Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-25T12:04:02Z No. of bitstreams: 1 williamcaneschi.pdf: 11580354 bytes, checksum: d5186d98d0e023e595e7509f1a08efff (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-25T13:32:21Z (GMT) No. of bitstreams: 1 williamcaneschi.pdf: 11580354 bytes, checksum: d5186d98d0e023e595e7509f1a08efff (MD5) / Made available in DSpace on 2017-05-25T13:32:21Z (GMT). No. of bitstreams: 1 williamcaneschi.pdf: 11580354 bytes, checksum: d5186d98d0e023e595e7509f1a08efff (MD5) Previous issue date: 2013-02-22 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Diversos análogos 1,3,4-oxadiazóis tem sido reportados na literatura uma vez que possuem grande espectro de atividade biológica, como: antiviral, antibacteriano, antitumoral, antioxidante, anti-inflamatório, anticonvulsivante, antimalarial, antifúngica e analgésica. Com o intuito de desenvolver moléculas ativas e seletivas a certas doenças, este trabalho descreve a síntese de derivados N-acilidrazonas e seus respectivos 2,3-diidro-1,3,4-oxadiazóis, o que resultou na obtenção de vinte e sete derivados inéditos, dentre eles: nove derivados de Nacilidrazonas e dezoito derivados 2,3-diidro-1,3,4-oxadiazóis. Foram obtidos vinte e oito derivados N-acilidrazonas a partir de uma reação de adição de aldeídos aromáticos e heteroaromáticos com a isoniazida. Para a obtenção das N-acilidrazonas com cadeia longa, foi proposto, inicialmente, a alquilação do 4-hidroxibenzaldeído com haletos de alquila com: 1-cloroexano, 1-cloro-octano, 1-bromononano, 1-clorodecano, 1-clorododecano, 1-lorotetradecano e 1-bromo-propargila. Foi proposta também a síntese de dois derivados sulfonados, sintetizados pela mesma metodologia de alquilação com os cloretos de mesila e tosila. Em seguida, os aldeídos alquilados e sulfonados sintetizados e dezenove aldeídos comerciais foram condensados a isoniazida gerando os derivados N-acilidrazonas, que por sua vez, foram ciclizados, em reação com anidrido acético, que gerou vinte e sete derivados 2,3-diidro-1,3,4-oxadiazólicos. As estruturas dos compostos obtidos foram elucidadas por espectroscopia na região do infravermelho, RMN de 1H e de 13C, técnicas de RMN 2D, espectrometria de massas. Os análogos oxadiazólicos foram submetidos a teste de citotoxicidade, antibacteriano, antioxidante e antimalarial. Os compostos se mostraram bastantes ativos contra células cancerígenas, com índices de seletividade superiores ao composto padrão utilizado. / Several analogs 1,3,4-oxadiazoles have been reported in the literature due to wide range biological activity, such as: antiviral, antibacterial, antitumoral, antioxidant, anti-inflammatory, anticonvulsivant, antimalarial, antifungal e analgesic activity. With the aim to develop molecules active and selective for some illness, this work describe the synthesis of some N-acyl-hydrazones derivatives and the respective 2,3-dihydro-1,3,4-oxadiazoles, that resulted the obtainment twenty seven inedited derivatives, among them: nine N-acyl-hydrazones derivatives and eighteen 2,3-dihydro-1,3,4-xadiazoles derivatives. At this work were obtainment twenty e eight N-acylhydrazones derivatives through the addition reaction of aromatics and heteroaromatics aldehydes with isoniazid. For the obtainment of N-acyl-hydrazones of long chain, the 4-hydroxybenzaldehyde was alkylated with several alkyl halides: 1-chlorohexane, 1-chlorooctane, 1-bromononane, 1-chlorodecane, 1-chlorododecane, 1-hlorotetradecane and 1-bromopropargyl. It was also proposed the synthesis of two sulfa derivatives, synthesized by the same alkylation methodology with the mesyl and tosyl chloride. These alkylated aldehydes and nineteen commercials aldehydes were condensed to the isoniazid that gave the N-acyl-hydrazones derivatives, which in turn were cyclized to obtain the 2,3-dihydro-1,3,4-oxadiazoles derivatives in reaction with acetic anhydride. The structures of the compounds were elucidated by in infrared, ¹H NMR, ¹³C NMR, 2D NMR techniques, mass spectrometry. The oxadiazoles analogs were submitted to citotoxicity, antibacterial, antioxidant and antimalarial activity. The compounds showed great activity against tumor cells, with selectivity index higher than standard compound used.

