Contexte: Bien que plusieurs algorithmes pharmacogénétiques de prédiction de doses de warfarine aient été publiés, peu d’études ont comparé la validité de ces algorithmes en pratique clinique réelle. Objectif: Évaluer trois algorithmes pharmacogénomiques dans une population de patients qui initient un traitement à la warfarine et qui souffrent de fibrillation auriculaire ou de problèmes de valves cardiaques. Analyser la performance des algorithmes de Gage et al., de Michaud et al. ainsi que de l’IWPC quant à la prédiction de la dose de warfarine permettant d’atteindre l’INR thérapeutique. Méthodes: Un devis de cohorte rétrospectif fut utilisé afin d’évaluer la validité des algorithmes chez 605 patients ayant débuté une thérapie de warfarine à l’Institut de Cardiologie de Montréal. Le coefficient de corrélation de Pearson ainsi que l’erreur absolue moyenne ont été utilisés pour évaluer la précision des algorithmes. L’exactitude clinique des prédictions de doses fut évaluée en calculant le nombre de patients pour qui la dose prédite était sous-estimée, idéalement estimée ou surestimée. Enfin, la régression linéaire multiple a été utilisée pour évaluer la validité d’un modèle de prédiction de doses de warfarine obtenu en ajoutant de nouvelles covariables. Résultats : L’algorithme de Gage a obtenu la proportion de variation expliquée la plus élevée (R2 ajusté = 44 %) ainsi que la plus faible erreur absolue moyenne (MAE = 1.41 ± 0.06). De plus, la comparaison des proportions de patients ayant une dose prédite à moins de 20 % de la dose observée a confirmé que l’algorithme de Gage était également le plus performant. Conclusion : Le modèle publié par Gage en 2008 est l’algorithme pharmacogénétique le plus exact dans notre population pour prédire des doses thérapeutiques de warfarine. / Background: Although numerous genotype-based warfarin dosing algorithms have been published, there is little data comparing the predictive ability of these algorithms in real clinical practice. Objectives: Our goal was to evaluate the performance of pharmacogenetic algorithms in an unselected patient population initiating warfarin treatment for atrial fibrillation or valve disease in a real-world clinical setting. The principal objective of the analysis was to determine if Gage’s, Michaud’s, and IWPC algorithms could predict the dose achieving the therapeutic International normalized ratio (INR). Methods: Data from a retrospective cohort study of 605 patients initiating warfarin therapy at the Montreal Heart Institute was used. We compared the dose predicted by the algorithms to the dose achieving the therapeutic INR. Pearson’s correlation coefficient and mean absolute error (MAE) were used to evaluate the predictive accuracy of the algorithms. Clinical accuracy of the predictions was assessed by computing the proportion of patients in which the predicted dose was under-estimated, ideally estimated, or overestimated. Finally, we used multiple linear regression analysis to evaluate the accuracy of a predictive model obtained by adding additional covariables in predicting therapeutic warfarin doses. Results: The proportion of variation explained (adjusted R2) was the highest for Gage’s algorithm (R2 = 44 %) and the mean absolute error was the smallest for the predictions made by Gage’s algorithm (MAE = 1.41 ± 0.06). Moreover, when we compared the proportion of patients whose predicted doses are within ± 20 % of the observed stable dose, Gage’s algorithm also performed the best overall. Conclusion: The algorithm published by Gage et al. in 2008 is the most accurate pharmacogenetically based equation in predicting therapeutic warfarin dose in our study population.

