Objetivos: objetivo geral deste trabalho foi avaliar a penetração cutânea da isotretinoína nanoencapsulada e livre utilizando técnicas de microdiálise e tape stripping in vitro. Métodos: A viabilidade da utilização da técnica de microdiálise para avaliar o perfil farmacocinético da isotretinoína após aplicação tópica foi investigada através da determinação da recuperação relativa (RR) in vitro por diálise e retrodiálise. A influência da concentração, do fluxo de perfusão e a ligação do fármaco à tubulação das sondas de microdiálise foram investigadas. A metodologia analítica para quantificação do fármaco em microdialisado foi validada. Em seguida, as penetrações cutâneas da isotretinoína livre e nanoencapsulada incorporada em géis hidrofílicos foram comparadas através da técnica de tape stripping in vitro em células de difusão de Franz utilizando pele humana e pele de porco. Para garantir a integridade das formulações, a estabilidade físico-química das mesmas foi avaliada. Os resultados de penetração cutânea foram comparados com os resultados in vivo obtidos em trabalho prévio do grupo de pesquisa. Resultados e Conclusões: Um método analítico simples e rápido para a determinação de isotretinoína em microdialisado foi validado de acordo com o FDA. A RR mostrou-se concentração independente e observou-se que há diferenças significativas entre as RR avaliadas pelos dois métodos utilizados, sendo a recuperação por retrodiálise 2,7 a 3,5 vezes superior que a obtida por diálise para os fluxos investigados. O fármaco aderiu às tubulações da sonda de microdiálise devido à sua lipofilicidade. Os hidrogéis de isotretinoína apresentaram estabilidade durante 2 meses de estocagem à 4 °C. Os experimentos de tape stripping in vitro mostraram que a isotretinoína não foi encontrada no compartimento receptor após 8 h, para ambas as formulações. A nanoencapsulação aumentou a penetração e prolongou a liberação da isotretinoína no estrato córneo de ambas as peles. A penetração cutânea em ambas as peles mostrou proporções similares para as duas formulações embora diferentes quantidades de fármaco tenham sido detectadas no estrato córneo. A pele de porco, mais permeável que a pele humana, é apropriada para prever a penetração cutânea da isotretinoína no estrato córneo humano in vitro (R = 0,79). O método in vitro não foi capaz de prever a penetração cutânea da isotretinoína in vivo. / Objectives: The aim of the present work was to assess the cutaneous penetration of isotretinoin free and loaded into polymeric nanocapsules using microdialysis and tape stripping in vitro. Methods: The feasibility of using microdialysis to determine the pharmacokinetic profile of isotretinoin after topical application was investigated through assessment of relative recovery (RR) in vitro by dialysis and retrodialysis. The influence of isotretinoin concentration, perfusion flow rate and drug binding to the probes were determined. The analytical method for quantification of microdialysate samples was validated. Furthermore the cutaneous penetration of isotretinoin free and loaded nanocapsules incorporated in hydrogel formulations were compared by tape stripping in vitro using Franz-type diffusion cells with excised human and pig skin. In order to ensure the integrity of the formulations used in this study, the chemical and physical stabilities were evaluated. The results of cutaneous penetration were compared with the results of tape stripping in vivo acquired in a previous study of our group. Results and Conclusions: A simple and rapid analytical method for quantification of isotretinoin in microdialysate samples was validated according to FDA. RR was concentration independent but method dependent under the conditions investigated being the retrodialysis recovery 3.5 to 2.7 times higher than the dialysis. Isotretinoin bound to the microdialysis tubing due to its high lipophilicity. The hydrogels showed storage stability for 2 months at 4 °C. In vitro tape stripping in human and pig skin showed that no isotretinoin reaches the receptor compartment for both formulations up to 8 h. Nanoencapsulation increased isotretinoin skin penetration for both stratum cornea and prolonged drug release. Similar proportion of cutaneous penetration for human and pig skin were observed although different amounts of drug were detected at the stratum corneum of both skin specimens. Pig skin, more permeable than human skin, is suitable for predicting cutaneous penetration of isotretinoin in humans in vitro (R = 0.79). The in vitro experiments were not suitable to reflect the in vivo results for percutaneous penetration of isotretinoin.

