Introdução: A torção testicular permanece como uma emergência urológica, despertando grande interesse em fármacos que podem minorar a lesão testicular e suas repercussões na fertilidade e produção hormonal. No entanto, não há fármaco aprovado para uso clínico rotineiro. Uma droga estudada em isquemia celular é a clorpromazina, sendo conhecidos seus efeitos protetores na função e estrutura da membrana celular e mitocondrial. Objetivos: Avaliar a diferença na lesão de células germinativas após 1 e 6 horas de torção e a ação da clorpromazina administrada previamente à resolução da torção no testículo isquêmico. Materiais e Métodos: 54 ratos Wistar, machos, com peso corporal entre 220 e 260 gramas distribuídos em 5 grupos: sham, controle com isquemia de 1 hora(A), controle com isquemia de 6 horas(B), experimental com isquemia de 1 hora(C) e experimental com isquemia de 6 horas(D). Em 48 animais foi realizada torção unilateral do cordão espermático com duas voltas em torno do seu eixo (720 graus), fixando-se o testículo nessa posição, após o que cada subgrupo foi separado em avaliação imediata (orquiectomia bilateral ao final do período de torção = 1) e tardia (orquiectomia bilateral, uma semana após a resolução da torção = 2). O grupo experimental recebeu 3 mg/kg de clorpromazina administrada via endovenosa, 30 minutos antes da resolução da torção. O grupo controle recebeu apenas solução salina a 0,9% por via endovenosa. Outros 6 animais formaram o grupo sham, onde foi realizada apenas a manipulação do cordão espermático. Após retiradas as gônadas, foram preparadas para análise histológica pela microscopia de luz e imunohistoquímica.Um pequeno fragmento de cada testículo foi separado para avaliação por microscopia eletrônica de transmissão (MET). Resultados: Na análise por microscopia de luz foram notadas alterações devido à isquemia como, necrose de coagulação e edema intersticial, principalmente nos grupos com isquemia mais prolongada (6h - B e D). Na avaliação por imunohistoquímica, houve maior expressão da caspase-3 nas células e túbulos dos testículos com 6 horas de isquemia, quando comparados com o grupo sham. No entanto, a expressão de bcl-2 não foi expressiva em nenhum grupo. Os grupos B e D também demonstraram alterações mais expressivas na análise por MET. Em nenhuma das avaliações foi observado superioridade do grupo da clorpromazina em relação ao grupo controle. Conclusão: As lesões celulares intratubulares induzidas pela isquemia e reperfusão testicular foram semelhantes após 1 e 6 horas, as diferenças foram relacionadas à sua maior intensidade no grupo com 6 horas e a clorpromazina não foi efetiva na prevenção da lesão por reperfusão. / Introdution: Testicular torsion remains as a urology emergency arousing interest about medicine which can reduce testicular injury and its impact on fertility and hormone production. However, there is no drug approved for routine clinical use. A drug studied in cell ischemia is chlorpromazine, being known its protective effects on the function and structure of cellular membrane and mitochondrial. Objective: To evaluate the difference in lesion of germ cells after 1 and 6 hours and the action of chlorpromazine administered before the resolution of ischemic testicle due torsion. Materials and methods: 54 male Wistar rats weighing between 220 to 260 grams divided into five groups: sham, control with one hour of ischemia (A) control with six hours of ischemia (B) experimental with one hour of ischemia (C) and experimental six hours of ischemia (D). In 48 animals was performed unilateral torsion of the spermatic cord with two laps around its axis (720 degrees), keeping the testicle in this position. After that, each subgroup was divided into immediate evaluation (bilateral orchiectomy at end of the torsion period = 1) or later (bilateral orchiectomy after one week of torsion resolution = 2). The experimental group received 3 mg / kg chlorpromazine administered intravenously 30 minutes before the resolution of torsion. The control group received only saline 0.9% intravenously. Other 6 animals were in the sham group, which was held just handling the spermatic cord. After withdrawal, the gonads were prepared for histological analysis by light microscopy and immunohistochemistry. A small piece of each testis was separated for evaluation by electron microscopy. Results: In analysis by light microscopy, ischemic changes were rated as coagulative necrosis and interstitial edema mainly in groups with prolonged ischemia (6h - B and D). When analyzed by immunohistochemistry, there was greater expression of caspase-3 in cells and tubules of the testes with 6 hour of ischemia compared to the sham group. However, bcl-2 expression was not impressive in either group. B and D groups also showed more significant changes in the analysis by electron microscopy. None of the ratings has been shown superiority of chlorpromazine group over the control group. Conclusion: The germ cell damage induced by ischemia and reperfusion was similar after 1 and 6 hours, the differences were related to its greatest intensity in the group with 6 hours and chlorpromazine was not effective in preventing reperfusion injury.