A talidomida foi amplamente prescrita entre 1950 e 1960, em quase 50 países, como hipnosedativo não-barbitúrico e antiemético para indisposição matinal durante a gravidez, sendo em seguida fortemente controlada em função da ocorrência de sérios problemas de teratogenicidade. Nos últimos anos, vários esforços foram realizados no sentido de buscar identificar e elucidar as propriedades antiinflamatórias, imunomodulatórias e antiangiogênicas da talidomida. Na mesma direção, investigações clínicas foram conduzidas em pacientes com diversas doenças, como mieloma múltiplo, carcinoma renal, câncer de próstata, Síndrome da rejeição paciente-enxerto, entre outras. A talidomida possui um centro quiral e dois anéis amida em sua estrutura e é sintetizada como racemato, constituída por dois enantiômeros ativos: (+)-(R)- e (+)-(S)-talidomida. A talidomida racêmica apresenta duas formas polimórficas, alfa ( ) e beta ( ). Assim, a variabilidade observada em relação ao polimorfismo representa um ponto crítico se considerarmos o potencial de alteração de propriedades biofarmacêuticas em decorrência da predominância de um ou outro polimorfo. No Brasil o medicamento talidomida é fabricado exclusivamente pela FUNED – Fundação Ezequiel Dias, laboratório público do Estado de Minas Gerais que integra o Sistema Oficial de produção de Medicamentos do País. Neste contexto, este trabalho tem como principal objetivo a avaliação de características físicas, físico-químicas e tecnológicas dos polimorfos da talidomida, com ênfase nos ensaios de cristalização, dissolução, degradação e compressão. Os resultados deste trabalho contribuíram para um melhor entendimento da relação entre características cristalográficas e propriedades farmacêuticas, agregando uma base científica capaz de avaliar as diferenças existentes destes polimorfos e garantir o controle de qualidade adequado do produto final produzido em um Laboratório Oficial. Estes estudos contribuíram para o cumprimento de exigências regulatórias. / Thalidomide was widely prescribed between 1950 and 1960, in nearly 50 countries, as sedative and antiemetic for morning sickness during pregnancy. After the occurrence of serious problems of teratogenicity it was heavily controlled. Growing interest has been observed in recent years to identify and elucidate the anti-inflammatory, immunomodulatory and anti-angiogenic properties of thalidomide. In the same direction, clinical investigations have been conducted in patients with various diseases such as myeloma, renal carcinoma, and prostate cancer, among others. Thalidomide possesses a chiral center and two amide rings in its structure and is synthesized as racemate, consisting of two active enantiomers: (+)-(R)- and (+)-(S)-thalidomide. It is known that the racemic thalidomide has two polymorphic forms, alpha ( ) and beta ( ). Thus, the observed variability in relation to the polymorphism represents a critical point considering the potential of changes in biopharmaceutical properties due to the predominance of one of them. In Brazil thalidomide tablets are manufactured exclusively by FUNED – Fundação Ezequiel Dias, the public laboratory of the State of Minas Gerais, which integrates the Country's Official System of drug production. In this context, the objectives of this work are the assessment of physical characteristics, physicalchemical and technological properties of polymorphic forms of thalidomide, with emphasis on crystallization, dissolution, degradation and compression. The results of this work contributed to a better understanding of the relationship between characteristic crystallographic properties and pharmaceutical properties, aggregating a scientific basis to assess the differences of these polymorphs and ensure the appropriate quality control of the final product produced in an official laboratory. These studies contributed to compliance with regulatory requirements.