A varfarina é o anticoagulante oral mais prescrito no mundo todo. Algoritmos farmacogenéticos têm sido desenvolvidos para estimar a dose de varfarina. Os principais objetivos deste estudo foram desenvolver um algoritmo farmacogenético estimador de dose de varfarina e comparar o algoritmo desenvolvido neste trabalho com algoritmos disponíveis na literatura. Para atingir os objetivos foram incluídos dois grupos de pacientes tratados com varfarina (primeira coorte, n = 832; e segunda coorte, n = 133). Foram realizadas as genotipagens dos polimorfismos CYP2C9*2, CYP2C9*3 e VKORC1 (c.G1639A). A derivação do algoritmo foi realizada utilizando os dados dos pacientes da primeira coorte com dose estável (n=368) e foi replicado utilizando os dados dos pacientes provenientes da segunda coorte (n=133). Como resultado o algoritmo desenvolvido neste trabalho alcançou um coeficiente de determinação de 40%, incluindo as variáveis: idade, sexo, peso, altura, raça autodeclarada, uso de amiodarona, uso de indutores enzimáticos, os genótipos na VKORC1 (c.G1639A) e os fenótipos de acordo com polimorfismos CYP2C9. Os dados sugerem que o nosso algoritmo desenvolvido é mais acurado do que o algoritmo IWPC (The International Warfarin Pharmacogenetics Consortium) quando a aplicação é focada em pacientes brasileiros. Os algoritmos farmacogenéticos estimadores de dose de varfarina desenvolvidos para uma população específica podem ser mais efetivos para a terapia com varfarina em comparação com o uso de algoritmos estimadores de dose atualmente disponíveis / Warfarin is the most prescribed oral anticoagulant in the world. Pharmacogenetic algorithms have been developed to estimate the dose of warfarin. The main aims of the present study were to develop a pharmacogenetic-based warfarin dosing algorithm and compare algorithm developed in this study with others algorithms available in the literature. We included two patient cohorts treated with warfarin (first cohort, n = 832; and second cohort, n = 133). Polymorphisms were genotyped in the CYP2C9 * 2, CYP2C9 * 3 and VKORC1 (c.G1639A). The derivation of the algorithm was performed using the data from the patients in the first cohort with a stable dose (n = 368) and was replicated using data from patients from the second cohort (n = 133). Our algorithm achieved a determination of coefficient of 40%, including variables: age, sex, weight, height, self-declared race, use of amiodarone, use of enzymatic inducers, genotypes in VKORC1 (c.G1639A) and phenotypes according to CYP2C9 polymorphisms. Our data suggest that the developed algorithm is more accurate than the IWPC algorithm when the application is focused on patients from the Brazilian population. Pharmacogenetics- based warfarin dosing algorithms developed for a specific population may lead to improved performance compared use dosing algorithms currently available

Grace S.N. Lau. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 335-362). / List of Abbreviations --- p.i / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction and Study Objectives / Chapter 1.1 --- Metabolic activation - role in drug toxicity and carcinogenesis --- p.5 / Chapter 1.2 --- Hepatocellular carcinoma --- p.12 / Chapter 1.2.1 --- Epidemiology --- p.12 / Chapter 1.2.2 --- Aetiological factors --- p.17 / Chapter 1.2.2.1 --- Hepatitis B virus infection --- p.17 / Chapter 1.2.2.2 --- Cirrhosis --- p.24 / Chapter 1.2.2.3 --- Aflatoxins --- p.25 / Chapter 1.2.2.4 --- Other factors --- p.26 / Chapter 1.2.2.5 --- Summary --- p.29 / Chapter 1.3 --- Study objectives --- p.30 / Chapter Chapter 2 --- The Metabolism of Paracetamol in Healthy Subjects andin Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.1.1. --- History of paracetamol --- p.34 / Chapter 2.1.2 --- Pharmacology of paracetamol --- p.37 / Chapter 2.1.3 --- "Absorption, Distribution, Metabolism and Excretion" --- p.38 / Chapter 2.1.3.1 --- Absorption --- p.38 / Chapter 2.1.3.2 --- Distribution --- p.41 / Chapter 2.1.3.3 --- Metabolism --- p.42 / Chapter 2.1.3.4 --- Excretion --- p.57 / Chapter 2.1.4 --- Toxicity and Overdosage --- p.59 / Chapter 2.2 --- Estimation of paracetamol and its metabolites in plasma and urine by high performance liquid chromatography --- p.72 / Chapter 2.2.1 --- Introduction --- p.72 / Chapter 2.2.2 --- Analytical method --- p.76 / Chapter 2.2.2.1 --- Materials --- p.76 / Chapter 2.2.2.2 --- Instrumentation --- p.77 / Chapter 2.2.2.3 --- Collection and storage of samples --- p.79 / Chapter 2.2.2.4 --- Chromatographic conditions --- p.79 / Chapter 2.2.3 --- Urine assay --- p.79 / Chapter 2.2.3.1 --- Preparation of standards and test samples for urine assay --- p.79 / Chapter 2.2.3.2 --- Calculation of results for urine assay --- p.80 / Chapter 2.2.3.3 --- Results of urine assay --- p.81 / Chapter 2.2.3.4 --- Validation of urine assay --- p.81 / Chapter 2.2.4 --- Plasma assay --- p.83 / Chapter 2.2.4.1 --- Preparation of standards and test samples for plasma assay --- p.83 / Chapter 2.2.4.2 --- Calculation of results for plasma assay --- p.91 / Chapter 2.2.4.3 --- Results of plasma assay --- p.91 / Chapter 2.2.4.4 --- Validation of plasma assay --- p.93 / Chapter 2.2.5 --- Summary --- p.99 / Chapter 2.3 --- The pharmacokinetics of paracetamol in healthy subjects --- p.103 / Chapter 2.3.1 --- Introduction --- p.103 / Chapter 2.3.2 --- Study protocol --- p.103 / Chapter 2.3.3 --- Methods --- p.103 / Chapter 2.3.3.1 --- Subjects --- p.103 / Chapter 2.3.3.2 --- Drug administration and sampling --- p.104 / Chapter 2.3.3.3 --- Drug analysis --- p.108 / Chapter 2.3.3.4 --- Calculations --- p.108 / Chapter 2.3.4 --- Pharmacokinetic analysis --- p.109 / Chapter 2.3.5 --- Statistical analysis --- p.113 / Chapter 2.3.6 --- Results --- p.114 / Chapter 2.3.6.1 --- Plasma Results --- p.114 / Chapter 2.3.6.2 --- Urine Results --- p.118 / Chapter 2.3.6.3 --- Pharmacokinetic Results --- p.125 / Chapter 2.3.6.4 --- Statistical Results --- p.134 / Chapter 2.3.7 --- Discussion --- p.145 / Chapter 2.4 --- "The pharmacokinetics of paracetamol in healthy subjects, patients with liver disease and hepatocellular carcinoma" --- p.155 / Chapter 2.4.1 --- Introduction --- p.155 / Chapter 2.4.2 --- Study protocol --- p.156 / Chapter 2.4.3 --- Methods --- p.156 / Chapter 2.4.3.1 --- Subjects --- p.156 / Chapter 2.4.3.2 --- Drug administration and sampling --- p.157 / Chapter 2.4.3.3 --- Drug analysis --- p.160 / Chapter 2.4.3.4 --- Calculations --- p.160 / Chapter 2.4.4 --- Pharmacokinetic analysis --- p.161 / Chapter 2.4.6 --- Results --- p.162 / Chapter 2.4.6.1 --- Plasma Results --- p.162 / Chapter 2.4.6.2 --- Urine Results --- p.162 / Chapter 2.4.6.3 --- Pharmacokinetic Results --- p.179 / Chapter 2.4.7 --- Discussion --- p.194 / Chapter 2.4.8 --- Summary --- p.203 / Chapter Chapter 3 --- Metabolic Activation of Aflatoxin B1 in Healthy Subjects and in Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 3.1 --- General introduction --- p.206 / Chapter 3.1.1 --- Chemical structures and properties --- p.207 / Chapter 3.1.2 --- Contamination of food by aflatoxins --- p.209 / Chapter 3.1.3 --- Metabolism of aflatoxins --- p.210 / Chapter 3.1.4 --- Human diseases possibly related to exposure to aflatoxins --- p.226 / Chapter 3.1.4.1 --- Acute aflatoxicosis --- p.226 / Chapter 3.1.4.2 --- Reye's syndrome --- p.227 / Chapter 3.1.4.3 --- Kwashiorkor --- p.228 / Chapter 3.1.4.4 --- Impaired immune function --- p.229 / Chapter 3.1.4.5 --- Hepatocellular carcinoma --- p.230 / Chapter 3.1.5 --- Biochemical and molecular epidemiology of aflatoxins --- p.232 / Chapter 3.2 --- Development of an ELISA method to monitor AFB1 exposure in human serum --- p.237 / Chapter 3.2.1 --- Introduction --- p.237 / Chapter 3.2.2 --- Preparation of all the components necessary for analysing AFB1-albumin adducts by ELISA --- p.243 / Chapter 3.2.2.1 --- Materials --- p.243 / Chapter 3.2.2.2 --- Preparation of rabbit AFB1 antiserum --- p.244 / Chapter 3.2.2.3 --- Preparation of the rat monoclonal antibody --- p.244 / Chapter 3.2.2.4 --- Concentration of cell culture supernatant by ammonium sulphate precipitation --- p.246 / Chapter 3.2.2.5 --- Preparation of the BSA-AFB1 conjugate --- p.248 / Chapter 3.2.2.6 --- Preparation of the immunoaffinity gel --- p.250 / Chapter 3.2.2.7 --- Preparation of the ELISA plates --- p.251 / Chapter 3.2.3 --- General procedures used in the analysis of AFB1- albumin adducts --- p.252 / Chapter 3.2.3.1 --- Competitive ELISA binding assay --- p.253 / Chapter 3.2.3.2 --- Sep-pak C18 cartridge --- p.254 / Chapter 3.2.3.3 --- Immunoaffinity column --- p.255 / Chapter 3.2.3.4 --- Evaporation process --- p.255 / Chapter 3.2.3.5 --- HPLC --- p.256 / Chapter 3.2.3.6 --- Radioactive counting --- p.256 / Chapter 3.2.3.7 --- Albumin isolation --- p.257 / Chapter 3.2.3.8 --- Digestion of albumin --- p.257 / Chapter 3.2.3.9 --- Animal procedures --- p.258 / Chapter 3.2.4 --- Validations --- p.259 / Chapter 3.2.4.1 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.259 / Chapter 3 2.4.2 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.259 / Chapter 3 2.4.3 --- Elution characteristics and capacity of the immunoaffinity column --- p.261 / Chapter 3.2.4.4 --- Comparison of immunoaffinity gels prepared with different affinity gels --- p.261 / Chapter 3.2.4.5 --- Immunoaffinity column experiment of AFB1-lysine --- p.263 / Chapter 3.2.4.6 --- HPLC Analysis of fractions from immunoaffinity column --- p.263 / Chapter 3.2.4.8 --- HPLC analysis of fractions from Sep- Pak C18 cartridge --- p.264 / Chapter 3.2.4.9 --- Digestion of serum albumin by proteinase K --- p.264 / Chapter 3.2.4.10 --- Effect of ethanol in samples to be loaded onto Sep-Pak C18 cartridge --- p.265 / Chapter 3.2.4.11 --- Effect of drying in a vacuum concentrator on recovery of radioactivity of 3H-AFB1 --- p.266 / Chapter 3.2.4.12 --- Evaluation of the overall procedure for the analysis of serum albumin adducts of AFB1 --- p.267 / Chapter 3.2.4.13 --- HPLC analysis of samples obtained after digestion and all clean-up procedures --- p.268 / Chapter 3.2.5 --- Results and discussion --- p.268 / Chapter 3.2.5.1 --- BSA-AFB1 conjugate --- p.268 / Chapter 3.2.5.2 --- Treatment of experimental animals with 3H-AFB1 --- p.270 / Chapter 3.2.5.3 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.272 / Chapter 3.2.5.4 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.275 / Chapter 3.2.5.5 --- Sep-Pak C18 cartridge - elution characteristics and capacity --- p.279 / Chapter 3.2.5.6 --- Elution characteristics of immunoaffinity columns --- p.282 / Chapter 3.2.5.7 --- Immunoaffinity column experiment of AFB1-lysine --- p.290 / Chapter 3.2.5.8 --- Digestion of serum albumin by proteinase K --- p.295 / Chapter 3.2.5.9 --- Effect of ethanol in samples to be applied onto Sep-Pak C18 cartridges --- p.297 / Chapter 3.2.5.10 --- Recovery of radioactivity after dryingin a vacuum concentrator --- p.300 / Chapter 3.2.5.11 --- Recovery of the overall clean-up procedure for the analysis of serum albumin adducts of AFB1 --- p.300 / Chapter 3.2.5.12 --- HPLC analysis of samples obtained after all clean-up procedures --- p.305 / Chapter 3.2.5.13 --- The use of rabbit anti-AFB1 anti-serum and rat anti-AFB1 monoclonal antibody --- p.308 / Chapter 3.2.6 --- Summary --- p.309 / Chapter 3.3 --- Monitoring of AFBralbumin adducts in plasma of patients with liver disease and hepatocellular carcinoma --- p.311 / Chapter 3.3.1 --- Introduction --- p.311 / Chapter 3.3.2 --- Material and methods --- p.314 / Chapter 3.3.2.1 --- Subject --- p.314 / Chapter 3.3.2.2 --- Sample collections --- p.315 / Chapter 3.3.2.4 --- Assay for AFB1-albumin adducts --- p.315 / Chapter 3.3.2.5 --- Statistical analysis --- p.318 / Chapter 3.3.3 --- Results and discussion --- p.318 / Chapter Chapter 4 --- Summary and Ideas for Further Studies --- p.330 / Acknowledgements --- p.333 / References --- p.335 / Appendices --- p.364

A heme oxigenase (HEOX) é uma enzima que converte o anel heme em quantidades equimolares de biliverdina, monóxido de carbono (CO) e Fe3+. Esta enzima tem um importante papel fisiológico, regulando os níveis de hemeproteínas e protegendo as células da agressão oxidativa do heme livre. A biliverdina é, subseqüentemente, transformada em bilirrubina que apresenta propriedades antioxidativas e o Fe3+ é seqüestrado pela ferritina. O CO ativa a guanilil ciclase, com resultante produção de 3?,5?-monofosfato de guanosina cíclico provocando relaxamento da musculatura lisa vascular. Em trabalho anterior, nós verificamos o efeito da inibição aguda da heme oxigenase com zinco protoporfirina (ZnPP IX) sobre a pressão arterial de ratos Wistar machos recebendo dietas hipossódica (0,15% de NaCl), normossódica (1,3% de NaCl) ou hipersódica (8% de NaCl) desde o desmame. Nos animais sob dieta hipersódica houve diminuição nos níveis pressóricos, enquanto que nos animais que recebiam dieta hipo e normossódica, ocorreu um aumento dos níveis pressóricos. A análise destes resultados mostrou a existência de uma correlação negativa entre a pressão arterial basal medida antes da injeção de ZnPP IX e o efeito deste tratamento sobre os níveis pressóricos. Esta correlação independeu do conteúdo de sal na dieta consumida pelos animais. Concluímos assim, que a resposta à inibição da HEOX é modulada pela pressão arterial basal. Recentemente, foi mostrado que a heme oxigenase é induzida por incrementos da pressão obtidos de maneiras diversas e que ao retornar os níveis pressóricos a valores basais esta indução da enzima é revertida. A estimulação da HEOX promove consumo de anel heme. O heme integra a estrutura molecular de enzimas importantes na regulação da pressão arterial, como por exemplo, guanilato ciclase, óxido nítrico sintase, hidroxilase e epóxigenase (enzimas pertencentes à família do citocromo P-450). Permitimo-nos conjecturar que a falta de heme repercute sobre a atuação destas enzimas, uma vez que o turnover destas enzimas é dependente de heme. O objetivo deste estudo foi confirmar em outros modelos de hipertensão arterial os achados anteriores e verificar se a enzima epoxigenase que metaboliza o ácido araquidônico formando compostos vasodilatadores, tais como EET e DiHTE, está envolvida na queda da pressão arterial dos animais sob dieta hipersódica. Neste estudo foi induzida hipertensão arterial crônica com angiotensina II (AII - 0,7 mg.kg-1.d-1, via minibomba osmótica durante 7 dias) ou com L?-nitro L-arginina metil ester (L-NAME ? 50 mg/L fornecido na água de beber ao longo de duas semanas) e hipertensão arterial aguda com fenilefrina (FE - 1 mg.kg-1.h-1 iv ). Hidralazina (15 mg.kg-1.dia-1 fornecido na água de beber durante 9 dias) e nitroprussiato de sódio (SNP - 7,7?g.kg-1.min-1 iv) foram utilizados para induzir diminuição crônica e aguda, respectivamente, da pressão arterial. A participação da via do citocromo P-450 no mecanismo de queda pressórica em resposta ao ZnPP IX nos animais sob dieta hipersódica (descrita no trabalho anterior), foi avaliada através da inibição desta via com clotrimazole (80 mg/kg/24 horas) em animais na vigência de sobrecarga de sal ou consumindo dieta normossódica. Grupos de ratos submetidos ao tratamento com AII, L-NAME, FE, SNP, hidralazina e clotrimazole foram submetidos ao teste de inibição da HEOX. A pressão arterial direta (artéria carótida) foi avaliada antes e após a administração de zinco protoporfirina IX (ZnPP IX ? 