that these drugs induced cell death via Ca2+-independent pathways. Immunoblotting showed that OC2 cells have background phospho-ERK, phospho-JNK and phospho-p38 MAPKs. It was found that cisplatin influenced the phosphorylation of ERK, JNK and p38 MAPKs at different time points.

　　Proliferative vitreoretinopathy (PVR) remains the major cause of failure of retinal detachment surgery. Retinal pigment epithelial (RPE) cells have been suggested to play a major role in the pathogenesis of PVR. Numerous studies have employed pharmacological means to modulate cellular activities in attempts to inhibit the process. Recent attempts using adjunctive therapy during PVR surgery that consisted of 5-fluorouracil (5-FU) and low molecular weight heparin showed some promise in preventing PVR but the concern is that prolonged 5-FU treatment may have a toxic effect. On the other hand, lectin from the edible mushroom Agaricus bisporus (ABL) was found to inhibit growth of RPE cells in a potent manner without apparent cytotoxicity. This lectin could be a candidate to modulate anomalous proliferation of RPE cells while the mechanism for the observed inhibition is unknown. 　　In our study, we investigated whether RPE cells treated with 5-FU or ABL would attenuate cellular proliferation, cell migration, cell adhesion and cell-mediated contraction rates. Further, we investigated if complementary inhibition for the above cellular activities could be obtained when RPE cells were treated with ABL after the short treatment using 5-FU. We also explored the possible mechanisms through which ABL inhibited RPE cell proliferation. 　　ARPE-19 and primary human RPE cells were treated with 5-FU or vehicle for 10 minutes. Cells were then maintained in culture medium supplemented with or without ABL. The rate of cellular proliferation was measured by a tetrazolium salt assay. Effects on cell adhesion were investigated through loading RPE cells onto the strips coated with collagen I or fibronectin. Cell migration was investigated using a scratch wound model. The effect on cell-mediated contraction was assessed using a free floating collagen I matrix. Cytotoxicity of 5-FU and ABL was determined by the live/dead assay. 　　To elucidate the mechanism through which ABL inhibited RPE cell proliferation, we investigated cell cycle distribution patterns using flow cytometry. Phosphorylation statuses of Erk, Jnk, p38, Akt as well as p53 and Cyclin D expression level were investigated by Western blotting. 　　Both 5-FU and ABL inhibited RPE cell proliferation. Only ABL promoted cell adhesion towards collagen I in hRPE3 cells. ABL was found to attenuate the rate of cell migration. Cell-mediated collagen gel contraction was attenuated by 5-FU only. Complementary inhibition in cellular proliferation and cell-mediated collagen gel contraction was observed when both 5-FU and ABL were applied. No significant cell death was observed after treatment with 5-FU, ABL or both. 　　ABL was found to reduce the amount of cells present at S phase. Akt and Erk were found to be hypo-phosphorylated and hyper-phosphorylated respectively after ABL treatment. The expression levels of phosphorylated-Jnk, phosphorylated-p38, p53, and Cyclin D1 were not altered when compared with the control. 　　These results showed that 5-FU and ABL complement with each other on inhibiting the wound healing activities of RPE cells in vitro without apparent cytotoxicity. They suggested a possible new treatment modality for PVR. ABL hypo-phosphorylated Akt and this observation is in line with the fact that ABL could attenuate cell proliferation. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

