Na Síndrome Metabólica, ações lipotóxicas e glicotóxicas contribuem para a aceleração do processo aterogênico cuja base é a inflamação. O tecido adiposo epicárdico vem sendo reconhecido como metabolicamente ativo e foi relacionado à elevação da produção de citocinas e adipocinas inflamatórias e aumento de DAC. Pioglitazona e Sinvastatina comprovadamente atuam como drogas pleiotrópicas na redução do processo inflamatório sistêmico. O presente estudo teve como objetivo principal avaliar possíveis correlações entre a presença de citocinas inflamatórias plasmáticas versus teciduais e a resposta de ambas à terapia medicamentosa. Para tanto, foram utilizadas monoterapia com Sinvastatina ou Pioglitazona e terapia combinada Pioglitazona+Sinvastatina e acompanhadas as variáveis lipídicas, glicêmicas e inflamatórias sistêmicas, células e citocinas inflamatórias em TAE, um tipo de tecido adiposo branco visceral instalado nas adjacências de focos ateroscleróticos em artérias coronárias de pacientes portadores de DAC e SMet. O estudo envolveu 73 pacientes com DAC multiarterial, avaliada pela cinecoronariografia e SMet que foram submetidos a revascularização e 20 pacientes submetidos à cirurgia valvar para substituição de valva mitral. Os pacientes com DAC eram incluídos de forma não aleatória a um dos 4 subgrupos: controle (n = 17), Simvastatina (20 mg / dia, n = 20), Pioglitazona (15 mg ou 30 mg / dia, n = 18) e Simvastatina + Pioglitazona (20 mg / dia + 15 mg ou 30 mg / dia, respectivamente, n = 18). Amostras de tecido adiposo epicárdico foram obtidas durante a cirurgia. Infiltração de macrófagos, linfócitos e secreção adipocitocinas foram investigados por coloração imunohistoquímica e comparados aos biomarcadores inflamatórios plasmáticos. Os resultados mostraram que a infiltração de macrófagos e a presença de citocinas pró-inflamatórias tais como TNF-, IL-6, leptina and resistina foram reduzidas em TAE de pacientes DAC/SMet após monoterapia com Pioglitazona. Os pacientes tratados apenas com Sinvastatina apresentaram os menores valores plasmáticos de leptina, resistina and MCP-1. Pioglitazona+Sinvastatina foram associadas aos menores valores plasmáticos de IL-6, TNF-, resistina, ADMA, MMP-9 em comparação ao grupo de pacientes não tratados. Além disso, a terapia combinada revelou a mais alta concentração de adiponectina plasmática concomitante ao menor valor de PCRus. Esses achados refletiram não só a condição plasmática como se correlacionaram positivamente à condição tecidual mostrada pela porcentagem média de área ocupada por macrófagos no TAE e a quantidade de PCRus presente no plasma após os tratamentos. Houve correlação positiva também entre citocinas sistêmicas e teciduais após os tratamentos, exceto para o TNF- após o tratamento com sinvastatina ( r = - 0,025, p = 0,33) e leptina após o tratamento com pioglitazona (r = -0,877, p <0,0001). Nos pacientes tratados com Sinvastatina, os fragmentos de TAE apresentaram agregados de linfócitos T, B e macrófagos concentrados na borda e ao redor de vasos sanguíneos / In the Metabolic Syndrome, the concentration of free fatty acids and the elevation of glycemia result in lipotoxic and glycotoxic actions, respectively, which contribute to accelerate the atherogenic process. (MS). Inapropriate secretion of adipocytokines plays a critical role in chronic inflammatory states associated with obesity-linked diseases, such as type 2 diabetes and atherosclerosis. The pleiotropic anti-inflammatory action of Simvastatin and/or Pioglitazone may counteract such systemic effects but its influence upon human epicardial adipose tissue is unknown. To assess the anti-inflammatory action of Simvastatin, Pioglitazone or both in epicardial adipose tissue in patients with coronary artery disease (CAD) and metabolic syndrome. The study comprised 73 patients with multivessel CAD, evaluated by cinecoronariography, and MS who underwent bypass grafting and 20 valvar patients who underwent surgery for mitral valve replacement. The 73 who underwent elective bypass grafting were non-randomly allocated to one of 4 subgroups: control (n=17), Simvastatin alone (20 mg/day, n=20), Pioglitazone alone (15 mg or 30 mg/day, n=18), or Simvastatin+Pioglitazone (20 mg/day + 15 mg or 30 mg/day, respectively, n=18). Epicardial adipose tissue sample was obtained during surgery. Infiltration of macrophages, lymphocytes and adipocytokines secretion were investigated by immunohistochemical staining and compared to plasma inflammatory biomarkers. Among CAD/MS patients, treatment with Simvastatin alone, Pioglitazone alone and Simvastatin+Pioglitazone significantly reduced plasma CRP and cytokines compared with control group. Monotherapy with Simvastatin significantly reduced plasma IL-6, leptin, resistin, MCP-1 (p<0.001 for all), whereas monotherapy with Pioglitazone reduced IL-6, TNF-, resistin and MMP-9 (p<0.001 for all) compared with control group. Simvastatin+Pioglitazone treatment reduced more plasmatic variables (IL-6, TNF-, resistin, ADMA and MMP-9 vs. control group (p<0.001). All treatments increased adiponectin plasma levels (p<0.001). In the combined treatment group, higher concentration in plasma adiponectin and lower hsCRP, were found simultaneously. There was positive correlation between mean percentage macrophages area in EAT and plasma hsCRP; also between systemic and tecidual citokynes after the treatments, except for TNF- after treatment with simvastatin (r = -0.025, p = 0.33) and leptin after treatment with pioglitazone (r = -0.877, p <0.0001). In fat fragments of patients treated with Simvastatin, T- and B-lymphocytes, and macrophages clusters concentrated near the edge or around blood vessels were observed by first time. In patients with CAD and MS treatment with Pioglitazone, Sinvastatin or combination can substantially reduce both epicardial tissue and plasma inflammatory markers. Such tissue effects may contribute to the control of coronary atherosclerosis progression

