Dissertations / Theses on the topic 'Diclofenac'

Os produtos farmacêuticos têm sido considerados como um problema ambiental devido à sua entrada contínua e persistência no ecossistema aquático, mesmo em baixas concentrações (µg L-1 e ng L-1). Muitos fármacos têm sido frequentemente determinados em Estações de Tratamento de Esgoto (ETEs), águas superficiais, subterrâneas e de abastecimento, devido à baixa eficiência dos sistemas convencionais de tratamento na eliminação destes compostos. O diclofenaco (DCF), é um Anti-Inflamatório Não Esteróide (AINE) comumente utilizado como analgésico, antiartrítico e antirreumático. Possui uma baixa biodegradabilidade e tem a capacidade de bioacumulação nos tecidos de seres vivos, podendo apresentar efeitos ecotóxicos. Como o DCF pode não ser eficientemente removido pelos tratamentos convencionais de água e esgoto, novos processos têm sido pesquisados para a sua remoção, entre eles os processos oxidativos avançados (POAs). O objetivo deste trabalho foi tratar o DCF sódico aquoso por fotocatálise heterogênea (TiO2/UV). Para isso foi realizado um planejamento fatorial 25 com a finalidade de se determinar os efeitos dos parâmetros reacionais -- dosagem e tipo de fotocatalisador, concentração inicial do fármaco, tipo de aceptor de elétrons e pH -- sobre o desempenho do tratamento. A variável-resposta observada foi a redução da área sob o espectro de absorção no UV. A condição ótima de tratamento foi: pH = 5; dosagem de TiO2 = 0,5 g L-1; concentração inicial de DCF = 20 mg L-1; aceptor de elétrons = oxigênio; e tipo de catalisador = P25 (Evonik). No estudo da cinética, a degradação do fármaco foi acompanhada por Cromatografia Líquida de Alta Eficiência (CLAE). Para a amostra tratada por fotocatálise heterogênea, a concentração de DCF foi reduzida a aproximadamente 40 µg L-1 em 30 min de irradiação e a degradação seguiu o modelo cinético de Langmuir-Hinshelwood (R2 = 0,95) com constante de velocidade k = (2,3 ± 0,070) × 103 µg L-1 min-1 e uma constante de adsorção K = (2,1 ± 0,17) × 10-4 L µg-1. No ensaio de fotólise a concentração foi reduzida a aproximadamente 70 µg L-1 em 12,5 min de tratamento e observou-se uma cinética de ordem zero (R2 = 0,96) com k = (2,7 ± 0,070) × 103 µg L-1 min-1. Foram identificados alguns intermediários de oxidação por cromatografia líquida acoplada a espectroscopia de massas (LC/MS-MS) e foram realizados ensaios ecotoxicológicos (Daphnia similis e Lactuca sativa) das amostras tratadas. Nenhum dos tratamentos (fotocatálise e fotólise) estudados gerou ecotoxidade nos organismos-teste. Indica-se a fotólise, nas condições deste trabalho, como mais eficiente para a degradação do diclofenaco.
Pharmaceuticals are considered an environmental problem due to their continuous input and persistence in aquatic ecosystems, even at low concentrations (µg L-1 e ng L-1). Many drugs are often detected in Sewage Treatment Plants (STPs), surface water, groundwater and drinking water due to the low efficiency of conventional treatment systems in removing those compounds. Diclofenac (DCF) is a nonsteroidal anti-inflammatory drug (NSAID) commonly used as an analgesic, antiarthritic and antirheumatic. It has low biodegradability and can bioaccumulate in the tissues of organisms, and may have ecotoxicological effects. Since DCF cannot be efficiently removed by conventional water and sewage treatments, new processes have been studied for its removal, including the advanced oxidation processes (AOPs). The aim of this study was to treat aqueous diclofenac by heterogeneous photocatalysis (TiO2/UV). For this purpose, a factorial design 25 was performed in order to determine the effects of the reaction parameters -- dosage and type of photocatalyst, initial concentration of the drug, type of electron acceptor and pH -- on the treatment performance. The response variable observed was the reduction in the area under the UV absorption spectrum. The optimal treatment condition was: pH = 5; TiO2 dosage = 0,5 g L-1; DCF initial concentration = 20 mg L-1; electron acceptor = oxygen; and type of catalyst = P25 (Evonik). In the kinetic study, the drug degradation was determined by High Performance Liquid Chromatography (HPLC). For the sample treated by heterogeneous photocatalysis, DCF initial concentration was reduced to approximately 40 µg L-1 in 30 min of irradiation, and the degradation followed the Langmuir-Hinshelwood kinetic model (R2 = 0,95) with rate constant k = (2,3 ± 0,070) × 103 µg L-1 min-1 and adsorption constant K = (2,1 ± 0,17) × 10-4 L µg -1. During photolysis, DCF initial concentration was reduced to approximately 70 µg L-1 in 12,5 min of treatment and followed a zero-order kinetics (R2 = 0,96) with k = (2,7 ± 0,070) × 103 µg L-1 min-1. Some oxidation intermediates were identified by liquid chromatography coupled to mass spectroscopy (LC/MS-MS) and ecotoxicological tests performed (Daphnia similis and Lactuca sativa) for the treated samples. None of the studied treatments (photocatalysis and photolysis) produced ecotoxicity to the test-organisms. Photolysis is preferable, within the present studied conditions, to degrade aqueous diclofenac.

Orientador: Jose Luiz Donato
Dissertação (mestrado] - Universidade Estadual de Campinas, Facu.dade de Ciencias Medicas
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Resumo: A barreira dérmica é representada por uma estrutura lipoprotéica e constitui um ambiente bioquímico altamente complexo. Trata-se de uma barreira muito eficiente ao ingresso de agentes químicos. Não há na literatura nenhum relato do uso de enzimas proteolíticas como agente potencializador da permeação de fármacos presentes em formulações para uso tópico. Neste trabalho desenvolvemos uma formulação de pomada contendo a enzima para potencializar a penetração de diclofenaco dietilamônio na pele, sendo realizado também o estudo da sua estabilidade na formulação através do acompanhamento da sua atividade catalítica, bem como a avaliação in vivo do seu potencial de alergenicidade cutânea. A atividade da papaína como potencializadora da penetração cutânea de diclofenaco dietilamônio em pomada foi avaliada in vitro através de células de difusão de Franz utilizando pele de porco, e in vivo, por meio de testes clínicos com voluntários humanos. O estudo de estabilidade, revelou que a atividade catalítica da papaina permaneceu estável na formulação quando a mesma foi armazenada a temperatura ambiente por pelo menos 24 meses, porém, sob condição acelerada (45°C e 70% U.R.), a enzima perdeu a sua atividade catalítica após seis meses de armazenamento. Não foi observada nos voluntários humanos, nenhuma reação adversa como eritema, pápulas ou vesículas durante o período de avaliação da alergenicidade cutânea da papaína, sendo a enzima aprovada para uso tópico. No teste de permeabilidade in vitro, foi observado um aumento de 50% no acúmulo de diclofenaco após quatro horas de permeação, quando a papaína esteve presente na formulação de diclofenaco dietilamônio em pomada. O teste de permeabilidade in vivo mostrou que a papaína aumentou a penetração de diclofenaco na pele quando a mesma foi tratada anteriormente com pomada contendo papaína e não quando a enzima foi administrada juntamente com o diclofenaco. Foi demonstrado o potencial da papaína como potencializadora da penetração de antiinflamatórios não-esteroidais (AINEs) em formulações de uso tópico, melhorando consideravelmente a eficiência das mesmas
Abstract: The skin barrier is represented by a lipoprotein structure and is a very complex biochemical environmental. It is considered a very efficient barrier to the absorption of many chemical compounds. There is no report about the use of proteolytic enzymes as a penetration enhancer of therapeutic drugs in topic formulations. In this study, an ointment formulation containing papain was developed to increase the diclofenac diethylammonium skin penetration. Stability studies were performed to verify the enzymatic activity after incorporation in the formulation. It was also evaluated the in vivo allergenecity potential of the papain. The penetration enhancing activity of this enzyme was investigated in vitro through Franz-type diffusion cell using porcine skin, and in vivo, through clinical tests with human volunteers. The stability study showed that papain remained stable in the formulation when it was stored at room temperature during 24 months, however, at accelerated condition ( 45°C and 70% of humidity) the enzyme lost its catalytic activity after 6 month of storage. Regarding in vivo allergenicity studies, the human volunteers no observed adverse reactions as eritema, papulas or vesicle during the period of evaluation, being the papain approved to topical use. On in vitro skin permeation study, an increase of about 50% in the diclofenac accumulation was observed after four hours of permeation when the papain was present in the formulation. The in vivo skin permeation study showed that papain increased diclofenac skin penetration when it was treated previously with ointment containing papain and no when the enzyme was administrated together diclofenac. It was demonstrated the papain potential as penetration enhancer in NSAIDs (non steroidal anti-inflammatory drugs) formulation for topical use improving its efficiency
Mestrado
Mestre em Farmacologia

Mestrado em Química Analítica e Controlo de Qualidade
O diclofenac de sódio é um composto utilizado pela indústria farmacêutica devido às suas propriedades anti-inflamatórias, analgésicas e anti-reumáticas. Em muitos casos o diclofenac é aplicado por via transdérmica, sendo necessário o seu controlo nas formulações, o que acontece pelo conhecimento da permeação do medicamento através da pele. Vários métodos são utilizados para estudar a permeação, mas estes necessitam da recolha da amostra da câmara receptora ao longo da experiência, alterando as condições de permeação. O objectivo deste trabalho é desenvolver um método analítico utilizando uma microbalança de cristais de quartzo para estudar a permeação através da pele, em tempo real. Para a realização do presente trabalho foram utilizadas membranas de celofane e pele humana, e foi estudada a permeação de duas emulsões comerciais: o Voltaren Emulgel® e o Diclofenac Gel Cinfa®. Para ambas, foram determinados o coeficiente de difusão e o fluxo do diclofenac de sódio, tendo-se obtido para a membrana de celofane, valores de D=3,63x10-7cm2/h e J=4,37x10-3mg/cm2.h, para o Voltaren Emulgel® e de D=4,53x10-7cm2/h e J=9,65x10-3mg/cm2.h para o Diclofenac Gel Cinfa®. Para a pele, obteve-se com o Voltaren Emulgel um coeficiente de difusão D=7,84x 10-7cm2/h e um fluxo J=2,36x10-3mg/cm2.h. Os coeficientes de difusão para ambas as membranas são da mesma ordem de grandeza (10-7), o que confirma a utilidade do celofane nestes estudos, como membrana modelo sempre que não é possível a utilização da pele. Quanto à média dos valores do fluxo obtida para a pele, e com o Voltaren Emulgel®, ela é igual ao valor encontrado na literatura, o que permite concluir que o novo método baseado na microbalança de cristais de quartzo conduz a resultados exactos e mesmo mais precisos do que os reportados com a célula de Franz na realização dos estudos de permeação.
Sodium diclofenac is a compound used by the pharmaceutical industry because of its anti-inflammatory, antipyretic and anti-rheumatic properties. In many cases, the diclofenac is applied transdermally, being necessary its control in the formulations, what happens upon the knowledge of the drug permeation through the skin. Several methods are used to study the permeation, but they require the collection of the sample of the receiving chamber throughout the experiment, what changes the conditions of permeation. The purpose of this study is to develop an analytical method using a quartz crystal microbalance to study the permeation through the skin in real time. In the realization of this work, were used cellophane and human skin as membranes, and the permeation of two commercial emulsions, Voltaren Emulgel® and Diclofenac Gel Cinfa®, were studied For both, we determined the diffusion coefficient and the flux of sodium diclofenac. For the cellophane membrane, the values were D=3,63x10-7cm2/h and J=4,37x10-3mg/cm2.h for Voltaren Emulgel®, and D=4,53x10-7cm2/h and J=9,65x 10-3mg/cm2.h for Diclofenac Gel Cinfa®. For the skin a diffusion coefficient of D=7,84x10-7cm2/h and a flux of J=2,36x10-3mg/cm2.h were obtained with Voltaren Emulgel®. The diffusion coefficients found are all in the 10-7 range, which confirms the usefulness of the studies in cellophane as a model membrane, whenever the use skin in not available. The average of the flux values obtained for the skin, with the Voltaren Emulgel®, is equal to the value found in the literature, which proves that the new method based on quartz crystal microbalance leads to accurate results. I fact the results were even more precise than those reported with the Franz cell.

Mestrado em Química - Química Analítica e Qualidade
Na última década, os fármacos têm sido apontados como importantes poluentes ambientais. Estes são compostos biologicamente ativos, podendo ser persistentes e portanto reconhecidos como uma ameaça contínua para o ambiente. Os compostos originais e/ou os respetivos metabolitos estão constantemente a ser depositados no ambiente, sendo a excreção pelo ser humano (urina e fezes) a principal forma de entrada, uma vez que as Estações de Tratamento de Águas Residuais (ETARs) não são, na generalidade, eficazes na sua eliminação. A fotodegradação é uma das principais vias de degradação abiótica de contaminantes nas águas naturais. Neste trabalho, irão ser abordados dois fármacos de classes diferentes, o diclofenac (anti-inflamatório) e o citalopram (antidepressivo). Estes fármacos foram submetidos a fotodegradação direta, através de radiação solar simulada, por forma a estudar a sua persistência no ambiente. Este estudo demonstrou que a velocidade de degradação do diclofenac é maior comparativamente com a velocidade de degradação do citalopram, sendo o tempo de meia vida de 0,057 SSD e 17 SSD, respetivamente. Adicionalmente aos estudos de fotodegradação direta, para o citalopram foi também efetuado o estudo do efeito do oxigénio dissolvido em solução aquosa. Estes estudos foram seguidos pela técnica de cromatografia líquida de alta eficiência (HPLC) e alguns produtos de fotodegradação foram identificados por espetrometria de massa (MS). Para este fármaco foi também efetuado o estudo com a porfirina H2TDMImP como fotossensibilizador por forma a avaliar o seu efeito na velocidade de degradação do citalopram, tendo esta aumentado em cerca de 6 vezes na presença de H2TDMImP.
During the last decade, pharmaceuticals have been identified as severe environmental pollutants. These are biologically active substances that can be persistent and are recognized as a continuous threat to the environment. The original substances and/or their metabolites are constantly thrown into the environment, being human excretions (urine and faeces) the main form of insertion, since Waste Water Treatment Plants (WWTP's) are, in general, not efficient in their elimination. Photodegradation is one of the main processes of abiotic degradation of contaminants in mineral water. This work will approach two pharmaceuticals of different classes: diclofenac (anti-inflammatory) and citalopram (antidepressive). These pharmaceuticals were subjected to direct photodegradation, through simulated solar irradiation, in order to assess their persistence in the environment. It was verified that the degradation rate of diclofenac is higher than that of citalopram, being their half lifetime of about 0.057 SSD and 17 SSD, respectively. Besides these direct photodegradation experiments with both pharmaceuticals, the effect of dissolved oxygen in the aqueous solution was also studied with citalopram. These tests were followed by High-Performance Liquid Chromatography (HPLC), and some of the products of photodegradation were identified by mass spectrometry (MS). Moreover, an experiment with the H2TDMImP porphyrin as a photosensitizer was also performed, in order to assess its effect on the degradation rate of citalopram, which increased over 6 times in the presence of the H2TDMImP.