Les inhibiteurs de tyrosine kinases (ITKs) sont une nouvelle classe thérapeutique ayant connu un grand essor ces dix dernières années. Inhibiteurs compétitifs de l’adénosine triphosphate (ATP), ils sont utilisés dans le traitement de nombreux cancers dans lesquels une dérégulation de tyrosine kinases a été mise en évidence. Malgré une efficacité prouvée, des cas de résistance sont rapportés, en particulier avec l’exemple de la leucémie myéloïde chronique (LMC) et le traitement par ITK. Cette variabilité inter-individuelle peut être due à des mécanismes de résistance propre de la cellule tumorale ou à des variations dans les paramètres pharmacocinétiques de la molécule. De nombreuses études ont analysé l’impact de polymorphismes (SNPs) dans des gènes codants pour les déterminants pharmacocinétiques et pharmacodynamiques des ITKs. Nous avons analysé l’impact de SNPs sur l’obtention de la réponse moléculaire majeure à 1 an dans 2 cohortes de patients atteints de LMC et traités par imatinib. C1236T, G2677T/A et C3435T, 3 SNPs du gène MDR-1 codant pour la glycoprotéine P et les SNPs de la région codante du gène SLC22A1 à l’origine du transporteur d’influx hOCT1. L’impact bénéfique de l’allèle 1236T ou haplotype *4 et l’impact péjoratif de l’allèle 2677G ou haplotype *1, retrouvés dans la 1ère cohorte n’ont pas été retrouvés dans la 2ième cohorte suggérant un impact mineur voire nul de ces derniers sur la réponse à l’imatinib. L’impact des SNPs de SLC22A1 observés dans la 2ième cohorte nécessite d’être confirmé. Des travaux supplémentaires à plus grande échelle, selon des critères nécessitant d’être harmonisés, sont nécessaires avant d’espérer pouvoir aboutir à une «médecine personnalisée» pour l’imatinib mais également de façon générale pour l’ensemble des ITKs. / Tyrosine kinases inhibitors (TKIs) are a new class of drugs having bloomed over the past decade. As competitive inhibitors of the adenosine triphosphate, they are used in the treatment of many cancers in which deregulation of tyrosine kinases has been demonstrated. In spite of dramatic efficacy, cases of resistance have been reported particularly with chronic myeloid leukemia (CML) and TKI treatment. This inter-individual variability may be due to mechanisms of intrinsic resistance of tumor cells or changes in the pharmacokinetic parameters of the molecule. Numerous studies have analyzed the impact of polymorphisms (SNPs) in genes coding for pharmacokinetic and pharmacodynamic determinants. We analyzed the impact of SNPs on major molecular response at 1 year in 2 cohorts of patients with CML treated with imatinib. C1236T, G2677T/A, C3435T, three SNPs in the MDR-1 gene encoding P-glycoprotein and SNPs in the coding region of the SLC22A1 gene encoding hOCT1. The protective impact of the 1236T allele or haplotype*4 and the pejorative impact of the 2677G allele or haplotype*1, found in the 1st cohort, were not replicated in the 2nd cohort, suggesting minor or no impact on the response to imatinib. The impact of SLC22A1 SNPs observed in the 2nd cohort needs to be confirmed. Further works on a larger cohort, according to criteria that need to be harmonized, are necessary before we reach a “personalized medicine” for imatinib but also for all TKIs

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jana Jeřábková Supervisor: Doc. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Interaction of selected anthocyanidins with farnesoid X receptor Human farnesoid X receptor (FXR) is a member of nuclear receptor superfamily that act as ligand-activated transcription factors. FXR binds to specific regulatory DNA regions and induces expression of many target genes. These regulated genes are involved in bile acid metabolism and transport, maintaining blood lipids, liporoteins and glucose homeostasis and also contribute to maintain intestinal bacterial balance, hepatoprotection and liver regeneration. The interest of recent studies is to test the range of FXR ligands for treatment and prevention of many diseases such as cholestais, cholesterol gallstone disease, steato-hepatitis, dyslipidemia, atherosclerosis, type 2 diabetes mellitus, metabolic syndrome, liver cancer and other forms of cancer such as breast cancer. In this experimental diploma thesis we are focused on testing of potencial ligands of human farnesoid X receptor from the group of natural plant pigments anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin) using the human hepatoma cell line...