90 ?mol/kg ip) ou veículo (carbonato de sódio ? 0,15mmol/kg ip). Nós verificamos nos modelos de hipertensão arterial crônicos ou no modelo agudo um resultado similar ao encontrado no estudo anterior, ou seja, em resposta à inibição da heme oxigenase houve uma queda importante da pressão arterial. Nos animais submetidos a hipotensão arterial não houve modificação da pressão arterial em resposta à administração de ZnPP IX. Nos grupos de animais submetidos ao tratamento com clotrimazole não houve modificação da pressão arterial após administração de ZnPP IX. Em conclusão, pressão basal elevada modifica o efeito da inibição da heme oxigenase sobre os níveis pressóricos em comparação com pressão arterial basal normal ou reduzida. A via do citocromo P-450 parece estar envolvida na queda pressórica após inibição da heme oxigenase nos animais submetidos a tratamento crônico com dieta hipersódica, uma vez que nos animais tratados com clotrimazole não houve modificação pressórica após a inibição da heme oxigenase. / Heme oxygenase (HEOX) is the rate-limiting enzyme that opens the heme ring, resulting in equimolar quantities of biliverdin, carbon monoxide (CO) and Fe3+. HEOX plays an important physiological role in the degradation of heme, regulating hemoprotein levels and therefore protecting cells from the deleterious effects of free heme. Biliverdin is subsequently reduced by biliverdin reductase to bilirubin that has antioxidant properties. Iron is sequestered by ferritin. CO activates soluble guanylate cyclase with resultant production of 3?,5?-cyclic guanosine monophosphate (cGMP) which produces relaxation of the vascular smooth muscle cells. Previously we demonstrated the effect of acute HEOX inhibition by zinc protoporphyrin IX (ZnPP IX) on blood pressure in male Wistar rats that were fed with low (LSD ? 0.15% NaCl), normal (NSD ? 1.3%) or high salt diet (HSD - 8% NaCl) from weaning (21 days-old animals). The blood pressure decreased in rats on HSD, and increased in the animals on NSD and LSD after ZnPP IX administration. A negative correlation was obtained between blood pressure measured before ZnPP IX and the area under the curve of the percentage of blood pressure changes induced by ZnPP IX. This correlation seems to be independent of the salt content in the diet. Our conclusion is that the response to heme oxygenase inhibition is modulated by basal arterial blood pressure. Recently it was showed that heme oxygenase is induced by high blood pressure and the levels of the enzyme returns to normal with blood pressure control. It is known that heme oxygenase stimulation provokes breakdown of heme ring. The heme integrates the molecular structure of several important enzymes that are involved in the regulation of blood pressure, such as, soluble guanylate cyclase, nitric oxide synthase, hydroxylase and epoxygenase (cytochrome P-450 family). We postulate that the deficiency in heme rings due to heme oxigenase induction might lead to disturbances in the activities of some of these enzymes and hence hypertension. The objective of this study was 1) to confirm the results observed previously in others models f hypertension and 2) to the role of epoxygenase in the regulation of blood pressure in response to HEOX inhibition in hypertensive animals. The chronic hypertension was induced by angiotensin II (AII - 0.7 mg.kg-1.d-1, by osmotic mini pumps during 7 days) or with L?-nitro L-arginine metyl ester (L-NAME ? 50 mg/L in the tap water was given during two weeks ) and acute hypertension with phenylephrine (FE - 1 mg.kg-1.h-1 iv ). Hidralazine (15 mg.kg-1.d-1 in the tap water was given during 9 days) and sodium nitropruside (SNP - 7.7?g.kg-1.min-1 iv) were used, respectively, to induce chronic and acute decreases of the blood pressure. The participation of the cytochrome P-450 pathway in the mechanism of blood pressure reduction in response to ZnPP IX was evaluated through the inhibition of this pathway by clorimazole (80 mg/kg/24 hours) in animals on high salt diet or nornal salt diet. Groups of rats treated with AII, L-NAME, FE, SNP, hidralazine and clotrimazole were submitted to HEOX inhibition. Direct blood pressure (carotid artery) measurements were undertaken before and after zinc protoprphyrin IX administration (ZnPP IX ? 90 ?mol/kg ip) or vehicle (sodium carbonate ? 0,15mmol/kg ip). In the chronic or acute hypertension models heme oxygenase inhibition induced a decrease of blood pressure. In the hypotension group there was no modification on blood pressure of these animals in response to ZnPP IX. Animals on high salt diet submitted to the clotrimazole treatment did not modify the blood pressure after ZnPP IX administration. Thus; the cytochrome P-450 pathway seems to be involved in the blood pressure decreases after HEOX inhibition in the animals under long term of high salt diet since there was no change in blood pressure in the clotrimazole-treated groups after heme oxygenase inhibition.