FundaÃÃo de Amparo à Pesquisa do Estado do CearÃ
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
A periodontite à definida como uma doenÃa inflamatÃria dos tecidos de suporte dos dentes, causada por microorganismos, caracterizada por infiltraÃÃo de leucÃcitos, destruiÃÃo progressiva dos ligamentos periodontais e osso alveolar. O esclarecimento do papel de prostaglandinas na reabsorÃÃo Ãssea periodontal tem contribuÃdo para a utilizaÃÃo mais racional de fÃrmacos inibidores das ciclooxigenases disponÃveis no mercado. Nesse sentido, torna-se necessÃrio o estudo da seguranÃa, nÃo obstante sua eficÃcia, destes agentes antiinflamatÃrios nÃo esteroidais, como celecoxib ou diclofenaco potÃssico. No presente estudo, utilizou-se um modelo de periodontite induzida por corpo estranho em ratos descrito na literatura, com o objetivo de avaliar a capacidade do celecoxib e do diclofenaco potÃssico. Observou-se que a inserÃÃo cirÃrgica do fio de nÃilon no modelo estudado induziu a perda Ãssea alveolar de forma significante aos 11 dias. Celecoxib nas doses diÃrias de 3, 9 e 27 mg/kg foi capaz de inibir a perda Ãssea alveolar de forma significante e dose-dependente (64%, 53% e 75,4%, respectivamente). O resultado com diclofenaco nas doses de 1 e 5 mg/kg tambÃm reduziu de forma significante, a perda Ãssea alveolar (41% e 54,5%, respectivamente). A anÃlise macroscÃpica dos estÃmagos mostrou que celecoxib e diclofenaco nÃo promoveram lesÃes gÃstricas de forma significante quando comparados aos animais nÃo tratados. O uso de celecoxib nÃo causou alteraÃÃes, de forma significante, no hemograma dos animais submetidos à periodontite. Com diclofenaco, verificou-se uma leucocitose em decorrÃncia do aumento do nÃmero de neutrÃfilos, com maior pico no perÃodo compreendido entre o 7 e 11 dia apÃs o procedimento cirÃrgico. A nÃo alteraÃÃo do nÃmero de plaquetas do sangue dos animais submetidos à periodontite, inclusive nos animais que receberam celecoxib ou diclofenaco pode sugerir que as doses utilizadas foram relativamente seguras sob o ponto de vista cardiovascular ou ainda, possivelmente, tais alteraÃÃes nÃo foram vistas por nÃo se tratar de um estudo prolongado. Tanto o celecoxib como o diclofenaco nÃo foram capazes de reverter, de forma significante, a perda de massa corpÃrea. As maiores doses de diclofenaco (10 e 25 mg/kg) causaram reduÃÃo significante da taxa de sobrevida dos animais a partir do primeiro dia pÃs-operatÃrio e atingindo 50% dos animais no terceiro dia. Os resultados mostraram que a induÃÃo da periodontite em ratos no grupo controle e no grupo tratado com celecoxib, nÃo alterou as funÃÃes renais ou hepÃticas avaliadas (urÃia e creatinina ou AST). Contudo, o uso de diclofenaco, tanto na dose de 1 mg/kg como tambÃm na dose de 5 mg/kg determinou alteraÃÃes em ambas as funÃÃes consideradas, induzindo um aumento significativo nos nÃveis sÃricos de creatinina e AST. O diclofenaco e o celecoxib apresentaram efeitos protetores semelhantes na perda Ãssea. O celecoxib, utilizado na periodontite induzida durante 11 dias, foi menos tÃxico que o diclofenaco, uma vez que este causou maior mortalidade de forma dose-dependente, e alteraÃÃes ao nÃvel de leucograma e das funÃÃes renal e hepÃtica
Periodontitis is an inflammatory disease of the tissue that supports the teeth. It is caused by microorganisms and is characterized by leukocyte infiltration, progressive destruction of the periodontal ligaments, and alveolar bone. The clear role of prostaglandins on periodontal bone resorption has contributed to the rational use of the cyclooxygenase inhibitors drugs available. In this sense, it becomes necessary to bare safety and efficacy studies of the non-steroidal anti-inflammatory agents, such as celecoxib and potassium diclofenac. For the present study, a foreign object induced periodontitis model in rats was used, such as described on specific literature, to evaluate the activity of celecoxib and potassium and diclofenac. A surgical insertion of a nylon tread induced significant alveolar bone loss after 11 days. Celecoxib, given daily at 3, 9 and 27 mg/kg, significantly reduced this loss in a dose-dependent manner (64%, 53% and 75.4%, respectively). Diclofenac produced a similar effect at 1 and 5 mg/kg, reducing the loss by 41% and 54.5%, respectively. Macroscopic analyses of stomachs indicated that neither celecoxib nor diclofenac promoted gastric lesions when compared with non-treated animals. Celecoxib-treated rats did not show significant hemogram parameters alterations when subjected to periodontitis. For diclofenac-treated animals, it was verified a leukocytosis due to the augmentation of neutrophil count, which peaked between the 7th and the 11th day post-surgery. Platelet number of periodontitis-subjected animals, including those that received celecoxib or diclofenac treatment, was not altered, which may suggest that the doses used are relatively safe from the cardiovascular point of view, or that this alterations was not seen due to the short period of the study. Celecoxib and diclofenac were not able to significantly reverse the loss of body mass. The higher doses of diclofenac (10 and 25 mg/kg/day) significantly reduced the survival rate since the first day after surgery, reaching 50% at day 3. The induction of periodontitis in control and celecoxib-treated rats did not alter renal or hepatic function according to the biochemical parameters evaluated (urea and creatinine or AST). However, diclofenac, at 1 and 5 mg/kg/day, determined alterations in both kidney and liver functions, with a significant increase of seric levels of creatinine and AST. Diclofenac and celecoxib presented similar effects on bone loss prevention. Celecoxib, used for 11 days to induce periodontitis, was less toxic than diclofenac, which caused a dose-dependent mortality and leukogram alterations along with disruption of renal and hepatic functions

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Biodegradable films prepared from biopolymers such as polysaccharides, proteins, lipids or combinations of those components have received great interest in recent years. Xylan is the major hemicellulose in the plant kingdom and it accounts for onethird of renewable biomass available on earth. The aim of this work was to develop and characterize biodegradable films of xylan from corn cobs with potential use as transdermal formulations. Films were produced by casting the film-forming dispersions with different concentrations of xylan and glycerol. In addition, the gelatin was incorporated to xylan films. According to macroscopic characterization, three films were selected to evaluate the thickness, solubility, biodegradability, opacity, water content and microscopic morphology of the films. The best formulation was selected to study the incorporation of the drug (drug content and release profile). The results show that the macroscopic aspect of xylan/gelatin films was better than films containing only xylan, probably due the high solubility of the gelatin in the films. The increase in the polysaccharide concentration affected the films solubility, opacity, water content and thickness of films. The drug content was 87.88% and the drug release occurred mainly in the first 4h, followed by a slow release until the end of the study.
Filmes biodegradáveis desenvolvidos a partir de biopolímeros como polissacarídeos, proteínas, lipídeos ou através da combinação destes polímeros tem recebido grande interesse nos últimos anos. A xilana é a principal hemicelulose presente no reino vegetal e é responsável por um terço da biomassa renovável disponível na terra. O objetivo deste trabalho foi desenvolver e caracterizar filmes biodegradáveis à base xilana de sabugo de milho com potencial uso em formulações transdérmicas. Os filmes foram produzidos através da secagem das dispersões filmogênicas com diferentes concentrações de xilana e glicerol. Adicionalmente, a gelatina foi incorporada aos filmes. De acordo com a caracterização macroscópica foram escolhidas três formulações para caracterização de espessura, solubilidade, biodegradabilidade, opacidade, conteúdo de água e análise microscópica. Com base nestes resultados foi escolhido o filme para os estudos de incorporação do fármaco (teor e perfil de liberação). Os resultados mostraram que o aspecto macroscópico dos filmes de xilana/gelatina foi melhor que o dos filmes de xilana, isso ocorreu provavelmente devido à alta solubilidade da gelatina. O aumento da concentração de xilana afetou a solubilidade, conteúdo de água, espessura e opacidade dos filmes. O teor de diclofenaco sódico presente no filme foi de 87,88% e a liberação do fármaco se deu principalmente nas primeiras 4 horas, seguida de uma liberação lenta até o final do estudo.

Para se garantir a eficácia, a segurança e a qualidade de um produto farmacêutico é necessário o conhecimento das propriedades físicas e físico-químicas do fármaco e dos excipientes empregados nas formulações, bem como dos procedimentos relacionados aos processos de produção. Conhecer as propriedades do estado sólido é de fundamental importância e relevância na área farmacêutica uma vez que estas propriedades têm um impacto profundo sobre a solubilidade, biodisponibilidade e estabilidade química dos fármacos. Os dados de difração de raio-X, ressonância quadrupolar nuclear, espectroscopia de infravermelho, análise térmica e microscopia foram usados para a caracterização e identificação das diferentes amostras de diclofenaco de sódio, duas comercializadas no Brasil (DPB1 e DPB3) e uma na Argentina (DPA2). Foi observada a presença das formas anidro, pentahidrato e desconhecida nas formulações comerciais DPB3, DPA2 e DPB1, respectivamente. A análise quantitativa do diclofenaco de sódio para os estudos in vitro de dissolução foi realizada por cromatografia líquida de alta eficiência empregando uma coluna de fase reversa C-18 e acetonitrila:ácido acético 0,7 mol/L (1:1, v/v) como fase móvel. No meio de dissolução composto por tampão fosfato de sódio 0,2 mol/L, pH 6,8 foi observada uma dissolução de 100% para as três formulações de diclofenaco de sódio em 1 hora. Para o meio de dissolução composto por tampão fosfato de sódio 0,2 mol/L, pH 4,5 a porcentagem do fármaco dissolvido foi de apenas 4% nas formulações avaliadas. Entretanto, diferenças na solubilidade entre as formulações avaliadas foram observadas, o que pode ser devido às diferenças na estrutura cristalina do diclofenaco de sódio. Também foram realizados estudos de dissolução apenas nas amostras anidro e hidrato, sem a interferência de revestimentos ou excipientes, nos meios de dissolução descritos acima e em solução de HCl 0,1 mol/L pH 1,2. Foi observado que a amostra anidra apresentou uma diferença estatisticamente significativa no perfil in vitro de dissolução comparada a forma hidratada em solução de pH 6,8. Para os demais valores de pH não foram observadas diferenças estatisticamente significativas nos perfis de dissolução. Também foi desenvolvido e validado um método para análise do diclofenaco de sódio em plasma, empregando a coluna RP-18 (125x4,6 mm, partículas de 5 m) protegida por uma coluna de guarda RP-18 (4,0x4,0 mm) e fase móvel composta por acetonitrila:ácido acético 0,7 mol/L (1:1, v/v). Foi realizada a extração líquido-líquido como procedimento de preparação da amostra usando uma mistura de hexano:éter (1:1, v/v) como solvente extrator. O método foi validado avaliando os parâmetros linearidade, recuperação, precisão e exatidão, limite de quantificação e estabilidade. Todos os parâmetros avaliados apresentaram resultados adequados e aceitos pela literatura. Posteriormente, o método desenvolvido e validado foi aplicado em um estudo piloto para avaliar a concentração plasmática do diclofenaco de sódio em coelhos. Também foi avaliada a influência das diferentes formas cristalinas do diclofenaco de sódio na resposta febril induzida por LPS em coelhos. Neste estudo não foi observada nenhuma diferença estatisticamente significativa na redução da resposta febril para as diferentes amostras avaliadas.
To assure effectiveness, security and quality of pharmaceutical products, the knowledge of the physical and chemical-physical properties of the drugs and the excipients used in formulations are necessary, as well as the proceeding related to the production processes. To know the properties of the solid state is important and relevant in the pharmaceutical area because they have a deep impact on the solubility, bioavailability and chemical stability of the drugs. Data of X-ray diffraction, nuclear quadrupole resonance, infrared spectroscopy, thermal analysis and scanning electron microscopy have been used for the characterization and identification of the different samples of diclofenac sodium. The presence of anhydrous, pentahydrate and unknown forms were observed in commercial formulations DPB3, DPA2 and DPB1, respectively. Quantitative analysis of the diclofenac sodium for studies in vitro of dissolution was carried out by high performance liquid chromatography using the column reverse phase C-18 and acetonitrila:acetic acid 0,7 mol/L (1:1, v/v) as mobile phase. Release profiles in vitro of dissolution of commercial diclofenac sodium formulations were evaluated using different dissolution medium. In the phosphate buffer solution 0.2 mol/L pH 6.8, it was observed dissolution of 100% for the three formulations of diclofenac sodium in 1 hour while in the phosphate buffer solution 0.2 mol/L pH 4.5, the percentage of drug dissolved was only 4%. However, differences in the solubility between the formulations evaluated were observed, which can be due to the differences in the crystalline structure of diclofenac sodium. Dissolution studies in the samples anhydrous and hydrate were carried out, without the interference of coverings or excipients, in the dissolution medium described above and in the solution 0.1 HCl mol/L pH 1.2. It was observed that the anhydrous sample showed a significant statistical difference in the in vitro dissolution profile when compared with hydrate form (pH 6.8). For the others values of pH, significant statistical differences in the dissolution profiles were not observed. It was also developed and validated a method of analysis of diclofenac sodium in plasma using the column RP-18 (125x4.6mm, particle size 5 µm) protected by a column of guard RP-18 (4.0x4.0 mm) and acetonitrila:acetic acid 0,7 mol/L (1:1, v/v) as mobile phase. Sample preparation was performed by liquidliquid extraction using hexano:ether as extracting solvent after acidification with 2.0 mol/L hydrochloric acid. The method was validated by evaluation of parameters such as linearity, recovery, precision and accuracy, limit of quantification and stability. All the evaluated parameters had presented results adequate and accepted in the literature. Subsequently, the developed and validated method was applied in a pilot study to evaluate the plasmatic concentration of the diclofenac sodium in rabbits. It was also evaluated the influence of the different crystalline forms of the diclofenac sodium in the LPS induced fever in rabbits. In this study no significant statistical difference was observed in the reduction of the febrile response within the different samples evaluated.

Os resíduos de fármacos presentes em matrizes ambientais têm sido foco em pesquisas no mundo todo. Este tema tem sido bastante discutido devido ao fato de que fármacos são freqüentemente encontrados em efluentes de estações de tratamento de esgotos (ETE´s), águas de abastecimento público e em outras matrizes ambientais, tais como solos, sedimentos e águas naturais em concentrações na faixa de µg L-1 e ng L-1. A grande preocupação da presença de resíduos de fármacos na água são os potenciais efeitos adversos para a saúde humana, animal e de organismos aquáticos. Neste trabalho estudou-se o diclofenaco sódico, por ser um dos antiinflamatórios mais prescritos pelos médicos. O método utilizado para a extração do diclofenaco sódico de amostras de efluentes domésticos da ETE de Araraquara-SP foi à extração em fase sólida, e subseqüentemente a determinação por cromatografia líquida de alta eficiência com detector UV. O método foi validado e a recuperação foi de 94-105%. Constatou-se a presença do diclofenaco sódico nas amostras do efluente doméstico da cidade de Araraquara-SP antes e após o tratamento e as concentrações foram 2,12 e 3,52 µg L-1 na coleta feita em março e 18,0 e 22,0µg L-1 na coleta feita em setembro.
The pharmacos residues that are present in the environmental matrices has been a focus of research all over the world. This subject has been discussed because the fact that pharmacos are frequently found in effluents of sewage treatment plants (STPs), public water supply and in others environmental matrices, such as the soil, sediments and water springs in concentrations between µg L-1 and ng L-1. The biggest concern of pharmacos residues in the water are the adverse effects for the human health and the other species too. So, in this research the sodium diclofenac was studied for being the most prescribed anti-inflammatory by the doctors. The method used for the extraction of the sodium diclofenac of samples from the domestic effluent at STP-Araraquara (SP) was the extraction in solid phase, and subsequently the determination by liquid chromatography of high efficiency with UV detector. The method was validated and the recovery was of 94 to 105%. The results of the research have shown the presence of sodium diclofenac in the samples of Araraquara\'s domestic effluent before and after the treatment and the concentrations were 2,12 and 3,52 µg L-1 in the collection made in March and 18,0 and 22,0µg L-1 in the collection made in September.

Over the last decade, three species of Gyps vultures on the Asian subcontinent have declined dramatically in population numbers, some as much as 97 to 99%. Although the initial cause was believed to be infectious, it was later shown to be due to an inadvertent exposure to diclofenac via the food chain. In order to protect the remaining wild vultures, diclofenac needed to be removed from the food chain. Unfortunately the Indian government was reluctant to ban diclofenac until an alternate veterinary non-steroidal anti-inflammatory drug (NSAID) that was both safe in vultures and effective in cattle could be identified. Although meloxicam was tentatively identified as this drug, toxicity testing still needed to be undertaken. Using a previously validated model, two studies were undertaken to determine the acute toxic effect of diclofenac in vulture as well as to ascertain if the drug had the potential to accumulate. In the first study, meloxicam in formulation was shown to be safe as a single oral dose up to 2mg/kg in African White Backed-Vultures (Gyps africanus). To further demonstrate the safety of food borne meloxicam, vultures were exposed to meat rich in meloxicam residues, with once again no signs of toxicity being evident. In the second study the drugs ability to accumulate was evaluated pharmacokinetically in Cape Griffon Vultures (Gyps corprotheres). From this study meloxicam was shown to have a very short half-life of elimination, making it unlikely that the drug could be a cumulative toxin. This was subsequently confirmed clinically by the absence of toxicity in birds receiving repeated doses of meloxicam. Although meloxicam was shown to be adequately safe, the safety of other veterinary NSAIDs still required elucidation. While further testing in vultures would have been possible, the small population size of the various vulture species made this unethical. Therefore a surrogate species needed to be identified. With the domestic chicken (Gallus domesticus) being commonly available, attempts were made to validate the chicken as a model. Although the dosed chickens did show similar toxicity patterns from clinical pathology to histopathology, a major problem was their higher tolerance making it impossible to use them as a surrogate. It was, however, concluded that the domestic chicken may be used in mechanistic studies in an attempt to establish an in vitro model. From the mechanistic studies both diclofenac and meloxicam were directly toxic to chicken and vulture renal tubular epithelial cells following 48h of incubation. It was later shown that this toxicity was associated with an increased production of reactive oxygen species (ROS), which could be temporarily ameliorated by pre-incubation with uric acid due to its anti-oxidant activity. When cultures were incubated with either drug for only two hours, meloxicam showed no toxicity in contrast to the cellular toxicity present for diclofenac. In both cases no increase in ROS production was evident. In addition diclofenac influenced the excretion of uric acid by interfering with p-amino-hippuric acid channels. The effect on uric acid excretion persisted after the removal of the diclofenac. It was therefore concluded that vulture susceptibility to diclofenac results from a combination of an increase in cellular ROS, a depletion of intracellular uric acid concentration and most importantly the drug’s long half-life in the vulture. Unfortunately the importance of the drug’s half-life in the toxicodynamics makes it unlikely that in vitro testing will be possible.
Thesis (PhD (Paraclinical Sciences))--University of Pretoria, 2007.
Paraclinical Sciences
unrestricted

