L’objectif principal de cette thèse a été le développement, la caractérisation et l’évaluation in vitro et in vivo de différents nanovecteurs plus spécifiquement les nanoparticules spécifiques pour le traitement de la mélanome et β-lapachone-lipossomale incorporée dans des hydrogels de biopolymère pour la cicatrisation de blessures topiques. La première étape de cette thèse a consisté en la révision de la littérature liées aux résentes avancées sur les nanoparticules pour le ciblage d’agents thérapeutiques pour des cellules circulantes et des mésenchymateuses de mélanomes. De plus, cette révision a approfondi la connaissance sur les principaux biomarcateurs qui étaient déjà identifiés dans ces cellules et quelles caractéristiques des nanovecteurs peuvent influencer leur performance in vivo. Dans la phase expérimentale, les nanoparticules ont été développées en utilisant la méthode de nanoprécipitation de polymères dérivés du poli(γ-benzyl-L-glutamate). Ensuite, des immuno-nanoparticules combinées avec l’anticorps MART-1 spécifique pour les cellules de mélanome, ont été obtenues par le lien streptavidine-biotine. La combinaison de l’anticorps sur la superficie des nanoparticules a été évaluée par western blot. Les nanoparticules ont été caractérisées et évaluées in vitro dans des cellules de mélanome B 16-GFP et des cellules endothéliales de la veine ombilicale humaine (HUVECs), l’activation de complément a été recherchée par la technique d’immunoélectrophorèse bidimensionnelle. Les nanoparticules ont présenté des tailles comprises entre 20 et 100 nm et une charge négative (-3 à -30 mV). La combinaison de l’anticorps sur la superficie des nanoparticules a été observée par la technique western blot et confirmée par les altérations de la taille et de la charge de superficie des particules. Les nanoparticules développées n’ont pas été capables d’activer le systhème complémentaire étant considérées de longue circulation sanguine. Pour l’analyse in vitro, les nanoparticules n’ont pas présenté de cytotoxixité lorsqu’elles ont été testées dans des cellules de mélanome ou dans des cellules normales endothéliales. Lors des tests de capture cellulaire, les immuno-nanoparicules, qui contenaient l’anticorps spécifique pour la reconnaissance de l’antigène surexprimé dans des cellules de mélanome, ont présenté une augmentation de 40 à 50% lors de la capture pour ces cellules, cela indiquant une plus grande spécificité de ce nanovecteurs. La deuxième partie de cette thèse a été le développement, la caractérisation et l’évaluation de l’activité cicatrisante in vivo de β-lapacho encapsulé dans des liposomes multilamellaires incorporés dans de l’hydrogel de biopolymère produit par la bactérie Zoogloea sp. (β-lap-Lipo/ZBP/HEC). β-lap-Lipo/ZBP/HEC a présenté un pH et un comportement rhéologique approprié pour l’application topique, ainsi qu’un profil de libération plus lent de la β-lapachone à partir de l’hydrogel. Les analyses hystopotologiques de l’activité cicatrisante in vivo ont montré que le véhicule hydrogel de biopolymère a été capable de stimuler les réparations du tissu, augmenter la cellularité locale, les fibroblastes, les cellules inflamatoires, les vaisseaux sanguins et les fibrilles de collagène pendant la phase proliférative de la cicatrisation. De plus, β-lap-Lipo/ZBP/HEC a favorisé une augmentation de l’angiogenèse locale et une dimminution de l’inflamation de la blessure. Ces résultats ont montré le potentiel de l’application topique de β-lap-Lipo/ZBP/HEC pour la thérapie des lésions. Pour conclure, cette thèse a contribué au développement de nanovecteurs promisseurs ayant différentes applications biologiques et formes d’administration, comme le traitement systémétique de la mélanome et l’action topique lors de la cicatrisation des blessures / This thesis had as general objective the development, characterization and evaluation in vitro or in vivo of different nanocarriers, specifically site-specific nanoparticles for the treatment of melanoma and liposomal-hydrogel containing β-lapachone for topical wound healing. The first part of this thesis consisted in a literature review about the recent advances in nanoparticles for the targeting of therapeutic agents to circulating and mesenchymal melanoma cells. In addition, this review deepened the knowledge about the main biomarkers identified in these cells and which characteristics of nanocarriers may influence on their in vivo performance. In the experimental phase, nanoparticles were developed through the nanoprecipitation method of polymers derived from poly (γ-benzyl-L-glutamate). Next, immunonanoparticles conjugated with MART-1 antibody specific for melanoma cells were obtained through the streptavidin-biotin binding. The conjugation of this antibody on the nanoparticles surface was evaluated by western blot. The nanoparticles were characterized and evaluated in vitro in B16-GFP melanoma cells and human umbilical vein endothelial cells (HUVECs) and the complement activation was investigated by bidimensional immunoelectrophoresis. The nanoparticles presented sizes between 20 and 100 nm and negative surface charge (-3 to -30 mV). The conjugation of antibody on the nanoparticle surfaces was detected by the western blot technique and confirmed by the changes in particle size and surface charge. The developed nanoparticles were not able to activate the complement system