Alterações morfológicas no tecido ósseo têm sido descritas nos usuários de hipoglicemiantes orais da classe das tiazolidinedionas (TZDs). Hipotetiza-se que alguns genes relacionados com a osteogênese e osteoclastogênese podem ser influenciados pelo tratamento farmacológico, entretanto, o exato mecanismo ainda não está bem esclarecido. O objetivo do estudo foi avaliar o efeito da pioglitazona no remodelamento ósseo através de genes envolvidos na osteoclastogênese em indivíduos recentemente diagnosticados com DM2 e modelos animais, com a finalidade de identificar marcadores genéticos sensíveis de alterações ósseas. Foram convidados para participar do estudo 199 indivíduos (100 diabéticos e 99 normoglicêmicos), no ambulatório de dislipidemias do Instituto Dante Pazzanese de Cardiologia. Os indivíduos diabéticos foram tratados com pioglitazona (15, 30, 45, 45 mg/ dia/ via oral) por 16 semanas. Foram colhidas amostras de sangue, antes e após o tratamento para avaliações laboratoriais, extração de DNA genômico e de RNA total. Os polimorfismos e a expressão do mRNA nas células sanguíneas foram determinados pela PCR em tempo real através do sistema TaqMan®. Para o estudo em modelo animal após a indução da dieta hiperlipídica por 32 semanas, foram utilizados 12 camundongos machos da linhagem C57BL/J6, os quais foram divididos em três grupos: controle (n=4); diabéticos induzidos pela dieta hiperlipídica (DH, n=4) e diabéticos induzidos pela dieta hiperlipídica e tratados com pioglitazona 35mg/Kg/dia por 16 semanas (DHP, n=4). Para os grupos experimentais foram colhidos: amostras de sangue, para exames laboratoriais; fêmures, para a extração do RNA total; e tíbias, para determinação dos parâmetros histomorfométricos. Os pacientes DM2 apresentaram diminuição nas concentrações séricas de osteocalcina e na expressão de OPG e aumento na expressão de VDR em comparação ao grupo NG (p<0,05). A expressão de RANKL e IL6 foi maior entre as mulheres, enquanto que a expressão de PPARG foi maior entre os homens com DM2 em comparação ao grupo NG (p=0,032). Pacientes DM2 antes do tratamento apresentaram glicemia e expressão do mRNA de IL6 negativamente associados ao cálcio ionizado, enquanto que as transcrições de TNFA e VDR foram associadas positivamente e negativamente com bALP respectivamente (p<0,05). O tratamento com pioglitazona reduziu a glicemia de jejum, glicemia pós-prandial, insulina, HOMA-IR, triglicerídeos, VLDL-C, tALP e bALP e aumentou a HDL, tACP, TNF-&#945; e a transcrição de OPG (p<0,05). A glicemia basal associou-se positivamente com o cálcio ionizado. A expressão basal de OPG foi associado negativamente com tALP, enquanto que a expressão basal de TNFA foi associada positivamente com tALP e negativamente com tACP. A expressão basal IL6 foi associada positivamente com tALP, enquanto que a expressão basal de VDR foi associada negativamente com osteocalcina e positivamente com bALP em resposta ao tratamento (p<0,05). O polimorfismo RANK rs1805034 foi associado com redução na transcrição do gene RANK nos indivíduos DM2 e com o remodelamento ósseo após o tratamento com pioglitazona (p<0,05). O polimorfismo RANKL rs9525641 foi associado com aumento da transcrição gênica de RANKL nos indivíduos NG e DM2 e melhora da resposta farmacológica nos indivíduos DM2 tratados com pioglitazona (p<0,05). O polimorfismo rs3102735 do gene OPG foi associado com aumento da formação óssea nos indivíduos DM2 antes e após o tratamento (p<0,05). O genótipo CG do polimorfismo OPG rs2073618 foi associado com alteração da transcrição de OPG no grupo DM2 pré e pós-tratamento (p<0,05). O polimorfismo PPARG rs1801282 foi associado com menor risco para o desenvolvimento de diabetes (p<0,05). O polimorfismo PPARG rs2972162 foi associado com melhora da resistência insulínica nos indivíduos DM2 tratados com pioglitazona (p=0,017). O polimorfismo ESRI rs9340799 foi associado com redução da formação óssea nos indivíduos DM2 (p=0,038). Nos camundongos, após a indução da dieta hiperlipídica por 32 semanas, observou-se aumento do peso, da glicemia, do colesterol total, da expressão do mRNA de RANK, RANKL, IL6 e TNFA em fêmures e aumento de Tb.Sp e diminuição de BV/TV em comparação ao grupo controle (p<0,05). O tratamento com pioglitazona diminuiu a expressão de TNFA (p=0,028). As medidas histomorfométricas não alteraram-se após o tratamento (p>0,05). Os resultados sugerem que o estado hiperglicêmico e o tratamento influenciam os marcadores bioquímicos e moleculares. Os polimorfismos dos genes RANK, RANKL, OPG e ESRI parecem estar envolvidos no remodelamento ósseo independentemente da hiperglicemia e do tratamento e os polimorfismos do gene PPARG parecem estar envolvidos com menor risco para desenvolver diabetes e com a melhora da resistência insulínica em resposta ao tratamento com pioglitazona. / Morphological changes in bone tissue have been reported in users of oral hypoglycemic class of thiazolidinediones (TZDs). It is hypothesized that some genes related to osteogenesis and osteoclastogenesis may be influenced by pharmacological treatment, however, was not aware exact mechanism. The study aims was to evaluate pioglitazone effect on bone remodeling through genes involved in osteoclastogenesis in individuals newly diagnosed with DM2 and animal models, in order to identify sensibles genetics markers of bone alterations. Were invited to participate in study 199 patients (100 diabetics and 99 normoglycemic), in dyslipidemia ambulatory of Institute Dante Pazzanese of Cardiology. Diabetic subjects were treated with pioglitazone (15, 30, 45 or 45 mg /day/oral) for 16 weeks. Blood samples were collected before and after treatment for laboratory evaluations, extraction of genomic DNA and total RNA. Polymorphisms and mRNA expression in blood cells was determined by real time PCR using TaqMan® system. For study in animal model after 32 weeks of fat diet induction, was used 12 male mice C57BL/J6, which were divided into three groups: control (n=4); induced diabetic fat diet (DH, n=4) and induced diabetic fat diet and treated with pioglitazone 35mg/Kg/day for 16 