Diclofenac has previously been shown to be toxic in three species of Gyps vultures (G. bengalensis, G. tenuirostris, and G. indicus) on the Indian subcontinent. Due to the devastating effect on the population of vultures (>99.9% species mortality), numerous efforts were initiated in order to protect the species. One such effort involved the removal of further threats to the species. At present the major threat identified has been the other non-steroidal drugs (NSAIDs) available for veterinary use. From research on ketoprofen and meloxicam (the former toxic and the latter safe), it was evident that toxicity was not general for the class of NSAIDs and that other factors played a role in toxicity. This unfortunately meant that each drug had to be tested individually in the vulture. While possible, the endangered status of vulture globally makes this approach unethical. As a result an alternate method of testing needed to be validated or sought. It was believed that a surrogate model could be the answer. The aim of this study was to establish if the crow could serve as such a surrogate model. The toxic effect of diclofenac in crows (n=6) was evaluated in a two cross over studies at doses of 0.8 and 10 mg/kg. No signs of toxicity were evident during the period of clinical monitoring, or necropsy or clinical pathology. In addition the drug was barely detectable in the birds and was described by a half-life of elimination of approximately 2.5 hours. To better explain the absence of observable toxicity, a follow-up study was initiated using freshly harvested renal tubular epithelial (RTE) cells and hepatocytes in a cell culture assay previously validated for cytotoxicity and reactive-oxidative generation. In general, the in vitro study results showed the hepatocytes and RTE cells to be tolerant to the presence of diclofenac, with cell viability remaining in the region of 80%. In contrast meloxicam appeared to be more toxic as previously seen with chicken primary RTE cells. Based on the in vivo and in vitro culture results, it was speculated that the absence of toxicity in the crow was due to a combination of rapid half-life of metabolism in combination with low susceptibility of the cells to toxicity. To further explain the role of metabolism in toxicity, meta-analysis of pharmacokinetic data for the domestic chicken (Gallus gallusDissertation (MSc)--University of Pretoria, 2011.
Paraclinical Sciences
unrestricted

Diclofenac toxicity in old world vultures is well described in the literature. As part of the toxicity of the drug, dead birds were generally found in the environment with signs of severe renal damage and gout. In birds that were tested before death, signs of hyperuricaemia and hyperkalaemia were also present. Although the clinical picture is very clear, the sequence of hyperuricaemia is not yet established with one possibility being drug induced renal damage leading to hyperuriceamia and the second being secondary kidney damage resulting from hyperuricaemic cellular damage. For this study we evaluated this sequence by assessing the effect of diclofenac on uric acid transporters in the chicken, a validated model for diclofenac toxicity. We speculated that diclofenac, a known uricosuric drug in people, inhibits the avian organic anionic transporters (OATs) with subsequent increase in plasma uric acid, precipitation and kidney damage. As a first step, the impact of diclofenac was evaluated in healthy chicken as it was not justifiable to kill vultures through diclofenac administration. For this two-phase study heathy chickens were treated intravenous with para-amino hippuric acid (PAH) and iohexol (IOH) in combination in phase 1 or this same combination with diclofenac (10 mg/kg) in phase 2. In both phases blood and faeces were sequentially collected. In phase 1, birds showed no signs of ill health, moreover PAH, IOH and uric acid clearance was rapid. In phase 2, two chickens eventually died with hyperuricaemia being present as soon as 8 hours after exposure. Necropsy showed classic signs of renal damage and hyperuricaemia. Pharmacokinetic analysis revealed a rapid half-life of elimination of less than 2 hours indicating that toxicity was due to irreversible inhibition of a physiological process. In phase 2 all the birds had decrease uric acid, PAH and IOH clearance. While tubular excretory rates of PAH were reduced in all birds in phase 2, they were 98% reduced in the two birds that died. Based on the global change in clearance parameters, it is concluded that diclofenac alters both renal perfusion (IOH measures glomerular filtration) and renal plasma flow. However death results from tubular secretion being reduced to negligible functionality for a prolonged period. With birds being highly reliant on tubular secretion of uric acid, we conclude that diclofenac hyperuricaemia is as a result of OAT inhibition and that cell death results secondarily from uric acid precipitation as described in gouty chicken. In the final conclusive study, we used next generation sequencing of the transcriptome of the renal tissue from one African white backed vulture (AWB), to establish if these tissues expressed OATs and multidrug resistance protein (MRP) transporters. Both these channels are known to be involved with uric acid transport either basolaterially or apically respectively. The Trinity assembled transcriptome, was used to create a local blast database from which predicted sequences of OAT1 and 2 and MRP2 and 4 from the Golden Eagle were evaluated for similarity. The golden eagle was selected as it was the closest related species phylogenetically that also had a complete genome sequence published. OAT3 was not included in this study as no avian sequence was available on the NCBI database. From the search, all four channels were identified in the AWB vulture kidney with high similarity to the golden eagle. Sanger sequencing confirmed the presence of the OAT 1, 2 and MRP2, 4 related mRNA. The predictive amino acid sequence and predictive protein channel (Swiss-Prot) was also used to provide some evidence that the proteins in question shared the required characteristic and function of OAT and MRP channels. After in silico analysis revealed the similarity of only AWB OAT2 gene with other species i.e chicken, expression study was carried out. It revealed that chicken OAT2 gene was expressed more than the vulture, this maybe the reason to vulture sensitivity to diclofenac. With the genes showing the presence of the requisite uric acid transport proteins in the kidney, the distribution of the OAT channels in the vulture was confirmed by immunohistochemistry (IHC) using mouse polyclonal OAT3 antibodies as sequence analysis showed high similarity between vulture OAT1 and mouse OAT3. As expected the IHC showed the presence of OAT1 with good distribution along the renal tubules.
Thesis (PhD)--University of Pretoria, 2019.
Paraclinical Sciences
PhD
Unrestricted

Diclofenac is commonly used 'off-label' for acute pain in children, and it has been shown to be effective for this indication. There is a five-fold range (0.5 to 2.5mg/kg) in dosing of diclofenac for acute pain in paediatric clinical studies, and little published safety information is available. The metabolism of diclofenac to 4'-hydroxydiclofenac is mediated by CYP2C9, the expression of which may differ during development. Three studies have been undertaken to answer the questions: What dose of diclofenac should be given to children with acute pain What are the adverse effects of diclofenac in children treated for acute pain Does the expression of CYP2C9 change with age in children aged one to 12 years The three studies carried out were: A population pharmacokinetic study on a paediatric day surgery ward investigating a new diclofenac oral suspension, results pooled with adult data supplied by the manufacturer and analysed with NONMEM to produce dosing guidelines a clinical safety study to ascertain common adverse reactions of diclofenac in children with acute pain, followed by a systematic literature review to investigate the type and incidence of rare adverse effects and an investigation of the influence of age and CYP2C9 genotype on the formation of 4'- hydroxydiclofenac in children aged one to 12 years using data collected during the pharmacokinetic study. The optimum dose of diclofenac for acute pain in children is lmg/kg. Diclofenac appeared to cause similar types of adverse reactions in children and adults, although the incidence of gastrointestinal bleeding is possibly lower in children. When diclofenac is used as part of the analgesic regimen in the peri-operative period, children suffer less nausea and vomiting, and no increase in operative bleeding. No differences were found in the expression of CYP2C9 estimated using diclofenac 4'-hydroxylation in children aged one to 12 years, which would appear to confirm in vitro findings in paediatric liver samples.

Residuos de fármacos estão presentes em diversas matrizes ambientais e estudos focados na determinação destes tem ganhado grande importância nos últimos anos, devido ao aumento do consumo de medicamentos pela população. A questão do controle de resíduos de compostos farmacologicamente ativos no meio ambiente aquático foi reconhecida como uma das questões emergentes na Química Ambiental, e tem-se dado maior importância visto que os fármacos são encontrados em matrizes em estudos em concentrações de μgL-1 e ngL-1. Nesta pesquisa estudou-se três fármacos antiflamatórios que são amplamente consumidos pela população: diclofenaco, nimesulida e paracetamol. O método analítico foi desenvolvido e validado para a determinação destes fármacos em amostras de águas superficiais da cidade de São Carlos (SP). Inicialmente foi feita a validação do método proposto segundo a Resolução DOQ-CGCRE-008 do INMETRO. Os limites de detecção, e de quantificação e inferior de quantificação do método para a determinação do diclofenaco, nimesulida e paracetamol, foram, respectivamente, 0,5; 1,1 e 1,1 μgL-1. A linearidade, desvio-padrão relativo, exatidão e recuperação média para o diclofenaco foram, respectivamente, R de 0,99, 3,03%, 100,55% e 97,94%. Para a nimesulida, os valores de linearidade, desvio-padrão relativo, exatidão e recuperação, foram, R de 0,98, 2,43%, 101,46% e 100,67%. Já para o paracetamol obteve-se os seguintes valores para linearidade, desvio-padrão relativo, exatidão e recuperação, R de 0,99, 3,50%, 97,94% e 93,17%, respectivamente. Na segunda etapa deste estudo aplicou-se o método validado na análise de amostras de águas coletadas na cidade de São Carlos (SP). Para o método de extração utilizou-se a extração em fase sólida (SPE) e como técnica analítica utilizou-se o HPLC/DAD. Os resultados não indicaram a presença dos fármacos diclofenaco, nimesulida e paracetamol até o limite de detecção do método empregado.
Residues of drugs are present in various environmental matrices and studies focused on the determination of these have gained in importance in recent years, due to increased drug consumption by the population. The issue of control of residues of pharmacologically active compounds in the aquatic environment was recognized as one of the emerging issues in environmental chemistry, and has given greater importance since the drugs are found in studies in matrices at concentrations μgL-1 and ngL-1. In this study was studied three drugs that are widely consumed by the population: diclofenac, nimesulide and acetaminophen. The analytical method was developed for the determination of these drugs in surface water samples from São Carlos (SP). Initially, made a validation of the method proposed second resolution DOQ-008-CGCRE INMETRO. The detection, quantification and lower quantification limits of method for determining of diclofenac, nimesulide and paracetamol were 0.5, 1.1 and 1.1 μgL-1, respectively. The linearity, relative standard , accuracy and average recovery of the method for diclofenac were, respectively, R equal to 0.99, 3.03%, 100.55% and 97.94%. For nimesulide, the values of linearity, relative standard, accuracy and recovery were R equal to 0.98, and 2.43%, 101.46% and 100.67%. For acetaminophen obtained the following values for linearity, relative standard, accuracy and recovery, R equal to 0.99, 3.50%, 97.94% and 93.17% respectively. In the second stage of the study applied the validated method in the analysis of water samples collected in the São Carlos (SP). For extracting the drugs, SPE cartridges were used followed by HPLC / DAD. The results indicate the absense of the studied drugs diclofenac, nimesulide and acetaminophen down to the detection limits of the method employed.

Orientador: Juliana Heloisa Pinê Américo-Pinheiro
Resumo: A qualidade da água é um assunto de crescente preocupação, especialmente devido à presença de fármacos que contaminam o ambiente aquático. Muitos compostos têm sido detectados em efluentes de estações de tratamento de esgoto (ETEs) e águas superficiais em todo o mundo. A ocorrência de fármacos no ambiente pode apresentar efeitos adversos aos ecossistemas aquáticos. O objetivo desse estudo foi avaliar a presença e a concentração dos fármacos diclofenaco, ibuprofeno e naproxeno durante março de 2017 a fevereiro de 2018 em 7 pontos de amostragem, sendo 5 pontos no Córrego das Marrecas - SP e 2 pontos na ETE do município de Dracena - SP. Em cada ponto do córrego foi mensurada a concentração de oxigênio dissolvido (OD), pH, temperatura e sólidos totais dissolvidos (SDT) com auxílio de uma Sonda Multiparamétrica Aquaread AP 2000. Para a identificação dos fármacos, as amostras foram preparadas por microextração líquido – líquido dispersiva e analisadas por cromatografia líquida de alta eficiência. Foi realizada uma análise descritiva para avaliação dos resultados de média, desvio padrão e coeficiente de variação dos parâmetros físico-químicos. Uma matriz de correlação foi aplicada para avaliar a interação entre os parâmetros físico-químicos da água e os fármacos dois a dois. Os três fármacos foram detectados em todos os pontos da ETE e do córrego. A maior concentração do diclofenaco (0,458 mg.L-1) foi registrada no ponto de lançamento do efluente da ETE no mês de março. Nesse ponto ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Water quality is a subject of increasing concern, especially due to the presence of drugs that contaminate the aquatic environment. Many compounds have been detected in effluents from sewage treatment plants (ETEs) and surface water worldwide. The occurrence of drugs in the environment may have adverse effects on aquatic ecosystems. The objective of this study was to evaluate the presence and concentration of the drugs diclofenac, ibuprofen and naproxen during March 2017 to February 2018 in 7 sampling points, 5 points in the Stream of Marrecas - SP and 2 points in the TTE of the municipality of Dracena - SP The concentrations of dissolved oxygen (DO), pH, temperature and total dissolved solids (TDS) were measured at each point of the stream using an Aquaread AP 2000 Multiparameter Probe. For drug identification, the samples were prepared by dispersive liquid-liquid microextraction and analyzed by chromatography high efficiency liquid. A descriptive analysis was performed to evaluate the results of mean, standard deviation and coefficient of variation of physical-chemical parameters. A correlation matrix was applied to evaluate the interaction between the physicochemical parameters of water and drugs two to two. All three drugs were detected at all points of the TEE and the stream. The highest concentration of diclofenac (0.458 mg.L-1) was recorded at the point of launch of the ETE effluent in March. At that point, the highest concentration of ibuprofen (0.120 mg.L-1) was obse... (Complete abstract click electronic access below)
Mestre

A inflamação é uma resposta protetora para livrar o organismo da causa inicial da lesão celular e suas consequências, porem se excessiva e não modulada, pode provocar a destruição progressiva do tecido. Este estudo pretende contribuir para o esclarecimento da influência de dois anti-inflamatórios, a dexametasona e o diclofenaco sódico, sobre a fase inflamatória do processo reparativo tecidual, bem como sobre o número de mastócitos nas diferentes fases do reparo. Foram utilizadas 60 ratas Wistar, divididas em 3 grupos (1, 2 e 3), medicadas 30 minutos antes de uma intervenção cirúrgica (lesão de 3mm de diâmetro no ventre lingual), com injeção intramuscular de 0,235mg/kg de dexametasona; 4,45mg/kg de diclofenaco sódico ou solução salina estéril, respectivamente. Os 3 grupos foram subdivididos em a, b, c, d, de acordo com o tempo de sacrifício: 6, 24, 48 e 120 horas após a cirurgia. As lesões foram fotografadas logo no momento cirúrgico e no sacrifício e as fotos utilizadas para análise clínica do processo de reparo e morfometria. As línguas foram extirpadas e enviadas para processamento histológico, os cortes foram direcionados para coloração em hematoxilina-eosina e em Azul de Toluidina para evidenciação e contagem do número de mastócitos. As características do processo reparativo foram descritas por meio de avaliação qualitativa dos seguintes componentes: extensão de áreas necróticas; intensidade de edema intersticial; tipo e intensidade do infiltrado inflamatório; graus de reepitelização, tecido de granulação e neovascularização. Os cortes corados em HE também tiveram seus campos digitalizados e analisados morfometricamente, para quantificação da reepitelização, mensurações do tecido de granulação, celularidade e edema. Os resultados da análise histológica semiquantitativa evidenciaram que o diclofenaco e a dexametasona acarretaram menor infiltrado inflamatório em relação ao controle na fase inflamatória do reparo, porém na fase produtiva a dexametasona exibiu menor intensidade de reepitelização e de neovascularização em relação aos demais grupos. Os resultados da análise histomorfométrica mostraram significativamente menor edema no grupo do diclofenaco em 6h (p=0,0041) e em 24h (p=0,0429), bem como menor porcentagem da celularidade em 6 horas no grupo da dexametasona (p<0,0001). O grupo diclofenaco ainda exibiu menor área de lesão em 120h do que os demais grupos (p=0,0060), indicando maior eficiência de reparo. Quanto à quantidade de mastócitos em 24, 48 e 120 horas, o grupo controle exibiu significativamente os maiores valores (p<0,0001). Com base nesses resultados, conclui-se que o grupo da dexametasona apresentou pior desempenho em relação à reparação; que o diclofenaco sódico apresentou um melhor efeito sobre o fechamento da ferida cirúrgica e redução mais intensa do infiltrado inflamatório, e que ambos provocam redução de mastócitos na área lesionada.
Inflammation is a protective response to rid the body of the initial cause of cell damage and its consequences, but when excessive and unmodulated, may cause progressive destruction of tissue. The aim of this study is to clarify the influence of two anti-inflammatory, diclofenac sodium and dexamethasone on the inflammatory phase of tissue repair process, as well as on the number of mast cells in different stages of repair. It was used 60 Wistar rats, divided into 3 groups (1, 2 and 3), medicated 30 minutes before surgery (lesion 3 mm in diameter in the ventral tongue), with intramuscular injection of 0.235 mg / kg dexamethasone, 4, 45mg/Kg of diclofenac sodium or sterile saline, respectively. The 3 groups were subdivided into a, b, c, d, according to the time of sacrifice, 6, 24, 48 and 120 hours after surgery. The lesions were photographed immediately at surgical time and at sacrifice, the photos were used for clinical analysis of the repair process and morphometry. The tongues were excised and sent for histological processing, the slices were directed for staining with hematoxylin-eosin and toluidine blue, for disclosure and count of the number of mast cells. The characteristics of the repair process were described through qualitative evaluation of the following components: extension of necrotic areas; intensity of interstitial edema, type and intensity of inflammatory infiltrate; degrees of reepithelialization, granulation tissue and neovascularization. Slices stained with HE also had their fields scanned and analyzed morphometrically to quantify reepithelialization, measurements of the granulation tissue, cellularity and edema. The results of semiquantitative histological analysis showed that diclofenac and dexamethasone led to lower inflammatory infiltrate compared to control in the inflammatory phase of repair, although in the productive phase dexamethasone showed less intensity of reepithelialization and neovascularization compared to the other groups. The results of the histomorphometric analysis showed significantly less edema in the diclofenac group at 6h (p = 0.0041) and 24 (p = 0.0429), as well as a lower percentage of cellularity in 6 hours in the dexamethasone group (p <0.0001). The diclofenac group also exhibited lower lesion area at 120h than the other groups (p = 0.0060), indicating greater efficiency of repair. Regarding the quantity of mast cells 24, 48 and 120 hours, the control group exhibited significantly higher values (p <0.0001). Based on these results, we conclude that the dexamethasone group showed the worst performance in relation to repair; that diclofenac sodium showed a better effect on surgical wound closure and greater reduction of inflammatory infiltration, and that both cause a reduction of mast cells in the injured area.

