<p>This thesis describes the design and synthesis of compounds that are</p><p>intended to inhibit serine and aspartic proteases. The first part of the text deals with preparation of inhibitors of the hepatitis C virus (HCV) NS3 serine protease. Hepatitis C is predominantly a chronic disease that afflicts about 170 million people worldwide. The NS3 protease, encoded by HCV, is essential for replication of the virus and has become one of the main targets when developing drugs to fight HCV. The inhibitors discussed here constitute surrogates for the widely used <em>N</em>-acyl-hydroxyproline isostere designated 4-hydroxy-cyclopentene. The stereochemistry of the 4-hydroxy-cyclopentene scaffold was determined by nuclear overhauser effect spectroscopy (NOESY) and the regiochemistry by heteronuclear multiple bond correlation (HMBC). The scaffold was decorated with different substituents to obtain both linear and macrocyclic HCV NS3 protease inhibitors that display low nanomolar activity. The second part of the thesis describes the design and synthesis of potential aspartic protease inhibitors. The hydroxyethylene motif was used as a noncleavable transition state isostere. The synthetic route yielded a pivotal intermediate with excellent stereochemical control, which was corroborated by NOESY experiments. This intermediate can be diversified with different substituents to furnish novel aspartic protease inhibitors.</p> / Report code: LIU-TEK-LIC-2006:45

Structure-based drug design and combinatorial chemistry play important roles in the search for new drugs, and both these elements of medicinal chemistry were included in the present studies. This thesis outlines the synthesis of protease inhibitors against thrombin and the HCV NS3 protease, as well as the synthesis of a combinatorial library using solid phase chemistry.In the current work potent thrombin inhibitors were generated based on the D-Phe-Pro-Arg motif incorporating cyclopentene and cyclohexene scaffolds that were synthesized by ring-closing metathesis chemistry. A structure-activity relationship study was carried out using the crystallographic results for one of the inhibitors co-crystallized with thrombin. HCV NS3 protease inhibitors comprising the proline bioisostere 4-hydroxy-cyclopent-2-ene-1,2-dicarbboxylic acid were synthesized displaying low nanomolar activity. The stereochemistry and regiochemistry of the scaffolds were determined by NOESY and HMBC spectra, respectively. The final diastereomeric target compounds were isolated and annotated by applying TOCSY and ROESY NMR experiments. Furthermore, a 4-phenyl-2-carboxypiperazine targeted combinatorial chemistry library was synthesized to be used early in the lead discovery phase. This was done using a scaffold that was synthesized by palladiumcatalyzed aromatic amination chemistry and subsequently derivatized with eight electrophiles and ten nucleophiles.

Hepatitis C is a global health problem that affects approximately 120–180 million people. This viral infection causes serious liver diseases and the therapy available suffers from low efficiency and severe side effects. Consequently, there is a huge unmet medical need for new therapeutic agents to combat the hepatitis C virus (HCV). Inhibition of the viral NS3 protease has recently emerged as a promising approach to defeat this infection, and the first HCV NS3 protease inhibitors have now entered clinical trials. In this project, several novel HCV NS3 protease inhibitors have been designed, synthesized and biochemically evaluated. Inhibitors with various P1 C-terminal functional groups intended as potential bioisosteres to the carboxylic acid found in product-based inhibitors have been revealed. Special focus has been placed on establishing structure–activity relationships of inhibitors containing the promising P1 C-terminal acyl sulfonamide group. The properties of the acyl sulfonamide functionality that are important for producing potent inhibitors have been identified. In addition, the advantages of the acyl sulfonamide group compared to the carboxylic acid have been demonstrated in both enzymatic and cell-based assays. Besides the acyl sulfonamide functionality, the acyl cyanamide and the acyl sulfinamide groups have been identified as new carboxylic acid bioisosteres in HCV NS3 protease inhibitors. The synthetic work included the development of a fast and convenient methodology for the preparation of aryl acyl sulfonamides. The use of microwave heating and Mo(CO)6 as a solid carbon monoxide source provided aryl acyl sulfonamides from aryl halides in excellent yields. This method was subsequently used in the decoration of novel HCV NS3 protease inhibitors comprising a non-natural P1 moiety. This new class of compounds can be used as lead structures in a future optimization process aimed at producing more drug-like HCV NS3 protease inhibitors.