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-01T12:09:03Z No. of bitstreams: 1 ingridestevampereira.pdf: 2004254 bytes, checksum: 326e3188dc98d9fc4ddc417af89ebc1e (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-06-02T15:12:44Z (GMT) No. of bitstreams: 1 ingridestevampereira.pdf: 2004254 bytes, checksum: 326e3188dc98d9fc4ddc417af89ebc1e (MD5) / Made available in DSpace on 2017-06-02T15:12:44Z (GMT). No. of bitstreams: 1 ingridestevampereira.pdf: 2004254 bytes, checksum: 326e3188dc98d9fc4ddc417af89ebc1e (MD5) Previous issue date: 2017-02-22 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A talidomida é uma potente droga anti-inflamatória empregada no tratamento de diversas patologias, incluindo Eritema Nodoso Leproso (ENL), câncer, doença de Crohn e outras desordens inflamatórias e vasculares. Entretanto, seus efeitos tóxicos e teratogênicos tornam sua utilização limitada e motivam pesquisas para a síntese de análogos que apresentem eficácia semelhante na imunomodulação, sem efeitos tóxicos. Diversos análogos da talidomida vêm sendo desenvolvidos no laboratório de química da UFJF. Em estudos anteriores, mostramos que a introdução de dois anidridos ftálicos na composição aumenta significativamente a atividade biológica e solubilidade em água do composto, sem aumento da toxicidade em modelos experimentais in vitro e in vivo. O presente trabalho visa a síntese de dois novos compostos análogos da talidomida, CAT-15 e CAT-16, formados por apenas um derivado anidrido ftálico aberto, mantendo um grupo amino livre, e a avaliação da sua atividade anti-inflamatória utilizando linhagem de célula HT-29 e células mononucleares de sangue periférico humano (PBMC) estimuladas com LPS. A citotoxicidade dos compostos foi avaliada pelo ensaio do MTT, com tratamento por 18 horas para células HT-29 e por 24 e 48 horas para PBMC, usando concentrações crescentes de talidomida, CAT-15, CAT-16. A dexametasona foi utilizada como controle positivo. A produção de TNF-α, CXCL-10, IL-6, IL-8 e IL-10 foi avaliada pelo método de ELISA. Os novos compostos não foram tóxicos para as células HT-29 e PBMC em nenhuma das concentrações testadas, com exceção de CAT-16 a 1600µM. Células HT-29 produziram grande quantidade de CXCL-10 em resposta ao LPS e os resultados deste trabalho mostram que a talidomida e os análogos CAT-16 e CAT-15 apresentam atividade inibitória sobre a produção desta quimiocina. O composto CAT-16 modulou a produção de CXCL-10 em concentrações menores que a talidomida em ambos os modelos de tratamento (simultâneo e prétratamento). Em contrapartida, a modulação por CAT-15 foi observada apenas no modelo de pré-tratamento. Com relação a IL-8, a talidomida e o CAT-16 inibiram a produção desta citocina por células HT-29 apenas na concentração de 100µM. Ao contrário das células HT-29, o PBMC produziu TNF-α em resposta ao LPS, tendo a talidomida e o análogo CAT-16 apresentado capacidade de inibição da produção do TNF-α em ambos os tempos de tratamento. O análogo CAT-15 não influenciou a produção de TNF-α por PBMC em nenhuma das concentrações e tempos de tratamento. Este estudo também mostra a atividade da talidomida e dos nos análogos sobre a produção de IL-6 e IL-10 por PBMC, havendo significativa inibição da produção de IL-6 por todos os compostos e tempos de tratamento e sobre IL-10 pelo composto CAT-15 após 48 horas de incubação. Nossos resultados sugerem a aplicabilidade dos novos compostos, CAT-15 e CAT-16, no controle de respostas inflamatórias uma vez que inibiram a produção de moléculas chave como TNF-α, IL6, IL-10, IL-8 e CXCL-10. Ainda, esses compostos possuem estruturas simplificadas, têm baixo custo de produção, são hidrossolúveis e não possuem centro quiral. Esses resultados podem contribuir no desenvolvimento de novas estratégias de tratamento para certas condições inflamatórias. / Thalidomide is a potent anti-inflammatory drug used in the treatment of various pathologies including Erythema Nodosum Leprosum (ENL), cancer, Crohn's disease and other inflammatory and vascular disorders. However, its toxic and teratogenic effects make its use limited and motivate research groups to synthesize analogues presenting similar immunomodulation efficacy, without toxic effects. Several analogs of thalidomide have been developed in the laboratory of chemistry of the UFJF. Previously, we have shown that introduction of two phthalic anhydrides into the composition significantly enhances biological activity and water solubility, without enhanced toxicity. The present work aims at the synthesis of two new analogues of thalidomide, CAT-15 and CAT-16, formed by only one open phthalic anhydride derivative, maintaining a free amino group, and the evaluation of its anti-inflammatory activity using HT- 29 and human peripheral blood mononuclear cells (PBMCs) stimulated with LPS. The cytotoxicity of the compounds was evaluated by the MTT assay, with 18 hours treatment for HT-29 