Thèses sur le sujet « Antitubercular »

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The author's objective was to prepare new solid phases of the antitubercular drug isoxyl [specifically polymorphs, solvates, cyclodextrin (CD) inclusion complexes and cocrystals] and to isolate and characterise a range of solvated forms of the rifamycin antibioticsrifampicin and rifaximin.

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Includes bibliographical references.
The pharmacokinetics of rifampicin, isoniazid, pyrazinamide and ethambutol in 78 patients with tuberculosis were described using non-linear mixed effects modeling. Pharmacodynamic data was comprised of weekly sputum liquid culture (using mycobacterial growth indicator tubes) time to detection results from 144 patients during the first 2 months of treatment. The effect of drug exposure on patient outcomes was investigated. To determine the adequacy of ofloxacin drug exposure, the probability of attaining the required area-under-the-curve to minimum inhibitory concentration ratio (AUC/MIC) of ofloxacin was determined in 65 patients on treatment for multidrug resistant tuberculosis. To improve efficiency in the clinical development of new drug regimens, clinical trial simulation was used to determine the optimal study design for a study investigating the efficacy of a new antitubercular drug regimen. The SLCO1B1 rs4149032 polymorphism existed at a high frequency of 0.70 in South Africans and resulted in a 28% decrease in bioavailability of rifampicin. The rifampicin peak concentration was a significant predictor of the 2 month treatment outcomes. A semimechanistic time to event model was developed to analyze days to positivity (time to detection) data. The model was comprised of a biexponential decay model describing bacillary decline in sputum from patients, followed by a logistic model with a lag time for growth of the mycobacteria in liquid culture. For the current 800 mg daily dose of ofloxacin, the probability of attaining an AUC/MIC target ratio of at least 100 was only 0.45. Based on clinical trial simulation, the optimum parallel study design was comprised of 125 study participants in each of 2 arms to achieve a study power of at least 80%. Increasing the study length beyond 42 days reduced study power perhaps due to increased amounts of censored data. Higher doses of rifampicin are required in the majority of South African patients with tuberculosis. A novel pharmacodynamic model of tuberculosis treatment is presented, which can be used for investigation of covariates such as drug exposure. Ofloxacin should be replaced with a more potent fluoroquinolone for treatment of multidrug resistant tuberculosis. Clinical trials should not be unduly long otherwise this may compromise study power.

Malaria and tuberculosis are listed among the major infectious diseases. They are responsible for severe morbidity and mortality especially in resource-poor settings where control interventions are inaccessible, unaffordable and plagued by widespread resistance. According to current estimates, malaria afflicts over 40% of the worldâs population and claims the lives of 1-3 million annually. The epidemiology of tuberculosis is just as grim. About one third of the world population is reported to be infected with Mycobacterium tuberculosis and it is responsible for 2-3 million deaths annually. Of particular interest to this project, is the fact that natural products have always been on the frontline in the battle against these diseases, that is, most of the clinically used drugs in antimalarial and antitubercular chemotherapy are of natural product-origin. In this project we therefore focussed on the design, synthesis, characterization and biological evaluation of novel antimalarial and antitubercular agents obtained by synthetically hybridizing and decorating scaffolds based on natural products or derivatives - with a history in the aforementioned disease models. Scaffolds selected include the thiolactone ring system, a key intermediate of the natural product thiolactomycin, the non-peptidic natural product isatin and the chalcone scaffold. In this way a series of hybrids were constructed which can be subdivided into three main groups: (i) thiolactone-isatin hybrids, (ii) -amino alcohol thiolactone-chalcone and isatin-chalcone hybrids, and (iii) dihydroartemisinin-isatin, dihydroartemisinin-chalcones and other miscellaneous hybrids. These were evaluated for antiplasmodial activity against the chloroquine resistant (W2) and chloroquine sensitive (D10) strains of Plasmodium falciparum as well as for inhibitory activity against cysteine proteases. Evaluation of antimycobacterial activity of the synthesized compounds against the drug sensitive H37Rv strain of M. tuberculosis was also undertaken. (i) For the first group of hybrids we used the C-4 hydroxyl group of the thiolactone ring as a handle for functionalization by attaching it via a variable, non-hydrolyzable alkyl linker to the isatin scaffold. Most striking, is the operational simplicity of the synthesis methodology employed and how it led to the discovery of a novel tetracyclic ring system. Identified from the latter is the compound 3.8p which is the most active antimalarial from this series with an IC50 of 6.92 μM in the W2 strain. Some of the hybrids (3.7 and 3.8) were more active than the monomers and the parent drug thiolactomycin, thus demonstrating the potential of hybridization as a drug discovery tool. Antimalarial structure activity relationships for the novel tetracycles 3.8 revealed the importance of substitution at C-5 of the isatin scaffold and vi the need for increased lipophilicity. Although the antitubercular activity of the hybrids was inferior compared to the control drugs, a number of advanced intermediates were identified which displayed promising activity against both fast growing and slow-growing, persistent forms of M. tuberculosis. (ii) The second group of hybrids consisted of a 36-member library obtained by the covalent linkage of methoxylated chalcones with the thiolactone ring and the isatin scaffold. Incorporated in their design is the -amino alcohol moiety, a known bioactiphore. For the synthesis of these hybrids we employed the copper-catalyzed Huisgen 1,3-dipolar cycloaddition reaction (also know as âclickâ chemistry) which in addition to expediting structure activity relationship studies yielded the 1,2,3-triazole ring system. The antiplasmodial results showed that the thiolactone-chalcones, with IC50s ranging from 0.68 to 6.08 μM, were more active against the W2 strain than the isatin-chalcones (IC50 = 2.09 - 14.90 μM). More so, structure activity relationships delineated for the former indicated the preference for triOMe substitution on ring A of the chalcone scaffold. The most active compound for this series 4.14f [IC50 = 0.68 μM (W2)] is 10-fold less active than chloroquine but has a greater efficacy than the parent natural product thiolactomycin. Results obtained for cysteine protease activity showed that the isatin-chalcone hybrids inhibited falcipain-2 activity, whereas the thiolactone-chalcone hybrids were devoid of enzyme inhibitory activity. With regard to antitubercular activity, the advanced intermediates were more active than the hybrid constructs. The most promising antitubercular agent identified is the acetylenic chalcone 4.10f (MIC = 13.1 μM) which is 2-fold more active than one of the controls, moxifloxacin (MIC = 31.1 μM) against the slow-growing persistent forms of M. tuberculosis. (iii) The final group of compounds is a limited series of semi-synthetic artemisinin analogues obtained by hybridizing the first generation analogue, dihydroartemisinin with previously mentioned scaffolds (isatin, chalcones, thiolactone) and other biologically relevant scaffolds such as the 4-aminoquinoline unit and azidovudine (AZT). As with the previous series we utilized the âclickâ reaction to effect the synthesis of these hybrids. The most active compound identified is the intermediate 5.4 [IC50 = 6.13 nM (W2)] which is more active than the parent natural product artemisinin [IC50 = 10.84 nM (W2)], 16 times more active than chloroquine and 2-times less active than dihydroartemisinin. The lack of antitubercular activity of compounds in this series moreover confirmed the antimalarial specificity of artemisinin analogues.

Glutamine synthetase (GS) plays an important role in nitrogen metabolism, where it catalyzes the ATP-dependent condensation of glutamate and ammonia to yield glutamine. Recent studies showed the importance of M. tuberculosis GS (MtGS) for growth and survival of the bacterium, and demonstrated its potential as a drug target. This thesis presents structural studies of MtGS and mammalian GSs, which are aimed at identifying and developing novel inhibitors against the mycobacterial target. The structure of MtGS was solved in complex with a phosphorylated form of the inhibitor methionine sulfoximine, magnesium and ADP. The complex structure provides a detailed picture of the active site, offering several insights into catalysis and inhibition, as well as forming a solid basis for structure-based drug design. The apo canine GS and liganded human GS structures described in this thesis represent the first structures of the mammalian enzymes. Comparison of the structures revealed substrate-induced conformational changes. Inspection of the nucleotide-binding site showed that it differs from that of MtGS, thus offering good opportunities to design specific and selective inhibitors of the mycobacterial enzyme. The amino acid-binding site of MtGS was evaluated as a target for inhibition, using a combination of a literature survey, structure-based virtual screening and the synthesis of a small library of compounds. As a result, several new inhibitors of MtGS could be identified. Finally, the structural basis for inhibition of MtGS by a purine analogue (PA) is provided. PA, an analogue of a class of compounds found to inhibit MtGS in a high-throughput screening assay, targets the nucleotide-binding site. The architecture of the HsGS nucleotide-binding site indicates that PA would not be able to bind to the human enzyme, offering good prospects for selective inhibition of MtGS.

There is a continuing need to make new antitubercular drugs due to development of resistance towards present drugs. To do this, series of pyruvate hydrazones (PVAHs) and their respective Cu(II) complexes and Zn(II) complexes were synthesized and fully characterised. Variable temperature NMR studies of PVAHs indicate the presence of E and Z isomers in the solution. Determination of the obtained single crystal X-ray structures reveals that Cu(II) ion binds to PVAH ligand in 1:1 ratio resulting in square pyramidal geometry in most of the Cu(II) complexes, whereas Zn(II) ion binds to two PVAH ligands in 1:2 ratio giving rise to octahedral geometry. The electrochemical studies of Cu(II) complexes of PVAH performed using cyclic voltammogram indicate the presence of quasi-reversible behaviour assigned to a Cu2+/Cu1+ peak potential. This indicates structural reorganisation of Cu(II) square pyramidal geometry towards Cu(I) tetrahedral geometry. The tetrahedral geometry of a synthesized Cu(I) complex of PVAHs was confirmed by X-ray crystal structure. The stability studies of selected PVAH ligands and their metal complex indicate that the investigated compounds were stable in extreme basic conditions, but they were unstable in extreme acidic conditions due hydrolysis of azomethine bond. However, stability of these compounds in physiological conditions, i.e. in PBS buffer, reveals that ligand hydrolyses slowly over a period of time, whereas the Cu(II) complex remains quite stable over a monitored period of 120 hours. Interestingly, dihydrazide analogue of PVAH was fairly stable in PBS buffer. EPR studies of investigated Cu(II) complexes in DMSO indicate that PVAHs remains strongly coordinated to Cu(II) centre. The evaluation of the antimycobacterial activity showed that the anionic PVAHs and Zn(II) complexes are essentially inactive. Some of the corresponding neutral Cu(II) complexes, however, exhibit promising antimycobacterial activities if tested under high iron (8 μg Fe per mL) conditions. As observed for the related antimycobacterial agent isoniazid, the activity of the complexes decreases if the M. tuberculosis cells are grown under low iron (0.02 μg Fe per mL) conditions. The Cu(II) complexes may thus have a similar mode of action and may require an iron-containing heme-dependent peroxidase for activation. A series hydrophobic cinnamaldehyde hydrazones (CAHs) and their Cu(II) complexes were also synthesized and tested for their antitubercular activity under similar conditions to that of PVAH series. But they failed to show any inhibitory activity due to their poor cellular uptake owing to their limited solubility in aqueous buffer.

In light of shrinking pharmaceutical drug pipelines and drug resistance, innovative drug discovery strategies are of imperative need. Drug repurposing and related strategies such as drug rescue and drug remodelling have garnered significant research interest. Various clinically approved non-antibiotics including phenothiazines hold promise as novel classes of therapeutics in other indications. However, in addition to inherent neuroleptic properties, phenothiazines and related antipsychotics elicit adverse side effects at clinically relevant doses thus precluding their extensive clinical application. Herein, it was postulated that the selectivity of phenothiazines and related drugs for nonneuroleptic indications could be enhanced through rationalized structural remodelling. Phenothiazine and related neuroleptics are known to obey a lipophilic chromophore/basic side chain paradigm. Deviation from this paradigm is expected to decrease potential for neuroleptic effects. Therefore, the remodelling strategies involved introduction of novel functionalities that are dissimilar to native phenothiazine structures. Prior to chemical synthesis, drug metabolism and pharmacokinetic related properties were predicted in silico to assess drug-likeness of the new chemical entities derived from phenothiazines and related antipsychotics. The in silico profiling also included prediction of blood/brain partition coefficients and CNS activity to determine their likelihood of exhibiting neuroleptic effects. The new chemical entities were then evaluated against drug-susceptible Mycobacterium tuberculosis-H37Rv. Furthermore, a selected series was screened for binding to dopamine and serotonin receptors to corroborate in silico CNS activity predictions. Moreover, pharmacokinetic studies were conducted with the selected series to determine in vitro microsomal stability, kinetic solubility and in vivo toxicity profiles. Another objective of this study was to evaluate the new chemical entities for their potential as anticancer agents. The key findings herein demonstrated that it is possible to abolish neuroleptic effects through rationalized structural manipulation and still retain bio-activities of interest. Several new chemical entities including N-alkylsulfonates (DS0031, DS0032, DS0034, DS0035, DS00366) and nitrobenzenesulfonamides (DS00325, DS00326, DS00329) of phenothiazines, displayed notable antitubercular (GAST/Fe MIC90 range: 9.9-125 µM; 7H9 MIC range 12.5- 25 µg/mL) and anticancer (IC50 range 4.51-12.43 µM) activities in comparison to native phenothiazine drugs. Furthermore, in vitro and in vivo preclinical evaluation revealed favourable pharmacokinetic profiles. Overall, this study presents novel subclasses of phenothiazines that hold promise for further development as non-neuroleptic agents in either tuberculosis or cancer treatment regimens.

Tuberculosis kills approximately 1.7 million people each year around the world making it one of the most lethal infectious diseases. This thesis concerns the development of two computational tools that can support the early stages of drug discovery, and their use in an anti-tubercular drug discovery program. One of the tools developed is a statistical molecular design (SMD) approach that generates information-rich libraries biased towards a lead structure. The other metod is a post-filtering technique to increase the success of virtual screening, has also been developed. Both methods have been validated using literature data. Ribonucleotide reductase (RNR) has been identified as a potential anti-tubercular target, and our focus has been to develop small-molecule inhibitors of this target. The enzyme consists of two subunits (a large R1 and a small R2 subunit) that have to associate in order to generate a bioactive complex. It had previously been shown that a heptapeptide corresponding to the small R2 subunits C-terminal inhibited the enzyme. In order to investigate the requirements for inhibitory effect of the peptide a library was designed using the developed SMD approach. The designed library was synthesized and evaluated for biological activity and an OPLS-DA model was derived to understand which positions were most important for activity. In order to identify small-molecule inhibitors of RNR a combined shape- and structure-based virtual screen was performed, employing ROCS, GlideXP and the developed post-filtering technique. Starting from a library of 1.5 million compounds 24 was acquired and evaluated for enzymatic activity. The best compounds were almost as potent as the starting peptide, but considerably more drug-like.

Bacterial membranes undergo dynamic rearrangements during cell division with lateral heterogeneity in phospholipid distribution. An understanding of the synthesis of the cell membrane can provide a basis for the rational design of new antibacterial compounds which will ultimately be developed towards potential new antibiotics. Mounting evidence is implicating the importance of phospholipid in the viability of mycobacteria. The genetically tractable and related organism, Streptomyces coelicolor represents an ideal model for analogous studies on Mycobacteria tuberculosis for the purpose of designing novel classes of anti-tuberculosis drugs targeting the phospholipid biosynthesis pathways. Genetic and biochemical studies carried out on phosphatidylserine synthase (Pss) and phosphatidylserine decarboxylase (Psd) demonstrated the essentiality of these membrane proteins in S. coelicolor. Alteration of pss expression affects the overall growth and morphology of S. coelicolor (i.e. hyphal growth, branching, septation and sporulation) therefore verified the potential of these proteins as drug targets. Although, the early stage "hit identification" approach using a modest collection of compounds was unsuccessful, further screening of relevant compounds should continue. Structural modifications should be carried out on some of the initial compounds which were devoid of antibacterial activity in order to address the possible pharmacodynamic issues. Protein X-ray crystallography or saturation transfer difference - nuclear magnetic resonance (STD-NMR) spectroscopy of these proteins should also be considered in the event of further futile attempts.