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Jà està estabelecido que a liberaÃÃo de produtos da ciclooxigenase e aminas simpatomimÃticas, medidores finais da hiperalgesia inflamatÃria à precedido pela geraÃÃo de uma cascata de citocinas prÃ-inflamatÃrias e nociceptivas. Dados anteriores demonstraram que a ativaÃÃo desta cascata de citocinas à dependente tambÃm da presenÃa de cÃlulas residentes como macrÃfagos e mastÃcitos no local da injÃria. Talidomida (TALD), pentoxifilina (PTX) e clorpromazina (CLP) sÃo drogas que dentre outras funÃÃes sÃo descritas como imunomoduladoras por modularem a produÃÃo de algumas citocinas e vem chamando atenÃÃo pelas suas propriedades antiinflamatÃrias na prÃtica clÃnica e em modelos experimentais. Com base nesses achados, o objetivo do presente trabalho foi estudar uma possÃvel atividade antinociceptiva de TALD, PTX e CLP correlacionando à essa atividade imunomodulatÃria. Para tanto injetou-se por via i.p. talidomida (TALD; 5-45 mg;kg), pentoxifilina (PTX; 0.5 â 45 mgkg) ou clorpromazina (CLP; 0.1 â 1 mgkg) 30 min antes da administraÃÃo de Ãcido acÃtico (AAc), zymosan (Zym) ou iloprost (ILO) para o teste de contorÃÃes abdominais em camundongos (CA), ou 30 min antes do Zym intrarticular no teste da incapacitaÃÃo articular (IA) em joelho de rato (teste de nocicepÃÃo articular). TALD, PTX e CLP tambÃm foram testadas em diferentes doses por via sistÃmica (i.p.) ou local (intraplantar) no teste da hiperalgesia (HYP) mecÃnica induzida por carragenina (Cg), bradicinina (Bk), fator de necrose tumoral (TNF), interleucina-1 (IL-1) ou prostaglandina E2 (PGE2) e no teste da placa quente (PQ). TALD, PTX ou CLP tambÃm foi injetada em camundongos 30 min antes do zymosan e apÃs 15 min foi feita a coleta do fluido peritoneal contendo cÃlulas residentes onde o mesmo foi posto bem cultura para avaliar a produÃÃo de citocinas por essas cÃlulas. O fluido articular de ratos tratados com TALD, PTX ou CLP e estimulados com Zym intrarticular tambÃm foi analisado no mesmo sentido. Nossos resultados demonstram que TALD, PTX e CLP sÃo antinociceptivas tanto no modelo de CA induzidas por zymosan (85.6, 82.9 e 63.7% de inibiÃÃo, respectivamente, p<0.001) ou AAc (60.3, 89.8 e 89% de inibiÃÃo, efeito mÃximo respectivamente, p<0.001), como tambÃm a IA induzida por Zym (75, 89 e 99% de inibiÃÃo, respectivamente, p<0.001), mas nÃo nas CA induzidas por ILO . TALD foi capaz de inibir o efeito hiperalgÃsico da Cg (78.6%) e Bk (82.4%), mas nÃo o de TNF e PGE2. PTX inibiu a HYP induzida por Cg (73.3%), Bk (55.2%), TNF (45.6%), mas nÃo a por IL-1 ou PGE2. CLP inibiu a HYP induzida por todos os estÃmulos, mas em graus diferentes (68.16, 58.5, 42, 38.8 e 21.1%de inibiÃÃo respectivamente para Cg, Bk, TNF, IL-1 e PGE2). Estes efeitos antinociceptivos parecem ser de domÃnio perifÃrico visto que as drogas nÃo modificaram o tempo de reaÃÃo na PQ e nÃo dependem da liberaÃÃo de opiÃides endÃgenos, pois naloxona nÃo reverteu a atividade antinociceptiva das drogas. Contudo a atividade antinociceptiva parece depender da inibiÃÃo da liberaÃÃo de citocinas prÃ-nociceptivas por cÃlulas residentes visto que TALD, PTX e CLP inibiram a liberaÃÃo de TNF (100, 85 e 54.4% de inibiÃÃo respectivamente p< 0.001) e IL-1 (97% de inibiÃÃo para PTX) por cÃlulas peritoneais residentes de camundongos como tambÃm a produÃÃo de TNF (77 e 87.5%de inibiÃÃo respectivamente para TALD e PTX) e IL-1 (47 e 32.6% de inibiÃÃo respectivamente para PTX e CLP) na cavidade articular de ratos estimulados com Zym. / The release of cyclo-oxygenase products and sympathomimetic amines, the final mediators of inflammatory pain, is preceded by the generation of pro-inflammatory and nociceptive cytokines by resident cells. Recently drugs such as thalidomide (TALD), pentoxifylline (PTX) and chlorpromazine (CLP), despite of other effects, have been associated with immunomodulatory activity in clinical practice mainly for their modulatory properties upon cytokine production. Since those drugs are able to inhibit the production of pro-inflammatory cytokines and the pivotal role of resident cells in the development of inflammatory pain we have decided to test the possibility of TALD, PTX and CLP, to modulate inflammatory pain. TALD (5 â 45mg/kg), PTX (0.5 â 45mg/kg) or CLP (0.1 â 1mg/kgl) was given 30 min before either acetic acid (AAc) or zymosan (Zym) or iloprost (ILO) administration in the writhing model. TALD, PTX or CLP, at the same doses were injected, i.p., 30 min before Zym (1 mg/animal; intra-articular) in the zymosan-induced rat knee joint incapacitation test (JI). Those drugs were also tested upon hyperalgesic effect of carrageenin (Cg), bradykinin (Bk), tumor necrosis factor (TNF), interleukin (IL) -1 and prostaglandin E2 (PGE2) on mechanical hyperalgesia test (HYP). Doses of those drugs that exerted maximum effect in the writhing test were also injected 30 min before the hot plate test. These same doses were injected ip before naloxone administration in the AAc-induced writhing model in mice. Cytokines levels (TNF, IL-1, IL-10 and IL-4) were determined in the supernatant of a macrophage culture, which were collected from peritoneal fluid of mice treated with Zym and pre-treated with the drugs under test and in the supernatant of articular fluid of rats pre-treated with the drugs and stimulated with Zym (TNF, IL-1 beta, IL-10, IL-6 and CINC-1). Our results showed that TALD, PTX and CLP inhibited the writhing response in mice induced by AAc or Zym up to 60.3, 89.8 e 89%, and up to 85.6, 82.9 and 63.7% respectively (p<0.001), but not the nociceptive response to ILO. Similar results were observed in the JI induced by Zym: 75, 89 e 99% of inhibition, respectively (p<0.01). TALD inhibited hyperalgesic effect of Cg (78.6%) and Bk (82.4%), but not the hyperalgesic effect of TNF or PGE2. PTX inhibited Cg, TNF or Bk-induced HYP in 73.3, 55.2 and 45.6% of inhibition respectively, but not IL-1 or PGE2 hyperalgesic activity. CLP inhibited a hyperalgesic effect of all stimuli in different levels (68.16, 58.5, 42, 38.8 and 21.1% of inhibition for Cg, Bk, TNF, IL-1 e PGE2, respectively). The antinociceptive activity of TALD, PTX and CLP seems to be peripheral, since these drugs presented no effect in the reaction time of the animals on hot plate test. This antinociceptive effect seems to have no relation with endogen opioid release since naloxone (opioid receptor antagonist) had no effect in reverting the antinociceptive effect of these drugs in the Zym-induced writhing in mice. The antinociceptive activities seems to be dependent of the release inhibition of pro-nociceptive cytokines by resident cells since TALD, PTX and CLP inhibited the release of TNF (100, 85 e 54.4%, respectively - p<0.005) and of IL-1 (97%, PTX effect - p<0,01) by mice peritoneal resident cells as well as the production of both TNF (77 e 87.5% by TALD and PTX respectively) and IL-1 (47 e 32.6% by PTX and CLP, respectively) in the articular cavity of ZYM stimulates rats.

