Most administered drugs are metabolised in the liver by Phase I enzymes and more importantly by the cytochrome P450 (CYP) system. The extent of first-pass metabolism is important in determining whether the drug will have therapeutic or adverse effects after being administered to a patient. To date the CYP family has been shown to consist of 74 families denoted as CYPl to CYP118, and only a few families are significantly involved in drug metabolism. CYP3A4 is the most important isoenzyme followed by CYP2D6, CYP2C9, and CYP2C19 with a small contribution by CYP2E1, CYP2A6, and CYPlA4. CYP2D6 and CYP3A4 enzyme isoforms have been well established to exhibit interethnic and interindividual variability with regard to drug metabolising capacity. Mutation on the gene coding for a metabolising enzyme is a major cause of variation in drug metabolism. This mutation gives rise to allelic variants producing enzymes with altered metabolising activity. The presence of an allele with decreased metabolic activity in an individual gives rise to the poor metabolising (PM) phenotype. When the PM phenotype occurs at a frequency of more than 1% within a given population, then the term genetic polymorphism applies. The aberrant metabolic capacity translates into variable drug responses of more than 20-fold, leading to different susceptibility to sub-therapeutic effects or adverse drug reactions. A significant number of drugs, such as the B-adrenergic blockers, antidepressants, antipsychotic and antiarrhythmic agents, are entirely or partly metabolised by CYP2D6 and CYP3A4. Genetic polymorphism is especially important for drugs with a narrow therapeutic/toxicity window. Phenotyping involves the use of a probe drug that is administered to the subject, followed by determination of the parent drug and its metabolites in the urine. The aim of this study was to develop and validate an HPLC method for phenotypic determination of the CYP3A4 and CYP2D6 enzymes, followed by the application of the assay in a random heterogeneous population of males. Dextromethorphan (DXM) was used as an in vivo probe for simultaneous determination of the phenotypic expression of CYP2D6 and CYP3A4. An HPLC method coupled with a fluorescence detector was developed for the phenotypic determination of CYP2D6 and CYP3A4 iso-enzymes as determined by the concentration of dextromethorphan/dextrophan (DXM/DX) and dextromethorphan/3methoxy-morphinan (DXM/3MM) metabolic ratios respectively. The compounds were separated on a phenyl column (150 x 4,6 mm, 5-um particle size) serially connected to nitrile column (250 x 4,6 mm, 5-um particle size) using mobile phase of 80% (1.5% glacial acetic acid and 0.1% triethyl amine in distilled water) and 20% acetonitrile. Solid phase extraction was used to extract the analytes from urine samples using silica cartridges. The suitability of the method was demonstrated in a preliminary study with sixteen healthy Caucasian males. After a single oral 30 mg DXM dose, the volunteers were required to collect all urine samples voided 8 hours post oral dose. DXM/3HM and DXM/DX metabolic ratios were determined from collected urine samples. The method was validated for DXM and DX at a concentration range of 0.25 - 30 ug/ml, and at 0.025 - 3 ug/ml for 3MM. Calibration curves were linear with R2 values of at-least 0.999 for all compounds of interest. Recoveries were 97%, 93%, and 65% for DX, DXM and 3MM, respectively. The method was reproducible with intra-day precision having coefficients of variation percentage (CV%) of less than 17% for all analytes. Inter-day precision had a CV% of less than 14% for all analytes. The limit of detection was 30 ug/ml for all compounds. All volunteers were classified with an extensive metaboliser (EM) phenotype. In conclusion the method described is suitable for polymorphic determination of CYP2D6 and CYP3A4 in a population study, and may have value in further studies planned at investigating the critical issue of racial genetic polymorphism in ethnic groups in South Africa. / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

