Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is a common and potentially lethal infectious human disease. Rifampicin is a front line anti-tuberculosis drug usually prescribed in combination with isoniazid, pyrazinamide and streptomycin for a period of six to seven months. When given orally for the treatment of MTB, rifampicin exhibits low bioavailability. Recent attempts to increase bioavailability and decrease dosage of anti-tuberculosis drugs have focused on creating polymer coated rifampicin nanoparticles. The research effort presented in this thesis evaluates the formation, characterization and relative bioavailability of rifampicin loaded carboxymethylcellulose acetate butyrate (CMCAB) particles using two different formulation techniques. Multi inlet vortex mixer (MIVM) and manual spray drying techniques were used to form the rifampicin containing CMCAB particles. Characterization studies and analyses of particles revealed differences in particle sizes, shapes and drug loading between the different particle formulation techniques. In vivo pharmacokinetic studies in BALB/c mice indicate that a single dose of rifampicin laden CMCAB spray dried particle formulations are able to improve pharmacokinetic parameters including relative bioavailability of rifampicin compared to that of the free drug form at the same concentration. / Master of Science

Tee PhD thesis presents the study of the response of Mycobacterium tuberculosis, the causative agent of tuberculosis, upon prolonged exposure to lethal concentrations of the first line anti-tuberculosis drug, rifampicin. The study shows that prolonged exposure to lethal concentration of rifampicin causes cell death initially by several log orders of cells, followed by a persistence phase, from where rifampicin resisters emerge carrying mutation in the RRDR locus of the rpoB gene. This phenomenon was found to occur even when the drug concentration is well above MBC levels. Luria-Delbruck experiment, in a modified format, showed that the resisters emerged as fresh mutants, and not due to the growth of pre-existing natural resisters to rifampicin. The per sister cells, which showed high levels of hydroxyl radical generation, were found to have thickened outer layer, unlike the mid-log phase cells, which restricts the permeability of a fluorescent-conjugate of rifampicin, 5-FAM-rifampicin, 10-fold less in per sister cells, as compared to mid-log phase cells. The thickened outer layer has high negative surface charge and is hydrophilic in nature. It is proposed that the hydrophilic-natured thickened outer layer might have restricted the permeability of lethal concentrations of hydrophobic-natured rifampicin. This, in turn, might have ensured the presence of sub-lethal concentration rifampicin inside the per sisters, which in turn might have generated the hydroxyl radical that caused mutagenesis to generate rifampicin resisters. The Chapter 1 is the Introduction to the thesis presented in 4 parts – Part 1.1, 1.2, 1.3 and 1.4, introducing briefly the history of antibiotics, antibiotic resistance, antibiotic persistence and a brief history of tuberculosis respectively. It is concluded with a rationale behind the present study. The Chapter 2 presents the entire Materials and Methods used in the experiments described in the thesis. The Chapter 3 presents the data on the response of M. tuberculosis to rifampicin upon extended exposure. Rifampicin exposure of susceptible M. tuberculosis H37Ra cells showed a decrease in their CFU/ml followed by a persistence phase wherein the CFU/ml remained constant. However, prolonged exposure of rifampicin even at higher concentrations showed regrowth in the culture, which was found to be due to the emergence of rifampicin-resistant bacteria. Screening of rifampicin-resistant mutants showed point mutations in the rifampicin resistance determining region (RRDR) of the rpoB gene in all the mutants. In parallel, using trans formants of M. tuberculosis expressing unstable GFP under the respective native ribosomal RNA promoter, the metabolic status of the per sister cells was determined. When actively growing highly fluorescing cells were exposed to lethal concentration of rifampicin, their metabolism diminished, as illustrated by the decrease in their fluorescence during persistence phase, followed by the emergence of a sub-population of bacteria which were again metabolically active. In order to verify whether the rifampicin resisters are freshly formed mutants or have come from the naturally existing resisters, Luria-Delbruck fluctuation test was performed in a modified manner. The number of rifampicin resisters that emerged from the persistent phase was found to vary amongst different cultures from different days and different times of exposure, showing fluctuation. However, the addition of theorem before persistence phase almost abolished the generation of the rifampicin-resistant bacilli, indicating the role of hydroxyl radical in the emergence