La tuberculose est l'une des maladies infectieuses les plus meurtrières au monde. Malgré l'existence d'un traitement polychimiothérapeutique efficace, le nombre de cas de tuberculose incurable augmente sensiblement en raison de l'apparition de souches de Mycobacterium tuberculosis résistantes aux traitements de 1ère, 2ème et 3ème intentions actuellement disponibles. Parmi les antibiotiques spécifiques de la tuberculose, nous nous intéressons plus particulièrement, dans le cadre de cette thèse, aux pro-drogues isoniazide et éthionamide. Ces deux médicaments ciblent l'enzyme InhA du Mycobacterium tuberculosis, qui est impliquée dans la synthèse de la paroi bactérienne. Les principales résistances de Mycobacterium tuberculosis à ces pro-drogues résident en un défaut des enzymes responsables de l'activation de ces médicaments à l'intérieur du pathogène. Le but de cette thèse est, dans un premier temps, d'étudier différentes approches originales visant à contourner ces résistances. La première stratégie consiste à concevoir des pro-drogues hybrides d'isoniazide et d'éthionamide qui pourraient être activées indifféremment par KatG et EthA. KatG est la catalase-peroxydase responsable de l'activation de l'isoniazide, et EthA la mono-oxygénase à flavine qui active l'éthionamide. Les chances de bio-activation de ces nouvelles molécules seraient donc supérieures même si l'une des deux enzymes est mutée. La deuxième stratégie examinée consiste à synthétiser des molécules capables d'être activées par l'enzyme KatG mutée qui reste fonctionnelle. Nous avons alors préparé des molécules analogues de l'isoniazide qui pourraient être éventuellement reconnues et activées par une KatG mutée montrant une modification du potentiel d'oxydation ou de la structure protéique. La dernière stratégie étudiée consiste à synthétiser des molécules qui ne nécessitent pas d'être activées par une enzyme pour exercer leur action mais simplement par des agents oxydants endogènes. En se basant sur une molécule décrite dans la littérature par nos collaborateurs brésiliens, le complexe d'isoniazide-fer(II) ((isoniazide)pentacyanoferrate(II) de sodium), nous avons synthétisé différents analogues de ce complexe en faisant varier le ligand et avons évalué par RPE leur capacité à générer des radicaux. Cette étude de relation structure-réactivité a permis de mieux comprendre le mécanisme d'activation de ces complexes en présence de H2O2. La deuxième partie de cette thèse est consacrée au mécanisme d'activation des pro-drogues isoniazide et éthionamide. Même si ces molécules sont utilisées depuis plus de 50 ans dans le traitement de la tuberculose, leur mécanisme d'activation d'un point de vue chimique est très mal décrit. Dans la mycobactérie, ces pro-drogues, une fois activées, forment un adduit avec le cofacteur NAD(H) donnant ainsi l'inhibiteur ultime de l'enzyme InhA. Dans le cas de l'isoniazide, nous avons utilisé le système biomimétique mis en place dans l'équipe pour étudier son mécanisme d'activation d'un point de vue moléculaire. Dans le cas de l'éthionamide, nous avons développé un système chimique biomimétique qui, pour la première fois, a conduit à la formation de l'adduit éthionamide-NAD+ in vitro. Grâce au succès de cette approche et à la caractérisation des intermédiaires et métabolites formés, nous avons pu proposer un mécanisme d'oxydation moléculaire de l'éthionamide entièrement original, s'affranchissant de l'intermédiaire clé acide sulfinique évoqué jusque-là dans la littérature sans aucune preuve expérimentale. / Tuberculosis is one of the leading causes of death in the world among infectious diseases. Despite the existence of efficient multidrug treatment, the number of incurable cases of tuberculosis substantially increases due to the emergence of Mycobacterium tuberculosis strains resistant to available 1st-, 2nd- and 3rd-lines-treatments. Among the specific drugs currently employed to treat tuberculosis, we particularly focus on pro-drugs (isoniazid and ethionamide) for which resistances mainly result in a default of their activation enzymes inside the pathogen. The aim of this thesis is, firstly, to study various innovative approaches to overcome the resistance. The first strategy consists in designing hybrid pro-drugs, by combination of isoniazid and ethionamide moieties, which could be activated by two different enzymes, KatG and EthA. KatG is the mycobacterial catalase peroxidase enzyme which activates isoniazid, and EthA the flavin monooxygenase responsible of ethionamide activation. Probability of bio-activation of these new molecules would therefore be higher even if one of the two enzymes is mutated. The second strategy discussed herein is the synthesis of molecules able to be activated by mutated KatG enzyme, which remains functional. We synthesized isoniazid derivatives, which might be recognized and activated by a mutated KatG enzyme showing a modification of its oxidation potential or in the protein structure. The last strategy is founded on the development of molecules that do not need to be activated by an enzyme but by a simple chemical oxidation. Based on a molecule described in the literature by our brazilian collaborators, an isoniazid-iron (II) complex (sodium (isoniazid)pentacyanoferrate(II)), we synthesized various analogues of this complex by varying the ligand structure and evaluated by ESR their ability to generate radicals in the presence of H2O2. The structure-reactivity relationship analysis led to better understanding of the molecular activation mechanism of these complexes in the presence of H2O2. The second part of this thesis is dedicated to the activation mechanisms of pro-drugs isoniazid and ethionamide. Even though these molecules have been used for more than 50 years for the treatment of tuberculosis, their activation mechanism on a molecular point of view is poorly described. In the mycobacterium, once activated these pro-drugs form an adduct with the NAD(H) cofactor, leading to the active metabolite. For isoniazid, we used the biomimetic system developed previously by our team to clarify the molecular activation mechanism. For ethionamide, we have developed a biomimetic system which, for the first time, leads to the formation of the ethionamide-NAD+ adduct in vitro. We used this method to study the molecular oxidation mechanism of ethionamide and to characterize intermediates and metabolites. We finally proposed a completely original mechanism, not involving the sulfinic acid intermediate, which has been mentioned in the literature without any experimental evidence.