Heme oxygenases (HO) catalyze the degradation of heme into biliverdin, carbon monoxide (CO) and free iron. The two major isoforms, HO-2 (constitutive) and HO-1 (inducible by various stressors such as heavy metals and reactive oxygen species) are involved in a variety of physiological functions, including anti-inflammation, antiapoptosis, neuromodulation, and vascular regulation. Major tools used in exploring these actions have been metalloporphyrin analogs of heme that inhibit the HOs. However, these tools are limited by their lack of selectivity; they affect other heme-dependent enzymes, such as cytochromes P450 (CYPs), soluble guanylyl cyclase (sGC), and nitric oxide synthase (NOS). Our laboratory has been able to successfully synthesize a series of small molecule non-porphyrin HO inhibitors (QC-xx) that have had little or no effect against sGC and NOS; however, their effects on various CYP isoforms has yet to be fully elucidated. In order to determine the effects on CYP enzyme activity, microsomal preparations of two CYP isoforms (2E1 and 3A1/3A2) were incubated with varying concentrations of HO inhibitor and the activity was determined via spectrophotometric analysis. Results indicated that some QC compounds demonstrated little to no inhibition of CYP2E1 and/or CYP3A1/2, while some others did inhibit these CYP isoforms. Four regions of interest were analyzed further and several structural changes were identified as conferring increased HO inhibition and decreased effect on both CYP2E1 and 3A1/2. Based on the information obtained, three putative compounds were designed and it is hypothesized that these compounds will be selective inhibitors for HO-1 over HO-2 and will display little effect on either CYP2E1 or 3A1/2 activities. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-11-20 11:19:48.841

Most administered drugs are metabolised in the liver by Phase I enzymes and more importantly by the cytochrome P450 (CYP) system. The extent of first-pass metabolism is important in determining whether the drug will have therapeutic or adverse effects after being administered to a patient. To date the CYP family has been shown to consist of 74 families denoted as CYPl to CYP118, and only a few families are significantly involved in drug metabolism. CYP3A4 is the most important isoenzyme followed by CYP2D6, CYP2C9, and CYP2C19 with a small contribution by CYP2E1, CYP2A6, and CYPlA4. CYP2D6 and CYP3A4 enzyme isoforms have been well established to exhibit interethnic and interindividual variability with regard to drug metabolising capacity. Mutation on the gene coding for a metabolising enzyme is a major cause of variation in drug metabolism. This mutation gives rise to allelic variants producing enzymes with altered metabolising activity. The presence of an allele with decreased metabolic activity in an individual gives rise to the poor metabolising (PM) phenotype. When the PM phenotype occurs at a frequency of more than 1% within a given population, then the term genetic polymorphism applies. The aberrant metabolic capacity translates into variable drug responses of more than 20-fold, leading to different susceptibility to sub-therapeutic effects or adverse drug reactions. A significant number of drugs, such as the B-adrenergic blockers, antidepressants, antipsychotic and antiarrhythmic agents, are entirely or partly metabolised by CYP2D6 and CYP3A4. Genetic polymorphism is especially important for drugs with a narrow therapeutic/toxicity window. Phenotyping involves the use of a probe drug that is administered to the subject, followed by determination of the parent drug and its metabolites in the urine. The aim of this study was to develop and validate an HPLC method for phenotypic determination of the CYP3A4 and CYP2D6 enzymes, followed by the application of the assay in a random heterogeneous population of males. Dextromethorphan (DXM) was used as an in vivo probe for simultaneous determination of the phenotypic expression of CYP2D6 and CYP3A4. An HPLC method coupled with a fluorescence detector was developed for the phenotypic determination of CYP2D6 and CYP3A4 iso-enzymes as determined by the concentration of dextromethorphan/dextrophan (DXM/DX) and dextromethorphan/3methoxy-morphinan (DXM/3MM) metabolic ratios respectively. The compounds were separated on a phenyl column (150 x 4,6 mm, 5-um particle size) serially connected to nitrile column (250 x 4,6 mm, 5-um particle size) using mobile phase of 80% (1.5% glacial acetic acid and 0.1% triethyl amine in distilled water) and 20% acetonitrile. Solid phase extraction was used to extract the analytes from urine samples using silica cartridges. The suitability of the method was demonstrated in a preliminary study with sixteen healthy Caucasian males. After a single oral 30 mg DXM dose, the volunteers were required to collect all urine samples voided 8 hours post oral dose. DXM/3HM and DXM/DX metabolic ratios were determined from collected urine samples. The method was validated for DXM and DX at a concentration range of 0.25 - 30 ug/ml, and at 0.025 - 3 ug/ml for 3MM. Calibration curves were linear with R2 values of at-least 0.999 for all compounds