An increasing number of pharmaceuticals have been found in the aquatic environment and the issue has become a human and environmental health concern. Many pharmaceuticals are not fully degraded in wastewater treatment plants (WWTPs) and are continuously released in the aquatic environment resulting in concentrations in the low µg/l range in the receiving waters. Diclofenac is a widely used non-steroidal anti-inflammatory drug (NSAID) and is persistent in the aquatic environment. This pharmaceutical has been frequently reported in wastewater effluents, surface waters, groundwaters and even drinking water. NSAIDs are known to inhibit the cyclooxygenase activity, an enzyme present in many species of the animal kingdom responsible for the synthesis of prostanoids, and chronic exposure to environmental diclofenac may have detrimental effects on metabolism of non-target organisms including microbes and fish. In this thesis, microbiology, genomics and metabolomics approaches were used to investigate the effects of diclofenac on aquatic microbes and fish. In the first study of the thesis (chapter 3), the biodegradation of selected NSAIDs was investigated, together with their potential toxicity to aquatic microbes. Aerobic biodegradation experiments were conducted using activated sludge and wastewater effluents as microbial inocula and diclofenac, ketoprofen or naproxen as sole carbon source (1-10 mg/l) in order to isolate and identify the bacterial degraders. Changes in the bacterial populations were monitored by optical density and PCR-DGGE. The analytical techniques solid phase extraction (SPE) and ultraperformance liquid chromatography-mass spectrometry (UPLC-TOF-MS) were optimised to quantify the pharmaceuticals in environmental samples. High recovery rates were obtained with 94% for diclofenac; 92% for ketoprofen and 85% for naproxen and with detection capabilities down to 3-7 ng/l. Results from the biodegradation experiments showed that ketoprofen and naproxen were eliminated at up to 99 and 55% respectively over a 40 days period. Consistently with previous studies, diclofenac showed no significant degradation. In all the enrichments, a significant decrease in the bacterial abundance was observed as a consequence of NSAIDs exposure and attempts to isolate the bacterial degrading populations were unsuccessful. Given the apparent micro-toxicity of these NSAIDs, the standardised test Microtox@ was carried out with Vibrio fischeri. The EC50 (15 min) estimated ranged from 13.5 mg/l + 2.3 for diclofenac to 42.1 mg/l + 3.9 for naproxen. Further toxicological tests were performed with diclofenac on bacterial strains isolated from activated sludge. Growth inhibitory effects were observed from 50-70 mg/l for Micrococcus luteus, Zoogloea ramigera and Comamonas denitrificans. Pseudomonas putida seemed more tolerant to diclofenac exposure and toxic effects were observed from 90 mg/l. These studies showed that diclofenac was the most toxic NSAID but toxicological effects in bacteria only occurred at concentrations at least 1,000 times higher than those found in the environment. However, chronic exposure to lower concentrations may cause similar interferences and affect the degradation potential of naturally occurring microbial populations. The second study (chapter 4) investigated the biological effects of sub-chronic exposure to waterborne diclofenac (0.5, 1, 5 and 25 µg/l) in female juvenile rainbow trout Oncorhynchus mykiss. After 21-day exposure, mRNA expression levels of cytochrome p450 1a1 (cyp1a1), cyclooxygenase (cox) 1 and 2, and p53 were investigated in the liver, kidney and gills using RT-PCR and QPCR. These genes were selected as they are likely targets for diclofenac in mammals. Histopathological investigations were carried out in the small intestine, liver and kidney because diclofenac has been reported to induce toxicity responses in these tissues. Fish bile was also analysed by SPE and UPLC-TOF-MS to evaluate the bioconcentration potential of diclofenac and look for evidences of diclofenac metabolism. Results showed a significant reduction of both cox1 and cox2 expression in the liver, gills and kidney from 1 μg diclofenac/l. In contrast diclofenac induced an increase in mRNA levels for cyp1a1 in the liver and gills but a significant reduction of cyp1a1 expression in the kidney from 1 µg/l. There were no clear effects of diclofenac on the mRNA levels of p53. Diclofenac exposure caused tissue damages at exposure concentrations as low as 1 µg/l. Histopathological injuries included inflammation, hyperplasia and fusion of the villi in the small intestine and tubule necrosis in the kidney. There were no obvious changes in the liver of diclofenac-exposed fish. The analysis of bile revealed a bioconcentration potential between 509 + 27 and 657 + 25. A reactive metabolite of diclofenac was also detected at the highest exposure concentration which may be responsible for the severe injuries found in those fish. Sub-chronic exposure to environmental concentrations of diclofenac altered gene expression and it is possible that long term exposure to environmental diclofenac lead to significant impacts on fish health. In the final part of this thesis (chapters 5 and 6) effects on the metabolite composition of biofluids were analysed in diclofenac-exposed fish. This work entailed developing and validating appropriate methodologies to analyse fish bile and blood plasma. Methanol extraction and UPLC-TOF-MS were optimised to analyse the plasma metabolome but the methodologies were not suitable to detect low abundance molecules such as eicosanoids due to the interferences (ion suppression) in the samples matrix. Multivariate data analysis failed to detect the endogenous metabolites of the plasma affected by the chemical exposure. The only discriminating metabolite was found after analysis of the plasma samples from control vs. 25 µg/l treatment groups and identified as the exogenous compound diclofenac. To analyse the bile, the developed SPE methodology was carried out in order to separate the metabolites between a free steroids (fatty acids, eicosanoids, etc.) fraction and a conjugated steroids (bile salts) fraction. Due to high levels of taurocholic acid masking other metabolites in the conjugated fraction, some bile samples were hydrolysed to deconjugate these metabolites. The non-hydrolysed and hydrolysed bile fractions were analysed by UPLC-TOF-MS in positive and negative ionization. Multivariate data analysis using principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) revealed significant perturbations in the bile metabolite profile of diclofenac-exposed rainbow from the lowest exposure concentration (0.5 µg/l). Over 50 metabolites were elevated or reduced as a result of the 21-day exposure, suggesting that diclofenac affected several metabolic pathways. One metabolite was identified as a lipooxygenase product. This suggests that the inhibition of prostanoids synthesis can cause a shift in the arachidonic cascade and increase the synthesis of other eicosanoids. Most of the other discriminative metabolites remain unidentified and FT-MS analysis will be performed to obtain a structural identity. The metabolomics study further highlights the concern of environmental diclofenac in non-target organisms and the need to investigate the metabolic pathways affected.

>Magister Scientiae - MSc
Liposomes as a drug carrier in the pharmaceutical industry has gained currency since its discovery in 1965 by Bangham A. D. Liposomes have been shown to improve bioavailability as they can be delivered to target sites and possess sustained release properties which could be used to mitigate certain weaknesses associated with current diclofenac sodium eye drops. Diclofenac sodium (DNa) eye drop is a sterile Nonsteroidal Anti-inflammatory Drug (NSAID) with diclofenac sodium as its active ingredient. It is indicated for the lessening of ocular pain, prevention of miosis in eye operations, easing of postoperative inflammation and cystoids macular edema. The residence time of eye drops after application has been found to be 1-2 minutes as a result of continuous production of tears diluting the active ingredient, draining the eye drops into the nasolacrimal path, and eliminating it during blinking. As a result of the active ingredient not residing at the target site for the required duration, more frequent administration and medication is required and the risk of non-compliance is increased. Given the aforementioned potential of liposomes to redress the above weaknesses of current eye drops (dosage form) available for diclofenac sodium ophthalmic application, this study sought to encapsulate diclofenac sodium into liposomes for ophthalmic application. The main components of liposomes (cholesterol and phosphotidylcholine) and incubation time were set as the independent variables while percentage encapsulation, polydispersity index (PDI) and drug release profile constituted the dependent variable. Using analysis of variance (ANOVA) and t-test statistics, the interaction between the independent variables and their effect on the dependent variables were tested.

Cette étude a eu pour objectif d’étudier l’influence de différents facteurs (concentration initiale de polluant, masse de TiO2, oxygène et H2O2) sur la vitesse de décomposition photocatalytique de polluants organiques. Un composé issu de l’industrie pharmaceutique, le diclofénac, et un composé phytosanitaire, le sulcotrione, ont été sélectionnés. Deux matériaux photocatalytique en poudre ont été utilisés : TiO2 Degussa P25 et TiO2 Millennium PC500. Par ailleurs, du papier Alströhm recouvert de PC500 a également été employé pour certaines expériences. Le modèle Langmuir-Hinshelwood a été utilisé avec succès dans les différentes expériences. Les résultats ont montré que le rendement de dégradation des composés organiques est le plus fort lors d’utilisation de P25. La photodégradation en utilisant le PC500 en poudre est 2 fois plus efficace que le PC500 en papier rapportée à la masse de TiO2. La nature et l’évolution des produits formés dans ces procédés photocatalytiques ont été déterminées par CLHP/MS. Le TiO2 déposé sur le papier peut constituer une solution technique qui évite une filtration finale limitante. Lors de séjours au Vietnam, des expériences ont ainsi été réalisées avec des eaux naturelles dopées et les résultats obtenus pour l’élimination de deux composés montrent une relativement bonne efficacité. Par conséquence, le procédé photocatalyse semble constituer une alternative prometteuse aux méthodes existantes de traitement chimique des eaux polluées; en effet ils permettent de détruire des composés organiques et d’obtenir, dans les conditions opératoires initiales, sa minéralisation complète en CO2 et H2O.

-Une grande variété de polluants émergents se trouvent dans les sources d’eau naturelles et traitées. Les méthodestraditionnelles de détection et de quantification de ces polluants font appel à la spectrométrie de masse, souventassociées à la chromatographie en phase gazeuse ou liquide. Ils sont coûteux, lents, nécessitent de gros appareillageset mobilisent des experts. Pour surmonter ces limitations, la mise au point de capteurs rapides, économiques etfaciles d’utilisation, destinés à l’analyse des polluants dans l’eau, revêt une importance capitale. Ces dernièresannées les biocapteurs électrochimiques font l’objet d’une attention considérable pour la détection et laquantification des polluants dans l’eau. Ils offrent l’avantage de détecter les contaminants à l’état de traces dansdifférentes matrices, telles que les échantillons d’eaux naturelles et traitées. Nous avons développé un nouveaubiocapteur basé sur l’utilisation d’un aptamère pour la reconnaissance moléculaire d'un polluant émergentpharmaceutique : le diclofénac (DCL). Une nouvelle classe de polymères a été utilisée comme matricebiocompatible pour l’immobilisation de l’aptamère. Il s’agit d’un film mince d'hydrogel greffé à la surface dutransducteur conducteur. L’immobilisation de l’aptamère sur l’hydrogel offre un environnement biodégradable quipermet de préserver la structure active et fonctionnelle de l’aptamère tout en permettant la détection du DCL. Legreffage de l’aptamère s’obtient par la formation de liaisons amides via l’activation des groupes acide carboxyliquede l’hydrogel Poly(Acide Acrylique) (PAA). La sensibilité du biocapteur est améliorée grâce à la densité de greffageélevée de l’apatmère et à la structure 3D de l’hydrogel. La spectroscopie d'impédance électrochimique (EIS) a étéutilisée pour détecter le DCL dans l’eau. La variation de la résistance au transfert de charge est linéaire avec uneconcentration cible comprise entre 30 pM et 1 μM. La limite de détection est de 0,02 nM
-Fabrication of Surface-Attached hydrogel immobilization matrix thin films with tunable and well controlled chemistry -Functionalization and immobilization of the biorecognition agent -Characterization and Performances of developed polymeric biosensing surfaces

O tratamento específico de patologias do cólon pode ser conseguido pelo desenvolvimento de profármacos resultantes da conjugação de um fármaco com ciclodextrinas (CDs). A formação destes conjugados permite que os fármacos atinjam o cólon de forma intacta, local no qual sofrem degradação enzimática por acção da vasta microflora existente no cólon, nomeadamente Bacteróides. Estas bactérias quebram as ligações glicosídicas das CDs originando pequenos sacáridos, permitindo a sua absorção bem como a do fármaco. A entrega direccionada de fármacos ao cólon permite o tratamento da doença no alvo terapêutico, e consequentemente, permite reduzir a dosagem administrada e os efeitos adversos associados. Entre os vários tipos de anti-inflamatórios não esteróides (AINEs), o diclofenac de sódio é um potencial candidato na terapêutica para libertação específica no cólon devido às suas propriedades anti-inflamatorias e quimiopreventivas do cancro do cólon. A conjugação do diclofenac com ciclodextrinas é um processo pouco explorado, tornando-se inovador com recurso ao microondas. Neste trabalho experimental, procedeu-se à variação da temperatura utilizada no microondas, entre os 100 e 200ºC, variando-se também os solventes utilizados, PEG-200 e DMF, mantendo uma potência de 75W e um tempo de reacção de 40 minutos. O principal objectivo do trabalho foi optimizar a síntese do conjugado βCD-Diclofenac. Os melhores resultados foram obtidos para as temperaturas de 140ºC e 160ºC no solvente DMF. Para as mesmas temperaturas em PEG-200, os rendimentos obtidos foram significativamente inferiores. Estes resultados sugerem a necessidade de continuar a explorar as melhores condições de reacção de síntese do conjugado βCD-Diclofenac de forma a obter maior rendimento, recorrendo a solventes polares apróticos (que favorecem reacções SN2), bem aquecidos pelo microondas e que apresentem baixa toxicidade.
The specific treatment of colon pathologies can be achieved by designing prodrugs in which a drug is covalently bound to cyclodextrins (CDs). The formation of these conjugates enables the drugs to reach the colon unmodified, where they undergo enzymatic degradation by the vast microflora present in the colon, mainly Bacteroides. These bacteria break the linkage between glucose units of the CDs forming small saccharides, allowing its absorption as well as the drugs’. Targeted drug delivery to the colon ensures the direct treatment at the disease site and consequently lowers the dosage and reduces the adverse side effects. Among the diverse nonsteroidal anti-inflammatory drugs (NSAIDs), sodium diclofenac is a potential candidate for specific drug delivery to the colon due to its anti-inflammatory and quimiopreventive properties of colon cancer. The conjugation of diclofenac with CDs is a process which has been poorly explored, and is considered novel using microwave heating. In this experiment, the temperature of the microwave varied between 100 and 200ºC, and the solvents used in the reaction also varied between PEG-200 and DMF. The microwave´s power and time reaction were maintained at 75W and 40 minutes, respectively. The aim of this experiment was to optimize the synthesis of the βCD-Diclofenac conjugate. The best results were achieved at temperatures of 140ºC and 160ºC in the solvent DMF. For the same temperatures in PEG-200, the yield was significantly lower. These results suggest the need to continue exploring the best reaction conditions for the synthesis of the βCD-Diclofenac conjugate in order to increase the yield, using polar aprotic solvents (that favor SN2 reactions) with well heated properties by microwave, and that present low toxicity.

