Malaria, Human Immunodeficiency Virus (HIV) and tuberculosis (TB) are global health problems having their worst situation in sub-Saharan Africa. Consequently, concomitant use of antimalarial, antiretroviral and antitubercular drugs may be needed, resulting in a potential risk of drug-drug interactions. Cytochrome P-450 (CYP) enzyme induction/inhibition may lead to drug-drug interactions and can be detected by probe drugs. An analytical method was developed for the quantitation of mephenytoin, CYP2B6 and CYP2C19 probe, and its metabolites. Induction/inhibition of principal CYP enzymes by the antimalarials; artemisinin, dihydroartemisinin, arteether, artemether and artesunate, was evaluated using the 4-hour plasma concentration ratios of probe drugs and their metabolites along with modelling the population pharmacokinetics of S-mephenytoin and its metabolites. The extent of change in enzymatic activities was different among the antimalarials, with artemisinin having strongest capacity for induction and inhibition, consequently, the strongest potential risk for drug-drug interactions. Drug-drug interactions between the antitubercular rifampicin and the antiretrovirals nevirapine and lopinavir were assessed, in TB/HIV patients, by developing population pharmacokinetic models. Rifampicin increased nevirapine oral clearance. Simulations suggested that increasing the nevirapine dose to 300 mg twice daily when co-administered with rifampicin, would result in nevirapine concentrations above subtherapeutic levels, with minimum exposure above the recommended maximum concentration. Lopinavir is co-formulated with ritonavir in the ratio of 4:1. In children, increasing ritonavir dose four times did not completely compensate the enhancement of lopinavir oral clearance caused by rifampicin. However, the predicted lopinavir trough concentration was above the recommended minimum therapeutic concentration. The work presented in this thesis followed an investigation line though not done for a particular drug. First the CYP enzymes involved in the interaction are identified. Afterwards, the expected drug-drug interaction is investigated where the potentially interacting drugs are concomitantly administered and an adjustment in the dose regimen is proposed that is subsequently evaluated.

Le mode vie autotrophique des plantes repose entièrement sur l’intégrité du chloroplaste et notamment l’étape de la biogénèse. La transcription des gènes chloroplastiques, assurée par une PEP (ARN polymérase encodée par le chloroplaste) et deux NEPs (ARN polymérase encodée par le noyau), est l’une des étapes primordiales dans le développement d’un chloroplaste photosynthétique. On distingue trois classes de gènes chloroplastiques : les gènes de classe I, transcrit par la PEP exclusivement; les gènes de classe II, transcrits par la PEP ou les NEPs; et les gènes de classe III, transcrits exclusivement par les NEPs. Pour assurer sa fonction, la PEP doit être associée à des facteurs sigmas. L’un de ceux-ci, la protéine SIG6, est un facteur sigma général et, associé à la PEP, assure la transcription de l’ensemble des gènes de classe I et II lors du développement du chloroplaste photosynthétique. Ainsi, le mutant sig6 présente un phénotype de cotylédons pâles, associé à un retard de biogénèse chloroplastique, ainsi qu’une diminution de la transcription des gènes de classe I, provoquant la diminution de la quantité de protéines de classe I. Dans le laboratoire, nous étudions les deux protéines WHIRLY chloroplastiques (WHY1 et WHY3) pour leur rôle dans le maintien de la stabilité génomique chloroplastique. Toutefois, peu de choses sont encore connues sur leur rôle potentiel dans la transcription ou la biogénèse chloroplastique. Par exemple, lorsque l’on tente de purifier la PEP, on obtient un gros complexe transcriptionnel nommé PTAC (Plastid Transcriptionally Active Chromosome) dans lequel sont retrouvées les deux protéines WHIRLY, suggérant qu’elles pourraient être impliquées dans la transcription chloroplastique. De plus, un possible rôle dans la biogénèse chloroplastique leur a été prêté, notamment chez le maïs. Dans cette étude, nous avons donc cherché à vérifier l’implication des protéines WHIRLY dans la biogénèse chloroplastique par une approche génétique de croisements entre les mutants sig6 et why1why3. Pour cela, nous avons isolé des doubles mutants sig6why1 et sig6why3, ainsi qu’un triple mutant sig6why1why3. À l’aide d’une caractérisation phénotypique et de la quantification de quelques protéines chloroplastiques, nous avons remarqué que la perte d’un des WHIRLY permet de complémenter le phénotype de cotylédons