The hepatitis C virus (HCV), with a global prevalence of roughly 2%, is among the most serious diseases today. Among the more promising HCV targets is the NS3 protease, for which several drug candidates have entered clinical trials. In this work, computational methods have been developed and applied to the design of inhibitors of the HCV NS3 protease. Comparative molecular field analysis (CoMFA) modeling and molecular docking are the two main computational tools used in this work. CoMFA is currently the most widely used 3D-QSAR method. Methodology for improving its predictive performance by evaluating 6120 combinations of non-default parameters has been developed. This methodology was tested on 9 data sets for various targets and found to consistently provide models of enhanced predictive accuracy. Validation was performed using q2, r2pred and response variable randomization. Molecular docking was used to develop SARs in two series of inhibitors of the HCV NS3 protease. In the first series, preliminary investigations indicated that replacement of P2 proline with phenylglycine would improve potency. Docking suggested that phenylglycine-based inhibitors may participate in two additional interactions but that the larger, more flexible phenylglycine group may result in worse ligand fit, explaining the loss in potency. In the second series, β-amino acids were explored as α-amino acid substitutes. Although β-amino acid substitution may reduce the negative attributes of peptide-like compounds, this study showed that β-amino acid substitution resulted in reduced potency. The P3 position was least sensitive to substitution and the study highlighted the importance of interactions in the oxyanion hole. Finally, docking was used to provide the conformations and alignment necessary for a CoMFA model. This CoMFA model, derived using default settings, had q2 = 0.31 and r2pred = 0.56. Application of the optimization methodology provided a more predictive model with q2 = 0.48 and r2pred = 0.68.

Since the first approved hepatitis C virus (HCV) NS3 protease inhibitors in 2011, numerous direct acting antivirals (DAAs) have reached late stages of clinical trials. Today, several combination therapies, based on different DAAs, with or without the need of pegylated interferon-α injection, are available for chronic HCV infections. The chemical foundation of the approved and late-stage HCV NS3 protease inhibitors is markedly similar. This could partly explain the cross-resistance that have emerged under the pressure of NS3 protease inhibitors. The first-generation NS3 protease inhibitors were developed to efficiently inhibit genotype 1 of the virus and were less potent against other genotypes. The main focus in this thesis was to design and synthesize a new class of 2(1H)-pyrazinone based HCV NS3 protease inhibitors, structurally dissimilar to the inhibitors evaluated in clinical trials or approved, potentially with a unique resistance profile and with a broad genotypic coverage. Successive modifications were performed around the pyrazinone core structure to clarify the structure-activity relationship; a P3 urea capping group was found valuable for inhibitory potency, as were elongated R6 residues possibly directed towards the S2 pocket. Dissimilar to previously developed inhibitors, the P1’ aryl acyl sulfonamide was not essential for inhibition as shown by equally good inhibitory potency for P1’ truncated inhibitors. In vitro pharmacokinetic (PK) evaluations disclosed a marked influence from the R6 moiety on the overall drug-properties and biochemical evaluation of the inhibitors against drug resistant enzyme variants showed retained inhibitory potency as compared to the wild-type enzyme. Initial evaluation against genotype 3a displayed micro-molar potencies. Lead optimization, with respect to improved PK properties, were also performed on an advanced class of HCV NS3 protease inhibitors, containing a P2 quinazoline substituent in combination with a macro-cyclic proline urea scaffold with nano-molar cell based activities. Moreover, an efficient Pd-catalyzed C-N urea arylation protocol, enabling high yielding introductions of advanced urea substituents to the C3 position of the pyrazinone, and a Pd-catalyzed carbonylation procedure, to obtain acyl sulfinamides, were developed. These methods can be generally applicable in the synthesis of bioactive compounds containing peptidomimetic scaffolds and carboxylic acid bioisosteres.