The skin is an amazingly resilient and relatively impermeable barrier that provides protective, perceptive and communication functions to the body (Ramachandran & Fleisher, 2000). The stratum corneum is widely accepted as the barrier of the skin - limiting the transport of molecules into and across the skin. One of the bottlenecks in the successful development of transdermal drug delivery devices is the fact that the skin (more accurately, the stratum corneum - SC) tends to control the rate of drug transport. This makes it very difficult to influence or regulate the transdermal drug absorption kinetics from outside, Le. by means of the vehicle. A possible, and even elegant, solution may be the use of so-called "penetration enhancers", thereby suppressing the dominant role of the stratum corneum penetration barrier (Bodde et al., 1990). For this study 5-fluorouracil (5-FU), a polar hydrophilic drug, was chosen as model drug to study its penetration through the stratum corneum. Terpenes used as possible penetration enhancers for 5-FU were menthol, isomenthol, menthone, l3-myrcene, limonene and 1,8-cineole. In previous studies, terpenes with low skin irritancy and low systemic toxicity, were found to be effective penetration enhancers for a number of hydrophilic and lipophillic drugs (Cornwell & Barry, 1994; Cornwell et a/., 1996; Godwin & Michniak, 1999). The objective of this study was to determine the different flux rates of 5-FU in the absence of any pre-treatment of the stratum corneum and also through ethanol and selected terpene pre-treated SC. The effect of each terpene on the penetration of 5-FU was determined. The penetration of the selected terpenes themselves through the human stratum corneum was also determined in vitro permeation studies were performed using vertical Franz diffusion cells with human skin (stratum corneum). A saturated aqueous solution of 5-fluorouracil in the absence and presence of pre-treatment of the SC was used as the donor phase. Pre-treatment was performed by applying a 5 % terpene solution or absolute ethanol to the SC half an hour before the saturated III solution was applied in the donor compartment. A 50/50 ethanol/water solution was used as the receptor phase. All the experiments were conducted over a 24 h period. The 37°C temperature was held constant by means of a water bath. For the analysis of 5-FU flux rates, samples from the receptor compartment were obtained and were analysed by means of high-pressure liquid chromatography (HPLC). In order to determine the cumulative percentage of terpenes penetrated through human stratum corneum, the samples were analysed by gas chromatography (GC). In this study, only menthol and isomenthol (both oxygen-containing terpenes) showed a statistically significant increase on the flux of 5-FU, with flux values of 1.13 +- 0.38 and 1.45 +- 0.68 ug/cm2/h, respectively, compared to untreated skin with a flux value of 0.54 +- 0.23 ug/cm2/h for 5-FU. It was also proved that ethanol itself had an enhancing effect on 5-FU and showed synergistic effects on the enhancement activities of all the terpenes. It was found that all the terpenes (applied as a 5 % solution in ethanol) penetrated through the stratum corneum in the absence of 5-fluorouracil. 5-Fluorouracil had either an increasing or decreasing effect on the penetration of the terpenes. From these findings, it could be concluded that oxygen-containing terpenes had the best penetration enhancing effect on 5-FU and that menthol and isomenthol were the most effective penetration enhancers, although the extend of penetration enhancement is not large enough for clinical application. All the terpenes have the ability to penetrate through human stratum corneum, and 5-FU either had an increasing or decreasing effect on their penetration. / Thesis (M.Sc.)--North-West University, Potchefstroom Campus, 2004.

The skin is the largest and most easily accessible organ of the human body thus making it the ideal route for systemic drug delivery. The transdermal route of drug delivery offers several advantages compared to the traditional routes including elimination of first pass metabolism and higher patient compliance. However, many drugs are topically and systemically ineffective when applied onto the skin, due to their almost complete failure to penetrate the skin. The main limitation lies in the stratum corneum, the barrier of the skin, which prevent the drug from reaching the deeper skin strata. 5-Fluorouracil is a polar hydrophilic drug and is therefore not a good penetrant through skin. A popular technique to increase transdermal permeation is to use a penetration enhancer, which reversibly reduce the permeability barrier of the stratum corneum. The primary aim of this study was to determine the effect of Brij 97 in the presence and absence of carrageenan on the transdermal delivery of 5-fluorouracil. The formulations were identified by means of confocal laser scanning microscopy and measurement of the particle size. The zeta-potential was measured to determine whether the formulations were stable and the pH was measured to determine if the internal structures of the formulations were affected by the drug. The drug released from the formulations was measured with a VanKel dissolution apparatus. In vitro transdermal diffusion studies were performed using vertical Franz diffusion cells with human epidermal skin. Histopathological studies were carried out on human epidermis skin to determine if the surfactant, Brij 97, had any effect on the skin. Through confocal laser scanning microscopy and particle size measurements, the 4 and 8% Brij 97 formulations without carrageenan could be identified as emulsions while the 15 and 25% Brij 97 formulations without carrageenan could be identified as microemulsions. The 4, 8, 15 and 25% Brij 97 formulations containing carrageenan could be identified as gels. The results obtained from the zeta-potential analysis indicated that the 4 and 8% Brij 97 formulations without carrageenan and 4% Brij 97 formulation with carrageenan are the most electronegative and thus the most stable. The pH measurements confirmed that the internal structure of the formulations was not influenced by the drug. 5-Fluorouracil was released from the formulations. The 4 and 8% Brij 97 formulations without carrageenan had an enhancing effect on the penetration of 5-fluorouracil while the 4, 8, 15 and 25% Brij 97 formulations with carrageenan and the 15 and 25% Brij 97 formulations without carrageenan had an hindering effect on the penetration of 5-fluorouracil. Although carrageenan led to good adhesiveness of the formulation on the skin, it did not lead to the enhancement of the penetration of 5-fluorouracil through the skin. When histopathological studies were carried out on female human abdominal skin, Brij 97, the surfactant, was found to have no damaging effect on the skin structure. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.