of rifampicin resisters. The generation of hydroxyl radical in mycobacteria exposed to rifampicin was confirmed using electron para-magnetic resonance spectrometry (EPR), with the spin trap agent, 5,5- Dimethyl-1-pyrroline N-oxide (DMPO) specific for hydroxyl radical. An increase in the formation DMPO-OH adduct in the persistence phase cells was observed, in comparison to mid-log phase cells. Exposure to theorem significantly diminished the adduct formation. The persistence phase cells also showed significantly high levels of signal specific to the hydroxyl-specific fluorescent dye, hydroxyphenyl fluorescein (HPF), as compared to the mid-log phase cells. In addition we have determined the oxidative stress in the bacilli upon rifampicin exposure using a redox biosensor (Mrx1-roGFP) which also showed high oxidative stress in the persistent phase. These observations confirmed the presence of high levels of oxidative stress and hydroxyl radical in the rifampicin persistent cells, in comparison with mid-log phase cells. Whole genome sequencing of the four independently isolated rifampicin resistant M. tuberculosis showed genome wide mutations having less common mutations with respect to the wild type genome, indicative of the occurrence of random mutagenesis. In addition mutation frequencies were comparable between the samples with respect to the wild type sample. About 69% of the mutations were A-C or T-G, followed by A-T or T-A, which is known to be due to oxidative stress in the cells. Variations in the colony morphology were also observed on the persistent phase cells, indicating the occurrence of mutagenesis in the bacterial genome during rifampicin treatment. The Chapter 4 is on the morphological and ultrastructural studies on rifampicin-exposed M. tuberculosis cells. Transmission electron micrographs of rifampicin per sisters showed significant thickening of the outermost capsular layer (OL), as compared to the mid-log phase cells. This observation was verified by staining the cells with ruthenium red, which specifically stains anionic polysaccharide of the OL. Zeta potential (ZP) measurement of the surface charge of the persistent cells showed high negative ZP, as compared to the mid-log phase cells. The negative ZP value was found to gradually increase during the course of rifampicin treatment and to decrease during the regrowth phase. Hexadecane assay showed larger proportion of per sister cells being retained in the aqueous phase, as compared to the mid-log phase cells. This indicated the higher hydrophilicity of the per sister cells, which was in agreement with the higher surface negative charge of the cells. The permeability of rifampicin per sister cells to 5-FAM-rifampicin (rifampicin conjugated to 5-carboxy fluorescein, which is as hydrophobic as rifampicin) was found to be 10-fold less than that of the mid-log phase cells. However, removal of the thick OL by bead beating (BB) with 4 mm glass beads significantly improved the permeability of per sister cells to 5-FAM-rifampicin. On the contrary, no difference in the 5-FAM-rifampicin uptake was observed in mid-log phase cells, with or without BB. These observations implied that the thick hydrophilic-natured OL with high negative surface charge may be playing a significant role in limiting the permeability of hydrophobic-natured rifampicin entry into the persisted cells. This in turn may ensure the presence of sub-lethal concentration of rifampicin inside the persisted cells that are exposed to lethal concentration of the antibiotic. Exposure of bacteria to sub-lethal concentration of antibiotics has been reported to generate oxidative stress in the bacteria, leading to mutagenesis. A model has been proposed based on these observations in which the persistent mycobacteria are protected from lethal concentrations of the rifampicin by the thick OL which in turn ensures sub-lethal intracellular antibiotic concentration, leading to the generation of hydroxyl radical mediated mutagenesis and thereby emergence of rifampicin resisters. This thesis is concluded with the list of salient findings, publications and references.

Made available in DSpace on 2014-12-17T14:16:37Z (GMT). No. of bitstreams: 1 DayanneLP_DISSERT.pdf: 9773593 bytes, checksum: c2be9b4f3b701218666100df5b460f30 (MD5) Previous issue date: 2014-03-24 / Since its synthesis over 48 years rifampicin has been extensively studied. The literature reports the characterization of thermal events for rifampicin in nitrogen atmosphere, however, no characterization in synthetic air atmosphere. This paper aims to contribute to the thermal study of rifampicin through thermal (TG / DTG, DTA, DSC and DSC - FOTOVISUAL ) and non-thermal (HPLC, XRPD , IR - FTIR , PCA) and its main degradation products ( rifampicin quinone , rifampicin N-oxide 3- formylrifamicin). Rifampicin study was characterized as polymorph form II from techniques DSC, IR and XRPD. TG curves for rifampicin in synthetic air atmosphere showed higher