Le virus de l'hépatite C (VHC) est une cause majeure d'affection hépatique chronique, notamment la cirrhose et le cancer du foie. On estime que 170 millions de personnes dans le monde sont des porteurs chroniques du VHC et que 3 à 4 millions de personnes sont infectées chaque année. Un des handicaps majeurs de la recherche sur le VHC est l'absence de systèmes de culture in vitro efficaces et de modèles animaux. Nous avons ainsi choisi une approche recombinante pour l'étude de protéines E1, E2 et p7 du VHC.Les protéines E1, E2 et p7 qui sont impliquées dans des étapes essentielles du cycle viral sont des protéines membranaires. Cependant, l'expression recombinante de cette classe de protéine est extrêmement complexe. En effet, la surexpression des protéines membranaires est souvent toxique pour les cellules hôtes. Ce phénomène est provoqué par l'agrégation ou la dégradation des protéines dans le cytoplasme dû à un manque de membrane disponible pour assurer leur intégration sur la cellule hôte. De plus, la surexpression de protéines membranaires induit la saturation de la machinerie cellulaire liée aux protéines membranaires. Ce détournement empêche le déroulement d'un cycle cellulaire normal et est ainsi fatal pour la cellule hôte. La forte concentration de protéines membranaires ou encore le fait que celles-ci soient hétérologues peut également provoquer la déstabilisation de la membrane de la cellule hôte et de son homéostasie. Afin de nous affranchir de ces limitations, nous avons utilisé une méthode de production des protéines membranaires sous forme native par un système acellulaire en présence de liposomes ; une technologie brevetée par l'université Joseph Fourier et exploitée par la société Synthelis. Dans un premier temps, nous avons procédé à la mise en place du système de production exploitant un lysat bactérien d'E. coli et d'un mélange énergétique complémentaire. Nous avons ensuite utilisé ce system pour étudier la viroporine p7. Cette protéine est essentielle pour la production de particules virales infectieuses et est impliquée dans l'assemblage viral ce qui en fait une cible thérapeutique intéressante. La production de protéoliposomes p7 en grande quantité nous a permis la caractérisation de la protéine par des techniques biochimiques et biophysiques. Nous avons mis en évidence l'inhibition de l'oligomérisation de p7 par le HMA qui ainsi inhibe sa fonction canal ionique. Grâce à la flexibilité du système d'expression acellulaire nous avons caractérisé la structure de la viroporine dans la membrane par réflectivité de neutron et avons confirmé la forme en entonnoir du complexe protéique. Des résultats préliminaires sur les proéoliposomes E1E2 quant à eux permettent d'espérer la production prochaine de particules virales mimant le VHC afin de mieux l'étudier et de lutter contre cette épidémie.L'ensemble de ces résultats confirment la pertinence de l'expression de protéines membranaires sous formes natives en système acellulaire en présence de liposomes. Les protéoliposomes produits constituent des nouveaux outils pour l'étude du VHC et permettent d'envisager de très grandes applications thérapeutiques ainsi que le développement de biomédicaments basés sur l'utilisation de protéines membranaires recombinantes. / The Hepatitis C virus (HCV) is a major cause of chronic liver disease, including cirrhosis and liver cancer. An estimated 170 million people worldwide are chronically infected with HCV and 3 to 4 million people are infected each year. One of the major handicaps of the HCV research is the lack of effective in vitro culture systems and animal models. To adress this issue, we chose a recombinant approach to study the E1, E2 and p7 proteins of HCV.The E1, E2 and p7 proteins are involved in critical steps of the viral cycle. They are membrane proteins, a class of protein that is extremely complex to express. Indeed, overexpression of membrane proteins is often toxic to the host cells. This phenomenon is caused by protein aggregation or degradation in the cytoplasm due