para diversos tipos de tratamento, como doenças auto-imunes, inflamatórias e até para pacientes submetidos a transplante de órgãos. Assim também, é capaz de causar efeitos adversos como toxicidade hepática e mais frequentemente a mielossupressão. Tiopurina Smetiltransferase (TPMT), uma enzima que catalisa a S-metilação dessas drogas, exibiu um polimorfismo genético em 10% de Caucasianos, sendo 1/300 indivíduos com completa deficiência. Pacientes intermediários ou completamente deficientes da atividade de TPMT estão em risco por excessiva toxicidade após receberem doses padrões de medicações tiopurínicas. O presente estudo visa a 1) pesquisar a frequência desses polimorfismos (TPMT*2, TPMT*3A, TPMT*3B e TPMT*3C) em populações de doadores de sangue em Hospital terciário Universitário e em pacientes que utilizem a Azatioprina para o tratamento da hepatite autoimune e transplantado renal, 2) padronizar os métodos para a identificação dos polimorfismos e associá-los com os níveis dos metabólitos da AZA. Esses achados podem explicar os efeitos benéficos da Azatioprina e ter importante implicação para o desenho de novas terapias específicas em doenças autoimunes e em transplante de orgãos / Thiopurine drugs such as azathioprine (AZA) are widely used for many types of treatment: autoimmune and inflammatory diseases and for patients who had undergone organ transplantation. AZA is capable of causing adverse effects such as hepatotoxicity and myelosuppression. Thiopurine S-methyltransferase (TPMT), which catalyzes the Smethylation of such drugs, exhibits a genetic polymorphism in 10% of Caucasians, and 1 / 300 individuals with complete deficiency. Patients who are intermediary or completely deficient in TPMT activity are at risk for excessive toxicity after receiving standard doses of thiopurine drugs. This recent study aims to 1) investigate the frequency of these polymorphisms (TPMT*2, TPMT*3A, TPMT * 3B and TPMT*3C) in blood donors from a terciary University Hospital and in patients using azathioprine for the treatment of autoimmune hepatitis and transplanted kidney; 2) standardize the methods for the identification of polymorphisms and associate with levels metabolites of AZA. These findings may explain the beneficial effects of azathioprine and have important implications to design new therapies for autoimmune diseases and organ transplant

Os sais de lítio são utilizados no tratamento dos transtornos do humor há mais de 50 anos. É a principal medicação prescrita para pacientes com diagnóstico de transtorno bipolar (TB), No entanto, aproximadamente 70% dos pacientes não respondem satisfatoriamente, ou seja, são refratários ao tratamento com lítio. Tendo em vista a importância do uso do lítio no tratamento de TB e a influência de fatores genéticos na variabilidade da biodisponibilidade do medicamento e em seus efeitos (95%), é de extrema importância esclarecer o papel da farmacogenética envolvida na resposta ao lítio. O objetivo deste trabalho foi detectar possíveis marcadores genéticos em pacientes com TB para melhor predição da resposta à terapia medicamentosa Para tanto, estudamos os polimorfismos dos genes relacionados ao mecanismo de ação do lítio: glicogênio- sintase quinase 3 - beta (GSK3-Beta), fator neurotrófico derivado do cérebro (BDNF), Receptor neurotrófico de tirosina quinase tipo 2 (NTRK2) e proteína ligada ao elemento de resposta cAMP (CREB) em pacientes bipolares respondedores e refratários ao tratamento com lítio com fenótipo determinado por escalas HDRS e YMRS, e análise dos prontuários, e escala ALDA. Na nossa amostra, os polimorfismos CREB1-1H (G > A), CREB1-7H (C > T), BDNF (rs6265 - G > A) e NTRK2 (rs1387923 - T > C) não apresentaram associação significativa dos genótipos com a resposta à terapia com lítio. Para os resultados obtidos na análise genotípica correspondente ao polimorfismo rs334558 (C > T) do gene GSK3-Beta, verificamos maior incidência do genótipo heterozigoto CT em pacientes respondedores e maior incidência do genótipo polimórfico homozigoto TT em pacientes refratários. Para a metodologia ALDA, não obtivemos diferenças que relacionassem os polimorfismos selecionados a refratariedade ao lítio. Acreditamos que o pequeno número amostral incluídos em nosso estudo pode ter prejudicado a determinação desses polimorfismos na resposta ao lítio / Lithium salts have been used in the treatment of mood disorders for more than 50 years. It is the main medication prescribed for patients diagnosed with bipolar disorder (BD). However, approximately 70% of the patients do not respond satisfactorily, that is, they are refractory to treatment with lithium. Given the importance of using lithium in the treatment of BD and the influence of genetic factors on the variability of the bioavailability of the drug and its effects (95%), it is extremely important to clarify the role of pharmacogenetics involved in the lithium response. The goal of this study was to detect possible genetic markers in BD patients to better predict the response to drug therapy. In order to do so, we studied the polymorphisms of genes related to the mechanism of action of lithium: glycogen synthase kinase 3 - beta (GSK3-Beta), brain-derived neutrophic factor (BDNF) tyrosine kinase type 2 neurotrophic receptor (NTRK2) and cAMP response element-binding protein (CREB) in responders and refractory bipolar patients that used lithium in the treatment. The phenotype was determined by HDRS and YMRS scales, and analysis of charts, and ALDA scale. In our sample, the polymorphisms CREB1-1H (G > A), CREB1-7H (C > T), BDNF (rs6265 - G > A) and NTRK2 (rs1387923 - T > C) had no significant association of genotypes with the response to lithium therapy. For the results obtained in the genotype analysis corresponding to the polymorphism rs334558 (C > T) of the GSK3-Beta gene, we verified a higher incidence of the heterozygous CT genotype in responding patients and a higher incidence of the polymorphic homozygous TT genotype in refractory patients. For the ALDA methodology, we did not obtain differences that related the selected polymorphisms to refractoriness to lithium. We believe that the small sample size included in our study may have impaired the determination of these polymorphisms in lithium response

