Avaliou-se a participação do óxido nítrico (NO), do fator hiperpolarizante derivado do endotélio (EDHF), dos produtos da ciclooxigenase (COX), das espécies reativas de oxigênio (EROs) e o efeito do tratamento com metformina (Met) nas alterações vasculares em ratos com obesidade induzida por glutamato monossódico (MSG). O tratamento com Met corrigiu alterações metabólicas em ratos MSG, reduzindo o acúmulo de gordura visceral, corrigindo a resistência à insulina, a hiperinsulinemia e a dislipidemia. Ratos MSG apresentaram aumentada resposta contrátil e diminuída sensibilidade à Ach, associadas a alterações na via do NO, do EDHF, dos produtos da COX e das EROs. Ratos MSG com 16 semamas apresentaram hiperresponsividade ao nitroprussiato de sódio, que foi mantida nos ratos tratados com Met. A Met corrige as alterações da resposta vascular atuando sobre o NO e o EDHF, reduzindo a geração de EROs e interferindo na resposta do músculo liso vascular, mantendo a hiperresponsividade ao NO. / The role of the nitric oxide (NO), the endothelium derived hyperpolarizing factor (EDHF), the ciclooxygenase (COX) products and the reactive oxygen species (ROS), as well as the effect of metformin (Met) treatment on the vascular alterations in rat model of obesity induced by monosodium glutamate (MSG) were evaluated. Met treatment corrected metabolic alterations in MSG, reducing fat accumulation, correcting dyslipidemia, insulin resistance and hyperinsulinemia. MSG rats had an increased response to norepinephrine and decreased sensitivity to acetilcholine, which were associated with alterations in NO, COX products and ROS. Sixteen-week-old MSG rats presented hyperresponsiveness to sodium nitroprusside, which was preserved in Met-treated group. Met corrects the alterations of the vascular reactivity acting on NO and EDHF, and decreasing the ROS generation, besides its effect on the vascular smooth muscle response, preserving the hyperresponsiveness to NO.

Orientadora: Profa. Dra. Ana Carolina Santos de Souza Galvão / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2018. / O hepatorcarcinoma é um dos tipos tumorais mais comuns e está entre as três malignidades que mais causam óbito. Embora grandes avanços terapêuticos tenham sido realizados nos últimos anos, ainda se faz grande a necessidade de desenvolvimento de novas terapias mais efetivas na eliminação desta doença e menos severas quanto aos efeitos adversos resultantes. Diante deste cenário, neste trabalho a metformina foi avaliada quanto à sua capacidade citotóxica, antiproliferativa e antimetastática sobre células de carcinoma hepático humano - HepG2. Desta forma, objetivou-se no presente trabalho avaliar as alterações na viabilidade, proliferação e potencial metastático de HepG2 após o tratamento com metformina, A viabilidade celular foi verificada através do teste de redução do MTT e incorporação do vermelho neutro. A progressão do ciclo celular foi avaliada por citometria de fluxo e a análise de marcadores de morte celular foi realizada por meio de avaliação de expressão/atividade das moléculas sinalizadoras caspase 3, PARP e PARP clivada através de Western blotting. Variações no potencial metastático foram verificadas através de estudo das atividades de adesão, via teste de redução do MTT, migração celular, por Scratch Assay, migração e invasão por ensaios em placas do tipo transwell e determinação da atividade das metaloproteinases MMP2 e MMP9, por Zimografia em gelatina. Os resultados indicam que a metformina atua de forma citotóxica e citostática sobre HepG2 de modo concentração e tempo-dependente, e sugerem também a ocorrência de morte celular envolvendo ativação da caspase 3. Adicionalmente, o tratamento com metformina reduziu as atividades de migração e invasão celular, redução esta provavelmente associada à diminuição nas atividades de MMP2 e MMP9, ao mesmo tempo em que aumentou a atividade de adesão celular. Conclui-se, assim, que a ação antitumoral da metformina está associada com a indução de efeito citotóxico e citostático em HepG2, e que o euglicemiante parece reduzir o potencial metastático dessas células. Estes achados indicam que, futuramente, a metformina poderá embasar uma nova estratégia terapêutica e preventiva contra o hepatocarcinoma humano. / Hepatocellular carcinoma is one of the most common types of tumor and represents the third leading cause of cancer-related death. In despite to the major progresses made in recent years, there is still a great needed for the development of new therapies. More efficient treatments should eliminate the disease while triggers few smoother adverse effects as possible. Metformin is a traditional hypoglycemic prescribed for the treatment of type II diabetes. It has received considerable attention in the past years due to its potential anti-tumor activity. Indeed, previous studies involving this drug showed interesting results not only for the treatment itself, but also in preventing the cancer development. In this project, effects of metformin over human hepatic carcinoma cells (HepG2 lineage) were evaluated, assessing changes on cell proliferation, viability and metastatic potential. HepG2 cells were exposed to different metformin concentrations for 24 and 48 hours, and the cell viability/proliferation was determined by MTT assay. The metastatic potential analysis, adhesive and migratory abilities of HepG2 cells were evaluated by the determination of metalloproteinases (MMP-2 and -9) activities by zymography, cell motility by scratch assay and cell adhesion by MTT, respectively. Analysis of cell death markers was performed by the evaluation of caspase 3, PARP and cleaved-PARP expression by Western blotting. Results suggested that metformin promotes the reduction of cell viability in a concentration and time-dependent manner. HepG2 cells had their viability reduced through the cytotoxic and cystostatic action of metformin. Regulation over caspase 3 activation and cleavage of its substrate, PARP, indicated occurrence of programmed cell death by apoptosis. Metformin increased the adhesion simultaneously to the reduction of HepG2 migration and invasion abilities. Finally, it seems that the drug was able to modulate MMP-2 and -9 activities. Thus, confirming literature data, metformin seems able to decrease the metastatic potential of HepG2 cells. These findings indicate that, in the future, metformin may support a new therapeutic and preventive strategy against human hepatocarcinoma.