to a lack of available membrane space for their integration into the host cell. Moreover, overexpression of membrane proteins induces saturation of the cellular machinery linked to membrane proteins. This diversion prevents the flow of a normal cell cycle and is fatal to the host cell. Destabilization of the host cell's membrane and its homeostatis may also be caused by the high concentration of membrane proteins or their heterologous nature. To circumvent these limitations, we used a method for producing membrane proteins in their native form by a cell-free system in the presence of liposomes; a technology patented by the University Joseph Fourier and licenced by the startup company Synthelis. First, we have set up the cell-free production system using a bacterial lysate from E. coli and a complementary energy mix. We then used this system to study the p7 viroporine. This protein is essential for the production of infectious virus particles and is involved in viral assembly making it an attractive therapeutic target. The production of a large quantity of p7 proteoliposomes allowed us to characterize the protein by biochemical and biophysical techniques. We have demonstrated the inhibition of oligomerization of p7 by HMA, which thereby inhibits its ion channel function. Thanks to the flexibility of the cell-free expression system we have characterized the structure of the viroporine within the membrane in a neutron reflectivity assay and have confirmed the funnel shape of the protein complex. Preliminary results on proteoliposomes E1E2 offer hope for the production viral particles mimicking the hepatitis C virus in order to better study the virus and fight against this epidemic.Together, these results confirm the suitability of the expression of membrane proteins in native forms using a cell-free system in the presence of liposomes. Proteoliposomes products are a new tool for the study of HCV and consideration for very broad therapeutic applications and the development of biopharmaceuticals based on the use of recombinant membrane proteins.

Le virus de l’hépatite C (VHC) est une des causes majeures de carcinome hépatocellulaire (CHC), dont les traitements ont une efficacité modéré. La transplantation hépatique (TH) est l’option thérapeutique de choix mais est limitée, chez les patients chroniquement infectés par le VHC, par une réinfection systématique du greffon. Nous proposons d’utiliser des lymphocytes génétiquement modifiés (LGM) issus de donneurs sains qui ont fait leurs preuves pour le traitement d’hémopathies malignes et qui,exprimant un gène suicide, peuvent être éliminés en cas d’effets secondaires. Nous montrons maintenant que ces LGM présentent d’une part une activité anti-tumorale vis-à-vis du CHC, et d’autre part un effet anti-viral envers le VHC. L’objectif est la création d’une banque de LGM allogéniques prêts-à-l’emploi, avec des avantages en termes de coût, de logistique et de disponibilité immédiate par rapport aux approches classiques d’immunothérapies, autologues pour la plupart. Parallèlement, nous avons étudié in vivo l’effet anti-viral d’un anticorps monoclonal anti-claudin-1 dirigé contre un co-récepteur duVHC et inhibant l’entrée du VHC dans les hépatocytes humains. Grâce à un modèle d’infection par VHC de souris présentant un foie chimérique humanisé, nous montrons que cet anticorps permet de prévenir efficacement l’infection par le VHC. Nos résultats apportent les preuves de concept de l’utilisation de deux produits, anticorps anti-claudin-1 et LGM, pour la prévention de la réinfection du greffon hépatique par le VHC, les LGM pouvant également être envisagés en association avec les thérapies actuelles du CHC. / The hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma (HCC),whose treatments are of limited efficacy. The liver transplantation (LT) is the optimal therapy but is limited by a very rapid and universal HCV reinfection of the liver graft. We propose to use healthy donor-derived suicide gene modified lymphocytes (GML), known to be efficient for