cells and for 24 and 48 hours for PBMC, using increasing concentrations of thalidomide, CAT-15, CAT-16. Dexamethasone was used as a positive control. Production of TNF-α, CXCL-10, IL-6, IL-8 and IL-10 was evaluated by the ELISA method. The novel compounds were not toxic to HT-29 and PBMC cells at any of the concentrations tested, with the exception of CAT-16 at 1600μM. HT-29 cells produced large amounts of CXCL-10 in response to LPS and the results of this work show that thalidomide and the CAT-15 and CAT-16 analogs exhibit inhibitory activity on the production of this chemokine. CAT-16 compound modulated the production of CXCL-10 at lower concentrations than thalidomide in both treatment models (simultaneous and pretreatment). In contrast, CAT-15 modulation was observed only in the pre-treatment model. Regarding IL-8, thalidomide and CAT-16 inhibited their production by HT-29 cells only at the concentration of 100 μM. Unlike HT-29 cells, PBMC produced TNF-α in response to LPS, with thalidomide and CAT-16 analog being able to inhibit TNF- production at both treatment times. The CAT-15 analogue did not influence the production of TNF-α by PBMC at any of the concentrations and treatment times. This study also shows the activity of thalidomide and the analogs on IL-6 and IL-10 production by PBMC, with significant inhibition of IL-6 production by all compounds and treatment times and on IL-10 by compound CAT-15 after 48 hours of incubation. Our results suggest the applicability of the new compounds, CAT-15 and CAT-16, in the control of inflammatory responses since they inhibited the production of key molecules such as TNF-α, IL-6, IL-10, IL-8 and CXCL-10. Furthermore, these compounds have simplified structures, and low cost of production, are water soluble and have no chiral center. These results may contribute to the development of novel treatment strategies for certain inflammatory conditions.

Mieloma Múltiplo é a segunda doença com maior prevalência nas doenças malignidades hematológica, incurável com média de sobrevivência de 3-5 anos. MM é uma malignidade das células do plasma caracterizada pela destruição e reabsorção óssea e supressão da formação do osso. A quimiocina SDF-1 (CXCL12) e seu receptor CXCR4 têm um importante papel direcional na migração, homing das células do plasma em mieloma múltiplo e mobilização das células de MM para fora da medula óssea. A talidomida tem sido usada com êxito no tratamento de pacientes com mieloma múltiplo. Neste estudo verificamos o efeito da talidomida na expressão da quimiocina SDF-1 e seu receptor CXCR4 em pacientes com mieloma múltiplo e em linhagem de células de mieloma múltiplo humano (RPMI-8226) tratados e sem tratamento de talidomida. Nossos resultamos mostraram uma expressão heterogênea na expressão da quimiocina SDF-1 e seu receptor CXCR4 nos pacientes com mieloma múltiplo estudado (n= 79). Entretanto, pacientes com mieloma múltiplo tratados com talidomida mostraram uma baixa expressão da quimiocina SDF-1 e seu receptor CXC4 quando comparados com pacientes recém diagnosticados para mieloma múltiplo e pacientes com mieloma múltiplo tratados com outros medicamentos. Nossos resultados sugerem que o tratamento com talidomida induz uma baixa regulação na expressão no ligante SDF-1 e seu receptor CXCR4 em pacientes com mieloma múltiplo / Multiple Myeloma (MM) is a second most prevalent hematological malignancy and remains incurable with a median survival of 3-5 years. MM is a plasma cell malignancy characterized by devastating bone destruction due to the enhanced bone resorption and suppressed bone formation. The chemokine stromal-derived factor-1 (SDF-1) and its receptor CXCR4 play an important role in directional migration, homing of plasma cells in multiple myeloma (MM) and mobilization of MM cells out of the bone marrow. The drug thalidomide has been successfully used in the treatment of patients with MM. In this study, we assessed the effect of thalidomide on SDF-1 and CXCR4 expression in MM patients and human myeloma-derived cell line, RPMI 8226 treated with or without thalidomide. A heterogeneous expression pattern of chemokines SDF-1 and CXCR4 receptor were observed for all MM patients studied. However, patients treated with thalidomide showed a significantly decrease in expression of SDF-1 and CXCR4 as compared to newly diagnosed MM patients and MM patients treated with other drugs. RPMI 8226 cell line treated with 10, 20 and 100µM thalidomide also demonstrated decrease in SDF-1 and CXCR4 expression as compared with cell control (RPMI-8226 without thalidomide). Ours results indicate that thalidomide therapy induces down-regulation of CXCR4 and its ligand SDF-1 in multiple myeloma