Orientadores: Osvaldir Pereira Taranto, Marcello Nitz
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
Made available in DSpace on 2018-08-19T18:12:35Z (GMT). No. of bitstreams: 1 Albanez_Roberta_M.pdf: 3222892 bytes, checksum: 3764421c1c9d6cfda6a9a56e6ed056e4 (MD5) Previous issue date: 2011
Resumo: Os pellets apresentam muitas vantagens biofarmacêuticas e são ideais para aplicação de recobrimento. Quando o recobrimento é funcional, um dos principais objetivos é formação de uma barreira que modifique o perfil de liberação da droga (liberação controlada ou gastrorresistente). Neste trabalho pellets de diclofenaco de sódio foram produzidos por extrusão/esferonização e, em seguida, foram recobertos em leito fluidizado tipo Wurster. Esse tipo de leito é um dos sistemas mais adequados para o recobrimento de partículas. Dentre suas vantagens destaca-se a não formação de zonas mortas. Este trabalho teve como objetivo estudar o recobrimento dos pellets produzidos com duas suspensões poliméricas aquosas comerciais entéricas, Advantia® Performance e Acryl-Eze® MP. O estudo do processo de recobrimento foi realizado por meio de um planejamento experimental 2³. As variáveis estudadas foram: temperatura do ar de entrada, vazão da suspensão e polímero de recobrimento. As variáveis de resposta foram: eficiência do processo, resultados acima de 78,2%, e fração de aglomerados, resultados inferiores a 8%. O efeito do tipo de polímero de recobrimento foi o que mais influenciou as variáveis de resposta, sendo que o Advantia® Performance resultou numa maior eficiência e uma maior fração de aglomerados. Determinou-se também o ganho de massa mínimo para atingir a gastrorresistência - Acryl-Eze® MP: 9,7% e Advantia® Performance: 8,6%. Os pellets revestidos passaram por testes de teor, dissolução e estabilidade. No teste de estabilidade os pellets recobertos com Advantia® Performance mantiveram seu perfil gastrorresistente. Porém, os pellets recobertos com Acryl-Eze® MP apresentaram um aumento da gastrorresistência após a exposição às condições de estabilidade, o que pode indicar que a coalescência das partículas do polímero aconteceu durante a estocagem. As suspensões foram caracterizadas quanto à sua reologia e ângulo de contato. O tempo de instantaneização do pó polimérico também foi testado
Abstract: Pellets have many biopharmaceutical advantages and are suitable for coating. When the coating is performed for functional purpose, one of the major goal is to form a barrier that modifies the drug release profile (controlled or enteric release). In this work, diclofenac sodium pellets were produced by the extrusion / spheronisation process and then coated in a fluidized bed coater column with a Wurster insert. This type of bed is one of the best suited systems for the coating of particles. One of the main advantages is that it avoids dead zones. This work aimed to study the coating of pellets produced with two commercial aqueous enteric polymer suspensions, Advantia® Performance and Acryl-Eze® MP. The study of the coating process was accomplished through a 2³ experimental design. The variables studied were: inlet air temperature, suspension flow rate and coating polymer. The response variables were: process efficiency, results above 78.2%, and agglomeration fraction, values below 8%. The effect of polymer coating type was the variable that influenced the response variables the most. The polymer Advantia ® Performance resulted in a better efficiency and increased the agglomerate fraction. The minimum mass gain to achieve the enteric profile was also determined - Acryl-Eze® MP: 9.7% and Advantia® Performance: 8.6%. The coated pellets were tested for content, dissolution and stability. In the stability test, pellets coated with Advantia® Performance maintained the enteric profile. However, the pellets coated with Acryl-Eze® MP presented a better enteric profile after the exposure to conditions of stability, which may indicate that coalescence of the polymer particles occurred during storage. The suspensions were characterized by rheology and contact angle. The static wettability of the polymeric powder was also tested
Mestrado
Engenharia de Processos
Mestre em Engenharia Química

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Novos fÃrmacos antiinflamatÃrios nÃo esteroidais, inibidores especÃficos de COX-2, tÃm chegado ao mercado com grande aceitaÃÃo por parte de clÃnicos e pacientes. Este trabalho testou a eficÃcia terapÃutica e o custo do tratamento do rofecoxibe 50 mg/dia,V.O. comparativamente com o diclofenaco sÃdico 50 mg, V.O., de 8 em 8 horas, no controle da dor, edema e do trismo pÃs-operatÃrio da exodontia de terceiros molares inferiores em 59 pacientes, 30 no grupo rofecoxibe e 29 no grupo diclofenaco sÃdico. As avaliaÃÃes foram feitas nos momentos do prÃ-operatÃrio, 30 minutos do pÃs-operatÃrio que coincidia com a administraÃÃo dos fÃrmacos e 0,5; 1,0; 2,0; 24; 48; 72 e 192 h pÃs-administraÃÃo. O rofecoxibe ofereceu um analgesia superior ao diclofenaco quando avaliado o sintoma dor por uma escala categorizada. Proporcionalmente, os pacientes que relataram analgesia completa nos intervalos de 1 e 2 h chegaram a 50 e 60,71% no grupo rofecoxibe, e 14,28 e 25,92% no grupo diclofenaco respectivamente. Essa diferenÃa foi estatisticamente significante (p<0,05). Quando a avaliaÃÃo feita foi pela escala visual analÃgica EVA, os escores de dor para o grupo diclofenaco foram superiores ao rofecoxibe nos momentos de 1 e 2 horas, sendo essa diferenÃa estatisticamente significante (p<0,05). O consumo de medicaÃÃo de resgate no grupo rofecoxibe foi significantemente menor nas primeiras 24 h do pÃs-operatÃrio em relaÃÃo ao diclofenaco (p<0,05.) Rofecoxibe, quando comparado ao diclofenaco, apresentou maiores mÃdias do edema, essa diferenÃa foi estatisticamente significante (p<0,05) no pico de 48 h do pÃs-operatÃrio. NÃo existiu diferenÃa estatisticamente significante no controle do trismo entre os medicamentos. Rofecoxibe apresentou um custo maior de 257,49% sobre o tratamento com o diclofenaco sÃdico genÃrico e de 73,06% sobre o VoltarenÂ. Quando indicado um protetor gÃstrico, associado ao diclofenaco o rofecoxibe pode ser a opÃÃo mais econÃmica isoladamente.
New selective COX-2 inhibitors non-steroidal antiinflammatory agents have been used with great approval by clinicians and patients. This work evaluated the therapeutic efficacy and the costs of the rofecoxib 50 mg/day, p.o. treatment compared to diclofenac sodium 50 mg, p.o 8/8h in the control of pain, swelling and trismus during the postoperative of the third lower molar exodontia in 59 patients, 30 in the rofecoxib group and 29 in the diclofenac sodium group. The assessment were made during the preoperative, 30 minutes after the surgical procedure, which corresponded with the administration of the drugs, and 0.5; 1.0; 2.0; 24; 48; 72 and 192h after the drugs administration. The rofecoxib provided a higher analgesia than diclofenac when the symptom pain was evaluated by a distinguished scale. In proportion, after the intervals of 1 and 2 hours, 50 and 60.71% of the patients in the rofecoxib group and 14.28 and 25.92% in the diclofenac group, respectively, referred complete analgesia. This difference was statistically significant (p<0.05). When the assessment was made by the EVA analogical visual scale, the diclofenac group pain scores were higher than rofecoxib after 1 and 2 hours. This difference was also statistically significant (p<0.05). The use of the rescue medication in the rofecoxib was significative lower compared with diclofenaco group (p<0.05). When Rofecoxib was compared to diclofenac, presented higher averages of swelling. This difference was statistically (p<0.05) 48h after the surgical procedure. There was no statistically significant difference in the trismus control with the use of the medications. Rofecoxib presents a higher cost of 257.49% if compared to the treatment of generic diclofenac and 73.06% if compared to Voltaren. When indicated a gastric protector associated to diclofenac, the rofecoxib is the most economic option isolated.

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Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil.
O escalonamento no processo de fabricação de comprimidos é um processo complexo e ainda bastante empírico, feito por método de tentativas e erros, cujos desafios são ainda maiores quando trata-se de formulação obtida por granulação úmida. Este trabalho realizou a transposição de escala de uma formulação de comprimidos revestidos contendo diclofenaco de sódio, fármaco de ação antiinflamatória,não esteroide e inibidor não seletivo das COX 1 e 2, avaliando os fatores relevantes para a obtenção tanto de uma formulação robusta, no que diz respeito ao processo de escalonamento em si, quanto de um medicamento que atendesse às exigências legais necessárias ao registro junto ao órgão regulador.Como resultado, o medicamento originário da formulação proposta apresentou-se aprovado em todos os ensaios exigidos, inclusive mostrando-se bioequivalente ao de referência. Este resultado ratificou a importância dos trabalhos preliminares,como a caracterização do ativo e os estudos referentes aos parâmetros a serem observados na transposição de escala. Na caracterização, as análises mostraram que a forma polimórfica do diclofenaco de sódio utilizado na formulação era a anidra,forma mais solúvel do fármaco; nas etapas de transposição de escala, identificou-se problemas com a alteração dos tempos de mistura do lote experimental para o lote piloto, pois não obteve-se uma boa compressibilidade para o lote piloto. Foi verificada, nas análises do granulado, uma diferença de granulometria que poderia justificar a diferença de desempenho entre os dois lotes. Um novo piloto foi manipulado, com a manutenção dos tempos de mistura do lote experimental, sendo que, dessa forma, os resultados satisfatórios do referido lote experimental foram reproduzidos para o segundo piloto, tanto em processabilidade quanto na avaliação granulométrica e demais ensaios.
Scaling up in the tabletting process is still a rather complex and empirical process carried out by trial and error methods, whose challenges are even greater when the formulation is obtained through wet granulation. Through this work, the development of large-scale production of a formulation of coated tablets containing diclofenac sodium, an anti-inflammatory drug, non-steroidal and non-selective inhibitor of COX 1 and 2, was conducted, evaluating the relevant factors for obtaining both a robust formulation with regard to the scale-up process itself and a drug which meets the legal requirements necessary for registration with the regulatory body. As a result, the product originating from the proposed formulation passed all of the required tests, and it was also proven to be bioequivalent to the reference drug. This result confirmed the importance of preliminary work, such as the characterization of active and studies concerning the parameters to be observed in the development of largescale production. In the characterization, the analysis showed that the polymorphic form of diclofenac sodium used in formulating was the anhydrous one, the more soluble form of the drug. In the steps of the development of large-scale production, there were problems with the alteration of the mixing times from the experimental batch to the pilot batch, as there was no good compressibility for the pilot batch. In the analyzes of the granulate, a difference of particle size was identified. It could explain the difference in performance between the two batches. Therefore, a new pilot batch was manipulated, with the maintenance of the mixing times of the experimental batch, and thus satisfactory results of the experimental batch were reproduced for the second pilot batch, regarding processability, particle assessment and other tests.

Desde hace unas décadas, los contaminantes farmacéuticos persistentes emergentes (EPPPs) han sido introducidos como un tipo de fuentes recalcitrantes de contaminantes en el agua. En este estudio, se ha realizado la síntesis y caracterización de polímeros con impresión molecular usando monómero funcional selectivo para la eliminación de diclofenaco e indometacina en medio acuoso a través del modo discontinuo. El siguiente procedimiento implicado fue la caracterización de la extracción en fase sólida molecularmente impresa usando monómero funcional selectivo para la eliminación y recuperación de diclofenaco e indometacina a partir de medios acuosos. A continuación, el experimento continuó con métodos analíticos para la aplicación de polímero impreso molecularmente usando monómero funcional selectivo para la recuperación de diclofenaco de agua y aguas residuales. En el estudio cinético se observó más del 95% de la eliminación de DCF e IDM dentro de los 3 primeros minutos, con una concentración inicial de 5 mg L-1 de DCF e IDM, agitando y a 25ºC. Del estudio de adsorción usando un cartucho empaquetado con 10 mg de MIP-IDM y MIP-DCF, se obtuvo una elevada capacidad de adsorción, concretamente de 600 mg IDM/g MIP y 200 mg DCP/g de MIP. Se utilizó el diagrama de Scatchard para estudiar la homogeneidad de MIP-IDM y MIP-DCF, y los resultados obtenidos mostraron que el proceso de sorción para MIP-DFC es homogéneo, mientras para MIP-IDM es heterogéneo. Durante el estudio de saturación mediante flujo continuo, se identificaron las curvas de ruptura. Para el estudio de selectividad, ambos MIPs se llevaron a cabo en modo “batch”. Se observó que el MIP preparado con AT como monómero se enlaza a moléculas de DCF. El desplazamiento en la señal observada se ha identificado como la interacción entre la amina del grupo AT con el ácido carboxílico del DCF. Se escogió el MIP-DCF para empaquetar en la columna de HPLC. Para el estudio de los grupos funcionales de tres tipos diferentes de MIP-DCF (MIP-DCF original, MIP-DCF cargado y MIP-DCF eluido después de la 10ª regeneración) se utilizó Fourier Transform Infrared-Attenuated Total Reflectance (FTIR-ATR). El estudio de la morfología se llevó a cabo microscopía electrónica de barrido (SEM). La pre-polimerización se estudió mediante 1H NMR. Se diseñaron tres disposiciones diferentes para aplicar los MIPs. En primer lugar, el modo de flujo continuo equipado con detección por espectrofotometría UV, en segundo lugar, la optimización de la extracción en fase sólida de impresión molecular (MISPE) utilizando muestras de agua real y en tercer lugar, utilizar la columna de MIP empaquetada en el equipo de cromatografía líquida de alta resolución con detección de ultravioleta (HPLC-UV) para determinación simultánea. La eficiencia en la eliminación y recuperación de EPPPs mejora cuando el MIP se utiliza como medio para empaquetar la columna de HPLC. En conclusión, el MIP funciona como un buen sorbente en la eliminación de DCF y IDM, y la tecnología de impresión molecular ha demostrado ser prometedora para la eliminación de EPPPs en agua.
Since a few decades ago, Emerging Persistent Pharmaceutical Pollutants (EPPPs) have been introduced as one type of recalcitrant pollutant sources in water. In this study, synthesis and characterization of molecularly imprinted polymer using selective functional monomer for diclofenac and indomethacin removal in aqueous media via batch mode has been done. Next procedure involved was the characterization of molecularly imprinted solid phase extraction using selective functional monomer for removal and recovery of diclofenac and indomethacin from aqueous media. Then the experimental continued with analytical methods for application of molecularly imprinted polymer using selective functional monomer for diclofenac recovery from water and wastewater. From the kinetic study, more than 95% of removal was observed for DCF and IDM, with an initial concentration of 5 mg L-1 of DCF and IDM within 3 min, agitated at 25 oC. From the total adsorption study using a cartridge pre-packed with 10 mg of MIP-IDM and MIP-DCF a high adsorption capacity of 600 mg IDM/g MIP and 200 mg DCF/g MIP respectively, were obtained. Scatchard plots were determined to study the homogeneity properties of MIPs finding that MIP-DCF differs to MIP-IDM. Breakthrough curves have been identified during the saturation study using continuous flow mode. Fourier transform infrared-attenuated total reflectance (FTIR-ATR) has been used in order to study the functional groups in three kinds of different MIP-DCF which were original MIP-DCF, MIP-DCF loaded and MIP- DCF eluting after 10 th times of regeneration. In order to study the morphology, scanning electron microscopy (SEM) was used. Pre-polymerization has been studied using 1H NMR. The shift in the signal observed has been identified with the interactions between amine of AT group with carboxylic acid on DCF. MIP-DCF was chosen for packing into the HPLC column. For the selectivity study, both MIPs were carried out in batch mode. The results show that MIP with AT as the monomer bind to DCF molecules. For application study, there were three methods has been designed in order to achieved the application objectives for this study. First, the continuous flow mode equipped to UV spectrophotometry detection; second, the optimization of molecularly imprinted solid phase extraction (MISPE) using real water samples; and thirdly, the MIP packed column equipped to high performance liquid chromatography with ultraviolet detection (HPLC-UV) for simultaneous determination. MIP enhance the efficiency in removal and recovery of EPPPs when the MIP has been used as packing media in column. As conclusion, the developed MIP works as a good sorbent in DCF and IDM removal. The molecularly imprinted technology has shown to be a promising technology for the removal of EPPPs in water. As conclusion, the developed MIP works as a good sorbent in DCF and IDM removal.

As evidências da contaminação das águas por resíduos de medicamentos e seus subprodutos levou esse grupo de resíduos a compor a lista de poluentes orgânicos emergentes, como consequência da expansão do uso de medicamentos, como o antidepressivo cloridrato de fluoxetina e o anti-inflamatório diclofenaco. Diversos Processos Oxidativos Avançados vêm sendo aplicados para a degradação destes compostos. Dentre eles, o processo de irradiação com feixe elétrons obteve bons resultados na remoção de toxicidade e degradação de fármacos. O presente estudo consistiu em aplicar radiação ionizante como uma possível tecnologia para degradar os fármacos em águas. A irradiação de solução aquosa contendo os fármacos foi aplicada usando acelerador de elétrons, cuja eficiência foi discutida mediante análises químicas (Cromatografia Líquida Ultra Rápida e Carbono Orgânico Total (COT)), ecotoxicológicas (ensaios de toxicidade com Vibrio fischeri e Daphnia similis) e biológicas (Ensaios Respirométricos). Os resultados de COT indicaram mineralização não significativa dos compostos, mesmo sendo observada degradação máxima de 99,9% para o diclofenaco e 55% para o cloridrato de fluoxetina na mistura (1:1) em 5.0 kGy. Foi observada toxicidade aguda dos fármacos, sendo mais acentuada para a fluoxetina, seguido do diclofenaco e, finalmente, da mistura para V. fischeri. Quando D. similis foram empregadas nessa avaliação, a ordem de toxicidade foi de fluoxetina, a mistura de ambos os medicamentos e do diclofenaco. Além disso, foi observada remoção de toxicidade nas amostras irradiadas em todas as doses aplicadas para a bactéria V. fischeri, com maior eficiência de remoção de toxicidade de 55%, em 5 kGy, na mistura dos dois fármacos. Para a D. similis, foi observada remoção significativa de toxicidade da mistura apenas na dose 2,5 kGy. Os ensaios respiroétricos não indicaram biodegradabilidade após o tratamento.
The evidence of water contamination by pharmaceuticals and byproducts residues took them to the list of wastewater emerging organic pollutants, as a result of expansion in drug usage. Fluoxetine hydrochloride antidepressant and diclofenac anti-inflammatory are good examplex. Several Advanced Oxidation Processes have been applied for degradation of these compounds. Among them, the electron beam irradiation process obtained good results in the removal of toxicity and degradation of pharmaceutical. The present study aimed to apply ionizing radiation as a possible technology to degrade the pharmaceutical in water. Irradiation of aqueous solution containing the pharmaceutical was applied using electron accelerator, whose efficiency was discussed through Chemical Analysis, COT, ecotoxicological (Toxicity Testing using Vibrio fischeri and Daphnia similis) and biological measurements (respirometric tests). The COT results indicated not significant mineralization of the compounds. It was observed maximum degradation of 99.9% for diclofenac and 55% for fluoxetine hydrochloride in a mixture solution (1:1) at 5.0 kGy. Regarding ecotoxicity, acute effects were more pronounced for fluoxetine, followed by diclofenac and finally the mixture, to Vibrio fischeri. When Daphnia similis were exposed fluoxetine was more toxic, followed by the mixture of both products and the third was diclofenac. Furthermore, radiation effects for removing toxicity was more effective with V. fischeri bacterium, all applied doses and > 55% removal of toxicity at 5 kGy (in the mixture). To D. similis, toxicity removal was effective when treated with 2.5 kGy ( mixture). No improvements in biodegradability was obtained by radiation ( respirometric tests).