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-09-13T17:47:07Z No. of bitstreams: 1 MauricioFernandes.pdf: 1547228 bytes, checksum: 87d9e63be597d8eaf8f7a783479e1294 (MD5) / Made available in DSpace on 2017-09-13T17:47:07Z (GMT). No. of bitstreams: 1 MauricioFernandes.pdf: 1547228 bytes, checksum: 87d9e63be597d8eaf8f7a783479e1294 (MD5) Previous issue date: 2017-04-28 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão / INTRODUCTION: In Brazil, it is reported that a great part of the patients who arrive for the primary care service have as main complaint: sadness (depression) and / or anxiety, also with more complex disorders such as problems related to abuse of alcohol and serious and persistent mental disorders, such as schizophrenia and affective psychoses (bipolar mood disorder). In the pharmacogenetic studies of antidepressants and antipsychotics, cytochrome P450 (CYPs) enzymes have been shown to be one of the most significant targets in the interindividual changes in drug response kinetic parameters. OBJECTIVE: To characterize the frequency of CYP2D6 polymorphisms (*4, *6 and *17) in users of psychotropic therapy, especially tricyclic antidepressants and antipsychotics. METHODS: Cross - sectional, descriptive study carried out in the city of São Luis – MA. RESULTS: From a total of 105 charts, a sample of 43 patients was collected. The mean age was 40.98 (± 11.04) years, of which 24 (55.81%) were male and 19 (44, 19%) of females. Regarding the pharmacotherapy, all participants (100%) were on daily use of the antipsychotic haloperidol, seventeen (39.53%) patients used risperidone and one (2.32%) patient used amitriptyline (tricyclic antidepressant), all three drugs are substrates of the enzyme CYP2D6. For the polymorphism of CYP2D6, we had a prevalence of 11 (25.48%) for allele 4, and all the patients presented with this polymorphism were heterozygous, that means they had one polymorphic allele and one normal allele. For allele 17, we had a prevalence of 4 (9.3%), also all patients being heterozygous for this allele. Allele 6 did not appear in any patient in our study. We also had two patients who presented the polymorphisms for alleles * 4 and * 17 at the same time. CONCLUSION: This study was able to characterize the frequency of CYP2D6 (* 4, * 6 and 17 *) polymorphisms in users of psychotropic therapy, including tricyclic antidepressants and antipsychotics, in addition to contributing to a higher dose / response do medication, ensuring greater safety and efficacy in the use of medicines, improving the quality of life of patients. / INTRODUÇÃO: No Brasil, tem-se o relato de que grande parte dos pacientes que chegam para o atendimento na atenção primária apresentam, como principal queixa, tristeza (depressão) e/ou ansiedade, aparecendo também transtornos mais complexos como os problemas relacionados ao abuso de álcool, assim como os transtornos mentais graves e persistentes, como a esquizofrenia e as psicoses afetivas (transtorno bipolar do humor). Nos estudos farmacogenéticos de antidepressivos e antipsicóticos, as enzimas do citrocromo P450 (CYPs) tem se mostrado um dos alvos mais significativos nas alterações interindividuais dos parâmetros cinéticos de resposta às drogas. OBJETIVO: caracterizar a frequência de polimorfismos do CYP2D6 (*4, *6 e 17*) em usuários da terapêutica com psicotrópicos, principalmente antidepressivos tricíclicos e antipsicóticos. MÉTODOS: Estudo transversal, descritivo realizado no Município de São Luís (MA). RESULTADO: De um total de 105 prontuários, coletou-se amostra de 43 pacientes, a média de idade foi de 40,98 (±11,04) anos, sendo 24 (55,81%) do sexo masculino e 19 (44,19%) do sexo feminino. Em relação à farmacoterapia, todos os participantes (100%) estavam em consumo diário do antipsicótico haloperidol, dezessete (39,53%) pacientes faziam uso de risperidona e um (2,32%) paciente utilizava amitriptilina (antidepressivo tricíclico), todos os três medicamentos são substratos da enzima CYP2D6. Para o polimorfismo da CYP2D6, tivemos para o alelo 4 uma prevalência de 11 (25,48%), sendo que todos os pacientes que apresentaram esse polimorfismo são heterozigotos, ou seja, apresentaram um alelo polimórfico e outro normal. Para o alelo 17, tivemos uma prevalência de 4 (9,3%), sendo também todos os pacientes heterozigotos para esse alelo. O alelo 6 não apareceu em nenhum paciente do nosso estudo. Tivemos também dois pacientes que apresentaram os polimorfismos para os alelos *4 e *17 ao mesmo tempo. CONCLUSÃO: Este estudo pôde caracterizar a frequência de polimorfismos do CYP2D6 (*4, *6 e 17*) em usuários da terapêutica com psicotrópicos, incluindo antidepressivos tricíclicos e antipsicóticos, além de contribuir para uma maior adequação da relação dose/resposta ao medicamento, garantindo maior segurança e eficácia quanto ao uso de medicamentos, melhorando a qualidade de vida dos pacientes.