weeks (DHP, n=4). For experimental groups were collected: blood samples for laboratory tests; femurs, for extraction of total RNA; and tibias, to determine histomorphometric parameters. DM2 patients showed decrease in serum osteocalcin and OPG expression and increased VDR expression compared to NG group (p<0.05). RANKL and IL6 expression were higher among women, whereas PPARG expression was higher among men with DM2 compared to NG group (p=0,032). DM2 patients before treatment showed blood glucose and IL6 mRNA expression negatively associated with ionized calcium, whereas TNFA and VDR transcription are positively and negatively associated with bALP respectively (p<0.05). Pioglitazone treatment reduced fasting glucose, postprandial glucose, insulin, HOMA-IR, triglycerides, VLDL-C, tALP and bALP and increased HDL, tACP, TNF-&#945; and OPG transcription (p<0.05). Basal blood glucose was positively associated with ionized calcium. Basal OPG expression was negatively associated with tALP, whereas basal TNFA expression was positively associated with tALP and negatively with tACP. Basal IL6 expression was positively associated with tALP, whereas basal VDR expression was negatively associated with osteocalcin and positively with bALP in response to treatment (p<0.05). RANK rs1805034 polymorphism was associated with RANK gene transcription reduction in subjects with DM2 and bone remodeling after treatment with pioglitazone (p<0.05). RANKL rs9525641 polymorphism was associated with increased RANKL gene transcription in NG and DM2 subjects and pharmacological response improvement in DM2 subjects treated with pioglitazone (p<0.05). OPG rs3102735polymorphism was associated with increased bone formation in DM2 subjects before and after treatment (p<0.05). CG genotype of OPG rs2073618 polymorphism was associated with OPG transcription change in DM2 group before and after treatment (p<0.05). PPARG rs1801282 polymorphism was associated with lower risk for diabetes development (p<0.05). PPARG rs2972162 polymorphism was associated with insulin resistance improvement in DM2 subjects treated with pioglitazone (p=0,017). ESRI rs9340799 polymorphism was associated with reduced bone formation in DM2 subjects (p=0,038). In mice, after 32 weeks of fat diet induction, was observed increase weight, blood glucose, total cholesterol and RANK, RANKL, IL6 and TNFA mRNA expression in femurs and Tb.Sp increase and BV/TV decrease compared to control group (p<0.05). Treatment with pioglitazone decrease TNFA (p=0,028). Histomorphometrics measurements not change after treatment (p>0.05). Results suggest that hyperglycemic state and treatment influence biochemical and molecular markers. RANK, RANKL, OPG and ESRI polymorphisms seens to be involved in bone remodeling regardless of hyperglycemia and treatment and PPARG gene polymorphisms seens to be associated with lower risk for diabetes development and with insulin resistance improvement in response to treatment with pioglitazone.

Alterações morfológicas no tecido ósseo têm sido descritas nos usuários de hipoglicemiantes orais da classe das tiazolidinedionas (TZDs). Hipotetiza-se que alguns genes relacionados com a osteogênese e osteoclastogênese podem ser influenciados pelo tratamento farmacológico, entretanto, o exato mecanismo ainda não está bem esclarecido. O objetivo do estudo foi avaliar o efeito da pioglitazona no remodelamento ósseo através de genes envolvidos na osteoclastogênese em indivíduos recentemente diagnosticados com DM2 e modelos animais, com a finalidade de identificar marcadores genéticos sensíveis de alterações ósseas. Foram convidados para participar do estudo 199 indivíduos (100 diabéticos e 99 normoglicêmicos), no ambulatório de dislipidemias do Instituto Dante Pazzanese de Cardiologia. Os indivíduos diabéticos foram tratados com pioglitazona (15, 30, 45, 45 mg/ dia/ via oral) por 16 semanas. Foram colhidas amostras de sangue, antes e após o tratamento para avaliações laboratoriais, extração de DNA genômico e de RNA total. Os polimorfismos e a expressão do mRNA nas células sanguíneas foram determinados pela PCR em tempo real através do sistema TaqMan®. Para o estudo em modelo animal após a indução da dieta hiperlipídica por 32 semanas, foram utilizados 12 camundongos machos da linhagem C57BL/J6, os quais foram divididos em três grupos: controle (n=4); diabéticos induzidos pela dieta hiperlipídica (DH, n=4) e diabéticos induzidos pela dieta hiperlipídica e tratados com pioglitazona 35mg/Kg/dia por 16 semanas (DHP, n=4). Para os grupos experimentais foram colhidos: amostras de sangue, para exames laboratoriais; fêmures, para a extração do RNA total; e tíbias, para determinação dos parâmetros histomorfométricos. Os pacientes DM2 apresentaram diminuição nas concentrações séricas de osteocalcina e na expressão de OPG e aumento na expressão de VDR em comparação ao grupo NG (p<0,05). A expressão de RANKL e IL6 foi maior entre as mulheres, enquanto que a expressão de PPARG foi maior entre os homens com DM2 em comparação ao grupo NG (p=0,032). Pacientes DM2 antes do tratamento apresentaram glicemia e expressão do mRNA de IL6 negativamente associados ao cálcio ionizado, enquanto que as transcrições de TNFA e VDR foram associadas positivamente e negativamente com bALP respectivamente (p<0,05). O tratamento com pioglitazona reduziu a glicemia de jejum, glicemia pós-prandial, insulina, HOMA-IR, triglicerídeos, VLDL-C, tALP e bALP e aumentou a HDL, tACP, TNF-&#945; e a transcrição de OPG (p<0,05). A glicemia basal associou-se positivamente com o cálcio ionizado. A expressão basal de OPG foi associado negativamente com tALP, enquanto que a expressão basal de TNFA foi associada positivamente com tALP e negativamente com tACP. A expressão basal IL6 foi associada positivamente com tALP, enquanto que a expressão basal de VDR foi associada negativamente com osteocalcina e positivamente com bALP em resposta ao tratamento (p<0,05). O polimorfismo RANK rs1805034 foi associado com redução na transcrição do gene RANK nos indivíduos