Esta tese se fundamenta na necessidade de novos tratamentos para o câncer do colo de útero visando o aumento da adesão dos pacientes aos tratamentos, assim como à qualidade de vida dos mesmos. Nesse sentido, formulações nanotecnológicas foram desenvolvidas com o objetivo de carrear o fármaco imiquimode para um local específico – a mucosa vaginal – esperando gerar melhores desempenhos nesse tratamento quando comparados com a formulação comercial. Três nanoestruturas com morfologias distintas foram propostas visando potencializar o efeito do fármaco em células de câncer cervical (SiHa). As formulações desenvolvidas compreenderam: nanoemulsões (NEimiq), nanocápsulas poliméricas (NCimiq) e nanocápsulas poliméricas revestidas com quitosana (NCimiq-chit). Observou-se que nanocápsulas poliméricas produzidas com poli(ε-caprolactona) apresentaram efeito mais pronunciado frente às células SiHa. Para tanto, essas formulações (NCimiq e NCimiq-chit) foram incorporadas em hidrogéis de quitosana e de hidroxietilcelulose a fim de possibilitar uma melhor futura aplicação para o paciente. Estudos envolvendo mucosa vaginal suína demonstraram que ambas as formulações são mucoadesivas e permeiam a mucosa vaginal. Porém, a formulação produzida com hidrogel de quitosana (NCimiq) apresentou maior desempenho. Esta foi a formulação escolhida para dar continuidade aos estudos deste trabalho, sendo objeto de estudo posterior em cultura de células SiHa a fim de elucidar o mecanismo de ação da mesma. Esses estudos demonstraram que há uma ocorrência de processos combinados de diminuição da viabilidade celular de maneira tempo-dependente e que mecanismos como apoptose, autofagia e parada de ciclo celular estão presentes. Essa formulação (NCimiq) apresentou porcentagens de morte celular significativas, mesmo utilizando baixas concentrações do fármaco. Portanto, os achados desta tese constataram que nanoestruturas modulam efetivamente a interação do fármaco com as células. / This thesis deals with the need of new treatments for cervical cancer in order to increase the adherence of patients to the treatment as well as to improve their quality of life. In this sense, nanotechnological formulations were developed to carry imiquimod to a specific site – the vaginal mucosa – expecting to obtain better performance than the commercial drug in the cervical cancer treatment. Three nanostructures with different morphologies were proposed to potentilize the drug effect on cervical cancer cells (SiHa). The developed formulations are: nanoemulsions (NEimiq), polymeric nanocapsules (NCimiq) and polymeric nanocapsules coated with chitosan (NCimiq-chit). It was observed that polymeric nanocapsules produced with poly(ε-caprolactone) presented a stronger effect against SiHa cells. Therefore, formulations NCimiq and NCimiq-chit were incorporated into hydrogels of chitosan and hydroxyethylcellulose to enable a better future application on patients. The studies of this thesis involving porcine vaginal mucosa demonstrated that both formulations are mucoadhesive and that they provided a good drug permeation. However, the formulation produced with chitosan hydrogel (NCimiq) showed a better performance. This formulation was therefore chosen to follow the next steps of this work, conducted in SiHa cell culture to elucidate its action mechanism. This study demonstrated that there is an occurrence of combined processes of decreasing cell viability in a time-dependent type. The study also showed that mechanisms such as apoptosis, autophagy and cell cycle arrest are simultaneously present. The formulation NCimiq presented a significantly percentage of cellular death, even when low concentrations of the drug were used. Consequently, the findings of this thesis indicate that nanostructures effectively modulate the interaction of the drug with the cancer cells.