Trabalho realizado na Universidade do Algarve
Tuberculosis (TB), despite being a completely curable disease, has reemerged due to drug resistance and deadly synergism with HIV infection, which limit the success of its management. Lung tuberculosis is the main manifestation of TB. Thus, exploring the inhalable route for a local delivery of antitubercular drugs seems a promising therapeutic approach. Partially hydrolyzed guar gum (PHGG) is a strong candidate as matrix material for antitubercular drug carriers. This is mainly due to its affinity for macrophages, the hosts of mycobacteria, which is mediated by the binding of sugar units to macrophage surface receptors. In this work, PHGG-based microparticle formulations were produced by spray-drying, evaluated for cristallinity pattern (X-ray diffraction) and ability for drug association, and in vitro drug release profiles were determined. The cytotoxicity of microparticles was also evaluated (MTT and LDH release assays). Additionally, the therapeutic effect of drug-loaded microparticles was evaluated in vitro on macrophage-like cells infected with mycobacteria strains. The results showed that microparticles exhibited suitable properties for pulmonary delivery (aerodynamic diameter between 1 and 3 μm). A favorable cytotoxic profile was evidenced, as no overt toxicity was detected in representative respiratory cell lines (A549 and Calu-3 cells), although a mild toxic effect was observed in macrophage-like cells. The in vitro response of infected macrophages to drug-loaded PHGG microparticles was considered promising, as only 20% of mycobacteria remained viable upon a single treatment with microparticles. This thesis addresses macrophages as therapeutic target, unraveling the unique role of polysaccharides on pulmonary drug delivery in the ambit of tuberculosis therapy.
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Hirsutellones are fungal polyketides with beautiful structures including a decahydrofluorene (tricyclic) core and a highly strained 12- or 13-membered macrocycle. Inspired by a biosynthetic hypothesis concerning their skeletons, we envisaged a biomimetic strategy for the total synthesis of hirsutellone B, via a coupling and a cascade cyclization from a tetramic acid moiety and a linear polyunsaturated precursor. In this dissertation, we will describe the synthesis of several tetramic acids and linear polyene building blocks, as well as the attempts on their coupling and macrocyclization. Furthermore, through a designed linear polyene precursor with both nucleophilic and electrophilic functional groups, we will present the biomimetic electrophilic cyclization followed by an IMDA reaction towards the tricyclic core of hirsutellone B and thus the achievement of the formal total synthesis of hirsutellones A, B and C. In the context of searching for biologically active compounds, the respiratory chain inhibitory isoprenoid quinoline alkaloids aurachins captured our attention for their synthesis, which was inspired by another biosynthetic hypothesis. Therefore, as an international collaboration project, the first total synthesis of the natural product aurachin D and its analogues with chain length or aromatic cycle variations will be presented, using a key Conrad-Limpach process. An interesting reductive epoxide-opening cascade will be described towards the synthesis of aurachin H analogues. A new biosynthetic hypothesis will also be presented from aurachin C to B. Furthermore, their biological activity was extensively studied by our collaborators and will be summarized
Les hirsutellones sont des polycétides fongiques possédant une structure intéressante avec un noyau decahydrofluorène (tricycliques) et un macrocycle particulièrement contraint à 12- ou 13- chaînons. S'inspirant d'une hypothèse de biosynthèse sur le squelette des hirsutellones, nous avons envisagé une stratégie biomimétique pour la synthèse totale de l'hirsutellone B, par l'intermédiaire d'un précurseur polyinsaturé linéaire. Dans cette thèse, nous décrirons la synthèse de plusieurs acides tétramiques et polyènes linéaires intermédiaires, ainsi que les tentatives de leur couplage et de macrocyclisation. En outre, grâce à un précurseur polyène linéaire porteur de groupes fonctionnels nucléophile et électrophile, nous présenterons la cyclisation électrophile biomimétique suivie d'une réaction IMDA vers le noyau tricyclique de hirsutellone B, et donc la réalisation d'une synthèse totale formelle des hirsutellones A, B et C. Dans le cadre de la recherche de composés biologiquement actifs, des alcaloïdes inhibiteurs de la chaîne respiratoire, les aurachines de type isoprénoïde-quinoléine, ont attiré notre attention pour réaliser leur synthèse qui a été inspiré par une autre hypothèse biosynthétique. Par conséquent, la première synthèse totale de l'aurachine D et de ses analogues de chaîne ou de cycle aromatique sera présentée. Une cascade d'ouverture d'époxyde suite à une hydrogénation sera décrite vers la synthèse d'analogues de l'aurachine H. Une nouvelle hypothèse de biosynthèse sera également présentée de l’aurachine C au B. Enfin, leurs activités biologiques ont été extensivement étudiées par nos collaborateurs et seront résumées

Includes bibliographical references.
Sub-Saharan Africa is overwhelmed by dual epidemics of human immunodeficiency virus (HIV) and tuberculosis (TB) infection. Non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART) is recommended for first-line treatment in adult HIV treatment programmes in resource-limited settings [1]. Many South African HIV-infected patients initiate ART while on TB treatment, 38 percent in one local study [2]. In addition, although ART reduces the incidence of TB, incidence in patients on ART is higher than in the HIV uninfected population [3], therefore incident TB on ART requiring concomitant treatment is very common. Efavirenz is regarded as the NNRTI of choice for TB co-infected patients [1] as outcomes are superior compared to those achieved with nevirapine-based ART [4] and concomitant TB treatment does not significantly reduce efavirenz concentrations [5]. However nevirapine is cheaper than efavirenz and is used extensively used in lower income countries with limited access to efavirenz [1]. Data characterising the extent to which concomitant rifampicin-based TB treatment decreases nevirapine plasma concentration therefore remain important.

La tuberculose est une infection humaine cosmopolite à transmission pulmonaire causée par Mycobactérium tuberculosis. Elle représente l'une des principales causes de mortalité dans le monde. La prise en charge thérapeutique de cette maladie est confrontée aujourd’hui à une forte pharmacorésistance des bacilles à la plupart des médicaments antituberculeux habituellement indiqués. Face à cette situation la recherche de nouvelles molécules plus efficaces échappant au phénomène de pharmacorésistance est encouragée par l'OMS. C'est dans ce contexte que nous avons conçu des dérivés de la 1,10-phénanthrolinone, analogues structuraux des quinolones connues pour leurs activités antituberculeuses. Ces dérivés ont été préparés par condensation de la 8-aminoquinoléine et de l'étoxyméthylènemalonate d'éthyle, suivit d'une cyclisation intramoléculaire pour conduire à la 1,10-phénanthrolinone-3-carboxylate d'éthyle. La modulation chimique de cette dernière a permis d'obtenir la plupart des dérivés de la 1,10-phénanthrolinone préparés. Les dérivés de la 1,10-phénanthrloninone synthétisés ont fait l'objet d'une évaluation de leurs activités antibacillaires. Parmi ceux-ci, certains dérivés 6-nitro-1,10-phénanthrolinones se sont particulièrement illustrés par leurs performances antibacillaires avec des CMI comprises entre 0,31 et 9,84 μM. Par ailleurs, cette activité est conservée sur les souches de mycobactéries sensibles et les souches résistantes aux quinolones ce qui fait penser à un probable mécanisme d'action différent. De plus, ces molécules se sont avérées non toxiques sur les cellules Vero avec des IC50> 100 μg/mL ( soit 16 à 64 fois leurs CMI). Notre approche pharmacochimique a conduit à l’élaboration de nouvelles molécules possédant une structure de type 1,10-phénantrolinone dont le profil chimique diffère de celui de toutes les classes chimiques d’antituberculeux existantes. Les résultats obtenus ouvrent de nouvelles voies d'investigations pour la recherche de nouveaux antituberculeux voire anti-infectieux en série chimique des 1,10-phénantrolinones
Tuberculosis is a cosmopolitan human lung infection caused by Mycobacterium tuberculosis. She represents one of the main reasons of mortality in the world. therapeutic management of this disease is confronted today with a strong pharmacoresistance of bacilli to most antituberculosis habitually used. Facing this situation the research of new more efficient molecules avoiding the phenomenon of pharmacoresistance is encouraged by the WHO. It is in this context that we conceived derivatives of the 1,10-phénanthrolinone, the structural analogues of quinolones known for their antituberculosis activity. These derivatives were prepared by condensation of 8-aminoquinoline and ethyl ethoxymethylenemalonate, followed by intramolecular cyclization to give the ethyl 1,10-phenanthrolinone-3-carboxylate. The chemical modulation of the latter have allowed to get most of the 1,10-phenanthrolinone derivatives. The 1,10-phenanthrloninone derivatives synthesized were evaluated for their antibacillary activities. Among these, some 6-nitro 1,10-phenanthrolinone derivatives are particularly illustrated by their antibacillary performance with MIC included between 0.31 and 9.84 μM. In addition, this activity is kept on susceptible strains of mycobacteria and the quinolone-resistant strains , suggesting a different mechanism of action. In addition, this activity is conserved on susceptible strains of mycobacteria and quinolone-resistant strains, suggesting a different mechanism of action. Moreover, these molecules proved to be not toxic on cells Vero with IC50> 100 μg / mL (16 to 64 times their MIC). Our pharmacochimique approach has led to the development of new molecules having a structure of type 1,10-phénantrolinone from which chemical profile differs from that of all chemical classes of antitubercular drugs existent. Got results open new ways of investigations for research of new anti-tuberculosis or anti-infectious drugs in the 1,10-phenantrolinone chemical series

Thesis (MNutr)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: Problem definition: Malnutrition increases the incidence and exacerbates the clinical manifestations of TB. Hepatotoxicity is one of the most serious and most frequent side-effects of anti-tuberculosis drugs and may be three times higher in malnourished patients. Objective: The influence of nutritional and retroviral status on the bio-availability and toxicity of anti-tuberculosis agents was studied and a possible relationship between abdominal lymph node enlargement and the occurrence of malnutrition investigated. Subjects and setting: The study subjects were 53 children, 19 HIV-infected and 34 HIV-uninfected, aged 3 months to 13 years with probable or confirmed tuberculosis admitted to the paediatric ward of Brooklyn Hospital for Chest Diseases in Cape Town, South Africa. The nutritional status of the children was assessed over the first four months of tuberculosis treatment by nutrient intake, anthropometric status and biochemical parameters. The relationship between abdominal lymph node enlargement and the occurrence of malnutrition was also evaluated. Pharmacokinetic studies were performed to evaluate the bio-availability of anti-tuberculosis agents and drug hepato-toxicity was evaluated by liver function. Results: Stunting (46.27%) and underweight (34.51%) were the most common types of malnutrition in the children studied. HIV-infection did not have a significant effect on stunting or wasting, but had a significant effect (p=0.003) on underweight for age with 31.5% HIV-infected compared to 2.9% HIV-uninfected at enrolment, but the effect was not statistically significant at month 4. There was no change in the number of stunted, wasted or underweight children from enrolment after 1 month of treatment to month 4 of treatment. HIV-infection did not have a significant effect on abdominal TB involvement (p=0.43354), and nutritional status was not significantly affected by abdominal lymph-node involvement. At enrolment weight for age had a significant effect on AST and ALT with p-values of 0.02166 and 0.02765 respectively and wasting had a significant effect on GGT at enrolment (p=0.03014). However on enrolment only two HIV-infected and two HIV-uninfected children had ALT values increased >X2 normal. Similarly AST values >X3 normal were found in only one HIV-infected child and two HIV-uninfected children. Stunting did not significantly affect liver enzymes. Anthopometric status did not have a significant effect on liver enzymes at month 4. None of the parameters used to determine nutritional status had a statistically significant effect on INH-levels or RMP-levels. HIV-infection had a significant negatve effect on selenium (p=0.030 and 0.012) and ferritin (p=0.026 and 0.002) at enrolment and month 4 and on IBC (p=0.025) at enrolment. At month 4 HIV-infection had a significant negative effect on the mean vitamin C-levels (p=0.005). Conclusions: HIV co-infection did not affect the extent or distribution of body composition changes in this study. Stunting was the most prevalent form of malnutrition in the study group, indicating longstanding undernutrition, which may be due to factors other than the present TB infection. Appropriate treatment of tuberculosis did not appear to affect the nutritional status over the four month period of the study.
AFRIKAANSE OPSOMMING: Probleemstelling: Wanvoeding verhoog die insidensie en vererger die kliniese beeld van TB. Hepatotoksisiteit is een van die ernstigste en algemeenste newe-effekte van anti-tuberkulose middels en mag tot drie keer hoër wees in wangevoede pasiënte. Doelwit: Die invloed van die kinders se voedings- en retrovirale status op die bio-beskikbaarheid en toksisiteit van anti-tuberkulose middels was ondersoek en 'n moontlike verband tussen vergrote abdominale limfnodes en die voorkoms van wanvoeding was ondersoek. Deelnemers en omgewing: Die deelnemers aan die studie was 53 kinders, 19 HIV-positief en 34 HIV-negatief, tussen die ouderdomme van 3 maande en 13 jaar met moontlike of bevestigde tuberkulose toegelaat tot die pediatriese saal van Brooklyn Hospitaal vir Borskwale in Kaapstad, Suid Afrika. Die voedingstatus van die kinders was bepaal oor die eerste vier maande van tuberkulose behandeling ten opsigte van nutriëntinname, antropometriese status en biochemiese parameters. Die verhouding tussen vergrootte abdominale limfnodes en die voorkoms van wanvoeding was ook geëvalueer. Farmakokinetiese studies was uitgevoer om die bio-beskikbaarheid van anti-tuberkulose middels te evalueer en hepatotoksisiteit was deur lewerfunksie geëvalueer. Resultate: Dwerggroei (46.27%) en ondergewig (34.51%) was die algemeenste tipes wanvoeding teenwoordig by die kinders bestudeer. HIV-infeksie het nie 'n noemenswaardige effek op dwerggroei of uittering gehad nie, maar het wel 'n noemenswaardige effek (p=0.003) getoon op ondergewig vir ouderdom met 31.5% HIV-positief vergeleke met 2.9% HIV-negatief by inskrywing, wat nie statisties noemenswaardig was teen maand 4 nie. Daar was geen verandering in die hoeveelheid kinders met dwerggroei, uittering of ondergewig vanaf inskrywing na 1 maand van behandeling tot maand 4 van behandeling nie. HIV-infeksie het nie 'n noemenswaardige effek op abdominale TB gehad nie (p=0.43354), en vergrootte abdominale limfnodes het nie 'n noemenswaardige effek op voedingstatus gehad nie. By inskrywing het gewig vir ouderdom 'n noemenswaardige effek op AST en ALT gehad met p-waardes van 0.02166 en 0.02765 onderskeidelik en uittering het 'n noemenswaardige effek op GGT by inskrywing gehad (p=0.03014). Dwerggroei het nie die lewerensieme noemenswaardig beïnvloed nie. Antropometriese status het nie 'n noemenswaardige effek op lewerensieme teen maand 4 gehad nie. Geen van die parameters wat gebruik is om voedingstatus te bepaal het 'n noemenswaardige statistiese effek op INH-vlakke of RMP-vlakke gehad nie. HIV-infeksie het 'n noemenswaardige effek op selenium (p=0.030 en 0.012) en ferritien (p=0.026 en 0.002) by inskrywing en maand 4 gehad en op IBC (p=0.025) by inskrywing. HIV-infeksie het 'n statisties noemenswaardige effek op die gemiddelde vitamien C-vlakke (p=0.005). Gevolgtrekking: HIV ko-infeksie het nie die verspreiding of mate van liggaamsamestelling veranderinge in hierdie studie geaffekteer nie. Dwerggroei was die algemeenste vorm van wanvoeding in die studiegroep, wat langstaande wanvoeding aandui en toegeskryf mag word aan faktore buiten die huidige TB infeksie. Toepaslike tuberkulose behandeling het nie 'n wesenlike effek op voedingstatus gehad tydens die vier maande periode van die studie nie.