A administração oral de isotretinoína (IST) é eficaz no tratamento da acne; no entanto, seu uso está associado a efeitos adversos, como ressecamento da pele, epistaxe, distúrbios imunológicos e psiquiátricos, enquanto a eficácia da aplicação tópica é prejudicada pela intensa irritação causada na pele. Proteger a IST do contato direto com a pele e direcionar sua liberação para os folículos pilosos, local onde a acne se desenvolve, pode reduzir a irritação e aumentar a eficácia do tratamento. O polímero Delonix (DLX), galactomanano natural derivado da semente de Delonix regia, pode ser manipulado para produzir nanopartículas para a liberação direcionada de IST na pele. Desta forma, o objetivo deste estudo foi desenvolver nanopartículas poliméricas de DLX (IST-DLX) contendo IST e avaliar seu potencial para o tratamento tópico da acne. O DLX foi coletado, purificado e caracterizado quanto a composição de monossacarídeos e massa molecular (MM). Nanopartículas DLX sem IST (branco), nanopartículas fluorescentes de DLX (nanopartículas contendo BODIPY (BOD-DLX)) e nanopartículas IST-DLX foram preparadas e caracterizadas por tamanho, índice de polidispersividade (PdI), potencial zeta (pz) e eficiência de encapsulação (EE). O estudo de liberação in vitro das nanopartículas IST-DLX foi realizado utilizando células de difusão e membrana sintética, enquanto a penetração cutânea das nanopartículas BOD-DLX e IST-DLX foi avaliada por microscopia confocal e tape stripping diferencial em pele de orelha de porco dermatomizada, respectivamente. A influência das nanopartículas IST-DLX na modulação de IL-6, IL-10 e TNF-? foi avaliada in vitro utilizando células de macrófagos AMJ-2 estimuladas com lipopolisacarídeos (LPS) e a atividade antimicrobiana foi avaliada em meio de cultura de Propionibacterium acnes. In vivo, as nanopartículas IST-DLX foram avaliadas quanto a penetração e irritação cutânea em ratos Wistar (protocolo de aprovação CEUA-FCFRP nº 17.5.213.60.3). A MM do polímero DLX foi de ~177 kDa, contendo predominantemente manose e galactose na proporção de 6,3:1. O tamanho das nanopartículas IST-DLX, determinado por dispersão de luz, foi entre 215 a 232 nm, PdI < 0,2, pz < -30 mV e EE > 25%. As nanopartículas IST-DLX sustentaram a liberação da IST, com cerca de 37% de IST sendo liberado em 48 h. As imagens de microscopia confocal mostraram que as nanopartículas BOD-DLX concentraram-se na epiderme e nos folículos pilosos em comparação com a solução de BODIPY livre, a qual permeou até a derme. Foi observada uma penetração de IST significativamente maior e descontrolada em todas as camadas da pele de orelha de porco quando a solução de polímero de IST-DLX foi usada em comparação com um acúmulo de IST menor e controlado quando as nanopartículas IST-DLX foram aplicadas. As nanopartículas IST-DLX direcionaram a IST para os folículos pilosos (26%) em comparação com a solução de IST (6,5%) que permeou por todas as camadas da pele. As nanopartículas IST-DLX reduziram significativamente a produção de IL-6 em células de macrófagos estimuladas por LPS, mesmo após 48 h, em comparação com a solução de IST livre, que reduziu significativamente a produção de IL-6 apenas até 24 h. Tanto as nanopartículas ISTDLX quanto a solução de IST livre não inibiram o crescimento de P. acnes. Não foi observado nenhum sinal de eritema nos ratos tratados com nanopartículas IST-DLX, com ou sem irradiação UVA, enquanto os tratados com solução de IST apresentaram eritema. A espessura da epiderme dos ratos tratados com as nanopartículas foi significativamente menor do que daqueles tratados com IST livre, com ou sem irradiação UVA. Conclui-se que as nanopartículas IST-DLX direcionam e sustentam a liberação de IST na pele em concentrações ótimas, modulam a resposta inflamatória da IL-6 e previnem o eritema inflamatório relacionado ao IST in vivo. Sendo assim, o polímero DLX pode ser considerado um polímero promissor para a liberação sustentada e direcionada de fármacos. / Oral isotretinoin (IST) has been effective in acne treatment; however, its use is associated with side effects such as skin dryness, epistaxis, immune disorder and psychiatric concerns while its topical gel application\'s effectiveness is hampered by irritancy. Protecting IST from direct skin contact and targeting its release to the hair follicles where the acne develops can reduce irritation and increase the effectiveness of treatment. Delonix (DLX) polymer is a natural galactomannan derived from Delonix regia seed that may be engineered to yield nanoparticles for IST skin targeting. Therefore, the aim of this study was to develop IST-loaded DLX (IST-DLX) polymeric nanoparticles and assess its skin targeting prospects for acne treatment. DLX was collected, purified and characterized by monosaccharide composition and molecular weight (Mw). Blank DLX nanoparticles, fluorescent DLX nanoparticles (BODIPY-loaded (BOD-DLX) nanoparticles) and IST-DLX nanoparticles were prepared and characterized by size, polydispersibility index (PdI), zeta potential (zp) and encapsulation efficiency (EE). In vitro release study of IST-DLX nanoparticles was performed using diffusion cells and synthetic membrane, while skin targeting of BOD-DLX and IST-DLX nanoparticles were assessed by confocal microscopy and differential tape stripping technique in dermatomed pig ear skin respectively. IST-DLX polymeric nanoparticles\' IL-6, IL-10 and TNF-? modulation was assessed in vitro using lipopolysaccharide (LPS) stimulated AMJ-2 macrophage cells and antimicrobial activity was assessed in Propionibacterium acnes culture medium. IST-DLX nanoparticles\' skin penetration and irritation were evaluated in vivo in Wistar rats (ethics committee approval protocol CEUA-FCFRP nº 17.5.213.60.3). DLX polymer Mw was ~177 kDa, containing predominantly mannose and galactose in ratio 6.3:1. IST-DLX nanoparticles\' size determined by light scattering was between 215 to 232 nm, PdI < 0.2, zp < -30 mV, EE > 25 %. IST-DLX nanoparticles sustained IST release, with about 37% IST being released in 48 h. Confocal images showed that BOD-DLX nanoparticles accumulated in the epidermis and hair follicles as compared to free BODIPY solution which permeated into the dermis. Significantly higher and uncontrolled IST penetration into all layers of pig ear skin with IST-DLX polymer solution was observed as compared to lower, controlled but optimum IST accumulation into the pig ear skin when IST-DLX nanoparticles were used. IST-DLX nanoparticles targeted the hair follicle (26 %) as compared to IST solution (6.5 %) which permeated through all skin layers. IST-DLX polymeric nanoparticles significantly reduced IL-6 production in LPS stimulated macrophage cells at 48 h as compared to free IST solution which significantly reduced IL-6 production only at 24 h. Both IST-DLX nanoparticles and free IST solution did not inhibit P. acnes growth. No sign of inflammatory erythema was observed in IST-DLX nanoparticles treated Wistar rats with or without UVA irradiation as compared to free IST treated ones. Epidermal thickness of rats treated with IST-DLX nanoparticles was significantly smaller than those treated with free IST with or without UVA irradiation. It can be concluded that IST-DLX nanoparticles can target, deliver and sustain IST release to the skin at optimum concentrations, modulate IL-6 inflammatory responses and may prevent IST related inflammatory erythema in vivo. DLX polymer could be promising polymer for drug delivery.