of interest. Recoveries were 97%, 93%, and 65% for DX, DXM and 3MM, respectively. The method was reproducible with intra-day precision having coefficients of variation percentage (CV%) of less than 17% for all analytes. Inter-day precision had a CV% of less than 14% for all analytes. The limit of detection was 30 ug/ml for all compounds. All volunteers were classified with an extensive metaboliser (EM) phenotype. In conclusion the method described is suitable for polymorphic determination of CYP2D6 and CYP3A4 in a population study, and may have value in further studies planned at investigating the critical issue of racial genetic polymorphism in ethnic groups in South Africa. / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

Notre objectif était d’identifier des cytochromes P450 capables d’oxygéner des mono et sesquiterpènes, pour produire des molécules aux propriétés organoleptiques intéressantes labellisés « naturelles » par l’industrie des arômes et du parfum.Nous avons identifié 7 couples P450-substrat catalysant une conversion in vitro supérieure ≥ 45 % et/ou formant un produit attendu. Les quantités de produit obtenu par bioconversion dans la levure restent insuffisantes pour un procédé industriel. Les facteurs limitants ont été identifiés dans le cas du valencène comme : 1) la toxicité induite par les produits, 2) l’accumulation du β-nootkatol dans les membranes, 3) l’inhibition de l’enzyme par les produits réactionnels. Trois cytochromes P450 d’Arabidopsis thaliana impliqués dans le métabolisme indolique oxydent activement le limonène. Ils s’expriment dans les inflorescences et constituent le premier exemple de P450s suceptibles de participer à deux voies métaboliques indépendantes chez les plantes. / Our aim was to identify cytochromes P450 catalyzing hydroxylation of mono-and sesquiterpenes to produce functionalized "natural" compounds with interesting organoleptic properties for the flavor and fragrance industry. We identified 7 P450-substrate pairs showing . 45 % in vitro conversion and/or forming an expected product. The amounts of products resulting from yeast bioconversion were however too low for implementation of an industrial process. Factors limiting the nootkatone production from the P450-dependent bioconversion of valencene were identified : 1) toxicity for yeast of the ƒÀ-nootkatol and nootkatone products, 2) ƒÀ-nootkatol accumulation in endomembranes, 3) products inhibition of valencene hydroxylation. Three previously characterized P450s from Arabidopsis thaliana in indolic metabolism were shown to actively oxidize limonene. They are expressed in inflorescences and may provide the first demonstrated case of multifunctional P450s involved in independent plant pathways.

Os gliomas representam o tipo de tumor primário mais comum do sistema nervoso central. Apesar dos avanços nas técnicas cirúrgicas e de radioterapia e nos protocolos de quimioterapia, não há tratamento eficiente disponível. O ácido retinóico na forma all-trans (atRA) é um agente anti-proliferativo utilizado para o tratamento de alguns tipos de lesões pré-malígnas e tumores, como a leucemia promielocítica. Entretanto, sua utilização no tratamento clínico de tumores sólidos, incluindo os gliomas, é limitada, devido ao rápido desenvolvimento de resistência aos efeitos anti-tumorais do atRA. Para endereçar este problema, foi proposto: a) clonar e analisar a função de cyp26b1 de rato, uma enzima da família dos citocromos P450 envolvida na metabolização de ácido retinóico, previamente descrito em nosso laboratório como potencialmente envolvido na resposta de células de glioma ao tratamento com atRA; b) gerar e caracterizar uma variante da linhagem celular C6 de glioma de rato resistente aos efeitos anti proliferativos de atRA. O cDNA de cyp26b1 de rato, até então desconhecido, foi amplificado, clonado e seqüenciado com sucesso. A seqüência protéica correspondente é conservada e agrupa-se no dendrograma com outras proteínas Cyp26B1, mesmo de organismos distantes como zebrafish, em detrimento de outras proteínas parálogas da família Cyp26. Cyp26b1 é fortemente induzida por atRA em células de glioma de rato e humano, de forma dose-dependente. Embora seja expresso em amostras de cérebros humanos normais e de glioma, não foram encontradas diferenças significativas entre os diferentes graus de matlignidade tumoral, ou em comparação com cérebro normal. A super-expressão de cyp26b1 de rato através de transfecção estável de células de glioma de rato e humano, bem como de células P19 de teratocarcinoma de camundongo, não alterou significativamente as características de crescimento