thermal stability than those in N2, when analyzed Ti and Ea. There was characterized as overlapping events melting and recrystallization under N2 with weight loss in the TG curve, suggesting concomitant decomposition. Images DSCFotovisual showed no fusion event and showed darkening of the sample during analysis. The DTA curve in synthetic air atmosphere was visually different from DTA and DSC curves under N2, suggesting the absence of recrystallization and melting or presence only decomposition. The IV - FTIR analysis along with PCA analysis and HPLC and thermal data suggest that rifampicin for their fusion is concomitant decomposition of the sample in N2 and fusion events and recrystallization do not occur in synthetic air atmosphere. Decomposition products studied in an air atmosphere showed no melting event and presented simultaneously to the decomposition initiation of heating after process loss of water and / or solvent, varying the Ti initiating events. The Coats - Redfern , Madsudhanan , Van Krevelen and Herwitz - Mertzger kinetic parameters for samples , through the methods of OZAWA , in an atmosphere of synthetic air and / or N2 rifampicin proved more stable than its degradation products . The kinetic data showed good correlation between the different models employed. In this way we contribute to obtaining information that may assist studies of pharmaceutical compatibility and stability of substances / estudada. H? relatos de estudos focando o desenvolvimento de metodologias anal?ticas, novas aplica??es farmac?uticas, bem como, desenvolvimento de novas formas farmac?uticas. A busca pelo entendimento dascaracter?sticas f?sico-qu?micas das subst?ncias tem auxiliado no desenvolvimento de novos produtos farmac?uticos, com seguran?a, efic?cia e qualidade,fornecendo informa??es ?teis sobre s?ntese e armazenamento. Dentre os produtos de decomposi??o j? conhecidos para rifampicina, temos a rifampicina quinona, rifampicina N-?xido e 3-formilrifampicina, para tais, dados t?rmicos s?o escassos na literatura. As t?cnicas t?rmicas v?m sendo utilizadas na ?rea farmac?utica em diversas aplica??es, como na caracteriza??o de f?rmacos, determina??o do grau de pureza, identifica??o de polimorfismo, estudos de estabilidade, compatibilidade e cin?tica de degrada??o. Este trabalho tem como objetivo contribuir com o estudo t?rmico da rifampicina atrav?s das t?cnicas t?rmicas (TG/DTG, DTA, DSC, DSC-Fotovisual)e n?o t?rmicas, e seus principais produtos de degrada??o (rifampicina quinona, rifampicina N-?xido 3-formilrifamicina). A partir de an?lises DSC, DRX e FTIR foi poss?vel caracterizar a rifampicina estudada como polimorfo II. O conjunto de t?cnicas t?rmicas e n?o t?rmicas auxiliaram a verificar que parte da rifamipicina ? decomposta durante o processo de fus?o, em atmosfera de nitrog?nio, bem como que, os eventos de fus?o e recristaliza??o n?o ocorrem em atmosfera de ar sint?tico passando a amostra diretamente a decomposi??o. Os produtos de decomposi??o estudados, quando em atmosfera de ar, n?o apresentaram evento de fus?o e, apresentaram v?rios passos de decomposi??o, com a ocorr?ncia de eventos exot?rmicos e endot?rmicos. A partir de curvas TG din?micas, foi poss?vel calcular os par?metros cin?ticos para as amostras, atrav?s dos m?todos de OZAWA, Coats-Redfern, Madsudhanan, Van Krevelen e Herwitz-Mertzger, em atmosfera de ar sint?tico e/ou nitrog?nio. Os dados cin?ticos mostraram boa correla??o entre os diferentes modelos empregados. Tanto para rifampicina quanto os produtos de degrada??o estudados, foi caracterizado rea??o de ordem um

Thesis (MSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Varying levels of Rifampicin (RIF) resistance in closely related clinical Mycobacterium tuberculosis isolates and in vitro generated mutants question the dogma that non-synonymous single nucleotide polymorphisms in the rpoB gene are the only mechanism explaining RIF resistance. This study aimed to identify biological mechanisms that define the level of RIF resistance in two closely related clinical M. tuberculosis isolates using proteomic, transcriptomic and genomic approaches. Two dimensional electrophoresis revealed an increase in the abundance of numerous membrane proteins in response to RIF at the critical concentration of 2g/ml. Fourty-one of these proteins were identified by mass spectrometry and could be grouped according to their cellular function (Energy metabolism, degradation, biosynthesis of cofactors, metabolic groups and carriers, lipid biosynthesis, central intermediate metabolism, synthesis and modification of macromolecules, chaperone/heat shock proteins). The identification of proteins responsible for ATP synthesis (atpA and atpH) suggests an ATP requirement to combat the toxic effect of RIF. These proteins are components of the FoF1 ATP synthase an enzyme which is involved in the oxidative phosphorylation pathway that generates ATP in the cell. QRT-PCR confirmed the up regulation of the transcription of the atpA and