A disfunção erétil (DE) é uma doença relacionada com a sinalização deficiente de óxido nítrico (NO). O NO é produzido a partir da L-arginina pelas enzimas óxido nítrico sintase neuronal (nNOS), endotelial (eNOS) e induzida (iNOS). A dimetilarginina assimétrica (ADMA) é um inibidor endógeno dos três subtipos existentes de NOS, e é metabolizada principalmente pelas enzimas dimetilarginina dimetilaminohidrolase 1 e 2 (DDAH1 e DDAH2). Vários estudos têm associado alterações em genes, expressão ou atividade das enzimas DDAH com distúrbios em que a sinalização de NO é prejudicada. O objetivo deste estudo foi avaliar se o nível de resposta ao tratamento da DE com sildenafil pode estar associado a polimorfismos dos genes DDAH1 (rs1554597 e rs18582) e DDAH2 (rs805304 e rs805305) e, também, aos haplótipos formados por estes polimorfismos. Foram selecionados 70 pacientes com DE pós-prostatectomia (DEPP) e 70 pacientes com DE clínica (DEC). A função erétil dos voluntários foi avaliada através do questionário Índice Internacional de Função Erétil (IIEF). Para avaliação da resposta ao sildenafil, foram calculadas a diferença entre as pontuações pré e pós-tratamento (?IIEF) e a percentagem atingida da máxima resposta possível (?IIEF%) de cada paciente. Também, os pacientes de cada grupo foram divididos em bons e maus respondedores ao sildenafil de acordo com a mediana dos valores de ?IIEF%. Os genótipos dos rs1554597, rs805304 e rs805305 foram obtidos pela técnica de reação em cadeia da polimerase (PCR) seguida de digestão enzimática, e do rs18582 pela técnica de PCR alelo específica. O software PHASE 2.1 foi utilizado para estimar os haplótipos em cada grupo. Os resultados mostraram que o alelo variante A do rs18582 apresentou tendência para associação com piores respostas ao sildenafil no grupo DEC (P=0,058). No grupo DEPP, portadores dos alelos variantes A do rs805304 e G do rs805305 foram associados a melhores respostas ao sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; e score IIEF pós-tratamento, P=0,014). Não foram encontradas outras associações significativas. Estes resultados mostram que os marcadores genéticos rs805304 e rs805305 do DDAH2 podem influenciar as respostas ao sildenafil em pacientes com DE. / Erectile dysfunction (ED) is a disease related to deficient nitric oxide (NO) signaling. NO is produced from L-arginine by three isoforms of the enzyme nitric oxide synthase: neuronal (nNOS), endothelial (eNOS) and induced (iNOS). Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the three existing NOS subtypes, and is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 and 2 (DDAH1 and DDAH2) enzymes. Several studies have associated changes in genes, expression or activity of DDAH enzymes with disorders in which NO signaling is impaired. The aim of this study was to evaluate whether the response to ED treatment with sildenafil may be associated with polymorphisms of the DDAH1 (rs1554597 and rs18582) and DDAH2 genes (rs805304 and rs805305), as well as the haplotypes formed by these polymorphisms. We selected 70 patients with postprostatectomy ED (PPED) and 70 patients with clinical ED (CED). The erectile function of the volunteers was assessed using the International Index for Erectile Function (IIEF) questionnaire. To evaluate the response to sildenafil, the difference between the pre- and post-treatment scores (?IIEF) and the percentage reached from the maximum possible response (?IIEF%) of each patient were calculated. Also, patients from each group were divided into good and bad responders to sildenafil according to the median values of ?IIEF%. The genotypes of rs1554597, rs805304 and rs805305 were obtained by the polymerase chain reaction (PCR) technique followed by enzymatic digestion, and rs18582 by the allele-specific PCR technique. The PHASE 2.1 software was used to estimate the haplotypes in each group. The results showed that the variant A allele of rs18582 showed a tendency to be associated with a greater chance of worse responses to sildenafil in the DEC group (P=0,058). In the DEPP group, carriers of the variant alleles A of rs805304 and G of rs805305 were associated with better responses to sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; and post-treatment IIEF score, P=0,014). No other significant associations were found. These results show that the genetic markers rs805304 and rs805305 of DDAH2 may influence the responses to sildenafil in patients with ED.