Mestrado em Toxicologia
O consumo de produtos farmacêuticos é, nas sociedades actuais, uma realidade em crescimento, originada em diversos factores. Este continuo e crescente aumento de consumo induz, que cada vez mais, o ambiente e em particular os sistemas aquáticos, sejam o destino de resíduos farmacologicamente activos, sendo esta contaminação uma preocupação pela incorporação de substâncias nos ecossistemas que podem interferir em sistemas biológicos específicos. Neste sentido o presente trabalho teve como objectivo identificar e avaliar os efeitos produzidos pelo Diclofenac de Sódio, substância activa presente em diversos medicamentos (e.g., Voltaren® e Flameril®), em organismos aquáticos não-alvo. Para este efeito realizaram-se ensaios agudos e crónicos de cladóceros em que se compararam a espécie padrão – Daphnia magna – e autóctone – Daphnia longispina – para avaliação da sobrevivência, reprodução e crescimento. D. magna, nos teste agudos, apresenta maior tolerância ao Diclofenac (EC50 = 134.087 mg/L) existindo uma maior sensibilidade de D. longispina (EC50 = 35.353 mg/L). O Diclofenac afecta significativamente a fecundidade (LOEC=38.745 mg/L) de D. magna e o crescimento somático (LOEC=57.713 mg/L), na concentração mais alta. Para D. longispina os “endpoints” sub-letais significativamente afectados pelo Diclofenac foram a fecundidade (LOEC = 5.0 mg/L), e a maturação (LOEC = 2.5 mg/L). Nos testes crónicos foi ainda possível observar uma inibição significativa, em ambas as espécies, no número e tamanho dos neonatos, na primeira ninhada. Diclofenac, como substância activa com efeito farmacológico em humanos, inibe de uma maneira geral a sobrevivência, a reprodução e o crescimento das espécies de cladóceros testados neste estudo – D. magna e D. longispina. Contudo, as concentrações utilizadas para produzir quer os efeitos agudos quer os efeitos crónicos são superiores às concentrações normalmente detectadas em meios aquáticos. Em conclusão, o Diclofenac afecta D. longispina em “endpoints” individuais (fecundidade e maturação), em oposição a D. magna, foi significativamente afectada na fecundidade e no crescimento somático, ao nível da concentração mais alta, utilizada no teste.
Consumption of pharmaceutical products is, in our societies, a reality in growth, originated in several factors. This continues increase in consumption induces, more and more, that the environment and in matter, the aquatic systems are the destiny of active pharmacological residues, being this contamination a concern for the incorporation of substances in the environment that can interfere in specific biological systems. In this sense the present work had as objective to identify and to evaluate the effects produced by Diclofenac Sodium, active substance present in several medicines (e.g. Voltaren® and Flameril®), in non-target aquatic organisms. For this goal we realize acute and chronic tests with cladocerans and in that we have compared the survival, reproduction and growth of Daphnia magna (a standard species) and Daphnia longispina (an autochthonous species). Diclofenac, are effective in the survivorship and reproduction of the two cladoceran species used in this study – D. magna and D. longispina. D. magna seems to be more tolerant to acute toxicity (EC50 = 134.087 mg/L) and we found more sensibility to Diclofenac in D. longispina (EC50 = 35.353 mg/L). Diclofenac significantly affected the fecundity (LOEC=38.745 mg/L) of D. magna and the somatic growth rate (LOEC=57.713 mg/L), at the last concentration tested. For D. longispina the sublethal endpoints significantly affected by Diclofenac was fecundity (LOEC = 5.0 mg/L), maturation (LOEC = 2.5 mg/L). In addition, in the chronic exposure, the number and size of neonates of first brood are also impaired, in both species. Diclofenac, as an active substance with pharmacological effect for humans, generally impairs in the survivorship, reproduction and growth of the cladoceran species used in this study – D. magna and D. longispina. However, the concentration levels used to produce these effects in acute and chronic tests are much higher, if we compare with the concentration levels detected in the aquatic environment. In conclusion, Diclofenac affect more D. longispina at individual-level endpoints (fecundity and maturation), in opposite to D. magna, where fecundity is impaired and the somatic growth rate is slightly, but significantly affected in the last concentration tested.

Mini-matrix multiple unit dosage forms (MUDFs) of diclofenac sodium and S(+) ibuprofen have been prepared. Normal tabletting techniques were used to form the mini-matrices prior to their enclosure in hard gelatin capsules. Four natural hydrophilic gums, namely xanthan, karaya, locust bean and carrageenan gums as well as hydroxypropyl methylcellulose (HPMC) were used as the principle release-retarding agents. Various excipients - lactose, Encompress®, cellulose acetate phthalate (CAP), Veegum F® and Avicel PH101® - were added in different proportions to further modify drug release. The diclofenac sodium mini-matrices (4.5 mm in diameter) were produced by the wet granulation method. The release profiles from several encapsulated minimatrices in phosphate buffer solution (pH 7.0) showed that xanthan, karaya and locust bean gums could sustain the release of diclofenac sodium while the carrageenan gum did not produce a satisfactory sustaining effect. The rank order of decreasing swelling rate in both axial and radial dimensions was xanthan > karaya > locust bean gum and each of these gums showed almost Fickian swelling behaviour. The solvent penetration rates were consistent with the swelling rates. However, the order of decreasing drug release and erosion rates was locust bean> xanthan > karaya gum. For each of these gums, the release behaviour was anomalous indicating that both Fickian drug diffusion and polymer relaxation were involved in the release process. The dominant mechanism depended on the nature and content of the gum, as well as the stage in the dissolution period. The study involving xanthan gum showed that the diclofenac sodium release rate declined linearly with a progressive increase in the gumcontent, without changing the release behaviour. However, for high drug: xanthan gum ratio (2:1), the release kinetics changed to Super Case II. Solubility differences between the excipients did not affect the release rate, but increasing proportions of each excipient produced a faster release rate with the release mechanism changing from anomalous to Case II and then to Super Case II transport. Mini-matrices containing HPMC produced faster drug release than those containing the three natural gums. There was no synergistic effect between xanthan and locust bean gums on the release of diclofenac sodium from mini-matrices. Variation in the stirring speed (used in the dissolution apparatus) and matrix volume had little effect on drug release, whereas the pH of the dissolution medium greatly affected the release of diclofenac sodium. Following on from the studies involving diclofenac sodium, xanthan and karaya gums were used to produce mini-matrices of S(+) ibuprofen. Excipients with good compressibility characteristics such as lactose, Encompress® and Avicel PH101® were needed in the formulations. At pH 7, higher drug release rates were obtained with karaya gum (Super Case II mechanism) compared with xanthan gum (anomalous behaviour). Solubility differences between the excipients slightly affected the release rate. Compression forces (11 - 26 kN) slightly affected the crushing strength. The minimatrices were relatively stable to variation in temperature (5 - 37°C) and relative humidity (10 - 75%) over a 2 month time period. These studies have shown that near zero-order release of diclofenac sodium and S(+) ibuprofen can be achieved using encapsulated mini-matrices formulations. The release mechanisms and release rates can be adjusted by variation of the type and content of gums and/or excipients.

Diclofenac sodium is a non-steroidal, anti-inflammatory drug used for the relief of pain and inflammation. Many patients have difficulty swallowing tablets and consequently do not take medication as prescribed. To achieve optimum benefit of a drug, it is desirable to present it in a formulation which can rapidly disperse in water. This formulation is easier to swallow, therefore enhancing patient compliance. The aim of this study was to develop a stable diclofenac sodium dispersible tablet for easier oral administration. The first step in the product development was an investigative study into the physico-chemical properties, indications, side-effects and contra-indications of diclofenac sodium. Diclofenac sodium - excipient compatibility studies were performed as part of a preformulation study. Methods of evaluation included differential scanning calorimetry (DSC) and high performance liquid chromatography (HPLC). Four dispersible tablet formulations were developed. Kollidon CL-M (crospovidone) and Disolcel (croscarmellose sodium) were used as disintegrants in concentrations of 2% and 5% of the tablet mass. Tabletting was performed using a Cadmach® (India) single-punch tabletting machine. The four formulations were put on accelerated stability according to ICH guidelines for three months at 25°C/60%RH, 30°C/65%RH and 40°C/75%RH. HPLC was used to determine the identification, chromatographic purity and concentration of diclofenac sodium. Other tests included uniformity of mass, hardness, friability, disintegration, fineness of dispersion, loss on drying and dissolution. Thermal compatibility studies revealed potential interactions between diclofenac sodium and the excipients. Since DSC results only serve as a rough indication of possible interactions, accelerated stability testing using HPLC was used as a more selective method to identify potential interactions between diclofenac sodium and excipients. The HPLC results revealed that no interactions exist between diclofenac sodium and the chosen excipients. At the end of the stability period, no change in the physical appearance of the tablets was observed, except for the samples stored at 40°C/75% RH which showed a colour change from white to a very light brown after 3 months. Uniformity of mass remained within specification and average tablet mass and diameter remained relatively constant during stability testing. There was an increase in average thickness, hardness, disintegration time and percentage loss on drying with time and increased stress conditions. This correlates with the decrease in friability observed with time. Differences in the disintegration times were noted between Kollidon CL-M® and Disolcel® formulations. The only formulation that disintegrated within 3 minutes was formulation B. Very few particles of formulation B were retained on the 710 urn sieve, indicating a homogeneous dispersion. Assay results for all four formulations were within specification throughout stability and no extra peaks ascribed to diclofenac related compound A or any other impurity were observed. After 30 minutes, more than 85% of diclofenac sodium in formulations A, B and D was dissolved. The diclofenac sodium in formulation C did not dissolve well. This correlates with the slow disintegration times of formulation C's tablets. Dissolution rates of formulations C and D decreased with time and increased stress conditions, with the effect more pronounced in the case of formulation C. It can be concluded from the stability results that 5% Disolcel® as disintegrant was superior to a 2% concentration and to Kollidon CL-M® in concentrations of 2% and 5% of the tablet mass. Formulation B (5% Disolcel®) was chosen as the most favourable formulation with the best marketing possibilities. Stability results were also used to determine storage conditions and set specifications for batch release and stability to ensure that all batches tested against these specifications, meet the requirements for quality, safety and efficacy.
Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

An arteriovenous fistula (AVF) is a vein graft which is created to permit vascular access allowing haemodialysis to be performed. AVFs are associated with failure rates as high as 50% at 6 months. Failure is principally due to vascular smooth muscle cell proliferation, leading to the development of neointima causing stenosis and impaired blood flow. The aims of this study were to; 1) explore the inflammatory and proliferative characteristics of AVF stenosis and the role of TLR-4, 2) assess the ability of anti-inflammatory diclofenac to inhibit VSM cell proliferation, 3) develop a novel model of AVF in the rabbit to assess the impact of cannulation injury and the effect of topical diclofenac and 4) investigate the mechanisms responsible for diclofenac mediated activity. Human stenotic AVF segments and cell explants taken from haemodialysis patients vs. healthy long saphenous vein controls were shown to have significantly higher TLR-4 expression and activation of the downstream kinase IRAK-4. Also associated with AVF stenosis was an increased expression of pro-inflammatory cytokines including MCP-1. VSM cell explants derived from stenosed AVF had a significantly increased capacity to proliferative vs. healthy controls, which was inhibited by diclofenac treatment. Using a novel rabbit AVF model, cannulation injury was shown for the first time to drive stenosis. Topical diclofenac significantly inhibited this injury response, reducing mean vein wall width from 46.8±5.7μM to 15.8±1.8μM, comparable to 16.7±1.6μM in the non-injured AVF. In addition to previously well defined COX inhibition, evidence was generated in this study to implicate AMPK in the anti-proliferative activity of diclofenac. Therefore, activation of TLR-4 in AVF stenosis appears to play a significant role in the generation of an inflammatory and proliferative VSM cell response. Cannulation injury, which undoubtedly causes a pro-inflammatory response, significantly contributes to AVF stenosis which is inhibited by prophylactic topical diclofenac via the activation of AMPK.