atpH genes in response to RIF, while DNA sequencing failed to identify mutations that could define the rate of transcription. To explain our findings we proposed that RIF induces a toxic response leading to the up regulation of a number of genes. The induction of metabolic enzymes, such as the FoF1 ATP synthase provides energy to activate ATP dependant mechanisms, including membrane ABC transporters. These ABC transporters actively pump RIF out of the cell thereby lowering the intracellular concentration of RIF to below its binding concentration with the rpoB protein leading to RIF resistance. Inhibition of efflux by the efflux pump inhibitors reserpine and verapamil leads to an accumulation of RIF within the cell and concurrent binding of RIF to rpoB, leading to inhibition of transcription and cell death (ongoing research in our laboratory). Similarly, we propose that the recently identified diarylquinoline compound (TMC207) inhibit ATP synthesis, thereby depleting the energy source necessary for active efflux. This will lead to an accumulation of anti-TB drug within the cell and subsequent cell death. In summary, this study provides the first evidence to suggest that the evolution of RIF resistance is a dynamic process involving a cascade of adaptive events which leads to a bacterial growth state where hydrophobic compounds are actively extruded from the cell. This has important ramifications for the treatment of RIF resistant TB and supports the need for the development of anti-TB drugs that target both efflux and ATP synthesis to improve the treatment outcome of MDR-TB and XDR-TB. / AFRIKAANSE OPSOMMING: Verskillende vlakke van Rifampisien (RIF) weerstandigheid, in naby verwante Mycobacterium tuberculosis kliniese isolate en in vitro mutante, bevraagteken die dogma dat nie-sinonieme enkel nukleotied polimorfismes in die rpoB geen die enigste verklaarbare meganisme vir RIF weerstandigheid is. Die doel van hierdie studie was om deur 'n proteomiese, transkriptomiese en genomiese benadering, biologiese meganismes te identifiseer wat die vlakke van RIF weerstandigheid in twee naby verwante kliniese M. tuberculosis isolate bepaal. Twee dimensionele elektroferese het gevind dat daar 'n verhoging in die hoeveelheid van verskeie proteïne is wanneer die isolate aan RIF by die 'n kritiese konsentrasie van 2μg/ml blootgestel is. Massa spektrometrie het 41 van hierdie proteine geïdentifiseer en die proteïne kan gegroepeer word in verskeie sellulêre funksies (Energie metabolism, degradering, biosintese van kofaktore, metaboliese groepe en draers, lipied biosintese, sentrale intemediêre metabolisme, sintese en modifisering van makromolekules, en “chaperone/heat shock” proteine). Die identifisering van proteïne verantwoordlik vir ATP sintese (atpA en atpH) stel voor dat ATP belangrik is om die toksiese effek van RIF te ontwyk. Hierdie proteïne is komponente van die FoF1 ATP sintase ensiem wat betrokke is in die oksidatiewe fosforilerings pad en wat lei tot die generering van ATP in die sel. Kwantitatiewe QRT-PCR het bevestig dat hierdie gene, atpA en atpH, opgereguleer word nadat die bakterium aan RIF blootgestel is. In teen deel kon DNA volgorde bepaling nie mutasies identifiseer wat die verandering in geen transkripsie kon verklaar nie. Om ons bevindings te verduidelik, stel ons voor dat RIF 'n toksiese effek in die sel induseer wat lei tot die opregulering van verskeie gene. Die indusering van metaboliese ensieme, soos die FoF1 ATP sintase, voorsien energie om ATP afhanklike meganismes, insluitende membraan ABC transporters, te aktiveer. Hierdie ABC transporters pomp RIF aktief uit die sel, wat daarvolgens die intrasellulêre konsentrasie van RIF verlaag tot 'n konsentrasie laer as die bindings konsentrasie met die rpoB protein en gevolglik lei tot weerstandigheid. Die onderdrukking van membraan pompe wat RIF uit die sel pomp deur middels soos reserpine en verapamil sal aanleiding gee lei tot akkumulering van RIF in die sel. Die verhoogde RIF in die sel versoorsaak dat RIF aan die rpoB protein gebind bly sodat dit transkripsie inhibeer, wat dan aanleiding gee tot seldood. (voortgesette navorsing in ons laboratorium). Soortgelyk, stel ons voor dat die onlangs geïdentifiseerde dairylquinoline verbinding (TMC207) ATP sintese inhibeer en daarvolgens die energie bron uitput wat noodsaaklik is vir aktiewe uitpomp van RIF. Dit sal aanleiding gee tot die ophoping van RIF in die sel en gevolglik lei tot seldood. In opsomming, hierdie studie voorsien die eerste bewys wat voorstel dat die evolusie van RIF weerstandighied 'n dinamiese proses is. Dit sluit 'n kaskade van aanpasbare gebeurtenisse in wat lei tot 'n bakteriële groei fase waar hidrofobiese verbindings aktief uit die sel gedryf word. Dit het rampspoedige gevolge vir die behandeling van RIF weerstandige TB en ondersteun die noodsaaklikheid om teen-TB middels te ontwikkel wat beide effluks pompe en ATP sintese teiken om die uikoms van behandeling vir MDR-TB en XDR-TB te verbeter.