As Ceratoses Actínicas (CA) são lesões hiperceratóticas, displásicas de pele. Estão comumente localizadas em áreas expostas ao sol como couro cabeludo, face e antebraços. Acredita-se que a radiação ultravioleta (RUV) cumulativa seja o maior fator etiológico, considerando-se, também, a imunossupressão e infecção pelo papilomavírus humano(HPV) fatores contribuintes importantes. Existe o potencial de uma CA se transformar em Carcinoma Espinocelular (CEC) e, dessa forma, indica-se tratamento para as referidas lesões. Terapias tradicionais como crioterapia, curetagem e eletrocoagulação, medicações tópicas como 5-fluorouracil (5-FU) são ainda habituais. Porém novas opções como terapia fotodinâmica, diclofenaco e imiquimod 5% creme apresentam boa eficácia e perfil menor de efeitos colaterais, embora possam representar custo maior. A Terapia Fotodinâmica (TFD) envolve o uso de um agente fotossensibilizante, oxigênio e luz de comprimento de onda específico para causar morte celular. O fotossensibilizante geralmente utilizado é o ALA (ácido aminodeltalevulínico) ou seu éster metilaminolevulinato (MAL). No tecido lesionado, esses são convertidos em porfirinas fotoativas (PFAs) por enzimas da via biossintética do heme. A ativação é realizada por meio de luzes de comprimentos de onda que variam de 405 nm a 635 nm. Células displásicas ou neoplásicas produzem maior quantidade de porfirinas que os queratinócitos normais, sendo destruídas durante a aplicação da luz. O imiquimod 5% creme é um imunomodulador que estimula a resposta imune inata através da indução, síntese e liberação de citocinas. Isso resulta em efeitos antitumorais e antivirais indiretos. Seu uso tópico é eficaz e liberado para o tratamento de CA, Carcinoma Basocelular (CBC) superficial, Doença de Bowen (DB) e verrugas vulgares. Este trabalho tem como objetivo comparar duas recentes opções de tratamento para CA, a Terapia Fotodinâmica com metilaminolevulinato e o imiquimod 5% creme, por não existirem, na literatura atual, estudos comparativos dessas duas opções de tratamento. Foram selecionadas 12 pacientes com CAs que foram submetidas primeiramente à 1 sessão de TFD com MAL num lado da face e, 1 mês após, iniciaram o tratamento com imiquimod 5% creme aplicado no lado contralateral, duas vezes na semana, durante 16 semanas. A randomização foi realizada para determinar a hemiface para cada tratamento. Na primeira semana após a realização da TFD e mensalmente durante o tratamento com imiquimod, as pacientes foram avaliadas em relação aos efeitos colaterais dos tratamentos. Seis meses após entrarem no estudo, ambos os tratamentos foram analisados por um investigador cego para sua eficácia, tolerabilidade e seu resultado cosmético. Previamente ao tratamento as pacientes apresentaram um total de 245 lesões de CAs, sendo 120 lesões no lado submetido à TFD e 125 no lado tratado com imiquimod. Após o tratamento o número total de lesões diminuiu para 34 no lado tratado com TFD e para 30 no lado tratado com imiquimod, respectivamente. Não foram observadas diferenças estatisticamente significativas na eficácia de ambos os tratamentos e na frequência de efeitos colaterais. Entretanto, os pacientes, significativamente preferiram o tratamento com a terapia fotodinâmica, talvez pela rapidez do método, comparado com a aplicação tópica do creme de imiquimod a 5%. / Background: Actinic keratosis (AK) represents an initial process that may lead to in situ or invasive squamous cell carcinoma. The importance of its early diagnosis and treatment is well-established. There are several effective options available for the treatment of actinic keratosis, including topical imiquimod 5% cream and photodynamic therapy (PDT). Objetive: To compare the efficacy and patient preference between topical MAL-PDT and imiquimod 5% cream for the treatment of AK. Methods: Twelve patients, with a total of 245 lesions, underwent treatment with MAL-PDT and imiquimod 5% cream. Randomization was performed to determine the hemiface (right or left) for each therapy. First, patients were submitted to MAL-PDT. After one month, they started to use imiquimod on the opposite side of the face, twice a week, for 16 weeks. Six months after entering the study, both treatments were analyzed by a blinded investigator for their effectiveness, tolerability and cosmetic result. Results: Both treatments showed a good therapeutic response. 72% of the lesions treated with MAL-PDT were completely cleared, and 76% of those treated with imiquimod. The mean size of the residual lesions after the treatments was similar. Ten patients (83%) preferred MAL-PDT rather than imiquimod. (p: 0.03). Conclusions: Both MAL-PDT and imiquimod are effective in clearing AKs. Our results showed similar efficacy and good cosmetic outcomes with both treatments. However, a significant percentage of the subjects preferred MAL – PDT.

