Previous work on the biosynthesis of mycophenolic acid is discussed. [1’-¹⁴C]-Orsellinic acid (22) and [1'-¹⁴C]-2,4-dihydroxy-5,6-dimethylbenzoic acid (23) were synthesized and administered to Penicillium brevi-compactum. The purpose of feeding these two acids was to determine the order or sequence of the reactions involved in the biosynthesis of mycophenolic acid. It was found that acid (22) was not incorporated into mycophenolic acid but that acid (23) was incorporated specifically to the extent of 11.5%. Degradation of the mycophenolic acid from the feeding of (23) showed that a completely specific incorporation had occurred and thus supported the role of this acid (23) as a precursor of mycophenolic acid. [1- ¹⁴C]-geraniol (24) was also fed in an attempt to determine the origin of the C₇ side chain of mycophenolic acid. (24) was incorporated but degradation of the mycophenolic acid showed that the geraniol was decomposed to acetate before incorporation, leaving the precursor status in doubt. Three attempts were made to synthesize mycophenolic acid using suspected biological intermediates with no success. This was presumably due to the difficulty encountered in trying to alkylate the aromatic skeleton of mycophenolic acid. A synthesis of lignans involving intramolecular phenolic oxidative coupling was attempted with little success, due to the inability to obtain the ester required to couple to give the lignan skeleton. / Science, Faculty of / Chemistry, Department of / Graduate

The thesis entitled "Synthesis of Natural Products Based on Cyclohexadienes" consists of two chapters. Chapter 1 is divided into two parts. Part I gives a brief introduction to the structure, synthesis, biosynthesis and biological activities of some naturally occurring phthalides (eg. mycophenolic acid 1, zinniol2, phthalides 3 & 4). A general strategy for the preparation of highly substituted phthalides is also described. Cycloaddition of 1,s-dimethoxycoclohexa-1, 4-diene with dimethylacetylenedicarboxylate(DMAD) followed by an Alder-Rickert reaction gave the diester 5 which upon hydrolysis with KOH and refluxing with acetic anhydride gave the phthalic anhydride 6. Regioselective reductions of the anhydride 6 gave the phthalides 7 and 8. Using a similar strategy the phthalides 11 & 12 were prepared from 2,6dimethoxytoluene through the intermediates 9 & 10. The aromatic ethers 13 & 14 upon Birch reduction followed by Diels-Alder reaction with maleic anhydride gave the bicyclic anhydrides 15 & 16 respectively. Attempts to dehydrogenate 15 using variety of conditions failed. But refluxing 15 in nitrobenzene gave a poor yield of 17 which is an important intermediate in the synthesis of mycophenolic acid. Part II describes the first total synthesis of zinniol 2, phthalide-1 3 & phthalide-2 4. Thus the diene 18, obtained from 2-methylcyclohexane-1,3dione, upon Diels-Alder and Alder-Rickert with DMAD gave the diester 19. Prenylation of 19 afforded the diester 20 which was convened into 21 upon hydrolysis and DCC treatment. DIBAL reduction of 20 gave Zinniol 2 which on oxidation provided the phthalides 3 & 4 (7:3 ratio respectively). The anhydride 21, on selective reduction, gave the same phthalides in 2:8 ratio which could be readily separated and characterized.

abstract / Microbiology / Master / Master of Philosophy

No description available.

No description available.