O câncer de mama representa um grave problema de saúde pública. Dentre os fármacos empregados, destaca-se o paclitaxel, um agente citotóxico eficaz, porém associado a severos efeitos colaterais. A metformina hidrocloreto tem obtido resultados promissores para o tratamento de neoplasias, porém é bastante hidrofílica, fator limitante da biodisponibilidade. A rapamicina tem demonstrado sinergismo com paclitaxel e potente atividade antitumoral. Todavia, é um fármaco lipofílico e possui desvantagens. Sistemas nanoestruturados de fármacos como lipossomas PEGlados são largamente empregados para a melhora da farmacocinética e potencialização da ação terapêutica. Ademais, a funcionalização de lipossomas com anticorpos monoclonais pode permitir a entrega seletiva do fármaco encapsulado à célula alvo. No presente trabalho objetivou-se desenvolver e caracterizar lipossomas e imunolipossomas funcionalizados com trastuzumabe, contendo paclitaxel, metformina hidrocloreto e/ou rapamicina, bem como avaliar as formulações através de estudos in vitro e in vivo. Os resultados mostraram que a metformina hidrocloreto foi encapsulada com baixa eficiência, menor que 20%, ao passo que paclitaxel e rapamicina puderam ser co-encapsulados com adequados valores de eficiência de encapsulação, equivalente a 56,32% para paclitaxel e 73,31% para rapamicina, e tamanho de partícula nanométrico, de 136,95 nm em composição biocompatível baseada em SPC:Col:DSPE-PEG(2000). Os dois fármacos apresentaram liberação lenta, e foram convertidos às formas molecular e amorfa, respectivamente para paclitaxel e rapamicina quando encapsulados. Os imunolipossomas foram funcionalizados com elevada eficiência com trastuzumabe e mantiveram o tamanho nanométrico, com adequados valores de encapsulação dos fármacos. Ainda, mostrou-se o sinergismo entre paclitaxel e rapamicina coencapsulados em lipossomas em células triplo negativas (4T1) e houve sinergismo entre os dois fármacos, mediado pelo anticorpo em imunolipossomas frente à linhagem celular HER2 positiva (SKBR3), em virtude do aumento do uptake celular mediado pelo trastuzumabe. Finalmente, os resultados obtidos in vitro foram confirmados in vivo, sendo que os lipossomas com paclitaxel e rapamicina coencapsulados foram capazes de controlar o crescimento tumoral em modelo de câncer de mama triplo negativo, ao passo que o imunolipossoma com os dois fármacos permitiu o controle do crescimento de tumores xenográficos HER2 positivos, cuja média de volume tumoral correspondeu a 25,27%, 44,38% e 47,78% das médias dos volumes tumorais de controle negativo, positivo e lipossoma, respectivamente. Portanto, a formulação desenvolvida nesse trabalho tem potencial para ser avaliada em estudos clínicos. / Breast cancer represents a severe public health problem. Among the drugs used in the treatment, paclitaxel is an effective cytotoxic drug, but associated with side effects. Hydrocloride metformin has shown promising results for cancer treatment, however it is very hydrophilic, a limiting factor for bioavailability. Rapamycin has demonstrated synergism with paclitaxel and potent anticancer activity, though it is a lipophilic drug with drawbacks that compromise its bioavailability. Nanostructured drug delivery systems, such as PEGylated liposomes are largely employed for pharmacokinetics improvement and enhancement of therapeutic effect. Furthermore, the functionalization of liposomes with monoclonal antibodies enables the selective delivery of the loaded drug to the target cell. In the present work, we aimed to develop and characterize liposomes and immunoliposomes functionalized with trastuzumab, containing paclitaxel, hydrocloride metformin and/or rapamycin, as well as to evaluate the formulations through in vitro and in vivo studies. The results showed that hydrocloride metformin was encapsulated with low efficiency, less than 20%, on the other hand paclitaxel and rapamycin could be co-loaded with suitable values of encapsulation efficiency, 56.32% for paclitaxel and 73.31% for rapamycin and nanometric particle size, 136.95 nm, based on a SPC:Chol:DSPE-PEG(2000) composition. The two drugs displayed slow release, and were converted to molecular and amorphous form, respectively for paclitaxel and rapamycin when encapsulated. The immunoliposomes were developed with high efficiency with trastuzumab and kept the nanometric size, with adequate encapsulation of drugs. Moreover, herein it was shown the synergism between paclitaxel and rapamycin co-loaded in liposomes in triple negative cells (4T1) and there was synergism between the two drugs mediated by the antibody in immunoliposomes in the HER2-positive cell line (SKBR3), due to the improved cell uptake mediated by trastuzumab. Finally, the results obtained in vitro were confirmed in vivo. Co-loaded paclitaxel and rapamycin were able to control tumor growth in a triple negative breast cancer animal model, while the immunoliposome containing the two drugs allowed for better control of tumor growth in a HER2-positive breast xenograft model, whose average tumor volume corresponded to 25.27%, 44.38% and 47.78% of the tumor volumes of positive control, negative control and liposome, respectively. Therefore, the formulation developed herein has potential to be evaluated in clinical trials.