DM2 e com o remodelamento ósseo após o tratamento com pioglitazona (p<0,05). O polimorfismo RANKL rs9525641 foi associado com aumento da transcrição gênica de RANKL nos indivíduos NG e DM2 e melhora da resposta farmacológica nos indivíduos DM2 tratados com pioglitazona (p<0,05). O polimorfismo rs3102735 do gene OPG foi associado com aumento da formação óssea nos indivíduos DM2 antes e após o tratamento (p<0,05). O genótipo CG do polimorfismo OPG rs2073618 foi associado com alteração da transcrição de OPG no grupo DM2 pré e pós-tratamento (p<0,05). O polimorfismo PPARG rs1801282 foi associado com menor risco para o desenvolvimento de diabetes (p<0,05). O polimorfismo PPARG rs2972162 foi associado com melhora da resistência insulínica nos indivíduos DM2 tratados com pioglitazona (p=0,017). O polimorfismo ESRI rs9340799 foi associado com redução da formação óssea nos indivíduos DM2 (p=0,038). Nos camundongos, após a indução da dieta hiperlipídica por 32 semanas, observou-se aumento do peso, da glicemia, do colesterol total, da expressão do mRNA de RANK, RANKL, IL6 e TNFA em fêmures e aumento de Tb.Sp e diminuição de BV/TV em comparação ao grupo controle (p<0,05). O tratamento com pioglitazona diminuiu a expressão de TNFA (p=0,028). As medidas histomorfométricas não alteraram-se após o tratamento (p>0,05). Os resultados sugerem que o estado hiperglicêmico e o tratamento influenciam os marcadores bioquímicos e moleculares. Os polimorfismos dos genes RANK, RANKL, OPG e ESRI parecem estar envolvidos no remodelamento ósseo independentemente da hiperglicemia e do tratamento e os polimorfismos do gene PPARG parecem estar envolvidos com menor risco para desenvolver diabetes e com a melhora da resistência insulínica em resposta ao tratamento com pioglitazona. / Morphological changes in bone tissue have been reported in users of oral hypoglycemic class of thiazolidinediones (TZDs). It is hypothesized that some genes related to osteogenesis and osteoclastogenesis may be influenced by pharmacological treatment, however, was not aware exact mechanism. The study aims was to evaluate pioglitazone effect on bone remodeling through genes involved in osteoclastogenesis in individuals newly diagnosed with DM2 and animal models, in order to identify sensibles genetics markers of bone alterations. Were invited to participate in study 199 patients (100 diabetics and 99 normoglycemic), in dyslipidemia ambulatory of Institute Dante Pazzanese of Cardiology. Diabetic subjects were treated with pioglitazone (15, 30, 45 or 45 mg /day/oral) for 16 weeks. Blood samples were collected before and after treatment for laboratory evaluations, extraction of genomic DNA and total RNA. Polymorphisms and mRNA expression in blood cells was determined by real time PCR using TaqMan® system. For study in animal model after 32 weeks of fat diet induction, was used 12 male mice C57BL/J6, which were divided into three groups: control (n=4); induced diabetic fat diet (DH, n=4) and induced diabetic fat diet and treated with pioglitazone 35mg/Kg/day for 16 weeks (DHP, n=4). For experimental groups were collected: blood samples for laboratory tests; femurs, for extraction of total RNA; and tibias, to determine histomorphometric parameters. DM2 patients showed decrease in serum osteocalcin and OPG expression and increased VDR expression compared to NG group (p<0.05). RANKL and IL6 expression were higher among women, whereas PPARG expression was higher among men with DM2 compared to NG group (p=0,032). DM2 patients before treatment showed blood glucose and IL6 mRNA expression negatively associated with ionized calcium, whereas TNFA and VDR transcription are positively and negatively associated with bALP respectively (p<0.05). Pioglitazone treatment reduced fasting glucose, postprandial glucose, insulin, HOMA-IR, triglycerides, VLDL-C, tALP and bALP and increased HDL, tACP, TNF-&#945; and OPG transcription (p<0.05). Basal blood glucose was positively associated with ionized calcium. Basal OPG expression was negatively associated with tALP, whereas basal TNFA expression was positively associated with tALP and negatively with tACP. Basal IL6 expression was positively associated with tALP, whereas basal VDR expression was negatively associated with osteocalcin and positively with bALP in response to treatment (p<0.05). RANK rs1805034 polymorphism was associated with RANK gene transcription reduction in subjects with DM2 and bone remodeling after treatment with pioglitazone (p<0.05). RANKL rs9525641 polymorphism was associated with increased RANKL gene transcription in NG and DM2 subjects and pharmacological response improvement in DM2 subjects treated with pioglitazone (p<0.05). OPG rs3102735polymorphism was associated with increased bone formation in DM2 subjects before and after treatment (p<0.05). CG genotype of OPG rs2073618 polymorphism was associated with OPG transcription change in DM2 group before and after treatment (p<0.05). PPARG rs1801282 polymorphism was associated with lower risk for diabetes development (p<0.05). PPARG rs2972162 polymorphism was associated with insulin resistance improvement in DM2 subjects treated with pioglitazone (p=0,017). ESRI rs9340799 polymorphism was associated with reduced bone formation in DM2 subjects (p=0,038). In mice, after 32 weeks of fat diet induction, was observed increase weight, blood glucose, total cholesterol and RANK, RANKL, IL6 and TNFA mRNA expression in femurs and Tb.Sp increase and BV/TV decrease compared to control group (p<0.05). Treatment with pioglitazone decrease TNFA (p=0,028). Histomorphometrics measurements not change after treatment (p>0.05). Results suggest that hyperglycemic state and treatment influence biochemical and molecular markers. RANK, RANKL, OPG and ESRI polymorphisms seens to be involved in bone remodeling regardless of hyperglycemia and treatment and PPARG gene polymorphisms seens to be associated with lower risk for diabetes development and with insulin resistance improvement in response to treatment with pioglitazone.