O presente trabalho utilizou o eletrorretinograma (ERG) em coelhos (Oryctolagus cuniculus) para avaliar uma forma alternativa de aplicação do ácido micofenólico no tratamento da uveíte, uma doença ocular inflamatória. Uveíte é o termo utilizado para definir a inflamação da íris, coróide e corpo ciliar. O ERG é um registro de potenciais elétricos originados na retina em resposta à estimulação luminosa. O objetivo desse estudo é analisar alterações em funções da retina em olhos tratados com diferentes doses intravítreas de ácido micofenólico. Foram medidas amplitude e latência das ondas a e b do ERG para a função de intensidade-resposta e de resolução temporal. O micofenolato de mofetila (MMF) é um potente medicamento imunossupressor e vem sendo empregado com sucesso no tratamento de uveítes. O MMF é uma pró-droga, que é transformada no fígado em ácido micofenólico (MPA), o imunossupressor ativo. O uso de MMF causa efeitos colaterais e, para diminuir esses efeitos, este estudo analisa a injeção intravítea de MPA em coelhos. Foram utilizados um total de quarenta animais, divididos em seis grupos. Primeiramente, foram realizados exames eletrorretinográficos em olhos intactos, sem administração de nenhuma substância, para determinação dos parâmetros dos registros. Foram testados também os olhos injetados apenas com solução salina balanceada (SSB). Após essa etapa, foram feitos testes com olhos injetados apenas com o veículo (Polissorbato 80). Esse procedimento foi necessário para estabelecer se o veículo pode ser utilizado ou se apresenta algum grau de toxicidade para a retina. As doses usadas para avaliação da toxicidade da droga foram cinco: 5 g/ml, 50 g/ml, 200 g/ml, 1.000 g/ml e 10.000 g/ml. Os exames eletrorretinográficos foram realizados 7 e 30 dias após injeção intravítrea de MPA. Os resultados indicam que não houve alteração nos parâmetros de ERG nos olhos que receberam apenas o veículo. Também não acusaram indícios de toxicidade os olhos tratados com as doses de 5, 50 e 200 g/ml da droga. Apenas com as doses mais altas, de 1.000 e 10.000 g/ml, os olhos apresentaram diminuição de sensibilidade ao estímulo luminoso. Não é possível afirmar que as células da retina não foram danificadas pela injeção intravítrea de MPA, pois o ERG de campo total não detecta lesões pontuais. O pequeno número de olhos analisados e a necessidade de utilização de anestésicos podem levar a interpretações equivocadas dos resultados. As substâncias anestésicas podem afetar a neurotransmissão e, consequentemente, os resultados obtidos nos registros do ERG. As análises dos dados de amplitude e tempo implícito das ondas a e b foram muito consistentes e apresentam variações compatíveis com a literatura / This work uses the application of the electroretinogram (ERG) in rabbits (Oryctolagus cuniculus) in order to evaluate an alternative way of using the mycophenolic acid for treatment of uveitis, an inflammatory eye disease. Uveitis is an inflammation of the iris, choroid and ciliary body. The ERG is the record of electrical responses originated in the retina to luminous stimulation. The purpose of this study is to analyse changes in the retinal functions of eyes treated with distinct intravitreous doses of mycophnolic acid. A and b waves amplitude and implicit times for the intensity-response and temporal resolution functions. Mofetil mycophenolate (MMF) is a powerful immunossupressant drug successfully used for treating the uveitis. It is a pro-drug that is transformed in the liver into mycophenolic acid (MPA), the active immunossupressor. MMF causes collateral effects and, in order to reduce these effects, the intravitreous MPA injection in rabbits is analysed in this study. Forty rabbits were used, divided in 6 groups. First of all, electroretinografic exams in control eyes were made, without the application of any substance, for determining the recording parameters. Then were tested the eyes injected with only balanced saline solution (BSS). After that, tests were made in eyes injected only with the vehicle (Polissorbato 80). This procedure was necessary for finding out whether the vehicle may be used or is in some degree toxic for the retina. The doses for evaluation of the drug toxicity were five: 5 g/ml, 50 g/ml, 200 g/ml, 1.000 g/ml e 10.000 g/ml. By the examination of the distinct parameters of the ERG, several visual functions can be evaluated (inner and outer retina) and possible impairments in the temporal resolution, in the relationship luminous intensity and amplitude of the response, and in other functions. The results show there were no changes in the ERG parameters for the eyes where only the vehicle was applied. Also, no toxicity signs were detected in 5, 50 or 200 g/ml doses. Sensitivity to the luminous stimulus was only shown in eyes treated with higher doses, or, 1.000 and 10.000 g/ml. It is not possible to assert that the retina cells were not injured by the MPA injection, as the total field ERG does not detect spot lesions. The small quantity of tested eyes and the usage of anesthetics may lead to erroneous interpretations of the results. The anesthetic substances can affect the neurotransmission and consequently the results of ERG records. The analysis of the a and b wave amplitude and implicit time were very consistent and show variations consistent with the pertinent literature