INTRODUÇÃO: Estudos observacionais e pré-clinicos sugerem o efeito antitumoral da metformina em tumores sólidos, incluindo o câncer colorretal. Entretanto, os efeitos da metformina no câncer colorretal ainda não foram testados em estudos prospectivos. PACIENTES E MÉTODOS: Este foi um estudo de fase II, unicêntrico, braço único de pacientes com câncer colorretal metastático em progressão e previamente tratados com 5-FU, irinotecano, oxaliplatina e anti-EGFR, se RAS selvagem. Os pacientes receberam metformina 850 mg VO duas vezes por dia e 5-FU 425 mg/m2 e leucovorin 50 mg IV semanal até progressão de doença, toxicidade inaceitável ou retirada de consentimento. O desfecho primário foi controle de doença em 8 semanas. RESULTADOS: Dos 50 pacientes incluídos, 11 (22%) alcançaram o desfecho primário. Para toda coorte, a SLP foi de 1,8 meses e a SG mediana de 7,9 meses. Quando avaliamos somente os 11 pacientes que alcançaram controle de doença na semana 8, a SLP foi de 5,6 meses e a SG foi de 16,2 meses. Houve tendência a maior sobrevida entre os obesos (12,4 vs 5,8 meses de acordo com IMC maior ou menor que 30) e aqueles que tiveram maior intervalo livre de 5FU antes de entrar no estudo. O tratamento foi bem tolerado e os principais efeitos colaterais de qualquer grau foram diarreia, náusea, vômito e mielotoxicidade. CONCLUSÃO: Neste estudo de fase II, a metformina combinada ao 5FU apresentou atividade modesta na população geral de pacientes com câncer colorretal metastático refratário. Em análise de subgrupo, obesos e maior intervalo livre de 5FU foram associados a controle de doença prolongado. Estudos prospectivos randomizados com metformina em câncer colorretal devem ser realizados / BACKGROUND: Observational and pre-clinical studies have suggested that metformin has antitumor effects in solid tumors, including colorectal cancer. However the effects of metformin in colorectal cancer have not been tested in clinical trials. PATIENTS AND METHODS: This was a single center, single-arm phase II clinical trial where histologically confirmed colorectal cancer patients with measurable and progressing metastatic disease previously treated with 5-FU, irinotecan, oxaliplatin and an anti-EGFR, if the tumor was RAS wild type, were enrolled to receive metformin 850 mg orally bid continuously plus 5-FU 425 mg/m2 and leucovorin 50 mg IV weekly until disease progression, unacceptable toxicity or consent withdrawn. The primary endpoint was disease control rate at 8 weeks. RESULTS: Among 50 patients included, 11 (22%) met the primary endpoint. The median progression free survival was 1.8 months and the median overall survival was 7.9 months. Analyzing only those 11 patients who achieved disease control rate at 8 weeks, their median progression-free survival was 5.6 months and their median overall survival was 16.2 months. There was a trend for prolonged median survival for obese patients (12.4 vs 5.8 months) and those longer off 5FU. The treatment was well tolerated and the main side effects were diarrhea, nausea, vomiting and myelotoxicity. CONCLUSION: Metformin and 5FU showed an overall modest but intriguing activity in refractory colorectal cancer patients in this phase II study. Some patients presented long-term disease control. Further trials are needed to confirm these results, particularly in obese patients with colorectal cancer

Orientador: Antonio Ari Gonçalves / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T15:30:30Z (GMT). No. of bitstreams: 1 Costa_EuniceCristinadaSilva_D.pdf: 2678840 bytes, checksum: 439eb1d320bc5d4828c77cb0e086e763 (MD5) Previous issue date: 2005 / Resumo: Objetivos: Metformina e glibenclamida são fármacos utilizados para diminuir a glicemia de diabéticos tipo 2. Metformina reduz a absorção gastrintestinal de glicose e a gliconeogênese hepática e aumenta a captação de glicose periférica. Por sua vez, glibenclamida aumenta a liberação de insulina após bloquear canais de K+. Apesar destes efeitos, metformina em altas concentrações e glibenclamida podem influenciar o sistema cardiovascular e acelerar a progressão de doenças vasculares, predispondo o coração à falência cardíaca ou infarto. Estas e outras mudanças fisiológicas podem ser associadas a um ECG anormal, mostrando aumento do intervalo QT e de sua dispersão (QTd). Estas mudanças podem ser associadas a um baixo limiar para arritmias ventriculares e provocar morte súbita durante isquemia. Neste estudo avaliamos os efeitos do tratamento com metformina e/ou glibenclamida em ratos diabéticos por aloxana sobre os intervalos do ECG: QT e suas derivadas QTc, QTd, e QTcd. Em outra série experimental avaliamos a pressão desenvolvida pelo ventrículo esquerdo (LVP) e as suas derivadas (DP/Dt+ e DP/Dt-), usando a preparação de Langendorff utilizando coração isolado de ratos diabéticos. A isquemia foi provocada pela perfusão (1 h) com noradrenalina (NE). Além disso, o glicogênio foi medido em coração de ratos antes e após perfusão com noradrenalina. As alterações histológicas no ventrículo também foram estudadas. Métodos: Ratos Wistar machos, diabéticos por aloxana, foram tratados com metformina (3,5, 30 