Le psoriasis est une dermatose inflammatoire chronique affectant 2 à 3 % de la population européenne. Les cytokines pro-inflammatoires ont un rôle crucial dans la pathogénèse du psoriasis. Parmi les voies de signalisation cytokinique, l'étude de la voie JAK/STAT a conduit au développement de traitements systémiques efficaces. Cependant, les essais cliniques évaluant les inhibiteurs JAK/STAT impliquent en grande majorité des administrations orales, la voie topique restant marginale. Le développement de formulations innovantes pour application topique contenant des inhibiteurs JAK/STAT semble être une stratégie prometteuse dans le cadre du psoriasis. Les nanoparticules de poly(acide lactique) (NP PLA) développées au laboratoire ont été étudiées pour la délivrance topique de principes actifs. Ce sont des vecteurs efficaces, qui s'accumulent dans les follicules pileux. De plus, l'encapsulation d'actifs dans des NP PLA permet une libération spécifique au niveau du site d'action, et donc une réduction de la toxicité. L'objectif de ce travail est d'élaborer des formulations semi-solides de NP PLA contenant un inhibiteur JAK/STAT pour le traitement topique du psoriasis, tout en caractérisant un modèle in vivo de psoriasis induit par applications d'Imiquimod. Cette caractérisation des lésions psoriasiformes permettra l'évaluation in vivo des nanoformulations topiques contenant des inhibiteurs JAK/STAT. Cinq formulations ont été élaborées puis caractérisées afin de répondre aux attentes galéniques pour une application topique. L'intégrité des NP PLA a été vérifiée, et la pénétration/perméation de molécules modèles à travers de la peau de souris inflammée a été évaluée / Psoriasis is a chronic inflammatory skin disease, affecting 2 to 3 % of European population. Inflammatory cytokines play a crucial role in the pathogenesis of psoriasis. Among cytokines signaling pathways, JAK/STAT pathway has been widely investigated, leading to the development of efficient systemic agents. However, current clinical trials evaluating JAK/Sat inhibitors mainly involve oral administrations, with few investigations on topical route. Developing innovative drug delivery systems for topical application of JAK/STAT inhibitors seems a promising strategy for psoriasis treatment. Poly(lactic acid) nanoparticles (PLA NPs) developed in the laboratory have been widely investigated for topical drug delivery and are efficient carriers for local dermatotherapy, especially through hair follicles. Moreover, drug encapsulation in PLA NPs for topical delivery allows a specific delivery to the site of action, and thus a decreased toxicity.The aim of this work was to elaborate semi-solid formulations of PLA NPs containing JAK/STAT inhibitors for topical treatment of psoriasis, while characterizing an in vivo model of Imiquimod-induced psoriasis in mice. This characterization of psoriasis-like skin lesions in Imiquimod treated mice provided key tools for in vivo evaluation of topical nanoformulations containing JAK/STAT inhibitors. Five formulations have been developed and then characterized in order to meet galenic criteria for topical drug administration. PLA NPs integrity was assessed, and penetration/permeation profiles of model dugs through inflamed mice skin were determined

Toxoplasma gondii est un parasite répandu, ayant un impact médical et vétérinaire. Chez les hôtes intermédiaires, les tachyzoïtes et les bradyzoïtes sont responsables de la toxoplasmose aiguë (TA) et chronique (TC), respectivement. Sous la réponse immunitaire, la TA évolue en TC, se manifestant par des kystes latents dans le cerveau et les muscles squelettiques. De plus, une forte corrélation existe entre la TC et plusieurs neuropathologies et cancers. Chez les patients immunodéprimés, la TC peut être réactivée et conduire à une maladie potentiellement fatale. Les traitements actuels ciblent principalement les TA, et présentent plusieurs effets secondaires. Nous nous sommes concentrés sur la TC et la compréhension de ses mécanismes moléculaires. Nous avons d’abord étudié l’efficacité de l’imiquimod contre la TA et la TC. Au cours de la TA, l'imiquimod a entraîné le recrutement de cellules T dans le péritoine et la rate de souris traitées et a considérablement diminué le nombre de kystes cérébraux lors de l'établissement de la TC. Remarquablement, le gavage de souris avec les kystes cérébraux restants chez des souris traitées à l'imiquimod n'a pas pu induire de TC. Après l'établissement de la TC, nous avons démontré que l'imiquimod réduisait considérablement le nombre de kystes cérébraux chez les souris chroniquement infectées et augmentait les récepteurs Toll-Like 11 et 12, qui se lient à une protéine du tachyzoïte, la profiline. Parallèlement, l’expression de TLR-7 augmentait, probablement par son agoniste, l'imiquimod. L'imiquimod induit une interconversion, comme l'indiquent la diminution du taux de protéine P21 et l'augmentation du taux de protéine P30, exprimées exclusivement et respectivement chez les bradyzoïtes et les tachyzoïtes. Les voies en aval de TLR-11/12 ont été activées via la voie MyD88 de signalisation, entraînant une induction ultérieure de la réponse immunitaire. In vitro, l'imiquimod n’affecte pas la souche Toxoplasma dépourvue de profiline, suggérant un rôle via le complexe Profilin/TLR-11/12. Enfin, le traitement par l'imiquimod a régulé positivement les transcrits des ligands 9 (CXCL9) et 10 (CXCL10), connus pour induire le recrutement de lymphocytes T dans des foyers réactivés du Toxoplasme afin d'éliminer l'infection.Ensuite, nous nous sommes concentrés sur les mécanismes moléculaires