Thesis (Ph. D.)--Michigan State University. Dept. of Biochemistry and Molecular Biology, 2008. / Title from PDF t.p. (viewed on Mar. 30, 2009). Includes bibliographical references (p. 204-227). Also issued in print.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O diclofenaco sÃdico à um fÃrmaco anti-inflamatÃrio nÃo esteroidal, que exerce seus efeitos por meio da inibiÃÃo da cicloxigenase e modulaÃÃo do Ãcido araquidÃnico; apresenta propriedades analgÃsicas, sendo utilizado para tratamento sintomÃtico de dores, principalmente relacionadas à inflamaÃÃo. à um fÃrmaco de amplo uso e de fÃcil acesso para o usuÃrio. Um estudo de bioequivalÃncia refere-se à comparaÃÃo estatÃstica das principais medidas farmacocinÃticas observadas no experimento, relativas aos produtos a serem testados. Este estudo teve o objetivo de avaliar a bioequivalÃncia entre uma formulaÃÃo de diclofenaco sÃdico cÃpsulas de liberaÃÃo prolongada de 100 mg, chamada formulaÃÃo teste, versus uma formulaÃÃo de diclofenaco sÃdico cÃpsulas de liberaÃÃo prolongada de 100 mg, produto de referÃncia, em voluntÃrios sadios de ambos os sexos, em jejum e alimentados, conforme recomendaÃÃo da Anvisa. Ensaio clÃnico do tipo aberto, randomizado, cruzado, com quatro perÃodos, duas sequÃncias, nos quais os voluntÃrios receberam em cada perÃodo distinto em jejum ou alimentados, 01 cÃpsula de liberaÃÃo prolongada da formulaÃÃo teste ou 01 cÃpsula de liberaÃÃo prolongada de diclofenaco sÃdico 100 mg da formulaÃÃo de referÃncia. Em cada internaÃÃo, os voluntÃrios receberam a formulaÃÃo teste ou referÃncia acompanhada ou nÃo de uma dieta padrÃo especÃfica. As formulaÃÃes foram administradas em dose Ãnica, via oral, seguida de coletas de sangue, de pelo menos quatro meias-vidas do fÃrmaco em estudo. Os perÃodos de tratamento obedeceram a um intervalo de sete meias-vidas, entre eles (washout). As concentraÃÃes em plasma do Diclofenaco foram dosadas por mÃtodo analÃtico especÃfico e validado, baseados em cromatografia lÃquida de alta eficiÃncia acoplada à espectrometria de massa (LC-MS/MS). Os resultados mostraram que em relaÃÃo à ASCinf (Ãrea sobre a curva), os fÃrmacos nÃo foram bioequivalentes para a extensÃo da absorÃÃo. O pico de concentraÃÃo plasmÃtica (concentraÃÃo mÃxima), que indica velocidade de absorÃÃo do fÃrmaco, nÃo foi bioequivalente entre a formulaÃÃo teste e a formulaÃÃo referÃncia, estando fora do intervalo de confianÃa de 80-125%. Considerando o uso amplamente aberto do diclofenaco, ressalta-se a importÃncia em avaliar custo-eficiÃncia versus custo-efetividade quando se orienta o uso de determinada formulaÃÃo do mercado. A nÃo equivalÃncia terapÃutica pode comprometer o tratamento de um determinado sintoma, ou mesmo de uma doenÃa, podendo levar ao descrÃdito o fÃrmaco escolhido. à relevante observar que cada fÃrmaco responde a um indivÃduo de maneiras distintas, de acordo com as variaÃÃes biolÃgicas do mesmo, podendo ambas as formulaÃÃes testadas ser eficaz, mesmo nÃo sendo bioequivalentes. / Diclofenac sodium is a non-steroidal anti- inflammatory drug that exerts its effects through inhibition of cyclooxygenase and modulation of arachidonic acid; it has analgesic properties and is used for symptomatic treatment of pain, mainly related to inflammation. It is a drug widely used and easily accessible to the user. A bioequivalence study refers to the statistical comparison of the main pharmacokinetic measures observed in the experiment concerning to the products to be tested. This study aimed to evaluate the bioequivalence between a formulation of diclofenac sodium extended-release 100 mg capsules, called test formulation, versus a formulation of diclofenac sodium extended-release 100 mg capsules reference product, in healthy volunteers of both sex, fasted and fed, as recommended by ANVISA. Open-type Clinical trial, randomized , crossover, with four periods, two sequences, in which participants received in each distinct period in fasting or fed, 01 extended-release capsule of the test formulation or 01 extended release 100 mg capsule of diclofenac sodium of the reference formulation. In each hospitalization, the volunteers received the test or reference formulation with or without a specific diet pattern. The formulations were administered in a single oral dose, followed by blood sampling, at least four half-lives of study drug. Treatment periods obey an interval of seven half-lives, between then (washout). The diclofenac concentrations in plasma were dosed by a specific and validated analytical method based on liquid chromatography high efficiency coupled to mass spectrometry (LC-MS/MS). The results showed that with regard to AUCinf (area under the curve), the drugs were not bioequivalent to the extent of absorption. The peak plasma concentration (maximum concentration), which indicates the rate of absorption of the drug, was not bioequivalent between the test formulation and reference formulation, being outside the confidence interval of 80-125%. Considering the wide open use of diclofenac, it emphazises the importance of evaluate cost-efficiency versus cost-effectiveness when it guides the use of certain formulation of the market. The non therapeutic equivalence can compromise the treatment of a particular symptom, or even a disease, which may lead to discrediting the drug chosen. It is important to note that each individual responds to a drug in different ways, according to biological variations thereof, both formulations tested may be effective, although not bioequivalent.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / 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