e 74 mg.g-1 de peso corporal ¿ p.c) ou glibenclamida (0,10 mg/g-1 p.c) e/ou glibenclamida e metformina (0,10 + 3,5 mg.g-1 p.c), simultaneamente durante 30 dias. O ECG foi registrado no 15o e 30o dia de tratamento. No 30º dia, sob anestesia, o coração foi isolado e perfundido com solução de Krebs-Henseleit em um aparelho de Langendorff. A isquemia foi induzida com noradrenalina 10-6 M (2 ml.min-1.g-1) mantida durante 1 h na solução perfusora. O glicogênio tecidual (mg.100.mg-1) foi extraído de fragmentos de ventrículo de ratos em repouso ou após a perfusão. O glicogênio foi medido pelo método do fenol sulfúrico. Em outros grupos de ratos, preparados de modo idêntico, os corações foram removidos sob anestesia e fixados em formoldeído em tampão PBS. Secções de ventrículo foram preparadas depois de embebidas em parafina e em seguida, os cortes foram fixados em lâminas e corados pelo método hematoxilina eosina (HE). O ensaio do glicogênio ventricular foi feito usando o método ácido de Schiff. Os núcleos foram contados e as suas áreas foram medidas (mm2). Os grânulos de glicogênio foram detectados pela coloração violeta do citoplasma, usando o método de schiff (PAS positivo) e fotografados. Resultados: Após 15 e 30 dias, a glicemia, o intervalo QT e as suas derivadas aumentaram nos ratos diabéticos. Após 30 dias, a glicemia diminuiu em ratos diabéticos que foram tratados com doses baixa ou intermediária de metformina (3,5 e 30 mg.g-1 p.c.), ou com glibenclamida e com a combinação glibenclamida + metformina (3,5 mg.g-1 p.c.). Entretanto, o grupo tratado com a dose mais alta de metformina (74 mg.g-1 p.c) não teve a sua glicemia diminuída. Por outro lado, nos ratos tratados com doses: baixa ou intermediária de metformina os intervalos do ECG: QTc, QTd e QTcd foram reduzidos, em relação ao grupo diabético tratado com a maior dose de metformina. Estes resultados também produziram melhores efeitos em comparação aos grupos diabéticos tratados com glibenclamida e nos grupos tratados com a associação glibenclamida e metformina. Doses baixas, intermediárias e altas (3,5, 30 e 74 mg.g-1 p.c.) de metformina aumentou o armazenamento de glicogênio no ventrículo de ratos diabéticos de 0,19 ± 0,007 (controle) para 0,38 ± 0,007 mg.100 mg-1, 0,5 ± 0,05 mg.100 mg-1 e 0,7 ± 0,04 mg.100 mg-1 (p< 0,05), respectivamente. Quanto à pressão sistólica ventricular, houve rápido aumento da pressão logo no inicio da perfusão com NE no grupo controle, com pico de pressão a 145 ± 9,7 mmHg), seguido de lenta queda até 99 ± 3 mmHg. Esta tendência foi observada também nas derivadas DP/Dt+ e DP/Dt-. Metformina (3,5 e 30 mg.g-1 p.c) e glibenclamida isoladamente ou em associação com metformina protegeram o músculo cardíaco durante a isquemia, não diferindo do grupo controle. Contudo, ratos diabéticos não tratados ou tratados com a maior dose de metformina, desenvolveram pressão sistólica máxima inferior a todos os grupos experimentais, revertendo aos níveis basais mais rapidamente que nos demais grupos. As derivadas DP/Dt+ e DP/Dt- mostraram curvas semelhantes. Após a isquemia, o glicogênio diminuiu em todos os grupos, sendo 0,09 ± 0,007 no grupo controle; 0,1 ± 0,006 nos diabéticos e 0,6 ± 0,005 nos diabéticos tratados com 74 mg.g-1 pc de metformina. O tratamento com glibenclamida e/ou metformina diminuiu o estoque de glicogênio de 0,62 ± 0,05 mg.100 mg-1 para 0,19 ± 0,05 mg.100 mg-1 e de 0,74 ± 0,03 mg.100 mg-1 para 0,22 ± 0,008 mg.100 mg-1, respectivamente. Entretanto, a utilização de glicogênio foi proporcional em todos os grupos. A análise morfológica demonstrou um aumento na quantidade dos núcleos no coração de ratos diabéticos de 21,33 ± 1.17 (no grupo controle) para 36,6 ± 5 (p< 0,05) e redução na média da área dos núcleos, de 0,16 ± 0,02 (controle) para 0,08 ± 0,01 (p< 0,05). No grupo tratado com a menor concentração de metformina (DM 3.5) diminuiu a quantidade de núcleos de 36,6 ± 5 (grupo diabético) para 22,8 ± 2 (p< 0,05), porém aumentou a média da área dos núcleos de 0,08 ± 0,01 mm2 para 0,17± 0,01 mm2 (p< 0,05). Nos grupos tratados com as maiores doses de metformina (30 e 74 mg.g-1 p.c.), a quantidade de núcleos aumentou para 34,16 ± 1,85 e 47,29 ± 2,92, respectivamente), e suas respectivas áreas aumentaram para 0,86 ± 0,05 e 0,5 ± 0,06, diferindo dos grupos controle e dos diabéticos não tratados. Nos grupos diabéticos tratados com glibenclamida e glibenclamida + metformina, as áreas dos núcleos aumentaram de 0,08 ± 0,01 mm2 para 0,71 ± 0,09 mm2 e 0,67 ± 0,01 mm2, respectivamente, (p< 0,001). Conclusões: O aumento na dispersão dos intervalos QT com o tratamento pode significar um risco de arritmia que predispõe ratos à morte súbita. Os resultados da pressão obtidos pelo método de Langendorff indicam que a força de contração diminuiu durante o período de isquemia por NE, sugerindo que o coração estava mais rígido. Estes resultados permitem-nos deduzir que as maiores doses de metformina, 74 mg.g-1 indicados como as máximas para humanos, podem causar sérios prejuízos ao trabalho cardíaco em caso de sobrecarga. Por outro lado, altas doses de metformina, de glibenclamida e a associação entre estas