Alkane monooxygenases (encoded by the alkB gene) are a group of microbial enzymes that catalyze the first reaction of alkane degradation. Studies to determine the diversity and prevalence of alkB genotypes in the environment have focused on culturable organisms. The goal of this research was to use culture-independent methods (DGGE, clone library) to identify and characterize alkB genes, and to determine their prevalence in Arctic contaminated soils. General alkB PCR degenerate primers (alkB-Mc) were designed using the conserved nucleotide sequences of the Histidine I Box and Histidine III Box. General alkB-Mc and alkM (Acinetobacter spp. alkane monooxygenase genes) primers were used to screen the soils for the presence of alkane monooxygenase genotypes. A predominance of the Rhodococcus spp. alkB genotypes and the absence of alkM genotypes in these soils was found. alkB PCR fragments amplified from the soils were analyzed by DGGE (Denaturing Gradient Gel Electrophoresis). BlastN and blastX results of the DGGE bands sequences showed that they were similar to Rhodococcus spp. alkB genotypes (~80-90% DNA identity and ~80-90% amino acid homology). An alkB clone library was built using the general alkB-Mc primer set, screened by RFLP (Restriction Fragment Length Polymorphism) and characterized by sequencing of alkB clones. BlastN and blastX results of the alkB clone sequences showed the presence of divergent alkB genotypes (≤ 70% DNA identity and ≤ 67% of amino acid homology to data base sequences). The alignment of the clone-derived amino acid sequences to confirm functional alkane monooxygenase sequences revealed the presence of Histidine Box II and the HYG motif in all of the deduced amino acid clone sequences. These results indicate that the alkB sequences from the clone library represent novel alkB sequences. Both alkB DGGE and clone library techniques were independently able to identify alkB genotypes from High G+C microorganisms as predominant in the 1A03 soil sample. Nevertheless, only the clone library approach identified putative novel alkB sequences. Mineralization of hexadecane and naphthalene was clearly observed at subzero temperatures (-5ºC) in Arctic contaminated soils, proving that the indigenous microbial communities could mineralize these representative hydrocarbons at subzero temperatures in an environment that is predominantly frozen for most of the year.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 83-100).

A varfarina é o anticoagulante oral mais prescrito no mundo todo. Algoritmos farmacogenéticos têm sido desenvolvidos para estimar a dose de varfarina. Os principais objetivos deste estudo foram desenvolver um algoritmo farmacogenético estimador de dose de varfarina e comparar o algoritmo desenvolvido neste trabalho com algoritmos disponíveis na literatura. Para atingir os objetivos foram incluídos dois grupos de pacientes tratados com varfarina (primeira coorte, n = 832; e segunda coorte, n = 133). Foram realizadas as genotipagens dos polimorfismos CYP2C9*2, CYP2C9*3 e VKORC1 (c.G1639A). A derivação do algoritmo foi realizada utilizando os dados dos pacientes da primeira coorte com dose estável (n=368) e foi replicado utilizando os dados dos pacientes provenientes da segunda coorte (n=133). Como resultado o algoritmo desenvolvido neste trabalho alcançou um coeficiente de determinação de 40%, incluindo as variáveis: idade, sexo, peso, altura, raça autodeclarada, uso de amiodarona, uso de indutores enzimáticos, os genótipos na VKORC1 (c.G1639A) e os fenótipos de acordo com polimorfismos CYP2C9. Os dados sugerem que o nosso algoritmo desenvolvido é mais acurado do que o algoritmo IWPC (The International Warfarin Pharmacogenetics Consortium) quando a aplicação é focada em pacientes brasileiros. Os algoritmos farmacogenéticos estimadores de dose de varfarina desenvolvidos para uma população específica podem ser mais efetivos para a terapia com varfarina em comparação com o uso de algoritmos estimadores de dose atualmente disponíveis / Warfarin is the most prescribed oral anticoagulant in the world. Pharmacogenetic algorithms have been developed to estimate the dose of warfarin. The main aims of the present study were to develop a pharmacogenetic-based warfarin dosing algorithm and compare algorithm developed in this study with others algorithms available in the literature. We included two patient cohorts treated with warfarin (first cohort, n = 832; and second cohort, n = 133). Polymorphisms were genotyped in the CYP2C9 * 2, CYP2C9 * 3 and VKORC1 (c.G1639A). The derivation of the algorithm was performed using the data from the patients in the first cohort with a stable dose (n = 368) and was replicated using data from patients from the second cohort (n = 133). Our algorithm achieved a determination of coefficient of 40%, including variables: age, sex, weight, height, self-declared race, use of amiodarone, use of enzymatic inducers, genotypes in VKORC1 (c.G1639A) and phenotypes according to CYP2C9 polymorphisms. Our data suggest that the developed algorithm is more accurate than the IWPC algorithm when the application is focused on patients from the Brazilian population. Pharmacogenetics- based warfarin dosing algorithms developed for a specific population may lead to improved performance compared use dosing algorithms currently available

