Dans ce travail de thèse, l'efficacité de la voie intrapulmonaire et les paramètres biopharmaceutiques influençant la diffusion pulmonaire après nébulisation d'antibiotiques ont été évalués. La présence et l'impact de certaines pompes d'efflux dans un modèle in vitro de cellules primaires épithéliales pulmonaires de rat ont été testés. Trois fluoroquinolones et la colistine ont été utilisées comme molécules de référence. La combinaison des molécules testées a permis d'obtenir une vue d'ensemble des caractéristiques de diffusion intrapulmonaire des antibiotiques. L'étude in vivo avec les fluoroquinolones a démontré que les concentrations pulmonaires de ces molécules sont plus importantes que dans le plasma, probablement dû à la présence des transporteurs comme la glycoprotéine-P. La présence de ces transporteurs a été confirmée dans le modèle de cellules pulmonaires de rats. L'étude in vivo avec la colistine a montré qu'une lente diffusion pourrait conférer un avantage à la nébulisation par rapport à l'administration intraveineuse. En conclusion, l'administration par nébulisation des molécules, qui traversent les tissus lentement (colistine), pourrait être avantageuse, alors que pour d'autres, qui traversent vite la barrière (fluoroquinolones), la voie nébulisée pourrait ne pas présenter des avantages par rapport à la voie intraveineuse. De plus, les résultats ont démontré qu'une faible perméabilité à travers le poumon (colistine) pourrait donner un avantage à la nébulisation des antibiotiques, tandis qu'une affinité pour des transporteurs (fluoroquinolones) semble présenter un intérêt aussi bien dans le cadre d'une nébulisation que d'une administration intraveineuse. / The aim of this study was to investigate the efficiency of intrapulmonary administration and the biopharmaceutical parameters regulating the pulmonary diffusion following nebulization. We examined whether certain efflux pumps were present in an in vitro model of rat lung cells and whether these efflux pumps could be beneficial by increasing lung concentrations in vivo. Fluoroquinolones and colistin were the molecules used as reference. These different molecules allowed an overview of the intrapulmonary diffusion characteristics of antibiotics. The in vivo study with fluoroquinolones showed that their lung concentrations are higher than in plasma, probably due to glycoprotein-P. The presence of this efflux pump was confirmed in the model with rat lung cells. The in vivo study with colistin showed that a slow diffusion may confer an advantage for nebulization over intravenous administration. In conclusion, the nebulization molecules passing slowly (colistin) across the tissues may be advantageous, whereas for others, with a fast passage across the barrier (fluoroquinolones), the pulmonary route may not provide an advantage over the intravenous administration. Moreover, the results showed that a slow permeability across the lung (colistin) may confer an advantage for the antibiotic nebulization, while affinity by transporters (fluoroquinolones) is beneficial for both nebulization and intravenous administration.

La fièvre boutonneuse Méditerranéenne (FBM) est due à Rickettsia conorii subsp. conorii, bactérie intracellulaire stricte. Cette maladie, initialement décrite comme bénigne, présente actuellement un taux de sévérité de l’ordre de 10% avec une augmentation de cette sévérité décrite dernièrement. Au cours d’une étude clinique rétrospective portant sur 161 cas de FBM, nous avons observé que le traitement par fluoroquinolones était associé à une évolution défavorable, ce qui à ce jour n’avait jamais été rapporté, alors que la doxycycline semble être protectrice. Nous avons également observé cet effet délétère des fluoroquinolones sur un modèle in vitro d’infection cellulaire à R. conorii, effet qui n’a pas été observé avec la doxycycline. Une des hypothèses pouvant expliquer cet effet est l’induction du module toxine-antitoxine par les fluoroquinolones. Ainsi nous avons montré que la ciprofloxacine modulait l’expression des gènes du couple toxine-antitoxine. Nous avons également montré que les statines pouvaient avoir un effet prophylactique au cours de l’infection par R. conorii. Enfin nous avons étudié la réponse de l’hôte au sein de l’escarre d’inoculation par une approche transcriptomique.Cette thèse a mis en évidence que les traitements reçus au cours de la FBM peuvent modifier le pronostic de cette maladie. Le choix des antibiotiques est donc crucial et doit faire l’objet d’études complémentaires. / Mediterranean spotted fever (MSF) is caused by Rickettsia conorii subsp. conorii, a strict intracellular bacterium. The disease initially described as benign presents currently rates of severity around 10% with an increase described recently. In a retrospective clinical study of 161 cases of MSF, we observed that treatment with fluoroquinolones was associated with an unfavourable outcome whereas doxycycline appeared to be protective. We also observed this deleterious effect of fluoroquinolones in vitro in a cellular model of R. conorii infection, which was not observed with doxycycline. One hypothesis that could explain this effect is the induction of toxin-antitoxin module by fluoroquinolones. Thus we have shown that ciprofloxacin modulates the gene expression of toxin-antitoxin module. We have also shown that statins may have a prophylactic effect during infection by R. conorii. Finally, we have studied the host response within the inoculation eschar by a transcriptomic approach. This thesis has shown that the treatment received during the MSF can change the prognosis of this disease. The choice of antibiotics is crucial, and should be subject to further studies.