pâles du mutant sig6 et favorise l’expression normale de protéines en principe sous-exprimées dans le mutant sig6. Toutefois, la perte des deux WHIRLY ne permet pas de compenser le phénotype de cotylédons pâles et provoque l’apparition d’un phénotype persistant associé à une expression anormale des protéines chloroplastiques. Ces résultats ne peuvent être expliqués par le rôle des WHIRLY dans le maintien de la stabilité génomique chloroplastique étant donné que le triple mutant sig6why1why3 présente moins de réarrangements que le double mutant why1why3. Finalement, nous montrons que les effets de la perte d’un WHIRLY sur le mutant sig6 peuvent être mimés par l’utilisation de la rifampicine, une drogue inhibant l’ARN polymérase chloroplastique de type bactérienne (PEP). Ensemble, ces résultats démontrent donc l’implication des protéines WHIRLY chloroplastiques dans la biogénèse chloroplastique en association avec la protéine SIG6. Nous proposons un modèle selon lequel les deux protéines WHIRLY permettraient de favoriser l’activité de l’ARN polymérase de type bactérienne, notamment lors du développement du chloroplaste photosynthétique. En cas d’absence d’une des deux protéines, cette diminution partielle d’activité de la PEP favoriserait la mise en place d’un mécanisme de complémentation par le NEPs, permettant finalement de rétablir la biogénèse chloroplastique dans un mutant sig6. En l’absence des deux WHIRLY, le mécanisme de complémentation par les NEPs serait incapable de compenser la forte inhibition de la PEP, se traduisant par une aggravation du retard de développement du chloroplaste dans le mutant sig6. / The autotrophic lifestyle of plants relies entirely on the integrity of chloroplasts and particularly on their biogenesis. Chloroplast gene transcription, performed by a Plastid-Encoded Polymerase (PEP) and two Nuclear-Encoded Polymerases (NEPs), is one of the key steps during the development of photosynthetic chloroplast. There are 3 classes of genes, one transcribed by PEP alone (class I), one by both PEP and NEPs (class II), and the third by NEPs alone (class III). To carry out transcription, PEP associates with plastid sigma factors including the general sigma factor SIG6. sig6 mutants have a pale cotyledon phenotype, a severe decrease in class I gene transcription and a reduction in the level of class I proteins. In our laboratory, we study the role of the two plastid WIHRLY proteins (WHY1 and WHY3) in maintaining plastid genome stability. However, little is known about any role these proteins may play in transcription or chloroplast biogenesis. It seems likely they are involved in plastid gene transcription since they are found in the Plastid Transcriptionally Active Chromosome (PTAC). Moreover, they have been implicated in chloroplast biogenesis in maize. In this study, we verified the implication of these proteins in plastid biogenesis using a genetic approach in which we crossed a sig6 mutant with a why1why3 mutant. We isolated sig6why1 and sig6why3 double mutants and a sig6why1why3 triple mutant. Using a phenotypic characterisation and quantification of some plastid proteins, we show that loss of one of the two Why genes complements the sig6 pale cotyledon phenotype and allows a more normal pattern of expression of plastid proteins that are under-expressed in the sig6 mutant. However, we also show that loss of the two Why genes does not alleviate the sig6 phenotype. Moreover, the triple mutant shows a second pale phenotype on true leaves, and the plastid protein expression pattern is abnormal compared to either sig6 or wild type plants. Those results cannot be explained by the role of WHIRLY proteins in plastid genome stability since the triple mutant shows fewer plastid genome rearrangements than the why1why3 mutant. Finally, we show that inhibition of the PEP polymerase using rifampicin elicits the same complementation of the sig6 phenotype as the loss of one of the two WHIRLY. Together, these results show the implication of WHIRLY proteins in plastid biogenesis in association with SIG6. We propose a model in which WHIRLY act as activators of PEP activity, particularly during the chloroplast biogenesis. Therefore, the absence of one of the WHIRLY would cause a weak inhibition of PEP, facilitating the set-up of a rescue mechanism by NEPs and, consequently, allowing the complementation of plastid biogenesis in the sig6 mutant. However, the absence of the two WHIRLY proteins would cause a strong inhibition of PEP, and the inability of the rescue mechanism by NEPs to compensate for this strong inhibition, resulting in a more severe phenotype in the sig6 mutant.