A ATP-binding cassette transporter A1 (ABCA1) é uma proteína transmembrana responsável pelo efluxo celular de colesterol e fosfolipídeos, que é um passo essencial para o transporte reverso do colesterol e para a biogênese da HDL. Polimorfismos do gene ABCA1 foram associados com risco de doença arterial coronariana, variações no perfil lipídico e diferenças na resposta a fármacos hipolipemiantes. Com a finalidade de avaliar os efeitos de polimorfismos do ABCA1 sobre a expressão gênica e a resposta a vastatinas, foram selecionados indivíduos normolipidemicos (NL, n=143) e hipercolesterolêmicos (HC, n=224). A resposta a atorvastatina (10 mg/dia/4 semanas) foi avaliada pelo perfil lipídico sérico em 141 indivíduos do grupo HC (ATORVA). DNA e RNA total foram extraídos de amostras de sangue periférico. Os polimorfismos de nucleotídeo único (SNP) G70943A (R219K), C-14T e C-105T, uma variante nova do ABCA1, foram detectados por PCR-RFLP e confirmados por seqüenciamento de DNA. A expressão de RNAm do ABCA1 em células mononucleares do sangue periférico (CMSP) foi analisada por PCR-duplex e PCR em tempo real, utilizando o gene GAPD como referência endógena. A freqüência do alelo -105T foi 1,4% em NL e 2,0% em HC. O alelo 70943A (genótipos GA+AA) foi associado com maior concentração sérica basal de apoAI (NL), de HDL-c (ATORVA) e com menores concentrações basais de triglicerídeos e VLDL-c e menor índice TG/HDL-c (HC e ATORVA) em comparação com o genótipo 70943GG (p<0,05). O polimorfismo C-105T está em desequilíbrio de ligação com o SNP C-14T (p=0,006). Portadores do alelo -105T (genótipos CT+TT), quando comparados aos portadores do genótipo -105CC, tiveram menores valores basais de triglicerídeos e VLDL-c, maior concentração de HDL-c e menor índice TG/HDL-c nos grupos HC e ATORVA e também maiores concentrações de apoAI e menor índice apoB/apoAI no grupo ATORVA (p<0,05). Nos grupos HC e ATORVA, os portadores do haplótipo -14CT+TT/-105CT+TT tiveram menores valores de triglicerídeos e VLDL-c basais, maiores concentrações de HDL-c e menor índice TG/HDL-c quando comparados aos portadores dos outros haplótipos (p<0,05). A expressão basal do ABCA1 foi menor nos HC que nos NL independentemente da taxa de expressão alta (GM1) ou baixa (GM2). Este efeito foi associado com os SNPs C-14T e G70943A SNPs. Após o tratamento com atorvastatina, a expressão de RNAm foi reduzida nos HC portadores do alelo - 14T em comparação com os portadores de alelo -14C. Esses resultados são sugestivos de que ABCA1 SNPs estão envolvidos na variação do perfil lipídico sérico e na expressão de RNAm em resposta a atorvastatina. / The ATP-binding cassette transporter A1 (ABCA1) is a transmembrane protein involved on cholesterol and phospholipid cellular efflux, which is an essential step for the reverse cholesterol transport and HDL biogenesis. Single nucleotide polymorphisms (SNPs) in the ABCA1 gene have been associated with increased risk of coronary heart disease, differences on serum lipid profile and response to lowering-cholesterol drugs. We have evaluated the influence of ABCA1 SNPs on mRNA expression and lipid-lowering response to atorvastatin. Normolipidemic (NL, n=143) hypercholesterolemic (HC, n=224) individuals were enrolled in this study and the response to atorvastatin (10 mg/day/4 weeks) was evaluated in HC individuals (ATORVA, n=141). Blood samples were collected for biochemical analyses, genomic DNA and total RNA extraction. SNPs G70943A (R219K), C-14T and C-105T, a novel variant of ABCA1, were detected by PCR-RFLP and confirmed for DNA sequencing. ABCA1 mRNA expression in peripheral blood mononuclear cells (PBMC) was analysed by PCR-duplex and Real Time PCR, using the GAPD as the endogenous reference. In HC and NL, the frequency of -105T allele was 2.0% and 1.4%, respectively. The 70943A allele (genotypes GA+AA) was associated with higher basal concentrations of apoAI (NL) and HDL-c (ATORVA) and lower triglyceride and VLDL-c and TG/HDL-c ratio (HC and ATORVA) than the 70943GG genotype (p<0.05). We found a linkage disequilibrium between C-14T and C-105T SNPs in HC group (p=0.006). Individuals carrying -105T allele (CT/TT genotypes), when compared with -105CC carriers, had lower basal concentrations of triglyceride and VLDL-c, higher concentration of HDL-c and lower TG/HDL-c ratio in HC and ATORVA groups and also higher concentration of apoAI and lower apoB/apoAI ratio in ATORVA group (p<0.05). In HC and ATORVA, individuals with -14CT+TT/-105CT+TT haplotype had lower basal values of triglyceride and VLDL-c, higher concentration of HDL-c and lower TG/HDL-c ratio than carries of others haplotypes (p<0,05). ABCA1 mRNA basal expression was lower in HC when compared to NL independently of high (GM1) or low (GM2) basal expression rate. This effect was associated with C-14T and G70943A SNPs. After atorvastatin treatment, mRNA expression was reduced in HC individuals carrying -14T allele in comparison with the -14C allele carriers. These results are sugestive that ABCA1 SNPs are involved on variation of serum lipid profile and mRNA expression in response to atorvastatin.