being considered long blood circulation. Regarding the in vitro analysis, the particles did not show cytotoxicity when tested in melanoma cells or normal endothelial cells. In the cell capture assays, the immunonanoparticles, containing a specific antibody for the recognition of the overexpressed antigen in melanoma cells, showed an increase of 40 to 50% in the uptake for these cells, indicating a specificity of this nanocarrier. The second part of this thesis consisted of the development, characterization and evaluation of the in vivo wound healing activity of β-lapachone encapsulated in multilamellar liposomes and incorporated in a biopolymer hydrogel produced by Zoogloea sp (β-lap-Lipo/ZBP/HEC). β-lap-Lipo/ZBP/HEC presented pH and rheological behavior suitable for topical application, as well as a slower release profile of β-lapachone through the hydrogel. Histopathological analyzes of the healing activity in vivo, showed that the biopolymer hydrogel vehicle was able to stimulate tissue repair, with the increase of local cellularity, fibroblasts, inflammatory cells, blood vessels and collagen fibers, during the proliferative phase of wound healing. In addition, β-lap-Lipo/ZBP/HEC promoted an increase in local angiogenesis and a decrease of inflammation at the wound site. These results demonstrate a promising topical application of β-lap-Lipo/ZBP/HEC for wound therapy. In conclusion, this thesis contributed for the development of promising nanocarriers with different biological applications and administration routes, such as systemic treatment of melanoma and topical action in wound healing.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / As quinonas sÃo metabÃlitos de ampla distribuiÃÃo na natureza que possuem diversas atividades farmacolÃgicas de importÃncia clÃnica. A naftoquinona α-lapachona demonstrou potencial como protÃtipo para o desenvolvimento de substÃncias com propriedades anticÃncer, como relatado pelo nosso grupo de pesquisa, em que o seu derivado tetrahidropirano (THP) apresentou citotoxicidade e seletividade significante contra a linhagem de melanoma MDA-MB-435. Assim, o objetivo deste trabalho foi avaliar o mecanismo de aÃÃo envolvido na citotoxicidade da α-lapachona e derivado THP em cÃlulas de melanoma MDA-MB-435. Inicialmente, avaliou-se a citotoxicidade da alfa-lapachona e derivado THP em 8 linhagens tumorais de mama e melanoma, atravÃs do ensaio do MTT, mostrando CI50 de 1,37 e 8,18 ÂM, respectivamente, apÃs 72 horas de incubaÃÃo. A seletividade do derivado THP no ensaio de Alamar Blue, demonstrou que este se apresentou 2,6 vezes menos citotÃxico para cÃlulas normais quando comparado Ãs cÃlulas tumorais. Estudos do mecanismo de morte celular na linhagem tumoral MDA-MB-435 indicaram que o derivado THP causou reduÃÃo de cÃlulas viÃveis associado com o aumento de cÃlulas nÃo-viÃveis por induÃÃo da perda de integridade da membrana plasmÃtica nas concentraÃÃes de 5 e 10 ÂM apÃs 24 horas de incubaÃÃo. A atividade citotÃxica do derivado THP nÃo està relacionada a uma fase especÃfica do ciclo celular, ativaÃÃo de caspases efetoras e formaÃÃo de espÃcies reativas de oxigÃnio, sugerindo a ocorrÃncia de um processo necrÃtico a partir de 6 horas de tratamento, demonstrado pela avaliaÃÃo da integridade de membrana. Assim, os resultados exibidos sugerem que a introduÃÃo do radical tetrahidropirano na molÃcula da α-lapachona aumenta a citotoxicidade em cÃlulas de melanoma MDA-MB-435, via necrose, o que reforÃa a importÃncia de naftoquinonas, como protÃtipo para o desenvolvimento de novos compostos sintÃticos com atividade antitumoral / Quinone metabolites are widely distributed in nature showing various pharmacological activities of clinical importance. The naphthoquinone α-lapachone has been shown to be suitable as a prototype for the development of substances with anticancer properties, as reported by our group to tetrahydropyran derivative (THP), which demonstrated significant cytotoxicity and selectivity against MDA-MB-435 melanoma line. The aim of the present work was to evaluate the mechanism of action involved in the cytotoxicity of α-lapachone and its THP derivative against MDA-MB-435 melanoma cells. Initially, we evaluated the cytotoxicity of α-lapachone and its THP derivative against 8 cell lines, by the MTT assay, showing IC50 of 1.37 and 8.18 ÂM to breast and melanoma lines, respectively, after 72 hours of incubation. The selectivity of the THP derivative in Alamar Blue assay, demonstrated that THP is 2.6 times less cytotoxic to normal cells as compared to tumor cells. Studies on the mechanism of cell death in MDA-MB-435 tumor line showed that the THP derivative caused a reduction on viable cells associated with an increase of non-viable cells by inducing loss of membrane integrity in concentrations of 5 and 10 ÂM. The cytotoxic activity of THP was independent of cell cycle, activation of effector caspases and formation of reactive oxygen species, suggesting the occurrence of a necrotic process after 6 hours of treatment, demonstrated by evaluation of membrane integrity. Thus, the data suggest that a tetrahydropyran group introduction in α-lapachone molecule enhances the cytotoxicity of MDA-MB-435 in melanoma cells, via necrosis, which reinforces the importance of naphthoquinones as prototypes for the development of new synthetic compounds with antitumor activity

No description available.