Submitted by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2014-10-21T17:51:39Z No. of bitstreams: 1 Dissertação PatríciaCondéde Lima.pdf: 2501003 bytes, checksum: ae9bd16a44582c654071a25e36e5497e (MD5) / Made available in DSpace on 2014-10-21T17:51:39Z (GMT). No. of bitstreams: 1 Dissertação PatríciaCondéde Lima.pdf: 2501003 bytes, checksum: ae9bd16a44582c654071a25e36e5497e (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Química. Rio de Janeiro, RJ, Brasil. / Oscomprimidosutilizadosno tratamento da tuberculose possuem quatro fármacos associados, Isoniazida(INH), Pirazinamida(PYA), Etambutol(EMB) e Rifampicina(RMP) e são distribuídos gratuitamente pelo Sistema Único de Saúde. Até o momento, os métodos analíticos oficiais para a análise dos teores dos fármacos presentes neste medicamento de dose fixa combinada são aqueles especificados na Farmacopeia Americana 36ª edição e na Farmacopeia Internacional 4ª edição. Porém, nenhum destes compêndios oficiais possui monografias para análise simultânea dos quatro fármacos. O desenvolvimento de métodos analíticos adequados para determinar simultaneamente as concentrações dos quatro fármacos em comprimidos dose fixa combinada é de grande importância para o controle da qualidade do medicamento em questão. O objetivo deste estudo foi desenvolver um método analítico para determinar simultaneamente todos os princípios ativos do comprimido para o tratamento da tuberculose por cromatografia a líquido de alta eficiência com detector de ultravioleta-visível. O método desenvolvido utilizou uma coluna cromatográfica com fase estacionária octadecilsila no, 250mm de comprimento, 4,6mm de diâmetro e5 μm de tamanho de partícula e fase móvel constituída de uma fase aquosa(85% tampão formiato de amônio 0,3 mol/L pH 5, 15%metanol e 0,005 mol/L de Cu2+ou 250 mg/L de CuSO4.5H2O) e fase orgânica(metanol, 0,1% de trietilamina e 0,2% de ácido fórmico). O fluxo empregado foi de 1,0 mL/min e comprimento de onda265 nm, quando o software do equipamento permitiu, para a INH, PYA e o EMB um comprimento de onda de 265 e de 335 nm para a RMP. Este estudo demonstrou que o método desenvolvido apresentou linearidade entre 0,05 a 0,10 mg/mL para INH, 0,27 a 0,52 mg/mL para PYA, 0,19 a 0,36 mg/mL para EMB e 0,05 a 0,1 mg/mL para RMPe mostrou-se satisfatório quanto ao parâmetro de precisão, com desvio padrão relativo inferior a 2,0% para os quatro fármacos estudados. / Tablets used to treat tuberculosis contain four drugs, isoniazid (INH), pyrazinamide (PYA), ethambutol (EMB) and rifampicin (RMP) and are freely distributed by the National Health System. So far, the official analytical methods for testing the amount of the ingredients present in the fixed-dose combination tablet are those described in the 36th editionof U.S. Pharmacopeia and in the 4th editionof International Pharmacopoeia. However, none of these official compendium present monographs for the simultaneous analysis of the four drugs. The development of suitable analytical methods for simultaneously determining the concentrations of the four drugs in fixed-dose combination tablets is of great importance to the quality control of the medicinal product concerned. The aim of this study was to develop an analytical method to simultaneously determine all the active ingredients of the tablet for tuberculosis treatment by high-performance liquid chromatography with ultraviolet-visible detector. The developed method used a chromatographic column with octadecylsilane stationary phase, with 250mm length, 4,6mm diameter and 5 μm particle size. The mobile phase was aqueous (85% ammonium formate buffer 0,3 mol/LpH 5, 15% methanol and 0,005 mol/Lof Cu2+or250 mg/LCuSO4.5H2O), and the organic phase(methanol, 0,1% triethylamine and 0,2 % formic acid). The used flow was 1,0mL/min, at a wavelength of 265 nm or when the equipment alloweda wavelength of 265 nm for INH, EMB, and PYA and 335nmfor RMP. This study showned that the developed method was linear from 0,05 to 0,10 mg/mL for INH, 0,27 to 0,52 mg/mL for PYA, 0,19 to 0,36 mg/mL for EMB and 0,05 to0,1 mg/mL for RMP and was satisfactory regarding the precision parameter, with relative standard deviation lower than 2,0 % for the four studied drugs.

Made available in DSpace on 2014-12-17T14:16:24Z (GMT). No. of bitstreams: 1 FatimaDF_resumo.pdf: 203448 bytes, checksum: 85b1056cddeab5f9a1555fe6c360a75b (MD5) Previous issue date: 2009-09-21 / Known for thousands of years, tuberculosis (TB) is the leading cause of mortality by a single infectious disease due to lack of patient adherence to available treatment regimens, the rising of multidrug resistant strains of TB (MDR-TB) and co-infection with HIV virus. Isoniazid and rifampicin are the most powerful bactericidal agents against M. tuberculosis. Because of that, this couple of drugs becomes unanimity in anti-TB treatment around the world. However, the rifampicin in acidic conditions in the stomach can be degraded rapidly, especially in the presence of isoniazid, which reduces the amount of available drug for absorption, as well as its bioavailability, contributing to the growing resistance to tuberculostatic drugs. Rifampicin is well absorbed in the stomach because of its high solubility between pH 1 and 2 and the gastric absorption of isoniazid is considered poor, therefore it is mostly intestinal. This work has