celular, tanto em substrato sólido quanto em substrato semi-sólido. O tratamento prolongado de células C6 de glioma de rato com atRA levou à obtenção de uma população policlonal resistente aos efeitos anti-proliferativos de atRA, a partir da qual uma linhagem celular clonal resistente foi isolada com sucesso. Esta variante clonal, denominada linhagem celular C6R, apresenta inibição de crescimento por atRA diminuída, quando comparada com a linhagem celular C6 parental e com a variante ST1. Além disso, esta variante também mostrou uma tendência, embora não significativa estatisticamente, de gerar tumores maiores quando injetados no subcutâneo de camundongos do tipo nude. A expressão de genes previamente descritos como induzidos pelo tratamento com atRA em células ST1 é fortemente inibida na linhagem celular C6R. Além disso, menor expressão de RARβ, RARγ e CRABP-1 foi também observada em células C6R, enquanto que expressão muito maior de RXRγ e CRABP-2 foi encontrada nas células resistentes a atRA, em comparação com as células C6 parentais. Como conclusões gerais deste trabalho, foi proposto que cyp26b1 parece estar envolvido no metabolismo e detoxificação de atRA e parece não ser efetor da inibição de crescimento induzida por atRA, nem estar relacionado com a resistência de células de glioma ao tratamento com atRA. Por outro lado, o isolamento e caracterização de uma linhagem celular resistente ao tratamento com atRA sugere que a resistência está relacionada à expressão diferencial de receptores de retinóides e de proteínas de ligação ao ácido retinóico. / Gliomas are among the most common primary tumors of the central nervous system. Despite the advances in surgical and radiotherapy procedures and chemotherapy protocols, efficient treatment is still not available. Retinoic acid is a anti-proliferative agent used for treatment of a number of pre-malignant lesions and tumors, as promyelocytic leukemia. However, its clinical use in treatment of solid tumors, including gliomas, is impaired by the rapid development of resistance to the anti-tumoral effects of atRA. In order to address this problem, we proposed: a) to clone and analyze the role of rat cyp26b1, a member of cytochrome P450 superfamily envolved in the metabolization of retinoic acid, which has previously been described by our group as being potentially involved in the response of glioma cells to retinoic acid treatment; b) to generate and characterize a variant of rat C6 glioma cell line resistant to anti proliferative effects of atRA. The previously unknown cDNA of rat cyp26b1 was successfully amplified, cloned and sequenced. The protein is conserved and clustered in dendrograms with other cyp26b1 proteins, even from distant organisms as zebrafish, in detriment of other cyp26 paralogs. Cyp26b1 is strongly induced by atRA in rat and human glioma cells, in a dose-dependent fashion. Although expressed in human normal brains and glioma samples, no significant differences were found among different tumor grades nor comparing to normal brain. Stable-transfection and overexpression of rat cyp26b1 in rat and human glioma cell lines, as well as P19 mouse teratocarcinoma cell line, did not significantly modified cell growth properties, either on solid or semi-solid substrates. Prolonged treatment of C6 rat glioma cell line with atRA leads to isolation of an atRA-resistant polyclonal cell population, from which a resistant clonal cell line was successfully isolated. This clonal variant, named C6R cell line, displayed diminished growth inhibition by atRA compared to the parental C6 cell line and the variant ST1 cell line. In addition, this variant also showed a trend, although not quite statistically significant, to generate larger tumors when xenografted s.c. into nude mice. Expression of genes previously described as being induced by atRA-treatment in ST1 cells is strongly impaired in the C6R resistant cell line. In addition, lower expression of RARβ, RAR&#947 and CRABP-1 was also observed in C6R cells, whereas a much higher expression of RXRγ and CRABP-2 was found in the resistant cells when compared to the parental C6 cells. As general conclusions of this work, we found that cyp26b1 is more likely to be involved in primary atRA metabolism and detoxification and does not seem to be an effector of atRA-induced cell growth arrest nor to be related to the resistance of glioma cells to atRA treatment. On the other hand, isolation and characterization of an atRA-resistant cell line suggests that atRA resistance is more likely to be due to differential expression of retinoid receptors and retinoic acid binding proteins.