La bêta-lapachone (b-lap) est une substance active présentant des activités trypanocides, anti-infectieuses et anticancéreuses, avec une sélectivité thérapeutique. Cependant, en raison de sa faible hydrosolubilité et de sa toxicité, b-lap n'est pas encore appliquée en clinique. Nous avons étudié son encapsulation dans des vésicules phospholipidiques, complexée ou non par des cyclodextrines. Nous avons d'abord analysé l'interaction de b-lap avec des excipients lipidiques par des mesures de pression de surface, DSC et SAXS. Elles ont montré que blap est insérée dans les bicouches lipidiques, proche des têtes polaires avec une solubilité maximale d'environ 3,5 mol%. Les résultats expérimentaux ont été confirmés par des simulations de dynamique moléculaire. Le taux d'encapsulation (ER%) de b-lap dans les liposomes s’est avéré en accord avec sa solubilité maximale dans les lipides. Des complexes b-lap:cyclodextrines ont été formés et incorporés dans le coeur aqueux de liposomes déjà chargés en b-lap. Des ER% plus élevés ont été obtenus, mais avec une efficacité d'encapsulation plus faible. Des tests in vitro sur des lignées cellulaires épithéliales et tumorales de prostate ont démontré la cytotoxicité élevée de b-lap, sans différence toutefois entre b-lap libre et formulée, ni entre les cellules normales et les cellules cancéreuses. / Beta-lapachone (b-lap) is a potential drug with trypanocidal, anti-infectious and anticancer activities with reported selectivity of effects. However, due to its poor water solubility and toxicity, b-lap is not yet applied in therapeutics. We have studied the encapsulation of b-lap in conventional phospholipid vesicles and in-cyclodextrin-in-liposomes. We first analyzed the interaction of b-lap with lipid excipients by surface pressure measurements, DSC and SAXS. They showed that b-lap inserts in lipid bilayers close to polar head groups with a maximum solubility of about 3.5 mol%. The experimental results were supported by molecular dynamics simulations. Encapsulation rates (ER%) of b-lap in liposomes were consistent with b-lap maximal solubility in lipids. B-lap:cyclodextrin complexes were formed and entrapped in the aqueous core of blap-loaded liposomes. Higher ER% were obtained, but with lower encapsulation efficiency. In vitro tests on prostate epithelial and tumor cell lines demonstrated the high cytotoxicity of b-lap, however, without difference between formulated and free b-lap, nor between normal and cancer cells.

<p><strong><em>Background</em></strong>: Chemotherapy and radiation resistance are major causes of failure in cancer treatment. The response to treatment in cancer cells depends on several mechanisms and pathways such as Janus kinases-signal transducers and activators of transcription JAK/STAT pathway. STAT1 was the first described transcription factor in the STAT family. STAT1 is activated by stimulation of signaling proteins such as type II interferon (IFN- γ) and the activated STAT1 translocates from cytoplasm to nucleus. The translocation of STAT1 would result in transcription and changes in the cell activity in terms of apoptosis, proliferation and angiogenesis. Overexpression of STAT1 is suggested to be involved in the development of resistance to chemotherapy and radiation. In this study, we were interested in finding an inhibitor of the STAT1 translocation. <strong><em>Material and methods</em></strong>: The cervix carcinoma cell line, HeLa, was exposed to test compounds for 2h and were then stimulated with IFN-γ to induce the translocation of STAT1. To detect STAT1-protein and the nucleus, the cells were stained with fluorescent antibodies and Hoescht 33324, respectively, using a STAT1 activator assay. The difference in fluorescence intensity between cytoplasm and nucleus was measured using a high-content microscope, ArrayScan^®. <strong><em>Results</em></strong>: β-lapachone and CRA-1 were found to be inhibitors of STAT1 translocation.</p>

Background: Chemotherapy and radiation resistance are major causes of failure in cancer treatment. The response to treatment in cancer cells depends on several mechanisms and pathways such as Janus kinases-signal transducers and activators of transcription JAK/STAT pathway. STAT1 was the first described transcription factor in the STAT family. STAT1 is activated by stimulation of signaling proteins such as type II interferon (IFN- γ) and the activated STAT1 translocates from cytoplasm to nucleus. The translocation of STAT1 would result in transcription and changes in the cell activity in terms of apoptosis, proliferation and angiogenesis. Overexpression of STAT1 is suggested to be involved in the development of resistance to chemotherapy and radiation. In this study, we were interested in finding an inhibitor of the STAT1 translocation. Material and methods: The cervix carcinoma cell line, HeLa, was exposed to test compounds for 2h and were then stimulated with IFN-γ to induce the translocation of STAT1. To detect STAT1-protein and the nucleus, the cells were stained with fluorescent antibodies and Hoescht 33324, respectively, using a STAT1 activator assay. The difference in fluorescence intensity between cytoplasm and nucleus was measured using a high-content microscope, ArrayScan®. Results: β-lapachone and CRA-1 were found to be inhibitors of STAT1 translocation.