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.

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.

OBJECTIVE: Utilization of nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, can produce gastrointestinal ulceration. Thus, cyclooxygenase-2-selective inhibitors, such as celecoxib, and protective agents (e.g. rebamipide) have been employed to alleviate harmful NSAID effects. This study sought to explore the influence of rebamipide on the hepatic outcomes following administration of two commonly prescribed NSAIDs. MATERIALS AND METHODS: Rats were given either vehicle or rebamipide (30 mg/kg) orally twice daily for two days, then on the third day respective groups were dosed with either vehicle, celecoxib (40 mg/kg), or diclofenac (10 mg/kg) in addition to a respective dose of vehicle or rebamipide. Livers were collected on day 4 following euthanasia. Hepatic tissue was examined via histopathology and assayed for oxidative stress and specific NSAID concentration. RESULTS: The liver sections were found to be free from structural changes. Oxidative stress biomarkers, reduced glutathione and malondialdehyde, were discovered to be unaltered among the groups tested. The hepatic NSAID concentrations were not significantly affected by the presence of rebamipide. CONCLUSIONS: The concomitant administration of rebamipide does not influence the hepatic condition of rats administered either celecoxib or diclofenac at the dosages and over the time course examined.

Nonsteroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal and renal side effects. Rebamipide is a mucoprotective agent that reduces gastrointenstinal side effects when administered concomitantly with NSAIDs. In this study, we investigated the pharmacokinetic drug interactions of rebamipide with two selected NSAIDs, celecoxib or diclofenac. Rats were randomly divided into five groups. Two groups received placebo and three groups were administered rebamipide (30 mg/kg) orally twice daily for two days. On day 3, the animals treated with placebo received celecoxib (40 mg/kg) or diclofenac (10 mg/kg) and rats receiving rebamipide were administerd rebamipide followed by a single dose of placebo, celecoxib, or diclofenac. To investigate drug protein interactions, blank rat plasma was spiked with known concentrations of rebamipide, diclofenac plus rebamipide, or celecoxib plus rebamipide then dialyzed through a Rapid Equilibrium Dialysis device. AUC (139.70 ± 24.97 μg h/mL), C max (42.99 ± 2.98 μg/mL), and CLoral (0.08 ± 0.02 L/h/kg) values of diclofenac in diclofenac plus rebamipide group altered when compared to those of diclofenac treated groups. Treatment with rebamipide showed no significant change in pharmacokinetic parameters of celecoxib treated rats. Cmax (7.80 ± 1.22 μg/mL), AUC (56.46 ± 7.30 μg h/mL), Vd/F (7.55 ± 1.37 L/kg), and CLoral (0.58 ± 0.09 L/h/kg) of rebamipide were significantly altered when diclofenac was co-administered with rebamipide. Pharmacokinetic parameters of rebamipide plus celecoxib group were not significantly different from those of rebamipide group. Plasma protein binding was not affected by concomitant administration of another drug. These results indicate alteration of pharmacokinetic parameters of both rebamipide and diclofenac when co-administered and cannot be explained by a variation in plasma protein binding.