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Introdução: Os problemas relacionados à interrupção e ao abandono do tratamento da tuberculose culminam em aumento da morbimortalidade. A ocorrência de reações adversas a medicamentos (RAM) é apontada como um dos principais fatores relacionados. Objetivo: Identificar evidência científica disponível sobre os fatores de risco associados às reações adversas decorrentes do uso de medicamentos antituberculose. Métodos: Trata-se de uma revisão sistemática em que se buscou estudos sobre fatores de risco associados às reações adversas aos medicamentos antituberculose nas bases Medical Literature Analysis and Retrieval System Online (MEDLINE), no período entre 1965 e 2013 e Literatura Científica e Técnica da América Latina e Caribe (LILACS), no período entre 1982 e 2013. Localizou-se 1389 artigos que passaram por uma triagem a partir da leitura dos títulos e resumos. A partir dessa análise, selecionou-se 85 estudos para serem lidos na íntegra. Ao final, 16 estudos foram incluídos na análise a partir dos critérios de elegibilidade adotados em cada etapa, que tiveram seus dados extraídos para os cálculos de Qui-quadrado, Mantel-haenszel, Odds ratio simples (OR) e combinada (ORc). Resultados: Os fatores de risco significantes para o desenvolvimento de RAM foram: idade (maior que 60 anos), esquemas de tratamento, alcoolismo, anemia, coinfecção pelo vírus da imunodeficiência humana ou vírus da hepatite, polimorfismo da N-acetiltransferase 2 (acetilador lento), além da deficiência de sódio, ferro e albumina. Enquanto as meta-análises evidenciaram que os fatores de proteção das RAM hepáticas são: sexo masculino (ORc=0,38; IC95%=0,20-0,72), idade >35 anos (ORc=0,38; IC95%=0,20-0,72), fenótipo acetilador rápido/intermediário da N-acetiltransferase 2 (ORc=0,41; IC95%=0,18-0,90). Conclusões: Há evidências para subsidiar o manejo de RAM antituberculose nos serviços de saúde pública.
Setting: The problems related to the interruption and the dropout of tuberculosis treatments lead to increased morbi-mortality. Drugs adverse effects are some of the main related reasons. Objective: To identify scientific evidence available about risk factors associated to adverse effects due to antituberculosis drugs usage. Design: A systematic review of studies about risk factors related to adverse effects of antituberculosis drugs selected in MEDLINE database from 1965 to 2013 and in LILACS database from 1982 to 2013. After screening papers by reading all titles and abstracts there were 1.389 approved papers. Based on this analysis, 85 papers were selected to be fully read. At the end, 16 papers were selected to be analyzed due to the eligibility criteria on each step, had their data extracted for calculation of Chi-square, Mantel-Haenszel, Odds ratio (OR) and combined Odds ratio (ORc). Results: Significant risk factors to the development of drugs adverse effects were: age over 60 years, treatment regimen, alcoholism, anemia, coinfection by human immunodeficiency or hepatitis viruses, phenotype slow acetylators of N-acetyltransferase 2 and the deficiency of sodium, iron and albumin. While, meta- analysis showed that protective factors of liver AED are: male (ORc = 0.38, 95%CI= 0.20 to 0.72), age > 35 years (ORc=0.38, CI95%=0.20 to 0.72), acetylator phenotype fast / intermediate of N-acetyltransferase 2 (ORc=0.41, 95% I= 0.18 to 0.90). Conclusion: There is evidence to support the management of antituberculosis AED in public health services.

The tuberculosis (TB) epidemic remains a major threat to public health globally, and is exacerbated by the escalating number of multi-drug resistant cases. These factors have highlighted the urgent need for new effective therapies or different approaches to augment the efficacy of current anti-TB drugs. Synergistic drug combinations present a feasible strategy towards expanding TB treatment options. Despite reported successes with combination screening, as well as the current reliance on combination therapy for TB, this approach remains largely underexplored. Evidence suggests that utilizing synergistic combinations might enable existing clinically-approved drugs to be readily re-purposed for TB treatment, including against multi-(MDR) and extensively- (XDR) drug resistant strains for current therapies are often ineffective. This thesis focused on the development and application of improved methods to identify and advance novel drug combinations for TB therapy. There were two key aspects to this work: firstly, exploring mechanisms of synergy between fusidic acid (FSA), a natural product antibiotic, and current anti-TB agents and, secondly, characterizing antibiotic action by delineating bacteriostatic and bactericidal compounds.

Mycobacterium tuberculosis (Mtb), the etiological agent of Tuberculosis (TB) is developing new multi drug-resistant (MDR) and extensively drug-resistant (XDR) strains to the current drugs used in therapy. Of particular concern the wide spreading of tuberculosis, the high rate of development of resistance, and the high mortality of the patients due to the lack of effective therapy against TB infections. In order to face this problem, two series of novel compounds were designed, synthesised and evaluated against a panel of mycobacterial strains. The first series of compounds includes analogues of the third line drug thioridazine (TZ). TZ is a known antipsychotic drug belonging to the phenothiazine drug group, which showed good activity against MDR-TB infections but causes severe side effects which limit its use in therapy. Among the first series of compounds, five new compounds showed anti-tubercular activity similar or higher than TZ. Moreover, two derivatives showed potent inhibition towards the whole-cell drug efflux pump activity of mycobacteria comparable to that of verapamil, and turning to be promising multi-drug resistance reversal agents. A second series of compounds consist of small molecules which have originally been designed as hybrids of the anti-tubercular drugs BM212 and SQ109. Computational studies revealed a perfect superposition of the structures of SQ109 and BM212 and showed that the two drugs share common features. Five of the resulting compounds showed micromolar anti-tubercular activity on pathogenic TB. Two of them proved to be highly active also against multi-drug resistant clinical isolates and one of these also showed minimal eukaryotic cell toxicity, and therefore would be an excellent lead candidate for preclinical trials. In parallel to the identification of novel compounds active against mycobacteria, new synthetic methodologies for the synthesis of antitubercular heterocyclic scaffolds have been developed. In particular two approaches for the synthesis of pyrrole compounds were developed. Both procedures involve an olefin or enyne metathesis reaction as a key step. The first approach involves the synthesis of 1,2,3-substituted pyrroles, through a tandem enyne cross metathesis-cyclization reaction of propargylamines with ethyl-vinyl ether. The reaction is rapid, procedurally simple and represents a facile entry to the synthetically challenging 4,5-unsubstituted pyrroles. The second methodology allows the synthesis of substituted pyrroles from diallyl-amines via a chemo-enzymatic cascade based on the combination of olefin metathesis together with monoamine oxidase (MAO) biocatalysts. These reactions were carried out in aqueous media and mild temperature leading to the formation of substituted pyrroles in a single step and in high yields.

Background: Tuberculosis (TB) is the most common opportunistic infection in children with human immunodeficiency virus (HIV) infection in developing countries, and co-treatment for HIV infection and TB is frequently indicated. Efavirenz and lopinavir/ritonavir (ratio 1:1) as part of antiretroviral therapy are used in combination with rifampicin-based antitubercular treatment in South African TB/HIV co-infected children. Adult studies show that concomitant rifampicin significantly reduces efavirenz and lopinavir plasma concentrations. However, the pharmacokinetics (PK) of efavirenz and lopinavir/ritonavir are poorly characterized in children, especially African children and no study has evaluated the effect of rifampicin-based antitubercular treatment on efavirenz and lopinavir/ritonavir plasma concentrations in children. Although therapeutic drug monitoring (TDM) is recommended in selected patients (including young children and patients receiving concomitant antitubercular treatment), TDM is seldom available in resource-constrained countries. There is an urgent need to develop a field friendly method which requires small volumes of blood, and inexpensive processing and storage conditions. Furthermore, because HIV replicates in the cells, efavirenz and lopinavir need to penetrate into these infected cells to inhibit viral replication. Therefore, directly measurement of intracellular concentrations of these drugs in HIV-infected children could provide better understanding of drug exposure at the action site. It is also important to evaluate the effects of frequently co-administered drugs on intracellular accumulation of efavirenz and lopinavir. Objectives: 1) To evaluate efavirenz and lopinavir/ritonavir plasma concentrations and determine the effects of rifampicin on efavirenz and lopinavir/ritonavir PK in HIV-infected African children with and without rifampicin-based antitubercular treatment. 2) To develop and validate the dried blood spot (DBS) method as an alternative to conventional plasma methods of drug concentration measurement in TDM. 3) To evaluate in vivo intracellular concentrations of efavirenz and lopinavir/ritonavir in HIV-infected children with and without concomitant antitubercular treatment. 4) To determine the in vitro modulation effects on the intracellular accumulation of efavirenz IV and lopinavir in human peripheral blood mononuclear cells (PBMCs) by drug efflux protein inhibitors, as well as frequently co-administered rifampicin and ritonavir (at low dose; as pharmacoenhancer). Methods: 1) Plasma efavirenz and lopinavir/ritonavir concentrations were measured by validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method in TB/HIV co-infected children during and after rifampicin-based antitubercular treatment as well as in a group of controls (HIV-infected children without TB). Children in the efavirenz study (n= 30) were receiving standard doses of efavirenz as part of antiretroviral treatment. Trough concentrations (Cmin) of efavirenz were estimated by extrapolation of the log-linear concentration-time line to 24 hours after the previous dose. Children in the lopinavir/ritonavir study were receiving additional ritonavir (lopinavir: ritonavir ratio 1:1) during antitubercular treatment (n= 15), and standard doses of lopinavir/ritonavir (LPV/r; ratio 4:1) after antitubercular treatment, and in controls (n= 15). The PK of lopinavir and ritonavir were characterized from concentration-time curves using WinNonlin version 4.1 by non-compartmental analysis. 2) Aliquots of 50 μ L of whole blood from the efavirenz and lopinavir/ritonavir studies were dried onto filter paper. The drug concentrations were analyzed using validated LC/MS/MS method. The effects of high temperature and direct sunlight on the stabilities of these antiretroviral drugs in DBS samples were tested. 3) Intracellular concentrations of efavirenz, lopinavir and ritonavir were measured in trough concentrations of 11 TB/HIV co-infected children using a validated LC/MS/MS method. Six children were receiving double dose of LPV/r (4:1) with concomitant rifampicin; 5 children were receiving standard doses of efavirenz with rifampicin-based antitubercular treatment, 3 of them had intracellular concentrations measured again after completing rifampicin-based antitubercular treatment. 4) in vitro intracellular accumulation of efavirenz and lopinavir were measured in human PBMCs in the absence and presence of P-glycoprotein inhibitors (verapamil at 50 μ M, V furosemide at 50 μ M and cyclosporine A at 20 μ M) and frequently co-administered drugs at levels representing the average concentrations found in patients (ritonavir at 5 mg/L and rifampicin at 4 mg/L). The concentrations of efavirenz and lopinavir in PBMCs were determined by LC/MS/MS. Results and Conclusions: 1) The co-administration of rifampicin did not significantly reduce efavirenz estimated Cmin concentrations. A high proportion of children with and without concomitant antitubercular treatment had sub-therapeutic efavirenz concentrations despite being correctly dosed according to the manufacturer's instructions, raising concerns about the adequacy of current efavirenz dosing recommendations in children. The lopinavir key PK parameter, Cmin, was not significantly different in same group of children during and after rifampicin-based antitubercular treatment or compared to HIV-infected children without tuberculosis. The recommended minimum therapeutic concentration was achieved in 87% of children during antitubercular treatment and in 92% without concomitant antitubercular treatment. Therefore, in the context of limited options, LPV/r with additional ritonavir (ratio 1:1) is an acceptable approach to treat young children receiving concomitant rifampicin-based antitubercular treatment, although safety remains a concern and hepatic alanine transaminase levels should be monitored regularly. 2) Plasma and DBS concentrations of efavirenz, lopinavir and ritonavir were strongly correlated. The median (interquartile range, IQR) DBS/plasma concentration ratios for efavirenz, lopinavir and ritonavir were 0.93 (IQR 0.83, 1.08), 0.73 (IQR 0.61, 0.90) and 1.05 (IQR 0.74, 1.21), respectively. PK parameters of efavirenz and ritonavir were closely similar between DBS and plasma; whereas lopinavir pre-dose and Cmin (at 12 hours after lopinavir intake) concentrations were 16% lower in DBS samples. The 3 antiretroviral drugs in DBS samples were stable at 37 deg C for 7 days and with exposure to direct sunlight for 2 hours. DBS can be used as an alternative field-friendly method for efavirenz, lopinavir and ritonavir concentration monitoring. However, pre-dose and Cmin concentrations of lopinavir in DBS samples need to be increased by 16% when used to predict plasma concentrations. VI 3) In vivo median intracellular/plasma concentration ratios for efavirenz, lopinavir and ritonavir amongst 11 TB/HIV co-infected children during antitubercular treatment were 0.91 (IQR 0.54, 1.19), 0.22 (IQR 0.09, 0.31) and 4.17 (IQR 1.30, 7.33), respectively. Two children had efavirenz intracellular/plasma concentration ratios during vs. after antitubercular treatment: 1.00 vs. 0.61 and 0.27 vs. 0.79. 4) Furosemide significantly increased efavirenz and lopinavir accumulation in healthy human PBMC samples by 1.2- 1.5 fold. Whereas, neither verapamil nor cyclosporin A had significant effects on efavirenz or lopinavir intracellular accumulation. Despite being an inducer of P-glycoprotein, rifampicin increased the accumulation of both efavirenz and lopinavir to different extents in all 3 PBMC samples. The low-dose ritonavir (at the concentration found in HIV-infected patients) had no effect on intracellular accumulation of efavirenz and lopinavir at therapeutic concentrations.