Fluoroquinolones (FQ) are broad-spectrum charged antimicrobials exhibiting excellent tissue/fluid permeation. Thus, FQ disposition depends essentially on active transport and facilitative diffusion. Although most early transporter studies investigating renal elimination of FQs have focused on apical efflux of FQs from renal proximal tubule cell (RPTC) into urine, their basolateral uptake mechanism(s) from blood into RPTC (i.e., first step to tubular secretion) has not yet been explored in detail. Renally expressed SLC22 members: organic anion (OATs) and cation (OCTs) transporters are known to transport such small organic ionic substrates (molecular weight ~400 Da). Hence it is of interest to explore the role of these basolateral transporters in renal elimination of FQs, and to further quantitatively assess their impact in clinically observed FQ drug-drug interactions (DDI). An initial systematic review of clinical literature for FQs (n=18) demonstrated substantial differences among their renal clearance (CLren~46-fold) and unbound renal clearance (CLrenu~20-fold), and suggested that tubular secretion and reabsorption could be major determinants of FQ half-life, efficacy, and DDIs. FQs (n=13) identified from the above review were investigated by in-vitro transport studies using stably transfected cell lines, for potential interactions with organic cation [human (h) OCT1, hOCT2 and hOCT3] and anion [mouse (m) and hOAT3, hOAT1; and hOAT4] transporters. Further, kinetic inhibition studies were conducted to determine inhibition potency (Ki/IC50 values) for those FQs exhibiting significant OCT/OAT inhibition in preliminary interaction experiments. Gatifloxacin, moxifloxacin, prulifloxacin, and sparfloxacin were determined to be competitive inhibitors of hOCT1 with Ki = 250±18, 161±19, 136±33, and 94±8 μM, respectively. Moxifloxacin competitively inhibited hOCT3-mediated uptake, Ki = 1,598±146 μM. Enoxacin, fleroxacin, levofloxacin, lomefloxacin, moxifloxacin, prulifloxacin, and sparfloxacin exhibited competitive inhibition for mOat3 with Ki = 396±15, 817±31, 515±22, 539±27, 1356±114, 299±35, 205±12 μM, respectively. Fleroxacin and pefloxacin were found to inhibit hOAT1 with IC50 = 2228±84 and 1819±144 respectively. Despite expression in enterocytes, hepatocytes, and RPTC, hOCT3 does not appear to contribute significantly to FQ disposition. However, due to hepatic and potential RPTC expression, hOCT1 could play an important role in elimination of these antimicrobials. Among renally expressed OATs in humans, hOAT1 and hOAT3 are likely to be involved in FQ elimination.

A presen&ccedil;a de antibi&oacute;ticos em &aacute;guas superficiais e subterr&acirc;neas &eacute; motivo de preocupa&ccedil;&atilde;o, devido ao surgimento de bact&eacute;rias resistentes. Contudo, a maioria dos trabalhos publicados na literatura cient&iacute;fica mant&eacute;m o foco nos antibi&oacute;ticos inalterados, sendo que as mol&eacute;culas degradadas tamb&eacute;m est&atilde;o no ambiente. Esses produtos de degrada&ccedil;&atilde;o podem ser t&atilde;o ou mais prejudiciais do que as mol&eacute;culas que lhes deram origem. Esse trabalho teve por objetivo desenvolver m&eacute;todos de extra&ccedil;&atilde;o utilizando a microextra&ccedil;&atilde;o por sorvente empacotado (MEPS) para pr&eacute;-concentrar e recuperar produtos de fotodegrada&ccedil;&atilde;o de representantes das sulfonamidas (sulfametazina) e fluoroquinlonas (ciprofloxacino), duas das mais importantes classes de antibi&oacute;ticos. MEPS &eacute; considerada uma t&eacute;cnica promissora utilizando pequenos volumes de amostra e de solventes, al&eacute;m de empregar pequenas quantidades de fase extratora, que ainda pode ser reutilizada. Foram comparadas as fases Oasis&reg; HLB e nanotubos de carbono, sendo que a primeira apresentou melhores resultados. Seis produtos de degrada&ccedil;&atilde;o da sulfametazina (SMZ) e oito produtos do ciprofloxacino (CIP), al&eacute;m das mol&eacute;culas inalteradas, foram pr&eacute;-concentrados e analisados por cromatografia l&iacute;quida acoplada &agrave; espectrometria de massas de alta resolu&ccedil;&atilde;o (LC-ESI-ToF). Inicialmente, as concentra&ccedil;&otilde;es de f&aacute;rmaco inalterado utilizadas na fotodegrada&ccedil;&atilde;o foram de 25 mg L-1 (SMZ) e 10mg L-1 (CIP), para que os produtos fossem identificados. As taxas de recupera&ccedil;&atilde;o por MEPS ficaram acima de 50%, o que &eacute; um resultado promissor, considerando-se as diferen&ccedil;as estruturais dos produtos de degrada&ccedil;&atilde;o. Em concentra&ccedil;&otilde;es 100 vezes menores, MEPS conseguiu pr&eacute;-concentrar todos os produtos da SMZ e do CIP, facilitando sua detec&ccedil;&atilde;o. Ap&oacute;s desenvolver a pr&eacute;-concentra&ccedil;&atilde;o por MEPS em &aacute;gua purificada, foram realizados estudos de efeito de matriz em esgoto sint&eacute;tico. Enquanto somente um produto de degrada&ccedil;&atilde;o da SMZ sofreu supress&atilde;o de ioniza&ccedil;&atilde;o, todos os outros (inclusive os de CIP) experimentaram um aumento de sinal devido &agrave; presen&ccedil;a dos interferentes de matriz. O m&eacute;todo desenvolvido para MEPS tamb&eacute;m foi testado para pr&eacute;-concentrar produtos de degrada&ccedil;&atilde;o anaer&oacute;bica da SMZ em reator biol&oacute;gico, obtendo-se &ecirc;xito. Dessa forma, MEPS desponta como uma t&eacute;cnica promissora para pr&eacute;-concentrar produtos de degrada&ccedil;&atilde;o e para que pesquisadores possam acompanhar a degrada&ccedil;&atilde;o de reatores biol&oacute;gicos, visto que requer pequenas quantidades de amostra, n&atilde;o alterando significativamente o volume do meio reacional. / Antibiotics are present both surface water and groundwater and this is motive of concern, due to their ability to cause bacterial resistance. Nevertheless, most of publications in scientific area focuses in unchanged compounds, even knowing the presence of degraded molecules in the environment. These degradation products might be so or more dangerous than unchanged compounds. In this work, extraction methods for degradation products were developed using microextraction by packed sorbent (MEPS). MEPS is an eco-friendly technique due to little consumption of sample, solvents and sorbent. The degradation products of sulfamethazine (SMZ) and ciprofloxacin (CIP) were generated by photodegradation at 25 mg L-1 and <br clear=\"all\" /> 10 mg L-1 of unchanged drug, respectively. The number of degradation products was six for SMZ and eight for CIP. Oasis&reg; HLB and carbon nanotubes were tested as sorbents and the first got better results in preconcentration. The chromatographic analysis was performed by liquid chromatography coupled to high resolution mass spectrometry (LC-ESI-ToF). Recovery rates obtained by MEPS were greater than 50%, which is a significant result, considering structural differences among degradation products. After setting extraction conditions, MEPS was able to recover degradation products at concentrations 100 times lower and more akin to those find in the environment. Using synthetic sewage as medium, matrix effect studies were performed. The prevalent effect was an increase of ionization in degradation products (both SMZ and CIP), just one SMZ product experienced suppression of ionization. At last, SMZ was degraded in an anaerobic reactor and the degradation products were preconcentrated by MEPS. Although the biological degradation products were not the same of photodegradation (except by one), MEPS was capable to preconcentrate them. Thereby, MEPS starts to dawn as a promising technique to preconcentrate degradation products, specially for researchers using biological reactors, since MEPS requires low volumes of sample and almost do not change the final bulk of reactor.

A tuberculose afeta mais de um bilhão de pessoas em todo o mundo. Desde a descoberta da rifampicina, em 1965, nenhum outro fármaco importante foi introduzido na terapêutica. A pirazinamida, um dos fármacos disponíveis na terapia da tuberculose, é atualmente considerada um bioprecursor do ácido pirazinóico, porque bactérias resistentes não expressam uma enzima, pirazinamidase, responsável pela conversão da pirazinamida no derivado ácido. Ésteres do ácido pirazinóico apresentam atividade antimicobacteriana, provavelmente por melhor penetração pela parede celular das micobactérias que o derivado ácido. Desta forma, podem ainda atuar em cepas resistentes por serem ativados por esterases. Algumas fluorquinolonas apresentam atividade antimicobacteriana, como o ciprofloxacino, ofloxacino e levofloxacino. O trabalho teve por objetivo obter formas latentes de ácido pirazinóico unindo-o a quinolonas com atividade antimicobacteriana, através de ligação éster, obtendo-se pró-fármacos recíprocos. Um dos compostos sintetizados apresentou atividade in vitro, com concentração inibitória mínima comparável ao ciprofloxacino, um dos fármacos mais ativos. / Tuberculosis affects over than one billion people around the world. Since the discovery of rifampin, in 1965, no one another important drug was introduced in therapeutics. Pyrazinamide, one of the drugs available in therapy of tuberculosis, is nowadays considered a bioprecursor of pyrazinoic acid, because resistant bacterias do not express an enzyme, pyrazinamidase, responsible by the conversion of pyrazinamide to pyrazinoic acid. Pyrazinoic acid esters exhibit antimycobacterial activity probably to better penetration through mycobacterial cell wall than the acid derivative. So, it could act in resistant strains because is activated by esterases. Some fluoroquinolones exhibit antimicobacterial activity, like ciprofloxacin, ofloxacin and levofloxacin. This work had as objective to obtain latent forms of pyrazinoic acid linking it to quinolones with antimycobacterial activity, through an ester bond, obtaining mutual prodrugs. One of the synthesized compounds showed activity in vitro, with minnimal inhibitory concentration comparable to ciprofloxacin, one of most active drugs.