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Nor-&#946;-lapachone (N&#946;L), a derivative compound obtained from nor-lapachol, is an important anti-cancer prototype. This semisynthetic naphthoquinone has been described as cytotoxic agent against several cancer cell lines. Regrettably, the use of this molecule has been limited due to the poor lipid solubility of compounds from the quinone class. In order to overcome this difficulty, we propose the synthesis of poly (lactide-co-glycolide) (PLGA) microparticles for N&#946;L delivery. In this work, we characterize N&#946;L-loaded microcapsules fabricated using the emulsification/solvent extraction technique. Features such as surface morphology, particle size distribution, zeta potential, optical absorption, Raman and Fourier transform infrared (FT-IR) spectroscopy, thermal analysis data, drug encapsulation efficiency, drug release kinetics and in vitro cytotoxicity were obtained. The microcapsules thus obtained showed appropriate morphological features (regular spherical shape, smooth surface and absence of pores). The presence of the compound inside the microcapsule was confirmed by Raman spectroscopy, and their release showed a biphasic profile. The first phase of the biphasic profile was due to dispersion of drug into the microcapsule surfaces. Quantum DFT calculations were also performed to estimate typical interaction energies between a single N&#946;L molecule and the surface of the microparticles, with predicted binding energies varying from 6 kcal/mol to 52 kcal/mol. Spherical microcapsules with size of 1.03  0.46 &#956;m were produced with encapsulation efficiency of approximately 19%. The N&#946;L-loaded PLGA microcapsules exhibited a pronounced initial burst release. After in vitro treatment with PLGA microcapsules loaded with N&#946;L, it can be seen the incorporation of the microcapsules in the first hour. The cytotoxic activity of N&#946;L against a set of cancer cell lines was investigated. In particular, the use of N&#946;L against prostate PC3M cells was more effective when delivered in PLGA microcapsules compared to the free drug. In vivo assay, Sarcoma 180, reduces tumor weight approximately 37% in treated group with microcapsules loaded with N&#946;L compared to negative control, without significant commitments in biochemical and hematological biomarkers. There was no free N&#946;L absorption by the intraperitoneal route for the group treated with the free drug. These results suggest that PLGA microcapsules loaded with N&#946;L can be used as an alternative delivery system to N&#946;L administration at the prostate cancer therapy. / Nor-&#946;-lapachona (N&#946;L), uma naftoquinona semissintÃtica derivada do nor-lapachol, à um importante protÃtipo anticÃncer descrito como agente citotÃxico contra diferentes tipos de cÃlulas neoplÃsicas. Entretanto, o seu uso tem sido limitado devido à sua baixa lipossolubilidade caracterÃstica de compostos da classe das quinona. A fim de impulsionar sua utilizaÃÃo como agente terapÃutico, foi proposta a preparaÃÃo de micropartÃculas de PLGA (poli-Ãcido lÃctico-co-Ãcido glicÃlido) como sistema de entrega de N&#946;L. Neste trabalho, caracterizamos as microcÃpsulas produzidas pela tÃcnica de emulsificaÃÃo e extraÃÃo por solvente, carregadas com N&#946;L. CaracterÃsticas como morfologia de superfÃcie, distribuiÃÃo de tamanho de partÃcula, potencial zeta, absorÃÃo Ãptica, espectroscopia Raman e infravermelho com transformada de Fourier (FT-IR), dados de anÃlise tÃrmica, eficiÃncia de encapsulaÃÃo, cinÃtica de liberaÃÃo do fÃrmaco e citotoxicidade foram obtidas. As microcÃpsulas assim obtidas apresentaram caracterÃsticas morfolÃgicas adequadas (forma esfÃrica regular, superfÃcie lisa e ausÃncia de poros em sua superfÃcie). A presenÃa de N&#946;L nas microcÃpsulas foi confirmada por espectroscopia Raman e a sua liberaÃÃo apresentou um perfil bifÃsico, sendo que a primeira fase deste perfil corresponde à dispersÃo do fÃrmaco nas superfÃcies de microcÃpsulas. CÃlculos DFT tambÃm foram realizados para estimar as