A heme oxigenase (HEOX) é uma enzima que converte o anel heme em quantidades equimolares de biliverdina, monóxido de carbono (CO) e Fe3+. Esta enzima tem um importante papel fisiológico, regulando os níveis de hemeproteínas e protegendo as células da agressão oxidativa do heme livre. A biliverdina é, subseqüentemente, transformada em bilirrubina que apresenta propriedades antioxidativas e o Fe3+ é seqüestrado pela ferritina. O CO ativa a guanilil ciclase, com resultante produção de 3?,5?-monofosfato de guanosina cíclico provocando relaxamento da musculatura lisa vascular. Em trabalho anterior, nós verificamos o efeito da inibição aguda da heme oxigenase com zinco protoporfirina (ZnPP IX) sobre a pressão arterial de ratos Wistar machos recebendo dietas hipossódica (0,15% de NaCl), normossódica (1,3% de NaCl) ou hipersódica (8% de NaCl) desde o desmame. Nos animais sob dieta hipersódica houve diminuição nos níveis pressóricos, enquanto que nos animais que recebiam dieta hipo e normossódica, ocorreu um aumento dos níveis pressóricos. A análise destes resultados mostrou a existência de uma correlação negativa entre a pressão arterial basal medida antes da injeção de ZnPP IX e o efeito deste tratamento sobre os níveis pressóricos. Esta correlação independeu do conteúdo de sal na dieta consumida pelos animais. Concluímos assim, que a resposta à inibição da HEOX é modulada pela pressão arterial basal. Recentemente, foi mostrado que a heme oxigenase é induzida por incrementos da pressão obtidos de maneiras diversas e que ao retornar os níveis pressóricos a valores basais esta indução da enzima é revertida. A estimulação da HEOX promove consumo de anel heme. O heme integra a estrutura molecular de enzimas importantes na regulação da pressão arterial, como por exemplo, guanilato ciclase, óxido nítrico sintase, hidroxilase e epóxigenase (enzimas pertencentes à família do citocromo P-450). Permitimo-nos conjecturar que a falta de heme repercute sobre a atuação destas enzimas, uma vez que o turnover destas enzimas é dependente de heme. O objetivo deste estudo foi confirmar em outros modelos de hipertensão arterial os achados anteriores e verificar se a enzima epoxigenase que metaboliza o ácido araquidônico formando compostos vasodilatadores, tais como EET e DiHTE, está envolvida na queda da pressão arterial dos animais sob dieta hipersódica. Neste estudo foi induzida hipertensão arterial crônica com angiotensina II (AII - 0,7 mg.kg-1.d-1, via minibomba osmótica durante 7 dias) ou com L?-nitro L-arginina metil ester (L-NAME ? 50 mg/L fornecido na água de beber ao longo de duas semanas) e hipertensão arterial aguda com fenilefrina (FE - 1 mg.kg-1.h-1 iv ). Hidralazina (15 mg.kg-1.dia-1 fornecido na água de beber durante 9 dias) e nitroprussiato de sódio (SNP - 7,7?g.kg-1.min-1 iv) foram utilizados para induzir diminuição crônica e aguda, respectivamente, da pressão arterial. A participação da via do citocromo P-450 no mecanismo de queda pressórica em resposta ao ZnPP IX nos animais sob dieta hipersódica (descrita no trabalho anterior), foi avaliada através da inibição desta via com clotrimazole (80 mg/kg/24 horas) em animais na vigência de sobrecarga de sal ou consumindo dieta normossódica. Grupos de ratos submetidos ao tratamento com AII, L-NAME, FE, SNP, hidralazina e clotrimazole foram submetidos ao teste de inibição da HEOX. A pressão arterial direta (artéria carótida) foi avaliada antes e após a administração de zinco protoporfirina IX (ZnPP IX ? 90 ?mol/kg ip) ou veículo (carbonato de sódio ? 0,15mmol/kg ip). Nós verificamos nos modelos de hipertensão arterial crônicos ou no modelo agudo um resultado similar ao encontrado no estudo anterior, ou seja, em resposta à inibição da heme oxigenase houve uma queda importante da pressão arterial. Nos animais submetidos a hipotensão arterial não houve modificação da pressão arterial em resposta à administração de ZnPP IX. Nos grupos de animais submetidos ao tratamento com clotrimazole não houve modificação da pressão arterial após administração de ZnPP IX. Em conclusão, pressão basal elevada modifica o efeito da inibição da heme oxigenase sobre os níveis pressóricos em comparação com pressão arterial basal normal ou reduzida. A via do citocromo P-450 parece estar envolvida na queda pressórica após inibição da heme oxigenase nos animais submetidos a tratamento crônico com dieta hipersódica, uma vez que nos animais tratados com clotrimazole não houve modificação pressórica após a inibição da heme oxigenase. / Heme oxygenase (HEOX) is the rate-limiting enzyme that opens the heme ring, resulting in equimolar quantities of biliverdin, carbon monoxide (CO) and Fe3+. HEOX plays an important physiological role in the degradation of heme, regulating hemoprotein levels and therefore protecting cells from the deleterious effects of free heme. Biliverdin is subsequently reduced by biliverdin reductase to bilirubin that has antioxidant properties. Iron is sequestered by ferritin. CO activates soluble guanylate cyclase with resultant production of 3?,5?