Philosophiae Doctor - PhD / Tuberculosis (TB) remains a global epidemic despite the fact that treatment has been available since the 1950’s. This disease is highly contagious and spreads via transmission of the Mycobacterium Tuberculosis (MTB) tubercle via coughing, sneezing and spitting. The disease has various side effects including weight loss, fatigue and even death. To date no cure has been found for TB and thus optimisation of treatment is a constant focus in health related research. TB is highly prevalent in South Africa due to the increased level of patients who are co-infected with HIV. Treatment for TB consists of first line drugs including isoniazid (INH), ethambutol (ETH), pyrazinamide (PYR) and rifampicin (RIF). These drugs are highly effective but also produce many adverse drug reactions (ADR’s) over the 6-month course of treatment. These reactions lead to patients not completing the course, losing quality of life and ultimately adding to the development of drug resistant strains of TB. A method of minimising these ADR’s is the development of a phenotype sensor, which is able to determine the metabolic profile of patients. Metabolic profiles play a huge role in the efficacy of treatment by tailoring treatment in order for patients to stay within the therapeutic range of treatment. This would in turn minimise both toxicity and ineffective treatment. Various methods for the quantification of drugs have been developed such as high performance liquid chromatography (HPLC), mass spectrometry (MS) and ultra-violet visible spectroscopy (UV-vis). / 2023-12-01

Fléau mondial depuis des millénaires, la tuberculose (TB) a régressé dans la deuxième moitié du 20ème siècle avant de connaitre une résurgence à partir des années 1980 à la faveur de la pandémie du VIH. Les deux maladies se potentialisent mutuellement et forment un « couple infernal ». En Afrique, la TB est la première cause de mortalité des adultes infectés par le VIH, quel que soit leur niveau d’immunité. Une des mesures pour lutter contre la TB associée au VIH est la chimioprophylaxie, consistant à traiter une infection tuberculeuse latente pour prévenir l’évolution vers une TB maladie. La mieux évaluée, consiste à prescrire 6 à 12 mois de monothérapie d’isoniazide (Isoniazid Preventive Therapy, IPT). Depuis 1993, l’OMS recommande la prescription de 6 mois d’IPT chez toutes les personnes infectées par le VIH sans signe de TB active. Malgré des preuves scientifiques solides à l’appui de cette recommandation, l’utilisation de l’IPT est toujours restée faible. Avant notre travail, trois raisons expliquaient cette faiblesse : (i) la crainte qu’une chimioprophylaxie mal appliquée ne favorise l’émergence de résistances ; (ii) le fait que les essais avaient démontré l’efficacité de l’IPT pour réduire l’incidence de TB, pas pour réduire la mortalité ; (iii) le fait que les essais d’IPT avaient eu lieu en majorité avant l’ère des antirétroviraux (ARV), chez des personnes très immunodéprimées. Les ARV permettant également de réduire le risque de TB en faisant régresser l’immunodépression, certains considéraient que l’IPT était devenue inutile. Dans cette thèse nous faisons d’abord un rappel des connaissances essentielles sur l’infection par le VIH, la TB, l’association TB/VIH, et le concept de chimioprophylaxie antituberculeuse. Puis nous exposons les résultats de l’analyse du suivi prolongé de l’essai randomisé Temprano ANRS 12136, qui s’est déroulé entre 2008 et 2015. Cet essai a suivi 2056 adultes infectés par le VIH dans 9 centres de soins à Abidjan. Les participants qui avaient des CD4 élevés (moyenne 477/mm3) étaient randomisés en 4 bras pour étudier deux interventions : 6 mois d’IPT (reçu vs. non reçu) et ARV (début immédiat vs. début différé). Les participants ont été suivis pendant 4,9 ans en moyenne. 