Made available in DSpace on 2015-05-14T12:59:47Z (GMT). No. of bitstreams: 1 parte1.pdf: 1499360 bytes, checksum: 73a1a71524a39b1fbaa99614b57d192f (MD5) Previous issue date: 2009-11-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The physiological and pharmacological effects of isotretinoin are yet to be completely understood despite its widespread use in the treatment of acne vulgaris and the known adverse effects that it may cause, dry eyes being one of the most common. The objective of this study was to evaluate the toxicity of oral isotretinoin on the conjunctiva of patients submitted to treatment with this drug, using impression cytology. A prospective cohort clinical trial was conducted at the Ophthalmological Referral Center of the Federal University of Paraíba and at the Laboratory of External Eye Diseases of the Federal University of São Paulo. Twentyeight patients with acne vulgaris were selected. A questionnaire on symptoms was applied and biomicroscopy, tear film break-up time (TBUT), fluorescein staining, Shirmer s test, rose bengal staining and impression cytology (IC) were performed prior to and after three months of treatment with oral isotretinoin. Samples for IC were collected from the temporal, superior, nasal and inferior bulbar conjunctiva of both eyes. The doses of isotretinoin varied from 0.35 to 0.88 mg/kg/day. Compared to pretreatment, burning, pruritus and gritty eye sensation were significantly more common during treatment with this drug, as were the biomicroscopic changes of hyperemia and blepharitis. The percentage of positive results for dry eyes according to TBUT and for rose bengal conjunctival staining was also greater during treatment. Regarding Shirmer s test and fluorescein staining of the cornea, no statistically significant changes were found with exposure to the drug. With respect to IC performed on the samples obtained from the superior and temporal quadrants, there was a reduction in the percentage of normal results from 100% to 82% and from 75% to 43%, respectively, and an increase in the percentage of borderline results from 0 to 14% and from 21% to 47%, respectively, during treatment compared to baseline results. For the samples from the nasal quadrant, an increase occurred in the percentage of abnormal findings from 0 to 11%, while in the samples taken from the inferior quadrant, no changes were found with the use of isotretinoin. The parameters affected by this treatment were cell-to-cell contact, nucleus-tocytoplasm ratio and the distribution of goblet cells, the scores of which increased significantly. No significant correlation was found between the results of IC, symptom score and tear function tests. Therefore, the present findings show that acne treatment with oral isotretinoin results in changes in the conjunctival epithelium in a significant percentage of patients. These changes are seen both in the exposed region of the bulbar conjunctiva (temporal and nasal) and in the unexposed conjunctiva (superior) and reflect a trend towards squamous metaplasia as an adaptive response of the conjunctival epithelium, which tends to become nonsecretory under the effect of the drug. / As ações fisiológicas e farmacológicas da isotretinoína ainda não são completamente conhecidas, apesar da sua ampla utilização no tratamento da acne vulgar e dos efeitos adversos que pode causar. Dentre esses, o quadro de olho seco é um dos mais frequentes. O objetivo deste estudo foi avaliar a toxicidade da isotretinoína oral sobre a conjuntiva de pacientes submetidos a tratamento com o fármaco, utilizando a citologia de impressão. Realizou-se estudo clínico prospectivo tipo coorte no Centro de Referência Oftalmológica da Universidade Federal da Paraíba e no Laboratório de Doenças Externas Oculares da Universidade Federal de São Paulo. Foram selecionados 28 pacientes portadores de acne vulgar. Realizou-se questionário de sintomas, biomicroscopia, teste do tempo de ruptura do filme lacrimal (TBUT), coloração por fluoresceína, teste de Shirmer, coloração por rosa bengala e citologia de impressão (CI) antes e aos 3 meses de tratamento com isotretinoína oral. Os espécimes para a CI foram coletados dos quadrantes temporal, superior, nasal e inferior da conjuntiva bulbar de ambos os olhos. As doses de isotretinoína variaram de 0,35 a 0,88 mg/kg/dia. Em relação ao pré-tratamento, os sintomas ardor ocular , prurido e sensação de areia nos olhos , assim como as alterações biomicroscópicas hiperemia e blefarite , ocorreram com uma frequência significantemente maior durante o tratamento com o fármaco. O percentual de resultados positivos para olho seco, para o TBUT e para a coloração conjuntival por rosa bengala também foi significantemente maior na vigência do tratamento. Para o teste de Shirmer e para a coloração da córnea por fluoresceína, não houve mudanças significantes com a exposição ao fármaco. Quanto à CI, para os espécimes obtidos dos quadrantes superior e temporal, houve diminuição do percentual de resultados normais (de 100 para 82 e de 75 para 43, respectivamente) e aumento do percentual de resultados limítrofes (de 0 para 14 e de 21 para 47, respectivamente) durante o tratamento, em relação aos resultados iniciais. Para os espécimes do quadrante nasal, houve aumento do percentual de resultados anormais (de 0 para 11), e para aqueles do quadrante inferior, não se observaram alterações com o uso de isotretinoína. Com o tratamento, foram afetados os parâmetros contato célula-célula , razão núcleo/citoplasma e distribuição das células caliciformes , que sofreram aumento significante em seus escores. Não houve uma correlação significante entre os resultados da CI, do escore de sintomas e dos testes da função lacrimal. Assim, de acordo com os resultados obtidos, o tratamento da acne com isotretinoína oral induz alterações no epitélio conjuntival de um percentual significante de pacientes. Essas alterações são observadas tanto na região exposta da conjuntiva bulbar (temporal e nasal), como na não exposta (superior) e representam uma tendência à metaplasia escamosa, como uma resposta adaptativa do epitélio conjuntival, que tende a se tornar não secretor sob efeito do fármaco.