A elevada introdu&ccedil;&atilde;o de antibi&oacute;ticos no ambiente aqu&aacute;tico tem aumentado a preocupa&ccedil;&atilde;o em rela&ccedil;&atilde;o ao desenvolvimento de microrganismos resistentes aos agentes antimicrobianos e seu potencial impacto na biota aqu&aacute;tica. Com o objetivo de monitorar a presen&ccedil;a, persist&ecirc;ncia e destina&ccedil;&atilde;o desses f&aacute;rmacos no meio ambiente, h&aacute; a necessidade de se desenvolver m&eacute;todos anal&iacute;ticos altamente sens&iacute;veis a baixas concentra&ccedil;&otilde;es desses antibi&oacute;ticos, na ordem de ng L-1 a &#956;g L-1. Nesse contexto o trabalho tem como objetivo a s&iacute;ntese de sorventes seletivos como pol&iacute;meros molecularmente impressos (MIP) e pol&iacute;meros molecularmente impressos com acesso restrito (RAMIP) para a an&aacute;lise de amostras aquosas complexas contendo fluoroquinolonas, empregando extra&ccedil;&atilde;o em fase s&oacute;lida (SPE) online acoplada por column-switching (CS) &agrave; LC-MS/MS. Dois MIPs (MIP1 e MIP2) foram sintetizados utilizando ofloxacino como mol&eacute;cula molde, &aacute;cido metacr&iacute;lico como mon&ocirc;mero e, etileno glicol dimetacrilato (MIP1) ou trimetilpropano trimetacrilato (MIP2) como agente de liga&ccedil;&atilde;o cruzada. O glicerol dimetacrilato e hidroximetil dimetacrilato foram utilizados para promover a forma&ccedil;&atilde;o de uma superf&iacute;cie hidrof&iacute;lica sobre os MIP1 e MIP2 (RAMIP1 e RAMIP2). Os pol&iacute;meros sintetizados (MIP1, MIP2, RAMIP1 e RAMIP2) foram acondicionados em colunas de 21,7 x 2,0 mm e empregados para an&aacute;lise direta das matrizes em um sistema de CS-LC-MS/MS online, para avalia&ccedil;&atilde;o da seletividade, exclus&atilde;o de macromol&eacute;culas e recupera&ccedil;&atilde;o relativa do processo. As fases sintetizadas foram recobertas com &aacute;cidos h&uacute;micos (MIP1-AH, MIP2-AH, RAMIP1-AH, RAMIP2-AH) e foram comparadas a fase extratora HLB-AH quanto a seletividade e capacidade de exclus&atilde;o de prote&iacute;nas e &aacute;cidos h&uacute;micos. Os pol&iacute;meros sintetizados mostraram-se seletivos para a an&aacute;lise de fluoroquinolonas quando comparados com poss&iacute;veis interferentes, em especial o MIP1 e MIP-AH, com seletividade maior em rela&ccedil;&atilde;o &agrave; apresentada pelas fases comerciais HLB Oasis&reg; e StrataX&reg;. Al&eacute;m disso, foi poss&iacute;vel eliminar cerca de 92-100% de prote&iacute;na quando percolada uma solu&ccedil;&atilde;o aquosa de 44 mg mL-1 de albumina s&eacute;rica bovina (BSA) e de 94-96% quando percolada uma solu&ccedil;&atilde;o aquosa de 6 mg mL-1 de &aacute;cido h&uacute;mico. A efici&ecirc;ncia relativa do processo de preparo de amostra apresentada pelos pol&iacute;meros sintetizados foi compar&aacute;vel &agrave; da fase comercial, no entanto o desvio padr&atilde;o relativo (DPR) apresentado pela fase HLB foi maior. O pol&iacute;mero MIP1-AH apresentou melhor efici&ecirc;ncia no processo de extra&ccedil;&atilde;o para a maioria dos analitos quando analisado em matriz de esgoto preparado em laborat&oacute;rio e esgoto sanit&aacute;rio enquanto que as fases MIP2 e RAMIP1 apresentaram um melhor desempenho na extra&ccedil;&atilde;o dos analitos em matriz de efluente de ETE. Em suma, o efeito matriz mostrou-se ainda persistente devido &agrave; complexidade das matrizes avaliadas, o que foge &agrave; idealidade para que se tenha detec&ccedil;&atilde;o de fluoroquinolonas em baix&iacute;ssimas concentra&ccedil;&otilde;es, como objetivo desse trabalho. Dessa forma, para que o m&eacute;todo possa ser utilizado para an&aacute;lise de amostras reais &eacute; necess&aacute;ria a compatibiliza&ccedil;&atilde;o das matrizes durante a valida&ccedil;&atilde;o do m&eacute;todo. / The increased use of antibiotics has led to a higher presence of these molecules into environment. Therefore, there is a raising concern on this matter, since antibiotics may yield microorganisms resistant to their effects. Aiming to monitoring the presence, persistence and destination of these drugs on the environment, there is an urge for the development of highly sensitive analytical methods, capable of detecting at the &#956;g L-1 to the ng L-1 concentration levels. In this fashion, this thesis focuses on the synthesis of selective sorbents such as molecular imprinted polymers (MIP) and restricted access molecular imprinted polymers (RAMIP) for the analysis of complex water samples containing fluoroquinolones. Additionally, it includes the use of these sorbents by online solid phase extraction (SPE) coupled to LC-MS/MS, using a column-switching (CS) setup. Two MIPs (MIP1 and MIP2) were synthesized using ofloxacin as template, methacrylic acid as monomer and ethylene glycol dimethacrylate (MIP1) or trimethylpropane trimethacrylate (MIP2) as cross-linking agent. The glycerol dimethacrylate and hydroximethyl dimethacrylate were employed to promote the formation of a hydrophilic surface layer on MIP1 and MIP2 (RAMIP1 and RAMIP2). The synthesized polymers (MIP1, MIP2, RAMIP1, RAMIP2) were packed into 21,7 x 2,0 mm columns and used for the direct analysis of complex water matrices using an online CS-LC-MS/MS system, for the evaluation of the