the treatment of hematological malignancies and that can be eliminated in case of adverse events. We show now that GML have a strong anti-tumoral activity against HCC and an anti-viral effect on HCV. Our objective is to create a bank of ready for-use allogeneic GML which could have numerous advantages in terms of costs,logistics and immediate availability, as compared with autologous immunotherapies. In parallel, we have studied the in vivo anti-viral effect of a monoclonal antibody (mAb) directed against an HCV coreceptor, claudin-1, inhibiting HCV entry in human hepatocytes. Using a human liver-chimeric mouse model of HCV infection, we show that this mAb can efficiently prevent HCV infection in vivo. Our results provide the proofs of concept that these two products, anti-claudin-1 mAb and GML, could be used for the prevention of liver graft HCV reinfection, while GML could further be used for the treatment of HCC, in combination with current HCC therapies

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-15T16:15:07Z No. of bitstreams: 1 Sione Passini Prevalência de infecção... 2013.pdf: 2410664 bytes, checksum: 9fd8b1765a81738455f4e2fc95fd769a (MD5) / Made available in DSpace on 2013-10-15T16:15:07Z (GMT). No. of bitstreams: 1 Sione Passini Prevalência de infecção... 2013.pdf: 2410664 bytes, checksum: 9fd8b1765a81738455f4e2fc95fd769a (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A transmissão materno-infantil (TMI) do vírus da hepatite C (VHC) ocorre em 4%-13% das gestações quando a mãe é infectada, e poucos estudos de prevalência foram realizados em gestantes no Brasil. O objetivo desta pesquisa foi determinar a prevalência de infecção e fatores associados à presença de infecção pelo VHC em gestantes, e determinar a taxa de TMI do VHC. O estudo foi realizado em Salvador, no período entre Maio de 2009 e Abril de 2011, na Maternidade Referência Professor José Maria Magalhães Netto. Todas as voluntárias que assinaram ao TCLE, tiveram seus prontuários revisados e tiveram amostra de soro coletada para a realização do teste rápido anti-VHC (Bioeasy), ELISA 3ª geração (Artech), e detecção do VHC-RNA Qualitativo e Quantitativo (AMPLICOR HCV TESTE, ROCHE versão 2.0). Em uma subamostra de gestantes selecionada aleatoriamente, realizouse entrevista para analisar fatores sócio-demográficos e de exposição ao VHC não disponíveis nos prontuários. A análise estatística descritiva e de associação foram realizadas utilizando o programa EPI Info 3.5.3 (CDC, Atlanta, GE, EUA). Foram incluídas no estudo 3.049 gestantes, sendo que 8 (0,26%; IC 95%: 0,12%-0,50%)foram soropositivos para o anti-VHC pelo ELISA e 6 (0,20%; IC 95%: 0,08%-0,41%) confirmaram viremia. Todos os VHC genotipados pertenceram ao genótipo 1, 1a ou 1b. Os principais fatores associados à infecção materna pelo VHC foram uso de drogas, tatuagem, e infecção por HIV (p < 0,05). Das 6 gestantes com HCV positivo, 2 (33,3%; IC 95%: 6,0%-73,8%) transmitiram o HCV para seus filhos. Em um dos casos de TMI, a mãe relatou ter feito uso de drogas injetáveis e inaláveis durante a gestação, possuía tatuagem e era portadora do HIV sob tratamento antiretroviral. O recém-nascido (RN) nasceu com idade gestacional (IG) de 38 semanas através de parto cesariano eletivo. No outro caso, a mãe relatou uso de drogas injetáveis e inaláveis, possuía piercing e tatuagem, mas não foi portadora de HIV. O RN nasceu com IG de 39 semanas, através de parto normal, com tempo de ruptura da bolsa de 15 horas. Concluímos que a prevalência de infecção pelo VHC entre gestantes é baixa quando comparada à da população em geral na mesma localidade, sendo associado a uso de drogas e tatuagem. A taxa de TMI do VHC encontrada foi maior do que o esperado. Assim, estudos mais amplos serão necessários. / The mother-to-child transmission (MTCT) of hepatitis C virus (HCV) occurs in 4%- 13% of pregnancies when the mothers are infected and very few studies were conducted to determine the HCV prevalence in pregnant women in Brazil. The aim of this work was to determine the