Estudos de metabolismo in vitro possuem o intuito de caracterizar e quantificar possíveis metabólitos, elucidar as vias metabólicas e sugerir modelos a serem seguidos para a realização de estudos in vivo. Com o intuito de estudar o metabolismo in vitro não estereosseletivo da rosiglitazona (RSG) empregando fração microssomal de fígado de ratos,foi desenvolvida uma metodologia por cromatografia líquida de alta eficiência (HPLC) com detecção UV em 245 nm, para analisar a RSG e seus principais metabólitos, p-hidroxi rosiglitazona (?-OH-R) e N-desmetil rosiglitazona (N-Dm-R). Os analitos foram separados em fase reversa, utilizando uma coluna X-Terra MS C-18 (partículas de 3,5 ?m) e fase móvel composta por água:acetonitrila:ácido acético (85:15:0,5, v/v/v), na vazão de 1 mL min-1. Matrizes biológicas contém um grande excesso de proteínas, lipídeos e outros materiais endógenos que interferem na análise de fármacos e metabólitos, tornando necessário um procedimento adequado de preparação das amostras antes da análise cromatográfica. A microextração em fase líquida com membrana cilíndrica oca (HF-LPME) é uma técnica promissora para a preparação de amostras em estudos de metabolismo in vitro, pois, além de promover o clean-up, promove também o enriquecimento dos analitos na amostra. A HF-LPME foi aplicada pela primeira vez para a extração simultânea desse fármaco e seus metabólitos. O sistema de três fases foi escolhido como o mais apropriado, empregando uma solução de ácido clorídrico como fase aceptora e 1-octanol como solvente orgânico. A otimização dos demais parâmetros foi realizada através de planejamento fatorial fracionário. O método foi validado e foi linear no intervalo de 50-6000 ng mL-1, apresentando limites de quantificação de 50 ng mL-1 e recuperações acima de 47 % para a RSG e seus metabólitos (?-OH-R e N-Dm-R). O método validado foi empregado em um estudo de metabolismo in vitro com fração microssomal de fígado de ratos. Nesse estudo, foi possível estimar as constantes de Michaelis-Menten (Km) e a velocidade inicial máxima (Vmax). N-Dm-R e ?-OH-R apresentaram valores de Vmax de 87,30 ± 8,04 e 51,64 ± 12,25 ?mol min-1 mg proteína-1, respectivamente, enquanto que os valores de Km foram de 58,14 ± 11,85 e 77,84 ± 36,77 mmol L-1, respectivamente. Outros metabólitos foram observados nos cromatogramas e a identificação foi feita por espectrometria de massas: ?rto-hidroxi-rosiglitazona e N-desmetil-hidroxi-rosiglitazona. A RSG é comercializada como uma mistura racêmica, apesar de possuir sua atividade antidiabética relacionada essencialmente com o enantiômero (S). O centro quiral desse fármaco possui um grupo carbonila, por isso, o enantiômero (R) pode se converter no enantiômero (S) ou vice-versa, via tautomerismo cetoenólico. Dados da literatura indicavam que essa racemização poderia ser lenta o suficiente para possibilitar o estudo dos enantiômeros isoladamente. Entretanto, até o momento não há dados sobre a disposição cinética ii e metabolismo enantiosseletivos desse fármaco. Sendo assim, propôs-se o desenvolvimento de metodologias analíticas para estudar a racemização da RSG e seus metabólitos e avaliar a possibilidade de estudar seu metabolismo in vitro de forma estereosseletiva. O método foi desenvolvido empregando HPLC com detecção em 245 nm. A separação dos enantiômeros do fármaco e metabólitos, também quirais, foi obtida empregando uma coluna Chiralcel OJ-H e fase móvel constituída por metanol:etanol (90:10; v/v), na vazão de 0,3 mL min-1. O estudo de racemização mostrou que o fármaco e seus metabólitos são racemizados nas condições em que o estudo de metabolismo é conduzido. Finalmente, para estudar o metabolismo in vitro da pioglitazona (PGZ), foi desenvolvido um método para análise desse fármaco e de seus principais metabólitos, a hidroxi-pioglitazona (M-IV) e a ceto-pioglitazona (M-III) empregando a eletroforese capilar (CE). As análises foram realizadas em capilar de sílica de 50 ?m de diâmetro interno e com comprimento efetivo de 40 cm, utilizando tampão fosfato de sódio 50 mmol L-1, pH 2,5, detecção em 190 nm, tensão de 30 kV e temperatura do capilar de 35 °C. A HF-LPME também foi empregada para a preparação das amostras. O sistema de três fases foi escolhido, empregando solução de ácido clorídrico como fase aceptora e o 1-octanol como solvente orgânico. A otimização dos demais parâmetros foi realizada através de planejamento fatorial fracionário. O método validado foi linear no intervalo de 200 - 25000 ng mL-1 para PGZ e 200 - 2000 ng mL-1 para os metabólitos, apresentando limites de quantificação de 200 ng mL-1 e recuperações acima de 19 % para a RSG e seus metabólitos M-IV e M-III. O método validado foi empregado em um estudo de metabolismo in vitro contendo fração microssomal de fígado de ratos, mas nesse estudo, não foi possível observar a formação dos metabólitos. Entretanto, esse método pode ser usado em outros modelos de metabolismo in vitro (microssomas humanos), nos quais se observa a formação desses metabólitos em concentrações maiores. / In vitro metabolism studies have been used to characterize and to quantify possible metabolites, to elucidate metabolic pathways and to suggest models to perform in vivo studies. So the purpose of this study was to evaluate the in vitro rosiglitazone (RSG) metabolism employing microsomal fraction obtained from rat livers. A high- performance liquid chromatography (HPLC) method with UV detection at 245 nm was developed to analyze RSG and the main metabolites, p-hydroxy rosiglitazone (?-OH-R) e N-desmethyl rosiglitazone (N-Dm-R). The analytes were separated under reversed phase conditions, using a X-Terra MS C-18 column (3.5 ?m particle size) and a mobile phase consisting of water:acetonitrile:acetic acid (85:15:0.5, v/v/v), at a flow rate of 1 mL min-1. Biological matrices contain a large excess of proteins, lipids and other endogenous compounds that interfere in the analysis of drugs and metabolites. So, a suitable sample preparation technique is required. Hollow-fiber liquid phase microextraction (HF-LPME) is a promising technique for the preparation of biological samples. Besides the clean-up, analytes enrichment is also achieved. HF-LPME for the simultaneous analysis of RSG and its main metabolites is described for the first time. The three-phase extraction was performed using hydrochloride acid solution as acceptor phase and 1-octanol as organic solvent. The other parameters were optimized by fractional factorial design. The method was validated and it was linear over the concentration range of 50-6000 ng mL-1, with quantification limits of 50 ng mL-1 and recoveries above 47 %. The validated method was used to estimate Michaelis-Menten (Km) constant and maximum initial velocity (Vmax). N-Dm-R e ?-OH-R showed Vmax values of 87.30 ± 8.04 and 51.64 ± 12.25 ?mol min-1 mg protein-1, respectively, while the Kmvalues were 58.14 ± 11.85 e 77.84 ± 36.77 mmol L-1, respectively. Other possible metabolites were observed in the chromatograms and they were identified by mass spectrometry: ?rtho-hydroxy-rosiglitazone e N-desmethyl-hydroxy-rosiglitazone. RSG is marketed as a racemic mixture although the antidiabetic activity is related essentially to the (S)-enantiomer. The chiral center has a carbonyl group, therefore the (R)-enantiomer could be transformed to the (S)-enantiomer or vice-versa by keto-enolic tautomerism. The literature indicates that this racemization is slow enough to allow the evaluation of the properties of the isolated enantiomers. However, there is no information in the literature about enantioselective RSG kinetic disposition and metabolism. Considering this facts, an analytical procedure was developed to study the racemization of RSG and its metabolites under different conditions and to determine if the enantioselective metabolism would be performed. The method was developed by HPLC with detection at 245 nm. The chiral separation of RSG and metabolites was achieved on a Chiralcel OJ-H column, with the mobile phase consisting of methanol:ethanol (90:10,v/v). The results obtained showed that the racemization occurs under the conditions used in in vitro iv metabolism studies. Finally, to study the in vitro metabolism of pioglitazone (PGZ), another method was developed by capillary electrophoresis (CE) to determine this drug and its main metabolites, hydroxy-pioglitazone (M-IV) and keto-pioglitazone (M-III). The analyses were conducted using a fused silica capillary (50 ?m inner diameter and 40 cm effective length), sodium phosphate buffer 50 mmol L-1, pH 2.5, detection at 190 nm, voltage of 30 kV and capillary temperature of 35°C. HF-LPME was also used for sample preparation with hydrochloride acid solution as acceptor phase and 1-octanol as organic solvent. The other parameters were optimized by fractional factorial design. The method was validated showing to be linear in the concentration range of 200 - 25000 ng mL-1 for PGZ and 200 - 2000 ng mL-1 for the metabolites. Quantification limits were 200 ng mL-1 for all analytes and the recoveries were higher than 19%. The validated method was used to study the in vitro metabolism of PGZ by rat liver microsomal fraction, but it was not possible to observe the formation of the metabolites in this study. However this method could be used in other in vitro metabolism models (human microssomes), in which higher concentrations of these metabolites are observed. Keywords:

No description available.