Studies in humans and laboratory animals have shown that inflammation and infectious processes are associated with changes in expression and activity of cytochrome P450 (CYP), which are responsible for the metabolism of most drugs in clinical use. In this context, the objectives of this work were: observe pharmacokinetic changes of CYP450 during human visceral leishmaniasis, describe the epidemiological, clinical and laboratory characteristics of patients with visceral leishmaniasis and evaluate the activity of the isoenzymes CYP3A4, CYP2C9 and CYP2C19 in patients with visceral leishmaniasis before and after treatment. This is a cross-sectional study, developed into an inpatient unit of a school hospital linked to the Federal University of Sergipe, reference in the diagnosis and treatment of visceral leishmaniasis in the state, which involved 24 patients. The study was conducted in four phases: before treatment, immediately after treatment, ninety and one hundred and eighty days after the end of treatment. In each phase, patients ingested the medication omeprazole (CYP2C19 substrate), losartan (CYP2C9 substrate) and midazolam (CYP3A4 substrate), which were used as evidence of the enzymes for which are substrates. Collecting blood samples (5 mL) of the patients before the administration of these drugs and 15, 30, 45 minutes and 1, 2, 3, 4, 5 and 6 hours after administration of those drugs. It was also collected all urine produced in 8 hours after the ingestion of drugs and separate a aliquot of 20 mL for analysis. Liquid chromatography coupled to mass spectrometry high efficiency (LC-MS) was used for the quantification of drugs and metabolites in the samples. The reason omeprazol/5-hidróxi-omeprazol plasma concentrations obtained 4 h after drug administration was used to evaluate the activity of CYP2C19, and the CYP2C9 enzyme activity was estimated by the ratio of concentrations losartano/E-3174 in urine collected 8h after administration of the drug. To estimate the activity of CYP3A4 was calculated the apparent clearance (CL / F) of midazolam. The results showed a prevalence of the disease in males and that most cases occurred in patients residing in urban areas. In relation to the clinical findings were present weight loss, fever, malaise and hepatomegaly in 100% of participants, and 50% were diagnosed between 90 and 180 days the onset of symptoms. On laboratory changes, anemia, leukopenia, thrombocytopenia, hypoalbuminemia, normal or slightly altered transaminases were found. As the enzymatic activity, the apparent clearance of midazolam found himself reduced during illness and increased after treatment; the metabolic ratio of omeprazole plasma concentration of 5-hydroxy-omeprazole and the ratio of the concentration urine losartano/E-3174 were increased during illness, and reduced after treatment, indicating a reduction in the activity of CYP3A4, 2C9 and 2C19 during LV and increase after treatment. The knowledge that occur changes on the activity of the cytochromes P450 and, consequently, on the pharmacokinetics of drugs during visceral leishmaniasis, may contribute to better understanding of the response to drugs during the disease. / Estudos realizados em seres humanos e animais de laboratório têm demonstrado que a inflamação e os processos infecciosos estão associados a alterações na expressão e na atividade dos citocromos P450 (CYP), os quais são responsáveis pelo metabolismo da maioria dos fármacos de uso clínico. Nesse contexto, os objetivos deste trabalho foram: observar as alterações farmacocinéticas dos CYP450 durante a LV humana, descrever as características epidemiológicas, clínicas e laboratoriais dos pacientes com leishmaniose visceral e avaliar a atividade das isoenzimas CYP3A4, CYP2C9 e CYP2C19 em pacientes com leishmaniose visceral antes e após o tratamento. Trata-se de um estudo transversal, desenvolvido em uma unidade de internamento de um hospital escola vinculado à Universidade Federal de Sergipe, referência no diagnóstico e tratamento da leishmaniose visceral no Estado, do qual participaram 24 pacientes. O estudo foi realizado em quatro fases: antes do início do tratamento, imediatamente após o término do tratamento, noventa e cento e oitenta dias após o término do tratamento. Em cada fase, os pacientes ingeriram os medicamentos omeprazol (substrato do CYP2C19), losartano (substrato do CYP2C9) e midazolam (substrato do CYP3A4), os quais foram usados como prova da atividade das enzimas para as quais são substratos. Foi realizada coleta de amostras de sangue (5 mL) dos pacientes antes da administração desses medicamentos, e 15, 30, 45 minutos e 1, 2, 3, 4, 5 e 6 horas após a administração dessas drogas. Também foi coletada toda a urina produzida nas 8h seguintes à ingestão dos medicamentos e separada uma alíquota de 20 mL para análise. A cromatografia líquida de alta eficiência acoplada à espectrometria de massas (LC-MS-MS) foi utilizada para quantificação dos medicamentos e seus metabólitos nas amostras coletadas. A razão das concentrações plasmáticas omeprazol/5-hidróxi-omeprazol obtidas 4h após a administração do fármaco foi usada para avaliar a atividade do CYP2C19, e a atividade da enzima CYP2C9 foi estimada pela razão das concentrações losartano/E-3174 na urina coletada nas 8h seguintes à administração do fármaco. Para estimar a atividade do CYP3A4 foi calculado o clearence aparente (CL/F) do midazolam. Os resultados mostraram que houve predomínio da doença no sexo masculino e que a maioria dos casos ocorreu em pacientes residentes na zona urbana. Em relação ao quadro clínico, estiveram presentes emagrecimento, febre, hepatomegalia e mal estar em 100% dos participantes, e 50% deles foram diagnosticados entre 90 e 180 dias do início dos sintomas. Sobre as alterações laboratoriais, foram encontradas anemia, leucopenia, plaquetopenia, hipoalbuminemia, transaminases normais ou levemente alteradas. Quanto à atividade enzimática, o clearence aparente do midazolam encontrava-se reduzido durante a doença e aumentado após o tratamento; a razão metabólica da concentração plasmática de omeprazol-5-hidróxi-omeprazol e a razão da concentração losartano/E-3174 na urina encontravam-se aumentadas durante a doença e reduzidas após o tratamento, indicando uma redução da atividade dos CYP3A4, 2C9 e 2C19 durante a LV e um aumento após o tratamento. O conhecimento de que ocorrem alterações na atividade dos citocromos P450 e, consequentemente, na farmacocinética das drogas durante a LV, pode contribuir para um melhor entendimento da resposta aos medicamentos durante a doença.