Abstract Acne is a common skin disease that affects nearly every teenager but in some cases it persists into adulthood as a chronic disease. Acne severity ranges from mild comedonal to severe cystic and scarring disease. The pathogenesis of acne is multifactorial: increased sebum production, hormonal influences, the hypercornification of pilosebaceous ducts, overgrowth of Propionibacterium acnes (P. acnes) and inflammation around pilosebaceous follicles are considered to be the main pathogenetic factors but few further details are known. The role of innate immunity in the pathogenesis of acne has been studied quite extensively, but the contribution of adaptive immune responses is not well characterized. Although isotretinoin has been the most potent medication for acne over 30 years, its mode of action is partially unknown. Furthermore, modern 16S ribosomal RNA (rRNA) gene amplicon sequencing based methods allow quantification of the abundance of skin bacteria, but these culture-independent techniques have not yet been used to assess the effect of systemic treatments, isotretinoin and tetracycline antibiotics on acne skin microbiota. In this study the innate and adaptive immune responses in the skin of acne patients were investigated. Skin biopsies were taken from early-stage acne lesions and from uninvolved skin of acne patients, and the samples were examined for the expression of cytokines, chemokines, transcription factors and antimicrobial peptides, using real-time polymerase chain reaction (PCR). In addition, the skin biopsies were examined by immunohistochemistry and Luminex technology, which was also used in the determination of cytokine levels in acne patients’ serum samples. The skin microbiota of swab samples was investigated using 16S rRNA gene amplicon sequencing. We found that innate and adaptive immune responses, in particular the activation of the IL-17/Th17 axis, are involved in early-stage acne lesions. Furthermore, the expression of effector cytokine TGF-&#946; and transcription factor of regulatory T cells (Tregs) FoxP3 mRNA was reduced in acne patients’ uninvolved skin compared to normal skin of healthy controls. Isotretinoin has no direct effect on innate and adaptive immune responses in newly formed acne lesions. It did, however, modify some innate immune responses: the expression of IL-1&#946; and toll-like receptor 2 (TLR2) mRNA was reduced in uninvolved skin of acne patients, and the number of CD68+ macrophages increased in acne patients’ skin with isotretinoin treatment. After six weeks’ treatment, the abundance of P. acnes was similarly reduced with either isotretinoin or lymecycline treatment. However, previous studies and clinical experience show that isotretinoin is more clinically effective against acne than tetracyclines, probably because of the effect of isotretinoin on sebaceous glands and sebum excretion. Acne is a disease, which at its worst results in significant scarring. Therefore it is important to understand the immunological factors behind its pathogenesis. Our findings show that both adaptive and innate immunity play an important role. Isotretinoin, although currently the most potent anti-acne drug, does not directly affect the adaptive immune response and therefore new medications are required to control the relevant immune responses, especially in severe cystic or fulminant acne. / Tiivistelmä Akne on yleinen ihosairaus, joka koskettaa lähes jokaista teini-iässä ja jatkuu osalla kroonisena sairautena läpi aikuisiän. Aknen vaikeusaste vaihtelee lievästä vaikeisiin, arpeuttaviin tautimuotoihin. Aknen patogeneesin tiedetään olevan monitekijäinen. Tärkeimmät aknen syntyyn vaikuttavat tekijät ovat lisääntynyt talineritys hormonien, erityisesti androgeenien vaikutuksesta, talirauhaskarvatuppitiehyen tukkeutuminen, Propionibacterium acnes (P. acnes) – bakteerin liikakasvu sekä tulehdusreaktio talirauhaskarvatupen