Hintergrund: Abdominale Adhäsionen entstehen vor allem durch Operationen. Sie können ernste Beschwerden auslösen, wie Dünndarmileus, Infertilität und chronische Schmerzen. Eine allgemein anerkannte Prophylaxe und/oder Therapie existiert trotz intensiver Forschung nicht. Steigende Lebenserwartung und erweiterte Operationsindikationen verschärfen das Problem. Ziel: Diese Studie soll zeigen, dass der Angiogeneseinhibitor Thalidomid postoperative Verwachsungen im Tiermodell hemmt ohne die Wundheilung zu gefährden. Methoden: 40 New Zealand White Kaninchen wurden bei einem operativen Eingriff einmalig intraperitoneal mit Thalidomid oder einem Placebo behandelt. Nach drei oder sieben Tagen wurden die Tiere erneut operiert. Danach wurden Adhäsionsbildung und Angiogenese beurteilt. Von TNF-alpha, einem wichtigen Mediator für Adhäsionen, wurden die Serumspiegel ermittelt. Die Wundheilung wurde durch visuelle Inspektion sowie durch Bestimmung von Berstungsdruck und –stelle kontrolliert. Ergebnisse: Thalidomid hemmt postoperative Adhäsionen. In der Therapiegruppe hatten 75 Prozent der Tiere keine und 25 Prozent minimale Adhäsionen. In der Kontrollgruppe dagegen traten bei knapp 50 Prozent der Tiere mäßige oder dichte Adhäsionen auf. Drei Mechanismen scheinen für die adhäsionsinhibierende Wirkung von Thalidomid verantwortlich zu sein: Hemmung der Angiogenese, Modulation der Fibrinolyse und Reduzierung der Entzündungsreaktion. Das Operationsergebnis gefährdet Thalidomid nicht. Bei der Inspektion wiesen alle Kaninchen regelgerechte Wundverhältnisse auf. Die ermittelten Berstungsdrücke und -stellen zeigten keine signifikanten Unterschiede zwischen Therapie- und Kontrollgruppe. Schlussfolgerungen: Wegen der entscheidenden Rolle der Angiogenese für die Wundheilung, aber auch wegen der teratogenen Effekte von Thalidomid, muss die adhäsionsinhibierende Wirkung von Thalidomid vor einem klinischen Einsatz in weiteren Tierversuchen verifiziert werden. Überprüft werden sollte dabei, ob neben den drei diskutierten Mechanismen, weitere vor allem immunmodulatorische Prozesse die Adhäsionshemmung bewirken. / Background: Abdominal adhesions mainly result from surgery. They can cause severe trouble like small bowel obstruction, female infertility and chronic pain. A generally recognised prevention and/or therapy does not exist despite intensive research. Increasing life expectancy and a wider range of indications for operations make matters worse. Objective: The purpose of the study is to demonstrate that the angiogenesis inhibitor thalidomide inhibits postsurgical adhesions in an animal model without impacting wound healing. Methods: 40 New Zealand White rabbits were treated once in an operation intraperitoneal with either thalidomide or with a placebo. After three or seven days the animals again underwent an operation. Thereafter the adhesions formation and angiogenesis was assessed. The level of TNF-alpha, an important mediator for adhesions, in blood was measured. The wound healing was controlled by visual inspection and the determination of the bursting pressure und location. Resuts: Thalidomid inhibits postsurgical adhesions. In the therapy sample 75 per cent of the animals had no and 25 per cent had minimal adhesions. In the control sample almost 50 per cent of the animals had moderate or dense adhesions. Three mechamisms appear to be responsible for thalidomide to inhibit adhesions: the inhibition of the angiogenesis, the modulation of the fibrinolysis and the reduction of the inflammation. The result of the operation was not impacted by thalidomide. All rabbits showed normal wound healing. The bursting pressure and location did not differ significantly between the therapy and the control sample. Conclusions: Because of the importance of the angiogenesis for wound healing as well as the teratogenic effects of thalidomide, thalidomide must be further analysed in animal tests before being applied in clinical practice. As part of this it should be examined whether in addition to the three mechansims discussed, other proceeses, in particular immune modulating processes contribute to inhibit adhesions.