drogas aumentam a quantidade e o tamanho dos núcleos. Conseqüentemente, o ventrículo hipertrofia, em decorrência do aumento da atividade celular, prejudicando de modo importante, a estrutura e a função cardíaca. Portanto, este aumento de glicogênio está associado à severidade e à duração do diabetes. Assim, o coração torna-se altamente susceptível à isquemia / Abstract: Metformin and glibenclamide are pharmacos used to decrease blood glucose on type 2 diabetics. Metformin decreases gastrointestinal absorption of glucose and gluconeogenesis and increases peripheric glucose uptake. Glibenclamide increases insulin secretion by blocking K+ channels. Besides these effects, metformin and glibenclamide may influence cardiovascular system, which accelerate the progression of vascular disease, predisposing heart to failure or infarct. These abnormalities associated to physiological changes may generate an abnormal ECG, with an increased QT interval and its correspondent dispersion (QTd). These changes could be associated to a lower threshold for malignant ventricular arrhythmias and a sudden death by ischemia. The aim of this study was to evaluate the effects of metformin and/or glibenclamide treatment on QT intervals and its derivatives: QTc, QTd, and QTcd. We also evaluated the pressure developed by left ventricle (LVP) and calculate the correspondents derivatives (DP/Dt+ and DP/Dt-) on heart isolated from diabetic rats, under ischemia caused by norepinephrine (NE). Glycogen was measured after ischemia and compared to control heart, non-submitted to NE. We also analyzed the histological changes in ventricle cells. Methods: Male Wistar diabetic rats were treated by metformin (3.5, 30 and 74 µg.g-1 b.w) or glibenclamide (0.13 µg.g-1 b.w) and its association to metformin (0.13 µg.g-1 b.w + 3.5 µg.g-1 b.w) during 30 days. A 6-lead ECG was recorded initially and after 15 and 30 days treatment. At the end, under anaesthesia, heart were isolated and perfused by Krebs-Henseleit solution in a Langendorff apparatus. Ischemia were induced by adding norepinephrine 10-6 M to the solution (2 ml.min-1.g) during 1 h. Glycogen (mg.100 mg-1 wet tissue) was measured on heart at rest or after perfusion, using the fenol sulfuric method. In another group, after anaesthesia hearts were removed, cleaned and fixed in phormoldheyde in PBS buffer. Thin ventricle sections were made and after paraffin embedding, fine slices were cut and stained with hematoxilin eosin (HE). Ventricle glycogen assay was performed on those slides using the acid Schiff process. The number of nuclei was counted out and nuclei area was measured (mm2). Glycogen granules were recognized the violet colored cytoplasm. Results: After 15 and 30 days, glycemia, QT interval and its derivates increased on diabetic rats. On the other hand, diabetic rats treated during 30 days by low and intermediate doses of metformin (3.5 and 30 µg.g-1 b.w.) or glibenclamide or glibenclamide plus metformin, all decreased glycemia. However, the group treated with the highest dose of metformin (74 µg.g-1 b.w) failed to reduce glycemia. On the other hand, the groups treated by low and intermediate doses reduced the ECG intervals: QTc, and QTd, and QTcd, in contrast to the diabetic group treated with the highest metformin dose and the groups treated by glibenclamide and glibenclamide associated to metformin. Metformin, in low and high doses (3.5, 30 and 74 mg.g-1 b.w.) increased glycogen storage on diabetic rat ventricle, from 0.19 ± 0.007 (control group) to 0.38 ± 0.007 mg.100 mg-1, 0.5 ± 0.05 mg.100 mg-1 and 0.7 ± 0.04 mg.100 mg-1, p< 0.05, respectively. The treatment with glibenclamide alone or associated to metformin increased glycogen, too. In the control group, isolate hearts showed a rapid increase on ventricular pressure, just initiation of NE perfusion (145 ± 9.7 mmHg), followed by a slow fall to 99 ± 3 mmHg. Similar changes was found on the derivates DP/Dt+ and DP/Dt-. Metformin (3.5 and 30 mg.g-1), glibenclamide and glibenclamide associated to metformin protected cardiac muscle during ischemia, similarly to the control group (p> 0.05). But, the non-treated diabetic group and the group treated by 74 mg.g-1 of metformin, produced a maximal pressure which were inferior to the control group and the reversion of the LVP, DP/Dt+ and DP/Dt- was faster than that of the control group. After ischemia, glycogen was reduced on all groups to 0.09 ± 0.007 mg.100 mg-1 on control group; 0.1 ± 0.006 mg.100 mg-1 on diabetic group and 0.06 ± 0.005 mg.100 mg-1 on DM74. However, this decrease was inferior to that of the group treated by the highest dose. The treatment with glibenclamide alone and associated to metformin diminished glycogen storage from 0.62 ± 0.05 mg.100 mg-1 to 0.19 ± 0.05 mg.100 mg-1 and 0.74 ± 0.03 mg.100 mg-1 to 0.22 ± 0.008 mg.100 mg-1. However its utilization was proportional for all groups. Heart submitted to ischemia decreases its reserve, (p< 0.05 compared to non-ischaemic). These results suggested that high doses metformin, in special 74 mg.g-1 b.w., indicated as maximal for humans, makes heart prompt to ischemia. Diabetic rat hearts