ympärillä. Synnynnäisen immuniteetin roolia aknen synnyssä on tutkittu paljon, sen sijaan hankittua immuniteettia huomattavasti vähemmän. Myöskään yli 30 vuotta käytössä olleen tehokkaimman aknelääkkeen, isotretinoiinin, vaikutusmekanismia ei täysin tiedetä. Lisäksi systeemilääkkeiden, isotretinoiinin ja tetrasykliiniryhmän antibioottien, vaikutusta akneihon bakteeristoon ei ole tutkittu moderneilla, polymeraasiketjututkimuksiin (PCR) perustuvilla menetelmillä. Tässä tutkimuksessa selvitettiin synnynnäisiä ja hankittuja immuunireaktiota aknea sairastavien ihossa ja isotretinoiinin vaikutusta näihin reaktioihin. Tutkimuksessa hyödynnettiin aknepotilaiden ihokoepaloja, joista tutkittiin sytokiinien, kemokiinien, transkriptiotekijöiden sekä antimikrobisten peptidien ilmentymistä reaaliaikaisella PCR-menetelmällä. Lisäksi ihokoepaloja tutkittiin immunohistokemiallisilla värjäyksillä ja Luminex-menetelmällä, jota hyödynnettiin myös sytokiini-tasojen määrittämiseen aknepotilaiden seeruminäytteistä. Aknea sairastavien ihon bakteeristoa tutkittiin 16S ribosomaalisen RNA:n geenien sekvensointiin pohjautuvalla menetelmällä. Väitöstutkimuksessa havaitsimme, että varhaisvaiheen aknemuutoksessa käynnistyvät sekä synnynnäisen että hankitun immuniteetin vasteet. Erityisesti IL-17/Th17-akseli on aktivoitunut. Lisäksi aknea sairastavien terveellä iholla tulehduksen välittäjäaineen TGF-1&#946; ja transkriptiotekijän FoxP3 ilmeneminen oli alentunut verrattuna terveihoisiin kontrolleihin. Isotretinoiinilla ei ole suoraa vaikutusta synnynnäisen ja hankitun immuniteetin reaktioihin jo olemassa olevissa varhaisen vaiheen aknemuutoksissa. Aknea sairastavien terveessä ihossa isotretinoiini muokkasi luontaisen immuniteetin vasteita mm. vähentämällä tollin kaltaisen reseptorin (TLR)- 2 ja tulehduksen välittäjäaineen IL-11&#946; ilmenemistä sekä lisäämällä makrofagien kertymistä tulehduspaikalle. Kuuden viikon hoito isotretinoiinilla tai lymesykliinillä vähentää yhtä tehokkaasti aknealueilla P. acnes -bakteerin suhteellista osuutta ihon bakteeristosta. Isotretinoiini on kuitenkin antibiootteja tehokkaampi aknelääke ja lisäksi hoitovaste saavutetaan pitemmäksi aikaa, mikä johtunee isotretinoiinin vaikutuksesta talirauhasiin ja talinerityksen vähentämisestä. Aknen tulehdusmekanismien ymmärtäminen on tärkeää taudissa, joka pahimmillaan johtaa merkittävään arpeutumiseen. Tutkimuksemme perusteella hankittu immuniteetti on tärkeässä roolissa synnynnäisen immuniteetin aktivoitumisen rinnalla aknen synnyssä. Tehokkain aknelääke, isotretinoiini ei vaikuta hankittuun immuunivasteeseen, minkä vuoksi jatkossa tarvitaan uusia lääkkeitä isotretinoiinin rinnalle tulehduksen nopeaan hillitsemiseen vaikeissa aknemuodoissa.

Approximately 7000 rare disorders exist, affecting 2 percent of Canadians and millions of people worldwide. Given that for many rare diseases only one allele is mutated, we hypothesize inducing expression of the remaining wild-type allele may have a therapeutic effect. SMAD3 heterozygosity results in Aneurysms-Osteoarthritis Syndrome (AOS) – an aortic aneurysm disorder also known as Loeys-Dietz Syndrome Type 3. We conducted a screen of FDA-approved compounds and found that 13-cis-retinoic acid (13-CRA) induces SMAD3 in normal human fibroblast cultures. Treatment with therapeutic concentrations of 13-CRA increased SMAD3 mRNA in normal human fibroblasts, patient fibroblasts, wild-type murine vascular smooth muscle cells and Smad3+/- murine vascular smooth muscle cells. Increases in SMAD3 protein were also observed in normal human fibroblasts, patient fibroblasts, and wild-type murine vascular smooth muscle cells. Immunofluorescent imaging revealed the primary site of protein induction to be nuclear. We report here the in vitro induction of SMAD3 mRNA and protein by therapeutic levels of 13-CRA and suggest further investigation of this modality for the treatment of AOS.