prevalence and associated factors of HCV infection in pregnant women, and the rate of the MTCT of HCV. It was conducted in Salvador, between March 2009 and April 2011, at the maternity hospital Prof. José Maria Magalhães Netto. All volunteers signed to informed consent, their medical records were reviewed and serum samples were collect for screening anti-HCV antibody by a rapid anti-HCV testing (Bioeasy), ELISA 3rd generation (Artech), and HCV-RNA qualitative and quantitative detection (AMPLICOR HCV TEST, ROCHE version 2.0). In a randomly selected pregnant women subsample, interviews were made to evaluate socio-demographic and exposure factors for HCV, not available in the records. The descriptive and association statistical analysis were performed using Epi Info 3.5.3 (CDC, Atlanta, GE, USA). The study included 3.049 pregnant women, 8 (0.26%; 95% CI: 0.12%-0.50%) were anti-HCV positive by ELISA and 6 (0.20%; 95% CI: 0.08%-0.41%) confirmed viremia. All genotypes HCV belonged to genotype 1, 1a or 1b. Injection and inhaled drug use, tattoo and piercing were the most important associated factors for the mother infection. Out of the 6 HCV infected mothers, 2 (33.3%; 95% CI: 6.0%-73.8%) transmitted HCV to their newborns. In one of the cases of MTCT, the mother reported to injection and inhaled drug user during pregnancy, had tattoo and was HIV carrier held under antiretroviral therapy. The newborn was born at gestational age (GA) 38 weeks by elective cesarean section. In the other case, the mother was also injection and inhaled drug user, had piercing and tattooing. Her newborn was born at GA 39 weeks by normal delivery, and time of rupture of membranes was estimated to be 15 hours. We conclude that the prevalence of HCV infection among pregnant women was lower than the prevalence in the general population in the same locality, been associated to drug use and tattoo. The rate of MTCT of HCV was higher than expected. Thus, larger studies are required.

Contexto: A suplementação de ácido fólico (AF) melhora a função endotelial de indivíduos infectados pelo HIV em uso contínuo de terapia antirretroviral (TARV). A literatura não demonstra com clareza esse benefício em indivíduos coinfectados HIV-HCV. Introdução: Indivíduos infectados pelo HIV ou em coinfecção pelo HIV-HCV apresentam um conjunto de fatores de risco que podem levar a disfunção endotelial. Estudos demonstram que a administração de AF possui efeitos benéficos sobre a função endotelial de diferentes populações com risco cardiovascular, inclusive em HIV monoinfectados. Objetivo: Determinar o efeito da suplementação de AF por quatro semanas sobre a dilatação mediada (FMD) pelo fluxo da artéria braquial em indivíduos infectados pelo HIV ou HIV-HCV em uso contínuo de TARV. Delineamento: Ensaio clínico randomizado (ECR), controlado por placebo. Local: Ambulatório de doenças infecciosas do Hospital Universitário de Santa Maria. População: Foram avaliados 69 indivíduos com idade entre 18-50 anos, de ambos os sexos, infectados pelo HIV com ou sem coinfecção pelo HCV, em TARV e com carga viral indetectável há mais de seis meses. Excluíram-se participantes com diabetes mellitus, infarto agudo do miocárdio, revascularização miocárdica, ou acidente vascular encefálico prévios, com creatinina >1,5 mg/dL, diagnóstico clínico, ecográfico, endoscópico ou laboratorial de cirrose hepática, em uso de estatinas, fibratos, terapia de reposição hormonal, sulfonamidas, suplementos vitamínicos, ou AF nos últimos 30 dias. Adicionalmente foram excluídas mulheres grávidas, e participantes de outro ECR. Intervenção: Indivíduos alocados para o grupo intervenção receberam AF, 5 mg, via oral, em dose única diária, pela manhã, durante quatro semanas. Os participantes alocados para grupo placebo receberam orientação para seguir a mesma posologia, sendo os comprimidos indistinguíveis em cor, aroma, sabor, forma e tamanho. Desfechos: Utilizaram-se as variações nos níveis de homocisteína, vitamina B12 e na FMD, aferida por Doppler, na artéria braquial, obtidos na