O diabete melito é uma doença metabólica com alta prevalência na população e quando no estado descompensado pode causar diversas complicações metabólicas e clínicas, entre elas a osteoporose. Entretanto, ainda não foram completamente esclarecidos os mecanismos pelos quais o diabete diminui a densidade mineral óssea e aumenta o risco a fraturas. Recentemente foram descritos alguns genes que estão envolvidos no turnover ósseo: OPG, RANK e RANKL. Além disso, o uso de hipoglicemiantes orais como as tiazolidinedionas (TZD), pode influenciar negativamente o metabolismo ósseo. Com a finalidade de identificar marcadores sensíveis de alteração do metabolismo ósseo foram investigadas as relações entre a expressão dos genes OPG, RANK e RANKL em células do sangue periférico e a resposta a TZDs em pacientes com DM2. Foram selecionados 52 indivíduos (36 diabéticos e 16 normoglicêmicos), no Instituto Dante Pazzanese de Cardiologia. Os indivíduos diabéticos foram tratados com pioglitazona (15, 30 e 45 mg/ dia/ via oral) por 16 semanas. Foram colhidas amostras de sangue, antes e após o tratamento para determinação de exames laboratoriais e extração de RNA total. A expressão de mRNA dos genes OPG, RANK e RANKL foi quantificada e avaliada por RT-PCR em tempo real, empregando-se o GAPD como controle endógeno. Observou-se que nos pacientes DM2 após o tratamento com pioglitazona, houve diminuição da glicemia de jejum, glicemia pós-prandial, insulina, Hb1Ac, índices HOMA-IR e HOMA-&#946; e aumento nas concentrações séricas de HDL, demonstrando a eficácia do tratamento. Ao comparar a expressão dos genes entre o grupo DM2 (sem tratamento) e o grupo normoglicêmico (NG), foi evidenciado um aumento da expressão de OPG no grupo NG em relação ao grupo DM2, e ao analisar a expressão entre as mulheres, constatou-se aumento da expressão de RANK no grupo DM2 em relação ao grupo NG. Além disso, ao correlacionar a expressão dos genes com as dosagens dos parâmetros bioquímicos, constatou-se que o aumento da expressão de RANK e RANKL está relacionado com o aumento das concentrações de cálcio ionizado e diminuição da expressão de OPG. Esses dados sugerem que a atividade osteoclástica está aumentada nos pacientes DM2 e com o tratamento o quadro osteoporótico pode ser agravado. / The diabetes mellitus is a metabolic disease with high prevalence in the population and can cause various metabolic and clinic complications, including osteoporosis, when it is decompensated. However, the mechanisms by which diabetes decreases bone mineral density and increases the risk of fractures are not completely clarified. Recently some genes which are involved in bone turnover were described: OPG, RANK and RANKL. Moreover, the treatment using oral hypoglycemic drugs such as thiazolidinediones (TZD), may negatively affect the bone metabolism. In order to identify sensitive markers related to the bone metabolism, were investigated the relationship between the expression of genes OPG, RANK and RANKL in peripheral blood leukocytes and the response to TZDs treatment in patients with DM2. Fifty-two individuals were selected (36 diabetics and 16 normoglycemics) at Dante Pazzanese Institute of Cardiology. Diabetic patients were treated with pioglitazone (15, 30 and 45 mg I day I oral) during 16 weeks. Blood samples were collected for biochemical analyses and total RNA extraction, before and after treatment. Gene expression of the genes OPG, RANK and RANKL in peripheral blood mononuclear cells was evaluated by Real Time PCR, using the GAPD housekeeping gene as the endogenous reference. In DM2 patients after treatment with pioglitazone there was reduction in their fasting glycemia, postprandial glycemia, insulin, Hb1Ac, HOMA-IR and HOMA-&#946; indices, and their serum concentrations of HDL increased, which demonstrates the effectiveness of the treatment. The bone profile markers have not altered after treatment, suggesting an anabolic action of the insulin in bone metabolism of these patients. Normoglycemics (NG) group gene expression, when compared with DM2 group (with no treatment), had increased OPG expression. Besides, RANK expression in group DM2 was higher than NG group when it was analyzed among women. Furthermore, having correlated the expression of the genes with the biochemical parameters data, the increase on RANK and RANKL gene expression is related to increased concentrations of ionized calcium and to decreased expression of OPG gene. These results are suggestive that osteoclastic activity is higher in DM2 patients, the treatment can exacerbate osteoporosis severity and the bone markers does not have enough sensibility to differentiate changes in individuals with type 2 diabetes mellitus.