Les maladies inflammatoires chroniques de l'intestin (MICI) désignent deux affections, la maladie de Crohn (MC) et la rectocolite hémorragique (RCH). Ces maladies sont caractérisées par une grande disparité de répartition dans le monde et une forte augmentation de leur incidence depuis 50 ans. Leur physiopathologie fait intervenir une composition anormale du microbiote intestinal (appelée dysbiose), une dysfonction de la barrière épithéliale, un déficit de l'immunité innée et une dérégulation de l'immunité adaptative. Plus de 163 gènes de prédisposition ont été identifiés, mais la plupart d'entre eux ne sont associés qu'à une augmentation modeste du risque de MICI (Odds Ratios compris entre 1,02 et 1,2). Les facteurs d'environnement semblent jouer un rôle important dans la survenue de ces maladies. Le tabagisme a un effet démontré, favorable dans la RCH, néfaste dans la MC. L'exposition solaire, la vitamine D, l'alimentation, les agents infectieux et les médicaments ont été associés aux MICI mais leur effet est moins bien démontré. Cette thèse d'épidémiologie est consacrée aux MICI; plus précisément, à l'étude de leurs facteurs d'environnement, et au risque de cancer associé aux médicaments des MICI. Elle est fondée sur l'étude de bases de données françaises et européennes. Un premier travail a exploré l'association entre l'exposition à l'isotrétinoïne (médicament prescrit pour traiter l'acné) et la survenue de RCH, rapportée par une étude américaine. Cette étude cas témoin a été menée à l'échelle de la France entière à partir des données du système national d'information inter-régimes de l'assurance maladie (SNIIRAM). Dans ce travail très peu de patients atteints de MICI ont reçu de l'isotrétinoïne durant l'année précédant le diagnostic de la maladie. La prise d'isotrétinoïne n'est pas associée au risque de RCH mais inversement associée au risque de MC.Dans un deuxième travail, nous avons étudié l'impact gobal de l'alimentation en modélisant les profils alimentaires associés à la survenue de RCH et de MC dans la cohorte prospective européenne EPIC (European Prospective Investigation Into Cancer). Un profil alimentaire riche “en produits sucrés et sodas” est associé à l'incidence de RCH dans le sous-groupe diagnostiqué au delà de 2 ans. Aucun profil alimentaire n'est associé au risque de MC. Le régime méditerranéen n'est ni associé à la RCH ni à la MCDans un troisième travail, nous nous sommes intéressés aux risques de cancer associés à l'exposition aux médicaments des MICI : immunosuppresseurs et anti-TNF. Leur prescription a beaucoup augmenté ces dernières années. Cependant ils ont été associés à un risque de lymphome, de cancer de la peau non mélanocytaire et de mélanome pour les thiopurines et les anti-TNF, respectivement. Nous avons évalué prospectivement le risque de cancer associé à ces médicaments, à l'échelle de la France entière dans des conditions de prescription courante, grâce aux données du SNIIRAM. Les résultats sont en en cours d'analyse.Notre thèse montre que l'étude des bases de données peut apporter une réponse à une question importante en pratique clinique , portant sur le lien entre isotrétinoïne et MICI. Notre travail a également permis de générer des hypothèses sur le lien entre les profils alimentaires et la survenue de MICI. Les limites de l'exercice (détaillées dans le manuscrit) ne doivent pas être sous estimées. Nos résultats suscitent des questions et appellent d'autres travaux, menés à une échelle encore plus vaste et avec d'autres méthodes statistiques. / Inflammatory bowel diseases refer to two conditions: Crohn's disease and ulcerative colitis. These diseases display an important geographic heterogeneity worldwide and an increase in incidence during the last fifty years. Their physiopathology is complex, involving anormal composition of gut microbiota (dysbiosis), dysfonction of epithelial barrier and dysregulation of innate and adaptative immune response. More than 163 predisposing genes have been identified, but most of them carry modest association with IBD (Odds ratios varrying from 1.02 to 1.3) (1–3). Environmental factors seem to play an important role in IBD onset. Smoking has a positive effect on UC and harmfull effect on CD. Sun exposure, vitamin D, diet, infections have been inconsistently associated with IBD. This epidemiology thesis is devoted to IBD and specifically, to environemental risk factors, and also to the risk of cancer associated to IBD drugs. It is based on French and European databases. Our first work, explored the association between isotretinoin and UC reported in a US study. Our study was performed in a case-control study in the whole French territory thanks to a medico-administrative database (SNIIRAM). In this work, only a few patients with IBD were exposed to isotetinoin in the year before disease onset. Exposure to isotretinoin was not associated with UC but negatively associated with CD.In a second study, we explored the global impact of diet on UC and CD in a European prospective study (European Prospective Investigation Into Cancer (EPIC)). A dietary pattern with “high sugars and soft drinks” was associated with UC risk when restricted to cases diagnosed at least two years after dietary assessment. No dietary pattern was associated with CD. Mediterranean diet had no effect on UC nor CD risks.In the third part of this work, we investigated the risk of cancer associated with IBD drugs: immunosuppresive agents and anti-TNF. These medications are more and more prescribed nowadays. Howevere, they are associated with an increased risk of cancer in observationnal studies: lymphoma and non melanoma skin cancer with thiopurines and anti-TNF agents respectively. Therefore, we aimed to investigate the cancer risks associated with thiopurine and/or anti-TNF exposure in the whole French territory in the real life , using a medico-adminstrative database (SNIRAM). Currently, we are still analyzing the results.Our work shows that studying large databases can answer an important issue in clinical practice related to a potential link between isotretinoin and IBD. Also, it has generated hypotheses about the link between dietary pattern and IBD. Limitations of our work (detailed in the manuscript) should be considered.. Otr studies using larger databases and other statistical methods should address these limitations.