Introdução: A torção testicular permanece como uma emergência urológica, despertando grande interesse em fármacos que podem minorar a lesão testicular e suas repercussões na fertilidade e produção hormonal. No entanto, não há fármaco aprovado para uso clínico rotineiro. Uma droga estudada em isquemia celular é a clorpromazina, sendo conhecidos seus efeitos protetores na função e estrutura da membrana celular e mitocondrial. Objetivos: Avaliar a diferença na lesão de células germinativas após 1 e 6 horas de torção e a ação da clorpromazina administrada previamente à resolução da torção no testículo isquêmico. Materiais e Métodos: 54 ratos Wistar, machos, com peso corporal entre 220 e 260 gramas distribuídos em 5 grupos: sham, controle com isquemia de 1 hora(A), controle com isquemia de 6 horas(B), experimental com isquemia de 1 hora(C) e experimental com isquemia de 6 horas(D). Em 48 animais foi realizada torção unilateral do cordão espermático com duas voltas em torno do seu eixo (720 graus), fixando-se o testículo nessa posição, após o que cada subgrupo foi separado em avaliação imediata (orquiectomia bilateral ao final do período de torção = 1) e tardia (orquiectomia bilateral, uma semana após a resolução da torção = 2). O grupo experimental recebeu 3 mg/kg de clorpromazina administrada via endovenosa, 30 minutos antes da resolução da torção. O grupo controle recebeu apenas solução salina a 0,9% por via endovenosa. Outros 6 animais formaram o grupo sham, onde foi realizada apenas a manipulação do cordão espermático. Após retiradas as gônadas, foram preparadas para análise histológica pela microscopia de luz e imunohistoquímica.Um pequeno fragmento de cada testículo foi separado para avaliação por microscopia eletrônica de transmissão (MET). Resultados: Na análise por microscopia de luz foram notadas alterações devido à isquemia como, necrose de coagulação e edema intersticial, principalmente nos grupos com isquemia mais prolongada (6h - B e D). Na avaliação por imunohistoquímica, houve maior expressão da caspase-3 nas células e túbulos dos testículos com 6 horas de isquemia, quando comparados com o grupo sham. No entanto, a expressão de bcl-2 não foi expressiva em nenhum grupo. Os grupos B e D também demonstraram alterações mais expressivas na análise por MET. Em nenhuma das avaliações foi observado superioridade do grupo da clorpromazina em relação ao grupo controle. Conclusão: As lesões celulares intratubulares induzidas pela isquemia e reperfusão testicular foram semelhantes após 1 e 6 horas, as diferenças foram relacionadas à sua maior intensidade no grupo com 6 horas e a clorpromazina não foi efetiva na prevenção da lesão por reperfusão. / Introdution: Testicular torsion remains as a urology emergency arousing interest about medicine which can reduce testicular injury and its impact on fertility and hormone production. However, there is no drug approved for routine clinical use. A drug studied in cell ischemia is chlorpromazine, being known its protective effects on the function and structure of cellular membrane and mitochondrial. Objective: To evaluate the difference in lesion of germ cells after 1 and 6 hours and the action of chlorpromazine administered before the resolution of ischemic testicle due torsion. Materials and methods: 54 male Wistar rats weighing between 220 to 260 grams divided into five groups: sham, control with one hour of ischemia (A) control with six hours of ischemia (B) experimental with one hour of ischemia (C) and experimental six hours of ischemia (D). In 48 animals was performed unilateral torsion of the spermatic cord with two laps around its axis (720 degrees), keeping the testicle in this position. After that, each subgroup was divided into immediate evaluation (bilateral orchiectomy at end of the torsion period = 1) or later (bilateral orchiectomy after one week of torsion resolution = 2). The experimental group received 3 mg / kg chlorpromazine administered intravenously 30 minutes before the resolution of torsion. The control group received only saline 0.9% intravenously. Other 6 animals were in the sham group, which was held just handling the spermatic cord. After withdrawal, the gonads were prepared for histological analysis by light microscopy and immunohistochemistry. A small piece of each testis was separated for evaluation by electron microscopy. Results: In analysis by light microscopy, ischemic changes were rated as coagulative necrosis and interstitial edema mainly in groups with prolonged ischemia (6h - B and D). When analyzed by immunohistochemistry, there was greater expression of caspase-3 in cells and tubules of the testes with 6 hour of ischemia compared to the sham group. However, bcl-2 expression was not impressive in either group. B and D groups also showed more significant changes in the analysis by electron microscopy. None of the ratings has been shown superiority of chlorpromazine group over the control group. Conclusion: The germ cell damage induced by ischemia and reperfusion was similar after 1 and 6 hours, the differences were related to its greatest intensity in the group with 6 hours and chlorpromazine was not effective in preventing reperfusion injury.