89% d’entre eux ont débuté des ARV. Pendant le suivi, il y a eu 86 décès, 34 dans le groupe avec IPT (probabilité à 6 ans : 4,1% ; IC95% 2,9–5,7) et 52 dans le groupe sans IPT (probabilité à 6 ans: 6,9% ; 5,1–9,2). Le Hazard ratio de décès dans le groupe avec IPT par rapport à l’autre groupe était 0,63 (95% CI 0,41-0,97). Il n’y avait pas d’interaction entre IPT et ARV précoce, ni entre IPT et le temps. Ces résultats ont été publiés dans The Lancet Global Health. Enfin nous discutons ces résultats avec ceux des essais d’IPT précédents, dans une revue critique de la littérature analysant les données d’efficacité et de tolérance, les déterminants de l’efficacité, et les risques de résistance. Nous montrons que l’essai Temprano complète et élargit le spectre des connaissances, et que les preuves scientifiques accumulées depuis 1993 jusqu’à l’essai Temprano inclus suggèrent que les ARV modifient certains paramètres de l’IPT qu’on pensait solidement établis. Avant l’ère des ARV on considérait que l’efficacité de l’IPT était forte chez les personnes avec IDR positive mais très faible voire inexistante chez les personnes avec IDR négative, qu’il y avait une perte d’efficacité de l’IPT au cours du temps et que l’IPT n’avait pas d’effet sur la mortalité. Avec les ARV, on voit que l’IPT est efficace quel que soit le résultat des tests tuberculiniques, que cette efficacité est prolongée, et qu’elle se traduit non seulement par une réduction de la TB mais aussi de la mortalité. L’IPT reste donc une intervention d’une grande actualité à l’ère des ARV. Ces résultats devraient convaincre les pays jusque-là réticents à appliquer les recommandations de l’OMS. / Tuberculosis (TB) has been a worldwide scourge for millennia. It has regressed in the second half of the 20th century before resurging in the 1980s because of the HIV pandemic. Both diseases potentiate each other and form a "cursed duet". In Africa, TB is the leading cause of mortality among HIV-infected adults, regardless of their level of immunity. One of the measures to fight HIV-associated TB is chemoprophylaxis, which consists in treating latent TB infection to prevent the progression to TB disease. The most evaluated chemoprophylaxis, referred to as "Isoniazid Preventive Therapy" (IPT), consists in prescribing 6 to 12 months of isoniazid monotherapy. Since 1993, WHO recommends the prescription of 6 months of IPT in all HIV-infected persons who do not have evidence of active TB. Despite strong scientific evidence to support this recommendation, the use of IPT has remained low. Before our work, there were three reasons for this:(i) people feared that chemoprophylaxis might favor the emergence of resistance to TB drug; (ii) the IPT trials demonstrated the effectiveness of IPT in reducing TB incidence, not in reducing mortality; (iii) most IPT trials took place before the antiretroviral treatment (ART) era, in highly immunocompromised individuals. As ART also reduces the risk of TB by decreasing immunosuppression, some people considered that IPT had become useless. In this work, we first go over the basic knowledge about HIV infection, TB, the combination of the two diseases, and the concept of antituberculous chemoprophylaxis. Then we present the results of the long-term follow-up of the Temprano ANRS 12136 randomized trial, which took place between 2008 and 2015. This trial followed 2056 HIV infected adults in 9 care centers in Abidjan. Participants with high CD4 counts (mean: 477 cells/mm3) were randomized into 4 arms to study two interventions: 6 months of IPT (received vs. not received) and early ART (immediate initiation vs. delayed initiation). Participants were followed for an average of 4.9 years. Eighty nine percent of participants received ART. During follow-up, there were 86 deaths, 34 in patients randomized to IPT (6-year probability: 4.1%, 95% CI 2.9-5.7) and 52 in those randomized to no-IPT (6-year probability: 6.9%, 5.1-9.2). The Hazard ratio of deaths among those randomized to IPT compared to others was 0.63 (95% CI 0.41-0.97). There was no interaction between IPT and early ART, nor between IPT and time. These results were published in The Lancet Global Health. Finally, we discuss these results with those of previous IPT trials, after reviewing all available randomized-controlled evidence on efficacy, safety, efficacy determinants and risks of resistance. We show that the Temprano trial complements and widens the spectrum of evidence accumulated since 1993 and that ART modifies some key parameters of IPT previously thought to be strongly established. Prior to the ART era, evidence suggested that the efficacy of IPT was high in people with positive Tuberculin Skin Test (TST) but very low in those with negative TST; that there was a loss of IPT efficacy over time; and that IPT had no effect on mortality. With ART, IPT appears to be effective regardless of TST results, have prolonged efficacy, and reduce not only TB but also mortality. IPT remains a very topical intervention in the ART era. These results should convince IPT-reluctant countries to implement WHO recommendations.