energias de interacÃÃo normal entre um Ãnico N&#946;L molÃcula e a superfÃcie das micropartÃculas, com energias de ligaÃÃo previstos variando de 6 kcal / mol a 52 kcal / mol. MicrocÃpsulas esfÃricas com um tamanho de 1,03  0,46 &#956;m foram produzidas com uma eficiÃncia de encapsulamento de aproximadamente 19% e uma rÃpida liberaÃÃo do conteÃdo encapsulado nas primeiras horas. ApÃs tratamento in vitro com microcÃpsulas de PLGA carregadas com N&#946;L, pode-se observar a incorporaÃÃo das microcÃpsulas na primeira hora. A atividade citotÃxica de microcÃpsulas de N&#946;L contra um painel de linhagens de cÃlulas neoplÃsicas foi investigada. Em particular, a utilizaÃÃo de microcÃpsula contendo N&#946;L contra cÃlulas tumorais de prÃstata da linhagem PC3M mostrou-se mais citotÃxica sendo entregue em microcÃpsulas de PLGA quando comparada com a molÃcula livre. Estudo in vivo em modelo murino, Sarcoma 180, apresenta uma reduÃÃo de aproximadamente 37% do peso do tumor no grupo tratado com microcÃpsulas de N&#946;L comparado ao controle negativo, sem relevantes comprometimentos nos marcadores bioquÃmicos e hematolÃgicos. NÃo houve absorÃÃo de N&#946;L livre por via intraperitoneal pelo grupo tratado com a droga livre. Tais resultados nos sugerem que as microcÃpsulas de PLGA carregadas com N&#946;L podem ser utilizadas como sistema de entrega alternativo na administraÃÃo N&#946;L no tratamento do cÃncer da prÃstata.

A B-lapachona é uma orto-naftoquinona consagrada por suas atividades farmacológicas, principalmente pela antitumoral, porém não há descrição de estudos de biotransformação microbiana da ?-lapachona. Tais estudos podem propiciar a obtenção de novos derivados dessa naftoquinona, além de fornecerem informações importantes sobre seu metabolismo. Muitos trabalhos descrevem que micro-organismos podem catalisar reações mimetizando enzimas humanas. Para o desenvolvimento dessa pesquisa, a ?-lapachona foi obtida por semissíntese a partir do lapachol. Nos processos de biotransformação foram utilizados os fungos filamentosos Mucor rouxii, Cunninghamella elegans, Cunninghamella echinulata, Penicillium crustosum e Papulaspora immersa e as bactérias gastrointestinais Escherichia coli, cultivada em aerobiose e anaerobiose, Lactobacillus acidophilus, Bifidobacterium sp. e cultura mista composta por Lactobacillus acidophilus, Bifidobacterium sp. e Streptococcus salivarius subesp. thermophilus. Com o intuito de estabelecer uma comparação entre o metabolismo microbiano da ?-lapachona com o do seu isômero ?-lapachona, estudos de biotransformação utilizando o fungo M. rouxii foram também conduzidos com a ?-lapachona. Sete derivados de biotransformação da ?-lapachona com o fungo M. rouxii foram identificados, sendo um inédito, cinco já descritos na literatura em um trabalho de metabolismo dessa naftoquinona utilizando sangue de mamíferos e humanos e uma espirobenzolactona relatada em um trabalho de síntese. Outros dois derivados inéditos da ?-lapachona, os quais são regioisômeros conjugados com glicose, foram produzidos após formação de hidroquinona no processo coduzido com o fungo C. elegans. O fungo P. immersa forneceu duas lactonas isoméricas também obtidas com a biotransformação com o fungo M. rouxii. Houve resultados positivos, com detecção de possíveis produtos de biotransformação da ?-lapachona por CLAE-DAD, com as bactérias E. coli em aerobiose e Bifidobacterium sp. No entanto, esses processos apresentaram um baixo rendimento, sendo possível a identificação de apenas um derivado com a E. coli, que também foi obtido com a biotransformação com o fungo M. rouxii. Um derivado glicosilado da ?-lapachona foi produzido após 24 horas de incubação no processo desenvolvido com o fungo M. rouxii, sendo posteriormente convertido em hidroxilapachol, que por sua vez originou a ?-lapachona novamente e também a ?-lapachona, a qual foi metabolizada também. O derivado glicosilado majoritário, obtido da biotransformação com a ?-lapachona com o fungo C. elegans, foi submetido à avaliação da atividade citotóxica frente à linhagem de câncer de mama humano SKBR-3 apresentando IC50 igual a 312,5 ?M, sendo o IC50 da ?-lapachona frente à mesma linhagem igual a 5,6 ?M. O derivado majoritário não apresentou citotoxidade frente à linhagem de fibroblastos normais humanos GM07492-A, enquanto a ?-lapachona foi altamente citotóxica (IC50 igual a 7,25 ?M). Esse mesmo derivado inédito foi também produzido em pequena quantidade no processo desenvolvido com o fungo C. echinulata. Na metabolização microbiana da ?