ABSTRACT Rifampicin is a major chemotherapeutic agent used against mycobacterial and nocardial infections. High level resistance is primarily due to mutational alterations in the rpoB gene encoding the β subunit of RNA polymerase. When challenged, these bacteria may inactivate rifampicin by one of four mechanisms: decomposition, ADP-ribosylation, glucosylation and phosphorylation. ADPribosylation occurs in many mycobacterial pathogens but nothing is known about the properties of the enzyme responsible. Consequently mutational analysis may be used to explore structure-function relationships in this protein. Three mutants with changes in the open reading frame were selected and studied. The altered arr gene in pMG1 was obtained by in vivo selection whilst in pMG2 and pMG4 by in vitro mutagenesis. The mutated arr gene in pMG1 and pMG2 conferred resistance to 50 μg/ml of rifampicin while in pMG4 to 200 μg/ml. This suggested that alterations near the N-terminus resulted in lowered activity because of closer proximity to the active site. This is the first successful report of induced arr gene expression. This over-expression of the Arr ADP-ribosyltransferase and its mutants assisted in their later purification by metal affinity chromatography.

A tuberculose (TB) ainda se apresenta como desafio para a Saúde Pública, a nível global. Essa doença negligenciada (DN) tem como tratamento de primeira escolha a rifampicina. Esse fármaco pertence à classe II, segundo o Sistema de Classificação Biofarmacêutica (SCB), apresentando baixa solubilidade em água. Tal característica constitui desafio no desenvolvimento de formas farmacêuticas eficazes e seguras. O uso de nanotecnologia tem se destacado como alternativa promissora para melhorar a solubilidade aquosa de fármacos. Nesse sentido, o presente trabalho teve como objetivo a preparação e a caracterização físico-química de nanocristais de rifampicina. A preparação dos nanocristais foi realizada empregando método de moagem de alta energia, homogeneização a alta pressão e moagem via úmida em escala reduzida. Os resultados referentes ao método de moagem de alta energia (MAE) demostraram formação de nanocristais, mas em quantidade reduzida seguida da formação de agregados (F1-M, F2-M e F3-M). A homogeneização a alta pressão (HAP) permitiu a formação de nanocristais (F1-H e F2-H). A formulação F1-H contendo o poloxâmero 188 não apresentou estabilidade após 24 horas da preparação. A F2-H obteve diâmetro hidrodinâmico médio (DHM) de 412,60 ± 4,12 nm, índice de polidispersividade igual a 0,12 ± 0,02 e potencial zeta igual a -9,94 ± 0,19 mV. A elevada concentração requerida do agente estabilizante para essa formulação foi fator limitante para o seu desenvolvimento. A moagem via úmida em escala reduzida permitiu a formação de nanocristais de rifampicina F1-MU e F2-MU, com DHM igual a 340,20 ± 5,44 nm e 364,2 ± 4,50 nm, respectivamente, e distribuição de tamanho uniforme. A avaliação do DHM, do IP e do PZ, por período de três meses, revelou a estabilidade dessas formulações. Essas formulações foram obtidas por meio de planejamento de experimentos por superfície de resposta tendo como variáreis a concentração de rifampicina, a concentração do agente estabilizante e a quantidade de esferas de zircônia. As medidas de distribuição de tamanho médio das partículas e a morfologia foram realizadas utilizando difração a laser (LD) e microscopia eletrônica de transmissão (MET), respectivamente. Adicionalmente, as avaliações empregando calorimetria exploratória diferencial (DSC) e difração de raio X (DRX) revelaram que não houve mudança na estrutura cristalina do polimorfo II de rifampicina e nem interação entre o fármaco e os excipientes. O presente trabalho permitiu a obtenção de de nanocristais de rifampicina estáveis e com solubilidade maior de até 1,92 vezes (F1-MU) e 1,66 vezes (F2-MU), em água, quando comparada à rifampicina matéria-prima. Os perfis de dissolução das formulações F1-MU e F2-MU demonstraram dissolução de 95% de rifampicina em aproximadamente 5 minutos. Esse resultado é significativamente superior àquele observado para o produto FURP-rifampicina suspensão oral 20 mg/mL que apresentou dissolução de 23,2% nesse mesmo intervalo de tempo. A avaliação da atividade antimicrobiana das nanosuspensões foi confirmada frente à rifampicina padrão por meio da determinação da sua concentração mínima inibitória. / Tuberculosis (TB) still presents a challenge for public health globally. This Neglected Tropical Disease (NTDs) has as the treatment of choice rifampicin. This drug belongs to the class II, according to Biopharmaceutics Classification System (BCS), with low water solubility. This characteristic is a challenge in the development of safe and effective dosage forms. The nanotechnology has emerged as a promising alternative to improve the aqueous solubility of drugs. Accordingly, the present work aimed to the preparation and physicochemical characterization of nanocrystals of rifampicin. The preparation of the nanocrystals was performed using high-energy ball milling method, high-pressure homogenization and wet grinding process on a