Many studies have reported low cure rates for multidrug-resistant tuberculosis (MDRTB) patients and MDR-TB patients co-infected with human immunodeficiency virus (HIV). However, little is 
known about the effect of HIV infection and antiretroviral therapy on the treatment outcomes of MDR-TB in South Africa. Therefore, the objectives of the study are: to find out whether HIV infection 
and interactions between ARVs and second line anti-TB drugs have an impact on the following MDR-TB treatment outcomes: cure rate and treatment failure at Brewelskloof Hospital. MDR-TB 
patients were treated for 18-24 months. The study was designed as a case-control retrospective study comparing MDR-TB treatment outcomes between HIV positive (cases) and HIV negative 
patients (controls). Patients were included in the study only if they complied with the following criteria: sensitivity to second line anti-TB drugs, MDR-TB infection, co-infection with HIV (for some 
of them), male and female patients, completion of treatment between 1 January 2006 and 31 December 2008. Any patients that presented with extreme drug-resistant tuberculosis (XDR-TB) 
were excluded from the study. Data were retrospectively collected from each patient&rsquo
s medical records. There were a total of 336 patients of which 242 (72%) were MDR-TB patients and 94 
(27.9%) MDRTB co-infected with HIV patients. Out of the 242 MDR-TB patients, 167 (69.2%) were males and 75 (30.7%) were females. Of the 94 patients with MDR-TB co-infected with HIV, 51 
(54.2%) males and 43 (45.7%) females. Patients with multidrug-resistant tuberculosis co-infected with HIV who qualify for antiretroviral therapy were treated with stavudine, lamivudine and 
efavirenz while all MDR-TB patients were given kanamycin, ethionamide, ofloxacin, cycloserine and pyrazinamide. The cure rate of MDR-TB in HIV (+) patients and in HIV (-) patients is 34.5% 
and 30 % respectively. There is no significant difference between both artes (pvalue = 0.80). The MDR-TB cure rate in HIV (+) patients taking antiretroviral drugs and in HIV (+) patients without 
antiretroviral therapy is 35% and 33% respectively. The difference between both rates is not statistically significant. The study shows that 65 (28.0%) patients completed MDR-TB treatment but 
could not be classified as cured or failure, 29 (12.5%) patients failed, 76 (32.7%) defaulted, 18 (7.7%) were transferred out and 44 (18.9%) died. As far as treatment completed and defaulted is concerned, 
there is no significant statistical difference between HIV (+) and HIV (-) The number of patients who failed the MDR-TB treatment and who were transferred out is significantly higher in the HIV (-) 
group than in the HIV (+) group. Finally the number of MDR-TB patients who died is significantly higher in the HIV (+) group). The median (range) duration of antiretroviral therapy before starting 
anti-tuberculosis drugs is 10.5 (1-60) months. According to this study results, the MDR-TB treatment cure rate at Brewelkloof hospital is similar to the cure rate at the national level. The study also 
hows that HIV infection and antiretroviral drugs do not influence any influence on MDR-TB treatment outcomes.

L’un des défis actuels de la lutte contre la tuberculose est de développer un traitement plus court et plus efficace. La modélisation mathématique constitue une approche qui peut nous aider à comprendre les problèmes actuels et favoriser les innovations thérapeutiques. L’objectif de ce travail est de construire un modèle thérapeutique mathématique de la tuberculose pulmonaire basé sur des éléments pharmacocinétiques, pharmacodynamiques et physiopathologiques. La mise en application du modèle pharmacodynamique a été précédée d’une étude théorique sur l’équation de Hill. Cette synthèse a permis de dégager les bases rationnelles de son utilisation en modélisation pharmacologique. En utilisant une approche de population, un modèle pharmacocinétique de diffusion pulmonaire a permis de décrire les concentrations en rifampicine dans le plasma et le poumon chez 34 sujets. Le modèle a ensuite été utilisé pour analyser la valeur d’indices pharmacodynamiques corrélés à l’effet chez 10 000 sujets fictifs, par simulation de Monte Carlo. Les résultats indiquent que la dose de standard de rifampicine conduit à des concentrations globalement peu efficaces et pouvant favoriser la résistance bactérienne. Un premier modèle mathématique du traitement de la tuberculose par la rifampicine, incluant un modèle physiopathologique formel, a enfin été construit. Il permet de simuler la dynamique bactérienne du premier jour de l’infection au dernier jour de traitement. L’ensemble des résultats conduit à une remise en question de la dose standard de rifampicine et suggère une nouvelle hypothèse sur les causes de la persistance de Mycobacterium tuberculosis au cours du traitement antituberculeux
There is a critical need for a shorter tuberculosis treatment to improve tuberculosis control. Mathematical models may be helpful to understand current problems associated with tuberculosis therapy and to suggest innovation resources. The objective of this study is to set up a full mathematical model of tuberculosis treatment by rifampin, based on pharmacokinetic, pharmacodynamic and physiological submodels. Prior to its application in the pharmacodynamic modeling framework, the Hill equation has been the focus of a theoretical study. The various properties of this equation have been reviewed and the rationale of its use in pharmacological modelling has been clarified. Rifampin pharmacokinetics in plasma and lungs was modelled in a population of 34 volunteers by use of a nonparametric population approach. Then, a 10,000 subject Monte Carlo simulation was performed to explore Mycobacterium tuberculosis killing effect and prevention of resistance by rifampin. The results suggest that rifampin pulmonary concentrations obtained with the standard dose are too low to be highly effective and prevent drug resistance in most subjects. Finally, a full mathematical model of tuberculosis treatment, including a physiological model, has been implemented. The model is able to simulate the time-course of bacterial counts from the first day of infection to the last day of treatment. Overall results of this modelling effort indicate that current dosage regimens of rifampin may be optimized. In addition, this work suggests a new hypothesis regarding the bacterial persistence during tuberculosis treatment

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2015.
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Proteínas tirosina fosfatases (PTP) têm um importante papel na transdução de sinal e no controle de diversas funções celulares. Bactérias patogênicas como as do gênero Yersinia e do complexo Mycobacterium tuberculosis (Mtb), utilizam suas PTP para subverter as células de defesa do hospedeiro. As bactérias patogênicas do gênero Yersinia possuem em comum a produção de uma PTP chamada YopH que modula a resposta inflamatória do hospedeiro à bactéria através de processos que envolvem a inibição da fagocitose, quebra de adesões focais e subversão da função dos linfócitos B e T prevenindo a resposta imune adaptativa. As doenças humanas causadas pelas espécies patogênicas de Yersinia variam desde síndromes gastrointestinais à peste bubônica, sendo esta última uma doença grave que ainda não foi erradicada e é associada a milhares de mortes no passado. O Mtb causa a tuberculose, doença que afeta principalmente o trato respiratório. Segundo dados da Organização Mundial da Saúde, a tuberculose foi responsável por 1,5 milhões de mortes somente no ano de 2013. Durantes a invasão das células de defesa, o Mtb produz duas PTP, a PtpA e a PtpB. Estas fosfatases, assim como a YopH, tem por função burlar o mecanismo de defesa do hospedeiro. A PtpA é responsável por modular a apoptose celular e impedir a acidificação do fagossomo e a fusão deste com o lisossomo. Já a PtpB, impede a produção de IL-6 e previne a morte do macrófago pela ativação da via de sinalização de Akt e bloqueio da atividade da caspase-3. A busca de inibidores da YopH de Yersinia spp. e das fosfatases do Mtb é de grande interesse para a produção de possíveis fármacos que poderiam ser utilizados no tratamento das doenças provocadas por estas bactérias. No presente trabalho, uma biblioteca de 398 compostos foi triada com o objetivo de identificar novos inibidores da YopH. Dentre os inibidores encontrados, 22 apresentaram valores de IC50 inferiores a 10 µM, sendo que 3 estão na faixa de nanomolar (o que os coloca entre os melhores inibidores descritos para esta fosfatase até o momento). Foram encontrados tanto inibidores competitivos (12 compostos) como não competitivos (6 compostos) da YopH e cinco compostos (delfinidina, AAA e os três complexos de vanádio) apresentaram valores de Ki na faixa de nanomolar. Avaliou-se também a citotoxicidade em células THP-1 e HepG2 além da atividade antitubercular de seis inibidores das fosfatases de Mtb (PtpA e PtpB) previamente relatados por nosso laboratório em 2012. Identificou-se dois compostos que não são citotóxicos (compostos 43 e 95) e dois compostos que apresentaram atividade em ensaios de infecção intracelular (compostos 95 e 96). De acordo com todos os resultados obtidos no presente trabalho, identificamos novas chalconas como eficientes inibidores da YopH além de 3 complexos de vanádio com uma marcante inibição em escala nanomolar. Quanto aos inibidores da PtpA e PtpB, o composto 95 mostrou-se não citotóxico e com atividade nos ensaios de infecção intracelular. Este composto poderia ser utilizado como molde na busca de outros compostos mais ativos ajudando assim no desenvolvimento de novas terapias contra o Mycobacterium tuberculosis.

Abstract : Protein tyrosine phosphatases (PTP) have an important role in signal transduction and in the control of many cell functions. Pathogenic bacteria from Yersinia genus and Mycobacterium tuberculosis (Mtb) complex, utilize their PTP to subvert host?s defense cells. Pathogenic bacteria from Yersinia genus have in common the production of a PTP named YopH, this enzyme modules the host inflammatory response against the bacteria through processes that evolves the phagocytosis inhibition, focal adhesion disruption and impairing the function of B and T lymphocytes preventing the adaptive immune response. Diseases caused by pathogenic species of Yersinia range from gastrointestinal syndromes to bubonic plague. Bubonic plague is a not eradicated disease that is associated with thousands of deaths in the past. Mtb causes tuberculosis, a disease that affects mainly the respiratory tract. According to World Health Organization data, only in 2013 tuberculosis caused 1.5 million deaths. During the defense cell invasion, Mtb produces two PTP, PtpA and PtpB. These phosphatases act like YopH, they help the bacteria to evade the host defense mechanism. PtpA modulates the cellular aptotosis and it also impairs the phagosome acidification and its fusion with lysosome. PtpB prevents the production of IL-6 and macrophage death by activation Akt signaling pathway and by blocking caspase 3 activity. The search for inhibitors of YopH from Yersinia and Mtb phosphatases is of great interest for the production of drugs that could be used in the treatment of the diseases caused by these bacteria. In this work, an in-house library of 398 compounds was screened with the objective to search new YopH inhibitors. Out of the inhibitors found, 22 presented IC50 values below 10 µM, three of those in nanomolar range (this characteristic put these three compounds between the best described inhibitors for this phosphatase). We found competitive inhibitors (12 compounds) and non-competitive inhibitors (6 compounds) for YopH and five compounds (Delphinidin, AAA and three vanadium complexes) presented Ki values in nanomolar range. Cytotoxicity assays were made with two human cell lines THP-1 and HepG2 we also assayed the antitubercular activity of six inhibitors of Mtb PTP. The inhibitory activity against PtpA and PtpB of these six compounds was previously described in 2012 by our lab. The cytotoxicity and antitubercular assays resulted in two non-cytotoxic compounds (compound 43 and 95) and two compounds that are activity in intracell infection assays (compounds 95 and 96). In this present work we show new chalcones as eficient inhibitors of YopH, we also identified 3 vanadium complex with remarkable inhibition in nanomolar scale. According to the results obtained to PtpA and PtpB inhibitors, we identified the compound 95 which is no cytotoxic and has activity in intracell assays. This compoud could be used for the design of new compound with improved activity helping in the development of new therapies against Mycobacterium tuberculosis.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2015.
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Proteínas tirosina fosfatases (PTP) têm um importante papel na transdução de sinal e no controle de diversas funções celulares. Bactérias patogênicas como as do gênero Yersinia e do complexo Mycobacterium tuberculosis (Mtb), utilizam suas PTP para subverter as células de defesa do hospedeiro. As bactérias patogênicas do gênero Yersinia possuem em comum a produção de uma PTP chamada YopH que modula a resposta inflamatória do hospedeiro à bactéria através de processos que envolvem a inibição da fagocitose, quebra de adesões focais e subversão da função dos linfócitos B e T prevenindo a resposta imune adaptativa. As doenças humanas causadas pelas espécies patogênicas de Yersinia variam desde síndromes gastrointestinais à peste bubônica, sendo esta última uma doença grave que ainda não foi erradicada e é associada a milhares de mortes no passado. O Mtb causa a tuberculose, doença que afeta principalmente o trato respiratório. Segundo dados da Organização Mundial da Saúde, a tuberculose foi responsável por 1,5 milhões de mortes somente no ano de 2013. Durantes a invasão das células de defesa, o Mtb produz duas PTP, a PtpA e a PtpB. Estas fosfatases, assim como a YopH, tem por função burlar o mecanismo de defesa do hospedeiro. A PtpA é responsável por modular a apoptose celular e impedir a acidificação do fagossomo e a fusão deste com o lisossomo. Já a PtpB, impede a produção de IL-6 e previne a morte do macrófago pela ativação da via de sinalização de Akt e bloqueio da atividade da caspase-3. A busca de inibidores da YopH de Yersinia spp. e das fosfatases do Mtb é de grande interesse para a produção de possíveis fármacos que poderiam ser utilizados no tratamento das doenças provocadas por estas bactérias. No presente trabalho, uma biblioteca de 398 compostos foi triada com o objetivo de identificar novos inibidores da YopH. Dentre os inibidores encontrados, 22 apresentaram valores de IC50 inferiores a 10 µM, sendo que 3 estão na faixa de nanomolar (o que os coloca entre os melhores inibidores descritos para esta fosfatase até o momento). Foram encontrados tanto inibidores competitivos (12 compostos) como não competitivos (6 compostos) da YopH e cinco compostos (delfinidina, AAA e os três complexos de vanádio) apresentaram valores de Ki na faixa de nanomolar. Avaliou-se também a citotoxicidade em células THP-1 e HepG2 além da atividade antitubercular de seis inibidores das fosfatases de Mtb (PtpA e PtpB) previamente relatados por nosso laboratório em 2012. Identificou-se dois compostos que não são citotóxicos (compostos 43 e 95) e dois compostos que apresentaram atividade em ensaios de infecção intracelular (compostos 95 e 96). De acordo com todos os resultados obtidos no presente trabalho, identificamos novas chalconas como eficientes inibidores da YopH além de 3 complexos de vanádio com uma marcante inibição em escala nanomolar. Quanto aos inibidores da PtpA e PtpB, o composto 95 mostrou-se não citotóxico e com atividade nos ensaios de infecção intracelular. Este composto poderia ser utilizado como molde na busca de outros compostos mais ativos ajudando assim no desenvolvimento de novas terapias contra o Mycobacterium tuberculosis.

Abstract : Protein tyrosine phosphatases (PTP) have an important role in signal transduction and in the control of many cell functions. Pathogenic bacteria from Yersinia genus and Mycobacterium tuberculosis (Mtb) complex, utilize their PTP to subvert host?s defense cells. Pathogenic bacteria from Yersinia genus have in common the production of a PTP named YopH, this enzyme modules the host inflammatory response against the bacteria through processes that evolves the phagocytosis inhibition, focal adhesion disruption and impairing the function of B and T lymphocytes preventing the adaptive immune response. Diseases caused by pathogenic species of Yersinia range from gastrointestinal syndromes to bubonic plague. Bubonic plague is a not eradicated disease that is associated with thousands of deaths in the past. Mtb causes tuberculosis, a disease that affects mainly the respiratory tract. According to World Health Organization data, only in 2013 tuberculosis caused 1.5 million deaths. During the defense cell invasion, Mtb produces two PTP, PtpA and PtpB. These phosphatases act like YopH, they help the bacteria to evade the host defense mechanism. PtpA modulates the cellular aptotosis and it also impairs the phagosome acidification and its fusion with lysosome. PtpB prevents the production of IL-6 and macrophage death by activation Akt signaling pathway and by blocking caspase 3 activity. The search for inhibitors of YopH from Yersinia and Mtb phosphatases is of great interest for the production of drugs that could be used in the treatment of the diseases caused by these bacteria. In this work, an in-house library of 398 compounds was screened with the objective to search new YopH inhibitors. Out of the inhibitors found, 22 presented IC50 values below 10 µM, three of those in nanomolar range (this characteristic put these three compounds between the best described inhibitors for this phosphatase). We found competitive inhibitors (12 compounds) and non-competitive inhibitors (6 compounds) for YopH and five compounds (Delphinidin, AAA and three vanadium complexes) presented Ki values in nanomolar range. Cytotoxicity assays were made with two human cell lines THP-1 and HepG2 we also assayed the antitubercular activity of six inhibitors of Mtb PTP. The inhibitory activity against PtpA and PtpB of these six compounds was previously described in 2012 by our lab. The cytotoxicity and antitubercular assays resulted in two non-cytotoxic compounds (compound 43 and 95) and two compounds that are activity in intracell infection assays (compounds 95 and 96). In this present work we show new chalcones as eficient inhibitors of YopH, we also identified 3 vanadium complex with remarkable inhibition in nanomolar scale. According to the results obtained to PtpA and PtpB inhibitors, we identified the compound 95 which is no cytotoxic and has activity in intracell assays. This compoud could be used for the design of new compound with improved activity helping in the development of new therapies against Mycobacterium tuberculosis.