-lapachona ocorreram tanto reações de fase I como de fase II, havendo mimetização do metabolismo de mamíferos, inclusive de humanos, como relatado em trabalhos da literatura. / B-lapachone is considered an important ortho-naphthoquinone by their pharmacological activities, mainly antitumor, but there is no description of microbial biotransformation studies of ?-lapachone. These researches may furnish new derivatives and significant information on its metabolism. Many studies describe that microorganisms can catalyze reactions mimicking human enzymes. ?-lapachone was obtained by semisynthetic procedure from lapachol. Biotransformation processes were carried out using the filamentous fungi Mucor rouxii, Cunninghamella elegans, Cunninghamella echinulata, Penicillium crustosum and Papulaspora immersa and the gastrointestinal bacteria Escherichia coli grown aerobically and anaerobically, Lactobacillus acidophilus, Bifidobacterium sp. and mixed culture with Lactobacillus acidophilus, Bifidobacterium sp. and Streptococcus salivarius subsp. thermophilus. In order to establish a comparison between ?-lapachone microbial transformation and those of its isomer ?-lapachone, biotransformation studies of ?-lapachone were also carried out using M. rouxii. Seven derivatives of ?-lapachone were produced in the process performed by M. rouxii, including one unpublished, five already described in a study of metabolism by mammalian and human blood and one spirobenzolactone reported in a syntetic study. Other two unpublished derivatives of ?-lapachone, which are regioisomers conjugated with glucose, were produced after formation of hydroquinone in the process carried out by C. elegans. P. immersa provided two isomeric lactones also obtained by biotransformation with M. rouxii. Possible biotransformation products were detected by using HPLC-DAD in the processes carried out by the bacteria E. coli under aerobic condition and Bifidobacterium sp. However, these processes exhibited a low yield, and it was possible to identify only one derivative produced by E. coli, which was also obtained in the process performed by M. rouxii. A glycosylated derivative of ?-lapachone was produced by biotransformation with M. rouxii after 24 hours of incubation and subsequently was converted in hydroxylapachol, which in turn gave rise to ?-lapachone again and also to ?-lapachone, which was also metabolized. The major derivative produced in the process carried out by C. elegans was submitted to cytotoxic activity evaluation using human breast cancer cell line SKBR3 showing IC50 312.5 ?M, being the ?-lapachone IC50 5.6 ?M against the same cell line. The major derivative did not show cytotoxicity to normal human fibroblast GM07492-A cell line, while ?-lapachone was highly cytotoxic (IC50 7.25 ?M). The same major derivative was also produced in smaller yield in the process performed by C. echinulata. In the ?-lapachone microbial transformation studies occurred phase I and phase II reactions, mimicking the metabolism of mammals, including humans, as reported in literature.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-05-04T17:34:06Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação-31 05 2016 correções da biblioteca (atualizada).pdf: 2843933 bytes, checksum: 1003152372d1c305370bd514f8eae798 (MD5) / Made available in DSpace on 2017-05-04T17:34:06Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação-31 05 2016 correções da biblioteca (atualizada).pdf: 2843933 bytes, checksum: 1003152372d1c305370bd514f8eae798 (MD5) Previous issue date: 2016-03-09 / A β-lapachona é uma naftoquinona (3,4-dihidro-2,2-dimetil-2H-naftol[1,2-b]pirano-5,6-diona) de peso molecular 243,3 g.mol-1 presente na Tabebuia avellanedae Lor, árvore conhecida como ipê roxo ou pau d’arco roxo. Esta naftoquinona é uma substância que pode ser obtida por uma semi-síntese a partir do lapachol bem como do lomatiol. Este bioativo apresenta múltiplos efeitos farmacológicos, como antibacteriano, antifúngico, antiviral, anti-inflamatório e anti-proliferativo. Segundo o sistema de classificação biofarmacêutica, a β-lapachona (BL ou β-lap) é classificada como classe II apresentando baixa solubilidade e alta permeabilidade. Desta forma, faz necessário um estudo de incremento de solubilidade aquosa. O objetivo deste trabalho foi caracterizar físico-quimicamente os complexos propostos utilizando dois polímeros: o dendrímero bis-MPA (DEN) e a cucurbit[6]urila (CUC). O presente estudo mostra duas metodologias para a formação de complexos entre a β-lapachona /dendrímero (BLD) e β-lapachona /cucurbiturila (BLC), visando o aumento da solubilidade deste fármaco através de interações intermoleculares e suas respectivas caracterizações físico-químicas. O complexo com o dendrímero foi preparado a partir de uma relação massa/massa de 3(fármaco):1 (carreador) variando o tempo de adsorção de 1, 3 