showed an increase on the amount of nuclei, from 21.33 ± 1.17 to 36.6 ± 5 (p< 0.05) and a reduction of its area, from 0.16 ± 0.02 mm2 to 0.08 ± 0.01 mm2 (p< 0.05) in comparison to the control group. The lowest dose of metformin (DM 3.5) diminished the amount of nuclei (36.6 ± 5 vs 22.8 ± 2; p< 0.05) and increased theirs size (0.08 ± 0.01 vs 0.17± 0.01). The amount of nuclei increased to 34.16 ± 1.85 and 47.29 ± 2.92 during the treatment with high metformin doses, (30 and 74 mg.g-1 b.w., respectively), and the nuclei area increased to 0.86 ± 0.05 mm2 and 0.5 ± 0.06 mm2, respectively, differing from control and non-treated diabetic groups. Similar result, was obtained on the group treated by glibenclamide and/or metformin, on cardiac cells, which the nuclei area increased to 0.71 ± 0.09 mm2 and 0.67 ± 0.01 mm2, respectively, (p< 0.001). Conclusions: The increased dispersion of QT intervals during treatment may be subjacent to the risks of arrhythmias that predispose humans to sudden death. Results shown on Langendorff methodology indicate that contraction force decreased, suggesting that ventricle muscle were prone to ischemia. Then, high metformin doses (74 mg.g-1), as indicated for humans, may cause damage to cardiac work during overload. High metformin doses, glibenclamide and glibenclamide associated to metformin increase the number of nuclei, as well, theirs size. Consequently, the ventricle hypertrophy due to an increased cellular activity may cause important injuries to cardiac structure and function. We can conclude that, the increased glycogen content on ventricle was associated to the severity and the duration of diabetes. Then, heart became more susceptible to the ischemia effects / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Les modifications physiopathologiques induites par l’obésité sont susceptibles de modifier la pharmacocinétique d’un grand nombre de médicaments. Toutes les étapes peuvent-être touchées : modification des compartiments de l’organisme, de la fonction rénale et de l’expression des protéines du métabolisme. L’intensité des variations induites reste difficile à anticiper en l’absence d’études spécifiques, ce qui pose également la question du choix du meilleur estimateur de poids dans l’expression du schéma posologique du médicament chez le sujet obèse. Nous avons évalué l’impact de différents descripteurs de poids sur les variabilités inter-individuelles de trois médicaments couramment utilisés en diabétologie, dans la douleur et en cancérologie. Une modélisation a été faite en pharmacocinétique de population (Monolix®) avec une évaluation des descripteurs de poids (poids de masse maigre, poids idéal, poids total, surface corporelle…). Pour la metformine (n=105 patients), molécule fortement hydrophile, CL/F et V/F augmentent avec le poids. Le poids de masse maigre est le meilleur descripteur de poids pour les deux paramètres et permet d’abaisser de manière importante la variabilité inter-individuelle. Dans le cas de la morphine (n=31), aucun descripteur n’est corrélé à CL/F ou V/F dans une population de sujets atteints d’obésité morbide. L’analyse de l’imatinib (n=54), de lipophilie plus importante, et de son métabolite actif montre que le poids idéal est la covariable la plus performante. Son impact n’explique cependant qu’une part minime de la variabilité de CL/F et ne remet pas en cause le principe d’une dose fixe. Les résultats confirment l’intérêt des méthodes de pharmacocinétique de population dans l’évaluation des covariables morphologiques, et indiquent qu’il n’existe pas de descripteur de poids universel. Ces résultats doivent nous encourager à évaluer systématiquement l’impact de l’obésité avec des modèles de population. Dans certains cas, des études de confirmation comparant différents schémas devraient-être mises en place. / Pharmacokinetic of drugs may be altered by pathophysiological changes associated with obesity: renal function, body compartments, expression of metabolism proteins. Real impact may be difficult to appreciate due to small number of specific studies. What is the best size-descriptor for optimal dosing in obesity remains a question of importance. Impact of different size descriptors was studied for three drugs currently used in diabetology, pain and oncology. Population pharmacokinetic modeling was done using Monolix® software to evaluate different covariates (LBW, TBW, BSA, …). CL/F and Vd/F of metformin, highly hydrophilic drug, increases positively with body weight (n=105). LBW was the best size-descriptor leading to substancial decrease in the between-subject variability BSV. No size-descriptors showed significant impact for morphine (n=31). Ideal body weight is the best size-descriptor for imatinib and its main metabolite (n=54), lipophilic drug. It explains only a small part of BSV and fixed dosing stays justified. Population PK analysis are the most formal assessment of morphological covariates and no single size descriptor can described all variabilities. Results must lead us to more systematic population PK analysis. Confirmation studies comparing different dosing regimens have to be done.