selectivity, macromolecules exclusion and relative process efficiency. The synthesized phases were coated with humic acids (MIP1-AH, MIP2-AH, RAMIP1-AH, RAMIP2-AH) and were compared to the extraction phase HLB-AH regarding its selectivity and capability of proteins and humic acid exclusion. The synthesized polymers displayed selectivity for the analysis of fluoroquionolones when compared to the possible interferents, specially the MIP1 and MIP1-AH, with a higher selectivity when compared with the commercial phases Oasis&reg; HLB and Strata X&reg;. Additionally, when percolating an aqueous solution containing 44 mg mL-1 of bovine seric albumin (BSA) it was possible to eliminate ca. 92-100% of the proteins and 94-96% of exclusion was obtained when percolating an aqueous solution containing 6 mg mL-1 of humic acid. The relative process efficiency of sample preparation resulted from the synthesized polymers was comparable to that of the commercial phase, however the relative standard deviation (RSD) for the HLB phase was higher. The polymer MIP1-AH displayed better efficiency for the extraction process for most of the analytes on both sewage matrices, the one prepared on the laboratory and the sanitary one. The phases MIP2 and RAMIP1 presented a better performance for the extraction of analytes in effluents of a wastewater treatment plant. In conclusion, the matrix effect showed to be a persistent effect, given the complexity of the matrices analyzed. This fact is far from an ideal condition of detecting fluoroquinolones in real-life application where they are present at very low concentrations. In this sense, for the application of this method for real samples, a strategy of matrix matching would be needed during the validation of the method.

Águas residuárias são continuamente contaminadas por fármacos. Dentre estes fármacos, os antimicrobianos causam grande preocupação pelos impactos sobre o desenvolvimento de resistência bacteriana. As principais fontes de contaminação destes fármacos são efluentes urbanos, hospitalares, de fazendas e de algumas indústrias. A complexidade das matrizes ambientais tais como águas residuárias é uma das principais dificuldades para extrair e detectar fármacos, fazendo-se necessário o uso de técnicas de preparo de amostra para a extração destes compostos de interesse. Técnicas clássicas como a extração líquido-líquido (LLE) e a extração em fase sólida (SPE) são largamente usadas para extração de fármacos nesse tipo de matriz, porém estas técnicas não atendem amplamente aos princípios da química verde. Dessa forma, novas técnicas, mais alinhadas à responsabilidade ambiental, têm sido desenvolvidas. Neste âmbito apresenta-se o desenvolvimento e a validação de um método de microextração líquido-líquido para extração e detecção de sulfonamidas e o desenvolvimento e otimização de um método utilizando planejamento experimental para a extração de fluoroquinolonas em águas residuárias. Foi possível obter-se o limite de detecção de 0,2 ng mL-1 para as sulfonamidas analisadas, este LD é relativamente baixo considerando que o detector que foi utilizado não possuía a possibilidade de fazer análises no modo MS/MS, o que certamente reduziria ainda mais o LD. Com os desenvolvimentos desse trabalho tornou-se possível a utilização de apenas 1 mL de solvente orgânico para a pré-concentração off-line, Esta etapa, adicionada a uma outra pré-concentração online (column switching) permitiu a extração dos analitos com a obtenção de um LD relativamente baixo, a partir de apenas 7 mL de amostra. / Drugs are continuously contaminating wastewater. Among these drugs antimicrobials cause great concern for the impacts on the development of bacterial resistance. The main sources of contamination by these drugs are urban effluents, hospitals, farms and some industries. The complexity of the environmental matrices such as wastewater is one of the main difficulties in extracting and detecting drugs, bringing up the need to use sample preparation techniques for the extraction of the interest compounds. Classical techniques such as liquid-liquid extraction (LLE) and solid phase extraction (SPE) are widely used for drug extraction in this type of matrix, but these techniques do not largely meet the principles of green chemistry. In this way, new techniques, more aligned with environmental responsibility, have been developed. In this context, this thesis presents the development and validation of a liquid-liquid microextraction method for sulfonamide extraction and detection and the development and optimization of a method using experimental design for the extraction of fluoroquinolones presented in wastewater. It was possible to obtain a limit of detection (LD) of 0.2 ng mL-1 for the sulfonamides analyzed, this LD is relatively low considering that the detector that was used did not have the possibility to perform analyzes in the MS/MS mode, which certainly would further reduce the LD. With the development of this thesis, it became possible to use only 1 mL of organic solvent for the off-line preconcentration of the analytes. This step, added to another online preconcentration (column switching) allowed the extraction of the analytes obtaining relatively low LDs, from just 7 mL of the sample.