randomização e ao final do seguimento. Resultados: Realizou-se o rastreamento de 239 participantes, sendo 72 elegíveis, 69 randomizados e 68 acompanhados, entre outubro de 2012 e julho de 2013. Em indivíduos infectados pelo HIV houve aumento significativo nos níveis de AF (12,8 ng/ml) no grupo intervenção em comparação ao placebo (P<0,001), acompanhada de variação negativa de homocisteína (-1,9 umol/l) no grupo intervenção e aumento mínimo no grupo placebo (P<0,001). Não houve variação significativa na FMD (P=0,9). Entre indivíduos coinfectados por HIV-HCV, a variação nos níveis de AF foi decorrente da elevação no grupo intervenção (12,6 ng/ml) e redução no grupo placebo (-0,7 ng/ml) (P<0,001). Os níveis de homocisteína aumentaram no grupo placebo (4,6 umol/l) e diminuíram no grupo intervenção (-1,0 umol/l) (P<0,0003). Em relação ao FMD, houve tendência à redução percentual no grupo intervenção e aumento no grupo placebo (P=0,007). As variações de vitamina B12 não foram significativas, independente do status de infecção para HCV. Conclusão: Esse estudo demonstrou que a suplementação de AF por um curto período de tempo, esteve associada com redução de homocisteína sérica, mas não modificou a FMD da artéria braquial, aferida por Doppler, em indivíduos adultos infectados pelo HIV ou HIV-HCV em uso de TARV. / Context: The supplementation of folic acid (FA) improves endothelial function in HIV-infected individuals in continuous use of highly active antiretroviral therapy (HAART). The literature does not clearly show this benefit in coinfected HIV-HCV. Introduction: Individuals infected with HIV or HIV-HCV coinfected presents a set of risk factors that can lead to endothelial dysfunction. Studies show that FA management has beneficial effects on endothelial function in different populations with cardiovascular risk, including HIV monoinfected. Objective: To determine the effect of FA supplementation for four weeks on the mediated dilation (FMD) by brachial artery flow in patients infected with HIV or HIV-HCV continuous HAART. Design: Randomized clinical trial (RCT), placebo-controlled study. Location: Division of Infectious Diseases, University Hospital of Santa Maria. Population: A total of 69 subjects aged 18-50 years, of both sex, HIV or HIV-HCV infected, on HAART, with undetectable viral load for more than six months. Patients presenting with diabetes mellitus, acute myocardial infarction, coronary revascularization, or stroke prior, with creatinine >1,5 mg/dL, clinical diagnosis, ultrasound, endoscopic or laboratory evidence of liver cirrhosis, on statins, fibrates, hormone replacement therapy, sulfonamides, vitamin supplements, or FA in the last 30 days. In addition pregnant women were excluded, and participants in a RCT. Intervention: Subjects allocated to the intervention group received FA, 5 mg orally once daily in the morning for four weeks. Participants allocated to placebo group were instructed to follow the same dosage, being indistinguishable tablets in color, aroma, taste, shape and size. Outcomes: changes were used in the levels of homocysteine, vitamin B12 and FMD, measured by Doppler, in the brachial artery obtained at randomization and at the end of follow-up. Results: We carried out the screening of 239 participants, 72 eligible, 69 randomized and 68 accompanied, between October 2012 and July 2013. In HIV-infected patients there was a significant increase in the levels of FA (12.8 ng/ml) in the intervention group compared to placebo (P <0.001), accompanied by negative variation of homocysteine (1.9 umol/L) in the group intervention and minimal increase in the placebo group (P <0.001). There was no significant change in FMD (P = 0.9). Between individuals coinfected with HIV-HCV, the variation in FA levels was due to the increase in the intervention group (12.6 ng/ml) and reduction in the placebo group (-0.7 ng / ml) (P <0.001). Homocysteine levels increased in the placebo group (4.6 umol/L) and decreased in the intervention group (-1.0 umol/L) (P <0.0003). Regarding the FMD, there was a tendency to percentage reduction in the intervention group and increased in the placebo group (P = 0.007). Variations of B12 were not significant, independent of HCV infection status. Conclusion: This study showed that AF supplementation for a short term, was associated with reduced serum homocysteine, but did not change the FMD of the brachial artery, measured by Doppler in adults infected with HIV or HIV-HCV in HAART.