Made available in DSpace on 2015-04-11T13:54:26Z (GMT). No. of bitstreams: 1 Jaquelane.pdf: 1247761 bytes, checksum: c3396b2f5aa59cae9b817bf74a866e28 (MD5) Previous issue date: 2013-08-30 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / High prevalence in Brazil, specifically in the Amazon Region, malária is a parasitic infectious disease with socioeconomic impact sin endemic areas. Plasmodium vivax is the most prevalent agent and has been associated with cases of severe malária before attributed only to Plasmodium falciparum. Genetic variability in host metabolism of antimalárial drugs, influenced by the polymorphism of cytochrome P450(CYP), could lead to significant changes in antimalárial treatment response. Little is known about the influence of genetic factors in the increase of the individual susceptibility of the host to development of severe disease. Therefore, this project aimed to determine the frequency of alleles CYP2B6*6, CYP2C8*3 and CYP2D6*4 in patients with vivax malária, to evaluate the relationship between the alleles found and the occurrence of severe malária and further establish the hematological and biochemical profile among carriers of alleles found. Therefore, we used peripheral blood samples of patients with vivax malária treated at a health care reference in Manaus-AM. Which were extracted leukocyte DNA, which in turn were amplified by real-time PCR using TaqMan ® system for allelic discrimination. The frequencies of the alleles CYP2B6*6, CYP2C8*3 and CYP2D6*4 were 28.26%, 6.7%, 8.87%, respectively. The CYP2D6*4 allele was more prevalent among patients diagnosed with severe vivax malária, and was associated with normal levels of reticulocytes, erythrocytes and hematocrit. Future studies are needed to understand the clinical implications of the polymorphisms found in patients with vivax malária. / De elevada prevalência no Brasil, especificamente na Região Amazônica, a malária é uma doença infecto-parasitária com impactos socioeconômicos em áreas endêmicas. O Plasmodium vivax é o agente de maior prevalência e tem sido associado a casos graves de malária antes atribuída apenas ao Plasmodium falciparum. A variabilidade genética do hospedeiro no metabolismo de fármacos antimaláricos, influenciada pelo polimorfismo de isoformas do citocromo P450 (CYP), poderia levar a alterações significativas na resposta terapêutica antimalárica. Pouco se conhece sobre a influência dos fatores genéticos no aumento da susceptibilidade individual do hospedeiro ao desenvolvimento de formas graves da doença. Portanto este projeto objetivou determinar a frequência dos alelos CYP2B6*6, CYP2C8*3 e CYP2D6*4 em pacientes com malária vivax, avaliar a relação entre os alelos encontrados e a ocorrência de malária grave e ainda estabelecer o perfil hematológico e bioquímico entre os portadores dos alelos encontrados. Para tanto foram utilizadas amostras de sangue periférico de pacientes com malária vivax atendidos numa unidade de saúde de referência na cidade de Manaus-AM. Dos quais foram extraídos o DNA leucocitário, que por sua vez foram amplificado por PCR em tempo real usando o sistema TaqMan® para a discriminação alélica. As freqüências dos alelos CYP2B6*6, CYP2C8*3 e CYP2D6*4 foram 28,26%, 6,7%, 8,87%, respectivamente. O alelo CYP2D6*4 foi o mais prevalente entre os pacientes diagnosticados com malária vivax grave, e também foi associado a níveis normais de reticulócitos, eritrócitos e hematócrito. Estudos futuros são necessários para compreender as implicações clínicas dos polimorfismos encontrados em pacientes com malária vivax.