Tuberculosis (TB) and fungal infections are two of the most lethal infectious diseases worldwide due to the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) and fungal strains that can resist the most potent antimicrobial drugs currently employed. Due to the rise of these drug resistant strains, effective treatment options for these two infections are limited. This dissertation aims at exploring novel drug scaffolds to help combat drug resistance in TB and fungal infections. TB caused by the pathogenic Mtb is, alongside with human immunodeficiency virus acquired immunodeficiency virus (HIV), the deadliest infectious disease worldwide with approximately 2-3 billion people infected yearly. The situation has become increasingly intensified due to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb strains.Aminoglycoside (AG) antibiotics such as amikacin and kanamycin A (KAN) are heavily relied upon for the treatment of MDR- and XDR-Mtb strains. However, the success rate for the treatment of these MDR- and XDR-TB cases is decreasing as a result of increased KAN resistance. It was reported by the Centers for Disease Control and Prevention (CDC) that upregulation of the enhanced intracellular survival (eis) gene was the cause of resistance to KAN in a large portion of Mtb clinical isolates. Our lab previously demonstrated that Eis is an AG acetyltransferase that can inactivate AGs via chemoenzymatic modification of the AG scaffolds. As Eis has been shown to acetylate a wide variety of AG scaffolds, the development of novel AGs that can completely escape the action of Eis remains highly challenging. Therefore, we suggested an alternative therapeutic approach involving inhibiting Eis enzyme and still employing the current FDA-approved KAN. As exemplified by the clinically successful combination of penicillin and b-lactamase inhibitors, we hypothesized that an Eis inhibitor may be used as adjuvant therapy in combination with KAN to treat MDR- and XDR-tuberculosis. Using high-throughput screening, we were able to identify several small-molecule scaffolds capable of inhibiting Eis. We performed structure activity relationship (SAR) studies using purified Eis enzyme and optimized lead compounds. Additionally, we also showed that co-administration of Eis lead inhibitors with KAN led to recovery of KAN activity against a KAN-resistant Mtb cell line that overexpressed Ei Invasive fungal infections are on the rise due to an increased population of critically ill patients as a result of HIV infections, chemotherapies, and organ transplantations. Unlike antibiotics that are greatly diverse in categories and mechanisms of action, our current antifungal drug repertoire is greatly limited and insufficient in addressing the problem of drug-resistant fungal infections. Thus, there is a growing need for novel antimycotics that are safe and effective. We report a number of lead compounds with potent antifungal activitiy. The MIC values of these compounds were as low as 0.02 mg/mL against the fungal strains tested. Our compounds are derived from the ebselen core structure, which has been shown to be safe in multicenter clinical trials. Notably, fungal cells treated with our compounds showed the accumulation of ROS, which may further contribute to the growth inhibitory effect against fungi. This study provides new lead compounds for the development of antimycotic agents.

Malgré la disponibilité d’un traitement curatif et un vaccin largement utilisé, l’OMS estime qu’approximativement un tiers de la population mondiale est infectée par Mycobacterium tuberculosis, l’agent étiologique de la tuberculose, et qu’environ 2 millions de personnes en meurent chaque année. La compréhension de l’épidémiologie de la tuberculose et les actions de contrôle de la maladie ont été, récemment, compliquées par l’émergence de bacilles tuberculeux résistants aux antibiotiques et par la synergie fournie par la co-infection avec le VIH. Une tendance alarmante pour la santé publique est l’émergence de souches résistantes à plusieurs antibiotiques (multi-résistantes, MDR), définies comme des isolats résistants au moins à l’isoniazide (INH) et la rifampicine (RIF), les deux agents anti-tuberculeux les plus puissants.

La sélection de mutants résistants se produit chez le patient lorsque les taux d’antibiotiques présents dans le corps sont sub-thérapeutiques ou lorsque la thérapie est inappropriée. Un des facteurs favorisant est l’exceptionnelle durée de la chémothérapie. Le besoin de maintenir des taux élevés d’antibiotiques pendant des mois, combiné avec la toxicité inhérente des agents, résultent en une observance incomplète du traitement par le patient et le risque d’acquérir des résistances. La résistance aux antibiotiques chez M. tuberculosis résulte d’altérations dans des gènes chromosomiques spécifiques. Les causes génétiques de la résistance ont été définies pour certains antibiotiques bien que plusieurs inconnues persistent.

Le présent travail a consisté en l’étude du problème de la résistance aux antibiotiques anti-tuberculeux par différentes approches :l’analyse génétique des mécanismes de résistance, l’évaluation de l’activité thérapeutique de nouvelles molécules et la caractérisation du profil de résistance de souches cliniques.

L’acide p-aminosalicylique (PAS) est un antibiotique bactériostatique de deuxième ligne dont le mécanisme d'action sur le bacille tuberculeux est incompris. Récemment, en utilisant la mutagenèse par transposon, la résistance au PAS fut associée à des mutations de la thymidylate synthase encodée par le gène thyA. Suite à cette découverte, nous avons entrepris une étude moléculaire de souches cliniques et de mutants spontanés résistants au PAS. Des mutations du gène thyA furent identifiées chez seulement 37% des souches. En tout, vingt-quatre mutations différentes furent identifiées dans le gène thyA. Les séquences nucléotidiques de cinq autres gènes de la voie de synthèse du folate et de la thymine (dfrA, folC, folP1, folP2, et thyX) ainsi que de 3 gènes encodant des N-acétyltransférases (nhoA, aac1 et aac2) furent également analysées mais aucune mutation associée à la résistance au PAS n’a pu être mise en évidence. L’utilisation de techniques bioinformatiques de prédiction structurelle révèle que les mutations identifiées affectent soit la structure soit le site fonctionnel de ThyA. L’étude des profils de croissance des organismes résistants au PAS nous permit de constater que les organismes porteurs d’une mutation de la protéine ThyA présentent un profil de croissance constant en présence de concentrations croissantes de PAS. Les organismes résistants au PAS possédant une protéine ThyA sauvage répondent, quant à eux, aux concentrations croissantes de PAS de façon dose-dépendante, indiquant que le(s) mécanisme(s) alternatif(s) de résistance au PAS est (sont) dose-dépendant(s).

La thymidylate synthase est également une des cibles du 5-fluorouracil (5-FU), l’agent chimiothérapeutique le plus largement utilisé pour le traitement du cancer colorectal avancé. Etant donné l’augmentation du nombre de souches résistantes de M. tuberculosis, de nouveaux composés anti-tuberculeux sont nécessaires de façon urgente. Ici, nous avons évalué l’efficacité in vitro et in vivo du 5-FU sur M. tuberculosis. La concentration minimale inhibitrice du 5-FU fut déterminée sur une collection de souches cliniques sensibles et multi-résistantes ainsi que sur des mutants spontanés résistants au PAS. Tous les isolats montrèrent une sensibilité au 5-FU à des concentrations allant de 0.4 à 1.8 µg/ml, et ce indépendamment de leur profil de sensibilité/résistance aux agents anti-tuberculeux actuels. Les études in vivo du 5-FU (sur un modèle murin de tuberculose active) montrèrent une efficacité de celui-ci durant les deux premières semaines de traitement puis une perte d’activité à la troisième semaine, vraisemblablement engendrée par les effets secondaires du 5-FU.

L’éthionamide (ETH) est un autre antibiotique de deuxième ligne dont l’utilisation est limitée aux tuberculoses multi-résistantes étant donné les effets secondaires qu’il engendre. Ces dernières années, les études ont montré que l’ETH est un pro-médicament, transformé en forme active par l’enzyme monooxygénase EthA dont l’expression est contrôlée par le répresseur transcriptionnel EthR. Notre étude décrit l’élaboration d’inhibiteur d’EthR capable d’augmenter la sensibilité de M. tuberculosis à l’ETH suite à l’amélioration de son activation. Les composés synthétisés et sélectionnés pour leur capacité à inhiber l’interaction EthR-ADN furent co-cristallisés avec EthR. Les structures tridimensionnelles des complexes furent utilisées pour la synthèse d’analogues capables d’améliorer la puissance de l’ETH en culture. Les molécules les plus prometteuses furent testées sur un modèle murin de tuberculose. Pour un des inhibiteurs d’EthR testés, nous avons montré que sa co-administration avec l’ETH permet une réduction de la dose d’ETH utilisée de 3 fois, pour l’obtention d’une même réduction de charge mycobactérienne pulmonaire. Ce travail démontre la possibilité d’augmenter l’index thérapeutique de l’éthionamide en agissant pharmacologiquement sur le mécanisme régulateur de son activation.

Dans certaines régions du monde, le problème de la multi-résistance devient très présent. Nous avons étudié l’état de la situation à Mourmansk (Fédération russe), une région à haute incidence de tuberculose. La résistance aux antibiotiques et l’épidémiologie moléculaire de la tuberculose furent étudiées sur des isolats collectés en 2003 et 2004 dans cette région. Une extrêmement haute prévalence de tuberculose multi-résistante (MDR-TB) fut constatée à la fois pour les nouveaux cas (primaires) (26%) et les cas précédemment traités (72.9%). Le typage des souches MDR primaires révèle une appartenance au génotype Beijing pour la plupart des isolats (79.8%) et l’homogénéité génétique des souches suggère une transmission active au sein de la population. L’analyse moléculaire des gènes impliqués dans la résistance à l’INH et à la RIF montre la présence des mutations katG codon 315 et rpoB codon 531 chez, respectivement, 98,2% et 76,3% des isolats MDR-TB primaires. La haute fréquence de ces mutations « communes » suggère la possible utilisation de tests moléculaires ciblant spécifiquement ces mutations pour détecter rapidement la plupart des cas de MDR-TB.