A disfunção erétil (DE) é uma doença relacionada com a sinalização deficiente de óxido nítrico (NO). O NO é produzido a partir da L-arginina pelas enzimas óxido nítrico sintase neuronal (nNOS), endotelial (eNOS) e induzida (iNOS). A dimetilarginina assimétrica (ADMA) é um inibidor endógeno dos três subtipos existentes de NOS, e é metabolizada principalmente pelas enzimas dimetilarginina dimetilaminohidrolase 1 e 2 (DDAH1 e DDAH2). Vários estudos têm associado alterações em genes, expressão ou atividade das enzimas DDAH com distúrbios em que a sinalização de NO é prejudicada. O objetivo deste estudo foi avaliar se o nível de resposta ao tratamento da DE com sildenafil pode estar associado a polimorfismos dos genes DDAH1 (rs1554597 e rs18582) e DDAH2 (rs805304 e rs805305) e, também, aos haplótipos formados por estes polimorfismos. Foram selecionados 70 pacientes com DE pós-prostatectomia (DEPP) e 70 pacientes com DE clínica (DEC). A função erétil dos voluntários foi avaliada através do questionário Índice Internacional de Função Erétil (IIEF). Para avaliação da resposta ao sildenafil, foram calculadas a diferença entre as pontuações pré e pós-tratamento (?IIEF) e a percentagem atingida da máxima resposta possível (?IIEF%) de cada paciente. Também, os pacientes de cada grupo foram divididos em bons e maus respondedores ao sildenafil de acordo com a mediana dos valores de ?IIEF%. Os genótipos dos rs1554597, rs805304 e rs805305 foram obtidos pela técnica de reação em cadeia da polimerase (PCR) seguida de digestão enzimática, e do rs18582 pela técnica de PCR alelo específica. O software PHASE 2.1 foi utilizado para estimar os haplótipos em cada grupo. Os resultados mostraram que o alelo variante A do rs18582 apresentou tendência para associação com piores respostas ao sildenafil no grupo DEC (P=0,058). No grupo DEPP, portadores dos alelos variantes A do rs805304 e G do rs805305 foram associados a melhores respostas ao sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; e score IIEF pós-tratamento, P=0,014). Não foram encontradas outras associações significativas. Estes resultados mostram que os marcadores genéticos rs805304 e rs805305 do DDAH2 podem influenciar as respostas ao sildenafil em pacientes com DE. / Erectile dysfunction (ED) is a disease related to deficient nitric oxide (NO) signaling. NO is produced from L-arginine by three isoforms of the enzyme nitric oxide synthase: neuronal (nNOS), endothelial (eNOS) and induced (iNOS). Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the three existing NOS subtypes, and is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 and 2 (DDAH1 and DDAH2) enzymes. Several studies have associated changes in genes, expression or activity of DDAH enzymes with disorders in which NO signaling is impaired. The aim of this study was to evaluate whether the response to ED treatment with sildenafil may be associated with polymorphisms of the DDAH1 (rs1554597 and rs18582) and DDAH2 genes (rs805304 and rs805305), as well as the haplotypes formed by these polymorphisms. We selected 70 patients with postprostatectomy ED (PPED) and 70 patients with clinical ED (CED). The erectile function of the volunteers was assessed using the International Index for Erectile Function (IIEF) questionnaire. To evaluate the response to sildenafil, the difference between the pre- and post-treatment scores (?IIEF) and the percentage reached from the maximum possible response (?IIEF%) of each patient were calculated. Also, patients from each group were divided into good and bad responders to sildenafil according to the median values of ?IIEF%. The genotypes of rs1554597, rs805304 and rs805305 were obtained by the polymerase chain reaction (PCR) technique followed by enzymatic digestion, and rs18582 by the allele-specific PCR technique. The PHASE 2.1 software was used to estimate the haplotypes in each group. The results showed that the variant A allele of rs18582 showed a tendency to be associated with a greater chance of worse responses to sildenafil in the DEC group (P=0,058). In the DEPP group, carriers of the variant alleles A of rs805304 and G of rs805305 were associated with better responses to sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; and post-treatment IIEF score, P=0,014). No other significant associations were found. These results show that the genetic markers rs805304 and rs805305 of DDAH2 may influence the responses to sildenafil in patients with ED.