as objective the development of gastro-resistant multiple-systems (granules and pellets) of isoniazid aiming to prevent the contact with rifampicin, with consequent degradation in acid stomach and modulate the release of isoniazid in the intestine. Granules of isoniazid were obtained by wet method using both alcoholic and aqueous solutions of PVP K-30 as aggregating and binder agent, at proportions of 5, 8 and 10%. The influence of the excipients (starch, cellulose or filler default) on the physical and technological properties of the granules was investigated. The pellets were produced by extrusionesferonization technique using isoniazid and microcrystalline cellulose MC 101 (at the proportion of 85:15) and aqueous solution of 1% Methocel as platelet. The pellets presented advantages over granular, such as: higher apparent density, smaller difference between apparent and compaction densities, smoother surface and, especially, smaller friability, and then were coated with an organic solution of Acrycoat L 100 ? in a fluidized bed. Different percentages of coating (15, 25 and 50%) were applied to the pellets which had their behavior evaluated in vitro by dissolution in acidic and basic medium. Rifampicin dissolution in the presence of uncoated and coated isoniazid pellets was evaluated too. The results indicate that the gastro resistance was only achieved with the greatest amount of coating and isoniazid is released successfully in basic step. The amount of rifampicin in the dissolution medium when the isoniazid pellets were not coated was lower than in the presence of enteric release pellets. Therefore, the polymer Acrycoat L 100 ? was efficient for coating with gastro-resistant function and can solve the problem of low bioavailability of rifampicin and help to reduce its dosage / Conhecida h? milhares de anos, a tuberculose (TB) ? a principal causa de mortalidade por uma ?nica doen?a infecciosa devido ? falta de ades?o dos pacientes aos esquemas terap?uticos dispon?veis, o aparecimento de cepas multiresistentes e a co-infec??o com o v?rus HIV. A isoniazida e a rifampicina possuem o maior poder bactericida frente ao M. tuberculosis, sendo por isso unanimidade no tratamento anti-TB em todo o mundo. No entanto, a rifampicina em condi??es ?cidas do est?mago degrada-se rapidamente, principalmente na presen?a da isoniazida, diminuindo a quantidade de f?rmaco dispon?vel para absor??o bem como a sua biodisponibilidade, contribuindo para a resist?ncia crescente aos f?rmacos tuberculost?ticos. A rifampicina ? bem absorvida no est?mago devido ? sua alta solubilidade entre pH 1 e 2 e a absor??o g?strica da isoniazida ? considerada pobre, sendo ent?o, majoritariamente intestinal. O presente trabalho teve como objetivo o desenvolvimento de sistemas multiparticulados (granulados e p?letes) de isoniazida gastrorresistentes visando evitar contato da rifampicina com isoniazida e conseq?ente degrada??o no meio ?cido estomacal bem como modular a libera??o da isoniazida no intestino. Granulados de isoniazida foram obtidos por via ?mida utilizando solu??o alco?lica e solu??o aquosa de PVP K-30 como agente agregante/aglutinante, nas propor??es 5, 8 e 10%. A influ?ncia do excipiente (amido, celulose ou aus?ncia de diluente) sobre as propriedades f?sicas e tecnol?gicas dos granulados foi investigada. Os p?letes foram produzidos pela t?cnica de extrus?o-esferoniza??o utilizando isoniazida e Celulose MC 101 (na propor??o 85:15) e solu??o aquosa de Methocel 1% como agregante. Os p?letes apresentaram vantagens em rela??o aos granulados, como: maior densidade aparente, menor diferen?a entre as densidades aparente e de compacta??o, superf?cie mais lisa e, principalmente, menor friabilidade, sendo ent?o revestidos com uma solu??o org?nica de Acrycoat L 100? em leito fluidizado. Diferentes percentuais de revestimento (15, 25 e 50%) foram aplicados aos p?letes, os quais tiveram seus comportamentos avaliados in vitro por dissolu??o em meio ?cido e b?sico. Em seguida, a dissolu??o da rifampicina em meio ?cido na presen?a da isoniazida em p?letes n?o revestidos e p?letes revestidos foi avaliada tamb?m. Os resultados indicam que a gastrorresist?ncia foi obtida somente com a maior quantidade de revestimento, sendo a isoniazida liberada com sucesso na etapa b?sica. A quantidade de rifampicina dissolvida quando associada a p?letes de isoniazida n?o revestidos foi menor do que a observada na presen?a de p?letes de libera??o ent?rica. O pol?mero Acrycoat L 100? mostrou-se eficiente para o recobrimento com a fun??o de gastrorresist?ncia, podendo resolver o problema da baixa biodisponibilidade da rifampicina assim como ajudar a diminuir a dosagem utilizada