A elevada introdu&ccedil;&atilde;o de antibi&oacute;ticos no ambiente aqu&aacute;tico tem aumentado a preocupa&ccedil;&atilde;o em rela&ccedil;&atilde;o ao desenvolvimento de microrganismos resistentes aos agentes antimicrobianos e seu potencial impacto na biota aqu&aacute;tica. Com o objetivo de monitorar a presen&ccedil;a, persist&ecirc;ncia e destina&ccedil;&atilde;o desses f&aacute;rmacos no meio ambiente, h&aacute; a necessidade de se desenvolver m&eacute;todos anal&iacute;ticos altamente sens&iacute;veis a baixas concentra&ccedil;&otilde;es desses antibi&oacute;ticos, na ordem de ng L-1 a &#956;g L-1. Nesse contexto o trabalho tem como objetivo a s&iacute;ntese de sorventes seletivos como pol&iacute;meros molecularmente impressos (MIP) e pol&iacute;meros molecularmente impressos com acesso restrito (RAMIP) para a an&aacute;lise de amostras aquosas complexas contendo fluoroquinolonas, empregando extra&ccedil;&atilde;o em fase s&oacute;lida (SPE) online acoplada por column-switching (CS) &agrave; LC-MS/MS. Dois MIPs (MIP1 e MIP2) foram sintetizados utilizando ofloxacino como mol&eacute;cula molde, &aacute;cido metacr&iacute;lico como mon&ocirc;mero e, etileno glicol dimetacrilato (MIP1) ou trimetilpropano trimetacrilato (MIP2) como agente de liga&ccedil;&atilde;o cruzada. O glicerol dimetacrilato e hidroximetil dimetacrilato foram utilizados para promover a forma&ccedil;&atilde;o de uma superf&iacute;cie hidrof&iacute;lica sobre os MIP1 e MIP2 (RAMIP1 e RAMIP2). Os pol&iacute;meros sintetizados (MIP1, MIP2, RAMIP1 e RAMIP2) foram acondicionados em colunas de 21,7 x 2,0 mm e empregados para an&aacute;lise direta das matrizes em um sistema de CS-LC-MS/MS online, para avalia&ccedil;&atilde;o da seletividade, exclus&atilde;o de macromol&eacute;culas e recupera&ccedil;&atilde;o relativa do processo. As fases sintetizadas foram recobertas com &aacute;cidos h&uacute;micos (MIP1-AH, MIP2-AH, RAMIP1-AH, RAMIP2-AH) e foram comparadas a fase extratora HLB-AH quanto a seletividade e capacidade de exclus&atilde;o de prote&iacute;nas e &aacute;cidos h&uacute;micos. Os pol&iacute;meros sintetizados mostraram-se seletivos para a an&aacute;lise de fluoroquinolonas quando comparados com poss&iacute;veis interferentes, em especial o MIP1 e MIP-AH, com seletividade maior em rela&ccedil;&atilde;o &agrave; apresentada pelas fases comerciais HLB Oasis&reg; e StrataX&reg;. Al&eacute;m disso, foi poss&iacute;vel eliminar cerca de 92-100% de prote&iacute;na quando percolada uma solu&ccedil;&atilde;o aquosa de 44 mg mL-1 de albumina s&eacute;rica bovina (BSA) e de 94-96% quando percolada uma solu&ccedil;&atilde;o aquosa de 6 mg mL-1 de &aacute;cido h&uacute;mico. A efici&ecirc;ncia relativa do processo de preparo de amostra apresentada pelos pol&iacute;meros sintetizados foi compar&aacute;vel &agrave; da fase comercial, no entanto o desvio padr&atilde;o relativo (DPR) apresentado pela fase HLB foi maior. O pol&iacute;mero MIP1-AH apresentou melhor efici&ecirc;ncia no processo de extra&ccedil;&atilde;o para a maioria dos analitos quando analisado em matriz de esgoto preparado em laborat&oacute;rio e esgoto sanit&aacute;rio enquanto que as fases MIP2 e RAMIP1 apresentaram um melhor desempenho na extra&ccedil;&atilde;o dos analitos em matriz de efluente de ETE. Em suma, o efeito matriz mostrou-se ainda persistente devido &agrave; complexidade das matrizes avaliadas, o que foge &agrave; idealidade para que se tenha detec&ccedil;&atilde;o de fluoroquinolonas em baix&iacute;ssimas concentra&ccedil;&otilde;es, como objetivo desse trabalho. Dessa forma, para que o m&eacute;todo possa ser utilizado para an&aacute;lise de amostras reais &eacute; necess&aacute;ria a compatibiliza&ccedil;&atilde;o das matrizes durante a valida&ccedil;&atilde;o do m&eacute;todo. / The increased use of antibiotics has led to a higher presence of these molecules into environment. Therefore, there is a raising concern on this matter, since antibiotics may yield microorganisms resistant to their effects. Aiming to monitoring the presence, persistence and destination of these drugs on the environment, there is an urge for the development of highly sensitive analytical methods, capable of detecting at the &#956;g L-1 to the ng L-1 concentration levels. In this fashion, this thesis focuses on the synthesis of selective sorbents such as molecular imprinted polymers (MIP) and restricted access molecular imprinted polymers (RAMIP) for the analysis of complex water samples containing fluoroquinolones. Additionally, it includes the use of these sorbents by online solid phase extraction (SPE) coupled to LC-MS/MS, using a column-switching (CS) setup. Two MIPs (MIP1 and MIP2) were synthesized using ofloxacin as template, methacrylic acid as monomer and ethylene glycol dimethacrylate (MIP1) or trimethylpropane trimethacrylate (MIP2) as cross-linking agent. The glycerol dimethacrylate and hydroximethyl dimethacrylate were employed to promote the formation of a hydrophilic surface layer on MIP1 and MIP2 (RAMIP1 and RAMIP2). The synthesized polymers (MIP1, MIP2, RAMIP1, RAMIP2) were packed into 21,7 x 2,0 mm columns and used for the direct analysis of complex water matrices using an online CS-LC-MS/MS system, for the evaluation of the