A isoenzima citocromo P450 3A5 (CYP3A5) tem um papel importante na biotransformação de compostos endógenos e xenobióticos. Polimorfismos de nucleotídeo único (SNPs) do gene CYP3A5 foram associados com variações na expressão gênica e atividade enzimática que podem modificar o metabolismo de vários fármacos. Com a finalidade de investigar a influência de SNPs do CYP3A5 sobre a expressão de RNAm e a resposta a atorvastatina, foram selecionados 99 indivíduos normolipidêmicos (NL) e 139 hipercolesterolêmicos (HC). Os pacientes HC foram tratados com atorvastatina (10 mg/dia/4 semanas). Amostras de sangue periférico foram coletadas antes e após o tratamento para análise do perfil lipídico sérico, e extração de DNA genômico e RNA total. Os SNPs CYP3A5*3C e CYP3A5*6 foram analisados por PCR-RFLP e o CYP3A5*1D por sequenciamento de DNA. A expressão de RNAm do CYP3A5 em células mononucleares do sangue periférico (CMSP) foi analisada por PCR em tempo real. No grupo HC, os alelos CYP3A5*3C e CYP3A5*1D foram mais frequentes em brancos (*3C: 84,9% e *1D 84,8%) que em afro-descendentes (*3C: 47,8% e *1D: 55,2%, p<0,01). Os alelos *3C e *1D estavam em desequilíbrio de ligação. A frequência do alelo CYP3A5*6 foi maior em negros (6,8%) que em brancos (0,6%, p=0,002). Não houve associação entre os SNPs do CYP3A5 e o perfil lipídico basal nos grupos NL e HC. Os HC brancos portadores do haplótipo CYP3A5*3A/*3A (alelos *3C e *1D combinados) apresentaram menor redução de colesterol total e LDL-c após o tratamento com atorvastatina do que os portadores dos haplótipos não-CYP3A5*3A/*3A (p<0,05). A expressão de RNAm de CYP3A5 em CMSP aumentou em resposta a atorvastatina (p<0,05) somente nos portadores de haplótipo CYP3A5*3A/*3A. Esses resultados são sugestivos de que os SNPs CYP3A5*3C e CYP3A5*1D são associados com a expressão gênica do CYP3A5 e a resposta de redução de colesterol a atorvastatina. / The cytochrome P450 isoenzyme 3A5 (CYP3A5) has an important role on biotransformation of endogenous compounds and xenobiotics. Single nucleotide polymorphisms (SNPs) of the CYP3A5 gene have been associated with variations on gene expression and enzyme activity that can modify the metabolism of several drugs. In order to evaluate the influence of CYP3A5 SNPs on RNAm expression and response to atorvastatin, 99 mormolipidemic (NL) and 139 hypercholesterolemic (HC) individuals have been enrolled in this study. HC patients were treated with atorvastatin (10 mg/day/four weeks). Blood samples were collected before and after treatment for serum lipids analyses, genomic DNA and total RNA extraction. CYP3A5*3C, and CYP3A5*6 SNPs were analyzed by PCR-RFLP. CYP3A5*1D was analyzed by direct sequencing. CYP3A5 RNAm expression in peripheral blood mononuclear cells (PBMC) was evaluated by Real Time PCR. In HC group, the frequencies of CYP3A5*3C and CYP3A5*1D alleles were higher in white individuals (*3C: 84.9% and *1D 84.8%) that in blacks (*3C: 47.8% and *1D: 55.2%, p<0.01). CYP3A5*3C and *1D alleles were in linkage desequilibrium. CYP3A5*6 allelle frequency was higher in blacks (6.8%) that in white individuals (0.6%, p=0.002). There was no association between CYP3A5 SNPs and baseline lipid profile in NL and HC groups. White HC carriers of CYP3A5*3A/*3A haplotype (*3C and *1D combined alleles) have lower total cholesterol and LDL-c response to atorvastatin than non-CYP3A5*3A/*3A carriers (p<0.05). CYP3A5 RNAm expression on PBMC increased in response to atorvastatin (p<0.05) only in CYP3A5*3A/*3A haplotype carriers. These results are suggestive that CYP3A5*3C and CYP3A5*1D SNPs are associated with CYP3A5 gene expression and cholesterol-lowering response to atorvastatin.