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-06T18:25:28Z No. of bitstreams: 2 Dissertação - Mariana Côrtes de Sousa - 2012.pdf: 1542160 bytes, checksum: 797a42ca723c95277c56015ce09b4be1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-06T18:30:44Z (GMT) No. of bitstreams: 2 Dissertação - Mariana Côrtes de Sousa - 2012.pdf: 1542160 bytes, checksum: 797a42ca723c95277c56015ce09b4be1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-06T18:30:44Z (GMT). No. of bitstreams: 2 Dissertação - Mariana Côrtes de Sousa - 2012.pdf: 1542160 bytes, checksum: 797a42ca723c95277c56015ce09b4be1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-02-28 / Flavonoids are an important class of natural products candidate drugs, and low molecular weight polyphenols, widely distributed throughout the plant kingdom. Investigations on its activities over the past 30 years, demonstrated a potential to prevent several diseases, among them cardiovascular diseases, inflammatory disorders, viral infections, diabetes and neurological disorders, in addition to its known antioxidant. The family of cytochrome P450 (CYP) is composed of monooxygenases, which play a crucial role in the metabolism of endogenous and exogenous substances, and participates in the metabolism of flavonoids. In this paper we describe the application of a methodology for exploring combined in silico prediction of sites of metabolism of quercetin, rutin, naringin and naringenin, found in abundance in nature. A methodology used was ligand based drug design (LBDD) to predict the sites of metabolism (SOM) and the program MetaPrint2D most likely estimate of the metabolites, combined with the method structure based drug design (SBDD) by using molecular docking and energy minimization, to predict the interaction of quercetin, rutin, naringin and naringenin with the isoforms CYP2C9 and CYP1A2. Metabolites were found several Phase I with catalytically active distance (<5 Å) and interaction sites described in the literature, with hydroxylation reactions of aliphatic, aromatic hydroxylation, dealkylation and O-dealkylation. The proposed in silico metabolic hydroxylation at the position corresponding to the C3' were consistent with studies in vitro and in vivo experiments described in the literature for naringin and naringenin. Amino acids of the active site of CYP isoforms have been identified as important in the positioning of the flavonoids quercetin, rutin, naringin and naringenin toward the heme, confirming the involvement of these isoforms in the metabolism of flavonoids. / Os flavonóides representam uma importante classe de produtos naturais candidatos à fármacos, sendo polifenóis de baixo peso molecular, amplamente distribuídos no reino vegetal. Investigações sobre suas atividades, nos últimos 30 anos, demonstraram uma prevenção potencial de diversas patologias, dentre elas doenças cardiovasculares, desordens inflamatórias, infecções virais, diabetes e desordens neurológicas, além de sua conhecida ação antioxidante. A família do citocromo P450 (CYP) é composta de monooxigenases, que desempenham um papel crucial no metabolismo de substâncias endógenas e exógenas, e participa do metabolismo de flavonóides. Neste trabalho, descrevemos a aplicação de uma metodologia in silico combinada para explorar a previsão dos sítios de metabolismo dos flavonóides quercetina, rutina, naringenina e naringina, encontrados em abundância na natureza. Utilizou-se uma metodologia baseada nos ligantes (LBDD) para previsão dos sítios de metabolismo (SOM) pelo programa MetaPrint2D e previsão dos metabólitos mais prováveis, combinado à metodologia baseada na estrutura do receptor (SBDD) através da utilização de docking molecular e minimização de energia, para prever a interação de quercetina, rutina, naringenina e naringina com as isoformas CYP2C9 e CYP1A2. Foram encontrados diversos metabólitos de Fase I, com distâncias cataliticamente ativas (< 5 Å) e sítios de interação descritos na literatura, apresentando reações de hidroxilação alifática, hidroxilação aromática, desalquilação e O-desalquilação. Os metabólitos propostos in silico correspondentes à hidroxilação na posição C3’ foram com coerentes com estudos in vitro e in vivo descritos na literatura para naringenina e naringina. Aminoácidos do sítio ativo das isoformas de CYP foram identificados como importantes no posicionamento dos flavonóides quercetina, rutina, naringenina e naringina em direção ao heme, confirmando a participação dessas isoformas no metabolismo de flavonóides.