selectivity, macromolecules exclusion and relative process efficiency. The synthesized phases were coated with humic acids (MIP1-AH, MIP2-AH, RAMIP1-AH, RAMIP2-AH) and were compared to the extraction phase HLB-AH regarding its selectivity and capability of proteins and humic acid exclusion. The synthesized polymers displayed selectivity for the analysis of fluoroquionolones when compared to the possible interferents, specially the MIP1 and MIP1-AH, with a higher selectivity when compared with the commercial phases Oasis&reg; HLB and Strata X&reg;. Additionally, when percolating an aqueous solution containing 44 mg mL-1 of bovine seric albumin (BSA) it was possible to eliminate ca. 92-100% of the proteins and 94-96% of exclusion was obtained when percolating an aqueous solution containing 6 mg mL-1 of humic acid. The relative process efficiency of sample preparation resulted from the synthesized polymers was comparable to that of the commercial phase, however the relative standard deviation (RSD) for the HLB phase was higher. The polymer MIP1-AH displayed better efficiency for the extraction process for most of the analytes on both sewage matrices, the one prepared on the laboratory and the sanitary one. The phases MIP2 and RAMIP1 presented a better performance for the extraction of analytes in effluents of a wastewater treatment plant. In conclusion, the matrix effect showed to be a persistent effect, given the complexity of the matrices analyzed. This fact is far from an ideal condition of detecting fluoroquinolones in real-life application where they are present at very low concentrations. In this sense, for the application of this method for real samples, a strategy of matrix matching would be needed during the validation of the method.

A presen&ccedil;a de antibi&oacute;ticos em &aacute;guas superficiais e subterr&acirc;neas &eacute; motivo de preocupa&ccedil;&atilde;o, devido ao surgimento de bact&eacute;rias resistentes. Contudo, a maioria dos trabalhos publicados na literatura cient&iacute;fica mant&eacute;m o foco nos antibi&oacute;ticos inalterados, sendo que as mol&eacute;culas degradadas tamb&eacute;m est&atilde;o no ambiente. Esses produtos de degrada&ccedil;&atilde;o podem ser t&atilde;o ou mais prejudiciais do que as mol&eacute;culas que lhes deram origem. Esse trabalho teve por objetivo desenvolver m&eacute;todos de extra&ccedil;&atilde;o utilizando a microextra&ccedil;&atilde;o por sorvente empacotado (MEPS) para pr&eacute;-concentrar e recuperar produtos de fotodegrada&ccedil;&atilde;o de representantes das sulfonamidas (sulfametazina) e fluoroquinlonas (ciprofloxacino), duas das mais importantes classes de antibi&oacute;ticos. MEPS &eacute; considerada uma t&eacute;cnica promissora utilizando pequenos volumes de amostra e de solventes, al&eacute;m de empregar pequenas quantidades de fase extratora, que ainda pode ser reutilizada. Foram comparadas as fases Oasis&reg; HLB e nanotubos de carbono, sendo que a primeira apresentou melhores resultados. Seis produtos de degrada&ccedil;&atilde;o da sulfametazina (SMZ) e oito produtos do ciprofloxacino (CIP), al&eacute;m das mol&eacute;culas inalteradas, foram pr&eacute;-concentrados e analisados por cromatografia l&iacute;quida acoplada &agrave; espectrometria de massas de alta resolu&ccedil;&atilde;o (LC-ESI-ToF). Inicialmente, as concentra&ccedil;&otilde;es de f&aacute;rmaco inalterado utilizadas na fotodegrada&ccedil;&atilde;o foram de 25 mg L-1 (SMZ) e 10mg L-1 (CIP), para que os produtos fossem identificados. As taxas de recupera&ccedil;&atilde;o por MEPS ficaram acima de 50%, o que &eacute; um resultado promissor, considerando-se as diferen&ccedil;as estruturais dos produtos de degrada&ccedil;&atilde;o. Em concentra&ccedil;&otilde;es 100 vezes menores, MEPS conseguiu pr&eacute;-concentrar todos os produtos da SMZ e do CIP, facilitando sua detec&ccedil;&atilde;o. Ap&oacute;s desenvolver a pr&eacute;-concentra&ccedil;&atilde;o por MEPS em &aacute;gua purificada, foram realizados estudos de efeito de matriz em esgoto sint&eacute;tico. Enquanto somente um produto de degrada&ccedil;&atilde;o da SMZ sofreu supress&atilde;o de ioniza&ccedil;&atilde;o, todos os outros (inclusive os de CIP) experimentaram um aumento de sinal devido &agrave; presen&ccedil;a dos interferentes de matriz. O m&eacute;todo desenvolvido para MEPS tamb&eacute;m foi testado para pr&eacute;-concentrar produtos de degrada&ccedil;&atilde;o anaer&oacute;bica da SMZ em reator biol&oacute;gico, obtendo-se &ecirc;xito. Dessa forma, MEPS desponta como uma t&eacute;cnica promissora para pr&eacute;-concentrar produtos de degrada&ccedil;&atilde;o e para que pesquisadores possam acompanhar a degrada&ccedil;&atilde;o de reatores biol&oacute;gicos, visto que requer pequenas quantidades de amostra, n&atilde;o alterando significativamente o volume do meio reacional. / Antibiotics are present both surface water and groundwater and this is motive of concern, due to their ability to cause bacterial resistance. Nevertheless, most of publications in scientific area focuses in unchanged compounds, even knowing the presence of degraded molecules in the environment. These degradation products might be so or more dangerous than unchanged compounds. In this work, extraction methods