Nos travaux illustrent les différentes voies à suivre pour maitriser le problème de la résistance aux antibiotiques :l’élucidation des mécanismes de résistance, le développement de nouveaux médicaments et la détection rapide des cas de résistance.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Uvod: Povezanost dijabetesa melitusa i  tuberkuloze je odavno primećena i bila je predmet ispitivanja mnogih studija. Dijagnoza šećerne bolesti pre otkrića insulina značila je smrtni ishod u roku od pet godina, a najčešći uzrok smrti su bile infekcije, uključujući tuberkulozu. Poslednjih godina incidenca tuberkuloze je u padu, ali je i dalje prisutan značajan broj obolelih od tuberkuloze u zemljama u razvoju. Sa druge strane incidenca dijabetes melitusa je u porastu, pre svega zbog tendencije porasta broja gojaznih osoba. Procenjeno je da će prevalencija obolelih od dijabetes melitusa dostići 438 miliona obolelih do 2030, a 80% svih slučajeva će biti stanovnici zemalja u razvoju gde je i dalje visoka prevalencija tuberkuloze. Kao rezultat ovakve epidemiološke situacije ove dve bolesti će se sve češće javljati uporedo, modifikujući tok jedna drugoj. Preduslov za uspešno lečenje dijabetičara obolelih od tuberkuloze je postizanje zadovoljavajuće metaboličke regulisanosti šećerne bolesti. smrtni ishod u roku od pet godina, a najčešći uzrok smrti su bile infekcije, uključujući tuberkulozu. Poslednjih godina incidenca tuberkuloze je u padu, ali je i dalje prisutan značajan broj obolelih od tuberkuloze u zemljama u razvoju. Sa druge strane incidenca dijabetes melitusa je u porastu, pre svega zbog tendencije porasta broja gojaznih osoba. Procenjeno je da će prevalencija obolelih od dijabetes melitusa dostići 438 miliona obolelih do 2030, a 80% svih slučajeva će biti stanovnici zemalja u razvoju gde je i dalje visoka prevalencija tuberkuloze. Kao rezultat ovakve epidemiološke situacije ove dve bolesti će se sve češće javljati uporedo, modifikujući tok jedna drugoj. Preduslov za uspešno lečenje dijabetičara obolelih od tuberkuloze je postizanje zadovoljavajuće metaboličke regulisanosti šećerne bolesti. Cilj istraživanja: Cilj rada je bilo ispitivanje uticaja dijabetesa melitusa na tok plućne tuberkuloze, prvenstveno na bakteriološki status, radiološku prezentaciju bolesti, dužinu terapijskog režima i učestalost recidiva bolesti. Materijal i metode: Ispitivanjem su obuhvaćene dve grupe od po pedeset bolesnika koji su hospitalizovani u Institutu za plućne bolesti Vojvodine. Prvu grupu činili su bolesnici sa plućnom tuberkulozom i pridruženom šećernom bolešću, a drugu grupu bolesnici sa plućnom tuberkulozom bez pridružene šećerne bolesti. Svi bolesnici su analizirani po sledećim karakteristikama: starost, pol, klinička slika, bakteriološki status, radiološka prezentacija, prisustvo neželjenih efekata antituberkulotika, prisustvo rezistencije M. tuberculosis na lekove, trajanje terapijskog režima, ishod lečenja, pojava recidiva i dužina hospitalizacije. Oboleli od šećerne bolesti bili su dodatno analizirani prema: tipu bolesti, dužini trajanja bolesti, metaboličkoj regulisanosti bolesti i prisustvu komplikacija. Svi bolesnici obuhvaćeni ispitivanjem bili su podvrgnuti standardnom dijagnostičkom algoritmu koji obuhvata: anamnezu i fizikalni pregled, direktnu mikroskopiju sputuma, kultivaciju sputuma, radiogram grudnog koša, CT grudnog koša u slučaju postavljenih kliničkih indikacija. Invazivna dijagnostika će se sprovesti kod bolesnika kod kojih dijagnoza nije mogla biti postavljena prethodno sprovedenom neinvazivnom dijagnostikom. Terapijski režim će biti započet tokom hospitalizacije u Institutu za plućne bolesti Vojvodine, a nastavljen ambulantno pod kontrolom Dispanzera za plućne bolesti. Po završetku terapijskog režima predviđena je kontrola u Institutu za plućne bolesti Vojvodine koja obuhvata procenu kliničke slike, bakteriološkog statusa, radiološkog nalaza i eventualnu potrebu za produženjem terapijskog režima. Rezultati: Istraživanje je pokazalo da je u grupi obolelih od tuberkuloze bez pridruženog dijabetes melitusa bio sličan broj bolesnika muškog i ženskog pola, a veći broj ispitanika se nalazio u starosnim kategorijama do 50 godina starosti, dok je u grupi obolelih od tuberkuloze sa dijabetes melitusom bio značajno više zastupljen muški pol i značajno više ispitanika se nalazilo u starosnim kategorijama preko 50 godina starosti. Beleži se statistički značajno veći broj recidiva u grupi obolelih od tuberkuloze sa dijabetes melitusom (p=0,001). Između ispitvanih grupa se ne beleži statistički značajna razlika u kliničkoj prezentaciji bolesti. U grupi obolelih od tuberkuloze sa dijabetes melitusom, statistički značajno je veći broj direktno pozitivnih nalaza sputuma (p=0,046). Utvrđeno je postojanje statistički značajne razlike u prosečnoj dužini vremena potrebnoj za direktnu konverziju sputuma (p=0,000) i prosečnoj dužini vremena potrebnoj za konverziju kulture sputuma (p=0,000). U oba slučaja je grupa obolelih od tuberkuloze sa pridruženim dijabetes melitusom imala duže prosečno vreme potrebno za konverziju. U grupi obolelih od tuberkuloze sa pridruženim dijabetes melitusom bilo je statistički značajno više bolesnika sa prisustvom kaverne (p=0,006) i lokalizacijom promena u sva tri režnja (p=0,000). Nije zapažena statistički značajna razlika u trajanju terapijskog režima, ispoljavanju neželjenih efekata lekova, pojavi rezistencije na lekove i ishodu lečenja između dve ispitivane grupe. Grupa obolelih od tuberkuloze sa pridruženim dijabetesom imala je statistički značajno veći broj bolničkih dana (p=0,000). Poređenjem grupa obolelih od tuberkuloze sa pridruženim zadovoljavajuće regulisanim dijabetesom i grupe obolelih od tuberkuloze sa loše regulisanim dijabetesom uočeno je statistički značajno duže trajanje terapijskog režima kod dijabetičara sa loše regulisanom bolešću (p=0,018). Nije bilo statistički značajne razlike u zastupljenosti recidiva, kliničke prezentacije bolesti, bakteriološkog i radiološkog statusa, ispoljavanju neželjenih efekata lekova, pojavi rezistencije na lekove, ishoda lečenja i broja bolničkih dana između dve ispitivane grupe. Dodatnim poređenjem grupa (oboleli od tuberkuloze bez pridruženog dijabetesa, oboleli od tuberkuloze sa pridruženim zadovoljavajuće regulisanim dijabetesom i oboleli od tuberkuloze sa pridruženim loše regulisanim dijabetesom) primećeno je da je grupi bolesnika obolelih od tuberkuloze sa loše regulisanim dijabetesom potrebno najduže vreme za direktnu konverziju i konverziju kultura sputuma na MT i da imaju najveći broj bolničkih dana. U grupi obolelih od tuberkuloze sa pridruženim loše regulisanim dijabetesom je bilo statistički značajno veći broj bolesnika koji su lečeni osam meseci u odnosu na druge dve grupe (p=0,011). Poređenjem grupa obolelih od tuberkuloze sa tipom 1 dijabetesa tipom 2 dijabetesa nije uočena statističk značajna razlika između grupa po svim ispitivanim varijablama. U grupi dijabetičara sa dobro regulisanim dijabetesom nalazi se veći broj onih koji imaju tip 2 bolesti, u odnosu na grupu bolesnika sa loše regulisanim dijabetesom. Grupa dijabetičara sa loše regulisanom bolesti ima statistički značajno veći broj komplikacija šećerne bolesti. Zaključak: Dokazano je da šećerna bolest značajno utiče na bakteriološki status, radiološku prezentaciju, dužinu terapijskog režima, učestalost recidiva tuberkuloze i broj bolničkih dana obolelih od tuberkuloze,kao i da je regulisanost šećerne bolesti imala značajan uticaj na dužinu terapijskog režima.
Introduction: The association of diabetes mellitus and tuberculosis has long been observed and has been the subject of many studies. The diagnosis of diabetes before the discovery of insulin meant death within five years, a leading cause of death were infections, including tuberculosis. Last years the incidence of tuberculosis has declined, but there is still a significant number of TB patients in developing countries. On the other hand, the incidence of diabetes is on the rise, primarily due to the tendency of an increasing number of obese people. It is estimated that the prevalence of patients with diabetes will reach 438 million sufferers by 2030, and 80% of all cases will be people in developing countries where it is still a high prevalence of tuberculosis. As a result of the epidemiological situation, these two diseases will increasingly occur in parallel, modifying the current one another. Aim: The aim of this study was to investigate the influence of diabetes mellitus on the course of pulmonary tuberculosis, primarily in the bacteriological status, radiological presentation of disease, duration of the treatment regimen and the frequency of disease relapse. Materials and Methods: The study included two groups of fifty patients who were hospitalized at the Institute for pulmonary diseases. The first group consisted of patients with pulmonary tuberculosis and concomitant diabetes mellitus, a second group consisted of patients with pulmonary tuberculosis without associated diabetes. All patients were analyzed by the following characteristics: age, gender, clinical picture, bacteriological status, radiological presentation, the presence of side effects of antituberculosis drugs, the presence of M. tuberculosis resistant to the drugs, the duration of the therapeutic regimen, treatment outcome, recurrence and length of hospitalization. Diabetics were further analyzed with respect to: the type of disease, duration of disease, a metabolic disease and the regulation for the presence of a complication. All patients completed this study were subjected to a diagnostic algorithm comprising: history and physical examination, direct microscopy of sputum, cultivation of sputum, radiographs of the chest, chest CT scan in case positioned on clinical indications. Invasive diagnostic will be performed in patients in whom the diagnosis could not be set previously conducted noninvasive diagnostics. The treatment regimen will be started during the hospitalization in the Institute of pulmonary diseases and is set under the control of ambulatory pulmonary dispensaries. Results: The study showed that in the group of TB patients without concomitant diabetes mellitus was a similar number of patients male and female, a greater number of respondents was in the age groups up to the age of 50, while in the group of TB patients with diabetes mellitus was significantly more frequent male half and significantly more respondents were in the age groups over 50 years of age. Significantly higher number of relapses is recorded in a group of TB patients with diabetes mellitus (p = 0,001). Between the two study groups was not significant difference in the clinical presentation of the disease. In the group of TB patients with diabetes mellitus, is statistically significant higher number of smear positive findings (p = 0,046). There is a statisticaly significant difference in the average length of time required for the smear conversion (p = 0,000) and average length of time needed for the conversion of sputum cultures (p = 0,000). In both cases, the group of TB patients with associated diabetes mellitus had a longer average time needed for the conversion. In the group of patients with tuberculosis associated with diabetes mellitus was statistically significantly more patients with the presence of the cavern (p = 0,006), and the localization of the pulmonary changes in all three lobes (p = 0,000). Between the two study groups was not observed a statistically significant difference in duration of the treatment regimen, the expression of adverse drug effects, develop resistance to the drugs, and the outcome of the treatment. Group of patients with tuberculosis associated with diabetes had a statistically significantly greater number of hospital days (p = 0,000). Between the groups of patients with tuberculosis associated with satisfactory controlled diabetes and the group of TB patients with poorly controlled diabetes was statistically significantly longer duration of the therapeutic regimen in diabetic patients with poor regulation of the disease (p = 0,018). There was no significant difference in the appearance of relapses, the clinical presentation of disease, the bacteriological status, radiology, the expression of adverse drug effects, develop resistance to the drugs, outcome of the treatment, number of hospital days between the two study groups. Comparing the three groups (tuberculosis without associated diabetes mellitus, TB patients with associated satisfactorily controlled diabetes and TB patients with associated poorly controlled diabetes), it was observed that the group of patients suffering from tuberculosis with poorly controlled diabetes takes the longest time to smear conversion and conversion of sputum culture and to have the highest number of hospital days. In the group of patients with tuberculosis associated with poorly controlled diabetes was significantly greater number of patients who were treated for eight months compared to the other two groups (p = 0,011). Comparing the group of TB patients with type 1 diabetes and type 2 diabetes is not a statistically significant difference between the groups in all variables. In the group of diabetic patients with satisfactorily controlled diabetes, there are a large number of those with type 2 disease, in comparison to the group of patients with poorly controlled diabetes. Group of diabetics with poorly regulated disease has a significantly greater number of diabetes comlications. Conclusion: It has been shown that diabetes mellitus has a significant effect on the bacteriological status, radiological presentation, the length of the treatment regimen, the frequency of recurrence of tuberculosis and the number of hospital days of patients with tuberculosis, and that the adjustment of diabetes had a significant effect on the length of the treatment regimen.

La tuberculose est une maladie infectieuse extrêmement contagieuse qui dans sa forme simple peut être soignée et guérie. Cependant, des formes multirésistantes aux traitements classiques ont récemment fait leur apparition et le développement de nouvelles molécules constitue donc un enjeu majeur de santé publique. Le 3,3-diméthyl-1,2-dihydro-3H-benzofuro[3,2-f][1]benzopyrane est un produit possédant une activité antituberculeuse marquée, y compris sur des lignées résistantes. L’objectif des travaux réalisés ici est la recherche de composés de puissance accrue et l’élucidation du mécanisme d’action de cette série. Dans une première partie, des modifications structurales portant sur le cycle A sont présentées. Les résultats des tests biologiques ont permis de sélectionner des composés qui possèdent un index de sélectivité favorable entre l’activité antituberculeuse et la toxicité. Un composé de structure linéaire a également montré un profil d’activité intéressant. Les études de mécanisme d’action ont mis en évidence que les composés synthétisés affectent les synthèses des époxy-mycolates ainsi que celle des α-mycolates de la paroi cellulaire. Dans une deuxième partie l’influence des groupements en position 3 du 3,3-diméthyl-1,2-dihydro-3H-benzofuro[3,2-f][1]benzopyrane a été étudiée. Des composés possédants des chaines de longueur croissante en cette position ont été synthétisés. L’activité antituberculeuse des premiers produits obtenus, a été mesurée et a montré une importante influence du type de chaine introduit sur l’activité biologique. Les études présentées dans ce travail ont donc permis d’une part de préciser la cible cellulaire de ces molécules et d’autre part de sélectionner des composés peu cytotoxiques
Tuberculosis is a highly contagious infectious disease which can be treated and cured in its simple form. However, multidrug resistant forms towards classic treatments have recently appeared and the development of new drugs is a major challenge for public health. The 3,3-dimethyl-1,2-dihydro-3H-benzofuro[3,2-f][1]benzopyran have a significant anti-TB activity, including resistant lines. The goal of this work is the research of more active compounds and the elucidation of the mechanism of action of this series. In a first part, modifications are introduced on A ring. The results of biological tests were permitted to select compounds that have a favorable selectivity index between tuberculosis activity and toxicity. A linear compound has also showed an interesting activity. Studies on mycolates biosynthesis revealed that the synthesized compounds affect the synthesis of epoxy-mycolates and α-mycolates of the cell wall. In the second part of this work the influence of groups introduced at position 3 of 3,3-dimethyl-1 ,2-dihydro-3H-benzofuro[3,2-f][1]benzopyran was studied. Compounds possessing different length chains at this position have been synthesized. Anti-TB activity of the first products obtained was measured and showed a significant influence of the type of chain introduced. The studies presented in this work permitted to define the cellular target of these compounds and on the other hand to select the compounds with lowest cytotoxicity

Introdução: O Tratamento Preventivo com Isoniazida (TPI) é indicado para os pacientes com HIV/aids e infecção latente por Micobacterium tuberculosis (ILMTb) para os quais não haja contraindicação à Isoniazida. Entretanto, barreiras de acesso podem impedir que os pacientes realizem este tratamento. Objetivos: O presente estudo avaliou a taxa de acesso à prescrição médica do TPI em sujeitos com HIV/aids e ILMTb em seguimento em um serviço especializado de HIV/aids no período de fevereiro de 2005 a dezembro de 2009. Para os sujeitos que não tiveram acesso à prescrição do TPI, buscou-se, em prontuário, justificativas para esta conduta. Também foi identificado o perfil epidemiológico, clínico e demográfico dos sujeitos com HIV/aids e ILMTb e foi descrita a característica do médico que solicitou o teste tuberculínico (TT) e do que prescreveu o TPI. Métodos: No período de 02 de fevereiro de 2005 a 31 de dezembro de 2009 que estavam em seguimento no SEAP HIV/Aids foram incluídos sujeitos com HIV/Aids e ILMTB, diagnosticada através do Teste Tuberculínico (TT). Informações referentes às variáveis analisadas foram coletadas nos prontuários médicos e através de consulta ao Sistema de Informação e Gestão Hospitalar (SIGH) - Módulo Farmácia. Resultados: Foram incluídos 238 sujeitos dentre os 310 que tiveram TT > 5 mm no período do estudo. Destes, 70,6% (168) eram do sexo masculino; a média de idade foi de 42,6 anos; 88,2% (210) dos sujeitos tiveram acesso à prescrição do TPI. O acesso à prescrição do TPI foi associado à idade, ao tamanho da resposta ao TT, ao nadir de Linfócitos TCD4+ dos sujeitos em TARV e à presença de cicatriz de BCG. Sujeitos mais jovens, com resposta ao TT igual ou maior do que 10 mm e com cicatriz de BCG tiveram maior acesso à prescrição do TPI. Uma das questões a ser explorada em futuros estudos se refere aos fatores que influenciam, ou não, a decisão do profissional de introduzir este tratamento na situação em que o mesmo está recomendado tecnicamente. Conclusão: Os sujeitos mais jovens, com melhor situação imunológica de base, maior valor de resposta ao TT e com presença da cicatriz de BCG, tiveram maior acesso ao TPI. Neste estudo foi evidenciada a necessidade de que as instituições de saúde invistam em educação continuada de seus profissionais para elevarem a cobertura de ações programáticas, como é o tratamento da ILMTB, previsto nos programas nacionais de tuberculose e de HIV/aids. Além disso, é necessário que as equipes interdisciplinares atuem de forma integrada e harmônica, para garantir o acesso às ações de saúde. É possível identificar, porém muitas barreiras que restam para a serem rompidas de modo que os cidadãos que vivem com HIV/aids tenham acesso a este e aos demais tratamentos de que tenham necessidade
Background: Isoniazid Preventive Treatment (IPT) is recommended for patients with HIV/AIDS and Latent Infection by Mycobacterium tuberculosis (ILMTb) and no contraindication to isoniazid. However, access barriers may prevent patients to undergo to this treatment. Objectives: This study evaluated the rate of access to the prescription of IPT in subjects with HIV/aids and ILMTb followed up in a specialized HIV/aids from February 2005 to December 2009. For subjects who did not have access to the prescription of IPT, we sought, on records, justification for this conduct. Also, the epidemiological, clinical and demographic profile of individuals with HIV/AIDS and ILMTb and the characteristic of the doctor who requested the tuberculin skin test (TST) and prescribed IPT were identified. Methods: from 02 February 2005 to 31 December 2009 subjects followed up at SEAP HIV/aids with HIV/aids and ILMTB, diagnosed by Tuberculin Test (TST) were included. Information was collected from the medical records and from the Hospital Information and Management System (SIGH) - Pharmacy Module. Results: 238 subjects were included, among the 310 who had TST > 5 mm during the study period. Of these, 70.6 % (168) were male and the average age was 42.6 years, 88.2 % (210) had access to the prescription of IPT. Access to IPT prescription was associated with age , size of response to TST, nadir of lymphocytes CD4 + in subjects on ART and presence of BCG scar: younger subjects with response to TST equal to or greater than 10 mm and BCG scar had higher access rate to IPT prescription. An issue to be explored in the future refers to variables that influence the professional\'s decision to prescribe this treatment when it is technically recommended. Conclusion: younger subjects with better immune status at baseline, greater response to TST and presence of BCG scar, had more access to IPT. This study highlighted the need of educational programs for health professionals, in order to improve the coverage of activities devoted to reduce morbidity and mortality in HIV/aids patients, as is the treatment of ILMTB, recommended in national tuberculosis and HIV/AIDS programs. Furthermore, it is crucial, for interdisciplinary health teams, to operate in an integrated and harmonious way, to ensure, for HIV/aids patients, a healthy and longer life

碩士
高雄醫學大學
天然藥物研究所
96
Fatoua pilosa Gaud. (Moraceae) is a small perennial herb, which distributed in Taiwan, Philippine, Moluccas and New Guinea. In a series of studies on the active constituents of Formosan plants, the methanolic extract of the whole plant of this species showed antitubercular activity against Mycobacterium tuberculosis H37Rv in vitro. There are only three species of Fatoua in the world. The chemical constituents and bioactivities of Fatoua plants have never been conducted. The methanolic extract of whole plant of this species was partitioned into EtOAc and H2O-soluble fractions. Investigation of EtOAc-soluble fraction led to the isolation of six new coumarins: (+)-fatouain A (1), (-)-fatouain B (2), (+)-fatouain C (3), (-)-fatouain D (4), (+)-fatouain E (5), (-)-fatouain F (6) and four new bis-coumarins: (+)-fatouain G (7), (+)-fatouain H (8), fatouain I (9), fatouapilosin (10), along with eighteen known compounds, including three simple coumarins: umbelliferone (11), scopoletin (12), phellodenol-A (13), six furanocoumarins: psoralen (14), bergapten (15), S-(+)-marmesin (16), S-(+)-rutaretin methy1 ether (17), S-(+)-rutaretin (18), 2’,3’-dehydromarmesin (19), one pyranocoumarin: xanthyletin (20), three chaclones: isobavachalcone (21), kanzonol C (22), (E)-1-[2,4-dihydroxy-3-(3-methyl-2-butenyl)phenyl]-3-(2,2-di- methyl-8-hydroxy-2H-benzopyran-6-yl)-2-propen-1-one (23), one benzenoid: syringaldehyde (24), one quinone: α-tocopheryl quinone (25), one triterpenoid: lupeol (26), two steroids: a mixture of β-sitosterol (27) and stigamasterol (28). The structures of these compounds were elucidated by spectral analysis or by chemical derivation. Scopoletin (12), isobavachalcone (21) and (E)-1-[2,4-dihydroxy-3-(3-methyl- 2-butenyl)-phenyl]-3-(2,2-dimethyl-8-hydroxy-2H-benzopyran-6-yl)-2-propen-1- one (23) showed antituburcular acivity aganist Mycobacterium tuberculosis H37Rv in vitro with MIC values: 42.1, 17.6 and 30.0 μg/ mL, respectively.