In den letzten Jahren erfuhr Thalidomid auf Grund seiner antiinflammatorischen, immunmodulatorischen und antiangiogenetischen Eigenschaften eine "Renaissance". Bei ophthalmologischen Erkrankungen, die durch Gefäßproliferation gekennzeichnet sind, wie bei der diabetischen Retinopathie und der altersbedingten Makuladegeneration (33) oder bei immunpathologische Erkrankungen wie die Uveitis (44) liegen Berichte zu günstigen Effekten vor. Aufgrund der erheblichen Nebenwirkungen bei der systemischen Verabreichung wurde der Versuch einer topischen Applikation angestrebt. Dieser Applikationsweg ist limitiert durch die schlechte Wasserlöslichkeit und hydrolytische Instabilität bei der Formulierung von wässrigen Augentropfen. Zur Erhöhung der intraokularen Bioverfügbarkeit von Thalidomid wurden Cyclodextrine, speziell HP-ß-CD, als Vehikel zur Verbesserung der physiko-chemischen Eigenschaften verwendet. Die experimentelle Arbeit wurde an 21 pigmentierten Kaninchen durchgeführt, die mit Thalidomid-HP-ß-CD haltigen Augentropfen oder mit einer Thalidomidsuspension behandelt wurden. Nach einer Einwirkungszeit von 30, 60, oder 240 Minuten wurden die Augen enukleiert, präpariert und eingefroren. Anschließend wurde mittels HPLC die jeweilige Thalidomid-Konzentration ermittelt. Unsere Arbeit hat signifikante (p / Background: Thalidomide, which has been rediscovered as an angiogenic, immunmodulatory and anti-inflammatory agent might be a treatment option for several eye diseases. The systemic application causes severe side effects. The water-insolubility and hydrolytic instability of thalidomide limits its ocular bioavailability during local application. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) is a cyclodextrin derivative, which has the ability to form inclusion complexes with lipophilic drugs increasing their stability and solubility. Methods: 21 pigmented male rabbits were treated with 5x50 mul 0,04 % thalidomide/12,5% HP-ß-CD (THA-CD) or 0,04 % thalidomide suspension (THA-SP). 30, 60 or 240 minutes post instillation the animals were sacrificed and the eyes were enucleated. The thalidomide concentrations where determined using HPLC. Results: A significant (p

Die antiangioneogenetischen Effekte in-vitro und in-vivo in Kombination mit der selektiven Inhibition der TNFalpha Produktion durch Thalidomid läßt dieses Medikament als geeignete Therapie für Krankheitszustände erscheinen, bei denen die TNFalpha Toxizität eine pathogenetische Rolle spielt, die Immunität jedoch intakt bleiben muß. Ziel dieser Monographie waren Untersuchungen der Wirkungen von Thalidomid in vitro, in vivo und klinische Studien. In einer prospektiv-randomisierten, doppel-blind Studie an 70 Patienten mit kleinzelligem Bronchialkarzinom konnte die Verlängerung der Überlebenszeit durch die additive Therapie mit Thalidomid in Kombination mit einer Standardpolychemotherapie und Strahlentherapie nachgewiesen werden. Vor dem additiven Einsatz des Angioneogenesehemmers Thalidomid in der Chirurgie muß unbedingt sichergestellt sein, daß die Therapie mit Thalidomid nicht zu einer erhöhten postoperativen Morbidität und Letalität führt. Durch in-vitro Untersuchungen konnte eine Proliferationshemmung von Kaninchenendothelzellen durch metabolisiertes Thalidomid bewiesen werden. In einer randomisierten Studie an New Zealand White Kaninchen konnte gezeigt werden, daß die intraperitoneale Gabe von Thalidomid den Berstungsdruck von Kolonanastomosen im Kaninchenmodell im Vergleich zu einer Kontrollgruppe nicht erniedrigt. Darüberhinaus zeigte sich in diesem Tiermodell, daß die Rate an postoperativen Verwachsungen durch die intraperitoneale Gabe von Thalidomid signifikant vermindert wird. Das Medikament könnte eine Rolle in der additiven Therapie von Patienten mit einem kleinzelligen Bronchialkarzinom spielen. Darüberhinaus wird die Heilung von Kolonanastomosen nicht durch die intraperitoneale Gabe von Thalidomid negativ beeinflusst. Somit könnte ein Einsatz in der perioperativen Therapie bei der Resektion gastrointestinaler Karzinome in der Zukunft erwogen werden. / The proven antiangiogenic effects in vivo and in vitro