Latent tuberculosis infection (LTBI) is a global health issue that affects approximately one quarter of the world’s population. It refers to a state of persistent immune response to Mycobacterium tuberculosis without clinical evidence of active tuberculosis (TB). Latent tuberculosis infected individuals are asymptomatic and not contagious to others, however 5-15% of all infected individuals are at risk of developing active tuberculosis and become contagious, severely ill, or worse, die from active TB. There are identified risk groups that are targeted for identification, diagnosis and treatment of latent tuberculosis infection. These are human immunodeficiency virus (HIV) patients, children and adolescents, household or close contacts of active TB cases, migrants, refugees, prisoners and health care workers. The standard treatment used for treating LTBI is the isoniazid monotherapy. It has a high proven efficacy rate but is linked to poor acceptance and low completion rates, basically due to its long treatment duration and poor tolerability. A newer treatment regimen is the rifapentine plus isoniazid combination therapy. It is an effective regimen against LTBI and has a shorter treatment duration. The aim of this literature study was to evaluate the efficacy of rifapentine plus isoniazid combination therapy compared with the isoniazid monotherapy as treatment of latent tuberculosis infection. This thesis was based on five randomized clinical trials collected from PubMed database. The studies should have entailed an efficacy comparison between isoniazid monotherapy and rifapentine plus isoniazid combination therapy for the treatment of patients with latent tuberculosis. The studies showed lower rates of active TB and death in the rifapentine plus isoniazid combination group in comparison with the isoniazid monotherapy. The studies also proved that rifapentine plus isoniazid combination therapy was noninferior to the standard isoniazid monotherapy. The completion rates were significantly higher in the combination therapy arm. The safety profile between the two treatment regimens was similar, but with an increased hepatotoxicity rates in the isoniazid-only arm. The rifapentine plus isoniazid combination therapy is as efficacious as the isoniazid monotherapy. This shorter regimen could be used as first hand therapy as well for latent tuberculosis patients with high-risk of developing active TB as it has shown good tolerability and higher completion rates that is important to successfully treat LTBI and help eliminate TB worldwide. / Latent tuberkulos är ett globalt hälsoproblem som drabbar ungefär en fjärdedel av världens befolkning. Den definieras som ett tillstånd av immunreaktion mot Mycobacterium tuberculosis utan kliniska tecken på aktiv tuberkulos (TB). De infekterade individerna är asymtomatiska och inte smittsamma för andra, men 5–15% av alla infekterade individer riskerar att utveckla aktiv tuberkulos och bli smittsamma, bli allvarligt sjuka, eller värre, dö av aktiv tuberkulos. Personer med latent tuberkulos som tillhör riskgrupperna prioriteras för identifiering, diagnos och behandling av latent tuberkulos. Dessa riskgrupper är humant immunbristvirus (HIV)-patienter, barn och ungdomar, nära kontakter till personer med aktiva TB-fall, migranter, flyktingar, fångar och vårdpersonal. Standardbehandlingen mot latent tuberkulos är isoniazid monoterapi. Den har en högt beprövad effektivitetsgrad men är kopplad till dålig acceptans och låga kompletteringsgrader, på grund av framförallt den långa behandlingstiden och dålig tolerans. En nyare form av behandling är rifapentin kombinerat med isoniazid. Den är en effektiv behandling mot latent tuberkulos med en kortare behandlingstid. Syftet med denna litteraturstudie var att utvärdera effekten av kombinationsterapi med rifapentin och isoniazid jämfört med isoniazid monoterapi för behandling av latent tuberkulos. Detta examensarbete baserades på fem randomiserade kliniska prövningar hämtade från PubMed-databasen. Samtliga fem studier innefattade effektivitetsjämförelse mellan isoniazid monoterapi och kombinationsterapi med rifapentin och isoniazid vid behandling av patienter med latent tuberkulos. Alla fem studier undersöktes visade lägre frekvens av aktiv TB och dödlighet i kombinationsterapi med rifapentin och isoniazid jämfört med isoniazid monoterapi. Resultatet bevisade också icke-underlägsenhet för kombinationsterapin jämfört med isoniazid monoterapin. Kompletteringsgraden var signifikant högre i kombinationsterapin. Säkerhetsprofilen mellan de två terapin var likartad, men med en ökad hepatotoxicitet i isoniazid monoterapi gruppen. Kombinationsterapi med rifapentin och isoniazid är lika effektiv som isoniazid monoterapi. Denna kortare behandling kan också användas som förstahandsbehandling för latent tuberkulos patienter med hög risk att utveckla till aktiv tuberkulos eftersom den har visat god tolerabilitet och högre kompletteringsgrad som är viktigt för att framgångsrikt behandla latent tuberkulos och hjälpa till att eliminera TB över hela världen.

Yes / We investigated whether companion drug resistance was associated with adverse outcome of the shorter MDR-TB regimen in Bangladesh, after adjusting for fluoroquinolone resistance. MDR/RR-TB patients registered for treatment with a standardized gatifloxacin-based shorter MDR-TB regimen were selected for the study. Drug resistance was determined using the proportion method, gatifloxacin and isoniazid minimum inhibitory concentration testing for selected isolates, and whole genome sequencing. Low-level and high-level fluoroquinolone resistance were the most important predictors of adverse outcomes, with pyrazinamide resistance having a significant yet lower impact. In patients with fluoroquinolone-/second-line injectable-susceptible TB, non-eligibility to the shorter MDR-TB regimen (initial resistance to either pyrazinamide, ethionamide, or ethambutol) was not associated with adverse outcome (aOR 1.01; 95%CI 0.4-2.8). Kanamycin resistance was uncommon (1.3%). Increasing levels of resistance to isoniazid predicted treatment failure, also in a subgroup of patients with high-level fluoroquinolone-resistant TB. Our results suggest that resistance to companion drugs of the shorter MDR-TB regimen, except kanamycin resistance, is of no clinical importance as long as fluoroquinolone susceptibility is preserved. Hence, contrary to current WHO guidelines, exclusions to the standard regimen are justified only in the case of fluoroquinolone, and possibly kanamycin resistance. / Damien Foundation Belgium for its financial and logistic support to run the project including its research activities. European Research Council (Starting Grant INTERRUPTB 311725).