碩士
高雄醫學大學
天然藥物研究所
96
Coptosapelta diffusa Steenis (Thysanospermum diffusum Champ., Rubiaceae) is a woody lianas, distributed in Southern China, Ryukyu, and Taiwan in broad-leaved forests. The MeOH extract of its whole plant showed significant antitubercular activity against Mycobacterium tuberculosis H37Rv. and was partitioned into EtOAc, n-BuOH and H2O-soluble parts. Investigation of the EtOAc-soluble part by repeated column chromatography led to the isolation of thirty-four compounds, including fourteen triterpenoids: thysanolactone (1), coptosalactone A (2), coptosalactone B (3), coptosalactone C (4), coptosalactone D (5), coptosalactone E (6), coptosalactone F (7), lupeol (8), lupenone (9), lupane (10), friedelin (11), β-amyrin (12), 3-acetyl ursolic acid (13), squalene (14); one naphthalene: methyl 1,4-dimethoxy-2-naphthoate (15); one benzophenone: 2-carbomethoxy-2''-hydroxy-5''-methyl-benzophenone (16); seven anthraquinones: 2-dimethoxymethylanthraquinone (17), 1-hydroxy-6-methylanthraquinone (18), 2-methylanthraquinone (19), 1-hydroxy-2-methylanthraquinone (20), 2-carbomethoxyanthraquinone (21), 2-carbomethoxy-1-hydroxyanthraquinone (22), 2-formylanthraquinone (23); three naphthoquinones: hydroxyhydrolapachol (24), lapachol (25), dehydro-α-lapachone (26); four steroids: β-sitostenone (27), ergosta- 4,6,8(14),22-tetraen-3-one (28), a mixture of β-sitosterol (29) and stigmasterol (30); one benzenoid: octadecyl ferulate (31); one quinone: α-tocopheryl quinone (32); one diterpenoid: trans-phytol (33); one fatty acid ester: methyl linoleate (34). The structures of these compounds were determined through spectral analysis. Among these isolates, 2, 4, 5, 6, 7 and 16 are new compounds, and compounds 3, 15, 17, 18 and 24 were first isolated from natural source. Compounds 23, 24, 25 and 26 showed antitubercular activity in vitro.

碩士
高雄醫學大學
天然藥物研究所碩士班
94
Maackia taiwanensis Hoshi & Ohashi (Leguminosae) is a deciduous tree, endemic in Taiwan. The MeOH extract of its leaves showed antitubercular activity against Mycobacterium tuberculosis 90-221387 in vitro. Isolation of the active components was performed by the bioassay- guided fractionation of the active n-hexane- and CHCl3-soluble parts, which were portioned from MeOH extract. Investigation of the CHCl3-soluble fraction of the leaves led to the isolation of 18 compounds, including 6 pterocarpans: maackiain (1), 2-hydroxypterocarpin (2), 3,4-dihydroxy-9-methoxypterocarpan (5), 3,4-dihydroxy-8,9-methylenedioxypterocarpan (6), (-)-sophoraptero- carpan A (9), trifolirhizin (11); 3 isoflavonids: 5,7,4’-trihydroxy-3’- methoxyisoflavone (7), licoagroisoflavone (8), wighteone (10); 2 benzenoids: vanillin (12), syringaldehyde (13); 6 chlorophyll compounds: the mixtures of methyl-132-hydroxy-(132-R)-pheorbide a (24) and methyl-132-hydroxy- (132-S)-pheorbide a (25), pheorbide a methyl ester (26), methyl-132-peroxy-(132-S)-pheorbide a (27), the mixtures of methyl-132-hydroxy-(132-S)-pheorbide b (28) and methyl-132-hydroxy- (132-R)-pheorbide b (29), 1 saccharide: D-3-O-methyl-chiro-inositol (33), respectively. Investigation of the n-hexane-fraction of the leaves led to the isolation of 15 compounds, including 2 pterocarpans: medicarpin (3), 3-hydroxy-4-methoxy-8,9-methylenedioxypterocarpan (4); 1 benzenoid: 4-hydroxybenzaldehyde (14); 3 steroids: β-sitosterol (15) and the mixtures of campesterol (16) and stigmasterol (17); 6 terpenoids: lupeol (18), lupenone (19), squalene (20), trans-phytol (21), lupeol palmitate (22)；1 apo-carotenoid: loliolide (23); 3 aliphatic compounds: palmitic acid (30), octacosane (31), octacosanol (32), respectively. Among these isolates, 27 is new compound. Compounds 1 and 14 exhibited significant antitubercular activity in vitro, and 2 and 32 showed weak antitubercular activity in vitro.

碩士
高雄醫學大學
天然藥物研究所
97
Ficus nervosa Heyne ex Roth. (Moraceae) is an evergreen tree, distributed in south China, India, Malaysia and at low altitudes throughout Taiwan. As an extension of our continuing studies on the active constituents of Formosan plants. The methanolic extracts of the root of this species showed antitubercular activity against Mycobacterium tuberculosis H37Rv in vitro. The aim of this study is the isolation of chemical constituents and antitubercular activity. Bioassay-guided fractionation of the active EtOAc soluble layer led to the isolation of two new pyranocoumarins, 3-hydroxyxanthyletin (1) and 3-methoxyxanthyletin (2), along with twenty-two known compounds, including four simple coumarins: xanthyletin (3), umbelliferone (4), scopoletin (5), S-(+)-marmesin (6), nine flavonoids: carpachromene (7), parvisoflavone B (8), alpinumisoflavone (9), genistein (10), 2´-hydroxygenistein (11), prunetin (12), cajanin (13), apigenin (14), (2S)-naringenin (15), five steroids: ergosterol peroxide (16), a mixture of 6??-hydroxystigmast-4-en-3-one (17) and 6??-hydroxystigmast-4,22-dien-3-one (18), a mixture of β-sitosterol (19) and stigmasterol(20), two benzenoids: 4-hydroxybenzaldehyde (21), vanillin (22), (S)-lasiodiplodin (23), one triterpenoid: oleanolic acid (24).The structures of these compounds were determined by spectroscopic analyses. Among these compounds, 3-hydroxyxanthyletin (1), genistein (10), prunetin (12), (2S)-naringenin (15), showed antitubercular activity against Mycobacterium tuberculosis H37RV in vitro with MIC values: 16, 35, 30, ≤ 2.8μg/ mL, respectively.

碩士
高雄醫學大學
天然藥物研究所
96
Abstract Beilschmiedia erythrophloia Hay. (Lauraceae) is an evergreen tree, distributed in south China, Hainan, Ryukyus, and throughout the Taiwan. There are only two species of Beilschmiedia in Taiwan, the methanolic extract from the root of B. erythrophloia showed antitubercular activity, and its chemical constituents have never been conducted. The aim of this study is the isolation of chemical constituents and antitubercular activity from the methanolic extract of the root, which was partitioned to get EtOAc layer, H2O layer and insoluble part. Examination of active EtOAc layer led to the isolation of 28 compounds︰10 new compounds, 1 firstly isolated from nature compound and 17 known compounds. 1. Nine endiandric acids︰erythrophloin A (1)~F (6), beilcyclone A (7), endiandric acids I (8) and J (9). All of them are new compounds. 2. Three benzopyrans︰oligandrol (10), oligandrol methyl ether (11), dehydrooligandrol methyl ether (12). Compound (12) is a new compound. 3. Two benzenoids︰farnesylol (13), vanillin (17). Compound (13) was firstly isolated from nature. 4. Two sesquiterpenes︰caryophyllene oxide (14), suberosol B (15). 5. One amide︰dioxamin (16). 6. Five steroids︰β-sitostenone (18), 6β-hydroxystigmast-4-en-3-one (19), 24(S)-3β-hydroxystigmast-5-en-7-one (20), β-sitosterol (21), stigmasterol (22). 7. Two triterpenes︰lupeol (23), 3-O-acetyl-epi-betulinic acid (24). 8. One benzoquinone︰α-tocopheryl quinone (25). 9. Three fatty acids︰methyl oleate (26), methyl palmitate (27), methyl linoleate (28). Compound 3 and 15 showed antitubercular activity.

碩士
高雄醫學大學
天然藥物研究所碩士班
93
Engelhardia roxburghiana Wall. (Juglandaceae) is a subtropical tree grown in India, Indochina, China and Taiwan. Its root showed antitubercular activity against Mycobacterium .tuberculosis 90-221387 strain in vitro. Bioassay-guided fraction has led to the isolation of engelhardione, 3-methoxyjuglone and (–)-4-hydroxy- 1-tetralone as antitubercular constituent. Continuing investigation of CHCl3-soluble layer of this plant led to the isolation of thirty compounds, including thirteen benzenoids: acetophenone (1), cinnamic acid (2), 4-hydroxybenzaldehyde (3), 2-(4-hydroxyphenyl)ethanol (4), 4-(2-hydroxyphenyl)-4-oxobutyric acid (5), methyl 4-(butyryloxy)benzoate (6), the mixture of docosyl trans-ferulate (7), hexacosyl trans-ferulate (8), tetracosyl trans-ferulate (9), and tricosyl trans-ferulate (10), 3,4,5-trimethoxy benzoic acid (11), vanillic acid (12) and vanillin (13); two naphthalenes: 5-methoxy-1-naphthalenol (14) and 1-methoxynaphthalene (15); two tetralones: (–)-5,8-hydroxy-4-methoxy-1-tetralone (16) and (–)-5-hydroxy-4-methoxy-1- tetralone (17); one benzoquinone: 2,6-dimethoxy-1,4-benzoquinone (18); three naphthoquinones: 5-hydroxy-2-hydroxymethyl-1,4-naphthoquinone (19), 2-methoxyjuglone (20) and 3-methoxyjuglone (21); two naphthoquinone dimers: engelharquinone (22) and engelharquinone epoxide (23); one diarylheptanoid: engelhardione (24); one butanolide: engelharolide (25); one sesquiterpenoid: engelhardic acid (26); two triterpenoids : 3-epi-betulinic acid (27) and 3-epi-betulinic acid acetate (28); two steroids: β-sitostenone (29) and β-sitosterol (30). The structures of these compounds were determined by spectroscopic analysis. Among these isolates, 6, 16, 22, 23, 25 and 26 were new compounds, and compounds 5, 14, and 19 were first isolated from natural sources. The active constituents 3-methoxyjuglone (21) and engelhardione (24) which have been isolated were obtained again this time. Compound 22 also showed significant antitubercular activity in vitro.

碩士
高雄醫學大學
天然藥物研究所碩士班
95
Pisonia umbellifera Seem. (Nyctaginaceae) is an evergreen tree, which distributed in Australia, Java, Malaysia, Hawaii, Madagascar, Micronesia, and Taiwan. The MeOH extract of the stem showed antitubercular activity against Mycobacterium tuberculosis H37RV, that was partitioned into n-hexane, EtOAc, n-BuOH and H2O-soluble parts. The EtOAc-soluble part showed significant antitubercular activity. Investigation of the n-hexane-soluble extract and EtOAc-soluble extract led to the isolation of thirty-four compounds, including one isoflavone: 6,8- dimethylisogenistein (1); one isoquinolinone: 3,4-dihydro-6-hydroxy-7-methoxyisoquinolin-1(2H)-one (2); two quinones: 3,4,4,5-tetramethoxy-cyclohexa-2,5-dienone (3) and 2,6-dimethoxy-1,4-benzoquinone (4); one quinol: 4-acetonyl-3,5-dimethoxy-p-quinol (5); fourteen benzenoids: (+)-ent-ficusol (6), goldfiussinol (7), pisoninol (8), pisoninol II (9), pavonisol (10), β-hydroxypropiovanillone (11), β-hydroxypropisosyringone (12), α-hydroxypropiovanillone (13), α-hydroxypropisosyringone (14), C-veratroylglycol (15), trans-methyl ferulate (16), vanillin (17), syringaldehyde (18) and methyl syringate (19); five triterpenoids: 24-methylene-3,4-seco-cycloart-4(28)-en-3-oic acid (20), secopisonic acid (21), oleanolic acid (22), oleanonic acid (23) and 29-hydroxy-3-oxoolean- 12-en-28-oic acid (24); two amides: N-trans-feruloyl-4’-Omethyldopamine (25), N-trans-feruloyltyramine (26); two lignans:(+)-syringaresinol (27) and (+)-glaberide I (28); four steroids: ergosterol peroxide (29), α-spinasterol (30), mixture of β-sitosterol (31) and stigmasterol (32); one chlorophyll: 132-hydroxy-(132S)-pheophytin-a (33), and β-tocopherol (34). The structures of these compounds were elucidated by the spectral analysis. Among these isolates, 1, 6 and 21 are new compounds, and compound 2, 3, 8 and 9 were first isolated from natural source. Compounds 9, 17, 25 and 26 showed antitubercular activity in vitro.