impliqués dans la TA et particulièrement dans la TC. Nous avons caractérisé P18, un membre de la superfamille SRS. Lorsque nous avons supprimé P18, la virulence était atténuée au cours de la TA, dû à un échappement plus rapide des tachyzoïtes du péritoine de souris, parallèle à un recrutement significatif de cellules dendritiques. De manière concomitante, moins de tachyzoïtes étaient détectés dans la rate, tandis que plus de parasites ont atteint le cerveau de souris infectées. L’élimination de P18 a augmenté le nombre de kystes de bradyzoïtes in vitro et dans le cerveau de souris infectées. Une expression induite de cytokines, notamment CXCL9 et 10, a également été observée. L’immunosuppression de souris KO P18 infectées a retardé la réactivation. L’infection orale de souris immunodéficientes ayant des macrophages fonctionnels a montré un prolongement de survie, contrairement aux souris n’ayant pas de macrophage, soulignant un rôle de l'IFN-g dans l’interconversion. Collectivement, ces données confirment le rôle de P18 dans la modulation de la réponse immunitaire, facilitant le passage des tachyzoïtes dans le cerveau et favorisant la formation de kystes. P18 joue également un rôle central dans la réactivation et la dissémination de parasites de manière dépendante de l'IFN-g. Dans l'ensemble, nous avons montré le potentiel thérapeutique prometteur de l'imiquimod contre la toxoplasmose et caractérisé le rôle de P18 dans l'immunomodulation afin de contrôler la dissémination et l'interconversion. Notre étude ouvre la voie à de nouvelles approches thérapeutiques contre la toxoplasmose, sa persistance et sa réactivation. / Toxoplasma gondii is a prevalent parasite of medical and veterinary impact. In intermediate hosts, tachyzoïtes and bradyzoïtes are responsible for acute and chronic toxoplasmosis (AT and CT), respectively. In immunocompetent patients, AT evolves, due to the host immunity, into a persistent CT, which manifests as latent tissue cysts in the brain and skeletal muscles. CT correlates with several neuro-pathologies and cancers. In immunocompromised patients, CT may reactivate and poses a life threatening condition. Current treatments primarily target AT, are limited to general anti-parasitic/anti-bacterial drugs, and associate with several limitations. Here, we focused on targeting CT and understanding its molecular mechanisms. First, we explored the efficacy of Imiquimod against AT and CT. During AT, Imiquimod led to recruitment of T cells to peritoneum and spleen of treated mice and significantly decreased the number of brain cysts upon establishment of CT. Remarkably, gavage of mice with the remaining brain cysts from Imiquimod treated mice, failed to induce CT. Post-establishment of CT, we demonstrated that Imiquimod sharply reduced the number of brain cysts in chronically infected mice, and significantly increased Toll-Like Receptors 11 and 12. These TLRs are usually expressed by dendritic cells and monocytes, and bind a tachyzoïte actin-binding protein, profilin. Concomitantly, TLR-7 was upregulated, likely by its agonist Imiquimod. Imiquimod induced interconversion as documented by the decreased protein levels of P21, and increased protein levels of P30, exclusively expressed in bradyzoïtes and tachyzoïtes respectively. Pathways downstream from TLR-11/12 were activated, through MyD88 dependent TLR signaling, which resulted in subsequent immune response induction. In vitro, Toxoplasma strain lacking profilin, does not respond to Imiquimod, suggesting a role through Profilin/TLR-11/12. Finally, Imiquimod treatment upregulated the transcript expression levels of Chemokine (C-X-C motif) ligand 9 (CXCL9) and 10 (CXCL10), known to induce T cell recruitment to reactivated Toxoplasma foci to clear the infection.Then, we focused on molecular mechanisms involved in AT and notably CT. We characterized P18, a Surface-Antigen 1 (SAG-1) Related Sequence (SRS) superfamily member. When we deleted P18, the virulence was attenuated during AT. Indeed, P18 depletion led to a faster clearance of the parasites from the peritoneum of mice, paralleled by a substantial recruitment of dendritic cells, presumably a vehicle for tachyzoïte dissemination. Concomitantly, a lower number of tachyzoïtes was detected in the spleens while a higher number of parasites reached the brains of infected mice. P18 depletion increased the number of bradyzoïte cysts, in vitro and in the brains of infected mice. An induced expression of cytokines/chemokines, including CXCL9 and 10 was also observed. Immunosuppression of infected mice with KO P18, delayed reactivation. Oral infection of Severe Combined Immunodeficiency (SCID) (with IFN-g secreting macrophages), and NOD/Shi-scid/IL-2Rgnull (NSG) mice (lacking IFN-g), showed a significant prolonged survival in infected SCID but not NSG mice. This underlines a role for IFN-g in the conversion from bradyzoïtes to tachyzoïtes. Collectively, these data support a role of P18 in orchestrating the immune response, which ultimately facilitates tachyzoïte trafficking to the brain and favors cyst formation. P18 plays also a central role in parasite reactivation and dissemination in an IFN-g dependent fashion.Altogether, we showed the promising therapeutic potential of Imiquimod against toxoplasmosis and characterized P18 role in immunomodulation to control dissemination and interconversion. Our study paves the path towards new therapeutic approaches against toxoplasmosis. It tackled key questions pertaining to establishment, maintenance and reactivation of CT and should result in a comprehensive solution to this endemic disease.