Introdução: A tuberculose é uma infecção curável causada pelo Mycobacterium tuberculosis. Requer tratamento prolongado e a combinação de vários fármacos implicando em efeitos colaterais que podem levar pacientes ao abandono do tratamento. Formulações de droga de liberação controlada como nanopartículas, nanoemulsões ou lipossomos têm tido sucesso contra doenças infecciosas. Em especial, brometo de dioctadecildimetilamônio (DODAB) disperso em água pode formar lipossomos ou vesículas grandes (LV) ou fragmentos de bicamada (BF) que podem carrear drogas hidrofóbicas ou hidrofílicas e ademais, podem atuar como microbicidas. Objetivos: determinar atividade do DODAB contra Mycobacterium tuberculosis e M. smegmatis tanto isoladamente como em combinação com duas drogas tuberculostáticas, rifampicina (RIF) e isoniazida (ISO); determinar a incorporação de RIF e ISO em dispersões de DODAB. Material e Métodos: Dispersões de DODAB foram obtidas por vortexação (LV) ou sonicação (BF) e sua interação com as drogas foi avaliada por espectros óticos das drogas, espalhamento de luz dinâmico para medida de distribuição de tamanhos e potencial-zeta e diálise para determinação de incorporação dos fármacos em DODAB LV ou BF. Viabilidade de M. smegmatis ou M. tuberculosis foi determinada por contagem de viáveis em função de concentração de DODAB. Combinações DODAB/droga contra micobactérias foram avaliadas por determinação de concentração inibitória minima (CIM), em µg/ml. Resultados: DODAB mata M. smegmatis a partir de 4 µM de concentração e 1 h de interação e M. tuberculosis, em 100 µM e 120 h de interação. ISO resultou permeável através da bicamada de DODAB em contraste com RIF que adsorveu irreversivelmente nas bicamadas, resultando em 75% de incorporação com 0.1 e 2 mM de droga e DODAB, respectivamente. CIM de RIF contra M. smegmatis foi 32 e, em combinação com 2 de DODAB caiu para 2. Para M. tuberculosis CIM de 0,015 caiu para 0,007 em combinação com 4 DODAB. A combinação foi sinérgica contra M. smegmatis e de ação independente contra M. tuberculosis. / Introduction: Tuberculosis is potentially curable but remains a serious public health problem with large numbers of infected people in several countries. The long time that the patient should receive medication, associated with a large number of adverse effects often cause treatment failure. Nanoparticles, liposomes and emulsions have been used successfully in antibacterial therapy. In particular, dioctadecyldimethylammonium bromide (DODAB) bilayers in form of bilayer fragments (BF) or vesicles (LV) provided adequate environment for solubilization and stabilization of several drugs with an additional advantage: they acted as antimicrobial agents themselves. Objectives: investigation of DODAB bactericidal activity against mycobacteria, determination of entrapment efficiency for rifampicin (RIF) and isoniazid (ISO) in DODAB dispersions and determination of the DODAB/drug activity against Mycobacterium smegmatis and tuberculosis. Material and Methods: DODAB dispersions were obtained by sonication of dioctadecyldimethylammonium bromide (DODAB) synthetic lipid (BF)or by vortexing (LV) the lipid powder in aqueous solution. The physic-chemical characteristics of drugs in DODAB dispersions were determined from optical spectra and dynamic light scattering for evaluating size distribution and zeta-potentials. Drug incorporation in DODAB dispersions was determined from dialysis. Cell viability was determined from plating and colony forming unities (CFU) counting as a function of [DODAB]. Minimal inhibitory concentration (MIC) was obtained for drug or DODAB/drug combinations. Results: DODAB killed M. smegmatis and tuberculosis from 4¨µM (1 h interaction) and 100 µM (120 h interaction), respectively. Rifampicin drug particles above its solubilization limit could be solubilized by BF at 0.5 mM lipid. LV was leaky to ISO whereas RIF could be incorporated in BF or LV bilayer at high percentiles (0.1 mM RIF in 2 mM DODAB BF or LV). MIC for combination DODAB/RIF was 2/2 or 4/0.007 µg/mL whereas synergism index was 0.5 or 1.0 against M. smegmatis or M. tuberculosis, respectively. DODAB and RIF acted synergistically when tested against M. smegmatis.