Major depression is a highly prevalent mood disorder with chronic debilitating effects. Additional to a rising rate in incidence, depression is highly co–morbid with other psychiatric disorders, but also chronic cardiometabolic illnesses that present with an inflammatory component. The exact aetiology of depression is still unknown, being multifactorial in its possible aetiology. Various hypotheses have attempted to shed light on both endogenous and exogenous risk factors as well as the underlying pathology that may lead to the development of the disease. This has led to a wide range of mediators being implicated, including biogenic amines, the HPA–axis, neurotrophic factors, inflammatory agents, the cholinergic system and circadian rhythm, to name a few. The mechanisms of action of current treatment strategies, except for a few atypical and novel treatment approaches, are limited to interactions with monoamines and are at best only 65% effective. Many of these are also plagued by troubling side–effects, relapse and recurrence. It has therefore become imperative to explore novel targets for the treatment of depression that may produce more rapid, robust and lasting antidepressant effects with a less daunting side–effect profile. The strong co–morbidity between depression and various cardiometabolic disorders, including cardiovascular disease, atherosclerosis, type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS) has led to the proposal that a metabolic disturbance may be a vital component that drives inflammatory and immunological dysfunction in depression. Supporting of this is evidence for a role of inflammatory cytokines and neurotrophic factors in the pathogenesis of depression. It has also been demonstrated that a link exists between insulin– and nitric oxide (NO)– mediated pathways in the brain, which further highlights the role of oxidative stress and cell damage. Furthermore, evidence supports a role for oxidative stress and NO in T2DM and/or insulin resistance. Insulin has also been implicated in various physiological processes in the central nervous system (CNS) and may also influence the release and reuptake of neurotransmitters. Preclinical and clinical evidence has provided support for the antidepressant–like effects of insulin–sensitizing peroxisome proliferator activated receptor (PPAR)– agonists, such as rosiglitazone and pioglitazone. In preclinical studies, however, these effects are limited to acute treatment with pioglitazone or sub–chronic (5 days) treatment with rosiglitazone. It is well–recognized that such findings need to be confirmed by chronic treatment paradigms. The aim of the current study was therefore to further investigate the proposed antidepressant–like effects of pioglitazone in a genetic animal model of depression, the Flinders sensitive line (FSL) rat, using a chronic treatment protocol. The FSL rat model was reaffirmed as presenting with inherent depressive–like behaviour compared to its more resilient counterpart, the Flinders resistant line (FRL) rat. Moreover, imipramine demonstrated a robust and reliable antidepressant–like effect in these animals using the forced swim test (FST), thus confirming the face and predictive validity of the FSL rat model for depression. In contrast to previous preclinical studies, acute dose–ranging studies with pioglitazone in Sprague Dawley rats delivered no significant anti–immobility effects in the FST, whereas results similar to that seen in the dose–ranging studies were observed following chronic treatment using FSL rats. Since altered pharmacokinetics could possibly influence the drug’s performance, another route of administration, viz. the subcutaneous route, was utilized as an additional measure to exclude this possibility. The results of the subcutaneous study, however, were congruent with that observed after oral treatment. In order to confirm an association between altered insulin sensitivity and antidepressant action and demonstration by recent studies that thiazolidinediones may augment the efficacy of existing antidepressants, we therefore investigated whether concomitant treatment with gliclazide (an insulin releaser and insulin desensitizer) or pioglitazone (an insulin sensitizer) may alter the antidepressant–like effects evoked by chronic treatment with imipramine. Pioglitazone did not positively or negatively affect the antidepressant effect of imipramine, although gliclazide tended to decrease the anti–immobility effects induced by this antidepressant. Taken together and considering the current available literature, this finding supports evidence linking the insulin–PPAR pathway to depression. However, further explorative studies are required to delineate the role of insulin sensitivity and glucose homeostasis in depression and antidepressant response. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2012.

Major depression is a highly prevalent mood disorder with chronic debilitating effects. Additional to a rising rate in incidence, depression is highly co–morbid with other psychiatric disorders, but also chronic cardiometabolic illnesses that present with an inflammatory component. The exact aetiology of depression is still unknown, being multifactorial in its possible aetiology. Various hypotheses have attempted to shed light on both endogenous and exogenous risk factors as well as the underlying pathology that may lead to the development of the disease. This has led to a wide range of mediators being implicated, including biogenic amines, the HPA–axis, neurotrophic factors, inflammatory agents, the cholinergic system and circadian rhythm, to name a few. The mechanisms of action of current treatment strategies, except for a few atypical and novel treatment approaches, are limited to interactions with monoamines and are at best only 65% effective. Many of these are also plagued by troubling side–effects, relapse and recurrence. It has therefore become imperative to explore novel targets for the treatment of depression that may produce more rapid, robust and lasting antidepressant effects with a less daunting side–effect profile. The strong co–morbidity between depression and various cardiometabolic disorders, including cardiovascular disease, atherosclerosis, type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS) has led to the proposal that a metabolic disturbance may be a vital component that drives inflammatory and immunological dysfunction in depression. Supporting of this is evidence for a role of inflammatory cytokines and neurotrophic factors in the pathogenesis of depression. It has also been demonstrated that a link exists between insulin– and nitric oxide (NO)– mediated pathways in the brain, which further highlights the role of oxidative stress and cell damage. Furthermore, evidence supports a role for oxidative stress and NO in T2DM and/or insulin resistance. Insulin has also been implicated in various physiological processes in the central nervous system (CNS) and may also influence the release and reuptake of neurotransmitters. Preclinical and clinical evidence has provided support for the antidepressant–like effects of insulin–sensitizing peroxisome proliferator activated receptor (PPAR)– agonists, such as rosiglitazone and pioglitazone. In preclinical studies, however, these effects are limited to acute treatment with pioglitazone or sub–chronic (5 days) treatment with rosiglitazone. It is well–recognized that such findings need to be confirmed by chronic treatment paradigms. The aim of the current study was therefore to further investigate the proposed antidepressant–like effects of pioglitazone in a genetic animal model of depression, the Flinders sensitive line (FSL) rat, using a chronic treatment protocol. The FSL rat model was reaffirmed as presenting with inherent depressive–like behaviour compared to its more resilient counterpart, the Flinders resistant line (FRL) rat. Moreover, imipramine demonstrated a robust and reliable antidepressant–like effect in these animals using the forced swim test (FST), thus confirming the face and predictive validity of the FSL rat model for depression. In contrast to previous preclinical studies, acute dose–ranging studies with pioglitazone in Sprague Dawley rats delivered no significant anti–immobility effects in the FST, whereas results similar to that seen in the dose–ranging studies were observed following chronic treatment using FSL rats. Since altered pharmacokinetics could possibly influence the drug’s performance, another route of administration, viz. the subcutaneous route, was utilized as an additional measure to exclude this possibility. The results of the subcutaneous study, however, were congruent with that observed after oral treatment. In order to confirm an association between altered insulin sensitivity and antidepressant action and demonstration by recent studies that thiazolidinediones may augment the efficacy of existing antidepressants, we therefore investigated whether concomitant treatment with gliclazide (an insulin releaser and insulin desensitizer) or pioglitazone (an insulin sensitizer) may alter the antidepressant–like effects evoked by chronic treatment with imipramine. Pioglitazone did not positively or negatively affect the antidepressant effect of imipramine, although gliclazide tended to decrease the anti–immobility effects induced by this antidepressant. Taken together and considering the current available literature, this finding supports evidence linking the insulin–PPAR pathway to depression. However, further explorative studies are required to delineate the role of insulin sensitivity and glucose homeostasis in depression and antidepressant response. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2012.