Made available in DSpace on 2014-12-17T14:16:26Z (GMT). No. of bitstreams: 1 EdilenePL_DISSERT.pdf: 1151865 bytes, checksum: 7a412187753da50d4030404745c567f7 (MD5) Previous issue date: 2010-02-26 / According to the global framework regarding new cases of tuberculosis, Brazil appears at the 18th place. Thus, the Ministry of Health has defined this disease as a priority in the governmental policies. As a consequence, studies concerning treatment and prevention have increased. Fixed-dose combination formulations (FDC) are recognized as beneficial and are recommended by WHO, but they present instability and loss on rifampicin bioavailability. The main purpose of this work was to carry out a pre-formulation study with the schedule 1 tuberculosis treatment drugs: rifampicin, isoniazid, pyrazinamide and ethambutol and pharmaceutical excipients (lactose, cellulose, magnesium stearate and talc), in order to develop an FDC product (150 mg of rifampicin + 75 mg of isoniazid + 400 mg of pyrazinamide + 250 mg of ethambutol). The studies consisted of the determination of particle size and distribution (Ferret s diameter) and shape through optical microscopy, as well as rheological and technological properties (bulk and tapped densities, Hausner Factor, Carr s Index, repose angle and flux rate) and interactions among drugs and drug excipient through thermal analysis (DSC, DTA, TG and your derivate). The results showed that, except isoniazid, the other drugs presented poor rheological properties, determined by the physical characteristics of the particles: small size and rod like particles shape for rifampicin; rectangular shape for pyrazinamide and ethambutol, beyond its low density. The 4 drug mixture also not presented flowability, particularly that one containing drug quantity indicated for the formulation of FDC products. In this mixture, isoniazid, that has the best flowability, was added in a lower concentration. The addition of microcrystalline cellulose, magnesium stearate and talc to the drug mixtures improved flowability properties. In DSC analysis probable interactions among drugs were found, supporting the hypothesis of ethambutol and pyrazinamide catalysis of the rifampicin-isoniazid reaction resulting in 3- formylrifamycin isonicotinyl hydrazone (HYD) as a degradation product. In the mixtures containing lactose Supertab? DSC curves evidenced incompatibility among drugs and excipient. In the DSC curves of mixtures containing cellulose MC101?, magnesium stearate and talc, no alterations were observed comparing to the drug profiles. The TG/DTG of the binary and ternary mixtures curves showed different thermogravimetrics profiles relating that observed to the drug isolated, with the thermal decomposition early supporting the evidences of incompatibilities showed in the DSC and DTA curves / De acordo com o quadro mundial da tuberculose, o Brasil ocupa o 18? lugar em n?mero de casos novos, assim o Minist?rio da Sa?de definiu a doen?a como prioridade entre as Pol?ticas Governamentais de Sa?de. Desde ent?o se intensificaram os estudos relacionados ao tratamento e preven??o desta doen?a. As formula??es em dose fixa combinada (DFC) s?o reconhecidas como ben?ficas e apoiadas pela OMS, mas apresentam problemas de instabilidade e queda na biodisponibilidade da rifampicina. O objetivo principal desse trabalho foi realizar estudo de pr?-formula??o com os f?rmacos que integram o esquema 1 para o tratamento da tuberculose: rifampicina, isoniazida, pirazinamida e etambutol e excipientes farmac?uticos (lactose, celulose, estearato de magn?sio e talco), visando o desenvolvimento de um produto em dose fixa combinada (150 mg de rifampicina + 75 mg de isoniazida + 400 mg de pirazinamida + 250 mg de etambutol). Os estudos consistiram na determina??o do tamanho e distribui??o das part?culas (di?metro de Ferret) e forma por microscopia ?ptica, al?m das propriedades reol?gicas e tecnol?gicas (densidades aparente e de compacta??o, Fator de Hausner, ?ndice de Carr, ?ngulo de repouso e velocidade de escoamento) e das intera??es entre os f?rmacos e f?rmacos-excipientes por an?lise t?rmica (DSC, DTA, TG e sua derivada). Os resultados mostraram que ? exce??o da isoniazida, os demais f?rmacos apresentaram propriedades reol?gicas pobres, determinadas pelas caracter?sticas f?sicas das part?culas: tamanho reduzido e forma de agulhas da rifampicina; forma retangular da pirazinamida e etambutol, al?m da baixa densidade deste ?ltimo. A mistura dos quatro ativos tamb?m n?o apresentou fluxo, especialmente a prepara??o contendo a quantidade de f?rmacos preconizada para a formula??o de produtos em dose fixa combinada, uma vez que nessa mistura, a isoniazida, que possui o melhor fluxo, foi adicionada a uma concentra??o menor. A adi??o de celulose microcristalina, estearato de magn?sio e talco ?s misturas dos f?rmacos, melhorou as propriedades de fluxo. Nas an?lises por DSC foram encontradas prov?veis intera??es entre as subst?ncias ativas, refor?ando a hip?tese de cat?lise por etambutol e pirazinamida para a rea??o entre rifampicina e isoniazida que resulta no produto de degrada??o 3-(isonicotinoilhidrazinometil)rifamicina. Nas curvas de DSC das misturas contendo lactose Supertab? foi evidenciada a ocorr?ncia de incompatibilidade entre os f?rmacos e o excipiente. As curvas de DSC das misturas contendo celulose MC101?, estearato de magn?sio e talco n?o apresentaram altera??es em rela??o ao perfil dos f?rmacos. As curvas de TG/DTG das misturas bin?rias e tern?rias apresentaram perfis termogravim?tricos diferentes em rela??o ao observado para os f?rmacos isoladamente, com in?cio da decomposi??o t?rmica antecipada, dando suporte as evid?ncias de incompatibilidades encontradas nas curvas de DSC e DTA