-cyclic guanosine monophosphate (cGMP) which produces relaxation of the vascular smooth muscle cells. Previously we demonstrated the effect of acute HEOX inhibition by zinc protoporphyrin IX (ZnPP IX) on blood pressure in male Wistar rats that were fed with low (LSD ? 0.15% NaCl), normal (NSD ? 1.3%) or high salt diet (HSD - 8% NaCl) from weaning (21 days-old animals). The blood pressure decreased in rats on HSD, and increased in the animals on NSD and LSD after ZnPP IX administration. A negative correlation was obtained between blood pressure measured before ZnPP IX and the area under the curve of the percentage of blood pressure changes induced by ZnPP IX. This correlation seems to be independent of the salt content in the diet. Our conclusion is that the response to heme oxygenase inhibition is modulated by basal arterial blood pressure. Recently it was showed that heme oxygenase is induced by high blood pressure and the levels of the enzyme returns to normal with blood pressure control. It is known that heme oxygenase stimulation provokes breakdown of heme ring. The heme integrates the molecular structure of several important enzymes that are involved in the regulation of blood pressure, such as, soluble guanylate cyclase, nitric oxide synthase, hydroxylase and epoxygenase (cytochrome P-450 family). We postulate that the deficiency in heme rings due to heme oxigenase induction might lead to disturbances in the activities of some of these enzymes and hence hypertension. The objective of this study was 1) to confirm the results observed previously in others models f hypertension and 2) to the role of epoxygenase in the regulation of blood pressure in response to HEOX inhibition in hypertensive animals. The chronic hypertension was induced by angiotensin II (AII - 0.7 mg.kg-1.d-1, by osmotic mini pumps during 7 days) or with L?-nitro L-arginine metyl ester (L-NAME ? 50 mg/L in the tap water was given during two weeks ) and acute hypertension with phenylephrine (FE - 1 mg.kg-1.h-1 iv ). Hidralazine (15 mg.kg-1.d-1 in the tap water was given during 9 days) and sodium nitropruside (SNP - 7.7?g.kg-1.min-1 iv) were used, respectively, to induce chronic and acute decreases of the blood pressure. The participation of the cytochrome P-450 pathway in the mechanism of blood pressure reduction in response to ZnPP IX was evaluated through the inhibition of this pathway by clorimazole (80 mg/kg/24 hours) in animals on high salt diet or nornal salt diet. Groups of rats treated with AII, L-NAME, FE, SNP, hidralazine and clotrimazole were submitted to HEOX inhibition. Direct blood pressure (carotid artery) measurements were undertaken before and after zinc protoprphyrin IX administration (ZnPP IX ? 90 ?mol/kg ip) or vehicle (sodium carbonate ? 0,15mmol/kg ip). In the chronic or acute hypertension models heme oxygenase inhibition induced a decrease of blood pressure. In the hypotension group there was no modification on blood pressure of these animals in response to ZnPP IX. Animals on high salt diet submitted to the clotrimazole treatment did not modify the blood pressure after ZnPP IX administration. Thus; the cytochrome P-450 pathway seems to be involved in the blood pressure decreases after HEOX inhibition in the animals under long term of high salt diet since there was no change in blood pressure in the clotrimazole-treated groups after heme oxygenase inhibition.

Verificou-se que microssomos de fígado apresentam uma atividade azo-redutásica completamente dependente de NADPH, usando DAB como substrato. Ao contrário de outros sistemas já descritos, a atividade azo-redutásica não pode ser identificada com a NADPH-citocromo c redutase, embora nossos resultados indiquem que esta enzima participa da reação. Não se pode excluir definitivamente a participação do citocromo b5 apesar de uma série de observações afastando essa possibilidade. Verificou-se que a atividade azo-redutásica pode ser induzida por tratamento com 3-MC, paralelamente à indução do citocromo P-450 tipo II. Isto sugere fortemente a participação desse citocromo na reação apesar desta não ser inibida por CO. O citocromo P-450 induzido por tratamento com 3-MC era funcionalmente ativo. A atividade azo-redutásica foi inibida especificamente pelo tratamento oral comoDAB, possibilitando a formulação de uma hipótese sobre a ação carcinogênica deste composto. Uma série de resultados mostra que a azo-redutase estudada é altamente específica podendo ser inibida por KCN e por mersalil. Face aos fatos expostos acima, as perspectivas do sistema parecem muito interessantes. A possibilidade do uso de novos métodos de fracionamento dos microssomos pode levar à resolução do sistema da azo-redutase. As semelhanças com a dessaturação de ácidos graxos, aliadas às indicações da existência de um novo tipo de citocromo P-450 que se combina com KCN, permitem descortinar maior amplitude para os limites do sistema da azo-redutase. Enfim, trata-se de uma reação de redução de um composto exógeno, com características até agora não relatadas, envolvendo processos biológicos de importância e cujo estudo terá prosseguimento. / Not available