A psoríase é uma doença inflamatória crônica, de alta incidência mundial, caracterizada por lesões de pele e prurido. Achados prévios do grupo mostraram que a administração i.p. do doador lento de sulfeto de hidrogênio (H2S), GYY4137, inibiu significativamente a inflamação cutânea e coceira em animais com psoríase. Assim, neste estudo avaliou-se o efeito terapêutico de uma nanodispersão tópica contendo GYY4137 sobre a psoríase induzida por imiquimode em camundongos e, comparou-se farmacologicamente o efeito do GYY4137 versus dexametasona. Animais controle ou psoríase foram tratados (1 e 2x/dia) com a nanodispersão (65 mg) contendo GYY4137, dexametasona ou apenas veículo. A aplicação tópica da nanodispersão com o GYY4137 (4%) 2x/dia, foi mais efetiva e reduziu de forma significativa o rubor, espessura, células sanguíneas totais, MPO e níveis de IL-6 e IL-1β. Os efeitos farmacológicos promovidos pelo H2S foram semelhantes aos da dexametasona, porém, a nanodispersão contendo GYY4137 demonstrou efeitos sistêmicos menos pronunciados quando comparada ao glicocorticóide. / Psoriasis is a chronic inflammatory disease, with high incidence worldwide and, characterized by skin lesions and intense itching. Previous findings of this group showed that systemic administration of the slow donor release of hydrogen sulfide (H2S), GYY4137, significantly inhibited skin inflammation and itching in mice with psoriasis. Thus, this study evaluated the therapeutic effect of a topical nanodispersion containing GYY4137 on the Imiquimod-induced psoriasis mice model and compared pharmacologically the effect of GYY4137 versus dexamethasone. Control or psoriasis animals were treated (1 and 2x/day) with nanodispersion (65 mg) containing GYY4137, dexamethasone, or just vehicle. Topical application of the nanodispersion with GYY4137 (4%) 2x/day was more effective and significantly reduced redness, thickness, total blood cells, MPO, and IL-6 and IL-1β levels. The pharmacological effects caused by H2S were similar to those of dexamethasone, however, the nanodispersion containing GYY4137 demonstrated less systemic effects when compared to glucocorticoid.

Mucoadhesive drug delivery systems which are being used from 1980’s to avoid first pass metabolism of drugs, commercially exist for only systemic drug delivery with fast erosion times (15-60 min), that may not be appropriate for local oral disorders. The goal of this research was to develop and characterize mucoadhesive films with flexibility of carrying different drugs and proteins and provide sustained release for local treatment of oral disorders. Mucoadhesive films composed of polyvinylpyrrolidone (PVP) and carboxymethlycellulose (CMC) were formulated with imiquimod, an immune response modifier. Problems such as solubilization of imiquimod to increase drug loading, uniformity in films and total amount of drug released into supernatants were addressed by use of acetate buffer after investigating multiple methods. Subsequently, other relevant properties of mucoadhesive systems, such as adhesion (shear, pull-off), tensile properties, swelling profiles, transport kinetics, and subsequent changes in release profiles as a function of film composition were characterized. The potential of the system for local retention of imiquimod, determined in oral mucosa of hamsters showed time dependent decrease in imiquimod amount through 12 hours, with no traces of drug in blood. Further testing in humans revealed that the residence time of the mucoadhesive films depended on the application site, increasing in the order of tongue < cheek < gingiva. In parallel, mucoadhesive films loaded with epidermal growth factor (EGF) were developed to promote treatment of oral mucosal wounds. Bioactivity was tested in vitro on buccal tissues by creating a wound followed by application of films. Although EGF-loaded films did not accelerate wound healing, but rather elicited a hyperparakeratotic response. In vitro buccal tissues may not be appropriate for testing the effects of EGF in wound healing without incorporation of other biochemical factors. Overall, a mucoadhesive system capable of delivering bioactive small molecules and proteins in sustained manner was developed in this work. A thorough understanding of the system properties was achieved to further tune for future applications. In vitro studies and in vivo studies in hamsters and humans clearly showed the potential and usefulness of the system to translate in to clinic for treatment of oral precancerous lesions.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Petr Tirala Supervisor: PharmDr. Barbora Švecová, Ph.D. Title of thesis: Formulation and (trans)dermal application of liposomes containing imiquimod Imiquimod (IMQ), a substance belonging to the class of heterocyclic imidazoquinolines, shows significant immunomodulatory effects after topical administration, which is used to treat a variety of viral and malignant diseases of the skin. IMQ is currently used in clinical practice in the form of cream Aldara® containing 5% of active substance, which is unstable and irritating and after removal from the skin IMQ poses an ecological load for the environment. The aim of this thesis was preparation of liposomes for topical administrativ containing lower - 1% amount of IMQ and evaluation of penetration of IMQ into human skin in vitro. To improve the properties of liposomes and promote patency of the active ingredient through the skin barrier to the deeper skin layers various additives were used. Permeation experiments were carried out in Franz diffusion cells on the human skin in order to create the conditions that are as physiological as possible. Amount of IMQ was determined in the uppermost layer of the skin, epidermis, dermis, acceptor phase...