e 7 dias. Para os complexos com a cucurbiturila uma relação molar 1:1 foi estabelecida para a formação dos complexos de inclusão com BL, variando o período de adsorção em 1, 3 e 7 dias. Após o período de agitação, os complexos BLD e BLC foram evaporados e secos a temperatura ambiente para posterior caracterização. Os complexos obtidos foram caracterizados por cromatografia líquida de alta eficiência, ultravioleta na região do visível, espectroscopia de absorção vibracional na região do infravermelho, espectrometria de ressonância magnética nuclear de hidrogênio, análise termogravimétrica, análise calorimétrica diferencial, difratometria de raio-X e microscopia eletrônica de varredura. Para o complexo BLD ficou evidente que a variação no tempo de adsorção não interferiu na quantidade de produto formado ou nas propriedades do complexo obtido. Os resultados das caracterizações mostram que o BLDD1 e o BLCD1 foram formados de acordo com os dados espectroscópicos e espectrométricos que demonstram variações nos sinais de BL quando complexado. Houve uma mudança na cristalinidade da BL, a qual era bastante cristalina, para um material mais amorfo com morfologia distinta do fármaco isolado. Os resultados das caracterizações térmicas exibiram, para os complexos, uma maior estabilidade térmica da β-lap complexada (BLDD e BLCD) e menor energia de fusão, quando comparado com β-lapachona livre. Essas informações também sugerem uma diminuição na cristalinidade do complexo o que é indicativo para o incremento de solubilidade. Desta forma propôs-se a caracterização físico-química como um indício de melhoria na solubilidade da β-lapachona através da formação de complexos entre BL e dois carreadores distintos e suas contribuições para estudos de uma possível formulação farmacêutica futura. / The β-naphthoquinone lapachol is a (3,4-dihydro-2,2-dimethyl-2H-naphthol [1,2-b] pyran-5,6-dione) molecular weight of 243,3g / mol present in Tabebuia avellanedae Lor, tree known as purple ipe or pau d'purple bow. This naphthoquinone is a substance that can be obtained by semi-synthesis from lapachol well as lomatiol. This bioactive has multiple pharmacological effects such as antibacterial, antifungal, antiviral, anti-inflammatory and anti-proliferative. According to biopharmaceutical classification system, the β-lapachone (BL or β-LAP) is classified as class II presenting low solubility and high permeability. Thus, an increase aqueous solubility study is necessary. The aim of this study was to characterize chemically-physical proposed complexes using two polymers: the bis-MPA dendrimer (DEN) and cucurbit[6]uril (CUC). This study shows two methods for the formation of complexes between β-lapachone / dendrimer (BLD) and β-lapachone / cucurbituril (BLC), in order to increase the solubility of the drug through intermolecular interactions and their physicochemical characterizations. The complex with the dendrimer was prepared from a mass / mass 3 (drug): 1 (carrier) varying the time for adsorption of 1, 3 and 7 days. For cucurbituril complexes with a molar ratio of 1: 1 was established for the formation of inclusion complexes with BL varying the adsorption period at 1, 3 and 7 days. After the stirring period, the BLC and BLD complexes were evaporated and dried at room temperature for further characterization. The obtained compounds were characterized by high-performance liquid chromatography, ultraviolet into the visible region spectroscopy, vibrational absorption in the infrared, nuclear magnetic resonance spectrometry of hydrogen, thermogravimetric analysis, differential scanning calorimetry, X-ray diffraction and electron microscopy scanning. For BLD complex it was evident that the variation in the adsorption time not interfere in the amount of complex formed or the product properties obtained. The results of the characterizations show that BLDD1 and BLCD1 were formed in accordance with spectroscopic and spectrometric data showing variations in signals when complexed BL. There was a change in the crystallinity of the BL, which was very clear, to a more amorphous material with distinct morphology of the drug alone. The results showed the thermal characterizations for the complex, greater thermal stability of the complexed β-LAP (BLDD and BLCD) and less fusion energy, compared with β-lapachone free. This information also suggests a decrease in crystallinity of the complex which is an indication to increase solubility. Thus it was proposed to physicochemical characterization as a better indication of the solubility of β-lapachone by forming complexes between BL and two different carriers and their contributions to studies of possible future pharmaceutical formulation.