small scale. The results of the high-energy ball milling method demonstrated formation of nanocrystals, but in small amounts followed by the formation of aggregates (F1-M, F2-M and F3-M). The high pressure homogenization (HPH) allowed the formation of nanocrystals (F1-H and F2-H). F1-M formulation containing Poloxamer 188 did not show stability after 24 hours preparation. F2-H obtained mean hydrodynamic diameter (DHM) of 412.60 ± 4.12 nm, polydispersity index of 0.12 ± 0.02 and zeta potential of -9.94 ± 0.19 mV. The high concentration of stabilizing agent required for this formulation was a limiting factor for the development. The wet grinding process on a small scale allowed the formation of rifampicin nanocrystal F1-MU and F2-MU with DHM of 340,20 ± 5,44 nm e 364,2 ± 4,50, respectively, and size distribution uniform. The evaluation of DHM, IP and PZ, for three months, showed stability of these formulations. These formulations were obtained by design of experiments using response surface having as variables the concentration of rifampicin, the concentration of the stabilizing agent and the amount of zirconia beads. The mean size distribution measurements of particles and morphology were performed using laser diffraction (LD) and transmission electron microscopy (TEM), respectively. Additionally, the evaluations using differential scanning calorimetry (DSC) and X-ray diffraction (XRD) revealed that there was no change in the crystalline structure of polymorph II of rifampicin and no interaction between the drug and excipients. This study allowed obtaining stable rifampicin nanocrystals and greater solubility of up to 1.92 times (F1-MU) and 1.66 times (F2-MU) in water compared to rifampicin feedstock. The dissolution profiles of F1-MU and F2-MU formulations showed 95% dissolution of rifampicin in approximately 5 minutes. This result is significantly higher than that observed for the rifampicin-FURP oral suspension product 20 mg / ml that had Dissolving 23.2% over the same time interval. The evaluation of the antimicrobial activity of nanosuspensions was confirmed against the standard rifampicin by determining its minimum inhibitory concentration.

Made available in DSpace on 2014-06-11T19:22:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-17Bitstream added on 2014-06-13T19:27:32Z : No. of bitstreams: 1 derosa_hj_me_arafcf.pdf: 478559 bytes, checksum: f1d5bcb78f493bd18ca8a97c083e3088 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / Propp / Este trabalho teve como objetivo estudar os efeitos da rifampicina (RMP) sobre os parâmetros farmacocinéticos da isoniazida (INH), sobre a produção de seus metabólitos e sobre a sua hepatotoxicidade. Foram utilizados 140 ratos (Wistar, machos, peso médio 250g) que receberam, por gavagem, durante 21 dias: Grupo I - água estéril (n = 20); Grupo II - INH (100mg/Kg) (n = 50); Grupo III - RMP (100mg/Kg) (n = 20) e Grupo IV- INH (100mg/Kg) + RMP (100mg/Kg) ( n= 50). Anteriormente ao início do experimento, o sangue de todos os animais foi coletado pela cauda para determinação da atividade sérica de AST e ALT, cujos valores foram considerados como basais. No 21o do experimento os animais foram sacrificados por decapitação e o material biológico obtido foi utilizado para a determinação da atividade de AST e ALT e para a análise dos parâmetros farmacocinéticos da INH nos grupos II e IV. A cinética da isoniazida e de seus metabólitos foi investigada com base na relação concentração plasmática x tempo a partir de amostras seriadas de sangue em 10 tempos diferentes (0; 15þ; 30þ; 45þ; 60þ; 1,5h; 3h; 6h; 12h; e 24h); para cada tempo de coleta foram empregados 5 ratos (5 replicatas). As amostras de soro foram desproteinizadas com ácido tricloroacético 10%, derivatizeda com cinamaldeído 1% e analisada por HPLC.... / The aim of the present study was to evaluate the hepatotoxicity, pharmacokinetic parameters and biotransformation of isoniazid when rats were treated with isoniazid (INH); rifampicin (RMP); and INH + RMP. Daily doses of the tuberculostatic drugs were administrated intragastrically to the animals (Wistar rats) for one period of 21 days as follow: sterile water (group I, control); INH (100mg/Kg) (group II), RMP (100mg/Kg) (group III); INH (100mg/Kg) + RMP (100mg/Kg) (group IV). The serum levels of the biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined before the administration of the drugs (basal) and after the 21 days treatments. On day 21, blood samples were obtained before and 15þ; 30þ; 45þ; 60þ; 1,5; 3h; 6h; 12h and 24 hours after the dose. (five animals for each point). The blood samples were deproteinized with 10% trichloroacetic acid, derivatized by 1% cinnamaldehyde and analyzed by liquid chromatograph. For the determination of the acetylated metabolites acetylisoniazid (AcINH) and acetylhydrazine (AcHz) a previous hydrolysis with 6 M hydrochloride acid was performed. The results are presented as mean and SEM. The pharmacokinetic parameter of the INH and its metabolites AcINH and hydrazine (Hz) were compared between the groups (p < 0.05, Student t-test)...(Complete abstract, click electronic address below).