Bakgrund: Akne är en av de vanligaste dermatologiska sjukdomarna och drabbar nästan alla människor under puberteten. Sjukdomen kan även fortsätta eller utvecklas i vuxen ålder där prevalensen av akne är högre bland kvinnor. Vid måttlig till svår akne förekommer inflammatoriska lesioner i ansikte och på övre bål som ofta lämnar ärr vid läkning. Isotretinoin (Iso) är det mest effektiva läkemedlet mot akne men kommer med många besvärliga och allvarliga biverkningar. Utifrån två fallpresentationer verkar Iso kunna påverka kroppens folatstatus negativt. Folat är ett essentiellt vitamin som har en viktig roll i DNA-syntesen och metabolismen av aminosyror. Syfte: Syftet med litteraturstudien var att undersöka om behandling med Iso har negativ effekt på kroppens folatstatus. Vid undersökning av detta söktes följande frågor att besvaras: Sänker Iso kroppens folatstatus? Ökar Iso homocysteinnivåerna i plasma? Sänker Iso kroppens B12-status? Metod: Arbetet har utförts genom systematisk litteratursökning i Pubmed och Scopus under januari och februari 2021. I båda databaserna har fyra olika fritextsökningar genomförts med isotretinoin som bestående sökord vidare tillsammans med folic acid, folate, homocysteine och vitamin B12. Elva artiklar har inkluderats för analys. Resultat: Effekten av Iso på kroppens folatstatus undersöktes i tio studier och i sex av dessa studier observerades signifikant minskade serumfolatnivåer. Homocysteinnivåer undersöktes i tio studier och i åtta av dessa observerades ökade homocysteinnivåer efter behandling med Iso. Åtta studier undersökte förändringar i serumkoncentrationen av B12 vid behandling med Iso och i tre av dessa studier observerades signifikant minskande B12-nivåer. Slutsats: Utifrån resultatet går det att tolka att omsättningen av folat och B12 kan ha ökat vid stigande homocysteinnivåer. Tillskott av folsyra och B12 kan vara att rekommendera till patienter med akne som behandlas med Iso och där homocysteinnivåerna i plasma stiger. Studier med längre behandlingsperiod bör utföras för att kunna avgöra hur en fullständig behandling med Iso påverkar vitaminstatusen för folat och B12. / Background: Acne is one of the most common dermatological diseases generally affecting people going through puberty. The prevalence of adult acne is increasing and especially among women. Clinical symptoms of acne in moderate and severe cases are inflammatory lesions located in the face and upper body that can often cause scarring. Isotretinoin (Iso) is the most effective therapeutic drug against acne but has many side-effects. Iso appears to adversely affect the body's folate status according to two case presentations. Folate is an essential vitamin and has an important role in DNA synthesis and amino acid metabolism. Aim: The aim of the literature study was to examine if treatment with Iso has a negative effect on the body's folate status. Following questions were searched to be answered: Does Iso lower the body's folate status? Dose Iso increase plasma homocysteine levels? Does Iso lower the body's vitamin B12 status?Method: Eleven articles examining the effect of Iso treatment on folate status, homocysteine and B12 levels were collected from a literature search in Pubmed and Scopus during January and February 2021. Four different free text searches were carried out with isotretinoin as the consisting key word together with folic acid, folate, homocysteine ​​and vitamin B12. Results: The effect of Iso on the body's folate status was examined by ten studies and in six of these studies significantly reduced serum folate levels were observed. Homocysteine levels were examined in ten studies and in eight of these were increased homocysteine levels observed after treatment with Iso. Eight studies examined changes in the serum concentration of B12 and in three of these studies significantly decreased B12 levels were observed after treatment with Iso. Conclusion: The results give an indication that the turnover of folate and B12 may have increased with rising homocysteine plasma levels. Supplements of folic acid and B12 may be recommended for patients undergoing treatment with Iso if plasma homocysteine levels are rising during treatment. Studies with a longer treatment period with Iso need to be done to determine how a complete treatment with Iso affects folate and B12 status.

A isotretinoína (ácido-13-cis-retinóico) é uma substância derivada da vitamina A. É indicada no tratamento de formas graves de acne nódulo císticas e resistentes a terapias anteriores. É uma substância termo e fotossensível, que requer cuidados especiais na formulação, produção e acondicionamento. Como atualmente existem várias preparações comerciais contendo essa substância, é importante que se estudem e validem métodos analíticos para sua determinação quantitativa, que possam ser aplicados no controle de qualidade destas formulações. O objetivo da pesquisa foi, portanto, a caracterização e a determinação quantitativa da isotretinoína, e avaliação do comportamento desta substância em três formulações farmacêuticas comercializadas no Brasil. Esta análise comparativa foi realizada através das técnicas de difração à laser, microscopia de luz polarizada, termogravimetria, e calorimetria exploratória diferencial, para caracterizar, respectivamente, a distribuição do tamanho de partícula e comportamento térmico. Não foi encontrada diferença entre o comportamento térmico das três amostras, mas na medida do tamanho de partícula, houve uma grande variação entre duas das três amostras. O tamanho de partícula é um fator conhecido que altera a absorção de moléculas no trato-gastrintestinal, podendo haver conseqüências na segurança e eficácia do produto. Foi desenvolvida e validada uma metodologia, para determinação quantitativa da isotretinoína nessas três amostras estudadas, um método de cromatografia líquida de alta eficiência, rápido e simples, empregando detector no UV. Duas das amostras ultrapassaram o limite de isotretinoína de 90 - 110% especificado pela Farmacopéia Americana. A apresentação, controle de qualidade e a caracterização de formulações farmacêuticas são importantes para assegurar a qualidade do produto final. Embora a cromatografia, análise térmica e determinação do tamanho de partícula sejam utilizadas pelas indústrias farmacêuticas, o tamanho de partícula ainda não é uma especificação definida pelas farmacopéias. Isso mostra a importância de futuras pesquisas, determinando o efeito dessas variações encontradas, possibilitando o estudo comparativo, entre a qualidade de produtos contendo isotretinoína em pacientes através de testes de bioequivalência. / Isotretinoin (13-cis retinoic acid), a vitamin A derivative is indicated for the treatment of several forms of acne, cystic nodules resistant to other therapies and also acts as an inhibitor of at proliferation neoplasic cells. It is a thermolabile substance, light and air sensitive and these physicochemical properties impose special cares in the formulation, development and packaging to avoid the isotretinoin degradation. Currently some preparations available on the stores contain this substance and it is important to study and validate quantitative methods to determine the quality control of these formulations. The purposes of research were to characterize, to determine quantitatively the isotretinoin and check the behavior of this substance in three pharmaceutical formulations available on the Brazilian stores. This comparative analysis was performed by laser diffraction/polarized light microscopy, thermogravimetry and differential scanning calorimetry techniques in order to characterize, respectively, the particle size distribution and the thermal behavior. The thermal stability was found to be none a variable parameter, the isotretinoin average particle size was significantly different in 2/3 formulations. Interestingly, the particle size is a facto r known to affect the absorption of the molecules from the gastrointestinal tract and, therefore, may have consequences for the safety and the efficiency of the product. Its was also developed and validated one analytical method for quantitative determination of isotretinoin in these three samples. A simple and rapid method using an isocratic high performance liquid chromatography and UV detection was developed. The average isotretinoin content of two ranged outside the 90 -110% date showed into United States Pharmacopeia specifications. Eventually, the design, the quality control and the characterization of the pharmaceutical formulations are important to ensure the quality of the final product. Although the chromatography, thermal and particle size analysis were currently used in the pharmaceutical industry, the particle size is not a specification stated in the pharmacopeias and should be considered with more interest. It would seem important; therefore, that future research determines the full effect of particle size variation and a comparative study with pharmaceutical quality of isotretinoin products on patient exposure to isotretinoin by comparative bioequivalence testes.