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2016-03-03T19:34:50Z No. of bitstreams: 2 Dissertação - Ayda Luz Malaver Slamanca - 2015.pdf: 2106945 bytes, checksum: dd89c61b0a1e8fd1299227d7513b4505 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2016-03-03T19:36:16Z (GMT) No. of bitstreams: 2 Dissertação - Ayda Luz Malaver Slamanca - 2015.pdf: 2106945 bytes, checksum: dd89c61b0a1e8fd1299227d7513b4505 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-03-03T19:36:16Z (GMT). No. of bitstreams: 2 Dissertação - Ayda Luz Malaver Slamanca - 2015.pdf: 2106945 bytes, checksum: dd89c61b0a1e8fd1299227d7513b4505 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-12-04 / Clozapine (CLZ) is the antipsychotic drug of choice in treatment refractory schizophrenia (TRS), however 30% of the patients with TRS don’t have full response to treatment with CLZ, these patients are considered to have super refractory schizophrenia (SRS). The response variability to treatment with CLZ may be associated with alterations of the CLZ plasma levels promoted by 1236C>T, 2677G>T/A e 3435C>T ABCB1 gene polymorphisms. ABCB1 is a transmembrane glycoprotein of transport, which export drugs from the intracellular to the extracellular space. ABCB1 polymorphisms cause structurally and functional protein changes that influence the intracellular CLZ levels and, consequently, promoting therapy failure. This work had the aim to establish the relation between the 1236C>T, 2677G>T/A e 3435C>T ABCB1 polymorphisms and the CLZ treatment response. This study included a total of 68 patients, 34 of whom were classified as TRS (CLZ responders) and 34 as SRS (CLZ non-responders). All patients were in use of CLZ for at least one year. For the genotype test was extracted genomic DNA, following for PCR and sequencing techniques. It was not observed any differences in allelic and genotype distribution between 1236C>T, 2677G>T/A and 3435C>T polymorphisms between RS and SRS groups. The polymorphisms not influenced the CLZ dose level, and percentage of BPRS change. In the same way, the results not showed relation between age, coffee intake, smoking behaviors and ethnicity and the CLZ treatment response, however, was evidenced a higher proportion of female patients in the SRS group when compared with the TRS group. In opposite, a lower proportion of male patients was observed in the SRS than the TRS group. Taken together, the results here obtained showed no association between 12361236C>T, 2677G>T/A e 3435C>T polymorphisms of ABCB1 gene and failure to CLZ treatment. / Clozapina (CLZ) é um antipsicótico de segunda geração, indicada no tratamento de esquizofrenia refratária (ER). Apesar de ser o fármaco de escolha nestes pacientes, 30% deles não respondem satisfatoriamente à terapia com CLZ, constituindo o grupo de indivíduos portadores de esquizofrenia super-refratária (ESR). A variabilidade na resposta ao tratamento com CLZ pode estar relacionada a alterações na farmacocinética da CLZ influenciadas pelos polimorfismos 1236C>T, 2677G>T/A e 3435C>T do gene ABCB1 . ABCB1 é uma proteína transportadora de membrana, que promove a passagem de fármacos do meio intra ao extracelular. Os polimorfismos de ABCB1 podem causar mudanças estruturais ou na expressão proteica, influenciando nas concentrações intracelulares de CLZ e contribuindo para o sucesso ou o fracasso da terapia. O presente trabalho teve como objetivo avaliar a associação entre os polimorfismos 1236C>T, 2677G>T/A e 3435C>T de ABCB1 com a resposta ao tratamento à CLZ. Um total de 68 pacientes em uso de CLZ, 34 portadores de ER e 34 de ESR, foram incluídos no estudo. Os testes de genotipagem foram realizados através da extração do DNA genômico, PCR e sequenciamento. Não foi observada diferença na distribuição alélica e genotípica dos polimorfismos de ABCB1, entre os grupos ER e ESR, assim mesmo não foi observada relação entre os polimorfismos estudados com a dosagem de CLZ e a gravidade psicopatológica avaliados pela escala BPRS. Fatores como consumo de café, cigarro, idade e etnia não influenciam na resposta ao tratamento com a CLZ. Foi evidenciada maior presença de pacientes do gênero feminino no grupo ESR em relação ao grupo ER e maior presença de indivíduos do gênero masculino no grupo ER quando comparado com o grupo ESR. O conjunto dos resultados aqui obtidos demonstram que não há associação entre os polimorfismos 1236C>T, 2677G>T/A e 3435C>T do gene ABCB1 com a refratariedade ao tratamento com CLZ.