A tuberculose (TB) ainda se apresenta como desafio para a Saúde Pública, a nível global. Essa doença negligenciada (DN) tem como tratamento de primeira escolha a rifampicina. Esse fármaco pertence à classe II, segundo o Sistema de Classificação Biofarmacêutica (SCB), apresentando baixa solubilidade em água. Tal característica constitui desafio no desenvolvimento de formas farmacêuticas eficazes e seguras. O uso de nanotecnologia tem se destacado como alternativa promissora para melhorar a solubilidade aquosa de fármacos. Nesse sentido, o presente trabalho teve como objetivo a preparação e a caracterização físico-química de nanocristais de rifampicina. A preparação dos nanocristais foi realizada empregando método de moagem de alta energia, homogeneização a alta pressão e moagem via úmida em escala reduzida. Os resultados referentes ao método de moagem de alta energia (MAE) demostraram formação de nanocristais, mas em quantidade reduzida seguida da formação de agregados (F1-M, F2-M e F3-M). A homogeneização a alta pressão (HAP) permitiu a formação de nanocristais (F1-H e F2-H). A formulação F1-H contendo o poloxâmero 188 não apresentou estabilidade após 24 horas da preparação. A F2-H obteve diâmetro hidrodinâmico médio (DHM) de 412,60 ± 4,12 nm, índice de polidispersividade igual a 0,12 ± 0,02 e potencial zeta igual a -9,94 ± 0,19 mV. A elevada concentração requerida do agente estabilizante para essa formulação foi fator limitante para o seu desenvolvimento. A moagem via úmida em escala reduzida permitiu a formação de nanocristais de rifampicina F1-MU e F2-MU, com DHM igual a 340,20 ± 5,44 nm e 364,2 ± 4,50 nm, respectivamente, e distribuição de tamanho uniforme. A avaliação do DHM, do IP e do PZ, por período de três meses, revelou a estabilidade dessas formulações. Essas formulações foram obtidas por meio de planejamento de experimentos por superfície de resposta tendo como variáreis a concentração de rifampicina, a concentração do agente estabilizante e a quantidade de esferas de zircônia. As medidas de distribuição de tamanho médio das partículas e a morfologia foram realizadas utilizando difração a laser (LD) e microscopia eletrônica de transmissão (MET), respectivamente. Adicionalmente, as avaliações empregando calorimetria exploratória diferencial (DSC) e difração de raio X (DRX) revelaram que não houve mudança na estrutura cristalina do polimorfo II de rifampicina e nem interação entre o fármaco e os excipientes. O presente trabalho permitiu a obtenção de de nanocristais de rifampicina estáveis e com solubilidade maior de até 1,92 vezes (F1-MU) e 1,66 vezes (F2-MU), em água, quando comparada à rifampicina matéria-prima. Os perfis de dissolução das formulações F1-MU e F2-MU demonstraram dissolução de 95% de rifampicina em aproximadamente 5 minutos. Esse resultado é significativamente superior àquele observado para o produto FURP-rifampicina suspensão oral 20 mg/mL que apresentou dissolução de 23,2% nesse mesmo intervalo de tempo. A avaliação da atividade antimicrobiana das nanosuspensões foi confirmada frente à rifampicina padrão por meio da determinação da sua concentração mínima inibitória. / Tuberculosis (TB) still presents a challenge for public health globally. This Neglected Tropical Disease (NTDs) has as the treatment of choice rifampicin. This drug belongs to the class II, according to Biopharmaceutics Classification System (BCS), with low water solubility. This characteristic is a challenge in the development of safe and effective dosage forms. The nanotechnology has emerged as a promising alternative to improve the aqueous solubility of drugs. Accordingly, the present work aimed to the preparation and physicochemical characterization of nanocrystals of rifampicin. The preparation of the nanocrystals was performed using high-energy ball milling method, high-pressure homogenization and wet grinding process on a small scale. The results of the high-energy ball milling method demonstrated formation of nanocrystals, but in small amounts followed by the formation of aggregates (F1-M, F2-M and F3-M). The high pressure homogenization (HPH) allowed the formation of nanocrystals (F1-H and F2-H). F1-M formulation containing Poloxamer 188 did not show stability after 24 hours preparation. F2-H obtained mean hydrodynamic diameter (DHM) of 412.60 ± 4.12 nm, polydispersity index of 0.12 ± 0.02 and zeta potential of -9.94 ± 0.19 mV. The high concentration of stabilizing agent required for this formulation was a limiting factor for the development. The wet grinding process on a small scale allowed the formation of rifampicin nanocrystal F1-MU and F2-MU with DHM of 340,20 ± 5,44 nm e 364,2 ± 4,50, respectively, and size distribution uniform. The evaluation of DHM, IP and PZ, for three months, showed stability of these formulations. These formulations were obtained by design of experiments using response surface having as variables the concentration of rifampicin, the concentration of the stabilizing agent and the amount of zirconia beads. The mean size distribution measurements of particles and morphology were performed using laser diffraction (LD) and transmission electron microscopy (TEM), respectively. Additionally, the evaluations using differential scanning calorimetry (DSC) and X-ray diffraction (XRD) revealed that there was no change in the crystalline structure of polymorph II of rifampicin and no interaction between the drug and excipients. This study allowed obtaining stable rifampicin nanocrystals and greater solubility of up to 1.92 times (F1-MU) and 1.66 times (F2-MU) in water compared to rifampicin feedstock. The dissolution profiles of F1-MU and F2-MU formulations showed 95% dissolution of rifampicin in approximately 5 minutes. This result is significantly higher than that observed for the rifampicin-FURP oral suspension product 20 mg / ml that had Dissolving 23.2% over the same time interval. The evaluation of the antimicrobial activity of nanosuspensions was confirmed against the standard rifampicin by determining its minimum inhibitory concentration.