in combination with a selective inhibition of the tnf alpha production seem to predestine thalidomide as an agens for diseases with a pathological elevated tnf alpha level. The theses of this monography were in vitro, in vivo and clinical effects of thalidomide. In a prospective randomized placebo controlled study on 70 patients with proven small cell lung cancer the additive treatment with oral thalidomide lead to a significant prolonged survival in combination with radio chemotherapy. Considering the treatment of thalidomide in combination with surgical therapy of patients it is essential to prove that this treatment does not lead to a higher postoperative morbidity of the patients. In an in vitro assay we could prove the antiproliferative effect of metabolized thalidomide in rabbit endothelial cells. We were than able to demonstrate that intraperitoneal administered thalidomide does not impair anastomotic healing of colonic anastomoses in a New Zealand white rabbit model compared to a control group. In addition to this significant less postoperative adhesions were found in the thalidomide group. In conclusion did the additive treatment with thalidomide in patients with small cell lung cancer lead to a prolonged survival. The antiangiogenic treatment with thalidomide in a perioperative setting does not impair the healing of colonic anastomoses in a rabbit model and may be possible in patients undergoing gastrointestinal resections in the future.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: O cloridrato de irinotecano (CPT-11) à um inibidor da topoisomerase I, clinicamente efetivo no tratamento de vÃrios tipos de cÃncer. Apesar da mucosite intestinal (MI) acompanhada de severa diarrÃia ser o efeito colateral mais limitante do uso terapÃutico do CPT-11, os exatos mecanismos que levam a estes efeitos nÃo sÃo estabelecidos. Objetivo: avaliar o envolvimento de mediadores inflamatÃrios (citocinas, Ãxido nÃtrico â NO e prostaglandinas â PGs) na patogÃnese dos eventos que acompanham a MI induzida pelo CPT-11; e estudar o efeito de inibidores da sÃntese e liberaÃÃo de citocinas, como pentoxifilina (PTX) e talidomida (TLD), e de um inibidor seletivo da ciclooxigenase-2 (COX-2), o celecoxibe (CLX), na lesÃo intestinal induzida pelo CPT-11. Material e MÃtodos: camundongos Swiss, machos, foram tratados durante quatro dias consecutivos com CPT-11 (50, 75 e 100 mg/kg, i.p.) ou veÃculo (0,5 mL, i.p.), a fim de se obter a melhor dose capaz de induzir injÃrias consistentes com o mÃnimo de letalidade. Os animais foram tratados com PTX (1,7, 5 e 15 mg/kg, s.c.), TLD (15, 30, 60 mg/kg, s.c), CLX (3, 10, 30 mg/kg, gavagem) ou veÃculo (0,5 mL, s.c. ou gavagem), um dia antes da primeira administraÃÃo do CPT-11 (75 mg/kg), e diariamente, atà o sacrifÃcio, no quinto ou sÃtimo dia. Os seguintes parÃmetros foram avaliados: diarrÃia, variaÃÃo de massa corpÃrea, leucograma, sobrevida, anÃlise histopatolÃgica, atividade de mieloperoxidase (MPO), dosagem de citocinas (TNF-α, IL-1β e KC) por ELISA e imunohistoquÃmica para TNF-α, IL-1β, iNOS e COX-2 nas mucosas duodenais. Resultados: CPT-11 induziu diarrÃia significante, acompanhada de perda acentuada de massa corpÃrea, leucopenia e reduÃÃo da sobrevida. As alteraÃÃes histopatolÃgicas intestinais induzidas pelo CPT-11 caracterizaram-se pela presenÃa de infiltrado inflamatÃrio nas cÃlulas da lÃmina prÃpria, perda da arquitetura das criptas e achatamento dos vilos. Observou-se ainda, aumento intestinal na atividade de MPO e dos nÃveis de TNF-α, IL-1β e KC, alÃm do aumento significativo na marcaÃÃo imunohistoquÃmica para TNF-α, IL-1β, iNOS e COX-2. O tratamento com PTX inibiu a diarrÃia tardia, reduziu as alteraÃÃes histopatolÃgicas, a atividade de MPO, e os nÃveis de TNF-α, IL-1β e KC, assim como a marcaÃÃo imunohistoquÃmica para TNF-α, IL-1β e iNOS na mucosa duodenal, entretanto, nÃo preveniu significativamente a perda de massa corpÃrea, a leucopenia e tampouco a mortalidade dos animais. O tratamento com TLD reduziu as lesÃes histopatolÃgicas induzidas pelo CPT-11 na mucosa intestinal, os nÃveis intestinais de MPO e TNF-α, bem como a marcaÃÃo imunohistoquÃmica de TNF-α, mas nÃo foi capaz de prevenir a diarrÃia, a perda de massa corpÃrea, a leucopenia e a sobrevida. O tratamento com CLX nÃo foi capaz de reduzir os parÃmetros inflamatÃrios e sistÃmicos observados nos animais tratados com CPT-11. ConclusÃo: Estes resultados sugerem o envolvimento de TNF-α, IL-1β, KC, NO e PGs na patogÃnese da MI induzida pelo CPT-11. PTX e TLD preveniram significativamente as alteraÃÃes histolÃgicas e inflamatÃrias induzidas pelo CPT-11, entretanto, somente PTX foi capaz de inibir o curso da diarrÃia / Introduction: Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established. Purpose: The aim of the present study was to elucidate the involvement of cytokines (TNF-α, IL-1β and KC), nitric oxide (NO) and prostaglandins (PGs) in the pathogenesis of CPT-11-induced mucositis and the effects of the cytokine production inhibitors, pentoxifylline (PTX) and thalidomide (TLD), as well as the effects of the selective cyclooxygenase (COX-2) inhibitor, celecoxib (CLX), in the CPT-11 induced intestinal mucositis, in mice. Materials and methods: the animals were treated with CPT-11 (50, 75 or 100 mg/kg, i.p.) or vehicle (0,5 ml, i.p.) daily for four days, in order to investigate the best dose able to induce intestinal mucositis without important mortality. In another set of experiments, the animals received PTX (1.7, 5, 15 mg/kg, s.c.), TLD (15, 30, 60 mg/kg, s.c.), CLX (3, 10, 30 mg/kg, oral gavage) or vehicle (0,5 ml, s.c. or oral gavage) one day before the 1st administration of CPT-11 (75 mg/kg; i.p.) and daily until the sacrifice, on the 5th or 7th day. The systemic parameters evaluated were: diarrhea, body mass variation, survival curve and leucogram. In addition, it was also performed histological analysis, myeloperoxidase (MPO) activity assay, duodenum levels of TNF-α, IL-1β and KC by ELISA and immunohistochemistry for TNF-α, IL-1β, iNOS and COX-2 in the duodenal segments. Results: CPT-11 induced an important diarrhea, weight loss, leucopenia and mortality increase. It was also observed histopathological changes, such as shortened villi, loss of the crypt architecture and inflammatory cells infiltration, observed in the lamina propria, as well as, an increase in MPO activity, TNF-α, IL-1β and KC tissue levels and a marked immuno-staining for TNF-α, IL-1β, iNOS and COX-2. The treatment with PTX inhibited the delayed diarrhea and reduced the following parameters: histopathological alterations, MPO activity, tissue levels of TNF-α, IL-1β and KC, and the immuno-staining for TNF-α, IL-1β and iNOS, however, did not prevent leucopenia, weight loss and mortality. TLD significantly reduced all the inflammatory parameters evaluated, but was not able to prevent diarrhea, leucopenia, weight loss and mortality. On the other hand, CLX did not inhibit the inflammatory nor the systemic alterations induced by CPT-11. Conclusion: These results suggest an important role of TNF-α, IL-1β, KC, NO and PGs in the pathogenesis of intestinal mucositis induced by CPT-11. PTX and TLD showed a protector effect in intestinal structures, however, only PTX reduced the severity of CPT-11-induced diarrhea

Το πολλαπλούν μυέλωμα είναι μια νεοπλασία η οποία, έως και σήμερα, παραμένει ανίατη. Στο ΠΜ, το ανοσοποιητικό σύστημα δε κατορθώνει να καταστρέψει τα κακοήθη πλασμοκύτταρα, ενώ υπάρχουν ενδείξεις ότι τα κύτταρα του όγκου παίζουν ενεργό ρόλο σε αυτό. Στην παρούσα διατριβή, ελέγξαμε ποικίλλες μυελωματικές σειρές σχετικά με την έκφραση των προ- και αντι-αποπτωτικών γονιδίων. Βρήκαμε ότι τα μυελωματικά κύτταρα εκφράζουν έναν μη φυσιολογικό φαινότυπο (fas high/bcl low). Μελετώντας τα κύτταρα του μυελού των οστών ενός ασθενούς με ΠΜ τύπου IgG/k σταδίου ΙΙΙΑ, διαπιστώσαμε ότι αυτά τα κύτταρα εξέφραζαν τα γονίδια granzyme B και perforin, τα οποία υπό κανονικές συνθήκες εκφράζονται μόνο από τα κυτταροτοξικά Τ λεμφοκύτταρα (CTLs) και τα φυσικά φονικά κύτταρα (ΝΚ). Προτείνουμε ότι πιθανόν το παραπάνω γεγονός αποτελεί έναν επιπλέον αμυντικό μηχανισμό των κυττάρων του όγκου εναντίον των CTLs και ΝΚ κυττάρων. Συγκεκριμένα υποθέτουμε ότι τα κύτταρα του όγκου προσελκύουν τα CTLs και τα ΝΚ κύτταρα και τα καταστρέφουν με τη διαδικασία της κυτταρόλυσης. Τα διφωσφονικά οξέα χρησιμοποιούνται ευρέως για τη θεραπεία του ΠΜ, ενώ η θαλιδομίδη χρησιμοποιείται σήμερα για τη θεραπεία πολλών τύπων ΠΜ. Μελετήσαμε την επίδραση της θαλιδομίδης, του ζολεδρονικού οξέος και το συνδυασμό τους στην επιβίωση των μυελωματικών κυττάρων. Βρήκαμε ένα συνεργιστικό αποτέλεσμα των δύο αυτών ουσιών. Όταν η θαλιδομίδη συγχοηγηθεί με το ζολεδρονικό οξύ σε ελάχιστους μη δραστικούς μηχανισμούς, μειώνει την τοξική επίδραση 50% του τελευταίου 3-4 φορές. / Multiple Myeloma (MM) is a plasma cell neoplasia which, to this day, remains incurable. In MM, the immune system fails to destroy the malignant plasmocytes, and evidence exists that the tumor plays an active part in this. In the present study, we tested various MM cell lines for the expression of pro- and anti-apoptotic genes. We found that MM cells express an abnormal phenotype, namely fas high/bcl low. By studying bone marrow cells from a patient suffering of MM IgG/k type stage IIIA, we found that those cells expressed granzyme B nad perforin, normally expressed by cytotoxic T cells (CTLs) and natural killer (NK) cells. We propose that this may constitute an additional acquired mechanism by the tumor cells to protect themselves against the host.

No description available.

Mieloma Múltiplo é a segunda doença com maior prevalência nas doenças malignidades hematológica, incurável com média de sobrevivência de 3-5 anos. MM é uma malignidade das células do plasma caracterizada pela destruição e reabsorção óssea e supressão da formação do osso. A quimiocina SDF-1 (CXCL12) e seu receptor CXCR4 têm um importante papel direcional na migração, homing das células do plasma em mieloma múltiplo e mobilização das células de MM para fora da medula óssea. A talidomida tem sido usada com êxito no tratamento de pacientes com mieloma múltiplo. Neste estudo verificamos o efeito da talidomida na expressão da quimiocina SDF-1 e seu receptor CXCR4 em pacientes com mieloma múltiplo e em linhagem de células de mieloma múltiplo humano (RPMI-8226) tratados e sem tratamento de talidomida. Nossos resultamos mostraram uma expressão heterogênea na expressão da quimiocina SDF-1 e seu receptor CXCR4 nos pacientes com mieloma múltiplo estudado (n= 79). Entretanto, pacientes com mieloma múltiplo tratados com talidomida mostraram uma baixa expressão da quimiocina SDF-1 e seu receptor CXC4 quando comparados com pacientes recém diagnosticados para mieloma múltiplo e pacientes com mieloma múltiplo tratados com outros medicamentos. Nossos resultados sugerem que o tratamento com talidomida induz uma baixa regulação na expressão no ligante SDF-1 e seu receptor CXCR4 em pacientes com mieloma múltiplo / Multiple Myeloma (MM) is a second most prevalent hematological malignancy and remains incurable with a median survival of 3-5 years. MM is a plasma cell malignancy characterized by devastating bone destruction due to the enhanced bone resorption and suppressed bone formation. The chemokine stromal-derived factor-1 (SDF-1) and its receptor CXCR4 play an important role in directional migration, homing of plasma cells in multiple myeloma (MM) and mobilization of MM cells out of the bone marrow. The drug thalidomide has been successfully used in the treatment of patients with MM. In this study, we assessed the effect of thalidomide on SDF-1 and CXCR4 expression in MM patients and human myeloma-derived cell line, RPMI 8226 treated with or without thalidomide. A heterogeneous expression pattern of chemokines SDF-1 and CXCR4 receptor were observed for all MM patients studied. However, patients treated with thalidomide showed a significantly decrease in expression of SDF-1 and CXCR4 as compared to newly diagnosed MM patients and MM patients treated with other drugs. RPMI 8226 cell line treated with 10, 20 and 100µM thalidomide also demonstrated decrease in SDF-1 and CXCR4 expression as compared with cell control (RPMI-8226 without thalidomide). Ours results indicate that thalidomide therapy induces down-regulation of CXCR4 and its ligand SDF-1 in multiple myeloma