A isotretinoína (ácido-13-cis-retinóico) é uma substância derivada da vitamina A. É indicada no tratamento de formas graves de acne nódulo císticas e resistentes a terapias anteriores. É uma substância termo e fotossensível, que requer cuidados especiais na formulação, produção e acondicionamento. Como atualmente existem várias preparações comerciais contendo essa substância, é importante que se estudem e validem métodos analíticos para sua determinação quantitativa, que possam ser aplicados no controle de qualidade destas formulações. O objetivo da pesquisa foi, portanto, a caracterização e a determinação quantitativa da isotretinoína, e avaliação do comportamento desta substância em três formulações farmacêuticas comercializadas no Brasil. Esta análise comparativa foi realizada através das técnicas de difração à laser, microscopia de luz polarizada, termogravimetria, e calorimetria exploratória diferencial, para caracterizar, respectivamente, a distribuição do tamanho de partícula e comportamento térmico. Não foi encontrada diferença entre o comportamento térmico das três amostras, mas na medida do tamanho de partícula, houve uma grande variação entre duas das três amostras. O tamanho de partícula é um fator conhecido que altera a absorção de moléculas no trato-gastrintestinal, podendo haver conseqüências na segurança e eficácia do produto. Foi desenvolvida e validada uma metodologia, para determinação quantitativa da isotretinoína nessas três amostras estudadas, um método de cromatografia líquida de alta eficiência, rápido e simples, empregando detector no UV. Duas das amostras ultrapassaram o limite de isotretinoína de 90 - 110% especificado pela Farmacopéia Americana. A apresentação, controle de qualidade e a caracterização de formulações farmacêuticas são importantes para assegurar a qualidade do produto final. Embora a cromatografia, análise térmica e determinação do tamanho de partícula sejam utilizadas pelas indústrias farmacêuticas, o tamanho de partícula ainda não é uma especificação definida pelas farmacopéias. Isso mostra a importância de futuras pesquisas, determinando o efeito dessas variações encontradas, possibilitando o estudo comparativo, entre a qualidade de produtos contendo isotretinoína em pacientes através de testes de bioequivalência. / Isotretinoin (13-cis retinoic acid), a vitamin A derivative is indicated for the treatment of several forms of acne, cystic nodules resistant to other therapies and also acts as an inhibitor of at proliferation neoplasic cells. It is a thermolabile substance, light and air sensitive and these physicochemical properties impose special cares in the formulation, development and packaging to avoid the isotretinoin degradation. Currently some preparations available on the stores contain this substance and it is important to study and validate quantitative methods to determine the quality control of these formulations. The purposes of research were to characterize, to determine quantitatively the isotretinoin and check the behavior of this substance in three pharmaceutical formulations available on the Brazilian stores. This comparative analysis was performed by laser diffraction/polarized light microscopy, thermogravimetry and differential scanning calorimetry techniques in order to characterize, respectively, the particle size distribution and the thermal behavior. The thermal stability was found to be none a variable parameter, the isotretinoin average particle size was significantly different in 2/3 formulations. Interestingly, the particle size is a facto r known to affect the absorption of the molecules from the gastrointestinal tract and, therefore, may have consequences for the safety and the efficiency of the product. Its was also developed and validated one analytical method for quantitative determination of isotretinoin in these three samples. A simple and rapid method using an isocratic high performance liquid chromatography and UV detection was developed. The average isotretinoin content of two ranged outside the 90 -110% date showed into United States Pharmacopeia specifications. Eventually, the design, the quality control and the characterization of the pharmaceutical formulations are important to ensure the quality of the final product. Although the chromatography, thermal and particle size analysis were currently used in the pharmaceutical industry, the particle size is not a specification stated in the pharmacopeias and should be considered with more interest. It would seem important; therefore, that future research determines the full effect of particle size variation and a comparative study with pharmaceutical quality of isotretinoin products on patient exposure to isotretinoin by comparative bioequivalence testes.