>Magister Scientiae - MSc / Engineered nanomaterials derived from various bulk materials are being developed in ever larger quantities and with very diverse chemical compositions. The physical and chemical properties of the smaller nanoparticles are very different compared to their larger bulk chemicals. Titanium dioxide nanoparticles (TiO2NPs) are an example of such an engineered nanomaterial. Titanium dioxide nanoparticles are mainly used as a pigment in many applications such as glazes, enamels, plastics, pharmaceuticals, cosmetics, and it is widely used in sunscreens. Human exposure to TiO2NPs can occur both during manufacturing and use.

Presynaptic and postsynaptic actions of dextroamphetamine (DEX) were studied on dopamine (DA) sensitive neurons of the subesophageal ganglion of the garden snail Helix aspersa utilizing standard microelectrode techniques. Dextroamphetamine (5.5 × 10-7-10-4 M) produced effects on DA-sensitive neurons similar to that caused by DA (5.5 × 10-7-10-4 M). On cells excited by DA, surfused DEX (5.5 × 10-7 M) caused an excitation that could be blocked by chlorpromazine (0.5-1 × 10-6 M) or haloperidol (0.5-1 × 10-6 M). Elevating the extracellular Mg2+ from 4 to 20 mM reduced the depolarization caused by DEX from 11 to 2.5 mV without affecting the response to DA. The response remaining is attributed to a direct response to DEX on DA receptors. Surfused DEX caused an inhibition of cells inhibited by DA. Both DA and DEX effects were selectively blocked by dihydroergotamine (0.5-1 × 10-6 M). Elevating the [Mg2+] decreased the hyperpolarization caused by DEX from 11 to 3 mV without affecting the DA response. The effect of elevated magnesium in decreasing responses to surfused DEX suggests that the primary action of DEX is at the nerve terminal to cause DA release. Iontophoretic application of DEX caused minimal excitation or inhibition of DA neurons. This is attributed to the fact that DA receptors at the site of drug application are not associated with synaptic innervation. The response obtained with iontophoretically applied DEX suggest a weak direct action on DA receptors.

La survenue de lésions hépatiques représente une cause majeure de retrait des médicaments au cours de leur développement et après leur mise sur le marché. La manifestation la plus fréquente des effets secondaires liés aux médicaments est lacholestase qui résulte d’un blocage de la sécrétion biliaire. Ils peuvent être prévisibles, généralement dépendants de la dose, ou dans certains cas n’être observés que chez un nombre restreint de patients traités, par exemple avec la chlorpromazine (CPZ), un neuroleptique. Il s’agit alors d’une hépatotoxicité idiosyncratique. Notre travail a eu pour but d’induire une cholestase avec ce médicament et d’étudier les mécanismes impliqués, en présence ou non d’un stress inflammatoire en utilisant comme modèle expérimental les cellules hépatiques différenciées HepaRG dérivées d’un cholangio-hépatocarcinome humain. Nous avons tout d’abord validé ce modèle en montrant que les principaux transporteurs d’influx et d’efflux canaliculaires et basolatéraux sont bien localizes dans les domaines membranaires appropriés, et que les canalicules biliaires sont fonctionnels et fermés comme dans les hépatocytes humains cultivés en sandwich, le modèle de référence. Le traitement par CPZ à une concentration élevée (50μM) entraine après 15min la génération d’un stress oxydant associé à une altération du potentiel membranaire mitochondrial et de la distribution péricanaliculaire des microfilaments de F-actine et à une inhibition de l’efflux canaliculaire de l’acide taurocholique. Après 24h, on observe notamment une inhibition de l’expression des deux principaux transporteurs canaliculaires, BSEP et MDR3, du transporter d’influx NTCP et une surexpression du transporteur basolatéral MRP4. Ces effets suggèrent une réponse compensatrice des cellules face à l’accumulation intracellulaire des acides biliaires. L’inflammation est considérée comme un facteur de susceptibilité dans l’hépatotoxicité idiosyncratique. Nous avons recherché si dans un context inflammatoire induit par l’IL-6 et l’IL-1β, les effets cytotoxiques et cholestatiques de CPZ sont aggravés. Après un prétraitement de 24h par les deux cytokines proinflammatoires, les cellules HepaRG, ont été co-exposées à 20μM CPZ pendant 1 à 5 jours. Bien que les cytokines aient induit un stress inflammatoire et inhibé le métabolisme de la CPZ et les transcrits de CYP3A4 et CYP1A2, deux principaux CYPs impliqués dans le métabolisme de ce médicament, la modulation des effets cytotoxiques et cholestatiques de la CPZ observés est restée limitée, y compris après 5 jours. Une cytotoxicité accrue de 20% et une amplification de l’inhibition des transcrits et de l’activité de NTCP ainsi que la dérégulation de l’expression d’autres gènes liés à la cholestase, ont été constatées suite au co-traitement à CPZ et aux cytokines. Au total, nos résultats montrent qu’il est possible d’induire une cholestase in vitro à partir des cellules HepaRG et que la cholestase induite par CPZ a pour origine l’induction d’un stress oxydant. Ils montrent en outre que l’étude de certains facteurs de susceptibilité peut être envisagée. / Drug-induced liver injury