Made available in DSpace on 2014-07-29T15:30:37Z (GMT). No. of bitstreams: 1 Dissertacao_sueli.pdf: 1117407 bytes, checksum: d50c0b1dad4e594bc8cd5d3880aadcab (MD5) Previous issue date: 2010-03-11 / Multidrug-resistant tuberculosis is a challenge worldwide. Rapid diagnosis by molecular techniques can provide a more aggressive and appropriate initial therapy. This study aimed to analyze the molecular basis of resistance to isoniazid (INH) and rifampin (R) of Mycobacterium tuberculosis strains isolated from cases of human tuberculosis in Goiás and to genetically determine the causes of the observed resistances. Of the 4.607 cultures for mycobacteria processed in the period of September of 2005 and December of 2007, 24 isolates from 16 patients resistant to at least H and/or R were analyzed. We compared the results obtained by phenotypic tests with mutations in key genes responsible for the development of resistance to these drugs, the rpoB gene for isolates resistant to R and katG gene for strains resistant to H. Seventy one percent of the isolates were resistant to H, and the mutations involved with resistance observed in the katG gene were in codon 315 (41%). The most frequent mutations observed in the rpoB gene of the R resistant isolates (71%) were in codons 456 (76.5%) and 451 (17.6%). Our findings are similar to those reported in the literature. We conclude that the percentage of agreement between genotypic and phenotypic tests was 41% for H and 94% for R considering the number of isolates and 40% and 91%, respectively considering the number of patients. / A tuberculose multidroga resistente representa um desafio em escala mundial. O diagnóstico rápido através de técnicas moleculares é capaz de proporcionar uma terapêutica inicial mais agressiva e adequada. Este trabalho teve como objetivo analisar as bases moleculares da resistência à isoniazida (H) e rifampicina (R) de cepas de Mycobacterium tuberculosis isoladas de casos de tuberculose em Goiás e determinar geneticamente as causas destas resistências. Do total de 4.607 culturas para micobactérias realizadas no período de setembro 2005 a dezembro de 2007, foram analisados 24 isolados de 16 pacientes resistentes a H e/ou R. Os resultados obtidos dos testes fenotípicos de sensibilidade aos antimicrobianos foram comparados às mutações observadas nos principais genes responsáveis pelo desenvolvimento de resistência a estas drogas, gene rpoB para isolados resistentes à R e gene katG para os isolados resistentes à H. Dentre os 24 isolados, 71% eram fenotípicamente resistentes a H e as únicas mutações envolvidas com resistência foram observadas no códon 315 (41%). Dos isolados resistentes a R (71%), foram observadas mutações nos códons 456 (76,5%), 451 (17,6%) e 447 (5,9%). Nossos achados estão em concordância com as principais mutações observadas nos isolados resistentes a R e/ou H descritos na literatura. O percentual de concordância entre os testes fenotípicos e genotípicos foi de 41% para H e 94% para R considerando o número de isolados e de 40% e 91% respectivamente considerando-se o número de pacientes.

Novos tipos de sílicas mesoporosas esféricas (SMEs) com tamanho de partícula entre 3 e 10 µm foram sintetizadas utilizando os copolímeros tribloco EO17PO60EO17 (P103) ou EO26PO39EO26 (P85) como direcionadores de estrutura. As SMEs foram preparadas via um processo de síntese com duas etapas de tratamento hidrotérmico (TH) em forno convencional, utilizando o ortosilicato de tetraetila como fonte de sílica, os surfatantes P103 ou P85 como moldes em combinação com o co-surfatante brometo de cetiltrimetilamônio (CTAB) e o co-solvente etanol, sob condições ácidas. As SMEs obtidas foram caracterizadas por microscopia eletrônica de varredura (MEV) e medidas de adsorção/dessorção de N2. O volume e o tamanho do poro das SMEs podem ser aumentados com o aumento da temperatura do TH. O volume e o tamanho do poro (0,41 cm3g-1; 2,84 nm) da amostra sintetizada com P103 aumentaram (1,20 cm3g-1; 4,32 nm) quando a temperatura do TH aumentou de 80°C para 120°C na segunda etapa do TH. O aumento do volume e do tamanho de poro também pode ser obtido utilizando um único tratamento hidrotérmico, porém empregando 1,3,5-trimetilbenzeno (TMB) como agente dilatador de poros. O volume e o tamanho do poro (0,34 cm3g-1; 2,02 nm) da amostra sintetizada com P85 sem TMB aumentaram (0,37 cm3g-1; 2,51 nm) na amostra preparada com P85 e TMB. As características texturais e de superfície dos materiais obtidos com P103 e P85 foram comparadas com um material sintetizado em paralelo, nas mesmas condições experimentais, utilizando o copolímero EO20PO70EO20 (P123) como direcionador de estrutura e CTAB como co-surfatante. A SME sintetizada com P103 foi usada como adsorvente de compostos orgânicos voláteis (COVs) oriundos de misturas padrões. Os componentes da mistura padrão foram então removidos do adsorvente (sílica) por dessorção térmica e introduzidos em uma coluna cromatográfica para separação por cromatografia a gás (CG) e identificação por espectrometria de massa (EM). Esta SME foi também testada como adsorvente de uma amostra de ar coletada em uma rua com significativo fluxo de veículos. O ar foi coletado paralelamente na SME e num adsorvente comercial (Tenax TA/Carbotrap). Os compostos n-hexano, benzeno, tolueno e o-xileno, oriundos de emissões veiculares, foram encontrados em ambos adsorventes (sílica e Tenax TA/Carbotrap). O fármaco Rifampicina foi encapsulado numa SME sintetizada com P123/CTAB e na sílica SBA-15 (poros ordenados hexagonalmente). A encapsulação do fármaco (cerca de 30%) em ambas as sílicas foi confirmada pelos resultados