Polycystisktovarialsyndrom (PCOS) förekommer hos 5-10% av alla kvinnor och är den vanligaste orsaken till anovulatorisk infertilitet. Andra delar av syndromet är hyperandrogena och metabola symtom. Infertilitet behandlas med klomifencitrat. Akne och hirsutism behandlas i första hand med kombinerade p-piller med östrogenprofil. Förhöjda blodsockernivåer, hypertoni, dyslipidemi och övriga komplikationer till syndromet behandlas farmakologiskt vid behov. Hyperandrogenism och insulinresistens tycks spela en huvudroll i uppkomsten av sjukdomen. Då metformin förbättrar insulinkänsligheten, och tros kunna påverka patofysiologin, har det föreslagits som en alternativ förstahandsbehandling. Denna litteraturstudie syftade till att undersöka vilket vetenskapligt underlag som finns för att ändra behandlingsrekommendationerna vid PCOS. De studier som jämfört resultatet av metformin och klomifencitrat vid anovulatorisk infertilitet visar att klomifencitrat mer effektivt framkallar ovulation och graviditet hos kvinnor med PCOS och övervikt, men att metformin är lika effektivt hos normalviktiga kvinnor. Medan metforminbehandling är en välbeprövad och säker behandling med få biverkningar har klomifencitrat allvarliga biverkningar i form av risk för flerbörd och ovarialthyperstimuleringssyndrom. De få studier som undersökt metformins påverkan på fostret finner inga belägg för teratogena effekter. Metformin har positiva effekter på hyperandrogena symtom vid PCOS, och man har inte kunnat se någon signifikant skillnad i effekt mellan metformin och p-piller då det gäller att minska akne och hirsutism. Dessutom finns det belägg för att metformin kan ha en positiv påverkan på BMI och blodtryck, förbättra lipidprofilen genom att sänka nivåerna av LDL kolesterol, samt minskar risken hos denna patientgrupp att utveckla typ 2 diabetes. Sammantaget kan dock sägas att det vetenskapliga underlaget ännu är för svagt för att man ska ändra den rådande behandlingsrekommendationen. Det finns behov av större, blindade studier där man jämför metforminbehandling med klomifencitrat och tittar på en kombination av faktorer och utfall kopplade till symtombilden vid PCOS.

Abstract Gestational diabetes mellitus (GDM) is defined as disturbed glucose metabolism first recognized during pregnancy. Untreated GDM increases the risk of obstetric and neonatal complications, such as fetal overgrowth (macrosomia). The first-line treatment of GDM includes diet therapy and the self-monitoring of blood glucose concentrations and, if needed, pharmacotherapy, which is most commonly accomplished with insulin. Oral anti-diabetic agents such as metformin have recently been under investigation. GDM increases the risk of developing overt diabetes, metabolic syndrome and cardiovascular diseases. The aim of the present study was to investigate the effect of metformin vs. insulin therapy on pregnancy and neonatal outcome as well as on later growth and development of the infant and to investigate the independent and concomitant effects of GDM and maternal overweight/obesity on pregnancy outcome and maternal long-term risks. In a randomized study of 100 women, metformin therapy was not associated with an increased risk of pregnancy or neonatal complications when compared with insulin treatment. However, 32% of the women treated with metformin needed additional insulin in the achievement of normoglycaemia. The need of additional insulin was associated with maternal obesity, an earlier need of pharmacotherapy and fasting hyperglycaemia in OGTT. Infants exposed to metformin were taller and heavier at the age of 18 months compared with infants exposed to insulin. There was no difference in the motor, social or linguistic development between these children when assessed at the age of 18 months. In an epidemiological study of 24,565 pregnancies, normal-weight women with GDM did not have an increased risk of macrosomia or Caesarean delivery when compared with normal-weight women without GDM. GDM was an independent risk factor of neonatal morbidity, especially hypoglycaemia. Maternal overweight and obesity were independent risk factors of macrosomia and obesity was also an independent risk factor of Caesarean delivery and neonatal morbidity. In a follow-up study (n = 116), women with a history of insulin-treated GDM had an increased risk of metabolic syndrome when compared with women without GDM 19 years after index pregnancy. However, maternal pre-pregnancy overweight as such was a stronger risk factor as regards the development of metabolic syndrome than previous GDM. / Tiivistelmä Raskausdiabetes on ensimmäisen kerran raskauden aikana ilmaantuva glukoosiaineenvaihdunnan häiriö. Hoitamattomana raskausdiabetes lisää raskaana olevan ja vastasyntyneen komplikaatioriskiä, erityisesti sikiön liiallista kasvua (makrosomiaa). Raskausdiabetestä hoidetaan ruokavaliolla, veren glukoosipitoisuuksien omaseurannalla sekä tarvittaessa lääkehoidolla, joka on useimmiten insuliinihoitoa. Muita diabeteslääkkeitä, kuten metformiinia, on tutkittu viime vuosina paljon. Raskausdiabetes lisää myöhemmällä iällä riskiä sairastua diabetekseen, metaboliseen oireyhtymään sekä sydän- ja verisuonisairauksiin. Tämän tutkimuksen tarkoituksena oli selvittää metformiinihoidon tehoa ja turvallisuutta verrattuna insuliiniin raskausdiabeteksen hoidossa. Lisäksi selvitettiin raskausdiabeteksen ja ylipainon itsenäistä vaikutusta raskauskomplikaatioiden esiintyvyyteen sekä naisen myöhempään