is the major cause of drug withdrawal during development and marketing process. The most common manifestation of adverse drug reactions is cholestasis, which results from alteration of bile flow. Adverse drug reactions are usually classified either as dose-dependent and reproducible (intrinsic) or unpredictable (idiosyncratic) occurring only in certain susceptible patients as observed with chlorpromazine (CPZ), a neuroleptic drug. Our work aimed to induce cholestasis with this drug and to study the mechanisms involved in the presence or absence of an inflammatory stress using differentiated HepaRG liver cells derived from a human cholangio-hepatocarcinoma as an experimental model. We firstly validated this cell model by demonstrating that the major canalicular and basolateral influx and efflux transporters are localized to the appropriate membrane domains, and that the bile canaliculi are functional and closed as in sandwichcultured human hepatocytes, the reference model. Treatment with CPZ at a high concentration (50μM) induces, as early as 15min, generation of oxidative stress which is associated with altered mitochondrial membrane potential, disruption of the pericanalicular F-actin cytoskeleton distribution and inhibition of canalicular efflux of taurocholic acid. After 24-hour treatment with CPZ, mRNA expression of the two main canalicular bile transporters, BSEP and MDR3, and of the main influx transporter, NTCP, was decreased. By contrast, expression of MRP4 mRNA, a basolateral transporter, was increased. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Inflammation is considered as a factor of susceptibility to idiosyncratic hepatotoxicity. We investigated whether in an inflammatory stress induced by IL-6 and IL-1β, cytotoxic and cholestatic effects of CPZ are exacerbated. After a 24 hour pre-treatment by either pro-inflammatory cytokines, HepaRG cells were co-exposed to 20μM CPZ for 1 to 5 days. Although cytokines have induced inflammatory stress and inhibited the metabolism of CPZ and transcripts of CYP3A4 and CYP1A2, two main CYPs involved in the metabolism of this drug, the modulation of cytotoxic and cholestatic effects of CPZ was limited, even after 5 daily treatments. Increased cytotoxicity by 20 %, amplification of NTCP mRNA and activity inhibition and deregulation of the expression of other genes associated with cholestasis, were observed in CPZ- and cytokine-co-treated cells. Altogether, our results show that it is possible to induce in vitro cholestasis using HepaRG cells and that CPZ-induced cholestasis depends on the generation of oxidative stress. They also show that the certain susceptibility factors may be investigated.

Les leucémies sont une famille de cancers issus de la prolifération maligne des cellules hématopoïétiques. La leucémie myéloïde aigüe (AML) représente 30% des leucémies chez les adultes et menace surtout les personnes âgées de 64 ans et plus. Actuellement, la chimiothérapie est la méthode principale de prise en charge de l’AML, mais celle-ci comporte des effets secondaires importants ainsi qu’un risque de récidive qui freinent son développement. Le redéploiement de molécules déjà utilisées pour d’autres maladies est une stratégie émergente dans le traitement du cancer. Par exemple, la chlorpromazine (CPZ), un antipsychotique, démontre une activité contre les lignées cellulaires issues d’AML, mais son activité au niveau du système nerveux central entraine des effets indésirables. Compte tenu de l’activité de CPZ contre les cellules leucémiques, nous avons mis au point un système de vectorisation des médicaments original pour le traitement de l’AML. La CPZ est d’abord incluse dans une cyclodextrine (CD) : molécule cage à base de glucose et ce complexe est ensuite encapsulé dans un liposome : vésicules à contenu aqueux délimitées par une ou plusieurs bicouches phospholipidiques. Ce système de “drug-in-CD-in-liposomes” (DCL) est conçu pour circuler longtemps dans le sang après injection intraveineuse mais ne pas passer la barrière hémato-encéphalique (BHE).Il a été nécessaire d’optimiser la formulation avec ses trois composants principaux : CD, CPZ et phospholipides. Ainsi, une évaluation physico-chimique approfondie a été réalisée pour les interactions CD/CPZ, CD/phospholipides et CPZ/phospholipides. La calorimétrie de titration isotherme (ITC) a permis de déterminer la stœchiométrie et la constante d’association pour les complexes d’inclusion CD/CPZ, démontrant les associations les plus importantes avec la Sugammadex ou la SBE-β-CD. La spectroscopie RMN 2D ROESY a mis en évidence des géométries d’inclusion différentes selon la taille et le type de substitution des CDs. Par ailleurs, l’inclusion de la CPZ dans les CDs accélère légèrement la photodégradation de l’antipsychotique. Quant à l’interaction CD/phospholipides, la SBE-Et-β-CD déstabilise les liposomes multilamellaires (MLVs) composés de palmitoylstearoylphosphatidylcholine (PSPC) pour des concentrations supérieures à 25 mM, tandis que les autres CDs sont sans effet. En outre, des études de turbidité ont démontré que les liposomes unilamellaires (SUV) ne sont pas perturbés en présence de la plupart des CDs. Une étude de l’interaction entre CPZ et PSPC a démontré que la quantité maximale