for degradation products were developed using microextraction by packed sorbent (MEPS). MEPS is an eco-friendly technique due to little consumption of sample, solvents and sorbent. The degradation products of sulfamethazine (SMZ) and ciprofloxacin (CIP) were generated by photodegradation at 25 mg L-1 and <br clear=\"all\" /> 10 mg L-1 of unchanged drug, respectively. The number of degradation products was six for SMZ and eight for CIP. Oasis&reg; HLB and carbon nanotubes were tested as sorbents and the first got better results in preconcentration. The chromatographic analysis was performed by liquid chromatography coupled to high resolution mass spectrometry (LC-ESI-ToF). Recovery rates obtained by MEPS were greater than 50%, which is a significant result, considering structural differences among degradation products. After setting extraction conditions, MEPS was able to recover degradation products at concentrations 100 times lower and more akin to those find in the environment. Using synthetic sewage as medium, matrix effect studies were performed. The prevalent effect was an increase of ionization in degradation products (both SMZ and CIP), just one SMZ product experienced suppression of ionization. At last, SMZ was degraded in an anaerobic reactor and the degradation products were preconcentrated by MEPS. Although the biological degradation products were not the same of photodegradation (except by one), MEPS was capable to preconcentrate them. Thereby, MEPS starts to dawn as a promising technique to preconcentrate degradation products, specially for researchers using biological reactors, since MEPS requires low volumes of sample and almost do not change the final bulk of reactor.

Fluoroquinolones are a class of antibiotics used clinically to treat a wide array of bacterial infections. These therapeutics act by targeting a bacterial enzyme required for cell viability, bacterial type-II topoisomerases. Fluoroquinolones act by forming a ternary complex with bacterial type II topoisomerases and cleaved DNA; religation of DNA is subsequently blocked, therefore leading to bacterial cell death. In ternary complex the keto-acid moiety of the fluoroquinolone is complexed with a divalent magnesium ion, forming a drug-magnesium-water bridge to a serine and an aspartate (or glutamate) residue on helix-4 of the topoisomerase enzyme. A major issue with fluoroquinolones is the rise in bacterial resistance. Resistance arises through substitutions of the serine or aspartate/glutamate residue, therefore preventing formation of the magnesium-water bridge and dramatically diminishing the overall antibiotic activity of the fluoroquinolone. Quinazoline-2,4-diones are structurally similar to fluoroquinolones; diones also form a ternary complex similar to fluoroquinolones, however, these complexes are less active due to lack of a potent magnesium-water bridge interaction in helix-4. While quinazoline-2,4-diones are therefore less potent antibiotics, their non-reliance on the magnesium water bridge generally affords equipotent activity with wild-type and fluoroquinolone-resistant strains of bacteria. The first objective of this work was to probe the helix-4 interaction of the bacterial type-II topoisomerase by quinazoline-2,4-dione modification, specifically at the N3 and C4 positions of the quinazoline-2,4-dione scaffold to afford potentially new binding contacts. These modified quinazoline-2,4-diones will provide deeper understanding of the helix-4 interaction and potentially afford potent novel quinazoline-2,4-dione scaffolds, against both wild-type and resistant bacteria, for iterative drug design. Metabolism is one of the primary sources of detoxification, inactivation, and clearance of drugs from the body and is a critical consideration for all early stage therapeutic development. Clinically used fluoroquinolones, i.e. Moxifloxacin and Ciprofloxacin, historically are metabolically stable, and are not known to be metabolized by Phase I and/or Phase II drug metabolizing enzymes. However, major modifications to the Moxifloxacin and Ciprofloxacin scaffolds, due to the development of next generation antibiotics, may display different metabolic stability profiles. Moreover, metabolism of quinazoline-2,4-diones, developed for fluoroquinolone-resistant bacteria, is not extensively studied and may be subject to different metabolic liabilities that may render the quinazoline-2,4-dione an ineffective potential antibiotic. The second objective of this work was to determine the in vitro Phase I and Phase II metabolic stabilities of fluoroquinolone and quinazoline-2,4-dione scaffolds to determine any structural features that render the potential therapeutic a metabolic liability. The results from these two objectives have led to the discovery of a novel bacterial type-II topoisomerase catalytic inhibitor and the acquisition of initial metabolic stability data of fluoroquinolone and quinazoline-2,4-dione scaffolds. These findings further promote research into quinazoline-2,4-diones as bacterial topoisomerase targets, and provide metabolic considerations for both fluoroquinolone and quinazoline-2,4-dione therapeutic development, which is severely underrepresented in the field of quinolone antibiotics.