Introdução: A tuberculose é uma infecção curável causada pelo Mycobacterium tuberculosis. Requer tratamento prolongado e a combinação de vários fármacos implicando em efeitos colaterais que podem levar pacientes ao abandono do tratamento. Formulações de droga de liberação controlada como nanopartículas, nanoemulsões ou lipossomos têm tido sucesso contra doenças infecciosas. Em especial, brometo de dioctadecildimetilamônio (DODAB) disperso em água pode formar lipossomos ou vesículas grandes (LV) ou fragmentos de bicamada (BF) que podem carrear drogas hidrofóbicas ou hidrofílicas e ademais, podem atuar como microbicidas. Objetivos: determinar atividade do DODAB contra Mycobacterium tuberculosis e M. smegmatis tanto isoladamente como em combinação com duas drogas tuberculostáticas, rifampicina (RIF) e isoniazida (ISO); determinar a incorporação de RIF e ISO em dispersões de DODAB. Material e Métodos: Dispersões de DODAB foram obtidas por vortexação (LV) ou sonicação (BF) e sua interação com as drogas foi avaliada por espectros óticos das drogas, espalhamento de luz dinâmico para medida de distribuição de tamanhos e potencial-zeta e diálise para determinação de incorporação dos fármacos em DODAB LV ou BF. Viabilidade de M. smegmatis ou M. tuberculosis foi determinada por contagem de viáveis em função de concentração de DODAB. Combinações DODAB/droga contra micobactérias foram avaliadas por determinação de concentração inibitória minima (CIM), em µg/ml. Resultados: DODAB mata M. smegmatis a partir de 4 µM de concentração e 1 h de interação e M. tuberculosis, em 100 µM e 120 h de interação. ISO resultou permeável através da bicamada de DODAB em contraste com RIF que adsorveu irreversivelmente nas bicamadas, resultando em 75% de incorporação com 0.1 e 2 mM de droga e DODAB, respectivamente. CIM de RIF contra M. smegmatis foi 32 e, em combinação com 2 de DODAB caiu para 2. Para M. tuberculosis CIM de 0,015 caiu para 0,007 em combinação com 4 DODAB. A combinação foi sinérgica contra M. smegmatis e de ação independente contra M. tuberculosis. / Introduction: Tuberculosis is potentially curable but remains a serious public health problem with large numbers of infected people in several countries. The long time that the patient should receive medication, associated with a large number of adverse effects often cause treatment failure. Nanoparticles, liposomes and emulsions have been used successfully in antibacterial therapy. In particular, dioctadecyldimethylammonium bromide (DODAB) bilayers in form of bilayer fragments (BF) or vesicles (LV) provided adequate environment for solubilization and stabilization of several drugs with an additional advantage: they acted as antimicrobial agents themselves. Objectives: investigation of DODAB bactericidal activity against mycobacteria, determination of entrapment efficiency for rifampicin (RIF) and isoniazid (ISO) in DODAB dispersions and determination of the DODAB/drug activity against Mycobacterium smegmatis and tuberculosis. Material and Methods: DODAB dispersions were obtained by sonication of dioctadecyldimethylammonium bromide (DODAB) synthetic lipid (BF)or by vortexing (LV) the lipid powder in aqueous solution. The physic-chemical characteristics of drugs in DODAB dispersions were determined from optical spectra and dynamic light scattering for evaluating size distribution and zeta-potentials. Drug incorporation in DODAB dispersions was determined from dialysis. Cell viability was determined from plating and colony forming unities (CFU) counting as a function of [DODAB]. Minimal inhibitory concentration (MIC) was obtained for drug or DODAB/drug combinations. Results: DODAB killed M. smegmatis and tuberculosis from 4¨µM (1 h interaction) and 100 µM (120 h interaction), respectively. Rifampicin drug particles above its solubilization limit could be solubilized by BF at 0.5 mM lipid. LV was leaky to ISO whereas RIF could be incorporated in BF or LV bilayer at high percentiles (0.1 mM RIF in 2 mM DODAB BF or LV). MIC for combination DODAB/RIF was 2/2 or 4/0.007 µg/mL whereas synergism index was 0.5 or 1.0 against M. smegmatis or M. tuberculosis, respectively. DODAB and RIF acted synergistically when tested against M. smegmatis.