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2018-06-22T19:23:21Z No. of bitstreams: 2 Tese - Marina França Dias - 2018.pdf: 4770115 bytes, checksum: 86e73a57e2ce0a02cb1666dfe5988ce3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-06-27T11:09:43Z (GMT) No. of bitstreams: 2 Tese - Marina França Dias - 2018.pdf: 4770115 bytes, checksum: 86e73a57e2ce0a02cb1666dfe5988ce3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-27T11:09:43Z (GMT). No. of bitstreams: 2 Tese - Marina França Dias - 2018.pdf: 4770115 bytes, checksum: 86e73a57e2ce0a02cb1666dfe5988ce3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-11 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Skin cancer has the highest incidence among all types of neoplasms and the tendency is the increasing number of new cases in next decades, wich makes necessary new modalities and treatment options. Chemotherapeutic agent imiquimod is used in treatment of disease, but the high occurrence of local and systemic adverse effects associated with its use as well as its low skin permeation impair adherence and therapeutical effectiveness, respectively. The aim of the present work was evaluate the antitumor activity of nanocapsules imiquimod-loaded compared to its commercial product in murine model of skin cancer. Polymeric nanocapsules containing imiquimod, in the absence and presence of chitosan coating, were obtained by the precipitation of preformed polymer technique and characterized by encapsulation efficiency, size, zeta potential, pH, morphology (transmission electron microscopy and scanning), optical scanning spectrophotometry and in vitro release through dialysis membrane in pH 5.6 buffer. Presence of chemical interactions between formulation components was evaluated by thermogravimetric analysis and infrared spectroscopy. Detection of crystalline structures was performed by X-ray diffractometry. The nanocapsules and commercial imiquimod formulation antiagiogenic activity was determined in a chicken embryo chorioallantoic membrane model. Cutaneous permeation of nanocapsules imiquimod-loaded and commercial imiquimod was determined in Swiss albino mice. The chemopreventive activity of colloidal dispersions and the commercial imiquimod was evaluated through the tumoral inhibition promoted by these treatments in a multi- stage model of chemical carcinogenesis in Swiss mice. Encapsulation efficiency, mean diameter, zeta potential and pH of uncoated nanocapsules imiquimod-loaded were 92.5% ± 0.4; 249 ± 22.4 nm; -40.1 mV ± 3.7 and 5.4 ± 0.01 respectively, whereas for nanocapsules with cationic coating the parameters found were: 88.6 ± 2.3%; 287.0 ± 12.6 nm; + 11.3 ± 0.5 mV and 3.7 ± 0.0, respectively. The formulations morphology obtained by scanning and transmission electron microscopy images confirmed the presence of nanocapsules. In the stability analysis by optical scanning spectrophotometry (Turbiscan), all dispersions obtained a backscattering variation less than 2% over 5 weeks and were considered stable. In the in vitro release assay, imiquimod-loaded nanoparticles obtained slower release of the drug compared to free and commercial imiquimod. There were chemical interactions between chitosan and other formulation components evaluated by thermogravimetric and infrared studies. No crystalline structure was detected by the X-ray diffraction technique for the coated and uncoated imiquimod formulation. The dispersion of nanocapsules containing imiquimod presented antiangiogenic activity superior than commercial formulation in chicken embryo chorioallantoic membrane model. Nanocapsules containing imiquimod both uncoated and coated with chitosan presented cutaneous permeation in deep layers of the skin and inhibition in the chemically induced carcinogenicity process superior than tumor control group and groups treated with placebo nanoparticles while the commercial formulation presented retention of the drug in superficial layers of the skin and did not obtain a statistically significant reduction in the number of papillomas formed compared to the carcinogenic control. These results allow to conclude, therefore, that stable nanocarreadores were obtained