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Aproximadamente 20% dos pacientes infectados pelo vírus da hepatite C (HCV) evoluem com clareamento viral espontâneo [3]. Variáveis como etnia e forma de contaminação tornam ainda mais difícil entender os fatores genéticos do hospedeiro que influenciam a evolução da hepatite C. Objetivo: Avaliar a relação entre o perfil de HLA e o clareamento espontâneo da infecção pelo vírus da hepatite C na população brasileira. Métodos: Foi realizada genotipagem dos HLAs -A, -B, -Cw, -DRB1 e -DQB1 em uma amostra de 135 pacientes infectados pelo HCV, sendo 45 pacientes com clareamento viral espontâneo (casos) e 90 pacientes com infecção crônica (controles). Os controles foram pareados por sexo, etnia (branco e não branco) e forma provável de contaminação (alta dose infectante ou baixa dose infectante). Foi realizada correção do p (pc) para múltiplas comparações usando a correção de Bonferroni. Resultados: quando analisamos os doentes em geral, nenhum alelo obteve significância estatística após a correção do p. Entretanto, uma nova associação protetora do HLA-DQB1*04 (p= 0,006; pc= 0,030) e uma associação protetora raramente descrita do HLA-DRB1*08 (p= 0,004; pc= 0,048) foram encontradas entre pacientes brancos, mas não entre não brancos. O alelo DRB1*11, previamente relatado em populações homogêneas, foi associado ao clareamento do HCV (p= 0,020) apenas entre pacientes com forma de contaminação provável por alta dose infectante. Conclusão: As bases imunogenéticas do clareamento do HCV diferem entre os grupos étnicos e as vias de contaminação. Mesmo em uma população etnicamente complexa como a brasileira é importante a realização de uma classificação étnica para parear os pacientes e para permitir uma análise estratificada para identificar diferentes associações para cada etnia. / Approximately 20% of Hepatitis C virus (HCV) infected individuals clear the virus [3]. The knowledge of host factors that influence the course of HCV infection is still limited. Factors such as ethnicity and route of infection make even more difficult to understand which host-dependent genetic factors influence the hepatitis C outcome. Aims: To investigate whether HLA alleles are associated with clearance of HCV infection in a highly admixed Brazilian population. Methods: HLA-A, -B, -Cw, -DRB1 and DQB1 genotyping were performed in a Brazilian cohort of 135 HCV infected subjects among which 45 cleared HCV infection (cases) and 90 had persistent viral infection (controls). Controls were matched by sex, ethnicity (withes and non whites) and expected route of infection (high infectious dose or low infectious dose). P-values were corrected for multiple comparisons using Bonferronis correction. Results: We didnt identify any significant association between HLA alleles and the outcome of HCV infection of all ethnic population. However, a new protective association of HLA-DQB1*04 (p= 0,006; pc= 0,030) and a rarely described protective association of HLA-DRB1*08 (p= 0,004; pc= 0,048) were found only among whites, but not among non whites patients. The DRB1*11 allele, previously reported in homogeneous population, was associated with HCV clearance (p= 0,020) only among patients with expected high-dose exposure. Conclusion: We conclude that the immunogenetic basis for HCV clearance differs between ethnic groups and route of infection. Even in an ethnically complex population it is important to establish an ethnic classification to match patients and homogenize groups and to allow stratified analysis to identify different associations for each ethnicity.