(Oberflächen-) Gewässer weltweit sind mit geringen Mengen (ng/L bis wenige µg/L) humaner Pharmazeutika belastet. Diclofenac (DCF; nicht-steroidal, entzündungshemmend) und Metoprolol (MTP; ß-Blocker) gehören entsprechend ihres hohen Verbrauchs zu den am häufigsten gefundenen Substanzen. Deren biologische Aktivität ist nicht auf den Menschen beschränkt. Gut konservierte Enzyme innerhalb der Vertebraten legen Auswirkungen auf Nicht-Zielorganismen wie Fische nahe, die bisher in Langzeituntersuchungen mit umweltrelevanten Konzentrationen unzureichend untersucht wurden. In der vorliegenden Arbeit wurden die physiologischen Effekte von DCF und MTP auf die Nil-Tilapie (Oreochromis niloticus), einem der wichtigsten Aquakulturfische weltweit, untersucht. In vitro konnte anhand primärer Hepatozyten gezeigt werden, dass bereits umweltrelevante Konzentrationen von DCF zu einer erhöhten Genexpression verschiedener Schlüsselenzyme der Detoxifizierung führten. Nach MTP-Exposition waren die Veränderungen weniger eindeutig. Beide Substanzen induzierten die Vitellogenin Genexpression, nur DCF jedoch bereits in umweltrelevanter Konzentration. In vivo wurden in zwei Langzeit-Expositionsversuchen die physiologischen Effekte vom befruchteten Ei bis 80 Tage nach Schlupf in O. niloticus untersucht. Beide Substanzen hatte keinen Einfluss auf Schlupferfolg und Überleben, das Wachstum war nach 80 Tagen nach Schlupf leicht reduziert. Die deutlichsten Auswirkungen waren histopathologische Veränderungen der Kiemen, veränderte Genexpressionen der Gonadotropine und eine erhöhte Expression von Vitellogenin. Die Ergebnisse legen eine stärkere östrogene Aktivität von DCF im Vergleich zu MTP nahe. Zusammenfassend sind die Bedenken gegenüber den Einzelsubstanzen eher gering, negative Auswirkungen auf die Reproduktion und sich verstärkende Effekte bei zeitgleicher Exposition gegenüber DCF und MTP lassen sich jedoch nicht ausschließen und sollten im Weiteren untersucht werden.
Surface waters worldwide are contaminated with low levels (ng/L up to few µg/L) of human pharmaceuticals. Diclofenac (DCF; non-steroidal, anti-inflammatory) and metoprolol (MTP; ß-blocker) are highly consumed and therefore commonly detected. Their biological activity is not restricted to humans. Well conserved enzymes within the vertebrates suggest effects on non-target organisms such as fish, poorly studied in long-term exposure experiments using environmentally relevant concentrations. In the presented work, physiological effects of DCF and MTP on the Nile tilapia (Oreochromis niloticus), an important aquaculture fish species, were studied. Using primary hepatocytes, it was shown in vitro that environmentally relevant concentrations of DCF increased the gene expression of different key enzymes of the detoxification, while MTP exposure had a less clear effect. Both substances induced vitellogenin gene expression, but only after DCF exposure this was significantly elevated already at the environmentally relevant concentration. In vivo, two long-term exposure studies on the physiological effects from the fertilized egg until 80 days post-hatch were evaluated. Both substances did not affect hatching success and survival, while growth was slightly reduced after 80 days post-hatch. Histopathological alterations of the gills, changed gene expression patterns of the gonadotropins and induced vitellogenin gene expression were the most dominant findings. The results indicate a stronger estrogenic mode of action of DCF compared to MTP. Overall, the risk due to a single substance exposure seems to be relatively low but adverse effects on reproduction and additive effects during simultaneous exposure to DCF and MTP cannot be excluded and should be investigated further.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The aim of the first study was to evaluate the plasma concentration of diclofenac sodium (DS) in dogs submitted to diclofenac phonophoresis and to evaluate if phonophoresis induces greater absorption of this drug. .Five dogs were used in eight different groups at different times: Group 1, application of ultrasound for six minutes, removal of the ultrasound gel and topical application of two grams of DS gel for six minutes; Group 2, topical application of two grams of DS gel for six minutes; Group 3 topical application of two grams of DS gel and then covering it with common gel to apply ultrasound for six minutes; Group 4, similar to Group 1, but the ultrasound device was switched off; Group 5, similar to Group 3, but the ultrasound device was switched off, Group 6, the application of ultrasound was performed using only two grams of DS; Group 7, similar to Group 6, but the ultrasound device was switched off and Group 8, oral administration of 40mg of DS. The application area was 20cm². It was used a frequency of 1MHz, continuous ultrasound and intensity of 0,4W cm-2. Blood collections were performed before treatment (T0), 1h (T1) and 4h (T2) after ultrasound application for all groups. DS concentrations in plasma were measured by high performance liquid choramatohraphy (HPLC). There was significant increase of DS plasma concentration only at T1 in the Group 8. It was no possible to detect any concentration of DS in the plasma of dogs after topical application of DS, even after DS phonophoresis. The facilitation of transdermal penetration by ultrasound has not been verified under the protocol specified in this research. The aim of the second study was to use medium frequency Neuromuscular Electrical Stimulation (NMES) in femoral quadriceps of dogs with induced muscular atrophy, evaluate the occurrence of gain in mass in these muscles and to compare NMES in different periods of treatment. Eight dogs, weighing between 15 and 25kg, were randomly placed in two groups: GI (NMES for 30min), GII, (NMES for 60min). For the induction of the muscular atrophy, the right femoral-tibial-patellar joint was immobilized for 30 days by the percutaneous transfixation type II method. NMES was carried out in the dogs of groups, three times a week, with an interval of 48h between each session, during 60 days. The parameters measured were: thigh perimetry, knee goniometry, creatine kinase (CK) enzyme activity and morphometry of the muscular fibers in transversal cuts of the vastus lateralis muscle, collected through a muscular biopsy. There was no significant difference regarding the values of thigh perimetry and CK enzyme activity. The goniometry presented a significant increase (P<0.05) in the groups GI and GII at 30 days from the surgical procedure for immobilization when compared with time zero. As for the morphometry of the fibers of the vastus lateralis, a significant increase (P<0.05) was observed in the transversal area of the treated groups GI e GII at 90 days from the surgical procedure for immobilization when compared with time zero. Thus, it can be concluded that NMES of medium frequency brings about hypertrophy of the vastus lateralis muscle in dogs after induced muscular atrophy. NMES for 60min (GII) presents a greater muscular gain related to the GI
Esta tese foi dividida em duas pesquisas distintas utilizando fonoforese e eletroterapia, na primeira pesquisa o objetivo foi constatar a concentração plasmática de diclofenaco sódico emulgel em cães com ou sem o uso de fonoforese e se a fonoforese induz maior absorção deste fármaco. Para a realização da fonoforese foram utilizados cinco cães em oito grupos distintos, denominados: Grupo1: aplicação de ultrassom (US) por 6 minutos, remoção do gel com papel toalha e após aplicação de dois gramas de diclofenaco sódico (DS) emulgel permanecendo por 6 minutos; Grupo2: aplicação de dois gramas de DS emulgel tópico permanecendo por 6 minutos; Grupo3 aplicação de dois gramas de DS emulgel, posteriormente recobrindo-o com gel comum para acoplamento e realizado US pelo tempo de 6 minutos; Grupo4: repetiu-se o protocolo do Grupo1 com o ultrassom desligado; Grupo5: repetiu-se o protocolo do Grupo3 com o ultrassom desligado; Grupo6: aplicação de dois gramas de DS emulgel tópico e realizado diretamente sobre este o US pelo tempo de 6 minutos; Grupo7: repetiu-se o protocolo do Grupo6 com o ultrassom desligado; Grupo8: administração oral de um comprimido de DS (40mg) por animal. A área de aplicação foi de 20cm². A frequência do ultrassom foi de 1MHz, modo contínuo, com intensidade de 0,4W cm-2. Realizou-se a coleta de amostra sanguínea antes de executar os protocolos (Tempo zero), após uma hora (Tempo 1) e após 4 horas da aplicação (Tempo 2) em todos os grupos e posterior análise das mesmas por Cromatografia Líquida de Alta Eficiência (CLAE). Houve diferença (P<0,05) apenas no Tempo 1 do Grupo8. Não foi possível verificar concentração plasmática de diclofenaco sódico com aplicação tópica, em cães submetidos ou não à fonoforese, apenas quantificou-se o diclofenaco sódico pela administração via oral. A facilitação da penetração transdérmica pelo ultrassom não foi verificada sob o protocolo especificado nesta pesquisa. Na segunda pesquisa o objetivo foi avaliar a ocorrência de ganho de massa muscular utilizando a estimulação elétrica neuromuscular de média freqüência (corrente de Kotz 2500Hz) no músculo quadríceps femoral de cães com atrofia muscular induzida e comparar a EENM sob diferentes tempos de tratamento. Para a realização da eletroterapia foram utilizados oito cães, pesando entre 15 e 25kg e distribuídos aleatoriamente em dois grupos denominados de GI (30minutos) e GII (60minutos). Para a indução da atrofia muscular, a articulação fêmoro-tíbio-patelar direita foi imobilizada por 30 dias por transfixação percutânea tipo II. Foi realizada a EENM nos cães dos grupos GI e GII três vezes por semana, com intervalo mínimo de 48 horas entre cada sessão, pelo período de 60 dias. Foram mensuradas a perimetria das coxas, goniometria dos joelhos, atividade da enzima creatina-quinase (CK) e morfometria das fibras musculares do vasto lateral em cortes transversais colhido mediante a biópsia muscular. Não houve diferença quanto aos valores da perimetria da coxa e atividade da enzima CK. A goniometria revelou significância (P<0,05) nos grupos GI e GII entre os tempos zero e 30. Os grupos GI e GII tiveram aumento significativo (P<0,05) da área de secção quando comparados com o dia zero e noventa. Pode-se concluir que a EENM de média freqüência ocasiona hipertrofia do músculo vasto lateral em cães após atrofia muscular induzida. A EENM com duração de 60minutos (GII) promove um maior ganho de massa muscular em relação ao GI.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Os antiinflamatÃrios podem apresentar efeitos diferenciados quanto a eficÃcia terapÃutica, podendo, alguns serem bons analgÃsicos e outros potentes antiinflamatÃrios. Neste estudo, foram avaliados e comparados os efeitos anti-edematogÃnicos de antiinflamatÃrios nÃo esteroidais seletivos COX-2, lumiracoxibe 5mg/Kg, 30mg/Kg, 100mg/Kg, relativamente seletivos, nimesulida 25mg/Kg, meloxicam 30mg/Kg, nÃo seletivos COX, diclofenaco de sÃdio 20mg/Kg, alÃm dos glicocorticÃides dexametasona (3mg/Kg) e hidrocortisona (4mg/Kg), no clÃssico modelo de edema em pata de rato induzido por carragenina (EPICg). Cada grupo com 4 animais, recebia uma hora antes da injeÃÃo subcutÃnea (s.c) intraplantar do estÃmulo inflamatÃrio a dose referente de cada droga a ser testada, sendo que o grupo-controle recebia soluÃÃo salina 0,9%. ApÃs uma hora da administraÃÃo destas doses, 0,1mL de carragenina a 1% era injetada na pata direita de cada animal. O volume da pata edemasiada foi aferido quatro vezes em intervalos de uma hora, em um pletismÃgrafo digital (Ugo Basile Â).O efeito antiedematogÃnico de cada droga testada foi determinado pela comparaÃÃo dos resultados com o grupo-controle atravÃs do teste ANOVA. Dexametasona (3mg/Kg) e diclofenaco (20mg/Kg) foram as drogas com melhor desempenho, com taxa de reduÃÃo significativa do edema, na 3Â hora, em 94,20% e 84,43%, respectivamente. JÃ o lumiracoxibe, nas trÃs concentraÃÃes utilizadas, 5mg/Kg, 30mg/Kg, 100mg/Kg, obteve efeito significativo na reduÃÃo do edema, com 47,49%, 61,21%, 47,76% respectivamente na 3Â hora. Meloxicam, nimesulida e hidrocortisona tambÃm demonstraram eficiÃncia, com taxas de 42,48%, 62,27% e 58,84%, respectivamente. O presente estudo demonstrou que dexametasona (3mg/Kg) e diclofenaco (20mg/Kg) apresentaram potente aÃÃo antiedematogÃnica e que drogas mais seletivas COX-2 nÃo mostraram eficÃcia comparÃvel ao diclofenaco
The anti-inflammatories might present several efects about de therapeutical efficacy being some powerful antiinflammatories, but others act as excellent analgesics. In this study were observed and compared the anti-edematogenic efects of selective COX-2 non-steroidal antiinflammatories, lumiracoxib 5mg/Kg, 30mg/Kg, 100mg/Kg, relatively selected, nimesulide 25mg/Kg, meloxicam 30mg/Kg, non selective COX, diclofenac 20mg/Kg, yet of the glucocorticoids dexamethasone (3mg/Kg) and hydrocortisone (4mg/Kg), on the classic model of the edema on rat paw induced by carrageenan. Each group of four animals got one hour before the subcutaneous injection (sc) of the inflamatory estimulation the dosage of each drug to be tested, being that the control group received saline solution 0.9%. After one hour of the management of those dosages, 0.1% to 1% of carrageenan was injected in the right paw of each animal. The volume of the edema paw was checked four times in between one hour breaks, in a digital pletismograph (Ugo Basile Â).The antiedematogenic effect of each tested drug was determined by the comparission of the results with the control group through the ANOVA test. Dexamethasone (3mg/Kg) and Diclofenac (20mg/Kg) were the drugs with best performance with significative reduction rate of the edema, on the third hour, in 94.20% and 84.43% respectively. But the lumiracoxib on the three concentrations used, 5mg/Kg, 30mg/Kg, 100mg/Kg got significative effect on the reduction of the edema with 47,49%, 61,21%, 47,76% respectively on the third hour. Meloxicam, nimesulide and hydrocortisone also demonstrated eficiency with rates of 42,48%, 62,27% and 58,84% respectively. The current study demonstrated that dexamethasone (3mg/Kg) and diclofenac (20mg/Kg) presented potencial antiedematogenic action and that more COX-2 selective drugs didnât show eficacy comparable to diclofenac

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International and national bodies require the development of analytical methods that are rapid, inexpensive and accurate for the determination of drugs. One way to meet these requirements a method of flow injection analysis employing multicommutation and spectrophotometric detection was used. In this paper an automated system was developed to determine diclofenac sodium in injectables. The reaction is based on the oxidation of diclofenac sodium with potassium permanganate in acid medium be-ing monitored at 465 nm this. The calibration curve was linear over the concentration range of 20-80 mg Lˉ¹ with a detection limit of 0.1 mg Lˉ¹ standard deviation of 0.9% (n = 20) and Sample Rate 80 samples per hour. The proposed method was applied successfully in three injectable solutions and the samples were compared with the official method and other automated methods employing the same reaction.
Os organismos internacionais e nacionais exigem o desenvolvimento de metodologi-as analíticas que sejam rápidas, baixo custo e precisas para a determinação de fár-macos. Uma forma de atender a essas exigências foi utilizado um método de análise por injeção em fluxo empregando multicomutação e com detecção espectrofotomé-trica. Neste trabalho um sistema automático foi desenvolvido para determinar o di-clofenaco de sódio em injetáveis. A reação é baseada na oxidação do diclofenaco de sódio com permanganato de potássio em meio ácido sendo esta monitorada a 465 nm. A curva de calibração foi linear no intervalo de concentração de 20-80 mg Lˉ¹, com um limite de detecção de 0,1 mg Lˉ¹, desvio padrão de 0,9% (n = 20) e taxa de amostragem de 80 determinações por hora. O método proposto foi aplicado com sucesso em três soluções injetáveis e as amostras foram comparadas com a meto-dologia oficial e outros métodos automáticos que empregam a mesma reação.

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Universidade Federal do Rio Grande do Norte
This work is combined with the potential of the technique of near infrared spectroscopy - NIR and chemometrics order to determine the content of diclofenac tablets, without destruction of the sample, to which was used as the reference method, ultraviolet spectroscopy, which is one of the official methods. In the construction of multivariate calibration models has been studied several types of pre-processing of NIR spectral data, such as scatter correction, first derivative. The regression method used in the construction of calibration models is the PLS (partial least squares) using NIR spectroscopic data of a set of 90 tablets were divided into two sets (calibration and prediction). 54 were used in the calibration samples and the prediction was used 36, since the calibration method used was crossvalidation method (full cross-validation) that eliminates the need for a validation set. The evaluation of the models was done by observing the values of correlation coefficient R 2 and RMSEC mean square error (calibration error) and RMSEP (forecast error). As the forecast values estimated for the remaining 36 samples, which the results were consistent with the values obtained by UV spectroscopy
Neste trabalho s?o combinadas as potencialidades da t?cnica de espectroscopia no infravermelho pr?ximo NIR e da quimiometria visando ? determina??o do teor de diclofenaco em comprimidos, sem destrui??o da amostra, para o qual utilizou-se como refer?ncia o m?todo de espectroscopia no ultravioleta, que ? um dos m?todos oficiais. Na constru??o dos modelos de calibra??o multivariada estudou-se v?rios tipos de pr?processamento dos dados espectrais NIR, como corre??o do espalhamento da luz, primeira derivada. O m?todo de regress?o usado na constru??o dos modelos de calibra??o foi o PLS (m?nimos quadrados parciais) utilizando dados espectrosc?picos do NIR de um conjunto de 90 comprimidos divididos em dois conjuntos (calibra??o e previs?o). Na calibra??o foram usadas 54 amostras e na previs?o foram usadas 36, uma vez que o m?todo de calibra??o utilizada foi o m?todo de valida??o cruzada (full cross validation) que dispensa a necessidade de um conjunto de valida??o. A avalia??o dos modelos foi feita observando os valores de coeficiente de correla??o R2 e os erros quadrados m?dios RMSEC (erro de calibra??o) e RMSEP (erro de previs?o). Sendo os valores de previs?o estimados para as demais 36 amostras, o qual os resultados se mostraram coerentes com os valores obtidos por espectroscopia no ultravioleta

The commonly used drug diclofenac is an important environmental anthropogenic pollutant. Currently, detection of diclofenac is mainly based on chemical and physical methods. Here we describe a yeast biosensor that drives the diclofenac-dependent expression of a recombinant fluorescent protein from the authentic promoter of the PDR5 gene. This key component of the pleiotropic drug response encodes a multidrug transporter that is involved in cellular detoxification. We analyse the effects on diclofenac sensitivity of artificial PDR5 promoter derivatives in wild-type and various yeast mutant strains. This approach enabled us to generate sensor strains with elevated drug sensitivity.

Dissertação (mestrado) - Pontifícia Universidade Católica do Paraná, Curitiba, 2008
Inclui bibliografia
A movimentação dentária induzida é baseada no princípio biológico que a pressão prolongada aplicada aos dentes resulta em remodelação óssea. O diclofenaco potássico é um antiinflamatório não esteroidal que inibe a ciclooxigenase 1 e 2, bloqueando a transf
Orthodontic tooth movement is based on the biologic principle that prolonged pressure on teeth results in bone remodeling. Potassium diclofenac is a nonsteroidal anti-inflammatory drug that inhibits cyclooxygenase 1 and 2, blocking the formation of prosta

Eficácia do anti-inflamatório não-esteroidal diclofenaco associado ou não ao opioide codeína para controle da dor, edema e trismo no modelo de extração bilateral de terceiros molares inferiores com alto grau de dificuldade O controle da dor e inflamação após cirurgias bucais é normalmente realizado através do uso de anti-inflamatórios não-esteroidais (AINEs); no entanto a combinação de opioides aos AINEs pode garantir uma melhor analgesia principalmente após cirurgias mais traumáticas. Apesar disso, poucos estudos têm comparado AINEs associados ou não aos opioides após cirurgias bucais e maxilofaciais. Este estudo cruzado, randomizado e duplo-cego comparou a eficácia clínica no controle da dor, edema e trismo pós-operatório em 46 voluntários que consumiram randomicamente os medicamentos diclofenaco sódico (50 mg) associado à codeína (50 mg) e apenas diclofenaco sódico (50 mg) após extrações dos dois terceiros molares em posições complexas como alto grau de dificuldade cirúrgica. Os voluntários enquanto em uso do diclofenaco associado à codeína relataram dor pós-operatória significativamente menor em vários momentos (90 minutos (p=0,043), 2 horas (p=0,014), 3 horas (p=0,001), 5 horas (p=0,010), 10 horas (p=0,005), 12 horas (p=0,006) e 24 horas (p=0,018)) dentro das primeiras 24 horas após a cirurgia e também consumiram significativamente menos (p=0,003) medicação de resgate (paracetamol) ao longo do estudo, comparados com os valores expressos pelos mesmos voluntários enquanto em uso do diclofenaco apenas. Em conclusão, o diclofenaco sódico associado à codeína foi mais eficaz no controle da dor pós-operatória, enquanto que o trismo e o edema não apresentaram diferenças quando comparado com o diclofenaco sem codeína.
Postoperative pain and inflammation after oral surgery is mostly managed using non steroidal anti-inflammatory drugs (NSAIDs); however, opioids combined with NSAIDs may improve pain management in patients especially after traumatic oral surgery. Despite this, few studies have compared NSAIDs with and without opioids after oral and maxillofacial surgery. This randomized double-blinded crossover study compared the clinical efficacy for managing postoperative pain in 46 volunteers consuming either sodium diclofenac (50 mg) plus codeine (50 mg) or only sodium diclofenac (50 mg) after invasive surgeries for extraction of both lower third molar surgeries in different appointments. Volunteers reported significantly less postoperative pain at various time points (90 minutes (p=0,043), 2 hours (p=0,014), 3 hours (p=0,001), 5 hours (p=0,010), 10 hours (p=0,005), 12 hours (p=0,006) e 24 hours (p=0,018)) within 24 hours after surgery and also consumed significantly less(p=0,003) rescue medication (acetaminophen) throughout the study while consuming diclofenac plus codeine when compared to only taking NSAIDs. In conclusion, despite no difference between inflammation aspects, oral sodium diclofenac with codeine was more effective for managing postoperative pain when compared to diclofenac without codeine.