de adsorção/dessorção de N2. / New types of mesoporous silica spheres with particle diameter of 3 - 10 µm were synthesized by using a triblock copolymer EO17PO60EO17 (P103) or EO26PO39EO26 (P85) as templates. The microspheres were prepared via a two-step hydrothermal treatment (HT) in an oven by using tetraethoxysilane as silica source, the surfactants P103 or P85 as templates in combination with a cosurfactant cetyltrimethylammonium bromide (CTAB) and a cosolvent ethanol, under acidic conditions. The obtained silica spheres in both procedures were characterized by scanning electron microscopy (SEM) and N2 sorption technique. The volume and the pore size of the silica spheres can become greater by increasing the temperature of the HT. The volume and the pore size (0.41 cm3g-1; 2.84 nm) of the sample prepared with P103 became greater (1.20 cm3g-1; 4.32 nm) when the temperature of HT increased by 80°C to 120°C in the second step of the HT. The volume and the pore size can also be increased using 1,3,5-trimethylbenzene (TMB) as a swelling agent, instead of raising the temperature of the HT. The volume and the pore size (0.34 cm3g-1; 2.02 nm) of the sample prepared with P85 without TMB became greater (0.37 cm3g-1; 2.51 nm) in the sample prepared with P85 and TMB. The characteristics of textures and surfaces of the materials synthesized by using P103 or P85 were compared with a material prepared with the copolymer EO20PO70EO20 (P123) as template using the same experimental conditions. The silica microspheres synthesized with P103 were used as adsorbents for volatile organic compounds (VOCs) from standard mixtures. The compounds of the standard mixture were then removed from the adsorbent (silica) by thermal desorption and introduced into a chromatographic column for separation by gas chromatography (GC) and identification by mass spectrometry (MS). This material was also used as adsorbent of an air sample collected on a street with a significant flow of motor vehicles. The air was collected on the silica and on a commercial adsorbent (Tenax TA/Carbotrap) one by one. The compounds n-hexane, benzene, toluene and o-xylene, resulted from the emissions from vehicles, were found in both adsorbents (silica and Tenax TA/Carbotrap). The drug Rifampicin was encapsulated in the mesoporous spherical silica, prepared with P123/CTAB and in the silica SBA-15 (hexagonally ordered pores). The encapsulation of the drug (about 30%) in both the silica was confirmed by measurements of adsorption/desorption of N2

A hanseníase, doença crônica, granulomatosa, infecto-contagiosa, transmitida pelo Mycobacterium leprae, ainda se mantém prevalente nos dias atuais, principalmente em países subdesenvolvidos. A OMS propôs aos países endêmicos um novo esquema terapêutico alternativo para pacientes com hanseníase paucibacilar com lesão única, que vem sendo implantado gradativamente no Brasil, constituindo-se da administração de rifampicina (600mg), ofloxacina (400mg) e minociclina (100mg), em dose única. Sendo uma estratégia recente, estudos são necessários para se avaliar a toxicidade principalmente na associação medicamentosa dos fármacos. O objetivo deste trabalho foi investigar a correlação dose/concentração plasmática versus alterações bioquímicas, hematológicas, hemostáticas e histopatológicas da administração da rifampicina, ofloxacina e minociclina a ratos machos Wistar, em regime de dose única e múltiplas em monoterapia e na associação medicamentosa. Após a administração intraperitoneal dos fármacos nos animais, foi determinada a concentração plasmática por cromatografia líquida de alta eficiência (CLAE), e avaliação dos parâmetros bioquímicos, hematológicos, hemostáticos e histopatológicos. A rifampicina e ofloxacina sofreram um aumento na concentração plasmática em esquema de dose única quando administradas associadas, e a minociclina uma redução. Em esquema de doses múltiplas, a rifampicina sofreu redução na concentração plasmática, a ofloxacina aumento, e a minociclina não sofreu alteração significativa, provavelmente por interferências em processos farmacocinéticos. Na análise bioquímica concluímos que houve alteração hepática e renal nos animais que receberam a rifampicina e o esquema ROM em dose única, fato não observado em regime de doses múltiplas. Com os parâmetros hematológicos e hemostáticos não obtivemos resultados suficientes para afirmar uma alteração clinicamente importante. A histopatologia confirmou a lesão hepática induzida por todos os fármacos em esquema de dose única e múltiplas, complementando de forma satisfatória o estudo proposto. Concluímos que a rifampicina provavelmente seja a principal responsável por alterações hepáticas e renais, e que as interações medicamentosas envolvendo o fármaco exigem estudos individualizados principalmente quando o fármaco é usado associado a ofloxacina e minociclina, que podem estar ligados a reações adversas de origem idiossincrásica. / Leprosy, a chronic granulomatous infectious-contagious disease transmitted by Mycobacterium leprae, continues to be prevalent today, especially in underdeveloped countries. The WHO has proposed a new alternative treatment scheme to endemic countries for patients with paucibacillary leprosy with a single lesion. This scheme is being gradually implanted in Brazil and consists of the administration of rifampicin (600 mg), ofloxacin (400 mg) and minocycline (100 mg) in a single dose. Since this is a recent strategy, studies are necessary to determine the toxicity of the treatment, especially regarding the medicamentous combination of the drugs. The objective of the present study was to investigate the dose/plasma concentration correlation regarding biochemical, hematologic, hemostatic and histopathological changes induced by the administration of rifampicin, ofloxacin and minocycline to male Wistar rats as a single dose or multiple doses in monotherapy and polytherapy. After intraperitoneal administration of the drugs, their plasma concentrations were determined by high performance liquid chromatography (HPLC) and the biochemical, hematologic, hemostatic and histopathological parameters were determined. Plasma rifampicin and ofloxacin concentrations were increased in the single dose scheme when administered in combination, whereas minocycline concentration was reduced. When administered in the multiple dose scheme, rifampicin showed reduction in plasma concentration, ofloxacin showed increase, and minocycline showed no significant change, probably due to interferences in the pharmacokinetic processes. Biochemical analysis revealed hepatic and renal changes in the animals that received rifampicin and the ROM scheme in a single dose, a fact not observed in the multiple dose regimen. Regarding the hematologic and hemostatic parameters, we did not obtain sufficient result to indicate a clinically important alteration. Histopathology confirmed the hepatic damage induced by all drugs administered as a single dose or multiple doses, satisfactorily complementing the proposed study. We conclude that rifampicin may probably be the drug mainly responsible for the hepatic and renal changes and that medicamentous combinations involving this drug require individualized studies, especially when the drug is used in combination with ofloxacin and minocycline, that may be linked to adverse reactions of idiosyncratic origin.

Thesis title: Formulation and characterization of oxime loaded PLGA nanoparticles Author: Ramin Hafezi Supervisor: PharmDr. Eva Šnejdrová, Ph.D. Advisor: PharmDr. Juraj Martiška, Ph.D. Department: Department of Pharmaceutical Technology The diploma thesis was focused on PLGA nanoparticles (NPs) which could be loaded with oximes, prepared by a double emulsion technique, and characterised by size, polydispersity and zeta potential. The theoretical part deals with the most common methods of the NPs preparation, the polymers and stabilizers employed, and drug delivery to brain. In the experimental part the effect of various formulation factors on NP characteristics were studied: linear or branched PLGA derivative, the concentrations of polymer, the volumes of primary emulsion. Dichloromethane (DCM) or Dimethyl sulfoxide (DMSO) as solvent for polymers were used and Poloxamer 407 or Didodecyldimethylammonium bromide (DDAB) as an outer phase stabilizer were employed. By comparison among the collected results, it seemed 1% A2 in DMSO and stabilization with poloxamer 407 could be best candidate for the oxime loaded drug delivery systems as it was possible to produce nanoparticles with size from 152 to 168 nm with PDI of below 0.15. Electrostatic stability in case of using DDAB was resulted excellent and...

Amorphous solid dispersion (ASD) is a popular method to increase drug solubility and consequently poor drug bioavailability. Cellulose ω-carboxyesters were designed and synthesized specifically for ASD preparations in Edgar lab that can meet the ASD expectations such as high Tg, recrystallization prevention and pH-triggered release due to the free -COOH groups. Rifampicin (Rif), Ritonavir (Rit), Efavirenz (Efa), Etravirine (Etra) and Quercetin (Que) cellulose ester ASDs were investigated in order to increase drug solubility, prevent release at low pH and controlled release of the drug at small intestine pH that can improve drug bioavailability, decrease needed drug content and medication price to make it affordable in third world countries, and extent pill efficiency period to improve patient quality of life and adherence to the treatment schedule. The studies were compared with cellulose based commercial polymers to prove the impact of the investigation and potential for the application. Furthermore, the in vitro results obtained were further supported by in vivo studies to prove the significant increase in bioavailability and show the extended release. The need of new cellulose derivatives for ASD applications extended the research area, the design and synthesis of a new class of polymers, alkyl cellulose ω-carboxyesters for ASD formulations investigated and the efficiency of the polymers were summarized to show that they have the anticipated properties. The polymers were synthesized by the reaction of commercial cellulose alkyl ethers with benzyl ester protected, monofunctional hydrocarbon chain acid chlorides, followed by removal of protecting group using palladium hydroxide catalyzed hydrogenolysis to form the alkyl cellulose wcarboxyalkanoate. Having been tested for ASD preparation, it was proven that the polymers were efficient in maintaining the drug in amorphous solid state, release the drug at neutral pH and prevent the recrystallization for hours, as predicted. / Ph. D.