O diabete melito é uma doença metabólica com alta prevalência na população e quando no estado descompensado pode causar diversas complicações metabólicas e clínicas, entre elas a osteoporose. Entretanto, ainda não foram completamente esclarecidos os mecanismos pelos quais o diabete diminui a densidade mineral óssea e aumenta o risco a fraturas. Recentemente foram descritos alguns genes que estão envolvidos no turnover ósseo: OPG, RANK e RANKL. Além disso, o uso de hipoglicemiantes orais como as tiazolidinedionas (TZD), pode influenciar negativamente o metabolismo ósseo. Com a finalidade de identificar marcadores sensíveis de alteração do metabolismo ósseo foram investigadas as relações entre a expressão dos genes OPG, RANK e RANKL em células do sangue periférico e a resposta a TZDs em pacientes com DM2. Foram selecionados 52 indivíduos (36 diabéticos e 16 normoglicêmicos), no Instituto Dante Pazzanese de Cardiologia. Os indivíduos diabéticos foram tratados com pioglitazona (15, 30 e 45 mg/ dia/ via oral) por 16 semanas. Foram colhidas amostras de sangue, antes e após o tratamento para determinação de exames laboratoriais e extração de RNA total. A expressão de mRNA dos genes OPG, RANK e RANKL foi quantificada e avaliada por RT-PCR em tempo real, empregando-se o GAPD como controle endógeno. Observou-se que nos pacientes DM2 após o tratamento com pioglitazona, houve diminuição da glicemia de jejum, glicemia pós-prandial, insulina, Hb1Ac, índices HOMA-IR e HOMA-&#946; e aumento nas concentrações séricas de HDL, demonstrando a eficácia do tratamento. Ao comparar a expressão dos genes entre o grupo DM2 (sem tratamento) e o grupo normoglicêmico (NG), foi evidenciado um aumento da expressão de OPG no grupo NG em relação ao grupo DM2, e ao analisar a expressão entre as mulheres, constatou-se aumento da expressão de RANK no grupo DM2 em relação ao grupo NG. Além disso, ao correlacionar a expressão dos genes com as dosagens dos parâmetros bioquímicos, constatou-se que o aumento da expressão de RANK e RANKL está relacionado com o aumento das concentrações de cálcio ionizado e diminuição da expressão de OPG. Esses dados sugerem que a atividade osteoclástica está aumentada nos pacientes DM2 e com o tratamento o quadro osteoporótico pode ser agravado. / The diabetes mellitus is a metabolic disease with high prevalence in the population and can cause various metabolic and clinic complications, including osteoporosis, when it is decompensated. However, the mechanisms by which diabetes decreases bone mineral density and increases the risk of fractures are not completely clarified. Recently some genes which are involved in bone turnover were described: OPG, RANK and RANKL. Moreover, the treatment using oral hypoglycemic drugs such as thiazolidinediones (TZD), may negatively affect the bone metabolism. In order to identify sensitive markers related to the bone metabolism, were investigated the relationship between the expression of genes OPG, RANK and RANKL in peripheral blood leukocytes and the response to TZDs treatment in patients with DM2. Fifty-two individuals were selected (36 diabetics and 16 normoglycemics) at Dante Pazzanese Institute of Cardiology. Diabetic patients were treated with pioglitazone (15, 30 and 45 mg I day I oral) during 16 weeks. Blood samples were collected for biochemical analyses and total RNA extraction, before and after treatment. Gene expression of the genes OPG, RANK and RANKL in peripheral blood mononuclear cells was evaluated by Real Time PCR, using the GAPD housekeeping gene as the endogenous reference. In DM2 patients after treatment with pioglitazone there was reduction in their fasting glycemia, postprandial glycemia, insulin, Hb1Ac, HOMA-IR and HOMA-&#946; indices, and their serum concentrations of HDL increased, which demonstrates the effectiveness of the treatment. The bone profile markers have not altered after treatment, suggesting an anabolic action of the insulin in bone metabolism of these patients. Normoglycemics (NG) group gene expression, when compared with DM2 group (with no treatment), had increased OPG expression. Besides, RANK expression in group DM2 was higher than NG group when it was analyzed among women. Furthermore, having correlated the expression of the genes with the biochemical parameters data, the increase on RANK and RANKL gene expression is related to increased concentrations of ionized calcium and to decreased expression of OPG gene. These results are suggestive that osteoclastic activity is higher in DM2 patients, the treatment can exacerbate osteoporosis severity and the bone markers does not have enough sensibility to differentiate changes in individuals with type 2 diabetes mellitus.

Objective: Despite the advanced therapy with statins, antithrombotics, and antihypertensive agents, the medical treatment of atherosclerotic disease is less than optimal. Therefore, additional therapeutic antiatherosclerotic options are desirable. This pilot study was performed to assess the potential antiatherogenic effect of the peroxisome proliferator-activated receptor-γ agonist pioglitazone in nondiabetic patients. Methods: A total of 54 nondiabetic patients were observed in a prospective, double-blind, placebo-controlled study. Patients were randomized to pioglitazone or placebo. The following efficacy parameters were determined by serial analyses: artery pulse wave analysis and carotid-femoral pulse wave velocity (PWV), static and dynamic retinal vessel function, and the common carotid intima-media thickness (IMT). The main secondary endpoint was the change in different biochemical markers. Results: After 9 months, no relevant differences could be determined in the two treatment groups in PWV (pioglitazone 14.3 ± 4.4 m/s vs. placebo 14.2 ± 4.2 m/s), retinal arterial diameter (pioglitazone 112.1 ± 23.3 μm vs. placebo 117.9 ± 21.5 μm) or IMT (pioglitazone 0.85 ± 0.30 mm vs. placebo 0.79 ± 0.15 mm). Additionally, there were no differences in the change in biochemical markers like cholesteryl ester transfer protein, lowdensity lipoprotein cholesterol, high-sensitivity C-reactive protein or white blood cell count. Conclusions : Treatment with a peroxisome proliferator-activated receptor-γ agonist in nondiabetic patients did not improve the function of large and small peripheral vessels (PPP Trial, clinicaltrialsregister. eu: 2006-000186-11).