Capítulo 1 O diclofenaco de sodio é um antiinflamatório não-esteroidal que, além de atividade antiinflamatória, apresenta também propriedades analgésica e antipirética. Seus efeitos farmacológicos estão relacionados à inibição da síntese de prostaglandinas. Vários métodos utilizando técnicas cromatográficas tem sido propostos para a determinação de diclofenaco em plasma, incluindo a cromatografia gás-líquido com detecção por captura de elétrons ou por ionização de chama, a cromatografia gás-líquido-espectrometria de massa e a cromatografia líquida de alta eficiência com detecção eletroquímica ou no ultravioleta. Descreve-se aqui um método rápido, sensível e específico para a determinação de diclofenaco em plasma usando apenas 200 µl de amostra e envolvendo extração simples e cromatografia líquida de alta eficiência (CLAE) com detecção no ultravioleta. Extraiu-se o diclofenaco adicionando-se 200 µl de plasma a tubos contendo 500 ng de padrão interno (ácido 2-(p-ciclo-hexen- 1\'-il-fenil)propiônico e 100 µl de ácido ortofosfórico 2,5 N. A seguir adicionaram-se 4 ml de diclorometano e extraiu-se a mistura em agitador de tubos durante 60 segundos. Após centrifugação a 3000 rpm por 20 minutos a fase aquosa foi desprezada e a fase orgânica foi filtrada em membrana Millipore® FHLP 01300 de 0,4 µm e evaporada em corrente de nitrogênio a 37°C. O resÍduo foi dissolvido em 100 a 1000 µl de fase móvel para injeção em CLAE. Empregou-se para a separação coluna Novapak® C18, 150 x 3,9 mm, 4 µm e fase móvel constituída por mistura de tampão acetato 0,75 M, pH 5,O e acetonitrila (55:45, v/v). A detecção foi feita em λ = 282 nm. O diclofenaco e seu padrão interno foram eluidos respectivamente a 3,3 e 6,5 minutos em fluxo de 0,9 ml/min. Os limites de confiança do método foram: 10-10000 ng/ml. linearidade; 1 ng/ml, sensibilidade; 95 %, recuperação relativa e 3,5 e 5,7 %, precisão intra e interdias, respectivamente. Este micrométodo mostrou-se suficientemente sensível, preciso e exato para estudos de disposição cinética e bioequivalêneia de fomulações contendo diclofenaco. Capítulo 2 Estudou-se a biodisponibilidade do diclofenaco nos produtos Voltaren® 50 (A), Voltaren Retard® 100 (B) e Artren® 100 (C) após administração de dose única peroral de um comprimido a oito voluntários de ambos os sexos, adultos e sadios. As formulações foram administradas seguindo protocolo de estudo estabelecido por sorteio. Os voluntários em jejum receberam os medicamentos em dose única pela manhã e as amostras de sangue foram colhidas 1, 2, 3, 4, 6, 8, 10, 12 e 24 horas após a administração. A concentração plasmática de diclofenaco foi determinada por técnica em cromatografia líquida de alta eficiência (CLAE) de fase reversa com detecção no ultravioleta em λ = 282 nm após extração com solvente orgânico em meio ácido. Com base nas curvas \"concentração plasmática (C) vs tempo\" e \"logC vs tempo\" foram detenninados os parâmetros da fase absortiva para os três produtos, em X-± EPM: Cmax= 741 ± 137 ng/ml e tmax = 2,6 ± 0,4 h para o produto A, Cmax = 1399 ± 326 ng/ml e tmax = 2,4 ± 0,2 h para o produto B, Cmax = 192 ± 70 ng/ml e tmax = 2,3 ± 0,2 h para o produto C. Os valores de AUCT, calculados pelo método dos trapezóides e extrapolação a infinito, foram, em X- ± EPM: 1603 ± 253 ng.h/ml para o produto A, 3177 ± 606 ng.h/ml para o produto B, 2237 ± 529 ng.h/ml para o produto C. A extensão da biodisponibilidade (EBA) do diclofenaco nos produtos Voltaren® 50 (A) e Artren® 100 (C) foi calculada usando-se como referência o produto Voltaren Retard® 100 (B). Foram obtidos os seguintes valores, em X- ± EPM (mediana): 138 ± 35 (123) % para o produto A, 102 ± 35 (68) % para o produto C. A partir dos resultados obtidos sugere-se que o produto B, citado como de liberação prolongada e usado como referência neste trabalho, apresentou características de rápida liberação, semelhantes às do produto A, sugerindo possível falha de impermeabilização no revestimento dos núcleos contendo diclofenaco de sódio durante o processamento industrial do lote do qual provieram os comprimidos aqui avaliados. Capítulo 3 O diclofenaco é um antiinflamatório não-esteroidal indicado para o tratamento de pacientes portadores de inflamações dolorosas de origem reumática ou não. É biotransformado por reações de oxidação seguidas de conjugação glicurônica. Uma pequena porcentagem do fármaco original sofre glicuronização direta. Vários métodos têm sido propostos para a determinação de diclofenaco e seus produtos de biotransformação em fluidos biológicos, incluindo cromatografia a gás com detecção por captura de elétrons e cromatografia líquida de alta eficiência (CLAE) com detecção eletroquímica ou no ultravioleta. Entretanto, nenhum dos métodos utilizando CLAE mostrou-se suficientemente seletivo para o uso em estudos de disposição cinética. Descreve-se aqui um método sensível e específico para a determinação de diclofenaco e seus produtos de biotransformação hidroxilados em urina por CLAE com detecção no ultravioleta precedida de hidrólise enzimática nas amostras e extração com solvente orgânico em meio ácido. Adicionaram-se 500 µl de urina a tubos contendo 10 mg de mistura de β-glicuronidase e aril-sulfatase e 500 µl de tampão acetato 0,75 M. A mistura foi incubada a 37°C por uma hora e, em seguida, transferiram-se 800 µl do hidrolisado para tubos contendo 3,3 µ de padrão interno para diclofenaco (ácido 2-(p-ciclo-hexen-1\'-fenil)propiônico) e 1,0 µ de padrão interno para produtos de biotransformação (fenacetina). Procedeu-se à extração com 2 ml de mistura de diclorometano e álcool isopropílico (9:1, v/v) e agitação seguida por centrifugação a 3000 rpm durante 30 minutos. Desprezou-se a fase aquosa e filtrou-se a fase orgânica em sistema Millipore® com membrana FHLP 01300 0,4 µm. O extrato orgânico foi evaporado em corrente de nitrogênio a 37°C e o resíduo, dissolvido em volumes de 500-2000 µl de fase móvel e injetado em CLAE. Usaram-se dois sistemas cromatográficos independentes e consecutivos para a separação do diclofenaco e seus metabólitos hidroxilados. Inicialmente utilizou-se coluna de fase reversa ODS-Shimadzu, 150 x 6,0 mm, 5 µm e fase móvel constituída por tampão acetato 0,01 M, pH 5,0, metanol e acetonitrila (50:40:5) a um fluxo de 1,0 ml/min para eluição do padrão interno (fenacetina) a 10 minutos, 4\',5-di-hidroxidiclofenaco a 12 minutos, 3\'-hidroxidiclofenaco a 34 minutos, 4\'-hidroxidiclofenaco a 40 minutos e 5-hidroxidiclofenaco a 44 minutos. Em seguida utilizou-se a coluna Novapak® C18, 150 x 3,9 mm, 4 µm e fase móvel constituída por tampão acetato 0,75 M, pH 5,0 e acetonitrila (55:45, v/v) e um fluxo de 0,9 ml/min para eluição do diclofenaco a 3,3 minutos e padrão interno (ácido 2-(p-ciclo-hexen-l\'-il-fenil)propiônico, a 6,5 minutos. Os limites de confiança do método foram: 0,4-10,0 µg/ml, linearidade; 0,1 µ/ml, sensibilidade, 75 %, recuperação relativa média da extração e precisão intra e interdias variando de 1,3 a 2,2 % e de 3,3 a 5,7 %, respectivamente. O método mostrou-se suficientemente sensível, preciso, exato e específico para o uso em estudos de excreção urinária de diclofenaco e seus produtos de biotransformação hidroxilados. Capítulo 4 Avaliou-se a existência ou não de interação farmacocinética entre a ranitidina e o diclofenaco em voluntários sadios após administração de dose peroral de Voltaren® 50. Selecionaram-se 15 e avaliaram-se oito voluntários adultos, de ambos os sexos, sadios, em estudo constituído por duas fases. Dos oito voluntários, um foi excluído do estudo. Na Fase I o diclofenaco foi administrado isoladamente aos voluntários em jejum pela manhã. Na Fase II, os voluntários foram submetidos a um tratamento de sete dias com ranitidina, depois do qual receberam nova dose de diclofenaco. A administração de ranitidina continuou por mais três dias. Foram colhidas amostras de sangue e urina no intervalo de 0-72 horas após a administração de diclofenaco nas duas fases do estudo. Determinou-se a concentração plasmática e urinária de diclofenaco e a concentração urinária de seus produtos de biotransformação hidroxilados por técnica de cromatografia líquida de alta eficiência, em dois perfis independentes e consecutivos para diclofenaco e produtos de biotransformação, precedida por extração com solvente orgânico em meio ácido e, no caso das amostras de urina, precedida também por hidrólise enzimática. Os valores de \"clearance\" de formação (Clf) dos produtos de biotransformação hidroxilados foram, em X ± EPM (mediana): 15,9 ± 2,9 (15,2) ml/min para o 4\',5-hidroxidiclofenaco, 3,3 ± 0,9 (2,6) ml/min para o 3\'-hidroxidiclofenaco, 22,1 ± 5,9 (23,5) ml/min para o 4\'-hidroxidiclofenaco e 8,74 ± 1,43 (7,4) ml/min para o 5-hidroxidiclofenaco quando se administrou diclofenaco isoladamente. Não houve alteração significativa destes valores pela associação com a ranitidina. Da mesma forma, o \"clearance\" renal (Clr) do diclofenaco, de 7,1 ± 2,8 (5,1) ml/min após administração de diclofenaco isolado, não sofreu alteração significativa pela associação com a ranitidina. A associação com a ranitidina provocou um aumento significativo de cerca de 43 % na meia-vida de eliminação do diclofenaco em plasma, que passou de 1,01 ± 0,13 h (X ± EPM) na Fase I (fármaco isolado) para 1,44 ± 0,34 h na Fase II (fármaco associado), além de causar redução da excreção urinária do produto de biotransformação 4\'-hidroxidiclofenaco, que teve sua fração eliminada total (FelT) reduzida de 5,85 ± 1,12 (5,06) % (X ± EPM (med)) na Fase I para 4,39 ± 0,75 (3,73) % na Fase II. Com base nestes resultados sugere-se que a ranitidina reduza a biotransformação do diclofenaco pela diminuição da excreção renal de seu principal produto de biotransformação hidroxilado (4\'-hidroxidiclofenaco) com conseqüente aumento da meia-vida de eliminação plasmática do diclofenaco. Apesar da interação farmacocinética aqui registrada, a associação diclofenaco-ranitidina é vantajosa para pacientes com história de úlcera péptica, suscetíveis a ulceração recorrente.
Capítulo 1 Diclofenac sodium is a non-steroid anti-inflammatory agent which shows a high degree of anti-inflammatory, analgesic and antipyretic activity . It inhibits prostaglandin biosynthesis in vitro and in vivo, and this inhibitory effect at least partly explains the mechanism of action of the drug. Several methods have been described for the determination of diclofenac in human plasma or serum, including gas chromatography with electron-capture or flame ionization detection, gas-chromatography-mass spectrometry, and high-performance liquid chromatography with UV-detection. We describe a rapid, sensitive and specific procedure for the determination of diclofenac in plasma, using only 200 µl of biological sample, involving single extraction and high-performance liquid chromatography (HPLC) with UV-detection. An extraction with dichlormethane was performed by adding 200 µl of plasma to a test tube containing 500 ng internal standard (2-(p-ciclohexen-1\'-fenil)propionic acid) and 100 µl 2,5 N phosphoric acid. HPLC grade dichlormethane (4 ml) was added and the mixture was agitated with a vortex mixer and eentrifuged at 3000 rpm for 20 minutes. The aqueous phase was discarded and the organic phase filtered through a 0,4 µm FHLP 01300 Millipore® filter and evaporated in a stream of nitrogen at 37°C. The residue was dissolved in 100-1000 µl mobile phase and injected into the liquid chromatograph. Analytical separation was performed in an isocratic system on a reverse-phase column (Novapk® C18, 150 x 3,9 mm, 4 µm). The mobile phase was 0,75 M acetate buffer, pH 5,0 and acetonitrile (55:45, v/v). Peaks were monitored at 955; = 282 nm. Diclofenac and its internal standard were eluted at 3,3 and 6,5 minutes, respeCtively, at a flow rate of 0,9 ml/min. Confidence limits for diclofenac measurements in plasma were: 10-10000 ng/ml, linearity; 1 ng/ml, sensitivity; 95 %, relative recovery, and intra and interassay precision of 3,5 and 5,7%, respectively. This micromethod proved to be sufficiently sensible, precise and accurate for kinetic disposition or bioequivalence studies. Capítulo 2 Bioavailability of diclofenac in two test formulations was compared to a reference after single oral dose to healthy adult volunteers of both sex. Formulations were administered after a fasting night following a randomized study protocol. Blood samples were collected at 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours after dose administration. Diclofenac plasma levels were measured by HPLC technique using a reversed phase system after single extraction with organic solvent in acidic medium.. Peaks were monitored at UV-282 nm. Based on \"plasma concentration vs time\" curves, parameters were determined,expressed as X-± SEM: Cmax = 741 ± 137 ng/ml and tmaxm = 2,6 ± 0,4 h, product A, Cmax = 1399 ± 326 ng/ml and tmax = 2,4 ± 0,2 h, product B and Cmax = 192 ± 70 ng/ml and tmax = 2,3 ± 0,2 h, product C AUCT values, determined by trapezoidal rule and integration to infinity, were, expressed as X- ± SEM: 1603 ± 253 ng.h/ml, product A, 3177 ± 606 ng.h/ml, product B and 2237 ± 529 ng.h/ml, product C. Bioavailability (EBA) of diclofenac in test products (A, B) was calculated against reference (B). EBA, expressed as X- ± SEM (median), were: 138 ±35(123) %, product A and 102 ±35 (68) %, product C. Based on this data formulation A and B are bioequivalents while C and B are not bioequivalents. There is a strong evidence that product B, slow-release formulations, as described by the manufacturer, presented fast-release properties. Capítulo 3 Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) advocated for use in painful and inflammatory rheumatic diseases and certain non-rheumatic conditions. Diclofenac is extensively metabolized mainly by oxidative pathways followed by glucuronide conjugation. A small percentage of the original drug is glucuronidated directly. Many methods have been described for the determination of diclofenac and its metabolites in biological fluids, including gas-chromatography with electron-capture detection and high-performance liquid chromatography (HPLC) with UV or electrochemical detection. However, none of the HPLC methods was sufficiently specific for use in kinetic disposition studies. We describe a sensitive and specific procedure for the determination of diclofenac and its hydroxy metabolites in urine, involving single extraction after enzymic hydrolysis of the glucuronic conjugates and HPLC with UV detection. The enzymic hydrolysis was performed by adding 500 µl of urine to test tubes containing 10 mg of a mixture of β-glucuronidase and aril-sulphatase and 500 µl of acetate buffer 0,75 M, pH 5,0. This mixture was incubated at 37°C for one hour. Then, 800 µl of the mixture were transferred to test tubes containing 3,3 µg internal standard for diclofenac (2-(p-ciclohexen-l\'-il-phenyl)propionic acid) and 1 µg internal standard for metabolites (phenacetine). Dichlormethane and isopropyl alcohol (9:1, v/v) (2 ml) was added and the mixture was agitated with a vortex mixer and centrifuged at 3000 rpm for 30 minutes. The aqueous phase was discarded and the organic phase filtered through a 0,4 µm FHLP 01300 Millipore® filter and evaporated in a stream of nitrogen at 37°C. The residue was dissolved in 500-2000 µl mobile phase and injected in the liquid chromatograph. Analytical separation was performed in two independent consecutives isocratic systems on reverse-phase column. In the first HPLC profile an ODS-Shimadzu column, 150 x 6,0 mm, 5 µm and a mobile phase constituted of acetate buffer 0,01 M, pH 5,0, methanol and acetonitrile (50:40:5, v/v) at a flow rate of 1,0