Zugl.: Köln, Univ., Diss., 2009

Orientador: Clarice Queico Fujimura Leite / Banca: Sylvia Luisa Pincherle Cardoso Leão / Banca: Sergio Akira Uyemura / Banca: Victor Marcelo Deflon / Banca: Alzir Azevedo Batista / Resumo: Tuberculose (TB) é uma doença infecciosa e curável transmitida pelo ar. A taxa de mortalidade reduziu globalmente em 2007, entretanto, TB-MDR, XDR e a coinfecção TB/HIV têm sufocado as tentativas de controle da TB, causando sofrimento e morte em todo o mundo. Adicionalmente, um terço da população mundial está infectada com o Mycobacterium tuberculosis (MTB) em estado de latência, o qual serve como reservatório para TB ativa. A rifampicina (RMP), descoberta há mais de 40 anos, representa a última classe de antibióticos introduzida como fármaco de primeira-linha no tratamento da TB. No esforço de sanar esta falha, é crescente a importância da Química Medicinal Inorgânica como um aliado na pesquisa de novos fármacos contra TB. É bem conhecido que alguns elementos metálicos desempenham papel fundamental nos seres vivos. Tem sido relatado desde 1970 que compostos de rutênio (Ru) exibem atividade anti-tumoral. Inclusive, alguns destes compostos se encontram na fase clínica de avaliação. Compostos de Ru foram avaliados também em nosso laboratório demonstrando atividade contra MTB. Os complexos com Ru(II) demonstraram ser mais ativos contra o MTB em relação a forma do ligante livre, em até 150 vezes, com baixa citotoxicidade e alta seletividade. Os resultados promissores inspiraram a procura de uma melhor abordagem para explorar o perfil pré-clínico in vitro e in vivo desses compostos. Assim, o objetivo deste trabalho foi desenvolver uma linha de pesquisa (pipeline) com ensaios fenotípicos rápidos, sensíveis, específicos e de baixo custo na pesquisa de um novo fármaco contra TB. Paralelamente, avaliou-se os melhores complexos heterolépticos de Ru(II) com fosfinas, diiminas e picolinato como ligantes, pré-selecionados, frente a esse pipeline. Os resultados obtidos com os complexos... (Resumo completo, clicar acesso eletrônico abaixo / Abstract: Tuberculosis (TB) is a preventable and curable infectious disease transmitted through the air. The mortality rates decreased globally in 2007, however, MDR and XDR TB, and co-infection TB/HIV have stifled attempts to control TB causing suffering and death worldwide. Additionally, a third of the world population is infected with Mycobacterium tuberculosis (MTB) in state of latency, which serves as a reservoir for active TB. Rifampicin (RMP), discovered more than 40 years ago, represents the last class of antibiotics introduced as first-line drug to treat TB. In an effort to correct this failure is an increasingly importance of Inorganic Medicinal Chemistry as an ally in the search for new drugs against TB. It is well known that some metallic elements play a fundamental role in living organisms. It has been reported since 1970 that compounds of ruthenium (Ru) exhibit anti-tumor activity. Even some of these compounds are in clinical phase of evaluation. Ru compounds were evaluated in our laboratory showing activity against MTB. Complexes with Ru(II) showed activity against MTB in relation to the free ligand up to 150 higher, with low cytotoxicity and high selectivity. The promising results inspired the search for a better approach to explore the pre-clinical in vitro and in vivo profile of these compounds. So, the objective of this study was to develop a research line (pipeline) fast, sensitive, specific and low-cost phenotypic testing in the search for a new drug against TB. At the same time the best complexes of heteroleptic Ru(II) phosphine/diimines/picolinate, pre-selected, against several biological in vitro and in vivo assays. Those evaluated assays were inserted into the pipeline developed at our laboratory. The in vitro results obtained with the Ru(II) (compounds SCAR) complexes are comparable and/or better than the first choice... (Complete abstract click electronic access below) / Doutor