and the chemopreventive activity and the antiangiogenic effect of these systems represent a promising alternative for the treatment of cutaneous neoplasias. / O câncer de pele é o de maior incidência dentre todos os tipos de neoplasias e a tendência é o aumento crescente do número de novos casos nas próximas décadas, tornando necessárias novas modalidades e opções de tratamento. O agente quimio-terápico imiquimode é utilizado no tratamento da doença, mas a elevada ocorrência de efeitos adversos locais e sistêmicos associados ao seu uso bem como sua baixa permeação cutânea prejudicam a adesão e a efetividade terapêutica, respectiva-mente. O objetivo do presente trabalho foi comparar a atividade quimiopreventiva do imiquimode veiculado em nanocápsulas com a da sua forma comercial, em modelo murino de câncer de pele. Nanocápsulas poliméricas contendo o imiquimode, na ausência e presença de revestimento de quitosana, foram obtidas pela técnica de precipitação do polímero pré-formado e caracterizadas quanto à eficiência de encap-sulação, ao tamanho, ao potencial zeta, ao pH, à morfologia (microscopia eletrônica de transição e varredura), à espectrofotometria de varredura óptica e à liberação in vitro através de membrana de diálise em tampão pH 5,6. A presença de interações entre componentes da formulação foi avaliada por meio de análises termogravimé- tricas e por espectroscopia na região do infravermelho. A detecção de estruturas cristalinas nas formulações foi realizada por difratometria de raios X. A atividade antiangiogênica das nanocápsulas obtidas, bem como da formulação comercial do imiquimode, foi determinada em modelo de membrana corioalantoica de embrião de galinha. A permeação cutânea das nanopartículas e do imiquimode comercial foi determinada em camundongos Swiss. A atividade quimiopreventiva das nanocápsulas obtidas e a do imiquimode comercial foi avaliada por meio da inibição tumoral promovida por esses tratamentos em modelo de carcinogênese química em camundongos Swiss. A eficiência de encapsulação, o diâmetro médio, o potencial zeta e o pH das nanocápsulas não revestidas contendo imiquimode foram respectivamente de 92,5% ± 0,4; 249 ± 22,4 nm; -40,1 mV ± 3,7 e 5,4 ± 0,01. Enquanto que, para as nanocápsulas com recobrimento catiônico os parâmetros encontrados foram 88,6 ± 2,3%; 287,0 ± 12,6 nm; +11,3 ± 0,5 mV e 3,7 ± 0,0, respectivamente. A morfologia das formulações, obtida pelas imagens de microscopia eletrônica de varredura e de transmissão, confirmou a presença de nanocápsulas. Nas análises de estabilidade por espectrofotometria de varredura óptica (Turbiscan), todas as dispersões obtiveram variação de retroespalhamento inferior a 2%, no período de cinco semanas, sendo consideradas estáveis. No ensaio de liberação in vitro, as nanopartículas contendo imiquimode obtiveram liberação mais lenta do fármaco comparativamente ao imiquimode livre (em solução) e ao imiquimode comercial. Foram verificadas interações entre a quitosana e os demais componentes das formulações nos estudos termogravimétricos e na espectroscopia na região do infravermelho. Não foi detectada presença de estrutura cristalina com emprego da técnica de difratometria de raios X para a formulações contendo imiquimode com e sem revestimento. A dispersão de nanocápsulas contendo imiquimode apresentou atividade antiangiogênica superior à da formulação comercial em modelo de membrana corioalantoica de embrião de galinha. As nanocápsulas contendo imi-quimode com e sem revestimento apresentaram permeação cutânea em camadas profundas da pele e inibição no processo de carcinogênese quimicamente induzido superior à do grupo-controle tumoral e à dos grupos tratados com nanopartículas placebo, enquanto a formulação comercial apresentou retenção do fármaco em camadas mais superficiais da pele e não obteve redução estatisticamente significativa no número de papilomas formados em relação ao controle cancerígeno. Esses resultados permitem concluir, portanto, que nanocarreadores estáveis foram obtidos e que a atividade quimiopreventiva e o efeito antiangiogênico desses sistemas representam uma alternativa promissora para o tratamento de neoplasias cutâneas.