qu’il est possible d’encapsuler était égale à 3,75 mol%. Par la suite, des DCL contenant CPZ ont été préparés par la méthode « dehydration-rehydration vesicles » (DRV) mais le pourcentage du principe actif encapsulé (EE) s’avérait faible (environ 10%). Ainsi la méthode DRV combinée à l’utilisation d’un gradient de concentration de sulfate d’ammonium a été mise en œuvre, ce qui a permis d’augmenter l’EE à 27% tout en gardant une taille compatible avec l’administration IV. Par la suite, la cytotoxicité de ces formulations a été évaluée sur 4 lignées cellulaires humaines issues d’AML (HL-60, MOLM13, MV4-11 and OCI-AML). Les liposomes contenant la CPZ ont tous démontré un effet cytotoxique tandis que les liposomes vides avaient tendance à stimuler la croissance cellulaire et les CD seules étaient sans effet. Pourtant, l’activité des DRVs avec CD était inférieure à celle des DRV/CPZ, selon l’ordre suivant : DRV-SGM/CPZ < DRV-SBE-β-CD/CPZ < DRV-HP-γ-CD/CPZ. L’activité était inversement proportionnelle à la constante d’affinité, suggérant une libération retardée. Cette hypothèse a été confortée par le fait que l’activité des DRV CD/CPZ était plus importante après une exposition de 72h qu’après 24h.Ces résultats sont prometteurs pour la mise au point de systèmes à libération contrôlée de CPZ pour le traitement d’AML. / Leukemia is a group of cancers caused by malignant clonal proliferation of hematopoietic stem cells. Accounting for 30% of all leukemias in adults, acute myeloid leukemia (AML) is especially a threat for older people with a median age of 64 years. Presently, chemotherapy is the main therapeutic method for AML but severe side-effects and possibility of relapse have hampered its development. “Repurposing” of drugs used in other diseases is a current trend for the treatment of cancer. For example, chlorpromazine (CPZ), a widely used anti-psychotic drug, shows effective activity in AML cell lines, but can also cause side effects in the central nervous system.Basing on the inhibiting capability of CPZ against leukemia-related cell lines, we have designed and developed a novel nanometric drug delivery system for AML treatment. CPZ is first entrapped in the hydrophobic internal cavity and forms inclusion complexes with cyclodextrin (CD), a cask-like molecule composed of glucose units, then the CD/CPZ inclusion complexes are encapsulated in liposomes, phospholipid vesicles consisting of a series of lipid bilayers enclosing aqueous compartments. The “drug-in-CD-in-liposomes” (DCL) system provides a promising strategy which is capable of achieving long circulation time in the blood after intravenous administration while circumventing the blood-brain barrier (BBB). It was necessary to optimize the formulation for its three main components: CD, CPZ and phospholipid. Therefore, a comprehensive physicochemical investigation of the interaction between CD/CPZ, CD/lipid and CPZ/lipid was performed. Isothermal titration calorimetry (ITC) was used to obtain the stoichiometry and association constant of the CPZ-CD inclusion complexes, showing that sugammadex and SBE-β-CD has the highest complexation ratio. 2D ROESY NMR revealed different inclusion processes depending on the size and chemical modification of CDs. Photodegradation experiments indicated that CDs can slightly accelerate CPZ's photodegradation. As far as liposome stability was concerned, SBE-Et-β-CD was found to have an obvious interaction with palmitoyl stearoyl phosphatidylcholine (PSPC) multilamellar liposomes (MLVs) above 25 mM, while the other CDs were without effect. However, turbidity measurements indicated that small unilamellar liposomes (SUVs) remain intact in presence of CDs. A study of the interaction between CPZ and PSPC showed that the maximum proportion of CPZ that could be incorporated into the lipid membrane was 3.75 mol%. Subsequently, DCL systems containing CPZ was prepared by the dehydration-rehydration vesicles (DRV) method. However, low drug encapsulation efficiency (EE) was obtained (about 10% of added drug). Therefore, an active loading strategy, the ammonium sulphate gradient method, was used in combination with the DRV method. As a result, the EE of CPZ increased to about 27% and yielded liposomal formulations with suitable size for IV administration.Finally, the cytotoxicity of CPZ, CDs, empty liposomes and liposomes containing CD/CPZ complexes was evaluated against a panel of leukemia cell lines. CPZ was found to show a growth-inhibiting effect on the human leukemia cell lines (HL-60, MOLM13, MV4-11 and OCI-AML), while empty liposomes were observed to slightly promote their growth and CDs alone had no significant cytotoxicity. However, a difference in activity was observed with the DRV containing CD/CPZ complexes, which were less active than DRV containing free CPZ, with the order: DRV-SGM/CPZ < DRV-SBE-β-CD/CPZ < DRV-HP-γ-CD/CPZ. The activity was inversely proportional to the formation constant of CD/CPZ complexes obtained by ITC, suggesting a delayed release from the complexes. This was confirmed by varying the exposure time; the activity of DRV CD/CPZ being relatively higher in a longer incubation.These results suggest that the DCL system could provide controlled release of CPZ for the treatment of AML.