碩士
高雄醫學大學
藥學研究所
99
Engelhardia roxburghiana Wall. (Juglandaceae) is a deciduous tree, distributed in India, Indochina, SW China, and low to middle altitudes of Taiwan. Approximately 1300 species of Formosan plants have been screened for antitubercular activity against Mycobacterium tuberculosis H37Rv in vitro, and E. roxburghiana was shown to be one of the active species. Previous investigations from our laboratory, eleven new compounds, and twenty-nine known compounds have been isolated from the root of this plant, among them, 2-methoxyjuglone, 3-methoxyjuglone, engelharquinone, engelhardione, and (–)-4-hydroxy-1-tetralone were antitubercular constituents. The stem of this plant also showed antitubercular activity. Bioassay-guided fractionation of the active EtOAc-soluble layer of the stem has led to the isolation of 32 compounds, including four new diarylheptanoids: (–)-engelheptanoxide A (1), engelheptanoxides B~D (2~4); two new cyclic diarylheptanoids: engelhardiols A~B (5~6); one new naphthoquinone dimer: engelharquinonol (7), one first isolated from nature: (4S)-4,6-dihydroxy-1-tetralone (8); along with twenty-four known compounds, including squamolone (9), rhoiptelol B (10), methyl gallate (11), (3R)-3'', 4''-epoxy-1-(4-hydroxyphenyl)-7-(3-methoxy- phenyl)heptan-3-ol (12), (2R)-3'', 4''-epoxy-2-hydroxy-1-(4-hydroxyphenyl)-7-(3-methoxy-phenyl)heptan-3-one (13), 1-(4-hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)heptane-3,5-diol (14), rhoiptelol C (15), (–)-syringaresinol (16), 7-hydroxycadalene (17), vomifoliol (18), 4-acetonyl-3,5-dimethoxy-p-quinol (19), (＋)-4-hydroxy-1-tetralone (20), 3-O-betulinic acid p-coumarate (21), engelhardione (3'',4''-epoxy-1-(4-hydroxyphenyl)-7-(3-hydroxyphenyl)heptan-3-one) (44), engeletin (47), katuranin (51), afzelin (57), 3-methoxyjuglone (63), engelharquinone epoxide (65), b-sitosterol (68), b-sitosterone (70), (–)-regiolone (74), 3-epi-betulinic acid (76) and 3-epi-betulinic acid acetate (77). Among the isolated, thirteen compounds (9~21) were first isolated from this plant .The structures of these compounds were determined by spectroscopic analysis. We also revised the structure of engelhardione from 3'',3''-epoxy-1-(4-hydroxyphenyl)- 7-(3-hydroxyphenyl)heptan-3- one to 3'',4''-epoxy-1-(4-hydroxyphenyl)-7-(3-hydroxy- phenyl)heptan-3-one. Among the isolates, (–)-engelheptanoxide A (1), engelhardiol A (5), engelhardiol B (6), squamolone (9), engelhardione (3'',4''-epoxy-1-(4-hydroxyphenyl)-7-(3-hydroxyphenyl)heptan-3-one) (44), katuranin (51), 3-methoxyjuglone (63) and engelharquinone epoxide (65) showed antitubercular activity with MIC 55.3, 25.0, 20.5, 20.5, 3.125, 27.0, 3.125 and 25.5 μg/mL, respectively.

碩士
高雄醫學大學
天然藥物研究所
97
Litsea hypophaea Hay. (syn.: Actinodaphne pedicellata Hay., Litsea kostermansii Chang) (Lauraceae) is a small evergreen tree, endemic to Taiwan, distributed below 1,000 m in the broad-leaved forests. The methanolic extract of the root of this plant showed antitubercular activity against Mycobacterium tuberculosis H37Rv in vitro. The chemical constituents have never been studied. Therefore, the aim of this study is the isolation of chemical constituents and its antitubercular activity. Investigation of the ethyl acetate-soluble layer of the root of this species led to the isolation of seven new butanolides: litseakolides H?{N (1?{7), one new biphenylpropanoid: hypophaone (8), two biphenylpropanoids: hypophaol (9)and hypophane (10), which are first isolated from nature, together with other fifteen known compounds, including six benzenoids: methyl syringate (11), 4-hydroxybenzaldehyde (12), vanillin (13), methyl 4-hydroxy-3-methoxybenzoate (14), methyl 4-hydroxy-benzoate (15), p-hydroxybenzoic acid (16), three amides: N-trans-feruloylmethoxytyramine (17), N-trans-feruloyltyramine (18), N-trans-sinapoyltyramine (19), two lignanamides:1,2- dihydro-6,8-dimethoxy-7-hydroxy-1-(3,5-dimethoxy- 4-hydroxylphenyl)-N1,N2-bis-[2-(4-hydroxyphenyl)ethyl]-2,3-naph-thalene dicarboxamide (20), and cannabisin D (21), one lignan: syringaresinol (22), one isoquinolone: northalifoline (23), a mixture ofβ-sitosterol (24)and stigmasterol (25). The structures of these compounds were determined by spectroscopic analysis. Among the isolates, litseakolide L (5), and N-trans-feruloylmethoxytyramine (17) exhibited antitubercular activity against Mycobacterium tuberculosis H37Rv in vitro, showing MIC values of 25.0 and ≤2.7 μg/mL, respectively.

碩士
高雄醫學大學
天然藥物研究所
97
Itea parviflora Hemsl. (Saxifragaceae) is a shrub, endemic to Taiwan, common in forest at low to medium altitudes. The methanolic extract from the root of this plant showed antitubercular activity against Mycobacterium tuberculosis H37Rv in vitro. Past chemical study reported the flavonoids, including orientin, isoorientin, vitexin, and isovitexin detected by HPLC method in Itea virginica, I. parviflora and I. japonica, respectively. But their biological activities have not been conducted. The aim of this study is the isolation of chemical and antitubercular constituents from this plant. The methanolic extract was partitioned into H2O, n-BuOH and EtOAc-soluble layer, and EtOAc-soluble layer showed antitubercular activity. Investigation of the EtOAc-soluble active layer from the root of this species led to the isolation of one new sesquiterpenoid, namely iteaphyllene (1), one new nor-diterpenoid, namely iteakaurenic acid (2), one benzoquinone, iteanone (5) firstly isolated from nature, together with thirteen known compounds: lupeol (3), 3-acetylbetulinal (4), (±)-pinoresinol (6), (+)-7S,8R-dihydrodehydroconiferyl alcohol (7), (–)-epicatechin (8), scopoletin (9), 7-(3,3-dimethylallyloxy)-6-methoxycoumarin (10), 3,4-dimethoxyphenol (11), syringaldehyde (12), 4-acetonyl-3,5-dimethoxy-p-quinol (13), α-tocopheryl quinone (14) and a mixture of β-sitosterol (15) and stigmasterol (16). The structures of these isolates were elucidated by spectral analysis, and iteakaurenic acid (2) was further confirmed by X-ray analysis. Among these isolates, iteaphyllene (1), scopoletin (9), 7-(3,3-dimethylallyloxy)-6-methoxycoumarin (10) and 3,4-dimethoxyphenol (11) showed antitubercular activity against Mycobacterium tuberculosis H37Rv in vitro with MIC values of 80.0, 42.1, 35.0, 55.0 μg/mL, respectively.

碩士
高雄醫學大學
天然藥物研究所碩士班
92
Cinnamomum kotoense Kanehira & Sasaki (Lauraceae) is an evergreen small tree, endemic in Lanyu Island of Taiwan, and recently was cultivated for ornamental purpose in Taiwan Island. The chemical constituents and biological activities of this plant have never been studied. In a screening program of antitubercular activity on Formosan plants, C. kotoense was shown to be one of the active species. Investigation on the MeOH extract of the stem wood of this species has led to the isolation of 41 compounds, including four butanolides: kotolactone A (1), kotolactone B (2), isoobtusilactone A (3), lincomolide B (4); one secobutanolide: secokotomolide (5); four flavonoids: apigenin (6), kaempferol (7), quercetin (8), genkwanin (9); three flavans: (+)-catechin (10), (-)-catechin (11), 4’-hydroxy-5,7,3’- trimethoxyflavan -3-ol (12); two lignans: (±)-syringaresinol (13), (-)-sesamin (14); eight benzenoids: trans-ferulic acid (15), trans-coumaric acid (16), 2,6-dimethoxy-1,4-benzoquinone (17), syringaldehyde (18), vanillin (19), 4-hydroxybenzaldehyde (20), protocatechuic acid (21), benzoic acid (22)；six steroids：the mixture of sitostenone (23) and stigmasta-4,22-dien-3-one (24), the mixture of β-sitosterol (25) and stigmasterol (26), the mixture of β-sitosteryl-3-O-β-D-glucoside (27) and stigmasteryl-3-O-β-D- glucoside (28)；one triterpenoid：squalene (29)；one diterpenoid ：trans-phytol (30)；two furans ：2-acetyl-5-methylfuran (31), 2-acetyl-5-dodecylfuran (32)； nine aliphatic compounds：the mixture of methyl palmitate (33) and methyl stearate (34), the mixture of palmitic acid (35), margaric acid (36) and stearic acid (37), lauric acid (38), docosanoic acid (39), tetracosane (40), kotodiol (41). The structures of these compounds were determined by spectroscopic analysis. Among these isolates, 1, 2, 5 and 41 were new compounds, and compound 32 was firstly isolated from plant origin, though it ever have been synthesized. Compound 3, the mixture of 25 and 26, and the mixture of 33 and 34 showed significant antitubercular activity in vitro.

碩士
高雄醫學大學
天然藥物研究所
98
Ehretia longiflora Champ. ex Benth. (Boraginaceae) is a medium sized deciduous tree, distributed in south China, Indochina, and in forests from low elevations to 750 m of Taiwan. The methanolic extract of the root of this plant showed antitubercular activity against Mycobacterium tuberculosis H37Rv in vitro. The aim of this study is to isolate the antitubercular active constituents from the root of this plant. Bioassay-guided fractionation of the ethyl acetate-soluble layer from the root of this species led to the isolation of thirty compounds, including one quinonoid: ehretiquinone (1); one coumarin: ehreticoumarin (2); one benzolactone: ehretilactone A (3); one butenolide: ehretilactone B (4); one amide: ehretiamide (5); two alkaloids: ehretine (6), ehretiquinolone (7); three triterpenoids: ehretiolide (8), O-acetyloleanolic acid (9), oleanolic acid (10); two chromanoids: (3S)-3,6-dihydroxy-2,2-dimethylchromane (11), 6-hydroxy-2,2-dimethyl-2H-chromene (12); five bezenoids: ehretiate (13), prenylhydroquinone (14), 4-hydroxy-3-prenylbenzoic acid (15), proglobeflowery acid (16), syringaldehyde (17); four diphenylbutanoid: ehretidiol A (18), ehretidiol B (19), a mixture of ehretidiol C (20) and (2R,3R)-1,4-diphenylbutan-2,3-diol (21); one hemiterpenoid: 3-methylbut-2-enoic acid (22); one sesquiterpenoid: (+)-(2E,6E)-methyl-10,11-dihydroxy-3,7,11-trimethyl-2,6-dodecadienoate (23); three glycerides: 2,3-dihydroxypropyl palmitate (24), (9Z,12Z)-2,3-dihydroxypropyl octadeca-9,12-dienoate (25), (9Z,12Z,15Z)-2,3-dihydroxypropyl octadeca-9,12,15-trienoate (26); one alkanoid derivative: methyl linoleate (27); two steroids: a mixture of β-sitosterol (28) and stigmasterol (29); one other skeleton: ehretilactone C (30)。The structures of these isolates were elucidated by spectral analysis. Among these isolates, compounds 1-6, 8, 11, 13 and 30 were new compounds, and compounds 7, 18-20 were first isolated from nature. Compounds 1, 2, 12, 14, 15, 18 and 22 exhibited antitubercular activities in vitro, and showing MIC values of 25.0, 50.0, 50.0, 26.2, 43.0, 30.0 and 36.0 µg/mL, respectively.

Dissertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015
Microparticles (MPs) of konjac glucomannan (KGM) are proposed in this thesis as an alternative therapeutic approach for antitubercular drug delivery to the lung, in the ambit of tuberculosis (TB) therapy. KGM is composed of mannose groups that might comprise an entry mechanism in macrophages infected with Mycobacterium tuberculosis, mimicking one of the input paths of this infectious agent causer of TB. For this purpose, KGM MPs need to have certain properties, in particular an aerodynamic diameter between 1 and 3 μm that enables reaching the alveolar zone. These properties might be attained by a production using spray-drying technology. The objective of this work was to produce MPs of KGM through this process and associate two first-line antitubercular drugs, isoniazid (INH) and rifabutin (RFB). Furthermore, the effect of incorporating mannitol or leucine in the microparticle formulations was evaluated, regarding MPs aerodynamic characteristics, drug association and release, and biocompatibility profile. After an initial optimization that required a hydrolysis of the original KGM polymer to enable spray-drying, several formulations of MPs were produced and characterised. The use of mannitol or leucine has demonstrated to improve the flow properties of some formulations, as did the association of RFB. However, the incorporation of excipients brought some disadvantages, especially regarding the in vitro release rate of the drug, which became faster. Additionally, the incorporation of mannitol was found to affect the association of INH, which became lower, suggesting a competing effect between INH and mannitol. Cytotoxicity assays in alveolar epithelial cells revealed that RFB-loaded KGM MPs may have a considerably toxic effect when used at a concentration of 1 mg/mL, in particular if MPs are also comprised of leucine. The obtained results indicate that KGM MPs are suitable inhalable delivery systems for antitubercular drugs. Considering that a combination of drugs is advised by WHO in TB treatment, no benefit was found to occur from the incorporation of either leucine or mannitol in the formulation of KGM MPs.

by Walubo Andrew.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1991.
Bibliography: leaves 148-174.
ABSTRACT --- p.i
declaration --- p.iv
acknowledgements --- p.v
Chapter chapter 1 --- general introduction --- p.1
Chapter chapter 2 --- literature review --- p.5
Chapter section 1 --- isoniazid --- p.5
Chapter section 2 --- rifampicin --- p.19
Chapter section 3 --- pyrazinamide --- p.27
Chapter section 4 --- an overview of the use of antituberculous drugs in uganda and hong kong --- p.30
Chapter section 5 --- review of analytical methods --- p.40
Chapter section 6 --- conclusions from the reviews --- p.44
Chapter chapter 3 --- some principles of pharmacokinetics --- p.47
Chapter section 1 --- introduction --- p.47
Chapter section 2 --- application of pharmacokinetics --- p.55
Chapter chapter 4. --- aims of the present project --- p.66
Chapter chapter 5 --- chromatographic methods --- p.68
Chapter section 1 --- a simultaneous assay for isoniazid and hydrazine metabolite in plasma and cerebrospinal fluid in the rabbit --- p.68
Chapter section 2 --- a simultaneous assay for rifampicin and pyrazinamide in human plasma --- p.89
Chapter chapter 6 --- the pharmacokinetics of isoniazid and hydrazine metabolite in the plasma and cerebrospinal fluid of rabbits --- p.91
Chapter chapter 7 --- the disposition of anti-tuberculous drugs in the plasma of elderly patients --- p.108
Chapter SECTION 1 --- METHODS --- p.109
Chapter SECTION 2 --- CLINICAL ASSESSMENT --- p.112
Chapter SECTION 3 --- GENERAL FINDINGS. --- p.117
Chapter 1)- --- THE DISPOSITION OF ISONIAZID AND HYDRAZINE METABOLITE IN THE PLASMA OF ELDERLY PATIENTS --- p.117
Chapter 2) - --- "THE DISPOSITION OF ISONIAZID, PYRAZINAMIDE AND RIFAMPICIN IN THE PLASMA OF ELDERLY PATIENTS" --- p.129
Chapter SECTION 4 --- CASE REPORT --- p.139
Chapter CHAPTER 8 --- p.145
Chapter SECTION 1 --- GENERAL CONCLUSIONS --- p.145
Chapter SECTION 2 --- FUTURE STUDIES --- p.147
REFERENCES --- p.148
APPENDICES: --- p.AO
Chapter APPENDIX A --- (WORK SHEETS) --- p.AO
Chapter APPENDIX B --- (SCIENTIFIC COMMUNICATIONS) --- p.BO