Orientador: Francisco Benedito Teixeira Pessine / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T03:10:58Z (GMT). No. of bitstreams: 1 Guedes_LucianadeSouza_M.pdf: 5716470 bytes, checksum: 363ce3e7be504393ef2dcd6d349ed421 (MD5) Previous issue date: 2014 / Resumo: O Imiquimod é um fármaco com propriedades imunomodulatórias, disponível na forma de creme para tratamento tópico de certas patologias da pele como carcinoma basocelular e ceratose actínica. O contato direto do medicamento com a pele pode causar desconforto durante o tratamento. Uma das alternativas para minimizar os efeitos adversos é a utilização de sistemas carreadores que evitam o contato direto do agente com a pele. A ?-ciclodextrina foi empregada como agente carreador neste estudo e o complexo de inclusão obtido foi caracterizado em solução e no estado sólido. As técnicas espectroscópicas de absorção UV/Vis e ressonância magnética nuclear foram utilizadas para caracterizar o complexo de inclusão em solução. Essas técnicas permitiram obter a constante de equilíbrio de associação (Kc), a estequiometria do complexo formado e evidências de sua geometria. A caracterização do complexo no estado sólido foi realizada por calorimetria diferencial de varredura, termogravimetria, difração de raios X e microscopia eletrônica de varredura. Os resultados indicaram a presença de estrutura amorfa que pode ser atribuída a amorfização dos componentes devido à liofilização do complexo e também à formação do complexo de inclusão. A estrutura tridimensional do complexo obtida por modelagem molecular apresentou concordância com os resultados experimentais observados pela técnica de RMN 1H ROESY 1D. Os valores baixos de Kc e da eficiência de complexação indicam fraca interação entre o Imiquimod e a ?-ciclodextrina. Os resultados demonstram que a ?-ciclodextrina não é o sistema carreador mais adequado para aumentar a solubilidade aquosa do Imiquimod / Abstract: Imiquimod, an immune response modifier, is used for topical treatment of basal cell carcinoma and actinic keratosis. Local skin reactions can occur in the treatment area. An alternative to minimize adverse events is to use drug delivery systems which avoid drug skin contact. ?-cyclodextrin was used as drug delivery system in this study and the inclusion complex was characterized in solution and in solid state. UV/Vis absorption spectroscopy and nuclear magnetic resonance were employed to study the inclusion complex in solution. Those techniques allowed to obtain the equilibrium constant (Kc), the complex stoichiometry and insight on the geometry. The inclusion complex characterization in solid state was performed by differential scanning calorimetry, thermogravimetry, X-ray diffraction and scanning electron microscopy. The results suggest the presence of amorphous structure which can be attributed not only to components amorphization due to lyophilization but also to complex formation. The complex tridimensional geometry was obtained by molecular modeling and agrees with the experimental results observed by NMR 1H ROESY 1D experiments. The low values of Kc and complex efficiency suggest weak interaction between Imiquimod and ?-cyclodextrin. In addition to those, the results showed that ?-cyclodextrin is not the most suitable drug delivery system to improve Imiquimod aqueous solubility / Mestrado / Físico-Química / Mestra em Química

Vulval intraepithelial neoplasia (VIN) is a distressing, premalignant condition, frequently associated with type HPV16 infection, with increasing incidence in younger women. Traditional surgical treatment is sub optimal. Several different clinical studies influencing local and/or systemic immunity to HPV have been reported. Imiquimod, an immune response modifier, can stimulate local innate immunity and also drive an adaptive immune response. Therapeutic HPV vaccines are designed to generate cell-mediated immunity against HPV infected cells. The rationale of this study was that local imiquimod treatment, in addition to having a direct effect on VIN could also provide an immunological platform for the therapeutic HPV vaccination to achieve an enhanced and durable response. In this phase II trial, we used a combination of imiquimod and vaccination with TA-CIN (HPV16 E6E7L2 fusion protein). Women with biopsy proven high-grade VIN were recruited. Imiquimod treatment for 8 weeks was followed by three i.m. injections of TA-CIN. The objectives were to measure lesion size, histology, lesion HPV status, symptoms, immune responses before and after treatment as well as safety, toxicity, and tolerability. Lymphoproliferation to HPV antigens was used to analyse immunity to HPV before and after treatment. Local immune factors (CD4, CD8 and T regulatory cells) were assessed by immunofluorescence. 74, 85 and 79% of women had a ≥50% reduction in the size of lesion and 32, 58 and 63% had regression of VIN on histology at weeks 10, 20 and 52 respectively. At baseline, 79% had moderate to severe symptoms compared to 21% at week 52(P=0.01). Of the women who showed histological responses at week 52, 5/10 also cleared their HPV 16 infection. Follow-up for an average of 15 months showed 84% of patients with a ≥50% reduction in lesion size. Treatment was well tolerated. A significant post vaccination increase in proliferation to TA-CIN and its components was associated with histological responders (P=0.008) but not the non-responders (P=0.7). In the group as a whole, significant increases in the number of CD4, CD8 and T regulatory cells (Treg) were evident by week 20 compared to baseline (P=0.03, P=0.01, P= 0.04 respectively); At week 20, the increased CD4 and CD8 density was significantly associated only with the histological responders (P=0.03; P= 0.03) while increased Treg density was associated with only non-responders (P=0.05). Intralesional Treg density was significantly higher in non-responders compared to responders pre and post treatment (P=0.01, 0.05 respectively). This study demonstrated that imiquimod followed by vaccination achieved histological clearance of VIN at 52 weeks in almost 60%. Higher pre-existing and post-treatment levels of Treg cells were associated with a lack of treatment response. Lymphoproliferation of PBMC established that the vaccination was immunogenic and HPV 16 antigen specific. Importantly, these systemic immune responses to HPV 16 antigens were significantly associated with treatment responders.