A síntese dos hormônios esteroides é feita através da ação de um complexo enzimático chamado citocromo P450. As enzimas 3&#946;-HSD (3&#946;-hidroxiesteroide desidrogenase), P450c17 (citocromo P450 17&#945;-hidroxilase/17,20-liase) e P450arom (citocromo P450 aromatase), fazem parte desse complexo e são responsáveis pela síntese dos hormônios progestágenos, andrógenos e estrógenos, espectivamente. Essas enzimas já foram identificadas em inúmeros tecidos, inclusive em embriões em período pré-implantação. Por isso, acredita-se que o blastocisto seja uma fonte de produção de progesterona e estrógeno, hormônios essenciais para implantação e manutenção da gestação. Em roedores, as informações sobre a capacidade esteroidogênica dos embriões são controversas e também restritas apenas a estudos com espécies laboratoriais. Para trazer mais informações sobre a capacidade esteroidogênica em blastocistos de roedores utilizamos o preá (Galea spixii), uma espécie de roedor silvestre encontrada nas regiões de Caatinga e Semiárido do Nordeste brasileiro e muito utilizado na alimentação de famílias de baixa renda como fonte proteica. Diante o exposto, o objetivo deste trabalho é fazer um apanhado histórico sobre os dados publicados a cerca da capacidade dos embriões de diversas espécies de produzir os hormônios esteroides além de identificar e quantificar a expressão das enzimas esteroidogênicas em embriões de preá através das técnicas de imunohistoquímica e PCR em tempo real. As análises morfológicas indicam que o desenvolvimento inicial do preá se assemelha com os demais roedores, porém o tempo de desenvolvimento difere das espécies usadas em laboratório. As marcações da imunohistoquímica foram positivas nos tecidos maternos, mas os embriões não tiveram marcação para nenhuma das três enzimas do estudo. No entanto, os dados obtidos pelo PCR indicam que os genes CYP11, CYP17 e CYP19 foram expressos nos embriões e, portanto, o concepto de G. spixii tem capacidade de produzir hormônios esteroides, assim como visto em várias outras espécies que já foram estudadas / The synthesis of steroid hormones is made through the action of an enzyme complex called cytochrome P450. 3&#946;-HSD (3&#946;-hydroxysteroid dehydrogenase), P450c17 (cytochrome P450 17 &#945;-hydroxylase/17,20-lyase) and P450arom (cytochrome P450 aromatase), are present in this complex and are are responsible for the synthesis of progestins, androgens and estrogens hormones, respectively. These enzymes have been identified in numerous tissues, including embryos in pre-implantation period. Therefore, it is believed that the blastocyst is a source of production of progesterone and estrogen, hormones essential for the implementation and maintenance of pregnancy. In rodents, the information about the steroidogenic capacity of embryos are controversial and confined to studies with laboratory species. To get more information about the steroidogenic capacity in blastocysts of rodents we used the spix yellow-toothed cavy (Galea spixii), a species of wild rodent found in the northeastern of Brazil, and used in the feeding of low-income families as a source of protein. On the above, the aim of this work is to make a historic about the data published about the ability of embryos of various species to produce steroid hormones and identify and quantify the expression of steroidogenics enzymes in embryos of spix yellow-toothed cavy by immunohistochemical and real-time PCR techniques. The morphological analyses indicate that the G. spixii early development resembles other rodents, but development time differs from the species used in laboratory. The immunohistochemical stainning was positive in maternal tissues, but the embryos did not have stainnig for any of the three enzymes of the study. However, the data obtained by PCR indicate that CYP11, CYP17 and CYP19 genes were expressed in embryos and, therefore, the concept of G. spixii has capacity to produce steroid hormones, as seen in several other species that have been studied

Mixed-function oxygenase systém (MFO systém) plays a vital role in the metabolism of a variety of both endogenous substrates and xenobiotics. This membrane systém consists of cytochrome P450s, NADPH:cytochrome P450 oxidoreductase (POR), cytochrome b5 and NADH:cytochrome b5 oxidoreductase (b5R). Cytochrome P450 catalyzes a monooxygenation of a substrate, while POR and cytochrome b5 represent its redox partners. Cytochrome b5, itself having a redox partner in b5R, effects the reactions catalyzed by the MFO system in various ways, through mechanisms that are not fully understood. This paper focuses on the purification of b5R and POR from rabbit liver. The microsomal fraction obtained by differential centrifugation contained 42 mg of protein per ml. From a portion of the microsomal fraction, b5R was obtained using chromatography on DEAE-Sepharose, CM-Sepharose and 5'-ADP agarose columns. The yield was 0,3 % of ferricynide-reductase activity and the product contained several contaminants in the molecular weight range of 50-70 kDa. A second purification of b5R from the microsomal fraction was carried out using a column of DEAE-Sepharose directly connected to a 5'-ADP agarose column. The b5R product was purified with a yield of 10,9 % and it once again contained several contaminants in the molecular...