Os materiais em estado sólido têm papel importante no desenvolvimento da tecnologia e na produção da forma farmacêutica, pois atuam como um dos principais constituintes das formulações. As dimensões das partículas sólidas normalmente influenciam a dissolução dos fármacos, visto que esta depende da área superficial exposta ao meio líquido. O presente trabalho estuda a influência do tamanho das partículas sólidas na liberação de fármacos contidos no sistema matricial. foram analisados comprimidos matriciais formados por hidroxipropilmetilcelulose (HPMC) (A) e fármacos (isoniazida (INH), fármaco hidrossolúvel (B) ou rifampicina (RMP) fármaco com baixa solubilidade(C)). Os pós para elaboração das formulações foram separados por tamisação, segundo classificação da USP XXIII, em quatro tamanho de partículas, partículas não homogêneas (branco), t≥0,425 , 0,177≤*t<0,250 e t<0,150 (t= tamanho em mm) e identificadas como A0, A1, A2, A3, B0, B1, B2, B3 e C0, C1, C2, C3, respectivamente. Por compressão direta foram preparados comprimidos matriciais de 250 mg, contendo, no primeiro tipo de formulação, 150 mg de isoniazida; 2,5 mg de estearato de magnésio e. 97,5 mg de HPMC e na segunda formulação mudou-se a constituição da fórmula, utilizando como fármaco 150 mg de rifampicina e os demais constituintes permaneceram os mesmos. As formulações foram obtidas pela combinação dos pós, mantendo-se constante a força de compressão, umidade do ambiente e demais variáveis, em A0B0, A1B1, A2B2, A3B3, A0C0, A0C1, A0C2, A0C3, A1C0, A1C1, A1C2, A1C3, A2C0, A2C1, A2C2, A2C3, A3C0, A3C1, A3C2 e A3C3. Os resultados das análises física e físico-química apresentaram claramente a influência do tamanho da partícula sólida sobre os parâmetros dos comprimidos matriciais estudados. Através da análise estatística dos parâmetros farmacocinéticos, Qmax e AUC, pode-se concluir que as formulações A1B1, A2B2, A3B3, A0C3, A1C1, A1C2, A1C3 e A2C1 apresentaram diferenças significativas entre as médias, quando comparada ao padrão. / The present work studies the solid particle size\'s influence of the rifampin and hydroxypropylmetylcellulose in kinetic release profile of the drug in matrix systems. The matrix tablets were formed by HPMC (A), isoniazid (B) and rifampin (C). The powders were classified by sieving in four different particle size distribution. (See table in file PDF) The twenty formulations were prepared by direct compression of the rifampin or isoniazid (150 mg), magnesium stearat (2,5 mg) and HPMC in order to obtain tablets of 250 mg. The formulations were obtained by the combination of the powders, in A0B0, A1,B1, A2B2, A3B3, A0C0, A0C1, A0C2, A0C3, A1C0, A1C1, A1C2, A1C3, A2C0, A2C1, A2C2, A2C3, A3C3, A3C1, A3C2 e A3C3. The drug release was studied by the dissolution and quantification assay. The dissolution curve represents the accumulated drug release during a period of 10 hours. The matrices dissolution profiles are upset by divergence the drugs and HPMC particle size in formulation. The estatistic analyse (teste t) the pharmacokinetic parameter (Qmax and AUC) show that the formulations A1B1, A2B2, A3B3, A1C1, A1C2, A1C3 and A2C1 are significantly differents when compare whith the standard tablets.

Made available in DSpace on 2014-12-17T15:42:17Z (GMT). No. of bitstreams: 1 SeverinaDSO_DISSERT.pdf: 2512071 bytes, checksum: 12a15e7529d5b018959d5e9c644f10cf (MD5) Previous issue date: 2013-07-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In this work a study was done using electrochemical cyclic voltammetry and differential pulse voltammetry for isoniazida (INH), ethambutol (EMB), rifampicina (RIF) and pyrazinamide (PZA) using the electrode boron-doped diamond (BDD) as working electrode. It also verified the applicability of the technique of differential pulse voltammetry in the quantification of the active compounds used in the treatment of tuberculosis, subsequently applying in samples of pharmaceutical formulation. Among the four active compounds studied, isoniazid showed the best results for the detection and quantification using differential pulse voltammetry. At pH 4 and pH 8, for the calibration curves to INH showed good linearity, with quantification limits of 6.15 mmol L-1 (0,844 ppm) and 4.08 mmol L-1 (0.560 ppm) for the respective pH. The proposed method can be used to determine drug isoniazid, for recovery values were obtained in approximately 100% / Neste trabalho foi feito um estudo eletroqu?mico utilizando a voltametria c?clica e voltametria de pulso diferencial para os f?rmacos isoniazida (INH), etambutol (EMB), rifampicina (RIF) e pirazinamida (PZA) usando o eletrodo de diamante dopado com boro (BDD) como eletrodo de trabalho. Foi tamb?m verificado a aplicabilidade da t?cnica de voltametria de pulso diferencial na quantifica??o dos princ?pios ativos usados no tratamento da tuberculose, posteriormente aplicando em amostras de formula??o farmac?utica. Dentre os quatro princ?pios ativos estudados, a isoniazida apresentou os melhores resultados de detec??o e quantifica??o com o uso da voltametria de pulso diferencial. Em pH 4 e pH 8, as curvas de calibra??o para a INH apresentaram boa linearidade, apresentando os limites de quantifica??o de 6,15 μmol L-1 (0,844 ppm) e 4,08 μmol L-1 (0,560 ppm), para os respectivos pH. O m?todo proposto pode ser usado para a determina??o de isoniazida em f?rmacos, pois foram obtidos valores de recupera??o em torno de 100%