A B-lapachona é uma orto-naftoquinona consagrada por suas atividades farmacológicas, principalmente pela antitumoral, porém não há descrição de estudos de biotransformação microbiana da ?-lapachona. Tais estudos podem propiciar a obtenção de novos derivados dessa naftoquinona, além de fornecerem informações importantes sobre seu metabolismo. Muitos trabalhos descrevem que micro-organismos podem catalisar reações mimetizando enzimas humanas. Para o desenvolvimento dessa pesquisa, a ?-lapachona foi obtida por semissíntese a partir do lapachol. Nos processos de biotransformação foram utilizados os fungos filamentosos Mucor rouxii, Cunninghamella elegans, Cunninghamella echinulata, Penicillium crustosum e Papulaspora immersa e as bactérias gastrointestinais Escherichia coli, cultivada em aerobiose e anaerobiose, Lactobacillus acidophilus, Bifidobacterium sp. e cultura mista composta por Lactobacillus acidophilus, Bifidobacterium sp. e Streptococcus salivarius subesp. thermophilus. Com o intuito de estabelecer uma comparação entre o metabolismo microbiano da ?-lapachona com o do seu isômero ?-lapachona, estudos de biotransformação utilizando o fungo M. rouxii foram também conduzidos com a ?-lapachona. Sete derivados de biotransformação da ?-lapachona com o fungo M. rouxii foram identificados, sendo um inédito, cinco já descritos na literatura em um trabalho de metabolismo dessa naftoquinona utilizando sangue de mamíferos e humanos e uma espirobenzolactona relatada em um trabalho de síntese. Outros dois derivados inéditos da ?-lapachona, os quais são regioisômeros conjugados com glicose, foram produzidos após formação de hidroquinona no processo coduzido com o fungo C. elegans. O fungo P. immersa forneceu duas lactonas isoméricas também obtidas com a biotransformação com o fungo M. rouxii. Houve resultados positivos, com detecção de possíveis produtos de biotransformação da ?-lapachona por CLAE-DAD, com as bactérias E. coli em aerobiose e Bifidobacterium sp. No entanto, esses processos apresentaram um baixo rendimento, sendo possível a identificação de apenas um derivado com a E. coli, que também foi obtido com a biotransformação com o fungo M. rouxii. Um derivado glicosilado da ?-lapachona foi produzido após 24 horas de incubação no processo desenvolvido com o fungo M. rouxii, sendo posteriormente convertido em hidroxilapachol, que por sua vez originou a ?-lapachona novamente e também a ?-lapachona, a qual foi metabolizada também. O derivado glicosilado majoritário, obtido da biotransformação com a ?-lapachona com o fungo C. elegans, foi submetido à avaliação da atividade citotóxica frente à linhagem de câncer de mama humano SKBR-3 apresentando IC50 igual a 312,5 ?M, sendo o IC50 da ?-lapachona frente à mesma linhagem igual a 5,6 ?M. O derivado majoritário não apresentou citotoxidade frente à linhagem de fibroblastos normais humanos GM07492-A, enquanto a ?-lapachona foi altamente citotóxica (IC50 igual a 7,25 ?M). Esse mesmo derivado inédito foi também produzido em pequena quantidade no processo desenvolvido com o fungo C. echinulata. Na metabolização microbiana da ?-lapachona ocorreram tanto reações de fase I como de fase II, havendo mimetização do metabolismo de mamíferos, inclusive de humanos, como relatado em trabalhos da literatura. / B-lapachone is considered an important ortho-naphthoquinone by their pharmacological activities, mainly antitumor, but there is no description of microbial biotransformation studies of ?-lapachone. These researches may furnish new derivatives and significant information on its metabolism. Many studies describe that microorganisms can catalyze reactions mimicking human enzymes. ?-lapachone was obtained by semisynthetic procedure from lapachol. Biotransformation processes were carried out using the filamentous fungi Mucor rouxii, Cunninghamella elegans, Cunninghamella echinulata, Penicillium crustosum and Papulaspora immersa and the gastrointestinal bacteria Escherichia coli grown aerobically and anaerobically, Lactobacillus acidophilus, Bifidobacterium sp. and mixed culture with Lactobacillus acidophilus, Bifidobacterium sp. and Streptococcus salivarius subsp. thermophilus. In order to establish a comparison between ?-lapachone microbial transformation and those of its isomer ?-lapachone, biotransformation studies of ?-lapachone were also carried out using M. rouxii. Seven derivatives of ?-lapachone were produced in the process performed by M. rouxii, including one unpublished, five already described in a study of metabolism by mammalian and human blood and one spirobenzolactone reported in a syntetic study. Other two unpublished derivatives of ?-lapachone, which are regioisomers conjugated with glucose, were produced after formation of hydroquinone in the process carried out by C. elegans. P. immersa provided two isomeric lactones also obtained by biotransformation with M. rouxii. Possible biotransformation products were detected by using HPLC-DAD in the processes carried out by the bacteria E. coli under aerobic condition and Bifidobacterium sp. However, these processes exhibited a low yield, and it was possible to identify only one derivative produced by E. coli, which was also obtained in the process performed by M. rouxii. A glycosylated derivative of ?-lapachone was produced by biotransformation with M. rouxii after 24 hours of incubation and subsequently was converted in hydroxylapachol, which in turn gave rise to ?-lapachone again and also to ?-lapachone, which was also metabolized. The major derivative produced in the process carried out by C. elegans was submitted to cytotoxic activity evaluation using human breast cancer cell line SKBR3 showing IC50 312.5 ?M, being the ?-lapachone IC50 5.6 ?M against the same cell line. The major derivative did not show cytotoxicity to normal human fibroblast GM07492-A cell line, while ?-lapachone was highly cytotoxic (IC50 7.25 ?M). The same major derivative was also produced in smaller yield in the process performed by C. echinulata. In the ?-lapachone microbial transformation studies occurred phase I and phase II reactions, mimicking the metabolism of mammals, including humans, as reported in literature.