Da die Arzneistoffpermeation durch okulare Gewebe einen entscheidenden Einfluss auf die Heilung vieler Augenleiden hat, wurde die in vitro-Permeation hydrophiler und lipophiler Arzneistoffe durch okulare Gewebe und Zellkulturen in dieser Arbeit untersucht. Die Dissertation befasst sich vorrangig mit der Permeation des hydrophilen Modellarzneistoffs Pilocarpinhydrochlorid (P-HCl) durch isolierte Schweinecornea (SC), Schweinesklera, Kaninchenkonjunktiva und corneale bzw. konjunktivale Kaninchenepithelzellkultur. Der Einfluss verschiedener Formulierungsparameter wie Benzalkoniumchlorid (BAC), Natriumedetat, pH-Wert und Tonizität auf die P-HCl-Permeation wurde untersucht. Dabei konnte eine gute Korrelation zwischen isolierten Geweben und Zellkulturen in Reaktion auf die Variation der Formulierungsparameter festgestellt werden. Unter den getesteten Parametern zeigte BAC den größten Enhancereffekt. Weiterhin wurden vergleichende Permeationsstudien an gelaserter SC mit P-HCl und dem relativ lipophilen Diclofenac-Natrium (D-Na) durchgeführt. Das Entfernen von Epithelschichten der SC mittels Excimer-Laser sollte den Heilungsverlauf, vor allem aber die zeitabhängige Reepithelisierung der Cornea nach erfolgter Photorefraktiver Keratektomie, simulieren. Die Ergebnisse dieser Studie zeigten, dass unterschiedliche Epitheldicken einen signifikanten Einfluss auf die P-HCl-Permeation haben. Im Gegensatz dazu blieb die D-Na-Permeation nahezu unbeeinflusst. Ein weiteres Anliegen dieser Arbeit bestand darin, eine okular applizierbare Formulierung des Immunsuppressivums Mycophenolatmofetil (MMF) zu entwickeln. Sowohl für das Prodrug MMF als auch für dessen aktiven Metaboliten Mycophenolsäure (MPA) wurde die Permeabilität von SC getestet. Ausgewählt wurde eine Zubereitung, die 1% MMF in Glutathion-Bicarbonat-Ringerlösung enthält und mit 10% Hydroxypropyl-beta-Cyclodextrin versetzt ist. Diese Suspension wurde bei 121° C und 200 kPa für 15 min autoklaviert, um das schwerlösliche MMF in Lösung zu bringen. Da der Ester MMF bei der Herstellung der Testlösung einer teilweisen Hydrolyse zu MPA unterliegt, außerdem eine minimale in vitro-Verfügbarkeit aufweist und sehr schwer wasserlöslich ist, sollte MPA in Augentropfenformulierungen der Vorzug gegeben werden. Die Korrelierbarkeit mit in vivo-Resultaten ist jedoch im Rahmen dieser Arbeit nicht untersucht worden, so dass die Ergebnisse als Grundlage für Permeationsstudien am in vivo-Modell zu bewerten sind. / The permeation of drugs through ocular tissues plays an important role in healing of various eye diseases. The objective of this work was to investigate the in vitro permeability of hydrophilic and lipophilic drugs through ocular tissues and cell cultures. Mainly, the permeability of the hydrophilic model drug pilocarpine hydrochloride (P-HCl) through isolated pig cornea (PCr) and sclera, rabbit conjunctiva, and rabbit conjunctival or corneal epithelial cell culture was compared. Additionally, the study included investigations about the influence of the formulation parameters benzalkonium chloride (BAC), ethylene diamine tetra acetic acid disodium salt, pH value and tonicity on the permeability of the small drug. In summary, a good correlation between the isolated tissues and cell cultures with regard to P-HCl transport could be observed. In general, BAC caused the most facilitated drug transport within the formulation parameters. Furthermore, the permeation of P-HCl and the lipophilic diclofenac-sodium (D-Na) through lasered PCr was studied. To investigate the effects of photorefractive keratectomy on drug permeation, excimer laser ablations with varying depths were performed on isolated pig eyes. As a result, P-HCl demonstrated a significant enhancement of permeation in relation to the ablation depth. In contrast, corneal epithelial thickness scarcely influenced the permeation rate of D-Na. Another aim of this work was to develop an appropriate application form for topical ocular use of the immunomodulating substance mycophenolate mofetil (MMF) and to investigate the in vitro permeability of PCr for the prodrug MMF and its parent substance mycophenolic acid (MPA). The test formulation consisted of Glutathion-bicarbonate-Ringer's solution, 10% hydroxypropyl- beta-cyclodextrin and 1% MMF. To reach a concentration of 1% of the poor soluble MMF a treatment under autoclaving conditions at 121° C and 200 kPa for 15 min was needed. MPA should be preferred in eye drops because of an higher in vitro availability compared to MMF, an hydrolysis of MMF to MPA in the cornea during permeation and the poor water solubility of MMF. The correlation with in vivo results was not studied in this work but the findings can be assumed as basis for investigations on in vivo models.

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.

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.