Orientador: Prof. Dr. Tiago Rodrigues / Among the main problem faced during the treatment of cancer patients is resistance to chemotherapy. Among the multiple drug resistance mechanisms (MDR), the best characterized involves the activity of P-glycoprotein (Pgp), the product of the MDR1 gene expression. The reversal of MDR due to Pgp inhibition has been observed by several drugs, including phenothiazines, which has currently been the focus of studies suggesting their use in antitumor therapy. Studies with copolymers of ethylene oxide and propylene oxide, termed poloxamers, demonstrated the ability to inhibit Pgp on MDR cells suggesting their use in antitumor formulations. Thus, this work aims to study cell death induced by phenothiazines inserted in nanostructured systems (liposomes, gold nanoparticles and polymeric micelles) in human myeloblastic leukemia cells K562 and Lucena 1, only the last one overexpressing the MDR1 gene. The results showed that the polymeric micellar systems consisting of poloxamers are more suitable for potentiation of the effect of phenothiazines. The characterization of micellar systems assays indicated that the structures are organized in a lamellar shape and the particle size is about 60 nm at 25°C and 25 nm at 37°C. The in vitro release assays showed that formulations were able to decrease the releasing by 80% when compared to phenothiazines in the free form. Formulations containing PL407/L81 (poloxamer 407 and Pluronic®L81) showed higher capacity to enhance the cytotoxic effect of phenothiazines. The formulation containing trifluoperazine (TFP) inserted in PL407/L81 system caused a significant decrease in the EC50 when compared to the free form only after 48 hours of incubation in K562 lines and Lucena 1. The formulation containing thioridazine (TR) significantly alter the EC50 for 24 hours in the cell line K562 and the both strains after 48 hours of incubation. For formulation containing chlorpromazine (CPZ) was significantly decreased upon incubation for 24 to 48 hours in both cell lines. The death induction profile was similar for all phenothiazines suggesting predominantly apoptosis and late apoptosis. The PL407 / L81 system was able to inhibit the activity of Pgp in resistant strain Lucena 1, this effect was also observed for TR and TFP which had the effect potentiated after insertion in the system containing PL407/L81. However, CPZ has not been capable of inhibiting Pgp in the absence of system PL407/L81 indicating that this system is essential for the enhancement of the cytotoxic effect of CPZ in the line Lucena 1. The tests on non-tumor cell line showed that the mononuclear PL407 / L81 system does not have cytotoxicity, and also is able to decrease the cytotoxic effects of phenothiazines. These data indicate that system containing PL407 and L81 has the greatest potential for application in anti-tumor therapy with the additional benefit of reduced cytotoxic effect on normal cells. / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2015. / Um dos principais problemas enfrentados no tratamento de pacientes com câncer é a resistência a quimioterápicos. Entre os mecanismos de resistência a múltiplas drogas (MDR), o melhor caracterizado envolve a atividade da glicoproteína P (Pgp), produto da expressão do gene MDR1. A reversão da MDR por inibição da Pgp foi observada por diversos fármacos, entre eles as fenotiazinas que atualmente tem sido alvo de estudos que sugerem sua utilização na terapia antitumoral. Estudos com co-polímeros constituídos por óxido de etileno e óxido de propileno, denominados poloxamers, demonstraram a capacidade de inibição da Pgp em células MDR sugerindo sua aplicação em formulações contendo antitumorais. Dessa forma, este trabalho tem como objetivo estudar a morte celular induzida por fenotiazinas inseridas em sistemas nanoestruturados (lipossomas, nanopartículas de ouro e micelas poliméricas) em células leucêmicas mieloblásticas humanas K562 e Lucena 1, sendo que apenas a última apresenta superexpressão do gene MDR1. Os resultados indicaram os sistemas micelares poliméricos constituídos de poloxamers são mais adequados para potencialização do efeito das fenotiazinas. Os ensaios de caracterização dos sistemas micelares indicaram que as estruturas se organizam de forma lamelar e possuem tamanho de partícula de aproximadamente 60 nm, a 25°C, e de 25 nm, a 37°C. Nos ensaios de liberação in vitro, as formulações diminuíram 80% da liberação quando comparadas a liberação das fenotiazinas na forma livre. As formulações contendo PL407/L81 (poloxamer 407 e Pluronic®L81) apresentaram maior capacidade de potencialização do efeito citotóxico das fenotiazinas. A formulação contendo trifluoperazina (TFP) inserida no sistema PL407/L81 promoveu diminuição significativa do EC50 em relação à forma livre apenas após 48 horas de incubação nas linhas K562 e Lucena 1. A formulação contendo tioridazina (TR) alterou significativamente o EC50 em 24 horas na linhagem K562 e em ambas a linhagens após 48 horas de incubação. Para a formulação contendo clorpromazina (CPZ) a diminuição foi significativa na incubação por 24 e 48 horas, em ambas as linhagens. O perfil de indução de morte foi semelhante para todas as fenotiazinas sugerindo, predominantemente, apoptose tardia e apoptose. O sistema PL407/L81 foi capaz de inibir a atividade da Pgp na linhagem resistente Lucena 1, este efeito também foi observado para TR e TFP que tiveram o efeito potencializado após a inserção no sistema contendo PL407/L81. Contudo, CPZ não foi capaz de inibir a Pgp na ausência do sistema PL407/L81 indicando que este sistema é essencial para a potencialização do efeito citotóxico da CPZ na linhagem Lucena 1. A avaliação na linhagem mononuclear normal mostrou que o sistema PL407/L81 não apresenta citotoxidade e, ainda, é capaz de diminuir o efeito citotóxico das fenotiazinas. Estes dados indicam que sistema contendo PL407 14,5% e L81 0,5%, possui maior potencial para aplicação na terapia antitumoral com o benefício adicional de diminuição do efeito citotóxico em células normais.