sairastuvuuteen. Satunnaistetussa tutkimuksessa (n = 100) metformiini ei lisännyt vastasyntyneen makrosomian eikä vastasyntyneen tai raskauskomplikaatioiden riskiä verrattuna insuliiniin. Metformiinilla hoidetuista naisista 32% tarvitsi lisäksi insuliinia normaalin glukoositasapainon saavuttamiseksi. Lisäinsuliinin tarvetta ennustivat äidin lihavuus, varhainen lääkehoidon tarve sekä kohollaan olevat glukoosin paastoarvot sokerirasituksessa. Metformiinille altistuneet lapset olivat sekä pidempiä että painavampia 18 kuukauden iässä kuin insuliinille altistuneet lapset, mutta heidän motorisessa, sosiaalisessa tai kielellisessä kehityksessään ei ollut eroja. Epidemiologisessa tutkimuksessa (n = 24,565) normaalipainoisen naisen raskausdiabetes ei lisännyt keisarileikkauksen tai sikiön makrosomian riskiä verrattuna normaalipainoisiin naisiin, joiden sokeriaineenvaihdunta oli normaali. Raskausdiabetes lisäsi itsenäisesti vastasyntyneen sairastavuuden ja hypoglykemian riskiä. Äidin ylipaino ja lihavuus lisäsivät itsenäisesti makrosomian riskiä ja lihavuus myös keisarileikkauksen ja vastasyntyneen sairastuvuuden riskiä. Seurantatutkimuksessa (n = 116) insuliinihoidettujen raskausdiabeetikoiden riski sairastua 19 vuotta raskauden jälkeen myöhempään metaboliseen oireyhtymään oli lisääntynyt verrattuna terveisiin verrokkeihin. Raskautta edeltävä ylipaino oli vahvempi riskitekijä metabolisen oireyhtymän kehittymiselle kuin aiempi raskausdiabetes.

Abstract Most women with polycystic ovary syndrome (PCOS) exhibit features of metabolic syndrome, including insulin resistance, abdominal obesity, dyslipidaemia, glucose intolerance and low-grade chronic inflammation, reflected in elevated levels of serum C-reactive protein (CRP), placing these women at increased risk of cardiovascular disease and type 2 diabetes (type 2 DM). The aim of this study was to investigate the effects of two well-known insulin-lowering drugs used in the treatment of type 2 DM, metformin and rosiglitazone, on traditional cardiovascular risk factors and inflammation in women with PCOS. In addition, the impact of rosiglitazone was evaluated as regards clinical, endocrine and metabolic aspects of PCOS. Six-months of metformin treatment in women with PCOS had beneficial effects on levels of CRP, lipid profile and blood pressure, expressed as increased levels of high-density lipoprotein cholesterol (HDL-C), and decreased levels of triglycerides (TGs), decreased ratio of total cholesterol/HDL-C, decreased levels of CRP, and decreased systolic and diastolic blood pressures. Four-month treatment with rosiglitazone in a randomised, double-blind, placebo-controlled study in overweight women with PCOS resulted in significant improvements in menstrual cyclicity, hyperandrogenism, insulin resistance and hyperinsulinaemia. In addition, rosiglitazone decreased levels of markers of low-grade inflammation, CRP and white blood cell (WBC) count, and the liver function marker alanine aminotransferase (ALAT), while having neutral effects on levels of lipids, and blood pressure. In conclusion, metformin treatment, in accordance with the known beneficial metabolic effects of this drug, could be useful in the prevention of cardiovascular complications in women with PCOS. Rosiglitazone represents an alternative treatment for overweight anovulatory women with PCOS. It could be useful in the prevention of type 2 DM in overweight women with PCOS and for those suffering from possible side-effects related to metformin treatment. In addition, alleviation of inflammation and improvement of liver function during rosiglitazone treatment may indicate decreased future risks of cardiovascular diseases and non-alcoholic fatty liver disease (NAFLD).

The robust glycolytic metabolism of glioblastoma multiforme (GBM) has proven them susceptible to increases in oxidative metabolism induced by the pyruvate mimetic dichloroacetate (DCA). Recent reports demonstrate that the anti-diabetic drug metformin enhances the damaging oxidative stress associated with DCA treatment in cancer cells. We sought to elucidate the role of metformin’s reported activity as a mitochondrial complex I inhibitor in the enhancement of DCA cytotoxicity in the VM-M3 model of GBM. We demonstrated that metformin potentiated DCA-induced superoxide production and that this was required for enhanced cytotoxicity towards VM-M3 cells with the combination. Similarly, rotenone enhanced oxidative stress resultant from DCA treatment and this too was required for the noted augmentation of cytotoxicity. Adenosine monophosphate kinase (AMPK) activation was not observed with the concentration of metformin required to enhance DCA activity. Moreover, addition of an activator of AMPK did not enhance DCA cytotoxicity, whereas an inhibitor of AMPK heightened the cytotoxicity of the combination. We also show that DCA and metformin reduce tumor burden and prolong survival in VM-M3 tumor-burdened mice as individual therapies. In contrast to our in vitro work, we did not observe synergy between DCA and metformin in vivo. Our data indicate that metformin enhancement of DCA cytotoxicity is dependent on complex I inhibition. Particularly, that complex I inhibition cooperates with DCA-induction of glucose oxidation to enhance cytotoxic oxidative stress in VM-M3 GBM cells. This work supports further investigation and optimization of a DCA/metformin combination as a potential pro-oxidant combinatorial therapy for GBM.