Solid organ transplant has emerged over the last half century as an important treatment for solid organ failure. Management has matured dramatically over the past two decades with improvements in acute rejection, but long-term graft survival has improved very little and current treatment is limited by the side-effects and toxicities of immunosuppressive medications. Nanoparticle delivery of therapeutics, improving transport characteristics and decreasing systemic and local toxicity has emerged as a dynamic treatment modality, but little work has been done using nanoparticles in transplantation. Our research examined the use of CD4-targeted nanoparticles encapsulated with mycophenolic acid (MPA), a commonly used immunosuppressant in organ transplantation. This work is the first to examine antigen-specific targeting of nanoparticles in any transplant model. MPA-loaded particles show a slow and continuous release profile and biodistribution suggested retention in the spleen. Targeting of nanoparticles to CD4 T cells was suggested using ex vivo and in vitro flow cytometry. In the fully allogeneic MHCII mismatch BALB/C to C57BL/6 mice we found improved graft survival in the non-targeted MPA group and even greater graft survival in the CD4-targeted group. Targeted and non-targeted particle groups showed equal delay in rejection in the less immunogenic single MHC mismatch B6.H-2bm12 to C57BL/6 model that we showed to be CD4 dependent. In both models, graft survival times were increased over free drug and controls with roughly one thousand fold lower dose of drug in the nanoparticles as compared with free MPA. Consistent with these findings were decreased proliferation with targeted and non-targeted MPA-nanoparticles using in vitro and ex vivo mixed lymphocyte reactions. We postulated that the similar rejection times in targeted and non-targeted groups was due to dendritic cell (DC) involvement and we found active uptake of nanoparticles in DCs, a decrease in inflammatory cytokine production and a decrease in treated DCs ability to stimulate T cells via mixed lymphocyte reactions. Furthermore we found a possible mechanism in the DC interaction with T cells through the upregulation of the inhibiting co-stimulatory molecules B7-DC and B7-H1 on DCs treated with MPA-nanoparticles. We also found possible upregulation of CD4+CD25+ Foxp3 expressing Tregs which may serve to increase graft acceptance. These results explore the involvement of dendritic cells in the process of nanoparticle-induced graft acceptance and suggest the feasibility of using nanoparticle drug vectors in clinical transplant.

The use of lung transplantation, a life saving intervention, has been increasing over the last thirty years with a disappointing median survival of only 4.8 years. Despite the progress made in immunosuppressive therapies, allograft rejection following transplantation is the leading cause of death. As part of the immunosuppressive therapy, mycophenolate mofetil (MMF), the ester prodrug of mycophenolic acid (MPA) has proven its efficacy among heart, liver, kidney as well as lung transplanted patients. However, due to its rapid excretion, high daily doses are necessary and lead to serious side effects, forcing the patient to stop and change their course of treatment. Administration of drugs to the lungs is known to minimize local and systemic side effects by employing a lower amount of drug, to increase patient compliance and to improve the efficacy of the treatment. Therefore, developing novel MMF formulations for targeted delivery to the lungs will broaden the therapeutic options against lung transplant rejection. Within the framework of this dissertation, the development of an inhaled formulation of MMF was investigated. MMF must be metabolized by carboxylesterases to become active and its metabolism suffers from high inter- and intra-patient variability. The first objective of this dissertation was to investigate the occurrence of MMF hydrolysis in the lung. The second objective was to study the in vivo deposition,metabolism and distribution in rats, of an inhaled micron-size MMF suspension in comparison to inhaled IV Cellcept® and oral Cellcept®, the currently marketed products. According to the in vitro results, MMF is metabolized in human lung cells by carboxylesterases. The in vivo results showed an incomplete metabolism of MMF when delivered as a suspension due to the limited dissolution of the drug in the lungs. Following inhalation, the MMF suspension achieved higher and more prolonged concentration of the total drug in the lungs and lymphoid tissues as compared to the inhaled IV Cellcept®. The pulmonary delivery of the MMF suspension was able to achieve similar levels of drug in the lungs, higher levels in the lymphoid tissues and significantly lower levels in the systemic circulation when compared to the levels obtained from the oral gavage of oral Cellcept®. Ultimately, this dissertation demonstrated that the administration of micron-size MMF suspension offers a great potential for pulmonary administration. / text

O presente trabalho utilizou o eletrorretinograma (ERG) em coelhos (Oryctolagus cuniculus) para avaliar uma forma alternativa de aplicação do ácido micofenólico no tratamento da uveíte, uma doença ocular inflamatória. Uveíte é o termo utilizado para definir a inflamação da íris, coróide e corpo ciliar. O ERG é um registro de potenciais elétricos originados na retina em resposta à estimulação luminosa. O objetivo desse estudo é analisar alterações em funções da retina em olhos tratados com diferentes doses intravítreas de ácido micofenólico. Foram medidas amplitude e latência das ondas a e b do ERG para a função de intensidade-resposta e de resolução temporal. O micofenolato de mofetila (MMF) é um potente medicamento imunossupressor e vem sendo empregado com sucesso no tratamento de uveítes. O MMF é uma pró-droga, que é transformada no fígado em ácido micofenólico (MPA), o imunossupressor ativo. O uso de MMF causa efeitos colaterais e, para diminuir esses efeitos, este estudo analisa a injeção intravítea de MPA em coelhos. Foram utilizados um total de quarenta animais, divididos em seis grupos. Primeiramente, foram realizados exames eletrorretinográficos em olhos intactos, sem administração de nenhuma substância, para determinação dos parâmetros dos registros. Foram testados também os olhos injetados apenas com solução salina balanceada (SSB). Após essa etapa, foram feitos testes com olhos injetados apenas com o veículo (Polissorbato 80). Esse procedimento foi necessário para estabelecer se o veículo pode ser utilizado ou se apresenta algum grau de toxicidade para a retina. As doses usadas para avaliação da toxicidade da droga foram cinco: 5 g/ml, 50 g/ml, 200 g/ml, 1.000 g/ml e 10.000 g/ml. Os exames eletrorretinográficos foram realizados 7 e 30 dias após injeção intravítrea de MPA. Os resultados indicam que não houve alteração nos parâmetros de ERG nos olhos que receberam apenas o veículo. Também não acusaram indícios de toxicidade os olhos tratados com as doses de 5, 50 e 200 g/ml da droga. Apenas com as doses mais altas, de 1.000 e 10.000 g/ml, os olhos apresentaram diminuição de sensibilidade ao estímulo luminoso. Não é possível afirmar que as células da retina não foram danificadas pela injeção intravítrea de MPA, pois o ERG de campo total não detecta lesões pontuais. O pequeno número de olhos analisados e a necessidade de utilização de anestésicos podem levar a interpretações equivocadas dos resultados. As substâncias anestésicas podem afetar a neurotransmissão e, consequentemente, os resultados obtidos nos registros do ERG. As análises dos dados de amplitude e tempo implícito das ondas a e b foram muito consistentes e apresentam variações compatíveis com a literatura / This work uses the application of the electroretinogram (ERG) in rabbits (Oryctolagus cuniculus) in order to evaluate an alternative way of using the mycophenolic acid for treatment of uveitis, an inflammatory eye disease. Uveitis is an inflammation of the iris, choroid and ciliary body. The ERG is the record of electrical responses originated in the retina to luminous stimulation. The purpose of this study is to analyse changes in the retinal functions of eyes treated with distinct intravitreous doses of mycophnolic acid. A and b waves amplitude and implicit times for the intensity-response and temporal resolution functions. Mofetil mycophenolate (MMF) is a powerful immunossupressant drug successfully used for treating the uveitis. It is a pro-drug that is transformed in the liver into mycophenolic acid (MPA), the active immunossupressor. MMF causes collateral effects and, in order to reduce these effects, the intravitreous MPA injection in rabbits is analysed in this study. Forty rabbits were used, divided in 6 groups. First of all, electroretinografic exams in control eyes were made, without the application of any substance, for determining the recording parameters. Then were tested the eyes injected with only balanced saline solution (BSS). After that, tests were made in eyes injected only with the vehicle (Polissorbato 80). This procedure was necessary for finding out whether the vehicle may be used or is in some degree toxic for the retina. The doses for evaluation of the drug toxicity were five: 5 g/ml, 50 g/ml, 200 g/ml, 1.000 g/ml e 10.000 g/ml. By the examination of the distinct parameters of the ERG, several visual functions can be evaluated (inner and outer retina) and possible impairments in the temporal resolution, in the relationship luminous intensity and amplitude of the response, and in other functions. The results show there were no changes in the ERG parameters for the eyes where only the vehicle was applied. Also, no toxicity signs were detected in 5, 50 or 200 g/ml doses. Sensitivity to the luminous stimulus was only shown in eyes treated with higher doses, or, 1.000 and 10.000 g/ml. It is not possible to assert that the retina cells were not injured by the MPA injection, as the total field ERG does not detect spot lesions. The small quantity of tested eyes and the usage of anesthetics may lead to erroneous interpretations of the results. The anesthetic substances can affect the neurotransmission and consequently the results of ERG records. The analysis of the a and b wave amplitude and implicit time were very consistent and show variations consistent with the pertinent literature

No description available.