Made available in DSpace on 2016-12-08T16:24:12Z (GMT). No. of bitstreams: 1 PGCV06MA009.pdf: 426326 bytes, checksum: 327decb1f96fe7fe958d4a50ff4d33d0 (MD5) Previous issue date: 2006-03-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study was designed to determine the hemodynamic and hemogasometric effects of the premedication with atropine in dogs submitted to chlorpromazine and dexmedetomidine administration under general anesthesia with isoflurane and kept in mechanic ventilation. Six dogs, weighing 17.9 ±3.9kg constituted the two groups of the study, with an interval of seven days between treatments. The animals were instrumentalized using isoflurane, proceeding the catheterization of the dorsal tibial artery and introduction of the SwanGanz catheter through the jugular vein. At the end of the instrumentalization the isoflurane concentration was adjusted to 1 CAM, initiating a 30 minute period of hemodynamic stabilization, when it was measured the parameters for the beginning of the protocol (M -15). It was administered atropine (0,04mg/kg/IM) (group atropine) or NaCl 0.9%/IM (saline group), and then M0 was measured 15 minutes after it. Immediately after this, chlorpromazine (0,5mg/kg) and dexmedetomidine (3&#956;g/kg) were administered intravenously. Five minutes (M5) later all parameters were measured. From this moment, all evaluations were carried through 15 minutes intervals (M20, M35, M50, M65). Data were analyzed one-way ANOVA. Mean comparisons were made by SNK test and t pareado (p&#8804;0,05). Intense bradycardia was observed in saline group and the systolic, diastolic and mean arterial pressure presented higher values in atropine group in relation to the values found in saline group five minutes after chlorpromazine and dexmedetomidine administration, however a lower cardiac index, mainly in M5, was observed in both groups. The total peripheral resistance index was higher in both groups from M5, being much higher and lasting in the atropine group. The left ventricular work index presented a superior value from M0 in atropine group in relation to the saline group. There were statistically significant differences in M20. The mechanical ventilation allowed the ventilatory stability and the hemogasometric parameters had no clinically significant differences. The results allowed concluding that chlorpromazine administration did not brighten up the severe and lasting arterial hypertension produced by the dexmedetomidine. The premedication with atropine kept the stability of the heart rate; however it aggravated the arterial hypertension produced by the alpha-2, increasing the cardiac work and the consumption of oxygen from myocardium / Objetivou-se avaliar os efeitos hemodinâmicos, hemogasométricos e cardiovasculares da prémedicação com atropina em cães tratados com clorpromazina e dexmedetomidina sob anestesia geral com isofluorano e mantidos em ventilação mecânica. Foram utilizados seis cães mestiços, pesando 17,9kg (±3,9), que respeitando-se intervalo de sete dias entre tratamentos, constituíram os dois grupos do estudo. Para instrumentação os animais foram anestesiados com isofluorano, procedendo-se a canulação da artéria tibial dorsal e introdução do cateter de Swan Ganz através da veia jugular direita. Ao término da instrumentação a concentração do isoflurano foi ajustada para 1 CAM, iniciando-se o período de estabilização hemodinâmica de 30 minutos, quando mensurou-se os parâmetros para início do protocolo experimental (M-15). Administrou-se, então, pela via intramuscular, atropina (0,04mg/kg) (grupo atropina) ou cloreto de sódio a 0,9% (grupo salina), após 15 minutos mensurou-se M0, quando subseqüentemente, aplicou-se por via intravenosa, em ambos os grupos, clorpromazina (0,5mg/kg) e dexmedetomidina (3&#956;g/kg). Decorridos cinco minutos (M5) repetiram-se as mensurações. A partir deste momento as avaliações foram realizadas em intervalos de 15 minutos (M20, M35, M50, M65). A análise estatística das médias entre grupos foi realizada através do teste t pareado e a avaliação entre tempo dentro de cada grupo através de ANOVA de 1 via do teste Student Newman Keuls (p&#8804;0,05). Observou-se intensa bradicardia no grupo salina e as pressões arteriais sistólica, diastólica e média apresentaram valores maiores no grupo atropina em relação aos encontrados no grupo salina a partir de M5, porém o índice cardíaco foi reduzido, principalmente em M5, nos dois grupos. No índice de resistência periférica total houve acréscimo a partir de M5 em ambos os grupos, sendo mais acentuado e duradouro no GAtropina. O índice do trabalho ventricular esquerdo apresentou valor superior a partir de M0 no grupo atropina em relação ao grupo salina, sendo a diferença estatisticamente significativa em M20. O controle da ventilação mecânica permitiu a estabilidade ventilatória e os parâmetros hemogasométricos não apresentaram diferença significativa clinicamente. Os resultados permitem concluir que a administração de clorpromazina não amenizou a hipertensão arterial severa e duradoura produzida pela dexmetomidina. A pré medicação com atropina manteve a freqüência cardíaca estável, porém agravou a hipertensão arterial produzida pelo alfa-2, aumentando o trabalho cardíaco e o consumo de oxigênio pelo miocárdio

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23408号 / 医博第4753号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 古川 壽亮, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM